The Preliminary Exploration Of Kasugamycin Biosynthesis And Kasugamycin Overproduction Mechanism

Kasugamycin,produced by Streptomyces kasugaensis,is a highly effective and general agricultural aminoglycoside antibiotic.Research progress on kasugamycin biosynthesis is very slow,and much attention is mainly focused on the optimization of the traditional fermentation process and mutagenesis techniques,which however has limited space in enhancing the kasugamycin yield.Kasugamycin biosynthetic gene cluster was first reported in 2006.However,the mechanism of kasugamycin biosynthesis has not been studied in detail.In 2016,several regulatory genes upstream of kasugamycin biosynthetic gene cluster were identified and their coding function was studied and confirmed.During the preliminary study,two knockout vectors targeting two unknown genes,kas B and kas I,were constructed and introduced into Streptomyces kasugaensis.The resultant kas B mutant cannot produce kasugamycin whereas kas I mutant did not show significant change in production of kasugamycin as compared to the wild type strain.The role of kas B in kasugamycin biosynthesis is discussed.As the genetic manipulation in S.microaureus CGMCC4.1057 is very difficult,the efficiency for gene knockout in this strain is very low.For this concern,the strategy of heterologous expression of the complete gene cluster is employed to study its biosynthesis.Firstly the genomic library of S.microaureus CGMCC4.1057 was constructed by using integrative vector,and 3360 clones with an average insertion of 30 kb,giving a genome coverage of 11.2 times.Based on the sequence of the known genes in kasugamycin biosynthetic cluster,two cosmids 15F10 and 26B1 were isolated by PCR.And then the kasugamycin gene cluster deletion strain of S.microaureus CGMCC4.1057 was obtained and could be used as a “heterologous host” for studying kasugamycin biosynthesis.Deletion of the interested gene on these two cosmids in E.coli and “heterologous expression” of the mutated kasugamycin gene clusters in kasugamycin gene cluster deletion strain would significantly affiliate the in vivo study of kasugamycin biosynthesis.On the other hand,engineering of kasugamycin high-yield strains either by increasing the copy number of the key genes or by doubling the complete gene cluster were conducted.Firstly,kas E encoding a hydrolase proposed to catalyze the myo-inositol monophosphate hydrolysis was overexpressed in kasugamycin high yielding strain.In parallel,additional copy of kasugamycin biosynthetic gene cluster was also introduced to high-yielding strain.The resultant strains were confirmed and the yields were compared with high-yielding strain in production of kasugamycin.Unfortunately,both mutants did not show obvious enhancement of kasugamycin yield.Finally we try to determine the potential genes that can affect the production of kasugamycin by comparing the transcriptome of the high-yielding strain and wild type strain.Several highly expressed genes possibly related to kasugamycin overproduction were identified,including those encoding glycolysis phosphofructokinase and pyruvate kinase,glucose-6-phosphate lactone enzyme and glucose 6-phosphate dehydrogenase that are possibly involved in the pentose phosphate pathway;acetyl coenzyme A carboxylase involved in fatty acid synthesis;aldehyde dehydrogenase and enoyl coenzyme A hydratase involved in fatty acid degradation.In particular,the key enzyme of inositol phosphate synthase contig2?522 related to the kasugamycin precursor synthesis was also identified indicating the importance of the myo-inositol supply in high yield strain.All these key enzymes will be the target for studying the mechanism of the high production of kasugamycin in future.