Genetic Engineering Of Validamycin A High-yielding Strains

Validamycin A(VAL-A)is the first independently developed antibiotic and produced in large scale in China.Validamycin A is produced by Streptomyces hygroscopicus var.jinggangensis and widely used as an antifungal agent against rice sheath blight disease in Asian countries.In addition,validamycin and its derived products can be used as the source materials for the chemical synthesis of acarbose and voglibose,both of which are important antidiabetic drugs.Given the rising marketing demand and economic value,we optimize the fermentation performance of validamycin high-yielding strain has been optimized via metabolic regulation and genomic engineering.Previous studies had found three afsA-arpA homologues for A-factor-like regulation in the genome of 5008.Compared with the wild-type 5008,combined shbR1 and shbR3 deletions could increase the transcriptional level of validamycin gene cluster and the yield of VAL-A by 40 %.Similarly,shbR1 and shbR3 genes were respectively deleted in high-yielding strain TL01 with increased yield of VAL-A by 14 % and 11 %.Combined deletions in TL01 resulted in an enhanced production of VAL-A from 22.5 g/L to 28 g/L.Commercial product of validamycin is composed of 70 % VAL-A,15 % VAL-B,and other similar components.Compared with VAL-A,VAL-B has relatively lower activity.Previous study indicated that ValE and ValJ proteins may catalyze the hydroxylation of VAL-A to VAL-B.Deletion The mutant of valE deletion produced trace amount of VAL-B,and the production of VAL-B was reduced by 35 % in the valJ deletion mutant.However,the yield of VAL-A was not changed in both mutants.In the validamycin fermentation broth,large amount of dark brown pigment(s)was produced and affected the color,yield and quality of the product.In order to eliminate the pigment(s),four putative gene sets were found in the genome and proposed to be responsible for pigment biosynthesis.Then the contribution of each pigment biosynthetic gene set to VAL-A yield,biomass accumulation,and the color of fermentation broth was evaluated via individual gene deletions.Compared with the parental strain,the deletion of DOPA melanin gene set increased the VAL-A titer by 12 % from 20.6 to 23.1 g/L,whereas the deletion of type III PKS melanin gene set showed no effect.The inactivation of both the type II PKS spore pigment and ochronotic pigment gene sets decreased the VAL-A production by 11.7 % and 17.2 %,respectively.All these mutant strains had no significant change in the transcription of val genes and the color of the fermentation broth.In order to construct a surrogate host for the heterologous production of other aminocyclitols,such as acarbose,the validamycin biosynthetic gene cluster was further replaced by the integration site attB for ?C31 in TL01(?shbR1/shbR3).The original acarbose biosynthetic gene cluster was cloned into plasmid pLQ666 and proved to be functional by restoring acarbose production in Actinoplanes sp.SE 50/110(?acb)with the whole cluster deleted.Alternatively,all the 17 putatively essential genes for acarbose biosynthesis were reassembled using strong PvalA promoter and optimized RBS.However,many attempts on introducing these integrative plasmids into TL01(?shbR1/shbR3)were failed due to unknown mechanism.When plasmid pLQ666 was introduced into S.coelicolor M145 and S.lividans 1326,low transcription was observed for acarbose biosynthetic genes,and no acarbose was detected.In conclusion,a few derivatives of the industrial strain TL01 with higher yield were constructed via genetic engineering,and the acarbose biosynthetic genes were reassembled for subsequent heterologous production in a TL01-derived surrogate host.