Utilization Of Intracellular Carbon Reservoir And Mechanism Of Carbon Flow Regulation Guided The High Yield Of Ascomycin

Ascomycin(FK520)is a natural 23-membered macrocyclic antibiotic produced by Streptomyces hygroscopicus var.ascomyceticus ATCC 14891,notable for its diverse biological and pharmacological activities,including antifungal,antimalarial,immunosuppressive and anticonvulsive effects.In this study,polyhydroxybutyrate(PHB)was used as an intracellular carbon reservoir to increase the available carbon supply for the synthesis of ascomycin,and the synthesis of ascomycin precursor was promoted through the regulation of carbon flow by the signal transduction protein belonging to PⅡ family,finally achieving the high yield of ascomycin in S.hygroscopicus var.ascomyceticu.The genetic analysis of S.hygroscopicus var.ascomyceticus showed that there is a PHB decomposition gene fkb U in the FK520 biosynthesis gene cluster.The PHB content in the parent strain FS35 showed that PHB can be accumulated in S.hygroscopicus var.ascomyceticus when carbon sources were abundant and degradated when carbon sources became insufficient.Further analysis of fermentation characteristics showed that in S.hygroscopicus var.ascomyceticus,the biomass accumulation was correlated with the polymerization of PHB,and the biosynthesis of FK520 was correlated with the degradation of PHB.The combined overexpression of exogenous PHB synthesis gene pha C and native PHB decomposition gene fkb U indicated that the polymerization of PHB in the exponential phase promoted the accumulation of biomass,and the degradation of PHB in the stationary phase increased the synthesis of FK520.The comparative transcription analysis between the parent strain FS35 and the cooverexpression strain Opha Cfkb U showed that the polymerization of PHB during the exponential phase accelerated the absorption and utilization of carbon sources and promoted the biosynthesis processes,thereby stimulating the growth of the strain,and the decomposition of PHB during the stationary phase enhanced the carbon flux in the primary metabolic pathways and increased the biosynthesis of FK520 precursors,thereby improving the output of FK520.This is the first time to report the carbon supply mechanism of PHB,which can be used for the biosynthesis of FK520 as an intracellular carbon reservoir.On this basis,the optimization of carbon sources addition in the fermentation medium further enhaced the carbon storage of PHB,thereby promoting the FK520 yield of strain Opha Cfkb U to626.30 mg/L,which was 2.11 times of the parent strain FS35 in the initial fermentation medium.The results of genome sequencing and amino acid sequence alignment showed that there are two PⅡ family signal transduction proteins,GlnB and GlnK in S.hygroscopicus var.ascomyceticus.The protein co-precipitation experiments and coupled enzyme assays showed that the GlnB protein could inhibit the activity of acetyl/propionyl-Co A carboxylase by binding to the α subunit of acetyl-Co A carboxylase,while relase the the activity of these two enzymes by binding with a sufficient concentration of 2-oxoglutarate(2-OG).GlnK protein can not affect the activityof these two enzymes because it can not interact with the α subunit.The concentrations of intracellular coenzyme A esters showed that the knockout of GlnB protein can release the activity of these two enzymes,thereby increasing the carbon flows of the pathways for precursor malonyl-Co A and methylmalonyl-Co A synthesis.The knockout of GlnB and GlnK proteins showed that the single deletion of either GlnB or GlnK protein did not affect cell growth,and the single deletion of GlnB significantly increased the production of FK520,while the double deletion of both GlnB and GlnK proteins severely impaired the strain growth and significantly reduced the FK520 yield.The complementation of GlnK and the electrophoretic mobility shift assays showed that when GlnB was deleted,GlnK could act as a substitute regulatory protein to maintain the normal strain growth under the control of the global regulatory GlnR.On this basis,the co-overexpression of the β-subunit and the ε-subunit of propionyl-Co A carboxylase further increased the supply of the precursor methylmalonylcoenzyme A,thereby increasing the FK520 yield to 546.38 mg/L,which was1.9 times of the parent strain FS35.In general,this study revealed the carbon supply mechanism of PHB as an intracellular carbon reservoir and the regulation mechanism of carbon flows by signal transduction protein GlnB.On this basis,the combination of genetic engineering and medium optimization achieved the high yield of FK520 in S.hygroscopicus var.ascomyceticus,providing a new strategy for the high yield of antibiotics in other Streptomyces.