Improvement of Thermotolerance of Zymomonas mobilis by Genes for Reactive Oxygen Species-Scavenging Enzymes and Heat Shock Proteins.

Thermotolerant genes, which are essential for survival at a high temperature, have been identified in three mesophilic microbes, including Zymomonas mobilis. Contrary to expectation, they include only a few genes for reactive oxygen species (ROS)-scavenging enzymes and heat shock proteins, which are assumed to play key roles at a critical high temperature (CHT) as an upper limit of survival. We thus examined the effects of increased expression of these genes on the cell growth of Z. mobilis strains at its CHT. When overexpressed, most of the genes increased the CHT by about one degree, and some of them enhanced tolerance against acetic acid. These findings suggest that ROS-damaged molecules or unfolded proteins that prevent cell growth are accumulated in cells at the CHT.

Sorbitol required for cell growth and ethanol production by Zymomonas mobilis under heat, ethanol, and osmotic stresses.

BACKGROUND: During ethanol fermentation, the ethanologenic bacterium, Zymomonas mobilis may encounter several environmental stresses such as heat, ethanol and osmotic stresses due to high sugar concentration. Although supplementation of the compatible solute sorbitol into culture medium enhances cell growth of Z. mobilis under osmotic stress, the protective function of this compound on cell growth and ethanol production by this organism under other stresses such as heat and ethanol has not been described yet. The formation of sorbitol in Z. mobilis was carried out by the action of the glucose-fructose oxidoreductase (GFOR) enzyme which is regulated by the gfo gene. Therefore, the gfo gene in Z. mobilis was disrupted by the fusion-PCR-based construction technique in the present study, and the protective function of sorbitol on cell growth, protein synthesis and ethanol production by Z. mobilis under heat, ethanol, and osmotic stresses was investigated. RESULTS: Based on the fusion-PCR-based construction technique, the gfo gene in Z. mobilis was disrupted. Disruption of the Z. mobilis gfo gene resulted in the reduction of cell growth and ethanol production not only under osmotic stress but also under heat and ethanol stresses. Under these stress conditions, the transcription level of pdc, adhA, and adhB genes involved in the pyruvate-to-ethanol (PE) pathway as well as the synthesis of proteins particularly in Z. mobilis disruptant strain were decreased compared to those of the parent. These findings suggest that sorbitol plays a crucial role not only on cell growth and ethanol production but also on the protection of cellular proteins from stress responses. CONCLUSION: We showed for the first time that supplementation of the compatible solute sorbitol not only promoted cell growth but also increased the ethanol fermentation capability of Z. mobilis under heat, ethanol, and osmotic stresses. Although the molecular mechanism involved in tolerance to stress conditions after sorbitol supplementation is still unclear, this research has provided useful information for the development of the effective ethanol fermentation process particularly under environmental conditions with high temperature or high ethanol and sugar concentration conditions.

Physiological importance of cytochrome c peroxidase in ethanologenic thermotolerant Zymomonas mobilis.

Zymomonas mobilis ZmCytC as a peroxidase bearing three heme c-binding motifs was investigated with ΔZmcytC constructed. The mutant exhibited filamentous shapes and reduction in growth under a shaking condition at a high temperature compared to the parental strain and became hypersensitive to exogenous H(2)O(2). Under the same condition, the mutation caused increased expression of genes for three other antioxidant enzymes. Peroxidase activity, which was detected in membrane fractions with ubiquinol-1 as a substrate but not with reduced horse heart cytochrome c, was almost abolished in ΔZmcytC. Peroxidase activity was also detected with NADH as a substrate, which was significantly inhibited by antimycin A. NADH oxidase activity of ΔZmcytC was found to be about 80% of that of the parental strain. The results suggest the involvement of ZmCytC in the aerobic respiratory chain via the cytochrome bc(1) complex in addition to the previously proposed direct interaction with ubiquinol and its contribution to protection against oxidative stress.

Analysis of the respiratory chain in Ethanologenic Zymomonas mobilis with a cyanide-resistant bd-type ubiquinol oxidase as the only terminal oxidase and its possible physiological roles.

The respiratory chain of the ethanologenic bacterium Zymomonas mobilis was investigated, in which the pyruvate-to-ethanol pathway has been demonstrated to be mainly responsible for NADH oxidation and the tricarboxylic acid cycle is incomplete. Membranes from cells cultivated under aerobic or anaerobic growth conditions showed dehydrogenase and oxidase activities for NADH, D-lactate and D-glucose and ubiquinol oxidase activity. Intriguingly, the NADH oxidase activity level of membrane fractions from cells grown aerobically was found to be higher than that of membrane fractions from Escherichia coli or Pseudomonas putida grown aerobically, indicating a crucial role of the respiratory chain in NADH oxidation in the organism. Cyanide-resistant terminal oxidase activity was observed and appeared to be due to a bd-type ubiquinol oxidase as the only terminal oxidase encoded by the entire genome. The terminal oxidase with a relatively strong ubiquinol oxidase activity exhibited remarkably weak signals of cytochrome d. Considering these findings and the presence of a type-II NADH dehydrogenase but not a type-I, a simple respiratory chain that generates less energymay have evolved in Z. mobilis.

Comparison of the gene expression patterns of alcohol dehydrogenase isozymes in the thermotolerant yeast Kluyveromyces marxianus and their physiological functions.

Four genes encoding alcohol dehydrogenase (Adh) isozymes in the thermotolerant yeast Kluyveromyces marxianus, a potent candidate for ethanol production at high temperatures, were investigated. Of these, KmADH3 and KmADH4 were cloned and sequenced, and their deduced amino acid sequences were compared with those of KmAdh1 and KmAdh2 and other Adhs of Kluyveromyces lactis and Saccharomyces cerevisiae. The four KmAdhs had high sequence similarity, though KmAdh3 and KmAdh4 possessed an amino-terminal extension as a mitochondrial targeting sequence, and appear to belong to the zinc-containing Adh family. These results and the results of Southern blot experiments suggest that there are at least four Adh isozymes in K. marxianus, two cytoplasmic enzymes and two mitochondrial enzymes. The expression profile revealed that KmADH genes are differently expressed depending on growth phase and carbon source, suggesting that these highly homologous Adhs play distinctive roles in cells.

Cloning and transcriptional analysis of groES and groEL in ethanol-producing bacterium Zymomonas mobilis TISTR 548.

Heat and ethanol had an affect not only on growth and cell viability of an obligatorily fermentative Gram-negative bacterium Zymomonas mobilis, but also on protein synthesis. Analysis by SDS-polyacrylamide gel electrophoresis revealed pronounced increasing of two dominant proteins designated as groES and groEL. Molecular cloning of the gene encoding groES and groEL was performed by PCR technique using specific primers synthesized based on the Z. mobilis groESL gene. Sequencing analysis of 2179 bp led to the detection of two open reading frames encoded for 95 and 549 amino acids, respectively. The deduced amino acid sequence of the Z. mobilis groES and groEL shows a high degree of identity with other. The strongly conserved carboxyl-terminus Gly-Gly-Met motif and two small segments, which appear more conserved between ethanol-producing organisms, were found, suggesting that their may be related to stability of protein under heat or ethanol stress. Induction of groES and groEL occurs in response to heat and ethanol, but not to salt stress.