Identification And Functional Study Of Mitochondrial Citrate Transporter In Aspergillus Niger

Aspergillus niger is an outstanding cell factory for citric acid production and the process of citrate export from the mitochondria to the cytoplasm is predicted to be one of rate-limiting steps in citric acid accumulation.There are currently three major rate-limiting steps known for citric acid accumulation in citric acid fermentation: 1.Glucose transporters on cell membranes;2.Citric acid transporters on cell membranes;Three carboxylic acid transporters on the mitochondrial membrane.Among them,glucose transporters and citric acid transporters on cell membranes have been studied in detail.Currently,the mitochondrial citrate carriers(Ctps)responsible for this process are not fully characterized.Here,six putative Ctp encoding genes(ctp1-ctp6)were identified to have homologies to a mitochondrial citrate transporter Sc Ctp1 from Saccharomyces cerevisiae.RT-PCR assay showed that the six genes were transcripted,but ctp1 transcripted only at the late stage.We found that individual disruption of(ctp1-ctp6)caused different degrees of decline in citric acid accumulation at different fermentation stages,while multiple disruptions of all the six Ctps in the mutant S1696 resulted in complete abolishment of citiric acid production.Moreover,S1696 exhibited the defects of delayed growth,reduced conidia formation and decreased pigmentogenesis.Colonial morphology analysis showed that exogenous addition of citrate partially restored the conidia formation and pigmentogenesis.Individual reintroduction of each ctp into S1696 at the same amy A locus showed that ctp1,ctp2 and ctp4 was found to restore the citric acid titers to 88.5%,93.8% and 94.6% of the parent strain,respectively.Additionally,the formation of conidia and pigment production could also be partially restored after ctp1,ctp2 or ctp4 reintroduction.Furthermore,individual overexpression of ctp1,ctp2 and ctp4 in the parent strain caused a significant increase in citric acid accumulation by32.8%,19.3% and 24.2%,respectively.These results suggest that multiple Ctps participate in citric acid transport across the mitochondrial membrane in a redundant manner and enhancement of this process can improve citric acid accumulation in A.niger.