WO2020181091A1 - Fatty acyl-acp reductases and their uses - Google Patents

Abstract

Fatty acyl-ACP reductase (FAR) enzymes are provided, along with their use in the production of fatty alcohol compositions in heterologous recombinant cells.

Description

FATTY ACYL-ACP REDUCTASES AND THEIR USES

FIELD OF THE INVENTION

The invention relates to fatty acyl-ACP reductase (FAR) enzymes and their use in the production of faty alcohol compositions.

BACKGROUND

Fatty alcohols (FOHs) are used for the commercial production of surfactants, soaps, detergents, cosmetics, lubricants, plastics, etc. More than 70% of commercial fatty alcohols are obtained from plant-derived oils, particularly palm oils. Between the years 2000 and 2010, the amount of fatty alcohols produced from palm and palm kernel oil grew' by 100% (Biennann, Bomscheuer et al. 2011).

In 2010, approximately 50 million tons of palm and palm kernel oil were produced, representing more than 1/3 of the annual global production of all plant-derived oils (Biennann, Bomscheuer et al. 2011) Continuous growth of this industry and concerns over rainforest destruction have resulted hr the implementation of more sustainable palm oil processes, as well as the development of more environmentally friendly approaches via production of fatty alcohols by fermentation of recombinant microorganisms. Several alternative biosynthetic routes have been developed (Reiser and Somerville 1997, Schirmer, Rude et al. 2010, Steen, Kang et al. 2010, Zheng, Li et al. 2012, Akhtar, Turner et al. 2013) and a summary of biosynthetic routes is presented in Figure 1. As can be seen, all four routes (corresponding to Figure 1, parts a-d) start with acyl-ACPs (also referred to herein as“fatty acyi- ACPs”). Acyl-ACPs are a common intermediary of the fatty acid biosynthetic pathway e.g. in bacteria. The differences between these four routes are the type, and number of enzymes required to produce fatty alcohols, as well as the energy (ATP), reducing equivalents (NAD(P)H) and tire requirement for acyl-CoA. One disadvantage of routes a-c is that multiple enzymes are required, and so to optimize the production process, each pathway must he optimized for the recombinant cell used. Furthermore, the routes shown in Figure 1 parts a-c lead to the production of free fatty aldehydes as intermediates and die routes shown in Figure 1 parts a and b also lead to the production of fatty acids as intermediates. These intermediates are undesirable because aldehydes are known to be very reactive, while fatty acids have been shown to cause membrane stress (Lennen, Kruziki et al. 2011). A more attractive and efficient route (shown in Figure 1 part d) involves the direct conversion of fatty acyl-ACPs into fatty alcohols using a single FAR enzyme which catalyzes the complete reduction to alcohol, without the liberation of undesirable intermediates.

Certain plant-derived and bacterial FARs can utilize E. coil fatty acyl-ACPs, and directly produce fatty alcohols (Metz, Pollard et al. 2000, Doan, Carlsson et al. 2009, Hofvander, Doan et al. 2011). However, plant FARs preferentially use acyl-CoA intermediates that are not nonnally present in bacteria and these enzymes utilize E. coli fatty acyl-ACPs with low efficiency. Plant plastid-iocated FAR enzymes that preferentially use fatty acyl-ACPs as substrates have now been reported (for a review see Rowland and Domergue 2012). In vitro characterization of bacterial FAR identified from Marinobacter aquaeolei VT8 suggests that bacterial FARs have a wider range of specificity and higher activity than plant FARs (Willis, Wahlen et ai 201 i).

Bacterial FARs are more active than plant FARs and therefore represent ideal candidates for the production of fatty alcohols from fatty acyl-ACPs. However, only a limited number of functional bacterial FARs have been reported. For example, US 8,216,815 mentions faty alcohol production by FARs identified in Marinobacter algicola and Oceanobacter sp. RED65 that directly produce fatty alcohols from faty acyl-ACP in E. coli. Liu et al. also mentioned the use of Marinobacter aquaeolei FAR enzyme (Liu, Chen et al. 2016). Previously known FARs (such as FAR identified in Marinobacter algicola, and reported in US 8,216,815) are capable of producing faty alcohols as short as C14, but these previously know_'ll FARs have not been demonstrated to produce highly desirable fatty alcohols having a shorter chain length. Considerable efforts have been dedicated to engineering known enzymes to produce shorter chain length fatty alcohols but, so far as the Inventors are aware, such attempts have not been successful enough to enable commercialization of a FAR-based process.

Based on the limited number of active FARs reported in the literature, there is an urgent and unmet need to identify new FAR enzymes that can be used for tire production of faty alcohols. There is likewise an urgent and unmet need to identify new FAR enzymes that can produce fatty- alcohols with a chain length of less than C14.

SUMMARY OF THE INVENTION

The present invention provides new fatty acyl-ACP reductase (FAR) enzymes. The FAR enzymes of the invention possess highly desirable characteristics including e.g. \ i) efficient production of fatty alcohols from acyl-ACPs involving a single enzyme (i.e. the polypeptide of the invention): (ii) production of faty alcohols without undesirable production of aldehyde and faty acid intermediates; (iii) production of a high yield of fatty alcohols; (iv) production of a high proportion of saturated fatty- alcohols; (v) production of a high proportion of highly desirable C 14:0 fatty alcohol (also known as Myristyl alcohol); and (vi) production of highly desirable Cl 2 faty- alcohol.

The invention provides a recombinant polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1.

The invention also provides an isolated nucleic acid which encodes a polypeptide of the invention. In some embodiments, the nucleic acid is codon optimized for expression in a prokaryotic cell. In some embodiments, the prokaryotic cell is selected from the group consisting of Acinetohacier, Agrobacterium, Escherichia, Cupriavidus, Clostridium, Rhodobacter, Marinobacter, Bacillus, Klebsiella, Taiumella, Pseudomonas, Ralstonia, Rhodococcus, Methylobacterium, Methylophilus, Methylococcus. Methylomicrobium, Methylomonas, Pantoea, Streptomyces, Parachlorella, Synechococcus, Synechocystis and Thermocynechococcus

In some embodiments, the prokaryotic cell is E. coli. In some embodiments, the nucleic acid sequence has at least 70% sequence identity to SEQ ID NO: 2

In some embodiments, the nucleic acid sequence is codon optimized for expression in a eukaryotic cell. In some embodiments, the eukaryotic cell is selected from the group consisting of a yeast cell, a fungal cell, an algal cell and a plant cell.

In some embodiments, the yeast cell is selected from the group consisting of Saccharomyces , Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Schefferomyces, Rhodosporidium, Hansenula, Klockera, Schwanniomyces, Issatchenkia, Yarrowia and Rhodotorula.

In some embodiments, the yeast cell is selected from the group consisting of S. cerevisiae, C. lipolytica, R gluiinis, S. bulderi, S. barnetti, S. exiguus , S. uvarum, S. diastaticus, K. lactis, K. marxianus K. fragile, P. kudriavzevii, S. siipitis and / orientalis.

In some embodiments, the fungal cell is a filamentous fungal cell. In some embodiments, the filamentous fungal ceil is selected from the group consisting of Aspergillus, Penicillium, Rhizopus, Chrysosporium, Myceliophthora, Trichoderma, Humicola, Acremonium and Fusarium. In some embodiments, the filamentous fungal cell is selected from the group consisting of A. niger, A. oryzae, T. reesei, P. chrysogenum, M. thermophila, and fit oryzae.

In some embodiments, the algal cell is selected from the group consisting of Botryococcus, Nannochloropsis, Chlorelia, Chlamydomonas, Dunaliella, Chaetoceros, Porphyridium, Scenedesmus and Pseudochlorococcum. In some embodiments, the algal cell is selected from the group consisting of B. braunii and N gaditana.

In some embodiments, the nucleic acid of the invention comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 3.

The invention also provides a vector comprising the nucleic acid of the invention. In some embodiments, the vector comprises: (a) an origin of replication: (b) a promoter sequence operably linked to said nucleic acid; and/or (c) a reporter gene. In some embodiments, the vector comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 4.

The invention also provides a recombinant cell engineered to express a polypeptide of the invention. In some embodiments, the recombinant ceil is a prokaryotic cell. In some embodiments, the prokaryotic cell is selected from Acinetobacter, Agrobacterium, Escherichia, Cupriavidus, Clostridium , Rhodobacter, Marinobacter , Bacillus, Klebsiella, Tatumella, Pseudomonas, Ralstonia, Rhodococcus, Methylobaclerium, Methylophilus, Methylococcus, Methylomicrohium, Methylomonas, Pantoea, Streptomyces, Pamchlorella, Synechococcus, Synechocystis and Thermocynechococcus .

In some embodiments, the prokaryotic cell is E. coti. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the eukaryotic ceil is selected from the group consisting of a yeast cell, a fungal ceil, an algal cell and a plant cell

In some embodiments, the yeast cell is selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Schefferomyces, Rhodosporidium, Hansenula, Klockera, Schwanniomyces, Issatchenkia, Yarrowia and Rhodotorula. In some embodiments, the yeast cell is selected from the group consisting of S. cerevisiae, C. lipolytica, R. glutinis, S. bulderi, S. barnetti, S. exiguus, S. uvanim, S. diastaticus, K. lactis, K. marxianus, K fragile, P. kudriavzevii, S. stipitis and /. orientalis.

In some embodiments, the fungal cell is a filamentous fungal cell. In some embodiments, the filamentous fungal ceil is selected from the group consisting of Aspergillus, Penicillium, Rhizopus, Chrysosporium. Myceliophthora, Trichoderma, Humicola, Acremonium and Fusarium. In some embodiments, the filamentous fungal cell is selected from the group consisting of A. niger, A. oryrrac. T. reesei, P. chrysogenum, M. thermophila, and /? oryzae.

In some embodiments, the algal cell is selected from the group consisting of Botryococcus, Nannochloropsis, Chlorella, Chlamydomonas, Dunaliella, Chaetoceros, Porpkyridium, Scenedesmus and Pseudochlorococcum. In some embodiments, the algal ceil is selected from the group consisting of B. hraunii and N gaditana.

In some embodiments, the recombinant cell of the invention comprises one or more genetic modifications resulting in at least one, any two, any three, any four or all five of the following phenotypes: (a) an increase in fatty acyl-ACP synthesis; (b) a decrease in fatty acyl-ACP degradation; (c) an upregulation of faty alcohol secretion; (d) an increase in flux through the fatty acid biosynthetic pathway; (e) an increase in tolerance to fatty alcohol.

In some embodiments, the recombinant cell comprises one or more genetic modifications resulting in a change in the degree of saturation of the fatty alcohols produced.

In some embodiments, the recombinant cell comprises one or more genetic modifications resulting in a change in the chain length of acyl-ACPs produced by the faty acid biosynthetic pathway.

The invention also provides a recombinant cell transformed with a vector of the invention. In some embodiments, the recombinant cell is stably transformed with the nucleic acid of the invention. In some embodiments, the recombinant cell is transiently transformed with a nucleic acid of the invention. The invention also provides a method of producing a faty alcohol composition comprising: a) culturing a recombinant cell in a suitable culture medium, wherein the recombinant cell comprises a gene encoding a heterologous FAR enzyme having at least 70% sequence identity to SEQ ID NO: 1; and b) allowing expression of said gene, wherein said expression results in the production of a faty' alcohol composition.

In some embodiments, the method further comprises purifying the faty alcohols from cell culture. In some embodiments, the method further comprises purifying the fatty alcohols from supernatant. In some embodiments, the method further comprises harvesting and lysing recombinant cell(s) to obtain faty alcohols. In some embodiments, the method further comprises purifying the fatty alcohols from lysate.

In one embodiment, the invention provides a method of producing a fatty alcohol composition comprising: a) culturing a recombinant cell in a suitable culture medium, wherein the recombinant cell comprises a gene encoding a heterologous FAR enzyme having at least 70% sequence identity to SEQ ID NO: 1; b) allowing expression of said gene, wherein said expression results in the production of a fatty alcohol composition; and e) purifying the faty' alcohols from cell culture.

In one embodiment, the invention provides a method of producing a faty alcohol composition comprising: a) culturing a recombinant cell a suitable culture medium, wherein the recombinant cell comprises a gene encoding a heterologous FAR enzyme having at least 70% sequence identity to SEQ ID NO: 1; b) allowing expression of said gene, wherein said expression results in the production of a fatty alcohol composition; and c) purifying the fatty alcohols from supernatant.

In one embodiment, the invention provides a method of producing a fatty alcohol composition comprising: a) culturing a recombinant cell in a suitable culture medium, wherein the recombinant cell comprises a gene encoding a heterologous FAR enzyme having at least 70% sequence identity to SEQ ID NO: 1; fa) allowing expression of said gene, wherein said expression results in the production of a fatty alcohol composition; and c) harvesting and lysing recombinant cell(s) to obtain fatty alcohols.

In one embodiment, the invention provides a method of producing a fatty alcohol composition comprising: a) culturing a recombinant cell in a suitable culture medium, wherein the recombinant cell comprises a gene encoding a heterologous FAR enzyme having at least 70% sequence identity to SEQ ID NO: 1 ; b) allowing expression of said gene, wherein said expression results in the production of a fatty alcohol composition; c) harvesting and lysing recombinant cell(s) to obtain fatty alcohols; and d) purifying the fatty alcohols from lysate.

In some embodiments, the recombinant cell is defined according to any one of claims 20 to 36. The invention also provides a cell-free method for producing a fatty alcohol composition, the method comprising incubating a polypeptide according to the invention with fatty acyl-ACP and a reductant. In some embodiments, the reductant is NADPH. In some embodiments, the method further comprises purifying the faty alcohol composition.

Typically, the faty alcohol composition produced by a method of the invention comprises C12 fatty alcohol.

In some embodiments, the method further comprises reducing the faty alcohol composition. In some embodiments, the method further comprises esterifying the fatty alcohol composition.

The invention also provides a fatty alcohol composition obtained by the method of the invention.

The invention also provides a composition comprising fatty alcohol composition or esterified faty alcohol composition obtained by a method of the invention.

The invention also provides use of a polypeptide of the invention in a method of producing a fatty alcohol composition.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages will become more readily appreciated as the same become beter understood by reference to the following detailed description, when taken m conjunction with the accompanying drawings.

FIGURE 1 summarizes current technologies for the production of fatty alcohols from fatty acyl-ACPs in E. coli. Parts a) and b) depict routes based on the use of thioesterases to produce fatty acids, in which fatty acids are converted into fatty aldehyde intermediates by different enzymes. Part c) depicts a route that directly produces fatty aldehydes, which are then converted to fatty alcohols by alcohol dehydrogenases. Part d) depicts a route that directly converts fatty acyl-ACPs into fatty alcohols using a FAR enzyme, which catalyzes the 2-step reduction of fatty acyl-ACP to fatty alcohols without liberating fatty aldehyde or fatty alcohol intermediates. FARs of the invention produce fatty alcohols via the route depicted in part d).

FIGURE 2 shows low copy expression vector, pCL-BP2 with speetinomycin resistance cassete and multiple cloning sites for FAR expression under the Trc inducible promoter.

FIGURE 3 shows the relative amount of total fatty alcohols produced by E. coli expressing a FAR gene from Aesturaiibacter salexigens (AsFAR), Massilia sp. Root335 (MaslFAR), Mannobacter (MarlFAR), Hahella ganghwensis (HgFAR), Marinobacter daepoensis (MdFAR), Duganella (DugFAR), Herbaspirillum (HerFAR), Spongii hacier marinus (SmFAR; strain 16), Zhongshania aliphaticivorans (ZaFAR), Marinobacter (Mar2FAR), Marinobacter lipolyticus (M1FAR), and Nanorana parkei (NpFAR) Data are relative to the total amount of fatty alcohols produced by SmFAR.

DETAILED DESCRIPTION OF THE INVENTION

Fatty Acyl-ACP Reductase Polypeptides

The present invention is based on the surprising discover}' of a previously unknown dehydrogenase enzyme with fatty acyl-ACP reductase (FAR) activity derived from Spongiibacter marinus (herein “SmFAR”) that catalyzes the direct reduction of fatty acyl-ACP into fatty alcohols, without the liberation of undesirable intermediates.

“Fatty acyl-ACP reductases” are sometimes referred to in the literature as“fatty acyl-CoA reductases” or“fatty acyl-CoA/ACP reductases” on the basis that they are frequently able to catalyze Acyl-CoA, as well as Acyl-ACP.

In comparison to previously reported FARs, the Spongiibacter marinus fatty acyl-ACP reductase (SmFAR) polypeptide of the invention produces a fatty alcohol composition having a distinct and advantageous fatty alcohol profile. The Inventors found that SmFAR can produce highly desirable shorter chain length fatty alcohols than previously reported FAR enzymes. The enzyme of the invention also produces a desirable and advantageously high proportion of saturated fatty alcohols, as compared to previously reported FAR enzymes. Advantageously, this can help simplify downstream processing when seeking to obtain saturated product.

SmFAR also comprises a unique polypeptide sequence that has low sequence identity to previously- reported FARs. Sequence identity comparisons between SmFAR and publicly available protein sequences identified the nearest homolog as having -65% sequence identity to SmFAR. Indeed, SmFAR has about -50% sequence homology to FAR derived from Marinobacter kydrocarbonoclasticus or from Marinobactoer algicola (“MaFAR”).

A FAR polypeptide of the invention comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, the FA polypeptide of the invention has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, the FAR polypeptide of the invention has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a functional fragment or catalytic domain of SEQ ID NO: i. The SmFAR polypeptide sequence is represented by SEQ ID NO: 1 :

M RDLNRDKSST!AALTGKQVLITGTTGFLGKVLLEKVLRTTPGVGGIYLL!Vi RGN RHFRDATDRFREEVISSSI FDTLR

HELGQEGFEALIASRVHCVTGEITAPRFGLTRTVFTALADKLDVVINSAASVNFREELDRALEINTFSLENVAKLCELG

GNI PLVHVSTCYVNGYRSGD!H ETSEGPVGLELPEGPGSFYETSAL! ERLQRLVEELRGQYEGRALKAKLI EHGAKEA

QAAGWNDTYTFTKWLGEQYLYKVM RGYSLTVVRPSII ESTLREPSPGWI EGVKVADAVLMAYARGKVAFFPGKRA

GVIDII PADLVANGVLLSTAEQLLEPGQH RIYQCCSGGRN PLM LGDFIDH LVAEASSH HGDYDKLFKAPPRRRFTAV

DKRVFSSVAMCVQVALTIVGAI LRRLGRRGKLRARRNIDTAVELSKIFSFYAQPNYCFHGDKLLSLSKSMGAVDQEL

FPVDAGVIDWQHYIRHVHM PGLN HYALQGQPSVRSQQRREEVKTAPKNLTATEA (SEQ ID NO: 1)

An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul et ctL, 1990, J . Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by- identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. These initial neighborhood word hits act as starting points to find longer HSPs containing them. The word hits are expanded in both directions along each of the two sequences being compared for as far as the cumulative alignment score can be increased. Extension of the word hits is stopped when: the cumulative alignment score falls off by the quantity X from a maximum achie ved value; the cumulative score goes to zero or below; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1992, Proc. Nat’l. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M'5, N'-4, and a comparison of both strands.

Any of the amino acid sequences described herein can be produced together or in conjunction with at least 1 , e.g., at least (or up to) 2, 3, 5, 10, or 20 heterologous amino acids flanking each of the C- and/or N-teiminal ends of the specified amino acid sequence, and or deletions of at least 1, e.g., at least (or up to) 2, 3, 5, 10, or 20 a mo acids from the C- and/or N-terminal ends.

FAR polypeptides of the invention may have one or more (e.g., up to 2, 3, 5, 10, 20, 30, 40, or 50) conservative amino acid substitutions relative to the polypeptide of SEQ ID NO: 1. Conservative substitutions can be chosen from among a group of amino acids having a similar side chain to the reference amino acid. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of am o acids having sulphur-containing side chains is cysteine and methionine. Accordingly, exemplary conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala: Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; He to Leu or Yak Leu to lie or Val; Lys to Arg; Gin or Glu; Met to Leu or He; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Val to He or Leu.

Hie invention also provides a fusion protein that includes at least a portion (e.g, a fragment or domain) of a FAR polypeptide of the invention attached to one or more fusion segments, which are typically heterologous to the FAR polypeptide. Suitable fusion segments include, without limitation, segments that can provide other desirable biological activity or facilitate purification of the FAR polypeptide (e.g, by affinity chromatography). Fusion segments can be joined to the amino or carboxy terminus of a FAR polypeptide . The fusion segments can be susceptible to cleavage.

Fatty Acyl-ACP Reductase Nucleic Acids

A FAR nucleic acid of the invention encodes a FAR polypeptide of the invention. The FAR nucleic acid may be isolated, synthetic or recombinant.

A FAR nucleic acid of the invention may be optimized for expression in a recombinant cell. Optimized” nucleic acid sequences encode an amino acid sequence using codons that are preferred in a recombinant cell. The optimized nucleic acid sequence is typically engineered to retain completely or as much as possible of the amino acid sequence originally encoded by the starting nucleic acid sequence, which is also known as the "‘parental” sequence. Several methods for codon optimization are known in the art. Preferably, codon optimized sequences avoid nucleotide repeats and restriction sites that are utilized in cloning the FAR nucleic acids, by adjusting the settings in commercial software or by manually altering the sequences to substitute codons that introduce undesired sequences, for example with highly utilized codons in the heterologous organism of interest.

In some embodiments, the nucleic acid is codon optimized for expression in a prokaryotic cell. In some embodiments, the nucleic acid is codon optimized for expression in Acinetohacter , Agrobacterium, Escherichia, Cupriavidus, Clostridium , Rhodohacter, Marinobacter, Bacillus, Klebsiella, Tatumella, Pseudomonas, Ralstonia, Rhodococcus, Methylobactenum, Meihylophilus, Methylococcus, Methylomicrobium Methylomonas, Pantoe a, Streptomyces, Parachlorella, Synechococcus, Synechocystis and Thermocynechococcus In some embodiments, the nucleic acid is codon optimized for expression in E. coli. In some embodiments, the nucleic acid sequence has at least 70% sequence identity to SEQ ID NO: 2. In some embodiments, the nucleic acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 2, over a region of at least about 10, e.g., at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, or 1557 nucleotides, or a catalytic domain thereof.

SEQ ID NO: 2 represents a nucleic acid sequence corresponding to the SmFAR polypeptide (SEQ ID NO: 1) codon optimized for expression in E. coir.

ATGCGTGATTTAAACCGCGATAAAAGTAGCACCATTGCCGCACTGACCGGCAAACAGGTGCTGATCACCGGC

ACCACCGGCTTTCTGGGTAAGGTGCTGCTGGAAAAAGTGCTGCGTACAACCCCGGGTGTTGGCGGCATTTATC

TGCTGATGCGCGGCAACCGTCACTTCCGCGACGCAACAGATCGTTTCCGCGAAGAAGTTATCAGTAGCAGCAT

CTTCGACACACTGCGCCATGAACTGGGTCAAGAAGGTTTTGAGGCCCTGATCGCAAGCCGCGTTCATTGTGTG

ACCGGCGAAATTACAGCCCCGCGCTTCGGTTTAACCCGCACCGTGTTTACCGCCCTGGCCGATAAACTGGACG

TGGTGATCAACAGCGCAGCCAGCGTGAACTTTCGTGAAGAACTGGATCGCGCCCTGGAGATCAATACCTTCA

GTCTGGAGAACGTGGCCAAACTGTGTGAGCTGGGCGGTAATATCCCTCTGGTGCACGTGAGCACCTGCTACG

TTAACGGCTACCGTAGCGGCGACATTCACGAAACTAGTGAAGGCCCGGTGGGTTTAGAATTACCGGAAGGCC

CGGGCAGCTTCTATGAAACTAGTGCCCTGATTGAACGCTTACAGCGTCTGGTGGAGGAACTGCGCGGCCAGT

ATGAGGGTCGTGCCCTGAAAGCCAAGCTGATTGAACATGGCGCCAAGGAAGCACAGGCAGCCGGCTGGAAC

GACACCTATACCTTCACAAAGTGGCTGGGTGAGCAGTACCTGTACAAGGTGATGCGCGGCTATAGTCTGACC

GTTGTGCGTCCGAGCATTATCGAGAGCACACTGCGTGAACCGAGTCCTGGTTGGATCGAAGGTGTGAAGGTT

GCAGACGCCGTGCTGATGGCATATGCCCGCGGCAAAGTGGCATTTTTTCCGGGCAAGCGCGCCGGTGTGATT

GATATCATCCCGGCAGATCTGGTGGCCAATGGTGTTCTGCTGAGCACAGCAGAACAGCTGCTGGAACCGGGC

CAACATCGCATCTATCAGTGTTGCAGTGGCGGTCGCAACCCGCTGATGCTGGGCGATTTTATTGACCACCTGG

TGGCCGAAGCCAGCAGCCATCACGGCGACTACGACAAACTGTTTAAAGCACCGCCGCGTCGCCGTTTTACCGC

CGTTGACAAGCGTGTGTTTAGCAGCGTGGCAATGTGCGTTCAGGTGGCCCTGACCATTGTTGGTGCAATCCTG

CGTCGTCTGGGCCGTCGTGGTAAGCTGCGTGCCCGTCGTAACATTGACACCGCAGTGGAGCTGAGCAAAATC

TTCAGCTTTTACGCCCAGCCGAACTACTGCTTTCACGGTGACAAACTGCTGAGCCTGAGCAAAAGCATGGGTG

CCGTGGATCAAGAACTGTTTCCGGTGGACGCAGGTGTGATCGACTGGCAGCACTATATTCGCCACGTTCACAT

GCCGGGCCTGAACCATTACGCACTGCAGGGTCAGCCGAGTGTGCGTAGCCAGCAACGCCGCGAGGAAGTGA

AAACCGCCCCTAAGAATCTGACCGCCACCGAGGCATGA (SEQ ID !MO: 2)

In some embodiments, the nucleic acid is codon optimized for expression in a eukaryotic cell. In some embodiments, the nucleic acid is codon optimized for expression in a yeast cell, a fungal cell, an algal cell and a plant cell. In some embodiments, the nucleic acid is codon optimized for expression in Saccharomyces, Kluyveromyces, Candida, Pichia, Sckizosaccharomyces, Schefferomyces, Rhodosporidium, Hamenula, Klockem, Schwanniomyces, Issatchenkia, Yarrow ia or Rhodotorula. In some embodiments, the nucleic acid is codon optimized for expression in S. cerevisiae, C. lipolytica, R. glutinis, S. bulderi, S. barnetti, S. exiguus, S uvarum, S. diastaticus, K lactis, K. marxianus K. fragile, P. kudriavzevii , S. stipitis or I. oriental! s.

In some embodiments, the nucleic acid is codon optimized for expression in a filamentous fungal cell. In some embodiments, the nucleic acid is codon optimized for expression in Aspergillus, Pemcillium, Rhizopus, Chrysosporium, Myceliophthora, Trichoderma, Humicola, Acremonium or Fusarium In some embodiments, the nucleic acid is codon optimized for expression in A. niger A. oryzae, T. reesei, P. chrysogenum, M. thermophila, or R. oryzae.

In some embodiments, the nucleic acid is codon optimized for expression in Botryococcus, Nannochloropsis, Chlorella, Chlamydomonas, Dunaliella, Chaetoceros, Porphyridium, Scenedesmus or Pseudochlorococcum. In some embodiments, the nucleic acid is codon optim ized for expression in B. braunii or N. gaditana.

In some embodiments, the nucleic acid has at least 70% sequence identity to SEQ ID NO: 3. In some embodiments, the nucleic acid sequence has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% sequence identity to SEQ ID NO: 3, over a region of at least about 10, e.g, at least about 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1 100, 1 150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, or 1557 nucleotides, or a catalytic domain thereof.

SEQ ID NO: 3 represents the native S. marinus FAR nucleic acid sequence corresponding to the StnFAR polypeptide sequence (SEQ ID NO: 1):

ATGCGGGATCTGAATCGAGATAAGTCGTCGACGATAGCGGCCCTGACGGGTAAGCAGGTGCTGATTACCGGT

ACAACGGGGTTCTTGGGCAAGGTGTTGTTAGAGAAGGTTCTGCGCACGACGCCGGGCGTGGGCGGTATTTAC

TTGCTCATGCGGGGCAATCGCCATTTTCGTGATGCCACTGACCGCTTTCGGGAAGAAGTGATCTCCTCCTCCAT

TTTTGACACCTTGCGGCATGAACTGGGGCAGGAGGGCTTTGAGGCGCTGATTGCAAGTCGCGTTCACTGTGT

GACGGGGGAAATTACCGCACCGCGGTTTGGTCTGACGCGCACAGTCTTTACGGCGTTGGCCGACAAGCTCGA

TGTGGTGATCAATTCCGCAGCGAGTGTAAATTTTCGTGAGGAGTTGGATCGAGCGCTGGAAATCAATACGTTC

TCCCTGGAAAACGTCGCGAAACTCTGTGAGTTGGGGGGGAATATACCACTTGTTCACGTGTCCACCTGCTACG

TAAACGGCTACCGGTCGGGTGATATCCACGAGACGTCCGAGGGCCCTGTTGGGCTGGAGTTACCTGAGGGGC

CGGGTAGTTTCTACGAAACCTCGGCCTTGATTGAGCGCTTGCAGCGCCTTGTTGAGGAGTTGCGCGGGCAATA

CGAGGGACGGGCGCTGAAGGCCAAGCTCATAGAGCATGGCGCGAAGGAAGCGCAAGCTGCGGGCTGGAAC

GATACCTATACCTTCACCAAGTGGCTGGGTGAGCAATATTTGTACAAGGTGATGCGAGGCTACTCGCTGACGG

TTGTTCGTCCGTCGATCATCGAGAGCACACTGCGCGAGCCAAGCCCAGGGTGGATTGAGGGAGTCAAGGTCG

CCGACGCGGTGTTGATGGCATATGCGCGCGGTAAGGTTGCGTTCTTTCCCGGTAAGCGGGCCGGGGTGATCG

ATATTATTCCGGCCGATCTCGTGGCCAATGGGGTATTGCTCTCGACAGCCGAGCAGTTGCTCGAACCGGGCCA

GCATCGCATCTATCAATGTTGTAGCGGTGGCCGTAACCCACTAATGCTGGGAGACTTTATTGACCACTTGGTG

GCAGAAGCCAGCAGTCATCACGGTGACTACGACAAGCTTTTTAAGGCGCCGCCCCGCCGGCGATTCACGGCG

GTGGATAAGCGTGTATTCAGTAGCGTCGCAATGTGCGTTCAGGTGGCGCTGACGATTGTTGGCGCGATATTG

CGTCGTCTCGGACGGCGCGGCAAGCTTAGAGCGCGGCGAAATATCGACACGGCTGTCGAGCTCTCCAAAATA

TTCTCTTTCTATGCTCAGCCCAATTACTGCTTTCACGGCGACAAGCTGCTTTCATTATCGAAGTCGATGGGCGCT

GTCGATCAGGAGCTGTTCCCTGTGGATGCCGGCGTGATTGATTGGCAGCACTATATTCGTCACGTTCATATGC

CGGGATTAAACCACTATGCGCTGCAGGGGCAGCCCTCAGTAAGGTCACAGCAGCGGCGCGAGGAAGTGAAA

ACGGCGCCGAAAAACCTGACGGCGACCGAGGCTTGA (SEQ ID ! !O: 3) Vectors

A vector of the invention comprises a FAR nucleic acid of the invention. A vector may comprise one or more of an origin of replication, a promoter sequence operably linked to a nucleic acid of the invention and a reporter gene or selectable marker.

Vectors according to the invention include, but are not limited to, pCL-BP2, pBR322, pBR327, pACYC184, pACYC177, pSC!Oi, pCL1920, and pCL1921.

Vectors of the invention comprising an origin of replication include, but are not limited to, ColEl, pMBi, pl 5A, pSClOl, R6K, RK2 and pRSFlOlO.

The promoter may be homologous or heterologous. The promoter may be constitutive or inducible.

In one embodiment, the promoter is inducible and is activated in the presence of an inducing agent. Inducing agents include, but are not limited to, sugars, metal salts, and antibiotics. In some cases, the promoter allows constitutive expression of the FAR polypeptide.

In one embodiment, promoters that are active at different stages of growth can be used.

The promoter sequence may be operable in a prokaryotic cell, for example an E. coli cell. In one embodiment the promoter is a Trc promoter.

The promoter sequence may be operable in a eukaryotic cell, for example a yeast cell, a fungal cell, an algal cell or a plant cell.

Where the recombinant cell is a fungal cell, the promoter can be a fungal promoter (including, but not limited to, a filamentous fungal promoter), a promoter operable in plant cells, or a promoter operable in mammalian cells. Mammalian, mammalian viral, plant and plant viral promoters can drive particularly high expression when the associated 5’ UTR sequence (i.e., the sequence which begins at the transcription start site and ends one nucleotide before the start codon), normally associated with the mammalian or mammalian viral promoter is replaced by a fungal 5' UTR sequence. The source of the 5’ UTR can vary provided it is operable in the filamentous fungal cell in various embodiments, the 5’ UTR can be derived from a yeast gene or a filamentous fungal gene. The 5’ UTR can be from the same species as the recombinant cell or from a different species.

Promoters for recombinant expression in yeast are known in the art. Suitable promoters for S. cerevisiae include, but are not limited to, the MFal promoter, galactose inducible promoters such as GAL1, GAL7, and GAL 10 promoters, glycolytic enzyme promoters including tire TPI and PGK promoters, the TDH3 promoter, the TEF1 promoter, the TRP1 promoter, the CYCI promoter, the CUP! promoter, the PFI05 promoter, the ADFIl promoter, and the HDP promoter A suitable promoter in the genus Pichia sp. is the AOXI promoter. Suitable reporter genes or selectable markers include, but are not limited to, a drug resistance gene, a metabolic enzyme, a factor required for survival of the recombinant cell, a fluorescent marker, or an enzyme that generates a detectable product. Cells transformed with the vector can be selected based on their ability to grow in the presence of inhibitors (e.g. antibiotics) or under conditions in which untransformed cells cannot grow .

The vector may be a high copy number vector, an intermediate copy number vector, or a low copy number vector. Preferably, the vector is a low copy number vector, e.g. vector pCL-BP2.

In some embodiments, the vector comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 4. In some embodiments, the vector comprises a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 4. Vector corresponding to SEQ ID NO: 4 is also referred to herein as pCl-BP2.

CGCGCGCGAAGGCGAAGCGGCATGCATTTACGTTGACACCATCGAATGGTGCAAAACCTT

TCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACC

AGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTG

GTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGC

GGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGCAAACAGTCGTTGCT

GATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATT

AAATCTCGCGCCGATCAACTGGGTGCCAGCGTGGTGGTGTCGATGGTAGAACGAAGCGGC

GTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATC

ATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTC

CGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAA

GACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAATCGCGCTG

TTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAATATC

TCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCG

GTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGC

CAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGG

TGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCC

GTCAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCT

GCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAA

AAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTC

ATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAA

TTAATGTGAGTTAGCGCGAATTGATCTGGTTTGACAGCTTATCATCGACTGCACGGTGCAC

CAATGCTTCTGGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAAT

CACTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGA

CATCATAACGGTTCTOGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCCGGCTCG

TATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCGCCGCTGAGAA

AAAGCGAAGCGGCACTGCTCTTTAACAATTTATCAGACAATCTGTGTGGGCACTCGACCG

GAATTATCGATTAACTTTATTATTAAAAATTAAAGAGGTATATATTAATGTATCGATTAAA

TAAGGAGGAATAAACCATGGATCCGAGCTCGAGATCTGCAGCTGGTACCATATGGGAATT

CGAAGCTTACGTAGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCA

TCATCATCATCATCATTGAGTTTAAACCAAATAAAACGAAAGGCTCAGTCGAAAGACTGG

GAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCA

TAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTC

TACAAACTCTTTATGTGCTIAGTGCATCTAACGCTTGAGTTAAGCCGCGCCGCGAAGCGGC GTCGGCTTGAACGAATTGTTAGACATTATTTGCCGACTACCTTGGTGATCTCGCCTTTCAC

GTAGTGGACAAATTCTTCCAACTGATCTGCGCGCGAGGCCAAGCGATCTTCTTCTTGTCCA

AGATAAGCCTGTCTAGCTTCAAGTATGACGGGCTGATACTGGGCCGGCAGGCGCTCCATT

GCCCAGTCGGCAGCGACATCCTTCGGCGCGATTTTGCCGGTTACTGCGCTGTACCAAATGC

CKJGACAACGTAAGCACTACATTTCGCTCATCGCCAGCCCAGTCGGGCGGCGAGTTCCATA

GCGTTAAGGTTTCATTTAGCGCCTCAAATAGATCCTGTTCAGGAACCGGATCAAAGAGTTC

AGATCAATGTCGATCGTGGCTGGCTCGAAGATACCTGCAAGAATGTCATTGCGCTGCCATT

CTCCAAATTGCAGTTCGCGCTTAGCTGGATAACGCCACGGAATGATGTCGTCGTGCACAAC

AATGGTGACTTCTACAGCGCGGAGAATCTCGCTCTCTCCAGGGGAAGCCGAAGTTTCCAA

AAGGTCGTTGATCAAAGCTCGCCGCGTTGTTTCATCAAGCCTTACGGTCACCGTAACCAGC

AAATCAATATCACTGTGTGGCTTCAGGCCGCCATCCACTGCGGAGCCGTACAAATGTACG

GCCAGCAACGTCGGTTCGAGATGGCGCTCGATGACGCCAACTACCTCTGATAGTTGAGTC

CTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCGCTTGC

TGCTTGGATGCCCGAGGCATAGACTGTACCCCAAAAAAACAGTCATAACAAGCCATGAAA

ACCGCCACTGCGCCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGC

GCATACGCTACTTGCATTACAGCTTACGAACCGAACAGGCTTATGTCCACTGGGTTCGTGC

CTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGGGCAGCAGCGAAGTCGAGGC

ATTTCTGTCCTGGCTGGCGAACGAGCGCAAGGTTTCGGTCTCCACGCATCGTCAGGCATTG

GCGGCCTTGCTGTTCTTCTACGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGA

TCGGAAGACCTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTTC

GCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGCTTCTGTATGGAACGGG

CATGCGGATCAGTGAGGGTTTGCAACTGCGGGTCAAGGATCTGGATTTCGATCACGGCAC

GATCATCGTGCGGGAGGGCAAGGGCTCCAAGGATCGGGCCTTGATGTTACCCGAGAGCTT

GGCACCCAGCCTGCGCGAGCAGGGGAATTAATTCCCACGGGTTTTGCTGCCCGCAAACGG

GCTGTrCTGGTGTTGCTAGTTTGTTATCAGAATCGCAGATCCGGCTTCAGCCGGTTTGCCG

GCTGAAAGCGCTATrrCTTCCAGAATTGCCATGAITTITTCCCCACGGGAGGCGTCACTGG

CTCCCGTGTTGTCGGCAGC1TTGATTCGATAAGCAGCATCGCCTGTTTCAGGCTGTCTATGT

GTGACTGTTGAGCTGTAACAAGTTGTCTCAGGTGTTCAATTTCATGTTCTAGTTGCTTTGTT

TTACTGGTTTCACCTGTTCTATTAGGTGTTACATGCTGTTCATCTGTTACATTGTCGATCTG

TTCATGGTGAACAGCTTTGAATGCACCAAAAACTCGTAAAAGCTCTGATGTATCTATCTTT

TTTACACCGTnTCATCTGTGCATATGGACAGTTTTCCCTTTGATATGTAACGGTGAACAGT

TGTrCTACTnTGTTTGTTAGTCTTGATGCTTCACTGATAGATACAAGAGCCATAAGAACCT

CAGATCCTTCCGTATTTAGCCAGTATGTTCTCTAGTGTGGTTCGTTGTTTTTGCGTGAGCCA

TGAGAACGAACCATTGAGATCATACTTACTTTGCATGTCACTCAAAAATTTTGCCTCAAAA

GTAGGAATCTGATGTAATGGTTGTTGGTATTTTGTCACCATTCA nTTATCTGGTTGTTCT

CAAGTTCGGTTACGAGATCCATTTGTCTATCTAGTTCAACTTGGAAAATCAACGTATCAGT

CGGGCGGCCTCGCTTATCAACCACCAATTTCATATTGCTGTAAGTGTTTAAATCTTTACTTA

TTGGTTTCAAAACCCATTGGTTAAGCCTTTTAAACTCATGGTAGTTATTTTCAAGCATTAAC

CCAGATTATATTTTATGAATTTTTTTAACTGGAAAAGATAAGGCAATATCTCTTCACTAAA

AACTAATTCTAATnTTCGCTTGAGAACTTGGCATAGTTTGTCCACTGGAAAATCTCAAAG

CC iTAACCAAAGGATTCCTGATITCCACAGTTCTCGTCATCAGCTCTCTGGTTGCTITAGC

TAATACACCATAAGCATTTTCCCTACTGATGTTCATCATCTGAGCGTATTGGTTATAAGTG

AACGATACCGTCCGTTCTTTCCTTGTAGGGTTTTCAATCGTGGGGTTGAGTAGTGCCACAC

AGCATAAAATTAGCTrGGTTTCATGCTCCGTTAAGTCATAGCGACTAATCGCTAGTTCATT

TGCnTGAAAACAACTAATTCAGACATACATCTCAATTGGTCTAGGTGATTTTAATCACTA

TACCAATTGAGATGGGCTAGTCAATGATAATTACTAGTCCTTTTCCnTGAGTTGTGGGTA

TCTGTAAATTCTGCTAGACCnTGCTGGAAAACTTGTAAATTCTGCTAGACCCTCTGTAAA

TTCCGCTAGACCTTTGTGTGTTTITTTTGTTTATATTCAAGTCKJTTATAATTTATAGAATAA

AGAAAGAATAAAAAAAGATAAAAAGAATAGATCCCAGCCCTGTGTATAACTCACTACTTT AGTCAGTTCCGCAGTATTACAAAAGGATGTCGCAAACGCTGTTTGCTCCTCTACAAAACAG

ACCTTAAAACCCTAAAGGCTTAAGTAGCACCCTCGCAAGCTCGGGCAAATCGCTGAATAT

TCCTTTTGTCTCCGACCATCAGGCACCTGAGTGGCTGTCTTTTTCGTGACATTCAGTTCGCT

GCGCTCACGGCTCTGGCAGTGAATGGGGGTAAATGGCACTACAGGCGCCmTATGGATT

CATGCAAGGAAACTACCCATAATACAAGAAAAGCCCGTCACGGGCTTCTCAGGGCGTTTT

ATCKJCGGGTCTGCTATGTGGTGCTATCTGACTTTTTGCTGITCAGCAGTTCCTGCCCTCTGA

TTTTCCAGTCTGACCACTTCGGATTATCCCGTGACAGGTCATTCAGACTGGCTAATGCACC

CAGTAAGGCAGCGGTATCATGAACAGGCTTACCCGTCTTACTGT (SEQ ID NO: 4)

Recombinant cells

The invention provides recombinant ceils engineered to express a polypeptide of the invention. The invention also provides recombinant cells transformed with a nucleic acid of the invention. The nucleic acid may be extrachromosomal, on a vector (typically a plasmid), which can be a low copy number vector, an intermediate copy number vector, or a high copy number vector. In some embodiments, the recombinant cell is transformed with a vector of the invention. In some embodiments, the recombinant cell is transiently transformed. The nucleic acid may be maintained episoma!ly and thus comprise a sequence for autonomous replication, such as an autosomal replication sequence. Alternatively, the recombinant cell may be stably transformed wherein the nucleic acid is integrated in one or more copies into the genome of the cell. Integration into the cell’s genome may occur at random by non-homologous recombination but preferably, the nucleic acid construct may be integrated into the cell’s genome by homologous recombination, as is well known the art.

In some embodiments, the recombinant cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is selected from Acinetobacter , Agrobacterium , Escherichia, Cupriavidus, Clostridium, Rhodobacter, Marmobacter, Bacillus, Klebsiella, Tatumella, Pseudomonas, Ralstoma, Rhodococcus, Methylobacterium, Methylophilus, Methylococcus, Methylomicrobium, Methylomonas, Pantoea, Streptomyces, Parachlorella, Syne choc occus, Synechocystis and Thermocynechococcus. In some embodiments, the prokaryotic cell is E. coli. Suitable cells of the bacterial genera include, but are not limited to, cells of Lactobacillus, Pseudomonas, and Streptomyces . Suitable cells of bacterial species include, but are not limited to, cells of Bacillus subtilis, Bacillus licheniformis, Lactobacillus brevis. Pseudomonas aeruginosa, and Streptomyces lividans. Typically, the recombinant cell is not Spongibacter marinus .

In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is selected from the group consisting of a yeast cell, a fungal cell, an algal cell and a plant cell. In some embodiments, the yeast cell is selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharornyces, Schefferomyces, Rhodosporidium, Hansenula, Klockera, Schwanniomyces, Issatchenkia, Yarrowia and Rhodotorula. In some embodiments, the yeast ceil is selected from the group consisting of S. cerevisiae, C. lipolytica, R. glutinis, S. bulderi, S. bametti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus, K. fragile, P. kudriavzevii, S. stipitis and I. orientalis. Suitable cells of yeast include, but are not limited to C. albicans, S. pombe, H. polymorpha, P. pastoris, P. canadensis, or P. rhodozyma.

In some embodiments, the fungal cell is a filamentous fungal cell. In some embodiments, the filamentous fungal cell is selected from the group consisting of Aspergillus, Penicillium, Rhizopus Chrysosporium, Myceliophthora, Trichoderma, Humicola, Acremonium and Fusarium. In some embodiments, the filamentous fungal cell is selected from the group consisting of A. niger , A. oryzae T. reesei. P. chrysogenum. M. thermophila, and R. oryzae. Suitable cells of filamentous fungal genera include, but are not limited to, cells of Aureobasidium, Bjerkandera, Ceriporiopsis, Coprinus, Coriolus, Corynascus, Chaetomium, Cryptococcus, Filobasidium, Gibberella, Hypocrea, Magnaporthe, Mucor, Neocallimastix, Neurospora, Paecilomyces, Phanerochaete, Phlebia, Piromyces, Pleurotus, Scytaldium, Schizopkyllum, Sporotnchum, Talaromyces, Thermoascus, Thielavia, tolypocladium and Trametes. In certain aspects, the recombinant cell is a Trichoderma sp. Penicillium sp., Humicola sp. (e.g., Humicola insolens); Aspergillus sp., Chrysosporium sp., Fusarium sp., or Hypocrea sp.. Suitable cells can also include cells of various anamotph and teleomorph forms of these filamentous fungal genera.

Suitable cells of filamentous fungal species include, but are not limited to, cells of Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger Aspergillus oryzae Chrysosporium lucknowense, Fusarium bactridioides, Fusarium cerealis Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium grammum, Fusariu heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides Fusarium sulphureum Fusarium torulosum Fusarium irichothecioides, Fusarium venenatum, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Neurospora intermedia, Penicillium purpurogenum, Penicillium canescens, Penicillium solitum, Penicillium fimiculosum, Phanerochaete chrysosporium, Phlebia radiate, Pleurotus eryngii, Talaromyces flavus, Thielavia terrestns, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii Trichoderma longibrachiatum Trichoderma reesei, and Trichoderma viride.

In some embodiments, the algal cell is selected from the group consisting of Botryococcus, Nannochloropsis, Chlorella, Chlamydomonas, Dunaliella, Chaetoceros, Porphyridium, Scenedesmus and Pseudochlorococcum. In some embodiments, the algal cell is selected from the group consisting of B. braunii and N. gaditana. Recombinant cells may be cultured in conventional nutrient media modified as appropriate for activating promoters (if an inducible promoter is present), selecting transformants, or amplifying the nucleic acid sequence encoding the FAR polypeptide. Culture conditions, such as temperature, pH and the like, are those previously used with the recombinant ceil selected for expression, and will he apparent to those skilled in the art. Many references are available for the culture and production of many cells, including cells of bacterial and fungal origin. Cell culture media in general are set forth in Atlas and Parks (eds.), 1993, Hie Handbook of Microbiological Media, CRC Press, Boca Raton, FL, which is incorporated herein by reference. Preferred culture conditions for a given recombinant cell may be found in the scientific literature and/or from the source of the recombinant cell such as the American Type Culture Collection (ATCC).

In cases where a FAR coding sequence is under the control of an inducible promoter, the inducing agent, e.g., a sugar, metal salt or antibiotic, may be added to the medium at a concentration effective to induce expression of FAR polypeptide.

A recombinant cell of the invention may compri se one or more genetic modifications to increase the yield and/or recovery of fatty alcohol. Such genetic modifications may result in at least one, any two, any three, any four or all five of the following phenotypes: (a) an increase in fatty acyl-ACP production; (b) a decrease in fatty acyl-ACP degradation; (c) an upregulation of fatty alcohol secretion; (d) an increase in flux through the fatty acid biosynthetic pathway; and (e) an increase in tolerance to fatty alcohol

(a) Increase in fatty acyl-ACP production

The yield of fatty alcohols may be improved by increasing the production of the SmFAR substrate, fatty acyl-ACP. Fatty acyl-ACP production may be increased by various modifications of die fatty acid biosynthetic (FAB) pathway in which fatty acyl-ACP is an intermediate. Hus may include overexpression of endogenous genes and/or expression of exogenous genes from different organisms. Methods for increasing fatly_' acyl-ACP production are known in the art (see for example, US 8,110,670, incorporated herein by reference). For example, increased fatty acyl-ACP production may be achieved in bacteria by overexpression of rnalonyl-CoA-ACP transacylase (fabD), b-ketoacyl- ACP synthase III (fabH), b-ketoacyl-ACP synthases (fabB and fabF), b-ketoacyi-ACP reductase (fabG), 3-hydrox-acyl-ACP dehydratase (fabA) and/or enoyl-ACP reductase (fabl).

(b) Decrease in acyl-ACP degradation

The yield of fatty alcohols may be improved by decreasing the degradation of the SmFAR substrate, fatty acyl-ACP. Methods to increase fatty alcohol yield by reducing acyl-ACP degradation are known in the art (see for example, Liu, Chen el al. 2016, incorporated herein by reference). In the FAB pathway, fatty acyl-ACP is converted to fatty acids by thioesterases. Reduced degradation of fatty acyi-ACP may therefore be achieved in bacteria by, for example, downregulation or deletion of thioesterases.

(c) Upregulation of fatty alcohol secretion

Increasing the recover}' of fatty alcohols may be achieved by increasing the rate of fatty alcohol secretion from the recombinant cell. Methods for increasing the secretion of fatty alcohols by microorganisms are known in the art, e.g. by overexpression of efflux pumps (see for example, Zhang et al. Biochem. Eng J. 133: 149-156 (2018), incorporated herein by reference). For example, prokaryotic multidrug resistance (MDR) transporters can export a wide variety of substrates, including fatty alcohols, and so recombinant upregulation of these transporters may lead to increased fatty alcohol secretion. Examples of MDR transporters in E. coli include MdtJ, Bcr and MdtH

(d) Increase in flux through the fatty acid biosynthetic pathway

The main precursors for the fatty acid biosynthetic pathway are malonyl-CoA and acetyl-CoA. Increasing the availability of these precursors may increase the rate of fatty acyl -A CP generation and therefore increase flux through the FAB pathway. Methods of increasing flux through the FAB pathway are known in the art (see for example, W02013/152051, incorporated herein by reference). In bacteria, overexpression of acetyl-CoA carboxylase (accA, accB, accC, and accD), which catalyzes the conversion of acetyl-CoA to malonyl-CoA, may increase the yield of fatty acyl-ACPs.

(e) Increase in tolerance to fatty alcohol

High levels of intracellular fatty alcohols could potentially lead to toxic effects in recombinant cells. If required, increased tolerance to fatty alcohols may be achieved by upregulating secretion of fatty alcohols from the recombinant ceil (see point (c) above).

The foregoing phenotypes may be introduced singly or in combinations of two, three, four, five or more modifications. One or more of the foregoing phenotypes may be obtained by increasing or decreasing expression of an endogenous protein (e.g., by at least a factor of about 1.1, about 1.2, about 1.5, about 2, about 5, about 10 or about 20) or a result of introducing expression of a heterologous polypeptide. For avoidance of doubt, “decreasing” or“reducing” gene expression encompasses eliminating expression. Decreasing (or reducing) the expression of an endogenous protein may be accomplished by inactivating one or more (or all) endogenous copies of a gene in a cell. A gene may be inactivated by deletion of at least part of the gene or by disruption of the gene e.g. by deleting some or all of a gene coding sequence or regulatory sequence whose deletion results in a reduction of gene expression in the cell Fatty alcohol production

The invention provides a method of producing a fatty alcohol composition comprising: a) culturing a recombinant cell in a suitable culture medium, wherein the recombinant cell comprises a gene encoding a heterologous FAR enzyme having at least 70% sequence identity to SEQ ID NO: 1; and b) allowing expression of said gene, wherein said expression results in the production of a fatty alcohol composition.

Recombinant cells of the invention may be cultured under suitable conditions in a liquid or solid medium. In some embodiments, recombinant cells of the invention undergo fermentation. Fermentation conditions include batch, fed-batch and continuous fermentation. Classical batch fermentation is a closed system, wherein the composition of the medium is not subject to artificial alterations during fermentation. In fed-batch fermentation, the substrate is added in increments as fermentation progresses. In both classical batch fermentation and batch-fed fermentation, the product(s) remain in the bioreactor until the end of the process. Batch and fed-batch fermentation are common and w ell -know n in the art. In continuous fermentation, a defined medium is added continuously to the bioreactor and an equal volume of product containing medium is removed simultaneously. Continuous fermentation aims to maintain steady state growth conditions. Methods for modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology. The fermentation process may be an aerobic or an anaerobic fermentation process.

The fermentation process is typically run at a temperature that is optimal for growth of the recombinant FAR-expressing cell. Fermentation is typically carried out at a temperature within the range of from about 10°C to about 70°C, from about 15°C to about 60°C, from about 20°C to about 50°C, or from about 25°C to about 40°C. Fermentation is typically carried out for a period of time w ithin the range of from about 8 to 240 hours, from about 12 hours to about 168 hours, from about 16 hours to about 144 hours, from about 20 hours to about 120 hours, or from about 24 hours to about 72 hours. Fermentation is typically carried out at a pH in the range of 4 to 8, in the range of 5 to 7, or the range of 5.5 to 6.5.

The method of the invention may comprise purifying fatty alcohols from cell culture to provide a purified faty alcohol composition.

The method of the invention may comprise purifying fatty alcohols from culture supernatant to provide a purified fatty alcohol composition.

The method of the invention may comprise harvesting and lysing recombinant cells to release intracellular fatty alcohols from the recombinant cell. Hie method of the invention may further comprise purifying the fatty alcohol composition from the lysate to provide a purified fatty alcohol composition.

The invention also provides a cell-free method for producing a fatty alcohol composition comprising incubating a FAR polypeptide of the invention with acyl-ACP and a reductant. In some embodiments, the reductant is NADPH. Typically a cell-free method of the invention involves incubating the FAR polypeptide of the invention under suitable conditions of temperature, pH, and ionic strength with a suitable substrate, e.g. acyl-ACP, and a suitable reductant, e.g. NAD(P)H. A cell-free method of the invention may comprise use of an immobilized FAR polypeptide of the invention. In one embodiment, an immobilized FAR polypeptide of the invention is immobilized on an inert, insoluble substrate. An immobilized FAR polypeptide may have greater stability' compared with a soluble form and may be more readily recovered from reaction mixtures. A cell-free method of the invention may further comprise purifying fatty alcohols from the cell-free reaction mixture to provide a purified fatty alcohol composition.

Wherein the faty alcohol composition was produced in a recombinant cell of the invention, puri fying the faty alcohol composition typically comprises separating fatty alcohols from components of the culture medium, fermentation process, and/or cellular material. Wherein the fatty alcohol composition was produced in a cell-free method of the invention, purifying the fatty alcohol composition typically comprises separating faty alcohols from the cell-free reaction mixture.

Fatty_' alcohol compositions may be purified using conventional methods known in the art. For example, fatty' alcohols may be purified by solvent extraction with a water immiscible solvent. In some embodiments, purification of fatty alcohols occurs contemporaneously with biosynthesis of fatty alcohols.

Fatty alcohol composition

Fatty alcohol compositions may be characterized using various analytical methods known in the art. Fatty alcohol characteristics described herein, such as % fatty alcohol distribution and % saturation, are typically determined using gas chromatography-mass spectrometry' (GCMS). An exemplary GCMS method is provided herein in Example 3

Fatty alcohol compositions produced by a method of the invention typically comprise 02 fatty alcohol. Fatty alcohol compositions produced by a method of the invention typically comprise saturated 02 fatty alcohol (i.e. “02:0” faty alcohol). In one embodiment, the fatty alcohol composition comprises at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of 02 fatty alcohol. In one embodiment, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the fatty alcohols in the fatty alcohol composition are saturated fatty alcohols. In one embodiment, at least 45% of the fatty alcohols in the fatty alcohol composition are saturated fatty alcohols.

FARs of the invention provide faty alcohol compositions with highly desirable characteristics (discussed herein). In some embodiments, modifieation(s) are introduced to the recombinant cell that alter the characteristics of fatty alcohol compositions produced by FARs of the invention. For example, the recombinant cell may be modified to change the proportion of saturation of the faty alcohol and/or modified to change the chain -length of faty acyl-ACPs produced by the FAB pathway.

The FAB pathway produces a range of saturated and unsaturated fatty acyl-ACPs which can be used as substrates by a FAR enzyme. Modifying the proportion of saturated and unsaturated fatty acyl-ACPs produced by the recombinant cell may allow the degree of saturation in the fatty alcohol composition to be altered. Methods to increase the proportion of saturated fatty acyl-ACPs produced by the FAB pathway are known in the art (see for example Heath and Rock, JBC, 271:27795-27801 (1996), incorporated herein by reference). As disclosed in WO 2013/096092 and US 9260727, in E. coli, overexpression of b-hydroxyacyl-ACP dehydratase enzymes FabA or FabZ, and/or enoyl-ACP reductase enzyme Fabl, increased the level of intermediates in the saturated branch of the FAB pathway and thereby increase the degree of saturated fatty acyl-ACPs produced.

The FAB pathway produces faty acyl-ACPs of varying chain lengths, with C14-C18 faty acyl-ACPs typically being the most abundant. The FAB pathway may be engineered to increase the proportion of shorter chain fatty acyl-ACPs produced thereby increasing the production of shorter chain fatty alcohols. Methods to alter the chain-length of fatty' acyl-ACPs produced by the FAB pathway are known in the art (see for example, Torella et al. PNAS, 110(28): 11290-11295 (2013), incorporated herein by reference). In E. coli, an increase in shorter chain faty acyl-ACP production may be achieved by altering the chain length specificity of FAB enzymes involved in chain length elongation (e.g. FabG, FabB or FabF).

In one embodiment, a method of the invention further comprises reducing the fatty alcohol composition to produce alkanes. Methods of reducing faty alcohols are known in the art. In one embodiment, reduction of faty alcohols is chemically -mediated. In one embodiment, reduction of fatty alcohols is biologically-mediated e.g. by a microorganism. In one embodiment, alkanes of the invention are purified from the faty alcohol composition from which they are derived.

In one embodiment, a method of the invention further comprises esterifying the fatty alcohol composition to produce esters. Methods of esterifying fatty alcohols are known in the art. In one embodiment, the fatty alcohols are reacted with a carboxylic acid to form esters. In one embodiment, esterification is carried out in the presence of a strong catalyst, e.g. sodium hydroxide. In one embodiment, esterification is catalyzed by an enzyme that catalyzes the conversion of fatty alcohols to esters. In one embodiment, esters are purified from the fatty alcohol composition from w'hich they are derived. In one embodiment, purified esters of the invention are used as biodiesel fuel.

In one embodiment, the method of the invention further comprises processing fatty alcohol compositions, alkane compositions and/or ester compositions to provide a fuel composition.

In one embodiment, the method of the invention further comprises processing fatty alcohol compositions, alkane compositions and/or ester compositions to provide a cleaning composition (e.g. detergent).

In one embodiment, the method of the invention further comprises processing fatty alcohol compositions, alkane compositions and/or ester compositions to provide an industrial composition (e.g. lubricant).

In one embodiment, the method of the invention further comprises processing fatty' alcohol compositions, alkane compositions and/or ester compositions to provide a cosmetic composition e.g. shampoo or soap.

The invention also provides fuel compositions, cleaning compositions (e.g. detergents), industrial compositions (e.g. lubricants), and cosmetic compositions (e.g. shampoos, soaps etc.) comprising or produced from fatty alcohol compositions, alkane compositions and/or ester compositions produced by the methods disclosed herein.

Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference in their entireties.

The following examples arc provided for the purpose of illustrating, not limiting, the material disclosed herein.

EXAMPLE 1

In the hope of identifying novel FAR enzymes, phylogenetic trees were constructed based on BLAST searches utilizing the known active FARs from M. aquaeolei, M. algicola and Oceanobacter sp. RED65 protein sequences. Based on these trees, a set of 24 protein sequences, distantly related to these 3 FARs were selected (see Table 1). FAR homologs were synthesized with codon optimized sequences for recombinant expression in E. coli by Genewiz (South Plainfield, NJ). The synthesized genes were cloned into vector pTrcHis2A (Invitrogen Inc. Carlsbad, CA) by Genewiz with the addition of restriction sites Ncol and Hindlll. Table 1. Identity of FARs selected for analysis.

EXAMPLE 2

Cloning of FAR into expression vector

To study FAR expression in vivo, the 24 FAR synthetic genes were cloned into the low-copy expression vector pCL-BP2 (see Figure 2). This vector was assembled from 4 synthetic DNA fragments (IDT, Caralville, lA), and is based on vector pCL1920 (Lerner and Inouye 1990), with some modifications, including the Trc promoter and optimized translation and termination sequences. The pCL-BP2 DNA sequence is shown in SEQ ID NO: 4. FAR genes were amplified by PCR using primers shown in Table 2 PCR products were cloned into vector pCL-BP2 using the In-Fusion cloning kit (Takara Bio) per manufacturer’s protocol using linearized pCL-BP2 by restriction enzymes Ncol and Hindlll. All DNA constructs were transformed into TOP 10 E. colt strain and sequence verified.

Table 2. PCR primer sequences used to amplify FAR for cloning into pCL-BP2

EXAMPLE 3

Evaluation of faty alcohol production

Recombinant TOP 10 E. colt strains expressing FAR were grown overnight at 30°C in SOB medium containing 100 ug/mL spectinomycin. Overnight cultures were used to inoculate 50 mL SOB medium with 100 ug/mL spectinomycin to a starting OD600 of 0.1. Cultures were incubated at 30°C shaking at 200 RPM until OD600 reached between 0.6 - 0.8, FAR expression was induced with isopropyl-b- D-thiogalaetoside (IPTG) to a final concentration of 1 mM Induced cultures were incubated under the same condition for about ~20 hours (overnight). 2 x 10 mL cultures were centrifuged at 4000 RPM, 24°C for 10 min. Supernatant and pellet were stored at -20°C or -80°C, respectively.

Fatty alcohols were extracted from both supernatant and pellets separately. Cell pellets were allowed to thaw at room temperature, followed by re-suspension in 0.5 mL heptane, vortexed for 30 seconds, incubated at 35°C for 1 min and vortexed again for 30 seconds. Extracts were centrifuged at 4000 RPM, i0°C for 5 min and the organic layers were analyzed by GC-MS. Supernatants were thawed at room temperature and vortexed to mix well. 1 mL supernatant was transferred to a different extraction vial, 2 mL of heptane were added and vortexed for 1 min at room temperature. The vials caps were carefully opened to allow any gas to vent out, before vortexing again for 1 min. Vials were centrifuged at 4000 RPM, 10°C for 1 min, and 1 mL of the organic heptane layers were transferred into clean vials. Samples were blow-dried with nitrogen and re-dissolved in 0.5 mL of heptane for GC-MS analysis.

2 mί samples were analyzed by GC-MS (Inlet: splitless or split ratio 1:2) on an HP-lnnow'ax column (30 m, 0.25 mm, 0.25 pm, Agilent P/N: 1909N-133). GC method started at 60°C (hold for 2 min), increased the temperature with a rate of 10°C/min to 250°C and held for 15 min with total flow, 41 mL/min with 0.5 min delay. Quantitative analysis was performed using a calibration curve made from reference fatty alcohol standards: C8:0-OH, C10:0-OH, C12:0-OH, C14:0-QH, C15:0-OH, 06:0- GH, C16: l-OH, C18:0~QH, C!8: l-OH, C18:2-OH, C18:3-OH. Calibration curve ranges were 0.5 mg/L to 100 mg/L with LOD: 0.1 mg/L and LOQ: 0.2 mg/L. Mass spec parameters set-up consisted of solvent delay at 3.5 min; mass scan range between 35 to 800 mu; collision energy at 70v. Compounds were identified by comparing observed spectral data with the NIST library. Agilent MassHunter software was used for quantitative analysis by calculating concentrations off of an established standard curve.

Evaluations of 24 FAR genes expressed in E. coli indicated that not all FAR enzymes were functional in E. coli. Surprisingly, of the 24 FAR genes expressed in E. coli, only 12 produced fatty alcohols (Figure 3). Analysis of the data showed that under the conditions used, FAR homologs that were more closely related to M. aquaeolei, M. algicola and Oceanobacter sp. RED65 protein sequences did not always produce fatty alcohols. Unexpectedly, this indicates that sequence homology to previously reported active FARs is not a good indicator when seeking to identify novel active FARs.

Based on level of fatty alcohols produced, some FAR enzymes were more active than others. Of the 12 tested, only the FARs Spongiibacter marinus (strain 16), Aestuariibacter salexigens (strain 1) and Zhongshania aliphaticivorans (strain 17), produced fatty alcohols to a level high enough to make them attractive for applied purposes. As shown in Figure 3, SmFAR (strain 16) markedly outperformed other FARs in the total level of fatty alcohols produced.

EXAMPLE 4

Production of fatty alcohols in E. coli strain W3110K

Recombinant W3110K strains expressing a gene encoding a FAR enzyme from either Spongiibacter marinus , Aestuariibacter salexigens or Zhongshania aliphaticivorans were grown overnight at 30 C in SOB medium containing 100 ug/mL spectinomycin. Overnight cultures were used to inoculate 50 niL LB medium with 1% glucose and 100 ug/mL spectinomycin to a starting ODsoo of 0.1. Cultures were incubated at 30^°C shaking at 200 RPM until ODsoo reached between 0.6 - 0.8, FAR expression was induced with isopropyl-P'-D-thiogalactoside (IPTG) to a final concentration of 1 niM. Induced cultures were incubated under the same conditions for about ~20 hours (overnight). 10 mL cultures were collected for fatty alcohol analysis. Extraction and quantification of fatty alcohols were performed as described in Example 3, except 5 ml of heptane was used for extraction.

Comparison of fatty alcohol production in SmFAR with MaFAR

US 8,216,815 describes characteristics of fatty alcohol compositions produced by FAR obtained from Marinobactoer algicola (denoted “MaFAR”). Table 3 provides a comparison between the fatty alcohol composition described in US 8,216,815 and the fatty alcohol composition produced using a FAR of the present invention. Table 3. Comparison between fatty alcohol compositions produced by SmFAR and MaFAR

ND = not detected

As set out in Table 3, under the conditions described in US 8,216,815, the fatty alcohol composition produced by MaFAR contained 8% C14 faty alcohol, 60% C16 fatty alcohol (30% 06:0, 30% 06: 1), and there was no detection of <04 faty alcohol. On the contrary, SmFAR yielded a faty alcohol composition containing 31% 04 fatty alcohol, 61 % 06 fatty alcohol (13% 06:0, 48% 06: 1), and, desirably, a detectable amount of 02 faty alcohol. Furthermore, the distribution of saturated vs. unsaturated fatty alcohol was different between MaFAR and SmFAR. Advantageously, total saturated fatty alcohol produced using SmFAR was -45%, whereas MaFAR achieved a total saturation level of -38%.

Distribution of faty alcohols produced in SmFAR. ZaFAR and AsFAR

As shown in Table 4, the recombinant E. coli strains produced between 228.5 mg/L to 930.3 rng/L of fatty alcohols. The fatty alcohols produced by the recombinant cells had varying levels of distribution between chain lengths. Analysis of the type of fatty alcohols produced by each of the FARs indicate that Cl 4:0 and Cl 6:0 fatty alcohols are produced at levels greater than 45% of total fatty alcohols produced.

Table 4. distribution of fatty alcohols produced by E. coli W31 10K overexpressing a FAR gene from S. marinus (SmFAR), Z aliphaticivorans (ZaFAR), or A. salexigens (AsFAR).

In conclusion, the FAR discovered in this work advantageously makes possible the direct production of fatty alcohols, without producing undesirable intermediates. In particular the S. marinus FAR produced a high proportion of 04:0 fatty alcohol, also known as Myristyl alcohol. This alcohol has numerous commercial applications, and is currently obtained from coconut and palm kernel oils. The FAR of the invention also produced highly desirable C12 faty alcohol winch, to the best of tire Inventors’ knowledge, has not been achieved by FARs in the literature. Moreover, the FAR of the invention provided a high yield of fatty alcohols, and which contain a high proportion of saturated fatty alcohols.

The FAR polypeptide of the invention is of considerable value for the production of faty alcohols. Moreover, as the FAR polypeptide is already capable of producing shorter chain faty alcohols (namely C l 2) than previously reported FARs, the Inventors believe that less strain engineering might be required (in comparison to strains expressing other FARs), when seeking to obtain even shorter chain fatty alcohols.

While illustrative embodiments have been illustrated and described herein, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the contents described herein.

REFERENCES

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Doan, T. T. P., A. S Carls son, M. Hamberg, L. Billow, S. Stymne and P. Olsson (2009) "Functional expression of five Arabidopsis fatty acyl-CoA reductase genes in Escherichia coli." Journal of Plant Physiology 166(8): 787-796.

Hofvander, P., T. T. P. Doan and M. Hamberg (2011). "A prokaryotic acyl -Co A reductase performing reduction of fatty acyl-CoA to fatty alcohol." FEES Letters 585(22): 3538-3543.

Lennen, R. M., M. A. Kruziki, K. Kumar, R. A. Zinkel, K. E. Burnurn, M S. Lipton, S W Hoover, D. R. Ranatunga, T. M. Wittkopp, W. D. Marner and B. F. Pfleger (2011).

"Membrane Stresses Induced by Overproduction of Free Fatty Acids in Escherichia coll.” Applied and Environmental Microbiology 77(22): 8114-8128.

Lerner, C. G. and M. Inouye (1990). "Low copy number plasmids for regulated low-level expression of cloned genes in Escherichia coli with blue/white insert screening capability." Nucleic acids research 18(15): 4631-4631

Liu, Y , S. Chen, J. Chen, J Zhou, Y Wang, M. Yang, X Qi, J. Xing, Q Wang and Y. Ma (2016) "High production of fatty alcohols in Escherichia coli with fatty acid starvation." Microbial Cell Factories 15(1): 129.

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Reiser, S. and C. Somerville (1997) "Isolation of mutants of Acinetobacter calcoaceticus deficient in wax ester synthesis and complementation of one mutation with a gene encoding a fatty acyl coenzyme A reductase " Journal of Bacteriology 179(9): 2969-2975.

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Catalyzing the Reduction of Fatty Acyl -Co A to Fatty Alcohol." Biochemistry 50(48): 10550- 10558.

Zheng, U.-N , L.-L. Li, Q. Liu, J.-M. Yang, X.-W. Wang, W. Liu, X. Xu, i s. Liu, G. Zhao and M. Xian (2012) "Optimization of fatty alcohol biosynthesis pathway for selectively enhanced production of C12/14 and 06/18 fatty alcohols in engineered Escherichia cofi." Microbial Cell Factories 11(1): 65.

Claims

1. A recombinant polypeptide comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1.

2. An isolated nucleic acid which encodes the polypeptide according to claim 1.

3. The nucleic acid according to claim 2, wherein the nucleic acid is codon optimized for expression in a prokaryotic cell.

4. The nucleic acid according to claim 3, wherein the prokaryotic cell is selected from the group consisting of Acinetobacter, Agrobacterium, Escherichia , Cupriavidus, Clostridium, Rhodobacter, Marinobacter, Bacillus, Klebsiella, Tatumella, Pseudomonas, Ralstonia, Rhodococcus,

Meihylobacterium, Meihylophilus, Methyiococcus, Methylomicrobium, Melhylomonas, Pantoea, Streptomyces, Parachlorella, Synechococcus, Synechocysiis and Therrnocynechococcus .

5. The nucleic acid according to claim 4, wherein the prokaryotic cell is E. coli.

6. The nucleic acid according to claim 5 comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 2.

7. The nucleic acid according to claim 2, wherein the nucleic acid sequence is codon optimized for expression in a eukaryotic cell.

8. The nucleic acid according to claim 7, wherein the eukaryotic cell is selected from the group consisting of a yeast cell, a fungal cell, an algal cell and a plant cell.

9. The nucleic acid according to claim 8, wherein the yeast cell is selected from the group consisting of Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Klockera, Schwanniomyces, Sche fferomyces, Rhodosporidium, Jssatchenkia, Yarrawia and

Rhodotonda.

10. The nucleic acid according to claim 9, wherein the yeast cell is selected from the group consisting of S. cerevisiae, C. lipolytica, R gluiinis, S. bulderi, S. harnetti, S. exiguus, S. uvarum, S. diastaticus, K lactis, K. marxianms K. fragile, P. kudriavzevii, S. stipitis, and / orientalis.

11. The nucleic acid according to claim 8, wherein the fungal cell is a filamentous fungal cell.

12. The nucleic acid according to claim 11, wherein the filamentous fungal cell is selected from the group consisting of Aspergillus, Pemcillium, Rhisopus, Chrysosporium, Afyceliophrfiora, Trichoderma, Humicoia, Acremomtm and Fusarium.

13. The nucleic acid according to claim 12, wherein the filamentous fungal cell is selected from the group consisting of A. niger, A. oryzae. T reesei, P. chrysogenum, M. thermophila, and R. oryzae.

14. The nucleic acid according to claim 8, wherein the algal cell is selected from the group comprising Botryococcus. Nannochloropsis. Chlorella , Chlamydomonas, Dunaliella, Chaetoceros, Porphyridium, Scenedesmus and Pseudochlorococcum.

15. The nucleic acid according to claim 14, wherein the algal cell is selected from the group comprising B. braunii and N gadiiana.

16. The nucleic acid according to claim 2 comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 3

17. A vector comprising the nucleic acid according to any one of claims 2 to 16.

18. The vector according to claim 17 comprising:

(a) an origin of replication:

(b) a promoter sequence operably linked to said nucleic acid; and/or

(c) a reporter gene.

19. The vector according to claim 17 or claim 18 comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 4.

20. A recombinant cell engineered to express the polypeptide according to claim 1.

21. The recombinant cell according to claim 20, wherein the recombinant cell is a prokaryotic cell .

22. The recombinant cell according to claim 21 , wherein die prokaryotic cell is selected from the group consisting of Acinetobacter, Agrobacterium , Escherichia , Cupriavidus, Clostridium. Rhodohacter, Marinohacter, Bacillus. Klebsiella. Tatumella, Pseudomonas. Ralstonia, Rhodococcus, Methylobacierium, Methylophilus, Methylococcus, Meihylomicrobiurn, Methylomonas, Panioea, Streptomyces, Parachlorella, Synechococcus, Synechocystis, and Thermocynechococcus.

23. Tire recombinant cell according to claim 22, wherein the prokaryotic cell is E. coli.

24. The recombinant cell according to claim 20, wherein the recombinant cell is a eukaryotic cell.

25. The recombinant cell according to claim 24, wherem the eukaryotic cell is selected from the group consisting of a yeast cell, a fungal cell, an algal cell and a plant cell.

26. The recombinant cell according to claim 25, wherein the yeast cell is selected from the group consisting of Saccharomyces, Kluyveromyces. Candida, Pichia, Schizosaccharomyces, Hansenula, Klockera, Schwanniomyces, Schefjeromyces, Rhodosporidium, Issatchenkia, Yarrowia and

Rhodotonda.

27. The recombinant cell according to claim 26, wherein the yeast cell is selected from the group consisting of S. cerevisiae, C. lipolytica, R glutinis, S. bulderi, S. harnetti, S. exiguus, S. uvarum,

S. diastaticus, K. lactis, K. marxiams, K. fragile, P. kudriavzevii, S stipites, and I. orientalis.

28. Tire recombinant cell according to claim 25, wherein the fungal cell is a filamentous fungal cell.

29. The recombinant cell according to claim 28, wherem the filamentous fungal cell is selected from the group consisting of Aspergillus, Petnailmm, Rhisopus, Chrysosporium, Myceliophthora , Trichoderma Humicoia . Acremonium and Fusarium.

30. The recombinant cell according to claim 29, wherein the filamentous fungal cell is selected from the group consi sting of A . mger , A. oryzae , 7 reesei, P. chrysogevum, M thennophila , and

R. oryzae.

31 . The recombinant cell according to claim 25, wherein the algal cell is selected from the group consisting of Botryococcus. Nannochloropsis, Chlorella, Chlamydomonas, Dunaliella, Chaetoceros, Porphyridium , Scenedesmus and Pseudochlorococcum.

32. The recombinant cell according to claim 31 , wherein the algal cell is selected from the group consisting of B. and N gaditana.

33. The recombinant cell according to any one of claims 20 to 32 comprising one or more genetic modifications resulting in at least one, any two, any three, any four or all five of the following phenotypes:

(a) an increase acyl-ACP synthesis;

(b) a decrease in acyl-ACP degradation;

(c) an upreguiation of fatty alcohol secretion;

(d) an increase in flux through the fatty acid biosynthetic pathway; and

(e) an increase in tolerance to fatty alcohol.

34. Tire recombinant cell according to any one of claims 20 to 33 comprising one or more genetic modifications resulting in a change in the degree of saturation of the fatty alcohols produced.

35. The recombinant cell according to any one of claims 20 to 34 comprising one or more genetic m odifications resulting in a change in the chain-length of the acyl-ACPs produced by the fatty acid biosynthetic pathway.

36. A recombinant ceil transformed with the vector according to any one of claims 17 to 19.

37. The recombinant cell according to claim 36, wherein the recombinant cell is stably transformed with the nucleic acid according to any one of claims 2 to 16.

38. The recombinant cell according to claim 36, wherein the recombinant cell is transiently transformed with the nucleic acid according to any one of claims 2 to 16.

39. A method of producing a fatty alcohol composition comprising:

a) culturing a recombinant cell in a suitable culture medium, wherein the recombinant cell comprises a gene encoding a heterologous fatty acyl-ACP reductase enzyme having at least 70% sequence identity to SEQ ID NO: 1 ; and

b) allowing expression of said gene, wherein said expression results in the production of a faty alcohol composition.

40. The method according to claim 39, wherein the recombinant cell is defined according to any one of claims 20 to 38.

41. The method according to claim 39 or claim 40, the method further comprising purifying the fatty alcohol composition from cell culture.

42. The method according to claims 39 or claim 41 , the method further comprising purifying the fatty alcohol composition from supernatant.

43. The method according to any one of claims 39 to 42, the method further compri sing harvesting and lysing recombinant ce!l(s) to obtain a fatty alcohol composition.

44. The method according to any one of claims 39 to 43, the method further comprising purifying the fatty alcohol composition from lysate.

45. The method according to any one of claims 39 to 44, wherein the fatty alcohol composition comprises C12 fatty alcohol.

46. The method according to any one of claims 39 to 45, wherein at least 45% of the fatty alcohols m the fatty alcohol composition are saturated fatty alcohols.

47. The method according to any one of the claims 39 to 46, the method further comprising reducing the fatty alcohol composition.

48. The method according to any one of claims 39 to 47, the method further comprising esterifying the fatty alcohol composition.

49. A fatty alcohol composition obtained by the method according to any one of claims 39 to 48.

50. A composition comprising the fatty alcohol composition or esterified fatty alcohol composition obtained by the method of any one of claims 39 to 49.

51. Use of a polypeptide according to claim 1 in a method of producing a fatty alcohol composition.