Method for coproducing succinic acid and poly beta-hydroxybutyrate using recombination of escherichia coli

A technology for recombining Escherichia coli and hydroxybutyrate, applied in the field of genetic engineering and microbial fermentation, can solve the problem of high cost of PHB

Inactive Publication Date: 2009-02-11
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cost of industrial production of PHB remains high

Method used

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  • Method for coproducing succinic acid and poly beta-hydroxybutyrate using recombination of escherichia coli
  • Method for coproducing succinic acid and poly beta-hydroxybutyrate using recombination of escherichia coli
  • Method for coproducing succinic acid and poly beta-hydroxybutyrate using recombination of escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, construction of succinic acid fermentation pathway (knockout sdhAB)

[0064] Bacteria: Escherichia coli MG1655

[0065] The LB medium is: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, ampicillin 100mg / L, kanamycin 50mg / L.

[0066] The ampicillin-resistant plate is an LB solid medium containing 100 mg / L ampicillin and 1.5% agar powder.

[0067] The kanamycin-resistant plate is an LB solid medium containing 50 mg / L ampicillin and 1.5% agar powder.

[0068] The SOC medium is: peptone 2g / L, yeast powder 0.5g / L, NaCl 0.0585g / L, KCl 0.0186g / L, MgCl 2 0.203g, MgSO 4 0.246g / L, glucose 20mmol / L.

[0069] (1) Cloning of homologous recombination fragments

[0070] The gene of interest was knocked out using the Red recombination system. Primers were designed according to the sdhAB gene sequence published by Genbank:

[0071] pKD-sdhAB primer1

[0072] 5′-GGGCCACACGCGAAACTGAAACTCGATCACCTGGGTAAAGTGTAGGCTGGAGCTGCTTC-3′

[0073] pKD-sdhAB primer2

[0074] 5′-G...

Embodiment 2

[0097] Embodiment 2, the construction of PHB synthetic metabolic pathway

[0098] Bacteria: Escherichia coli DH5α

[0099] The medium LB: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, ampicillin, 100mg / L.

[0100] The PHB synthetase gene is derived from Alcaligenes eutrophus (A.eutrophus).

[0101] The ampicillin-resistant plates contained 1.5% agar and 100 mg / L ampicillin.

[0102] (1) Construction of PHB synthetase expression vector

[0103] (1) Alcaligenes eutropha was inoculated in LB medium, and the genome was extracted using a universal bacterial genome extraction kit.

[0104] (II) According to the genome sequence of Alcaligenes eutropha published by Genbank, design primers:

[0105] phbCAB primer 1: 5′-ATCCCCGGGGCGACCGGCAAAGGCGCGGCAGCTTCCA-3′

[0106] phbCAB primer 2: 5′-ATGGAATTCCAGCCCATATGCAGGCCGCCGTTGAGC-3′

[0107] (II) Cloning of PHB synthase gene

[0108] Using the genome of Alcaligenes eutropha as a template, the phbCAB gene was amplified by PCR. PCR rea...

Embodiment 3

[0128] Example 3, Construction of recombinant Escherichia coli co-producing fermentative succinic acid and PHB

[0129] 1. Construction of succinate synthesis pathway (knockout of iclR gene):

[0130] Bacteria: Escherichia coli MG1655△sdhAB

[0131] (I) Cloning of homologous recombination fragments

[0132] Primers were designed according to the Escherichia coli genome sequence published by GenBank

[0133] pKD-iclR primer1:

[0134] 5′-ATGAAAATGATTTCCACGATACAGAAAAAAGACTGTCGTGTAGGCTGGAGCTGCTTC-3′

[0135] pKD-iclR primer2:

[0136] 5′-TATGATGGGCAGAATATTGCCTCTGCCCGCCAGAAAAAGATGGGAATTAGCCATGGTCC-3′, the recombinant fragment with kanamycin resistance was amplified in vitro by PCR (polymerase chain reaction) using pKD4 plasmid as a template. PCR reaction system is as follows: (primer concentration is 20 μ mol / L)

[0137] 5 μl of 10× buffer;

[0138] 25mmol / LMgCl2 4μl;

[0139] 1μl of 10mmol / L four kinds of dNTP mixture;

[0140] 1 μl each of upstream and downstream primer...

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Abstract

The invention discloses a method for co-producing butanedioic acid and PHB by utilizing colon bacillus for the first time, which is used for knocking out sdhAB and iclR genes in the colon bacillus, and for constructing aerobic fermentation way of the butanedioic acid, so that butanedioic acid fermentation technology bacterial strains MG1655 delta sdhAB delta iclR::kan; on that basis, a PHB biosynthetic pathway is educed into colon bacillus MG1655 delta sdhAB delta iclR::kan, thereby successfully constructing the butanedioic acid and PHB fermentation way in the same colon bacillus. The fermentation and test results prove that the engineering bacterial strain can effectively transform glucose to generate the butanedioic acid and the PHB. 75g / L of glucose is consumed, and 37.5g / L of butanedioic acid is generated; at the same time, PHB is accumulated by thalli to be weighed as 28% of the dry weight of the thalli. The engineering bacterial strain has important application prospect.

Description

technical field [0001] The invention relates to the fields of genetic engineering and microbial fermentation, in particular to a method for co-producing succinic acid and poly-β-hydroxybutyrate (PHB) by using recombinant Escherichia coli. Background technique [0002] Succinic acid, scientific name succinic acid, is a four-carbon dicarboxylic acid with important application value, which is widely used as a precursor in the synthesis of drugs and biodegradable polymers. Due to environmental concerns, many studies have turned to microbial fermentation to produce succinic acid. The succinic acid fermentation method using renewable resources as raw materials will gradually replace the traditional chemical synthesis method. It has great application potential to increase the production of microbial succinic acid and reduce the cost of succinic acid fermentation through metabolic engineering. At the same time, there is also the challenge of constructing a succinic acid fermentatio...

Claims

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Application Information

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IPC IPC(8): C12P7/46C12P7/62C12N15/52C12N15/63C12N1/21C12R1/19
Inventor 祁庆生康振
Owner SHANDONG UNIV
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