2H and 13C metabolic flux analysis elucidates in vivo thermodynamics of the ED pathway in Zymomonas mobilis

Jacobson, Tyler B.; Adamczyk, Paul; Stevenson, David; Regner, Matthew; Ralph, John; Reed, Jennifer L.; Amador‐Noguez, Daniel

doi:10.1016/j.ymben.2019.05.006

Cited by 52 publications

(60 citation statements)

References 81 publications

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“…Energy generation through increased glycolysis and energy conservation through decreased cell motility for acidic-pH resistance: It is reported that Streptococcus mutans altered its metabolism by increasing the glycolytic activity to produce ATP at acidic-pH conditions [64,65], and ATP utilization was further derived from cell growth for acid tolerance [66]. Although the Entner-Doudoroff (ED) pathway only produces one mole of ATP per single mole of glucose, it is reported that the ED pathway in Z. mobilis is nearly twice as thermodynamically favorable as the Embden-Meyerhof-Parnas (EMP) pathway in E. coli or S. cerevisiae [67]. Our RNA-Seq results demonstrated that four genes involved in the glycolysis pathway, ZMO1478 (pgl), ZMO1240 (gpmA), ZMO1596 (adhB), and ZMO1236 (adhA), were signi cantly upregulated at the acidic pH 3.8 compared to a neutral pH 6.2 in ZM4 and 3.6M.…”

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

Tang

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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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

Tang

et al. 2020

Preprint

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“…cerevisiae [72]. Our RNA-Seq result demonstrated that 6 genes involved in glycolysis pathway (ZMO1478 (pgl), ZMO0997 (eda), ZMO0177 (gap), ZMO1240 (gpmA), ZMO1608 (eno) and ZMO0152 (pyk)) were significantly upregulated at acidic pH compared with neutral pH in ZM4 and 3.6M, which could help produce more ATP for acidic pH tolerance ( Fig.…”

Section: Energy Generation Through Increased Glycolysis and Energy Comentioning

confidence: 77%

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

Yang

Wang

et al. 2020

Preprint

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Background: Acid pretreatment is a common strategy used to break down lignocellulosic biomass as substrate for biochemical production, which however generates inhibitory compounds and results in acidic pH condition. Although the natural ethanologenic bacterium Zymomonas mobilis can grow in a broad pH range, cell growth and ethanol fermentation are still affected at 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 condition. Two mutant strains named 3.6M and 3.5M with enhanced acidic-pH tolerance were selected and confirmed. Mutant strains 3.6M and 3.5M exhibited 50~130% enhancement on growth rate, 4~9 h reduction on fermentation time, 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 mutations in four genes ZMO0421 (Ala67Thr), ZMO0712 (Gly539Asp), ZMO1432 (Pro480Leu), and ZMO1733 (Thr7Lys) with non-synonymous amino acid changes might be related with the acidic-pH tolerance. Additionally, RNA-Seq result showed that the upregulation of genes involved in glycolysis and the downregulation of mobility related genes would help generate and redistribute cellular energy to help resist acidic pH while keep 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 acidic condition compared with ZM4, which could help maintain the pH homeostasis in mutant strains for low acidic-pH resistance. Furthermore, our results also demonstrated that genes related to branch amino acid biosynthesis from threonine to isoleucine were significantly upregulated in mutant 3.6M under acidic condition compared with ZM4, and genes encoding F 1 F 0 ATPase to pump excess protons out of cells were upregulated in mutant 3.6M under pH 3.8 compared with pH 6.2. These differences could help mutant 3.6M manage acidic condition better than ZM4. A few gene targets were then selected for genetics study to confirm their role on acidic pH tolerance, and our results demonstrated that the expression of two operons in the shuttle plasmids could help Z. mobilis tolerate acidic pH condition, which are ZMO0956-ZMO0958 encoding cytochrome bc1 complex and ZMO1428-ZMO1432 encoding RND efflux pump.Conclusion: Two acidic-pH tolerant mutants 3.6M and 3.5M obtained through this study especially 3.6M can be used as candidate strains for commercial bioethanol production under acidic fermentation conditions, and 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. In addition, 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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“…2C). We did not detect Pyr labeled at the first and second carbons (i.e., [1,2-13 C 2 ]Pyr), which would have been indicative of ED pathway activity (27). Finally, as shown in Table S1 in the supplemental material, high intracellular levels of the EMP intermediates FBP and dihydroxyactone phosphate (DHAP), which were comparable to those in anaerobic E. coli, a known EMP-utilizing organism, together with nondetectable levels of the ED pathway intermediates 6PG and KDPG, which are present in the ED pathway, utilizing organisms such as Z. mobilis, was consistent with the existence of an active EMP pathway and an inactive ED pathway in T. saccharolyticum (27,44).…”

mentioning

confidence: 76%

“…2A and B). If the ED pathway was the exclusive glycolytic pathway, 3PG and PEP would have been 100% M ϩ 0 in [1- 13 C 1 ]glucose or 100% M ϩ 1 in [6][7][8][9][10][11][12][13] C 1 ]glucose (27). If both the EMP and ED pathways were active, labeling of 3PG and PEP would have been Ͻ50% M ϩ 1 in [1- 13 activity, when T. saccharolyticum was fed [1,2-13 C 2 ]glucose, Pyr was labeled only at the second and third carbon (i.e., [2,[3][4][5][6][7][8][9][10][11][12][13] C 2 ]Pyr), consistent with its production solely via the EMP pathway ( Fig.…”

mentioning

confidence: 99%

In VivoThermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using¹³C and²H Tracers

et al. 2020

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Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are thermophilic anaerobic bacteria with complementary metabolic capabilities that utilize distinct glycolytic pathways for the conversion of cellulosic sugars to biofuels. We integrated quantitative metabolomics with 2H and 13C metabolic flux analysis to investigate the in vivo reversibility and thermodynamics of the central metabolic networks of these two microbes. We found that the glycolytic pathway in C. thermocellum operates remarkably close to thermodynamic equilibrium, with an overall drop in Gibbs free energy 5-fold lower than that of T. saccharolyticum or anaerobically grown Escherichia coli. The limited thermodynamic driving force of glycolysis in C. thermocellum could be attributed in large part to the small free energy of the phosphofructokinase reaction producing fructose bisphosphate. The ethanol fermentation pathway was also substantially more reversible in C. thermocellum than in T. saccharolyticum. These observations help explain the comparatively low ethanol titers of C. thermocellum and suggest engineering interventions that can be used to increase its ethanol productivity and glycolytic rate. In addition to thermodynamic analysis, we used our isotope tracer data to reconstruct the T. saccharolyticum central metabolic network, revealing exclusive use of the Embden-Meyerhof-Parnas (EMP) pathway for glycolysis, a bifurcated tricarboxylic acid (TCA) cycle, and a sedoheptulose bisphosphate bypass active within the pentose phosphate pathway. IMPORTANCE Thermodynamics constitutes a key determinant of flux and enzyme efficiency in metabolic networks. Here, we provide new insights into the divergent thermodynamics of the glycolytic pathways of C. thermocellum and T. saccharolyticum, two industrially relevant thermophilic bacteria whose metabolism still is not well understood. We report that while the glycolytic pathway in T. saccharolyticum is as thermodynamically favorable as that found in model organisms, such as E. coli or Saccharomyces cerevisiae, the glycolytic pathway of C. thermocellum operates near equilibrium. The use of a near-equilibrium glycolytic pathway, with potentially increased ATP yield, by this cellulolytic microbe may represent an evolutionary adaptation to growth on cellulose, but it has the drawback of being highly susceptible to product feedback inhibition. The results of this study will facilitate future engineering of high-performance strains capable of transforming cellulosic biomass to biofuels at high yields and titers.

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2H and 13C metabolic flux analysis elucidates in vivo thermodynamics of the ED pathway in Zymomonas mobilis

Cited by 52 publications

References 81 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

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

In VivoThermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using¹³C and²H Tracers

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2H and 13C metabolic flux analysis elucidates in vivo thermodynamics of the ED pathway in Zymomonas mobilis

Cited by 52 publications

References 81 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

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

In VivoThermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using13C and2H Tracers

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In VivoThermodynamic Analysis of Glycolysis in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Using¹³C and²H Tracers