US20150064782A1 - Production of fatty acid derivatives - Google Patents

Production of fatty acid derivatives Download PDF

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US20150064782A1
US20150064782A1 US14/390,378 US201314390378A US2015064782A1 US 20150064782 A1 US20150064782 A1 US 20150064782A1 US 201314390378 A US201314390378 A US 201314390378A US 2015064782 A1 US2015064782 A1 US 2015064782A1
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host cell
fatty acid
fatty
acid derivative
activity
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Derek L. Greenfield
Andreas W. Schirmer
Elizabeth J. Clarke
Eli S. Groban
Bernardo M. da Costa
Zhihao Hu
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Genomatica Inc
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REG Life Sciences LLC
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Definitions

  • the invention relates to engineered host cells together with vector and strain modifications effective to improve the titer, yield and productivity of fatty acid derivatives relative to “wild-type” or non-engineered host cells.
  • the invention further relates to methods of making and using such modified vectors and strains for the fermentative production of fatty acid derivatives and fatty acid derivative compositions.
  • Fatty acid derivatives including fatty aldehydes, fatty alcohols, hydrocarbons (alkanes and olefins), fatty esters (e.g., waxes, fatty acid esters, or fatty esters) and ketones comprise important categories of industrial chemicals and fuels. These molecules and their derivatives have numerous applications including use as surfactants, lubricants, plasticizers, solvents, emulsifiers, emollients, thickeners, flavors, fragrances, and fuels.
  • Crude petroleum is currently a primary source of raw materials for producing petrochemicals and fuels.
  • the two main classes of raw materials derived from petroleum are short chain olefins (e.g., ethylene and propylene) and aromatics (e.g., benzene and xylene isomers). These raw materials are derived from longer chain hydrocarbons in crude petroleum by cracking it at considerable expense using a variety of methods, such as catalytic cracking, steam cracking, or catalytic reforming. These raw materials can be used to make petrochemicals such as monomers, solvents, detergents, and adhesives, which otherwise cannot be directly refined from crude petroleum.
  • Petrochemicals in turn, can be used to make specialty chemicals, such as plastics, resins, fibers, elastomers, pharmaceuticals, lubricants, and gels.
  • specialty chemicals such as plastics, resins, fibers, elastomers, pharmaceuticals, lubricants, and gels.
  • specialty chemicals that can be produced from petrochemical raw materials include fatty acids, hydrocarbons, fatty aldehydes, fatty alcohols, esters, ketones, etc.
  • Hydrocarbons have many commercial uses. For example, shorter chain alkanes and alkenes are used in transportation fuels. Longer chain alkenes are used in plastics, lubricants, and synthetic lubricants. In addition, alkenes are used as a feedstock to produce alcohols, esters, plasticizers, surfactants, tertiary amines, enhanced oil recovery agents, fatty acids, thiols, alkenylsuccinic anhydrides, epoxides, chlorinated alkanes, chlorinated alkenes, waxes, fuel additives, and drag flow reducers.
  • Esters have many commercial uses.
  • biodiesel an alternative fuel, is comprised of esters (e.g., fatty acid methyl ester, fatty acid ethyl esters, etc.).
  • esters are volatile with a pleasant odor which makes them useful as fragrances or flavoring agents.
  • esters are used as solvents for lacquers, paints, and varnishes.
  • some naturally occurring substances such as waxes, fats, and oils are comprised of esters.
  • Esters are also used as softening agents in resins and plastics, plasticizers, flame retardants, and additives in gasoline and oil.
  • esters can be used in the manufacture of polymers, films, textiles, dyes, and pharmaceuticals.
  • Aldehydes are used to produce many specialty chemicals. For example, aldehydes are used to produce polymers, resins (e.g., Bakelite), dyes, flavorings, plasticizers, perfumes, pharmaceuticals, and other chemicals, some of which may be used as solvents, preservatives, or disinfectants. In addition, certain natural and synthetic compounds, such as vitamins and hormones, are aldehydes, and many sugars contain aldehyde groups. Fatty aldehydes can be converted to fatty alcohols by chemical or enzymatic reduction.
  • Fatty alcohols have many commercial uses.
  • the shorter chain fatty alcohols are used in the cosmetic and food industries as emulsifiers, emollients, and thickeners. Due to their amphiphilic nature, fatty alcohols behave as nonionic surfactants, which are useful in personal care and household products, such as, for example, detergents.
  • fatty alcohols are used in waxes, gums, resins, pharmaceutical salves and lotions, lubricating oil additives, textile antistatic and finishing agents, plasticizers, cosmetics, industrial solvents, and solvents for fats.
  • Fatty alcohols are aliphatic alcohols consisting of a chain of 8 to 22 carbon atoms. Fatty alcohols usually have even number of carbon atoms and a single alcohol group (—OH) attached to the terminal carbon. Some are unsaturated and some are branched. They are widely used in industrial chemistry. Most fatty alcohols in nature are found as waxes which are esters with fatty acids and fatty alcohols. They are produced by bacteria, plants and animals.
  • fatty alcohols are produced via catalytic hydrogenation of fatty acids produced from natural sources, such as coconut oil, palm oil, palm kernel oil, tallow and lard, or by chemical hydration of alpha-olefins produced from petrochemical feedstocks.
  • natural sources such as coconut oil, palm oil, palm kernel oil, tallow and lard
  • alpha-olefins produced from petrochemical feedstocks.
  • Fatty alcohols derived from natural sources have varying chain lengths. The chain length of fatty alcohols is important and specific to particular applications. Dehydration of fatty alcohols to alpha-olefins can also be accomplished by chemical catalysis.
  • One method of producing renewable petroleum is by engineering host cells to produce renewable petroleum products.
  • Biologically derived fuels and chemicals offer advantages over petroleum based fuels.
  • Biologically derived chemicals such as hydrocarbons (e.g., alkanes, alkenes, or alkynes), fatty alcohols, esters, fatty acids, fatty aldehydes, and ketones are directly converted from biomass to the desired chemical product.
  • the present invention provides novel genetically engineered host cells which produce fatty acid derivative compositions at a high titer, yield or productivity; cell cultures comprising such novel genetically engineered host cells and methods of using the same.
  • the invention also provides methods of making compositions comprising fatty acid derivatives by culturing the genetically engineered host cells of the invention, compositions made by such methods, and other features apparent upon further review.
  • the invention provides a cultured genetically engineered host cell comprising (a) a polynucleotide sequence encoding one or more of: (i) an acetyl-CoA carboxylase (EC 6.4.1.2) polypeptide, (ii) a FadR polypeptide, (iii) a heterologous iFAB polypeptide, (iv) a sequence having a transposon insertion in the yijP gene, and (v) a heterologous ACP protein; as well as (b) a polynucleotide sequence encoding a fatty acid derivative biosynthetic polypeptide, wherein the genetically engineered host cell produces a fatty acid derivative composition at a higher titer, yield or productivity when cultured in medium containing a carbon source under conditions effective to overexpress the polynucleotide(s) relative to a corresponding wild type host cell propagated under the same conditions as the genetically engineered host cell.
  • the fatty acid derivative composition includes one or more of a fatty acid, a fatty aldehyde, a fatty alcohol, a fatty ester, an alkane, a terminal olefin, an internal olefin and a ketone.
  • the genetically engineered host cell produces a fatty acid derivative composition with a titer, yield or productivity that is at least 3 times greater, at least 5 times greater, at least 8 times greater, or at least 10 times greater than the titer of a fatty acid derivative composition produced by a corresponding wild type (non-engineered) host cell propagated under the same conditions as the genetically engineered host cell (e.g., a titer of from 30 g/L to 250 g/L, a yield of from 10% to 40%, or a productivity of 0.7 mg/L/hr to 3 g/L/hr).
  • a titer of from 30 g/L to 250 g/L, a yield of from 10% to 40%, or a productivity of 0.7 mg/L/hr to 3 g/L/hr.
  • the fatty acid derivative composition is produced extracellularly.
  • the host cell is further engineered to comprise a heterologous acp sequence with or without an introduced sfp gene.
  • polypeptide sequence encoding a fatty acid derivative biosynthetic polypeptide is selected from the group consisting of a polypeptide:
  • AAR acyl-CoA reductase
  • FIG. 1 presents an exemplary biosynthetic pathway for use in production of acyl CoA as a precursor to fatty acid derivatives in a recombinant microorganism.
  • the cycle is initiated by condensation of malonyl-ACP and acetyl-CoA.
  • FIG. 2 presents an exemplary fatty acid biosynthetic cycle, where malonyl-ACP is produced by the transacylation of malonyl-CoA to malonyl-ACP (catalyzed by malonyl-CoA:ACP transacylase; fabD), then ⁇ -ketoacyl-ACP synthase III (fabH) initiates condensation of malonyl-ACP with acetyl-CoA.
  • malonyl-ACP is produced by the transacylation of malonyl-CoA to malonyl-ACP (catalyzed by malonyl-CoA:ACP transacylase; fabD), then ⁇ -ketoacyl-ACP synthase III (fabH) initiates condensation of malonyl-ACP with acetyl-CoA.
  • Elongation cycles begin with the condensation of malonyl-ACP and an acyl-ACP catalyzed by ⁇ -ketoacyl-ACP synthase I (fabB) and ⁇ -ketoacyl-ACP synthase II (fabF) to produce a ⁇ -keto-acyl-ACP, then the ⁇ -keto-acyl-ACP is reduced by ⁇ -ketoacyl-ACP reductase (fabG) to produce a ⁇ -hydroxy-acyl-ACP, which is dehydrated to a trans-2-enoyl-acyl-ACP by ⁇ -hydroxyacyl-ACP dehydratase (fabA or fabZ).
  • fabricG ⁇ -ketoacyl-ACP reductase
  • FabA can also isomerize trans-2-enoyl-acyl-ACP to cis-3-enoyl-acyl-ACP, which can bypass fabI and can used by fabB (typically for up to an aliphatic chain length of C16) to produce ⁇ -keto-acyl-ACP.
  • the final step in each cycle is catalyzed by enoyl-ACP reductase (fabI) that converts trans-2-enoyl-acyl-ACP to acyl-ACP.
  • enoyl-ACP reductase fabricI
  • termination of fatty acid synthesis occurs by thioesterase removal of the acyl group from acyl-ACP to release free fatty acids (FFA).
  • Thioesterases e.g., tesA
  • hydrolyze thioester bonds which occur between acyl chains and ACP through sulfhydryl bonds.
  • FIG. 3 illustrates the structure and function of the acetyl-CoA carboxylase (accABCD) enzyme complex.
  • FIG. 4 presents an overview of an exemplary biosynthetic pathway for production of fatty alcohol starting with acyl-ACP, where the production of fatty aldehyde is catalyzed by the enzymatic activity of acyl-ACP reductase (AAR) or thioesterase and carboxylic acid reductase (Car).
  • AAR acyl-ACP reductase
  • Car carboxylic acid reductase
  • the fatty aldehyde is converted to fatty alcohol by aldehyde reductase (also referred to as alcohol dehydrogenase).
  • aldehyde reductase also referred to as alcohol dehydrogenase
  • FIG. 5 presents an overview of two exemplary biosynthetic pathways for production of fatty esters starting with acyl-ACP, where the production of fatty esters is accomplished by a one enzyme system or a three enzyme system.
  • FIG. 6 presents an overview of exemplary biosynthetic pathways for production of hydrocarbons starting with acyl-ACP, where the production of ketones is catalyzed by the enzymatic activity of OleA; the production of internal olefins is catalyzed by the enzymatic activity of OleABCD; the production of alkanes is catalyzed by the enzymatic conversion of fatty aldehydes to alkanes by way of aldehyde decarbonylase to; and the production of terminal olefins is catalyzed by the enzymatic conversion of fatty acids to terminal olefins by a decarboxylase
  • FIG. 7 illustrates fatty acid derivative (“Total Fatty Species”) production by the MG1655 E. coli strain with the fadE gene attenuated (i.e., deleted) compared to fatty acid derivative production by E. coli MG1655.
  • the data presented in FIG. 7 shows that attenuation of the fadE gene did not affect fatty acid derivative production.
  • FIG. 8 shows malonyl-CoA levels in DAM1_i377 in log phase expressing eight different C. glutamicum acetyl-CoA carboxylase (Acc) operon constructs.
  • FIG. 9 shows intracellular short chain-CoA levels in E. coli DAM1 — ⁇ 377 in log phase expressing ptrc1/3_accDACB-birA ⁇ panK operon constructs. “accDACB+birA” is also referred to herein as “accD+”.
  • FIG. 10 shows fatty acid methyl ester (FAME) production in E. coli strain DV2 expressing ester synthase 9 from M. hydrocarbonoclasticus and components of an acetyl-CoA carboxylase complex from C. glutamicum.
  • FAME fatty acid methyl ester
  • FIG. 11 shows production of fatty alcohols by E. coli expressing the Synechococcus elongatus PCC7942 AAR together with the accD+operon” from C. glutamicum on a pCL plasmid. Triplicate samples are shown for the accD+strains.
  • FIGS. 12A and B show data for production of “Total Fatty Species” (mg/L) from duplicate plate screens when plasmid pCL-WT TRC WT TesA was transformed into each of the iFAB-containing strains shown in the figures and a fermentation was run in FA2 media with 20 hours from induction to harvest at both 32° C. ( FIG. 12A ) and 37° C. ( FIG. 12B ).
  • FIG. 13 shows FAME production of E. coli DAM1 with plasmid pDS57 and integrated fabHI operons.
  • the fabH/I genes are from Marinobacter aquaeoli VT8 or from Acinetobacter baylyi ADP1. See Table 7 for a more details on the fabH/I operons in these strains.
  • FIG. 14 shows FAME production of E. coli DAM 1 with plasmid pDS57 and different configurations of the C. glutamicum acc genes as well as integrated fabHI operons.
  • the strains contain the fabH/I genes from Rhodococcus opacus or Acinetobacter baylyi ADP1. See Table 7 for more details on the fabH/I and acc operons.
  • FIG. 15 shows FAME and FFA titers of two E. coli DAM1 pDS57 strains with integrated fabH/I genes strains selected from FIG. 13 compared to the control strain E. coli DAM1 pDS57.
  • FIG. 16 is a diagrammatic depiction of the iFAB 138 locus, including a diagram of cat-loxP-T5 promoter integrated in front of FAB 138 ( 16 A); and a diagram of iT5 — 138 ( 16 B).
  • the sequence of cat-loxP-T5 promoter integrated in front of FAB138 with 50 base pair of homology shown on each side of cat-loxP-T5 promoter region is provided as SEQ ID NO:1 and the sequence of the iT5 — 138 promoter region with 50 base pair homology on each side is provided as SEQ ID NO:2.
  • FIG. 17 shows that correcting the rph and ilvG genes in the EG149 strain allows for a higher level of FFA production than in the V668 strain where the rph and ilvG genes were not corrected.
  • FIG. 18 is a diagrammatic depiction of a transposon cassette insertion in the yijP gene of strain LC535 (transposon hit 68F11). Promoters internal to the transposon cassette are shown, and may have effects on adjacent gene expression.
  • FIG. 19 illustrates fatty alcohol production in E. coli DV2 expressing Synechococcus elongatus acyl-ACP reductase (AAR) and coexpressing various cyanobacterial acyl carrier proteins (ACPs). (Details regarding the source of the ACPs are provided in Table 13).
  • FIG. 20 illustrates fatty acid production in E. coli DV2 expressing leaderless E. coli thioesterase 'tesA and coexpressing a cyanobacterial acyl carrier protein (cACP) and B. subtilis sfp.
  • cACP cyanobacterial acyl carrier protein
  • the invention is based, at least in part, on the discovery that modification of various aspects of the fatty acid biosynthetic pathway in a recombinant host cell facilitates enhanced production of fatty acid derivatives by the host cell.
  • the invention relates to compositions of fatty acid derivatives having desired characteristics and methods for producing the same. Further, the invention relates to recombinant host cells (e.g., microorganisms), cultures of recombinant host cells, methods of making and using recombinant host cells, for example, use of cultured recombinant host cells in the fermentative production of fatty acid derivatives having desired characteristics.
  • recombinant host cells e.g., microorganisms
  • cultures of recombinant host cells e.g., microorganisms
  • methods of making and using recombinant host cells for example, use of cultured recombinant host cells in the fermentative production of fatty acid derivatives having desired characteristics.
  • a recombinant host cell includes two or more such recombinant host cells
  • reference to “a fatty alcohol” includes one or more fatty alcohols, or mixtures of fatty alcohols
  • reference to “a nucleic acid coding sequence” includes one or more nucleic acid coding sequences
  • reference to “an enzyme” includes one or more enzymes, and the like.
  • NCBI Accession Numbers are identified herein as “NCBI Accession Numbers” or alternatively as “GenBank Accession Numbers”.
  • UniProtKB Accession Numbers are identified herein as “UniProtKB Accession Numbers”.
  • EC numbers are established by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB), description of which is available on the IUBMB Enzyme Nomenclature website on the World Wide Web. EC numbers classify enzymes according to the reaction catalyzed.
  • nucleotide refers to a monomeric unit of a polynucleotide that consists of a heterocyclic base, a sugar, and one or more phosphate groups.
  • the naturally occurring bases (guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) are typically derivatives of purine or pyrimidine, though it should be understood that naturally and non-naturally occurring base analogs are also included.
  • the naturally occurring sugar is the pentose (five-carbon sugar) deoxyribose (which forms DNA) or ribose (which forms RNA), though it should be understood that naturally and non-naturally occurring sugar analogs are also included.
  • Nucleic acids are typically linked via phosphate bonds to form nucleic acids or polynucleotides, though many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, and the like).
  • polynucleotide refers to a polymer of ribonucleotides (RNA) or deoxyribonucleotides (DNA), which can be single-stranded or double-stranded and which can contain non-natural or altered nucleotides.
  • RNA ribonucleotides
  • DNA deoxyribonucleotides
  • polynucleotide refers to a polymeric form of nucleotides of any length, either RNA or DNA. These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and single-stranded RNA.
  • RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to methylated and/or capped polynucleotides.
  • the polynucleotide can be in any form, including but not limited to, plasmid, viral, chromosomal, EST, cDNA, mRNA, and rRNA.
  • polypeptide and “protein” are used interchangeably to refer to a polymer of amino acid residues.
  • recombinant polypeptide refers to a polypeptide that is produced by recombinant techniques, wherein generally DNA or RNA encoding the expressed protein is inserted into a suitable expression vector that is in turn used to transform a host cell to produce the polypeptide.
  • homologous polynucleotides or polypeptides have polynucleotide sequences or amino acid sequences that have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least about 99% homology to the corresponding amino acid sequence or polynucleotide sequence.
  • sequence “homology” and sequence “identity” are used interchangeably.
  • the length of a first sequence that is aligned for comparison purposes is at least about 30%, preferably at least about 40%, more preferably at least about 50%, even more preferably at least about 60%, and even more preferably at least about 70%, at least about 80%, at least about 90%, or about 100% of the length of a second sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions of the first and second sequences are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent homology between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, that need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm, such as BLAST (Altschul et al., J. Mol. Biol., 215(3): 403-410 (1990)).
  • the percent homology between two amino acid sequences also can be determined using the Needleman and Wunsch algorithm that has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 (Needleman and Wunsch, J. Mol. Biol., 48: 444-453 (1970)).
  • the percent homology between two nucleotide sequences also can be determined using the GAP program in the GCG software package, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • One of ordinary skill in the art can perform initial homology calculations and adjust the algorithm parameters accordingly.
  • a preferred set of parameters are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions describes conditions for hybridization and washing.
  • Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference and either method can be used.
  • Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions—6 ⁇ sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2 ⁇ SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C.
  • SSC sodium chloride/sodium citrate
  • low stringency conditions 1) medium stringency hybridization conditions—6 ⁇ SSC at about 45° C., followed by one or more washes in 0.2 ⁇ SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditions—6 ⁇ SSC at about 45° C., followed by one or more washes in 0.2. ⁇ SSC, 0.1% SDS at 65° C.; and 4) very high stringency hybridization conditions—0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2 ⁇ SSC, 1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions unless otherwise specified.
  • an “endogenous” polypeptide refers to a polypeptide encoded by the genome of the parental microbial cell (also termed “host cell”) from which the recombinant cell is engineered (or “derived”).
  • exogenous polypeptide refers to a polypeptide which is not encoded by the genome of the parental microbial cell.
  • variant (i.e., mutant) polypeptide is an example of an exogenous polypeptide.
  • heterologous typically refers to a nucleotide sequence or a protein not naturally present in an organism.
  • a polynucleotide sequence endogenous to a plant can be introduced into a host cell by recombinant methods, and the plant polynucleotide is then a heterologous polynucleotide in a recombinant host cell.
  • fragment of a polypeptide refers to a shorter portion of a full-length polypeptide or protein ranging in size from four amino acid residues to the entire amino acid sequence minus one amino acid residue. In certain embodiments of the invention, a fragment refers to the entire amino acid sequence of a domain of a polypeptide or protein (e.g., a substrate binding domain or a catalytic domain).
  • mutagenesis refers to a process by which the genetic information of an organism is changed in a stable manner. Mutagenesis of a protein coding nucleic acid sequence produces a mutant protein. Mutagenesis also refers to changes in non-coding nucleic acid sequences that result in modified protein activity.
  • the term “gene” refers to nucleic acid sequences encoding either an RNA product or a protein product, as well as operably-linked nucleic acid sequences affecting the expression of the RNA or protein (e.g., such sequences include but are not limited to promoter or enhancer sequences) or operably-linked nucleic acid sequences encoding sequences that affect the expression of the RNA or protein (e.g., such sequences include but are not limited to ribosome binding sites or translational control sequences).
  • Expression control sequences are known in the art and include, for example, promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the polynucleotide sequence in a host cell.
  • Expression control sequences interact specifically with cellular proteins involved in transcription (Maniatis et al., Science, 236: 1237-1245 (1987)).
  • Exemplary expression control sequences are described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).
  • an expression control sequence is operably linked to a polynucleotide sequence.
  • operably linked is meant that a polynucleotide sequence and an expression control sequence(s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the expression control sequence(s).
  • Operably linked promoters are located upstream of the selected polynucleotide sequence in terms of the direction of transcription and translation.
  • Operably linked enhancers can be located upstream, within, or downstream of the selected polynucleotide.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid, i.e., a polynucleotide sequence, to which it has been linked
  • an episome i.e., a nucleic acid capable of extra-chromosomal replication
  • Useful vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.”
  • expression vectors of utility in recombinant DNA techniques are often in the form of “plasmids,” which refer generally to circular double stranded DNA loops that, in their vector form, are not bound to the chromosome.
  • plasmid and vector are used interchangeably herein, in as much as a plasmid is the most commonly used form of vector.
  • vectors that serve equivalent functions and that become known in the art subsequently hereto.
  • a recombinant vector further comprises a promoter operably linked to the polynucleotide sequence.
  • the promoter is a developmentally-regulated, an organelle-specific, a tissue-specific, an inducible, a constitutive, or a cell-specific promoter.
  • the recombinant vector typically comprises at least one sequence selected from the group consisting of (a) an expression control sequence operatively coupled to the polynucleotide sequence; (b) a selection marker operatively coupled to the polynucleotide sequence; (c) a marker sequence operatively coupled to the polynucleotide sequence; (d) a purification moiety operatively coupled to the polynucleotide sequence; (e) a secretion sequence operatively coupled to the polynucleotide sequence; and (f) a targeting sequence operatively coupled to the polynucleotide sequence.
  • the nucleotide sequence is stably incorporated into the genomic DNA of the host cell, and the expression of the nucleotide sequence is under the control of a regulated promoter region.
  • the expression vectors described herein include a polynucleotide sequence described herein in a form suitable for expression of the polynucleotide sequence in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc.
  • the expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the polynucleotide sequences as described herein. Expression of genes encoding polypeptides in prokaryotes, for example, E.
  • Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino- or carboxy-terminus of the recombinant polypeptide.
  • Such fusion vectors typically serve one or more of the following three purposes: (1) to increase expression of the recombinant polypeptide; (2) to increase the solubility of the recombinant polypeptide; and (3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide. This enables separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide.
  • a polynucleotide sequence of the invention is operably linked to a promoter derived from bacteriophage T5.
  • the host cell is a yeast cell
  • the expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari et al., EMBO J., 6: 229-234 (1987)), pMFa (Kurjan et al., Cell, 30: 933-943 (1982)), pJRY88 (Schultz et al., Gene, 54: 113-123 (1987)), pYES2 (Invitrogen Corp., San Diego, Calif.), and picZ (Invitrogen Corp., San Diego, Calif.).
  • the host cell is an insect cell
  • the expression vector is a baculovirus expression vector.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include, for example, the pAc series (Smith et al., Mol. Cell. Biol., 3: 2156-2165 (1983)) and the pVL series (Lucklow et al., Virology, 170: 31-39 (1989)).
  • polynucleotide sequences described herein can be expressed in mammalian cells using a mammalian expression vector.
  • suitable expression systems for both prokaryotic and eukaryotic cells are well known in the art; see, e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual,” second edition, Cold Spring Harbor Laboratory, (1989).
  • acyl-CoA refers to an acyl thioester formed between the carbonyl carbon of alkyl chain and the sulfhydryl group of the 4′-phosphopantethionyl moiety of coenzyme A (CoA), which has the formula R—C(O)S—CoA, where R is any alkyl group having at least 4 carbon atoms.
  • acyl-ACP refers to an acyl thioester formed between the carbonyl carbon of alkyl chain and the sulfhydryl group of the phosphopantetheinyl moiety of an acyl carrier protein (ACP).
  • ACP acyl carrier protein
  • the phosphopantetheinyl moiety is post-translationally attached to a conserved serine residue on the ACP by the action of holo-acyl carrier protein synthase (ACPS), a phosphopantetheinyl transferase.
  • ACPS holo-acyl carrier protein synthase
  • an acyl-ACP is an intermediate in the synthesis of fully saturated acyl-ACPs.
  • an acyl-ACP is an intermediate in the synthesis of unsaturated acyl-ACPs.
  • the carbon chain will have about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbons.
  • Each of these acyl-ACPs are substrates for enzymes that convert them to fatty acid derivatives such as those described in FIGS. 4-6
  • fatty acid derivative means a “fatty acid” or a “fatty acid derivative”, which may be referred to as a “fatty acid or derivative thereof”.
  • fatty acid means a carboxylic acid having the formula RCOOH.
  • R represents an aliphatic group, preferably an alkyl group.
  • R can comprise between about 4 and about 22 carbon atoms.
  • Fatty acids can be saturated, monounsaturated, or polyunsaturated.
  • a “fatty acid derivative” is a product made in part from the fatty acid biosynthetic pathway of the production host organism.
  • “Fatty acid derivatives” includes products made in part from acyl-ACP or acyl-ACP derivatives.
  • Exemplary fatty acid derivatives include, for example, acyl-CoA, fatty acids, fatty aldehydes, short and long chain alcohols, fatty alcohols, hydrocarbons, esters (e.g., waxes, fatty acid esters, or fatty esters), terminal olefins, internal olefins, and ketones.
  • esters e.g., waxes, fatty acid esters, or fatty esters
  • terminal olefins e.g., waxes, fatty acid esters, or fatty esters
  • terminal olefins e.g., waxes, fatty acid esters, or fatty esters
  • ketones e.g., ketones.
  • a “fatty acid derivative composition” as referred to herein is produced by a recombinant host cell and typically comprises a mixture of fatty acid derivative.
  • the mixture includes more than one type of product (e.g., fatty acids and fatty alcohols, fatty acids and fatty acid esters or alkanes and olefins).
  • the fatty acid derivative compositions may comprise, for example, a mixture of fatty alcohols (or another fatty acid derivative) with various chain lengths and saturation or branching characteristics.
  • the fatty acid derivative composition comprises a mixture of both more than one type of product and products with various chain lengths and saturation or branching characteristics.
  • fatty acid biosynthetic pathway means a biosynthetic pathway that produces fatty acids and derivatives thereof.
  • the fatty acid biosynthetic pathway may include additional enzymes to produce fatty acids derivatives having desired characteristics.
  • fatty aldehyde means an aldehyde having the formula RCHO characterized by a carbonyl group (C ⁇ O).
  • the fatty aldehyde is any aldehyde made from a fatty alcohol.
  • the R group is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19, carbons in length.
  • the R group is 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, or 6 or less carbons in length.
  • the R group can have an R group bounded by any two of the above endpoints.
  • the R group can be 6-16 carbons in length, 10-14 carbons in length, or 12-18 carbons in length.
  • the fatty aldehyde is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, or a C26 fatty aldehyde.
  • the fatty aldehyde is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, or C18 fatty aldehyde.
  • fatty alcohol means an alcohol having the formula ROH.
  • the R group is at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19, carbons in length.
  • the R group is 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, or 6 or less carbons in length.
  • the R group can have an R group bounded by any two of the above endpoints.
  • the R group can be 6-16 carbons in length, 10-14 carbons in length, or 12-18 carbons in length.
  • the fatty alcohol is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, or a C26 fatty alcohol.
  • the fatty alcohol is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, or C18 fatty alcohol.
  • the R group of a fatty acid derivative for example a fatty alcohol
  • a fatty acid derivative can be a straight chain or a branched chain.
  • Branched chains may have more than one point of branching and may include cyclic branches.
  • the branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, C24, C25, or a C 2-6 branched fatty acid, branched fatty aldehyde, or branched fatty alcohol.
  • the branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is a C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, or C18 branched fatty acid, branched fatty aldehyde, or branched fatty alcohol.
  • the hydroxyl group of the branched fatty acid, branched fatty aldehyde, or branched fatty alcohol is in the primary (C1) position.
  • the branched fatty acid derivative is an iso-fatty acid derivative, for example an iso-fatty aldehyde, an iso-fatty alcohol, or an antesio-fatty acid derivative, an anteiso-fatty aldehyde, or an anteiso-fatty alcohol.
  • the branched fatty acid derivative is selected from iso-C7:0, iso-C8:0, iso-C9:0, iso-C10:0, iso-C11:0, iso-C12:0, iso-C13:0, iso-C14:0, iso-C15:0, iso-C16:0, iso-C17:0, iso-C18:0, iso-C19:0, anteiso-C7:0, anteiso-C8:0, anteiso-C9:0, anteiso-C10:0, anteiso-C11:0, anteiso-C12:0, anteiso-C13:0, anteiso-C14:0, anteiso-C15:0, anteiso-C16:0, anteiso-C17:0, anteiso-C18:0, and an anteiso-C19:0 branched
  • the R group of a branched or unbranched fatty acid derivative can be saturated or unsaturated. If unsaturated, the R group can have one or more than one point of unsaturation.
  • the unsaturated fatty acid derivative is a monounsaturated fatty acid derivative.
  • the unsaturated fatty acid derivative is a C6:1, C7:1, C8:1, C9:1, C10:1, C11:1, C12:1, C13:1, C14:1, C15:1, C16:1, C17:1, C18:1, C19:1, C20:1, C21:1, C22:1, C23:1, C24:1, C25:1, or a C26:1 unsaturated fatty acid derivative.
  • the unsaturated fatty acid derivative is a C10:1, C12:1, C14:1, C16:1, or C18:1 unsaturated fatty acid derivative. In other embodiments, the unsaturated fatty acid derivative is unsaturated at the omega-7 position. In certain embodiments, the unsaturated fatty acid derivative comprises a cis double bond.
  • a recombinant or engineered “host cell” is a host cell, e.g., a microorganism used to produce one or more of fatty acid derivatives include, for example, acyl-CoA, fatty acids, fatty aldehydes, short and long chain alcohols, hydrocarbons, fatty alcohols, esters (e.g., waxes, fatty acid esters, or fatty esters), terminal olefins, internal olefins, and ketones.
  • the recombinant host cell comprises one or more polynucleotides, each polynucleotide encoding a polypeptide having fatty acid biosynthetic enzyme activity, wherein the recombinant host cell produces a fatty acid derivative composition when cultured in the presence of a carbon source under conditions effective to express the polynucleotides.
  • clone typically refers to a cell or group of cells descended from and essentially genetically identical to a single common ancestor, for example, the bacteria of a cloned bacterial colony arose from a single bacterial cell.
  • a culture typically refers to a liquid media comprising viable cells.
  • a culture comprises cells reproducing in a predetermined culture media under controlled conditions, for example, a culture of recombinant host cells grown in liquid media comprising a selected carbon source and nitrogen.
  • “Culturing” or “cultivation” refers to growing a population of recombinant host cells under suitable conditions in a liquid or solid medium.
  • culturing refers to the fermentative bioconversion of a substrate to an end-product.
  • Culturing media are well known and individual components of such culture media are available from commercial sources, e.g., under the DifcoTM and BBLTM trademarks.
  • the aqueous nutrient medium is a “rich medium” comprising complex sources of nitrogen, salts, and carbon, such as YP medium, comprising 10 g/L of peptone and 10 g/L yeast extract of such a medium.
  • the host cell can be additionally engineered to assimilate carbon efficiently and use cellulosic materials as carbon sources according to methods described in U.S. Pat. Nos. 5,000,000; 5,028,539; 5,424,202; 5,482,846; 5,602,030; WO 2010127318.
  • the host cell is engineered to express an invertase so that sucrose can be used as a carbon source.
  • the term “under conditions effective to express said heterologous nucleotide sequence(s)” means any conditions that allow a host cell to produce a desired fatty acid derivative. Suitable conditions include, for example, fermentation conditions.
  • modified or an “altered level of” activity of a protein, for example an enzyme, in a recombinant host cell refers to a difference in one or more characteristics in the activity determined relative to the parent or native host cell. Typically differences in activity are determined between a recombinant host cell, having modified activity, and the corresponding wild-type host cell (e.g., comparison of a culture of a recombinant host cell relative to the corresponding wild-type host cell).
  • Modified activities can be the result of, for example, modified amounts of protein expressed by a recombinant host cell (e.g., as the result of increased or decreased number of copies of DNA sequences encoding the protein, increased or decreased number of mRNA transcripts encoding the protein, and/or increased or decreased amounts of protein translation of the protein from mRNA); changes in the structure of the protein (e.g., changes to the primary structure, such as, changes to the protein's coding sequence that result in changes in substrate specificity, changes in observed kinetic parameters); and changes in protein stability (e.g., increased or decreased degradation of the protein).
  • the polypeptide is a mutant or a variant of any of the polypeptides described herein.
  • the coding sequence for the polypeptides described herein are codon optimized for expression in a particular host cell.
  • one or more codons can be optimized as described in, e.g., Grosjean et al., Gene 18:199-209 (1982).
  • regulatory sequences typically refers to a sequence of bases in DNA, operably-linked to DNA sequences encoding a protein that ultimately controls the expression of the protein.
  • regulatory sequences include, but are not limited to, RNA promoter sequences, transcription factor binding sequences, transcription termination sequences, modulators of transcription (such as enhancer elements), nucleotide sequences that affect RNA stability, and translational regulatory sequences (such as, ribosome binding sites (e.g., Shine-Dalgarno sequences in prokaryotes or Kozak sequences in eukaryotes), initiation codons, termination codons).
  • the phrase “the expression of said nucleotide sequence is modified relative to the wild type nucleotide sequence,” means an increase or decrease in the level of expression and/or activity of an endogenous nucleotide sequence or the expression and/or activity of a heterologous or non-native polypeptide-encoding nucleotide sequence.
  • the term “express” with respect to a polynucleotide is to cause it to function.
  • a polynucleotide which encodes a polypeptide (or protein) will, when expressed, be transcribed and translated to produce that polypeptide (or protein).
  • the term “overexpress” means to express or cause to be expressed a polynucleotide or polypeptide in a cell at a greater concentration than is normally expressed in a corresponding wild-type cell under the same conditions.
  • altered level of expression and “modified level of expression” are used interchangeably and mean that a polynucleotide, polypeptide, or hydrocarbon is present in a different concentration in an engineered host cell as compared to its concentration in a corresponding wild-type cell under the same conditions.
  • titer refers to the quantity of fatty acid derivative produced per unit volume of host cell culture.
  • a fatty acid derivative is produced at a titer of about 25 mg/L, about 50 mg/L, about 75 mg/L, about 100 mg/L, about 125 mg/L, about 150 mg/L, about 175 mg/L, about 200 mg/L, about 225 mg/L, about 250 mg/L, about 275 mg/L, about 300 mg/L, about 325 mg/L, about 350 mg/L, about 375 mg/L, about 400 mg/L, about 425 mg/L, about 450 mg/L, about 475 mg/L, about 500 mg/L, about 525 mg/L, about 550 mg/L, about 575 mg/L, about 600 mg/L, about 625 mg/L, about 650 mg/L, about 675 mg/L, about 700 mg/L, about 725 mg/L, about 750 mg/
  • a fatty acid derivative is produced at a titer of more than 100 g/L, more than 200 g/L, more than 300 g/L, or higher, such as 500 g/L, 700 g/L, 1000 g/L, 1200 g/L, 1500 g/L, or 2000 g/L.
  • the preferred titer of fatty acid derivative produced by a recombinant host cell according to the methods of the invention is from 5 g/L to 200 g/L, 10 g/L to 150 g/L, 20 g/L to 120 g/L and 30 g/L to 100 g/L.
  • the titer may refer to a particular fatty acid derivative or a combination of fatty acid derivatives produced by a given recombinant host cell culture.
  • the “yield of fatty acid derivative produced by a host cell” refers to the efficiency by which an input carbon source is converted to product (i.e., fatty alcohol or fatty aldehyde) in a host cell.
  • Host cells engineered to produce fatty acid derivatives according to the methods of the invention have a yield of at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, or at least 30% or a range bounded by any two of the foregoing values.
  • a fatty acid derivative or derivatives is produced at a yield of more than 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.
  • the yield is about 30% or less, about 27% or less, about 25% or less, or about 22% or less.
  • the yield can be bounded by any two of the above endpoints.
  • the yield of a fatty acid derivative or derivatives produced by the recombinant host cell according to the methods of the invention can be 5% to 15%, 10% to 25%, 10% to 22%, 15% to 27%, 18% to 22%, 20% to 28%, or 20% to 30%.
  • the yield may refer to a particular fatty acid derivative or a combination of fatty acid derivatives produced by a given recombinant host cell culture.
  • the productivity of a fatty acid derivative or derivatives produced by a recombinant host cell is at least 100 mg/L/hour, at least 200 mg/L/hour, at least 300 mg/L/hour, at least 400 mg/L/hour, at least 500 mg/L/hour, at least 600 mg/L/hour, at least 700 mg/L/hour, at least 800 mg/L/hour, at least 900 mg/L/hour, at least 1000 mg/L/hour, at least 1100 mg/L/hour, at least 1200 mg/L/hour, at least 1300 mg/L/hour, at least 1400 mg/L/hour, at least 1500 mg/L/hour, at least 1600 mg/L/hour, at least 1700 mg/L/hour, at least 1800 mg/L/hour, at least 1900 mg/L/hour, at least 2000
  • the productivity of a fatty acid derivative or derivatives produced by a recombinant host cell according to the methods of the may be from 500 mg/L/hour to 2500 mg/L/hour, or from 700 mg/L/hour to 2000 mg/L/hour.
  • the productivity may refer to a particular fatty acid derivative or a combination of fatty acid derivatives produced by a given recombinant host cell culture.
  • total fatty species and “total fatty acid product” may be used interchangeably herein with reference to the amount of fatty alcohols, fatty aldehydes and fatty acids, as evaluated by GC-FID as described in International Patent Application Publication WO 2008/119082. The same terms may be used to mean fatty esters and free fatty acids when referring to a fatty ester analysis.
  • glucose utilization rate means the amount of glucose used by the culture per unit time, reported as grams/liter/hour (g/L/hr).
  • carbon source refers to a substrate or compound suitable to be used as a source of carbon for prokaryotic or simple eukaryotic cell growth.
  • Carbon sources can be in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO 2 ).
  • Exemplary carbon sources include, but are not limited to, monosaccharides, such as glucose, fructose, mannose, galactose, xylose, and arabinose; oligosaccharides, such as fructo-oligosaccharide and galacto-oligosaccharide; polysaccharides such as starch, cellulose, pectin, and xylan; disaccharides, such as sucrose, maltose, cellobiose, and turanose; cellulosic material and variants such as hemicelluloses, methyl cellulose and sodium carboxymethyl cellulose; saturated or unsaturated fatty acids, succinate, lactate, and acetate; alcohols, such as ethanol, methanol, and glycerol, or mixtures thereof.
  • the carbon source can also be a product of photosynthesis, such as glucose.
  • the carbon source is biomass.
  • the carbon source is glucose.
  • the carbon source is sucrose.
  • biomass refers to any biological material from which a carbon source is derived.
  • a biomass is processed into a carbon source, which is suitable for bioconversion.
  • the biomass does not require further processing into a carbon source.
  • the carbon source can be converted into a biofuel.
  • An exemplary source of biomass is plant matter or vegetation, such as corn, sugar cane, or switchgrass.
  • Another exemplary source of biomass is metabolic waste products, such as animal matter (e.g., cow manure).
  • Further exemplary sources of biomass include algae and other marine plants.
  • Biomass also includes waste products from industry, agriculture, forestry, and households, including, but not limited to, fermentation waste, ensilage, straw, lumber, sewage, garbage, cellulosic urban waste, and food leftovers.
  • biomass also can refer to sources of carbon, such as carbohydrates (e.g., monosaccharides, disaccharides, or polysaccharides).
  • the term “isolated,” with respect to products refers to products that are separated from cellular components, cell culture media, or chemical or synthetic precursors.
  • the fatty acids and derivatives thereof produced by the methods described herein can be relatively immiscible in the fermentation broth, as well as in the cytoplasm. Therefore, the fatty acids and derivatives thereof can collect in an organic phase either intracellularly or extracellularly.
  • the terms “purify,” “purified,” or “purification” mean the removal or isolation of a molecule from its environment by, for example, isolation or separation. “Substantially purified” molecules are at least about 60% free (e.g., at least about 70% free, at least about 75% free, at least about 85% free, at least about 90% free, at least about 95% free, at least about 97% free, at least about 99% free) from other components with which they are associated. As used herein, these terms also refer to the removal of contaminants from a sample. For example, the removal of contaminants can result in an increase in the percentage of fatty acid derivatives in a sample.
  • a fatty acid derivative when a fatty acid derivative is produced in a recombinant host cell, the fatty acid derivative can be purified by the removal of host cell proteins. After purification, the percentage of fatty acid derivative in the sample is increased.
  • the terms “purify,” “purified,” and “purification” are relative terms which do not require absolute purity.
  • a purified fatty acid derivative when a fatty acid derivative is produced in recombinant host cells, a purified fatty acid derivative is a fatty acid derivative that is substantially separated from other cellular components (e.g., nucleic acids, polypeptides, lipids, carbohydrates, or other hydrocarbons).
  • a desired fatty acid derivative composition e.g., acyl-CoA, fatty acids, fatty aldehydes, short and long chain alcohols, hydrocarbons, fatty alcohols, esters (e.g., waxes, fatty acid esters, or fatty esters), terminal olefins, internal olefins, and ketones
  • a desired fatty acid derivative composition e.g., acyl-CoA, fatty acids, fatty aldehydes, short and long chain alcohols, hydrocarbons, fatty alcohols, esters (e.g., waxes, fatty acid esters, or fatty esters), terminal olefins, internal olefins, and ketones.
  • the invention provides recombinant host cells which have been engineered to provide enhanced fatty acid biosynthesis relative to non-engineered or native host cells (for example by strain improvements, as further described herein below).
  • polynucleotides useful in the recombinant host cells, methods, and compositions of the invention identifies polynucleotides useful in the recombinant host cells, methods, and compositions of the invention; however it will be recognized that absolute sequence identity to such polynucleotides is not necessary.
  • changes in a particular polynucleotide sequence can be made and the encoded polypeptide screened for activity. Such changes typically comprise conservative mutations and silent mutations (such as, for example, codon optimization).
  • Modified or mutated (i.e., mutant) polynucleotides and encoded variant polypeptides can be screened for a desired function, such as, an improved function compared to the parent polypeptide, including but not limited to increased catalytic activity, increased stability, or decreased inhibition (e.g., decreased feedback inhibition), using methods known in the art.
  • the disclosure identifies enzymatic activities involved in various steps (i.e., reactions) of the fatty acid biosynthetic pathways described herein according to Enzyme Classification (EC) number, and provides exemplary polypeptides (i.e., enzymes) categorized by such EC numbers, and exemplary polynucleotides encoding such polypeptides.
  • polypeptides and polynucleotides which are identified herein by Accession Numbers and/or Sequence Identifier Numbers (SEQ ID NOs), are useful for engineering fatty acid pathways in parental host cells to obtain the recombinant host cells described herein. It is to be understood, however, that polypeptides and polynucleotides described herein are exemplary and non-limiting.
  • sequences of homologues of exemplary polypeptides described herein are available to those of skill in the art using databases such as, for example, the Entrez databases provided by the National Center for Biotechnology Information (NCBI), the ExPasy databases provided by the Swiss Institute of Bioinformatics, the BRENDA database provided by the Technical University of Braunschweig, and the KEGG database provided by the Bioinformatics Center of Kyoto University and University of Tokyo, all which are available on the World Wide Web.
  • NCBI National Center for Biotechnology Information
  • ExPasy databases provided by the Swiss Institute of Bioinformatics
  • BRENDA database provided by the Technical University of Braunschweig
  • KEGG database provided by the Bioinformatics Center of Kyoto University and University of Tokyo, all which are available on the World Wide Web.
  • a variety of host cells can be modified to contain a fatty acid biosynthetic pathway such as those described herein, resulting in recombinant host cells suitable for the production of fatty acid derivatives. It is understood that a variety of cells can provide sources of genetic material, including polynucleotide sequences that encode polypeptides suitable for use in a recombinant host cell provided herein.
  • FadR is a key regulatory factor involved in fatty acid degradation and fatty acid biosynthetic pathways (Cronan et al., Mol. Microbiol., 29(4): 937-943 (1998)).
  • the E. coli ACS enzyme FadD and the fatty acid transport protein FadL are essential components of a fatty acid uptake system. FadL mediates transport of fatty acids into the bacterial cell, and FadD mediates formation of acyl-CoA esters.
  • acyl-CoA esters When no other carbon source is available, exogenous fatty acids are taken up by bacteria and converted to acyl-CoA esters, which can bind to the transcription factor FadR and derepress the expression of the fad genes that encode proteins responsible for fatty acid transport (FadL), activation (FadD), and ⁇ -oxidation (FadA, FadB, FadE, and FadH).
  • FadL fatty acid transport
  • FadD activation
  • FadA, FadB, FadE, and FadH FadA, FadB, FadE, and FadH.
  • FadA, FadB, FadE, and FadH When alternative sources of carbon are available, bacteria synthesize fatty acids as acyl-ACPs, which are used for phospholipid synthesis, but are not substrates for ⁇ -oxidation.
  • acyl-CoA and acyl-ACP are both independent sources of fatty acids can result in different end-products (Caviglia et al., J. Biol. Chem., 279(12): 1163-1169 (2004)).
  • U.S. Provisional Application No. 61/470,989 describes improved methods of producing fatty acid derivatives in a host cell which is genetically engineered to have an altered level of expression of a FadR polypeptide as compared to the level of expression of the FadR polypeptide in a corresponding wild-type host cell.
  • acyl-ACPs from acetyl-CoA via the acetyl-CoA carboxylase (acc) complex and fatty acid biosynthetic (fab) pathway is another step that may limit the rate of fatty acid derivative production ( FIG. 3 ).
  • acc acetyl-CoA carboxylase
  • fab fatty acid biosynthetic pathway
  • transposon mutagenesis and high-throughput screening was carried out to find beneficial mutations that increase the titer or yield.
  • Example 5 describes studies where it was observed that a transposon insertion in the yijP gene can improve the fatty alcohol yield in shake flask and fed-batch fermentations.
  • polypeptides i.e., enzymes
  • fatty acid biosynthetic polypeptides i.e., enzymes
  • fatty acid pathway polypeptides suitable for use in recombinant host cells of the invention are provided herein.
  • the invention includes a recombinant host cell comprising a polynucleotide sequence (also referred to herein as a “fatty acid biosynthetic polynucleotide” sequence) which encodes a fatty acid biosynthetic polypeptide.
  • a polynucleotide sequence also referred to herein as a “fatty acid biosynthetic polynucleotide” sequence
  • fatty acid biosynthetic polynucleotide also referred to herein as a “fatty acid biosynthetic polynucleotide” sequence
  • the polynucleotide sequence which comprises an open reading frame encoding a fatty acid biosynthetic polypeptide and operably-linked regulatory sequences, can be integrated into a chromosome of the recombinant host cells, incorporated in one or more plasmid expression systems resident in the recombinant host cell, or both.
  • both plasmid expression systems and integration into the host genome are used to illustrate different embodiments of the present invention.
  • a fatty acid biosynthetic polynucleotide sequence encodes a polypeptide which is endogenous to the parental host cell of the recombinant cell being engineered. Some such endogenous polypeptides are overexpressed in the recombinant host cell.
  • An “endogenous polypeptide”, as used herein, refers to a polypeptide which is encoded by the genome of the parental (e.g., wild-type) cell that is engineered to produce the recombinant host cell.
  • the fatty acid biosynthetic polynucleotide sequence encodes an exogenous or heterologous polypeptide.
  • the polypeptide encoded by the polynucleotide is exogenous to the parental host cell.
  • An “exogenous” (or “heterologous”) polypeptide refers to a polypeptide not encoded by the genome of the parental (e.g., wild-type) host cell that is being engineered to produce the recombinant host cell.
  • Such a polypeptide can also be referred to as a “non-native” polypeptide.
  • a variant (that is, a mutant) polypeptide is an example of a heterologous polypeptide.
  • the genetically modified host cell overexpresses a gene encoding a polypeptide (protein) that increases the rate at which the host cell produces the substrate of a fatty acid biosynthetic enzyme, i.e., a fatty acyl-thioester substrate.
  • a fatty acid biosynthetic enzyme i.e., a fatty acyl-thioester substrate.
  • the enzyme encoded by the over expressed gene is directly involved in fatty acid biosynthesis.
  • Such recombinant host cells may be further engineered to comprise a polynucleotide sequence encoding one or more “fatty acid biosynthetic polypeptides”, (enzymes involved in fatty acid biosynthesis), for example, a polypeptide:
  • AAR acyl-CoA reductase
  • AAR acyl-CoA reductase
  • At least one polypeptide encoded by a fatty acid biosynthetic polynucleotide is an exogenous (or heterologous) polypeptide (for example, a polypeptide originating from an organism other than the parental host cell, or, a variant of a polypeptide native to the parental microbial cell) or an endogenous polypeptide (that is, a polypeptide native to the parental host cell) wherein the endogenous polypeptide is overexpressed in the recombinant host cell.
  • exogenous (or heterologous) polypeptide for example, a polypeptide originating from an organism other than the parental host cell, or, a variant of a polypeptide native to the parental microbial cell
  • an endogenous polypeptide that is, a polypeptide native to the parental host cell
  • Table 1 provides a listing of exemplary proteins which can be expressed in recombinant host cells to facilitate production of particular fatty acid derivatives.
  • coli K12 ⁇ -hydroxydecanoyl NP_415474 4.2.1.60 increase fatty thioester acyl- dehydratase/isomerase ACP/CoA production fabB
  • E. coli 3-oxoacyl-[acyl- BAA16180 2.3.1.41 increase fatty carrier-protein] acyl- synthase I ACP/CoA production fabD
  • E. coli K12 [acyl-carrier-protein] AAC74176 2.3.1.39 increase fatty S-malonyltransferase acyl- ACP/CoA production fabF E.
  • coli K12 3-oxoacyl-[acyl- AAC74179 2.3.1.179 increase fatty carrier-protein] acyl- synthase II ACP/CoA production fabG
  • E. coli K12 3-oxoacyl-[acyl-carrier AAC74177 1.1.1.100 increase fatty protein] reductase acyl- ACP/CoA production fabH
  • E. coli K12 3-oxoacyl-[acyl- AAC74175 2.3.1.180 increase fatty carrier-protein] acyl- synthase III ACP/CoA production fabI E.
  • coli K12 ⁇ -hydroxydecanoyl NP_415474 4.2.1.60 produce thioester unsaturated dehydratase/isomerase fatty acids
  • GnsA E. coli suppressors of the ABD18647.1 none increase secG null mutation unsaturated fatty acid esters
  • GnsB E. coli suppressors of the AAC74076.1 none increase secG null mutation unsaturated fatty acid esters fabB
  • E. coli 3-oxoacyl-[acyl- BAA16180 2.3.1.41 modulate carrier-protein] unsaturated synthase I fatty acid production des Bacillus subtilis D5 fatty acyl O34653 1.14.19 modulate desaturase unsaturated fatty acid production 4.
  • Fatty Alcohol Output thioesterases see increase fatty above) acid/fatty alcohol production
  • BmFAR Bombyxmori FAR (fatty alcohol BAC79425 1.1.1.— convert acyl- forming acyl-CoA CoA to fatty reductase) alcohol acr1 Acinetobacter sp.
  • acyl-CoA reductase YP_047869 1.2.1.42 reduce fatty ADP1 acyl-CoA to fatty aldehydes yqhD
  • coli K12 acyl-CoA synthetase NP_416319 6.2.1.3 activates fatty acids to fatty acyl-CoAs atoB Erwinia carotovora acetyl-CoA YP_049388 2.3.1.9 production of acetyltransferase butanol hbd Butyrivibrio fibrisolvens Beta-hydroxybutyryl- BAD51424 1.1.1.157 production of CoA dehydrogenase butanol CPE0095 Clostridium crotonasebutyryl-CoA BAB79801 4.2.1.55 production of perfringens dehydryogenase butanol bcd Clostridium butyryl-CoA AAM14583 1.3.99.2 production of beijerinckii dehydryogenase butanol ALDH Clostridium coenzyme A-acylating AAT66436 1.2.1.3 production of beijerincki
  • AtMRP5 Arabidopsis Arabidopsis thaliana NP_171908 none modify thaliana multidrug resistance- product associated export amount AmiS2 Rhodococcus sp. ABC transporter JC5491 none modify AmiS2 product export amount AtPGP1 Arabidopsis Arabidopsis thaliana p NP_181228 none modify thaliana glycoprotein 1 product export amount AcrA Candidatus Protochlamydia amoebophila putative multidrug- CAF23274 none modify UWE25 efflux transport protein product acrA export amount AcrB Candidatus Protochlamydia amoebophila probable multidrug- CAF23275 none modify UWE25 efflux transport product protein, acrB export amount TolC Francisella tularensis Outer membrane ABD59001 none modify subsp.
  • Fermentation replication increase checkpoint output genes efficiency umuD Shigella sonnei DNA polymerase V, YP_310132 3.4.21.— increase Ss046 subunit output efficiency umuC E. coli DNA polymerase V, ABC42261 2.7.7.7 increase subunit output efficiency pntA, pntB Shigella flexneri NADH:NADPH P07001, 1.6.1.2 increase transhydrogenase P0AB70 output (alpha and beta efficiency subunits) 9.
  • the recombinant host cells may comprise one or more polynucleotide sequences that comprise an open reading frame encoding a thioesterase, e.g., having an Enzyme Commission number of EC 3.1.1.5 or EC 3.1.2.—(for example, EC 3.1.2.14), together with operably-linked regulatory sequences that facilitate expression of the protein in the recombinant host cells.
  • the open reading frame coding sequences and/or the regulatory sequences are modified relative to the corresponding wild-type gene encoding the thioesterase.
  • a fatty acid derivative composition comprising fatty acids is produced by culturing a recombinant cell in the presence of a carbon source under conditions effective to express the thioesterase.
  • the recombinant host cell comprises a polynucleotide encoding a polypeptide having thioesterase activity, and one or more additional polynucleotides encoding polypeptides having other fatty acid biosynthetic enzyme activities.
  • the fatty acid produced by the action of the thioesterase is converted by one or more enzymes having a different fatty acid biosynthetic enzyme activity to another fatty acid derivative, such as, for example, a fatty ester, fatty aldehyde, fatty alcohol, or a hydrocarbon.
  • the chain length of a fatty acid, or a fatty acid derivative made therefrom, can be selected for by modifying the expression of particular thioesterases.
  • the thioesterase will influence the chain length of fatty acid derivatives produced.
  • the chain length of a fatty acid derivative substrate can be selected for by modifying the expression of selected thioesterases (EC 3.1.2.14 or LC 3, 1.1.5).
  • host cells can be engineered to express, overexpress, have attenuated expression, or not express one or more selected thioesterases to increase the production of a preferred fatty acid derivative substrate.
  • C 10 fatty acids can be produced by expressing a thioesterase that has a preference for producing C 10 fatty acids and attenuating thioesterases that have a preference for producing fatty acids other than C 10 fatty acids (e.g., a thioesterase which prefers to produce C 14 fatty acids). This would result in a relatively homogeneous population of fatty acids that have a carbon chain length of 10.
  • C 14 fatty acids can be produced by attenuating endogenous thioesterases that produce non-C 14 fatty acids and expressing the thioesterases that use C 14 -ACP.
  • C 12 fatty acids can be produced by expressing thioesterases that use C 12 -ACP and attenuating thioesterases that produce non-C 12 fatty acids.
  • C12 fatty acids can be produced by expressing a thioesterase that has a preference for producing C12 fatty acids and attenuating thioesterases that have a preference for producing fatty acids other than C12 fatty acids. This would result in a relatively homogeneous population of fatty acids that have a carbon chain length of 12.
  • the fatty acid derivatives are recovered from the culture medium with substantially all of the fatty acid derivatives produced extracellularly.
  • the fatty acid derivative composition produced by a recombinant host cell can be analyzed using methods known in the art, for example, GC-FID, in order to determine the distribution of particular fatty acid derivatives as well as chain lengths and degree of saturation of the components of the fatty acid derivative composition.
  • Acetyl-CoA, malonyl-CoA, and fatty acid overproduction can be verified using methods known in the art, for example, by using radioactive precursors, HPLC, or GC-MS subsequent to cell lysis.
  • thioesterases and polynucleotides encoding them for use in the fatty acid pathway are provided in PCT Publication No. WO 2010/075483, expressly incorporated by reference herein.
  • the recombinant host cell produces a fatty aldehyde.
  • a fatty acid produced by the recombinant host cell is converted into a fatty aldehyde.
  • the fatty aldehyde produced by the recombinant host cell is then converted into a fatty alcohol or a hydrocarbon.
  • native (endogenous) fatty aldehyde biosynthetic polypeptides such as aldehyde reductases, are present in the host cell (e.g., E. coli ) and are effective to convert fatty aldehydes to fatty alcohols.
  • a native (endogenous) fatty aldehyde biosynthetic polypeptide is overexpressed.
  • an exogenous fatty aldehyde biosynthetic polypeptide is introduced into a recombinant host cell and expressed or overexpressed.
  • a native or recombinant host cell may comprise a polynucleotide encoding an enzyme having fatty aldehyde biosynthesis activity (also referred to herein as a “fatty aldehyde biosynthetic polypeptide” or a “fatty aldehyde biosynthetic polypeptide” or enzyme).
  • fatty aldehyde biosynthetic polypeptide also referred to herein as a “fatty aldehyde biosynthetic polypeptide” or a “fatty aldehyde biosynthetic polypeptide” or enzyme.
  • a recombinant host cell engineered to produce a fatty aldehyde will typically convert some of the fatty aldehyde to a fatty alcohol.
  • a fatty aldehyde is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a polypeptide having fatty aldehyde biosynthetic activity such as carboxylic acid reductase (CAR) activity.
  • CAR carboxylic acid reductase
  • CarB is an exemplary carboxylic acid reductase.
  • a gene encoding a carboxylic acid reductase polypeptide may be expressed or overexpressed in the host cell.
  • the CarB polypeptide has the amino acid sequence of SEQ ID NO: 7.
  • the CarB polypeptide is a variant or mutant of SEQ ID NO: 7.
  • carboxylic acid reductase (CAR) polypeptides and polynucleotides encoding them include, but are not limited to FadD9 (EC 6.2.1.-, UniProtKB Q50631, GenBank NP — 217106, SEQ ID NO: 34), CarA (GenBank ABK75684), CarB (GenBank YP889972; SEQ ID NO: 33) and related polypeptides described in PCT Publication No. WO 2010/042664 and U.S. Pat. No. 8,097,439, each of which is expressly incorporated by reference herein.
  • the recombinant host cell further comprises a polynucleotide encoding a thioesterase.
  • the fatty aldehyde is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a fatty aldehyde biosynthetic polypeptide, such as a polypeptide having acyl-ACP reductase (AAR) activity.
  • a fatty aldehyde biosynthetic polypeptide such as a polypeptide having acyl-ACP reductase (AAR) activity.
  • AAR acyl-ACP reductase
  • Native (endogenous) aldehyde reductases present in a recombinant host cell e.g., E. coli
  • Exemplary acyl-ACP reductase polypeptides are described in PCT Publication Nos. WO2009/140695 and WO/2009/140696, both of which are expressly incorporated by reference herein.
  • a composition comprising fatty aldehydes (“a fatty aldehyde composition”) is produced by culturing a host cell in the presence of a carbon source under conditions effective to express the fatty aldehyde biosynthetic enzyme.
  • the fatty aldehyde composition comprises fatty aldehydes and fatty alcohols.
  • the fatty aldehyde composition is recovered from the extracellular environment of the recombinant host cell, i.e., the cell culture medium.
  • the recombinant host cell comprises a polynucleotide encoding a polypeptide (an enzyme) having fatty alcohol biosynthetic activity (also referred to herein as a “fatty alcohol biosynthetic polypeptide” or a “fatty alcohol biosynthetic enzyme”), and a fatty alcohol is produced by the recombinant host cell.
  • a composition comprising fatty alcohols (“a fatty alcohol composition”) may be produced by culturing the recombinant host cell in the presence of a carbon source under conditions effective to express a fatty alcohol biosynthetic enzyme.
  • the fatty alcohol composition comprises fatty alcohols, however, a fatty alcohol composition may comprise other fatty acid derivatives.
  • the fatty alcohol composition is recovered from the extracellular environment of the recombinant host cell, i.e., the cell culture medium.
  • recombinant host cells have been engineered to produce fatty alcohols by expressing a thioesterase, which catalyzes the conversion of acyl-ACPs into free fatty acids (FFAs) and a carboxylic acid reductase (CAR), which converts free fatty acids into fatty aldehydes.
  • Native (endogenous) aldehyde reductases present in the host cell e.g., E. coli
  • the fatty alcohol is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a polypeptide having fatty alcohol biosynthetic activity which converts a fatty aldehyde to a fatty alcohol.
  • a polynucleotide encoding a polypeptide having fatty alcohol biosynthetic activity which converts a fatty aldehyde to a fatty alcohol.
  • an “alcohol dehydrogenase” also referred to herein as an “aldehyde reductase”, e.g., EC 1.1.1.1
  • the term “alcohol dehydrogenase” refers to a polypeptide capable of catalyzing the conversion of a fatty aldehyde to an alcohol (e.g., a fatty alcohol).
  • coli alcohol dehydrogenases such as YjgB, (AAC77226) (SEQ ID NO: 5), DkgA (NP — 417485), DkgB (NP — 414743), YdjL (AAC74846), YdjJ (NP — 416288), AdhP (NP — 415995), YhdH (NP — 417719), YahK (NP — 414859), YphC (AAC75598), YqhD (446856) and YbbO [AAC73595.1]. Additional examples are described in International Patent Application Publication Nos.
  • the fatty alcohol biosynthetic polypeptide has aldehyde reductase or alcohol dehydrogenase activity (EC 1.1.1.1).
  • Fatty alcohol may be produced via an acyl-CoA dependent pathway utilizing fatty acyl-ACP and fatty acyl-CoA intermediates and an acyl-CoA independent pathway utilizing fatty acyl-ACP intermediates but not a fatty acyl-CoA intermediate.
  • the enzyme encoded by the over expressed gene is selected from a fatty acid synthase, an acyl-ACP thioesterase, a fatty acyl-CoA synthase and an acetyl-CoA carboxylase.
  • the protein encoded by the over expressed gene is endogenous to the host cell. In other embodiments, the protein encoded by the overexpressed gene is heterologous to the host cell.
  • Fatty alcohols are also made in nature by enzymes that are able to reduce various acyl-ACP or acyl-CoA molecules to the corresponding primary alcohols. See also, U.S. Patent Publication Nos. 20100105963, and 20110206630 and U.S. Pat. No. 8,097,439, expressly incorporated by reference herein.
  • Strategies to increase production of fatty alcohols by recombinant host cells include increased flux through the fatty acid biosynthetic pathway by overexpression of native fatty acid biosynthetic genes and/or expression of exogenous fatty acid biosynthetic genes from different organisms in the production host such that fatty alcohol biosynthesis is increased.
  • fatty ester may be used with reference to an ester.
  • a fatty ester as referred to herein can be any ester made from a fatty acid, for example a fatty acid ester.
  • a fatty ester contains an A side and a B side.
  • an “A side” of an ester refers to the carbon chain attached to the carboxylate oxygen of the ester.
  • a “B side” of an ester refers to the carbon chain comprising the parent carboxylate of the ester.
  • the A side is contributed by an alcohol
  • the B side is contributed by a fatty acid.
  • any alcohol can be used to form the A side of the fatty esters.
  • the alcohol can be derived from the fatty acid biosynthetic pathway.
  • the alcohol can be produced through non-fatty acid biosynthetic pathways.
  • the alcohol can be provided exogenously.
  • the alcohol can be supplied in the fermentation broth in instances where the fatty ester is produced by an organism.
  • a carboxylic acid such as a fatty acid or acetic acid, can be supplied exogenously in instances where the fatty ester is produced by an organism that can also produce alcohol.
  • the carbon chains comprising the A side or B side can be of any length.
  • the A side of the ester is at least about 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, or 18 carbons in length.
  • the A side of the ester is 1 carbon in length.
  • the A side of the ester is 2 carbons in length.
  • the B side of the ester can be at least about 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 carbons in length.
  • the A side and/or the B side can be straight or branched chain.
  • the branched chains can have one or more points of branching.
  • the branched chains can include cyclic branches.
  • the A side and/or B side can be saturated or unsaturated. If unsaturated, the A side and/or B side can have one or more points of unsaturation.
  • the fatty ester is produced biosynthetically.
  • first the fatty acid is “activated.”
  • “activated” fatty acids are acyl-CoA, acyl ACP, and acyl phosphate.
  • Acyl-CoA can be a direct product of fatty acid biosynthesis or degradation.
  • acyl-CoA can be synthesized from a free fatty acid, a CoA, and an adenosine nucleotide triphosphate (ATP).
  • ATP adenosine nucleotide triphosphate
  • An example of an enzyme which produces acyl-CoA is acyl-CoA synthase.
  • the recombinant host cell comprises a polynucleotide encoding a polypeptide, e.g., an enzyme having ester synthase activity, (also referred to herein as an “ester synthase polypeptide” or an “ester synthase”).
  • a fatty ester is produced by a reaction catalyzed by the ester synthase polypeptide expressed or overexpressed in the recombinant host cell.
  • a composition comprising fatty esters also referred to herein as a “fatty ester composition” comprising fatty esters is produced by culturing the recombinant cell in the presence of a carbon source under conditions effective to express an ester synthase.
  • the fatty ester composition is recovered from the cell culture.
  • Ester synthase polypeptides include, for example, an ester synthase polypeptide classified as EC 2.3.1.75, or any other polypeptide which catalyzes the conversion of an acyl-thioester to a fatty ester, including, without limitation, a thioesterase, an ester synthase, an acyl-CoA:alcohol transacylase, an acyltransferase, or a fatty acyl-CoA:fatty alcohol acyltransferase.
  • the polynucleotide may encode wax/dgat, a bifunctional ester synthase/acyl-CoA:diacylglycerol acyltransferase from Simmondsia chinensis, Acinetobacter sp. Strain ADP, Alcanivorax borkumensis, Pseudomonas aeruginosa, Fundibacter jadensis, Arabidopsis thaliana , or Alkaligenes eutrophus .
  • the ester synthase polypeptide is an Acinetobacter sp.
  • ester synthase polypeptide is for example ES9 (a wax ester synthase from Marinobacter hydrocarbonoclasticus DSM 8798, UniProtKB A3RE51 (SEQ ID NO: 6); ES8 of Marinobacter hydrocarbonoclasticus DSM8789 (GenBank Accession No.
  • the polynucleotide encoding the ester synthase polypeptide is overexpressed in the recombinant host cell.
  • a fatty acid ester is produced by a recombinant host cell engineered to express three fatty acid biosynthetic enzymes: a thioesterase enzyme, an acyl-CoA synthetase (fadD) enzyme and an ester synthase enzyme (“three enzyme system”; FIG. 5 ).
  • a fatty acid ester is produced by a recombinant host cell engineered to express one fatty acid biosynthetic enzyme, an ester synthase enzyme (“one enzyme system”; FIG. 5 ).
  • ester synthase polypeptides and polynucleotides encoding them suitable for use in these embodiments include those described in PCT Publication Nos. WO 2007/136762 and WO2008/119082, and WO/2011/038134 (“three enzyme system”) and WO/2011/038132 (“one enzyme system”), each of which is expressly incorporated by reference herein.
  • the recombinant host cell may produce a fatty ester, such as a fatty acid methyl ester, a fatty acid ethyl ester or a wax ester in the extracellular environment of the host cells.
  • a fatty ester such as a fatty acid methyl ester, a fatty acid ethyl ester or a wax ester in the extracellular environment of the host cells.
  • This aspect of the invention is based, at least in part, on the discovery that altering the level of expression of a fatty aldehyde biosynthetic polypeptide, for example, an acyl-ACP reductase polypeptide (EC 6.4.1.2) and a hydrocarbon biosynthetic polypeptide, e.g., a decarbonylase in a recombinant host cell facilitates enhanced production of hydrocarbons by the recombinant host cell.
  • a fatty aldehyde biosynthetic polypeptide for example, an acyl-ACP reductase polypeptide (EC 6.4.1.2) and a hydrocarbon biosynthetic polypeptide, e.g., a decarbonylase in a recombinant host cell.
  • the recombinant host cell produces a hydrocarbon, such as an alkane or an alkene (e.g., a terminal olefin or an internal olefin) or a ketone.
  • a fatty aldehyde produced by a recombinant host cell is converted by decarboxylation, removing a carbon atom to form a hydrocarbon.
  • a fatty acid produced by a recombinant host cell is converted by decarboxylation, removing a carbon atom to form a terminal olefin.
  • an acyl-ACP intermediate is converted by decarboxylation, removing a carbon atom to form an internal olefin or a ketone. See, FIG. 6 .
  • An alkane biosynthetic pathway from cyanobacteria consisting of an acyl-acyl carrier protein reductase (AAR) and an aldehyde decarbonylase (ADC), which together convert intermediates of fatty acid metabolism to alkanes and alkenes has been used to engineer recombinant host cells for the production of hydrocarbons ( FIG. 6 ).
  • AAR acyl-acyl carrier protein reductase
  • ADC aldehyde decarbonylase
  • the second of two reactions in the pathway through which saturated acyl-ACPs are converted to alkanes in cyanobacteria entails scission of the C1-C2 bond of a fatty aldehyde intermediate by the enzyme aldehyde decarbonylase (ADC), a ferritin-like protein with a binuclear metal cofactor of unknown composition.
  • ADC aldehyde decarbonylase
  • the hydrocarbon is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a polypeptide having hydrocarbon biosynthetic activity such as an aldehyde decarbonylase (ADC) activity (e.g., EC 4.1.99.5).
  • ADC aldehyde decarbonylase
  • exemplary polynucleotides encoding an aldehyde decarbonylase useful in accordance with this embodiment include, but are not limited to, those described in PCT Publication Nos. WO 2008/119082 and WO 2009/140695 which are expressly incorporated by reference herein and those sequences presented in Table 2, below.
  • the recombinant host cell further comprises a polynucleotide encoding a fatty aldehyde biosynthesis polypeptide. In some embodiments the recombinant host cell further comprises a polynucleotide encoding an acyl-ACP reductase. See, for example Table 2, below.
  • a composition comprising hydrocarbons (also referred to herein as a “hydrocarbon composition”) is produced by culturing the recombinant cell in the presence of a carbon source under conditions effective to express the Acyl-CoA reductase and decarbonylase polynucleotides.
  • the hydrocarbon composition comprises saturated and unsaturated hydrocarbons, however, a hydrocarbon composition may comprise other fatty acid derivatives.
  • the hydrocarbon composition is recovered from the extracellular environment of the recombinant host cell, i.e., the cell culture medium.
  • alkane means saturated hydrocarbons or compounds that consist only of carbon (C) and hydrogen (H), wherein these atoms are linked together by single bonds (i.e., they are saturated compounds).
  • olefin and “alkene” are used interchangeably herein, and refer to hydrocarbons containing at least one carbon-to-carbon double bond (i.e., they are unsaturated compounds).
  • terminal olefin ⁇ -olefin
  • terminal alkene ⁇ -olefin
  • 1-alkene alkenes with a chemical formula CxH2x, distinguished from other olefins with a similar molecular formula by linearity of the hydrocarbon chain and the position of the double bond at the primary or alpha position.
  • a terminal olefin is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a hydrocarbon biosynthetic polypeptide, such as a polypeptide having decarboxylase activity as described, for example, in PCT Publication No. WO 2009/085278 which is expressly incorporated by reference herein.
  • the recombinant host cell further comprises a polynucleotide encoding a thioesterase.
  • an internal olefin is produced by expressing or overexpressing in the recombinant host cell a polynucleotide encoding a hydrocarbon biosynthetic polypeptide, such as a polypeptide having OleCD or OleBCD activity together with a polypeptide having OleA activity as described, for example, in PCT Publication No. WO 2008/147781, expressly incorporated by reference herein.
  • a hydrocarbon biosynthetic polypeptide such as a polypeptide having OleCD or OleBCD activity together with a polypeptide having OleA activity as described, for example, in PCT Publication No. WO 2008/147781, expressly incorporated by reference herein.
  • Strategies to increase production of fatty acid derivatives by recombinant host cells include increased flux through the fatty acid biosynthetic pathway by overexpression of native fatty acid biosynthetic genes and expression of exogenous fatty acid biosynthetic genes from different organisms in the production host.
  • the term “recombinant host cell” or “engineered host cell” refers to a host cell whose genetic makeup has been altered relative to the corresponding wild-type host cell, for example, by deliberate introduction of new genetic elements and/or deliberate modification of genetic elements naturally present in the host cell. The offspring of such recombinant host cells also contain these new and/or modified genetic elements.
  • the host cell can be selected from the group consisting of a plant cell, insect cell, fungus cell (e.g., a filamentous fungus, such as Candida sp., or a budding yeast, such as Saccharomyces sp.), an algal cell and a bacterial cell.
  • recombinant host cells are “recombinant microorganisms.”
  • host cells that are microorganisms, include but are not limited to cells from the genus Escherichia, Bacillus, Lactobacillus, Zymomonas, Rhodococcus, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces, Yarrowia , or Streptomyces .
  • the host cell is a Gram-positive bacterial cell. In other embodiments, the host cell is a Gram-negative bacterial cell.
  • the host cell is an E. coli cell.
  • the host cell is a Bacillus lentus cell, a Bacillus brevis cell, a Bacillus stearothermophilus cell, a Bacillus lichenoformis cell, a Bacillus alkalophilus cell, a Bacillus coagulans cell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillus thuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell, a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell.
  • the host cell is a Trichoderma koningii cell, a Trichoderma viride cell, a Trichoderma reesei cell, a Trichoderma longibrachiatum cell, an Aspergillus awamori cell, an Aspergillus fumigates cell, an Aspergillus foetidus cell, an Aspergillus nidulans cell, an Aspergillus niger cell, an Aspergillus oryzae cell, a Humicola insolens cell, a Humicola lanuginose cell, a Rhodococcus opacus cell, a Rhizomucor miehei cell, or a Mucor michei cell.
  • the host cell is a Streptomyces lividans cell or a Streptomyces murinus cell.
  • the host cell is an Actinomycetes cell.
  • the host cell is a Saccharomyces cerevisiae cell.
  • the host cell is a cell from a eukaryotic plant, algae, cyanobacterium, green-sulfur bacterium, green non-sulfur bacterium, purple sulfur bacterium, purple non-sulfur bacterium, extremophile, yeast, fungus, an engineered organism thereof, or a synthetic organism.
  • the host cell is light-dependent or fixes carbon.
  • the host cell has autotrophic activity.
  • the host cell has photoautotrophic activity, such as in the presence of light.
  • the host cell is heterotrophic or mixotrophic in the absence of light.
  • the host cell is a cell from Arabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, Zea mays, Botryococcuse braunii, Chlamydomonas reinhardtii, Dunaliela sauna, Synechococcus Sp. PCC 7002, Synechococcus Sp. PCC 7942, Synechocystis Sp.
  • PCC 6803 Thermosynechococcus elongates BP-1, Chlorobium tepidum, Chlorojlexus auranticus, Chromatiumm vinosum, Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas palusris, Clostridium ljungdahlii, Clostridiuthermocellum, Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pseudomonas fluorescens , or Zymomonas mobilis.
  • fatty acid derivatives can be produced by recombinant host cells comprising strain improvements as described herein, including, but not limited to, fatty acids, acyl-CoA, fatty aldehydes, short and long chain alcohols, hydrocarbons (e.g., alkanes, alkenes or olefins, such as terminal or internal olefins), fatty alcohols, esters (e.g., wax esters, fatty acid esters (e.g., methyl or ethyl esters)), and ketones.
  • the higher titer of fatty acid derivatives in a particular composition is a higher titer of a particular type of fatty acid derivative (e.g., fatty alcohols, fatty acid esters, or hydrocarbons) produced by a recombinant host cell culture relative to the titer of the same fatty acid derivatives produced by a control culture of a corresponding wild-type host cell.
  • the fatty acid derivative compositions may comprise, for example, a mixture of the fatty alcohols with a variety of chain lengths and saturation or branching characteristics.
  • the higher titer of fatty acid derivatives in a particular compositions is a higher titer of a combination of different fatty acid derivatives (for example, fatty aldehydes and alcohols, or fatty acids and esters) relative to the titer of the same fatty acid derivative produced by a control culture of a corresponding wild-type host cell.
  • different fatty acid derivatives for example, fatty aldehydes and alcohols, or fatty acids and esters
  • a polynucleotide (or gene) sequence is provided to the host cell by way of a recombinant vector, which comprises a promoter operably linked to the polynucleotide sequence.
  • the promoter is a developmentally-regulated, an organelle-specific, a tissue-specific, an inducible, a constitutive, or a cell-specific promoter.
  • the recombinant vector comprises at least one sequence selected from the group consisting of (a) an expression control sequence operatively coupled to the polynucleotide sequence; (b) a selection marker operatively coupled to the polynucleotide sequence; (c) a marker sequence operatively coupled to the polynucleotide sequence; (d) a purification moiety operatively coupled to the polynucleotide sequence; (e) a secretion sequence operatively coupled to the polynucleotide sequence; and (f) a targeting sequence operatively coupled to the polynucleotide sequence.
  • the expression vectors described herein include a polynucleotide sequence described herein in a form suitable for expression of the polynucleotide sequence in a host cell. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, etc.
  • the expression vectors described herein can be introduced into host cells to produce polypeptides, including fusion polypeptides, encoded by the polynucleotide sequences as described herein.
  • Fusion vectors add a number of amino acids to a polypeptide encoded therein, usually to the amino- or carboxy-terminus of the recombinant polypeptide.
  • Such fusion vectors typically serve one or more of the following three purposes: (1) to increase expression of the recombinant polypeptide; (2) to increase the solubility of the recombinant polypeptide; and (3) to aid in the purification of the recombinant polypeptide by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant polypeptide. This enables separation of the recombinant polypeptide from the fusion moiety after purification of the fusion polypeptide.
  • enzymes include Factor Xa, thrombin, and enterokinase.
  • Exemplary fusion expression vectors include pGEX (Pharmacia Biotech, Inc., Piscataway, N.J.; Smith et al., Gene, 67: 31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.), and pRITS (Pharmacia Biotech, Inc., Piscataway, N.J.), which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant polypeptide.
  • GST glutathione S-transferase
  • inducible, non-fusion E. coli expression vectors examples include pTrc (Amann et al., Gene (1988) 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gni). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident 2 prophage harboring a T7 gni gene under the transcriptional control of the lacUV 5 promoter.
  • Suitable expression systems for both prokaryotic and eukaryotic cells are well known in the art; see, e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual,” second edition, Cold Spring Harbor Laboratory, (1989).
  • Examples of inducible, non-fusion E. coli expression vectors include pTrc (Amann et al., Gene, 69: 301-315 (1988)) and PET 11 d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., pp. 60-89 (1990)).
  • a polynucleotide sequence of the invention is operably linked to a promoter derived from bacteriophage T5.
  • the host cell is a yeast cell.
  • the expression vector is a yeast expression vector.
  • Vectors can be introduced into prokaryotic or eukaryotic cells via a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell. Suitable methods for transforming or transfecting host cells can be found in, for example, Sambrook et al. (supra).
  • a gene that encodes a selectable marker e.g., resistance to an antibiotic
  • selectable markers include those that confer resistance to drugs such as, but not limited to, ampicillin, kanamycin, chloramphenicol, or tetracycline.
  • Nucleic acids encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a polypeptide described herein or can be introduced on a separate vector. Cells stably transformed with the introduced nucleic acid can be identified by growth in the presence of an appropriate selection drug.
  • an engineered or recombinant “host cell” is a cell used to produce a fatty acid derivative composition as further described herein.
  • a host cell is referred to as an “engineered host cell” or a “recombinant host cell” if the expression of one or more polynucleotides or polypeptides in the host cell are altered or modified as compared to their expression in a corresponding wild-type (or “native”) host cell under the same conditions.
  • the host cell can be selected from the group consisting of a eukaryotic plant, algae, cyanobacterium, green-sulfur bacterium, green non-sulfur bacterium, purple sulfur bacterium, purple non-sulfur bacterium, extremophile, yeast, fungus, engineered organisms thereof, or a synthetic organism.
  • the host cell is light dependent or fixes carbon.
  • the host cell has autotrophic activity.
  • Various host cells can be used to produce fatty acid derivatives, as described herein.
  • the polypeptide is a mutant or a variant of any of the polypeptides described herein.
  • mutant and variant refer to a polypeptide having an amino acid sequence that differs from a wild-type polypeptide by at least one amino acid.
  • the mutant can comprise one or more of the following conservative amino acid substitutions: replacement of an aliphatic amino acid, such as alanine, valine, leucine, and isoleucine, with another aliphatic amino acid; replacement of a serine with a threonine; replacement of a threonine with a serine; replacement of an acidic residue, such as aspartic acid and glutamic acid, with another acidic residue; replacement of a residue bearing an amide group, such as asparagine and glutamine, with another residue bearing an amide group; exchange of a basic residue, such as lysine and arginine, with another basic residue; and replacement of an aromatic residue, such as phenylalanine and tyrosine, with another aromatic residue.
  • the mutant polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid substitutions, additions, insertions, or deletions.
  • Preferred fragments or mutants of a polypeptide retain some or all of the biological function (e.g., enzymatic activity) of the corresponding wild-type polypeptide. In some embodiments, the fragment or mutant retains at least 75%, at least 80%, at least 90%, at least 95%, or at least 98% or more of the biological function of the corresponding wild-type polypeptide. In other embodiments, the fragment or mutant retains about 100% of the biological function of the corresponding wild-type polypeptide. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without affecting biological activity may be found using computer programs well known in the art, for example, LASERGENETM software (DNASTAR, Inc., Madison, Wis.).
  • a fragment or mutant exhibits increased biological function as compared to a corresponding wild-type polypeptide.
  • a fragment or mutant may display at least a 10%, at least a 25%, at least a 50%, at least a 75%, or at least a 90% improvement in enzymatic activity as compared to the corresponding wild-type polypeptide.
  • the fragment or mutant displays at least 100% (e.g., at least 200%, or at least 500%) improvement in enzymatic activity as compared to the corresponding wild-type polypeptide.
  • polypeptides described herein may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on the polypeptide function. Whether or not a particular substitution will be tolerated (i.e., will not adversely affect desired biological function, such as carboxylic acid reductase activity) can be determined as described in Bowie et al. (Science, 247: 1306-1310 (1990)).
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • Variants can be naturally occurring or created in vitro.
  • such variants can be created using genetic engineering techniques, such as site directed mutagenesis, random chemical mutagenesis, Exonuclease III deletion procedures, or standard cloning techniques.
  • such variants, fragments, analogs, or derivatives can be created using chemical synthesis or modification procedures.
  • variants are well known in the art. These include procedures in which nucleic acid sequences obtained from natural isolates are modified to generate nucleic acids that encode polypeptides having characteristics that enhance their value in industrial or laboratory applications. In such procedures, a large number of variant sequences having one or more nucleotide differences with respect to the sequence obtained from the natural isolate are generated and characterized. Typically, these nucleotide differences result in amino acid changes with respect to the polypeptides encoded by the nucleic acids from the natural isolates.
  • variants can be prepared by using random and site-directed mutagenesis. Random and site-directed mutagenesis are described in, for example, Arnold, Curr. Opin. Biotech., 4: 450-455 (1993).
  • Random mutagenesis can be achieved using error prone PCR (see, e.g., Leung et al., Technique, 1: 11-15 (1989); and Caldwell et al., PCR Methods Applic., 2: 28-33 (1992)).
  • error prone PCR PCR is performed 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.
  • nucleic acids to be mutagenized e.g., a polynucleotide sequence encoding a carboxylic reductase enzyme
  • PCR primers e.g., a polynucleotide sequence encoding a carboxylic reductase enzyme
  • reaction buffer MgCl 2 , MnCl 2 , Taq polymerase, and an appropriate concentration of dNTPs for achieving a high rate of point mutation along the entire length of the PCR product.
  • the reaction can be performed using 20 fmoles of nucleic acid to be mutagenized, 30 pmole of each PCR primer, a reaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH 8.3), 0.01% gelatin, 7 mM MgCl 2 , 0.5 mM MnCl 2 , 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP.
  • PCR can be performed for 30 cycles of 94° C. for 1 min, 45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciated that these parameters can be varied as appropriate.
  • the mutagenized nucleic acids are then cloned into an appropriate vector, and the activities of the polypeptides encoded by the mutagenized nucleic acids are evaluated.
  • Site-directed mutagenesis can be achieved using oligonucleotide-directed mutagenesis to generate site-specific mutations in any cloned DNA of interest.
  • Oligonucleotide mutagenesis is described in, for example, Reidhaar-Olson et al., Science, 241: 53-57 (1988). Briefly, in such procedures a plurality of double stranded oligonucleotides bearing one or more mutations to be introduced into the cloned DNA are synthesized and inserted into the cloned DNA to be mutagenized (e.g., a polynucleotide sequence encoding a CAR polypeptide). Clones containing the mutagenized DNA are recovered, and the activities of the polypeptides they encode are assessed.
  • Assembly PCR involves the assembly of a PCR product from a mixture of small DNA fragments. A large number of different PCR reactions occur in parallel in the same vial, with the products of one reaction priming the products of another reaction. Assembly PCR is described in, for example, U.S. Pat. No. 5,965,408.
  • Still another method of generating variants is sexual PCR mutagenesis.
  • sexual PCR mutagenesis forced homologous recombination occurs between DNA molecules of different, but highly related, DNA sequences in vitro as a result of random fragmentation of the DNA molecule based on sequence homology. This is followed by fixation of the crossover by primer extension in a PCR reaction.
  • Sexual PCR mutagenesis is described in, for example, Stemmer, Proc. Natl. Acad. Sci., U.S.A., 91: 10747-10751 (1994).
  • Variants can also be created by in vivo mutagenesis.
  • random mutations in a nucleic acid sequence are generated by propagating the sequence in a bacterial strain, such as an E. coli strain, which carries mutations in one or more of the DNA repair pathways.
  • a bacterial strain such as an E. coli strain
  • Such “mutator” strains have a higher random mutation rate than that of a wild-type strain.
  • Propagating a DNA sequence e.g., a polynucleotide sequence encoding a CAR polypeptide
  • Mutator strains suitable for use for in vivo mutagenesis are described in, for example, International Patent Application Publication No. WO 1991/016427.
  • cassette mutagenesis a small region of a double-stranded DNA molecule is replaced with a synthetic oligonucleotide “cassette” that differs from the native sequence.
  • the oligonucleotide often contains a completely and/or partially randomized native sequence.
  • Recursive ensemble mutagenesis can also be used to generate variants.
  • Recursive ensemble mutagenesis is an algorithm for protein engineering (i.e., protein mutagenesis) developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis.
  • Recursive ensemble mutagenesis is described in, for example, Arkin et al., Proc. Natl. Acad. Sci., U.S.A., 89: 7811-7815 (1992).
  • variants are created using exponential ensemble mutagenesis.
  • Exponential ensemble mutagenesis is a process for generating combinatorial libraries with a high percentage of unique and functional mutants, wherein small groups of residues are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins.
  • Exponential ensemble mutagenesis is described in, for example, Delegrave et al., Biotech. Res, 11: 1548-1552 (1993).
  • variants are created using shuffling procedures wherein portions of a plurality of nucleic acids that encode distinct polypeptides are fused together to create chimeric nucleic acid sequences that encode chimeric polypeptides as described in, for example, U.S. Pat. Nos. 5,965,408 and 5,939,250.
  • Insertional mutagenesis is mutagenesis of DNA by the insertion of one or more bases. Insertional mutations can occur naturally, mediated by virus or transposon, or can be artificially created for research purposes in the lab, e.g., by transposon mutagenesis. When exogenous DNA is integrated into that of the host, the severity of any ensuing mutation depends entirely on the location within the host's genome wherein the DNA is inserted. For example, significant effects may be evident if a transposon inserts in the middle of an essential gene, in a promoter region, or into a repressor or an enhancer region. Transposon mutagenesis and high-throughput screening was done to find beneficial mutations that increase the titer or yield of a fatty acid derivative or derivatives.
  • the term “fermentation” broadly refers to the conversion of organic materials into target substances by host cells, for example, the conversion of a carbon source by recombinant host cells into fatty acids or derivatives thereof by propagating a culture of the recombinant host cells in a media comprising the carbon source.
  • condition permissive for the production means any conditions that allow a host cell to produce a desired product, such as a fatty acid or a fatty acid derivative.
  • condition in which the polynucleotide sequence of a vector is expressed means any conditions that allow a host cell to synthesize a polypeptide. Suitable conditions include, for example, fermentation conditions. Fermentation conditions can comprise many parameters, including but not limited to temperature ranges, levels of aeration, feed rates and media composition. Each of these conditions, individually and in combination, allows the host cell to grow. Fermentation can be aerobic, anaerobic, or variations thereof (such as micro-aerobic). Exemplary culture media include broths or gels.
  • the medium includes a carbon source that can be metabolized by a host cell directly.
  • enzymes can be used in the medium to facilitate the mobilization (e.g., the depolymerization of starch or cellulose to fermentable sugars) and subsequent metabolism of the carbon source.
  • the engineered host cells can be grown in batches of, for example, about 100 mL, 500 mL, 1 L, 2 L, 5 L, or 10 L; fermented; and induced to express a desired polynucleotide sequence, such as a polynucleotide sequence encoding a CAR polypeptide.
  • a desired polynucleotide sequence such as a polynucleotide sequence encoding a CAR polypeptide.
  • the engineered host cells can be grown in batches of about 10 L, 100 L, 1000 L, 10,000 L, 100,000 L, and 1,000,000 L or larger; fermented; and induced to express a desired polynucleotide sequence.
  • large scale fed-batch fermentation may be carried out.
  • the fatty acid derivative compositions described herein are found in the extracellular environment of the recombinant host cell culture and can be readily isolated from the culture medium.
  • a fatty acid derivative may be secreted by the recombinant host cell, transported into the extracellular environment or passively transferred into the extracellular environment of the recombinant host cell culture.
  • the fatty acid derivative is isolated from a recombinant host cell culture using routine methods known in the art.
  • fraction of modem carbon or fM has the same meaning as defined by National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs4990B and 4990C, known as oxalic acids standards HOxI and HOxII, respectively.
  • SRMs4990B and 4990C Standard Reference Materials
  • HOxI and HOxII oxalic acids standards
  • the fundamental definition relates to 0.95 times the 14C/12C isotope ratio HOxI (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-Industrial Revolution wood.
  • fM is approximately 1.1.
  • Bioproducts e.g., the fatty acid derivatives produced in accordance with the present disclosure
  • Bioproducts comprising biologically produced organic compounds, and in particular, the fatty acid derivatives produced using the fatty acid biosynthetic pathway herein
  • These new bioproducts can be distinguished from organic compounds derived from petrochemical carbon on the basis of dual carbon-isotopic fingerprinting or 14 C dating.
  • the specific source of biosourced carbon e.g., glucose vs. glycerol
  • dual carbon-isotopic fingerprinting see, e.g., U.S. Pat. No. 7,169,588, which is herein incorporated by reference).
  • bioproducts from petroleum based organic compounds is beneficial in tracking these materials in commerce.
  • organic compounds or chemicals comprising both biologically based and petroleum based carbon isotope profiles may be distinguished from organic compounds and chemicals made only of petroleum based materials.
  • the bioproducts herein can be followed or tracked in commerce on the basis of their unique carbon isotope profile.
  • Bioproducts can be distinguished from petroleum based organic compounds by comparing the stable carbon isotope ratio ( 13 C/ 12 C) in each sample.
  • the 13 C/ 12 C ratio in a given bioproduct is a consequence of the 13 C/ 12 C ratio in atmospheric carbon dioxide at the time the carbon dioxide is fixed.
  • the major cause of differences in the carbon isotope ratio in plants is closely associated with differences in the pathway of photosynthetic carbon metabolism in the plants, particularly the reaction occurring during the primary carboxylation (i.e., the initial fixation of atmospheric CO 2 ).
  • Two large classes of vegetation are those that incorporate the “C3” (or Calvin-Benson) photosynthetic cycle and those that incorporate the “C4” (or Hatch-Slack) photosynthetic cycle.
  • C3 plants the primary CO 2 fixation or carboxylation reaction involves the enzyme ribulose-1,5-diphosphate carboxylase, and the first stable product is a 3-carbon compound.
  • C3 plants such as hardwoods and conifers, are dominant in the temperate climate zones.
  • C4 plants an additional carboxylation reaction involving another enzyme, phosphoenolpyruvate carboxylase, is the primary carboxylation reaction.
  • the first stable carbon compound is a 4-carbon acid that is subsequently decarboxylated.
  • the CO 2 thus released is refixed by the C3 cycle.
  • Examples of C4 plants are tropical grasses, corn, and sugar cane.
  • Both C4 and C3 plants exhibit a range of 13 C/ 12 C isotopic ratios, but typical values are about ⁇ 7 to about ⁇ 13 per mil for C4 plants and about ⁇ 19 to about ⁇ 27 per mil for C3 plants (see, e.g., Stuiver et al., Radiocarbon 19:355 (1977)). Coal and petroleum fall generally in this latter range.
  • the 13C measurement scale was originally defined by a zero set by Pee Dee Belemnite (PDB) limestone, where values are given in parts per thousand deviations from this material.
  • the “613C” values are expressed in parts per thousand (per mil), abbreviated, % o, and are calculated as follows:
  • ⁇ 1 3C(%) [( 13 C/ 12 C)sample ⁇ ( 13 C/ 12 C)standard]/( 13 C/ 12 C) standard ⁇ 1000
  • compositions described herein include bioproducts produced by any of the methods described herein, including, for example, fatty aldehyde and alcohol products.
  • the bioproduct can have a ⁇ 13 C of about ⁇ 28 or greater, about ⁇ 27 or greater, ⁇ 20 or greater, ⁇ 18 or greater, ⁇ 15 or greater, ⁇ 13 or greater, ⁇ 10 or greater, or ⁇ 8 or greater.
  • the bioproduct can have a ⁇ 13 C of about ⁇ 30 to about ⁇ 15, about ⁇ 27 to about ⁇ 19, about ⁇ 25 to about ⁇ 21, about ⁇ 15 to about ⁇ 5, about ⁇ 13 to about ⁇ 7, or about ⁇ 13 to about ⁇ 10.
  • the bioproduct can have a ⁇ 13 C of about ⁇ 10, ⁇ 11, ⁇ 12, or ⁇ 12.3.
  • Bioproducts produced in accordance with the disclosure herein can also be distinguished from petroleum based organic compounds by comparing the amount of 14 C in each compound. Because 14 C has a nuclear half-life of 5730 years, petroleum based fuels containing “older” carbon can be distinguished from bioproducts which contain “newer” carbon (see, e.g., Currie, “Source Apportionment of Atmospheric Particles”, Characterization of Environmental Particles, J. Buffle and H. P. van Leeuwen, Eds., 1 of Vol. I of the IUPAC Environmental Analytical Chemistry Series (Lewis Publishers, Inc.) 3-74, (1992)).
  • the fundamental definition relates to 0.95 times the 14 C/ 12 C isotope ratio HOxI (referenced to AD 1950). This is roughly equivalent to decay-corrected pre-Industrial Revolution wood.
  • fM is approximately 1.1.
  • compositions described herein include bioproducts that can have an fM 14 C of at least about 1.
  • the bioproduct of the invention can have an fM 14 C of at least about 1.01, an fM 14 C of about 1 to about 1.5, an fM 14 C of about 1.04 to about 1.18, or an fM 14 C of about 1.111 to about 1.124.
  • a biologically based carbon content is derived by assigning “100%” equal to 107.5 pMC and “0%” equal to 0 pMC. For example, a sample measuring 99 pMC will give an equivalent biologically based carbon content of 93%. This value is referred to as the mean biologically based carbon result and assumes all the components within the analyzed material originated either from present day biological material or petroleum based material.
  • a bioproduct comprising one or more fatty acid derivatives as described herein can have a pMC of at least about 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100.
  • a fatty acid derivative described herein can have a pMC of between about 50 and about 100; about 60 and about 100; about 70 and about 100; about 80 and about 100; about 85 and about 100; about 87 and about 98; or about 90 and about 95.
  • a fatty acid derivative described herein can have a pMC of about 90, 91, 92, 93, 94, or 94.2.
  • a host cell can be cultured, for example, for about 4, 8, 12, 24, 36, or 48 hours. During and/or after culturing, samples can be obtained and analyzed to determine if the conditions allow expression. For example, the host cells in the sample or the medium in which the host cells were grown can be tested for the presence of a desired product. When testing for the presence of a product, assays, such as, but not limited to, TLC, HPLC, GC/FID, GC/MS, LC/MS, MS, can be used. Recombinant host cell cultures are screened at the 96 well plate level, 1 liter and 5 liter tank level and in a 1000 L pilot plant using a GC/FID assay for “total fatty species”.
  • a fatty acid is a carboxylic acid with a long aliphatic tail (chain), which is either saturated or unsaturated. Most naturally occurring fatty acids have a chain of an even number of carbon atoms, from 4 to 28. Fatty acids are usually derived from triglycerides. When they are not attached to other molecules, they are known as “free” fatty acids. Fatty acids are usually produced industrially by the hydrolysis of triglycerides, with the removal of glycerol.
  • Palm, soybean, rapeseed, coconut oil and sunflower oil are currently the most common sources of fatty acids. The majority of fatty acids derived from such sources are used in human food products.
  • coconut oil and palm kernel oil (consist mainly of 12 and 14 carbon fatty acids). These are particularly suitable for further processing to surfactants for washing and cleansing agents as well as cosmetics.
  • Palm, soybean, rapeseed, and sunflower oil, as well as animal fats such as tallow contain mainly long-chain fatty acids (e.g., C18, saturated and unsaturated) which are used as raw materials for polymer applications and lubricants. Ecological and toxicological studies suggest that fatty acid-derived products based on renewable resources have more favorable properties than petrochemical-based substances.
  • Fatty aldehydes are used to produce many specialty chemicals. For example, aldehydes are used to produce polymers, resins (e.g., Bakelite), dyes, flavorings, plasticizers, perfumes, pharmaceuticals, and other chemicals, some of which may be used as solvents, preservatives, or disinfectants. In addition, certain natural and synthetic compounds, such as vitamins and hormones, are aldehydes, and many sugars contain aldehyde groups. Fatty aldehydes can be converted to fatty alcohols by chemical or enzymatic reduction.
  • Fatty alcohols have many commercial uses. Worldwide annual sales of fatty alcohols and their derivatives are in excess of U.S. $1 billion.
  • the shorter chain fatty alcohols are used in the cosmetic and food industries as emulsifiers, emollients, and thickeners. Due to their amphiphilic nature, fatty alcohols behave as nonionic surfactants, which are useful in personal care and household products, such as, for example, detergents.
  • fatty alcohols are used in waxes, gums, resins, pharmaceutical salves and lotions, lubricating oil additives, textile antistatic and finishing agents, plasticizers, cosmetics, industrial solvents, and solvents for fats.
  • the invention also provides a surfactant composition or a detergent composition comprising a fatty alcohol produced by any of the methods described herein.
  • a surfactant composition or a detergent composition comprising a fatty alcohol produced by any of the methods described herein.
  • fatty alcohols can be produced and used.
  • the characteristics of the fatty alcohol feedstock will affect the characteristics of the surfactant or detergent composition produced.
  • the characteristics of the surfactant or detergent composition can be selected for by producing particular fatty alcohols for use as a feedstock.
  • a fatty alcohol-based surfactant and/or detergent composition described herein can be mixed with other surfactants and/or detergents well known in the art.
  • the mixture can include at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or a range bounded by any two of the foregoing values, by weight of the fatty alcohol.
  • a surfactant or detergent composition can be made that includes at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or a range bounded by any two of the foregoing values, by weight of a fatty alcohol that includes a carbon chain that is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 carbons in length.
  • Such surfactant or detergent compositions also can include at least one additive, such as a microemulsion or a surfactant or detergent from non-microbial sources such as plant oils or petroleum, which can be present in the amount of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or a range bounded by any two of the foregoing values, by weight of the fatty alcohol.
  • a microemulsion or a surfactant or detergent from non-microbial sources such as plant oils or petroleum
  • Esters have many commercial uses.
  • biodiesel an alternative fuel, is comprised of esters (e.g., fatty acid methyl esters, fatty acid ethyl esters, etc.).
  • esters are volatile with a pleasant odor, which makes them useful as fragrances or flavoring agents.
  • esters are used as solvents for lacquers, paints, and varnishes.
  • some naturally occurring substances such as waxes, fats, and oils are comprised of esters.
  • Esters are also used as softening agents in resins and plasticizers, flame retardants, and additives in gasoline and oil.
  • esters can be used in the manufacture of polymers, films, textiles, dyes, and pharmaceuticals.
  • Hydrocarbons have many commercial uses. For example, shorter chain alkanes are used as fuels. Longer chain alkanes (e.g., from five to sixteen carbons) are used as transportation fuels (e.g., gasoline, diesel, or aviation fuel). Alkanes having more than sixteen carbon atoms are important components of fuel oils and lubricating oils. Even longer alkanes, which are solid at room temperature, can be used, for example, as a paraffin wax. In addition, longer chain alkanes can be cracked to produce commercially valuable shorter chain hydrocarbons.
  • shorter chain alkanes are used as fuels. Longer chain alkanes (e.g., from five to sixteen carbons) are used as transportation fuels (e.g., gasoline, diesel, or aviation fuel). Alkanes having more than sixteen carbon atoms are important components of fuel oils and lubricating oils. Even longer alkanes, which are solid at room temperature, can be used, for example, as a paraffin wax. In addition, longer chain alkane
  • short chain alkenes are used in transportation fuels.
  • Longer chain alkenes are used in plastics, lubricants, and synthetic lubricants.
  • alkenes are used as a feedstock to produce alcohols, esters, plasticizers, surfactants, tertiary amines, enhanced oil recovery agents, fatty acids, thiols, alkenylsuccinic anhydrides, epoxides, chlorinated alkanes, chlorinated alkenes, waxes, fuel additives, and drag flow reducers.
  • Ketones are used commercially as solvents. For example, acetone is frequently used as a solvent, but it is also a raw material for making polymers. Ketones are also used in lacquers, paints, explosives, perfumes, and textile processing. In addition, ketones are used to produce alcohols, alkenes, alkanes, imines, and enamines.
  • Lubricants are typically composed of olefins, particularly polyolefins and alpha-olefins. Lubricants can either be refined from crude petroleum or manufactured using raw materials refined from crude petroleum. Obtaining these specialty chemicals from crude petroleum requires a significant financial investment as well as a great deal of energy. It is also an inefficient process because frequently the long chain hydrocarbons in crude petroleum are cracked to produce smaller monomers. These monomers are then used as the raw material to manufacture the more complex specialty chemicals.
  • This example describes the construction of a genetically engineered host cell wherein the expression of a fatty acid degradation enzyme is attenuated.
  • E. coli MG1655 an E. coli K strain
  • Lambda Red also known as the Red-Driven Integration
  • Del-fadE-F (SEQ ID NO: 9) 5′-AAAAACAGCAACAATGTGAGCTTTGTTGTAATTATATTGTAAACATA TTGATTCCGGGGATCCGTCGACC; and Del-fadE-R (SEQ ID NO: 10) 5′-AAACGGAGCCTTTCGGCTCCGTTATTCATTTACGCGGCTTCAACTTT CCTGTAGGCTGGAGCTGCTTC
  • the Del-fadE-F and Del-fadE-R primers were used to amplify the kanamycin resistance (KmR) cassette from plasmid pKD13 (described by Datsenko et al., supra) by PCR.
  • the PCR product was then used to transform electrocompetent E. coli MG1655 cells containing pKD46 (described in Datsenko et al., supra) that had been previously induced with arabinose for 3-4 hours. Following a 3-hour outgrowth in a super optimal broth with catabolite repression (SOC) medium at 37° C., the cells were plated on Luria agar plates containing 50 ⁇ g/mL of Kanamycin.
  • SOC catabolite repression
  • Resistant colonies were identified and isolated after an overnight incubation at 37° C. Disruption of the fadE gene was confirmed by PCR amplification using primers fadE-L2 and fadE-R1, which were designed to flank the E. coli fadE gene.
  • the fadE deletion confirmation primers were:
  • E. coli MG1655 AfadE E. coli MG 1655 D1.
  • Fatty acid derivative (“total fatty species”) production by the MG1655 E. coli strain with the fadE gene deleted was compared to fatty acid derivative production by E. coli MG1655.
  • the data presented in FIG. 7 shows that deletion of the fadE gene did not affect fatty acid derivative production.
  • the main precursors for fatty acid biosynthesis are malonyl-CoA and acetyl-CoA ( FIG. 1 ). It has been suggested that these precursors limit the rate of fatty acid biosynthesis in E. coli .
  • synthetic acc operons [ Corynebacterium glutamicum accABCD ( ⁇ birA)] were overexpressed and the genetic modifications led to increased acetyl-coA and malonyl-CoA production in E. coli.
  • C. glutamicum acetyl-CoA carboxylase enzyme complex from Corynebacterium glutamicum
  • acc consists of four discrete subunits, accA, accB, accC and accD ( FIG. 3 ).
  • C. glutamicum acc The advantage of C. glutamicum acc is that two subunits are expressed as fusion proteins, accCB and accDA, respectively, which facilitates its balanced expression.
  • C. glutamicum birA which biotinylates the accB subunit ( FIG. 3 ).
  • Exemplary C. glutamicum bir DNA sequences are presented as SEQ ID NO:55 and SEQ ID NO:56.
  • a C. glutamicum bir protein sequence is presented as SEQ ID NO:57.
  • the synthetic operons of the C. glutamicum acc genes were cloned in the following way in OP80: Ptrc1-accDACB, Ptrc3-accDACB, Ptrc1-accCBDA and Ptrc3-CBDA.
  • Ptrc1 and Ptrc3 are derivatives of the commonly used Ptrc promoter, which allow attenuated transcription of target genes. Note that the native sequences were amplified from the chromosomal DNA as they showed favorable codon usage (only the codon for Arg6 in accCB was changed).
  • the C. glutamicum birA gene was codon optimized and obtained by gene synthesis. It was cloned then downstream of the acc genes in all four operon constructs. Below we refer to the operon configuration accDACB as accD- and the operon configuration accDACB+birA as accD+.
  • E. coli DAM1_i377 contains integrated copies (i) of leaderless thioesterease 'tesA and acyl-CoA synthetase fadD from E. coli and Ester synthase 9 (ES9) from Marinobacter hydrocarbonoclasticus . All genes are controlled by Ptrc promoters.
  • the strains were grown in 5NBT media in shake flasks and were analyzed for malonyl-CoA using the method described above.
  • FIG. 9 shows that six of the eight C. glutamicum acc ⁇ birA constructs showed elevated levels of malonyl-CoA in logarithmic phase demonstrating their functionality in E. coli .
  • panK In order to test the effect of combining panK and acc-birA overexpression, the optimized panK gene was cloned downstream of birA in ptrc1/3_accDACB-birA.
  • Pantothenate kinase panK (or CoaA) catalyzes the first step in the biosynthesis of coenzyme A, an essential cofactor that is involved in many reactions, e.g. the formation of acetyl-CoA, the substrate for acetyl-CoA carboxylase.
  • the resulting plasmids were transformed into DAM1_i377, grown in 5NBT (+TVS1) media in shake flasks, and the strains were analyzed for short-chain-CoAs using the method described above.
  • panK in log phase panK coexpression further increased malonyl-CoA levels and also increased acetyl-CoA levels demonstrating that panK can further increase the malonyl-CoA levels
  • ester synthase 9 (SEQ ID NO:6) with and without acc genes in another E. coli production host. More specifically, plasmids OP80 (vector control), pDS57 (with ES9), pDS57-accD- (with ES9 and accDACB) or pDS57-accD+(with ES9 and accDACB-birA; SEQ ID NO:63) were transformed into E. coli strain DV2 and the corresponding transformants were selected on LB plates supplemented with 100 mg/L of spectinomycin.
  • Two transformants of each plasmid were independently inoculated into LB medium supplemented with 100 mg/L of spectinomycin and grown for 5-8 hours at 32C.
  • the cultures were diluted 30-fold into a minimal medium with the following composition: 0.5 g/L NaCl, 1 mM MgSO4 ⁇ 7H2O, 0.1 mM CaCl2, 2 g/L NH4C1, 3 g/L KH2PO4, 6 g/L Na2HPO4, 1 mg/L thiamine, 1 ⁇ trace metal solution, 10 mg/L ferric citrate, 100 mM Bis-Tris (pH7.0), 30 g/L glucose and 100 mg/L spectinomycin.
  • the cultures were diluted 10-fold in quadruplicate into minimal medium of the same composition except that the media contained 1 g/L instead of 2 g/L NH4Cl and was supplemented with 1 mM IPTG and 2% (v/v) methanol.
  • the resulting cultures were then grown at 32° C. in a shaker.
  • FAMEs fatty acid methyl esters
  • GC-FID flame ionization detector
  • the analysis conditions were as follows: instrument: Trace GC Ultra, Thermo Electron Corporation with Flame ionization detector (FID) detector; column: DB-1 (1% diphenyl siloxane; 99% dimethyl siloxane) CO1 UFM 1/0.1/5 01 DET from Thermo Electron Corporation, phase pH 5, FT: 0.4 ⁇ m, length 5 m, id: 0.1 mm; inlet conditions: 250° C.
  • instrument Trace GC Ultra, Thermo Electron Corporation with Flame ionization detector (FID) detector
  • column DB-1 (1% diphenyl siloxane; 99% dimethyl siloxane) CO1 UFM 1/0.1/5 01 DET from Thermo Electron Corporation, phase pH 5, FT: 0.4 ⁇ m, length 5 m, id: 0.1 mm; inlet conditions: 250° C.
  • FID Flame ionization detector
  • splitless, 3.8 m 1/25 split method used depending upon sample concentration with split flow of 75 mL/m; carrier gas, flow rate: Helium, 3.0 mL/m; block temperature: 330° C.; oven temperature: 0.5 m hold at 50° C., 100° C./m to 330° C., 0.5 m hold at 330° C.; detector temperature: 300° C.; injection volume: 2 ⁇ L; run time/flow rate: 6.3 m/3.0 mL/m (splitless method), 3.8 m/1.5 mL/m (split 1/25 method), 3.04 m/1.2 mL/m (split 1/50 method).
  • FAMEs produced are shown in FIG. 10 .
  • the expression of ES9 by itself in E. coli DV2 led to FAME production above the control DV20P80.
  • Coexpression of the C. glutamicum acetyl-CoA carboxylase complex led to an approx. 1.5-fold increase in FAMEs and the additional expression of the C. glutamicum biotin protein ligase led to an approx. 5-fold increase in FAMEs.
  • the accDABC-birA operon was cloned downstream from the aar gene in pLS9185, a pCL1920 derivative) using Infusion technology, the resulting plasmid was transformed into E. coli DV2 and the corresponding transformants were selected on LB plates supplemented with 100 mg/L of spectinomycin.
  • FIG. 11 Fatty alcohols produced are shown in FIG. 11 .
  • the data were reproducible (triplicate samples were shown).
  • Example 3 describes co-expression of acc genes together with entire fab operons.
  • Strategies to increase the flux through the fatty acid synthesis pathway in recombinant host cells include both overexpression of native E. coli fatty acid biosynthesis genes and expression of exogenous fatty acid biosynthesis genes from different organisms in E. coli.
  • E. coli DV2 has the following genetic characterization: F-, ⁇ -, ilvG-, rfb-50, rph-1, ⁇ fhuA::FRT, ⁇ fadE::FRT.
  • iFABs 130-145 Sixteen strains containing iFABs 130-145 were evaluated. The detailed structure of iFABs 130-145 is presented in iFABs Table 4, below.
  • Each “iFAB” comprises various components in the following order: BS_fabI, BS_FabL, Vc_FabV, or Ec_FabI. All constructs contain St_H, St_D, and St_G, yet half of them have a synthetic RBS in front of St_H. All constructs contain either St_fabA or St_fabZ.
  • the plasmid pCL-WT TRC WT TesA was transformed into each of the strains shown above and a fermentation was run in FA2 media with 20 hours from induction to harvest at both 32° C. and 37° C. Data for production of “Total Fatty Species” from duplicate plate screens is shown in FIGS. 12A and 12B .
  • a full synthetic fab operon was integrated into the E. coli chromosome and evaluated for increased FAME production by expression in E. coli DAM1 pDS57.
  • four synthetic acc operons from Corynebaterium glutamicum were coexpressed and evaluated for improved FAME productivity.
  • Several strains were obtained that produced FAMEs at a faster rate and higher titers.
  • Genotype of integrated fab operons DAM1- IS5-11::PlacUV5 BsfabI (natRBS) StfabHDG StfabA ifab130 CacfabF::FRT DAM1- IS5-11::PlacUV5 BsfabI (natRBS) StfabHDG StfabZ ifab131 CacfabF::FRT DAM1- IS5-11::PlacUV5 BsfabI (synRBS) StfabHDG ifab132 StfabART CacfabF::F DAM1- IS5-11::PlacUV5 BsfabI (synRBS) StfabHDG StfabZ ifab133 CacfabF::FRT DAM1- IS5-11::PlacUV5 BsfabL (natRBS) StfabHDG StfabA ifab134 CacfabF::FRT DAM1- IS5-11::PlacUV5 BsfabL (natRBS) StfabHDG StfabA ifab134
  • the DAM1 ifab strains were analyzed in 96-well plates (4NBT medium), shake flasks (5NBT medium) and in fermenters at 32° C. The best results were obtained in 96-well plates and in shake flasks, where several DAM1 ifab strains with pDS57-acc-birA plasmids showed higher FAME titers.
  • DAM1 ifab131, ifab135, ifab137, ifab138 and ifab143 with pDS57-accDACB-birA showed 20-40% improved titers indicating that in these strains a higher flux through the fatty acid pathway was achieved, which apparently resulted in a better product formation rate (these results were reproducible in several independent experiments).
  • FabH and fabI are two fatty acid biosynthetic enzymes that have been shown to be feedback inhibited. A study was conducted to determine if FabH and FabI might be limiting the rate of FAME production.
  • FabH and fabI homologues (from E. coli, B. subtilis, Acinetobacter baylyi ADP1, Marinobacter aquaeoli VT8, and Rhodococcus opacus ) were overexpressed as a synthetic operon and evaluated in E. coli DAM1 pDS57 (a strain observed to be a good FAME producer).
  • fabHfabI operons were constructed from organisms that accumulate waxes ( A. baylyi, M. aquaeoli ) or triacylglycerides ( R. opacus ) and integrated into the chromosome of E. coli DAM1 pDS57.
  • a synthetic acc operons from C. glutamicum were co-expressed (as described in Example 2, above).
  • Genotype of integrated fabHI operons Strain Genotype of additional fab operon plasmid StEP117 DAM 1 IS5-11 ::PlacUV5 (synRBS) EcfabH (synRBS) bsfabI::kan pDS57 StEP118 DAM 1 IS5-11 ::PlacUV5 (synRBS) EcfabH (synRBS) BsfabL::kan pDS57 StEP127 DAM 1 IS5-11 ::PlacUV5 (ecRBS) ecfabH (ecRBS) bsfabI::kan pDS57 StEP128 DAM 1 IS5-11 ::PlacUV5 (ecRBS) EcfabH (ecRBS) BsfabL:kan pDS57 StEP129 DAM 1 IS5-11 ::PlacUV5 (ecRBS) ADP1fabH (ecRBS) ADP1fabI::kan pDS57 StEP130 DAM 1
  • the DAM1 ifabHI strains were analyzed in 96-well plates (4NBT medium), shake flasks (5NBT medium) and in fermenters at 32° C.
  • the lacUV5 promoter of FAB138 was replaced by a T5 promoter leading to higher levels of expression of FAB138, as confirmed by mRNA analysis.
  • the expression of FAB138 from the T5 promoter resulted in a higher titer, yield and productivity of fatty esters.
  • Primers DG405 and DG406 were used to amplify a cat-loxP and T5 promoter cassette adding 50 bp homology to each end of the PCR product, such that it could be integrated into any strain replacing the lacUV5 promoter regulating expression of the FAB138 operon.
  • the cat-loxP-T5 promoter was transformed into BD64/pKD46 strain. Transformants were recovered on LB+chloramphenicol plates at 37° C. overnight, patched to a fresh LB+chloramphenicol plate, and verified by colony PCR using primers DG422 and DG423.
  • Plasmid pJW168 was transformed into strain BD64 i-cat-loxP-T5 — 138 and selected on LB+carbenicillin plates at 32° C. In order to remove the cat marker, expression of the cre-recombinase was induced by IPTG. The plasmid pHW168 was removed by growing cultures at 42° C. Colonies were patched on LB+chloramphenicol and LB+carbenicillin to verify loss of pJW168 and removal of cat marker, respectively. The colony was also patched into LB as a positive control, all patched plates were incubated at 32° C. The removal of the cat marker was confirmed by colony PCR using primers DG422 and DG423. The resulting PCR product was verified by sequencing with primers EG744, EG749 and oTREE047, the strain was called shu.002.
  • FIGS. 16A and B provides a map of the strains generated.
  • FIG. 16 shows the FAB138 locus: a diagram of the cat-loxP-T5 promoter integrated in front of FAB138 ( FIG. 16A ) and a diagram of the iT5 — 138 promoter region ( FIG. 16B ).
  • the sequence of the cat-loxP-T5 promoter integrated in front of FAB138 with 50 base pair of homology shown in each side of cat-loxP-T5 promoter region is presented as SEQ ID NO:1 and the sequence of the iT5 — 138 promoter region with 50 base pair homology in each side is presented as SEQ ID NO:2.
  • a frozen cell bank vial of the selected E. coli strain was used to inoculate 20 mL of LB broth in a 125 mL baffled shake flask containing spectinomycin antibiotic at a concentration of 115 ⁇ g/mL. This shake flask was incubated in an orbital shaker at 32° C.
  • the pH of the culture was maintained at 6.9 using 28% w/v ammonia water, the temperature at 33° C., the aeration rate at 1 lpm (0.5 v/v/m), and the dissolved oxygen tension at 30% of saturation, utilizing the agitation loop cascaded to the DO controller and oxygen supplementation.
  • Foaming was controlled by the automated addition of a silicone emulsion based antifoam (Dow Corning 1410).
  • a nutrient feed composed of 3.9 g/L MgSO 4 heptahydrate and 600 g/L glucose was started when the glucose in the initial medium was almost depleted (approximately 4-6 hours following inoculation) under an exponential feed rate of 0.3 hr-1 to a constant maximal glucose feed rate of 10-12 g/L/hr, based on the nominal fermentation volume of 2 L.
  • Production of fatty alcohol in the bioreactor was induced when the culture attained an OD of 5 AU (approximately 3-4 hours following inoculation) by the addition of a 1M IPTG stock solution to a final concentration of 1 mM.
  • the bioreactor was sampled twice per day thereafter, and harvested approximately 72 hours following inoculation.
  • a 0.5 mL sample of the well-mixed fermentation broth was transferred into a 15 mL conical tube (VWR), and thoroughly mixed with 5 mL of butyl acetate. The tube was inverted several times to mix, then vortexed vigorously for approximately two minutes. The tube was then centrifuged for five minutes to separate the organic and aqueous layers, and a portion of the organic layer transferred into a glass vial for gas chromatographic analysis.
  • VWR conical tube
  • transposon mutagenesis was carried out and beneficial mutations were sequenced.
  • a transposon insertion in the yijP strain was shown to improve the strain's fatty alcohol yield in both shake flask and fed-batch fermentations.
  • the SL313 strain produces fatty alcohols.
  • the genotype of this strain is provided in Table **.
  • the genotype of this strain is MG1655 ( ⁇ fadE::FRT ⁇ fhuA::FRT fabBA329V ⁇ entD::T5-entD ⁇ insH-11:: PlacUV5 fab138 rph+lacI::PA1_tesA) containing the plasmid pDG109 (pCL1920_PTRC_carBopt — 12H08_alrAadp1_fabB[A329G]_fadR).
  • transposon mutagenesis was carried out by preparation of transposon DNA was prepared by cloning a DNA fragment into the plasmid EZ-Tn5TM pMODTM ⁇ R6K ori/MCS> (Epicentre Biotechnologies).
  • the DNA fragment contains a T5 promoter and the cat gene flanked by loxP sites.
  • the resulting plasmid was named p100.38 and the sequence is listed in Appendix I.
  • This plasmid was digested with PshAI restriction enzyme, incubated with EZ-Tn5TM Transposase enzyme (Epicentre Biotechnologies), and electroporated into electrocompetent SL313 cells as per the manufacturer's instructions.
  • the resulting colonies contained the transposon DNA inserted randomly into the chromosome of SL313.
  • Transposon clones were then subjected to high-throughput screening to measure production of fatty alcohols. Briefly, colonies were picked into deep-well plates containing LB, grown overnight, inoculated into fresh LB and grown for 3 hours, inoculated into fresh FA-2.1 media, grown for 16 hours, then extracted using butyl acetate. The crude extract was derivatized with BSTFA (N,O-bis[Trimethylsilyl]trifluoroacetamide) and analyzed using GC/FID. Spectinomycin (100 ug/mL) was included in all media to maintain selection of the pDG109 plasmid.
  • BSTFA N,O-bis[Trimethylsilyl]trifluoroacetamide
  • Hits were selected by choosing clones that produced a similar total fatty species as the control strain SL313, but that had a higher percent of fatty alcohol species and a lower percent of free fatty acids than the control.
  • Strain 68F11 was identified as a hit and was validated in a shake flask fermentation, according to the shake flask fermentation method described below. A comparison of transposon hit 68F11 to control strain SL313 indicated that 68F11 produces a higher percentage of fatty alcohol species than the control, while both strains produce similar titers of total fatty species.
  • DG150 (SEQ ID NO: 27) 5′-GCAGTTATTGGTGCCCTTAAACGCCTGGTTGCTACGCCTG-3′
  • DG131 (SEQ ID NO: 28) 5′-GAGCCAATATGCGAGAACACCCGAGAA-3′
  • Strain LC535 was determined to have a transposon insertion in the yijP gene ( FIG. 18 ).
  • yijP encodes a conserved inner membrane protein whose function is unclear.
  • the yijP gene is in an operon and co-transcribed with the ppc gene, encoding phosphoenolpyruvate carboxylase, and the yijO gene, encoding a predicted DNA-binding transcriptional regulator of unknown function. Promoters internal to the transposon likely have effects on the level and timing of transcription of yijP, ppc and yijO, and may also have effects on adjacent genes frwD, pflC, pfld, and argE. Promoters internal to the transposon cassette are shown, and may have effects on adjacent gene expression.
  • the yijP transposon cassette was further evaluated in a different strain V940, which produces fatty alcohol at a higher yield than strain SL313.
  • the yijP::Tn5-cat cassette was amplified from strain LC535 using primers:
  • LC277 (SEQ ID NO: 29) 5′-CGCTGAACGTATTGCAGGCCGAGTTGCTGCACCGCTCCCGCCAGGCA G-3′
  • LC278 (SEQ ID NO: 30) 5′-GGAATTGCCACGGTGCGGCAGGCTCCATACGCGAGGCCAGGTTATCC AACG-3′ This linear DNA was electroporated into strain SL571 and integrated into the chromosome using the lambda red recombination system. Colonies were screened using primers outside the transposon region:
  • a colony with the correct yijP transposon cassette was transformed with the production plasmid pV171.1 to produce strain D851.
  • D851 (V940 yijP::Tn5-cat) was tested in a shake-flask fermentation against isogenic strain V940 that does not contain the yijP transposon cassette. The result of this fermentation showed that the yijP transposon cassette confers production of a higher percent of fatty alcohol by the D851 strain relative to the V940 strain and produces similar titers of total fatty species as the V940 control strain.
  • Strain D851 was evaluated in a fed-batch fermentation on two different dates. Data from these fermentations is shown in Table 9 which illustrates that in 5-liter fed-batch fermentations, strains with the yijP::Tn5-cat transposon insertion had an increased total fatty species (“FAS”) yield and an increase in percent fatty alcohol (“FALC”).
  • FAS total fatty species
  • FALC percent fatty alcohol
  • total fatty species and “total fatty acid product” may be used interchangeably herein with reference to the amount of fatty alcohols, fatty aldehydes and free fatty acids, as evaluated by GC-FID as described in International Patent Application Publication WO 2008/119082. The same terms may be used to mean fatty esters and free fatty acids when referring to a fatty ester analysis.
  • fatty esters includes beta hydroxy esters.
  • a glycerol vial of desired strain was used to inoculate 20 mL LB+spectinomycin in shake flask and incubated at 32° C. for approximately six hours. 4 mL of LB culture was used to inoculate 125 mL Low PFA Seed Media (below), which was then incubated at 32° C. shaker overnight. 50 mL of the overnight culture was used to inoculate 1 L of Tank Media. Tanks were run at pH 7.2 and 30.5° C. under pH stat conditions with a maximum feed rate of 16 g/L/hr (glucose or methanol).
  • Ppc activity was measured in cells grown in a shake flask fermentation (as detailed above) and harvested 12-16 hours after induction. Approximately 5 mL of cells were centrifuged and the cell paste was suspended in BugBuster Protein Extraction Reagent (Novagen) with a protease inhibitor cocktail solution. The cell suspension was incubated with gentle shaking on a shaker for 20 min. Insoluble cell debris was removed by centrifugation at 16,000 ⁇ g for 20 min at 4° C. followed by transferring the supernatant to a new tube.
  • Ppc activity in the cell lysate was determined by a coupling reaction with citrate synthase using following reaction mixture: 0.4 mM acetyl-CoA, 10 mM phosphoenolpyruvate, 0.5 mM monobromobimane, 5 mM MgCl 2 , 10 mM NaHCO 3 , and 10 units citrate synthase from porcine heart in 100 mM Tris-HCl (pH 8.0).
  • the formation of CoA in the reaction with citrate synthase using oxaloacetate and acetyl-CoA was monitored photometrically using fluorescent derivatization of CoA with monobromobimane.
  • the Ppc assay results showed that the yijP::Tn5-cat transposon cassette decreased the Ppc activity in the cell. The results also indicate that the highest yield of fatty alcohol production requires a level of Ppc expression lower than the wild-type level.
  • Proteomics data was also collected to assess the abundance of the Ppc protein in two strains with and without the yijP::Tn5-cat transposon cassette. Protein samples were collected from strains V940 and D851 grown in bioreactors under standard fatty alcohol production conditions. Samples were taken at three different time points: 32, 48, 56 hours and prepared for analysis.
  • Proteomics data showed a significant reduction in the relative abundance of Ppc protein in D851 strain when compared to V940 at 36 hours and 48 hours). These data show that the yijP::Tn5-cat transposon cassette results in a significant reduction in Ppc abundance in the cell. This suggests that the observed benefits to fatty alcohol production by strains harboring the yijP::Tn5-cat transposon hit is due to reducing the amount of Ppc protein.
  • a strain designated, “shu.010” was developed which is isogenic to strain BD64 except that it contains the yijP::Tn5-cat transposon cassette.
  • the cassette containing the yijP::(Tn5) transposon DNA was amplified from strain DG851 using primers DG408 and DG407 (Table 12).
  • the cassette was transformed into BD64/pKD46. Transformants were recovered on LB+chloramphenicol plates at 37° C. overnight, patched to a fresh LB+chloramphenicol plate, and verified by colony PCR using primers DG131, DG407, and DG408.
  • Plasmid pKEV022 was transformed into shu.010. After selection in LB+spectinomycin plates, one colony was selected and called shu.015. Strain shu.015 was grown in tanks using standard conditions (see Appendix I for media and tank conditions). The tank performance of shu.015 was compared to strains KEV006.1 (BD64 pKEV018) and KEV075 (BD64 pKEV022) for Total Fatty Acid Product, Total Product Yield and glucose utilization rate.
  • acp genes from several cyanobacteria were cloned downstream from the Synechococcus elongatus PCC7942 acyl-ACP reductase (AAR) present in pLS9-185, which is a pCL1920 derivative (3-5 copies/cell).
  • AAR acyl-ACP reductase
  • the sfp gene accesion no. X63158; SEQ ID NO:53
  • Bacillus subtilis encoding a phosphopantetheinyl transferase with broad substrate specificity, was cloned downstream of the respective acp genes. This enzyme is involved in conversion of the inactive apo-ACP to the active holo-ACP.
  • the plasmids constructed are described in Table 13.
  • one cyanobacterial ACP gene with sfp was amplified from pDS171s (Table 13) and cloned downstream from 'tesA into a pCL vector. The resulting operon was under the control of the Ptrc3 promoter, which provides slightly lower transcription levels than the Ptrc wildtype promoter. The construct was cloned into E. coli DV2 and evaluated for fatty acid production. The control strain contained the identical plasmid but without cyanobacterial ACP and B. subtilis sfp.
  • FIG. 20 The results from a standard microtiter plate fermentation experiment are shown in FIG. 20 .
  • Significant improvement in fatty acid titer was observed in the strain coexpressing the heterologous ACP demonstrating that ACP overexpression can be beneficial for fatty acid production, in this case presumably by increasing the flux through the fatty acid biosynthetic pathway.
  • polypeptide 49 Synechococcus elongatu ATGAGCCAAGAAGACATCTTCAGCAAAGTCAAAGACATTGTGGCTGAGCAGCTGAGTGTGGATGTGGCTG PCC 7942_acp AAGTCAAGCCAGAATCCAGCTTCCAAAACGATCTGGGAGCGGACTCGCTGGACACCGTGGAACTGGTGAT Accession# YP_399555 GGCTCTGGAAGAGGCTTTCGATATCGAAATCCCCGATGAAGCCGCTGAAGGCATTGCGACCGTTCAAGAC (DNA) GCCGTCGATTTCATCGCTAGCAAAGCTGCCTAG 50 Synechococcus elongatu MSQEDIFSKVKDIVAEQLSVDVAEVKPESSFQNDLGADSLDTVELVMALEEAFDIEIPDEAAEGIATVQD PCC 7942_acp AVDFIASKAA Accession# YP_399555 (polypeptide) 51 Nostoc sp.
  • FT /note “aadA1- aminoglycoside 3′- adenylyltransferase”
  • FT /translation “MRSRNWSRTLTERSGGNGAVAVFMACYDCFFGVQSMPRASKQQA FT RYAVGRCLMLWSSNDVTQQGSRPKTKLNIMREAVIAEVSTQLSEVVGVIERHLEPTLL FT AVHLYGSAVDGGLKPHSDIDLLVTVTVTVRLDETTRRALINDLLETSASPGESEILRAVE FT VTIVVHDDIIPWRYPAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVAL FT VGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWAG
  • FT /note “repA protein”
  • FT /translation “MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRK FT ERTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAK FT FSSEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHK FT ANIEISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTL FT IFQVELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTS FT FEAKFLSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI” FT vector join(1 .
  • FT /source “pCL1920revised”
  • FT /type “Custom cloned insert”
  • FT /note “TERM rrnB T1 and T2 transcriptional terminators” FT misc_feature 2037 . . . 2063
  • FT /note “mini cistron ORF” FT misc_feature 2052 . . .
  • FT /note “g10 RBS (gene 10 region)”
  • FT /note “RBS”
  • FT /note “Lac Repressor lacIq ORF”
  • FT /translation “VKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAE FT LNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERS FT GVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII FT FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREG FT DWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDT
  • FT /note “ C. glutamicum accCB”
  • FT /translation “MSVETRKITKVLVANRGEIAIRVFRAARDEGIGSVAVYAEPDAD FT
  • NEGLIWIGPSPESIRSLGDKVTARHIADTAKAPMAPGTKEPVKDAAEVVAFAEEFGLP IAIKAAFGGGGRGMKVAYKMEEVADLFESATREATAAFGRGECFVERYLDKARHVEAQ FT
  • FT /note “rare Arg codon, change to CGT or CGC”
  • FT /note “GTG start codon, change to ATG” FT gene 5507 . . .
  • FT /note “aadA1- aminoglycoside 3′- adenylyltransferase”
  • FT /translation “MRSRNWSRTLTERSGGNGAVAVFMACYDCFFGVQSMPRASKQQA FT RYAVGRCLMLWSSNDVTQQGSRPKTKLNIMREAVIAEVSTQLSEVVGVIERHLEPTLL FT AVHLYGSAVDGGLKPHSDIDLLVTVTVTVRLDETTRRALINDLLETSASPGESEILRAVE FT VTIVVHDDIIPWRYPAKRELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVAL FT VGPAAEELFDPVPEQDLFEALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIA FT PKDVAADWAMERLPAQYQPVILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVV FT GK”
  • misc_feature “a
  • FT /note “repA protein”
  • FT /translation “MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRK FT ERTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAK FT FSSEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHK FT ANIEISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTL FT IFQVELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLHNGLRKTLHDALTAKIQLTS FT FEAKFLSDMQSKYDLNGSFSWLTQKQRTTLENILAKYGRI” FT vector join(1 .
  • FT /source “pCL1920revised”
  • FT /type “Custom cloned vector” FT insert join(330 . . . 1840, 3500 . . . 8061)
  • FT /source “pCL1920Ptrc”
  • FT /type “Custom cloned insert”
  • FT /note “TERM rrnB T1 and T2 transcriptional terminators” FT misc_feature 543 . . .
  • FT /note “Lac Repressor lacI ORF”
  • FT /note “dtsR1(accDA1)”
  • FT /translation “MTISSPLIDVANLPDINTTAGKIADLKARRAEAHFPMGEKAVEK FT
  • FT /note “ C. glutamicum accCB”
  • FT /translation “MSVETRKITKVLVANRGEIAIRVFRAARDEGIGSVAVYAEPDAD FT
  • NEGLIWIGPSPESIRSLGDKVTARHIADTAKAPMAPGTKEPVKDAAEVVAFAEEFGLP IAIKAAFGGGGRGMKVAYKMEEVADLFESATREATAAFGRGECFVERYLDKARHVEAQ FT
  • FT /note “rare Arg codon, change to CGT or CGC”
  • FT /note “GTG start codon, change to ATG” FT gene 6957 . . .
  • FT /note “birA_Cg_opt”
  • FT /translation “MNVDISRSREPLNVELLKEKLLQNGDFGQVIYEKVTGSTNADLL FT ALAGSGAPNWTVKTVEFQDHARGRLGRPWSAPEGSQTIVSVLVQLSIDQVDRIGTIPL FT AAGLAVMDALNDLGVEGAGLKWPNDVQIHGKKLCGILVEATGFDSTPTVVIGWGTNIS FT LTKEELPVPHATSLALEGVEVDRTTFLINMLTHLHTRLDQWQGPSVDWLDDYRAVCSS FT IGQDVRVLLPGDKELLGEAIGVATGGEIRVRDASGTVHTLNAGEITHLRLQ” FT misc_feature 6943 .

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