The Response Of Rhizopus Oryzae To Stress During Fumaric Acid Production

Enviroment facts play important roles in microorganism fermentation,like substrates,temperature,pH.Thus,study of the metabolic characteristics of micrrorganisms with different environmental factors would provide the most direct guidance for the subsequent fermentation process control.In the present study,transcriptomic and metabolomic analyses were applied to comprehensively understand the global difference of Rhizopus oryzae under different environment conditions.The main results are listed as follows:(1)RNA-Seq was applied to generate two transcriptional maps of Rhizopus oryzae to investigate the effects of acid stress on Rhizopus oryzae and the metabolic characteristics of Rhizopus oryzae with different morphologies.The results showed a total of 11,340 coding genes could uniquely mapped to the Rhizopus oryzae genome,among them,615 genes were up-regulated in mycelia sample and 814 genes were down-regulated in pellet sample.The KEGG pathway analysis indicated that genes related to unsaturated fatty acids metabolism,amino acids metabolism,sugar and sugar alcohols synthesis pathway,chitin and cellulose degradation,and N-acetyl glucosamine amine synthesis were up-regulated.(2)The metabolic profile of Rhizopus oryzae under different morphologies was analyzed.The results showed that the ROS level in pellet sample was higher than the mycelia sample.To reduce the reactive oxygen species-mediated damage,Rhizopus oryzae increased the metabolites concentration of unsaturated fatty acids,amino acids,sugar and sugar alcohols,as well as the cell wall components,which was benefited for pellet formation.Base on these observations,we increased the dissolved oxygen concentration during Rhizopus oryzae cultivation,pellet was obtained.(3)The metabolic profile of Rhizopus oryzae with different carbon source was analyzed.The results showed that,“carbon starvation” was presented in Rhizopus oryzae during xylose fermentation,and induced oxidative-mediated damage.To adapt to this unfavorable condition,Rhizopus oryzae increased the abundance of unsaturated fatty acid,amino acid,and sugar alcohol or sugars.These responses might divert the carbon flow for fumaric acid production.Further,we found that during xylose fermentation,increasing nitrogen concentration could enhance the stress resistance of cells and benefit for fumaric acid production.While during glucose fermentation,increasing nitrogen concentration resulted in energy surplus,and decreased the carbon flow to the fumaric acid synthesis pathway.(4)To decrease the surplus energy,we developed a strain selection method with neomycin,which could inhibit the enzyme of ATPase to generate ATP.After a series of selection,finally we found a strain named N6 showed a higher fumaric acid production and productivity.In summary,the metabolic mechanisms of Rhizopus oryzae under different environment conditions were discovered by transcriptomic and metabolomic analyses,and based on these findings,a Rhizopus oryzae mutant with higher fumaric acid production were selected out.