Study On The Furfural Tolerance Mechanism Of Zymomonas Mobilis F211

Bioethanol production from lignocellulose is a hot research topic in renewable energy development.However,inhibitors generated during lignocellulose pretreatment are toxic to fermentation strains.Removal of inhibitors will increase fermentation costs,and it is a more economical way to improve the tolerance of strains to inhibitors.Therefore,it is necessary to explore the tolerance mechanism of fermentation strains to lignocellulose hydrolysis inhibitors and this will provide a theoretical basis for the improvement of strain tolerance.Furfural is a major inhibitor in lignocellulosic hydrolysate.In the early stage of our laboratory,a high furfural tolerance Z.mobilis strain F211 was obtained by error-PCR whole-genome shuffling and resequenced.In this study,the deletion and overexpression strains of the mutant genes in the F211 were constructed to explore the furfural tolerance function of the mutant gene through gene knockout/overexpression complemental experiments.We found that mutation of ZCP4＿0270,encoding di-guanylate cyclase/phosphodiesterase,and mutation of ZCP4＿0970,encoding Tol C outer membrane protein,could improve the furfural tolerance of Z.mobilis.The flocculation rate of F211 and ZCP4＿0270 mutant gene overexpression strain Δ0270(p HW-M0270)was 600% and470% higher than that of the control CP4,respectively,indicating that the ZCP4＿0270gene mutation gene mutation can improve furfurl tolerance by causing cell flocculation.The mutation of ZCP4＿0970 gene leads to the inactivation of the protein encoded by this gene,and the inactivation of this protein can improve furfural tolerance.The RNA-Seq experiment was used to explore the transcriptome differences between F211 and the starting strain CP4 under furfural stress.A total of 139 differentially expressed genes were revealed,of which 77 were up-regulated and 62 were down-regulated.Through transcriptome analysis and function complementation experiments,the potential furfural tolerance mechanism of F211 is revealed: down-regulation of genes related to sulfate assimilation pathway and up-regulation of sulfite exporter gene both can reduce NAD(P)H consumption to provide reducing power for furfural detoxification and further improve furfural tolerance;down-regulation of the genes related to energy-consuming efflux pump reduces energy consumption and provides energy for cell growth;up-regulation of succinate semialdehyde dehydrogenase gene promotes the regeneration of NAD(P)H and enhances furfural detoxification;up-regulation of stress protein genes strengthens the response of the stress system and resists furfural stress.This study provides a new theoretical basis for the rational design of highly furfural-tolerant industrial strains.