WO2015191422A1 - Acides carboxyliques oméga-hydroxylés - Google Patents

Acides carboxyliques oméga-hydroxylés Download PDF

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WO2015191422A1
WO2015191422A1 PCT/US2015/034629 US2015034629W WO2015191422A1 WO 2015191422 A1 WO2015191422 A1 WO 2015191422A1 US 2015034629 W US2015034629 W US 2015034629W WO 2015191422 A1 WO2015191422 A1 WO 2015191422A1
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coa
omega
overexpressed
microorganism
group
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Ramon Gonzalez
James M. CLOMBURG
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William Marsh Rice University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids

Definitions

  • the disclosure relates to biological synthesis of various chemicals through a reverse beta oxidation pathway.
  • 61/531/911, filed 9/7/2011 used one of 14 primers, none of them being acetyl-CoA or propionyl-CoA (although acetyl-coA does condense with the primer, acting as an extender unit, to add two carbon units thereto).
  • This invention takes the development of the reverse beta-oxidation cycle even further, elaborating significantly on the production of omega-hydroxylated carboxylic acids.
  • omega carbon of n-alcohols and carboxylic acids generated by the ⁇ -oxidation reversal can be functionalized by introducing carboxylic or alcohol groups.
  • Examples of potential products to be generated include ⁇ - hydroxylated carboxylic acids, ⁇ -carboxylated n-alcohols, dicarboxylic acids, and diols.
  • products of different chain lengths can be obtained: i.e. products with an internal/spacer chain between the alpha and omega ends of different lengths, depending on the number of turns of the cycle, and containing different functionalities, depending on the ⁇ -oxidation intermediate used as precursor for their synthesis.
  • the latter can include a hydroxy or keto group in the beta carbon or an ⁇ , ⁇ unsaturation.
  • the priming step is engineered to use a primer or starter with a functionalized (hydroxylated or carboxylated) omega carbon (examples illustrated in FIG. 2).
  • Omega-functionalized intermediates of varying chain length are generated from one or multiple turns of a beta-oxidation reversal, which can be converted to various products through the use of different terminations pathways (examples illustrated in FIG. 1).
  • Specific combinations of priming molecules and termination pathway leading to the synthesis of omega-hydroxy carboxylic acids are illustrated in FIGS. 3-5.
  • alternate termination pathways are engineered to functionalize (hydroxylate or carboxylate) the omega carbon of an intermediate or a product of the engineered reversal of the ⁇ -oxidation cycle made with a traditional primer (illustrated by the omega-oxidation of carboxylic acids in FIG. 6).
  • a traditional primer illustrated by the omega-oxidation of carboxylic acids in FIG. 6.
  • the latter could take place before or after the intermediates of the engineered reversal of the ⁇ -oxidation cycle have been converted to carboxylic acids and n-alcohols by the appropriate termination enzymes.
  • acetyl-CoA "normal/standard” starter or primer used in the engineered reversal of the ⁇ -oxidation cycle is acetyl-CoA, which leads to the synthesis of even-chain n-alcohols and carboxylic acids.
  • Propionyl-CoA can also be used as starter unit/primer by thiolase(s), thus enabling the synthesis of odd-chain carboxylic acids and n-alcohols.
  • a methyl group is always found at the omega end of both of the aforementioned starter/primer molecules.
  • the use of starter/primer molecules with an omega hydroxylated or omega carboxylated carbon i.e. a functionalized omega end
  • FIG. 2 illustrates the first reaction of the ⁇ -oxidation reversal (i.e. non-decarboxylative condensation catalyzed by thiolases) for the use of representative ⁇ -functionalized primers with carboxylated and hydroxylated omega carbons.
  • the functionalized priming molecule can be generated either internally or for the purposes of proof of concept studies can be exogenously supplied as the acid form. In the latter case, and in certain instances through internal generation, the activation of the acid form of the functionalized primer to a CoA intermediate is required before subsequent condensation with acetyl-CoA can take place (FIG. 2).
  • This approach requires: 1) identification/engineering of appropriate activation enzymes for the conversion of the ⁇ - functionalized acid to its CoA intermediate, 2) a thiolase enzyme(s) capable of condensing an ⁇ -functionalized acyl-CoA with acetyl-CoA, 3) enzymes for the dehydrogenation, dehydration, and reduction steps of the core ⁇ -oxidation reversal that are active on corresponding ⁇ -functionalized substrates, 4) appropriate termination pathways leading to product synthesis (FIG. 1).
  • ⁇ -hydroxylation and further oxidation to the carboxylic acid group will be achieved by using the ⁇ -oxidation pathway.
  • This pathway is used by industrially important yeasts and bacteria during the degradation of alkanes and long chain fatty acids.
  • the methyl group at the omega carbon is first oxidized to a hydroxy 1 group, then to an oxo group, and finally to a carboxyl group.
  • the long chain dicarboxylates derived from omega-oxidation then enter the ⁇ -oxidation cycle for further degradation (WIREs System Biology and Medicine 5, 575-585, 2013).
  • This ⁇ -oxidation pathway can be used in conjunction with a functional reversal of the ⁇ -oxidation pathway to generate carboxylic acids and n-alcohols with hydroxylated or carboxylated omega carbons (producing dicarboxylic acids, ⁇ -hydroxyacids, or diols depending on the starting product and the extent of omega-oxidation).
  • This approach for the synthesis of omega-hydroxy carboxylic acids is illustrated in FIG. 6 with termination from a beta-oxidation reversal leading to carboxylic acids followed by omega-oxidation resulting in the desired product.
  • Bacteria from a wide range of species have been successfully modified, and may be the easiest to transform and culture, since the methods were invented in the 70 's and are now so commonplace, that even school children perform genetic engineering experiments using bacteria.
  • Such species include e.g., Bacillus, Streptomyces, Azotobacter, Trichoderma, Rhizobium, Pseudomonas, Micrococcus, Nitrobacter, Proteus, Lactobacillus, Pediococcus, Lactococcus, Salmonella, and Streptococcus, or any of the completely sequenced bacterial species.
  • yeasts are a common species used for microbial manufacturing, and many species can be successfully transformed.
  • rat acyl ACP thioesterase has already been successfully expressed in yeast Saccharomyces and functional reversal of the beta oxidation cycle has also been achieved in Saccharomyces, demonstrating that this method has wide applicability to microbes, as expected since the beta oxidation pathway is ubiquitous (Lian 2015).
  • Candida Aspergillus, Arxula adeninivorans, Candida boidinii, Hansenula polymorpha (Pichia angusta), Kluyveromyces lactis, Pichia pastoris, Saccharomyces cerevisiae and Yarrowia lipolytica, to name a few.
  • Spirulina Apergillus, Chlamydomonas, Laminaria japonica, Undaria pinnatifida, Porphyra, Eucheuma, Kappaphycus, Gracilaria, Monostroma, Enteromorpha, Arthrospira, Chlorella, Dunaliella, Aphanizomenon, Isochrysis, Pavlova, Phaeodactylum, Ulkenia, Haematococcus, Chaetoceros, Nannochloropsis, Skeletonema, Thalassiosira, and Laminaria japonica, plus any of the algal species named above.
  • microalga Pavlova lutheri is already being used as a source of economically valuable docosahexaenoic (DHA) and eicosapentaenoic acids (EPA), and Crypthecodinium cohnii is the heterotrophic algal species that is currently used to produce the DHA used in many infant formulas.
  • DHA docosahexaenoic
  • EPA eicosapentaenoic acids
  • Crypthecodinium cohnii is the heterotrophic algal species that is currently used to produce the DHA used in many infant formulas.
  • a number of databases include vector information and/or a repository of vectors that can be selected for use in these various microbes. See e.g., Addgene.org, which provides both a repository and a searchable database allowing vectors to be easily located and obtained from colleagues. See also Plasmid Information Database (PlasmID) and DNASU having over 191,000 plasmids.
  • Plasmid Information Database PlasmID
  • DNASU having over 191,000 plasmids.
  • a collection of cloning vectors of E. coli is also kept at the National Institute of Genetics as a resource for the biological research community.
  • vectors including particular ORFS therein
  • fatty acids means any saturated or unsaturated aliphatic acids having the common formulae of CnH2n ⁇ xCOOH, wherein x ⁇ n, which contains a single carboxyl group.
  • Acid and base names are used interchangeably herein, e.g., succinic acid and succinate.
  • reduced activity is defined herein to be at least a 75% reduction in protein activity, as compared with an appropriate control species. Preferably, at least 80, 85, 90, 95% reduction in activity is attained, and in the most preferred embodiment, the activity is eliminated (100%). Proteins can be inactivated with inhibitors, by mutation, or by suppression of expression or translation, by knock-out, by adding stop codons, by frame shift mutation, and the like. Reduction in activity is indicated by a negative superscript, e.g., FadD " .
  • a gene can be completely (100%) reduced by knockout or removal of part of all of the gene sequence.
  • Use of a frame shift mutation, early stop codon, point mutations of critical residues, or deletions or insertions, and the like, can also completely inactivate (100%) gene product by completely preventing transcription and/or translation of active protein. All knockout mutants herein are signified by Agene.
  • overexpression or “overexpressed” is defined herein to be at least 150% of protein activity as compared with an appropriate control species, or any expression in a species that otherwise lacks the activity. Preferably, the activity is increased 200-500%.
  • Overexpression can be achieved by mutating the protein to produce a more active form or a form that is resistant to inhibition, by removing inhibitors, or adding activators, and the like. Overexpression can also be achieved by removing repressors, adding multiple copies of the gene to the cell, or up-regulating the endogenous gene, and the like.
  • Overexpressed genes or proteins can be signified herein by "+".
  • accession numbers are to GenBank or UniProt unless indicated otherwise.
  • Exemplary gene or protein species are provided herein.
  • gene and enzyme nomenclature varies widely (esp. in bacteria), thus any protein (or gene encoding same) that catalyzes the same reaction can be substituted for a named protein herein.
  • exemplary protein sequence accession numbers are provided herein, each is linked to the corresponding DNA sequence, and to related sequences. Further, related sequences can be identified easily by homology search and requisite activities confirmed as by enzyme assay, as is known in the art.
  • E. coli gene and protein names can be ascertained through ecoliwiki.net/ and enzymes can be searched through brenda- enzymes.info/. ecoliwiki.net/ in particular provides a list of alternate nomenclature for each enzyme/gene.
  • Many similar databases are available including UNIPROTKB, PROSITE; 5 EC2PDB; ExplorEnz; PRIAM; KEGG Ligand; IUBMB Enzyme Nomenclature; IntEnz; MEDLINE; and MetaCyc, to name a few.
  • fadD is the gene encoding FadD or acyl-CoA synthetase.
  • FIG. 1 Reverse beta-oxidation for the synthesis of functionalized products.
  • Omega- hydroxyacids can be produced through the condensation of acetyl-CoA with co-carboxylated CoA (A) or co-hydroxylated CoA (B) priming molecules and subsequent steps of a ⁇ - oxidation reversal and appropriate termination enzymes.
  • FIG. 3 Synthesis of omega-hydroxy carboxylic acids and their alpha, beta functionalized derivatives through omega-hydroxylated priming. Initial priming of a functional beta-oxidation reversal with an omega-hydroxylated primer and n elongation cycles generates omega-functionalized CoA intermediates that can be converted to omega- hydroxy carboxylic acids through the termination pathways depicted. [0041] FIG. 4. Synthesis of omega-hydroxy carboxylic acids and their alpha, beta functionalized derivatives with omega-carboxylated priming molecules.
  • FIG. 5 Synthesis of omega-hydroxy carboxylic acids and their alpha, beta functionalized derivatives with omega-hydroxylated priming molecules.
  • Initial priming of a functional beta-oxidation reversal with an omega-hydroxylated primer and n elongation cycles generates omega-functionalized CoA intermediates that can be converted to omega- hydroxy carboxylic acids through the termination pathways depicted.
  • the alpha and beta carbons are named according to the initial CoA intermediate (and not the final product) resulting in different products for the case of alpha, beta functionalized derivatives depending on the termination pathway selected.
  • FIG. 6 Synthesis of omega-hydroxy carboxylic acids and their alpha, beta functionalized derivatives through omega-functionalization termination.
  • Initial priming of a functional beta-oxidation reversal with acetyl-CoA and n elongation cycles generates CoA intermediates that can be converted to omega-hydroxy carboxylic acids through the termination pathways depicted.
  • FIG. 7. co-hydroxyacid production through omega-hydroxylated priming.
  • MG1655 (DE3) AglcD (pET-Pl-bktB-phaBl-P2-acPhaJ) (pCDF-Pl-mePCT-P2- tdTER) grown at 30°C in LB media with 10 g/L Glucose and 40 mM Glycolate.
  • FIG. 8. ⁇ -hydroxyacid production through omega-carboxylated priming. 6- hydroxyhexanoic acid production from succinyl-CoA priming with overexpression of genes encoding thiolase (PaaJ), 3-hydroxyacyl-CoA dehydrogenase (PaaH), enoyl-CoA hydratase (PaaF), and trans-enoyl-CoA reductase (tdTER) components, along with activation enzyme for succinate to succinyl-CoA conversion ⁇ catl).
  • PaaJ thiolase
  • PaaH 3-hydroxyacyl-CoA dehydrogenase
  • PaaF enoyl-CoA hydratase
  • tdTER trans-enoyl-CoA reductase
  • FIG. 9. co-hydroxyacid production through omega-carboxylated priming. 7- hydroxyheptanoic acid production from glutaryl-CoA priming with overexpression of genes encoding thiolase (PaaJ), 3-hydroxyacyl-CoA dehydrogenase (PaaH), enoyl-CoA hydratase (PaaF), and trans-enoyl-CoA reductase (tdTER) components, along with activation enzyme for glutarate to glutaryl-CoA conversion (Catl).
  • PaaJ thiolase
  • PaaH 3-hydroxyacyl-CoA dehydrogenase
  • PaaF enoyl-CoA hydratase
  • tdTER trans-enoyl-CoA reductase
  • FIG. 10 Synthesis of co-hydroxyacids through the ⁇ -oxidation of carboxylic acids generated from a ⁇ -oxidation reversal. 6-Hydroxyhexanoic acid, 8-hydroxyoctanoic acid, and 10-hydroxydecanoic acid production shown from 72 hr fermentations with JCOl (DE3) bktB CJ5 fadB CJ5 AfadA egTER CT5 ydiI M AtesB expressing either alkBGT or CPR2 from pETDuet vector using rich (LB) medium with glycerol as the carbon source.
  • JCOl DE3
  • bktB CJ5 fadB CJ5 AfadA egTER CT5 ydiI M
  • alkBGT or CPR2 from pETDuet vector using rich (LB) medium with glycerol as the carbon source.
  • FIG. 1 1A-C. co-hydroxyacid production with JCOl (DE3) bktB CT5 fadB CT5
  • AfadA egTER CT5 ydiI M AtesB (pETDuet-l-Pl-P2-a/£5G7) in minimal media.
  • FIG. 12 Relevant genes for activation, priming, core/elongation, termination, and ⁇ -oxidation modules of a functional reversal of the ⁇ -oxidation cycle for co-hydroxyacid synthesis (See FIG. 1 for pathway details).
  • FIG. 13 Genotypes of strains resulting in co-hydroxyacid synthesis from the use of co-hydroxylated primers in combination with carboxylic acid forming termination pathways through a reversal of the ⁇ -oxidation cycle (See FIG. 12 for details/source of genes).
  • FIG. 14 Genotypes of strains resulting in co-hydroxyacid synthesis from the use of ⁇ -carboxylated primers in combination with alcohol forming termination pathways through a reversal of the ⁇ -oxidation cycle (See FIG. 12 for details/source of genes).
  • FIG. 15 Genotypes of strains resulting in ⁇ -hydroxyacid synthesis from the omega-oxidation of carboxylic acids generated from a functional reversal of the ⁇ -oxidation cycle (See FIG. 12 for details/source of genes).
  • glycolate as a functionalize primer for the ⁇ -oxidation reversal, we first identified and characterized enzymes capable of converting glycolate acid to glycolyl-CoA.
  • the propionyl-CoA transferase from Megasphaera elsdenii was selected due to its reported activity with a variety of hydroxylated short chain carboxylic acids (Journal or Bacteriology 124, 1462-1474, 1975) as well as for the conversion of glycolate to glycolyl-CoA (Journal of Biotechnology 156, 214-217, 2011; Nature Communications 4, 1414, 2013).
  • mePCT utilizes acetyl-CoA as a donor for the transfer of CoA to glycolate resulting in the conversion of acetyl-CoA and glycolate to acetate and glycolyl-CoA.
  • mePCT was purified and characterized through HPLC-MS analysis of reaction substrates/products.
  • mePCT resulted in the formation of glycolyl-CoA with an associated decrease in acetyl-CoA, confirming its ability to activate glycolate to glycolyl-CoA (data not shown).
  • mePCT as an enzyme for activation of the functionalized priming molecule
  • the identification of core/elongation modules of the ⁇ - oxidation reversal capable of working on ⁇ -hydroxylated intermediates was then required.
  • the thiolase enzyme represents perhaps the most critical as its selectivity for condensation of a functionalized primer with acetyl-CoA compared to the condensation of two acetyl-CoA molecules is a significant determining factor in the control of product synthesis.
  • the 3-ketoacyl-CoA thiolase encoded by bktB from Ralstonia eutropha was a promising candidate owning to its reported ability to function with hydroxylated molecules (Nature Communications 4, 1414, 2013) as well as its potential for condensing longer chain acyl-CoA compounds (JACS 133, 11399-11401, 2011).
  • the 3-hydroxybutyryl-CoA dehydrogenase PhaBl from Ralstonia eutropha was included, as the reduction of the 3-oxo-acyl-CoA with the consumption of NADH makes the overall reaction more thermodynamically favored.
  • Enzymes for potential use as the reductase module focused on NADH-dependent trans-2-enoyl-CoA reductases (TER), a class of enzymes that has been extensively studied in recent years for the reduction of the 2,3 double bond of various chain length enoyl- CoA molecules.
  • TER enzymes from Idiomarina loihiensis, Cytophaga hutchinsonii, Methylobacillus flagellates, and Treponema denticola were selected and their associated genes cloned into the Duet vector framework for testing.
  • denticola (tdlL ; FEBS Letters 581, 1561-1566, 2007) as functional enzymes for the thiolase, 3- hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydratase, and acyl-CoA dehydrogenase/trans-enoyl-CoA reductase steps a beta-oxidation reversal with omega-carboxylated intermediates.
  • acyl-CoA reductase Aid from Clostridium beijerinckii (cbjALD; Applied and Environmental Microbiology 65, 4973-4980, 1999) was selected as a potential termination enzyme given its role in the production of butanol in C. beijerinckii.
  • alkane hydroxylase system of P. putida encoded by alkBGT, is part of the pathway that enables growth on linear alkanes C6-C16 and has been recently shown to ⁇ -hydroxylate medium chain length fatty acid methyl esters (Advanced Synthesis & Catalysis 353, 3485-3495, 2011).
  • gracilis TER, egTER modules along with independent chromosomal expression of thioestarase (ydil) termination resulted in the ability to produce C6, C8, and CIO chain length carboxylic acids, providing products generated from a ⁇ - oxidation reversal that through ⁇ -functionalization will enable the synthesis of our target products.
  • alkBGT resulted in the synthesis of 6-hydroxyhexanoic acid, 8-hydroxyoctanoic acid, and 10- hydroxydecanoic acid
  • CPR2 expression enabled the synthesis of 10-hydroxydecanoic acid as the sole omega-hydroxyacid produced (FIG. 10).
  • alkBGT resulted in the synthesis of more than 800 mg/L of C 6 - Cio co-hydroxyacids, including 271 ⁇ 20 mg/L 6-hydroxyhexanoic acid, 403 ⁇ 24 mg/L 8- hydroxyoctanoic acid, and 150 ⁇ 8 mg/L 10-hydroxydecanoic acid after 96 hours (Fig. 11).
  • the production of these compounds was verified via GC-MS (Fig. 11), with comparison of fragmentation patterns of peaks to that of analytical standards confirming their identity (data not shown).

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Abstract

L'invention concerne des acides carboxyliques omega-hydroxylés préparés au moyen d'un cycle de bêta-oxydation inverse soit en commençant avec des amorces de thioester CoA oméga-fonctionnalisés soit par oméga fonctionnalisation de produit(s)/intermédiaire(s) de bêta-oxydation. L'invention concerne également des bactéries et des procédés associés.
PCT/US2015/034629 2014-06-12 2015-06-08 Acides carboxyliques oméga-hydroxylés Ceased WO2015191422A1 (fr)

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EP3099763A4 (fr) * 2014-01-27 2017-08-02 William Marsh Rice University Enzymes responsables de la synthèse d'acides gras du type ii par b-oxydation inverse
WO2017161041A1 (fr) 2016-03-16 2017-09-21 William Marsh Rice University Synthèse microbienne de précurseurs d'isoprénoïdes, d'isoprénoïdes et de dérivés comprenant des composés aromatiques prénylés
CN110713962A (zh) * 2019-09-06 2020-01-21 南京农业大学 一株高产丙二酰辅酶a的基因工程菌及其构建方法和应用
CN111593079A (zh) * 2019-02-21 2020-08-28 上海凯赛生物技术股份有限公司 一种提高长链二元酸发酵转化率的方法
US12163177B2 (en) 2015-04-15 2024-12-10 Ramon Gonzalez Modified fatty acid biosynthesis with ACP-dependent thiolases

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WO2017096310A1 (fr) 2015-12-04 2017-06-08 Invista North America S.A.R.L. Procédés et matériaux pour la production de monomères à 7 atomes de carbone
US20180066296A9 (en) 2015-12-04 2018-03-08 Invista North America S.A.R.L. Methods and materials for producing 7-carbon monomers
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