Pigment-free low molecular weight welan gum production strain and its construction method and application

A construction method and a technology for sorting enzymes, which are applied in the field of pigment-free and low-molecular-weight welan gum production strains and their construction, can solve the problems of difficult separation of bacterial gums and large amounts of ethanol, and achieve the effects of improving fermentation levels and good application value

Active Publication Date: 2021-03-26
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Purpose of the invention: In order to solve the problems of the large amount of ethanol used in the existing welan gum production process and the difficulty in separating the bacteria gum, the present invention provides a genetically engineered bacterium of Sphingomonas that removes the encapsulation structure and has pigment defects as a pigment-free Production Strain of Low Molecular Weight Welan Gum

Method used

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  • Pigment-free low molecular weight welan gum production strain and its construction method and application
  • Pigment-free low molecular weight welan gum production strain and its construction method and application
  • Pigment-free low molecular weight welan gum production strain and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Construction of pigment-deficient recombinant Sphingomonas genetically engineered bacteria.

[0060] Step 1. Identification of key enzyme genes of carotenoid synthesis pathway in S.sp.HT-1

[0061] (1)Degenerate primer design

[0062] According to the whole genome sequence of two strains with high homology, Sphingomonas sp.ATCC31555 (producing welan gum) and Sphingomonas elodea ATCC31461 (producing gellan gum). The gene sequences related to key enzymes in the carotenoid synthesis pathway of the two strains were searched in GenBank, and degenerate primers were designed by Vector NTI 11.5.1 software (Table 1).

[0063] Table 1 Degenerate primers for PCR amplification of genes related to key enzymes in carotenoid synthesis pathway

[0064]

[0065] (2) Cloning and identification of key enzyme genes

[0066] Genome extraction kit from TaKaRa Company was used for S.sp.HT-1 genome extraction. For specific methods, please refer to the instruction manual. Using...

Embodiment 2

[0092] Example 2 Construction of Pigment-Deficient and Encapsulation-Removed Recombinant Sphingomonas Genetically Engineered Bacteria

[0093]The construction method is the same as in Example 1, except that the recipient strain is S.sp.-△crtB in Example 1, the knockout gene is the srtW gene, the nucleotide sequence is shown in SEQ ID NO: 2, and the primer sequence is shown in In Table 7 and Table 8, the sequence of the upstream / downstream genes of the sortase srtW gene was overlapped by PCR and constructed on the suicide vector pJQ200SK, and transformed into E.coli S17-1 competent cells. Using the method of conjugative transfer, under the action of the helper bacteria E.coli HB101(pRK2013), the suicide plasmid pJQ-△srtW carrying the upstream and downstream gene fragments of srtW was transformed into the welan gum producing strain S.sp.-△crtB . Single exchange clones were picked for expansion. In the second step, single exchange clones were inoculated into welan gum seed medi...

Embodiment 3

[0099] Example 3 Construction of recombinant sphingomonas genetic engineering bacteria with pigment deficiency and degradative enzyme gene knockout.

[0100] The construction method is the same as in Example 1, except that the recipient strain is S.sp.-△crtB in Example 1, the knockout gene is the gelR gene, the nucleotide sequence is shown in SEQ ID NO: 3, and the primer sequence list See Table 9, the obtained S.sp.-ΔcrtB-ΔgelR strain was named S.sp.WG-2.

[0101] Table 9 Primers required for constructing gelR knockout strains

[0102]

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Abstract

The invention discloses a pigment-free low-molecular weight Welan gum production strain. Sphingomonas sp. HT-1 (CCTCC NO: M2012062) is taken as an original strain to respectively construct a pigment-deficient and decapsulated-structure sphingomonas genetically engineered strain WG-1, a pigment and degrading enzyme gene-deficient sphingomonas engineered strain WG-2, and a pigment and degrading enzyme gene-deficient and decapsulated-structure sphingomonas genetically engineered strain WG-3. The yield of the Welan gum produced by the sphingomonas strain WG-2 in the invention is increased by 10% to 30% compared with the original strain, reaching 35 to 45g / L, and the molecular weight is not significantly changed (Mn: 10000 to 20000kDa). The sphingomonas strain WG-1 and the sphingomonas strain WG-3 in the invention are fermented to produce low-molecular weight Welan gum (Mn: 500 to 1000kDa), and the yield of the Welan gum produced by the decapsulated-structure strain WG-3 is increased by 20%to 30% compared with the strain WG-1, reaching 20 to 25g / L. The strain constructed by the invention can obtain Welan gum products of different molecular weight ranges, and significantly improves thefermentation level of Welan gum.

Description

technical field [0001] The invention relates to the field of microbial genetic engineering bacteria, in particular to a pigmentless low molecular weight welan gum production strain and its construction method and application. Background technique [0002] Microbial polysaccharides are a kind of environmentally friendly and green products that can be obtained through biological fermentation, and play an important role in human life and industrial and agricultural production applications. The success of microbial polysaccharides in scientific research and industrial production is mainly based on the following points: (1) the properties of polysaccharides and their application value; (2) they can be produced under controlled conditions by selecting suitable strains; ( 3) the feasibility of the extraction process; (4) no toxin; (5) different microorganisms can synthesize many very special ionic and neutral polysaccharides, these microbial polysaccharides have a wide range of app...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12P19/04C12R1/01
CPCC12N9/1085C12N15/74C12P19/04C12Y205/01032C12Y205/01096C12Y205/01099
Inventor 李莎赵明徐虹薛瑞刘晓柳冯小海朱萍
Owner NANJING TECH UNIV
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