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. 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 in 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. 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.

中文翻译：

---

通过适应和基于下一代测序的基因组重测序和 RNA-Seq 开发和表征运动发酵单胞菌的酸性 pH 耐受突变体。

酸预处理是用于分解木质纤维素生物质的半纤维素成分以释放戊糖的常用策略，随后的酶水解步骤通常用于从纤维素中释放己糖。预处理和酶解后的水解产物含有己糖和戊糖，然后可以用作生化生产的底物。然而，酸预处理的液体也可以直接用作微生物发酵的底物，其具有酸性pH并含有在预处理过程中产生的抑制性化合物。虽然天然产乙醇细菌运动发酵单胞菌可以在 pH 3.5 ~ 7.5 的广泛范围内生长，但在低于 pH 4.0 的酸性 pH 条件下，细胞生长和乙醇发酵仍然受到影响。在这项研究中，应用适应性实验室进化 (ALE) 策略来适应酸性 pH 条件下的运动发酵单胞菌。选择并确认了 3.6M 和 3.5M 两个具有增强的酸性 pH 耐受性的突变菌株，其中 3.5M 在酸性 pH 条件下的生长优于 ZM4，但低于 3.6M，作为 3.6M 和 ZM4 之间的参考菌株帮助解开酸性-pH耐受机制。与 pH 3.8 的野生型 ZM4 相比，突变菌株 3.5M 和 3.6M 的生长速率提高了 50~130%，消耗葡萄糖的发酵时间缩短了 4~9 小时，乙醇生产率提高了 20~63%。应用基于下一代测序 (NGS) 的全基因组重测序 (WGR) 和 RNA-Seq 技术来揭示突变菌株的酸性 pH 耐受机制。WGR结果表明，与野生型ZM4、3.5M和3相比。6M 分别具有 7 个和 5 个单核苷酸多态性 (SNP)，其中 4 个是共有的。此外，RNA-Seq 结果表明，糖酵解相关基因的上调以及鞭毛和运动相关基因的下调将有助于产生和重新分配细胞能量以抵抗酸性 pH，同时保持运动发酵单胞菌的正常生物过程。此外，与 ZM4 相比，与 ZM4 相比，在酸性 pH 条件下，突变体中涉及 RND 流出泵、ATP 结合盒 (ABC) 转运蛋白、质子消耗和碱性代谢产物产生的基因显着上调，这有助于维持突变菌株的 pH 稳态用于耐酸性 pH 值。此外，我们的结果表明，在突变体 3.6M 中，与 pH 6.2 相比，编码 F1F0 ATPase 以将多余质子泵出细胞的基因在 pH 3.8 下被上调。这种差异可能有助于突变体 3.6M 比 ZM4 和 3.5M 更好地管理酸性条件。然后选择一些基因靶点进行遗传学研究，以探索它们在酸性 pH 耐受性中的作用，我们的结果表明，穿梭质粒中两个操纵子的表达，编码细胞色素 bc1 复合物的 ZMO0956-ZMO0958 和编码 RND 外排泵的 ZMO1428-ZMO1432，可以帮助运动发酵单胞菌耐受酸性 pH 条件。通过本研究获得的耐酸性 pH 突变体 3.6M 可用于酸性发酵条件下的商业生物乙醇生产。此外，进一步提出了运动发酵单胞菌耐酸性pH的分子机制，这可以促进未来对具有增强的对酸性-pH条件的耐受性的合成微生物的合理设计的研究。此外，本研究开发的策略结合了 ALE、基因组重测序、RNA-Seq 和经典遗传学研究的方法，用于突变体进化和表征，可应用于其他工业微生物。