WO2013096475A1 - Transport extracellulaire d'hydrocarbures biosynthétiques et d'autres molécules - Google Patents

Transport extracellulaire d'hydrocarbures biosynthétiques et d'autres molécules Download PDF

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WO2013096475A1
WO2013096475A1 PCT/US2012/070666 US2012070666W WO2013096475A1 WO 2013096475 A1 WO2013096475 A1 WO 2013096475A1 US 2012070666 W US2012070666 W US 2012070666W WO 2013096475 A1 WO2013096475 A1 WO 2013096475A1
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microorganism
engineered
alkanes
alkenes
recombinant
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Kevin M. Smith
Christian Perry Ridley
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Joule Unlimited Technologies, Inc.
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    • 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
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • C12P5/026—Unsaturated compounds, i.e. alkenes, alkynes or allenes
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
    • C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
    • C12Y102/0108—Long-chain acyl-[acyl-carrier-protein] reductase (1.2.1.80)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/99—Other Carbon-Carbon Lyases (1.4.99)
    • C12Y401/99005—Octadecanal decarbonylase (4.1.99.5)

Definitions

  • This application includes a Sequence Listing submitted electronically as a text file named X_PCT_sequencelisting.txt, created on X, with a size of X bytes. The sequence listing is incorporated by reference.
  • Recombinant photosynthetic microorganisms have been engineered to produce hydrocarbons, including alkanes, in amounts that exceed the levels produced naturally by the organism.
  • compositions and methods for increasing the amount of hydrocarbons e.g., n-alkanes and n-alkenes
  • engineered microorganisms comprising recombinant enzymes for producing hydrocarbons are provided, wherein said microorganisms are further modified to secrete said hydrocarbons in greater amounts than otherwise identical hydrocarbon-producing microorganisms lacking the modifications.
  • an engineered microorganism comprising (i) one or more recombinant genes encoding enzymes which catalyze the production of alkanes and/or alkenes, and (ii) one or more recombinant genes encoding one or more tripartite transporter proteins selected from the group consisting of EmrA, EmrB, AcrE, AcrF, EmrK, EmrY, MacA, MacB, MdtA, MdtB, MdtC, MdtE, MdtF, SdsR, SdsQ, and SdsP.
  • said one or more recombinant genes encoding one or more tripartite transporter proteins is EmrA and/or EmrB or a homologue thereof. In some aspects, said one or more recombinant genes encoding one or more tripartite transporter proteins is EmrA and EmrB.
  • said microorganism is a bacterium. In some aspects, said microorganism is a gram-negative bacterium. In some aspects, said microorganism is E. coli.
  • expression of an operon comprising the one or more recombinant genes encoding tripartite transporter proteins is controlled by a recombinant promoter, and wherein the promoter is constitutive or inducible.
  • said operon is integrated into the genome of said microorganism. In some aspects, said operon is extrachromosomal.
  • said microorganism is a photosynthetic microorganism. In some aspects, said microorganism is a cyanobacterium. In some aspects, said microorganism is a Synechococcus species.
  • said one or more tripartite transporter proteins are selected from the group consisting of EmrA and EmrB, and wherein the native leader sequences of said proteins are replaced with leader sequences native to said photosynthetic microorganism.
  • said one or more recombinant genes encoding one or more tripartite transporter proteins are E. coli genes. In some aspects, the expression of said one or more tripartite transporter proteins is increased relative to an otherwise identical
  • microorganism lacking said one or more tripartite transporter proteins lacking said one or more tripartite transporter proteins.
  • the activity of said one or more tripartite transporter proteins is increased relative to an otherwise identical microorganism lacking said one or more tripartite transporter proteins.
  • said microorganism is a photosynthetic organism, wherein said recombinant genes encoding enzymes which catalyze the production of alkanes and/or alkenes comprise recombinant AAR and recombinant ADM, and wherein said one or more recombinant genes encoding one or more tripartite transporter proteins comprise EmrA and EmrB.
  • said expression of said one or more tripartite transporter proteins is driven by a T5 promoter.
  • said microorganism comprises a recombinant TolC gene or homologue thereof.
  • said enzymes which catalyze the production of alkanes and/or alkenes are selected from the group consisting of a recombinant acyl-ACP reductase (AAR) enzyme and a recombinant alkanal deformylative monooxygenase (ADM) enzyme.
  • said one or more recombinant genes encode enzymes which catalyze the production of alkanes and/or alkenes are Synechococcus AAR and ADM genes.
  • said one or more recombinant genes encoding enzymes which catalyze the production of alkanes and/or alkenes are Synechococcus elongatus AAR and ADM genes.
  • Also described herein is a method for producing hydrocarbons, comprising:
  • said culture medium does not include a surfactant. In some aspects, said culture medium does not include EDTA. In some aspects, said culture medium does not include Tris buffer.
  • said engineered microorganism secretes at least 2.5-fold more n- alkanes and n-alkenes relative to an otherwise identical microorganism, cultured under identical conditions, but lacking said recombinant genes for efflux of n-alkanes or n-alkenes.
  • said engineered microorganism secretes 2-4 fold more n-alkanes and n- alkenes relative to an otherwise identical microorganism, cultured under identical conditions, but lacking said recombinant genes for efflux of n-alkanes or n-alkenes.
  • said engineered microorganism is an engineered E.
  • said engineered microorganism is an engineered E. coli, and wherein at least 95% of said n-alkanes or n- alkenes are secreted into the culture medium. In some aspects, said engineered
  • microorganism produces at least 0.1 mg/L/OD/hr of said n-alka/enes.
  • Also described herein is a method for producing hydrocarbons, comprising:
  • culturing an engineered photosynthetic microorganism disclosed herein in a culture medium and exposing said engineered photosynthetic microorganism to light and carbon dioxide, wherein said exposure results in the conversion of said carbon dioxide by said microorganism into n-alkanes, wherein said n-alkanes are secreted into said culture medium in an amount greater than that secreted by an otherwise identical microorganism, cultured under identical conditions, but lacking said recombinant genes.
  • said engineered photosynthetic microorganism further produces at least one n-alkene or n-alkanol.
  • said engineered photosynthetic microorganism produces at least one n-alkene or n-alkanol selected from the group consisting of ft-pentadecene, n-heptadecene, and 1-octadecanol.
  • said n-alkanes comprise predominantly n-heptadecane, n-pentadecane or a combination thereof.
  • the above method further comprises isolating at least one n- alkane, n-alkene or n-alkanol from said culture medium.
  • At least one of said recombinant genes is encoded on a plasmid. In some aspects, at least one of said recombinant genes is incorporated into the genome of said engineered photosynthetic microorganism. In some aspects, at least one of said recombinant genes is present in multiple copies in said engineered photosynthetic
  • At least two of said recombinant genes are part of an operon, and wherein the expression of said genes is controlled by a single promoter.
  • At least 50 to 95% of said n-alkanes are n-pentadecane, n- heptadecane, and/or n-heptadecene. In some aspects, at least 95% of said n-alkanes are n- pentadecane and n-heptadecane.
  • the expression of at least one of said recombinant genes is controlled by one or more inducible promoters.
  • at least one promoter is a urea-repressible, nitrate-inducible promoter.
  • Also described herein is a method of identifying a cyanobacteria capable of effluxing n-alkane and/or n-alkene, comprising determining that the cyanobacteria is resistant to an antibiotic applied to the cyanobacteria, wherein the antibiotic resistance is conferred by the presence of a tripartite transporter complex comprising EmrA, EmrB, and TolC, and wherein the complex is capable of mediating efflux of n-alkane and/or n-alkene.
  • the antibiotic is SDS, deoxycholate, CCCP, Rhomadine 6G, nalidixic acid, tetrachlorosalicyl anilide, 2-chlorophenylhydrazine, thiolactomycin or methylviologen.
  • Also described herein is a method of producing an engineered cyanobacteria capable of effluxing n-alkane and/or n-alkene, comprising contacting the cyanobacteria with an engineered outer membrane protein capable of forming a tripartite transporter complex with endogenous EmrA and EmrB, wherein the complex is capable of mediating efflux of n- alkane and/or n-alkene.
  • Also described herein is a method of producing an engineered cyanobacteria capable of effluxing n-alkane and/or n-alkene, comprising contacting the cyanobacteria with an engineered EmrA and/or an engineered EmrB protein capable of forming a tripartite transporter complex with one or more endogenous outer membrane proteins, wherein the complex is capable of mediating efflux of n-alkane and/or n-alkene.
  • an engineered photosynthetic microbe comprising a recombinant nucleic acid or recombinant protein comprising a sequence selected from SEQ ID NOs shown in the sequence listing.
  • said engineered microbe is a photosynthetic microbe.
  • said engineered photosynthetic microbe is a cyanobacterium.
  • Cyanobacteria contain not only a plasma membrane (PM) like non-photosynthetic prokaryotic hosts (as well as an outer membrane like their Gram-negative non-photosynthetic counterparts), but also, typically, an intracellular thylakoid membrane (TM) system that serves as the site for photosynthetic electron transfer and proton pumping.
  • PM plasma membrane
  • TM thylakoid membrane
  • heterologous integral plasma membrane proteins both prokaryotic and eukaryotic in origin that must be targeted to the plasma membrane of the cyanobacterial host in order to function as desired.
  • HIPMPs heterologous integral plasma membrane proteins
  • HIPMPs of particular interest with respect to the efflux of n-alkanes and n-alkenes are the integral plasma membrane subunits, EmrA and EmrB.
  • membrane proteins that are not HIPMPs, i.e., proteins that are derived from membranes other than the plasma membrane.
  • Such alternative membranes include: the thylakoid membrane, the endoplasmic reticulum membrane, the chloroplast inner membrane, and the mitochondrial inner membrane.
  • the disclosure provides methods for designing a protein comprising a pseudo-leader sequence (PLS) of defined sequence fused to the N-terminus of an HIPMP of interest, wherein the resulting chimeric protein is expressed in a cyanobacterial host cell, e.g. , JCC138 (Synechocystis sp. PCC 7002) or an engineered derivative thereof.
  • PLS pseudo-leader sequence
  • the expression of the chimeric protein will increase the amount of hydrocarbon products of interest (e.g., alkanes, alkenes, alkyl alkanoates, etc.) exported from the cynanobacterial host cell.
  • the PLS encodes a contiguous polypeptide sub-fragment of a protein from a different thylakoid- membrane-containing cyanobacterial host, e.g., JCC160 (Synechococcus sp. PCC 6803), that localizes as uniquely as possible to the plasma membrane of that host.
  • JCC160 Synechococcus sp. PCC 6803
  • the mechanism that this non-JCC138 host natively employs to effect the localization of the protein to the plasma membrane should be conserved in order for the localization to occur in the recipient host.
  • PLSs are designed to ensure, or at least bias, the targeting of HIPMPs to the plasma membrane of the heterologous cyanobacterial host, they may not always be required. This is because sufficient levels of functional HIPMP may become embedded in the plasma membrane if the cyanobacterial host does, in fact, mechanistically recognize the protein as a native plasma membrane protein - even if some fraction of the protein is targeted to the thylakoid membrane or ends up in neither membrane (e.g. , as inclusion bodies).
  • the PLS is derived from a plasma-membrane-resident protein that is naturally anchored in the membrane of a different cyanobacterial species (i.e.
  • said plasma- membrane -resident protein naturally has its N-terminus within the cytoplasm and its C-terminus within the cytoplasm (Ni n /Ci n ), spanning the plasma membrane via an in-to-out transmembrane a helix, followed by an (ideally short) periplasmic loop sequence, followed by an out-to-in transmembrane a helix.
  • the PLS is derived from a plasma-membrane-resident protein that is naturally anchored in the membrane of a different cyanobacterial species via one transmembrane a helix, and (ii) said plasma- membrane-resident protein naturally has its N-terminus within the cytoplasm and its C- terminus within the periplasm (Ni n /C ou t).
  • PLSs are derived from host proteins that have most of their mass in either the periplasmic and/or cytoplasmic spaces.
  • said PLSs should contain only two a helices with Ni n /Q n topology (for creating Ni n HIPMPs) and only one a helix with Ni n /C out topology (for creating N out HIPMPs).
  • the potential for intermolecular homomultimerization among the transmembrane helices of the PLSs is minimized.
  • fused refers to the joining of one functional protein or protein subunit (e.g. , a pseudo- leader sequence) to another functional protein or protein subunit (e.g. , an integral plasma membrane protein). Fusing can occur by any method which results in the covalent attachment of the C-terminus of one such protein molecule to the N-terminus of another. For example, one skilled in the art will recognize that fusing occurs when the two proteins to be fused are encoded by a recombinant nucleic acid under control of a promoter and expressed as a single structural gene in vivo or in vitro.
  • non-target refers to a protein or nucleic acid that is native to a species that is different than the species that will be used to recombinantly express the protein or nucleic acid.
  • Alkanes also known as paraffins, are chemical compounds that consist only of the elements carbon (C) and hydrogen (H) (i.e., hydrocarbons), wherein these atoms are linked together exclusively by single bonds (i.e., they are saturated compounds) without any cyclic structure.
  • n-Alkanes are linear, i.e., unbranched, alkanes.
  • AAR or ADM enzymes are referred to herein as Aar genes (aar) or Adm genes (adm), respectively. Together, AAR and ADM enzymes function to synthesize n-alkanes from acyl-ACP molecules.
  • an AAR enzyme refers to an enzyme with the amino acid sequence of the SYNPCC7942 1594 protein or a homolog thereof, wherein a SYNPCC7942 1594 homolog is a protein whose BLAST alignment (i) covers >90% length of SYNPCC7942 1594, (ii) covers >90% of the length of the matching protein, and (iii) has >50% identity with SYNPCC7942_1594 (when optimally aligned using the parameters provided herein), and retains the functional activity of SYNPCC7942_1594, i.e., the conversion of an acyl-ACP (acyl-acyl carrier protein) to an n-alkanal.
  • acyl-ACP acyl-acyl carrier protein
  • An ADM enzyme refers to an enzyme with the amino acid sequence of the SYNPCC7942 1593 protein or a homolog thereof, wherein a SYNPCC7942 1593 homolog is defined as a protein whose amino acid sequence alignment (i) covers >90% length of SYNPCC7942_1593, (ii) covers >90% of the length of the matching protein, and (iii) has >50% identity with
  • SYNPCC7942_1593 (when aligned using the parameters provided herein), and retains the functional activity of SYNPCC7942 1593, i.e., the conversion of an n-alkanal to an (n-X)- alkane.
  • Exemplary AAR and ADM enzymes are listed in Table 1 and Table 2, respectively, of U.S. utility application 12/759,657, filed April 13, 2010 (now U.S. Pat. No. 7,794,969), and U.S. utility application 12/833,821, filed July 9, 2010.
  • Other ADM activities are described in U.S. Pat. App. No. 12/620,328, filed November 17, 2009. Applicants note that in previous related applications, this enzyme was referred to as an alkanal decarboxylative monooxygenase.
  • the protein is referred to herein as an alkanal deformylative
  • ADM monooxygenase
  • parameters for BLASTp are: Expectation value: 10 (default); Filter: none; Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Maximum alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.
  • Functional homologs of other proteins described herein may share significant amino acid identity (e.g., >50%) with the named proteins whose sequences are presented herein.
  • Such homo logs may be obtained from other organisms where the proteins are known to share structural and functional characteristics with the named proteins. For example, an outer membrane protein that is at least 95% identical to E. coli TolC is considered a TolC homolog.
  • a functional outer membrane protein that is at least 95% identical to TolC except for the replacement/addition of leader sequences, C-terminal sequences or other modifications intended to increase its functionality in a particular environment (e.g., a non-native host) are also considered functional homo logs of TolC.
  • the same definitions apply to other protein homologs referred to herein.
  • EmrA, EmrB, and their homologs are members of the Major Facilitator
  • EmrAB Superfamily of proteins
  • the criteria used to identify EmrAB homologs are based on analysis of the sequence of the entire protein(s) and (1) The EmrAB homolog is >30% identical to E. coli EmrB or EmrA; (2) The EmrAB homolog classifies based on its amino acid sequence as a member of the drug:proton antiporter-2 subfamily (containing 14 transmembrane alpha helices) of the MFS based on the Transporter Classification Database (TCDB 2.
  • TCDB 2 Transporter Classification Database
  • the gene encoding the EmrB homolog protein is found in an operon also containing a gene encoding an EmrA homolog protein that classifies based on its amino acid sequence as a member of the Membrane Fusion Protein (MFP) family of proteins (TCDB 8.A.1).
  • MFP Membrane Fusion Protein
  • cyanobacteria are also applicable other organisms, e.g., gram-negative bacteria such as E. coli.
  • a chimeric integral plasma membrane protein for facilitating alkane efflux in E. coli could be designed by fusing a pseudo leader sequence derived from E. coli or a related bacterium to a heterologous integral plasma membrane protein.
  • nucleic acid 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 internucleoside 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: l 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, alkene, or alkanal
  • 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.
  • the term “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 mR As 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.
  • percent sequence identity or “identical” in the context of nucleic acid sequences 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 in some instances at least about 36 or more nucleotides.
  • FASTA FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis.
  • GCG Genetics Computer Group
  • percent sequence identity between nucleic acid sequences can be determined using FASTA 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 hybridizes 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
  • 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 disclosure 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 et al., Technique, 1 : 11-15 (1989) and Caldwell and Joyce, PCR Methods App lie.
  • 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 et al., Technique, 1 : 11-15 (1989) and Caldwell and Joyce, PCR Methods App lie.
  • 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. In some instances 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 refers to 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.
  • the term "knock out” refers to 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. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors" (or simply "expression vectors").
  • “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 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
  • 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 disclosure have particular utility.
  • the heterologous polypeptide included within the fusion protein of the present disclosure 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. Alternatively, a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein.
  • GFP green fluorescent protein
  • antibody refers to a polypeptide, at least a portion of which is encoded by at least one immunoglobulin gene, or fragment thereof, and that can bind specifically to a desired target molecule.
  • the term includes naturally-occurring forms, as well as fragments and derivatives.
  • fragments within the scope of the term "antibody” include those produced by digestion with various proteases, those produced by chemical cleavage and/or chemical dissociation and those produced recombinantly, so long as the fragment remains capable of specific binding to a target molecule.
  • fragments include Fab, Fab', Fv, F(ab') 2 , and single chain Fv (scFv) fragments.
  • Derivatives within the scope of the term include antibodies (or fragments thereof) that have been modified in sequence, but remain capable of specific binding to a target molecule, including: interspecies chimeric and humanized antibodies; antibody fusions; heteromeric antibody complexes and antibody fusions, such as diabodies (bispecific antibodies), single-chain diabodies, and intrabodies (see, e.g., Intracellular Antibodies:
  • antibodies can be produced by any known technique, including harvest from cell culture of native B lymphocytes, harvest from culture of hybridomas, recombinant expression systems and phage display.
  • 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.
  • Examples of unconventional amino acids include: 4-hydroxyproline, ⁇ -carboxyglutamate, ⁇ - ⁇ , ⁇ , ⁇ -trimethyllysine, ⁇ - ⁇ -acetyllysine, O-phosphoserine, N-acetylserine, N- formylmethionine, 3-methylhistidine, 5 -hydroxy lysine, 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 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.
  • 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.
  • An algorithm that can be used 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 (1997)).
  • 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.
  • database searching using amino acid sequences can be measured by algorithms other than blastp known in the art.
  • 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).
  • Percent dry cell weight refers to a measurement of hydrocarbon production obtained as follows: a defined volume of culture is centrifuged to pellet the cells. Cells are washed then dewetted by at least one cycle of microcentrifugation and aspiration. Cell pellets are lyophilized overnight, and the tube containing the dry cell mass is weighed again such that the mass of the cell pellet can be calculated within ⁇ 0.1 mg. At the same time cells are processed for dry cell weight determination, a second sample of the culture in question is harvested, washed, and dewetted.
  • the resulting cell pellet corresponding to 1-3 mg of dry cell weight, is then extracted by vortexing in approximately 1 ml acetone plus butylated hydroxytolune (BHT) as antioxidant and an internal standard, e.g., n-eicosane.
  • BHT butylated hydroxytolune
  • Cell debris is then pelleted by centrifugation and the supernatant (extractant) is taken for analysis by GC.
  • flame ionization detection FID
  • n-Alkane concentrations in the biological extracts are calculated using calibration relationships between GC-FID peak area and known concentrations of authentic n-alkane standards. Knowing the volume of the extractant, the resulting concentrations of the n-alkane species in the extractant, and the dry cell weight of the cell pellet extracted, the percentage of dry cell weight that comprised n-alkanes can be determined.
  • 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, 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 nucleic acid molecule of the present disclosure encodes a polypeptide having the amino acid sequence of any of the protein sequences provided in the SEQ ID NOs of the sequence listing.
  • the nucleic acid molecule of the present disclosure encodes a polypeptide sequence of at least 50%, 60, 70%, 80%>, 85%, 90% or 95%) identity to one of the protein sequences shown in the SEQ ID NOs in the sequence listing and the identity can even more preferably be 96%, 97%, 98%>, 99%, 99.9% or even higher.
  • a nucleic acid molecule has at least 50%, 60, 70%, 80%, 85%, 90% or 95% identity to one of the sequences shown in SEQ ID NOs 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 21 , 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, or 58 and the identity can even more preferably be 96%, 97%, 98%, 99%, 99.9% or even higher.
  • a nucleic acid molecule of the present disclosure encodes a polypeptide sequence of at least 50%, 60, 70%, 80%, 85%, 90% or 95% identity to one of the protein sequences shown in SEQ ID NOs 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, or 47 in the sequence listing and the identity can even more preferably be 96%, 97%, 98%, 99%, 99.9% or even higher.
  • the present disclosure 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 disclosure 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
  • nucleic acid probes of known sequence find utility in determining chromosomal structure (e.g., by
  • 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 disclosure 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, NY: Marcel Dekker. (ISBN:
  • 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 magnetic resonance
  • NIR near-infrared
  • Chem. 340(3): 186 can be used to analyze the levels and the identity of the product produced by the organisms of the present disclosure.
  • Other methods and techniques may also be suitable for the measurement of enzyme activity, as would be known by one of skill in the art.
  • vectors including expression vectors, which comprise the above nucleic acid molecules of the present disclosure, as described further herein.
  • the vectors include the isolated nucleic acid molecules described above.
  • the vectors of the present disclosure include the above-described nucleic acid molecules operably linked to one or more expression control sequences.
  • the vectors of the instant disclosure may thus be used to express an Aar and/or Adm polypeptide contributing to n-alkane producing activity by a host cell, and/or a chimeric efflux protein for effluxing n-alkanes and other hydrocarbons out of the cell.
  • host cells transformed with the nucleic acid molecules or vectors of the present disclosure, and descendants thereof, are provided.
  • these cells carry the nucleic acid sequences of the present disclosure on vectors, which may but need not be freely replicating vectors.
  • the nucleic acids have been integrated into the genome of the host cells.
  • the host cell comprises one or more AAR or ADM encoding nucleic acids which express AAR or ADM in the host cell.
  • the host cells of the present disclosure can be mutated by recombination with a disruption, deletion or mutation of the isolated nucleic acid of the present disclosure so that the activity of the AAR and/or ADM protein(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 Eucarya, 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 bacteria.
  • 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 Synechococcus 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) and 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 Natronobacterium, 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: Acanthoceras, Acanthococcus, Acaryochloris, Achnanthes, Achnanthidium, Actinastrum, Actinochloris, Actinocyclus, Actinotaenium, Amphichrysis, Amphidinium, Amphikrikos, Amphipleura, Amphiprora, Amphithrix, Amphora, Anabaena, Anabaenopsis, Aneumastus, Ankistrodesmus, Ankyra, Anomoeoneis, Apatococcus, Aphanizomenon, Aphanocapsa, Aphanochaete, Aphanothece, Apiocystis, Apistonema, Arthrodesmus, Artherospira, Ascochloris, Asterionella, Asterococcus, Audouinella, Aulacoseira, Bacillaria, Balbiania, Bambusina,
  • Chrysostephanosphaera Clodophora, Clastidium, Closteriopsis, Closterium, Coccomyxa, Cocconeis, Coelastrella, Coelastrum, Coelosphaerium, Coenochloris, Coenococcus, Coenocystis, Colacium, Coleochaete, Collodictyon, Compsogonopsis, Compsopogon, Conjugatophyta, Conochaete, Coronastrum, Cosmarium, Cosmioneis, Cosmocladium, Crateriportula, Craticula, Crinalium, Crucigenia, Crucigeniella, Cryptoaulax, Cryptomonas, Cryptophyta, Ctenophora, Cyanodictyon, Cyanonephron, Cyanophora, Cyanophyta, Cyanothece, Cyanothomonas, Cyclonexis, Cyclostephanos, Cyclotella, Cy
  • Cymbellonitzschia Cystodinium Dactylococcopsis, Debarya, Denticula, Dermatochrysis, Dermocarpa, Dermocarpella, Desmatractum, Desmidium, Desmococcus, Desmonema, Desmosiphon, Diacanthos, Diacronema, Diadesmis, Diatoma, Diatomella, Dicellula, Dichothrix, Dichotomococcus, Dicranochaete, Dictyochloris, Dictyococcus,
  • Distrionella Docidium, Draparnaldia, Dunaliella, Dysmorphococcus, Ecballocystis,
  • Eucocconeis Eudorina, Euglena, Euglenophyta, Eunotia, Eustigmatophyta, Eutreptia, Fallacia, Fischerella, Fragilaria, Fragilariforma, Franceia, Frustulia, Curcilla, Geminella, Genicularia, Glaucocystis, 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, Granulochloris, Granulocystopsis, Groenbladia, Gymnodinium, Gymnozyga, Gyrosigma, Haematococcus, Hafniomonas, Hallassia, Hammatoidea, Hannaea, Hantzschia, Hapalosiphon, Haplotaenium, Haptophyta, Haslea, Hemidinium, Hemitoma, Heribaudiella, Heteromastix, Heterothrix, Hibberdia, Hildenbrandia,
  • Hyalobrachion Hyalocardium, Hyalodiscus, Hyalogonium, Hyalotheca, Hydrianum, Hydrococcus, Hydrocoleum, Hydrocoryne, Hydrodictyon, Hydrosera, Hydrurus, Hyella, Hymenomonas, Isthmochloron, Johannesbaptistia, Juranyiella, Karayevia, Kathablepharis, Katodinium, Kephyrion, Keratococcus, Kirchneriella, Klebsormidium, Kolbesia, Koliella, Komarekia, Korshikoviella, Kraskella, Lagerheimia, Lagynion, Lamprothamnium, Lemanea, Lepocinclis, Leptosira, Lobococcus, Lobocystis, Lobomonas, Luticola, Lyngbya,
  • Gastogloia Melosira, Merismopedia, Mesostigma, Mesotaenium, Micractinium,
  • mice Micrasterias, Microchaete, Microcoleus, Microcystis, Microglena, Micromonas, Microspora, Microthamnion, Mischococcus, Monochrysis, Monodus, Monomastix, Monoraphidium, Monostroma, Mougeotia, Mougeotiopsis, Myochloris, Myromecia, Myxosarcina,
  • Rhoicosphenia Rhopalodia, Rivularia, Rosenvingiella, Rossithidium, Roya, Scenedesmus, Scherffelia, Schizochlamydella, Schizochlamys, Schizomeris, Schizothrix, Schroederia, Scolioneis, Scotiella, Scotiellopsis, Scourfieldia, Scytonema, Selenastrum, Selenochloris, Sellaphora, Semiorbis, Siderocelis, Diderocystopsis, Dimonsenia, Siphononema,
  • Sirocladium Sirogonium, Skeletonema, Sorastrum, Spermatozopsis, Sphaerellocystis, Sphaerellopsis, Sphaerodinium, Sphaeroplea, Sphaerozosma, Spiniferomonas, Spirogyra, Spirotaenia, Spirulina, Spondylomorum, Spondylosium, Sporotetras, Spumella, Staurastrum, Stauerodesmus, Stauroneis, Staurosira, Staurosirella, Stenopterobia, Stephanocostis,
  • Stephanodiscus Stephanoporos, Stephanosphaera, Stichococcus, Stichogloea, Stigeoclonium, Stigonema, Stipitococcus, Stokesiella, Strombomonas, Stylochrysalis, Stylodinium,
  • Styloyxis Stylosphaeridium, Surirella, Sykidion, Symploca, Synechococcus, Synechocystis, Synedra, Synochromonas, Synura, Tabellaria, Tabularia, Molingia, Temnogametum,
  • Tetraselmis Tetraspora, Tetrastrum, Thalassiosira, Thamniochaete, Thorakochloris, Thorea, Tolypella, Tolypothrix, Trachelomonas, Trachydiscus, Trebouxia, Trentepholia, Treubaria, Tribonema, Trichodesmium, Trichodiscus, Trochiscia, Tryblionella, Ulothrix, Uroglena, Uronema, Urosolenia, Urospora, Uva, Vacuolaria, Vaucheria, Volvox, Volvulina, Westella, Woloszynskia, Xanthidium, Xanthophyta, Xenococcus, Zygnema, Zygnemopsis, and Zygonium.
  • a partial list of cyanobacteria that can be engineered to express the recombinant described herein include members of the genus Chamaesiphon, Chro
  • 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 Thermochromatium, Thiocapsa, Thiorhodococcus, and Thiocystis
  • Purple non-sulfur bacteria include but are not limited to the following genera: Phaeospirillum, 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, Thiovulum sp., Thiobacillus sp.,
  • Archaeobacteria include but are not limited to methanogenic archaeobacteria such as Methanobacterium sp., Methanobrevibacter sp., Methanothermus sp., Methanococcus sp., Methanomicrobium sp., Methanospirillum 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 sp
  • microorganisms such as, Bacillus subtilis, Saccharomyces cerevisiae, Streptomyces sp., Ralstonia sp., Rhodococcus sp., Corymb acteria sp., Brevibacteria sp., Mycobacteria sp., and oleaginous yeast.
  • Preferred organisms for the manufacture of n-alkanes according to the methods discloused herein 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 Chromatium vinosum (purple sulfur bacteria); Rhodospirillum rubrum,
  • Rhodobacter capsulatus and Rhodopseudomonas palusris (purple non- sulfur bacteria).
  • 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 pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonas mobilis.
  • carbon dioxide bacteria such as Escherichia coli, Acetobacter aceti, Bacillus subtilis, yeast and fungi such as Clostridium ljungdahlii, Clostridium thermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens, or Zymomonas mobilis.
  • a suitable organism for selecting or engineering is autotrophic fixation of C0 2 to products. This would cover photosynthesis and methanogenesis. Acetogenesis,
  • cyanobacteria represents the C0 2 fixation pathway in almost all aerobic autotrophic bacteria, for example, the cyanobacteria.
  • an engineered cyanobacterium e.g., a Synechococcus or Thermosynechococcus species.
  • Other preferred organisms include Synechocystis, Klebsiella oxytoca, Escherichia coli or Saccharomyces cerevisiae.
  • Other prokaryotic, archaeal and eukaryotic host cells are also encompassed within the scope of the present disclosure.
  • 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: 1-dodecanol, 1- tetradecanol, 1-pentadecanol, n-tridecane, n-tetradecane, 15: 1 n-pentadecene, n-pentadecane, 16: 1 n-hexadecene, n-hexadecane, 17: 1 n-heptadecene, n-heptadecane, 16: 1 n-hexadecen-ol, n-hexadecan-l-ol and n-octadecen-l-ol, as shown in the Examples herein.
  • the carbon chain length ranges from C 10 to C 20 . Accordingly, the disclosure provides production of various chain lengths of al
  • the methods of the present disclosure include 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 disclosure enables production of various defined chain length of hydrocarbons and alcohols, the secreted products are easily recovered or separated. The products of the disclosure, therefore, can be used directly or used with minimal processing.
  • the methods disclosed herein include producing a material from the microorganisms disclosed herein such as a hydrocarbon (e.g., an alkane or an alkene).
  • the methods disclosed herein include recovering the material.
  • the methods disclosed herein include extracting the material.
  • the methods disclosed herein include processing the material, e.g., the hydrocarbon.
  • the processing of the material produces a processed material.
  • processed material refers to a carbon-based material produced using one or more hydrocarbons as a raw starting material, wherein the one or more hydrocarbons are produced via one or more methods disclosed herein.
  • Such processed materials can include fuel, biodiesel, plastic, rubber, a cosmetic, a pharmaceutical agent, a specialty chemical, and a surfactant.
  • Other processed materials are generally known to one of skill in the art.
  • Various methods for making processed materials from hydrocarbons are generally known to one of skill in the art. Such methods can include, e.g., cracking, distillation, hydrotreating, reforming, and resid processing.
  • a method for producing hydrocarbons comprising: culturing one or more engineered microorganisms disclosed herein in a culture medium, wherein said engineered microorganism secretes increased amounts of n-alkanes or n-alkenes into the culture medium relative to an otherwise identical microorganism, cultured under identical conditions, but lacking said recombinant genes; and processing of the n- alkanes or n-alkenes to produce a processed material.
  • compositions produced by the methods of the disclosure are used as fuels.
  • Such 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
  • 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 disclosure produce an array of hydrocarbons, such as C 13 -C 17 or C 10 -C 15 to alter cloud point.
  • 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 Construction of pJB2166 for tetracycline-inducible hydrocarbon production in E. coli
  • pJB2166 was constructed using molecular biology techniques. Table 1 shows the regions and sequences of pJB2166.
  • emrAB was PCR amplified from E. coli MG1655 genomic DNA using primers KS313 (5' aataCATATGAGCGCAAATGCGGAGACTCAAA 3'; SEQ ID NO:21) and KS314 (5' aataGCATGCTTAGTGCGCACCGCCTCCGC 3'; SEQ ID NO:22) and Phusion HF DNA polymerase (NEB).
  • the resulting emrAB PCR product was digested with Ndel and Sphl, and ligated into pJB1719 ( ⁇ r)l5A_ori ⁇ P(T5)> ⁇ acrAB> ⁇ cat ⁇ ) cut with the same restriction enzymes to remove acrAB.
  • the resulting plasmid, pJB1849 (
  • Table 2 shows the regions and sequences of pJB1849.
  • Example 3 Hji/rocarbon secretion in E. coli by emrAB overexpression
  • a plasmid for anhydrotetracycline inducible expression of adm-aar, pJB2166, and another plasmid for expression of emrAB were introduced into an E. coli strain (JCC2264) lacking the only other hydrocarbon transporter found to date (YbhGFSR). emrAB expression rescued cell growth, and promoted hydrocarbon production and secretion in this strain.
  • E. coli strain JCC2264 (AfadEAybhGFSR) was contructed using a gene knockout vector system obtained from Yale (See Coli Genetic Stock Center website on November 9, 2011). JCC2264 was co-transformed with pJB2166 + pJB1849 and pJB2166 + pJB1720 (pJB1720 does not have any genes downstream of the T5 promoter). The latter strain served as a negative control.
  • Transformants were isolated on LB plates containing carbenicillin (100 ⁇ g/ml) and chloramphenicol (25 ⁇ g/ml), picked from colonies into 3 ml LB seed cultures with carbenicillin (100 ⁇ g/ml) and chloramphenicol (25 ⁇ g/ml)and incubated for 16 hours at 37°C, 260 rpm.
  • Table 3 shows the effect of emrAB overexpression on hydrocarbons production and secretion in E. coli strain JCC2264 harboring pJB2166.
  • vectors disclosed herein are re-engineered for use in cyanobacteria.
  • regions of DNA homologous to PCC 7002 genomic DNA
  • UHR and DHR upstream and downstream of the genetic elements of interest. This allows homologous recombination to occur once the vector is transformed into a bacterial strain (e.g., a cyanobacteria strain, e.g., JCC 138 and/or JCC2055), resulting in integration of the foreign DNA.
  • the genetic elements of interest can include a promoter that is known to function in PCC 7002, EmrAB-TolC (or other functional outer membrane protein), and/or adm-aar, and an antibiotic marker.
  • a panel of promoters are tested and whichever provides the best phenotype is selected for use. See, e.g., Huang H-H et al. (2010). Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology. NAR 38:2577-2593; and Dexter J and Fu P (2009). Metabolic engineering of cyanobacteria for ethanol production. Energy and Environ. Sci. 8:857-864; and Lan EI and Liao JC (201 1).
  • a photosynthetic bacterial strain e.g., a cyanobacteria strain such as JCC138
  • plasmids e.g. pJB1849 vector re-engineered for use in cyanobacteria
  • EmrA and/or EmrB e.g., SEQ ID NO: 15-16 and 19- 20
  • the strain can also be transformed with one or more plasmids expressing recombinant TolC (e.g., SEQ ID NO:21-22) or homologues thereof.
  • the strain can also be transformed with one or more plasmids (e.g., pJB2166 vector re-engineered for use in cyanobacteria) expressing ADM and/or AAR (e.g., SEQ ID NO:7-8) or homologues thereof.
  • Negative controls include the photosynthetic bacterial strain transformed with identical plasmids as above but without emrAB (e.g., pJB1720 vector re-engineered for use in cyanobacteria). Transformants are isolated on plates with appropriate antibiotics, picked from colonies into seed cultures with appropriate antibiotics, and incubated.
  • Hydrocarbon production and efflux of each strain is tested. Cells are harvested from the seed cultures and used to inoculate shake cultures. Following inoculation, cultures are incubated. In some instances, the hydrocarbon pathway is activated as described in the Examples above. Hydrocarbon production and efflux of each strain can be determined at various time points under various conditions. Overexpression of emrAB in the test strain(s) allows for continuous cell growth and increases total hydrocarbon production and % secretion in comparison with the negative control strain(s).
  • the hydrocarbons are recovered. In some instances, the hydrocarbons are extracted. In some instances, the hydrocarbons are processed to produce a processed material.
  • vectors disclosed herein are re-engineered for use in cyanobacteria as described above.
  • a photosynthetic bacterial strain e.g., a cyanobacteria strain, such as JCC138
  • one or more plasmids e.g.
  • EmrA EmrB
  • EmrK e.g., SEQ ID NO: 15-16 and 19-20
  • EmrE e.g., SEQ ID NO: 15-16 and 19-20
  • AcrE AcrF
  • EmrK e.g., SEQ ID NO: 15-16 and 19-20
  • EmrY e.g., SEQ ID NO: 15-16 and 19-20
  • MacA e.g., SEQ ID NO: 15-16 and 19-20
  • the amino acid sequences of AcrE, AcrF, EmrK, EmrY, MacA, MacB, MdtA, MdtB, MdtC, MdtE, MdtF, SdsR, SdsQ, and SdsP are shown in SEQ ID NOs 34-47.
  • the strain can also be transformed with one or more plasmids expressing recombinant TolC (e.g., SEQ ID NO :21-22) or homologues thereof.
  • the strain can also be transformed with one or more plasmids (e.g., pJB2166 vector re-engineered for use in cyanobacteria) expressing ADM and/or AAR (e.g., SEQ ID NO:7-8) or homologues thereof.
  • Negative controls include the photosynthetic bacterial strain transformed with identical plasmids as above but without the recombinant tripartite transporter protein(s) (e.g., pJB1720 vector re- engineered for use in cyanobacteria). Transformants are isolated on plates with appropriate antibiotics, picked from colonies into seed cultures with appropriate antibiotics, and incubated.
  • Hydrocarbon production and efflux of each strain is tested. Cells are harvested from the seed cultures and used to inoculate shake cultures. Following inoculation, cultures are incubated. In some instances, the hydrocarbon pathway is activated as described in the Examples above. Hydrocarbon production and efflux of each strain can be determined at various time points under various conditions. Overexpression of the recombinant tripartite transporter protein(s) in the test strain(s) allows for continuous cell growth and increases total hydrocarbon production and % secretion in comparison with the negative control strain(s).
  • the hydrocarbons are recovered. In some instances, the hydrocarbons are extracted. In some instances, the hydrocarbons are processed to produce a processed material.
  • JCC138 alkanogen such as JCC2055
  • cyanobacterial e.g., Nostoc punctiforme PCC 73102 (Npun) or Cyanothetce PCC 7822 (Cyan7822)
  • EmrA and EmrB homologs. e.g., Nostoc punctiforme PCC 73102 (Npun) or Cyanothetce PCC 7822 (Cyan7822)
  • P(xxx)-Npun_F6382-Npun_F6383-Npun_F6384-Npun_F638 (ybhG-macBl-macB2- omp) where P(xxx) indicates a multiplicity of promoters operably linked to the indicated operons, and the sequences in parantheses indicate the corresponding gene identities, omp representing a gene encoding an outer membrane protein. Dashes indicate separations between promoter and/or coding sequences. emrA, emrB, entS, ybhG, macBI, and macB2 sequences from Npun and/or Cyan7822 are shown in the sequence listing at SEQ ID NOs:23-33. [0148] Transformants are isolated on plates with appropriate antibiotics, picked from colonies into seed cultures with appropriate antibiotics, and incubated.
  • Hydrocarbon production and efflux of each strain is tested. Cells are harvested from the seed cultures and used to inoculate shake cultures. Following inoculation, cultures are incubated. In some instances, the hydrocarbon pathway is activated as described in the Examples above. Hydrocarbon production and efflux of each strain can be determined at various time points under various conditions. Overexpression of the recombinant tripartite transporter protein(s) in the test strain(s) allows for continuous cell growth and increases total hydrocarbon production and % secretion in comparison with the negative control strain(s).
  • the hydrocarbons are recovered. In some instances, the hydrocarbons are extracted. In some instances, the hydrocarbons are processed to produce a processed material.
  • Example 7 Hydrocarbon secretion by emrAB overexpression
  • pJB2302 was constructed using molecular biology techniques. Table 4 shows the regions and sequences of pJB2302. This vector integrates into the A2208 locus.
  • pJB2303 was constructed using molecular biology techniques. This vector included the same sequences as pJB2302, except that the P ⁇ psaA ⁇ promter of pJB2302 was exchanged for the P ⁇ tsr2142 ⁇ promoter from cyanobacteria BP-1 shown in SEQ ID NO:55. This vector integrates into the A2208 locus.
  • pJB2304 was constructed using molecular biology techniques. This vector included the same sequences as pJB2302, except that the P ⁇ psaA ⁇ promter of pJB2302 was exchanged for the P ⁇ aphII ⁇ promoter shown in SEQ ID NO:56. This vector integrates into the A2208 locus.
  • pJB2305 was constructed using molecular biology techniques. This vector included the same sequences as pJB2302, except that the P ⁇ psaA ⁇ promter of pJB2302 was exchanged for the P ⁇ ompR ⁇ promoter shown in SEQ ID NO:57. This vector integrates into the A2208 locus.
  • pJB2306 was constructed using molecular biology techniques. This vector included the same sequences as pJB2302, except that the P ⁇ psaA ⁇ promter of pJB2302 was exchanged for the P ⁇ nir_07_PnirA_PCC7942 v2 ⁇ promoter shown in SEQ ID NO:58. This vector integrates into the A2208 locus.
  • a photosynthetic bacterial strain e.g., a cyanobacteria strain such as JCC138 or JCC2055
  • one or more plasmids e.g. pJB2302, pJB2303, pJB2304, pJB2305, or pJB2306
  • EmrA and EmrB e.g., SEQ ID NO: 15-16 and 19-20 and 51-52
  • the strain can also be transformed with one or more plasmids expressing recombinant TolC (e.g., SEQ ID NO:21-22) or homologues thereof.
  • the strain can also be transformed with one or more plasmids (e.g., pJB2166 vector re-engineered for use in cyanobacteria) expressing ADM and/or AAR (e.g., SEQ ID NO:7-8) or homologues thereof.
  • Negative controls include the photosynthetic bacterial strain transformed with identical plasmids as above but without emrAB (e.g., pJB1720 vector re-engineered for use in cyanobacteria). Transformants are isolated on plates with appropriate antibiotics, picked from colonies into seed cultures with appropriate antibiotics, and incubated.
  • Hydrocarbon production and efflux of each strain is tested. Cells are harvested from the seed cultures and used to inoculate shake cultures. Following inoculation, cultures are incubated. In some instances, the hydrocarbon pathway is activated as described in the Examples above. Hydrocarbon production and efflux of each strain can be determined at various time points under various conditions. Overexpression of emrAB in the test strain(s) allows for continuous cell growth and increases total hydrocarbon production and % secretion in comparison with the negative control strain(s).
  • the hydrocarbons are recovered. In some instances, the hydrocarbons are extracted. In some instances, the hydrocarbons are processed to produce a processed material.
  • Cyan7822 22 MATNQFSSKTKLSKKSKDLVYDAGGYVQGPRKWAIAVTASLGAILEVIDTSI INVALT 43 DIQTTLGATITEIAWVATGYAIANVILIPLSAWLGDFFGKKTYFVFSMVGFTFASVLC
  • YPTIQVVTLY PGASPDVMTS AVTAPLERQF GQMSGLKQMS SQSSGGASVI TLQFQLTLPL DVAEQEVQAA INAATNLLPS DLPNPPVYSK VNPADPPIMT LAVTSTAMPM TQVEDMVETR VAQKISQISG VGLVTLSGGQ RPAVRVKLNA QAIAALGLTS ETVRTAITGA NVNSAKGSLD GPSRAVTLSA NDQMQSAEEY RQLI IAYQNG APIRLGDVAT VEQGAENSWL GAWANKEQAI VMNVQRQPGA NIISTADSIR QMLPQLTESL PKSVKVTVLS DRTTNIRASV DDTQFELMMA IALVVMIIYL FLRNIPATI I PGVAVPLSLI GTFAVMVFLD FSINNLTLMA LTIATGFVVD DAIVVIENIS RYIEKGEKPL AAALKGAGEI GFTI ISLTFS LIAVLIPLLF MGDIV
  • EIPFVALSLA VLFYGIQSNA FYTKFVAILF VVATVLEIGS LFLIYKWSYG EPLIRLI IAG PILMGCMFLM RTHRLGLVFF AVAIVAIYGQ TFPAMLDYPE VVVRLTLWCI VVGLYPTLLM TLIGVLWFPS RAISQMHQAL NDRLDDAISH LTDSLAPLPE TRIEREALAL QKLNVFCLAD DANWRTQNAW WQSCVATVTY IYSTLNRYDP TSFADSQAI I EFRQKLASEI NKLQHAVAEG QCWQSDWRIS ESEAMAAREC NLENICQTLL QLGQMDPNTP PTPAAKPPSM AADAFTNPDY MRYAVKTLLA CLICYTFYSG VDWEGIHTCM LTCVIVANPN VGSSYQKMVL RFGGAFCGAI LALLFTLLVM PWLDNIVELL FVLAPIFLLG AWIATSSERS SYIGTQMVVT F
  • promoter gcccctatattatgcatttatacccccacaatcatgtcaagaattcaagcatcttaa P ⁇ psaA ⁇ taatgttaattatcggcaaagtctgtgctccccttctataatgctgaattgagcattc gcctcctgaacggtctttattcttccattgtgggtctttagattcacgattcttcaca atcattgatctaaggatctttgtagattctctcTGTACA

Abstract

La présente invention concerne des procédés et des compositions permettant de modifier des organismes photoautotrophes pour obtenir des hôtes, de façon que les organismes convertissent de manière efficace le dioxyde de carbone et la lumière en hydrocarbures, par exemple des n-alcanes et des n-alcènes, les n-alcanes étant sécrétés dans le milieu de culture par l'intermédiaire de protéines de transporteur exprimées de façon recombinante. L'utilisation de ces organismes pour la production commerciale de n-alcanes et de molécules apparentées est envisagée.
PCT/US2012/070666 2011-12-19 2012-12-19 Transport extracellulaire d'hydrocarbures biosynthétiques et d'autres molécules WO2013096475A1 (fr)

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US10041098B2 (en) 2013-05-10 2018-08-07 Cj Cheiljedang Corporation O-phosphoserine export protein and the method of producing O-phosphoserine using the same
WO2021125493A1 (fr) * 2019-12-20 2021-06-24 씨제이제일제당(주) Micro-organisme comprenant une modification génétique qui augmente l'activité d'un transporteur de médicaments multiples et procédé de production de métabolites de tryptophane à l'aide de celui-ci
KR20210079889A (ko) * 2019-12-20 2021-06-30 씨제이제일제당 (주) 다중약물 수송체의 활성을 증가시키는 유전적 변형을 포함하는 미생물, 및 그를 이용한 트립토판 대사체의 생산 방법
KR102370964B1 (ko) 2019-12-20 2022-03-10 씨제이제일제당 주식회사 다중약물 수송체의 활성을 증가시키는 유전적 변형을 포함하는 미생물, 및 그를 이용한 트립토판 대사체의 생산 방법

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