Development and Characterization of Acidic-pH Tolerant Mutants of Zymomonas mobilis through Adaptation and Next Generation Sequencing based Genome Resequencing and RNA-Seq

Abstract

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.

Molecular Mechanism of Engineered Zymomonas Mobilis Response to Furfural and Acetic acid Stress

“…The transcriptomics results demonstrated that genes encoding ABC transporters such as ZMO_RS08315, ZMO_RS04690, ZMO_RS01550, ZMO_RS02545 and ZMO_RS01130 were signi cantly up-regulated in ZM532 (Additional le 7, 8: Table S7, S8). The up-regulation of ABC transporter genes were consistent with results from acidic-pH treatment [41,42]. The up-regulation of ABC transporters may enhance and sustain the cytoplasmic pH homeostasis in mutant strain ZM532 under AF conditions.…”

“…6C; Additional le 7, 8: Table S7, S8). The up-regulation of these genes stimulate more ATPs for acidic tolerance as established by previous reports [41,42].…”

“…Proteins associated with posttranslational alteration, protein turnover, and molecular chaperone complex responsive to stress shock could react to various stress conditions, including extreme temperature, depletion of cellular resources, concentrations of ions and toxic substances [73]. Transcriptional level of ZMO_RS02930, Novel00004 (dnaK) and ZMO_RS08760 encode GroEL protein for adaptation to acidic stress [41]. This protein (GroEL protein) was however, highly expressed in ZM532 than in ZM4.…”

“…6B; Additional le 7, 8: Table S7, S8). These may be involved in protein remodeling and reactivation [41,[76][77][78] to enhance the expression of these proteins to protect DNA and protein from damage in acidic cytoplasm. However, our transcriptomics results demonstrated that sulfur encoding genes (ZMO_RS00040, ZMO_RS06540, ZMO_RS00045 ZMO_RS03345, and ZMO_RS00025)…”

QTL Mapping of a Brazilian Bioethanol Strain Unravels the Cell Wall Protein-Encoding Gene GAS1 as a Major Contributor to Low Ph Tolerance in S. Cerevisiae

“…The combination of low pH and inhibitory by-products perturb the intracellular pH homeostasis, inducing cell-death and consequent loss of productivity [9,10]. Therefore, an additional step of pH neutralization is often required before proceeding to the fermentation process increasing the operational costs [11].…”