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Polymer grade Lactic acid monomer producing strain, construction method thereof and lactic acid manufacturing technique

A technology of lactic acid monomer and manufacturing technology, applied in the field of microbial application, can solve the problems of complex culture medium, unsatisfactory optical purity of lactic acid, incapable of large-scale production of D-lactic acid and L-lactic acid of extremely high optical purity, etc., and achieve efficient preparation effect of the process

Active Publication Date: 2016-06-22
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Traditional lactic acid producing strains such as Rhizopus oryzae and Lactobacillus thermophilus cannot be used for extremely optically pure D-lactic acid and L lactic acid due to their own characteristics and the complexity of the required medium. - Large-scale production of lactic acid

Method used

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  • Polymer grade Lactic acid monomer producing strain, construction method thereof and lactic acid manufacturing technique
  • Polymer grade Lactic acid monomer producing strain, construction method thereof and lactic acid manufacturing technique
  • Polymer grade Lactic acid monomer producing strain, construction method thereof and lactic acid manufacturing technique

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0118] Example 1 - Mutation Cassette ldhA::kan-cI ts 857-p R -p L build

[0119] The ldhA gene fragment ldhA' on the B0013 chromosome was amplified by PCR with primers ldhA1 and ldhA2, and cloned into pUC18 vector to obtain the recombinant plasmid pUC-ldhA'. Plasmid pPL451 was amplified by inverse PCR with primers PPL1 and PPL2, and combined with the kanamycin resistance gene fragment (plasmid pSK sym Km was digested with SmaI, and the 966bp fragment was recovered by gel) for ligation to obtain the recombinant plasmid pPL-Kan. Plasmid pPL-Kan was amplified by PCR with primers PPL3 and PPL4, and the product was double-digested with EcoRI and EcoRV, and then reverse-PCR amplified with primers Ec-RlA1 and Ec-RlA2 using pUC-ldhA' as a template and used EcoRI digested products were ligated to obtain the recombinant plasmid pUC-ldhAp::kan-cI ts 857-p R -p L . The physical map of the resulting recombinant plasmid is attached figure 1 shown. The recombinant plasmid pUC-ldhAp...

Embodiment 2

[0120] Example 2 - Construction of Mutation Cassette thiE::difGm

[0121] Using E. coliB0013 chromosomal DNA as template, ThiE1p and ThiE2p as primers, the partial sequence of thiE gene with a length of 0.6kb was amplified. This was cloned into pUC18 to obtain the recombinant plasmid pUC-thiE. The 1.2kb difGm fragment was cloned into the StuI site in the middle of thiE to obtain the recombinant plasmid pUC-thiE::difGm. The physical map of the resulting recombinant plasmid is attached figure 2 shown. The recombinant plasmid pUC-thiE::difGm was digested with ApaLI, and the linearized plasmid was recovered by gel, and PCR amplification was performed with primers ThiE1p and ThiE2p to obtain the thiE::difGm gene fragment.

Embodiment 3

[0122] Example 3 - Construction of Mutation Cassette dld::difGm

[0123] Using the chromosomal DNA of bacterial strain B0013 as a template, the dld' gene fragment (0.9kb) was amplified by PCR with primers Dld1 and Dld2, and the PCR product was cloned into the vector pUC18SmaI site to obtain the recombinant plasmid pUC-dld'. Digest the recombinant plasmid with EcoRV, remove the 0.4 kb gene fragment in the middle of the dld' gene, and clone into the dif-Gm-dif fragment to obtain the recombinant plasmid pUC-dld'::difGm. The physical map of the resulting recombinant plasmid is attached image 3 shown. The recombinant plasmid pUC-dld'::difGm was digested with EcoRI, and the linearized plasmid was recovered by gel, and PCR amplification was performed with primers Dld1 and Dld2 to obtain the dld'::difGm gene fragment.

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Abstract

Provided are an extremely high optical voidness D-lactic acid and L-lactic acid fermentation producing strain, a construction method thereof and a method of manufacturing extremely high optical voidness D-lactic, wherein the preservation number of a D-lactic fermentation producing strain is CGMCC No.11059, and the preservation number of an L-lactic acid fermentation producing strain is CGMCC No.11060.

Description

technical field [0001] The invention relates to the field of microbial application, in particular to a polymerization-grade lactic acid monomer production bacterium, a construction method thereof and a lactic acid production technology. Background technique [0002] Biodegradable materials refer to a class of novel materials whose waste after use can be degraded and utilized by environmental (micro) organisms. The new generation of biodegradable materials is represented by polylactic acid, which is divided into poly L-lactic acid and poly D-lactic acid, which are obtained by the polymerization of its monomer L-lactic acid or D-lactic acid, respectively. It is estimated that by 2020, the global annual demand for polylactic acid will reach 15 million tons, and the annual demand for polyethylene terephthalate (PET) and polystyrene (Polystyrene, PS) will reach 50 million tons. the most likely substitute for . [0003] The poly D-lactic acid material polymerized from high-quali...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P7/56C12R1/19
CPCC07K14/245C12N9/0006C12P7/56
Inventor 王正祥田康明牛丹丹路福平
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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