Genetically engineered strain for producing gel-state xantham gum as well as construction method and application of genetically engineered strain
A technology of genetically engineered bacteria and xanthan gum, applied in the field of genetically engineered bacteria and its construction for the production of gel-state xanthan gum, can solve the problems of quality analysis and sorting of xanthan gum products, and affect the rheology of xanthan gum characteristics, increase the production cost and instability of xanthan gum, and achieve superior thickening and gel properties and a wide range of industrial application prospects
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[0042] The present invention also provides a method for preparing genetically engineered bacteria, comprising the following steps: 1) knocking out the gumF gene and gumG gene in the starting strain to obtain a bacterial strain XC (ΔFG) in which the gumF gene and the gumG gene are deleted; The genetically engineered bacteria were obtained by overexpressing the ketolyltransferase gene gumL in the strain XC (ΔFG) in which the gumF gene and gumG gene were deleted in step 1).
[0043] In the present invention, the gumF gene and the gumG gene in the starting strain preferably include the following steps: 1.1) Using the genome of the starting strain as a template, perform PCR amplification to obtain the upstream homology arm fragment of the gene gumF and the gene gumG The downstream homology arm fragment of the gene gumF; the nucleotide sequence of the upstream homology arm fragment of the gene gumF is shown in SEQ ID No: 1; the nucleotide sequence of the downstream homology arm fragm...
Embodiment 1
[0087] Construction of the Scarless Knockout Plasmid p1O3-FG
[0088] Genome kit was used to extract the genome of XC, using it as a template, using fgsu / fgsl and fgxu / fgxl primers, and amplifying the upstream homology arm fragment of gumF gene and gumG gene respectively by PrimeSTAR DNA polymerase (Takara Bio, Tokyo, Japan) The downstream homology arm fragments; overlap PCR recombination upstream and downstream fragments, agarose gel electrophoresis separation, gel recovery kit recovery target fragments. Use restriction endonucleases PacI and XbaI to digest the recombinant target fragment and plasmid pLO3 (from Dr. Oliver Lenz, the plasmid map is as follows: Figure 8 shown), connect overnight at 16°C, and transform into E.coli S17 competent cells; use primer fg1 / fg2 for colony PCR verification to screen the correct transformant, and save the glycerol tube for future use.
[0089] The PCR and amplification system of gene gumF and gene gumG are as follows:
[0090] 25 μl of ...
Embodiment 2
[0133] Fermentation of genetically engineered bacteria constructed in Example 1 to produce gelatinous xanthan gum
[0134] Pick a single colony of genetically engineered bacteria and inoculate it into 5ml of seed medium, cultivate it at 200rpm and 30°C for 20h, and then inoculate it into 100ml of seed medium with 1% inoculum to make the bacterial concentration OD 600 Reach 0.5, cultivate at 200rpm, 30°C for 18h, then inoculate with 10% inoculum size into a 500ml shake flask containing 100ml medium, and cultivate at 30°C, 200rpm for 72h.
[0135] Among them, seed medium (g / L): sucrose, 20g; peptone, 3g; yeast powder, 1g; beef extract, 5g; pH 7.0±0.02; fermentation medium (g / L): cornstarch, 50g; fish peptone , 4g; calcium carbonate, 4g.
[0136] Add 2 to 3 times the volume of ethanol (v / v) to the fermentation broth, stir to obtain a xanthan gum precipitate, filter, put the precipitate in an oven at 90°C for 6 hours, crush it to a particle size of 80 mesh, and store it for later...
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