Construction and application of multiple gene coexpression system containing angolosamine glycosylsynthetase and glycosyltransferase

Abstract

The invention provides construction and application of a multiple gene co-expression system containing angolosamine glycosylsynthetase and glycosyltransferase. The invention is characterized by taking a streptomyces genome bank plasmid as a template; amplifying six angolosamine synthetase genes and a glycosyltransferase gene through PCR and connecting the genes in sequence and placing the genes in the lower reaches of a streptomyces promoter PactIII-actI to form a transcription unit; transferring the transcription unit to a streptomyces plasmid carrier pSET152, thus constructing a streptomyces expression plasmid pAYT55 co-expressed by multiple genes; leading the pAYT55 into a host cell streptomyces coelicolor CH999, mixedly culturing obtained engineering bacteria and streptomyces B135 of accumulated polyketide kalafungin, thus realizing bioconversion of kalafungin into a novel antibiotic with angolosamine; or directly leading pAYT55 into the streptomyces B135 and carrying out single culture to realize glycosylation of kalafungin. Therefore, by adopting the system, rare angolosamine can be synthesized in the cell and angolosamine modification can be carried out on polyketide by adopting the low substrate recognition specificity of antibiotic glycosyltransferases.

Description

Technical field: [0001] The present invention relates to a multi-gene co-expression system related to antibiotic rare glycosyl synthesis and transfer and antibiotic structural modification (glycosylation), especially a co-expression plasmid containing angolan glycosamine synthetase and glycosyltransferase genes Application of constructing and expressing engineering bacteria. Background technique: [0002] Many natural active substances, such as antibiotics, contain sugar groups in their structures. These sugar groups are connected to specific positions on the mother nucleus of the antibiotic molecule, and are crucial to the biological activity of the compound, and are even essential functional groups. The differences in the sugar structure, the number of sugar groups, and the connection position and connection method on the sugar receptors will have a great impact on the biological activity of antibiotics. The glycosyl structures of antibiotics are diverse, and the correspo...

AI Technical Summary

Problems solved by technology

[0003] Because the in vitro chemical synthesis of deoxysugar groups contained in the natural structure of antibiotics is often complicated and the yield is very low

The source of these deoxyglycosyl groups largely limits the modification of antibiotics in vitro, so there are not many examples of glycosylation studies on antibiotics using in vitro glycosylation experiments.

[0004] Angolosamine (angolosamine) is a rare deoxyhexosamine, which is only found in the secondary metabolite structure of a limited number of Streptomyces, and is not commercialized at present. Its chemical synthesis requires more than ten steps and the yield is very low. [Fraser-reid B, Kelly DR, Tulshian DB. And Prasad SR, Routes From "Triacetyl Glucal" to 6-Deoxy-Hex-2-Enopyranosides, J Carbohydrate Chemistry, 1983, 2(2): 105-114], this This limits the use of this glycosyl group in the study of Angora glycosylation of antibiotics in vitro

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