Molecular Mechanism Of Engineered Zymomonas Mobilis ZM532 Response To Furfural And Acetic Acid Stress

Acetic acid and furfural are two major inhibitors of microorganisms during lignocellulosic ethanol production.In our previous study,we successfully constructed an engineered Zymomonas mobilis ZM532 strain tolerant these double inhibitors by genome shuffling,but the molecular mechanisms of tolerance to these inhibitors are still unknown.This study investigated the responses of ZM532 and wild-type ZM4 to acetic acid and furfural using both transcriptomics and label free quantitative proteome.The main results are as follows:1.The mutant ZM532 was constructed by genome shuffling.The ZM532 exhibited superior performance with a shortened time of fermentation(30 hours)and higher ethanol production(0.463 g/L/h)compared to the parental strain AQ8-1 in 7.0 g/L acetic acid medium.We identified a total of 23 single nucleotide polymorphisms(SNPs)in the coding sequence(CDS;4)and intergenic region(19)via Sanger sequencing technology.Six SNPs were however novel in this study.These SNPs related to ZMO?RS09095,Alpha/betahydrolase/Trna Met,ZMO?RS04295,ZMO?RS09160 and ZMO?RS0165genes.May be these genes involved in the same mutations on the parental and mutant strains have been reported previously and currently may be play critical role against acids stressor.2.In this study ZM4 and ZM532 were cultured in RM and AF media.Based on preliminary experiment,the concentrations of acetic acid and furfural combinations were set at 5.0 g/L and 3.0 g/L to study the responses of ZM4 and mutant ZM532.By transcriptome,we identified a total of 1865 and 14 novel differentially expressed genes(DEGs)in ZM532 and wild-type ZM4.Among these,352 DEGs were upregulated;while and 393 were down-regulated in AF?ZM532vs RM?532,respectively.However,442 DEGs were up-regulated while 463 were down-regulated in AF?ZM4 vs RM?ZM4.Moreover,2 up and8 down-regulated genes identified in AF?ZM532vs AF?ZM4;while 7 up and 1 down-regulated genes were found in RM?ZM532vs RM?ZM.ZMO?RS06525,ZMO?RS03765,ZMO?RS08315,ZMO?RS00235,ZMO?RS02740,ZMO?RS04690,ZMO?RS01550,ZMO?RS02545 and ZMO?RS01130,ZMO?RS04890,ZMO?RS01205,(dnak,ZMO?RS02930),Gro EL(Novel00014,Novel00013,ZMO?RS08760),ZMO?RS03400,ZMO?RS03395,ZMO?RS05565,ZMO?RS05445,ZMO?RS03970,ZMO?RS05445 ZMO?RS05290 and ZMO?RS02495 were exclusively found in ZM532.These genes are involved in amino acid biosynthesis,macromolecules repair,glycolysis,flagella assembly,ABC transporter,fermentation,ATP synthesis etc pathways.In addition,purin encoded up-regulated ZMO?RS08390 gene play important role against acid resistance.Moreover,these mentioned genes confirmed and help to unravel the acetic acid and furfural tolerance mechanism between ZM532 and wildtype ZM4.3.In our proteome data analysis,we also identified 1,532 proteins among 107 up and 204 downregulated proteins detected in ZM4?AF.vs.ZM4?RM,123 up and 205 down regulated proteins were identified in ZM532?AF.vs.ZM532?RM,respectively.In addition,a total of 16 up and 5 down-regulated proteins were identified out of 1462 in ZM4?AF.vs.ZM532?AF,while 8 up and 5 down-regulated proteins were observed out of 1491 in ZM4?RM.vs.ZM532?RM.ZMO?RS07620,ZMO?RS06825,ZMO?RS07005,ZMO?RS06600,ZMO?RS02795,ZMO?RS06410 ZMO?RS02890,ZMO?RS02760,ZMO?RS02720,ZMO?RS02685,ZMO?RS01435,transcriptional response regulatory proteins such as ZMO?RS05270,Yeb C/Pmp R family,ZMO?RS00645,ZMO?RS06920,ZMO?RS05215,ZMO?RS06945,ZMO?RS05270,ybe Y(ZMO?RS00305),ZMO?RS04930,ZMO?RS04435(Hsp20family protein),mutm,ZMO?RS09070,ZMO?RS03810(peptidylprolyl isomerase),ZMO?RS04910,Pgk,gpm A and ZMO?RS07905,ZMO?RS03400,ZMO?RS03395,ZMO?RS05565,ZMO?RS05445,ZMO?RS03970,ZMO?RS05445,ZMO?RS05290,ZMO?RS04295 and ZMO?RS02495 were exclusively found in ZM532 and absent ZM4.These proteins are involved in amino acid biosynthesis,macromolecules repair,glycolysis,flagella assembly,ABC transporter,fermentation,and ATP synthesis pathways and stress response.May be these proteins play key roles in ZM532 regulation with strong expressions under acids stress conditions.Hence,the effect of acids on mentioned proteins involved in these pathways are poorly studied needs to be studied their function in detail.In conclusion,ZM532 grew better than wild-type ZM4 unraveling the acetic acid and furfural tolerance mechanism between ZM532 and wild-type ZM4.4.Furthermore,we knocked-out and overexpressed two differentially expressed genes(DEGs)on the base of their highest log2 fold change,ZMO?RS02740 up-regulated and ZMO?RS06525 downregulated in RNA-Seq data to investigate their roles in acetic acid and furfural tolerance.Our knockout results demonstrated that growth activity and glucose consumption of mutant strains ZM532?ZMO?RS02740 and ZM4?ZMO?RS02740 were decreased and thus,increasing fermentation time from 42 h in ZM532 to 55 h.Ethanol production was 58 % higher in ZM532 than that of ZM532?ZMO?RS02740.However,in ZMO?RS06525 knockout in ZM4,fermentation time were shortened significantly from 60 h for ZM4 to 42 h for ZM4?ZMO?RS06525,which contributed to 45.54 %increase ethanol productivity.The results demonstrated that growth activity and glucose consumption of mutant strains and ZM4 were lower than ZM532 because mutant ZM532 has more ability to convert sugar to ethanol and to withstand toxic conditions.Further more,The results of complementary experiments revealed that the stress-resistant phenotype was restored by substituting the corresponding genes in the knockout mutants.As a result,the up-regulated expression gene ZMO RS02740 and the down-regulated expression gene ZMO-RS06525 play important roles in dealing with Furfural and acetic acid stress.In this present study,we can concluded that ZM532 can be used to substitute ZM4 as a biocatalyst for bioethanol under acetic acid and furfural condition,with a shorter fermentation time and higher productivity.Further studies may be required to clarify the relationship between the acid resistance and the genetic disparity of mutant strains.