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Corynebacterium glutamicum for producing high-yield succinic acid by utilizing straw hydrolysate, and construction and applicaitons

A Corynebacterium glutamicum and succinic acid technology, applied in the direction of bacteria, microbial-based methods, and the use of vectors to introduce foreign genetic material, can solve the problems of no transformation of xylose metabolism ability and low production rate of succinic acid, and achieve good results. The effect of industrial potential

Inactive Publication Date: 2017-08-04
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the xylose metabolism ability of the strain was not further modified, and the lower xylose consumption rate resulted in a lower succinate production rate.

Method used

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  • Corynebacterium glutamicum for producing high-yield succinic acid by utilizing straw hydrolysate, and construction and applicaitons
  • Corynebacterium glutamicum for producing high-yield succinic acid by utilizing straw hydrolysate, and construction and applicaitons
  • Corynebacterium glutamicum for producing high-yield succinic acid by utilizing straw hydrolysate, and construction and applicaitons

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: pD-sacB knockout vector construction and Corynebacterium glutamicum seamless knockout operation

[0035] (1) Construction of pD-sacB knockout vector

[0036] 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.

[0037] Connect the sacB gene digested by MunI / EcoRV to the pECXK99E digested by EcoRI / SmaI, and then use the following primers trcsacB1 / trcsacB2 to amplify the trcsacB fragment containing the trc promoter with the pECXK99E-sacB plasmid as a template, 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 1 shown.

[0038] (2) Corynebacterium glutamicum traceless knockout operatio...

Embodiment 2

[0047] Example 2: Knockout of by-product acetic acid production pathway pta-ackA gene operon, by-product acetic acid production pathway actA gene and by-product lactic acid production pathway ldh gene

[0048] (1) The specific operation of knocking out the pta-ackA gene operon of the acetate production pathway is as follows:

[0049] Using two pairs of primers pta1, pta2, pta3, pta4, and using C. glutamicum ATCC 13032 as a template, KOD-plus high-fidelity DNA polymerase was used to amplify the upstream and downstream homology arms with sizes of 870bp and 929bp, respectively. After gel cutting and recovery, primers pta5 and pta6 were used to perform fusion PCR amplification using KOD-plus high-fidelity DNA polymerase to obtain a splicing product of about 1.6 kb in the upstream and downstream homology arms. Then the fusion product and the pD-sacB plasmid were digested with Thermo Fast digestXbaI / SalI, and after ligation and transformation, the pta-ackA gene operon knockout vecto...

Embodiment 3

[0054] Example 3: Introduction of P before the start codon of the phosphoenolpyruvate carboxylase ppc gene on the chromosome sod Strong promoter, P sod Strong promoter (different from 001)

[0055] (1) On the chromosome, P sod The specific operation of the strong promoter is as follows:

[0056] Using the C. glutamicum ATCC 13032 genome as a template, PCR amplification was performed with the following primers. sodppc1 and sodppc2 were used to amplify the upstream fragment (557bp) of gene ppc. sodppc3 and sodppc4 are used to amplify the promoter of sod gene (192bp). sodppc5 and sodppc6 were used to amplify the initial fragment (563bp) of gene ppc. After the three fragments were gel-cut, purified and recovered, the equimolar proportion of the fragments was used as a template, and sodppc1 and sodppc6 were used as primers to amplify the fusion product (1.3 kb) of the three fragments. Then the fusion product and pD-sacB plasmid were digested with Thermo Fast digest XbaI / HindI...

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Abstract

The invention discloses a corynebacterium glutamicum for producing high-yield succinic acid by utilizing straw hydrolysate, and a construction and applications. The method comprises the following steps: (1) knocking out side products acetic acid and lactic acid formation pathway related genes from corynebacterium glutamicum ATCC 13032, and introducing anaplerotic pathway phosphoenolpyruvate carboxylase and pyruvate carboxylase and phosphopentose pathway transketoaldehydase and transketolase on a strong promoter overexpression chromosome; and introducing xylose transport gene on the chromosome; and (2) expressing pyruvate carboxylase, succinic acid export protein and citrate synthase as well as xylose isomerase and xylulokinase on the corynebacterium glutamicum obtained in the step (1). 98.6gL<-1> of succinic acid can be anaerobically produced after 22.5h by utilizing mixed glucose and xylose in straw hydrolysate, with the yield of 0.98g succinic acid / g total sugar. The final concentration, yield and productivity of succinic acid achieve higher level, and the corynebacterium glutamicum has good industrial potential.

Description

technical field [0001] The invention belongs to the field of bioengineering technology and application, and in particular relates to a Corynebacterium glutamicum strain capable of high-yielding succinic acid by using straw hydrolyzate and its construction and application. Background technique [0002] In 2004, succinic acid (also known as succinic acid) was listed by the U.S. Department of Energy as one of the twelve most valuable platform compounds, and because it is widely used as a biodegradable plastic, food umami agent and pharmaceutical intermediate As a synthetic raw material for body, it has been regarded as a high value-added product and studied by researchers. At present, the chemical method is still the main process for the synthesis of succinic acid, but with the increasing environmental hazards of the traditional chemical industry, the use of microbial fermentation to convert renewable resources has received more and more attention. The microbial fermentation m...

Claims

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Application Information

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IPC IPC(8): C12N15/77C12N1/21C12P7/46C12R1/15
Inventor 王智文毛雨丰常志帅陈涛赵学明
Owner TIANJIN UNIV
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