Construction of corynebacterium glutamicum engineering bacteria for high-yielding production of L-valine and method for fermentation production of L-valine

A glutamic acid rod, valine technology, applied in the direction of microorganism-based methods, fermentation, biochemical equipment and methods, etc., can solve the problems of expression combinations that have not been studied

Inactive Publication Date: 2014-08-27
JIANGNAN UNIV
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Problems solved by technology

Gene lrp , brnFE and w...

Method used

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  • Construction of corynebacterium glutamicum engineering bacteria for high-yielding production of L-valine and method for fermentation production of L-valine
  • Construction of corynebacterium glutamicum engineering bacteria for high-yielding production of L-valine and method for fermentation production of L-valine
  • Construction of corynebacterium glutamicum engineering bacteria for high-yielding production of L-valine and method for fermentation production of L-valine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Construction of knockout combined strain WCC003

[0027] Departure strain ATCC13869. for aceE To knock out the gene, first construct the aceE Gene upstream and downstream homology arms and containing loxp Kanamycin resistance gene selectable marker for recombinase recognition site ( loxp-kan-loxp ) knockout vector pDTW301. Then transfer the knockout vector into ATCC13869, and use resistance selection markers (if available, auxotrophs can be used at the same time) to screen out loxp-kan-loxp with the genome aceE A transformant undergoing homologous recombination between the upstream and downstream homologous arms of the gene. site-specific cre The temperature-sensitive plasmid pDTW109 of the recombinase was screened out by using the resistance selection marker to remove the can fragment transformants. Finally, change the culture temperature to remove pDTW109, and obtain ATCC13869Δ through genotype and phenotype verification aceE Mutant strains (J...

Embodiment 2

[0029] Example 2 Overexpression of genes in WCC003 lrp 1 , brnFE with wxya 1

[0030] Firstly, separate expression vectors for each gene were constructed. Genes derived from ATCC13869 and VWB-1 lrp and lrp 1 Linked to pJYW-4 to construct the expression vector pJYW-4- lrp and pJYW-4- lrp 1 , found that the expression effect of the latter was weaker in growth inhibition and higher in L-valine production. VWB-1 derived gene brnFE and wxya 1 respectively connected to pJYW-4 to obtain the expression vector pJYW-4- brnFE and pJYW-4- wxya 1 .

[0031] Then pJYW-4- brnFE middle belt tac Promoter brnFE Gene fragment tac-brnFE Connected to pJYW-4- lrp 1 , to obtain the expression vector pJYW-4- lrp 1 - brnFE ; tac-lrp 1 -tac-brnFE The fragment was ligated into pJYW-4- wxya 1 , to obtain the final expression vector pJYW-4- wxya 1 - lrp 1 - brnFE (attached image 3 ).

[0032] Finally pJYW-4- wxya 1 - lrp 1 - brnFE Trans...

Embodiment 3

[0033] Example 3 Engineering bacteria WCC003 / pJYW-4- wxya 1 - lrp 1 - brnFE tank fermentation

[0034] After the strain was activated, it was inoculated in 200 mL of seed medium, fermented in a reciprocating shake flask for 18 hours, and then inserted into a 5L fermenter with 2L of fermentation medium. Ammonia water is used to control the pH at 7.2, the temperature is 30°C, the dissolved oxygen is maintained at 30% through the coupling of rotation speed and ventilation, and glucose is added when the residual sugar content is lower than 60g / L, and the fermentation time is 96h. The final L-valine yield reached 36.6g / L.

[0035] The formula of activation medium is: 91g / L sorbitol, 18.5g / L brain heart extract, 5g / L peptone, 5g / L sodium chloride, 5g / L glucose, 2.5g / L yeast extract, 0.4g / L iso Leucine, 30 mg / L kanamycin, prepared with deionized water.

[0036] The seed culture medium is a fermentation medium with appropriately reduced glucose and amm...

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Abstract

The invention discloses construction of a strain of corynebacterium glutamicum engineering bacteria for high-yielding production of L-valine and a method for fermentation production of L-valine, and belongs to the technical field of food biological engineering. Corynebacterium glutamicum ATCC13869 is used as an original bacterial strain, knockout combination of genes of aceE, alaT and ilvA is carried out, and an ATCC13869[delta]aceE[delta]alaT[delta]ilvA mutant bacterial strain is obtained and named as WCC003; expression combination of genes of lrp1, brnFE and ilvBNC1 is carried out on the WCC003, and expression-combined engineering bacteria WCC003/pJYW-4-(ilvBNC1)-lrp1-brnFE are obtained and preserved in China center for type culture collection with the preservation number of CCTCC NO:M2014149; the expression-combined engineering bacteria are used for fermentation production of L-valine. The corynebacterium glutamicum engineering bacteria based on the expression regulating protein Lrp, the transfer protein BrnFE and the L-valine synthetic route key gene ilvBNC1 and used for high-yielding production of L-valine are provided, advantageous mutation Arg39Trp of Lrp in corynebacterium glutamicum is clear and definite for the first time, a fact that Lrp and BrnFE are used for fermentation production of L-valine is also reported for the first time, and the study on the expression combination of the genes of lrp1, brnFE and ilvBNC1 is also created for the first time.

Description

technical field [0001] The construction of a Corynebacterium glutamicum engineering bacterium with high L-valine production and the method for producing L-valine by fermentation of the present invention involve the use of Corynebacterium glutamicum ATCC13869 as the starting strain, and the genetic aceE , alaT and ilvA The knockout combination of ATCC13869Δ was obtained aceE Δ alaT Δ ilvA Mutant strain, named WCC003; genes were carried out on WCC003 lrp 1 , brnFE and wxya 1 The expression combination was obtained to obtain the expression combination engineering bacteria WCC003 / pJYW-4- wxya 1 - lrp 1 - brnFE, It has been preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M2014149; and the expression combination engineering bacteria are used to ferment and produce L-valine, which belongs to the technical field of food bioengineering. Background technique [0002] L-valine (L-valine) is a branched chain amino...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N15/77C12P13/08C12R1/15
Inventor 王小元陈诚李颜颜胡瑾瑜
Owner JIANGNAN UNIV
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