Method for the preparation of 2,4-dihydroxybutyrate
First Claim
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1. A method for the preparation of 2,4-dihydroxybutyrate (2,4-DHB) from homoserine comprising:
- deaminating homoserine to form 2-oxo-4-hydroxybutyrate (OHB), where the deamination of homoserine is catalyzed by an enzyme having homoserine transaminase activity, whereinthe enzyme having homoserine transaminase activity is produced via a transformed host microorganism that comprises a first chimeric gene including a first nucleic acid sequence encoding the enzyme having homoserine transaminase activity for converting the primary amino group of homoserine to a carbonyl group to obtain OHB, andthe enzyme having homoserine transaminase activity is selected from the group consisting of aspC from Escherichia coli (SEQ ID NO;
64), ilvE from Escherichia coli (SEQ ID NO;
60), bcaT from Lactococcus lactis (SEQ ID NO;
68), tyrB from Escherichia coli (SEQ ID NO;
62), araT from Lactococcus lactis (SEQ ID NO;
66), and ARO8 from Saccharomyces cerevisiae (SEQ ID NO;
70), or is selected from any sequence sharing a sequence identity of at least 90% with at least one of the sequences of said enzymes having homoserine transaminase activity; and
reducing the OHB to form 2,4-DHB, where the reduction of OHB is catalyzed by an enzyme having OHB reductase activity, whereinthe enzyme having OHB reductase activity is produced via the transformed host microorganism, which further comprises a second chimeric gene including a second nucleic acid sequence encoding the enzyme having OHB reductase activity for reducing OHB to 2,4-DHB, andthe enzyme having OHB reductase activity is selected from the group consisting of (L)-malate dehydrogenase from Escherichia coli (SEQ ID NO;
2), (D)-lactate dehydrogenase from Escherichia coli (SEQ ID NO;
4), (L)-lactate dehydrogenase from Lactococcus lactis (SEQ ID NO;
6), (L)-lactate dehydrogenase from Bacillus subtilis (SEQ ID NO;
8), (L)-lactate dehydrogenase from Geobacillus stearothermophilus (SEQ ID NO;
10), the two isoforms of (L)-lactate dehydrogenase from Oryctalagus cuniculus (SEQ ID NO;
12 and SEQ ID NO;
14), or is selected from any sequence sharing a sequence identity of at least 90% with at least one of said enzymes having OHB reductase activity.
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Abstract
A method for the preparation of 2,4-dihydroxybutyric acid from homoserine includes a first step of conversion of the primary amino group of homoserine to a carbonyl group to obtain 2-oxo-4-hydroxybutyrate, and a second step of reduction of the obtained 2-oxo-4-hydroxybutyrate (OHB) to 2,4-dihydroxybutyrate.
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Citations
13 Claims
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1. A method for the preparation of 2,4-dihydroxybutyrate (2,4-DHB) from homoserine comprising:
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deaminating homoserine to form 2-oxo-4-hydroxybutyrate (OHB), where the deamination of homoserine is catalyzed by an enzyme having homoserine transaminase activity, wherein the enzyme having homoserine transaminase activity is produced via a transformed host microorganism that comprises a first chimeric gene including a first nucleic acid sequence encoding the enzyme having homoserine transaminase activity for converting the primary amino group of homoserine to a carbonyl group to obtain OHB, and the enzyme having homoserine transaminase activity is selected from the group consisting of aspC from Escherichia coli (SEQ ID NO;
64), ilvE from Escherichia coli (SEQ ID NO;
60), bcaT from Lactococcus lactis (SEQ ID NO;
68), tyrB from Escherichia coli (SEQ ID NO;
62), araT from Lactococcus lactis (SEQ ID NO;
66), and ARO8 from Saccharomyces cerevisiae (SEQ ID NO;
70), or is selected from any sequence sharing a sequence identity of at least 90% with at least one of the sequences of said enzymes having homoserine transaminase activity; andreducing the OHB to form 2,4-DHB, where the reduction of OHB is catalyzed by an enzyme having OHB reductase activity, wherein the enzyme having OHB reductase activity is produced via the transformed host microorganism, which further comprises a second chimeric gene including a second nucleic acid sequence encoding the enzyme having OHB reductase activity for reducing OHB to 2,4-DHB, and the enzyme having OHB reductase activity is selected from the group consisting of (L)-malate dehydrogenase from Escherichia coli (SEQ ID NO;
2), (D)-lactate dehydrogenase from Escherichia coli (SEQ ID NO;
4), (L)-lactate dehydrogenase from Lactococcus lactis (SEQ ID NO;
6), (L)-lactate dehydrogenase from Bacillus subtilis (SEQ ID NO;
8), (L)-lactate dehydrogenase from Geobacillus stearothermophilus (SEQ ID NO;
10), the two isoforms of (L)-lactate dehydrogenase from Oryctalagus cuniculus (SEQ ID NO;
12 and SEQ ID NO;
14), or is selected from any sequence sharing a sequence identity of at least 90% with at least one of said enzymes having OHB reductase activity. - View Dependent Claims (2, 3, 4)
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5. A modified microorganism for the preparation of 2,4-dihydroxybutyrate (2,4-DHB) from homoserine via a two-step pathway comprising:
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deaminating homoserine to form 2-oxo-4-hydroxybutyrate (OHB), where the deamination of homoserine is catalyzed by an enzyme having homoserine transaminase activity selected from the group consisting of aspC from Escherichia coli (SEQ ID NO;
64), ilvE from Escherichia coli (SEQ ID NO;
60), bcaT from Lactococcus lactis (SEQ ID NO;
68), tyrB from Escherichia coli (SEQ ID NO;
62), araT from Lactococcus lactis (SEQ ID NO;
66), and ARO8 from Saccharomyces cerevisiae (SEQ ID NO;
70), or is selected from any sequence sharing a sequence identity of at least 90% with at least one of the sequences of said enzymes having homoserine transaminase activity, andreducing the OHB to form 2,4-DHB, where the reduction of OHB is catalyzed by an enzyme having OHB reductase activity, wherein the enzyme having OHB reductase activity is selected from the group consisting of (L)-malate dehydrogenase from Escherichia coli (SEQ ID NO;
2), (D)-lactate dehydrogenase from Escherichia coli (SEQ ID NO;
4), (L)-lactate dehydrogenase from Lactococcus lactis (SEQ ID NO;
6), (L)-lactate dehydrogenase from Bacillus subtilis (SEQ ID NO;
8), (L)-lactate dehydrogenase from Geobacillus stearothermophilus (SEQ ID NO;
10), and the two isoforms of (L)-lactate dehydrogenase from Oryctalagus cuniculus (SEQ ID NO;
12 and SEQ ID NO;
14), or is selected from any sequence sharing a sequence identity of at least 90% with at least one of said enzymes having homoserine transaminase activity;wherein the modified microorganism is a host microorganism that has been transformed to enhance production of 2,4-DHB compared to a non-transformed host microorganism, the transformed host microorganism comprising; a first chimeric gene including a first nucleic acid sequence encoding the enzyme having homoserine transaminase activity for converting the primary amino group of homoserine to a carbonyl group to obtain OHB, and a second chimeric gene including a second nucleic acid sequence encoding the enzyme having OHB reductase activity for reducing OHB in 2,4-DHB. - View Dependent Claims (6, 7, 8, 9, 10, 11, 12, 13)
has at least one of the genes deleted chosen among ldhA, adhE, ackA, pta, poxB, focA, pfIB, sad, gabABC, sfcA, maeB, ppc, pykA, pykF, mgsA, sucAB, ptsl, ptsG, pgi, fumABCaldA, HdD, iclR, metA, thrB, lysA, eda, rthA, rthB, and rthC.
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11. The modified microorganism of claim 5, wherein the enzyme having homoserine transaminase activity is selected from the group consisting of aspC from Escherichia coli (SEQ ID NO:
- 64), ilvE from Escherichia coli (SEQ ID NO;
60), bcaT from Lactococcus lactis (SEQ ID NO;
68), tyrB from Escherichia coli (SEQ ID NO;
62), araT from Lactococcus lactis (SEQ ID NO;
66), and ARO8 from Saccharomyces cerevisiae (SEQ ID NO;
70), andwherein the enzyme having OHB reductase activity is selected from the group consisting of (L)-malate dehydrogenase from Escherichia coli (SEQ ID NO;
2), (D)-lactate dehydrogenase from Escherichia coli (SEQ ID NO;
4), (L)-lactate dehydrogenase from Lactococcus lactis (SEQ ID NO;
6), (L)-lactate dehydrogenase from Bacillus subtilis (SEQ ID NO;
8), (L)-lactate dehydrogenase from Geobacillus stearothermophilus (SEQ ID NO;
10), and the two isoforms of (L)-lactate dehydrogenase from Oryctalagus cuniculus (SEQ ID NO;
12 and SEQ ID NO;
14).
- 64), ilvE from Escherichia coli (SEQ ID NO;
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12. A method of production of 2,4-DHB comprising the steps of
culturing the modified microorganism of claim 5 in an appropriate culture medium, recovering 2,4-DHB from the culture medium. -
13. The method of claim 12 wherein the 2,4-DHB is further purified.
Specification