Materials and methods utilizing biotin producing mutant hosts for the production of 7-carbon chemicals
10947570 ยท 2021-03-16
Assignee
Inventors
- Alexander Brett Foster (Yarm, GB)
- Stephen Thomas Cartman (Stockton-on-Tees, GB)
- Jonathan Kennedy (North Yorkshire, GB)
Cpc classification
C12P13/005
CHEMISTRY; METALLURGY
C12Y206/01018
CHEMISTRY; METALLURGY
International classification
C12P13/00
CHEMISTRY; METALLURGY
Abstract
Disclosed are methods for regulating biosynthesis of at least one of pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine, 7-aminoheptanoland 1,7-heptanediol (C7 building blocks) using a pathway having a pimeloyl-ACP intermediate, the method including the step of downregulating the activity of BioF. Also disclosed are recombinant hosts by fermentation in which the above methods are performed. Further disclosed are recombinant hosts for producing pimeloyl-ACP, the recombinant host including a deletion of a bioF gene.
Claims
1. A method for regulating biosynthesis of at least one C7 building block chosen from pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, 7-aminoheptanol, and 1,7-heptanediol in a recombinant host using a pathway having a pimeloyl-ACP intermediate, said method comprising: deleting the activity of an ACP-dependent BioF enzyme; overexpressing a BioW enzyme and a CoA-specific BioF enzyme, wherein: the BioW enzyme is a pimeloyl-CoA ligase having at least 85% sequence identity or homology to the amino acid sequence set forth in SEQ ID NO: 18; and the CoA-specific BioF enzyme is an 8-amino-7-oxononanoate synthase having at least 85% sequence identity or homology to the amino acid sequence set forth in SEQ ID NO: 19; and wherein the recombinant host is a prokaryote chosen from the genera Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Delftia, Bacillus, Lactobacillus, Lactococcus, and Rhodococcus or the recombinant host is a eukaryote chosen from the genera Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula, and Kluyveromyces; and wherein the deletion of the activity of an ACP-dependent BioF enzyme occurs through a knockout of the BioF gene.
2. The method of claim 1, wherein: said BioW enzyme enzymatically converts pimelic acid to pimeloyl-CoA; or said CoA-specific BioF enzyme enzymatically converts pimeloyl-CoA to 8-amino-7-oxo-nonanoic acid.
3. The method of claim 1, wherein said pathway comprises: enzymatically synthesizing a C7 aliphatic backbone from malonyl-ACP via two cycles of methyl-ester shielded carbon chain elongation; and enzymatically forming two terminal functional groups in said backbone, thereby forming the C7 building block.
4. The method of claim 3, wherein: a S-adenosyl-L-methionine (SAM)-dependent methyltransferase converts malonyl-ACP to malonyl-ACP methyl ester; and said two cycles of methyl-ester shielded carbon chain elongation produce pimeloyl-ACP methyl ester from malonyl-ACP methyl ester using a trans-2-enoyl-CoA reductase.
5. The method of claim 3, wherein each of said two cycles of methyl-ester shielded carbon chain elongation comprises using (i) a -ketoacyl-ACP synthase, (ii) a 3-oxoacyl-ACP reductase, (iii) a 3-hydroxyacyl-ACP dehydratase, and (iv) an enoyl-ACP reductase.
6. The method of claim 3, wherein: a hydroxyl terminal group is enzymatically formed by a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 4-hydroxybutyrate dehydratase, or an alcohol dehydrogenase; a carboxyl terminal group is enzymatically formed by a thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a glutaconate CoA-transferase, or a reversible succinyl-CoA ligase; or an amine terminal group is enzymatically formed by a -transaminase or a deacetylase.
7. The method of claim 1, wherein an intermediate with a terminal aldehyde group is formed by a carboxylate reductase and enhanced by a phosphopantetheinyl transferase during the biosynthesis of said at least one C7 building block.
8. The method of claim 1, wherein said method is performed in said recombinant host by fermentation.
9. The method of claim 8, wherein: said recombinant host is subjected to a cultivation condition under aerobic, anaerobic, micro-aerobic or mixed oxygen/denitrification cultivation conditions; said recombinant host is cultured under conditions of nutrient limitation; said recombinant host is retained using a ceramic hollow fiber membrane to maintain a high cell density during fermentation; or said recombinant host's tolerance to high concentrations of the at least one C7 building block is improved through continuous cultivation in a selective environment.
10. The method of claim 8, wherein: the principal carbon source fed to the fermentation is a biological feedstock that is, or derives from, at least one feedstock chosen from monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid, formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, and municipal waste; or the principal carbon source fed to the fermentation is a non-biological feedstock that is, or derives from, at least one feedstock chosen from natural gas, syngas, CO.sub.2/H.sub.2, methanol, ethanol, benzoate, non-volatile residue (NVR) caustic wash waste stream from cyclohexane oxidation processes, and terephthalic acid/isophthalic acid mixture waste streams.
11. The method of claim 1, wherein: said prokaryote is Escherichia coli, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium kluyveri, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus metallidurans, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas oleavorans, Delftia acidovorans, Bacillus subtillis, Lactobacillus delbrueckii, or Lactococcus lactis; said eukaryote is Aspergillus niger, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Issathenkia orientalis, Debaryomyces hansenii, Arxula adenoinivorans, or Kluyveromyces lactis.
12. The method of claim 1, wherein the knockout of the BioF gene occurs through homologous recombination.
Description
BRIEF DESCRIPTION OF DRAWING(S)
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DETAILED DESCRIPTION
(22) This disclosure provides enzymes, non-natural pathways, cultivation strategies, feedstocks, host microorganisms and attenuations to the host's biochemical network, which generates a C7 aliphatic backbone from central metabolites in which two terminal functional groups may be formed leading to the synthesis of pimelic acid, 7-aminoheptanoic acid, heptamethylenediamine, 7-aminoheptanol or 1,7-heptanediol (referred to as C7 building blocks herein). As used herein, the term central precursor is used to denote any metabolite in any metabolic pathway shown herein leading to the synthesis of a C7 building block. The term central metabolite is used herein to denote a metabolite that is produced in all microorganisms to support growth.
(23) The term bioF refers to any one of a number of bioF genes well known in the art and present in multiple organisms. The term BioF refers to a protein encoded by any one of such genes. Numerous bioF genes and BioF proteins from different organisms are well known in the art and can be easily identified in public databases such as GenBank, ExPASy, and via Enzyme Commission numbers.
(24) Host microorganisms described herein can include endogenous pathways that can be manipulated such that one or more C7 building blocks can be produced. In an endogenous pathway, the host microorganism naturally expresses all of the enzymes catalyzing the reactions within the pathway. A host microorganism containing an engineered pathway does not naturally express all of the enzymes catalyzing the reactions within the pathway but has been engineered such that all of the enzymes within the pathway are expressed in the host.
(25) The term exogenous as used herein with reference to a nucleic acid (or a protein) and a host refers to a nucleic acid that does not occur in (and cannot be obtained from) a cell of that particular type as it is found in nature or a protein encoded by such a nucleic acid. Thus, a non-naturally-occurring nucleic acid is considered to be exogenous to a host once in the host. It is important to note that non-naturally-occurring nucleic acids can contain nucleic acid subsequences or fragments of nucleic acid sequences that are found in nature provided the nucleic acid as a whole does not exist in nature. For example, a nucleic acid molecule containing a genomic DNA sequence within an expression vector is non-naturally-occurring nucleic acid, and thus is exogenous to a host cell once introduced into the host, since that nucleic acid molecule as a whole (genomic DNA plus vector DNA) does not exist in nature. Thus, any vector, autonomously replicating plasmid, or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a whole does not exist in nature is considered to be non-naturally-occurring nucleic acid. It follows that genomic DNA fragments produced by PCR or restriction endonuclease treatment as well as cDNAs are considered to be non-naturally-occurring nucleic acid since they exist as separate molecules not found in nature. It also follows that any nucleic acid containing a promoter sequence and polypeptide-encoding sequence (e.g., cDNA or genomic DNA) in an arrangement not found in nature is non-naturally-occurring nucleic acid. A nucleic acid that is naturally-occurring can be exogenous to a particular host microorganism. For example, an entire chromosome isolated from a cell of yeast x is an exogenous nucleic acid with respect to a cell of yeast y once that chromosome is introduced into a cell of yeast y.
(26) In contrast, the term endogenous as used herein with reference to a nucleic acid (e.g., a gene) (or a protein) and a host refers to a nucleic acid (or protein) that does occur in (and can be obtained from) that particular host as it is found in nature. Moreover, a cell endogenously expressing a nucleic acid (or protein) expresses that nucleic acid (or protein) as does a host of the same particular type as it is found in nature. Moreover, a host endogenously producing or that endogenously produces a nucleic acid, protein, or other compound produces that nucleic acid, protein, or compound as does a host of the same particular type as it is found in nature.
(27) For example, depending on the host and the compounds produced by the host, one or more of the following enzymes may be expressed in the host in addition to a malonyl-ACP O-methyltransferase; a -ketoacyl-[acp] synthase, a -ketothiolase, a 3-oxoacyl-[acp] reductase, acetoacetyl-CoA reductase, a 3-hydroxyacyl-CoA dehydrogenase, a 3-hydroxybutyryl-CoA dehydrogenase, an enoyl-CoA hydratase, 3-hydroxyacyl-ACP dehydratase, an enoyl-ACP reductase, a trans-2-enoyl-CoA reductase, a thioesterase, a reversible CoA ligase, a CoA-transferase, an acetylating aldehyde dehydrogenase, a 6-oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, an aldehyde dehydrogenase, a carboxylate reductase, a -transaminase, a N-acetyl transferase, an alcohol dehydrogenase, a deacetylase, a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, or a 4-hydroxybutyrate dehydrogenase. In recombinant hosts expressing a carboxylate reductase, a phosphopantetheinyl transferase also can be expressed as it enhances activity of the carboxylate reductase.
(28) For example, a recombinant host can include at least one exogenous nucleic acid encoding (i) a malonyl-ACP O-methyltransferase, (ii) a 3-ketoacyl-ACP synthase or a -ketothiolase, (iii) a 3-oxoacyl-ACP reductase, acetoacetyl-CoA reductase, a 3-hydroxyacyl-CoA dehydrogenase or a 3-hydroxybutyryl-CoA dehydrogenase, (iv) an enoyl-CoA hydratase or 3-hydroxyacyl-ACP dehydratase, (v) an enoyl-ACP reductase or a trans-2-enoyl-CoA reductase and produce pimeloyl-ACP or pimeloyl-CoA.
(29) Such recombinant hosts producing pimeloyl-ACP or pimeloyl-CoA further can include at least one exogenous nucleic acid encoding one or more of a thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a glutaconate CoA-transferase, a reversible succinyl-CoA ligase, an acetylating aldehyde dehydrogenase, or a carboxylate reductase and produce pimelic acid or pimelate semialdehyde. For example, a recombinant host producing pimeloyl-ACP or pimeloyl-CoA further can include a thioesterase, a reversible Co-ligase (e.g., a reversible succinyl-CoA ligase), or a CoA transferase (e.g., a glutaconate CoA-transferase) and produce pimelic acid. For example, a recombinant host producing pimeloyl-CoA further can include an acetylating aldehyde dehydrogenase and produce pimelate semilaldehyde. For example, a recombinant host producing pimelate further can include a carboxylate reductase and produce pimelate semialdehyde.
(30) In some embodiments, the recombinant host can comprise at least one exogenous nucleic acid encoding bioW and a CoA-specific bioF.
(31) A recombinant host producing pimelate semialdehyde further can include at least one exogenous nucleic acid encoding a -transaminase and produce 7-aminoheptanoate.
(32) In some embodiments, a recombinant host producing pimeloyl-CoA includes a carboxylate reductase and a -transaminase to produce 7-aminoheptanoate.
(33) A recombinant host producing pimelate or pimelate semialdehyde further can include at least one exogenous nucleic acid encoding a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase or a 4-hydroxybutyrate dehydrogenase, and produce 7-hydroxyheptanoic acid. In some embodiments, a recombinant host producing pimeloyl-CoA includes an acetylating aldehyde dehydrogenase, and a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase or a 4-hydroxybutyrate dehydrogenase to produce 7-hydroxyheptanoate. In some embodiments, a recombinant host producing pimelate includes a carboxylate reductase and a 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase or a 4-hydroxybutyrate dehydrogenase to produce 7-hydroxyheptanoate.
(34) A recombinant host producing 7-aminoheptanoate, 7-hydroxyheptanoate or pimelate semialdehyde further can include at least one exogenous nucleic acid encoding a -transaminase, a deacetylase, a N-acetyl transferase, or an alcohol dehydrogenase, and produce heptamethylenediamine. For example, a recombinant host producing 7-hydroxyheptanoate can include a carboxylate reductase with a phosphopantetheine transferase enhancer, a -transaminase and an alcohol dehydrogenase.
(35) A recombinant host producing 7-hydroxyheptanoic acid further can include one or more of a carboxylate reductase with a phosphopantetheine transferase enhancer and an alcohol dehydrogenase, and produce 1,7-heptanediol.
(36) Within an engineered pathway, the enzymes can be from a single source, i.e., from one species or genus, or can be from multiple sources, i.e., different species or genera. Nucleic acids encoding the enzymes described herein have been identified from various organisms and are readily available in publicly available databases such as GenBank or EMBL.
(37) Any of the enzymes described herein that can be used for production of one or more C7 building blocks can have at least 50%, 60% or 70% sequence identity or homology (e.g., at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of the corresponding wild-type enzyme. It will be appreciated that the sequence identity can be determined on the basis of the mature enzyme (e.g., with any signal sequence removed).
(38) The percent identity and homology between two amino acid sequences can be determined as follows. First, the amino acid sequences are aligned using the BLAST 2 Sequences (Bl2seq) program from the stand-alone version of BLASTZ containing BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained from www.fr.com/blast/ or the U.S. government's National Center for Biotechnology Information web site (www.ncbi.nlm.nih.gov). Instructions explaining how to use the Bl2seq program can be found in the readme file accompanying BLASTZ. Bl2seq performs a comparison between two amino acid sequences using the BLASTP algorithm. To compare two amino acid sequences, the options of Bl2seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\Bl2seq c:\seq1.txt-j c:\seq2.txt-p blastp-o c:\output.txt. If the two compared sequences share homology (identity), then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology (identity), then the designated output file will not present aligned sequences. Similar procedures can be following for nucleic acid sequences except that blastn is used.
(39) Once aligned, the number of matches is determined by counting the number of positions where an identical amino acid residue is presented in both sequences. The percent identity is determined by dividing the number of matches by the length of the full-length polypeptide amino acid sequence followed by multiplying the resulting value by 100. It is noted that the percent identity value is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2. It also is noted that the length value will always be an integer.
(40) When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution and this process results in sequence homology of, e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer. Applic. Biol. Sci., 1988, 4, 11-17, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Cailf., USA). This alignment and the percent homology or identity can be determined using any suitable software program known in the art, for example those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al. (eds) 1987, Supplement 30, section 7.7.18). Such programs may include the GCG Pileup program, FASTA (Pearson et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 2444-2448), and BLAST (BLAST Manual, Altschul et al., Nat'l Cent. Biotechnol. Inf., Nat'l Lib. Med. (NCIB NLM NIH), Bethesda, Md., and Altschul et al., NAR, 1997, 25, 3389-3402). Another alignment program is ALIGN Plus (Scientific and Educational Software, Pa.), using default parameters. Another sequence software program that finds use is the TFASTA Data Searching Program available in the Sequence Software Package Version 6.0 (Genetics Computer Group, University of Wisconsin, Madison, Wis.).
(41) A conservative substitution is a substitution of one amino acid for another with similar characteristics. Conservative substitutions include substitutions within the following groups: valine, alanine, and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above mentioned polar, basic, or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a non-conservative substitution is a substitution of one amino acid for another with dissimilar characteristics.
(42) For example, a 8-amino-7-oxononanoate synthase (BioF, or ACP-dependent BioF) described herein can have at least 70% sequence identity or homology (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of an Escherichia coli 8-amino-7-oxononanoate synthase (BioF) (EC:2.3.1.47, see GenBank Accession No. AAA23516.1, SEQ ID NO: 23), See
(43) For example, a thioesterase (TE) described herein can have at least 70% sequence identity or homology (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of an Escherichia coli thioesterase encoded by tesB (see GenBank Accession No. AAA24665.1, SEQ ID NO: 1), or the gene products encoded by tesA or fatB (see GenBank Accession No. ABJ63754.1, SEQ ID NO:21; see GenBank Accession No. CCC78182.1, SEQ ID NO:22). See
(44) For example, a carboxylate reductase described herein can have at least 70% sequence identity or homology (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Mycobacterium marinum (see Genbank Accession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatis (see Genbank Accession No. ABK71854.1, SEQ ID NO: 3), a Segniliparus rugosus (see Genbank Accession No. EFV11917.1, SEQ ID NO: 4), a Mycobacterium smegmatis (see Genbank Accession No. ABK75684.1, SEQ ID NO: 5), a Mycobacterium massiliense (see Genbank Accession No. EIV11143.1, SEQ ID NO: 6), or a Segniliparus rotundus (see Genbank Accession No. ADG98140.1, SEQ ID NO: 7) carboxylate reductase. See,
(45) For example, a -transaminase described herein can have at least 70% sequence identity or homology (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Chromobacterium violaceum (see Genbank Accession No. AAQ59697.1, SEQ ID NO: 8), a Pseudomonas aeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO: 9), a Pseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ ID NO: 10), a Rhodobacter sphaeroides (see Genbank Accession No. ABA81135.1, SEQ ID NO: 11), an Escherichia coli (see RefSeq Accession No. NP 417544.5, SEQ ID NO: 12), or a Vibrio fluvialis (see Genbank Accession No. AEA39183.1, SEQ ID NO: 13) -transaminase. Some of these -transaminases are diamine -transaminases. See,
(46) For example, a phosphopantetheinyl transferase described herein can have at least 70% sequence identity or homology (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Bacillus subtilis phosphopantetheinyl transferase (see RefSeq Accession No. WP 003234549.1, SEQ ID NO:14) or a Nocardia sp. NRRL 5646 phosphopantetheinyl transferase (see Genbank Accession No. ABI83656.1, SEQ ID NO:15). See
(47) For example, a pimeloyl-ACP methyl ester esterase described herein can have at least 70% sequence identity or homology (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of an Escherichia coli pimeloyl-ACP methyl ester esterase (BioH) (see GenBank Accession No. AAC76437.1, SEQ ID NO:17). See,
(48) For example, a 6-carboxyhexanoate-CoA ligase described herein can have at least 70% sequence identity or homology (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of an Bacillus subtilis (strain 168) 6-carboxyhexanoate-CoA ligase (BioW) (EC:6.2.1.14, see GenBank Accession No. AAB17457.1, SEQ ID NO:18). See,
(49) For example, a 8-amino-7-oxononanoate synthase described herein can have at least 70% sequence identity or homology (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of an Bacillus subtilis (strain 168) 8-amino-7-oxononanoate synthase (BioF) (EC:2.3.1.47, see GenBank Accession No. AAB17459.1, SEQ ID NO: 19). See,
(50) For example, a SAM-dependent malonyl-ACP O-methyltransferase described herein can have at least 70% sequence identity or homology (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Escherichia coli malonyl-ACP O-methyltransferase (BioC) (EC:2.1.1.197, see RefSeq Accession No. NP_415298.1, SEQ ID NO: 20). See,
(51) It will be appreciated that a number of nucleic acids can encode a polypeptide having a particular amino acid sequence. The degeneracy of the genetic code is well known to the art; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. For example, codons in the coding sequence for a given enzyme can be modified such that optimal expression in a particular species (e.g., bacteria or fungus) is obtained, using appropriate codon bias tables for that species.
(52) Functional fragments of any of the enzymes described herein can also be used in the methods of the disclosure. The term functional fragment as used herein refers to a peptide fragment of a protein that has at least 25% (e.g., at least: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 95%; 98%; 99%; 100%; or even greater than 100%) of the activity of the corresponding mature, full-length, wild-type protein. The functional fragment can generally, but not always, be comprised of a continuous region of the protein, wherein the region has functional activity.
(53) This disclosure also provides (i) functional variants of the enzymes used in the methods of the disclosure and (ii) functional variants of the functional fragments described above. Functional variants of the enzymes and functional fragments can contain additions, deletions, or substitutions relative to the corresponding wild-type sequences. Enzymes with substitutions will generally have not more than 50 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, or 50) amino acid substitutions (e.g., conservative substitutions). This applies to any of the enzymes described herein and functional fragments. A conservative substitution is a substitution of one amino acid for another with similar characteristics. Conservative substitutions include substitutions within the following groups: valine, alanine and glycine; leucine, valine, and isoleucine; aspartic acid and glutamic acid; asparagine and glutamine; serine, cysteine, and threonine; lysine and arginine; and phenylalanine and tyrosine. The nonpolar hydrophobic amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Any substitution of one member of the above-mentioned polar, basic or acidic groups by another member of the same group can be deemed a conservative substitution. By contrast, a nonconservative substitution is a substitution of one amino acid for another with dissimilar characteristics.
(54) Deletion variants can lack one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid segments (of two or more amino acids) or non-contiguous single amino acids. Additions (addition variants) include fusion proteins containing: (a) any of the enzymes described herein or a fragment thereof; and (b) internal or terminal (C or N) irrelevant or heterologous amino acid sequences. In the context of such fusion proteins, the term heterologous amino acid sequences refers to an amino acid sequence other than (a). A heterologous sequence can be, for example a sequence used for purification of the recombinant protein (e.g., FLAG, polyhistidine (e.g., hexahistidine (SEQ ID NO: 24)), hemagglutinin (HA), glutathione-S-transferase (GST), or maltosebinding protein (MBP)). Heterologous sequences also can be proteins useful as detectable markers, for example, luciferase, green fluorescent protein (GFP), or chloramphenicol acetyl transferase (CAT). In some embodiments, the fusion protein contains a signal sequence from another protein. In certain host cells (e.g., yeast host cells), expression and/or secretion of the target protein can be increased through use of a heterologous signal sequence. In some embodiments, the fusion protein can contain a carrier (e.g., KLH) useful, e.g., in eliciting an immune response for antibody generation) or ER or Golgi apparatus retention signals. Heterologous sequences can be of varying length and in some cases can be a longer sequences than the full-length target proteins to which the heterologous sequences are attached.
(55) Engineered hosts can naturally express none or some (e.g., one or more, two or more, three or more, four or more, five or more, or six or more) of the enzymes of the pathways described herein. Thus, a pathway within an engineered host can include all exogenous enzymes, or can include both endogenous and exogenous enzymes. Endogenous genes of the engineered hosts also can be disrupted to prevent the formation of undesirable metabolites or prevent the loss of intermediates in the pathway through other enzymes acting on such intermediates. Engineered hosts can be referred to as recombinant hosts or recombinant host cells. As described herein recombinant hosts can include nucleic acids encoding one or more of a methyltransferase, a synthase, -ketothiolase, a dehydratase, a hydratase, a dehydrogenase, a methylesterase, a thioesterase, a reversible CoA-ligase, a CoA-transferase, a reductase, deacetylase, N-acetyl transferase or a -transaminase as described in more detail below.
(56) In addition, the production of one or more C7 building blocks can be performed in vitro using the isolated enzymes described herein, using a lysate (e.g., a cell lysate) from a host microorganism as a source of the enzymes, or using a plurality of lysates from different host microorganisms as the source of the enzymes.
(57) Enzymes Generating the C7 Aliphatic Backbone for Conversion to C7 Building Blocks
(58) As depicted in
(59) The native biotin pathway thus provides a potential source of the pimelic acid intermediate that can be subsequently metabolized to 7-AHA. As biotin is only required in trace amounts the natural flux through this pathway is very low. Initial attempts to increase the flux to pimelic acid in E. coli have yielded only trace amounts of pimelic acid (<0.1 ppm), due in part to complex regulation of the biotin pathway and the toxicity associated with overexpression of pathway enzymes. In order to bypass these issues strains of E. coli with increased flux through the biotin pathway were sourced. A series of strains, generated by multiple rounds of random mutagenesis, have been reported to produce almost 1 g/L of biotin (U.S. Pat. No. 6,284,500 B1). These strains were used in order to provide a genetic background in which flux through the native biotin pathway is high with feedback and other negative regulatory mechanisms non-functional or much reduced.
(60) In some embodiments, a methyl ester shielded carbon chain elongation associated with biotin biosynthesis route comprises using a malonyl-ACP O-methyltransferase to form a malonyl-ACP methyl ester, and then performing two cycles of carbon chain elongation using a -ketoacyl-ACP synthase, a 3-oxoacyl-ACP reductase, a 3-hydroxyacyl-ACP dehydratase, and an enoyl-ACP reductase. A pimeloyl-ACP methyl ester esterase can be used to cleave the resulting pimeloyl-ACP methyl ester.
(61) In some embodiments, a methyltransferase can be a malonyl-ACP O-methyltransferase classified, for example, under EC 2.1.1.197 such as the gene product of bioC from Bacillus cereus (see Genbank Accession No. AAS43086.1, SEQ ID NO:16) (see, for example, Lin, 2012, Biotin Synthesis in Escherichia coli, Ph.D. Dissertation, University of Illinois at Urbana-Champaign).
(62) In some embodiments, a -ketoacyl-ACP synthase may be classified, for example, under EC 2.3.1.(e.g., EC 2.3.1.41, EC 2.3.1.179 or EC 2.3.1.180) such as the gene product of fabB, fabF, or fabH.
(63) In some embodiments, a 3-oxoacyl-ACP reductase may be classified under EC 1.1.1.100, such as the gene product of fabG.
(64) In some embodiments, an enoyl-ACP dehydratase such as a 3-hydroxyacyl-ACP dehydratase may be classified under EC 4.2.1.59, such as the gene product of fabZ.
(65) In some embodiments, a trans-2-enoyl-CoA reductase may be classified under EC 1.3.1.(e.g., EC 1.3.1.38, EC 1.3.1.8, EC 1.3.1.44), such as the gene product of ter (Nishimaki et al., J. Biochem., 1984, 95, 1315-1321; Shen et al., 2011, supra) or tdter (Bond-Watts et al., Biochemistry, 2012, 51, 6827-6837).
(66) In some embodiments, an enoyl-ACP reductase may be classified under EC 1.3.1.10 such as the gene product of fabI.
(67) In some embodiments, a pimeloyl-ACP methyl ester esterase may be classified, for example, under EC 3.1.1.85 such as the gene product of bioH from E. coli. See Genbank Accession No. AAC76437.1, SEQ ID NO:17.
(68) Enzymes Generating the Terminal Carboxyl Groups in the Biosynthesis of C7 Building Blocks
(69) As depicted in
(70) In some embodiments, the second terminal carboxyl group leading to the synthesis of a C7 building block is enzymatically formed by a thioesterase classified, for example, under EC 3.1.2.-, such as the gene product of YciA, tesB (Genbank Accession No. AAA24665.1, SEQ ID NO: 1) or the gene products encoded by tesA or fatB (see GenBank Accession No. ABJ63754.1, SEQ ID NO:21; see GenBank Accession No. CCC78182.1, SEQ ID NO:22) (see, for example, Cantu et al., Protein Science, 2010, 19, 1281-1295; Zhuang et al., Biochemistry, 2008, 47(9), 2789-2796; or Naggert et al., J. Biol. Chem., 1991, 266(17), 11044 11050).
(71) Enzymes Generating the Terminal Amine Groups in the Biosynthesis of C7 Building Blocks
(72) As depicted in
(73) In some embodiments, the first or second terminal amine group leading to the synthesis of 7-aminoheptanoic acid is enzymatically formed by a -transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as that obtained from Chromobacterium violaceum (Genbank Accession No. AAQ59697.1, SEQ ID NO: 8), Pseudomonas aeruginosa (Genbank Accession No. AAG08191.1, SEQ ID NO: 9), Pseudomonas syringae (Genbank Accession No. AAY39893.1, SEQ ID NO: 10), Rhodobacter sphaeroides (Genbank Accession No. ABA81135.1, SEQ ID NO: 11), Escherichia coli (RefSeq Accession No. NP 417544.5, SEQ ID NO: 12), Vibrio Fluvialis (Genbank Accession No. AEA39183.1, SEQ ID NO: 13), Streptomyces griseus, or Clostridium viride. Some of these -transaminases are diamine -transaminases (e.g., SEQ ID NO: 12). For example, the -transaminases classified, for example, under EC 2.6.1.29 or EC 2.6.1.82 may be diamine -transaminases.
(74) The reversible -transaminase from Chromobacterium violaceum (Genbank Accession No. AAQ59697.1, SEQ ID NO: 8) has demonstrated analogous activity accepting 6-aminohexanoic acid as amino donor, thus forming the first terminal amine group in adipate semialdehyde (Kaulmann et al., Enzyme and Microbial Technology, 2007, 41, 628-637).
(75) The reversible 4-aminobubyrate:2-oxoglutarate transaminase from Streptomyces griseus has demonstrated analogous activity for the conversion of 6-aminohexanoate to adipate semialdehyde (Yonaha et al., Eur. J. Biochem., 1985, 146:101-106).
(76) The reversible 5-aminovalerate transaminase from Clostridium viride has demonstrated analogous activity for the conversion of 6-aminohexanoate to adipate semialdehyde (Barker et al., J. Biol. Chem., 1987, 262(19), 8994-9003).
(77) In some embodiments, a terminal amine group leading to the synthesis of 7-aminoheptanoate or heptamethylenediamine is enzymatically formed by a diamine -transaminase. For example, the second terminal amino group can be enzymatically formed by a diamine -transaminase classified, for example, under EC 2.6.1.29 or classified, for example, under EC 2.6.1.82, such as the gene product of YgjG from E. coli (RefSeq Accession No. NP_417544.5, SEQ ID NO: 12).
(78) The gene product of ygjG accepts a broad range of diamine carbon chain length substrates, such as putrescine, cadaverine and spermidine (see, for example, Samsonova et al., BMC Microbiology, 2003, 3:2).
(79) The diamine -transaminase from E. coli strain B has demonstrated activity for 1,7 diaminoheptane (Kim, The Journal of Chemistry, 1964, 239(3), 783-786).
(80) In some embodiments, the second terminal amine group leading to the synthesis of heptamethylenediamine is enzymatically formed by a deacetylase such as acetylputrescine deacetylase classified, for example, under EC 3.5.1.62. The acetylputrescine deacetylase from Micrococcus luteus K-11 accepts a broad range of carbon chain length substrates, such as acetylputrescine, acetylcadaverine and N.sup.8_acetylspermidine (see, for example, Suzuki et al., 1986, BBAGeneral Subjects, 882(1):140-142).
(81) Enzymes Generating the Terminal Hydroxyl Groups in the Biosynthesis of C7 Building Blocks
(82) As depicted in
(83) In some embodiments, a terminal hydroxyl group leading to the synthesis of 1,7 heptanediol is enzymatically formed by an alcohol dehydrogenase classified, for example, under EC 1.1.1.(e.g., 1, 2, 21, or 184) such as the gene product of YMR318C (classified, for example, under EC 1.1.1.2, see Genbank Accession No. CAA90836.1) (Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172), the gene product of YghD, the gene product of cpnD (Iwaki et al., 2002, Appl. Environ. Microbiol., 68(11):5671-5684), the gene product of gbd, or a 6-hydroxyhexanoate dehydrogenase classified, for example, under EC 1.1.1.258 such as the gene product of ChnD (Iwaki et al., Appl. Environ. Microbiol., 1999, supra).
Biochemical Pathways
(84) Pathways from Malonyl-ACP to Pimeloyl-ACP as Central Precursor Leading to C7 Building Block
(85) As depicted in
(86) The native biotin pathway thus provides a potential source of the pimelic acid intermediate that can be subsequently metabolized to 7-AHA. As biotin is only required in trace amounts the natural flux through this pathway is very low. Initial attempts to increase the flux to pimelic acid in E. coli have yielded only trace amounts of pimelic acid (<0.1 ppm), due in part to complex regulation of the biotin pathway and the toxicity associated with overexpression of pathway enzymes. In order to bypass these issues strains of E. coli with increased flux through the biotin pathway were sourced. A series of strains, generated by multiple rounds of random mutagenesis, have been reported to produce almost 1 g/L of biotin (U.S. Pat. No. 6,284,500 B1). These strains were used in order to provide a genetic background in which flux through the native biotin pathway is high with feedback and other negative regulatory mechanisms non-functional or much reduced.
(87) Pathways Using Pimeloyl-ACP as Central Precursor to Pimelic Acid
(88) In some embodiments, pimelic acid is synthesized from the central precursor, pimeloyl-ACP, by conversion of pimeloyl-ACP to pimelate by a thioesterase classified, for example, under EC 3.1.2.such as the gene products encoded by tesA orfatB (Genbank Accession No. ABJ63754.1, SEQ ID NO:21; Genbank Accession No. CCC78182.1, SEQ ID NO:22). See
(89) Pathways Using Pimelate Semialdehyde as Precursor to 7-Aminoheptanoate
(90) In some embodiments, 7-aminoheptanoate is synthesized from the central precursor, pimelate, by conversion of pimelate to pimelate semialdehyde by a carboxylate reductase classified, for example, under EC 1.2.99.6 such as the gene product of car in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sfp (RefSeq Accession No. WP 003234549.1, SEQ ID NO:14) gene from Bacillus subtilis or npt (Genbank Accession No. ABI83656.1, SEQ ID NO:15) gene from Nocardia) or the gene products of GriC and GriD from Streptomyces griseus (Suzuki et al., J. Antibiot., 2007, 60(6), 380-387); followed by conversion of pimelate semialdehyde to 7-aminoheptanoate by a -transaminase (e.g., EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.48, EC 2.6.1.29, EC 2.6.1.82 such as SEQ ID NOs:8-13). The carboxylate reductase can be obtained, for example, from Mycobacterium marinum (Genbank Accession No. ACC40567.1, SEQ ID NO: 2), Mycobacterium smegmatis (Genbank Accession No. ABK71854.1, SEQ ID NO: 3), Segniliparus rugosus (Genbank Accession No. EFV11917.1, SEQ ID NO: 4), Mycobacterium smegmatis (Genbank Accession No. ABK75684.1, SEQ ID NO: 5), Mycobacterium massiliense (Genbank Accession No. EIV11143.1, SEQ ID NO: 6), or Segniliparus rotundus (Genbank Accession No. ADG98140.1, SEQ ID NO: 7). See
(91) Pathway Using 7-Aminoheptanoate, 7-Hydroxyheptanoate or Pimelate Semialdehyde as a Precursor to Heptamethylenediamine
(92) In some embodiments, heptamethylenediamine is synthesized from the central precursor, 7-aminoheptanoate, by conversion of 7-aminoheptanoate to 7-aminoheptanal by a carboxylate reductase classified, for example, under EC 1.2.99.6 such as the gene product of car (see above) in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sfp (RefSeq Accession No. WP 003234549.1, SEQ ID NO:14) gene from Bacillus subtilis or npt (Genbank Accession No. ABI83656.1, SEQ ID NO:15) gene from Nocardia) or the gene product of GriC & GriD; followed by conversion of 7-aminoheptanal to heptamethylenediamine by a -transaminase (e.g., classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as SEQ ID NOs:8-13, see above). See
(93) The carboxylate reductase encoded by the gene product of car and the phosphopantetheine transferase enhancer npt or sfp has broad substrate specificity, including terminal difunctional C4 and C5 carboxylic acids (Venkitasubramanian et al., Enzyme and Microbial Technology, 2008, 42, 130-137).
(94) In some embodiments, heptamethylenediamine is synthesized from the central precursor, 7-hydroxyheptanoate (which can be produced as described in
(95) In some embodiments, heptamethylenediamine is synthesized from the central precursor, 7-aminoheptanoate, by conversion of 7-aminoheptanoate to N7-acetyl-7-aminoheptanoate by a N-acetyltransferase such as a lysine N-acetyltransferase classified, for example, under EC 2.3.1.32; followed by conversion to N7-acetyl-7-aminoheptanal by a carboxylate reductase classified, for example, under EC 1.2.99.6 such as the gene product of car (see above) in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia) or the gene product of GriC & GriD; followed by conversion to N7-acetyl-1,7-diaminoheptane by a -transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, EC 2.6.1.46, or EC 2.6.1.82 such as SEQ ID NOs:8-13, see above; followed by conversion to heptamethylenediamine by an acetylputrescine deacylase classified, for example, under EC 3.5.1.62. See,
(96) In some embodiments, heptamethylenediamine is synthesized from the central precursor, pimelate semialdehyde, by conversion of pimelate semialdehyde to heptanedial by a carboxylate reductase classified, for example, under EC 1.2.99.6 such as the gene product of car (see above) in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia) or the gene product of GriC & GriD; followed by conversion to 7-aminoheptanal by a -transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, or EC 2.6.1.48; followed by conversion to heptamethylenediamine by a -transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, EC 2.6.1.46, or EC 2.6.1.82 such as SEQ ID NOs:8-13, see above. See
(97) Pathways Using Pimelate or Pimelate Semialdehyde as Central Precursor to 1,7-Heptanediol
(98) In some embodiments, 7-hydroxyheptanoate is synthesized from the central precursor, pimelate, by conversion of pimelate to pimelate semialdehyde by a carboxylate reductase classified, for example, under EC 1.2.99.6 such as the gene product of car (see above) in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia) or the gene product of GriC & GriD; followed by conversion to 7-hydroxyheptanoate by a dehydrogenase classified, for example, under EC 1.1.1.such as a 6-hydroxyhexanoate dehydrogenase classified, for example, under EC 1.1.1.258 such as the gene from of ChnD or a 5-hydroxypentanoate dehydrogenase classified, for example, under EC 1.1.1.such as the gene product of CpnD (see, for example, Iwaki et al., 2002, Appl. Environ. Microbiol., 68(11):5671-5684) or a 4-hydroxybutyrate dehydrogenase such as gbd. See
(99) In some embodiments, 1,7 heptanediol is synthesized from the central precursor, 7-hydroxyheptanoate, by conversion of 7-hydroxyheptanoate to 7-hydroxyheptanal by a carboxylate reductase classified, for example, under EC 1.2.99.6 such as the gene product of car (see above) in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia) or the gene product of GriC & GriD; followed by conversion of 7-hydroxyheptanal to 1,7 heptanediol by an alcohol dehydrogenase classified, for example, under EC 1.1.1.such as EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the gene product of YMR318C or YqhD (see, e.g., Liu et al., Microbiology, 2009, 155, 2078-2085; Larroy et al., 2002, Biochem 361(Pt 1), 163-172; or Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the protein having GenBank Accession No. CAA81612.1 (from Geobacillus stearothermophilus). See,
(100) Cultivation Strategy
(101) In some embodiments, the cultivation strategy entails achieving an aerobic, anaerobic or micro-aerobic cultivation condition.
(102) In some embodiments, the cultivation strategy entails nutrient limitation such as nitrogen, phosphate or oxygen limitation.
(103) In some embodiments, a cell retention strategy using, for example, ceramic hollow fiber membranes can be employed to achieve and maintain a high cell density during either fed-batch or continuous fermentation.
(104) In some embodiments, the principal carbon source fed to the fermentation in the synthesis of one or more C7 building blocks can derive from biological or non-biological feedstocks.
(105) In some embodiments, the biological feedstock can be, can include, or can derive from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid and formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, or municipal waste.
(106) The efficient catabolism of crude glycerol stemming from the production of biodiesel has been demonstrated in several microorganisms such as Escherichia coli, Cupriavidus necator, Pseudomonas oleavorans, Pseudomonas putida and Yarrowia lipolytica (Lee et al., Appl. Biochem. Biotechnol., 2012, 166, 1801-1813; Yang et al., Biotechnology for Biofuels, 2012, 5:13; Meijnen et al., Appl. Microbiol. Biotechnol., 2011, 90, 885-893).
(107) The efficient catabolism of lignocellulosic-derived levulinic acid has been demonstrated in several organisms such as Cupriavidus necator and Pseudomonas putida in the synthesis of 3-hydroxyvalerate via the precursor propanoyl-CoA (Jaremko and Yu, Journal of Biotechnology, 2011, 155, 2011, 293-298; Martin and Prather, Journal of Biotechnology, 2009, 139, 61-67).
(108) The efficient catabolism of lignin-derived aromatic compounds such as benzoate analogues has been demonstrated in several microorganisms such as Pseudomonas putida, Cupriavidus necator (Bugg et al., Current Opinion in Biotechnology, 2011, 22, 394-400; Perez-Pantoja et al., FEMS Microbiol. Rev., 2008, 32, 736-794).
(109) The efficient utilization of agricultural waste, such as olive mill waste water has been demonstrated in several microorganisms, including Yarrowia lipolytica (Papanikolaou et al., Bioresour. Technol., 2008, 99(7), 2419-2428).
(110) The efficient utilization of fermentable sugars such as monosaccharides and disaccharides derived from cellulosic, hemicellulosic, cane and beet molasses, cassava, corn and other agricultural sources has been demonstrated for several microorganism such as Escherichia coli, Corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcus lactis (see, e.g., Hermann et al, Journal of Biotechnology, 2003, 104, 155-172; Wee et al., Food Technol. Biotechnol., 2006, 44(2), 163-172; Ohashi et al., Journal of Bioscience and Bioengineering, 1999, 87(5), 647-654).
(111) The efficient utilization of furfural, derived from a variety of agricultural lignocellulosic sources, has been demonstrated for Cupriavidus necator (Li et al., Biodegradation, 2011, 22, 1215-1225).
(112) In some embodiments, the non-biological feedstock can be or can derive from natural gas, syngas, CO.sub.2/H.sub.2, methanol, ethanol, benzoic acid, non-volatile residue (NVR), a caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams.
(113) The efficient catabolism of methanol has been demonstrated for the methylotrophic yeast Pichia pastoris.
(114) The efficient catabolism of ethanol has been demonstrated for Clostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008, 105(6) 2128-2133).
(115) The efficient catabolism of CO.sub.2 and H.sub.2, which may be derived from natural gas and other chemical and petrochemical sources, has been demonstrated for Cupriavidus necator (Prybylski et al., Energy, Sustainability and Society, 2012, 2:11).
(116) The efficient catabolism of syngas has been demonstrated for numerous microorganisms, such as Clostridium ljungdahlii and Clostridium autoethanogenum (Kopke et al., Applied and Environmental Microbiology, 2011, 77(15), 5467-5475).
(117) The efficient catabolism of the non-volatile residue waste stream from cyclohexane processes has been demonstrated for numerous microorganisms, such as Delftia acidovorans and Cupriavidus necator (Ramsay et al., Applied and Environmental Microbiology, 1986, 52(1), 152-156).
(118) In some embodiments, the host microorganism is a prokaryote. For example, the prokaryote can be a bacterium from the genus Escherichia such as Escherichia coli; from the genus Clostridia such as Clostridium ljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; from the genus Corynebacteria such as Corynebacterium glutamicum; from the genus Cupriavidus such as Cupriavidus necator or Cupriavidus metallidurans; from the genus Pseudomonas such as Pseudomonas fluorescens, Pseudomonas putida or Pseudomonas oleavorans; from the genus Delftia such as Delftia acidovorans; from the genus Bacillus such as Bacillus subtillis; from the genus Lactobacillus such as Lactobacillus delbrueckii; or from the genus Lactococcus such as Lactococcus lactis. Such prokaryotes also can be a source of genes to construct recombinant host cells described herein that are capable of producing one or more C7 building blocks.
(119) In some embodiments, the host microorganism is a eukaryote. For example, the eukaryote can be a filamentous fungus, e.g., one from the genus Aspergillus such as Aspergillus niger. Alternatively, the eukaryote can be a yeast, e.g., one from the genus Saccharomyces such as Saccharomyces cerevisiae; from the genus Pichia such as Pichia pastoris; or from the genus Yarrowia such as Yarrowia lipolytica; from the genus Issatchenkia such as Issathenkia orientalis; from the genus Debaryomyces such as Debaryomyces hansenii; from the genus Arxula such as Arxula adenoinivorans; or from the genus Kluyveromyces such as Kluyveromyces lactis. Such eukaryotes also can be a source of genes to construct recombinant host cells described herein that are capable of producing one or more C7 building blocks.
(120) Metabolic Engineering
(121) The present disclosure provides methods involving less than all the steps described for all the above pathways. Such methods can involve, for example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve or more of such steps. Where less than all the steps are included in such a method, the first, and in some embodiments the only, step can be any one of the steps listed.
(122) Furthermore, recombinant hosts described herein can include any combination of the above enzymes such that one or more of the steps, e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more of such steps, can be performed within a recombinant host. This disclosure provides host cells of any of the genera and species listed and genetically engineered to express one or more (e.g., two, three, four, five, six, seven, eight, nine, 10, 11, 12 or more) recombinant forms of any of the enzymes recited in the disclosure. Thus, for example, the host cells can contain exogenous nucleic acids encoding enzymes catalyzing one or more of the steps of any of the pathways described herein.
(123) In addition, this disclosure recognizes that where enzymes have been described as accepting CoA-activated substrates, analogous enzyme activities associated with ACP-bound substrates exist that are not necessarily in the same enzyme class.
(124) Also, this disclosure recognizes that where enzymes have been described accepting (R)-enantiomers of substrate, analogous enzyme activities associated with (S)-enantiomer substrates exist that are not necessarily in the same enzyme class.
(125) This disclosure also recognizes that where an enzyme is shown to accept a particular co-factor, such as NADPH, or a co-substrate, such as acetyl-CoA, many enzymes are promiscuous in terms of accepting a number of different co-factors or co-substrates in catalyzing a particular enzyme activity. Also, this disclosure recognizes that where enzymes have high specificity for e.g., a particular co-factor such as NADH, an enzyme with similar or identical activity that has high specificity for the co-factor NADPH may be in a different enzyme class.
(126) In some embodiments, the enzymes in the pathways outlined herein are the result of enzyme engineering via non-direct or rational enzyme design approaches with aims of improving activity, improving specificity, reducing feedback inhibition, reducing repression, improving enzyme solubility, changing stereo-specificity, or changing co-factor specificity.
(127) In some embodiments, the enzymes in the pathways outlined herein can be gene dosed (i.e., overexpressed by having a plurality of copies of the gene in the host organism), into the resulting genetically modified organism via episomal or chromosomal integration approaches.
(128) In some embodiments, genome-scale system biology techniques such as Flux Balance Analysis can be utilized to devise genome scale attenuation or knockout strategies for directing carbon flux to a C7 building block.
(129) Attenuation strategies include, but are not limited to; the use of transposons, homologous recombination (double cross-over approach), mutagenesis, enzyme inhibitors and RNA interference (RNAi).
(130) In some embodiments, fluxomic, metabolomic and transcriptomal data can be utilized to inform or support genome-scale system biology techniques, thereby devising genome scale attenuation or knockout strategies in directing carbon flux to a C7 building block.
(131) In some embodiments, the host microorganism's tolerance to high concentrations of at least one C7 building block can be improved through continuous cultivation in a selective environment.
(132) In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, a recombinant glucose dehydrogenase such as the gene product of gdh can be overexpressed in the host organism (Satoh et al., J. Bioscience and Bioengineering, 2003, 95(4):335-341).
(133) In some embodiments, endogenous enzymes facilitating the conversion of NADPH to NADH can be attenuated, such as the NADH generation cycle that may be generated via inter-conversion of glutamate dehydrogenases classified under EC 1.4.1.2 (NADH-specific) and EC 1.4.1.4 (NADPH-specific). For example, avoiding dissipation of an NADH imbalance towards C7 building blocks, a NADPH-specific glutamate dehydrogenase can be attenuated.
(134) In some embodiments, an endogenous glutamate dehydrogenase (EC 1.4.1.3) that utilizes both NADH and NADPH as co-factors can be attenuated.
(135) In some embodiments, a membrane-bound enoyl-CoA reductases can be solubilized via expression as a fusion protein to a small soluble protein such as a maltose binding protein (Gloerich et al., FEBS Letters, 2006, 580, 2092-2096).
(136) In some embodiments using hosts that naturally accumulate polyhydroxyalkanoates, the endogenous polymer synthase enzymes can be attenuated in the host strain.
(137) In some embodiments, a L-alanine dehydrogenase can be overexpressed in the host to regenerate L-alanine from pyruvate as an amino donor for -transaminase reactions.
(138) In some embodiments, a L-glutamate dehydrogenase specific for the co-factor used to achieve co-factor imbalance can be overexpressed in the host to regenerate L-glutamate from 2-oxoglutarate as an amino donor for -transaminase reactions. For example, promoting dissipation of the NADH imbalance towards C7 building blocks, a NADH-specific glutamate dehydrogenase can be overexpressed.
(139) In some embodiments, enzymes such as pimeloyl-CoA dehydrogenase classified under, EC 1.3.1.62; an acyl-CoA dehydrogenase classified, for example, under EC 1.3.8.7 or EC 1.3.8.1; and/or a glutaryl-CoA dehydrogenase classified, for example, under EC 1.3.8.6 that degrade central metabolites and central precursors leading to and including C7 building blocks can be attenuated.
(140) In some embodiments, endogenous enzymes activating C7 building blocks via Coenzyme A esterification such as CoA-ligases (e.g., a pimeloyl-CoA synthetase) classified under, for example, EC 6.2.1.14 can be attenuated.
(141) In some embodiments, a methanol dehydrogenase and a formaldehyde dehydrogenase can be overexpressed in the host to allow methanol catabolism via formate.
(142) In some embodiments, a S-adenosylmethionine synthetase can be overexpressed in the host to generate S-Adenosyl-L-methionine as a co-factor for malonyl-ACP O-methyltransferase.
(143) In some embodiments, the efflux of a C7 building block across the cell membrane to the extracellular media can be enhanced or amplified by genetically engineering structural modifications to the cell membrane or increasing any associated transporter activity for a C7 building block.
(144) The efflux of heptamethylenediamine can be enhanced or amplified by overexpressing broad substrate range multidrug transporters such as Blt from Bacillus subtilis (Woolridge et al., 1997, J. Biol. Chem., 272(14):8864-8866); AcrB and AcrD from Escherichia coli (Elkins & Nikaido, 2002, J. Bacteriol., 184(23), 6490-6499), NorA from Staphylococcus aereus (Ng et al., 1994, Antimicrob Agents Chemother, 38(6), 1345-1355), or Bmr from Bacillus subtilis (Neyfakh, 1992, Antimicrob Agents Chemother, 36(2), 484-485).
(145) The efflux of 7-aminoheptanoate and heptamethylenediamine can be enhanced or amplified by overexpressing the solute transporters such as the lysE transporter from Corynebacterium glutamicum (Bellmann et al., 2001, Microbiology, 147, 1765-1774).
(146) The efflux of pimelic acid can be enhanced or amplified by overexpressing a dicarboxylate transporter such as the SucE transporter from Corynebacterium glutamicum (Huhn et al., Appl. Microbiol. & Biotech., 89(2), 327-335).
(147) Producing C7 Building Blocks Using a Recombinant Host
(148) Typically, one or more C7 building blocks can be produced by providing a host microorganism and culturing the provided microorganism with a culture medium containing a suitable carbon source as described above. In general, the culture media and/or culture conditions can be such that the microorganisms grow to an adequate density and produce a C7 building block efficiently. For large-scale production processes, any method can be used such as those described elsewhere (Manual of Industrial Microbiology and Biotechnology, 2.sup.nd Edition, Editors: A. L. Demain and J. E. Davies, ASM Press; and Principles of Fermentation Technology, P. F. Stanbury and A. Whitaker, Pergamon). Briefly, a large tank (e.g., a 100 gallon, 200 gallon, 500 gallon, or more tank) containing an appropriate culture medium is inoculated with a particular microorganism. After inoculation, the microorganism is incubated to allow biomass to be produced. Once a desired biomass is reached, the broth containing the microorganisms can be transferred to a second tank. This second tank can be any size. For example, the second tank can be larger, smaller, or the same size as the first tank. Typically, the second tank is larger than the first such that additional culture medium can be added to the broth from the first tank. In addition, the culture medium within this second tank can be the same as, or different from, that used in the first tank.
(149) Once transferred, the microorganisms can be incubated to allow for the production of a C7 building block. Once produced, any method can be used to isolate C7 building blocks. For example, C7 building blocks can be recovered selectively from the fermentation broth via adsorption processes. In the case of pimelic acid and 7-aminoheptanoic acid, the resulting eluate can be further concentrated via evaporation, crystallized via evaporative and/or cooling crystallization, and the crystals recovered via centrifugation. In the case of heptamethylenediamine and 1,7-heptanediol, distillation may be employed to achieve the desired product purity.
(150) The present disclosure is further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1
(151) Strain Selection
(152) A series of six biotin overproducing strains of E. coli were obtained, these were assessed for biotin productivity in small scale experiments and, based on initial biotin productivity, two of the strains were selected for further analysis, FERM BP 4928 and FERM BP 5667. These two strains were also analysed in high density fermentations with titres of biotin metabolites being >200 mg/L in chemically defined growth medium.
(153) The biotin overproducing strains were modified to facilitate further manipulation and analysis; the plasmids bearing extra copies of the biotin operon were removed and; the T7 RNA polymerase gene was introduced to allow high level expression from the T7 promoter. This generated strains FERM BP-4928-P2(DE3)-4 and FERM BP-5667-P2(DE3)-A7.
Example 2
(154) Expression of Thioesterases
(155) The release of pimelic acid from pimelyl-ACP is catalysed by acyl-ACP thioesterases. The expression of a suitable thioesterase would be predicted to result in the release of pimelic acid. Nine different thioesterases were selected on the basis of diversity and in vitro activity and, following induction of expression, culture supernatants were analysed for pimelic acid. Pimelic acid levels were in all cases below the level of quantification and in some cases induction of gene expression also resulted in the cessation of cell growth. See Table 1.
Example 3
(156) Deletion of Biotin Biosynthesis Genes
(157) It was sought to increase the potential pool of pimelyl-ACP by deleting specific biotin biosynthesis genes from the chromosome. The bioF gene was the major target as the product of this gene catalyses the conversion of pimelyl-ACP to 8-amino-7-oxo-nonanoic acid (KAPA) (
(158) The strains carrying deletions of bioF and bioH were then analysed, and all were biotin auxotrophs. The bioF mutant strains were found to produce detectable levels of pimelic acid in shake flask experiments (2.5 mg/L), while the bioH mutant strains were found to produce both pimelic acid methyl ester (0.6 mg/L) and pimelic acid (0.2 mg/L) in the culture supernatant. The production of both pimelic acid and pimelic acid methyl ester in the absence of an additional thioesterase indicates that native E. coli thioesterase activities are able to release ACP bound pimelic acid (and methyl ester). Additional thioesterase was expressed in the bioF strains, however it did not resulted in an increase in pimelic acid levels above those found without an additional thioesterase. See Table 1.
(159) In Table 1, pimelate production was analyzed in biotin mutant strains. Strains were cultured in chemically defined medium and were grown for 24 to 48 hours following induction. Culture supernatants were then analysed by LC-MS for pimelic acid and pimelic acid methyl ester.
(160) TABLE-US-00001 TABLE 1 pimelic acid pimelic acid methyl ester Strain bioF bioH thioesterase (ppm) (ppm) FERM BP-4928-P2(DE3)-4 WT WT None 0 0 FERM BP-4928-P2(DE3)-4 WT WT 9 different 0, trace 0, trace TEs.sup.1 amount - TE2, amount - TE5 (<0.1 TE11 ppm) FERM BP-4928-P2(DE3)-4 WT None 2.5 0 FERM BP-4928-P2(DE3)-4 WT None 0.2 0.6 FERM BP-4928-P2(DE3)-4 None 0.5 1.5 FERM BP-4928-P2(DE3)-4 WT 7 different 0.1-0.8 0, 0.2 with TEs.sup.2 TE11 .sup.1TEs utilised were TE2, TE5, TE8, TE11, TE14, TE17, YciA, AA077182, tesA. .sup.2TEs utilised were TE2, TE5, TE8, TE11, TE14, TE17, tesA.
Example 4
(161) Production of 7-AHA in E. coli
(162) Conversion of pimelic acid to 7-AHA requires the activity of two enzymes, CAR and -TAM (
(163) As shown in
Example 5
(164) Enzyme Activity of -Transaminase Using Pimelate Semialdehyde as Substrate and Forming 7-Aminoheptanoate
(165) A sequence encoding an N-terminal His-tag was added to the genes from Chromobacterium violaceum, Pseudomonas syringae, Rhodobacter sphaeroides, and Vibrio Fluvialis encoding the -transaminases of SEQ ID NOs: 8, 10, 11 and 13, respectively (see
(166) The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was resuspended and lysed via sonication. The cell debris was separated from the supernatant via centrifugation and the cell free extract was used immediately in enzyme activity assays.
(167) Enzyme activity assays in the reverse direction (i.e., 7-aminoheptanoate to pimelate semialdehyde) were performed in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mM 7-aminoheptanoate, 10 mM pyruvate and 100 M pyridoxyl 5 phosphate. Each enzyme activity assay reaction was initiated by adding cell free extract of the -transaminase gene product or the empty vector control to the assay buffer containing the 7-aminoheptanoate and incubated at 25 C. for 4 h, with shaking at 250 rpm. The formation of L-alanine from pyruvate was quantified via RP-HPLC.
(168) Each enzyme only control without 7-aminoheptanoate demonstrated low base line conversion of pyruvate to L-alanine. See
(169) Enzyme activity in the forward direction (i.e., pimelate semialdehyde to 7-aminoheptanoate) was confirmed for the transaminases of SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 13. Enzyme activity assays were performed in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mM pimelate semialdehyde, 10 mM L-alanine and 100 M pyridoxyl 5 phosphate. Each enzyme activity assay reaction was initiated by adding a cell free extract of the -transaminase gene product or the empty vector control to the assay buffer containing the pimelate semialdehyde and incubated at 25 C. for 4 h, with shaking at 250 rpm. The formation of pyruvate was quantified via RP-HPLC.
(170) The gene product of SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 13 accepted pimelate semialdehyde as substrate as confirmed against the empty vector control. See
Example 6
(171) Enzyme Activity of Carboxylate Reductase Using Pimelate as Substrate and Forming Pimelate Semialdehyde
(172) A sequence encoding a HIS-tag was added to the genes from Segniliparus rugosus and Segniliparus rotundus that encode the carboxylate reductases of SEQ ID NOs: 4 and 7, respectively (see
(173) The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was resuspended and lysed via sonication, and the cell debris was separated from the supernatant via centrifugation. The carboxylate reductases and phosphopantetheine transferases were purified from the supernatant using Ni-affinity chromatography, diluted 10-fold into 50 mM HEPES buffer (pH=7.5), and concentrated via ultrafiltration.
(174) Enzyme activity assays (i.e., from pimelate to pimelate semialdehyde) were performed in triplicate in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 2 mM pimelate, 10 mM MgCl.sub.2, 1 mM ATP and 1 mM NADPH. Each enzyme activity assay reaction was initiated by adding purified carboxylate reductase and phosphopantetheine transferase gene products or the empty vector control to the assay buffer containing the pimelate and then incubated at room temperature for 20 min. The consumption of NADPH was monitored by absorbance at 340 nm. Each enzyme only control without pimelate demonstrated low base line consumption of NADPH. See
(175) The gene products of SEQ ID NO 4 and SEQ ID NO 7, enhanced by the gene product of sfp, accepted pimelate as substrate, as confirmed against the empty vector control (see
Example 7
(176) Enzyme Activity of Carboxylate Reductase Using 7-Hydroxyheptanoate as Substrate and Forming 7-Hydroxyheptanal
(177) A sequence encoding a His-tag was added to the genes from Mycobacterium marinum, Mycobacterium smegmatis, Segniliparus rugosus, Mycobacterium smegmatis, Mycobacterium massiliense, and Segniliparus rotundus that encode the carboxylate reductases of SEQ ID NOs: 2-7respectively (see
(178) Each expression vector was transformed into a BL21[DE3] E. coli host and the resulting recombinant E. coli strains were cultivated at 37 C. in a 250 mL shake flask culture containing 50 mL LB media and antibiotic selection pressure, with shaking at 230 rpm. Each culture was induced overnight at 37 C. using an auto-induction media.
(179) The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was resuspended and lysed via sonication. The cell debris was separated from the supernatant via centrifugation. The carboxylate reductases and phosphopantetheine transferase were purified from the supernatant using Ni-affinity chromatography, diluted 10-fold into 50 mM HEPES buffer (pH=7.5) and concentrated via ultrafiltration.
(180) Enzyme activity (i.e., 7-hydroxyheptanoate to 7-hydroxyheptanal) assays were performed in triplicate in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 2 mM 7-hydroxyheptanal, 10 mM MgCl.sub.2, 1 mM ATP, and 1 mM NADPH. Each enzyme activity assay reaction was initiated by adding purified carboxylate reductase and phosphopantetheine transferase or the empty vector control to the assay buffer containing the 7-hydroxyheptanoate and then incubated at room temperature for 20 min. The consumption of NADPH was monitored by absorbance at 340 nm. Each enzyme only control without 7-hydroxyheptanoate demonstrated low base line consumption of NADPH. See
(181) The gene products of SEQ ID NO 2-7, enhanced by the gene product of sfp, accepted 7-hydroxyheptanoate as substrate as confirmed against the empty vector control (see
Example 8
(182) Enzyme Activity of -Transaminase for 7-Aminoheptanol, Forming 7-Oxoheptanol
(183) A nucleotide sequence encoding an N-terminal His-tag was added to the Chromobacterium violaceum, Pseudomonas syringae and Rhodobacter sphaeroides genes encoding the -transaminases of SEQ ID NOs: 8, 10 and 11, respectively (see
(184) The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was resuspended and lysed via sonication. The cell debris was separated from the supernatant via centrifugation and the cell free extract was used immediately in enzyme activity assays.
(185) Enzyme activity assays in the reverse direction (i.e., 7-aminoheptanol to 7-oxoheptanol) were performed in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mM 7-aminoheptanol, 10 mM pyruvate, and 100 M pyridoxyl 5 phosphate. Each enzyme activity assay reaction was initiated by adding cell free extract of the -transaminase gene product or the empty vector control to the assay buffer containing the 7-aminoheptanol and then incubated at 25 C. for 4 h, with shaking at 250 rpm. The formation of L-alanine was quantified via RP-HPLC.
(186) Each enzyme only control without 7-aminoheptanol had low base line conversion of pyruvate to L-alanine. See
(187) The gene products of SEQ ID NO 8, 10 & 11 accepted 7-aminoheptanol as substrate as confirmed against the empty vector control (see
Example 9
(188) Enzyme Activity of -Transaminase Using Heptamethylenediamine as Substrate and Forming 7-Aminoheptanal
(189) A sequence encoding an N-terminal His-tag was added to the Chromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae, Rhodobacter sphaeroides, Escherichia coli, and Vibrio fluvialis genes encoding the -transaminases of SEQ ID NOs: 8-13, respectively (see
(190) The pellet from each induced shake flask culture was harvested via centrifugation. Each pellet was resuspended and lysed via sonication. The cell debris was separated from the supernatant via centrifugation and the cell free extract was used immediately in enzyme activity assays.
(191) Enzyme activity assays in the reverse direction (i.e., heptamethylenediamine to 7-aminoheptanal) were performed in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mM heptamethylenediamine, 10 mM pyruvate, and 100 pyridoxyl 5 phosphate. Each enzyme activity assay reaction was initiated by adding cell free extract of the -transaminase gene product or the empty vector control to the assay buffer containing the heptamethylenediamine and then incubated at 25 C. for 4 h, with shaking at 250 rpm. The formation of L-alanine was quantified via RP-HPLC.
(192) Each enzyme only control without heptamethylenediamine had low base line conversion of pyruvate to L-alanine. See
(193) The gene products of SEQ ID NO 8-13 accepted heptamethylenediamine as substrate as confirmed against the empty vector control (see
Example 10
(194) Enzyme Activity of Carboxylate Reductase for N7-Acetyl-7-Aminoheptanoate, Forming N7-Acetyl-7-Aminoheptanal
(195) The activity of each of the N-terminal His-tagged carboxylate reductases of SEQ ID NOs: 3, 6, and 7 (see Examples 7, and
(196) The gene products of SEQ ID NO 3, 6, and 7, enhanced by the gene product of sfp, accepted N7-acetyl-7-aminoheptanoate as substrate as confirmed against the empty vector control (see
Example 11
(197) Enzyme Activity of -Transaminase Using N7-Acetyl-1,7-Diaminoheptane, and Forming N7-Acetyl-7-Aminoheptanal
(198) The activity of the N-terminal His-tagged -transaminases of SEQ ID NOs: 8-13 (see Example 9, and
(199) Each enzyme only control without N7-acetyl-1,7-diaminoheptane demonstrated low base line conversion of pyruvate to L-alanine. See
(200) The gene product of SEQ ID NO 8-13 accepted N7-acetyl-1,7-diaminoheptane as substrate as confirmed against the empty vector control (see
(201) Given the reversibility of the -transaminase activity (see example 2), the gene products of SEQ ID 8-13 accept N7-acetyl-7-aminoheptanal as substrate forming N7-acetyl-1,7-diaminoheptane.
Example 12
(202) Enzyme Activity of Carboxylate Reductase Using Pimelate Semialdehyde as Substrate and Forming Heptanedial
(203) The N-terminal His-tagged carboxylate reductase of SEQ ID NO 7 (see Example 7 and
(204) The gene product of SEQ ID NO 7, enhanced by the gene product of sfp, accepted pimelate semialdehyde as substrate as confirmed against the empty vector control (see
OTHER EMBODIMENTS
(205) It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.