Production of 3-hydroxybutyrate

A hydroxybutyric acid, wild-type technology, applied in the biological field of producing 3-hydroxybutyric acid and/or its variants, can solve the problems of energy consumption in the process required for extraction, not utilizing the photosynthetic potential of cyanobacteria, etc. Waste, no material loss, increased production efficiency

Active Publication Date: 2018-02-16
EVONIK OPERATIONS GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite efforts to increase PHB biosynthesis through both genetic engineering strategies and optimization of culture conditions, PHB biosynthesis in cyanobacteria is a multistage culture process that involves nitrogen starvation followed by supplementation of fructose or acetate, which do not utilize Important photosynthetic potential of cyanobacteria
Most importantly, since the cells secrete neither lipid nor PHB, the process required for its extraction is energy-intensive and remains one of the major obstacles to commercial application.

Method used

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  • Production of 3-hydroxybutyrate

Examples

Experimental program
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Effect test

Embodiment 1

[0072] Generation of acetogens genetically modified to form 3HB via acetoacetate

[0073] Thiolase (thl) from Clostridium acetobutylicum ATTC 824, acetoacetate transferase (ctfAB) from Clostridium acetobutylicum ATTC 824 and secondary alcohol dehydrogenase (sadh) from Clostridium beijerinckii DSM 6423 Gene insertion vector pEmpty. This plasmid is based on the plasmid backbone pSOS95 ( figure 1 ). To use pSOS95, it was digested with BamHI and Kasl. This removed the operon ctfA-ctfB-adc, but left the thl promoter and rho-independent terminator of adc. Clostridium ljungdahlii and C. autoethanogenum The transformation of was performed as described in Leang et al. 2013. The nucleotide sequences of the enzymes used are SEQ ID NO: 1, 3 (ctfA), 5 (ctfB) and 7, respectively. These sequences were transformed to be under the control of the thiolase promoter and integrated into the vector backbone as a single operator. The created vector was named pTCtS. The vector pTCts was...

Embodiment 2

[0075] 3HB strain in H 2 and CO 2 Upper fermentation showing acetoacetate and 3HB production

[0076] For cell culture of Clostridium ljungdahlii pTCts, grow 5 mL of culture anaerobically in a medium with approximately 400 mg / L L-cysteine ​​hydrochloride and 400 mg / L Na 2 S×9H 2 O, 100 mg / L erythromycin in 500 ml medium (ATCC1754 medium: pH 6.0; 20 g / L MES; 1 g / L yeast extract, 0.8 g / L NaCl, 1 g / L NH 4 Cl, 0.1 g / L KCl, 0.1 g / L KH 2 PO 4 , 0.2 g / L MgSO 4 ×7H 2 O; 0.02 g / L CaCl 2 ×2H 2 O; 20 mg / L nitrilotriacetic acid 10 mg / LMnSO 4 ×H 2 O; 8 mg / L (NH 4 ) 2 Fe(SO 4 ) 2 ×6H 2 O; 2 mg / L CoCl 2 ×6H 2 O; 2 mg / L ZnSO 4 ×7H 2 O; 0.2mg / L CuCl 2 ×2H 2 O; 0.2 mg / L Na 2 MoO 4 ×2H 2 O; 0.2 mg / L NiCl 2 ×6H 2 O; 0.2 mg / / L Na 2 SeO 4 ; 0.2 mg / L Na 2 WO 4 ×2H 2 O; 20 μg / L d-biotin, 20 μg / L folic acid, 100 g / L pyridoxine-HCl; 50 μg / L thiamine-HCl × H 2 O; 50 μg / L riboflavin; 50 μg / L niacin, 50 μg / L calcium pantothenate, 1 μg / L vitamin B12; 50 μg / L p-amin...

Embodiment 3

[0079] Fermentation of the vector control strain did not produce acetoacetate or 3HB

[0080] For cell culture of Clostridium ljungdahlii pEmpty, grow a 5 mL culture anaerobically in a medium with approximately 400 mg / L L-cysteine ​​hydrochloride and 400 mg / L Na 2 S×9H 2 O, 100 mg / L erythromycin in 500 ml medium (ATCC1754 medium: pH 6.0; 20 g / L MES; 1 g / L yeast extract, 0.8 g / L NaCl, 1 g / L NH 4 Cl, 0.1 g / L KCl, 0.1 g / L KH2 PO 4 , 0.2 g / L MgSO 4 ×7H 2 O; 0.02 g / L CaCl 2 ×2H 2 O; 20 mg / L nitrilotriacetic acid 10 mg / LMnSO 4 ×H 2 O; 8 mg / L (NH 4 ) 2 Fe(SO 4 ) 2 ×6H 2 O; 2 mg / L CoCl 2 ×6H 2 O; 2 mg / L ZnSO 4 ×7H 2 O; 0.2mg / L CuCl 2 ×2H 2 O; 0.2 mg / L Na 2 MoO 4 ×2H 2 O; 0.2 mg / L NiCl 2 ×6H 2 O; 0.2 mg / / L Na 2 SeO 4 ; 0.2 mg / L Na 2 WO 4 ×2H 2 O; 20 μg / L d-biotin, 20 μg / L folic acid, 100 g / L pyridoxine-HCl; 50 μg / L thiamine-HCl × H 2 O; 50 μg / L riboflavin; 50 μg / L niacin, 50 μg / L calcium pantothenate, 1 μg / L vitamin B12; 50 μg / L p-aminobenzoate; 50 μg / L l...

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Abstract

There is provided amicrobial cell which is capable of producing acetoacetate, 3-hydroxybutyrate and / or 3-hydroxybutyrate variants, wherein the cell is genetically modified to comprise an increased expression relative to its wild type cell of: - an enzyme E1 capable of catalysing the conversion of acetyl-CoA to acetoacetyl-CoA; - an enzyme E2 capable of catalysing the conversion of acetoacetyl-CoAto acetoacetate; and -an enzyme E3 capable of catalysing the conversion of acetoacetate to 3-hydroxybutyrate and / or variants thereof.

Description

technical field [0001] The present invention relates to a biotechnological process for the production of 3-hydroxybutyric acid and / or variants thereof. In particular, the method involves biotechnological production of 3-hydroxybutyrate from carbon sources via acetoacetyl-CoA and acetoacetate. Background technique [0002] 3-Hydroxybutyric acid, also known as β-hydroxybutyric acid, has the formula CH 3 CH(OH)CH 2 CO 2 H organic compounds. It is a beta hydroxy acid and is a chiral compound with two enantiomers D-3-hydroxybutyric acid and L-3-hydroxybutyric acid. Its oxidized and polymeric derivatives are widely found in nature. 3-Hydroxybutyrate is a precursor to polyesters, which are biodegradable plastics including poly(3-hydroxybutyrate). 3-Hydroxybutyric acid is also a precursor for the production of 2-Hydroxyisobutyric acid and polyhydroxyalkanoates containing 2-Hydroxyisobutyric acid monomer units, including methacrylic acid. Methacrylic acid, its esters, and poly...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/42C12P7/40C12R1/145
CPCC12N9/0006C12N9/1029C12N9/13C12N9/88C12P7/40C12P7/42C12Y101/01001C12Y203/01009C12Y208/03008C12Y401/01004C12N15/52
Inventor T.哈斯A.赫克M.珀特T.比尔特
Owner EVONIK OPERATIONS GMBH
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