The Effect Of Over-expression Hemoglobin On Cit-Ric Acid Fermentation By Aspergillus Niger

Citric acid has extensive application in food,pharmaceutical and chemical industry.The main working horse for citric acid production uses a strictly aerobic microorganism Aspergillus niger,which is very sensitive to low oxygen condition during the citric acid fermentation process,and even a short-time low oxygen supply can dramatically decrease the citric acid production.Therefore,dissolved oxygen is an essential element for citric acid fermentation.Vitreoscilla strains inductively express an oxygen-binding soluable protein Vitreoscilla hemoglobin(VHb)under low oxygen level.Since VHb binds more oxygen to improve oxygen utilization efficiency,it hs been cloned into various heterologous aerobic microorganisms to improve oxygen uptake and increase production and yield of target products.In this study,VHb was expressed in an industrial citric acid-producing A.niger strain to study the effect of VHb overexpression on citric acid production.First,the recombinant VHb was expressed in Escherichia coli BL21(DE3)and its biological function was confirmed by CO difference spectrum.Then,a vector carrying the vgb gene and glaA promoter was constructed.PEG-mediated protoplast transformation method was thoroughly studied and improved in the industrial citric acid-producing A.niger.By using the transformation method,and subsequent strain puring,genomic PCR testing,a VHb-overexpressing industrial Aspergillus niger transformant was successfully obtained.Western-blot and CO difference spectra analysis showed that the VHb was actively expressed in the A.niger strain.The effect of overexpressing VHb on A.niger citric acid fermentation was investigated under different dissolved oxygen conditions.Under normal oxygen supply,VHb significantly increased the citric acid production by 7.75%and improved the yield by 7.94%.With the decrease of dissolved oxygen,VHb alleviated the effects of hypoxia on the strain.Aslo in short off oxygen conditions under fermenter level,VHb make citric acid accumulation upgrade about 6%.However under the tested condition of serious oxygen shortage or interrupted oxygen supply for long time,citric acid production in the VHb-overexpressiong strain decreased,which could be caused by metabolic burden or carbon distribution by overexpression of VHb.In this study,we thoroughly improved the protoplast-based transformation system of an industrial citric acid producing strain,which lays the foundation for molecular biological research and genetic engineering of industrial A.niger strains.We also carried out a preliminary study on the effect of VHb on citric acid production,and suggested that further optimization of VHb overexpression may benefit for constructing a robust industrial citric acid-producing strain under harsh oxygen supply condition in the industrial citric acid production process.