WO2019094447A2 - Production d'acides aminés de type mycosporine dans des cyanobactéries - Google Patents

Production d'acides aminés de type mycosporine dans des cyanobactéries Download PDF

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WO2019094447A2
WO2019094447A2 PCT/US2018/059595 US2018059595W WO2019094447A2 WO 2019094447 A2 WO2019094447 A2 WO 2019094447A2 US 2018059595 W US2018059595 W US 2018059595W WO 2019094447 A2 WO2019094447 A2 WO 2019094447A2
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seq
enzyme
genetically modified
mycosporine
cell
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Frank ULICZKA-OPITZ
Marco SCHOTTKOWSKI
Ulf Duhring
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Algenol Biotech LLC
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    • C12N9/10—Transferases (2.)
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Definitions

  • This application contains a sequence listing submitted by EFS-Web, thereby satisfying the requirements of 37 C.F.R. ⁇ 1.821-1.825.
  • the present invention relates generally to the recombinant production of mycosporine-like amino acids (MAAs) in cyanobacterial host cells.
  • MAAs have UV protective ability and can be used as natural skin protectants, such as sunscreens.
  • Cyanobacteria can be utilized as bio-factories to produce compounds of interest from sunlight, CO2, and nutrients.
  • the transformation of the cyanobacterial genus Synechococcus with genes of interest has been described (U.S. Patent Nos. 6,699,696 and 6,306,639, both to Woods et al.).
  • the transformation of the cyanobacterial genus Synechocystis has been described, for example, in PCT/EP2009/000892 and in PCT/EP2009/060526.
  • the transformation of the cyanobacterial genus Cyanobacterium sp. has been described (U.S. Patent No. 8,846,369, U.S. Patent No. 9,315,832, and PCT/US2013/077364).
  • UV-A is regarded as the main cause of skin aging and wrinkling of human skin. Since both UV-A and UV-B are harmful, protection for both kinds of rays are recommended.
  • the FDA has approved a list of active chemical/physical ingredients for use in sunscreens which absorb into the top layers of skin. For some people, however, the ingredients can lead to skin irritation, allergic reactions and even skin damage or aging.
  • sunscreen component oxybenzone has recently been found to contribute to damage to coral reefs by bleaching of corals and disruption of coral reproduction and growth (Downs et al., "Toxicopathological Effects of the Sunscreen UV Filter, Oxybenzone (Benzophenone-3), on Coral Planulae and Cultured Primary Cells and Its Environmental Contamination in Hawaii and the U.S. Virgin Islands", Arch Environ Contam Toxicol., 70:265-88 (2016).
  • Mycosporine-like amino acids are natural, water-soluble, carbonaceous, nitrogenous compounds that absorb light in the UV-A/UV-B range between 310 and 362 nm.
  • MAAs are naturally produced by a number of organisms, including certain algae species, cyanobacteria, dinoflagellates and corals. MAAs apparently act as natural sunscreens in these organisms. In addition to their ability to act as a sunscreen, MAAs also have antioxidant activity (Wada et al. "Mycosporine-Like Amino Acids and Their Derivatives as Natural Antioxidants” Antioxidants 4:603-646 (2015)).
  • MAAs such as mycosporine-glycine, shinorine, porphyra-334, mycosporine-2-glycine, palythine, asterina, mycosporine-glutamic acid-glycine, mycosporine-ornithine, usujirene, and palythene (for a review, see Carreto et al., "Mycosporine- Like Amino Acids: Relevant Secondary Metabolites", Chemical and Ecological Aspects Mar Drugs, 9:387-446 (2011)).
  • MAA-based bio-sunscreen compounds are exclusively sourced from marine macroalgae, such as Porphyra umbilicalis, containing MAAs such as porphyra-334 and shinorine. Purified MAAs are not currently commercially available, but extracts from the red alga Porphyra umbilicalis are sold and used in cosmetic products (Helioguard® and Helionori®).
  • the Porphyra extracts are reported to contain the MAA porphyra-334 and shinorine with absorption coefficients ( ⁇ molar) of 42'300 and 44'700 at 334 nm.
  • concentration of the MAAs in the extract is low: in the range of 1 % of total dry weight (Hartmann et al. "Quantitative analysis of mycosporine- like amino acids in marine algae by capillary electrophoresis with diode-array detection", Jour. Pharm. Biomed. Analysis, 138: 153-157 (2017)).
  • cyanobacteria naturally produce lower amounts of MAAs (0.03 to 0.98 mg MAA/g dry cell weight) compared to P. umbilicalis (10 mg/g dry cell weight), complicating the industrial benefit of cyanobacteria (Garcia-Pichel et al., "The phylogeny of unicellular, extremely halotolerant cyanobacteria", Archives of Microbiology, 169:469-482 (1998)).
  • cyanobacterial host cells Provided herein are modified cyanobacterial host cells, plasmid constructs, and methods to produce high levels of various MAAs from cyanobacterial cultures.
  • a genetically modified cyanobacterial cell having at least one heterologous gene that encodes an enzyme involved in the production of a mycosporine-like amino acid is provided.
  • the MAA can be selected, for example, from the group consisting of mycosporine-glycine, shinorine, mycosporine-2-glycine, 4-deoxygadusol, usujirene, porphyra- 334, asterina, palythene, palythine, my cosporine-2-(4-deoxygadusolyl-orni thine), mycosporine- ornithine, mycosporine-lysine, mycosporine-alanine, gadusol, 4-deoxygadusol, mycosporine- taurine, palythine-threonine, palythine-serine, mycosporine-methylamine-glycine, mycosporine- methylamine-threonine, my
  • the enzyme involved in the production of an MAA can be selected from the group consisting of 4-deoxygadusol (DDG) synthase, O- methyltransferase, ATP-grasp family protein, D-Ala-D-Ala-ligase, nonribosomal peptide synthetase ( RPS), clavaminic acid-like synthetase and 3-hydroxyisobutyrate-like dehydrogenase.
  • DDG 4-deoxygadusol
  • O- methyltransferase O- methyltransferase
  • ATP-grasp family protein ATP-grasp family protein
  • D-Ala-D-Ala-ligase nonribosomal peptide synthetase
  • RPS nonribosomal peptide synthetase
  • clavaminic acid-like synthetase 3-hydroxyisobutyrate-like dehydrogenase.
  • more than one MAA can be produced by said cyanobacterial cell.
  • the cell can be, for example, a non-filamentous cyanobacterial cell.
  • the cyanobacterial cell can be grown photoautotrophically or photomixotrophically.
  • the heterologous gene can be from an organism such as, for example, cyanobacteria, bacteria, fungi, dinoflagellates, cnidaria, or algae.
  • the enzyme involved in the production of an MAA has at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, and SEQ ID NO: 164 and can be codon optimized for optimal expression in the cyanobacterial cell.
  • the genetically modified cyanobacterial cell comprises at least one heterologous gene that encodes an enzyme involved in the production of an MAA having at least 70%, preferably at least 80%, most preferably at least 90% identity to at least one amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15 , SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162 , SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, and SEQ ID NO: 170.
  • These enzymes are from the mysABCD gene combinations from Anabaena/Nasfoc and Anabaena/Aphanaothece, the mysABCDl/D2 gene set from Trichodesmium, and the mysABCDl/D2 gene set from Cyanobacterium HL-69 for the production of any of the compounds mycosporine-glycine, mycosporine-2-glycine, shinorine, porhyra-344, palythine.
  • the genetically modified cyanobacterial cell for the production of mycosporine-glycine comprises at least three heterologous genes, wherein the first heterologous gene codes for a first enzyme, which is at least 70%), preferably at least 80%, most preferably at least 90% identical to either one of the SEQ ID NO: 3, SEQ ID NO: 158, or to SEQ ID NO: 164, the second heterologous gene codes for a second enzyme, which is at least 70%, preferably at least 80%, most preferably at least 90% identical to either one of the SEQ ID NO: 6, SEQ ID NO: 160, or SEQ ID NO: 166, the third heterologous gene codes for a third enzyme, which is at least 70%, preferably at least 80%, most preferably at least 90% identical to either one of the SEQ ID NO: 9, SEQ ID NO: 162, or SEQ ID NO: 168.
  • the first heterologous gene codes for a first enzyme, which is at least 70%), preferably at least 80%, most preferably at least 90% identical to either one of the
  • the first enzyme can therefore be selected from the mysA genes from Anabaena, Trichodesmium and Cyanobacterium HL 69
  • the second enzyme can be selected from the mysB genes from Anabaena, Trichodesmium and Cyanobacterium HL 69
  • the third enzyme can be selected from the mysC genes from Anabaena, Trichodesmium and Cyanobacterium HL 69.
  • These three mysABC genes code for enzymes, which produce mycosporine-glycine (see for example Fig. 1).
  • a further embodiment of the invention is directed to a genetically modified cyanobacterial for the production of mycosporine-2-glycine, additionally including a fourth heterologous gene coding for a fourth enzyme which is at least 70%, preferably at least 80%>, most preferably at least 90% identical to SEQ ID NO: 15 or 93.
  • This forth enzyme can therefore be selected from the enzyme for MAA production encoded by the gene Ap3855 or the Enzyme for MAA production encoded by the gene Tery_2970 [Trichodesmium erythraeum IMS101] (see also Fig. 1 and 40).
  • Another inventive embodiment is directed to a genetically modified cyanobacterial cell for the production of shinorine/porphyra-334 and mycosporine-glycine-alanine, additionally including a fourth heterologous gene coding for a fourth enzyme which is at least 70%, preferably at least 80%, most preferably at least 90% identical to either one of the SEQ ID NO: 12, 15, 85, 93, 95, 170, 172 or 174.
  • the forth enzyme is therefore selected from at least one enzyme out of a group consisting of the enzyme for MAA production encoded by the gene NpF5597, an enzyme for MAA production encoded by the gene Ap3855, an enzyme for MAA production encoded by the gene MysD [Nostoc commune var.
  • flagelliforme QSY 1 an enzyme for MAA production encoded by the gene Tery_2971 [Trichodesmium erythraeum IMS 101], an enzyme for MAA production encoded by the gene Tery_2970 [Trichodesmium erythraeum IMS 101], an enzyme for MAA production encoded by the gene mysD [Cyanobacterium stanieri HL-69], an enzyme for MAA production encoded by the gene mysD [Nostoc verrucosum KU005], and an enzyme for D- alanine— ligase [Actinosynnema mirum DSM 43827].
  • the promoter can be a regulatable promoter or a constitutive promoter.
  • the promoter has at least 80%> identity to a promoter selected from the group consisting of PrbcL, PntcA, PnblA, PisiA, PpetJ, PpetE, PcorT, PsmtA, PziaA, PsigB, PlrtA, PhtpG, PhspA, PclpB l, PhliB, PggpS, PpsbA2, PpsaA, PnirA, PnarB, PnrtA, PisiB, PnrsB, PmrgA, PpstS, and PcrhC, , PpsbD, PnblA, PrpoA, PrnpA, PrpsL, , PcpcB, PnirA*2, PnirA*3, PnirA*4,
  • At least one heterologous gene can be located on a modified endogenous or a heterologous extrachromosomal plasmid.
  • the heterologous genes are located on more than one modified endogenous or heterologous plasmid.
  • the least one heterologous gene can be integrated into the chromosome.
  • the genus of the cell can be selected from the group consisting of Arthrospira sp., Synechocystis, Synechococcus, Acaryochloris, Anabaena, Aphanothece, thermosynechococcus, Chamae siphon, Chroococcus, Cyanobacterium, Cyanobium, Dactylococcopsis, Euhalothece, Gloeobacter, Gloeocapsa, Gloeothece, Microcystis, Prochlorococcus, Prochloron, Chroococcidiopsis, Cyanocystis, Dermocarpella, Myxosarcina, Pleurocapsa, Stanieria, Xenococcus, Arthrospira, Borzia, Crinalium, Geitlerinema, Halospirulina, Leptolyngbya, Limnothrix, Lyngbya, Microcoleus, Cyano
  • a method of producing an MAA in a cyanobacterial cell by growing the genetically modified cyanobacterial cell as described herein in a culture medium suitable for production of the MAA in a photobioreactor with adequate light and CO2 addition; and then isolating the MAA from the cells and/or the culture medium.
  • a pharmaceutical composition or a cosmetic composition such as a UV sunscreen agent, wherein the UV sunscreen agent can be an MAA that has been produced in a genetically modified cyanobacterial cell as described herein.
  • the composition comprises at least 2 different MAAs, and further wherein the wavelength range of UV protection can be broader than with a single MAA.
  • the composition can be a skin care product or a sunscreen.
  • the UV protection can occur over at least a portion of UV-A and at least a portion of UV-B.
  • FIG. 1 is a general biosynthetic pathway for the synthesis of several MAAs in cyanobacteria.
  • FIG. 2 is a diagram of the biosynthetic pathway for the production of the MAA "mycosporine-glycine" in cyanobacteria.
  • FIG. 3 is a table showing several plasmid constructs for the production of mycosporine- glycine, mycosporine-2-glycine and shinorine, respectively, in cyanobacteria.
  • FIG. 4 is a map of the endogenous 6.8 kb plasmid isolated from Cyanobacterium sp. ABICyanol (SEQ ID NO: 16).
  • FIG. 5 A depicts a map of the 13121 bp circular plasmid construct and sequence annotation of plasmid #2737 (pABICyanol_6.8: :Porf0223*-Ava_3858(ABICyanolopt)-IScpcBA* l- Ava_3857(ABICyanolopt)-IScpcBA* l-Ava_3856(ABICyanolopt)). (SEQ ID NO: 54).
  • FIG. 5B depicts a map of the 13059 bp circular plasmid construct and sequence annotation of plasmid #2742 (pABl_6.8: :PsmtA-Ava_3858(ABICyanolopt)-IScpcBA* l- Ava_3857(ABICyanolopt)-IScpcBA* l-Ava_3856(ABICyanolopt)-TpsaB) (SEQ ID NO: 17).
  • FIG. 6 depicts a map of the 13209 bp circular plasmid construct and sequence annotation of plasmid #2752 (pAB l_6.8: :PcpcB*3-Ava_3858(ABICyanolopt)-IScpcBA* l- Ava_3857(ABICyanolopt)-IScpcBA* l-Ava_3856(ABICyanolopt)-TpsaB) (SEQ ID NO: 18)
  • FIG. 7 depicts a map of the 10040 bp circular plasmid construct and sequence annotation of plasmid #2796 (pAB lB_Rep::PrpsL-Gm**-TB0014-oriVT-PsmtA-
  • FIG. 8 depicts a map of the 10013 bp circular plasmid construct and sequence annotation of plasmid #2798 (pABlB_Rep: :PrpsL-Gm**-TB0014-oriVT-PsmtA-ap3855(ABICyanolopt)) (SEQ ID NO: 20).
  • FIG. 9 depicts a map of the 14443 bp circular plasmid construct and sequence annotation of plasmid #2840 (pABl_6.8: :PsmtA-Ava_3858(ABICyanolopt)-IScpcBA* l- Ava_3857(ABICyano 1 opt)-IScpcB A* 1 - Ava_3856(ABICyano 1 opt)-TpsaB-Porf0221 - ap3855(ABICyanolopt)) (SEQ ID NO: 21).
  • FIG. 10A is an absorbance spectrum of a culture of strain AB 1224 (ABICyanol strain harboring the plasmid #2737 (FIG. 5 A; SEQ ID NO: 54), which allows Cu-inducible expression of the MAA mycosporine-glycine), after 24 hours of growth in a GC vial.
  • Mycosporine-glycine absorbance maximum is at 310 nm.
  • FIG. 10B is an absorbance spectrum of cultures of either strain AB1225 (Zn-inducible) or AB1226 (constitutive) expression of the MAA mycosporine-glycine, after 24 hours of growth in a GC vial in comparison to the wild type control.
  • the mycosporine-glycine absorbance maximum is 310 nm.
  • the graph shows that cultures of several of the clones having the constitutive promoter had a higher absorbance at 310 than a culture of the clone that was Zn-inducible. In comparison, a culture of the wild type did not show a peak at 310 nm.
  • FIG. IOC is a line graph showing that the production of mycosporine-glycine (mg/L) made from strain #AB 1226 (constitutive expression) was stable over at least a 35 day period, resulting in a final production rate of about 290 mg/L after 40 days.
  • FIG. 10D is a line graph of the culture described in FIG. IOC, shown on a dry weight basis (mg MAA/g dry weight.)
  • FIG. 11A is a line graph showing the amount of mycosporine-glycine (mg/L) made from strain #AB1225 (ABICyanol strain transformed with plasmid #2742, which has a Zn-inducible promoter), in 1.2 L indoor photobioreactors with induction by either ZnS0 4 (at 2 ⁇ per unit OD750 increase) or Zn-EDTA (added at 20 ⁇ , 30 ⁇ and 30 ⁇ ), compared to the control with no Zn added, over a 27 day period. After 27 days, the Zn-EDTA culture was diluted and re-grown to confirm the stability of production over a 50 day period.
  • XyM in Fig. 1 1 A stands for multiple zinc feeding throughout the cultivation as follows: 20 ⁇ ZnEDTA on day 1, 13, 22 and 27; 30 ⁇ ZnEDTA day 2 and 28; 6 ⁇ ZnS04 on day 34 and 36.
  • FIG. 1 IB is a line graph showing the percentage of mycosporine-glycine from FIG. 11A that was found in the culture medium, over the same 50 day period. In general, about 80% of the mycosporine-glycine produced was excreted from the cell.
  • Fig. l lC shows the production of mycosporine-glycine by the strain AB1225 with optimized cultivation conditions.
  • the strain was cultivated as described in example 27.
  • FIG. 12A is a comparison map of MAA synthesis genes from 3 cyanobacterial species: Aphanothece halophytica, Nostoc punctiforme ATCC 29133, and Anabaena variabilis ATCC 29413.
  • FIG. 12B is a diagram showing the chemical structure of shinorine, mycosporine-glycine, and mycosporine-2-glycine, as well the genes that can be utilized to convert mycosporine-glycine into either shinorine or mycosporine-2-glycine.
  • FIG. 12C is an absorbance spectrum showing the increased production of shinorine when the additional genes NpF5597 (mysD from N. punctiforme) and Ava3855 were transformed to AB1225, using a separate plasmid, pAB lB. Also shown is the increased production of mycosporine-2-glycine when the additional gene Ap3855 (mysD from A. halophytica), on a separate plasmid, pAB lB, was transformed to the host cell in addition to the mycosporine-glycine encoding genes.
  • FIG. 13 A is a line graph showing the production (in mg/L) of several MAAs (mycosporine- glycine, shinorine, and mycosporine-2-glycine) made by the strains AB 1225, AB1252 and AB1253 over a 26 day period in 1.2 L indoor photobioreactors.
  • MAAs mycosporine- glycine, shinorine, and mycosporine-2-glycine
  • FIG. 13B is a line graph showing the production of several MAAs (mycosporine-glycine, shinorine, and mycosporine-2-glycine), as a percentage of dry weight, over a 26 day period in 1.2 L indoor photobioreactors.
  • MAAs mycosporine-glycine, shinorine, and mycosporine-2-glycine
  • FIG. 13C is a line graph from the same experiment as in FIG. 13A and FIG. 13B, showing the percentage of each MAA that was located in the culture medium over the 26 day period.
  • Fig. 13D is a line graph showing the production of shinorine by the strain AB 1252 over the course of 3 days under improved cultivation conditions as described in example 27.
  • FIG. 14 is a line graph showing the production of the MAA mycosporine-2-glycine (in mg/L) from strain #AB 1277 (ABICyanol transformed with the plasmid #2840, as shown in FIG. 3).
  • FIG. 15A is a plasmid map, sequence annotation, and complete sequence of the 11649 bp plasmid construct#2794:pABCyanolB_Rep: :P sL-Gm**-TB0014-oriVT-PsmtA-Ava_3855 (ABICyanolopt)-TB0050 (SEQ ID NO: 55).
  • FIG. 15B is a plasmid map, sequence annotation, and complete sequence of the 11657 bp plasmid construct #2880: pABICyanolB_Rep: :PrpsL-Gm**-TB0014-oriVT-PsmtA- mysAB_Pum(ABICyanolopt)-TpsaB (SEQ ID NO: 56).
  • FIG. 15C is a plasmid map, sequence annotation, and complete sequence of the 11777 bp plasmid construct #2881 : pABCyanolB_Rep: :PrpsL-Gm**-TB0014-oriVT-PsmtA- mysCD_Pum(ABICyanolopt)-TpsaB (SEQ ID NO: 57).
  • FIG. 15C is a plasmid map, sequence annotation, and complete sequence of the 11777 bp plasmid construct #2881 : pABCyanolB_Rep: :PrpsL-Gm**-TB0014-oriVT-PsmtA- mysCD_Pum(ABICyanolopt)-TpsaB (SEQ ID NO: 57).
  • FIG. 15C is a plasmid map, sequence annotation, and complete sequence of the 11777 bp plasmid construct #2881 : pABCyan
  • 15D is a plasmid map, sequence annotation, and complete sequence of the 11732 bp plasmid construct #2882: pABICyanolB_Rep: :PrpsL-Gm**-TB0014-oriVT-PsmtA- mysCD_Chc(ABICyanolopt)-TpsaB (SEQ ID NO: 58).
  • Fig. 16A shows the dry weight production rate of ABICyanol and ABCyano4 under different light intensities of 350 ⁇ m ' V 1 and of 1000 ⁇ m ' V 1 .
  • Fig. 16B shows the total carbon fixation rate of ABICyanol and ABCyano4 under different illumination conditions.
  • Fig. 17A shows the dry weight productivity and turbidostat cultivation for ABICyanol and ABCyano4.
  • Fig. 17B shows the total carbon fixation rate and turbidostat cultivation for ABICyanol and ABCyano4.
  • Fig. 18A shows a microscopic image of ink-negative stained ABCyano4 cells.
  • Fig. 18B shows a microscopic image of ink-negative stained ABICyanol cells.
  • Fig. 19A shows a comparison between the growth of two different ABCyano4 strains, AB4092 and AB4094 over a time period of cultivation of nearly 10 days.
  • Fig. 19B shows the concentration of MAAs shinorine/porphyra-334 for both ABCyano4 strains AB4092 and AB4094.
  • Fig. 19C shows the carbon-partitioning for the cultivation, shown in Fig. 19A and Fig. 19 B.
  • Fig. 20A shows a comparison of the growth of an ABICyanol and ABCyano4 mycosporine-glycine producing cell lines.
  • Fig. 20B shows the mycosporine-glycine production of ABICyanol and ABCyano4 strains.
  • Fig. 20C shows the carbon-partitioning of ABCyano4 strains in comparison to
  • Fig. 21 A depicts the growth of mycosporine-2-glycine producing cell lines of
  • Fig. 21B shows the production of mycosporine-2-glycine of ABICyanol and ABCyano4 producing strains.
  • Fig. 21C shows the carbon-partitioning for mycosporine-2-glycine producing strains of ABICyanol and ABCyano4.
  • Fig. 22A and 22B show the whole cell absorption spectrum and the HPLC profile of the ABICyanol strain AB1356. After overexpression of so far undescribed enzymes from
  • Trichodesmium erythraeum IMS101 in the mycosporine-glycine (M-Gly) producer strain AB1225 which results in the strain AB1356, a shift from 310 nm (M-Gly) to 320 nm (palythine) can be observed in the whole cell absorption spectrum.
  • FIG. 23 A and 23B show the whole cell absorption spectrum and the HPLC profile of the ABICyanol strain AB1358. This strain was produced by transformation of the M-Gly producer strain AB1225 with plasmids carrying genes for so far undescribed enzymes from
  • Trichodesmium erythraeum EVIS101 resulting in the production of mycosporine-methylamine- glycine and mycosporine-2-glycine (M-2-Gly).
  • Fig. 24A and 24B show the whole cell absorption spectrum and the HPLC profile of the ABICyanol strain AB1360. This strain was produced by transformation of the M-Gly producer strain AB1225 with plasmids carrying genes for so far undescribed enzymes from
  • Trichodesmium erythraeum FMS101 resulting in the production of Tery-364, an MAA with yet unknown structure.
  • Fig. 25A and 25B show the whole cell absorption spectrum and the HPLC profile of the ABICyanol strain AB4033. This strain was produced by transformation of the M-Gly producer strain AB1225 with the plasmid carrying the genes mylCDE from Nostoc commune var.
  • Fig. 26A, and 26B show the production of 4-deoxygadusol (4-DG) over a time period of 2 days, and the whole cell absorption spectrum of the ABCyano4 strain AB 1312. This strain was produced by transformation of ABCyano4 with genes encoding the two enzymes DHQS
  • Fig. 27 A, 27B and 27C show the production of M-Gly over a time period of 2 days, the whole cell absorption spectrum and the HPLC profile for the ABCyano4 strains AB1322 and AB1361.
  • Fig. 28A, 28B and 28C show the production of shinorine/porphyra-334 in a ratio of 60%/40% over a time period of nearly 3 days, the whole cell absorption spectrum and the HPLC profile for the ABCyano4 strain AB 1333.
  • Fig. 29A, 29B and 29C show the production of mycosporine-2-glycine over a time period of nearly 3 days, the whole cell absorption spectrum and the HPLC profile for the ABCyano4 strain AB 1334.
  • Fig. 30A, 30B and 30C show the production of shinorine/porphyra-334 in a ratio of 45%/55% over a time period of 3 days, the whole cell absorption spectrum and the FIPLC profile for the ABCyano4 strain AB4016.
  • Fig. 31 A, 3 IB and 31C show the production of shinorine/porphyra-334 in a ratio of 10%/90% over a time period of 3 days, the whole cell absorption spectrum and the FIPLC profile for the ABCyano4 strain AB4046.
  • Fig. 32A, and 32B show the whole cell absorption spectrum and the FIPLC profile for the ABCyano4 strain AB4055, producing palythine.
  • the strain overexpresses thus far undescribed enzymes from Trichodesmium erythraeum EVIS101 (Tery_2972, a Clavaminic acid synthetase (CAS)-like enzyme and a D-ala-D-ala ligase) in the genetic background of the M-Gly producer strain AB 1322.
  • Trichodesmium erythraeum EVIS101 Tery_2972, a Clavaminic acid synthetase (CAS)-like enzyme and a D-ala-D-ala ligase
  • Fig. 33 A, and 33B show the whole cell absorption spectrum and the HPLC profile for the ABCyano4 strain AB4031, producing mycosporine-methylamine:threonine.
  • the strain overexpresses a thus far undescribed enzyme from Trichodesmium erythraeum EVIS101
  • Fig. 34A, and 34B show the whole cell absorption spectrum and the HPLC profile for the ABCyano4 strain AB4074, producing Tery-322.
  • the strain overexpresses a thus far undescribed enzyme from Trichodesmium erythraeum IMS 101 in the genetic background of the M-Gly producer strain AB1322.
  • Fig. 35 A shows a comparison between the production of M-Gly by different ABCyano4 strains transformed with genes operably linked to a constitutive promoter PcpcB (strain AB 1365) and the strain AB1322 wherein the heterologous genes for MAA production are operably linked to a zinc-inducible promoter PsmtA.
  • Fig. 35B shows a comparison between the production of M-Gly by different ABCyano4 strains transformed with genes operably linked to a constitutive promoter PpsaL (strain
  • Fig. 35C shows the copper-dependent production of M-Gly by an ABCyano4 strain transformed with genes operably linked to a copper-inducible promoter PpetE (strain AB4095).
  • Fig. 36A, 36B and 36C show a comparison between the production of MAA, the accumulation of biomass and the percentage of carbon partitioning for the different ABCyano4 strains AB 1333 and the dual mysA strain AB4028 over the time period of 6 days of cultivation.
  • the dual mysA strain AB4028 contains a second mysA copy from Anabaena variabilis ATCC 29413, operably linked to a zinc-inducible promoter PsmtA.
  • Fig. 37 shows the fractionation of the MAAs between the intracellular space of the host cells, cell-associated MAAs with the capsular EPS and secreted MAAs into the culture medium for two different ABCyano4 strains AB1333 and AB 1334 over the course of 3 days producing either shinorine/porphyra-334 or mycosporine-2-glycine.
  • Fig. 38 shows the fractionation of the MAAs between cell-associated MAAs with the capsular EPS and secreted MAAs into the culture medium for the dual mysA strain AB4028 over the course of 20 days producing a mixture of shinorine and porphyra-334.
  • Fig. 39 shows an overview of the MAA biosynthesis gene cluster in Trichodesmium erythraeum IMS 101.
  • Fig. 40 depicts a proposed pathway for the synthesis of palythine, mycosporine- methylamine:threonine, mycosporine-methylamine:glycine, Tery-322 and Tery-364. with the respective heterologous genes encoding the enzymes involved in these pathways
  • Fig. 41 depicts the pathway for the synthesis of mycosporine-2-(4-deoxygadusolyl- ornithine) starting with sedoheptulose-7-phosphate.
  • Fig. 42A shows a protein sequence alignment of the MysA enzymes from different cyanobacterial species, including the consensus sequence. The first nine MysA variants
  • the divergent clade has some conserved motifs (e.g., YxxxEY(G)-xNxxET and QC(D)RPHA(G)YGHTWSP) distinct from the classical clade.
  • Fig. 42B and 42C show the production of mycosporine-glycine and the C-partitioning (M-Gly produced per dry weight) involving different mysA genes.
  • the AB1322 contains the mysABC operon from Anabaena variabilis ATCC 29413 operably linked to a zinc-inducible promoter PsmtA
  • the second strain AB4015 contains the same PsmtA-controlled mysABC operon from Anabaena variabilis ATCC 29413 plus a second mysA copy from Anabaena variabilis ATCC 29413, operably linked to a zinc-inducible promoter PsmtA.
  • the third strain contains the mysA gene from Cyanobacterium sp. HL-69 and mysBC genes from Anabaena variabilis ATCC 29413 all operably linked to a zinc-inducible promoter PsmtA.
  • Fig. 43 shows the plasmid map of one endogenous ABCyano4 plasmid, pABCyano4B, having an approximate copy number of 30-50 copies per cell in ABCyano4.
  • the integration site for the plasmids used for transformation of ABCyano4 via homologous recombination is indicated by an arrow.
  • Fig. 44 shows the plasmid map of one endogenous ABCyano4 plasmid, pABCyano4C, having an approximate copy number of 12-20 copies per cell in ABCyano4.
  • the integration site for the plasmids used for transformation of ABcyano4 via homologous recombination is indicated by an arrow.
  • Fig 45 A depicts a plasmid map of the 8763 bp circular plasmid construct and sequence annotation of plasmid #2870 (pJetl.2: :lacI-Ptac-Ava_3858(ABICyanolopt)-IScpcBA* l- Ava_3857(ABICyanolopt)-IScpcBA* l-Ava_3856(ABICyanolopt)-TB0011).
  • Fig. 45B shows the FIPLC profile of E.
  • Fig. 46 depicts the HPLC profile ofE. coli BL21 transformed with plasmid # 3119, producing mycosporine-methylamine-threonine.
  • Fig. 47 shows the HPLC profile of £ coli BL21 transformed with plasmid # 3117, producing palythine.
  • Fig. 48 shows the HPLC profile of £ coli BL21 transformed with plasmid # 3120, producing Tery-322.
  • Fig. 49 depicts the HPLC profile of £ coli BL21 transformed with plasmid # 3129, producing Tery-364.
  • Fig. 50 shows a sequence alignment of Tery_2966 (O-methyltransferase) homologs from different cyanobacterial species.
  • Fig. 51 to 86 show the plasmid maps of various plasmids included in genetically modified host cells of the present invention.
  • MAA mycosporine-like amino acid
  • cyanobacterium refers to a member from the group of photoautotrophic prokaryotic microorganisms which can utilize solar energy and fix carbon dioxide. Cyanobacteria are also referred to as blue-green algae.
  • host cell and "recombinant host cell” are intended to include a cell suitable for metabolic manipulation, e.g., which can incorporate heterologous polynucleotide sequences, e.g., which can be transformed.
  • the term is intended to include progeny of the cell originally transformed.
  • the cell is a prokaryotic cell, e.g., a cyanobacterial cell.
  • recombinant host cell is intended to include a cell that has already been selected or engineered to have certain desirable properties and to be suitable for further genetic enhancement.
  • Competent to express refers to a host cell that provides a sufficient cellular environment for expression of endogenous and/or exogenous polynucleotides.
  • the term "genetically modified” refers to any change in the endogenous genome of a wild type cell or to the addition of non-endogenous genetic code to a wild type cell, e.g., the introduction of a heterologous gene. More specifically, such changes are made by the hand of man through the use of recombinant DNA technology or mutagenesis.
  • the changes can involve protein coding sequences or non-protein coding sequences, including regulatory sequences such as promoters or enhancers.
  • polynucleotide and “nucleic acid” also refer to a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof.
  • nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs.
  • nucleotide sequence when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T.”
  • nucleic acids of this present invention may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages, charged linkages, alkylators, intercalators, pendent moieties, modified linkages, and chelators. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.
  • nucleic acid also referred to as polynucleotide
  • nucleic acid molecules having an open reading frame encoding a polypeptide, and can further include non-coding regulatory sequences and introns.
  • the terms are intended to include one or more genes that map to a functional locus.
  • the terms are intended to include a specific gene for a selected purpose. The gene can be endogenous to the host cell or can be recombinantly introduced into the host cell.
  • the invention also provides nucleic acids which are at least 60%, 70%, 80% 90%, 95%, 99%, or 99.5% identical to the nucleic acids disclosed herein.
  • the percentage of identity of two nucleic acid sequences or two amino acid sequences can be determined using the algorithm of Thompson et al. (CLUSTALW, 1994, Nucleic Acids Research 22: 4673-4680).
  • a nucleotide sequence or an amino acid sequence can also be used as a so-called "query sequence” to perform a search against public nucleic acid or protein sequence databases in order, for example, to identify further unknown homologous promoters, which can also be used in embodiments of this invention.
  • any nucleic acid sequences or protein sequences disclosed in this patent application can also be used as a "query sequence" in order to identify yet unknown sequences in public databases, which can encode for example new enzymes, which could be useful in this invention.
  • Such searches can be performed using the algorithm of Karlin and Altschul (1990, Proceedings of the National Academy of Sciences U.S.A. 87: 2,264 to 2,268), modified as in Karlin and Altschul (1993, Proceedings of the National Academy of Sciences U.S.A. 90: 5,873 to 5,877).
  • Such an algorithm is incorporated in the NBLAST and XBLAST programs of Altschul et al. (1990, Journal of Molecular Biology 215: 403 to 410).
  • Suitable parameters for these database searches with these programs are, for example, a score of 100 and a word length of 12 for BLAST nucleotide searches as performed with the NBLAST program.
  • BLAST protein searches are performed with the XBLAST program with a score of 50 and a word length of 3.
  • the enzyme commission numbers (EC numbers) cited throughout this patent application are numbers which are a numerical classification scheme for enzymes based on the chemical reactions which are catalyzed by the enzymes.
  • Recombinant refers to polynucleotides synthesized or otherwise manipulated in vitro ("recombinant polynucleotides”) and to methods of using recombinant polynucleotides to produce gene products encoded by those polynucleotides in cells or other biological systems.
  • a cloned polynucleotide may be inserted into a suitable expression vector, such as a bacterial plasmid, and the plasmid can be used to transform a suitable host cell.
  • a host cell that comprises the recombinant polynucleotide is referred to as a "recombinant host cell” or a “recombinant bacterium” or a “recombinant cyanobacterium .”
  • the gene is then expressed in the recombinant host cell to produce, e.g., a "recombinant protein.”
  • a recombinant polynucleotide may serve a non-coding function (e.g., promoter, origin of replication, ribosome-binding site, etc.) as well.
  • homologous recombination refers to the process of recombination between two nucleic acid molecules based on nucleic acid sequence similarity.
  • the term embraces both reciprocal and nonreciprocal recombination (also referred to as gene conversion).
  • the recombination can be the result of equivalent or non- equivalent cross-over events. Equivalent crossing over occurs between two equivalent sequences or chromosome regions, whereas nonequivalent crossing over occurs between identical (or substantially identical) segments of nonequivalent sequences or chromosome regions. Unequal crossing over typically results in gene duplications and deletions.
  • non-homologous or random integration refers to any process by which
  • DNA is integrated into the genome that does not involve homologous recombination. It appears to be a random process in which incorporation can occur at any of a large number of genomic locations.
  • exogenously refers to polynucleotides that are native to the host cell and are naturally expressed in the host cell.
  • operably linked refers to a functional relationship between two parts in which the activity of one part (e.g., the ability to regulate transcription) results in an action on the other part (e.g., transcription of the sequence).
  • a polynucleotide is "operably linked to a promoter" when there is a functional linkage between a polynucleotide expression control sequence (such as a promoter or other transcription regulation sequences) and a second polynucleotide sequence (e.g., a native or a heterologous polynucleotide), where the expression control sequence directs transcription of the polynucleotide.
  • vector as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid generally refers to a circular double stranded DNA molecule into which additional DNA segments may be ligated, but also includes linear double-stranded molecules such as those resulting from amplification by the polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors" (or simply "expression vectors").
  • a “promoter” is an array of nucleic acid control sequences that direct transcription of an associated polynucleotide, which may be a heterologous or native polynucleotide.
  • a promoter includes nucleic acid sequences near the start site of transcription, such as a polymerase binding site. The promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.
  • the term “promoter” is intended to include a polynucleotide segment that can transcriptionally control a gene of interest, e.g., a pyruvate decarboxylase gene that it does or does not transcriptionally control in nature.
  • the transcriptional control of a promoter results in an increase in expression of the gene of interest.
  • a promoter is placed 5' to the gene of interest.
  • a heterologous promoter can be used to replace the natural promoter, or can be used in addition to the natural promoter.
  • a promoter can be endogenous with regard to the host cell in which it is used or it can be a heterologous polynucleotide sequence introduced into the host cell, e.g., exogenous with regard to the host cell in which it is used.
  • Promoters of the invention may also be inducible, meaning that certain exogenous stimuli (e.g., nutrient starvation, heat shock, mechanical stress, light exposure, etc.) will induce the promoter leading to the transcription of the gene.
  • recombinant nucleic acid molecule includes a nucleic acid molecule (e.g., a DNA molecule) that has been altered, modified or engineered such that it differs in nucleotide sequence from the native or natural nucleic acid molecule from which the recombinant nucleic acid molecule was derived (e.g., by addition, deletion or substitution of one or more nucleotides).
  • the recombinant nucleic acid molecule e.g., a recombinant DNA molecule
  • Gene refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids. “Gene” also refers to a nucleic acid fragment that expresses a specific protein or polypeptide, including regulatory sequences preceding (5' non- coding sequences) and following (3' non-coding sequences) the coding sequence.
  • endogenous gene refers to a native gene in its natural location in the genome of an organism.
  • a “foreign” gene or “heterologous” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer.
  • Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.
  • a “transgene” is a gene that has been introduced into the genome by a transformation procedure.
  • fragment refers to a nucleotide sequence of reduced length relative to the reference nucleic acid and comprising, over the common portion, a nucleotide sequence substantially identical to the reference nucleic acid.
  • a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.
  • Such fragments comprise, or alternatively consist of, oligonucleotides ranging in length from at least about 6, 50, 100, 200, 500, 1,000, to about 1,500 or more consecutive nucleotides of a polynucleotide according to the invention.
  • ORF open reading frame
  • upstream refers to a nucleotide sequence that is located 5' to reference nucleotide sequence.
  • upstream nucleotide sequences generally relate to sequences that are located on the 5' side of a coding sequence or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.
  • downstream refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence.
  • downstream nucleotide sequences generally relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.
  • homology refers to the percent of identity between two polynucleotide or two polypeptide moieties.
  • the correspondence between the sequence from one moiety to another can be determined by techniques known to the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single- stranded-specific nuclease(s) and size determination of the digested fragments.
  • substantially similar refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence.
  • substantially similar also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript.
  • restriction endonuclease and “restriction enzyme” refer to an enzyme that binds and cuts within a specific nucleotide sequence within double stranded DNA.
  • expression refers to the transcription and stable accumulation mRNA derived from a nucleic acid or polynucleotide. Expression may also refer to translation of mRNA into a protein or polypeptide.
  • an "expression cassette” or “construct” refers to a series of polynucleotide elements that permit transcription of a gene in a host cell.
  • the expression cassette includes a promoter and a heterologous or native polynucleotide sequence that is transcribed.
  • Expression cassettes or constructs may also include, e.g., transcription termination signals, polyadenylation signals, and enhancer elements.
  • codon refers to a triplet of nucleotides coding for a single amino acid.
  • codon-anticodon recognition refers to the interaction between a codon on an mRNA molecule and the corresponding anticodon on a tRNA molecule.
  • codon bias refers to the fact that different organisms use different codon frequencies.
  • codon optimization refers to the modification of at least some of the codons present in a heterologous gene sequence from a triplet code that is not generally used in the host organism to a triplet code that is more common in the particular host organism. This can result in a higher expression level of the gene of interest.
  • transformation is used herein to mean the insertion of heterologous genetic material into the host cell.
  • the genetic material is DNA on a plasmid vector, but other means can also be employed.
  • General transformation methods and selectable markers for bacteria and cyanobacteria are known in the art (Wirth, Mol Gen Genet. 216: 175-177 (1989); Koksharova, Appl Microbiol Biotechnol 58: 123-137 (2002). Additionally, transformation methods and selectable markers for use in bacteria are well known (see, e.g., Sambrook et al, supra).
  • selectable marker means an identifying factor, usually an antibiotic or chemical resistance gene, that is able to be selected for based upon the marker gene's effect, i.e., resistance to an antibiotic, resistance to a herbicide, colorimetric markers, enzymes, fluorescent markers, and the like, wherein the effect is used to track the inheritance of a nucleic acid of interest and/or to identify a cell or organism that has inherited the nucleic acid of interest.
  • selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, spectinomycin, kanamycin, hygromycin, zeocin, chloramphenicol, and the like.
  • a "polypeptide” is a polymeric compound comprised of covalently linked amino acid residues.
  • a “protein” is a polypeptide that performs a structural or functional role in a living cell.
  • a "heterologous protein” refers to a protein not naturally produced in the cell.
  • An "isolated polypeptide” or “isolated protein” is a polypeptide or protein that is substantially free of those compounds that are normally associated therewith in its natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids).
  • fragment of a polypeptide refers to a polypeptide whose amino acid sequence is shorter than that of the reference polypeptide. Such fragments of a polypeptide according to the invention may have a length of at least about 2, 50, 100, 200, or 300 or more amino acids.
  • a "variant" of a polypeptide or protein is any analogue, fragment, derivative, or mutant which is derived from a polypeptide or protein and which retains at least one biological property of the polypeptide or protein.
  • Different variants of the polypeptide or protein may exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequences of the structural gene coding for the protein, or may involve differential splicing or post- translational modification. The skilled artisan can produce variants having single or multiple amino acid substitutions, deletions, additions, or replacements.
  • primer is an oligonucleotide that hybridizes to a target nucleic acid sequence to create a double stranded nucleic acid region that can serve as an initiation point for DNA synthesis under suitable conditions. Such primers may be used in a polymerase chain reaction.
  • PCR polymerase chain reaction
  • PCR refers to an in vitro method for enzymatically amplifying specific nucleic acid sequences. PCR involves a repetitive series of temperature cycles with each cycle comprising three stages: denaturation of the template nucleic acid to separate the strands of the target molecule, annealing a single stranded PCR oligonucleotide primer to the template nucleic acid, and extension of the annealed primer(s) by DNA polymerase. PCR provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the starting pool of nucleic acids.
  • MAA mycosporine-like amino acid
  • exemplary MAAs include, for example, mycosporine-glycine, shinorine, and mycosporine-2-glycine. These molecules absorb UV light, and exhibit photoprotection functions.
  • UV refers to light between the wavelengths of 290 - 400. This is divided between “UV-A” and "UV-B”.
  • UV-A refers to light between the wavelengths of 320-400 nm.
  • UV-B refers to light between the wavelengths of 290 to 320 nm.
  • selection-free refers to a growth medium that does not include a selection agent, such as an antibiotic, that would allow only cells having a functional selectable marker gene to survive.
  • heterologous gene in the context of genes coding for enzymes involved in the production of MAA and the term “MAA gene” will be used interchangeably. If more than one heterologous gene coding for enzymes involved in the production of MAA is present in a genetically modified cyanobacterial cell, then these genes will be referred to as the first heterologous gene, the second heterologous gene and so on.
  • extracellular polysaccharides EPS
  • capsule polysaccharides CPS
  • EPS extracellular polysaccharides
  • CPS capsule polysaccharides
  • cyanobacterial cells can be modified to produce various MAAs.
  • the modified cyanobacterial cells are relatively easy to cultivate, in comparison, for example, to red macroalgae from the genus Porphyra.
  • the heterologous genes can be linked to regulatable or constitutive promoters.
  • a commercially suitable strain of cyanobacteria already known for its ability to grow well in both indoor and outdoor commercially relevant conditions, and also for the production of ethanol and other compounds, can be utilized to carry the MAA gene clusters.
  • endogenous promoters from this strain, as well as their derivatives can be utilized to produce high amounts of MAAs using sunlight, CO2, and inorganic nutrients.
  • heterologous systems have been utilized by other researchers to produce larger amounts of MAAs than can typically be found in nature. Cyanobacterial genes have been transferred to heterologous bacteria, where they can then be grown in fermentor systems without light. For example, heterotrophic production of heterologous MAAs in E.
  • heterotrophic bacteria also contain MAA synthesis gene clusters.
  • MAA synthesis gene clusters One example is Actinosynnema mirum DSM 43827 whose mysABCD gene cluster was expressed in Streptomyces avermitilis SUKA22. The major product was shinorine and minor products were porphyra-334 and mycosporine-glycine-alanine (Miyamoto et al., Discovery of Gene Cluster for Mycosporine-Like Amino Acid Biosynthesis from Actinomycetales Microorganisms and Production of a Novel Mycosporine-Like Amino Acid by Heterologous Expression" Appl Environ Microbiol. 80:5028-5036 (2014)).
  • Patent application EP20150792252 describes a method for producing a mycosporine-like amino acid by using the genes amir_4256, amir_4257, amir_4258 and amir_4259 genes from A. mirum, expressing them in Streptomyces species, Corynebacterium, Aurantiochytrium sp. E.
  • the extracellular production amount of shinorine in Streptomyces lividans 1326 was 510 mg/L and 25 mg/L mycosporine-glycine-alanine after two weeks of cultivation.
  • MAA biosynthesis starts with the conversion of sedoheptulose- 7-phosphate (from the Calvin-Benson cycle) to desmethyl-4-deoxygadusol, a cyclohexenone natural product that resembles the mycosporine core, by the DHQS homolog 4-deoxygadusol synthase (Ava_3858).
  • the gene Ava_3857 encodes an O-methyltransf erase, converting desmethyl-4-deoxygadusol in 4-deoxygadusol.
  • a similar cluster of three genes in N. punctiforme ATCC 29133 (NpR5600, NpR5599, NpR5598) has also been shown to catalyze the same reactions (Gao, supra).
  • Mycosporine-glycine is thought to be the intermediate MAA for attachment of additional amino acids to the core via imine linkages.
  • Ava_3855 encoding a nonribosomal peptide synthetase was described to attach a serine, yielding shinorine (Balskus et al., supra).
  • the genetic basis for the pathway leading to the formation of the shinorine does not apparently share consistency among cyanobacteria. Cyanobacteria that lack homologues of Ava_3855 tend to have a homologue of an ATP-grasp proteins similar to D-Ala-D-Ala ligase involved in peptidoglycan synthesis (Gao, supra).
  • the NpF5597 gene of N. punctiforme encodes a D-Ala-D-Ala ligase, attaching a serine to mycosporine-glycine, producing shinorine (Waditee-Sirisattha et al, "Identification and upregulation of biosynthetic genes required for accumulation of mycosporine- 2-glycine under salt stress conditions in the halotolerant cyanobacterium Aphanothece halophyticd Appl Environ Microbiol, 80: 1763-1769 (2013).
  • AB 1252 (ABICyanol #2742 #2796) additionally contains a codon-adapted NpF5597 gene (coding for a D-Ala-D-Ala ligase) under control of a zinc-inducible promoter and yields 110 mg/L shinorine (5 % per dry weight).
  • the third and fourth strains AB1253 (ABICyanol #2742 #2798) / AB1277 (ABICyanol #2840) contain instead of NpF5597 the homologous, codon-adapted D-Ala-D-Ala ligase gene from A. halophytica (Ap3855) and produce 200 mg/L mycosporine-2-glycine (5 - 6% per dry weight).
  • the genetically modified cyanobacterial cell is for the production of mycosporine-glycine, and comprises at least three heterologous genes, namely a first heterologous gene coding for a first enzyme, which is at least 70%, preferably at least 80%, most preferably at least 90% identical to SEQ ID NO: 3 or which is at least 70%, preferably at least 80%, most preferably at least 90% identical to SEQ ID NO 164, a second heterologous gene coding for a second enzyme, which is at least 70%, preferably at least 80%, most preferably at least 90% identical to SEQ ID NO: 6, and a third heterologous gene coding for a third enzyme, which is at least 70%, preferably at least 80%, most preferably at least 90%) identical to SEQ ID NO: 9.
  • a first heterologous gene coding for a first enzyme which is at least 70%, preferably at least 80%, most preferably at least 90% identical to SEQ ID NO: 3 or which is at least 70%, preferably at least 80%, most preferably at least
  • the first heterologous genes codes for an enzyme, which is highly homologous to an DHQS-like protein, such as the enzyme encoded by the gene Ava_3858, a MysA homologue, converting sedoheptoluse-7-phosphate to desmethyl-4-deoxygadudusol.
  • the second heterologous genes code for an O-methyltransferase, such as the gene Ava-3857, a MysB homologue, which converts desmethyl-4-deoxygadudusol to 4-deoxygadudusol.
  • the third heterologous genes codes for an ATP -grasp protein, such as the gene Ava-3856, a MysC homologue, which converts 4-deoxygadudusol to mycosporine-glycine (see also Fig. 1 for the mode of action of enzymes coded by MysA to MysC).
  • Ava-3856 a MysC homologue, which converts 4-deoxygadudusol to mycosporine-glycine
  • Fig. 1 for the mode of action of enzymes coded by MysA to MysC.
  • the homologues disclosed therein can be employed.
  • the genetically modified cyanobacterial cell of the previous claim exhibits a low pigment phenotype compared to the wildtype, in particular with respect to the chlorophyll and/or the phycocyanin content of the cell.
  • a low pigment type cyanobacterial cell can lead to higher production of MAAs, because cells with a lower amount of pigment do not shade other cells to a great extent, so that the incident light can penetrate deeper into a cell culture and more light can reach the cells.
  • the genetically modified cyanob acted al cell can exhibit a low chlorophyll genotype, due to a Aycf37 gene inactivation associated with a low chlorophyll phenotype.
  • Another embodiment of the invention is directed to a genetically modified cyanobacterial cell for the production of mycosporine-2-glycine, additionally including a fourth heterologous gene coding for a fourth enzyme which is at least 70%, preferably at least 80%>, most preferably at least 90% identical to SEQ ID NO: 15.
  • the fourth heterologous gene codes for an MysD homologous protein, such as the protein encoded by the gene Ap_3855, which converts mycosporine-glycine to mycosporine-2-glycine.
  • Another embodiment is directed to a genetically modified cyanobacterial cell for the production of shinorine/porphyra-334, additionally including a fourth heterologous gene coding for a fourth enzyme which is at least 70%, preferably at least 80%, most preferably at least 90%) identical to the enzyme of SEQ ID NO: 1111.
  • This gene is highly homologous to the gene NpF5597 from Nostoc punctiforme, which codes for a D-ala-D-ala ligase protein (MysD homolog), converting mycosporine-glycine to shinorine and phorphyra-334, and to some extent also mycosporine-glycine-alanine (see also Fig. 28C and Fig. 40)
  • Another embodiment is directed to a genetically modified cyanobacterial cell for the production of shinorine, additionally including a fourth heterologous gene coding for a fourth enzyme which is at least 70%, preferably at least 80%, most preferably at least 90% identical to SEQ ID NO: 61.
  • This gene is highly homologous to the gene Ava_3855, which codes for an NRPS-like protein, converting mycosporine-glycine to shinorine in the presence of a phosphopantethiene-protein transferase (see also Fig. 1).
  • the genetically modified cyanobacterial cell apart from a first copy of a mysA homologous gene, can further include a second copy of the first heterologous gene which codes for a first enzyme, which is at least 70%, preferably at least 80%, most preferably at least 90%) identical to SEQ ID NO: 3.
  • This additional mysA copy can lead to a higher expression of MysA, higher conversion rate of sedoheptulose-7-phosphate to desmethyl-4-deoxygadusol and finally to higher MAA productivity of e.g. shinorine and porphyra-334 productivity shown in Fig. 42 B and C.
  • the genetically modified cyanobacterial cell apart from a first copy of a classical mysA homologous gene, can further include a second copy of a mysA heterologous gene from the divergent clade which codes for a first enzyme, which is at least 80%, preferably 90% identical to SEQ ID NO: 164.
  • This divergent clade mysA copy can lead to a higher conversion rate of sedoheptulose-7-phosphate to desmethyl-4-deoxygadusol and finally to higher MAA productivity of e.g. shinorine and porphyra-334 productivity shown in Fig. 42 B, C and 19 A, B, C.
  • the genetically modified cyanobacterial cells of the present invention can have productivities of 50-100mg/L*d, preferably 120-200mg/L*d over a period of 3-10 days for mycosporine-glycine, mycosporine-2-glycine and shinorine/porphyra-334 under cultivation conditions as described in example 27.
  • the new MAA compounds Tery-322 and Tery-364 are produced by a genetically modified host cell comprising at least one heterologous gene that encodes an enzyme involved in the production of the compounds Tery-322 or Tery-364.
  • the heterologous genes can be mysA, mysB and mysC from any of the host cells disclosed in this application.
  • the mysA gene (for example the genes Ava_3858, Tery 2977, mysA_HL-69, NpR5600) encodes a DHQS-like enzyme, which converts sedoheptulose-7-phosphate to desmethyl-4-deoxygadusol.
  • the mysB gene (for example the genes Ava_3857, Tery 2976, mysB_HL-69, NpR5599), codes for an O-MT enzyme, which further converts the desmethyl-4-deoxygadusol to 4-deoxygudasol.
  • the mysC gene (for example the genes Ava_3856, Tery_2975, mysC_HL-69, NpR5598), encodes an enzyme that is responsible for the conversion of 4-deoxygudasol to mycosporine-glycine (see also Fig. 1 and Fig. 40). Mycosporine-glycine is then the branching point for a variety of different MAAs dependent on the enzyme involved and the amino acid participating in the conversion (see Fig. 40).
  • mysD or mysD homologous genes encoding a D-ala-D-ala ligase can catalyze the reaction of mycosporine-glycine with glycine to form mycosporine-2-glycine, with mycosporine-glycine and threonine to form porphyra-334, with mycosporine-glycine and alanine to form mycosporine- glycine-alanine or with mycosporine-glycine and serine to result in shinorine (see Fig. 40).
  • a D-ala-D-ala ligase for example NpF5597, Ap3855, mysD_HL-69, mysD_K005, Tery 2971, Tery 2970, Amir_4256
  • Tery-322 in particular can be produced by a genetically modified host cell comprising at least one heterologous gene encoding an enzyme having at least 80 % sequence identify, preferably at least 90 % sequence identity with the enzyme of SEQ ID NO: 103, which is the Tery -2966 homolog of O-methyltransferase from the TERY IMS 101 gene cluster from Trichodesmium erythraeum IMS 101 for production of Tery-322.
  • An exemplary gene encoding the enzyme of SEQ ID NO: 103 is the nucleic acid sequence with the SEQ ID NO: 102.
  • Tery-322 can be produced employing mycosporine-glycine as a starting compound.
  • a genetically modified host cell transformed with the above-mentioned enzyme of SEQ ID NO: 103 should be used, which naturally produces mycosporine-glycine, or additional heterologous genes encoding enzymes involved in the production of mycosporine-glycine, for example mysA, mysB and/or mysC have to be present in the host cell.
  • Homologous genes of Tery 2966 with high degree of amino acid sequence similarity can be found in multiple cyanobacterial species.
  • FIG. 50 shows the O-methyltransferase-like amino acid sequences from Synechococcus sp. PCC7335, Euhalothece sp. KZN001, Croococcidiopsis sp. TS821, Trichodesmium erythraeum FMS101, and Calothrix sp. NIES2100.
  • Respective O- methyltransferases from Synechococcus sp. PCC7335, Euhalothece sp. KZN001, and Croococcidiopsis sp. TS821 have been proven to catalyze the same reaction as Tery 2966 from Trichodesmium erythraeum IMS 101 with mycosporine-glycine as substrate to form Tery-322 as product.
  • a further variant of the invention is directed to a genetically modified host cell which further comprises another heterologous gene encoding an enzyme having at least 80 % sequence identify, preferably at least 90 % sequence identity with the enzyme of SEQ ID NO: 99, which is the Tery-2968 gene homolog of NRPS from the TERY IMS 101 gene cluster from Trichodesmium erythraeum IMS101 for production of Tery-364 employing Tery-322 as starting compound.
  • An exemplary gene encoding the enzyme of SEQ ID NO 99 is the nucleic acid sequence with the SEQ ID NO: 89.
  • the genetically modified host cell further comprises
  • a first enzyme having at least 80 % sequence identity, preferably at least 90 % sequence identity with either the enzymes of SEQ ID NO: 3 or having at least 80 % sequence identity, preferably at least 90 % sequence identity with the enzyme of SEQ ID NO: 164 (Ava-3858 mysA variant or distant clade mysA variant),
  • a third enzyme having at least 80 % sequence identity, preferably at least 90 % sequence identity with the enzyme of SEQ ID NO: 9 (Ava-3856 mysC variant). These enzymes are able to produce the starting compounds 4-deoxygudasol, desmethyl-4-deoxygadusol or mycosporine-glycine for the production of Tery-322 and Tery-364 (see Fig. 40).
  • Another embodiment of the invention is directed to a genetically modified host cell, comprising at least one heterologous gene encoding a protein having at least 80 % sequence identify, preferably at least 90 % sequence identity with the protein of SEQ ID NO: 95 and SEQ ID NO: 93.
  • An exemplary gene encoding the enzyme of SEQ ID NO: 93 Tery 2971 (mysDl EVISlOl) is the nucleic acid sequence with the SEQ ID NO: 92. This enzyme is responsible for the formation of mycosporine-2-glycine, employing glycine as the reaction partner and using mycosporine-glycine as the starting compound (see Fig. 40).
  • An exemplary gene encoding the enzyme of SEQ ID NO: 95 Tery 2970 is the nucleic acid sequence with the SEQ ID NO: 94. This enzyme is responsible for the formation of shinorine employing serine as reaction partner and using mycosporine-glycine as starting compound.
  • the genetically modified host cell of the invention can further comprise at least one further heterologous gene encoding an enzyme having at least 80 % sequence identify, preferably at least 90 % sequence identity with an enzyme encoded by either one of the nucleic acids of SEQ ID NO: 91or SEQ ID NO: 97. It is possible that only one, two or all three of the enzymes are present in the host cell. These enzymes are responsible for the formation of mycosporine- methylamine-glycine, mycosporine-methylamine-threonine or palythine, employing either porphyra-334 or mycosporine-2-glycine as starting compounds (see Fig. 40).
  • the genetically modified host cell of any of the above described embodiments can be selected from a group consisting of bacteria and eukaryotic cells.
  • enterobacteria such as E. coli
  • cyanobacteria such as Cyanobacterium strains like Cyanobacterium sp. ABICyanol or Cyanobacterium sp. ABCyano4, Anabaena PCC7120, Synechocystis PCC6803, Synechococcus PCC7942, or Synechococcus PCC7002
  • enterobacteria such as E. coli
  • cyanobacteria such as Cyanobacterium strains like Cyanobacterium sp. ABICyanol or Cyanobacterium sp. ABCyano4, Anabaena PCC7120, Synechocystis PCC6803, Synechococcus PCC7942, or Synechococcus PCC7002
  • Another embodiment of the invention is directed to the compound Tery-322, which is a mycosporine-like amino acid having an absorption maximum at 322 nm, being producible by culturing a genetically modified host cell including four heterologous genes encoding four enzymes,
  • a first enzyme having at least 80 % sequence identity, preferably at least 90 % sequence identity with either the enzyme of SEQ ID NO: 3 or having at least 80 % sequence identity, preferably at least 90 % sequence identity with the enzyme of SEQ ID NO: 164 (Ava-3858 mysA variant or distant clade mysA variant),
  • a third enzyme having at least 80 % sequence identity, preferably at least 90 % sequence identity with the enzyme of SEQ ID NO: 9 (Ava-3856 mysC variant), and
  • a fourth enzyme having at least 80 % sequence identify, preferably at least 90 % sequence identity with the enzyme of SEQ ID NO: 103 (Tery_2966).
  • Tery-322 Apart from the unique absorption at 322 nm, Tery-322 also exhibits a retention time, not observed for other MAAs. For example on a HPLC system with a HILIC column (as described in Hartmann et al., 2015) the retention time for Tery-322 is at ⁇ 6.6 min, for M-Gly is at - 6.9 min, for Palythine at ⁇ 9.0 min, for porphyra-334 at ⁇ 9,7 min, for Tery-364 at ⁇ 10,1 min, and for shinorine 10,7 min.
  • a further embodiment of the invention is directed to the compound Tery-364, which is a mycosporine-like amino acid having an absorption maximum at 364 nm, being producible by culturing a genetically modified host cell including at least five heterologous genes encoding five enzymes,
  • a first enzyme having at least 80 % sequence identity, preferably at least 90 % sequence identity with either the enzyme of SEQ ID NO: 3 or having at least 80 % sequence identity, preferably at least 90 % sequence identity with the enzyme of SEQ ID NO: 164 (Ava-3858 mysA variant or distant clade mysA variant),
  • a second enzyme having at least 80 % sequence identity, preferably at least 90 % sequence identity with the enzyme of SEQ ID NO: 6 (Ava-3857 mysB variant)
  • a third enzyme having at least 80 % sequence identity, preferably at least 90 % sequence identity with the enzyme of SEQ ID NO: 9 (Ava-3856 mysC variant)
  • the genetically modified host cell can further include an enzyme having at least 80
  • % sequence identify preferably at least 90 % sequence identity with the enzyme of SEQ ID NO: 176, which is important for the activity of the enzyme with SEQ ID NO: 99. (Phosphopantethiene- protein transferase).
  • Tery-364 Apart from the unique absorption at 364 nm, Tery-364 also exhibits a retention time, not observed for other MAAs. For example on a HPLC system with a HILIC column (as described in Hartmann et al., 2015) the retention time for Tery-322 is at ⁇ 6.6 min, for M-Gly is at ⁇ 6.9 min, for Palythine at ⁇ 9.0 min, for porphyra-334 at ⁇ 9,7 min, for Tery-364 at ⁇ 10,1 min, and for shinorine 10,7 min.
  • genetically modified phototrophic cyanobacteria can produce large amounts of a desired MAA or mixture of MAAs.
  • the MAA productivities of phototrophically grown Cyanobacterium sp. ABICyanol and Cyanobacterium sp. ABCyano4, on a dry weight basis, can be in the range of from about 40, 60, 80, 100, 120, 140, 160, 180, 200, or more mg / g dry weight.
  • the modified cyanobacterial cells do not require the addition of an organic carbon source, as they are able to utilize sunlight and CO2 to make the MAA compounds.
  • sustainable production of high amounts for example, between about 100, 150, 200, and about 250, 300, 350 or more, in particular 400, 600, 800, 1000, 1200, 1400 or more mg/L of the MAAs mycosporine-glycine, shinorine and mycosporine-2-glycine, and others, such as porphyra-334, mycosporine-glycine-alanine and palythine, can be produced, for example, by heterologous expression of the MAA synthesis pathway genes from A. variabilis ATCC 29413, N. punctiforme ATCC 29133, Nostoc commune var. flagelliforme QSY 1, Nostoc verrucosum KU005 P.
  • the cyanobacterial host cell is Cyanobacterium sp. ATCC accession number PTA-13311 ("ABICyanol", also termed “ABl”).
  • ABICyanol also termed "ABl”
  • This strain has been found to grow well under various indoor and outdoor conditions, and can be genetically modified to produce a compound of interest.
  • the strain, as well as its endogenous plasmid p6.8 (FIG. 4, SEQ ID NO: 16) has been described, for example, in U.S. Patent NO: 8,846,369, U.S. 9,315,832, and U.S. Patent NO: 9,157, 101, all of which are hereby incorporated by reference in their entireties.
  • the cyanobacterial host cell is
  • CPS layer capsular exopoly saccharide layer
  • the strain as well as its endogenous plasmids pABCyano4-B and pABCyano4-C (FIG. 43 and 44), has been applied for heterologous MAA production by insertion of MAA biosynthesis genes of interest from different cyanobacterial origin.
  • a deposit of the Algenol Biotech LLC's proprietary strain of Cyanobacterium sp., strain ABCyano4, disclosed above and recited in the appended claims has been made with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110.
  • ATCC Accession Number is PTA-125253.
  • the 16S rDNA of Cyanobacterium sp. ABCyano4 shows a high sequence identity of around 99% to the 16S rDNA sequences of different cyanobacterial species of the genus Cyanobacterium, including Cyanobacterium IHB-410, Cyanobacterium aponinum ETS-03, and Cyanobacterium sp. MBIC10216.
  • the 16S ribosomal RNA (rRNA) gene sequences (16S rDNA) of ABICyanol was predicted from the genome sequence with the RNAmmer program (Lagesen K, et al. (2007) RNAmmer: consistent and rapid annotation of ribosomal RNA genes.
  • a culture of the cyanobacterial cell is grown in an outdoor photobioreactor system, and the MAA that is produced can be isolated, for example, from the culture medium or from the cells.
  • a culture of the cyanobacterial cells is grown in an indoor photobioreactor system.
  • Cyanobacteria can be modified via addition of MAA biosynthesis genes as shown herein in order to produce various MAAs.
  • the DNA sequences encoding the genes can be amplified by the polymerase chain reaction (PCR) using specific primers.
  • PCR polymerase chain reaction
  • the amplified PCR fragments can then be digested with the appropriate restriction enzymes and cloned into either a self-replicating plasmid or an integrative plasmid.
  • An antibiotic resistance cassette for selection of positive clones can be present on the appropriate plasmid.
  • the nucleic acids of interest can be amplified from nucleic acid samples using amplification techniques.
  • PCR can be used to amplify the sequences of the genes directly from DNA, from mRNA, from cDNA, from genomic libraries or cDNA libraries.
  • PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, and for nucleic acid sequencing.
  • DNA vectors suitable for transformation of cyanobacteria can be prepared. Techniques for transformation are well known and described in the technical and scientific literature. For example, a DNA sequence encoding one or more of the genes described herein can be combined with transcriptional and other regulatory sequences which will direct the transcription of the sequence from the gene in the transformed cyanobacteria.
  • the MAA biosynthesis genes of interest are inserted into the cyanobacterial chromosome.
  • the gene insertions can be present in all of the copies of the chromosome, or in some of the copies of the chromosome.
  • the inserted MAA biosynthesis genes are present on an extrachromosomal plasmid.
  • the extrachromosomal plasmid can be derived from an outside source, such as, for example, RSFlOlO-based plasmid vectors, or it can be derived from an endogenous plasmid from the cyanobacterial cell or from another species of cyanobacteria.
  • the inserted MAA genes are present on one or several extrachromosomal plasmids, wherein the plasmids have multiple copies per cell.
  • the plasmids can be present, for example, at about 1, 3, 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or more copies per host cyanobacterial cell.
  • the plasmids are fully segregated.
  • the inserted MAA genes are present on one cassette driven by one promoter.
  • the inserted genes are present on separate plasmids, or on different cassettes.
  • the cassette can be part of an operon.
  • the cell includes an operon including the first heterologous gene and a second heterologous gene involved in the production of a MAA, the operon including an intergenic sequence between the first heterologous gene and the second heterologous gene.
  • an operon allows for a simple overexpression of more than one enzyme involved in the production of MAA, wherein the plurality of heterologous genes of the operon are controlled by one promoter.
  • the operon can include the first heterologous gene, and at least one further gene, the second heterologous gene.
  • the second heterologous gene can code for an enzyme which further acts on the molecule produced by the enzyme coded by the first heterologous gene.
  • a consecutive chain of enzymes for MAA production can be overexpressed in one operon, wherein for example a second enzyme for MAA production converts the product of a first enzyme for MAA production into another product, on which then a third enzyme can act.
  • heterologous gene and a fourth heterologous gene coding for a third and a fourth enzyme for producing MAA, respectively, can be present in the operon.
  • shinorine can be produced from sedoheptulose-7-phosphate by overexpressing a 3-dehydroquinate synthase homolog enzyme (DHQS-homolog), an O-methyltransferase (O-MT), an ATP-grasp family protein and a nonribosomal peptide synthetase ( RPS-like protein) or a D-Ala-D-Ala ligase, which subsequently convert sedoheptulose-7-phosphate into shinorine (see also Fig. 1).
  • the intergenic sequence between the respective heterologous genes contains a ribosomal binding site and further nucleotide sequences, which can influence the overexpression of the enzymes encoded by the heterologous genes.
  • the intergenic sequence is derived from an intergenic sequence located between two genes coding for a heterodimeric protein.
  • proteins coded by genes arranged in an operon exhibit different levels of expression, wherein proteins whose genes are further downstream in the operon show a lower level of expression compared to the enzymes whose genes are located upstream in the operon.
  • These different levels of expression of different enzymes involved in the production of an MAA can negatively affect the overall yield of the MAA. Therefore, employing the intergenic sequence of an operon coding for two proteins, which are roughly expressed at the same levels can ensure a higher and more equal level of expression of enzymes for MAA production, leading to a higher overall yield of MAAs.
  • the intergenic sequence can be derived from the cpcB-cpcA operon coding for phycocyanin.
  • Phycocyanin is a highly expressed, abundant pigment-protein complex from the light-harvesting phycobiliprotein family, which contains two subunits, the alpha and beta subunit.
  • Employing the intergenic sequence of this operon can ensure a higher yield of MAA.
  • the intergenic sequence is at least 90 % identical to the sequence with the SEQ ID NO: 177, which is the native IScpcBA sequence of the cpcB-cpcA operon.
  • a further variant of the genetically modified cyanobacterial cell of the invention includes an intergenic sequence having a ribosomal binding side and wherein the intergenic sequence exhibits nucleotide changes in the ribosomal binding site and in between the ribosomal binding site and a start codon of either the first or second heterologous gene in comparison to the native intergenic sequence.
  • These nucleotide changes can be changes of the nucleotides or even nucleotide additions or deletions in comparison to the native sequence of the intergenic sequence, such as the sequence of SEQ ID NO: 177.
  • These nucleotide changes can further increase in the yield of MAA.
  • One particular example of a modified intergenic sequence is the IScpcBA* 1 sequence of SEQ ID NO: 178.
  • the operon comprises a plurality of heterologous genes encoding a plurality of enzymes for producing MAA, any of the above discussed intergenic sequence being present between all the heterologous genes.
  • Such a design of an operon can ensure roughly the same level of expression for the plurality of the enzymes, thereby enhancing the overall MAA yield.
  • the inserted MAA genes are modified for optimal expression by modifying the nucleic acid sequence to accommodate the cyanobacterial cell's protein translation system. Modifying the nucleic acid sequences in this manner can result in an increased expression of the genes.
  • the genetically modified cyanobacterial cell is able to produce high amounts of MAAs over an extended period of time, for example at least 25 mg L _1 d _1 , preferably at least 50 mg L _1 d _1 more preferably at least 75 mg L _1 d " 1 , most preferred at least 100 mg L _1 d _1 , or at least 150 mg L _1 d _1 or even 200 mg L _1 d _1 over a period of 1-2 weeks.
  • the productivity rates can be in the range from 25 mg L "1 d "1 to 250 mg L "1 d "1 .
  • the ratio of the MAAs produced in comparison to the dry weight of the cells can be at least 4%, preferably 6%, most preferred at least 8%.
  • Genetically modified cyanobacterial cells able to produce the above mentioned high overall yields comprise at least one or a combination of at least two or three of the following features responsible for the high yield:
  • the promoter operably linked to the at least one heterologous gene that encodes an enzyme involved in the production of MAA is a promoter inducible by a change of a metal-ion concentration, in particular the smtA promoter or the petE promoter.
  • These promoters can be induced by addition of Zn-ions or Cu-ions, respectively, and allow for a tight control of the expression of the enzymes for MAA production. In particular, only traces of MAA production can be observed in the uninduced state of the cells, so that a high cell density can be reached before inducing the cells and then producing a high yield of MAAs.
  • these metal-inducible promoters are easily titratable by a change of a metal ion concentration and allow for high induction.
  • an intergenic sequence between different heterologous genes coding for enzymes of MAA production which is derived from an intergenic sequence located between two genes coding for a heterodimer protein.
  • an intergenic region can ensure that a plurality of heterologous genes included in an operon operably linked to one promoter can be expressed at roughly the same levels.
  • heterologous genes coding for enzymes whose predicted function are included in parentheses can be codon-optimized mysA (dimethyl-4-deoxygadusol (DDG) synthase), mysB (O-methyltransferase), mysC (ATP-grasp family protein) and mysD (D-Ala ligase homolog) or RPS gene (nonribosomal peptide synthetase).
  • DDG dimethyl-4-deoxygadusol
  • mysB O-methyltransferase
  • mysC ATP-grasp family protein
  • mysD D-Ala ligase homolog
  • RPS gene nonribosomal peptide synthetase
  • heterologous genes or operons containing the heterologous genes located on extrachromosomal high copy plasmids are present.
  • These high copy plasmids ensure a replication independent of the chromosomes and therefore result in a high copy number of the heterologous genes, resulting in high expression of the enzymes encoded by the heterologous genes.
  • the high copy plasmids are present in a cell in at least 10, preferably at least 20, most preferably at least 40 copies per cell. 30 - 50 copies should be present for the pABCyano4B and 12-20 copies of the plasmid pABCyano4C into which heterologous genes for MAA production can be inserted via homologous recombination.
  • cyanobacterial strains which can reach a high amount of biomass during batch cultivation, such as ABICyanol and ABCyano4.
  • the amount of biomass can be assessed within a specified time period by employing standard lab conditions, such as cultivation in a tubular plastic film or rigid plastic photobioreactor, for example a 1.2L LvPBR (lab-scale vertical photobioreactor); Start OD: 0.5; Cultivation pH: 7.3; CO2 supply: 15% pH dependent into liquid phase; Aeration: 38 mL min "1 ; Light: 350 ⁇ fluorescent light from one side, 12h photoperiod; Temperature day 35°C/ night 25°C; Media: ASW BG11 (artificial seawater medium BG11).
  • cyanobacterial strains having an extracellular capsular polysaccharide layer (CPS) with a thickness of between 1 and 3 ⁇ , preferably between 1.1 to 2 ⁇ .
  • CPS capsular polysaccharide layer
  • Such cyanobacterial strains, such as ABCyano4 can greatly ease the purification of the MAA.
  • the MAAs are often associated with the CPS and therefore can more easily be purified than when they are secreted into the culture medium. Additionally, a purification of the MAAs from the CPS results in higher yields.
  • Enzyme for MAA production encoded by the gene 61
  • Enzyme for MAA production encoded by the gene mysCD 67
  • Enzyme for MAA production encoded by the gene mysCD 69
  • Enzyme for MAA production encoded by the gene 71 dehydroquinate synthase MylA [Cylindrospermum stagnale
  • Enzyme for MAA production encoded by the gene MylB 73
  • Enzyme for MAA production encoded by the gene MylC 75 [Cylindrospermum stagnale PCC 7417] GenBank:
  • Enzyme for MAA production encoded by the gene MylD 77
  • Enzyme for MAA production encoded by the gene MylE 79
  • Enzyme for MAA production encoded by the gene MysA 81 [Nostoc commune var ⁇ . flagelliforme QSY 1] GenBank:
  • Enzyme for MAA production encoded by the gene MysB 83
  • Enzyme for MAA production encoded by the gene MysD 85 [Nostoc commune var ⁇ . flagelliforme QSY 1] GenBank:
  • Enzyme for MAA production encoded by the gene MysC2 87
  • Enzyme for MAA production encoded by the gene MysCl 89
  • Enzyme for MAA production encoded by the gene Tery 2972 91 [Trichodesmium erythraeum IMSlOl] GenBank: ABG52130.1
  • Enzyme for MAA production encoded by the gene Tery 2971 93
  • Enzyme for MAA production encoded by the gene Tery 2970 95 [Trichodesmium erythraeum IMSlOl] GenBank: ABG52128.1
  • Enzyme for MAA production encoded by the gene Tery 2969 97
  • Enzyme for MAA production encoded by the gene Tery 2967 101 [Trichodesmium erythraeum IMSlOl] GenBank: ABG52125.1
  • Enzyme for MAA production encoded by the gene Tery 2966 103 [Trichodesmium erythraeum IMSlOl] GenBank: ABG52124.1
  • Enzyme for MAA production encoded by the gene Tery 2977 158 [Trichodesmium erythraeum IMSlOl] GenBank: ABG52135.1
  • Heterologous gene encoding O-methyltransferase Tery 2976 159 [Trichodesmium erythraeum IMSlOl] GenBank: ABG52134.1
  • Enzyme for MAA production encoded by the gene Tery 2976 160 [Trichodesmium erythraeum IMSlOl] GenBank: ABG52134.1
  • Heterologous gene encoding ATP -grasp ligase Tery 2975 161 [Trichodesmium erythraeum IMSlOl] GenBank: ABG52133.1 Description SEQ ID NO:
  • Enzyme for MAA production encoded by the gene Tery 2975 162 [Trichodesmium erythraeum IMSlOl] GenBank: ABG52133.1
  • Enzyme for MAA production encoded by the gene mysA 164 [Cyanobacterium stanieri HL-69] GenBank: AUC61936.1
  • Enzyme for MAA production encoded by the gene mysB 166 [Cyanobacterium stanieri HL-69] GenBank: AUC61935.1
  • Enzyme for MAA production encoded by the gene mysC 168 [Cyanobacterium stanieri HL-69] GenBank: AUC61933.1
  • Enzyme for MAA production encoded by the gene mysD 170 [Cyanobacterium stanieri HL-69] GenBank: AUC61934.1
  • Enzyme for MAA production encoded by the gene mysD 172 [Nostoc verrucosum KU005] GenBank: BBC27544.1
  • At least some of the heterologous MAA genes to be expressed in cyanobacterial host cells can be codon improved for optimal expression in the target cyanobacterial strain.
  • the underlying rationale is that the codon usage frequency of highly expressed genes is generally correlated to the host cognate tRNA abundance. (Bulmer, Nature 325:728-730; 1987). Codon improvement (sometimes referred to as codon optimization or codon adaptation) can be performed to increase the expression level of foreign genes.
  • Codon improvement of the gene of interest can be performed for improved expression in the cyanobacterial host cell. Codon improvement can also be performed by adapting the codon usage of the at least one recombinant gene to the codon usage in Cyanobacterium sp., in particular ABICyanol and ABCyano4.
  • the G and/or C wobble bases in the codons for the amino acids in the at least one recombinant gene can be replaced by A and/or T because the GC content of the genome of ABICyanol and ABCyano4 is relatively low at about 36%, respectively.
  • codon improved In an embodiment, only 2% to 6% or 1% to 10% of the codons of variants of recombinant genes are codon improved. In another embodiment, highly codon improved variants of recombinant genes, at least 25%, to at least 50%, 65% or even at least 70% of the codons have been changed. In another embodiment, recombinant genes are used which are not codon improved.
  • Codon improvement of heterologously derived genes was conducted using the software Gene Designer (DNA 2.0, Menlo Park, CA), and was guided by the ABICyanol and ABCyano4 codon usage table derived from ribosomal proteins and highly expressed genes (such as photosynthesis genes).
  • OPTIMIZER a web server for optimizing the codon usage of DNA sequences Nucleic Acids Research 35(suppl 2):W126-W131) based on the codon-usage table derived from ABICyanol and ABCyano4 genomes.
  • the pre-optimized sequences were further modified with Gene Designer 2.0 (available at dna20.com /genedesigner2/) to ensure that their codon adaptation index is similar to the ABICyanol and ABCyano4 codon usage table (Sharp PM & Li W-H (1987).
  • the codon adaptation index is a measure of directional synonymous codon usage bias, and its potential applications, (see Nucleic Acids Research 15(3): 1281-1295).
  • the effective number of codons (see, Wright F (1990) Gene 87(l):23-29) are designed match those of highly expressed genes (such as ribosomal proteins) in the ABICyanol and the ABCyano4 genome.
  • the resulting polynucleotides using improved codons were further modified and optimized to avoid the presence of any known or predicted putative ABICyanol and ABCyano4 endonuclease restriction sites ⁇ Aval, BsaHI, Kasl, Xhol HgiOI, Hpall, EcoKY etc.); internal Shine-Dalgarno sequence and RNA destabilizing sequences; an internal terminator sequence; and a repeat sequence of greater than about 10 bp (see, Welch et al., PLOS One 4, e7002; 2009; and Welch et al., Journal of the Royal Society; Interface 6 (Suppl 4), S467-S476; 2009).
  • the selectable marker genes are also modified so that they will have improved expression in cyanobacteria.
  • the selectable marker gene that confers gentamycin or kanamycin resistance was codon optimized for higher expression in cyanobacteria.
  • the selectable marker gene that confers kanamycin resistance was codon optimized for higher expression in cyanobacteria.
  • the GC % of the antibiotic resistance genes decreased from 40-53% to 33-40%, which is similar to that of ABICyanol and to that of ABCyano4 coding genes (about 36% on average).
  • codon adaptation index of the codon improved antibiotic resistance genes is significantly improved from less than 0.4 to greater than 0.8, which is similar to that of ABICyanol and to that of ABCyano4 endogenous genes.
  • Table 2 depicts the codon usage statistics within the cyanobacterial strain ABICyanol, which is also valid for ABCyano4.
  • Table 2 Codon Usage - ABICyanol
  • the inserted genes can be controlled by one promoter, or they can be controlled by different individual promoters.
  • the promoters can be constitutive or regulatable.
  • the promoters can be, for example, inducible.
  • the promoter sequences can be derived, for example, from the host cell, from another organism, or can be synthetically derived.
  • Any desired promoter can be used to regulate the expression of the inserted MAA biosynthesis genes.
  • Exemplary promoter types include but are not limited to, for example, constitutive promoters, regulatable promoters such as inducible promoters (e.g., by nutrient source, nutrient starvation, heat shock, mechanical stress, environmental stress, metal concentration, specific metabolites, light exposure, etc.), endogenous promoters, heterologous promoters, and the like.
  • Suitable promoter sequences are also disclosed, for example, in U.S. Patent No. 9,315, 820, U. S. Patent No. 9,551,014, PCT/EP2012/067534, U.S. Patent No. 9,476,067, U. S. Patent No. 9, 157,101, PCT/US2013/077364, U.S. Patent No. 9,493,794, and PCT/US2015/000210, all of which are hereby incorporated by reference in their entireties.
  • the recombinant MAA biosynthesis gene(s) can be under the transcriptional control of a constitutive promoter.
  • a constitutive promoter can be endogenous to the cyanobacterial cell. This has the advantage that no recombinant transcription factor has to be present in the host cell.
  • the endogenous promoter is usually well-recognized by the metabolically enhanced cyanobacterial cell without the need to introduce further genetic modifications.
  • Suitable constitutive promoters include, without limitation, the PrpsL promoter
  • Suitable endogenous constitutive promoters from genes with unknown function exhibiting appropriate transcriptional activity include, without limitation, the promoters of Gene IDs ABICyano_orfl924, ABICyano_orfl997, ABICyano_orf3446, ABICyano_orf0865, ABICyano_orfl919, ABICyano_orf3278, ABICyano orfl 181, ABICyano_orfl627, ABICyano_orf0265 and ABICyano_orf2536, ABICyano_orf0615, and variants thereof.
  • the promoters can be derived from the cyanobacterial strain Cyanobacterium sp. PTA-13311, or they can be derived from another cyanobacterium or from another organism. In an embodiment, the promoters can be about 60%, 70%, 75%, 80%, 85%, 90%), 95%), 97%), 99%), or 100 % identical to the promoter sequences described herein.
  • the promoters can be regulatable promoters, such as inducible promoters.
  • inducible promoters For example, certain promoters are up-regulated by the presence of a compound, while other promoters can be up-regulated by the absence of a compound (also termed "repressible").
  • promoters that can be used include promoters that are regulatable by the presence (or in other promoters, by the absence) of inductors such as different metal ions, different nutrient sources, different metabolites, different external stimuli such as heat, cold, salinity or light.
  • the regulatable or inducible promoters are induced under conditions such as nutrient starvation, stationary growth phase, heat shock, cold shock, oxidative stress, salt stress, light, darkness, metal ions, organic chemical compounds, and combinations thereof.
  • a particularly tight control of the expression of gene can be achieved if a gene is under the transcriptional control of a Zn-, Ni-, Cu-, or Co-inducible promoter.
  • Exemplary Zn-regulatable promoters and their variants are described, for example, in International Application No. PCT/EP2013/077496.
  • Exemplary Zn, Ni, and Co-regulatable promoters are described, for example, in International Application No. PCT/2012/076790, both of which are incorporated by reference herein in their entireties.
  • the regulatable or inducible promoter is inducible by a change of a metal-ion concentration.
  • a change of metal-ion concentration includes for instance the addition or depletion of certain metal ions.
  • Suitable inducible promoters include, without limitation, the PziaA promoter, the PsmtA promoter, PaztA promoter, the PcorT promoter, the PnrsB promoter, the PpetJ promoter, the PpetE promoter, the Porf0316, the Porf01460 promoter, the Porf0221 promoter, the Porf0223 promoter, the Porf3126 promoter, the PmntC promoter, and variations thereof.
  • the promoter being inducible by a change of a metal-ion concentration can be inducible by Zinc-ions and/or Cu-ions, such as the PsmtA promoter or the petE promoter.
  • the regulatable or inducible promoter is endogenous to the cyanobacterial cell.
  • An endogenous inducible promoter is usually well-recognized by the metabolically enhanced cyanobacterial cell without the need to introduce further genetic modifications.
  • the choice of regulatable or inducible promoters can include, but are not limited to, PntcA, PnblA, PisiA, PpetJ, PpetE, PggpS, PpsbA2, PsigB, PlrtA, PhtpG, PnirA, PnarB, PnrtA, PhspA, PclpB l, PhliB, PcrhC, PziaA, PsmtA, PcorT, PnrsB, PnrsB916, PaztA, PbmtA, Pbxal, PzntA, PczrB, PnmtA, PpstS, and the like.
  • the regulatable or inducible promoter can, for instance, also be a nitrate inducible promoter.
  • Suitable nitrate inducible promoters include, without limitation, the PnirA promoter, the PnrtA promoter, the PnarB promoter, and variations thereof.
  • truncated or partially truncated versions of these promoters including only a small portion of the native promoters upstream of the transcription start point, such as the region ranging from -35 to the transcription start can often be used.
  • introducing nucleotide changes into the promoter sequence e.g. into the TATA box, the operator sequence, 5 '-untranslated region and/or the ribosomal binding site (RBS) can be used to tailor or optimize the promoter strength and/or its induction conditions, e.g. the concentration of inductor required for induction.
  • the different inducible promoters are inducible by different metal ions.
  • the promoters PhspA, PclpBl, and PhliB can be induced by heat shock (raising the growth temperature of the host cell culture from 30°C to 40°C), cold shock (such as, for example, reducing the growth temperature of the cell culture from 30°C to 20°C), oxidative stress (for example by adding oxidants such as hydrogen peroxide to the culture), or osmotic stress (for example by increasing the salinity).
  • the promoter PsigB can be induced by stationary growth, heat shock, and osmotic stress.
  • the promoters PntcA and PnblA can be induced by decreasing the concentration of nitrogen in the growth medium and the promoters PpsaA and PpsbA2 can be induced by low light or high light conditions.
  • the promoter PhtpG can be induced by osmotic stress and heat shock.
  • the promoter PcrhC can be induced by cold shock.
  • An increase in copper concentration can be used in order to induce the promoter PpetE, whereas the promoter PpetJ is induced by decreasing the copper concentration. Additional details of these promoters can be found, for example, in PCT/EP2009/060526, which is incorporated by reference herein in its entirety.
  • the promoters of any of the above embodiments may be selected from the endogenous inducible promoters identified in Cyanobacterium sp. with the ATCC accession number PTA-13311 ("ABICyanol”) as listed in table 3 A below, and variants thereof.
  • PrnpA AATAGTTGATAATTACTCGTTACTCATTACTCACTTAAACCTGCCACCTG 46
  • PrpsL CTCCGCTTAAAAAATTTCATTTTTCGATCAAAAAAGACAAATTATTACTA 47
  • PcpcB AACTTTAGATATTCGTAGTTGGCAATGTCGTAAATGCGGAACAATACAT 52
  • PcpcB*3 AACTTTAGATATTCGTAGTTGGCAATGTCGTAAATGCGGAACAATACAT 134
  • the promoters of any of the above embodiments may be selected from the endogenous inducible promoters identified in Cyanobacterium sp. with the ATCC accession number PTA-125253 ("ABCyano4") as listed in table 3B below, and variants thereof.
  • Table 3B Cyanohacterium sp. ABCyano4 endogenous promoter sequences
  • truncated or partially truncated versions of these promoters including only a small portion of the native promoters upstream of the transcription start point, such as the region ranging from -35 to the transcription start can often be used.
  • introducing nucleotide changes into the promoter sequence e.g. into the TATA box, the operator sequence and/or the ribosomal binding site (RBS) can be used to tailor or optimize the promoter strength and/or its induction conditions, e.g. the concentration of inductor required for induction.
  • Table 3C shows the nucleotide sequences of two intergenic regions IScpcBA and IScpcBA* 1 employed to enhance the expression of the enzymes involved in the production of MAAs as described herein.
  • the cyanobacterial strain ABICyanol or ABCyano4 is transformed with heterologous genes in order to produce the MAA.
  • Methods for producing a genetically enhanced, non-naturally occurring Cyanobacterium sp. and ABICyanol as well as ABCyano4 host cells are disclosed herein.
  • methods include introducing a recombinant nucleic acid sequence into a cyanobacterial host cell. At least one recombinant gene can be introduced into the host cells through the transformation of the host cell by an extrachromosomal plasmid.
  • the extrachromosomal plasmid can independently replicate in the host cell.
  • At least one recombinant gene can be introduced into the genome of the host cell. In yet another embodiment, at least one recombinant gene is introduced into the genome of the host cell by homologous recombination. In another embodiment, at least one recombinant gene can be introduced into the endogenous plasmids of the host cell. In yet another embodiment, at least one recombinant gene is introduced into the endogenous plasmids of the host cell by homologous recombination.
  • a recombinant gene is present on the chromosomal DNA of the cyanobacterial cell.
  • a recombinant gene is present on an extrachromosomal plasmid that can replicate independently from the chromosomes of the cyanobacterial cell.
  • the extrachromosomal plasmids described herein are present in high numbers in the host cells so that a compound of interest can be produced in a high yield.
  • Genetically enhanced cyanobacterial cells for example ABICyanol or ABCyano4 host cells, can include further genetic enhancements such as partial deletions of endogenous genes or other recombinant genes which can increase the overall yield of the compound being produced by the host cells. Examples of such genetic enhancements are described in PCT patent publication number WO 2009/098089 A2, which is hereby incorporated by reference in its entirety.
  • genetic enhancements of the genes encoding enzymes of the carbon fixation and subsequent carbohydrate metabolism can be genetically enhanced to further increase the production of a compound of interest.
  • Genetic enhancement targets include, but are not limited to, components of the photosystems (antennas and pigment modification), and components of the photosynthetic and respiratory electron transport systems.
  • Genetic enhancement targets include local and global regulatory factors including, but not limited to, the two component system, sigma factors, small regulating RNAs and antisense RNAs.
  • a recombinant nucleic acid sequence can be provided as part of an extrachromosomal plasmid containing cyanobacterial nucleic acid sequences in order to increase the likelihood of success for the transformation.
  • Cyanobacterium sp. host cell uses an extrachromosomal plasmid derived from an endogenous plasmid of the host cell to introduce a recombinant nucleic acid sequence into the host cell.
  • This endogenous plasmid can be, for example, an extrachromosomal plasmid derived from the 6.8 kb endogenous plasmid of ABICyanol .
  • the cyanobacterial strain ABICyanol is transformed with at least one heterologous gene in order to produce the desired MAA. It has been found that the use of modified endogenous plasmids improves the stability of the plasmid in the host cell.
  • the cyanobacterial strain ABICyanol contains three endogenous plasmids. In combination with other genotypic and phenotypic attributes, these endogenous plasmids differentiate ABICyanol from other cyanobacterial species.
  • One plasmid is 6,826 base pairs (SEQ ID NO: 16), another is 39,702 base pairs, and a third plasmid is 28,554 base pairs.
  • the 6,826 bp endogenous plasmid is alternatively referred to herein as pABICyanol, p6.8 or 6.8.
  • plasmid 6.8 has been modified in vivo and in vitro for use as a plasmid vector containing genes of interest for the production of compounds of interest.
  • a modified endogenous vector derived from p6.8 from ABICyanol was developed.
  • the modified endogenous vector from ABICyanol can be used to transform cyanobacteria from a broad range of genera, including ABICyanol itself.
  • the cyanobacterial strain ABICyano4 is transformed with at least one heterologous gene in order to produce the desired MAA. It has been found that the integration of heterologous genes into the endogenous plasmids improves expression of the respective enzymes.
  • the cyanobacterial strain ABCyano4 comprises three endogenous plasmids, which, in combination with other genotypic and phenotypic attributes, differentiate ABICyano4 from other Cyanobacterium species.
  • One plasmid herein termed "pABCyano4B” is 37990 base pairs.
  • a second, smaller plasmid "pABCyano4C” is 31678 base pairs.
  • both plasmids can be used for integration of heterologous genes via homologous recombination.
  • the present invention includes the p6.8 plasmid or the pABICyanolB plasmid, and modified vectors comprising sequences of these plasmids.
  • the modified endogenous vector contains at least one of the following: a recombinant gene that encodes an enzyme involved in MAA production; and an origin of replication suitable for replication in ABICyanol .
  • plasmids that can be used to carry genes of interest in several strains of cyanobacteria (such as, for example, Synechocystis, Synechococcus PCC7002 and Anabaena), are the RSFlOlO-based shuttle plasmids (extrachromosomal broad-host vector) such as pVZ321- 6 series (described, for example, in PCT/EP2009/060526 and in PCT/EP2009/000892, both of which are incorporated herein by reference in their entireties). Further, the plasmid vector described in U.S. Patent NO: 9,476,067 can be utilized to carry heterologous genes of interest in many cyanobacterial species.
  • a gene coding for a replication initiation factor that binds to the origin of replication can either be present on the modified endogenous vector or can be present in the chromosomes or other extrachromosomal plasmids of ABICyanol .
  • An origin of replication suitable for replication in ABICyanol and the gene coding for the replication initiation factor binding to that origin of replication ensure that the modified endogenous vector can be replicated in ABICyanol .
  • the nucleotide sequence of an origin of replication of the modified endogenous plasmid vector can have at least 80%, 90%, and 95% identity or can be identical to the nucleotides 3375 to 3408 of the sequence of the endogenous 6.8 kb plasmid (SEQ ID NO: 16).
  • the sequence of the gene coding for the replication initiation factor has at least 80%, 90%, and 95% identity or is identical to nucleotides 594 to 3779 of the sequence of the endogenous 6.8 kb plasmid (SEQ ID NO: 16).
  • the gene coding for the replication initiation factor codes for a protein having at least 80%, 90%, and 95% sequence identity or is identical to the protein coded by nucleotides 594 to 3779 of the sequence of the endogenous 6.8 kb plasmid (SEQ ID NO: 16) of ABICyanol .
  • This putative initiation replication factor is thought to bind to the putative origin of replication, thereby ensuring the replication of a plasmid containing the initiation factor in ABICyanol .
  • a modified endogenous plasmid vector can contain a sequence having at least 50%, at least 55 %, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%), at least 85%, at least 90% or at least 95% identity to the sequence of the endogenous 6.8 kb plasmid (SEQ ID NO: 16).
  • the modified endogenous vector contains the entire p6.8 endogenous plasmid from ABICyanol .
  • gene delivery vehicles that are developed using the endogenous 6.8 kb plasmid (or a portion of the plasmid) containing characteristic portions of the endogenous 6.8 kb plasmid may be able to be efficiently transformed into a wide range of cyanobacteria.
  • characteristic portions of the 6.8 kb endogenous plasmid from ABICyanol include portions that enable it to replicate in a host cell (origin of replication and replication initiation factor, for example) and can be referred to as the backbone of the endogenous 6.8 kb plasmid.
  • Such vectors may also be able to efficiently produce heterologous proteins and other compounds of interest in cyanobacterial cultures.
  • modifications starting with the backbone of the 6.8 kb endogenous plasmid from ABICyanol are performed individually or together to increase transformation efficiency, increase the replication rate within the cell, and to increase the production of a desired product from the cyanobacterial cell.
  • Suitable modifications include, for example, insertion of selection markers (such as antibiotic resistance genes), recombinant genes or cassettes for the production of a desired compound, and other modifications to increase the expression or stability of the plasmid in the cyanobacterial cell.
  • the invention includes cyanobacteria, e.g. ABICyanol, comprising a modified p6.8 plasmid having any of these improved characteristics.
  • the modular design of the p6.8 derived vector allows complex sequence manipulation in cyanobacteria.
  • Table 4A ABICyanol p6.8 and pABICyanolB derived plasmids for production of an MAA
  • Table 4C Plasmids for production of a MAAs in Escherichia coli
  • the method for producing a genetically enhanced cyanobacterial cell involves protecting the recombinant nucleic acid sequence, for example a plasmid, against endogenous restriction endonucleases of the host cell by methylating at least a part of the recombinant nucleic acid sequence or modifying and/or eliminating the recognition sequences of the endogenous restriction endonucleases.
  • endogenous restriction endonucleases of ABICyano 1 and ABCyano4 for example, can cut an extrachromosomal plasmid carrying recombinant genes, thereby preventing genetic transformation of this host cell.
  • methyltransferases for example M. Aval, M. Acy ⁇ and M. Mspl can be used to protect recombinant vector extrachromosomal plasmids.
  • the plasmids can either be incubated with the methyltransferases in vitro or a helper plasmid can be present in a helper E. coli strain in order to methylate the extrachromosomal plasmids in vivo before conjugation takes place during the transformation of ABICyanol, ABCyano4 or other cyanobacterium.
  • recognition sequences for the restriction enzymes can be modified or deleted.
  • plasmid pRL528 can be used as a helper plasmid for conjugal transfer.
  • the indicated genes are M. Aval coding for the methyltransferase protecting against the restriction endonuclease Aval and the respective gene coding for M. Avail. The latter is not required for transformation of ABICyanol, as it lacks any endonuclease activity of Avail.
  • M. Mspl from Moraxella sp. ATCC 49670 is required for transformation of ABCyano4.
  • the vector to be transformed into Cyanobacterium sp. host cells can be modified to integrate into the cyanobacterial chromosome by adding an appropriate DNA sequence homologous to the target region of the host genome.
  • the vector to be transformed can be modified to integrate into the cyanobacterial chromosome through in vivo transposition by introducing mosaic ends to the vector. Once the plasmid is established in the host cell, it can be present, for example, at a range of from 1 to many copies per cell.
  • an endogenous plasmid derived from ABICyanol can be modified, either in vivo or in vitro, to be a plasmid vector capable of introducing exogenous genes encoding enzymes for the production of a compound or compounds of interest into a wide range of host cyanobacterial cells such as ABICyanol, Cyanobacterium sp., or other cyanobacterial genera such as Synechocystis and Synechococcus.
  • the ABICyanol 6.8 kb endogenous plasmid is used as a backbone for a plasmid vector used for transformation of Cyanobacterium sp. Since this is the endogenous vector from the species, it is likely to be more stable when transformed into the cell than plasmids derived from completely different organisms.
  • the entire p6.8 endogenous plasmid is inserted into a vector used for transformation.
  • a sequence of about 50%, 70%, 75%, 80% 85%, 90%, 95%, 98%, 99%, or 99.5% identity to the entire endogenous plasmid sequence is inserted into the extrachromosomal plasmid vector.
  • a modified p6.8 vector is designed to have several modular units that can be swapped out using specific restriction enzymes. Promoters, genes of interest, selectable markers, and other desired sequences can be moved in and out of the vector as desired. This modular design makes genetic experiments faster and more efficient.
  • the modified p6.8 vector can replicate in both cyanobacteria and in E. coli.
  • the vector contains a replication unit that can function in a broad range of cyanobacterial genera.
  • the vector also contains a replicon for propagation in E. coli for ease of cloning and genetic manipulation using E. coli.
  • a plasmid shuttle vector which is characterized by being replicable in both E. coli and cyanobacterial species.
  • the plasmid comprises a promoter capable of functioning in cyanobacteria and E. coli and a DNA sequence encoding a sequence capable of functioning as a selective marker for both E. coli and cyanobacteria.
  • the shuttle vector includes two different promoter systems, one functioning in cyanobacteria and the other one functional in E. coli.
  • the plasmid shuttle vector contains at least 50% of the p6.8 plasmid. The plasmid shuttle vector enables the efficient transformation of cyanobacteria and the expression of recombinant genes of interest.
  • the p6.8 derived plasmid vector also contains an origin of transfer (oriT) which is suitable for conjugation.
  • the plasmid vector can contain a combined origin of replication and an origin of transfer (oriVT), which enables replication in Enterobacteriaceae, in particular E. coli, and which also enables conjugation with, for example, an E. coli donor strain and Cyanobacterium sp., in particular ABICyanol as a recipient strain.
  • Such an plasmid vector can be used for bi- or triparental mating wherein a conjugative plasmid present in one bacterial strain assists the transfer of a mobilizable plasmid, for example a plasmid vector disclosed herein, present in a second bacterial strain into a third recipient bacterial strain, which can be ABICyanol.
  • a non-naturally occurring plasmid vector in which a gene encoding an enzyme involved in MAA production is operably linked to a shuttle vector.
  • cyanobacterial cells are transformed with the recombinant shuttle vector.
  • the recombinant shuttle vector is relatively small in size, relatively stable in a cyanobacterial host cell, and can replicate in a variety of cyanobacterial species. This recombinant vector is useful for expressing a variety of heterologous genes in cyanobacteria.
  • a shuttle vector expresses a codon-optimized antibiotic resistance gene (Ab R ), such as codon improved kanamycin or gentamycin resistance genes.
  • the shuttle vector is constructed based on a modular basis so that all of the key elements (replication origin, Ab R gene and reporter gene) are exchangeable via unique restriction sites thus providing versatile cloning options and facilitating the delivery of genes of interest to target organisms.
  • Other antibiotic resistance genes can be used if desired. For example, genes conferring resistance to ampicillin, chloramphenicol, erythromycin, zeocin, spectinomycin or other antibiotics can be inserted into the vector, under the control of a suitable promoter.
  • the vector contains more than one antibiotic resistance gene.
  • the plasmid is modified by several factors so that it is capable of efficient replication in multiple types of cyanobacterial species.
  • the vector has also been organized so that various sequences can be easily replaced with other desired sequences as needed.
  • a construct having a different gene (or genes) of interest, a different antibiotic, a different promoter, etc. can be made with relative ease.
  • the modified vector allows for rapid testing of various heterologous constructs in a cyanobacterial cell.
  • RSF1010 The transfer of exogenous genes into cyanobacteria often involves the construction of vectors having a backbone from a broad-host range bacterial plasmid, such as RSF1010.
  • the RSFlOlO-based vector has been widely used as a conjugation vector for transforming bacteria, including cyanobacteria (Mermet-Bouvier et al. (1993) "Transfer and Replication of RSF1010- derived Plasmids in Several Cyanobacteria of the Genera Synechocystis and Synechococcus" Current Microbiology 27:323-327).
  • RSF1010 has a E. coli origin of replication, but does not have a cyanobacterial origin of replication.
  • cyanobacteria disclosed herein can be transformed to add biochemical pathways to produce the MAAs of interest.
  • Recombinant DNA sequences encoding genes can be amplified by PCR using specific primers or produced by gene synthesis.
  • the amplified PCR fragments can then be digested with the appropriate restriction enzymes and cloned into either a self-replicating plasmid or into an integrative plasmid.
  • An antibiotic resistance cassette for selection of positive clones can be present on the appropriate plasmid.
  • the recombinant nucleic acids of interest can be amplified from nucleic acid samples using known amplification techniques.
  • PCR can be used to amplify the sequences of the genes directly from mRNA, from cDNA, from genomic libraries or cDNA libraries.
  • PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, and for nucleic acid sequencing.
  • recombinant DNA vectors suitable for transformation of cyanobacteria can be prepared.
  • a DNA sequence encoding one or more of the genes described herein can be combined with transcriptional and other regulatory sequences which will direct the transcription of the sequence from the gene in the transformed cyanobacteria.
  • recombinant genes of interest are inserted into the cyanobacterial chromosome.
  • the gene insertions can be present in all of the copies of the chromosome, or in some of the copies of the chromosome.
  • recombinant genes are present on an extrachromosomal plasmid.
  • the extrachromosomal plasmid can be derived from an outside source such as RSF1010- based plasmid vectors, for example, or can be derived from an endogenous plasmid from Cyanobacterium sp. host cells or from other cyanobacteria.
  • recombinant genes are present on an extrachromosomal plasmid having multiple copies per cell.
  • the plasmid can be present, for example, at about 1, 3, 5, 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or more copies per cyanobacterial host cell.
  • the recombinant plasmids are fully segregated from the non-recombinant plasmids.
  • recombinant genes are present on one cassette driven by one promoter. In another embodiment, the recombinant genes are present on separate plasmids, or on different cassettes.
  • recombinant genes are modified for optimal expression by modifying the nucleic acid sequence to accommodate the cyanobacterial cell's protein translation system. Modifying the nucleic acid sequences in this manner can result in an increased expression of the genes. Transformation of Cyanobacterial Cells
  • Some strains of cyanobacteria can be transformed through natural uptake of exogenous DNA.
  • Other cyanobacterial strains can be transformed, for example, by the use of conjugation or electroporation.
  • Some cyanobacterial strains are difficult to transform by any known means. For many of these types of difficult to transform strains, specific methods of preparing the cells for transformation, as well as specific methods of allowing entry of the foreign DNA into the cells, need to be designed de novo.
  • Exemplary cyanobacteria that can be transformed include, but are not limited to,
  • Synechocystis Synechocystis, Synechococcus, Acaryochloris, Anabaena, Arthrospira, Thermosynechococcus, Chamae siphon, Chroococcus, Cyanobacterium, Cyanobium, Dactylococcopsis, Gloeobacter, Gloeocapsa, Gloeothece, Microcystis, Prochlorococcus, Prochloron, Chroococcidiopsis, Cyanocystis, Dermocarpella, Myxosarcina, Pleurocapsa, Stanieria, Xenococcus, Arthrospira, Borzia, Crinalium, Geitlerinema, Halospirulina, Leptolyngbya, Limnothrix, Lyngbya, Microcoleus, Cyanodictyon, Aphanocapsa, Oscillatoria, Planktothrix, Prochlorothr
  • Exemplary methods suitable for transformation of cyanobacteria include, as nonlimiting examples, natural DNA uptake (Chung, et al. (1998) FEMS Microbiol. Lett. 164: 353- 361; Frigaard, et al. (2004) Methods Mol. Biol. 274: 325-40; Zang, et al. (2007) J. Microbiol. 45: 241-245), conjugation, transduction, glass bead transformation (Kindle, et al. (1989) J. Cell Biol. 109: 2589-601; Feng, et al. (2009) Mol. Biol. Rep. 36: 1433-9; U.S. Pat. No.
  • Biotechnol. 78: 729-35 laser-mediated transformation, or incubation with DNA in the presence of or after pre-treatment with any of poly(amidoamine) dendrimers (Pasupathy, et al. (2008) Biotechnol. J. 3 : 1078-82), polyethylene glycol (Ohnuma, et al. (2008) Plant Cell Physiol. 49: 117-120), cationic lipids (Muradawa, et al. (2008) J. Biosci. Bioeng. 105: 77-80), dextran, calcium phosphate, or calcium chloride (Mendez-Alvarez, et al. (1994) J. Bacterid.
  • the cyanobacterial strains ABICyanol and ABCyano4 are used as the host cell for production of the MAA.
  • electroporation can also be used for successful transformation of ABICyanol and ABCyano4, although at a somewhat lower efficiency.
  • Strain-specific adaptations of standard electroporation protocols may be made to avoid DNA digestion by endogenous restriction enzymes and to allow DNA entry through the extracellular polysaccharide layer (CPS layer) of ABICyanol host cells.
  • CPS layer extracellular polysaccharide layer
  • DNA may be protected against endogenous restriction enzymes by methylation.
  • ABICyanol cells Prior to electroporation using techniques well known in the art, ABICyanol cells may be pretreated with positively charged polyaminoacids such as poly-L-lysine hydrobromide or poly-L-ornithine hydrochloride or combinations thereof (in particular poly-L-lysine hydrobromide) in order to increase the DNA uptake efficiency.
  • positively charged polyaminoacids such as poly-L-lysine hydrobromide or poly-L-ornithine hydrochloride or combinations thereof (in particular poly-L-lysine hydrobromide) in order to increase the DNA uptake efficiency.
  • the presence of a foreign gene encoding antibiotic resistance in a cell is selected by placing putatively transformed cells into a media containing an amount of the corresponding antibiotic and selecting cells that survive. The selected cells are then grown in the appropriate culture medium to allow for further testing.
  • colony PCR methods are used to confirm transformants.
  • three primer sets are used and are directed against parts of the shuttle vectors to detect specific fragments of the shuttle vector.
  • Transformants exhibiting the predicted PCR products are analyzed further by plasmid rescue.
  • plasmid rescue a 25 mL liquid culture is subjected to DNA isolation.
  • plasmid rescue 500 ng to 1 ⁇ g of isolated DNA from transformants containing the transformed plasmids is re-transformed into E. coli and usually results in approximately 10-20 transformants per transformation implemented. Plasmid DNA of ten E. coli colonies is isolated and analyzed by PCR using specific primers for the transformed plasmids. The plasmid DNA is further analyzed with specific restriction enzymes and then sequenced.
  • a method of producing an MAA in a cyanobacterial cell or any host cell disclosed herein comprises:
  • step a) further comprises adding an amino acid to the culture medium.
  • an amino acid such as glycine, serine, threonine, alanine for the production of mycosporine-2-glycine, shinorine, porphyra-334, and mycosporine-glycine-alanine (See Fig. 40).
  • step b) at least 50%, preferably at least 60% more preferred at least 65% of the MAA produced is associated with the cyanobacterial cell and wherein in step b) the MAA is isolated from the cells by separating the cells from the culture medium and isolating the MAA from the cells.
  • the cyanobacterial cells employed in the methods have a CPS and the MAA is associated with the CPS of the host cells.
  • some cyanobacterial host cells such as Cyanobacterium sp. ABCyano4 or to a lesser extent Cyanobacterium sp. ABICyanol have a thick CPS sheath, wherein a large fraction of the MAA produced stays associated with the host cells and only a minor fraction is secreted into the culture medium.
  • step b) the
  • CPS is removed from the cells, thereby removing the MAA from the cells. This can be done for example by employing methanol-water mixtures.
  • an enzyme involved in MAA production is synthesized in cyanobacterial cultures by preparing host cyanobacterial cells having the gene constructs discussed herein, and growing cultures of the cells.
  • Methods of growing cyanobacteria in liquid media and on agarose-containing plates are well known to those skilled in the art (see, e.g., websites associated with ATCC). Any of these methods or media maybe used to culture the cyanobacterial cells.
  • a number of known recipes for cyanobacterial growth medium can be used.
  • BG11 medium is used, see Stanier, R.Y., et al., Bacteriol. Rev. 1971, 35: 171-205, which is hereby incorporated by reference.
  • the cyanobacterial strain is a fresh water strain, and BG11 is used.
  • the cyanobacteria culture grows best in a marine (salt water) medium, by adding an amount of salt to the BG11 medium.
  • marine BG11 mBGl 1 contains about 35 practical salinity units (psu), see Unesco, The Practical Salinity Scale 1978 and the International Equation of State of Seawater 1980. Tech. Pap. Mar. Sci., 1981, 36: 25 which is hereby incorporated by reference.
  • the cells are grown autotrophically, and the only carbon source is CO2 or bicarbonate. In another embodiment, the cells are grown mixotrophically, for example with the addition of another carbon source such as glycerol or glucose.
  • the cultures can be grown indoors or outdoors.
  • the light cycle can be set as for continuous light, or for periodic exposure to light, e.g., 16 hours on and 8 hours off, or 14 hours on and 10 hours off, or 12 hours on and 2 hours off, or any alternative variation of on and off hours of light comprising about a day.
  • the cultures can be axenic, or the cultures can also contain other contaminating species.
  • the cyanobacteria are grown in enclosed bioreactors in quantities of at least about 1 L, 20L, 50L, 100 L, 500 L, 1000 L, 2000 L, 5000 L, or more. In a preferred embodiment the bioreactors are about 20 L to about 100 L. In an embodiment, the cyanobacterial cell cultures are grown in disposable, flexible, and tubular photobioreactors made of a clear plastic material.
  • cultures are grown indoors or outdoors with continuous light, in a sterile environment.
  • the cultures are grown outdoors in an open pond type of photobioreactor.
  • the choice of culture medium can depend on the cyanobacterial species.
  • the following BG11 medium for growing cyanobacteria can be used.
  • artificial seawater recipe Table 5
  • ASW BG11 artificial seawater recipe
  • Restriction endonucleases were purchased from Thermo Fisher Scientific, unless otherwise noted. PCR was performed using a Biometra thermocycler (Biometra, Germany), using Phire Plant Direct PCR Master Mix polymerase or Taq DNA polymerase (Thermo Fisher Scientific) for diagnostic amplifications, and Phusion polymerase for high fidelity amplifications. PCR temperature profiles were set up as recommended by the polymerase manufacturer. Cloning was performed in E. coli using NEB 10-beta or NEB Turbo competent cells (New England Biolabs) following the manufacturer's protocol. NEBuilder HiFi assembly kits were purchased from New England Biolabs. Gene synthesis was conducted by GeneArt Gene Synthesis (Thermo Fisher Scientific).
  • E. coli strains HB 101 Promega
  • NEB 10 and NEB Turbo New Endland Biolabs
  • Epicentre E. coli strains HB 101 (Promega), NEB 10 and NEB Turbo (New Endland Biolabs), andEClOOD (Epicentre)) were grown in Luria-Bertani (LB) medium at 37 °C.
  • Carbencillin 100 ⁇ g/mL
  • kanamycin 50 ⁇ g/mL
  • chloramphenicol 34 ⁇ g/mL
  • zeocin 25 ⁇ g/ml
  • streptomycin 25 ⁇ g/ml
  • ABICyanol and ABCyano4 were cultured at from 28 °C to 37 °C in liquid BGl l fresh water medium in bubbled bottles under continuous illumination or 12h/12h day/night cycles of approximately 30 - 40 ⁇ photons*m "2 *sec _1 .
  • Plasmid DNA from E. coli strains was isolated using a GeneJet Plasmid Miniprep
  • Kit Fermentas according to the manufacture's protocol.
  • total DNA was prepared according to Saha et al. (2005), World Jour. Microbiol Biotechnol 21 : 877-881.
  • BG-11 stock solution was purchased from Sigma
  • BG-11 Marine BG-11 (MBG-11) was prepared by dissolving 35 g Instant Ocean (United Pet Group, Inc, Cincinnati, OH) in 1 L water and supplementing with BG-11 stock solution. Vitamin B 12 (Sigma Aldrich) was supplemented to MBG-1 1 to achieve a final concentration of 1 ⁇ g/L, as needed. Stock solutions of the antibiotics were purchased from Sigma Aldrich or Carl Roth GmbH).
  • the ABICyanol and ABCyano4 transformants were selected on solid BG11 medium containing 10 - 20 ⁇ g/mLof the appropriate antibiotic.
  • the endogenous 6.8 kb plasmid of ABICyanol (p6.8) can be used as a means of shuttling exogenous DNA to cyanobacterial host cells.
  • an origin of replication that is effective in E. coli such as R6Kori or oriVT
  • the p6.8 kb plasmid DNA can be manipulated in bacteria, such as E. coli, to incorporate genes and sequences of interest into a recombinant p6.8 kb.
  • modifications to decrease the effectiveness of endogenous restriction systems that are present in ABICyanol, such as methylation can be performed.
  • ABICyanol prior to conjugation.
  • the method involves several steps: treatment of cells with N- acetylcysteine (NAC), washing steps that utilize NaCl, treatment with lysozyme, and subsequent washing followed by a conjugation procedure.
  • NAC N- acetylcysteine
  • the cell pellet was resuspended in 0.5 M sucrose and incubated 60 min at room temperature (RT) with slow shaking (85 rpm). Then, cells were centrifuged and resuspended in 40 mL of a solution containing 50mM Tris (pH 8.0), 10 mM EDTA (pH 8.0), 4% sucrose, and 20-40 ⁇ g/mL lysozyme.
  • Triparental mating was performed as follows: E. coli strain J53 bearing a conjugative RP4 plasmid and E. coli strain HB101 bearing the cargo to be introduced into ABICyanol and the pRL528 helper plasmid (for in vivo methylation) were used. E. coli strains were grown in LB broth supplemented with the appropriate antibiotics overnight at 37 °C with shaking at 100 rpm. An aliquot of 3 - 5 mL of each culture was centrifuged, washed twice with LB medium and resuspended in 200 ⁇ LB medium. Subsequently, t e E.
  • coli strains were mixed, centrifuged and resuspended in 100 ⁇ BGl 1 medium.
  • Two hundred mL of exponentially growing cyanobacterial culture (OD750nm of greater than 0.5 and less than 1.0) was centrifuged (3000 rpm, 10 min), pretreated as described in Example 4, and subsequently washed and resuspended in 400 ⁇ BGl l culture medium containing Tris/sucrose buffer (Example 4).
  • a 100 ⁇ aliquot of resuspended cyanobacterial and E. coli cultures was mixed and applied onto a membrane filter (Millipore GVWP, 0.22 ⁇ pore size) placed on the surface of solid BGl 1 medium supplemented with 5% LB.
  • Petri dishes were incubated under dim light (5 ⁇ / ⁇ 2 - ⁇ & ⁇ ) for 2 days. Cells were then resuspended in fresh BGl l medium and plated onto selective medium containing 10 and 15 ⁇ g/mL kanamycin, respectively. The following selection conditions were used: light intensity of approximately 20 - 40 ⁇ / ⁇ 2 - ⁇ & ⁇ at a temperature of approximately 28 °C. Transformants were visible after approximately 7-10 days. The transformant colonies were then plated on BGl 1 media containing 15 ⁇ g/mL kanamycin and then transferred stepwise to higher kanamycin concentrations (up to kanamycin 60 ⁇ g/mL) to aid in the selection process.
  • Electroporation can also be used for successful transformation of Cyanobacterium sp. ABICyanol and ABCyano4, or other strains such as Arthrospira, Synechococcus, and Synechocystis, using, for example, the same plasmids as for conjugation, but with lower efficiency.
  • strain-specific adaptations of standard electroporation protocols can be made to avoid DNA digestion by endogenous restriction enzymes and to allow DNA entry through the CPS layer. To achieve successful electroporation, DNA is protected against endogenous restriction enzymes by methylation.
  • ABICyanol and ABCyano4 cells Prior to electroporation, ABICyanol and ABCyano4 cells are pretreated with positively charged polyaminoacids such as poly-L-lysine hydrobromide or poly-L-ornithine hydrochloride or combinations thereof (in particular poly-L-lysine hydrobromide) in order to increase the DNA uptake efficiency.
  • positively charged polyaminoacids such as poly-L-lysine hydrobromide or poly-L-ornithine hydrochloride or combinations thereof (in particular poly-L-lysine hydrobromide) in order to increase the DNA uptake efficiency.
  • ABICyanol or ABCyano4 cultures were harvested, washed and resuspended in 0.9 % NaCl containing 25 mM Tris-HCl (pH 8.0). Poly-L-lysine hydrobromide was added to the resuspended cells to obtain a final concentration of 50 ⁇ g/mL.
  • ABICyanol or ABCyano4 cells were then incubated for several hours or overnight before electroporation.
  • the cells were washed sequentially once more with 1 mM HEPES and ETMT buffer containing 0.1 mM HEPES, 0.2 mM K2HPO4 and 0.2 mM MgCk.
  • the cells were harvested by centrifugation at 15,000 x g for 5 min. All of the washes and centrifugations were carried out on ice or in a pre-chilled centrifuge (4 °C). For each electroporation procedure, 3 ⁇ g methylated DNA is added to 100 ⁇ of concentrated cells.
  • BGl 1 medium was immediately added to the cyanobacterial suspension, which was subsequently transferred to a 50 mL flask containing 15 mL fresh BGl 1 medium.
  • E. coli strains were generally grown in LB broth supplemented with the appropriate antibiotics overnight at 37 °C with shaking at 100 rpm. An aliquot of 3-5 ml of each culture was centrifuged, washed twice with LB medium and resuspended in 200 ⁇ LB medium.
  • the E. coli strains were mixed, centrifuged and resuspended in 100 ⁇ BGl l medium.
  • a 100 ⁇ aliquot of the resuspended cyanobacterial cells and the E. coli cultures was mixed and applied onto a membrane filter (Millipore GVWP, 0.22 ⁇ pore size) placed on the surface of solid BGl 1 medium supplemented with 5% LB.
  • Petri dishes were incubated under dim light of 5 ⁇ photons m "2 s "1 for two days.
  • Cells were then resuspended in fresh BGl 1 medium and plated onto selective medium containing > 10 ⁇ g/ml of the respective antibiotic.
  • the following selection conditions were used: light intensity approximately 20 - 40 ⁇ photons m "2 sec "1 at a temperature of approximately 30 °C. Transformants were visible after approximately 3- 4 days. The transformant colonies were then stepwise transferred to higher antibiotic concentrations.
  • a plasmid construct was prepared by inserting 3 genes from Anabaena variabilis
  • ABICyanol strain # AB 1226 The ABICyanol strain containing this plasmid is named ABICyanol strain # AB 1226.
  • the plasmid was transformed to ABICyanol host cells, and the transformation was confirmed. Cultures of the modified cells were grown in a GC vial experiment to confirm that the MAA was being produced (FIG. 10B).
  • the plasmids were transformed to ABICyanol host cells, and the transformation was confirmed by PCR.
  • the Cu inducible strain AB 1224, the Zn inducible strain AB1225 and the constitutive MAA expressing strain AB 1226 (previous example) were grown in GC vials for 24 hours, and the absorbance spectra of the cultures were examined (FIG. 10A and 10B).
  • Several AB 1226 clones were found to produce high MAA peaks at 310 nm, while the strains AB1224 (Cu inducible) and AB1225 (Zn-inducible) appeared to produce less MAA.
  • AB1226 was grown in small (375 mL) indoor vertical photobioreactors under a 12 hour on/12 hour off light cycle, using a light level set at 350 ⁇ /m 2 - sec. AB 1226 was shown to produce mycosporine-glycine over a cultivation period of 40 days, resulting in the production of 250 mg/L mycosporine-glycine after 30 days (Fig. IOC). Over the whole production period up to 300 mg/L mycosporine-glycine was produced.
  • Zn was present (about 30 mg/L throughout the run), the addition of Zn (as Zn-EDTA) resulted in a high production of mycosporine-glycine: by 15 days, about 140 mg/L product was produced. At about 20 days, about 160 mg/L of the mycosporine-glycine product was produced. Although the induction with ZnS0 4 appeared to produce less mycosporine-glycine than induction with Zn- EDTA, the production was almost the same by the end of the run. This resulted in a productivity of about 6% MAA per dry weight of algae.
  • Zn added without the EDTA produced about 83 mg/L at 15 days, and about 130 mg/L by 20 days.
  • the Zn-EDTA culture was allowed to continue growing to better understand the stability of the system.
  • the culture was diluted with fresh medium, and the Zn- EDTA culture continued to produce high amounts of mycosporine-glycine through the 50 day test period.
  • Another MAA "mycosporine-2-glycine", which has 2 amino acid moieties attached to the central ring (see FIG. 12A and FIG. 12B), was prepared by starting with a plasmid that contained the genes for the production of mycosporine-glycine (such as shown above in Examples 6-9), and further inserting an additional gene encoding the mysD gene (ap_3855) from Aphanothece halophytica, linked to a zinc-inducible promoter, and localized on a second endogenous plasmid pABCyanolB from the ABICyanol strain.
  • AB1277 and AB 1324 by locating the several MAA pathway genes on one extrachromosomal plasmid (plasmid #2840 and #2893, as shown in FIG. 3 and Table 4) instead of divided between two plasmids as shown in the above example.
  • the MAAs "shinorine” and “porphyra-334 were produced in cyanobacterial host cells using the following method.
  • the modified cyanobacterial host cells that already have the mysABC genes for the production of mycosporine-glycine (such as shown above in Examples 6- 9), were then transformed with a second endogenous plasmid pABICyanolB that contains one additional gene: either mysD from Nostoc punctiforme (NpF5597) or Ava_3855, which encodes a nonribosomal peptide synthetase ( RPS) from Anabaena variabilis (see FIG. 12A and FIG. 12B).
  • Cyanobacterium sp. ABICyanol cells harboring the genes for mycosporine, shinorine/porphyra-334, or mycosporine-2-glycine were grown in ASW-BG11 (35 psu) medium in 1.2 liter indoor vertical photobioreactors under a 12 hour on/12 hour off light cycle, using a light level set at 350 The starting OD750 was 0.2.
  • the culture was aerated, and the pH was set at 7.3, with automated adjustment of pH by the addition of 15% CO2 as needed.
  • the nitrogen source was 17.3 mM nitrate.
  • the graph shows that the mycosporine-glycine producing culture produced the most MAA over the length of the run (FIG. 13 A, FIG. 13B), although the mycosporine-2-glycine producing culture was about the same by the end of the run, at about 200 mg/L at 26 days.
  • the mycosporine-glycine producing culture produced about 6% of the cell dry weight at about day 15.
  • the shinorine/porphyra-334 producing culture produced about 110 mg/L at 26 days.
  • FIG. 13C shows that over time, the MAAs accumulate in the culture medium. By the end of the run at 26 days, about 85% to 90 % of the MAA was present in the culture medium.
  • the mycosporine-glycine producing cells (transformed with a single plasmid, #2840; Strain #AB1277; see Table 4) from Example 11 were also examined for MAA production.
  • the cells were grown in BASW (35 psu) medium in a 1.2 liter indoor vertical photobioreactors under a 12 hour on/12 hour off light cycle, using a light level set at 350 The starting OD750 was 0.2.
  • the culture was aerated, and the pH was set and maintained at 7.3, with adjustment by the addition of 15% CO2 as needed.
  • the nitrogen source was 17.3 mM nitrate.
  • the resulting MAA production over time is shown in FIG. 14.
  • another MAA is produced in cyanobacteria by the following method.
  • the biosynthetic pathway for the MAA is determined, and suitable enzymes are identified by analysis of the genome sequence from another cyanobacterial species.
  • the genes, linked to constitutive or inducible promoters, are used to transform a suitable cyanobacterial strain, such as ABICyanol .
  • the modified cells are grown and the production of the desired MAA is confirmed.
  • the cells are then scaled-up to a desired culture volume, and grown for a suitable number of days.
  • the MAA is then isolated, purified, and quantified.
  • the MAA so produced is used in skin care and cosmetic products.
  • a culture of modified cyanobacteria is scaled-up to flasks, then 1 liter containers, then 5 liter containers, then to outdoor or indoor photobioreactors, in a suitable culture medium, with the pH set at 7.3 by use of air bubbling with CO2 addition on demand. After two days, the host cells are induced to produce the MAA and production is allowed to proceed for a specified period of time, usually not more than 30 days of continuous growth under production conditions.
  • a culture of MAA-producing cyanobacterial cells is grown and the production of the MAA of interest is induced in an indoor or outdoor photobioreactor. After a selected time period of growth, the culture is harvested by centrifugation or other means, and the MAA is collected from the cell-free medium using crossflow ultrafiltration combined with bulk ion exchange chromatography, or alternatively through absorption/de-absorption cycling using an organic substrate such as activated charcoal as an isolation matrix. The concentrated MAA is quantified and further purified, if desired. If desired, other products of interest are isolated from the culture in addition to the MAA, such as biomass, pigments, proteins, lipids, etc.
  • MAAs can be isolated from the concentrated culture biomass.
  • the cell biomass is harvested by centrifugation or other means of harvesting, with the result that the cells are separated from the culture medium.
  • the concentrated cell slurry is dried and the cells disrupted by grinding or other means.
  • the cellular material is resuspended in water, mixed for 30 minutes at room temperature, and centrifuged to separate the insoluble solid biomass from the soluble MAA.
  • the MAA-enriched cell extract can then be used without further purification, or it can be further purified, such as, for example, using crossflow ultrafiltration, centrifugation, and ion exchange, as needed.
  • the purity of the MAA is determined using mass spectrometry.
  • the amount (and type) of MAA produced is quantified by Capillary
  • Electrophoresis following the method described in Hartmann et al. "Quantitative analysis of mycosporine-like amino acids in marine algae by capillary electrophoresis with diode-array detection", Jour. Pharm. Biomed. Analysis, 138: 153-157 (2017).
  • An MAA is produced in an indoor or outdoor photobioreactor containing modified cyanobacterial strain according to the above examples.
  • the MAA is obtained from the culture, and is further purified using either a tangential cross-flow filtration unit or a batch addition of an ion exchange medium.
  • the MAA obtained via ion exchange method has a purity of at least 95% by HPLC analysis.
  • the MAA is further purified to about 99% using additional chromatography methods.
  • the purified MAA is mixed with one or more different MAAs (of different absorbance maxima) that have also been prepared from cyanobacteria. This mixture of various MAAs increases the breadth of the UV protection over a wider absorbance range.
  • the MAA mixture is then mixed with a naturally obtained, suitable carrier for application to human skin (such as an oil or a lotion) to result in a spreadable material that protects the skin from sun damage.
  • MAA "porphyra-334" which has two amino acid moieties attached to the central ring, is prepared by starting with a strain that contains the genes for the production of mycosporine-glycine (such as shown above in Examples 6-9), and further inserting an additional gene encoding the mysCD fusion-gene from Porphyra umbilicalis or Chondrus crispus, linked to a Zn inducible promoter, and localized on a second endogenous plasmid pAB lB from the ABICyanol strain.
  • MAAs like mycosporine-ornithine, mycosporine- lysine and mycosporine-2-(4-deoxygadusolyl-ornithine) were produced by starting with a strain that contained the genes for the production of mycosporine-glycine (such as shown above in Examples 6-9), and further inserting the additional mylCDE gene cluster from Cylindrospermum stagnate PCC 7417 or the mysD-Cl-C2 cluster from Nostoc commune var. flagelliforme QSY, linked to a Zn inducible promoter, and localized on a second endogenous plasmid pABICyanolB from the ABICyanol strain.
  • Fig. 25A and 25B show the whole cell absorption spectrum and the HPLC profile of the ABICyanol strain AB4033.
  • This strain was produced by transformation of the M-Gly producer strain AB1225 with the plasmid carrying the genes mylCDE from Nostoc commune var. flagelliforme QSY 1, resulting in the production of mycosporine-ornithine-4-deoxygadusolyl- ornithine (M-2-DO).
  • M-2-DO mycosporine-ornithine-4-deoxygadusolyl- ornithine
  • the heterologous genes mylD, mylE and mylC were introduced in the strain as codon optimized variants under the transcriptional control of the PsmtA promoter.
  • Fig. 25A shows the shift in the absorbance spectrum and Fig.
  • 25B shows the large HPLC peak for mycosporine-glycine and smaller peaks for mycosporine-ornithine-4-deoxygadusolyl-ornithine, mycosporine-2-(4-deoxygadusolyl-ornithine) (M-2-DO) and mycosporine-ornithine.
  • FIG. 23A and 23B show the whole cell absorption spectrum and the HPLC profile of the ABICyanol strain AB1358.
  • This strain was produced by transformation of the M-Gly producer strain AB1225 with plasmids carrying genes for thus far undescribed enzymes from Trichodesmium erythraeum EVIS101, resulting in the production of mycosporine-methylamine-glycine and mycosporine-2-glycine (M-2-Gly).
  • Fig. 23A shows the shift in the absorbance spectrum when producing mycosporine-methylamine- glycine and mycosporine-2-glycine (AB1358) instead of mycosporine-glycine (AB1225).
  • Fig. 23B shows the HPLC spectrum identifying the considerably larger peaks for mycosporine- methylamine-glycine and mycosporine-2-glycine and the much smaller peak for the remaining mycosporine-glycine.
  • Fig. 24A and 24B show the whole cell absorption spectrum and the HPLC profile of the ABICyanol strain AB 1360.
  • This strain was produced by transformation of the M-Gly producer strain AB 1225 with plasmids carrying genes for so far undescribed enzymes from Trichodesmium erythraeum IMS101, resulting in the production of Tery-364, an MAA with yet unknown structure.
  • the heterologous genes Tery_4684, Tery_2968 and Tery_2966 were introduced under the transcriptional control of the PsmtA promoter.
  • Fig. 24A shows the difference in the absorbance spectrum between the initial AB1225 strain and AB 1360.
  • Fig. 24B identifies the HPLC peaks for mycosporine-glycine and Tery-364 with an absorbance maximum at 364 nm.
  • the strains can contain the genes for the production of mycosporine-glycine (such as shown above in Examples 6-9), and further inserting parts of the additional genes from the MAA gene cluster (Tery_2966 - Tery_2972 from Trichodesmium erythraeum IMS 101), linked to inducible or constitutive promoters, and localized at the same or on a second endogenous plasmid from the ABICyanol strain.
  • MAA gene cluster Teery_2966 - Tery_2972 from Trichodesmium erythraeum IMS 101
  • the minimum MAA biosynthesis gene cluster consists of the three genes mysABC, which is widely distributed in the five subsections of cyanobacteria.
  • the MAA gene cluster can contain additional genes, which encode for a D-Ala-D-Ala-Ligase (MysD) or an RPS (non- ribosomal peptide synthase).
  • Some cyanobacterial strains have even more, partially unknown, genes associated within in the MAA gene cluster.
  • One example is the MAA gene cluster in the filamentous cyanobacterial strain Trichodesmium erythraeum FMS101. The genome sequence can be found on NCBI (GenBank: CP000393.1).
  • erythraeum FMS101 consists of twelve genes in total. Besides mysABC (Tery_2977 - Tery_2975), two mysD genes mysDl and mysD2 (Tery_2971, Tery_2970) and a NRPS (Tery_2968) are present.
  • the operon moreover contains two genes which encode for a transporter/permease (Tery_2974, Tery_2973), an additional mysB-like O-methyltransferase (Tery_2966) and three hypothetical genes (Tery_2972, Tery_2969 and Tery_2967) of yet unknown function in conjunction with MAA biosynthesis.
  • the amino acid sequence of Tery 2972 exhibits homology to a clavaminic acid synthetase, whereas Tery 2969 shows homology to a hydroxyl-isobutyrate dehydrogenase.
  • Fig. 32A, and 32B show the whole cell absorption spectrum and the HPLC profile for the ABCyano4 strain AB4055, producing palythine.
  • the strain overexpressed so far undescribed enzymes from Trichodesmium erythraeum IMS 101 (Tery 2972, a Clavaminic acid synthetase (CAS)-like enzyme and Tery_2971, a D-ala-D-ala ligase).
  • the strain was created by transformation of the mycosporine-glycine producer strain AB1322 with the heterologous codon optimized genes Tery 2972 and mysDI IMSlOl (Tery 2971), both of which were under the transcriptional control of the PsmtA promoter.
  • Fig. 32A shows the difference in the absorbance spectrum between the initial AB 1322 strain and AB4050.
  • Fig. 32B shows the HPLC profile with the peaks for mycosporine-glycine and palythine.
  • Fig. 33A, and 33B show the whole cell absorption spectrum and the HPLC profile for the ABCyano4 strain AB4031, producing mycosporine-methylamine-threonine.
  • the strain overexpressed a so far undescribed enzyme from Trichodesmium erythraeum IMS 101
  • Fig. 33 A shows the huge difference in the absorbance spectrum between the initial AB1322 strain and AB4031.
  • Fig. 33B depicts the HPLC profile with the peaks for mycosporine-methylamine-threonine, porphyra-334 and shinorine.
  • Fig. 34A, and 34B show the whole cell absorption spectrum and the HPLC profile for the ABCyano4 strain AB4074, producing Tery-322, an MAA with yet unknown structure, with an absorbance maximum at 322 nm.
  • the strain overexpressed a thus far undescribed enzyme from Trichodesmium erythraeum IMS 101, Tery_2966 under the transcriptional control of the PsmtA promoter.
  • the strain was created by transformation of the M-Gly producer strain AB 1322 with the plasmid #3094.
  • Fig. 34A shows the difference in the absorbance spectrum between the initial AB1322 strain and AB4074.
  • Fig. 34B shows the FIPLC profile with the peak designating Tery- 322.
  • the strains were cultivated at pH 7.3 ⁇ 0.01, controlled by CO2 (15 % CO2 in air) injection into the liquid phase. Mixing of the culture was ensured by continuous aeration at 38 mL / min. Biomass dry weight was determined as follows. A homogenous biomass sample was filtered through a glass fiber filter to collect all organic particulates. After filtration, the filter was dried for a suitable period in a drying oven at 70°C. Particulate mass was determined by subtracting the initial filter mass from the dry mass of the filter plus the particulates. Measurements were reported in units of dry weight mass per volume water, i.e., g D.W.*L-1.
  • Fig. 16A shows that at both light intensities ABCyano4 produced more dry weight in comparison to ABICyanol . Additionally, as evidenced by Fig. 16B, ABCyano4 also had a higher organic carbon-fixation rate in comparison to ABICyanol . Therefore, a skilled person also would expect genetically modified ABCyano4 cells to produce a higher amount of a compound of interest, for example ethanol or MAAs.
  • DIR combustion catalytic oxidation/non-dispersive infrared
  • Wild type ABICyanol and ABCyano4 cells were cultivated under the following conditions: in 1.2L LvPBRs for continuous cultivation; Operational OD at 750nm: set point sOD750nm at 1pm: 1, 2, 3, 4, 5, and 6; feed medium 35 psu, ASW BG11 with 4mM urea as sole nitrogen source, cultivation pH: 7.3 via pH dependent CO2 supply (10% in air into the liquid phase; aeration: at a continuous flow of 38 ml/min, mixed via bubbling and illuminated with 350 ⁇ m "2 s "1 homogenous light field, from one side with fluorescent light, at a 12h photoperiod over a time period of at least 7 days per operational optical density (sOD set point).
  • Fig. 17A and 17B show that the dry weight productivity as well as the total carbon fixation rate of ABCyano4 is higher in comparison to ABICyanol, so that a skilled person would expect a higher productivity for any compounds of interest for genetically modified variants of ABCyano4 in comparison to ABICyanol .

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Abstract

L'invention concerne des cellules cyanobactériennes modifiées capables de produire divers acides de type mycosporine (MAA), utiles en tant que composés absorbant les UV naturels.
PCT/US2018/059595 2017-11-07 2018-11-07 Production d'acides aminés de type mycosporine dans des cyanobactéries Ceased WO2019094447A2 (fr)

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WO2020231426A1 (fr) * 2019-05-15 2020-11-19 Algenol Biotech LLC Production d'acides aminés de type mycosporine utilisant des souches de production améliorées et de nouvelles enzymes
CN113980918A (zh) * 2021-10-18 2022-01-28 中国海洋大学 一种南极冰藻MAAs合成酶及其编码基因和应用
KR20220018155A (ko) * 2020-08-06 2022-02-15 한국생명공학연구원 방사무늬김 유래의 유전자를 이용한 마이크로스포린 유사 아미노산을 대량 생산하는 난노클로롭시스 형질전환체의 제조 방법
CN114395053A (zh) * 2022-03-25 2022-04-26 中国科学院南海海洋研究所 一种虫黄藻来源的DHQS-OMT融合基因及其在高效合成MAAs中的应用
CN115851807A (zh) * 2022-10-10 2023-03-28 深圳市灵蛛科技有限公司 一种重组生物体及生产多种类环孢素氨基酸的方法
US11739337B2 (en) 2019-09-06 2023-08-29 Oregon State University Gadusol derivative production in bacteria
KR102603304B1 (ko) * 2023-06-28 2023-11-16 전남대학교산학협력단 포피라-334 생산용 조성물 및 생산방법
KR102637622B1 (ko) * 2023-06-28 2024-02-20 전남대학교산학협력단 시노린 생산용 조성물 및 생산 방법
CN119193457A (zh) * 2024-11-29 2024-12-27 浙江大学海南研究院 一种生产类菌胞素代谢物的链霉菌重组工程菌株及其构建方法与应用
WO2025184401A1 (fr) * 2024-02-29 2025-09-04 Heliobiosys, Inc. Procédé de purification d'acides aminés analogues de la mycosporine à partir d'organismes pour produits cosmétiques et écrans solaires

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WO2020231426A1 (fr) * 2019-05-15 2020-11-19 Algenol Biotech LLC Production d'acides aminés de type mycosporine utilisant des souches de production améliorées et de nouvelles enzymes
US12378562B2 (en) 2019-09-06 2025-08-05 Oregon State University Gadusol derivative production in bacteria
US11739337B2 (en) 2019-09-06 2023-08-29 Oregon State University Gadusol derivative production in bacteria
KR102388144B1 (ko) 2020-08-06 2022-04-19 한국생명공학연구원 방사무늬김 유래의 유전자를 이용한 마이크로스포린 유사 아미노산을 대량 생산하는 난노클로롭시스 형질전환체의 제조 방법
KR20220018155A (ko) * 2020-08-06 2022-02-15 한국생명공학연구원 방사무늬김 유래의 유전자를 이용한 마이크로스포린 유사 아미노산을 대량 생산하는 난노클로롭시스 형질전환체의 제조 방법
CN113980918B (zh) * 2021-10-18 2023-11-24 自然资源部第一海洋研究所 一种南极冰藻MAAs合成酶及其编码基因和应用
CN113980918A (zh) * 2021-10-18 2022-01-28 中国海洋大学 一种南极冰藻MAAs合成酶及其编码基因和应用
CN114395053A (zh) * 2022-03-25 2022-04-26 中国科学院南海海洋研究所 一种虫黄藻来源的DHQS-OMT融合基因及其在高效合成MAAs中的应用
CN115851807A (zh) * 2022-10-10 2023-03-28 深圳市灵蛛科技有限公司 一种重组生物体及生产多种类环孢素氨基酸的方法
CN115851807B (zh) * 2022-10-10 2024-04-30 深圳市灵蛛科技有限公司 一种重组生物体及生产多种类环孢素氨基酸的方法
KR102603304B1 (ko) * 2023-06-28 2023-11-16 전남대학교산학협력단 포피라-334 생산용 조성물 및 생산방법
KR102637622B1 (ko) * 2023-06-28 2024-02-20 전남대학교산학협력단 시노린 생산용 조성물 및 생산 방법
WO2025184401A1 (fr) * 2024-02-29 2025-09-04 Heliobiosys, Inc. Procédé de purification d'acides aminés analogues de la mycosporine à partir d'organismes pour produits cosmétiques et écrans solaires
CN119193457A (zh) * 2024-11-29 2024-12-27 浙江大学海南研究院 一种生产类菌胞素代谢物的链霉菌重组工程菌株及其构建方法与应用

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