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

Investigation of the impact of a broad range of temperatures on the physiological and transcriptional profiles of Zymomonas mobilis ZM4 for high-temperature-tolerant recombinant strain development

“…The transcriptomics study was followed previous work [ 37 , 42 – 49 ]. Briefly, cell culture samples at different temperatures were collected during the mid-exponential phase followed by total RNA extraction and rRNA depletion using TRIzol reagent (Invitrogen, USA) and Ribo-Zero rRNA Removal Kit (Illumina, USA), respectively.…”

Revitalizing the ethanologenic bacterium Zymomonas mobilis for sugar reduction in high-sugar-content fruits and commercial products

“…mobilis ZM4 (ATCC 31821) (Seo et al 2005 ) and Saccharomyces cerevisiae BY4743 were used in this study. Z. mobilis ZM4 was cultured in Rich Medium (RM: 10 g/L yeast extract, 2 g/L KH 2 PO 4 , with different concentration of glucose or fructose, pH 5.8) at 30 ℃ without shaking as previously described (Yang et al 2020 ). The sugars used in RMG5, RMG10, RMF5.5, RMF11, RMG5F5.5, and RMG10F11 were 50 g/L glucose, 100 g/L glucose, 55 g/L fructose, 110 g/L fructose, 50 g/L glucose and 55 g/L fructose, 100 g/L glucose and 110 g/L fructose, respectively.…”

Metabolic Remodeling during Nitrogen Fixation in Zymomonas mobilis

“…Bacterial flagellar motors are powered by the translocation of ions across the inner membrane ( 43 , 44 ). Others have observed decreased expression of flagellar genes in Z. mobilis under stress conditions that disrupt the maintenance of an electrochemical gradient across the membrane, such as low pH, osmotic stress, and high ethanol concentrations ( 45 – 47 ). This is in contrast to other environmental inhibitors such as oxygen and lignocellulosic toxins, which have been shown to increase expression of motility-related genes in Z. mobilis ( 48 – 50 ).…”