Corynebacterium glutamicum engineering strain for producing 5-aminolevulinic acid
A technology of Corynebacterium glutamicum and aminolevulinic acid, applied in the direction of microorganism-based methods, bacteria, microorganisms, etc., to achieve the effect of promoting transportation and increasing yield
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Embodiment 1
[0025] Example 1: Construction of knockout plasmid pD-sacB and construction of overexpression 5-aminolevulinic acid synthase gene plasmid pXA
[0026] Construction of pD-sacB plasmid
[0027] Firstly, the linear fragment of pK18mobsacB cut by HindIII was used as a template, and the sacB gene was amplified with the following primers sacB-1 / sacB-2. The sacB gene fragment was ligated with the plasmid pEC-XK99E after the MunI / EcoRV double digestion and the EcoRI / SmalI double digestion to obtain the plasmid pEC-XK99E-sacB. Use the following primers trcsacB-1 / trcsacB-2 to amplify the trcsacB fragment containing the trc promoter using the pEC-XK99E-sacB plasmid as a template.
[0028] Use the following primers pD-1 / pD-2, and use the pK18mobsacB plasmid as a template to amplify the pD fragment containing kanamycin resistance and E. The pD fragments were ligated to obtain plasmid pD-sacB.
[0029] Construction of pXA plasmid
[0030] The 5-aminolevulinic acid synthase gene hemA of ...
Embodiment 2
[0031] Example 2: Knockout of the lactate dehydrogenase encoding gene ldhA and knockout of the acetate production pathway genes pta-ackA, pqo and cat
[0032] Knockout of the gene ldhA encoding lactate dehydrogenase:
[0033] Using the genome of Corynebacterium glutamicum (C. glutamicum) ATCC 13032 as a template, using ldh-1 / ldh-2 as primers to amplify the upstream fragment of gene ldhA, and ldh-3 / ldh-4 as primers to amplify the downstream of gene ldhA fragment. After the two fragments were recovered by gel cutting, the fusion product of the two fragments was amplified using the equimolar proportion of the fragments as a template and ldh-1 / ldh-4 as primers. The fused fragment was digested with EcoRI / HindIII and ligated with pD-sacB after the same double digestion to obtain plasmid pD-ldhA.
[0034] Transfer the pD-ldhA plasmid into C.glutamicum ATCC 13032, and use kanamycin to screen the positive clones with successful recombination. The selected transformants were inoculate...
Embodiment 3
[0047] Example 3: Insertion of a strong sod promoter in front of the ppc gene and knockout of the gene pck encoding phosphoenolpyruvate carboxykinase
[0048] insert sod promoter in front of ppc gene
[0049] Using C. glutamicum ATCC13032 genome as a template and ppc-1 / ppc-2 as primers to amplify the upstream fragment of gene ppc. sod-1 / sod-2 are used to amplify the promoter of the sod gene. ppc-3 / ppc-4 is used to amplify the downstream fragment of the ppc gene. After the three fragments are cut and recovered, the equimolar proportion of the fragment is used as a template, and ppc-1 / ppc-4 is used as a primer to amplify the obtained Fusion product of three fragments. The fused fragment was digested with XbaI / HindIII and ligated with the plasmid vector pD-sacB after the same double digestion. The plasmid pD-ppc was obtained.
[0050] The constructed plasmid pD-ppc was transformed into the strain CG4 by electroporation, the positive clones with successful recombination were s...
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