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Recombinant Corynebacterium glutamicum producing 3-hydroxypropionic acid, its construction method and application

A technology of Corynebacterium glutamicum and hydroxypropionic acid, which is applied in the field of genetic engineering and microbial fermentation, can solve the problems of many by-products, poor acid tolerance, and high price of glycerin, and achieve simplification of the separation process, less by-products, and solution Biosecurity Effects

Active Publication Date: 2019-09-20
GUANGDONG TSINGDA SMART BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this technical route is that the price of glycerin is relatively high, and this process produces a large amount of by-products such as 1,3-propanediol, etc.
[0008] In view of the technical problems existing in the current 3-hydroxypropionic acid production process, especially the many by-products, the high cost of raw materials, and the poor tolerance of bacterial strains to acids, it is urgent to develop a new method for producing 3-hydroxypropionic acid

Method used

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  • Recombinant Corynebacterium glutamicum producing 3-hydroxypropionic acid, its construction method and application
  • Recombinant Corynebacterium glutamicum producing 3-hydroxypropionic acid, its construction method and application
  • Recombinant Corynebacterium glutamicum producing 3-hydroxypropionic acid, its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Overexpression of the dihydroxyacetone phosphate phosphatase gene hdpA and the glycerol dehydrogenase gene gldA derived from Escherichia coli in Corynebacterium glutamicum

[0037] Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, PCR was performed with primers hdpA-F (5'-cagctatgaccatgattacgATAAGGCTGATTAGCGGGAAAATTTCG-3') and hdpA-R (5'-CCAGTCGTGTCTAGTCAGTGAACTGCTGCTCATCT-3') to obtain the hdpA gene (hdpA The gene sequence is about 0.9kb (as shown in SEQ ID NO: 1) and the PCR product is purified. Using the genome of Escherichia coli MG1655 as a template, with primers gldA-F (5'-CACTGACTAGACACGACTGGAATGCCGC-3') and gldA-R (5'-atccccgggtaccgagctcgttaTTCCCACTCTTGCAGGAAACGC-3') as primers, PCR was carried out to obtain the gldA gene (gldA gene sequence as SEQ ID NO: 2) about 1.2kb and PCR product purification. The Escherichia coli-Corynebacterium glutamicum shuttle plasmid pEC-K18mob2 (Journal of Biotechnology 104 (2003) 287-299) was diges...

Embodiment 2

[0038] Example 2 Overexpression of glycerol dehydratase and its activator gene pduCDEGH from Klebsiella pneumoniae and 3-hydroxypropionaldehyde dehydrogenase gene aldH from Escherichia coli

[0039] Using the genome of Klebsiella pneumoniae HR526 as a template, PCR was performed with primers pdu-F (5'-GGAGGCCtgaAAGGAGATATACCATGAGATCGAAAAGATTTG-3') and pdu-R (5'-gctcggtacccggggatcctTTAAGCATGGCGATCCCGAAAT-3') to obtain glycerol dehydratase The pduCDEGH operator fragment (sequence shown in SEQ ID NO: 3) and its activator were purified by PCR. Using the genome of Escherichia coli HR526 as a template, PCR was performed with primers aldH-F (5′-tgcatgcctgcaggtcgactCTGACGTTCACAAACTGCATATATCTGATAGAC-3′) and aldH-R (5′-TATATCTCCTTtcaGGCCTCCAGGCTTATCCA-3′) to obtain 3-hydroxypropionate dehydrogenase Gene aldH fragment (sequence shown in SEQ ID NO: 4) and purified. The Escherichia coli-Corynebacterium glutamicum shuttle plasmid pXMJ19 was digested with XbaI, and the pduCDEGH operon fragm...

Embodiment 3

[0040] Example 3 Fermentative production of 3-hydroxypropionic acid by recombinant Corynebacterium glutamicum

[0041] Corynebacterium glutamicum wild strain ATCC 13032 and recombinant bacteria C. glutamicum / pEC-hdpA-gldA, C. glutamicum / pEC-hdpA-gldA / pXMJ-aldH-pdu were cultured on LB plates overnight. Single bacterium colony was inoculated from this fresh flat board to the 250ml belt baffle shake flask that contains 30ml seed culture medium, 32 ℃ 200rpm cultivated 12 hours, the OD of gained seed solution 600 The value is 10.

[0042] The formulation of the seed medium includes (g / L): Glucose 25, (NH 4 ) 2 SO 4 5.0,K 2 HPO 4 1.5, MgSO 4 1.0, MnSO 4 0.005, FeSO 4 0.005, corn steep liquor 30, kanamycin 0.025.

[0043] Inoculate the seed liquid into 2L fermentation medium with an inoculation amount of 10% (v / v), use a 5L fermenter for fermentation, control the temperature at 32°C, and the ventilation rate at 1vvm, adjust the speed to keep the dissolved oxygen level a...

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Abstract

The invention relates to a 3-hydracrylic-acid-producing recombinant Corynebacterium glutamicum strain, and a construction method and application thereof. The construction method of the recombinant Corynebacterium glutamicum strain comprises the following steps: in a Corynebacterium glutamicum strain, overexpressing an endogenous dihydroxyacetone phosphate phosphatase gene hdpA, and overexpressing a glyceroldehydrogenase gene gldA, a glycerol anhydrase and activating factor gene pduCDEGH and a 3-hydroxypropylaldehyde dehydrogenase gene aldH. When the recombinant strain is used for producing 3-hydracrylic acid by fermentation, the Corynebacterium glutamicum strain can perform fermentation by using different cheap raw materials, thereby further lowering the raw material cost. The 3-hydracrylic-acid-producing recombinant Corynebacterium glutamicum strain solves the problems of biosafety and acid tolerance. The strain in the fermentation process can be used in a feed additive as a product. The method can generate fewer byproducts, so that the separation process of the end product 3-hydracrylic acid is simplified.

Description

technical field [0001] The invention relates to the field of genetic engineering and microbial fermentation, in particular to a recombinant Corynebacterium glutamicum producing 3-hydroxypropionic acid, its construction method and application. Background technique [0002] 3-Hydroxypropionic acid is an important potential platform compound. In 2004, the U.S. Department of Energy listed the 12 most potential bio-based chemical products in the world, and 3-hydroxypropionic acid ranked third. 3-Hydroxypropionic acid is an important functional organic acid, which can be directly used as food or feed additive and preservative. Its more important potential use is as a C3 platform compound, which can be converted into a variety of important chemical products by coupling existing mature catalytic technologies such as dehydration, oxidation, reduction, esterification, polymerization, etc. Diacid, 1,3-propanediol, acrylamide, acrylocyanide, poly-3-hydroxypropionic acid, etc. As a pl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/77C12P7/42C12R1/15
Inventor 陈振刘德华
Owner GUANGDONG TSINGDA SMART BIOTECH CO LTD
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