Construction method of pediococcus acidilactici for producing L-lactic acid through co-fermentation of glucose and xylose

A technology for the construction of Pediococcus lactis, which is applied in the field of Pediococcus lactis construction, can solve the problems of no actual proof of engineering strains and increased fermentation costs

Active Publication Date: 2019-01-22
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the xylRAB in this document is expressed in a plasmid and is not integrated into the genome. The resulting engineering strain is not a stable engineering strain. During the fermentation process, antibiotics need to be added to maintain the normal growth of the recombinant bacteria. Addition will increase the cost of fermentation, which is an important drawback for large-scale industrial applications
In addition, the literature and the author's later research did not use lignocel

Method used

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  • Construction method of pediococcus acidilactici for producing L-lactic acid through co-fermentation of glucose and xylose
  • Construction method of pediococcus acidilactici for producing L-lactic acid through co-fermentation of glucose and xylose
  • Construction method of pediococcus acidilactici for producing L-lactic acid through co-fermentation of glucose and xylose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0031] Implementation case 1: Implementation of xylose metabolism in P.acidilactici TY112.

[0032] (1) Construction of expression plasmid pMG36e-PldhD-xylAB_2911

[0033] First, using the P.acidilactici DSM20284 genome as a template, using xylAB_2911-F (SEQ ID NO:1) and xylAB_2911-R (SEQ ID NO:2) as primers to amplify to obtain xylAB_2911, using P.acidilactici TY112 as a template, and PldhD- F (SEQ ID NO: 3) and PldhD-R (SEQ ID NO: 4) were used as primers to amplify the promoter sequence PldhD about 300 bp upstream of the ldhD start codon, and then the expression box PldhD_xylAB_2911 was obtained by fusion PCR technology, and then passed EcoR I and Xba I double enzyme digestion, the P32 promoter of pMG36e itself was replaced with the expression cassette PldhD_xylAB_2911, and the expression plasmid pMG36e-PldhD_xylAB_2911 was obtained.

[0034] (2) Verification of xylose fermentation ability of recombinant strain P.acidilactici TY112 (pMG36e-PldhD_xylAB_2911)

[0035] The ex...

Embodiment example 2

[0036] Implementation Case 2: Blocking of the Phosphoketolase Pathway (PK Pathway)

[0037] (1) Construction of knockout plasmid pSET4E-Δpkt

[0038] Using the P. acidilactici TY112 genome as a template, using up-pkt-F (SEQ ID NO:5) and up-pkt-R (SEQ ID NO:6) as primers to amplify the upstream homology arm of about 1,000 bp of the pkt gene Fragment (up-pkt), using down-pkt-F (SEQ ID NO: 7) and down-pkt-R (SEQ ID NO: 8) as primers to amplify the downstream about 1,000bp homology arm fragment sequence of the pkt gene (down-pkt). The down-pkt fragment was inserted between the BamH I and Sac I sites of pSET4E, and then the up-pkt fragment was inserted between the Pst I and Sal I sites to obtain the plasmid pSET4E-Δpkt for pkt gene knockout.

[0039] (2) Knockout of phosphoketolase gene pkt

[0040] The knockout plasmid pSET4E-Δpkt was electrotransformed into P. acidilactici TY112 to obtain the recombinant strain P. acidilactici TY112 (pSET4E-Δpkt), and the recombinant strain wa...

Embodiment example 3

[0043] Implementation Case 3: Construction of the Pentose Phosphate Pathway (PP Pathway)

[0044] (1) Construction of integrated plasmid pSET4E-Δpkt::(tkt_tal)

[0045] Using the P.acidillactici TY112 genome as a template, using PldhD-F (SEQ ID NO:3) and PldhD-R (SEQ ID NO:4) as primers to amplify the promoter PldhD; using P.acidillactici DSM20284 as a template, design primers tkt_tal-F (SEQ ID NO: 11) and tkt_tal-R (SEQ ID NO: 12) were amplified to obtain tkt_tal. Then the cloned PldhD and tkt_tal were obtained by fusion PCR to obtain the expression cassette PldhD_tkt_tal, and inserted between Sal I and BamH I of the knockout plasmid pSET4E-Δpkt obtained in Case 2 to obtain the integrated plasmid pSET4E-Δpkt::(tkt_tal).

[0046] (2) Genomic integration of tkt_tal

[0047] The integrated plasmid pSET4E-Δpkt::(tkt_tal) was electrotransformed into the engineering strain P. acidilactici TY112-Δpkt obtained in Case 2 to obtain the recombinant strain P. acidilactici TY112-Δpkt (pSE...

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Abstract

The invention discloses a construction method of pediococcus acidilactici for producing L-lactic acid through co-fermentation of glucose and xylose, and belongs to the technical field of gene engineering. The construction method comprises the following construction steps: integrating heterogenous xylose isomerase, xylulokinase, transketolase and transaldolase coding genes on a genome of a strain pediococcus acidilactici TY112 (with the accession number of CGMCC NO.8664) for producing L-lactic acid by utilizing a thermosensitive knockout system; knocking out phosphoketolase and acetokinase coding genes; and carrying out adaptive evolution for improving the capacity of co-fermentation of glucose and xylose. With the construction method, an engineering strain capable of efficiently producingoptically pure L-lactic acid through co-fermentation of glucose and xylose is successfully obtained, the engineering strain is named P.acidilactici ZY271, and the accession number is CGMCC NO.13611.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a construction method of Pediococcus lactis producing L-lactic acid by co-fermenting glucose and xylose using a heat-sensitive knockout system based on the principle of homologous recombination. Background technique [0002] Lactic acid is an important industrial chemical, which is widely used in food, medicine, leather and textile industries. In recent years, the use of optically pure L-lactic acid as a precursor to produce biodegradable plastic polylactic acid has greatly increased the demand for optically pure L-lactic acid. At present, lactic acid production mainly includes two routes: chemical synthesis and microbial fermentation. The main disadvantage of chemical synthesis is that the lactic acid it produces is D / L mixed lactic acid, while the advantage of microbial fermentation is that it can produce optically pure lactic acid, low production temperature and...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N15/90C12N15/61C12N15/54C12N1/21C12R1/01
CPCC12N9/1022C12N9/1205C12N9/1217C12N9/92C12N15/74C12N15/902C12Y202/01001C12Y202/01002C12Y207/01017C12Y207/02001C12Y503/01005Y02E50/10
Inventor 鲍杰邱忠洋高秋强
Owner EAST CHINA UNIV OF SCI & TECH
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