WO2016181205A2 - Production régulée de produits d'intérêt à base de carbone - Google Patents

Production régulée de produits d'intérêt à base de carbone Download PDF

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Publication number
WO2016181205A2
WO2016181205A2 PCT/IB2016/000544 IB2016000544W WO2016181205A2 WO 2016181205 A2 WO2016181205 A2 WO 2016181205A2 IB 2016000544 W IB2016000544 W IB 2016000544W WO 2016181205 A2 WO2016181205 A2 WO 2016181205A2
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Prior art keywords
microorganism
carbon
interest
engineered
based product
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WO2016181205A3 (fr
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Christian Perry PERRY
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Hocking Jason
Joule Unlimited Technologies Inc
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Hocking Jason
Joule Unlimited Technologies Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • an engineered photosynthetic microorganism modified to increase internal storage of one or more nutrients relati ve to an otherwise identical control microorganism that is not modified to increase internal storage of the one or more nutrients, optionally wherein the one or more nutrients comprises phosphorus, sulfur, nitrogen, carbon, iron, manganese, cobalt, zinc, molybdenum, copper, boron, or combinations thereof, optionally wherein the engineered photosynthetic microorganism comprises one or more recombinant nucleotide sequences encoding one or more proteins capable of producing one or more carbon-based produces) of interest, and optionally wherein the modification to increase internal storage of one or more nutrients is one or more modifications to the expression or regulation of phoU gene(s) or one or more modifications to the expression or regulation of phoU protein(s) of the microorganism, wherein the expression or activity' of the protein encoded by the modified phoU gene(s) or the expression or activity of the phoU protein(s) can
  • the one or more recombinant nucleotide sequences encoding one or more proteins capable of producing carbon-based produces) of interest comprise adh, pdc, aar, and/or adm. In some aspects, the one or more recombinant nucleotide sequences encoding one or more proteins capable of producing carbon-based product(s) of interest comprise adh and pdc. In some aspects, the one or more recombinant nucleotide sequences encoding one or more proteins capable of producing carbon-based product(s) of interest comprise aar and adm.
  • the one or more recombinant nucleotide sequences encoding one or more proteins capable of producing carbon-based product(s) of interest are described in any of the following patent applications, each of which is herein incorporated by reference, in its entirety, for all purposes: PCT/US2008/075899 (filed 9/10/2008);
  • PCT/US2008/083056 (filed 11/10/2008); PCT/US2009/035937 (filed 3/3/2009);
  • PCT/US2009/055949 (filed 9/3/2009); PCT US2010/039558 (filed 6/22/2010);
  • PCT/US2010/041619 (filed 7/9/2010); U.S. Pat. No. 7,7949,69 (application filed 4/13/2010); PCT/US201 1/051648 (filed 9/14/201 1); U.S. Prov. App. No. 61/756,973 (filed 1/25/2013); U.S. Prov. App. No. 61/826,637 (filed 5/23/2013); U.S. Pat. Nos.
  • said one or more recombinant nucleotide sequences are integrated into the genome of said microorganism. In some aspects, said one or more recombinant nucleotide sequences are extrachromosomal.
  • an inducible or repressible promoter operably linked to the coding sequence of the phoU gene(s).
  • the expression profile of the phoU gen e(s) can be altered via the promoter relative to the otherwise identical microorganism.
  • the expression profile of the phoU gen e(s) can be temporally altered via the promoter relative to the otherwise identical microorganism.
  • the expression profile of the phoU gene(s) can be altered via one or more modifications.
  • the one or more modifications comprise one or more mutations in the modified phoU gene(s).
  • the modified/? ?of/gene is a deleted phoU gene(s).
  • the one or modifications comprise cleavage or silencing of the mRNA product(s) of the phoU gene(s) via RNA interference (RNAi).
  • RNAi RNA interference
  • the amount of mRNA product(s) of the phoU gene(s) is reduced.
  • the microorganism further comprises one or more recombinant nucleotide sequences encoding a catalase or wherein the microorganism further comprises a catalase.
  • the carbon-based product(s) of interest comprises or is ethanol or an alkane.
  • Other carbon-based product(s) of interest are decribed herein.
  • Other carbon-based product(s) of interest are decribed in PCT/US2008/075899 (filed 9/10/2008);
  • PCT/US2008/083056 (filed 11/10/2008); PCT/US2009/035937 (filed 3/3/2009);
  • PCT/US2009/055949 (filed 9/3/2009); PCT/US2010/039558 (filed 6/22/2010);
  • PCT/US2010/041619 (filed 7/9/2010); U.S. Pat. No. 7,7949,69 (application filed 4/13/2010); PCT/US2011/051648 (filed 9/14/2011); U.S. Prov. App. No. 61/756,973 (filed 1/25/2013); U.S. Prov. App. No. 61/826,637 (filed 5/23/2013); U.S. Pat. Nos.
  • the phosphate uptake rate of the microorganism is about 2, 3, 4, 5, 6, 7, 8, or 9 times the rate of the phosphate uptake in an otherwise identical microorganism cultured under identical conditions. In some aspects, the phosphate uptake rate of the microorganism is about 9 times the rate of the phosphate uptake in an otherwi se identical microorganism cultured under identical conditions.
  • the phosphate uptake rate of the microorganism is greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ⁇ h -1 . In some aspects, the phosphate uptake rate of the microorganism is about 80 to about 300 nM h -1 .
  • the microorganism is capable of producing an equivalent rate and/or amount of carbon-based product(s) of interest in both the presence and absence of a distinct microorganism.
  • the distinct microorganism is a carbon-based product(s) of interest catabolizing microorganism.
  • the distinct microorganism is a carbon-based product(s) of interest catabolizing microorganism.
  • microorganism comprises Acinetobacter baylyi ADPl, Bacillus thuringiensis, and/or Pseudomonas stutzeri.
  • the distinct microorganism is present at 10 or greater than 10 colony forming units (CFU)/ml. In some aspects, the distinct microorganism is present at 10 3 CFU/ml.
  • the microorganism is capable of producing carbon-based product(s) of interest over an increased period of time in the presence of acetate. In some aspects, the microorganism is capable of producing carbon-based produces) of interest over an increased period of time in the presence of about 10 mmol acetate.
  • said microorganism is a cyanobacterium. In some aspects, said microorganism is a thermophillic or thermotolerant cyanobacterium. In some aspects, said microorganism is a Synechococcus species.
  • a cell culture comprising a culture medium and a microorganism disclosed herein.
  • the culture further comprises acetate.
  • the culture further comrpsies one or more distinct microorganisms.
  • the one or more distinct microorganisms are carbon-based product(s) of interest catabolizing microorganisms.
  • the distinct microorganism is present at 10 or greater than 10 CFU/ml. In some aspects, the distinct microorganism is present at 10 3 CFU/ml.
  • Also described herein is a method for producing carbon-based product(s) of interest, comprising: culturing an engineered photsynthetic microorganism described herein in a culture medium, wherein said engineered microorganism produces equivalent or increased amounts of carbon-based product(s) of interest relative to an otherwise identical microorganism, cultured under identical conditions.
  • the method further comprises allowing the carbon-based product(s) of interest to accumulate in the culture.
  • the method further comprises isolating at least a portion of the carbon-based product(s) of interest from said culture.
  • the method further comprises processing the isolated carbon-based product(s) of interest to produce a processed compound.
  • Also described herein is a method for producing carbon-based product(s) of interest, comprising: (i) culturing an engineered photosynthetic microorganism disclosed herein in a culture medium; and (ii) exposing said engineered photosynthetic microorganism to light and inorganic carbon, wherein said exposure results in the conversion of said inorganic carbon by said microorganism into carbon-based product(s) of interest in an amount greater than or equal to that converted by an otherwise identical photosynthetic microorganism, cultured under identical conditions.
  • the method further comprises allowing the carbon-based product(s) of interest to accumulate in the culture.
  • the method further comprises isolating at least a portion of the carbon-based product(s) of interest from said culture.
  • the method further comprises processing the isolated carbon-based product(s) of interest to produce a processed compound.
  • composition comprising carbon-based product(s) of interest, wherein said carbon-based product(s) of interest are produced by a method described herein.
  • the composition comprises at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% carbon-based product(s) of interest.
  • Figure 1 Growth of JCC5048 and JCC5473 in phosphate containing JB3.0 medium containing urea.
  • Figure 2 Phosphate uptake by JCC5048 and JCC5473 in phosphate containing JB3.0 medium containing urea.
  • FIG. 3 Growth of JCC5048 and JCC5473 in phosphate containing JB3.0 medium containing urea.
  • the black arrow indicates the time of addition of 50 ⁇ cumate to the induced flasks.
  • FIG. 4 Phosphate consumption by JCC5048 and JCC5473 in phosphate containing JB3.0 medium containing urea.
  • the black arrow indicates the time of addition of 50 ⁇ cumate to the induced flasks.
  • FIG. 5 Ethanol production by JCC5048 and JCC5473 in phosphate containing JB3.0 medium containing urea.
  • the black arrow indicates the time of addition of 50 ⁇ cumate to the induced flasks.
  • FIG. 6 Growth of JCC5048 and JCC5473 in phosphate containing JB3.0 medium containing urea.
  • the black arrow indicates the time of addition of 20 ⁇ cumate to all the cultures. Those flasks which received contaminants are indicated with the starting contaminant inoculation concentration in CFU/ml.
  • FIG. 7 Ethanol production of JCC5048 and JCC5473 in phosphate containing JB3.0 medium containing urea.
  • the black arrow indicates the time of addition of 20 ⁇ cumate to all the cultures. Those flasks which received contaminants are indicated with the starting contaminant inoculation concentration in CFU/ml.
  • FIG. 8 Ethanol production of JCC5473 in JB3.0 medium urea (base medium).
  • the black arrow indicates the time of addition of 20 ⁇ cumate to both cultures and 10 mM acetate (final concentration) to one flask.
  • Figure 9 Growth of JCC5473 in JB3.0 medium containing urea (base medium). The black arrow indicates the time of addition of 20 ⁇ cumate to both cultures and 10 mM acetate (final concentration) to one flask. [0029] Figure 10. Concentration of phosphate in the medium detected over time following inoculation of JCC5473 in IB3.0 media with either lx, 2x, 3x, or 4x the standard phosphate level of JB3.0.
  • Figure 1 Growth of JCC5473 in JB3.0 base medium containing either lx, 2x, 3x, or 4x the phosphate concentration of JB3.0.
  • FIG. 12 Growth of JCC5473 in JB3.0 lacking phosphate. Prior to inoculation, cultures were grown in media containing either lx, 2x, 3x, or 4x the phosphate of JB3.0 (source indicated in legend). The flask cultured in 4x phosphate was washed with JB3.0 lacking phosphate to ensure no phosphate carryover.
  • FIG. 13 Ethanol productivity of JCC5473 in JB3.0 lacking phosphate. Prior to inoculation, cultures were grown in media containing either lx, 2x, 3x, or 4x the phosphate of JB3.0 (source indicated in legend). The flask cultured in 4x phosphate was washed with JB3.0 lacking phosphate to ensure no phosphate carryover. The black arrow indicates the time of addition of 20 ⁇ cumate.
  • FIG. 14 Growth of JCC5473 in JB3.0 lacking phosphate. Prior to inoculation, cultures were grown in media containing either lx, 2x, 3x, or 4x the phosphate of JB3.0 (source indicated in legend). The flask cultured in 4x phosphate was washed with JB3.0 lacking phosphate to ensure no phosphate carryover. The black arrow indicates the time of addition of 20 ⁇ cumate.
  • Figure 15 Ethanol production of the cultures in JB3.0 medium containing urea.
  • the black arrow indicates the time of addition of 20 ⁇ cumate to both cultures and 10 mM acetate (final concentration) to one flask as indicated in the legend by NaAc.
  • Both cultures of JCC5473 died before the end of the experiment and the final timepoint for the flasks indicates when they had turned white and were removed from the incubator.
  • FIG. 16 Growth of the cultures in JB3.0 medium containing urea.
  • the black arrow indicates the time of addition of 20 ⁇ cumate to both cultures and 10 mM acetate (final concentration) to one flask as indicated in the legend by NaAc.
  • Both cultures of JCC5473 died before the end of the experiment and the final timepoint for the flasks indicates when they had turned white and were removed from the incubator.
  • FIG. 17 Phosphate consumption by JCC5290 and JCC5476 in JB3.0 medium containing urea over the first 5 days of the experiment. The black arrow indicates the time of addition of 20 ⁇ cumate to the flasks.
  • Figure 18. Growth of JCC5048 and JCC5473 in JB3.0 media containing the indicated phosphate and lOmM acetate in an outdoor photobioreactor. The black arrow indicates the time of addition of 5 uM cumate.
  • FIG. 19 Ethanol productivities of JCC5048 and JCC5473 in JB3.0 media containing the indicated phosphate and lOmM acetate in an outdoor photobioreactor.
  • the black arrow indicates the time of addition of 5 ⁇ cumate.
  • FIG. 20 Growth of JCC5048 and JCC6082 in the FMT photobioreactor under diurnal light conditions of 200-2000 uE/m2/s. In the indicated culture JCC6082 was induced with 100 uM DOC following phosphate depletion. The black arrow indicates the time of addition of 5 uM cumate.
  • FIG. 22 Daily ethanol productivity of JCC5048 and JCC6082 in the FMT photobioreactor under diurnal light conditions of 200-2000 uE/m2/s. In the indicated culture JCC6082 was induced with 100 uM DOC following phosphate depletion. The black arrow indicates the time of addition of 5 ⁇ cumate.
  • nucleic acid or “nucleic acid molecule” or “nucleotide sequence” refers to a polymeric form of nucleotides of at least 10 bases in length.
  • the term includes DNA molecules ⁇ e.g., cDNA or genomic or synthetic DNA) and RNA molecules ⁇ e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native intemucleoside bonds, or both.
  • the nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single-stranded, double- stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation.
  • nucleic acid comprising SEQ ID NO: 1 refers to a nucleic acid, at least a portion of which has either (i) the sequence of SEQ ID NO: 1, or (ii) a sequence complementary to SEQ ID NO: 1.
  • the choice between the two is dictated by the context. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target.
  • RNA, DNA or a mixed polymer is one which is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, e.g., ribosomes, polymerases and genomic sequences with which it is naturally associated.
  • an "isolated" organic molecule e.g., an alkane or ethanol
  • an "isolated" organic molecule is one which is substantially separated from the cellular components (membrane lipids,
  • chromosomes, proteins of the host cell from which it originated, or from the medium in which the host cell was cultured.
  • the term does not require that the biomolecule has been separated from all other chemicals, although certain isolated biomolecules may be purified to near homogeneity.
  • the term "recombinant" refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature.
  • recombinant can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and/or mRNAs encoded by such nucleic acids.
  • an endogenous nucleic acid sequence in the genome of an organism is deemed "recombinant” herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered.
  • a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous (originating from the same host cell or progeny thereof) or exogenous (originating from a different host cell or progeny thereof).
  • a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that this gene has an altered expression pattern. This gene would now become
  • a nucleic acid is also considered “recombinant” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome.
  • an endogenous coding sequence is considered “recombinant” if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention.
  • a "recombinant nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.
  • the phrase "degenerate variant" of a reference nucleic acid sequence encompasses nucleic acid sequences that can be translated, according to the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence.
  • the term "degenerate oligonucleotide” or “degenerate primer” is used to signify an oligonucleotide capable of hybridizing with target nucleic acid sequences that are not necessarily identical in sequence but that are homologous to one another within one or more particular segments.
  • sequence identity refers to the residues in the two sequences which are the same when aligned for maximum correspondence.
  • the length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.
  • polynucleotide sequences can be compared using FAST A, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis.
  • FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (hereby incorporated by reference in its entirety).
  • percent sequence identity between nucleic acid sequences can be determined using F ASTA with its default parameters (a word size of 6 and the NOP AM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference.
  • sequences can be compared using the computer program, BLAST (Altschul et al, J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al, Meth. Enzymol. 266: 131-141 (1996); Altschul et al. Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al, Nucleic Acids Res. 25:3389-3402 (1997)).
  • nucleic acid or fragment thereof indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 76%, 80%, 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.
  • nucleic acid or fragment thereof hybri dizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under stringent hybridization conditions.
  • Stringent hybridization conditions and “stringent wash conditions” in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of hybridization.
  • stringent hybridization is performed at about 25°C below the thermal melting point (T m ) for the specific DNA hybrid under a particular set of conditions.
  • stringent conditions are defined for solution phase hybridization as aqueous hybridization (i.e., free of formamide) in 6xSSC (where 20xSSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65°C for 8-12 hours, followed by two washes in 0.2xSSC, 0.1% SDS at 65°C for 20 minutes. It will be appreciated by the skilled worker that hybridization at 65°C will occur at different rates depending on a number of factors including the length and percent identity of the sequences which are hybridizing.
  • the nucleic acids (also referred to as polynucleotides) of this present invention may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They 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,
  • intemucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g.,
  • phosphorothioates phosphorodithioates, etc.
  • pendent moieties e.g., polypeptides
  • intercalators e.g., acridine, psoralen, etc.
  • chelators e.g., alkylators
  • modified linkages e.g., alpha anomeric nucleic acids, etc.
  • synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.
  • Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
  • Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as the modifications found in "locked" nucleic acids.
  • mutated when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence may be inserted, deleted or changed compared to a reference nucleic acid sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence.
  • a nucleic acid sequence may be mutated by any method known in the art including but not limited to mutagenesis techniques such as "error-prone PCR" (a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product; see, e.g., Leung ei ah, Technique, 1 : 11-15 (1989) and Caldwell and Joyce, PCR Methods Applic.
  • mutagenesis techniques such as "error-prone PCR” (a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product; see, e.g., Leung ei ah, Technique, 1 : 11-15 (1989) and Caldwell and Joyce, PCR Methods Applic.
  • oligonucleotide-directed mutagenesis a process which enables the generation of site-specific mutations in any cloned DNA segment of interest; see, e.g., Reidhaar-Olson and Sauer, Science 241 :53-57 (1988)).
  • Attenuate generally refers to a functional deletion, including a mutation, partial or complete deletion, insertion, or other variation made to a gene sequence or a sequence controlling the transcription of a gene sequence, which reduces or inhibits production of the gene product, or renders the gene product non-functional.
  • a functional deletion is described as a knockout mutation.
  • Attenuation also includes amino acid sequence changes by altering the nucleic acid sequence, placing the gene under the control of a less active promoter, down-regulation, expressing interfering RNA, ribozymes or antisense sequences that target the gene of interest, or through any other technique known in the art.
  • the sensitivity of a particular enzyme to feedback inhibition or inhibition caused by a composition that is not a product or a reactant is lessened such that the enzyme activity is not impacted by the presence of a compound.
  • an enzyme that has been altered to be less active can be referred to as attenuated.
  • Deletion The removal of one or more nucleotides from a nucleic acid molecule or one or more amino acids from a protein, the regions on either side being joined together.
  • Knock-out A gene whose level of expression or activity has been reduced to zero.
  • a gene is knocked-out via deletion of some or all of its coding sequence.
  • a gene is knocked-out via introduction of one or more nucleotides into its open reading frame, which results in translation of a non-sense or otherwise non-functional protein product.
  • 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 loop 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.
  • PCR polymerase chain reaction
  • Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC).
  • BAC bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome (discussed in more detail below).
  • 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.
  • certain preferred vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as
  • “Operatively linked” or “operably linked” expression control sequences refers to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest.
  • expression control sequence refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences.
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.
  • control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence.
  • control sequences is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • recombinant host cell (or simply "host cell”), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell” as used herein.
  • a recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism.
  • peptide refers to a short polypeptide, e.g., one that is typically less than about 50 amino acids long and more typically less than about 30 amino acids long.
  • the term as used herein encompasses analogs and mimetics that mimic structural and thus biological function.
  • polypeptide encompasses both naturally-occurring and non-naturally- occurring proteins, and fragments, mutants, derivatives and analogs thereof.
  • a polypeptide may be monomeric or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities.
  • isolated protein or "isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material (e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds).
  • polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components.
  • a polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
  • isolated does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from its native environment.
  • polypeptide fragment refers to a polypeptide that has a deletion, e.g., an amino-terminal and/or carboxy-terminal deletion compared to a full-length polypeptide.
  • the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally -occurring sequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45, amino acids, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long.
  • a “modified derivative” refers to polypeptides or fragments thereof that are substantially homologous in primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate amino acids that are not found in the native polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with
  • radionuclides and various enzymatic modifications, as will be readily appreciated by those skilled in the art.
  • a variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well known in the art, and include radioactive isotopes such as and 3 H ligands which bind to labeled antiligands (e.g., antibodies),
  • fluorophores fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand.
  • the choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation. Methods for labeling polypeptides are well known in the art. See, e.g., Ausubel et al.. Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002) (hereby incorporated by reference).
  • fusion protein refers to a polypeptide comprising a polypeptide or fragment coupled to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins.
  • a fusion protein comprises at least 10 contiguous amino acids from a polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50 or 60 amino acids, yet more preferably at least 75, 100 or 125 amino acids. Fusions that include the entirety of the proteins of the present invention have particular utility.
  • the heterologous polypeptide included within the fusion protein of the present invention is at least 6 amino acids in length, often at least 8 amino acids in length, and usefully at least 15, 20, and 25 amino acids in length. Fusions that include larger
  • polypeptides such as an IgG Fc region, and even entire proteins, such as the green fluorescent protein (“GFP") chromophore-containing proteins, have particular utility.
  • Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein.
  • a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein.
  • non-peptide analog refers to a compound with properties that are analogous to those of a reference polypeptide.
  • a non-peptide compound may also be termed a "peptide mimetic” or a "peptidomimetic.” See, e.g., Jones, Amino Acid and Peptide Synthesis, Oxford University Press (1992); Jung, Combinatorial Peptide and Nonpeptide Libraries: A Handbook, John Wiley (1997); Bodanszky et al., Peptide Chemistry— A
  • a "polypeptide mutant” or “mutein” refers to a polypeptide whose sequence contains an insertion, duplication, deletion, rearrangement or substitution of one or more amino acids compared to the amino acid sequence of a native or wild-type protein.
  • a mutein may have one or more amino acid point substitutions, in which a single amino acid at a position has been changed to another amino acid, one or more insertions and/or deletions, in which one or more amino acids are inserted or deleted, respectively, in the sequence of the naturally-occurring protein, and/or truncations of the amino acid sequence at either or both the amino or carboxy termini.
  • a mutein may have the same but preferably has a different biological activity compared to the naturally-occurring protein.
  • a mutein has at least 85% overall sequence homology to its wild-type counterpart. Even more preferred are muteins having at least 90% overall sequence homology to the wild- type protein.
  • a mutein exhibits at least 95% sequence identity, even more preferably 98%, even more preferably 99% and even more preferably 99.9% overall sequence identity.
  • Sequence homology may be measured by any common sequence analysis algorithm, such as Gap or Bestfit.
  • Amino acid substitutions can include those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity or enzymatic activity, and (5) confer or modify other physicochemical or functional properties of such analogs.
  • the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2 nd ed. 1991), which is incorporated herein by reference.
  • Stereoisomers ⁇ e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as ⁇ -, ⁇ -di substituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present invention.
  • unconventional amino acids include: 4-hydroxyproline, ⁇ -carboxyglutamate, ⁇ - ⁇ , ⁇ , ⁇ - trimethyllysine, ⁇ - ⁇ -acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3- methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids ⁇ e.g., 4-hydroxyproline).
  • the left-hand end corresponds to the amino terminal end and the right-hand end corresponds to the carboxy- terminal end, in accordance with standard usage and convention.
  • a protein has "homology” or is “homologous” to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein.
  • a protein has homology to a second protein if the two proteins have "similar” amino acid sequences.
  • homology 7 between two regions of amino acid sequence is interpreted as implying similarity in function.
  • the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson, 1994, Methods Mol. Biol.
  • the following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
  • Sequence homology for polypeptides is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using a measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap” and "Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1.
  • a preferred algorithm when comparing a particular polypeptide sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul et al, J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266: 131-141 (1996); Altschul et al, Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al, Nucleic Acids Res. 25:3389-3402
  • Preferred parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 1 1 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.
  • the length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues.
  • polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1.
  • FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (incorporated by reference herein).
  • percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1 , herein incorporated by reference.
  • Specific binding refers to the ability of two molecules to bind to each other in preference to binding to other molecules in the environment.
  • “specific binding” discriminates over adventitious binding in a reaction by at least two-fold, more typically by at least 10-fold, often at least 100-fold.
  • the affinity or avidity of a specific binding reaction, as quantified by a dissociation constant is about 10 -7 M or stronger ⁇ e.g., about 10 -8 M, 10 -9 M or even stronger).
  • region refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein.
  • domain refers to a structure of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be coextensive with regions or portions thereof; domains may also include distinct, non-contiguous regions of a biomolecule. Examples of protein domains include, but are not limited to, an Ig domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain.
  • molecule means any compound, including, but not limited to, a small molecule, peptide, protein, sugar, nucleotide, nucleic acid, lipid, etc., and such a compound can be natural or synthetic.
  • Carbon-based Products of Interest include alcohols such as ethanol, propanol, isopropanol, butanol, fatty alcohols, fatty acid esters, wax esters; hydrocarbons and alkanes such as propane, octane, diesel, Jet Propellant 8 (JP8); polymers such as terephthalate, 1,3-propanediol, 1,4-butanediol, polyols, Polyhydroxyalkanoates (PHA), poly-beta- hydroxybutyrate (PHB), acrylate, adipic acid, ⁇ -caprolactone, isoprene, caprolactam, rubber; commodity chemicals such as lactate, Docosahexaenoic acid (DHA), 3-hydroxypropionate, ⁇ -valerolactone, lysine, serine, aspartate, aspartic acid, sorbitol, ascorbate, ascorbic acid, isopentenol, lanos
  • Biofuel refers to any fuel that derives from a biological source.
  • Biofuel can refer to one or more hydrocarbons, one or more alcohols (such as ethanol), one or more fatty esters, or a mixture thereof.
  • Hydrocarbon The term generally refers to a chemical compound that consists of the elements carbon (C), hydrogen (H) and optionally oxygen (O). There are essentially three types of hydrocarbons, e.g., aromatic hydrocarbons, saturated hydrocarbons and unsaturated hydrocarbons such as alkenes, alkynes, and dienes. The term also includes fuels, biofuels, plastics, waxes, solvents and oils. Hydrocarbons encompass biofuels, as well as plastics, waxes, solvents and oils.
  • the term "nutrient” refers to a component of a cell culture (e.g., one or more cells in a culture medium) or cell that can be used for growth and/or survival of the cell or cell culture.
  • nutrients can be phosphorus (e.g., Phosphate, phosphite, phosphonates), sulfur (e.g., Sulfate, sulfite), nitrogen (e.g., Nitrate, nitrite, Ammonium or ammonia, urea), carbon (e.g., Inorganic carbon like carbon dioxide or bicarbonate/organic forms of carbon like sugars), iron (e.g., Iron salts), or trace metal components (e.g., manganese, cobalt, zinc, molybdenum, copper or boron); or combinations thereof.
  • phosphorus e.g., Phosphate, phosphite, phosphonates
  • sulfur e.g., Sulfate, sulfite
  • the present invention provides isolated nucleic acid molecules for genes encoding enzymes, and variants thereof Exemplary full-length nucleic acid sequences for genes encoding enzymes and the corresponding amino acid sequences are presented in the Table(s).
  • the nucleic acid sequence can be preferably greater than 80%, 85%, 90%, 95%, 98%, 99%, 99.9% or even higher identity to the wild-type gene.
  • the nucleic acid molecule of the present invention encodes a polypeptide having an amino acid sequence disclosed in the Table(s).
  • the nucleic acid molecule of the present invention encodes a polypeptide sequence of at least 50%, 60, 70%, 80%, 85%, 90% or 95% identity to the amino acid sequences shown in the Table(s)and the identity can even more preferably be 96%, 97%, 98%, 99%, 99.9% or even higher.
  • the present invention also provides nucleic acid molecules that hybridize under stringent conditions to the above-described nucleic acid molecules.
  • stringent hybridizations are performed at about 25°C below the thermal melting point (T m ) for the specific DNA hybrid under a particular set of conditions, where the T m is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Stringent washing is performed at temperatures about 5°C lower than the T m for the specific DNA hybrid under a particular set of conditions.
  • Nucleic acid molecules comprising a fragment of any one of the above-described nucleic acid sequences are also provided. These fragments preferably contain at least 20 contiguous nucleotides. More preferably the fragments of the nucleic acid sequences contain at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguous nucleotides.
  • the nucleic acid sequence fragments of the present invention display utility in a variety of systems and methods.
  • the fragments may be used as probes in various hybridization techniques.
  • the target nucleic acid sequences may be either DNA or RNA.
  • the target nucleic acid sequences may be fractionated ⁇ e.g., by gel electrophoresis) prior to the hybridization, or the hybridization may be performed on samples in situ.
  • nucleic acid probes of known sequence find utility in determining chromosomal structure ⁇ e.g., by Southern blotting) and in measuring gene expression ⁇ e.g., by Northern blotting).
  • sequence fragments are preferably detectably labeled, so that their specific hydridization to target sequences can be detected and optionally quantified.
  • nucleic acid fragments of the present invention may be used in a wide variety of blotting techniques not specifically described herein.
  • nucleic acid sequence fragments disclosed herein also find utility as probes when immobilized on microarrays.
  • Methods for creating microarrays by deposition and fixation of nucleic acids onto support substrates are well known in the art. Reviewed in DNA Microarrays: A Practical Approach (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet. 21(l)(suppl): l-60 (1999); Microarray Biochip: Tools and Technology, Schena (ed.), Eaton Publishing Company/BioTechniques Books Division (2000) (ISBN: 1881299376), the disclosures of which are incorporated herein by reference in their entireties.
  • microarrays comprising nucleic acid sequence fragments, such as the nucleic acid sequence fragments disclosed herein, are well-established utility for sequence fragments in the field of cell and molecular biology.
  • sequence fragments immobilized on microarrays are described in Gerhold et al., Trends Biochem. Sci. 24: 168-173 (1999) and Zweiger, Trends Biotechnol. 17:429-436 (1999); DNA Microarrays: A Practical Approach (Practical Approach Series), Schena (ed.), Oxford University Press (1999) (ISBN: 0199637768); Nature Genet.
  • enzyme activities can be measured in various ways. For example, the pyrophosphorolysis of OMP may be followed spectroscopically
  • the activity of the enzyme can be followed using chromatographic techniques, such as by high performance liquid chromatography (Chung and Sloan, (1986) J. Chromatogr. 371 :71-81).
  • the activity can be indirectly measured by determining the levels of product made from the enzyme activity. These levels can be measured with techniques including aqueous chloroform/methanol extraction as known and described in the art (Cf M. Kates (1986) Techniques ofLipidology; Isolation, analysis and identification of Lipids. Elsevier Science Publishers, New York (ISBN: 0444807322)).
  • More modern techniques include using gas chromatography linked to mass spectrometry (Niessen, W. M. A. (2001). Current practice of gas chromatography— mass spectrometry. New York, N.Y: Marcel Dekker. (ISBN: 0824704738)). Additional modern techniques for identification of recombinant protein activity and products including liquid chromatography-mass spectrometry (LCMS), high performance liquid chromatography (HPLC), capillary electrophoresis, Matrix- Assisted Laser Desorption Ionization time of flight-mass spectrometry (MALDI-TOF MS), nuclear magnetic resonance (NMR), near-infrared (NIR) spectroscopy, viscometry (Knothe, G (1997) Am. Chem. Soc. Symp.
  • LCMS liquid chromatography-mass spectrometry
  • HPLC high performance liquid chromatography
  • MALDI-TOF MS Matrix- Assisted Laser Desorption Ionization time of flight-mass spectrometry
  • NMR nuclear
  • vectors including expression vectors, which comprise the above nucleic acid molecules of the present invention, as described further herein.
  • the vectors include the isolated nucleic acid molecules described above.
  • the vectors of the present invention include the above-described nucleic acid molecules operably linked to one or more expression control sequences. The vectors of the instant invention may thus be used to express a polypeptide contributing to a carbon-based product of interest producing activity by a host cell.
  • isolated polypeptides (including muteins, allelic variants, fragments, derivatives, and analogs) encoded by the nucleic acid molecules of the present invention are provided.
  • the isolated polypeptide comprises the polypeptide sequence corresponding to a polypeptide sequence shown in the Table(s).
  • the isolated polypeptide comprises a polypeptide sequence at least 85% identical to a polypeptide sequence shown in the Table(s).
  • the isolated polypeptide of the present invention has at least 50%, 60, 70%, 80%, 85%, 90%, 95%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7?/o, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or even higher identity to a polypeptide sequence shown in the Table(s).
  • isolated polypeptides comprising a fragment of the above-described polypeptide sequences are provided. These fragments preferably include at least 20 contiguous amino acids, more preferably at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or even more contiguous amino acids.
  • the polypeptides of the present invention also include fusions between the above- described polypeptide sequences and heterologous polypeptides.
  • the heterologous sequences can, for example, include sequences designed to facilitate purification, e.g. histidine tags, and/or visualization of recombinantly-expressed proteins.
  • Other non-limiting examples of protein fusions include those that permit display of the encoded protein on the surface of a phage or a cell, fusions to intrinsically fluorescent proteins, such as green fluorescent protein (GFP), and fusions to the IgG Fc region.
  • GFP green fluorescent protein
  • host cells transformed with the nucleic acid molecules or vectors of the present invention, and descendants thereof, are provided.
  • these cells carry the nucleic acid sequences of the present invention on vectors, which may but need not be freely replicating vectors.
  • the nucleic acids have been integrated into the genom e of the host cells.
  • the host cells of the present invention can be mutated by recombination with a disruption, deletion or mutation of the isolated nucleic acid of the present invention so that the activity of one or more enzyme(s) in the host cell is reduced or eliminated compared to a host cell lacking the mutation.
  • Microorganism Includes prokaryotic and eukaryotic microbial species from the Domains Archaea, Bacteria and Eiicarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista.
  • microbial cells and “microbes” are used interchangeably with the term microorganism.
  • Photoautotrophic organisms include eukaryotic plants and algae, as well as prokaryotic cyanobacteria, green-sulfur bacteria, green non-sulfur bacteria, purple sulfur bacteria, and purple non-sulfur bacteri a.
  • Extremophiles are also contemplated as suitable organisms. Such organisms withstand various environmental parameters such as temperature, radiation, pressure, gravity, vacuum, desiccation, salinity, pH, oxygen tension, and chemicals. They include
  • hyperthermophiles which grow at or above 80°C such as Pyrolobus fumarii; thermophiles, which grow between 60-80°C such as Synechococciis lividis; mesophiles, which grow between 15-60°C and psychrophiles, which grow at or below 15°C such as Psychrobacter and some insects.
  • Radiation tolerant organisms include Deinococcus radiodurans.
  • Pressure- tolerant organisms include piezophiles, which tolerate pressure of 130 MPa.
  • Weight-tolerant organisms include barophiles.
  • Hypergravity e.g., >lg
  • hypogravity e.g., ⁇ lg
  • tolerant organisms are also contemplated.
  • Vacuum tolerant organisms include tardigrades, insects, microbes and seeds.
  • Dessicant tolerant and anhydrobiotic organisms include xerophiles such as Artemia salina; nematodes, microbes, fungi and lichens.
  • Salt-tolerant organisms include halophiles (e.g., 2-5 M NaCl) Halobacteriacea and Dunaliella salina.
  • pH-tolerant organisms include alkaliphiles such as Natronohacter mm, Bacillus firmus OF4, Spirulina spp. (e.g., pH > 9) and acidophiles such as Cyanidium caldarium, Ferroplasma sp. (e.g., low pH).
  • Anaerobes which cannot tolerate 0 2 such as Methanococcus jannaschii; microaerophils, which tolerate some 0 2 such as Clostridium and aerobes, which require 0 2 are also contemplated.
  • Gas-tolerant organisms, which tolerate pure C0 2 include Cyanidium caldarium and metal tolerant organisms include metalotolerants such as Ferroplasma acidarmanus (e.g., Cu, As, Cd, Zn), Ralstonia sp. CH34 (e.g., Zn, Co, Cd, Hg, Pb). Gross, Michael. Life on the Edge: Amazing Creatures Thriving in Extreme Environments. New York: Plenum (1998) and Seckbach, J.
  • Plants include but are not limited to the following genera: Arabidopsis, Beta, Glycine, Jatropha, Miscanthus, Panicum, Phalaris, Populus, Saccharum, Salix, Simmondsia and Zea.
  • Algae and cyanobacteria include but are not limited to the following genera:
  • Chrysonebida Chrysophyta, Chrysopyxis, Chrysosaccus, Chrysophaerella,
  • Chrysostephanosphaera Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis, Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus,
  • Coenocystis Colacium, Coleochaete, Collodictyon, Compsogonopsis, Compsopogon, Conjugatophyta, Conochaete, Coronastrurn, Cosmarium, Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta, Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta,
  • Cyanothece Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella, Cylindrocapsa, Cylindrocystis, Cylindrospermum, Cylindrotheca, Cymatopleura, Cymbella,
  • Cymbellonitzschia Cystodinium Dactylococcopsis, Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella, Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon, Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicelhda, Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus,
  • Distrionella Docidium, Draparnaldia, Lhmaliella, Dysmorphococciis, Ecballocystis, Elakatothrix, Ellerbeckia, Encyonema, Enteromorpha, Entocladia, Entomoneis, Entophysalis, Epichrysis, Epipyxis, Epithemia, Eremosphaera, Euastropsis, Euastrum, Eucapsis, Encocconeis, Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta, Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, Genicularia, Glancocystis, Glaucophyta, Glenodiniopsis, Glenodinium,
  • Gloeocapsa Gloeochaete, Gloeochrysis, Gloeococcus, Gloeocystis, Gloeodendron,
  • Gloeomonas Gloeoplax, Gloeothece, Gloeotila, Gloeotrichia, Gloiodictyon, Golenkinia, Golenkiniopsis, Gomontia, Gomphocymbella, Gomphonema, Gomphosphaeria,
  • Gonatozygon Gongrosia, Gongrosira, Goniochloris, Gonium, Gonyostomum,
  • Granulochlo s Granulocystopsis, Groenhladia, Gymnodinium, Gymnozyga, Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea, Hantzschia,
  • Hapalosiphon Haplotaenium, Haptophyta, Haslea, Hemidinium, Hemitonia, Heribaudiella, Heteromastix, Heterothrix, Hibberdia, Hildenbrandia, Hillea, Holopedium, Homoeothrix, Hormanthonema, Hormotila, Hyalobrachion, Hyalocardium, Hyalodiscus, Hyalogonium, Hyalotheca, Hydrianum, Hydrococciis, Hydrocoleum, Hydrocoryne, Hydrodictyon,
  • Microglena Micromonas, Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus, Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis,
  • Myochloris Myromecia, Myxosarcina, Naegeliella, Nannochloris, Nautococcus, Navicula, Neglectella, Neidium, Nep roclamys, Nephrocytium, Nephrodiella, Nephroselmis, Netrium, Nitella, Nitellopsis, Nitzschia, Nodularia, Nostoc, Ochromonas, Oedogonium,
  • Pseudoncobyrsa Pseiidoquadrigula, Pseudosphaerocystis, Pseudostaurastrum,
  • Rhabdoderma Rhabdomonas, Rhizoclonium, Rhodomonas, Rhodophyta, Rhoicosphenia, Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmiis, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizolhrix, Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia, Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis, Siderocelis, Diderocystopsis, Dirnonsenia, Siphononema, Sirocladium,
  • Sirogonhim Skeletonema, Sorastrum, Spermatozopsis, Sphaerellocystis, Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma, Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum, Spondyloshim, Sporotetras, Spnmella, Staurastrum,
  • Stauerodesmns Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis, Slephanodisciis, Slephanoporos, Stephanosphaera, Stichococcus, Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella, Strombomonas, Stylochrysalis, Stylodinium, Styloyxis, Stylosphaeridiurn, Siirirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra, Synochromonas, Syrtura, Tabellaria, Tabularia, Molingia, Temnogametum, Tetmemorus, Tetrachlorella, Tetracyclus, Tetradesmus, Tetraedriella, Tetraedron, Tetraselmis,
  • Tetraspora Tetrastrum
  • Thalassiosira Thamniochaete
  • Thorakochloris Thorea
  • Tolypella Tolypothrix
  • Trachelomonas Trachydiscus, Trebonxia, Trentepholia, Trenbaria, Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella, Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vaciiolaria, Vancheria, Volvox, Volvulina, Westella,
  • Cyanobacteria include members of the genus Chamaesiphon, Chroococcus, Cyanobacteriiim, Cyanobiiim, Cyanothece, Dactylococcopsis, Gloeobacter, Gloeocapsa, Gloeothece, Microcystis, Prochlorococcus, Prochloron, Synechococcus, Synechocystis, Cyanocystis, Dermocarpella, Stanieria, Xenococcus, Chroococcidiopsis, Myxosarcina, Arthrospira, Borzia, Crinalium, Geitlerinemia, Leptolyngbya, Limnothrix, Lyngbya, Microcoleus, Oscillatoria, Planktothrix, Prochiorothrix, Pseudanabaena, Splegilina, Starria, Symploca, Trichodesmium, Tychonema, Anaba
  • Cyanospira Cylindrospermopsis, Cylindrospermum, Nodularia, Nostoc, Scylonema,
  • Green non-sulfur bacteria include but are not limited to the following genera:
  • Chloroflexus Chloronema, Oscillochloris, Heliothrix, Herpetosiphon, Roseiflexus, and Thermomicrobium.
  • Green sulfur bacteria include but are not limited to the following genera:
  • Purple sulfur bacteria include but are not limited to the following genera:
  • Rhodovulum Ihermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis
  • Purple non-sulfur bacteria include but are not limited to the following genera: Phaeospir ilium, Rhodobaca, Rhodobacter, Rhodomicrobium, Rhodopila,
  • Rhodopseudomonas Rhodothalassium, Rhodospirillum, Rodovibrio, and Roseospira.
  • Aerobic chemolithotrophic bacteria include but are not limited to nitrifying bacteria such as Nitrobacteraceae sp., Nitrobacter sp., Nitrospina sp., Nitrococcus sp., Nitrospira sp., Nitrosomonas sp., Nitrosococcus sp., Nitrosospira sp., Nitrosolobus sp., Nitrosovibrio sp.; colorless sulfur bacteria such as, Thi ⁇ udum sp., Thiobacillus sp.,
  • Archaeobacteria include but are not limited to methanogenic archaeobacteria such as Methanobacterium sp., Methanobrevibacter sp., Methanothermus sp., Methanococcus sp., Methanomicrobium sp., Methanospir ilium sp., Methanogenium sp., Methanosarcina sp., Methanolobus sp., Methanothrix sp., Methanococcoides sp., Methanoplanus sp.; extremely thermophilic S-Metabolizers such as Thermoproteus sp., Pyrodictium sp., Sulfolobus sp., Acidianus sp.
  • methanogenic archaeobacteria such as Methanobacterium sp., Methanobrevibacter sp., Methanothermus sp., Methanococcus s
  • microorganisms such as, Bacillus sublilis, Saccharomyces cerevisiae, Streptomyces sp., Ralstonia sp., Rhodococcus sp., Corynebacteria sp., Brevibacteria sp., Mycobacteria sp., and oleaginous yeast.
  • Preferred organi sms for the manufacture of carbon-based products of interest include: Arabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, and Zea mays (plants); Botryococcus braunii, Chlamydomonas reinhardtii and Dunaliela salina (algae); Synechococcus sp PCC 7002, Synechococcus sp. PCC 7942, Synechocystis sp. PCC 6803, Thermosynechococcus elongatus BP-1
  • cyanobacteria Chlorobium tepidum (green sulfur bacteria), Chloroflexus auranticus (green non-sulfur bacteria); Chromatium tepidum and Chromalium vinosum (purple sulfur bacteria); Rhodospirillum rubrum, Rhodobacter capsulattis, and Rhodopseudomonas palusris (purple non-sulfur bacteria).
  • Still other suitable organisms include synthetic cells or cells produced by synthetic genomes as described in Venter et al. US Pat. Pub. No. 2007/0264688, and cell-like systems or synthetic cells as described in Glass et al. US Pat. Pub. No. 2007/0269862.
  • microorganisms that can be engineered to fix carbon dioxide bacteria such as Escherichia coli, Acetobacter aceti, Bacillus subtilis, yeast and fungi such as Clostridium ljungdahlii, Clostridium thermocellum, Penicillium
  • chrysogenum Pichia pastor is, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonas mobilis.
  • a suitable organism for selecting or engineering is capable of autotrophic fixation of C0 2 to products. This would cover photosynthesis and methanogenesis. Acetogenesis, encompassing the three types of C0 2 fixation; Calvin cycle, acetyl-CoA pathway and reductive TCA pathway is also covered. The capability to use carbon dioxide as the sole source of cell carbon (autotrophy) is found in almost all major groups of prokaryotes. The C0 2 fixation pathways differ between groups, and there is no clear distribution pattern of the four presently-known autotrophic pathways. See, e.g., Fuchs, G. 1989. Alternative pathways of autotrophic CO 2 fixation, p. 365-382. In H. G. Schlegel, and B. Bowien (ed.), Autotrophic bacteria. Springer- Verlag, Berlin, Germany. The reductive pentose phosphate cycle
  • Carbon-based product of interest production via engineered cyanobacteria e.g., a Synechococcus or Thermosynechococcus species, is preferred.
  • Other preferred organisms include Synechocystis, Klebsiella oxytoca, Escherichia coli or Saccharomyces cerevisiae.
  • Other prokaryotic, archaea and eukaryotic host cells are also encompassed within the scope of the present invention.
  • carbon-based product of interest production via a photosynthetic organism can be carried out using the compositions, materials, and methods described in: PCT/US2009/035937 (filed March 3, 2009); and PCT/US2009/055949 (filed September 3, 2009); each of which is herein incorporated by reference in its entirety, for all purposes.
  • Carbon-Based Products of Interest Hydrocarbons & Alcohols
  • desired hydrocarbons and/or alcohols of certain chain length or a mixture thereof can be produced.
  • the host cell produces at least one of the following carbon-based products of interest: alkanes such as heptane, nonane, tridecane, pentadecane, and/or undecane and/or alcohols such as ethanol.
  • alkanes such as heptane, nonane, tridecane, pentadecane, and/or undecane and/or alcohols such as ethanol.
  • the carbon chain length ranges from
  • the methods provide culturing host cells for direct product secretion for easy recovery without the need to extract biomass. These carbon-based products of interest are secreted directly into the medium. Since the invention enables production of various defined chain length of hydrocarbons and alcohols, the secreted products are easily recovered or separated. The products of the invention, therefore, can be used directly or used with minimal processing.
  • compositions produced by the methods of the invention are used as fuels.
  • fuels comply with ASTM standards, for instance, standard specifications for diesel fuel oils D 975-09b, and Jet A, Jet A-l and Jet B as specified in ASTM Specification D. 1655-68.
  • Fuel compositions may require blending of several products to produce a uniform product. The blending process is relatively straightforward, but the determination of the amount of each component to include in a blend is much more difficult.
  • Fuel compositions may, therefore, include aromatic and/or branched hydrocarbons, for instance, 75% saturated and 25% aromatic, wherein some of the saturated hydrocarbons are branched and some are cyclic.
  • the methods of the invention produce an array of hydrocarbons, such as to alter cloud point.
  • the methods of the invention produce an array of hydrocarbons, such as to alter cloud point. Furthermore, the
  • compositions may comprise fuel additives, which are used to enhance the performance of a fuel or engine.
  • fuel additives can be used to alter the freezing/gelling point, cloud point, lubricity, viscosity, oxidative stability, ignition quality, octane level, and flash point.
  • Fuels compositions may also comprise, among others, antioxidants, static dissipater, corrosion inhibitor, icing inhibitor, biocide, metal deactivator and thermal stability improver.
  • Example 1 Increased rate of phosphate uptake in AphoU ethanologen with a maintained rate of ethanol production.
  • JCC1581 The construction of urea-repressible ethanologen JCC1581 was detailed in patent application PCT/US1.2/71250, herein incorporated by reference. JCC5048 was prepared in the same manner with the sole difference that the P(cum02) promoter (Table A) regulates the expression of the pdc-adh operon instead of P(nir07).
  • a chromosomal targeting vector was constructed containing 500 bp of upstream and downstream sequence homology to phoU allowing insertion of an erythromycin marker in place of the phoU gene (SYNPCC7002 A1708; Table A).
  • SYNPCC7002 A1708 The sequence and annotation of this plasmid (pJB3730) is provided in Table A.
  • This plasmid was naturally transformed into JCC138 using a standard cyanobacterial transformation and segregation protocol.
  • the genotypes of the two strains of cyanobacteria referenced in this example are provided in Table 1.
  • JB3.0 medium (pH -8.2) consists of 18.0 g/1 sodium chloride, 2.5 g/1 magnesium sulfate
  • heptahydrate 4.0 g/1 sodium nitrate, 1.0 g/1 Tris, 0.6 g/1 potassium chloride, 0.14 g/1 calcium chloride (anhydrous), 0.2 g/1 potassium phosphate monobasic, 34.3 mg/1 boric acid, 29.4 mg/1 EDTA (disodium salt dihydrate), 21.1 mg/1 EDTA iron (III) sodium salt, 4.3 mg/1 manganese chloride tetrahydrate, 315.0 g/l zinc chloride, 30.0 g/l molybdenum (VI) oxide, 12.2 g/l cobalt ( ⁇ ) chloride hexahydrate, 10.0 ⁇ g/l vitamin B 12 , and 3.0 ⁇ g/l copper (II) sulfate pentahydrate.
  • the cultures were incubated in a Multitron ⁇ (Infors) shaking photoincubator under the same conditions as the inoculum culture. Samples were taken for growth by taking OD 730 measurements and supernatant samples were taken to quantify the phosphate and ethanol concentrations in the medium.
  • phosphate assay kit from Abeam (ab65622) was used following the provided protocol with the following exceptions.
  • a phosphate calibration curve was prepared in JB3.0 medium between 0-60 ⁇ (5-60 ⁇ is the linear range of the assay in this medium, limit of detection ⁇ 1 ⁇ ) and culture supernatant dilutions were carried out in phosphate-free medium when necessary for quantification. 100 ⁇ of sample was added and 10 ⁇ of phosphate reagent to each well in the 96-well plate. The room temperature incubation time period was 1 hour instead of 30 min.
  • ethanol quantification and workup was performed as previously described (PCT/US12/71250). Briefly, the ethanol titre plots indicate the actual ethanol concentration in the flasks, and the cumulative ethanol plots indicate the total amount of ethanol produced by the strain under each condition (ethanol concentrations are adjusted to account for loss due to stripping from the flasks).
  • JCC5048 and JCC5473 were similar in JB3.0 medium containing 0.25x phosphate (figure 1), but the phosphate uptake rate by JCC5473 was much faster (figure 2).
  • the rate of phosphate uptake by JCC5473 was approximately 9-fold faster than JCC5048 (table 2), leading to phosphate depletion by the 4 hour timepoint whereas JCC5048 took over 24 hours to accomplish phosphate depletion in the medium.
  • JCC5473 grew comparably to JCC5048 (figure 3) and consumed all the phosphate by the 24 h timepoint (figure 4) but JCC5048 did not until day 6 (if ethanol production was not induced) or until day 8 (if ethanol production was induced). Through day 10, the ethanol production of JCC5473 matches that of JCC5048 (figure 5).
  • Example 2 Contaminated JCC5473 cultures produce ethanol at same rate as clean cultures.
  • Table 3 three strains of ethanol -catabolizing heterotrophs (Table 3) were added to concentration of 10 colony- forming units (CFUs) per ml to one flask for both strains, and 10 a3 CFU/ml was added to another flask of JCC5473.
  • CFUs colony- forming units
  • One flask for each strain received no addition of contamination for ethanol production rate comparison. Cultures were monitored for growth by taking OD 7 0 measurements. Culture supernatant samples were used to quantify the ethanol and phosphate concentrations.
  • JCC5473 and JCC5048 grew similarly and JCC5473 was able to sustain the same rate of ethanol production as the control flasks (figures 6-7, Table 4) even in the presence of a high concentration of contaminant cells at the beginning of the experiment (10 3 CFU/ml).
  • JCC5048 was not able to produce any significant amount ethanol even with the lower starting contaminant inoculation of 10 CFU/ml (figure 7).
  • Example 3 Improved ethanol production timespan of JCC5473 supplemented with acetate
  • JCC5473 was capable of taking up greater than 3x the phosphate normally present in JB3.0 (figure 10). As the biomass accumulation or phosphate uptake rate was unaffected by increased phosphate uptake (figure 10-11), JCC5473 can continue to accumulate phosphate if it is present in the medium. The cells with higher intracellular phosphate concentrations were capable of growing to a higher OD 730 (figure 12) in media lacking phosphate, indicating that JCC5473 is capable of utilizing increased stores of phosphate.
  • the JCC5473 cultures that had been incubated with 2x or higher phosphate were also able to produce 6-fold more ethanol in medium lacking phosphate than the JCC5473 culture incubated in lx phosphate (figure 13), despite displaying similar growth after induction (figure 14).
  • Example 5 Improved ethanol production timespan of phoU strains via expression of a catalase
  • JCC5290 was prepared in the same manner as JCC3351 with the sole difference that P(cum02) promoter (see example 1) regulates the expression of the pdc-adh operon instead of P(nir07).
  • P(cum02) promoter see example 1
  • the derivitive of JCC5290 was prepared as described in example 1 to yield JCC5476. The genotypes of these two strains are provided in table 5a.
  • Cultures were monitored for growth by taking OD 730 measurements and culture supernatant samples were taken for ethanol quantification and phosphate uptake.
  • JCC5476 was able to produce ethanol for the same time period as JCC5290, and acetate addition did not extend the longevity of ethanol production (Figure 15). Therefore, expression of katG appears to mitigate the negative impact of the phoU knockout, indicating that the shortened lifespan of JCC5473 may be due to peroxide generation. Ethanol production was not enhanced by acetate addition to JCC5290 and JCC5476, but those cultures with acetate did reach a higher cell density ( Figure 16). JCC5476 was able to remove all phosphate from the media by the day 1 timepoint ( Figure 17), showing that the strain maintains a high rate of phosphate uptake with katG expression.
  • JCC5048 and JCC5473 were grown in JB3.0 and JB3.0 with 3x phosphate.
  • the photobioreactor setup utilized a 50 foot length of clear flex 70-1 culture tubing, with an inner diameter of 0.75 inches, and a tube wall of 0.125 inches.
  • the reactor was sparged with 2% C0 2 at a rate of 0.80 liters per minute, circulated with a Watson Marlow 720u pump set at 180 revolutions per minute, and operated at pH of 7.2 ⁇ .2 and 35°C.
  • JCC5473 was capable of using solely internal phosphate stores such that growth (figure 18) and ethanol productivity (figure 19) matched that of the culture with additional phosphate input. Additionally, the contaminated culture of JCC5473 was capable of controlling contamination throughout the run such that productivity was indistinguishable from that of the axenic JCC5473 reactor.
  • JCC5048 shows AP activity, but only when depleted for phosphate.
  • JCC5473 shows AP activity independent of the phosphate concentration in the media.
  • JCC6082 shows AP activity independent of phosphate concentration when repressed for phoU, but only in the absence of phosphate when induced with for phoU expression with the addition of DOC (table 6). Together this indicates that sufficient control of phoU exists to allow temporal control of the pho regulon.
  • Example 7 Increased ethanol productivity with induced expression of phoU after phosphate depletion in the culture medium.
  • JCC5048 was grown in JB3.0, and JCC6082 was grown in JB3.0 with 0.6x phosphate with lOmM acetate.
  • Cells were cultured in an indoor photobioreactor mimicking outdoor insolation with a peak insolation of 2000 uE/m2/s. At the time indicated by the arrow below, 20 uM cumate was added to the reactors.
  • Example 8 Preventing contaminant catabolism of extracellular products in
  • strains of E. coli can be engineered to rapidly deplete the medium of phosphate similar to the previous examples given for Synechococcus sp. PCC7002. It has previously been shown that a phoU knockout of E. coli uptakes phosphate at a much higher rate as well and accumulates much more polyphosphate than the respective wild type strains (Morohoshi et. al. 2002). Inducible or modified constitutive regulation of phoU may also increase phosphate uptake and strain stability.
  • ppk polyphosphate kinase
  • ackA acetate kinase
  • pst operon phosphate transporter systems
  • E. coli strains co-expressing pdc and adh and showing improved phosphate uptake via some or all the genetic manipulations detailed above and also in Table 7 is evaluated for their ability to prevent contaminant catabolism of ethanol under fermentation conditions when three ethanol consuming heterotrophs are present (Table 3).
  • Table 7 Representative E. coli genes and respective manipulations to increase phosphate uptake from medium.
  • Example 9 Preventing contaminant catabolism of extracellular products in
  • strains of yeast like Saccharomyces cerevisiae can also be engineered to rapidly deplete the medium of phosphate. They store phosphate in the cell as polyphosphate similar to prokaryotes and also are capable of rapid uptake of phosphate.
  • the phosphate uptake regulatory system is more complex (Ljundahl and Daignan-Fornier 2012), with 22 ORFs being involved in the rapid phosphate uptake response (Ogawa et. al. 2000).
  • the transcription factor Pho4 plays an important role in the transcriptional regulation of the phosphate uptake response including control of high affinity phosphate transporters and polyphosphate biosynthetic genes. When wild-type S.
  • Pho4 is phosphorylated by cyclin/kinase pair Pho80/Pho85 causing Pho4 to be exported from the nuclease to the cytoplasm. This prevents Pho4 from complexing with the transcriptional activator Pho2 and catalyzing increased expression of the phosphate uptake pathways.
  • Knocking out the native Pho4 and expressing a mutant form named Pho4 SAl234PA6 that has four phosphorylation sites removed through serine -> alanine swaps and one site with a proline -> alanine swap has been shown to allow full induction of the phosphate-uptake genes in phosphate-replete media.
  • expression of the native Pho4 in S. cerevisiae in a ⁇ pho4 ⁇ pho80 also allows high expression of the phosphate pathway when grown in replete phosphate conditions (Komeili and O' Shea 1999).
  • Regulating Pho4, pho4 SA1234PA6 or Pho80 expression under inducible promoters or other alternate promoters may allow optimal regulation of the pathway. Ensuring strong expression of the phosphate uptake pathway genes may not be enough however to deplete the phosphate in the medium, as post-translational regulation of phosphate uptake is known to exist is S. cerevisiae.
  • S. cerevisiae the primary high-affinity phosphate transporter under acidic conditions is Pho84, which appears to be removed from the membrane under high phosphate conditions even if the Pho84 gene is being strongly transcribed (Pratt et. al. 2004). Therefore, mutants may need to be identified where the existing post-transcriptional mechanisms of phosphate control in S.
  • cerevisiae are knocked out in order to allow high rates of phosphate uptake when phosphate is replete in the medium in addition to having the necessary transcriptional control of phosphate-uptake associated genes. It may also be necessary to express alternate/additional phosphate uptake transporters from different organisms or over-express native phosphate transporters to ensure optimal phosphate uptake kinetics.

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Abstract

La présente invention identifie des procédés et des compositions pour la modification d'organismes photoautotrophes en tant qu'hôtes, de sorte que les organismes produisent efficacement des produits d'intérêt à base de carbone, et en particulier l'utilisation de tels organismes pour la production commerciale d'éthanol et de molécules associées à base de carbone en présence de micro-organismes contaminants qui peuvent utiliser les utiliser. L'invention concerne d'autres matériaux, procédés et compositions.
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CN108359617A (zh) * 2017-12-29 2018-08-03 浙江双良商达环保有限公司 不动杆菌cl05及其在村镇污水除磷处理中的应用
US10138489B2 (en) 2016-10-20 2018-11-27 Algenol Biotech LLC Cyanobacterial strains capable of utilizing phosphite
CN109439571A (zh) * 2018-10-31 2019-03-08 广州小众环保科技有限公司 一种氨氮去除菌剂

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PL389665A1 (pl) * 2007-02-09 2010-07-05 The Regents of the University of CaliforniaThe Regents of the University of California Wytwarzanie biopaliwa przez rekombinowane mikroorganizmy
EP2615164A1 (fr) * 2007-11-10 2013-07-17 Joule Unlimited Technologies, Inc. Organismes hyperphotosynthétiques
SG10201401472YA (en) * 2009-04-14 2014-08-28 Solazyme Inc Methods Of Microbial Oil Extraction And Separation
US9523096B2 (en) * 2010-12-20 2016-12-20 Matrix Genetics, Llc Modified photosynthetic microorganisms for producing lipids
US10065876B2 (en) * 2013-06-12 2018-09-04 Malcolm A Barnard Algal system for improving water quality

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10138489B2 (en) 2016-10-20 2018-11-27 Algenol Biotech LLC Cyanobacterial strains capable of utilizing phosphite
CN108359617A (zh) * 2017-12-29 2018-08-03 浙江双良商达环保有限公司 不动杆菌cl05及其在村镇污水除磷处理中的应用
CN109439571A (zh) * 2018-10-31 2019-03-08 广州小众环保科技有限公司 一种氨氮去除菌剂

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