Methods, reagents and cells for biosynthesizing compounds
09957535 ยท 2018-05-01
Assignee
Inventors
Cpc classification
C12N9/00
CHEMISTRY; METALLURGY
C12P7/40
CHEMISTRY; METALLURGY
C12N9/0008
CHEMISTRY; METALLURGY
C12Y206/01018
CHEMISTRY; METALLURGY
C08G69/26
CHEMISTRY; METALLURGY
International classification
C12P13/00
CHEMISTRY; METALLURGY
C12P7/40
CHEMISTRY; METALLURGY
C08G69/26
CHEMISTRY; METALLURGY
Abstract
This document describes biochemical pathways for producing glutaric acid, 5-aminopentanoic acid, 5-hydroxypentanoic acid, cadaverine or 1,5-pentanediol by forming one or two terminal functional groups, comprised of carboxyl, amine or hydroxyl group, in a C5 backbone substrate such as 2-oxoglutarate.
Claims
1. A method of biosynthesizing a C5 building block selected from the group consisting of glutaric acid, 5-hydroxypentanoic acid, 5-aminopentanoic acid, cadaverine, and 1,5-pentanediol, in a recombinant host, the method comprising: (a) (i) enzymatically converting 2-oxo-adipate to 2-amino-adipate using at least one polypeptide having the activity of an alpha-aminotransaminase classified under EC 2.6.1.7 or EC 2.6.1.39, (ii) enzymatically converting 2-oxo-adipate to 5-oxopentanoate using at least one polypeptide having the activity of a 2-oxoacid decarboxylase classified under EC 4.1.1.71, EC 4.1.1.72, EC 4.1.1.43 or EC 4.1.1.74, or (iii) enzymatically converting 2-oxo-adipate to glutaryl-CoA using at least one polypeptide having the activity of a 2-oxoglutarate dehydrogenase complex classified under EC 1.2.4.2, EC 1.8.1.4 and EC 2.3.1.61; (b) enzymatically converting the 2-amino-adipate, 5-oxopentanoate, or glutaryl-CoA to the C5 building block, wherein: at least one polypeptide having the activity of a thioesterase classified under EC 3.1.2.-, a glutaconate CoA-transferase classified under EC 2.8.3.12, a succinate-CoA ligase classified under EC 6.2.1.5, an aldehyde dehydrogenase classified under EC 1.2.1.-, an acyl-[acp] thioesterase classified under EC 3.1.2.-, a 5-oxopentanoate dehydrogenase classified under EC 1.2.1.-, a 7-oxoheptanoate dehydrogenase classified under EC 1.2.1.-, or a 6-oxohexanoate dehydrogenase classified under EC 1.2.1.63 enzymatically forms a terminal carboxyl group of the C5 building block; at least one polypeptide having the activity of an alpha-amino decarboxylase classified under EC 4.1.1.-, a -transaminase classified under EC 2.6.1.-, or a deacetylase classified under EC 3.5.1.17 or EC 3.5.1.62 enzymatically forms a terminal amine group of the C5 building block; and/or at least one polypeptide having the activity of a 6-hydroxyhexanoate dehydrogenase classified under EC 1.1.1.258, a 5-hydroxypentanoate dehydrogenase classified under EC 1.1.1-, a 4-hydroxybutyrate dehydratase classified under EC 1.1.1-, or an alcohol dehydrogenase classified under EC 1.1.1- enzymatically forms a terminal hydroxyl group of the C5 building block.
2. The method of claim 1, wherein the polypeptide having the activity of a 2-oxoacid decarboxylase has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1.
3. The method of claim 1, wherein the polypeptide having the activity of a 2-oxoglutarate dehydrogenase complex has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16.
4. The method of claim 1, wherein the polypeptide having the activity of a thioesterase activity has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 17 or 18.
5. The method of claim 1, wherein the polypeptide having the activity of a -transaminase activity has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 8-13.
6. The method of claim 1, wherein the polypeptide having the activity of an alpha-amino decarboxylase has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 19-22.
7. The method of claim 1, wherein a polypeptide having the activity of a carboxylate reductase classified under EC 1.2.99.6 enzymatically forms a terminal aldehyde group as an intermediate in forming the C5 building block.
8. The method of claim 7, wherein the polypeptide having the activity of a carboxylate reductase has at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 2-7.
9. The method of claim 7, wherein the polypeptide having the activity of a carboxylate reductase is used in combination with a polypeptide having the activity of a phosphopantetheine transferase, wherein the polypeptide having the activity of a phosphopantetheine transferase has at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14 or 15.
10. The method of claim 1, wherein the 2-oxo-adipate is obtained by enzymatically converting 2-oxo-glutarate to 2-oxo-adipate using at least one polypeptide having an activity selected from the group consisting of a 2-isopropylmalate synthase or homocitrate synthase classified under EC 2.3.3.14 or EC 2.3.3.13, a homoaconitate hydratase or 3-isopropylmalate dehydratase classified under EC 4.2.1.114, EC 4.2.1.36 or EC 4.2.1.33, and a homoisocitrate dehydrogenase or 3-isopropylmalate dehydrogenase classified under EC 1.1.1.85, EC 1.1.1.87 or EC 1.1.1.286.
11. The method of claim 1, wherein glutaryl-CoA is enzymatically converted to glutaric acid using at least one polypeptide having an activity selected from the group consisting of (i) a thioesterase classified under EC 3.1.2.-, (ii) a succinate-CoA-ligase classified under EC 6.2.1.5, (iii) a CoA-transferase classified under EC 2.8.3.-, (iv) an acylating dehydrogenase classified under EC 1.2.1.10 or EC 1.2.1.76 and an aldehyde dehydrogenase classified under EC 1.2.1.-, and (v) an aldehyde dehydrogenase classified under EC 1.2.1.3.
12. The method of claim 1, wherein 2-amino-adipate is enzymatically converted to 5-aminopentanoic acid using a polypeptide having the activity of an alpha-amino decarboxylase classified under EC 4.1.1.-.
13. The method of claim 1, wherein 5-oxopentanoic acid is enzymatically converted to 5-hydroxypentanoic acid using at least one polypeptide having an activity selected from the group consisting of a 5-hydroxypentanoate dehydrogenase classified under EC 1.1.1.-, a 6-hydroxyhexanoate dehydrogenase classified under EC 1.1.1.258, and an alcohol dehydrogenase classified under EC 1.1.1.-.
14. The method of claim 1, wherein 5-oxopentanoic acid is enzymatically converted to cadaverine using at least one polypeptide having an activity selected from the group consisting of a carboxylate reductase classified under EC 1.2.99.6 and a -transaminase classified under EC 2.6.1.-.
15. The method of claim 1, wherein the C5 building block is glutaric acid and the method further comprises enzymatically converting glutaric acid to 5-aminopentanoic acid using at least one polypeptide having an activity selected from the group consisting of a carboxylate reductase classified under EC 1.2.99.6 and a -transaminase classified under EC 2.6.1.-.
16. The method of claim 1, wherein the C5 building block is glutaric acid and the method further comprises enzymatically converting glutaric acid to 5-hydroxypentainoic acid using at least one polypeptide having an activity selected from the group consisting of a carboxylate reductase classified under EC 1.2.99.6 and an alcohol dehydrogenase classified under EC 1.1.1.-.
17. The method of claim 1, wherein the C5 building block is 5-aminopentanoic acid and the method further comprises enzymatically converting 5-aminopentanoic acid to cadaverine using at least one polypeptide having an activity selected from the group consisting of a carboxylate reductase classified under EC 1.2.99.6 and a -transaminase classified under EC 2.6.1.-.
18. The method of claim 1, wherein the C5 building block is 5-hydroxypentanoic acid and the method further comprises enzymatically converting 5-hydroxypentanoic acid to cadaverine using at least one polypeptide having an activity selected from the group consisting of a carboxylate reductase classified under EC 1.2.99.6, a -transaminase classified under EC 2.6.1.-, and an alcohol dehydrogenase classified under EC 1.1.1.-.
19. The method of claim 1, wherein the C5 building block is 5-hydroxypentanoic acid and the method further comprises enzymatically converting the 5-hydroxypentanoic acid to 1,5-pentanediol using at least one polypeptide having an activity selected from the group consisting of a carboxylate reductase classified under EC 1.2.99.6 and an alcohol dehydrogenase classified under EC 1.1.1.-.
20. The method of claim 1, wherein the C5 building block is 5-aminopentanoic acid and the method further comprises enzymatically converting the 5-aminopentanoic acid to cadaverine using at least one polypeptide having an activity selected from the group consisting of an N-acetyltransferase classified under EC 2.3.1.32, a carboxylate reductase classified under EC 1.2.99.6, a -transaminase classified under EC 2.6.1.-, and a deacetylase classified under EC 3.5.1.17 or EC 3.5.1.62.
21. A method for producing a bioderived 5-carbon compound comprising performing the method according to claim 1 under conditions and for a sufficient period of time to produce the bioderived 5-carbon compound, wherein, optionally, the bioderived 5-carbon compound is selected from the group consisting of glutaric acid, 5-hydroxypentanoic acid, 5-aminopentanoic acid, cadaverine, and 1,5-pentanediol, and combinations thereof.
Description
DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION
(20) This document provides enzymes, non-natural pathways, cultivation strategies, feedstocks, host microorganisms and attenuations to the host's biochemical network, which generates a five carbon chain backbone such as glutaryl-CoA or 5-oxopentanoate (also known as glutarate semialdehyde) from central metabolites in which one or two terminal functional groups may be formed leading to the synthesis of one or more of glutaric acid, 5-aminopentanoic acid, cadaverine (also known as 1,5 pentanediamine), 5-hydroxypentanoic acid, or 1,5-pentanediol (hereafter C5 building blocks). Glutarate semialdehyde (also known as 5-oxopentanoate) can be produced as an intermediate to other products. 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 C5 building block. The term central metabolite is used herein to denote a metabolite that is produced in all microorganisms to support growth.
(21) Host microorganisms described herein can include endogenous pathways that can be manipulated such that one or more C5 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.
(22) 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.
(23) 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.
(24) 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 including (i) a homocitrate synthase or a 2-isopropylmalate synthase, (ii) a homoaconitate hydratase or a 3-isopropylmalate dehydratase (iii) a homoisocitrate dehydrogenase or 3-isopropylmalate dehydrogenase, (iv) a 2-oxoacid decarboxylase, a 2-oxoglutarate dehydrogenase complex or an alpha-amino decarboxylase, a 4-hydroxybutyrate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, an alcohol dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, an aldehyde dehydrogenase, a -transaminase, or a carboxylate reductase. In recombinant hosts expressing a carboxylate reductase, a phosphopantetheinyl transferase also can be expressed as it enhances activity of the carboxylate reductase.
(25) In some embodiments, a recombinant host can include at least one exogenous nucleic acid encoding one or more of a (i) a homocitrate synthase or a 2-isopropylmalate synthase, (ii) a homoaconitate hydratase or a 3-isopropylmalate dehydratase (iii) a homoisocitrate dehydrogenase or 3-isopropylmalate dehydrogenase, and produce 2-oxoadipate.
(26) In some embodiments, a recombinant host that produces 2-oxoadipate can include at least one exogenous nucleic acid encoding a 2-oxoacid decarboxylase or a 2-oxoglutarate dehydrogenase complex, and further produce 5-oxopentanoate or glutaryl-CoA.
(27) In some embodiments, a recombinant host producing 5-oxopentanoate includes at least one exogenous nucleic acid encoding an aldehyde dehydrogenase such as a succinate semialdehyde dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, or a 7-oxoheptanoate dehydrogenase and produces glutarate.
(28) In some embodiments, a recombinant host producing glutaryl-CoA includes at least one exogenous nucleic acid encoding a (i) a thioesterase, (ii) a reversible CoA-ligase, (iii) a CoA-transferase, or (iv) an acylating dehydrogenase, and (v) an aldehyde dehydrogenase and further produce glutaric acid or 5-oxopentanoate.
(29) In some embodiments, a recombinant host that produces 2-oxoadipate can include at least one exogenous nucleic acid encoding an alpha-amino transaminase and an alpha-amino decarboxylase, and further produce 5-aminopentanoate.
(30) In some embodiments, a recombinant host that produces 5-oxopentanoate can include at least one exogenous nucleic acid encoding (i) a reversible -transaminase (e.g., a 5-aminovalerate transaminase) and produce 5-aminopentanoate.
(31) In some embodiments, a recombinant host that produces 5-oxopentanoate can include at least one exogenous nucleic acid encoding an alcohol dehydrogenase such as 4-hydroxybutyrate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, and further produce 5-hydroxypentanoate.
(32) A recombinant host producing 5-hydroxypentanoic acid further can include one or more of (i) a carboxylate reductase and (ii) an alcohol dehydrogenase, and produce 1,5-pentanediol.
(33) A recombinant host producing 5-aminopentanoate, 5-hydroxypentanoate, 1,5-pentanediol or glutarate semialdehyde further can include one or more of (i) a carboxylate reductase, (ii) a -transaminase, (iii) a N-acetyltransferase, (iv) an alcohol dehydrogenase and (v) a deacetylase, and produce cadaverine.
(34) 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.
(35) Any of the enzymes described herein that can be used for production of one or more C5 building blocks can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 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) or on the basis of the immature enzyme (e.g., with any signal sequence included). It also will be appreciated that the initial methionine residue may or may not be present on any of the enzyme sequences described herein.
(36) For example, a 2-oxoacid decarboxylase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Salmonella typhimurium indolepyruvate decarboxylase (see Genbank Accession No. CAC48239.1, SEQ ID NO: 1). See,
(37) For example, a carboxylate reductase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 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,
(38) For example, a -transaminase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 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 Genbank Accession No. AAA57874.1, 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,
(39) For example, a phosphopantetheinyl transferase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Bacillus subtilis phosphopantetheinyl transferase (see Genbank Accession No. CAA44858.1, SEQ ID NO: 14) or a Nocardia sp. NRRL 5646 phosphopantetheinyl transferase (see Genbank Accession No. ABI83656.1, SEQ ID NO: 15). See
(40) For example, a 2-oxoglutaryl-CoA dehydrogenase complex described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of an Azotobacter vinelandii 2-oxoglutarate dehydrogenase complex (see Genbank Accession Nos. CAA36680.1 & CAA36678.1 & CAA36679.1, SEQ ID NO: 16). See
(41) For example, a thioesterase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of a Lactobacillus brevis acyl-[acp] thioesterase (see Genbank Accession Nos. ABJ63754.1, SEQ ID NO: 17), a Lactobacillus plantarum acyl-[acp] thioesterase (see Genbank Accession Nos. CCC78182.1, SEQ ID NO: 18). See
(42) For example, an alpha-amino decarboxylase described herein can have at least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of an Escherichia coli glutamate decarboxylase (see Genbank Accession No. AAA23833.1, SEQ ID: 19), an Escherichia coli lysine decarboxylase (see Genbank Accession No. AAA23536.1, SEQ ID: 20), an Escherichia coli ornithine decarboxylase (see Genbank Accession No. AAA62785.1, SEQ ID: 21), an Escherichia coli lysine decarboxylase (see Genbank Accession No. BAA21656.1, SEQ ID: 22). See
(43) The percent identity (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 Fish & Richardson's web site (e.g., 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-i 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.
(44) 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 (homology) 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 (homology) value is rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2. It also is noted that the length value will always be an integer.
(45) 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.
(46) Functional fragments of any of the enzymes described herein can also be used in the methods of the document. 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.
(47) This document also provides (i) functional variants of the enzymes used in the methods of the document 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.
(48) 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), 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.
(49) 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 reductase, deacetylase, N-acetyltransferase, synthase, hydratase, dehydrogenase, decarboxylase, or -transaminase as described herein.
(50) In addition, the production of one or more C5 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.
(51) Enzymes Generating the Terminal Carboxyl Groups in the Biosynthesis of a C5 Building Block
(52) As depicted in
(53) In some embodiments, a terminal carboxyl group leading to the synthesis of glutarate is enzymatically formed by a thioesterase classified under EC 3.1.2.-, such as the gene product of YciA, tesB (Genbank Accession No. AAA24665.1) or Acot13 (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).
(54) In some embodiments, the second terminal carboxyl group leading to the synthesis of glutaric acid is enzymatically formed by a CoA-transferase such as a glutaconate CoA-transferase classified, for example, under EC 2.8.3.12 such as from Acidaminococcus fermentans. See, for example, Buckel et al., 1981, Eur. J. Biochem., 118:315-321. See, e.g.,
(55) In some embodiments, the second terminal carboxyl group leading to the synthesis of glutaric acid is enzymatically formed by a reversible CoA-ligase such as a succinate-CoA ligase classified, for example, under EC 6.2.1.5 such as from Thermococcus kodakaraensis. See, for example, Shikata et al., 2007, J. Biol. Chem., 282(37):26963-26970. See, e.g.,
(56) In some embodiments, the second terminal carboxyl group leading to the synthesis of glutaric acid is enzymatically formed by an acyl-[acp] thioesterase classified under EC 3.1.2.-, such as the acyl-[acp] thioesterase from Lactobacillus brevis (GenBank Accession No. ABJ63754.1, SEQ ID NO: 17) or from Lactobacillus plantarum (GenBank Accession No. CCC78182.1, SEQ ID NO: 18). Such acyl-[acp] thioesterases have C6-C8 chain length specificity (see, for example, Jing et al., 2011, BMC Biochemistry, 12(44)). See, e.g.,
(57) In some embodiments, the second terminal carboxyl group leading to the synthesis of glutaric acid is enzymatically formed by an aldehyde dehydrogenase classified, for example, under EC 1.2.1.3 (see, Guerrillot & Vandecasteele, Eur. J. Biochem., 1977, 81, 185-192). See, e.g.,
(58) In some embodiments, the second terminal carboxyl group leading to the synthesis of glutaric acid is enzymatically formed by an aldehyde dehydrogenase classified under EC 1.2.1.- such as a glutarate semialdehyde dehydrogenase classified, for example, under EC 1.2.1.20, a succinate-semialdehyde dehydrogenase classified, for example, under EC 1.2.1.16 or EC 1.2.1.79, or an aldehyde dehydrogenase classified under EC 1.2.1.3. For example, an aldehyde dehydrogenase classified under EC 1.2.1.- can be a 5-oxopentanoate dehydrogenase such as the gene product of CpnE, a 6-oxohexanoate dehydrogenase (e.g., the gene product of ChnE from Acinetobacter sp.), or a 7-oxoheptanoate dehydrogenase (e.g., the gene product of ThnG from Sphingomonas macrogolitabida) (Iwaki et al., Appl. Environ. Microbiol., 1999, 65(11), 5158-5162; Lopez-Sanchez et al., Appl. Environ. Microbiol., 2010, 76(1), 110-118). For example, a 6-oxohexanoate dehydrogenase can be classified under EC 1.2.1.63 such as the gene product of ChnE. For example, a 7-oxoheptanoate dehydrogenase can be classified under EC 1.2.1.-. See, e.g.,
(59) Enzymes Generating the Terminal Amine Groups in the Biosynthesis of a C5 Building Block
(60) As depicted in
(61) In some embodiments, the first terminal carboxyl group is formed by an alpha-amino decarboxylase classified, for example, under EC 4.1.1.- such as EC 4.1.1.15, EC 4.1.1.17, EC 4.1.1.18 or EC 4.1.1.19.
(62) In some embodiments, the first terminal carboxyl group is formed by a 5-aminovalerate transaminase classified, for example, under EC 2.6.1.48, such as obtained from Clostridium viride. 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).
(63) In some embodiments, one terminal amine group leading to the synthesis of 5-aminopentanol or 5-aminopentanal can be 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 (Genbank Accession No. AEA39183.1, SEQ ID NO: 13), Vibrio fluvialis (Genbank Accession No. AAA57874.1, SEQ ID NO: 12), or Streptomyces griseus. Some of the -transaminases classified, for example, under EC 2.6.1.29 or EC 2.6.1.82 are diamine -transaminases (e.g., SEQ ID NO: 11). See,
(64) 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, forming the first terminal amine group in adipate semialdehyde (Kaulmann et al., Enzyme and Microbial Technology, 2007, 41, 628-637).
(65) 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).
(66) In some embodiments, the second terminal amine group leading to the synthesis of cadaverine 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 (Genbank Accession No. AAA57874.1, SEQ ID NO: 12).
(67) The gene product of ygjG accepts a broad range of diamine carbon chain length substrates, such as putrescine, cadaverine and spermidine (Samsonova et al., BMC Microbiology, 2003, 3:2).
(68) The diamine transaminase from E. coli strain B has demonstrated activity for 1,5 diaminopentane (Kim, The Journal of Chemistry, 1964, 239(3), 783-786).
(69) In some embodiments, the second terminal amine group leading to the synthesis of hexamethylenediamine is enzymatically formed by a deacetylase such as an acyl-lysine deacylase classified, for example, under EC 3.5.1.17 or 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).
(70) Enzymes Generating the Terminal Hydroxyl Groups in the Biosynthesis of a C5 Building Block
(71) As depicted in
(72) For example, a terminal hydroxyl group leading to the synthesis of 5-hydroxypentanoate can be enzymatically formed 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 (e.g., the gene from of ChnD), 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), a 5-hydroxypentanoate dehydrogenase from Clostridium viride, or a 4-hydroxybutyrate dehydrogenase such as gabD (see, for example, Ltke-Eversloh & Steinbchel, 1999, FEMS Microbiology Letters, 181(1):63-71). See,
(73) A terminal hydroxyl group leading to the synthesis of 1,5 pentanediol can be enzymatically formed by an alcohol dehydrogenase classified under EC 1.1.1.- (e.g., EC 1.1.1.1, 1.1.1.2, 1.1.1.21, or 1.1.1.184). See
(74) Biochemical Pathways
(75) Pathway to 2-Oxoadipate
(76) As depicted in
(77) Pathway to 5-Oxopentanoate, Glutaryl-CoA, or Glutarate Using 2-Oxoadipate as a Central Precursor
(78) As depicted in
(79) As depicted in
(80) As depicted in
(81) Pathway to 5-Aminopentanoate Using 5-Oxopentanoate, Glutarate and 2-Aminoadipate as a Central Precursor
(82) As depicted in
(83) As depicted in
(84) Pathway to 5-Hydroxypentanoate Using 5-Oxopentanoate or Glutaric Acid as a Central Precursor
(85) As depicted in
(86) As depicted in
(87) Pathway Using 5-Aminopentanoate, 5-Hydroxypentanoate, or Glutarate Semialdehyde as Central Precursor to Cadaverine
(88) As depicted in
(89) The carboxylate reductase encoded by the gene product of car and 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).
(90) In some embodiments, cadaverine is synthesized from the central precursor 5-hydroxypentanoate (which can be produced as described in
(91) In some embodiments, cadaverine is synthesized from the central precursor 5-aminopentanoate by conversion of 5-aminopentanoate to N5-acetyl-5-aminopentanoate by an N-acetyltransferase such as a lysine N-acetyltransferase classified, for example, under EC 2.3.1.32; followed by conversion to N5-acetyl-5-aminopentanal by a carboxylate reductase such as the gene product of car 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 products of GriC and GriD from Streptomyces griseus; followed by conversion to N5-acetyl-1,5-diaminopentane by a -transaminase classified, for example, under EC 2.6.1.- such as 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 from 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 Genbank Accession No. AAA57874.1, SEQ ID NO: 12), or a Vibrio fluvialis (see Genbank Accession No. AEA39183.1, SEQ ID NO: 13); followed by conversion to cadaverine by an acetylputrescine deacetylase classified, for example, under EC 3.5.1.17 or EC 3.5.1.62. See,
(92) In some embodiments, cadaverine is synthesized from the central precursor glutarate semialdehyde by conversion of glutarate semialdehyde to pentanedial by a carboxylate reductase classified, for example, under EC 1.2.99.6 such as from a Segniliparus rotundus (see Genbank Accession No. ADG98140.1, SEQ ID NO: 7), in combination with a phosphopantetheine transferase enhancer (e.g., encoded by a sfp (Genbank Accession No. CAA44858.1, SEQ ID NO:21) gene from Bacillus subtilis or npt (Genbank Accession No. ABI83656.1, SEQ ID NO:22) gene from Nocardia), or the gene product of GriC & GriD (Suzuki et al., J. Antibiot., 2007, 60(6), 380-387); followed by conversion to 5-aminopentanal by a -transaminase classified, for example, under EC 2.6.1.- such as 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; followed by conversion to cadaverine by a -transaminase classified, for example, under EC 2.6.1.- such as 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 from 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), or an Escherichia coli (see Genbank Accession No. AAA57874.1, SEQ ID NO: 12). See
(93) In some embodiments, cadaverine is synthesized from the central precursor 1,5-pentanediol by conversion of 1,5-pentanediol to 5-hydroxypentanal by an alcohol dehydrogenase classified, for example, under EC 1.1.1.- (e.g., 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 (Genbank Accession No. CAA90836.1) or YqhD (from E. coli, GenBank Accession No. AAA69178.1) (Liu et al., Microbiology, 2009, 155, 2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the protein having GenBank Accession No. CAA81612.1; followed by conversion of 5-oxopentanal to 5-aminopentanol by a -transaminase classified, for example, under EC 2.6.1.- such as 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 from 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 Genbank Accession No. AAA57874.1, SEQ ID NO: 12), or a Vibrio fluvialis (see Genbank Accession No. AEA39183.1, SEQ ID NO: 13); followed by conversion to 5-aminopentanal by an alcohol dehydrogenase classified, for example, under EC 1.1.1.- (e.g., 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 (Genbank Accession No. CAA90836.1) or YqhD (from E. coli, GenBank Accession No. AAA69178.1) (Liu et al., Microbiology, 2009, 155, 2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; Jarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the protein having GenBank Accession No. CAA81612.1; followed by conversion to cadaverine by a -transaminase classified, for example, under EC 2.6.1.- such as 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 from 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), or an Escherichia coli (see Genbank Accession No. AAA57874.1, SEQ ID NO: 12). See
(94) Pathways Using 5-Hydroxypentanoate as Central Precursor to 1,5-Pentanediol
(95) As depicted in
(96) Cultivation Strategy
(97) In some embodiments, the cultivation strategy entails achieving an aerobic, anaerobic, micro-aerobic, or mixed oxygen/denitrification cultivation condition. Enzymes characterized in vitro as being oxygen sensitive require a micro-aerobic cultivation strategy maintaining a very low dissolved oxygen concentration (See, for example, Chayabatra & Lu-Kwang, Appl. Environ. Microbiol., 2000, 66(2), 493 0 498; Wilson and Bouwer, 1997, Journal of Industrial Microbiology and Biotechnology, 18(2-3), 116-130).
(98) In some embodiments, a cyclical cultivation strategy entails alternating between achieving an anaerobic cultivation condition and achieving an aerobic cultivation condition.
(99) In some embodiments, the cultivation strategy entails nutrient limitation such as nitrogen, phosphate or oxygen limitation.
(100) In some embodiments, a final electron acceptor other than oxygen such as nitrates can be utilized. In some embodiments, a cell retention strategy using, for example, ceramic membranes can be employed to achieve and maintain a high cell density during either fed-batch or continuous fermentation.
(101) In some embodiments, the principal carbon source fed to the fermentation in the synthesis of one or more C5 building blocks can derive from biological or non-biological feedstocks.
(102) In some embodiments, the biological feedstock can be 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.
(103) 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).
(104) 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, 2011, supra; Martin and Prather, J. Biotechnol., 2009, 139:61-67).
(105) 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; Prez-Pantoja et al., FEMS Microbiol. Rev., 2008, 32, 736-794).
(106) 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).
(107) 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, J. Biotechnol., 2003, 104:155-172; Wee et al., Food Technol. Biotechnol., 2006, 44(2):163-172; Ohashi et al., J. Bioscience and Bioengineering, 1999, 87(5):647-654).
(108) 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).
(109) In some embodiments, the non-biological feedstock can be or can derive from natural gas, syngas, CO.sub.2/H.sub.2, methanol, ethanol, benzoate, non-volatile residue (NVR) or a caustic wash waste stream from cyclohexane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams.
(110) The efficient catabolism of methanol has been demonstrated for the methylotrophic yeast Pichia pastoris.
(111) The efficient catabolism of ethanol has been demonstrated for Clostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008, 105(6) 2128-2133).
(112) 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).
(113) 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).
(114) 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).
(115) 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 C5 building blocks.
(116) 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 C5 building blocks.
(117) Metabolic Engineering
(118) The present document 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.
(119) 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 document 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 document. 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.
(120) In addition, this document 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.
(121) Also, this document 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.
(122) This document also recognizes that where an enzyme is shown to accept a particular co-factor, such as NADPH, or 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 document 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.
(123) 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.
(124) In some embodiments, the enzymes in the pathways outlined here can be gene dosed, i.e., overexpressed, into the resulting genetically modified organism via episomal or chromosomal integration approaches.
(125) 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 C5 building block.
(126) Attenuation strategies include, but are not limited to; the use of transposons, homologous recombination (double cross-over approach), mutagenesis, enzyme inhibitors and RNAi interference.
(127) 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 C5 building block.
(128) In some embodiments, the host microorganism's tolerance to high concentrations of a C5 building block can be improved through continuous cultivation in a selective environment.
(129) In some embodiments, the host microorganism's endogenous biochemical network can be attenuated or augmented to (1) ensure the intracellular availability of 2-oxoglutarate, (2) create a NADPH imbalance that may be balanced via the formation of one or more C5 building blocks, (3) prevent degradation of central metabolites, central precursors leading to and including one or more C5 building blocks and/or (4) ensure efficient efflux from the cell.
(130) In some embodiments requiring the intracellular availability of L-glutamate for C5 building block synthesis, the enzymes catalyzing anaplerotic reactions supplementing the citric acid cycle intermediates are amplified.
(131) In some embodiments requiring the intracellular availability of 2-oxoglutarate, a PEP carboxykinase or PEP carboxylase can be overexpressed in the host to generate anaplerotic carbon flux into the Krebs cycle towards 2-oxo-glutarate (Schwartz et al., 2009, Proteomics, 9, 5132-5142).
(132) In some embodiments requiring the intracellular availability of 2-oxo-glutarate, a pyruvate carboxylase can be overexpressed in the host to generated anaplerotic carbon flux into the Krebs cycle towards 2-oxoglutarate (Schwartz et al., 2009, Proteomics, 9, 5132-5142).
(133) In some embodiments requiring the intracellular availability of 2-oxo-glutarate, a PEP synthase can be overexpressed in the host to enhance the flux from pyruvate to PEP, thus increasing the carbon flux into the Krebs cycle via PEP carboxykinase or PEP carboxylase (Schwartz et al., 2009, Proteomics, 9, 5132-5142).
(134) In some embodiments requiring the intracellular availability of 2-oxo-glutarate for C5 building block synthesis, anaplerotic reactions enzymes such as phosphoenolpyruvate carboxylase (e.g., the gene product of pck), phosphoenolpyruvate carboxykinase (e.g., the gene product of ppc), the malic enzyme (e.g., the gene product of sfcA) and/or pyruvate carboxylase are overexpressed in the host organisms (Song and Lee, 2006, Enzyme Micr. Technol., 39, 352-361).
(135) In some embodiments, carbon flux can be directed into the pentose phosphate cycle to increase the supply of NADPH by attenuating an endogenous glucose-6-phosphate isomerase (EC 5.3.1.9).
(136) In some embodiments, carbon flux can be redirected into the pentose phosphate cycle to increase the supply of NADPH by overexpression a 6-phosphogluconate dehydrogenase and/or a transketolase (Lee et al., 2003, Biotechnology Progress, 19(5), 1444-1449).
(137) In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C5 building block, a gene such as UdhA encoding a puridine nucleotide transhydrogenase can be overexpressed in the host organisms (Brigham et al., Advanced Biofuels and Bioproducts, 2012, Chapter 39, 1065-1090).
(138) In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C5 Building Block, a recombinant glyceraldehyde-3-phosphate-dehydrogenase gene such as GapN can be overexpressed in the host organisms (Brigham et al., 2012, supra).
(139) In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C5 building block, a recombinant malic enzyme gene such as maeA or maeB can be overexpressed in the host organisms (Brigham et al., 2012, supra).
(140) In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C5 building block, a recombinant glucose-6-phosphate dehydrogenase gene such as zwf can be overexpressed in the host organisms (Lim et al., J. Bioscience and Bioengineering, 2002, 93(6), 543-549).
(141) In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C5 building block, a recombinant fructose 1,6 diphosphatase gene such as fbp can be overexpressed in the host organisms (Becker et al., J. Biotechnol., 2007, 132:99-109).
(142) In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C5 building block, endogenous triose phosphate isomerase (EC 5.3.1.1) can be attenuated.
(143) In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C5 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).
(144) 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).
(145) In some embodiments, an endogenous glutamate dehydrogenase (EC 1.4.1.3) that utilizes both NADH and NADPH as co-factors can be attenuated.
(146) In some embodiments using hosts that naturally accumulate polyhydroxyalkanoates, the endogenous polyhydroxyalkanoate synthase enzymes can be attenuated in the host strain.
(147) 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.
(148) In some embodiments, a L-glutamate dehydrogenase, a L-glutamine synthetase, or a glutamate synthase can be overexpressed in the host to regenerate L-glutamate from 2-oxoglutarate as an amino donor for -transaminase reactions.
(149) In some embodiments, enzymes such as; 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 or EC 1.3.99.7 that degrade central metabolites and central precursors leading to and including C5 building blocks can be attenuated.
(150) In some embodiments, endogenous enzymes activating C5 building blocks via Coenzyme A esterification such as CoA-ligases (e.g., a glutaryl-CoA synthetase) classified under, for example, EC 6.2.1.6 can be attenuated.
(151) In some embodiments, the efflux of a C5 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 C5 building block.
(152) The efflux of cadaverine 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).
(153) The efflux of 5-aminopentanoate and cadaverine 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).
(154) The efflux of glutaric 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).
(155) Producing C5 Building Blocks Using a Recombinant Host
(156) Typically, one or more C5 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 C5 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.
(157) Once transferred, the microorganisms can be incubated to allow for the production of a C5 building block. Once produced, any method can be used to isolate C5 building blocks. For example, C5 building blocks can be recovered selectively from the fermentation broth via adsorption processes. In the case of glutaric acid and 5-aminopentanoic 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 cadaverine and 1,5-pentanediol, distillation may be employed to achieve the desired product purity. The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1
(158) Enzyme Activity of -Transaminase Using Glutarate Semialdehyde as Substrate and Forming 5-Aminopentanoate
(159) A nucleotide sequence encoding an N-terminal His-tag was added to the genes from Chromobacterium violaceum and Rhodobacter sphaeroides encoding the -transaminases of SEQ ID NOs: 8 and 10 respectively (see
(160) 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.
(161) Enzyme activity assays in the reverse direction (i.e., 5-aminopentanoate to glutarate semialdehyde) were performed in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mM 5-aminopentanoate, 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 5-aminopentanoate 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.
(162) Each enzyme only control without 5-aminopentanoate demonstrated low base line conversion of pyruvate to L-alanine See
(163) Enzyme activity in the forward direction (i.e., glutarate semialdehyde to 5-aminopentanoate) was confirmed for the transaminase of SEQ ID NO 10. Enzyme activity assays were performed in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mM glutarate 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 glutarate semialdehyde and incubated at 25 C. for 4 h, with shaking at 250 rpm. The formation of pyruvate was quantified via RP-HPLC.
(164) The gene product of SEQ ID NO 10 accepted glutarate semialdehyde as substrate as confirmed against the empty vector control. See
Example 2
(165) Enzyme Activity of Carboxylate Reductase Using 5-Hydroxypentanoate as Substrate and Forming 5-Hydroxypentanal
(166) A nucleotide sequence encoding a His-tag was added to the genes from Mycobacterium marinum, Mycobacterium smegmatis, Segniliparus rugosus, Mycobacterium massiliense, and Segniliparus rotundus that encode the carboxylate reductases of SEQ ID NOs: 2-4, 6 and 7, respectively (GenBank Accession Nos. ACC40567.1, ABK71854.1, EFV11917.1, EIV11143.1, and ADG98140.1, respectively) (see
(167) 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.
(168) Enzyme activity (i.e., 5-hydroxypentanoate to 5-hydroxypentanal) assays were performed in triplicate in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 2 mM 5-hydroxypentanal, 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 5-hydroxypentanoate 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 5-hydroxypentanoate demonstrated low base line consumption of NADPH. See
(169) The gene products of SEQ ID NOs: 2-4, 6 and 7, enhanced by the gene product of sfp, accepted 5-hydroxypentanoate as substrate as confirmed against the empty vector control (see
Example 3
(170) Enzyme Activity of -Transaminase for 5-Aminopentanol, Forming 5-Oxopentanol
(171) A nucleotide sequence encoding an N-terminal His-tag was added to the Chromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae, Rhodobacter sphaeroide, Escherichia coli and Vibrio fluvialis genes encoding the -transaminases of SEQ ID NOs: 8-13, respectively (see
(172) 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.
(173) Enzyme activity assays in the reverse direction (i.e., 5-aminopentanol to 5-oxopentanol) were performed in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mM 5-aminopentanol, 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 5-aminopentanol and then incubated at 25 C. for 4 h, with shaking at 250 rpm. The formation of L-alanine was quantified via RP-HPLC.
(174) Each enzyme only control without 5-aminopentanol had low base line conversion of pyruvate to L-alanine See
(175) The gene products of SEQ ID NOs: 8-13 accepted 5-aminopentanol as substrate as confirmed against the empty vector control (see
Example 4
(176) Enzyme Activity of -Transaminase Using Cadaverine as Substrate and Forming 5-Aminopentanal
(177) A nucleotide sequence encoding an N-terminal His-tag was added to the Chromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae, and Escherichia coli genes encoding the -transaminases of SEQ ID NOs: 8-10 and 12, respectively (see
(178) 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.
(179) Enzyme activity assays in the reverse direction (i.e., cadaverine to 5-aminopentanal) were performed in a buffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mM cadaverine, 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 cadaverine and then incubated at 25 C. for 4 h, with shaking at 250 rpm. The formation of L-alanine was quantified via RP-HPLC.
(180) Each enzyme only control without cadaverine had low base line conversion of pyruvate to L-alanine See
(181) The gene products of SEQ ID NOs: 8-10 and 12 accepted cadaverine as substrate as confirmed against the empty vector control (see
Example 5
(182) Enzyme Activity of -Transaminase Using N5-Acetyl-1,5-Diaminopentane, and Forming N5-Acetyl-5-Aminopentanal
(183) The activity of the N-terminal His-tagged -transaminases of SEQ ID NOs: 8, 10-13 (see Example 3, and
(184) Each enzyme only control without N5-acetyl-1,5-diaminopentane demonstrated low base line conversion of pyruvate to L-alanine See
(185) The gene product of SEQ ID NOs: 8, 10 accepted N5-acetyl-1,5-diaminopentane as substrate as confirmed against the empty vector control (see
(186) Given the reversibility of the -transaminase activity (see Example 1), the gene products of SEQ ID NOs: 8, 10 accept N5-acetyl-5-aminopentanal as substrate forming N5-acetyl-1,5-diaminopentane.
Example 6
(187) Enzyme Activity of Carboxylate Reductase Using Glutarate Semialdehyde as Substrate and Forming Pentanedial
(188) The N-terminal His-tagged carboxylate reductase of SEQ ID NO 7 (see Example 3 and
(189) The gene product of SEQ ID NO 7, enhanced by the gene product of sfp, accepted glutarate semialdehyde as substrate as confirmed against the empty vector control (see
Other Embodiments
(190) 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.