Metabolic Engineering of Bacterial Respiration: High vs. Low P/O and the Case of Zymomonas mobilis

Kalnenieks, Uldis; Balodite, Elina; Rutkis, Reinis

doi:10.3389/fbioe.2019.00327

“…In addition, the upregulated expression of cytochrome bc1 complex impacted the growth in neutral pH condition (Fig. 6B), which may indicate a potential role of low expression of this "dead-end" respiration branch in cell growth, since the function of cytochrome bc1 in electron transport is still unknown in Z. mobilis [89]. Similarly, with the increase of tetracycline inducer concentration from 0 to 0.8 μg/mL, the growth advantage of recombinant strain containing pEZ-Tc5(M) decreased in acidic-pH condition, but there was still an advantage with 0.8 μg/mL tetracycline (Fig.…”

Section: Resultsmentioning

confidence: 98%

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

Yang

¹

,

Yang

²

,

Tang

³

et al. 2020

Preprint

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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.

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“…Another fundamental function of the cytoplasmic membrane is cellular respiration. The respiratory chain plays an important role in the energetic metabolism of a cell, the maintenance of intracellular redox balance, or the protection against oxidative stress [ 55 ]. The membranes harbor the electron transport chain, a well-known system with oxygen as the final electron acceptor in aerobic respiration processes.…”

Section: Resultsmentioning

confidence: 99%

Antimicrobial Activity and Mode of Action of Celastrol, a Nortriterpen Quinone Isolated from Natural Sources

Padilla-Montaño

¹

,

Guerra

²

,

Moujir

³

2021

Foods

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Species of the Celastraceae family are traditionally consumed in different world regions for their stimulating properties. Celastrol, a triterpene methylene quinone isolated from plants of celastraceas, specifically activates satiety centers in the brain that play an important role in controlling body weight. In this work, the antimicrobial activity and mechanism of action of celastrol and a natural derivative, pristimerin, were investigated in Bacillus subtilis. Celastrol showed a higher antimicrobial activity compared with pristimerin, being active against Gram-positive bacteria with minimum inhibitory concentrations (MICs) that ranged between 0.16 and 2.5 µg/mL. Killing curves displayed a bactericidal effect that was dependent on the inoculum size. Monitoring of macromolecular synthesis in bacterial populations treated with these compounds revealed inhibition in the incorporation of all radiolabeled precursors, but not simultaneously. Celastrol at 3 µg/mL and pristimerin at 10 µg/mL affected DNA and RNA synthesis first, followed by protein synthesis, although the inhibitory action on the uptake of radiolabeled precursors was more dramatic with celastrol. This compound also caused cytoplasmic membrane disruption observed by potassium leakage and formation of mesosome-like structures. The inhibition of oxygen consumption of whole and disrupted cells after treatments with both quinones indicates damage in the cellular structure, suggesting the cytoplasmic membrane as a potential target. These findings indicate that celastrol could be considered as an interesting alternative to control outbreaks caused by spore-forming bacteria.

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“…In addition, the upregulated expression of cytochrome bc1 complex impacted the growth in neutral pH condition (Fig. 6 b), which may indicate a potential role of low expression of this “dead-end” respiration branch in cell growth, since the function of cytochrome bc1 in electron transport is still unknown in Z. mobilis [ 88 ]. Similarly, with the increase of tetracycline inducer concentration from 0 to 0.8 μg/mL, the growth advantage of recombinant strain containing pEZ-Tc5(M) decreased in acidic-pH condition, but there was still an advantage with 0.8 μg/mL tetracycline (Fig.…”

Section: Resultsmentioning

confidence: 99%

Development and characterization of acidic-pH-tolerant mutants of Zymomonas mobilis through adaptation and next-generation sequencing-based genome resequencing and RNA-Seq

Yang

¹

,

Yang

²

,

Tang

³

et al. 2020

Biotechnol Biofuels

View full text Add to dashboard Cite

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

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Metabolic Engineering of Bacterial Respiration: High vs. Low P/O and the Case of Zymomonas mobilis

Cited by 25 publications

References 115 publications

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

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

Antimicrobial Activity and Mode of Action of Celastrol, a Nortriterpen Quinone Isolated from Natural Sources

Development and characterization of acidic-pH-tolerant mutants of Zymomonas mobilis through adaptation and next-generation sequencing-based genome resequencing and RNA-Seq

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