Study On Biodetoxification Mechanism Of Furan Aldehyde Inhibitors And The Construction Robust Biorefinery Fermenting Strains

Furfural and 5-hydroxymethylfurfural?HMF?are the two major furan aldehyde inhibitors derived from the over-degradation of pentose and hexose respectively during the lignocellulose pretreatment process,which can severely inhibit consequent microbial cell growth and fermentation metabolism.It is strongly required to fast and complete removal of furfural and HMF from pretreated lignocellulose for efficient biorefinery fermentation,owing to their high abundance and strong toxicity.Biological detoxification method using specific microorganisms to convert furfural and HMF into non-toxic substances prior to fermentation process was proposed and considered as an efficient strategy to overcome the inhibition.Alternatively,development of robust fermenting strains with high tolerance to the furan aldehydes was another attractive method and has been the subject of extensive investigation.Amorphotheca resinae ZN1 is a specific powerful biodetoxification fungus isolated in our previous work and has been practically applied for the high performance of ethanol,lipid,lactic acid,citric acid,gluconic acid and xylonic acid production.In order to identify the degradation mechanism of furan aldehydes in A.resinae ZN1,the real-time quantitative PCR?qRT-PCR?method was firstly applied to investigate the transcription levels of 137 putative genes involved in the degradation of inhibitors in A.resinae ZN1 under the stress of furfural and HMF,as well as the stress of their secondary metabolites,furfuryl alcohol and HMF alcohol.Several alcohol dehydrogenase genes and aldo-keto reductase/aldehyde reductase genes were found to be responsible for the furfural and HMF conversion to their corresponding alcohols.For the further conversion of the two furan alcohols to the corresponding acids,different alcohol dehydrogenase genes,aldehyde dehydrogenase gene,and oxidase genes were identified.In addition,the RNA-Seq technology was employed to analyze the genome-wide transcriptional response to furfural and HMF.A.resinae ZN1 triggered not only the expression of corresponding oxidoreductase genes,but also the expression of relevant genes involved in redox and cofactors regeneration?TCA cycle?and energy production?respiratory chain?.The significantly differentially expressed genes also included genes related to transporters and oxidative stress response process which played important roles in response to furan aldehyde inhibitors in A.resinae ZN1.Zymomonas mobilis ZM4 is an important industrial strain for cellulosic ethanol fermentation.Previous study showed that Z.mobilis ZM4 contains its native oxidoreductases to catalyze the reduction of furfural and HMF,but it can be still severely inhibited by furfural and?or?HMF.Over-expression of the exogenous aldo-keto reductase gene ARZ₁3395_T1 from A.resinae ZN1,or over-expression of the endogenous alcohol dehydrogenase gene ZM01771 from Z.mobilis ZM4,the conversion rate to both furfural and HMF by Z.mobilis ZM4 was significantly enhanced,and resulted in an accelerated cell growth and improved ethanol productivity in corn stover hydrolysate.The in vitro enzymatic analysis confirmed that the ZM01771 encoding enzyme is NADPH dependent for furfural and HMF reduction,the ethanol fermentation performance of Z.mobilis ZM4 was enhanced again by co-expression of the transhydrogenase gene udhA from E.coli with ZM01771 by elevating the NADPH availability.Corynebacterium glutamicum S9114-128 is a stable strain for glutamic acid production with high tolerance to furan aldehyde inhibitors evolved by a long-term evolutionary adaptation strategy using undetoxified corn stover hydrolysate.In order to determine the genetic changes responsible for the improved inhibitor tolerance in C.glutamicum S9114-128,the genome re-sequence analysis was performed using the high-throughput sequencing technology.The study found that the intergenic mutation between gene CGS9114 RS11050 and CGS9114_RS11055 increased the transcriptional level of gene CGS9114 RS11050 which involved in the transport of glucose in C.glutamicum S9114-128.The enhancement in sugar transport in C.glutamicum S9114-128 resulted in an accelerated glucose metabolism and also led to the flux distribution of glucose towards lactic acid production.However,the relationship between the reprogrammed pathways and the improved phenotype in C.glutamicum S9114-128 remained unclear and required further extensive study.In order to overcome the inhibitory effects of furan aldehydes on microbial metabolism,the biodetoxification method and the use of the robust fermenting strains were investigated in the thesis.Based on the studies above,the furan aldehydes degradation metabolism in A.resinae ZN1 was elucidated at the molecular level and the key candidate genes responsible the inhibitors transformation was identified as well.With the rational genetic modification strategy,the tolerance to furan aldehyde inhibitors was effectively improved in Z.mobilis ZM4.The molecular mechanism of the enhanced inhibitor tolerance to furan aldehydes in the adapted robust strain C.glutamicum S9114-128 was analyzed by the genome re-sequencing analysis.These results in present study will pave the way for the industrial development of lignocellulose biorefinery.