Background: Acid pretreatment is a common strategy used to break down the hemicellulose component of the lignocellulosic biomass to release pentoses, and a subsequent enzymatic hydrolysis step is usually applied to release hexoses from the cellulose. The hydrolysate after pretreatment and enzymatic hydrolysis containing both hexoses and pentoses can then be used as substrates for biochemical production. However, the acid-pretreated liquor can also be directly used as the substrate for microbial fermentation, which has an acidic pH and contains inhibitory compounds generated during pretreatment. Although the natural ethanologenic bacterium Zymomonas mobilis can grow in a broad range of pH 3.5~7.5, cell growth and ethanol fermentation are still affected under acidic-pH conditions below pH 4.0. Results: In this study, adaptive laboratory evolution (ALE) strategy was applied to adapt Z. mobilis under acidic-pH conditions. Two mutant strains named 3.6M and 3.5M with enhanced acidic-pH tolerance were selected and confirmed, of which 3.5M grew better than ZM4 but worse than 3.6M in acidic-pH conditions that is served as a reference strain between 3.6M and ZM4 to help unravel the acidic-pH tolerance mechanism. Mutant strains 3.5M and 3.6M exhibited 50~130% enhancement on growth rate, 4~9 h reduction on fermentation time to consume glucose, and 20~63% improvement on ethanol productivity than wild-type ZM4 at pH 3.8. Next-generation sequencing (NGS)-based whole genome resequencing (WGR) and RNA-Seq technologies were applied to unravel the acidic-pH tolerance mechanism of mutant strains. WGR result indicated that compared to wild-type ZM4, 3.5M and 3.6M have seven and five single nucleotide polymorphisms (SNPs) respectively, among which four are shared in common. Additionally, RNA-Seq result showed that the upregulation of genes involved in glycolysis and the downregulation of flagellar and mobility related genes would help generate and redistribute cellular energy to resist acidic pH while keeping normal biological processes in Z. mobilis. Moreover, genes involved in RND efflux pump, ATP-binding cassette (ABC) transporter, proton consumption, and alkaline metabolite production were significantly upregulated in mutants under the acidic-pH condition compared with ZM4, which could help maintain the pH homeostasis in mutant strains for acidic-pH resistance. Furthermore, our results demonstrated that in mutant 3.6M, genes encoding F1F0 ATPase to pump excess protons out of cells were upregulated under pH 3.8 compared to pH 6.2. This difference might help mutant 3.6M manage acidic conditions better than ZM4 and 3.5M. A few gene targets were then selected for genetics study to explore their role on acidic-pH tolerance, and our results demonstrated that the expression of two operons in the shuttle plasmids, ZMO0956-ZMO0958 encoding cytochrome bc1 complex and ZMO1428-ZMO1432 encoding RND efflux pump, could help Z. mobilis tolerate acidic-pH conditions. Conclusion: An acidic-pH tolerant mutant 3.6M obtained through this study can be used for commercial bioethanol production under acidic fermentation conditions. In addition, the molecular mechanism of acidic-pH tolerance of Z. mobilis was further proposed, which can facilitate future research on rational design of synthetic microorganisms with enhanced tolerance against acidic-pH conditions. Moreover, the strategy developed in this study combining approaches of ALE, genome resequencing, RNA-Seq, and classical genetics study for mutant evolution and characterization can be applied in other industrial microorganisms.

Background: Acid pretreatment is a common strategy used to break down lignocellulosic biomass as substrate for biochemical production, which however generates inhibitory compounds and results in acidic pH condition. Although the natural ethanologenic bacterium Zymomonas mobilis can grow in a broad pH range, cell growth and ethanol fermentation are still affected at acidic pH conditions below pH 4.0.Results: In this study, adaptive laboratory evolution (ALE) strategy was applied to adapt Z. mobilis under acidic pH condition. Two mutant strains named 3.6M and 3.5M with enhanced acidic-pH tolerance were selected and confirmed. Mutant strains 3.6M and 3.5M exhibited 50~130% enhancement on growth rate, 4~9 h reduction on fermentation time, and 20~63% improvement on ethanol productivity than wild-type ZM4 at pH 3.8. Next-generation sequencing (NGS)-based whole genome resequencing (WGR) and RNA-Seq technologies were applied to unravel the acidic pH tolerance mechanism of mutant strains. WGR result indicated that mutations in four genes ZMO0421 (Ala67Thr), ZMO0712 (Gly539Asp), ZMO1432 (Pro480Leu), and ZMO1733 (Thr7Lys) with non-synonymous amino acid changes might be related with the acidic-pH tolerance. Additionally, RNA-Seq result showed that the upregulation of genes involved in glycolysis and the downregulation of mobility related genes would help generate and redistribute cellular energy to help resist acidic pH while keep normal biological processes in Z. mobilis . Moreover, genes involved in RND efflux pump, ATP-binding cassette (ABC) transporter, proton consumption, and alkaline metabolite production were significantly upregulated in mutants under acidic condition compared with ZM4, which could help maintain the pH homeostasis in mutant strains for low acidic-pH resistance. Furthermore, our results also demonstrated that genes related to branch amino acid biosynthesis from threonine to isoleucine were significantly upregulated in mutant 3.6M under acidic condition compared with ZM4, and genes encoding F 1 F 0 ATPase to pump excess protons out of cells were upregulated in mutant 3.6M under pH 3.8 compared with pH 6.2. These differences could help mutant 3.6M manage acidic condition better than ZM4. A few gene targets were then selected for genetics study to confirm their role on acidic pH tolerance, and our results demonstrated that the expression of two operons in the shuttle plasmids could help Z. mobilis tolerate acidic pH condition, which are ZMO0956-ZMO0958 encoding cytochrome bc1 complex and ZMO1428-ZMO1432 encoding RND efflux pump.Conclusion: Two acidic-pH tolerant mutants 3.6M and 3.5M obtained through this study especially 3.6M can be used as candidate strains for commercial bioethanol production under acidic fermentation conditions, and molecular mechanism of acidic pH tolerance of Z. mobilis \was further proposed, which can facilitate future research on rational design of synthetic microorganisms with enhanced tolerance against acidic pH conditions. In addition, the strategy developed in this study combining approaches of ALE, genome resequencing, RNA-Seq and classical genetics study for mutant evolution and characterization can be applied in other industrial microorganisms.