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A method of production of 2,4-dihydroxybutyric acid

A technology of dihydroxybutyric acid and malic acid, applied in biochemical equipment and methods, carbon-carbon lyase, enzymes, etc., can solve problems such as uncertainty

Active Publication Date: 2014-12-31
AVEATIS ANIMAL NUTRITION SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Only a few studies have reported the production of 2,4-DHB in patients with succinate semialdehyde dehydrogenase deficiency (Shinka et al. 2002), however the enzyme reactions involved in 2,4-DHB production have not been identified

Method used

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  • A method of production of 2,4-dihydroxybutyric acid
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  • A method of production of 2,4-dihydroxybutyric acid

Examples

Experimental program
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Effect test

Embodiment 1

[0102] Example 1: Demonstration of malyl-CoA lyase activity

[0103] Construction of plasmids containing wild-type genes encoding malyl-CoA lyase: Encoding of malyl-CoA lyase in outer chain Methylobacterium (Arps et al., 1993) and Rhodobacter capsulata (Meister et al., 2005) The DNA sequence of the mcl gene was optimized for expression in E. coli using GENEius software (Eurofins). Optimized sequence by Eurofins MWG For synthesis, NheI and EcoRI were added at restriction sites upstream of the start codon and mcl was added downstream of the stop codon, which enabled clones of synthetic DNA fragments to directly enter pET28a+ vector (Novagen) using T4 DNA ligase (Biolabs). Bound products were expanded by transfection into E. coli DH5α cells, and the plasmids pET28-Mex-mcl (expressing malyl-CoA lyase from outer chain Methylobacterium) and pET28-Mex were isolated using standard genetic protocols (Sambrook et al., 1989). Rca-mcl (from M. capsulatus expressing malyl-CoA lyase). N...

Embodiment 2

[0118] Example 2: Demonstration of Malyl-CoA Reductase Activity

[0119] Construction of plasmids containing wild-type genes encoding malyl-CoA reductase and succinyl-CoA reductase: encoding of malyl-CoA reductase mcr in the hyperthermophilic archaea str 7 (Alber et al., 2006) The DNA sequence of the gene was optimized for expression in E. coli using the GENEius software (Eurofins). Optimized mcr sequence and native DNA sequence of the sucD gene encoding succinyl-CoA reductase in Porphyrophyllum gingivalis W83 via Eurofins MWG For synthesis, NheI and EcoRI were added upstream of the start codon and mcl downstream of the stop codon, respectively, which enabled direct clones of synthetic DNA fragments into pET28a+ vector (Novagen) using T4 DNA ligase (Biolabs) . Bound products were expanded by transshipment into E. coli DH5α cells, and the plasmids pET28-St-mcr (expressing malyl-CoA reductase from hyperthermophilic archaea) and pET28 were isolated using standard genetic proto...

Embodiment 3

[0141] Example 3: Demonstration of DHB reductase activity

[0142] To identify suitable 2,4DHB reductases, β-hydroxycarboxylic acid dehydrogenases of different biological origins were tested for their ability to reduce malate semialdehyde. The enzymes tested were: methylbutyraldehyde reductase Ypr1 from Saccharomyces cerevisiae (Ford & Ellis, 2002) (SEQ ID No.14), 4-hydroxybutyrate dehydrogenase from Porphyrophyllum gingivalis, 4hbdh (SEQ ID No.187 ), alcohol dehydrogenase YqhD from Escherichia coli (SEQ ID no.185), and succinate semialdehyde reductase Ms-Ssr from Metallococcus (Kockelkorn & Fuchs, 2009) (SEQ ID No.16). Use the primers listed in Table 5 to amplify the genes YPR1, 4hbdh, yqhD, and Ms-SSR and clone them into the vector pET28 (see Table 5 for restriction enzymes) to obtain plasmids pET28-Sce-YPR1 and pET28-Pgi-4hbdh respectively , pET28-Eco-yqhd and pET28-Mse-SSR. The protein was expressed and purified as described in Example 1.

[0143]

[0144] table 5:...

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Abstract

The present invention deals with a method for the preparation of 2,4- dihydroxybutyric acid (2,4-DHB) comprising the successive steps of converting malate, succinyl-CoA and / or glyoxylate into malyl-CoA, converting malyl-CoA previously obtained into malate-4-semialdehyde, and converting malate-4- semialdehyde into 2,4-DHB using a DHB dehydrogenase.

Description

technical field [0001] The present invention relates to a novel process for the production of 2,4-dihydroxybutyrate from malate and / or glyoxylate and / or succinyl-CoA by implementing a synthetic route to convert malate and / or glyoxylate and / or conversion of succinyl-CoA to malyl-CoA, conversion of malyl-CoA to malate-4-semialdehyde, and conversion of said malate-4-semialdehyde to 2,4-di Hydroxybutyric acid (2,4-DHB). Background technique [0002] The carboxylic acids mentioned in the present invention are named equivalently under their salt form (eg 2,4-dihydroxybutyrate) or acid form (eg 2,4-dihydroxybutyric acid). [0003] 2,4-Dihydroxybutyric acid (equivalent to 2,4-DHB or DHB) is a compound with considerable economic benefits. DHB can be easily converted to α-hydroxy-γ-butyrolactone in aqueous medium by adjusting the appropriate pH value. α-Hydroxy-γ-butyrolactone is an important precursor for the production of methionine substitute 2-hydroxy-4-(methylthio)-butyrate (H...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12N9/00C12N9/04C12N9/02C12N9/10C12N9/88C07C59/10
CPCC12Y102/01075C12Y102/01076C12Y602/01009C12Y401/03024C12N9/0006C12N9/0008C12N9/13C12Y101/01002C12Y101/01061C12N9/88C12N9/93C12Y208/03C12P7/52C12N9/001C12Y103/01028C12P7/42
Inventor 托马斯·瓦尔特克莱芒蒂娜·德雷赛尔伊莲娜·科迪尔让-玛丽·弗朗索瓦
Owner AVEATIS ANIMAL NUTRITION SA
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