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Corynebacterium glutamicum strain for high-yield production of chiral D-(-)-acetoin as well as construction method and application of strain

A technology of Corynebacterium glutamicum and acetoin, which is applied in the field of high-yield chiral D--acetoin Corynebacterium glutamicum strains and its construction and application, and can solve the problem of acetoin yield and yield of engineering strains , unsatisfactory yield, etc.

Active Publication Date: 2017-06-20
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] After searching, there are currently published literatures reporting on the production of acetoin by Corynebacterium glutamicum, but the yield, yield, and yield of acetoin produced by the final engineering strain are not ideal.

Method used

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  • Corynebacterium glutamicum strain for high-yield production of chiral D-(-)-acetoin as well as construction method and application of strain
  • Corynebacterium glutamicum strain for high-yield production of chiral D-(-)-acetoin as well as construction method and application of strain
  • Corynebacterium glutamicum strain for high-yield production of chiral D-(-)-acetoin as well as construction method and application of strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: pD-sacB knockout vector construction and Corynebacterium glutamicum seamless knockout operation

[0036] (1) Construction of pD-sacB knockout vector

[0037] Firstly, the linear fragment of pK18mobsacB cut by HindIII was used as a template, and the primer sacB1 / sacB2 was used to amplify the sacB gene, which was about 1.6kb.

[0038] Connect the MunI / EcoRV double-digested sacB gene to the EcoRI / SmaI double-digested pECXK99E, and then use the following primers trcsacB1 / trcsacB2, and use the pECXK99E-sacB plasmid as a template to amplify the trcsacB fragment containing the trc promoter, about 1.8 kb. Using primers pD1 / pD2, the pK18mobsacB plasmid was used as a template to amplify the pD fragment containing kanamycin resistance and E. coli replicon, about 2.6kb. Finally, the AatII digested fragment trcsacB and pD were ligated to obtain pD-sacB. The final pD-sacB plasmid map is as follows figure 2 shown.

[0039](2) Corynebacterium glutamicum traceless knock...

Embodiment 2

[0048] Example 2: Knockout of the gene ldh encoding lactate dehydrogenase in the by-product lactic acid production pathway

[0049] The specific operation of knocking out the gene ldh gene encoding the by-product lactic acid production pathway lactate dehydrogenase is as follows:

[0050] Using two pairs of primers D-ldh-FU, D-ldh-FL and D-ldh-BU, D-ldh-BL, using C. glutamicum ATCC13032 as a template, using KOD-plus high-fidelity DNA polymerase to amplify the obtained Upstream and downstream homology arms ldh-F and ldh-B. After gel cutting and recovery, primers D-ldh-FU and D-ldh-BL were used, and KOD-plus high-fidelity DNA polymerase was also used for fusion PCR amplification to obtain the 1461bp splicing product ldh-FB of the upstream and downstream homology arms. Then the fusion product ldh-FB and pD-sacB plasmids were digested with Thermo Fast digest SalI and HindIII, and after ligation and transformation, the ldh gene knockout vector pD-sacB-ldh-FB was obtained (see Fi...

Embodiment 3

[0051] Example 3: Knockout of the pta-ack encoding gene operon of the by-product acetate production pathway acetate kinase and phosphotransacetylase

[0052] The specific operation of knocking out the pta-ack gene operon of the by-product acetate production pathway acetate kinase and phosphotransacetylase encoding gene is as follows:

[0053] Using two pairs of primers D-pta-ack-FU, D-pta-ack-FL and D-pta-ack-BU, D-pta-ack-BL, using C. glutamicum ATCC 13032 as a template, using KOD-plus high-definition The upstream and downstream homology arms pta-ack-F and pta-ack-B were amplified by true DNA polymerase, respectively. After gel cutting and recovery, use primers D-pta-ack-FU and D-pta-ack-BL, and also use KOD-plus high-fidelity DNA polymerase to perform fusion PCR amplification to obtain a 1703bp splicing product of the upstream and downstream homology arms pta-ack-fb. Then the fusion product pta-ack-FB and pD-sacB plasmids were digested with Thermo Fast digest XbaI and SalI...

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Abstract

The invention discloses a corynebacterium glutamicum strain for high-yield production of chiral D-(-)-acetoin as well as a construction method and an application of the strain. The construction method comprises the following steps: (1) knocking out an ldh gene, a ptaack gene operon, a nagD gene and a butA gene from corynebacterium glutamicum ATCC 13032; generating a ptaack site on acetic acid of chromosome, and interposing a synthesis path alsSD operon with over-expression of a Ptuf strong promoter; and (2) linking a vector pECXK99E to the synthesis path alsSD operon so as to obtain pECXK99EalsSD plasmid, deleting an lacIq region so as to obtain pECXK99ESD[delta]lacIq, and introducing the pECXK99ESD[delta]lacIq into the corynebacterium glutamicum obtained in the step (1). The strain provided by the invention is safe; and the acetoin is produced by virtue of glucose under an aerobic condition.

Description

technical field [0001] The invention belongs to the field of bioengineering technology and application, and in particular relates to a high-yield chiral D-(-)-acetoin Corynebacterium glutamicum strain and its construction and application. Background technique [0002] The chemical name of acetoin is 3-hydroxy-2-butanone, also known as methyl acetylmethanol. Acetoin exists in many foods and has a milky smell. It is a commonly used food-grade spice. Acetoin is also a kind of 4-C platform compound, which is listed as one of the 30 platform compounds worthy of priority development by the US Department of Energy. Acetoin has a chiral carbon atom, so there are two chiral isomers, D-(-)- and L-(+)-. Chiral acetoin has high added value and can be used in the synthesis of pharmaceutical intermediates and chiral compounds. For a long time, chemical synthesis has been the main method for the industrial production of acetoin, including the oxidation of 2,3-butanediol, the chlorinatio...

Claims

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Application Information

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IPC IPC(8): C12N15/77C12N1/21C12P7/26C12R1/15
CPCC12N9/0006C12N9/1217C12N15/77C12P7/26C12Y207/02001
Inventor 陈涛陶然付晶毛雨丰黄灿王智文赵学明
Owner TIANJIN UNIV
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