Sugar chain elongation glycosyltransferase mutant and its coding gene as well as genetically engineered bacteria and their application
A technology of glycosyltransferase and genetically engineered bacteria, which is applied in the field of genetic engineering, can solve the problems of many chemical synthesis steps, high cost, and serious environmental pollution, and achieve important scientific research value and social benefits
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[0047] According to a preferred embodiment of the present invention, the gene encoding phenylalanine ammonia lyase PAL is from Arabidopsis thaliana AtPAL Gene, GenBank: AY133595.1.
[0048] According to a preferred embodiment of the present invention, the gene encoding 4-coumaric acid coenzyme A ligase 4CL is shown in SEQ ID No. 5, or the gene from parsley Pc4CL Gene, GenBank: X13325.1, preferably as shown in SEQ ID No.5.
[0049] According to a preferred embodiment of the present invention, the gene encoding cinnamoyl-CoA reductase CCR is from Arabidopsis thaliana AtCCR Gene, GenBank: AF332459.1.
[0050] According to a preferred embodiment of the present invention, the gene encoding UDP glucosyltransferase UGT73C5 is shown in SEQID No. 4, or the gene from Arabidopsis thaliana AtUGT73C5 Gene, GenBank: KJ138865.1, preferably as shown in SEQ ID No.4.
[0051] According to a preferred embodiment of the present invention, the gene encoding UDP-glucose dehydrogenase is end...
Embodiment 1
[0085] This example is used to illustrate the acquisition of the sugar chain extension glycosyltransferase mutant CaUGT3-TN
[0086] synthetic CaUGT3 The gene (SEQ ID No. 1) was used as the template, and the primers CaUGT3-5FPNde (SEQ ID No. 7) and CaUGT3-3RPBam (SEQ ID No. 8) were used for PCR amplification. Nde I and Bam After HI digestion, the Nde I and Bam The plasmid pET28a digested by HI was transferred into E. coli DH5α, and the plasmid was extracted after amplification. The sequence of CaUGT3 was verified by sequencing, and pET28a-CaUGT3 was constructed.
[0087] Through the Tang method (Tang, L., Gao, H., Zhu, X., Wang, X., Zhou, M., Jiang, R., 2012. Construction of “small-intelligent” focused mutagenesislibraries using well-designed combinatorial degenerate primers. BioTechniques52, 149-158.), designed four mutation primers to introduce 20 natural amino acids into the mutation library uniformly and without redundancy, and constructed a saturated mutant libra...
Embodiment 2
[0104] This example is used to illustrate the remodeling of the synthetic pathway of rosavide E in high-producing phenylalanine Escherichia coli
[0105] High phenylalanine-producing Escherichia coli strain BPHE does not express tyrR (DNA-binding transcriptionaldual regulator TyrR), tyrA (fused chorismate mutase / prephenate dehydrogenase) , trpE (anthranilate synthase subunit TrpE) gene, and its construction method refers to patent application number 201910403327.6 (Liu Tao, Bi Huiping, Wang Shuai, Zhuang Yibin, Ma Yanhe. Loxavir analogs and genetically engineered bacteria producing the Loxavir analogs and applications thereof. ).
[0106] Escherichia coli expression vector V1 is pCDFDuet-4CL-PAL-CCR. The preparation method of this vector refers to patent application number 201611179577.9 (Liu Tao, Zhou Wei, Bi Huiping, Zhuang Yibin, Yin Hua, Ma Yanhe. Recombinant Escherichia coli for the production of cinnamyl alcohol and Luosai , construction methods and applications)....
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