Development And Characterization Of Efficient Xylose Utilization Strains Of Zymomonas Mobilis

Lignocellulosic biomass contains three main components of cellulose,hemicellulose,and lignin.The efficient use of glucose in cellulose and xylose in hemicellulose is the key to the economic production of biofuels and biochemical products.The ethanologenic bacterium Zymomonas mobilis can efficiently ferment glucose into ethanol,but it cannot utilize pentoses in the hemicellulose.Although recombinant strains 8b which have successfully constructed by engineering xylose isomerase and xylulose kinase(Xyl A,B)as well as two enzymes(Tal,Tkt)of pentose phosphate pathway(PPP)of E.coli into Z.mobilis ZM4 to utilize pentose and achieved significant progress,there is still a need to increase the efficiency of xylose utilization.In this work,the strategy of combining metabolic engineering with adaptive laboratory evolution(ALE)was employed to develop recombinant Z.mobilis strains that can utilize xylose efficiently at high xylose concentrations.We first expressed heterologous xylose isomerase and xylulose kinase in Z.mobilis ZM4,and found that the strains can grow with xylose.We then tried to express these two enzymes in xyloseutilizing recombinant strain Z.mobilis 8b to enhance its capacity of xylose utilization.The results showed that xylose utilization capacity of the recombinant strain 8b-Rs XIxyl B was enhanced,and the maximum OD was 1.40 times of that of the control strain,which indicates that the increase of copy number of xylose metabolism genes can improve xylose utilization.After continuous adaption over 100 days with 38 transfers of strain 8b-Rs XI-xyl B in medium containing 50 g/L xylose,a series of strains with improved xylose utilization were acquired,and the one with highest xylose-utilization was named as 8b-S38.The fermentation experiment showed that the xylose consumption rate of 8b-S38 was accelerated compared with that of 8b in medium containing different concentrations of xylose ranging from 50 g/L to 150 g/L,and ethanol production was also increased by 16?40%.When the xylose concentrations in the medium reached 50 g/L and 100 g/L,only 8b-S38 can completely consume xylose.Mixed sugar fermentation test results show that 8b-S38 has a higher xylose consumption rate than 8b,and its maximum ethanol production rate is 1.2-1.4 times higher than 8b and 8b-S8.Combining next-generation sequencing(NGS)-based approaches of genome resequencing and RNA-Seq transcriptomics,we explored the molecular mechanism of efficient xylose utilization by investigating the differences of 8b,8b-S8,and 8b-S38 at the gene and transcriptional levels in media containing the substrate of glucose or xylose.The whole-genome sequencing results showed that there are three common genetic changes between 8b and improved xylose-utilization mutants of 8b-S8 and 8bS38,which include two synonymous changes and one genetic change of Ala812 Thr in the plasmid gene p ZYM39?027.In addition,there are two other changes in 8b-S38 including a synonymous change in ZMO0975 and a Glu207* change in Dna J encoded by gene ZMO0661.Besides the changes at genomic level,the expression of p ZYM39?027 was also significantly upregulated when these strains were grown in media containing xylose compared to that in RMG especially for mutant strain 8b-S38 in medium containing a high concentration of xylose.Although further investigation is still needed to understand the specific mechanism,the overexpression of p ZYM39?027 encoding autotransporter probably can facilitate the production of bacterial adhesin to promote the formation of biofilm,and therefore help bacteria resist environmental pressures.Our transcriptomics study also demonstrated that the expression levels of genes encoding Gro EL/ES chaperone proteins,ATP-dependent protease Hsl U and Hsl V,phage shock proteins,Ton B-dependent receptor proteins,and ABC transporter proteins were significantly increased in xylose medium for the mutant strain 8b-S38.The upregulated expression of these genes helps repair misfolded proteins and enhance their resistance to external stresses,while at the same time enhance the transportation of xylose and other substances.This may therefore contribute to the efficient xylose utilization of mutant strain 8b-S38 by maintaining the normal cell metabolism and growth,repairing cellular damages,preserving the redox balance,and thus help resist the pressure caused by the external environment and maintain the homeostasis of the cells for efficient xylose utilization and normal cell growth.In summary,this work demonstrated that the introduction of additional xylose isomerase and xylulose kinase can enhance the xylose utilization.Combining the approaches of metabolic engineering and adaptive laboratory evolution,we obtained a series of mutant strains of Z.mobilis that can efficiently utilize xylose.Of which,8bS38 was the best among these mutants that can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations,which has practical significance for the production of cellulosic biofuels and biochemicals.In addition,we also revealed the molecular mechanism of efficient xylose utilization through NGS-based genome resequencing and RNA-Seq,which lays a solid foundation and provides genetic candidates for subsequent genetic engineering and the construction of synthetic cell factories for efficient and effective xylose utilization in the future.