Omics-based Approaches Analyzing The Response Of Rhizopus Oryzae To Furfural Stress For Fumaric Acid-production From Xylose

Fumaric acid,a typical platform chemical with four-carbon dicarboxylic acid,was mainly produced by the fermentation of Rhizopus oryzae.As R.oryzae could utilize xylose naturally,hemicellulose hydrolysate was being developed for the production of fumaric acid.However,some by-products(especially for furfural)generated in the process of hemicellulose acid-hydrolysis limit the fermentability of hydrolysate.The impact of furfural on R.oryzae and fumaric acid production was investigated firstly.It was found that the growth of R.oryzae was not seriously influenced by 1.0 g/L furfural addition at 0 h,but with a decrease of fumaric acid production,from 15.1 g/L to 5.78 g/L.However,when 1.0 g/L furfural was added at 8 h,the growth of cells and the synthesis of fumaric acid were nearly stopped.In order to alleviate the toxicity of furfural on Rhizopus oryzae fermentation,the molecular mechanism of R.oryzae responding to furfural stress for fumaric acidproduction was investigated by omics-based approaches.The metabolism of R.oryzae responded to furfural stress was characterized by GC-TOF-MS-based metabolic profiling analysis.Obvious changes of 29 metabolites involved in xylose and amino acid metabolism,as well as sugars,polyols and fatty acids biosynthesis were analyzed.It was found that the intracellular metabolism of R.oryzae was seriously disturbed when furfural stress was suddenly exerted on the growing cell,but it maintained the basic cell metabolism when furfural was added at the beginning of fermentation.To further uncover the survival mechanism,LC-TOF-MS/MS-based lipidomics was carried out.Combined with the PLS-DA analysis and Pearson correlation analysis,it was revealled that phosphatidylcholine,phosphatidylglycerol,phosphatidylinositol and polyunsaturated acyl chains might be closely correlated with R.oryzae's adapting to furfural stress.Based on the above omics analysis,2.4 mg lecithin,5.4 mg inositol and 2.4 ?L soybean oil were exogenously supplemented into 250 ml shake flaskes separately with an optimized concentration in the presence of furfural,which increased fumaric acid titer from 5.78 g/L to 10.03 g/L,10.05 g/L and 12.13 g/L after 92 h,35 °C fermentation,verified the omics analysis.