Mining,Identification And Application Of Key Factors For Reducing Cr(Ⅵ) By Aspergillus Niger

Wastewater discharged from industries such as chromium plating,dyestuff and leather contain large amounts of heavy metals,leading to a polluted environment and threating the life safety of organisms.In order to remediate chromium contamination in the environment,the reduction of Cr（Ⅵ）,which is highly oxidizing and bioavailable,to Cr（III）,which is less toxic and less soluble,has become a direction of interest for many researchers.Although Aspergillus niger has been reported to have the ability to reduce Cr（Ⅵ）,the specific molecular mechanism is still unclear.In this study,the results of previous experiments showed that A.niger S469 could tolerate 100 mg/L of Cr（Ⅵ）and that soluble chromate reductase might exist in its cytoplasm.To investigate the molecular mechanism of Cr（Ⅵ）reduction in A.niger and enhance its Cr（Ⅵ）reduction ability,the genes related to chromate reductase,sulfate transporter protein and superoxide dismutase in A.niger were retrieved by bioinformatics tools in this study,and their functions were identified.The functions of chromate reductase Chr A,sulfate transporter protein Sul B and superoxide dismutase Sod A in Cr（Ⅵ）reduction in A.niger were finally determined.Subsequently,the Cr（Ⅵ）repair ability of A.niger was enhanced by overexpression of chr A,sul B and sod A,which also further confirmed the functions of these genes.The main results of this study are as follows,（1）To identify chromate reductase in A.niger,two suspected chromate reductase genes chr A and chr B were obtained by sequence alignment analysis from chromate reductase Yie F of Escherichia coli and putative chromate reductase（XP＿001388504.1）of A.niger in A.niger ATCC 1015.The functional characterization of related genes using molecular biology techniques and fermentation process showed that the expression of chr A was up-regulated177.9-fold in S469 under Cr（Ⅵ）stress;knockdown of the suspected genes in S469 was performed independently,and it was found that the Cr（Ⅵ）reduction efficiency was significantly lower in chr A knockdown strain than in chr B knockdown strain（P<0.001）.After overexpression of the putative genes in S469,respectively,it was found that the Cr（Ⅵ）reduction ability of chr A overexpression strain was more significantly enhanced,and the reduction efficiency could reach 42.9%.In conclusion,Chr A is a chromate reductase in A.niger.（2）Sulfate transporter proteins can transport Cr（Ⅵ）into the cell,which is mainly in the form of Cr O₄^2-.To identify sulfate transporter proteins in A.niger,sequence alignment and functional domain analysis of sulfate transporter proteins Sul1p and Sul2p from Saccharomyces cerevisiae were performed to identify the putative proteins Sul A and Sul B responsible for sulfate transport in A.niger.The relative expression of sul A and sul B under Cr（Ⅵ）induction and the efficiency of Cr（Ⅵ）reduction in knockout strains were analyzed,and Sul B was found to be the main sulfate transporter protein mediating the uptake of Cr（Ⅵ）by A.niger.Overexpression of sul B on strains overexpressing chr A using genetic engineering technology further increased the Cr（Ⅵ）reduction efficiency by 23.5%.In conclusion,Sul B is a sulfate transporter protein of A.niger,and the results indicate that enhanced Cr（Ⅵ）transport can further enhance the reduction of Cr（Ⅵ）by A.niger.（3）In A.niger,the ROS produced along with the reduction of Cr（Ⅵ）will cause oxidative stress in the fungus.To reduce the oxidative damage caused by ROS,this study also identified the candidate superoxide dismutase（SODs）in A.niger.The superoxide dismutase Sod A,Sod B and Sod C in A.niger were conducted to assess their roles in Cr（Ⅵ）reduction.The results showed that Sod A plays a major role in the scavenging of ROS,and overexpression of sod A can further enhance the Cr（Ⅵ）reduction ability of A.niger.