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Bacillus subtilis strain for producing high-purity chiral meso-2,3-butanediol as well as construction method and application of strain

A technology of meso-2 and Bacillus subtilis, applied in the direction of microorganism-based methods, bacteria, microorganisms, etc., can solve the problems that there is no transformation of Bacillus subtilis

Active Publication Date: 2014-11-26
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] After searching, there is currently no report on the use of metabolic engineering to transform Bacillus subtilis and use it to produce chiral meso-2,3-butanediol

Method used

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  • Bacillus subtilis strain for producing high-purity chiral meso-2,3-butanediol as well as construction method and application of strain
  • Bacillus subtilis strain for producing high-purity chiral meso-2,3-butanediol as well as construction method and application of strain
  • Bacillus subtilis strain for producing high-purity chiral meso-2,3-butanediol as well as construction method and application of strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 The construction of the starting strain for screening the Bacillus subtilis system without antibiotic markers and the construction of the basic operation plasmid pCU

[0031] (1) Construction of starting strains for Bacillus subtilis screening without antibiotic markers

[0032] The strain obtained by knocking out the upp gene in Bacillus subtilis B. subtilis168 was used as the starting strain of the Bacillus subtilis system without antibiotic markers, and was named BSF1.

[0033] The steps for constructing the starting strains of the Bacillus subtilis system without antibiotic markers are as follows:

[0034] Use a plasmid containing the upper and lower homology arms of the upp gene and the neo resistance marker to transform into Bacillus subtilis B. subtilis 168 through Spizizen, and select transformants on an LB plate containing 5ug / mL of kanamycin, and colonize After PCR verification, the starting strain BSF1 with upp gene knockout was obtained. The meth...

Embodiment 2

[0037] Example 2: Knockout of the acoA gene in the degradation pathway of the direct substrate acetoin of 2,3-butanediol

[0038] The specific operation of knocking out the acoA gene in the degradation pathway of acetoin (the direct substrate of 2,3-butanediol) is as follows:

[0039] Using two pairs of primers D-acoA-FU, D-acoA-FL and D-acoA-BU, D-acoA-BL, using B. subtilis 168 as a template, using KOD-plus high-fidelity DNA polymerase to amplify the size The upstream and downstream homology arms acoA-F and acoA-B are 1143bp and 1208bp. After gel cutting and recovery, primers D-acoA-FsnU and D-acoA-FsnL were used, and KOD-plus high-fidelity DNA polymerase was also used for fusion PCR amplification to obtain the 2073bp splicing product acoA-FB of the upstream and downstream homology arms. Then the fusion product acoA-FB and pCU plasmid were digested with Thermo Fast digest SalI and KpnI, and after ligation and transformation, the acoA gene knockout vector pCU-acoA-FB was obtain...

Embodiment 3

[0042] Example 3: Knockout of the bdhA gene encoding acetoin reductase

[0043] Using two pairs of primers D-bdhA-FU, D-bdhA-FL and D-bdhA-BU, D-bdhA-BL, using B.subtilis 168 as a template, using KOD-plus high-fidelity DNA polymerase to amplify the size The upstream and downstream homology arms bdhA-F and bdhA-B are 1096 and 973bp. After gel cutting and recovery, use primers D-bdhA-FsnU and D-bdhA-FsnL, and KOD-plus high-fidelity DNA polymerase to perform fusion PCR amplification to obtain the 1796bp splicing product bdhA-FB of the upstream and downstream homology arms. Then the fusion product bdhAFB and pCU plasmids were digested with Thermo Fast digest SphI and KpnI, and after ligation and transformation, the bdhA gene knockout vector pCU-bdhA-FB was obtained (see Figure 4 ). The plasmid with the correct sequencing result was transformed into Bacillus subtilis BSF2 by Spizizen, the positive clones with successful recombination were screened with chloramphenicol, and verif...

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Abstract

The invention discloses a bacillus subtilis strain for producing high-purity chiral meso-2,3-butanediol as well as a construction method and application of the strain. The construction method comprises the steps of knocking out an acoA gene of a direct substrate of 2,3-butanediol, acetoin in a degradation pathway, from bacillus subtilis, knocking out an acetoin reductase coding gene bdhA, knocking out a pta gene in a byproduct acetic acid formation path and knocking out a 1dh gene in a byproduct lactic acid formation path, over-expressing a synthesis branch a1sSD promoter through a P43 strong promoter, over-expressing meso-2,3-butanediol dehydrogenase coding gene budC through a P43 strong promoter, and over-expressing a transhydrogenase coding gene udhA in a reducing force equilibrium process through a P43 strong promoter. The constructed bacillus subtilis engineering strain is safe and harmless, and can produce meso-2,3-butanediol with chiral purity greater than 99% by taking glucose as a substrate.

Description

technical field [0001] The invention belongs to the field of bioengineering technology and application, and in particular relates to a bacillus subtilis strain for producing high-purity chiral butanediol and its construction and application. Background technique [0002] 2,3-Butanediol (2,3-Butanediol) molecule contains 2 chiral carbon atoms, and there are 3 kinds of optical isomers, which are D-(-)-2,3-Butanediol, L- (+)-2,3-Butanediol and meso-2,3-Butanediol. As an important chemical raw material and liquid fuel, 2,3-butanediol is widely used in chemical, food and other fields. The combustion value of 2,3-butanediol is 27.2kJ / g, which is comparable to methanol (22.1kJ / g) and ethanol (29.0kJ / g). It has a high octane number and is a potential value fuel additives. 2,3-Butanediol and its derivatives also have many potential applications, such as the production of inks, perfumes, fumigants, spandex, moisturizers, softeners, plasticizers, and drug carriers. 2,3-butanediol c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P7/18C12R1/125
Inventor 陈涛付晶王智文赵学明
Owner TIANJIN UNIV
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