Methods and Materials for Producing 7-Carbon Monomers
20170218406 · 2017-08-03
Assignee
Inventors
- Alexander Van Eck CONRADIE (Eaglescliffe, GB)
- Adriana L. Botes (Rosedale East, GB)
- Alec Foster (Wilton, GB)
- Changlin Chen (Ingleby Barwick, GB)
Cpc classification
C12P17/182
CHEMISTRY; METALLURGY
C07C47/21
CHEMISTRY; METALLURGY
C12P7/46
CHEMISTRY; METALLURGY
C12P7/6427
CHEMISTRY; METALLURGY
C12N9/1029
CHEMISTRY; METALLURGY
C12P7/40
CHEMISTRY; METALLURGY
C12N9/80
CHEMISTRY; METALLURGY
C12N9/0008
CHEMISTRY; METALLURGY
C12Y206/01018
CHEMISTRY; METALLURGY
C08G69/26
CHEMISTRY; METALLURGY
C07C59/147
CHEMISTRY; METALLURGY
International classification
C12P7/40
CHEMISTRY; METALLURGY
C12P17/18
CHEMISTRY; METALLURGY
C08G69/26
CHEMISTRY; METALLURGY
C07C47/21
CHEMISTRY; METALLURGY
C12N9/80
CHEMISTRY; METALLURGY
Abstract
This document describes biochemical pathways for producing pimeloyl-CoA using a polypeptide having the enzymatic activity of a hydroperoxide lyase to form non-3-enal and 9-oxononanoate from 9-hydroxyperoxyoctadec-10,12-dienoate. Non-3-enal and 9-oxononanoate can be enzymatically converted to pimeloyl-CoA or a salt thereof using one or more polypeptides having the activity of a dehydrogenase, a CoA ligase, an isomerase, a reductase, a thioesterase, a monooxygenase, a hydratase, and/or a thiolase. Pimeloyl-CoA can be enzymatically converted to pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine, or 1,7-heptanediol, or corresponding salts thereof. This document also describes recombinant microorganisms producing pimeloyl-CoA, as well as pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine, and 1,7-heptanediol, or corresponding salts thereof.
Claims
1. A method of producing non-3-enal and 9-oxononanoate in a recombinant microorganism, said method comprising enzymatically converting 9-hydroxyperoxyoctadec-10,12-dienoate to non-3-enal and 9-oxononanoate using an exogenous polypeptide having the activity of a hydroperoxide lyase classified under EC 4.2.99.-, said method optionally further comprising enzymatically converting non-3-enal to azelaic acid using one or more polypeptides comprising at least one polypeptide having the activity of a dodecenoyl-CoA isomerase classified under EC 5.3.3.8 or at least one polypeptide having the activity of an enoate reductase classified under EC 1.3.1.31.
2. (canceled)
3. (canceled)
4. The method of claim 1, said method comprising enzymatically converting non-3-enal to azelaic acid using one or more polypeptides, comprising: at least one polypeptide having the activity of a dodecenoyl-CoA isomerase classified under EC 5.3.3.8, wherein said at least one polypeptide having the activity of a dodecenoyl-CoA isomerase classified under EC 5.3.3.8 enzymatically converts non-3-enoyl-CoA to non-2-enoyl-CoA; or at least one polypeptide having the activity of an enoate reductase classified under EC 1.3.1.31, wherein said at least one polypeptide having the activity of an enoate reductase classified under EC 1.3.1.31 enzymatically converts non-2-enal to nonanal, and optionally a polypeptide having the activity of a monooxygenase classified under EC 1.14.14.- or EC 1.14.15.-, wherein said polypeptide having the activity of a monooxygenase enzymatically converts nonanoic acid to 9-hydroxynonanoic acid.
5-8. (canceled)
9. The method of claim 1, said method further comprising producing azelaic acid in the recombinant microorganism, wherein the method comprises the steps of enzymatically converting 9-hydroxyperoxyoctadec-10,12-dienoate to non-3-enal and 9-oxononanoate using an exogenous polypeptide having the activity of a hydroperoxide lyase classified under EC 4.2.99.- and enzymatically converting non-3-enal to azelaic acid using one or more polypeptides, including at least one polypeptide having the activity of a dodecenoyl-CoA isomerase classified under EC 5.3.3.8 or at least one polypeptide having the activity of an enoate reductase classified under EC 1.3.1.31.
10. (canceled)
11. The method of claim 9, wherein said non-3-enal is converted to azelaic acid using one or more polypeptides having the enzymatic activities of a monooxygenase, an enal isomerase, an aldehyde dehydrogenase, a CoA ligase, a dodecenoyl-CoA isomerase, a trans-2-enoyl-CoA reductase, a thioesterase, a monooxygenase, and/or an alcohol dehydrogenase.
12-17. (canceled)
18. The method of claim 9, wherein said non-3-enal is converted to azelaic acid using one or more polypeptides having the enzymatic activities of a monooxygenase, an enoate reductase, an aldehyde dehydrogenase, a monooxygenase, and/or an alcohol dehydrogenase; or said 9-oxononanoate is converted to azelaic acid using a polypeptide having the enzymatic activity of an aldehyde dehydrogenase.
19-23. (canceled)
24. The method of claim 9, said method further comprising enzymatically converting azelaic acid to pimeloyl-CoA using one or more polypeptides having the enzymatic activities of a CoA ligase, an acyl-CoA dehydrogenase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, and/or a β-ketothiolase.
25-35. (canceled)
36. The method of claim 24, said method further comprising enzymatically converting pimeloyl-CoA to one or more of pimelate semialdehyde, pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, or 1,7-heptanediol, or corresponding salts thereof, in one or more steps.
37-45. (canceled)
46. The method of claim 36, wherein said pimeloyl-CoA is converted to pimelic acid using a polypeptide having the activity of a CoA ligase classified under EC 6.2.1.-., a polypeptide having the activity of a CoA transferase classified under EC 2.8.3.-, or a polypeptide having the activity of a thioesterase classified under EC 3.1.2.-.
47-49. (canceled)
50. The method of claim 36, wherein said pimeloyl-CoA is converted to pimelate semialdehyde using one or more polypeptides having the activity of an acetylating aldehyde dehydrogenase; said method further comprising optionally converting pimelate semialdehyde to 7-aminoheptanoate using one or more polypeptides having the activity of a ω-transaminase classified under EC 2.6.1.-; converting pimelate semialdehyde to 7-hydroxyheptanoate using one or more polypeptides having the activity of an alcohol dehydrogenase, wherein said alcohol dehydrogenase is a 4-hydroxybutanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase, or a 6-hydroxyhexanoate dehydrogenase; or converting pimelate semialdehyde to heptanedial using one or more polypeptides having the activity of a carboxylase reductase classified under EC 1.2.99.6; converting heptanedial to 7-aminoheptanal using one or more polypeptides having the activity of a ω-transaminase classified under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.48, EC 2.6.1.29, or EC 2.6.1.82; and converting 7-aminoheptanal to heptamethylenediamine using one or more polypeptides having the activity of a ω-transaminase classified under EC 2.6.1.-.
51-54. (canceled)
55. The method of claim 50, wherein pimelate semialdehyde is converted to 7-aminoheptanoate, said method further comprising converting said 7-aminoheptanoate to 7-aminoheptanal using one or more polypeptides having the activity of a carboxylase reductase classified under EC 1.2.99.6, and converting said 7-aminoheptanal to heptamethylenediamine using one or more polypeptides having the activity of a ω-transaminase classified under EC 2.6.1.-; or converting 7-aminoheptanoate to N7-acetyl-7-aminoheptanoate using one or more polypeptides having the activity of an N-acetyltransferase classified under EC 2.3.1.32; converting N7-acetyl-7-aminoheptanoate to N7-acetyl-7-aminoheptanal using one or more polypeptides having the activity of a carboxylase reductase classified under EC 1.2.99.6; converting N7-acetyl-7-aminoheptanal is converted to N7-acetyl-1,7-diaminoheptane using one or more polypeptides having the activity of a ω-transaminase classified under EC 2.6.1.-; and converting N7-acetyl-1,7-diaminoheptane to heptamethylenediamine using one or more polypeptides having the activity of a deacylase classified under EC 3.5.1.-.
56-59. (canceled)
60. The method of claim 50, wherein pimelate semialdehyde is converted to 7-hydroxyheptanoate, said method further comprising converting said 7-hydroxyheptanoate to 7-hydroxyheptanal using one or more polypeptides having the activity of a carboxylase reductase classified under EC 1.2.99.6; converting 7-hydroxyheptanal to 7-aminoheptanol using one or more polypeptides having the activity of a ω-transaminase classified under EC 2.6.1.18, EC 2.6.1.19, or EC 2.6.1.48; converting 7-aminoheptanol to 7-aminoheptanal using one or more polypeptides having the activity of an alcohol dehydrogenase classified under EC 1.1.1.-; and converting 7-aminoheptanal to heptamethylenediamine using one or more polypeptides having the activity of a ω-transaminase classified under EC 2.6.1.-; or converting 7-hydroxyheptanoate to 7-hydroxyheptanal using a carboxylase reductase classified under EC 1.2.99.6; and converting 7-hydroxyheptanal to 1,7 heptanediol using one more polypeptides having the activity of an alcohol dehydrogenase classified under EC 1.1.1.-.
61-65. (canceled)
66. The method of claim 65, wherein said 9-hydroxyperoxyoctadec-10,12-dienoate is enzymatically produced from octadecanoyl-CoA using one or more polypeptides having the activity of a delta9-desaturase, a delta12-desaturase, a thioesterase, and/or a 9-lipoxygenase.
67-70. (canceled)
71. The method of claim 1, wherein said method is performed in a recombinant microorganism.
72. The method of claim 71, wherein said microorganism is subjected to a cultivation strategy under aerobic, anaerobic, or micro-aerobic cultivation conditions and/or said microorganism is cultured under conditions of nutrient limitation.
73. (canceled)
74. (canceled)
75. The method of claim 71, wherein the principal carbon source fed to the fermentation derives from a biological feedstock.
76. The method of claim 75, wherein the biological feedstock is, or derives from, monosaccharides, disaccharides, lignocellulose, hemicellulose, cellulose, lignin, levulinic acid, formic acid, triglycerides, glycerol, fatty acids, agricultural waste, condensed distillers' solubles, plant oils, or municipal waste.
77. The method of claim 71, wherein the principal carbon source fed to the fermentation derives from a non-biological feedstock.
78. The method of claim 77, wherein the non-biological feedstock is, or derives from, natural gas, syngas, CO.sub.2/H.sub.2, methanol, ethanol, benzoate, non-volatile residue (NVR) caustic wash waste stream from cycloheptane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams.
79. The method of claim 71, wherein the microorganism is a prokaryote.
80. The method of claim 79, wherein said prokaryote is from a genus selected from Escherichia, Clostridia, Corynebacteria, Cupriavidus, Pseudomonas, Delftia, Bacillus, Lactobacillus, Lactococcus, and Rhodococcus.
81. (canceled)
82. The method of claim 71, wherein the microorganism is a eukaryote.
83. The method of claim 82, wherein said eukaryote is from a genus selected from Aspergillus, Saccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula, and Kluyveromyces.
84-86. (canceled)
87. The method of claim 71, wherein said microorganism comprises an attenuation to one or more of the following enzymes: a polyhydroxyalkanoate synthase, an acetyl-CoA thioesterase, a phosphotransacetylase forming acetate, an acetate kinase, a lactate dehydrogenase, a menaquinol-fumarate oxidoreductase, an alcohol dehydrogenase forming ethanol, a triose phosphate isomerase, a pyruvate decarboxylase, a glucose-6-phosphate isomerase, an NADH-consuming transhydrogenase, an NADH-specific glutamate dehydrogenase, an NADH/NADPH-utilizing glutamate dehydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoA dehydrogenase accepting C7 building blocks and central precursors as substrates; a butaryl-CoA dehydrogenase; or an adipyl-CoA synthetase.
88. The method of claim 71, wherein said microorganism overexpresses one or more genes encoding: an acetyl-CoA synthetase, a 6-phosphogluconate dehydrogenase; a transketolase; a puridine nucleotide transhydrogenase; a glyceraldehyde-3P-dehydrogenase; a malic enzyme; a glucose-6-phosphate dehydrogenase; a glucose dehydrogenase; a fructose 1,6 diphosphatase; a L-alanine dehydrogenase; a L-glutamate dehydrogenase; a formate dehydrogenase; a L-glutamine synthetase; a diamine transporter, a dicarboxylate transporter, and/or a multidrug transporter.
89. A recombinant microorganism producing azelaic acid, said microorganism comprising one or more nucleic acids encoding a polypeptide having the enzymatic activities of any one or more of the following enzymes: (i) a hydroperoxide lyase, (ii) an aldehyde dehydrogenase, (iii) a CoA ligase, (iv) a dodecenoyl-CoA isomerase, (v) a trans-2-enoyl-CoA reductase, (vi) a thioesterase, (vii) a monooxygenase, and/or (viii) an alcohol dehydrogenase; (ix) a hydroperoxide lyase, (x) an enoate reductase, (xi) an aldehyde dehydrogenase, (xii) a monooxygenase, and/or (xiii) an alcohol dehydrogenase; or (xiv) a hydroperoxide lyase, and/or (xv) an aldehyde dehydrogenase, wherein at least one of the nucleic acids is exogenous.
90. (canceled)
91. (canceled)
92. The recombinant microorganism of claim 89, said microorganism further comprising one or more exogenous nucleic acids encoding a polypeptide having one or more of the enzymatic activities of: (i) a CoA ligase, (ii) an acyl-CoA dehydrogenase, (iii) an enoyl-CoA hydratase, (iv) a 3-hydroxyacyl-CoA dehydrogenase or a 3-oxoacyl ACP reductase, and/or (v) a β-ketothiolase, said microorganism further producing pimeloyl-CoA.
93. The recombinant microorganism of claim 92, said microorganism further comprising one or more exogenous nucleic acids encoding a polypeptide having one or more of the enzymatic activities of a thioesterase, a CoA ligase, a CoA transferase, an acetylating aldehyde dehydrogenase, an alcohol dehydrogenase, an N-acetyltransferase, a deacylase, a ω-transaminase, a carboxylate reductase, and/or an aldehyde dehydrogenase, said microorganism further producing one or more of pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoate, heptamethylenediamine, and 1,7-heptanediol.
94-100. (canceled)
101. The recombinant microorganism of claim 89, said microorganism further comprising one or more nucleic acids encoding a polypeptide having one or more of the enzymatic activities of a delta9-desaturase, a delta12-desaturase, a thioesterase, or a 9-lipoxygenase.
102. (canceled)
103. A nucleic acid construct or expression vector comprising a polynucleotide encoding a polypeptide having carboxylate reductase activity or a polynucleotide encoding a polypeptide having ω-transaminase activity, wherein the polynucleotide encoding a polypeptide having carboxylate reductase activity is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having carboxylate reductase activity is selected from: (a) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 1; (b) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 2; (c) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 3; (d) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 4, (e) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 5 and (f) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 6; and the polynucleotide encoding a polypeptide having ω-transaminase activity is operably linked to one or more heterologous control sequences that direct production of the polypeptide and wherein the polypeptide having ω-transaminase activity is selected from: (g) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 7; (h) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 8; (i) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 9; (i) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 10; (k) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 11 or SEQ ID NO: 48; and (l) a polypeptide having at least 70% sequence identity to the polypeptide of SEQ ID NO: 12.
104. (canceled)
105. A composition comprising the nucleic acid construct or expression vector of claim 103.
106. A culture medium comprising the nucleic acid construct or expression vector of claim 103.
107-110. (canceled)
111. Means for producing pimeloyl-CoA, comprising culturing a non-naturally occurring microorganism comprising at least one exogenous nucleic acid encoding a polypeptide having the enzymatic activity of (i) a hydroperoxide lyase, (ii) an aldehyde dehydrogenase, (iii) a CoA ligase, (iv) a dodecenoyl-CoA isomerase, (iv) a trans-2-enoyl-CoA reductase, (v) a thioesterase, (vi) an enoate reductase, (vii) a monooxygenase, (viii) an alcohol dehydrogenase, (ix) an acyl-CoA dehydrogenase, (x) an enoyl-CoA hydratase, (xi) a 3-hydroxyacyl-CoA dehydrogenase and (xii) a β-ketothiolase, expressed in a sufficient amount in said microorganism to produce pimeloyl-CoA.
112. A bio-derived, bio-based, or fermentation-derived product, wherein said product comprises: (i) a composition comprising at least one bio-derived, bio-based, or fermentation-derived compound produced by a method according to claim 1 or any combination thereof, (ii) a bio-derived, bio-based, or fermentation-derived polymer comprising the bio-derived, bio-based or fermentation-derived composition or compound of (i), or any combination thereof, (iii) a bio-derived, bio-based, or fermentation-derived resin comprising the bio-derived, bio-based, or fermentation-derived compound or bio-derived, bio-based, or fermentation-derived composition of (i) or any combination thereof or the bio-derived, bio-based, or fermentation-derived polymer of (ii) or any combination thereof, (iv) a molded substance obtained by molding the bio-derived, bio-based, or fermentation-derived polymer of (ii) or the bio-derived, bio-based, or fermentation-derived resin of (iii), or any combination thereof, (v) a bio-derived, bio-based, or fermentation-derived formulation comprising the bio-derived, bio-based, or fermentation-derived composition of (i), bio-derived, bio-based, or fermentation-derived compound of (i), bio-derived, bio-based, or fermentation-derived polymer of (ii), bio-derived, bio-based, or fermentation-derived resin of (iii), or bio-derived, bio-based, or fermentation-derived molded substance of (v), or any combination thereof, or (vi) a bio-derived, bio-based, or fermentation-derived semi-solid or a non-semi-solid stream, comprising the bio-derived, bio-based, or fermentation-derived composition of (i), bio-derived, bio-based, or fermentation-derived compound of (i), bio-derived, bio-based, or fermentation-derived polymer of (ii), bio-derived, bio-based, or fermentation-derived resin of (iii), bio-derived, bio-based, or fermentation-derived formulation of (v), or bio-derived, bio-based, or fermentation-derived molded substance of (iv), or any combination thereof.
Description
DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION
[0224] In general, this document provides enzymes, non-natural pathways, cultivation strategies, feedstocks, microorganisms, and attenuations to the microorganism's biochemical network, for producing pimeloyl-CoA or one or more of pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoic acid, heptamethylenediamine, or 1,7-heptanediol, or corresponding salts thereof, all of which are referred to as C7 building blocks herein.
[0225] As used herein, a “bio-based product” is a product in which both the feedstock (e.g., sugars from sugar cane, corn, wood; biomass; waste streams from agricultural processes) and the conversion process to the product are biologically based (e.g., fermentation/enzymatic transformation involving a biological host/organism/enzyme). As used herein, a “bio-derived product” is a product in which one of the feedstocks (e.g., sugars from sugar cane, corn, wood; biomass; waste streams from agricultural processes) or the conversion process to the product is biologically based (e.g., fermentation/enzymatic transformation involving a biological host/organism/enzyme).
[0226] As used herein, a “fermentation-derived product” is a product produced by fermentation involving a biological host or organism.
[0227] The term “C7 building block” is used to denote a seven (7) carbon chain aliphatic backbone. 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.
[0228] Microorganisms described herein can include endogenous pathways that can be manipulated such that pimeloyl-CoA or one or more other C7 building blocks can be produced. In an endogenous pathway, the microorganism naturally expresses all of the enzymes catalyzing the reactions within the pathway. A 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 microorganism.
[0229] The term “exogenous” as used herein with reference to a nucleic acid (or a protein) and a microorganism 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 microorganism once in the microorganism. 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 a non-naturally-occurring nucleic acid, and thus is exogenous to a microorganism once introduced into the microorganism, 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 a non-naturally-occurring nucleic acid. A nucleic as acid that is naturally-occurring can be exogenous to a particular 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.
[0230] In contrast, the term “endogenous” as used herein with reference to a nucleic acid (e.g., a gene) (or a protein) and a microorganism refers to a nucleic acid (or protein) that does occur in (and can be obtained from) that particular microorganism 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 microorganism of the same particular type as it is found in nature. Moreover, a microorganism “endogenously producing” or that a “endogenously produces” a nucleic acid, protein, or other compound produces that nucleic acid, protein, or compound as does a microorganism of the same particular type as it is found in nature.
[0231] For example, depending on the microorganism and the compounds produced by the microorganism, one or more polypeptides having the following specific enzymatic activities may be expressed in the microorganism in addition to a hydroperoxide lyase: an acetylating aldehyde dehydrogenase, a CoA ligase, a dodecenoyl-CoA isomerase or an enoate reductase, a trans-2-enoyl-CoA reductase, a thioesterase, a monooxygenase, an enoyl-CoA hydratase, a deacetylase, an acyl-CoA dehydrogenase, an enoyl-CoA hydratase, an alcohol dehydrogenase, a 4-hydroxybutanoate dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, an aldehyde dehydrogenase, a succinate-semialdehyde dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, an acyl-CoA dehydrogenase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a 3-oxoacyl ACP reductase, a fi-ketothiolase, a CoA transferase, a carboxylate reductase, a ω-transaminase, an N-acetyltransferase, and/or a deacylase. In recombinant microorganisms expressing a polypeptide having the activity of a carboxylate reductase, a polypeptide having the activity of a phosphopantetheinyl transferase also can be expressed as it enhances activity of the carboxylate reductase.
[0232] For example, a recombinant microorganism can include a polypeptide having the activity of an exogenous hydroperoxide lyase and produce non-3-enal and 9-oxononanoate from 9-hydroxyperoxyoctadec-10,12-dienoate. The non-3-enal and 9-oxononanoate can be converted enzymatically to pimeloyl-CoA and subsequently to one or more of pimelic acid, 7-aminoheptanoate, 7-hydroxyheptanoic acid, heptamethylenediamine, or 1,7-heptanediol, or corresponding salts thereof.
[0233] For example, a recombinant microorganism producing pimeloyl-CoA can include one or more of exogenous polypeptides having the enzymatic activity of: a thioesterase, a CoA ligase, a CoA transferase, an acetylating aldehyde dehydrogenase, a succinate semialdehyde dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, and/or a 7-oxoheptanoate dehydrogenase, and further produce pimelic acid. See
[0234] For example, a recombinant microorganism producing pimeloyl-CoA can include an exogenous polypeptide having the activity of a thioesterase and produce pimelic acid. For example, a recombinant microorganism producing pimeloyl-CoA can include an exogenous polypeptide having the activity of a CoA ligase or a CoA transferase, and further produce pimelic acid. For example, a recombinant microorganism producing pimeloyl-CoA can include an exogenous polypeptide having the activity of an acetylating aldehyde dehydrogenase and one or more polypeptides having the enzymatic activity of: an aldehyde dehydrogenase, a succinate-semialdehyde dehydrogenase, a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, and/or a 7-oxoheptanoate dehydrogenase, and produce pimelic acid. See
[0235] For example, a recombinant microorganism can include one or more exogenous polypeptides having the enzymatic activity of an aldehyde dehydrogenase, a ω-transaminase, and/or a carboxylate reductase, and produce 7-aminoheptanoate. See
[0236] For example, a recombinant microorganism producing pimeloyl-CoA can include an exogenous polypeptide having the activity of an acetylating aldehyde dehydrogenase and an exogenous polypeptide having the activity of a ω-transaminase, and produce 7-aminoheptanoate. For example, a recombinant microorganism producing pimelate (see
[0237] For example, a recombinant microorganism producing pimeloyl-CoA can include one or more exogenous polypeptides having the enzymatic activity of a carboxylate reductase, an alcohol dehydrogenase, a 4-hydroxybutanoate dehydrogenase, a 5, hydroxypentanoate dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, and/or an so aldehyde dehydrogenase and further produce 7-hydroxyheptanoate. See
[0238] For example, a recombinant microorganism producing pimeloyl-CoA can include an exogenous polypeptide having the activity of a carboxylate reductase and an exogenous polypeptide having the activity of a 4-hydroxybutanoate dehydrogenase, and produce 7-hydroxyheptanoate. For example, a recombinant microorganism producing pimeloyl-CoA can include an exogenous polypeptide having the activity of a carboxylate reductase and an exogenous polypeptide having the activity of a 5-hydroxypentanoate dehydrogenase, and produce 7-hydroxyheptanoate. For example, a recombinant microorganism producing pimeloyl-CoA can include an exogenous polypeptide having the activity of a carboxylate reductase and an exogenous polypeptide having the activity of a 6-hydroxyhexanoate dehydrogenase, and produce 7-hydroxyheptanoate. For example, a recombinant microorganism producing pimelate (see
[0239] For example, a recombinant microorganism producing pimeloyl-CoA can include one or more exogenous polypeptides to produce 7-aminoheptanoate or 7-hydroxyheptanoate. See
[0240] For example, a recombinant microorganism producing pimeloyl-CoA can include the polypeptides necessary to convert pimeloyl-CoA to 7-aminoheptanoate and can 2 include an exogenous polypeptide having the activity of a carboxylate reductase and one or more exogenous polypeptides having the activity of ω-transaminases (e.g., one transaminase or two different transaminases) and produce heptamethylenediamine. For example, a recombinant microorganism producing pimeloyl-CoA can include the polypeptides necessary to convert pimeloyl-CoA to 7-aminoheptanoate and can include so one or more exogenous polypeptides having the activity of an N-acetyltransferase, a carboxylate reductase, a ω-transaminase, and/or a deacylase, and produce heptamethylenediamine. For example, a recombinant microorganism producing pimeloyl-CoA can include the polypeptides necessary to convert pimeloyl-CoA to 7-hydroxyheptanoate and can include one or more exogenous polypeptides having the 6 activity of a carboxylate reductase, a ω-transaminase (e.g., one transaminase or two different transaminases), and/or an alcohol dehydrogenase, and produce heptamethylenediamine. See
[0241] For example, a recombinant microorganism producing pimeloyl-CoA can include the polypeptides having the necessary enzymatic activity for conversion of pimeloyl-CoA to 7-hydroxyheptanoate (see
[0242] In any of the recombinant microorganisms, the recombinant microorganism also can include one or more (e.g., one, two, or three) of the following exogenous enzymes used to convert either octadecanoyl-CoA to 9-hydroxyperoxyoctadec-10,12-dienoate: a delta9-desaturase, a delta12-desaturase, a thioesterase, or a 9-lipoxygenase. For example, a recombinant microorganism can include a delta9-desaturase, a delta12-desaturase, a thioesterase, and a 9-lipoxygenase.
[0243] Within an engineered pathway, the enzymes can be from a single source, i.e., from one species or genera, 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. Enzyme Commission (EC) numbers for many enzymes are also provided. EC numbers are well known in the art and provide a numerical classification scheme for enzymes based on the chemical reactions they catalyze. An enzyme classified with an EC number to the fourth level is discretely and specifically classified on the basis of the reactions that its members are able to perform. Well known nomenclature databases such as ENZYME, maintained by the Swiss Institute of Bioinformatics, so provide examples of specific enzymes corresponding to specific EC numbers.
[0244] Any of the enzymes described herein that can be used for production of one or more C7 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.
[0245] For example, a polypeptide having the activity of 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: 1), a Mycobacterium smegmatis (see GenBank Accession No. ABK71854.1, SEQ ID NO: 2), a Segniliparus rugosus (see GenBank Accession No. EFV11917.1, SEQ ID NO: 3), a Mycobacterium smegmatis (see GenBank Accession No. ABK75684.1, SEQ ID NO: 4), a Mycobacterium massiliense (see GenBank Accession No. EIV11143.1, SEQ ID NO: 5), or a Segniliparus rotundus (see GenBank Accession No. ADG98140.1, SEQ ID NO: 6) carboxylate reductase. See
[0246] For example, a polypeptide having the activity of a ω-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: 7), a Pseudomonas aeruginosa (see GenBank Accession No. AAG08191.1, SEQ ID NO: 8), a Pseudomonas syringae (see GenBank Accession No. AAY39893.1, SEQ ID NO: 9), a Rhodobacter sphaeroides (see GenBank Accession No. ABA81135.1, SEQ ID NO: 10), an Escherichia coli (see GenBank Accession No. AAA57874.1, SEQ ID NO: 11, SEQ ID NO: 48), or a Vibrio fluvialis (see GenBank Accession No. AEA39183.1, SEQ ID NO: 12) ω-transaminase. Some of these ω-transaminases are diamine ω-transaminases. See
[0247] For example, a polypeptide having the activity of a hydroperoxide lyase 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 Cucumis sativus (see GenBank Accession No. AAF64041.1, SEQ ID NO: 13) or a Oryza sativa hydroperoxide lyase (see GenBank Accession No. BAG97978.1, SEQ ID NO: 14). See
[0248] For example, a polypeptide having the activity of an enoate 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 Lactobacillus casei (see GenBank Accession No. AGP69310.1, SEQ ID NO: 15) or a Pseudomonas putida enoate reductase (see GenBank Accession No. AAN66878.1, SEQ ID NO: 16). See
[0249] For example, a polypeptide having the activity of an isomerase 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 Saccharomyces cerevisiae isomerase (see GenBank Accession No. AAC83700.1, SEQ ID NO: 17 and SEQ ID NO: 19). See
[0250] For example, a polypeptide having the activity of 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 Clostridium perfringens (see GenBank Accession No. ABG82470.1, SEQ ID NO: 18), a Bacteroides thetaiotaomicron VPI-5482 (see GenBank Accession No. AAO77182.1, SEQ ID NO: 20), a Lactobacillus plantarum WCFS1 (see GenBank Accession No. CCC78182.1, SEQ ID NO: 22), or a Anaerococcus tetradius ATCC 35098 (see GenBank Accession No. EEI82564.1, SEQ ID NO: 23). See
[0251] For example, a polypeptide having the activity of an alcohol dehydrogenase 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 Geobacillus stearothermophilus (see GenBank Accession No. CAA81612.1, SEQ ID NO: 21). See
[0252] 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 (B12seq) 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 B12seq program can be found in the readme file accompanying BLASTZ. B12seq performs a comparison between two amino acid sequences using the BLASTP algorithm. To compare two amino acid sequences, the options of B12seq 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:\B12seq -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.
[0253] 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 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.
[0254] 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.
[0255] 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.
[0256] 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 100 (e.g., not more than one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100) 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.
[0257] Deletion variants can lack one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 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., heptahistidine (SEQ ID NO: 47)), 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 microorganisms (e.g., yeast 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.
[0258] Engineered microorganisms 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 microorganism can include all exogenous enzymes, or can include both endogenous and exogenous enzymes. Endogenous genes of the engineered microorganisms 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 microorganisms can be referred to as recombinant microorganisms or recombinant cells. As described herein recombinant microorganisms can include nucleic acids encoding one or more of a hydroperoxide lyase, an aldehyde dehydrogenase, a CoA ligase, a dodecenoyl-CoA isomerase or an enoate reductase, a trans-2-enoyl-CoA reductase, a thioesterase, a monooxygenase, an enoyl-CoA hydratase, a deacetylase, an acyl-CoA dehydrogenase, an enoyl-GoA hydratase, an alcohol dehydrogenase, a 4-hydroxybutanoate dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, an aldehyde dehydrogenase, a succinate-semialdehyde dehydrogenase a 5-oxopentanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, a 7-oxoheptanoate dehydrogenase, an acyl-CoA dehydrogenase, an enoyl-CoA hydratase, a 3-hydroxyacyl-CoA dehydrogenase, a 3-oxoacyl ACP reductase, a β-ketothiolase, a delta9-desaturase, a delta12-desaturase, a thioesterase, or a 9-lipoxygenase, as described herein.
[0259] In addition, the production of C7 building blocks can be performed in vitro using the isolated enzymes described herein, using a lysate (e.g., a cell lysate) from a microorganism as a source of the enzymes, or using one or more lysates from different microorganisms as the source of the enzymes.
[0260] The reactions of the pathways described herein can be performed in one or more microorganisms (a) naturally expressing one or more relevant enzymes, (b) genetically engineered to express one or more relevant enzymes, or (c) naturally expressing one or more relevant enzymes and genetically engineered to express one or more relevant enzymes. Alternatively, relevant enzymes can be isolated, purified or extracted from of the above types of microorganism cells and used in a purified or semi-purified form. Moreover, such extracts include lysates (e.g., cell lysates) that can be used as sources of relevant enzymes. In the methods provided by the document, all the steps can be performed in microorganism cells, all the steps can be performed using extracted enzymes, or some of the steps can be performed in cells and others can be performed using extracted enzymes.
Enzymes
Enzymes Generating Pimeloyl-CoA
[0261] As depicted in
[0262] In some embodiments, a polypeptide having the activity of a delta9-desaturase may be classified under EC 1.14.19.1, such as, for example, the gene product of Le-FAD1 from Lentinula edodes (UniProtKB Accession No. Q76C19), the gene product of SCD1 from Mesocricetus auratus (UniProtKB Accession No. A7LCI9), an acyl-CoA-delta9-3a-desaturase from Dendrolimus punctatus (UniProtKB Accession No. B7SB75), the gene product of scd1 from Rattus norvegicus (UniProtKB Accession No. P07308), the gene product of PF3D70511200 from Plasmodium falciparum (UniProtKB Accession No. Q8I0W9), or the gene product of desB1 from Bombus lucorum (UniProtKB Accession No. A5CKEI).
[0263] A polypeptide having the activity of a delta12-desaturase may be classified under EC 1.14.19.6, such as, for example, the gene product of D12Des from Acheta domesticus (UniProtKB Accession No. B7SB91), the gene product of FAD2 from Gossypium hirsutum (UniProtKB Accession No. Q8W2B9), the gene product of CFad6 from Chlorella vulgaris (UniProtKB Accession No. D3U658), a delta12 fatty acid desaturase from Triadica sebifera (UniProtKB Accession No. A5J295), the gene product of Pc-fad2 from Phanerochaete chrysosporium (UniProtKB Accession No. D4Q8H2), the gene product of Cs-fad2 from Ceriporiopsis subvermispora (UniProtKB Accession No. D4Q8S6), or the gene product of AN1037.2 from Emericella nidulans (UniProtKB Accession No. Q5BEJ3).
[0264] A polypeptide having the activity of a thioesterase may be classified under EC 3.1.2.-, such as, for example, the gene product of BT_2075 from Bacteroides thetaiotaomicron (strain ATCC 29148/DSM 2079/NCTC 10582/E50/VPI-5482) (GenBank Accession No. AAO77182.1, SEQ ID NO: 20), the gene product of lp_0708 from Lactobacillus plantarum (strain ATCC BAA-793/NCIMB 8826/WCFS1) (GenBank Accession No. CCC78182.1, SEQ ID NO: 22), the gene product of HMPREF0077_1317 from Anaerococcus tetradius ATCC 35098 (GenBank Accession No. EE182564.1, SEQ ID NO: 23), or the gene product of CPF_2954 from Clostridium perfringens (strain ATCC 13124/DSM 756/JCM 1290/NCIMB 6125/NCTC 8237/Type A) (GenBank Accession No. ABG82470.1, SEQ ID NO: 18).
[0265] A polypeptide having the activity of a 9-lipoxygenase may be classified, for example, under EC 1.13.11.58, EC 1.13.11.60, EC 1.13.11.61, or EC 1.13.11.62, such as, for example, an allene oxide synthase-lipoxygenase protein from Plexaura homomalla (UniProtKB Accession No. O16025), a Psi-producing oxygenase A from Emericella nidulans (UniProtKB Accession No. Q6RET3), a 5,8-linoleate dial synthase from Aspergillus fumigatus (UniProtKB Accession No. C1KH66), or a linoleate diol synthase from Gaeunmannomyces graminis (UniProtKB Accession No. Q9UUS2).
[0266] As further depicted in
[0267] As shown in
[0268] As shown in
[0269] As shown in
[0270] In some embodiments, a polypeptide having the activity of an aldehyde dehydrogenase may be classified under EC 1.2.1.-, such as EC 1.2.1.3, EC 1.2.1.4, EC 1.2.1.5, or EC 1.2.1.48, such as, for example, the gene product of Bt-aldh from Geobacillus thermoleovorans B23 (UniProtKB Accession No. Q9FAB1), the gene product of dhaS from Bacillus subtilis (UniProtKB Accession No. O34660), the gene product of ALD5 from Saccharomyces cerevisiae (UniProtKB Accession No. A6ZR27), the gene product of ALDH2C4 from Arabidopsis thaliana (UniProtKB Accession No. Q56YU0), the gene product of aldh7 from Rhodococcus ruber (UniProtKB Accession No. Q840S9), the gene product of alkH from Pseudomonas oleovorans (UniProtKB Accession No. P12693), the gene product of ald1 from Acinetobacter sp. M-1 (UniProtKB Accession No. Q9FDS1), or the gene product of acoD from Ralstonia eutropha (UniProtKB Accession No. P46368).
[0271] In some embodiments, a polypeptide having the activity of an enoate reductase may be classified, for example, under EC 1.3.1.31, such as, for example, the gene product of xenA from Pseudomonas putida (GenBank Accession No. AAN66878.1, SEQ ID NO: 16) or the gene product of LOCK919_2632 from Lactobacillus casei (GenBank Accession No. AGP69310.1, SEQ ID NO: 15).
[0272] In some embodiments, a polypeptide having the activity of a CoA ligase may be classified under EC 6.2.1.-, such as, for example, the gene product of acs6 from Brassica napus (UniProtKB Accession No. Q9FNT6), the gene product of PCS60 from Saccharomyces cerevisiae (UniProtKB Accession No. P38137), the gene product of alkK from Pseudomonas oleovorans (UniProtKB Accession No. Q00594), the gene product of ACSM5 from Homo sapiens (UniProtKB Accession No. Q6NUN0), or the gene product of alkK from Aeropyrum pernix (UniProtKB Accession No. Q9YF45).
[0273] In some embodiments, a polypeptide having the activity of a dodecenoyl-CoA isomerase may be classified under EC 5.3.3.8, such as, for example, the gene product of ECI1 from Saccharomyces cerevisiae (GenBank Accession No. AAC83700.1, SEQ ID NO: 17 and SEQ ID NO: 19, Geisbrecht et al J. Biol. Chem, 1998 273 (50) 33184-33191).
[0274] In some embodiments, a polypeptide having the activity of a trans-2-enoyl-CoA reductase may be classified under EC 1.3.1.38 or EC 1.3.1.44, such as, for example, the gene product of ter from Escherichia coli, Fibrobacter succinogenes, or Treponema denticola (Nishimaki et al., J. Biochem., 1984, 95:1315-1321; Shen et al., 2011, supra) or tdter from Treponema denticola (Bond-Watts et al., Biochemistry, 2012, 51:6827-6837) or EC 1.3.1.8 (Inui et al, Eur. J. Biochem., 1984, 142, 121-126).
[0275] In some embodiments, a polypeptide having the activity of a thioesterase may be classified under EC 3.1.2.-, such as, for example, the gene product of BT_2075 from Bacteroides thetaiotaomicron (strain ATCC 29148/DSM 2079/NCTC 10582/E50/VPI-5482) (GenBank Accession No. AAO77182.1, SEQ ID NO: 20), the gene product of lp_0708 from Lactobacillus plantarum (strain ATCC BAA-793/NCIMB 8826/WCFS1) (GenBank Accession No. CCC78182.1, SEQ ID NO: 22), the gene product of HMPREF0077_1317 from Anaerococcus tetradius ATCC 35098 (GenBank Accession No. EE182564.1, SEQ ID NO: 23), or the gene product of CPF_2954 from Clostridium perfringens (strain ATCC 13124/DSM 756/JCM 1290/NCIMB 6125/NCTC 8237/Type A) (GenBank Accession No. ABG82470.1, SEQ ID NO: 18).
[0276] In some embodiments, a polypeptide having the activity of a monooxygenase may be classified in the cytochrome P450 family under EC 1.14.14.- or EC 1.14.15.-, such as EC 1.14.14.1, EC 1.14.14.3, EC 1.14.15.1, or EC 1.14.15.3 or as the gene products of alkBGT from Pseudomonas putida, CYP153A from Polaromonas sp., or CYP52A3 from Saccharomyces cerevisiae.
[0277] In some embodiments, a polypeptide having the activity of an alcohol dehydrogenase may be classified under EC 1.1.1., such as a 6-hydroxyhexanoate dehydrogenase classified, for example, under EC 1.1.1.258, such as, for example, the gene product of chnD from Acinetobacter sp. NCIMB9871 (Donoghue et al., Eur. J. Biochem, 1975, 60: 1-7); or a 4-hydroxybutanoate dehydrogenase classified, for example, under EC 1.1.1.61 such as, for example, the gene product of gbd (e.g., from Sorangium cellulosum) or gabD from, for example, Escherichia coli (Bartsch et al., J. Bacteriol., 1990, 172(12), 7035). In some embodiments, a polypeptide having the activity of an aldehyde dehydrogenase may be classified under, for example, EC 1.2.1.-, such as a 7-oxoheptanoate dehydrogenase (e.g., the gene product of thnG from Sphingomonas macrogolitabida), a 6-oxohexanoate dehydrogenase (e.g., the gene product of chnE from Acinetobacter sp.) classified, for example, under EC 1.2.1.63, a 5-oxopentanoate dehydrogenase classified, for example, under EC 1.2.1.20 (e.g., the gene product of cpnE Comamonas sp.), a succinate-semialdehyde dehydrogenase classified, for example, under EC 1.2.1.16, EC 1.2.1.24, or EC 1.2.1.79 (e.g., the gene product of ALDH5F1 from Arabidopsis thaliana (UniProtKB Accession No. Q9SAK4), the gene product of araE from Azospirillum brasilense (UniProtKB Accession No. Q1JUP4), the gene product of Ssadh from Drosophila melanogaster (UniProtKB Accession No. Q9VBP6), the gene product of ALDH5A1 from Gorilla gorilla (UniProtKB Accession No. Q6A2H1), the gene product of ALDH5A1 from Hylobates lar (UniProtKB Accession No. Q3MSM3), the gene product of ssadh from Lucilia cuprina (UniProtKB Accession No. B0JFD4), the gene product of ALDH5A1 from Pan paniscus (UniProtKB Accession No. Q3MSM4), the gene product of ALDH5A1 from Pan troglodytes (UniProtKB Accession No. Q6A2H0), the gene product of ALDH5A1 from Pongo abelii (UniProtKB Accession No. Q6A2H2), the gene product of ALDH5A1 from Pongo pygmaeus (UniProtKB Accession No. Q6A2H2), or the gene product of gapN-1 from Sulfolobus solfataricus (UniProtKB Accession No. Q97XS9)), or an aldehyde dehydrogenase classified under EC 1.2.1.3.
[0278] As shown in
[0279] As shown in
[0280] In some embodiments, a polypeptide having the activity of an acyl-CoA dehydrogenase may be classified under, for example, EC 1.3.8.-, such as EC 1.3.8.6, EC 1.3.8.7, or EC 1.3.8.8.
[0281] In some embodiments, a polypeptide having the activity of an enoyl-CoA hydratase may be classified under, for example, EC 4.2.1.17, such as, for example, the gene product of crt from Clostridium acetobutylicum, or classified under EC 4.2.1.119, such as, for example, the gene product of phaJ from Pseudomonas aeruginosa. In some embodiments, a polypeptide having the activity of a 3-hydroxyacyl-CoA dehydrogenase may be classified for example, under EC 1.1.1.-, such as EC 1.1.1.35 (e.g., the gene product of fadB from Escherichia coli), EC 1.1.1.36 (e.g., the gene product of phaB from Cupriavidus necator), or EC 1.1.1.157 (e.g., the gene product of hbd from Clostridium acetobutylicum), and a polypeptide having the activity of a 3-oxoacyl-ACP reductase may be classified, for example, under EC 1.1.1.100, such as, for example, the gene product of fabG from Escherichia coli.
[0282] In some embodiments, a polypeptide having the activity of a β-ketothiolase may be classified, for example, under EC 2.3.1.16 or EC 2.3.1.174 such as, for example, the gene product of bktB from Cupriavidus necator or paaJ from Escherichia coli.
Enzymes Generating the Terminal Carboxyl Groups in the Biosynthesis of Pimelic Acid
[0283] As depicted in
[0284] In some embodiments, the second terminal carboxyl group leading to the synthesis of pimelic acid can be enzymatically formed in pimeloyl-CoA by a polypeptide having the activity of a thioesterase classified under EC 3.1.2.-. The polypeptide having the activity of a thioesterase can be, for example, the gene product of yciA from Escherichia coli or acot13 from Mus musculus (Cantu et al., Protein Science, 2010, 19, 1281-1295; Zhuang et al., Biochemistry, 2008, 47(9):2789-2796; Naggert et al., J. Biol. Chem., 1991, 266(17): 11044-11050), or tesB from Escherichia coli, or the gene product of BT_2075 from Bacteroides thetaiotaomicron (strain ATCC 29148/DSM 2079/NCTC 10582/E50/VPI-5482) (GenBank Accession No. AAO77182.1, SEQ ID NO: 20), the gene product of lp_0708 from Lactobacillus plantarum (strain ATCC BAA-793/NCIMB 8826/WCFS1) (GenBank Accession No. CCC78182.1, SEQ ID NO: 22), the gene product of HMPREF0077_1317 from Anaerococcus tetradius ATCC 35098 (GenBank Accession No. EE182564.1, SEQ ID NO: 23), or the gene product of CPF_2954 from Clostridium perfringens (strain ATCC 13124/DSM 756/JCM 1290/NCIMB 6125/NCTC 8237/Type A) (GenBank Accession No. ABG82470.1, SEQ ID NO: 18)).
[0285] In some embodiments, the second terminal carboxyl group leading to the synthesis of pimelic acid can be enzymatically formed in pimeloyl-CoA by a polypeptide having the activity of a CoA ligase classified under EC 6.2.1.-, such as EC 6.2.1.5 or EC 6.2.1.15, or a polypeptide having the activity of a CoA transferase classified under EC 2.8.3.-, such as EC 2.8.3.8 or EC 2.8.3.12 (e.g., a succinyl-CoA:acetate CoA-transferase from Acetobacter aceti (UniProtKB Accession No. B3EY95), the gene product of ANACAC_01149 from Anaerostipes caccae (UniProtKB Accession No. B0MC58), a butyryl-CoA:acetate CoA-transferase from Butyrivibrio fibrisolvens (UniProtKB Accession No. D2WEY7), a butyryl-CoA:acetate CoA-transferase from Eubacterium hallii (UniProtKB Accession No. D2WEY8), the gene product of FAEPRAA2165_01575 from Faecalibacterium prausnitzii (UniProtKB Accession No. C7H5K4), a butyryl-CoA:acetate CoA-transferase from Faecalibacterium prausnitzii (UniProtKB Accession No. D2WEZ2), the gene product of FAEPRAM212_02812 from Faecalibacterium prausnitzii (UniProtKB Accession No. A8SFP6), a butyryl-CoA transferase from Roseburia hominis (UniProtKB Accession No. Q2TME9), or a butyryl-CoA:acetate CoA-transferase from Roseburia inulinivorans (UniProtKB Accession No. D2WEY6)).
[0286] In some embodiments, pimeloyl-CoA can be enzymatically converted to pimelate semialdehyde by a polypeptide having the activity of an aldehyde dehydrogenase classified under, for example, EC 1.2.1.10, such as an acetaldehyde dehydrogenase encoded by pduB from Salmonella typhimurium. The second terminal carboxyl group leading to the synthesis of pimelic acid can be enzymatically formed in pimelate semialdehyde by a polypeptide having the activity of an aldehyde dehydrogenase classified under EC 1.2.1.3 (Guerrillot & Vandecasteele, Eur. J. Biochem., 1977, 81, 185-192); a 7-oxoheptanoate dehydrogenase (e.g., the gene product of thnG from Sphingomonas macrogolitabida; López-Sánchez et al., Appl. Environ. Microbiol., 2010, 76(1), 110-118) classified under EC 1.2.1.-; a 6-oxohextanoate dehydrogenase (e.g., the gene product of chnE from Acinetobacter sp.) classified, for example, under EC 1.2.1.63; a 5-oxopentanoate dehydrogenase classified, for example, under EC 1.2.1.20 (e.g., the gene product of cpnE from Comamonas sp.) or a succinate-semialdehyde dehydrogenase classified, for example, under EC 1.2.1.16, EC 1.2.1.24, or EC 1.2.1.79 (e.g., the gene product of ALDH5F1 from Arabidopsis thaliana (UniProtKB Accession No. Q9SAK4), the gene product of araE from Azospirillum brasilense (UniProtKB Accession No. Q1JUP4), the gene product of Ssadh from Drosophila melanogaster (UniProtKB Accession No. Q9VBP6), the gene product of ALDH5A1 from Gorilla gorilla (UniProtKB Accession No. Q6A2H1), the gene product of ALDH5A1 from Hylobates lar (UniProtKB Accession No. Q3MSM3), the gene product of ssadh from Lucilia cuprina (UniProtKB Accession No. B0JFD4), the gene product of ALDH5A1 from Pan paniscus (UniProtKB Accession No. Q3MSM4), the gene product of ALDH5A1 from Pan troglodytes (UniProtKB Accession No. Q6A2H0), the gene product of ALDH5A1 from Pongo abelii (UniProtKB Accession No. Q6A2H2), the gene product of ALDH5A1 from Pongo pygmaeus (UniProtKB Accession No. Q6A2H2), or the gene product of gapN-1 from Sulfolobus solfataricus (UniProtKB Accession No. Q97XS9)).
Enzymes Generating 7-Aminoheptanoate
[0287] As depicted in
[0288] Alternatively, pimelate (pimelic acid) as shown in
[0289] An additional ω-transaminase that can be used in the methods and microorganisms described herein is from Escherichia coli (GenBank Accession No. AAA57874.1, SEQ ID NO: 11, SEQ ID NO: 48). Some of the ω-transaminases classified, for example, under EC 2.6.1.29 or EC 2.6.1.82 are diamine ω-transaminases (e.g., SEQ ID NO: 11, SEQ ID NO: 48).
[0290] The reversible ω-transaminase from Chromobacterium violaceum (GenBank Accession No. AAQ59697.1, SEQ ID NO: 7) has demonstrated analogous activity accepting 7-aminoheptanoic acid as amino donor, thus forming the first terminal amine group in pimelate semialdehyde (Kaulmann et al., Enzyme and Microbial Technology, 2007, 41, 628-637).
Enzymes Generating the Terminal Amine Groups in the Biosynthesis of Heptamethylenediamine
[0291] As depicted in
[0292] In some embodiments, a terminal amine group leading to the synthesis of 7-aminoheptanoic acid is enzymatically formed in 7-aminoheptanal by a polypeptide having the activity of a ω-transaminase classified, for example, under EC 2.6.1.-, e.g., 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, for example, from Chromobacterium violaceum (GenBank Accession No. AAQ59697.1, SEQ ID NO: 7), Pseudomonas aeruginosa (GenBank Accession No. AAG08191.1, SEQ ID NO: 8), Pseudomonas syringae (GenBank Accession No. AAY39893.1, SEQ ID NO: 9), Rhodobacter sphaeroides (GenBank Accession No. ABA81135.1, SEQ ID NO: 10), Vibrio fluvialis (GenBank Accession No. AEA39183.1, SEQ ID NO: 12), Streptomyces griseus, or Clostridium viride. See
[0293] An additional polypeptide having the activity of a ω-transaminase that can be used in the methods and microorganisms described herein is from Escherichia coli (GenBank Accession No. AAA57874.1, SEQ ID NO: 11, SEQ ID NO: 48). Some of the polypeptides having the activity of ω-transaminases classified, for example, under EC 2.6.1.29 or EC 2.6.1.82 are diamine ω-transaminases (e.g., SEQ ID NO: 11, SEQ ID NO: 48).
[0294] The reversible ω-transaminase from Chromobacterium violaceum (GenBank Accession No. AAQ59697.1, SEQ ID NO: 7) has demonstrated analogous activity accepting 7-aminoheptanoic acid as amino donor, thus forming the first terminal amine group in pimelate semialdehyde (Kaulmann et al., Enzyme and Microbial Technology, 2007, 41, 628-637).
[0295] The reversible 4-aminobubyrate:2-oxoadipate transaminase from Streptomyces griseus has demonstrated activity for the conversion of 7-aminoheptanoate to pimelate semialdehyde (Yonaha et al., Eur. J. Biochem., 1985, 146, 101-106).
[0296] The reversible 5-aminovalerate transaminase from Clostridium viride has demonstrated activity for the conversion of 7-aminoheptanoate to pimelate semialdehyde (Barker et al., J. Biol. Chem., 1987, 262(19), 8994-9003).
[0297] In some embodiments, the second terminal amine group leading to the synthesis of heptamethylenediamine is enzymatically formed in 7-aminoheptanal by a polypeptide having the activity of a diamine transaminase classified, for example, under EC 2.6.1.29 or classified, for example, under EC 2.6.1.82, such as, for example, the gene product of ygjG from E. coli (GenBank Accession No. AAA57874.1, SEQ ID NO: 11, SEQ ID NO: 48). The polypeptides having the activity of a transaminase set forth in SEQ ID NOs: 7-10 and 12 also can be used to produce heptamethylenediamine. See
[0298] The gene product of ygjG from Escherichia coli accepts a broad range of diamine carbon chain length substrates, such as putrescine, cadaverine and spermidine (Samsonova et al, BMC Microbiology, 2003, 3:2).
[0299] 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).
[0300] In some embodiments, the second terminal amine group leading to the synthesis of heptamethylenediamine is enzymatically formed in N7-acetyl-1,7%-diaminoheptane by a polypeptide having the activity of a deacylase classified, for example, under EC 3.5.1.-, such as, for example, EC 3.5.1.62 or EC 3.5.1.82.
Enzymes Generating the Terminal Hydroxyl Groups in the Biosynthesis of 7-Hydroxyheptanoate
[0301] As depicted in
[0302] Alternatively, as shown in
Enzymes Generating the Terminal Hydroxyl Groups in the Biosynthesis of 1,7 Heptanediol
[0303] As depicted in
Biochemical Pathways
Pathways to Pimeloyl-CoA
[0304] In some embodiments, and as shown in
[0305] In some embodiments, and as shown in
[0306] In some embodiments, and as shown in
[0307] In some embodiments, and as shown in
[0308] In some embodiments, and as shown in
[0309] In some embodiments, and as shown in
Pathways Using Pimeloyl-CoA as Central Precursor to Pimelic Acid
[0310] In some embodiments, pimelic acid is synthesized from pimeloyl-CoA by a polypeptide having the activity of a thioesterase classified under, for example, EC 3.1.2.-. The polypeptide having the activity of a thioesterase can be the gene product of yciA from Escherichia coli or acot13 from Mus musculus (Cantu et al., Protein Science, 2010, 19, 1281-1295; Zhuang et al., Biochemistry, 2008, 47(9):2789-2796; Naggert et al., J. Biol. Chem., 1991, 266(17):11044-11050), or tesB from Escherichia coli or a polypeptide represented by one of the following GenBank accession numbers: AAO77182.1 (SEQ ID NO: 20); CCC78182.1 (SEQ ID NO: 22); EE182564.1 (SEQ ID NO: 23); or ABG82470.1 (SEQ ID NO: 18).
[0311] In some embodiments, pimelic acid is synthesized from pimeloyl-CoA by a polypeptide having the activity of a CoA ligase classified under, for example, EC 6.2.1.-, such as EC 6.2.1.5 or EC 6.2.1.15, or a CoA transferase classified under, for example, EC 2.8.3.-, such as EC 2.8.3.8 or EC 2.8.3.12 (e.g., a succinyl-CoA:acetate CoA-transferase from Acetobacter aceti (UniProtKB Accession No. B3EY95), the gene product of ANACAC_01149 from Anaerostipes caccae (UniProtKB Accession No. B0MC58), a butyryl-CoA:acetate CoA-transferase from Butyrivibrio fibrisolvens (UniProtKB Accession No. D2WEY7), a butyryl-CoA:acetate CoA-transferase from Eubacterium hallii (UniProtKB Accession No. D2WEY8), the gene product of FAEPRAA2165_01575 from Faecalibacterium prausnitzii (UniProtKB Accession No. C7H5K4), a butyryl-CoA:acetate CoA-transferase from Faecalibacterium prausnitzii (UniProtKB Accession No. D2WEZ2), the gene product of FAEPRAM212.02812 from Faecalibacterium prausnitzii (UniProtKB Accession No. A8SFP6), a butyryl-CoA transferase from Roseburia hominis (UniProtKB Accession No. Q2TME9), or a butyryl-CoA:acetate CoA-transferase from Roseburia inulinivorans (UniProtKB Accession No. D2WEY6)).
[0312] In some embodiments, pimeloyl-CoA is converted to pimelate semialdehyde by a polypeptide having the activity of an aldehyde dehydrogenase, such as an acetaldehyde dehydrogenase classified under, for example, EC 1.2.1.10, such as that encoded by pduB from Salmonella typhimurium.
[0313] Pimelate semialdehyde is then converted to pimelic acid by a polypeptide having the activity of an aldehyde dehydrogenase classified under, for example, EC 1.2.1.-, such as a 7-oxoheptanoate dehydrogenase (e.g., the gene product of thnG from Sphingomonas macrogolitabida), a 6-oxohextanoate dehydrogenase (e.g., the gene product of chnE from Acinetobacter sp.) classified, for example, under EC 1.2.1.63, a 5-oxopentanoate dehydrogenase classified, for example, under EC 1.2.1.20 (e.g., the gene product of cpnE from Comamonas sp.), a succinate-semialdehyde dehydrogenase classified, for example, under EC 1.2.1.16, EC 1.2.1.24, or EC 1.2.1.79, or an aldehyde dehydrogenase classified under EC 1.2.1.3. See
Pathways Using Pimeloyl-CoA as Central Precursor to 7-Aminoheptanoate
[0314] In some embodiments, pimeloyl-CoA is converted to pimelate semialdehyde using a polypeptide having the enzymatic activity of an aldehyde dehydrogenase classified under, for example, EC 1.2.1.10, such as an acetaldehyde dehydrogenase encoded by pduB from Salmonella typhimurium or pduP from Klebsiella pneumoniae. Pimelate semialdehyde is then converted to 7-aminoheptanoate using a polypeptide having the enzymatic activity of a ω-transaminase classified, for example, under EC 2.6.1.-, e.g., 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, for example, that obtained from Chromobacterium violaceum (GenBank Accession No. AAQ59697.1, SEQ ID NO: 7), Pseudomonas aeruginosa (GenBank Accession No. AAG08191.1, SEQ ID NO: 8), Pseudomonas syringae (GenBank Accession No. AAY39893.1, SEQ ID NO: 9), Rhodobacter sphaeroides (GenBank Accession No. ABA81135.1, SEQ ID NO: 10), Vibrio fluvialis (GenBank Accession No. AEA39183.1, SEQ ID NO: 12), Streptomyces griseus, or Clostridium viride.
[0315] In some embodiments, pimelate (see
Pathways Using Pimeloyl-CoA as Central Precursor to 7-Hydroxyheptanoate
[0316] In some embodiments, 7-hydroxyheptanoate is synthesized from the central precursor, pimeloyl-CoA using a polypeptide having the enzymatic activity of an aldehyde dehydrogenase classified under, for example, EC 1.2.1.10, such as an acetaldehyde dehydrogenase encoded by pduB from Salmonella typhimurium or pduP from Klebsiella pneumoniae; followed by conversion of pimelate semialdehyde to 7-hydroxyheptanoate by a polypeptide having the activity of an alcohol dehydrogenase classified, for example, under EC 1.1.1.2 such as, for example, the gene product of YMR318C from Saccharomyces cerevisiae, a 6-hydroxyhexanoate dehydrogenase classified, for example, under EC 1.1.1.258, a 5-hydroxypentanoate dehydrogenase classified, for example, under EC 1.1.1.-, such as, for example, the gene product of cpnD from Comamonas sp. (Iwaki et al., Appl. Environ. Microbiol., 1999, 65(11):5158-5162), or a 4-hydroxybutanoate dehydrogenase classified, for example, under EC 1.1.1.- such as, for example, the gene product of gabD from Escherichia coli (Bartsch et al., J. Bacteriol., 1990, 172(12), 7035). The alcohol dehydrogenase encoded by YMR318C has broad substrate specificity, including the oxidation of C7 alcohols. See
Pathway Using 7-Aminoheptanoate, 7-Hydroxyheptanoate, Pimelate Semialdehyde, or as 1,7-Heptanediol as a Central Precursor to Heptamethylenediamine
[0317] In some embodiments, heptamethylenediamine is synthesized from the central precursor, 7-aminoheptanoate (which can be produced as described in
[0318] The carboxylate reductase encoded by the gene product of car and enhancer npt from Nocardia or sfp from Bacillus subtilis has broad substrate specificity, including terminal difunctional C4 and C5 carboxylic acids (Venkitasubramanian et al., Enzyme and Microbial Technology, 2008, 42, 130-137).
[0319] In some embodiments, heptamethylenediamine is synthesized from the central precursor, 7-hydroxyheptanoate (which can be produced as described in
[0320] In some embodiments, heptamethylenediamine is synthesized from the central precursor, 7-aminoheptanoate (which can be produced as described in
[0321] In some embodiments, heptamethylenediamine is synthesized from the central precursor, pimelate semialdehyde, by conversion of pimelate semialdehyde to heptanedial by a polypeptide having the activity of a carboxylate reductase classified, for example, under EC 1.2.99.6 such as, for example, the gene product of car (see above, e.g., SEQ ID NO: 5) 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 from Streptomyces griseus (Suzuki et al., J. Antibiot., 2007, 60(6), 380-387) followed by conversion to 7-aminoheptanal by a polypeptide having the activity of a ω-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; followed by conversion to heptamethylenediamine by a polypeptide having the activity of a ω-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, for example, SEQ ID NOs: 7-12. See
Pathways Using 7-Hydroxyheptanoate as Central Precursor to 1,7-Heptanediol
[0322] In some embodiments, 1,7 heptanediol is synthesized from the central precursor, 7-hydroxyheptanoate (which can be produced as described in
Cultivation Strategy
[0323] In some embodiments, one or more C7 building blocks are biosynthesized in a recombinant microorganism using anaerobic, aerobic or micro-aerobic cultivation conditions. In some embodiments, the cultivation strategy entails nutrient limitation such as, for example, nitrogen, phosphate, or oxygen limitation.
[0324] 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.
[0325] 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.
[0326] 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, plant oils, or municipal waste.
[0327] 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. Bio. 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).
[0328] The efficient catabolism of lignocellulosic-derived levulinic acid has been 2 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).
[0329] 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; Pérez-Pantoja et al., FEMS Microbiol. Rev., 2008, 32, 736-794).
[0330] 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).
[0331] 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, 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).
[0332] 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).
[0333] 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 cycloheptane oxidation processes, or terephthalic acid/isophthalic acid mixture waste streams.
[0334] The efficient catabolism of methanol has been demonstrated for the methylotrophic yeast Pichia pastoris.
[0335] The efficient catabolism of ethanol has been demonstrated for Clostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008, 105(6) 2128-2133).
[0336] 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).
[0337] The efficient catabolism of syngas has been demonstrated for numerous microorganisms, such as Clostridium ljungdahlii and Clostridium autoethanogenum (Köpke et al., Applied and Environmental Microbiology, 2011, 77(15):5467-5475).
[0338] The efficient catabolism of the non-volatile residue waste stream from cycloheptane 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).
[0339] In some embodiments, the 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 microorganisms described herein that are capable of producing one or more C7 building blocks.
[0340] In some embodiments, the 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 microorganisms described herein that are capable of producing one or more C7 building blocks.
Metabolic Engineering
[0341] 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.
[0342] Furthermore, recombinant microorganisms 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 microorganism. This document provides cells of any of the genera and species listed and genetically engineered to express one or more (e.g., one, 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 cells can contain exogenous nucleic acids encoding enzymes catalyzing one or more of the steps of any of the pathways described herein.
[0343] 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.
[0344] Also, this document recognizes that where enzymes have been described as accepting (R)-enantiomers of substrate, analogous enzyme activities associated with (S)-enantiomer substrates exist that are not necessarily in the same enzyme class.
[0345] 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.
[0346] 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.
[0347] 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.
[0348] 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.
[0349] Attenuation strategies include, but are not limited to; the use of transposons, homologous recombination (double cross-over approach), mutagenesis, enzyme inhibitors and RNAi interference.
[0350] Attenuation strategies have been employed to increase the yield of desired end products of engineered metabolic pathways. For example, genetic manipulations previously studied to make succinate the major fermentation product in E. coli include deletion of the fermentative lactate dehydrogenase (LDH) pathway (Mat-Jan et al., 1989), deletion of both the LDH and pyruvate formate lyase (PFL) pathways (Bunch et al., 1997), and deletion of multiple pathways including PFL and LDH pathways with an additional ptsG mutation (Donnelly et al., 1998; Chatterjee et al., 2001). Overexpression of phosphoenolpyruvate carboxylase (PEPC) (Millard et al., 1996), overexpression of the malic enzyme (Stols and Donnelly, 1997; Hong and Lee, 2000), overexpression of pyruvate carboxylase (PYC) (Gokarn et al., 1998; Gokarn et al., 2000; Vemuri et al., 2002a), and overexpression of the heterologous Actinobacillus succinogenes phosphoenolpyruvate carboxykinase in a PEPC E. coli mutant (Kim et al., 2004) have also been studied to improve succinate yield from recombinant E. coli.
[0351] 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.
[0352] In some embodiments, the microorganism's tolerance to high concentrations of a C7 building block can be improved through continuous cultivation in a selective environment.
[0353] In some embodiments, the microorganism's endogenous biochemical network can be attenuated or augmented to (1) ensure the intracellular availability of acetyl-CoA or malonyl-CoA, (2) create an NADH or NADPH imbalance that may only be balanced via the formation of one or more C7 building blocks, (3) prevent degradation of central metabolites, central precursors leading to and including one or more C7 building blocks, and/or (4) ensure efficient efflux from the cell.
[0354] In some embodiments requiring intracellular availability of acetyl-CoA for C7 building block synthesis, endogenous enzymes catalyzing the hydrolysis of acetyl-CoA such as short-chain length thioesterase can be attenuated in the microorganism.
[0355] In some embodiments requiring the intracellular availability of acetyl-CoA for C7 building block synthesis, an endogenous gene encoding a phosphotransacetylase generating acetate such as pta can be attenuated (Shen et al., Appl. Environ. Microbiol., 2011, 77(9):2905-2915).
[0356] In some embodiments requiring the intracellular availability of acetyl-CoA for C7 building block synthesis, an endogenous gene in an acetate synthesis pathway encoding an acetate kinase, such as ack, can be attenuated.
[0357] In some embodiments requiring the intracellular availability of acetyl-CoA and NADH for C7 building block synthesis, an endogenous gene encoding an enzyme that catalyzes the degradation of pyruvate to lactate such as lactate dehydrogenase encoded by IdhA can be attenuated (Shen et al., 2011, supra).
[0358] In some embodiments, enzymes that catalyze anapleurotic reactions such as PEP carboxylase and/or pyruvate carboxylase can be overexpressed in the microorganism.
[0359] In some embodiments requiring the intracellular availability of acetyl-CoA and NADH for C7 building block synthesis, endogenous genes encoding enzymes, such as menaquinol-fumarate oxidoreductase, that catalyze the degradation of phophoenolpyruvate to succinate such as frdBC can be attenuated (see, e.g., Shen et al., 2011, supra).
[0360] In some embodiments requiring the intracellular availability of acetyl-CoA and NADH for C7 building block synthesis, an endogenous gene encoding an enzyme that catalyzes the degradation of acetyl-CoA to ethanol such as, for example, the alcohol dehydrogenase encoded by adhE from Clostridium acetobutylicum can be attenuated (Shen et al., 2011, supra). In some embodiments, where pathways require excess NADH co-factor for C7 building block synthesis, a recombinant formate dehydrogenase gene, e.g., fdh1 from Candida boidinii, can be overexpressed in the microorganism (Shen et al., 2011, supra).
[0361] In some embodiments, where pathways require excess NADH co-factor for C7 building block synthesis, a recombinant NADH-consuming transhydrogenase can be attenuated.
[0362] In some embodiments, an endogenous gene encoding an enzyme that catalyzes go the degradation of pyruvate to ethanol such as pyruvate decarboxylase can be attenuated.
[0363] In some embodiments requiring the intracellular availability of acetyl-CoA for C7 building block synthesis, a recombinant acetyl-CoA synthetase such as, for example, the gene product of acsA from Cupriavidus necator can be overexpressed in the microorganism (Satoh et al., J. Bioscience and Bioengineering, 2003, 95(4):335-341).
[0364] 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).
[0365] 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) from, for example, Escherichia coli.
[0366] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, a gene such as udhA from Escherichia coli encoding a puridine nucleotide transhydrogenase can be overexpressed in the microorganisms (Brigham et al., Advanced Biofuels and Bioproducts, 2012, Chapter 39, 1065-1090).
[0367] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 Building Block, a recombinant glyceraldehyde-3-phosphate-dehydrogenase gene such as gapN from Sulfolobus solfataricus can be overexpressed in the microorganisms (Brigham et al., 2012, supra).
[0368] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, a recombinant malic enzyme gene such as maeA or maeB from Cupriavidus necator can be overexpressed in the microorganisms (Brigham et al., 2012, supra).
[0369] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, a recombinant glucose-6-phosphate dehydrogenase gene such as zwf from Escherichia coli can be overexpressed in the microorganisms (Lim et al., J. Bioscience and Bioengineering, 2002, 93(6), 543-549).
[0370] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, a recombinant fructose 1,6 diphosphatase gene such as fbp from Corynebacterium glutamicum can be overexpressed in the microorganisms (Becker et al, J. Biotechnol., 2007, 132:99-109).
[0371] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, endogenous triose phosphate isomerase (EC 5.3.1.1) can be attenuated.
[0372] In some embodiments, where pathways require excess NADPH co-factor in the synthesis of a C7 building block, a recombinant glucose dehydrogenase such as, for example, the gene product of gdh from Bacillus subtilis can be overexpressed in the microorganism (Satoh et al., J. Bioscience and Bioengineering, 2003, 95(4):335-341).
[0373] In some embodiments, endogenous enzymes facilitating the conversion of NADPH to NADH can be attenuated, such as, for example, 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).
[0374] In some embodiments, an endogenous glutamate dehydrogenase (EC 1.4.1.3) that utilizes both NADH and NADPH as co-factors can be attenuated.
[0375] In some embodiments, a membrane-bound cytochrome P450 such as CYP4F3B can be solubilized by only expressing the cytosolic domain and not the N-terminal region that anchors the P450 to the endoplasmic reticulum (Scheller et al., J. Biol. Chem., 1994, 269(17):12779-12783).
[0376] In some embodiments, an enoyl-CoA reductase can be solubilized via expression as a fusion protein with a small soluble protein, for example, the maltose binding protein (Gloerich et al., FEBS Letters, 2006, 580, 2092-2096).
[0377] In some embodiments using microorganisms that naturally accumulate polyhydroxyalkanoates, the endogenous polymer synthase enzymes can be attenuated in the microorganism strain.
[0378] In some embodiments, a L-alanine dehydrogenase can be overexpressed in the microorganism to regenerate L-alanine from pyruvate as an amino donor for ω-transaminase catalyzed reactions. For example, the L-alanine dehydrogenase may be from Escherichia coli.
[0379] In some embodiments, an L-glutamate dehydrogenase, a L-glutamine synthetase, or an alpha-aminotransaminase can be overexpressed in the microorganism to regenerate L-glutamate from 2-oxoglutarate as an amino donor for ω-transaminase catalyzed reactions. For example, the L-glutamate dehydrogenase, the L-glutamine synthetase, or the alpha-aminotransaminase may be from Escherichia coli.
[0380] In some embodiments, enzymes such as a pimeloyl-CoA dehydrogenase classified, for example, under EC 1.3.1.62; an acyl-CoA dehydrogenase classified, for example, under EC 1.3.8.7, EC 1.3.8.1, or EC 1.3.99.-; and/or a butyryl-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. Examples of polypeptides having the activity of an acyl-CoA dehydrogenase classified under EC 1.3.99.- include, but are not limited to, the gene product of atuD from Pseudomonas aeruginosa (UniProtKB Accession No. Q9HZV8), the gene product of scu from Drosophila melanogaster (UniProtKB Accession No. O18404), the gene product of fadE26 from Mycobacterium tuberculosis (UniProtKB Accession No. I6YCA3), the gene product of aidB from Escherichia coli (UniProtKB Accession No. P33224), the gene product of acdh-11 from Caenorhabditis elegans (UniProtKB Accession No. Q9XWZ2), and the gene product of Acad11 from Mus musculus (UniProtKB Accession No. Q80XL6).
[0381] In some embodiments, endogenous enzymes activating C7 building blocks via Coenzyme A esterification such as CoA-ligases (e.g., an adipyl-CoA synthetase) classified under, for example, EC 6.2.1.- can be attenuated.
[0382] 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.
[0383] The efflux of heptamethylenediamine can be enhanced or amplified by overexpressing broad substrate range multidrug transporters such as Bit 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 Bmhr from Bacillus subtilis (Neyfakh, 1992, Antimicrob Agents Chemother, 36(2), 484-485).
[0384] The efflux of 7-aminoheptanoate and heptamethylenediamine can be enhanced or amplified by overexpressing the solute transporters such as, for example, the lysE transporter from Corynebacterium glutamicum (Bellmann et al., 2001, Microbiology, 147, 1765-1774).
[0385] The efflux of pimelic acid can be enhanced or amplified by overexpressing a dicarboxylate transporter such as, for example, the SucE transporter from Corynebacterium glutamicum (Huhn et al, Appl. Microbiol. & Biotech., 89(2), 327-335).
[0386] Metabolically engineering recombinant hosts with various enzymes to produce final products has been successfully demonstrated by several groups. See, e.g., Blombach B et al., Bioeng Bugs., 2011, 2(6):346-50 (teaching successful metabolic engineering of the last two steps of the Ehrlich pathway (by expression of genes encoding a broad range 2-ketoacid decarboxylase and an alcohol dehydrogenase) in recombinant hosts for the production of higher isobutanol); Adkins, J. et al, Front Microbiol., 2012, 3:313 (summarizing numerous biomonomers (such as polyester building-blocks) that can be produced as a result of metabolic and pathway engineering in various recombinant hosts); Chan, S. et al., Bioresour Technol., 2012, 103(1):329-36 (teaching production of succinic acid from sucrose and sugarcane molasses by metabolically engineering E. coli with sucrose-utilizing genes (cscKB and cscA)); Lee, S. et al., Appl Biochem Biotechnol., 2012, 167(1):24-38 (teaching successful metabolic engineering of P. aeruginosa and E. coli for improving long-chain fatty acid production by co-expressing essential enzymes that are involved in the fatty acid synthesis metabolic pathway (accA and fabD) as well as fatty acyl-acyl carrier protein thioesterase gene); Rathnasingh, C. et al., Biotechnol Bioeng., 2009, 104(4):729-39 (teaching successful metabolic engineering of E. coli for producing 3-hydroxypropionic acid from glycerol by overexpression of glycerol dehydratase (DhaB) and aldehyde dehydrogenase (AldH) along with glycerol dehydratase reactivase (GDR)).
Producing C7 Building Blocks Using a Recombinant Microorganism
[0387] Typically, one or more C7 building blocks can be produced by providing a 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 greater than 500 gallon 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.
[0388] 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.
[0389] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1
Enzyme Activity of ω-Transaminase Using Pimelate Semialdehyde as Substrate and Forming 7-Aminoheptanoate
[0390] A nucleotide sequence encoding an N-terminal His-tag was added to the nucleic acid sequences from Chromobacterium violaceum, Pseudomonas syringae, Rhodobacter sphaeroides, and Vibrio fluvialis encoding the ω-transaminases of SEQ ID NOs: 7, 9, 10, and 12, respectively (see
[0391] 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.
[0392] 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 μM pyridoxyl 5′ phosphate. Each enzyme activity assay reaction was initiated by adding cell free extract of the ω-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.
[0393] Each enzyme only control without 7-aminoheptanoate demonstrated low base line conversion of pyruvate to L-alanine. See
[0394] Enzyme activity in the forward direction (i.e., pimelate semialdehyde to 7-aminoheptanoate) was confirmed for the transaminases of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 12. See
[0395] The gene products represented by SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 12 accepted pimelate semialdehyde as substrate as confirmed against the empty vector control. See
Example 2
Enzyme Activity of Carboxylate Reductase Using Pimelate as Substrate and Forming Pimelate Semialdehyde
[0396] A nucleotide sequence encoding a HIS-tag was added to the nucleic acid sequences from Segniliparus rugosus and Segniliparus rotundus that encode the carboxylate reductases of SEQ ID NOs: 3 (EFV111917.1) and 6 (ADG98140.1), respectively (see
[0397] 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.
[0398] 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 bars for EFV11917.1 and ADG98140.1 in
[0399] The gene products represented by SEQ ID NO: 3 (EFV11917.1) and SEQ ID NO: 6 (ADG98140.1), enhanced by the gene product of sfp from Bacillus subtilis, accepted pimelate as a substrate, as confirmed against the empty vector control (see
Example 3
Enzyme Activity of Carboxylate Reductase Using 7-Hydroxyheptanoate as Substrate and Forming 7-Hydroxyheptanal
[0400] A nucleotide sequence encoding a His-tag was added to the nucleic acids from Mycobacterium marinum, Mycobacterium smegmatis, Segniliparus rugosus, Mycobacterium massiliense, and Segniliparus rotundus that encode the carboxylate reductases of SEQ ID NOs: 1-6 respectively (see
[0401] 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.
[0402] 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
[0403] The gene products represented by SEQ ID NO 1-6 enhanced by the gene product of sfp, accepted 7-hydroxyheptanoate as substrate as confirmed against the empty vector control (see
Example 4
Enzyme Activity of ω-Transaminase for 7-Aminoheptanol, Forming 7-Oxoheptanol
[0404] A nucleotide sequence encoding an N-terminal His-tag was added to the Chromobacterium violaceum, Pseudomonas syringae, and Rhodobacter sphaeroides nucleic acids encoding the ω-transaminases of SEQ ID NOs: 7, 9, and 10, respectively (see
[0405] 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.
[0406] 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 μM pyridoxyl 5′ phosphate. Each enzyme activity assay reaction was initiated by adding cell free extract of the ω-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.
[0407] Each enzyme only control without 7-aminoheptanol had low base line conversion of pyruvate to L-alanine. See
[0408] The gene products represented by SEQ ID NOs: 7, 9, and 10 accepted 7-aminoheptanol as substrate as confirmed against the empty vector control (see
Example 5
Enzyme Activity of ω-Transaminase Using Heptamethylenediamine as Substrate and Forming 7-Aminoheptanal
[0409] A nucleotide sequence encoding an N-terminal His-tag was added to the Chromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae, Rhodobacter sphaeroides, Escherichia coli, and Vibrio fluvialis nucleic acids encoding the ω-transaminases of SEQ ID NOs: 7-12, respectively (see
[0410] 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.
[0411] 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 μM pyridoxyl 5′ phosphate. Each enzyme activity assay reaction was initiated by adding cell free extract of the ω-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.
[0412] Each enzyme only control without heptamethylenediamine had low base line conversion of pyruvate to L-alanine. See
[0413] The gene products of SEQ ID NOs: 7-12 accepted heptamethylenediamine as substrate as confirmed against the empty vector control (see
Example 6
Enzyme Activity of Carboxylate Reductase for N7-Acetyl-7-Aminoheptanoate, Forming N7-Acetyl-7-Aminoheptanal
[0414] The activity of each of the N-terminal His-tagged carboxylate reductases of SEQ ID NOs: 2, 5, and 6 (see Examples 2 and 3, and
[0415] The gene products of SEQ ID NOs: 2, 5, and 6, enhanced by the gene product of sfp, accepted N7-acetyl-7-aminoheptanoate as substrate as confirmed against the empty vector control (see
Example 7
Enzyme Activity of ω-Transaminase Using N7-Acetyl-1,7-Diaminoheptane, and Forming N7-Acetyl-7-Aminoheptanal
[0416] The activity of the N-terminal His-tagged ω-transaminases of SEQ ID NOs: 7-12 (see Example 5, and
[0417] Each enzyme only control without N7-acetyl-1,7-diaminoheptane demonstrated low base line conversion of pyruvate to L-alanine. See
[0418] The gene product of SEQ ID NOs: 7-12 accepted N7-acetyl-1,7-diaminoheptane as substrate as confirmed against the empty vector control (see
[0419] Given the reversibility of the ω-transaminase activity (see Example 1), the gene products represented by SEQ ID NOs: 7-12 accept N7-acetyl-7-aminoheptanal as substrate forming N7-acetyl-1,7-diaminoheptane.
Example 8
Enzyme Activity of Carboxylate Reductase Using Pimelate Semialdehyde as Substrate and Forming Heptanedial
[0420] The N-terminal His-tagged carboxylate reductase of SEQ ID NO: 6 (see Example 3 and
[0421] The gene product of SEQ ID NO: 6, enhanced by the gene product of sfp from Bacillus subtilis, accepted pimelate semialdehyde as substrate as confirmed against the empty vector control (see
OTHER EMBODIMENTS
[0422] 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.