Effects Of Methylmalonyl-CoA Node On The Production Of Ansamitocin In Actinosynnema Pretiosum

Ansamitocins are important secondary metabolites produced by Actinosynnema pretiosumssp.auranticum ATCC 31565.Ansamitocins are potent antitumor agents whose productionimprovement could be achieved through metabolic engineering and fermentation processoptimization.According to the R group on C-3 position,the ansamitocins could be sorted asAP-1,AP-2,AP-3 and AP-4 etc,and the yield of ansamitocins could be indicated by the amountof AP-3,the majority of ansamitocins in fermentation medium.In A.pretiosum,methylmalonyl-CoA is not only the precursor for ansamitocin synthesis,but might also act asone of critical metabolic nodes in the secondary metabolism.In this study,we have investigatedseveral genetic and metabolic engineering manipulations in order to understand thecharacteristics of methylmalonyl-CoA node and enhance the production of ansamitocins.Methylmalonyl-CoA is converted from propionyl-CoA catalyzed by propionyl-CoAcarboxylase,while propionyl-CoA is usually formed during several amino acids,fatty acids andcholesterol metabolisms.Three genes in Actinosynnema mirum,namely amir₆390,amir₆396,and amir₆229,were found to encode propionyl-CoA carboxylase.We also found thatmethylmalonyl-CoA was mainly metabolized through two different ways:?1?entering into typeI PKS system to be carbon chain extension part of proansamitocin;?2?being transformed intosuccinyl-CoA by methylmalonyl-CoA mutase and then entering into the TCA cycle.Threegenes amir₂277,amir₆418,amir₆419 in A.mirum work together to encode methylmalonyl-CoA mutase?amir₂277 and amir₆418 code the big subunits,and armir₆419 codes the smallsubunit of the mutase?.The AP-3 production of amir₂277 single knockout mutant strain was70 mg/L at day 7 of fermentation,about 3-fold compared with that of the wild-type strain.However,the AP-3 production of amir₆418 single knockout strain was slightly reduced,whileAP-2 production was significantly increased by more than 3-fold.In this study,we also foundthat heterogeneous expression of gene amir₆390 in A.pretiosum could improve the productionof AP-3 by 1.6-fold.The AP-3 production of the mutant that heterogeneous express propionyl-CoA carboxylase system was 51.5 mg/L at day 7 of fermentation,about 2.5-fold compared withthe wild-type strain,and 3-fold compared with wild-type strain with empty plasmid pIB139.