Research On Adaptability Of Heterologous Aminotransferase And Chassis Of Valienamine Artificial Synthetic Pathways

Valienamine belongs to the group of C₇N aminocyclols and is the core structure of glycoside hydrolase inhibitors.As a common precursor of acarbose and voglibose,the therapeutic drugs for type II diabetes,valienamine has attracted much attention in the durg research and development for diabetes and other diabetes-related diseases.At present,valienamine is mainly produced through chemical synthesis and semi-synthetic methods in microorganisms,which exist the disadvantages such as cumbersome procedures,low yield,high cost,etc.The efficient synthesis of valienamine has always been a hot spot in chemical and biological synthesis process.In our lab previous work,the concept of synthetic biology was used to synthsize the precursors of valienone catalyzed by ValA,ValD and ValK derived from the validamycin biosynthetic pathway,and the stereoselectivity and catalytic efficiency of heterologous aminotransferases were examined,finally,the synthesis of valienamine was achieved.In this study,the metabolic network and fermentation products of valC knockout strain S5008?valC?Streptomyces hygroscopicus 5008,S5008?was systematically analyzed.The supply capacity of glutamate and glutamine from amino acid donors was examined.The glutamic acid dehydrogenase?SHJG₇666?and glutamine synthetase?SHJG₃687?in their nitrogen metabolism pathways were characterized and the catalytic functions of SHJG₇666 and SHJG₃687 were confirmed.These work laid the foundation for increasing the supply of amino donors through gene overexpression and other metabolic engineering methods and increasing the valienamine production by engineered strains.At the same time,we investigated the effect of four different promoters:valAp,ermEp*,kasOp*,and SF14 on the regulation of the expression of the exogenous aminotransferase VarB in the S5008?valC cells.The transcription intensity was kasOp*>SF14>ermEp*>valAp.In the presence of strong promoter kasOp*,the transcription level of the aminotransferase varB gene was increased 10⁵ folds.To further investigate the spatial adaptability of exogenous aminotransferases and the metabolic pathway of the chassis,a gene replacement system was constructed in this study.The exogenous aminotransferase gene varB,kasOp*-varB,and the valienone phosphatase gene valC were achieved in situ replacement in the genome.The varB and downstream gene valD transcription levels of varB in the position of the natural promoter of the genome and the presence of a strong kasOp*promoter in this position were examined.The real-time reverse transcription PCR?qRT-PCR?results showed that in the presence of strong promoter kasOp*,the transcription amount of the aminotransferase varB gene was 3-4 times higher than that of the natural initiation state,and did not affect the expression of the downstream gene valD.The fermentation yield of valienamine in the genetically engineered strains S5008?valC::varB?in situ?was achieved at 160.3?g/L.In summary,this study systematically investigated the supply of precursors,the adaptability of exogenous genes and the adaptability of the chassis cells in the design synthesis route of the valienamine,for the purpose to optimize the metabolic flux of the design pathway,increase the metabolic flux and efficiently synthesize the target product.