Bacillus subtilis strain for producing high-purity chiral D-(-)-2,3-butanediol, and construction and applications

A technology of Bacillus subtilis and butanediol, applied in the directions of microorganism-based methods, bacteria, microorganisms, etc., can solve problems such as no modification of Bacillus subtilis.

Active Publication Date: 2013-10-23
TIANJIN UNIV
View PDF4 Cites 11 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 D-(-)-2,3-butanediol

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Bacillus subtilis strain for producing high-purity chiral D-(-)-2,3-butanediol, and construction and applications
  • Bacillus subtilis strain for producing high-purity chiral D-(-)-2,3-butanediol, and construction and applications
  • Bacillus subtilis strain for producing high-purity chiral D-(-)-2,3-butanediol, and construction and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0032] The strain obtained by knocking out the upp gene in Bacillus subtilis B. subtilis168 was selected 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 the 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 by Spizizen, select the transformant on the LB plate containing 5ug / mL kanamycin, and pass through the colony After PCR verification, the starting strain BSF1 with upp gene knockout was obtained. ...

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 operations for knocking out the acoA gene in the degradation pathway of acetoin (the direct substrate of 2,3-butanediol) are 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 obt...

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 plasmid 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 verifi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a bacillus subtilis strain for producing high-purity chiral D-(-)-2,3-butanediol, and construction and applications. The construction method comprises the steps of: in bacillus subtilis, knocking out acoA gene in a degradation path of direct substrate acetoin of 2,3-butanediol, knocking out encoding gene bdhA of acetoin reductase, knocking out pta gene in a side product acetic acid formation path, knocking out ldh gene in a side product lactic acid formation path, overexpressing a synthetic branch alsSD operon by using a P43 strong promoter, overexpressing dehydrogenase encoding gene bdhA of D-(-)-2,3-butanediol by using a P43 strong promoter, and overexpressing transhydrogenase encoding gene udhA in reducing power balancing process by using a P43 strong promoter. The constructed bacillus subtilis strain is safe and harmless, and the D-(-)-2,3-butanediol, with the chiral purity of more than 99%, can be produced by utilizing 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P7/18C12R1/125
Inventor 陈涛付晶王智文赵学明
Owner TIANJIN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap