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Method for co-production of D-1,2,4-butanetriol and PHB by metabolic modification of escherichia coli, and application

A technology of Escherichia coli and butanetriol is applied in the field of genetic engineering to achieve the effect of reducing the generation and discharge of three wastes and reducing the cost of fermentation

Pending Publication Date: 2020-12-18
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Since the products of 1,2,4-butanetriol and polyhydroxybutyrate are extracellular and intracellular respectively, theoretically speaking, strains can be used to ferment and produce both substances at the same time. If a strain producing two kinds of products can be obtained, it will lay the foundation for converting a variety of cheap substrates into a variety of high value-added products

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  • Method for co-production of D-1,2,4-butanetriol and PHB by metabolic modification of escherichia coli, and application
  • Method for co-production of D-1,2,4-butanetriol and PHB by metabolic modification of escherichia coli, and application

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Embodiment 1

[0033] Example 1: Construction of a recombinant strain that co-produces PHB and D-1,2,4-butanetriol

[0034]phbCAB comes from Alcaligenes eutropha, its nucleotide sequence is shown in GenBank: MH558939.1, phbCAB is linked to the restriction site SacI and SalI of pRSFDuet-tac vector through homologous recombinase (ABclonal company) enzyme ( The construction method of the pRSFDuet-tac vector is as follows: the nucleotide sequence tac shown in SEQ ID NO.1 is connected to the BamHIII and SalI sites of the pRSFDuet vector to construct the pRSFDuet-tac-phbCAB expression vector, and the expression vector Transform into Escherichia coli BL21, grow single clones at 37°C, pick single clones and sequence verification, obtain positive transformants, extract plasmids from positive transformants, and obtain pRSFDuet-tac-phbCAB expression vector, express The vector pRSFDuet-tac-phbCAB was transformed into E.coli KXW3009, and the recombinant strain E.coli KXW3009A was obtained through sequenc...

Embodiment 2

[0038] Example 2: Fermentative production of PHB and D-1,2,4-butanetriol by recombinant strains

[0039] Inoculate E.coli KXW3009 and recombinant strains E.coli KXW3009A, E.coliKXW3009AK, E.coli KXW3009AP, and E.coli KXW3009AKP in liquid LB medium respectively, and cultivate them with shaking at 37°C and 200r / min according to 1mL / 100mL inoculum was transferred to fermentation medium, 37°C, 200r / min, culture OD 600 =0.6~0.8, add 0.5mmol·L -1 IPTG, continue to induce culture for 48 hours, and the fermentation ends. After the fermentation was completed, the yields of PHB and D-1,2,4-butanetriol were measured.

[0040] The results showed that overexpression of phbCAB of Alcaligenes eutropha in E.coli KXW3009 could realize co-production of 1,2,4-butane in E.coli KXW3009A, E.coli KXW3009AK, E.coli KXW3009AP, E.coli KXW3009AKP Alcohol and PHB. And the PHB yield of the strain E.coli KXW3009AK overexpressing the glk gene can reach 19.83%, which is significantly higher than that of ...

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Abstract

The invention discloses a method for co-production of D-1,2,4-butanetriol and PHB by metabolic modification of escherichia coli, and application, and belongs to the field of gene engineering. According to the invention, a genetic engineering method is applied; and plasmids with over-expression of beta-keto thiolase PhbA, acetoacetyl coenzyme A reductase PhbB and PHA synthase PhbC from alcaligeneseutrophus are transferred into engineering bacteria E.coli KXM3009 to construct a synthetic route of PHB. Furthermore, the gene glk related to glycolysis is over-expressed, so that co-production of 1,2,4-butantriol and PHB is realized; and a foundation is laid for co-production of multiple added-value products.

Description

technical field [0001] The invention relates to a method and application for metabolically transforming Escherichia coli to co-produce D-1,2,4-butanetriol and PHB, and belongs to the field of genetic engineering. Background technique [0002] 1,2,4-butanetriol (1,2,4-butanetriol, BT) is a non-natural, high-value four-carbon compound, its properties are similar to glycerin, it is a colorless, odorless, transparent Syrup-like liquid, easily soluble in water and alcohols, has certain stability and hygroscopicity, and is often used as a solvent, wetting agent, intermediate of medicine and explosives. At present, the solid propellants and plasticizers used in the military are gradually changing from the traditional nitroglycerin (NG) to the high-energy material 1,2,4-butanetriol trinitrate (1,2,4-butanetriol trinitrate, BTTN ) is excessive, the compound is produced after nitration of BT as a precursor, and has the advantages of strong thermal stability, small impact, non-volatil...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/53C12N15/54C12P7/18C12P7/62C12R1/19
CPCC12N15/52C12N9/0006C12N9/1029C12N15/70C12P7/18C12P7/62C12Y101/01036C12Y203/01009
Inventor 诸葛斌狄莹莹杨欣雨郭超陆信曜宗红
Owner JIANGNAN UNIV
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