Method for the preparation of 2,4-dihydroxybutyrate

A method for the preparation of 2,4-dihydroxybutyrate 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, wherein
• the enzyme having homoserine transaminase activity either
• is encoded by the sequence set forth in SEQ ID NO:
• 59, SEQ ID NO:
• 61, SEQ ID NO:
• 63, SEQ ID NO:
• 65, SEQ ID NO:
• 67 or SEQ ID NO:
• 69, or
• is selected from SEQ ID NO:
• 60, SEQ ID NO:
• 62, SEQ ID NO:
• 64, SEQ ID NO:
• 66, SEQ ID NO:
• 68 or SEQ ID NO:
• 70 or is selected from any sequence sharing a sequence identity of at least 90% with SEQ ID NO:
• 60, SEQ ID NO:
• 62, SEQ ID NO:
• 64, SEQ ID NO:
• 66, SEQ ID NO:
• 68 or SEQ ID NO:
• 70, and
• 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 acid group of homoserine to a carbonyl group to obtain OHB;
• and
• reducing the OHB to form 2,4-dihydroxybutyrate (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 an OHB reductase that either
• is selected from SEQ ID NO:
• 2, SEQ ID NO:
• 4, SEQ ID NO:
• 6, SEQ ID NO:
• 8, SEQ ID NO:
• 10, SEQ ID NO:
• 12, SEQ ID NO:
• 14, SEQ ID NO:
• 288, SEQ ID NO:
• 30, SEQ ID NO:
• 32, SEQ ID NO:
• 102, SEQ ID NO:
• 104, SEQ ID NO:
• 106, SEQ ID NO:
• 108, SEQ ID NO:
• 110, SEQ ID NO:
• 112, SEQ ID NO:
• 114, SEQ ID NO:
• 116 or SEQ ID NO:
• 118 or is selected from any sequence sharing a sequence identity of at least 90% with SEQ ID NO:
• 2, SEQ ID NO:
• 4, SEQ ID NO:
• 6, SEQ ID NO:
• 8, SEQ ID NO:
• 10, SEQ ID NO:
• 12, SEQ ID NO:
• 14, SEQ ID NO:
• 288, SEQ ID NO:
• 30, SEQ ID NO:
• 32, SEQ ID NO:
• 102, SEQ ID NO:
• 104, SEQ ID NO:
• 106, SEQ ID NO:
• 108, SEQ ID NO:
• 110, SEQ ID NO:
• 112, SEQ ID NO:
• 114, SEQ ID NO:
• 116 or SEQ ID NO:
• 118, or
• is encoded by a nucleic acid sequence selected from SEQ ID NO:
• 1, SEQ ID NO:
• 3, SEQ ID NO:
• 5, SEQ ID NO:
• 7, SEQ ID NO:
• 9, SEQ ID NO:
• 11, SEQ ID NO:
• 13, SEQ ID NO:
• 287, SEQ ID NO:
• 29, SEQ ID NO:
• 31, SEQ ID NO:
• 101, SEQ ID NO:
• 103, SEQ ID NO:
• 105, SEQ ID NO:
• 107, SEQ ID NO:
• 109, SEQ ID NO:
• 111, SEQ ID NO:
• 113, SEQ ID NO:
• 115 or SEQ ID NO:
• 117.

A modified microorganism for the preparation of 2,4-DHB (2,4-dihydroxybutyrate) from homoserine via a two-step pathway comprising:

• 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 either
• is encoded by the sequence set forth in SEQ ID NO:
• 59, SEQ ID NO:
• 61, SEQ ID NO:
• 63, SEQ ID NO:
• 65, SEQ ID NO:
• 67 or SEQ ID NO:
• 69, or
• is selected from SEQ ID NO:
• 60, SEQ ID NO:
• 62, SEQ ID NO:
• 64, SEQ ID NO:
• 66, SEQ ID NO:
• 68 or SEQ ID NO:
• 70 or is selected from any sequence sharing a sequence identity of at least 90% with SEQ ID NO:
• 60, SEQ ID NO:
• 62, SEQ ID NO:
• 64, SEQ ID NO:
• 66, SEQ ID NO:
• 68 or SEQ ID NO:
• 70, and
• reducing 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 an OHB reductase that is selected from the group consisting of (D)-lactate dehydrogenase from Escherichia coli (SEQ ID NO:
• 4), (L)-lactate dehydrogenase from Lactococcus lactis (SEQ ID NO:
• 6), the two isoforms of (L)-lactate dehydrogenase from Oryctalagus cuniculus (SEQ ID NO:
• 12 and SEQ ID NO:
• 14), (L)-lactate dehydrogenase from Geobacillus stearothermophilus (SEQ ID NO:
• 10), (L)-lactate dehydrogenase from Bacillus subtilis (SEQ ID NO:
• 8), (L)-malate dehydrogenase from Escherichia coli (SEQ ID NO:
• 2), branched chain (D)-2-hydroxyacid dehydrogenase from Lactococcus lactis, and dehydrogenases having an amino acid sequence sharing a sequence identity of at least 90% with at least one of said sequences;
• 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 microorganism comprising:
• a first chimeric gene including a first nucleic acid sequence encoding the enzyme having homoserine transaminase activity for converting the primary amino acid 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 to 2,4-DHB.