A kind of recombinant microorganism producing sialic acid and its application
A technology for recombinant microorganisms and sialic acid, applied in microorganisms, microorganism-based methods, recombinant DNA technology, etc., can solve the problems of low yield of sialic acid and difficult to meet industrial production, and achieve lower production costs, efficient transformation, and important industrial applications. effect of value
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Embodiment 1
[0075] The construction of the starting strain of embodiment 1
[0076] In this example, taking Escherichia coli BL21 as an example, a starting strain capable of synthesizing sialic acid was constructed, specifically: knocking out the nanATEK gene cluster in Escherichia coli BL21 (purchased from China Industrial Microorganism Culture Collection Center), and introducing the overexpression plasmid pXMJ at the same time -neuBC, to obtain the starting strain BL21ΔnanATEK / pXMJ-neuBC capable of producing sialic acid.
[0077] 1. Knockout of nanATEK
[0078] 利用Red重组方法敲除nanATEK(基因簇序列如SEQ ID NO.16所示),具体方法如下:以nan-F(ggtataacaggtataaaggtatatcgtttatcagacaagcatcacttcagaggtatttgtgtaggctggagctgcttc)和nan-R(tcataatttttctccctgggccaacagcgcagccccaagtaaacctgcatcatggcggtaatgcgccgccctgtcaaacatgagaattaa)为引物,以质粒pKD13(购自Addgene) A 1.3Kb PCR fragment was obtained for template amplification, which was transferred into E. coli BL21 containing plasmid pSIJ8 (purchased from Addgene) by electroporation, and ...
Embodiment 2
[0083] Example 2 Construction of recombinant bacteria overexpressing glucosamine 6-phosphate synthase or its mutant
[0084] On the basis of the starting strain constructed in Example 1, further overexpress glucosamine 6-phosphate synthase (the amino acid sequence is shown in SEQ ID NO.1, and the coding gene sequence is shown in SEQ ID NO.2) or its mutant E15K+D387V+S450P+E525G (the amino acid sequence is shown in SEQ ID NO.11, and the coding gene sequence is shown in SEQ ID NO.12), the specific method is as follows:
[0085] 1. Construction of a plasmid overexpressing glucosamine-6-phosphate synthase glmS or its mutant glmS*
[0086] Artificially synthesized glmS* mutant gene, the sequence is shown in SEQ ID NO.12, the sequence contains the following mutation E15K compared with glmS (wild-type glmS, sequence shown in SEQ ID NO.2) derived from Escherichia coli W3110, D387V, S450P, E525G, the enzyme activity is higher and not inhibited by acetylglucosamine compared with wild-t...
Embodiment 3
[0089] Example 3 Construction of recombinant bacteria overexpressing glucosamine 6-phosphate synthase and fructose-1,6-bisphosphatase simultaneously
[0090] Fructose-1,6-bisphosphatase can catalyze the dephosphorylation of fructose-1,6-bisphosphate to produce fructose 6-phosphate. The present invention finds that on the basis of overexpressing glucosamine 6-phosphate synthase or its mutant, further Overexpression of fructose-1,6-bisphosphatase glpX could further increase the production of sialic acid.
[0091] Simultaneously overexpress Escherichia coli 6-phosphate glucosamine synthase (the amino acid sequence is shown in SEQ ID NO.1, the coding gene sequence is shown in SEQ ID NO.2) or its mutant (the amino acid sequence is shown in SEQ ID NO.11) shown, the coding gene sequence is shown in SEQ ID NO.12) and fructose-1,6-bisphosphatase (the amino acid sequence is shown in SEQ ID NO.3, the coding gene sequence is shown in SEQ ID NO.4) The construction method of recombinant ba...
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