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Building method of pediococcus acidilactici ZP26 for producing D-lactic acid by co-fermenting glucose and xylose

A technology of Pediococcus lactis and glucose, applied in the field of genetic engineering

Active Publication Date: 2018-11-23
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no report on the construction of the xylose metabolic pathway of Pediococcus lactis to produce D-lactic acid. Therefore, a method for the construction of the xylose metabolic pathway of Pediococcus lactis is established, and D - Lactic acid production is very necessary

Method used

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  • Building method of pediococcus acidilactici ZP26 for producing D-lactic acid by co-fermenting glucose and xylose
  • Building method of pediococcus acidilactici ZP26 for producing D-lactic acid by co-fermenting glucose and xylose
  • Building method of pediococcus acidilactici ZP26 for producing D-lactic acid by co-fermenting glucose and xylose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1

[0038] Implementation case 1: Comparison of xylose fermentation performance of P. acidilactici ZP26 carrying different xylAB expression cassettes.

[0039] (1) PCR amplification of different promoters and different xylAB operons

[0040] The genomes of P. acidilactici ZP26, P. acidilactici DSM20284 and P. pentosaceus ATCC25745 were extracted according to the method described in the Tiangen Genome Extraction Kit. Using the P.acidilacticiZP26 genome as a template, design primers Pldh-F (SEQ ID NO: 1) and Pldh-R (SEQ ID NO: 2) to amplify the ldh promoter Pldh (SEQ ID NO: 3), and design primer PldhD -F (SEQ ID NO: 4) and PldhD-R (SEQ ID NO: 5) were amplified to obtain the ldhD promoter PldhD (SEQ ID NO: 6); using the P. acidilactici DSM20284 genome as a template, the primer xylAB_2911-F was designed (SEQ ID NO: 7) and xylAB_2911-R (SEQ ID NO: 8) were amplified to obtain xylAB_2911 (SEQ ID NO: 9); using the P.acidilactici DSM20284 genome as a template, amplified to obtain xylAB_30...

Embodiment example 2

[0048] Example 2: The xylAB expression cassette was integrated into the ldh gene locus of P.acidillactici ZP26

[0049] (1) Construction of integrated plasmid pSET4E-Δldh::xylAB

[0050] First, using the P. acidilactici ZP26 genome as a template, the gene sequence up-ldh about 1,000 bp upstream of ldh was amplified, and the gene sequence down-ldh about 1,000 bp downstream of ldh was amplified. Using the expression plasmid pMG36e-PldhD_xylAB_2911 obtained in Example 1 as a template, PldhD_xylAB_2911 was amplified. Then by restriction endonuclease ligation, the up-ldh fragment was inserted between the Pst I and BamH I sites of pSET4E, and the down-ldh fragment was inserted between the BamH I and EcoR I sites, to obtain the ldh gene knockout Plasmid pSET4E-Δldh, and then the expression cassette PldhD_xylAB_2911 was inserted between Xho I and BamH I of pSET4E-Δldh to obtain the integrated plasmid pSET4E-Δldh::xylAB.

[0051] (2) Genomic integration of xylAB

[0052]The integrat...

Embodiment example 3

[0055] Example 3: Knockout of the phosphoketolase gene pkt

[0056] (1) Construction of knockout plasmid pSET4E-Δpkt

[0057] Using the P. acidilactici ZP26 genome as a template, the upstream about 1,000 bp fragment (up-pkt) and the downstream about 1,000 bp fragment sequence (down-pkt) of the pkt gene were amplified. 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 for pkt gene knockout-for get.

[0058] (2) Knockout of pkt

[0059] The knockout plasmid pSET4E- was used to obtain electrotransformation into the bacterial strain P.acidilactici ZP26::xylAB obtained in Case 2 to obtain the recombinant strain P.acidilactici ZP26::xylAB (pSET4E-Δpkt), and then the recombinant strain was placed at 42°C, After 12 hours of culture in the MRS medium added with erythromycin, the bacterial solution was diluted 10 6 Spread it on the MRS plate contain...

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Abstract

The invention discloses a building method of pediococcus acidilactici ZP26 for producing D-lactic acid by co-fermenting glucose and xylose, and belongs to the field of gene engineering. The method comprises the following building steps of integrating heterogenous xylose isomerase, xylulokinase, transketolase and transaldolase encoding genes onto pediococcus acidilactici ZP26 (with the preservationnumber being CGMCC NO.8665) genomes by using a thermosensitive knock-out system; knocking out phosphotransferase encoding genes so as to block the generation of byproducts of acetic acids; the capability of the engineering strain for co-fermenting glucose and xylose is obviously improved through an adaptive domestication strategy. The method provided by the invention has the advantages that the building of the xylose metabolism path is successfully realized for the first time; the engineering strain for producing optical pure D-lactic acid by co-fermenting glucose and xylose at high efficiency is obtained, is named as P.acidilactici ZY15, and has the preservation number of CGMCC NO.13612.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a construction method of Pediococcus lactis for co-fermenting glucose and xylose to produce D-lactic acid based on the principle of homologous recombination by using a thermosensitive knockout system. Background technique [0002] D-lactic acid is an important industrial chemical, which is widely used in food, medicine, leather and textile industries. In recent years, the use of D-lactic acid as a precursor to produce biodegradable plastic polylactic acid has greatly increased the demand for optically pure D-lactic acid. However, at present, 90% of the world's D-lactic acid production is obtained by fermentation of starch raw materials, which has the problem of "competing with others for food". Finding cheaper raw materials for D-lactic acid production will help reduce the food crisis. Lignocellulosic raw material is a kind of renewable biomass energy with low pri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N1/21C12R1/01
CPCC12N9/1022C12N9/1205C12N9/92C12N15/74C12Y202/01001C12Y202/01002C12Y207/01017C12Y503/01005C12N2800/22Y02E50/10
Inventor 鲍杰邱忠洋高秋强
Owner EAST CHINA UNIV OF SCI & TECH
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