Ethanologenic Bacterium Zymomonas Mobilis Research Articles

Efficient genome-editing tools to engineer the recalcitrant non-model industrial microorganism Zymomonas mobilis

Current biotechnology relies on a few well-studied model organisms, such as Escherichia coli and Saccharomyces cerevisiae, for which abundant information and efficient toolkits are available for genetic manipulation, but which lack industrially favorable characteristics. Non-model industrial microorganisms usually do not have effective and/or efficient genome-engineering toolkits, which hampers the development of microbial cell factories to meet the fast-growing bioeconomy. In this study, using the non-model ethanologenic bacterium Zymomonas mobilis as an example, we developed a workflow to mine and temper the elements of restriction–modification (R-M), CRISPR/Cas, toxin–antitoxin (T-A) systems, and native plasmids, which are hidden within industrial microorganisms themselves, as efficient genome-editing toolkits, and established a genome-wide iterative and continuous editing (GW-ICE) system for continuous genome editing with high efficiency. This research not only provides tools and pipelines for engineering the non-model polyploid industrial microorganism Z. mobilis efficiently, but also sets a paradigm to overcome biotechnological limitations in other genetically recalcitrant non-model industrial microorganisms.

Eminent Antimicrobial Peptide Resistance in Zymomonas mobilis: A Novel Advantage of Intrinsically Uncoupled Energetics

Relative to several model bacteria, the ethanologenic bacterium Zymomonas mobilis is shown here to have elevated resistance to exogenous antimicrobial peptides (AMPs)— with regard to both peptide bulk concentration in the medium and the numbers of peptide molecules per cell. By monitoring the integration of AMPs in the bacterial cell membrane and observing the resulting effect on membrane energy coupling, it is concluded that the membranotropic effects of the tested AMPs in Z. mobilis and in Escherichia coli are comparable. The advantage of Z. mobilis over E. coli apparently results from its uncoupled mode of energy metabolism that, in contrast to E. coli, does not rely on oxidative phosphorylation, and hence, is less vulnerable to the disruption of its energy-coupling membrane by AMPs. It is concluded that the high resistance to antimicrobial peptides (AMPs) observed in Z. mobilis not only proves crucial for its survival in its natural environment but also offers a promising platform for AMP production and sheds light on potential strategies for novel resistance development in clinical settings.

Metabolic engineering of an industrial bacterium Zymomonas mobilis for anaerobic l-serine production

Due to the complicated metabolic and regulatory networks of l-serine biosynthesis and degradation, microbial cell factories for l-serine production using non-model microorganisms have not been reported. In this study, a combination of synthetic biology and process optimization were applied in an ethanologenic bacterium Zymomonas mobilis for l-serine production. By blocking the degradation pathway while introducing an exporter EceamA from E. coli, l-serine titer in recombinant Z. mobilis was increased from 15.30 mg/L to 62.67 mg/L. It was further increased to 260.33 mg/L after enhancing the l-serine biosynthesis pathway. Then, 536.70 mg/L l-serine was achieved by removing feedback inhibition with a SerA mutant, and an elevated titer of 687.67 mg/L was further obtained through increasing serB copies while enhancing the precursors. Finally, 855.66 mg/L l-serine can be accumulated with the supplementation of the glutamate precursor. This work thus not only constructed an l-serine producer to help understand the bottlenecks limiting l-serine production in Z. mobilis for further improvement, but also provides guidance on engineering non-model microbes to produce biochemicals with complicated pathways such as amino acids or terpenoids.

Lipid membrane remodeling and metabolic response during isobutanol and ethanol exposure in Zymomonas mobilis

BackgroundRecent engineering efforts have targeted the ethanologenic bacterium Zymomonas mobilis for isobutanol production. However, significant hurdles remain due this organism’s vulnerability to isobutanol toxicity, adversely affecting its growth and productivity. The limited understanding of the physiological impacts of isobutanol on Z. mobilis constrains our ability to overcome these production barriers.ResultsWe utilized a systems-level approach comprising LC–MS/MS-based lipidomics, metabolomics, and shotgun proteomics, to investigate how exposure to ethanol and isobutanol impact the lipid membrane composition and overall physiology of Z. mobilis. Our analysis revealed significant and distinct alterations in membrane phospholipid and fatty acid composition resulting from ethanol and isobutanol exposure. Notably, ethanol exposure increased membrane cyclopropane fatty acid content and expression of cyclopropane fatty acid (CFA) synthase. Surprisingly, isobutanol decreased cyclopropane fatty acid content despite robust upregulation of CFA synthase. Overexpression of the native Z. mobilis’ CFA synthase increased cyclopropane fatty acid content in all phospholipid classes and was associated with a significant improvement in growth rates in the presence of added ethanol and isobutanol. Heterologous expression of CFA synthase from Clostridium acetobutylicum resulted in a near complete replacement of unsaturated fatty acids with cyclopropane fatty acids, affecting all lipid classes. However, this did not translate to improved growth rates under isobutanol exposure.Correlating with its greater susceptibility to isobutanol, Z. mobilis exhibited more pronounced alterations in its proteome, metabolome, and overall cell morphology—including cell swelling and formation of intracellular protein aggregates —when exposed to isobutanol compared to ethanol. Isobutanol triggered a broad stress response marked by the upregulation of heat shock proteins, efflux transporters, DNA repair systems, and the downregulation of cell motility proteins. Isobutanol also elicited widespread dysregulation of Z. mobilis’ primary metabolism evidenced by increased levels of nucleotide degradation intermediates and the depletion of biosynthetic and glycolytic intermediates.ConclusionsThis study provides a comprehensive, systems-level evaluation of the impact of ethanol and isobutanol exposure on the lipid membrane composition and overall physiology of Z. mobilis. These findings will guide engineering of Z. mobilis towards the creation of isobutanol-tolerant strains that can serve as robust platforms for the industrial production of isobutanol from lignocellulosic sugars.

Adaptive Laboratory Evolution and Metabolic Engineering of Zymomonas mobilis for Bioethanol Production Using Molasses.

Molasses with abundant sugars is widely used for bioethanol production. Although the ethanologenic bacterium Zymomonas mobilis can use glucose, fructose, and sucrose for ethanol production, levan production from sucrose reduces the ethanol yield of molasses fermentation. To increase ethanol production from sucrose-rich molasses, Z. mobilis was adapted in molasses, sucrose, and fructose in parallel. Adaptation in fructose is the most effective route to generate an evolved strain F74 with improved molasses utilization, which is majorly due to a G99S mutation in Glf for enhanced fructose import. Subsequent sacB deletion and sacC overexpression in F74 to divert sucrose metabolism from levan production to ethanol production further enhanced ethanol productivity 28.6% to 1.35 g/L/h. The efficient utilization of molasses by diverting sucrose metabolic flux through adaptation and genome engineering not only generated an excellent ethanol producer using molasses but also provided the strategy for developing microbial cell factories.

In vitro characterization of a pAgo nuclease TtdAgo from Thermococcus thioreducens and evaluation of its effect in vivo.

In spite of the development of genome-editing tools using CRISPR-Cas systems, highly efficient and effective genome-editing tools are still needed that use novel programmable nucleases such as Argonaute (Ago) proteins to accelerate the construction of microbial cell factories. In this study, a prokaryotic Ago (pAgo) from a hyperthermophilic archaeon Thermococcus thioreducens (TtdAgo) was characterized in vitro. Our results showed that TtdAgo has a typical DNA-guided DNA endonuclease activity, and the efficiency and accuracy of cleavage are modulated by temperature, divalent ions, and the phosphorylation and length of gDNAs and their complementarity to the DNA targets. TtdAgo can utilize 5'-phosphorylated (5'-P) or 5'- hydroxylated (5'-OH) DNA guides to cleave single-stranded DNA (ssDNA) at temperatures ranging from 30°C to 95°C in the presence of Mn2+ or Mg2+ and displayed no obvious preference for the 5'-end-nucleotide of the guide. In addition, single-nucleotide mismatches had little effects on cleavage efficiency, except for mismatches at position 4 or 8 that dramatically reduced target cleavage. Moreover, TtdAgo performed programmable cleavage of double-stranded DNA at 75°C. We further introduced TtdAgo into an industrial ethanologenic bacterium Zymomonas mobilis to evaluate its effect in vivo. Our preliminary results indicated that TtdAgo showed cell toxicity toward Z. mobilis, resulting in a reduced growth rate and final biomass. In conclusion, we characterized TtdAgo in vitro and investigated its effect on Z. mobilis in this study, which lays a foundation to develop Ago-based genome-editing tools for recalcitrant industrial microorganisms in the future.

Improved Hydrogen Peroxide Stress Resistance of Zymomonas mobilis NADH Dehydrogenase (ndh) and Alcohol Dehydrogenase (adhB) Mutants

Unintended shifts in stress resistance of microbial strains with engineered central metabolism may impact their growth and production performance under oxidative, lignocellulosic, solvent, and other stress conditions, and as such, must be taken into account in bioprocess design. In the present work, we studied oxidative stress resistance in mutant strains of the facultatively anaerobic, ethanologenic bacterium Zymomonas mobilis with modified respiratory (inactivated NADH dehydrogenase Ndh, by disruption of ndh) and ethanologenic (inactivated iron-containing alcohol dehydrogenase isoenzyme ADH II, by disruption of adhB) catabolism, using exogenously added H2O2 in the concentration range of 2–6 mM as the oxidative stressor. Both mutations improved H2O2 resistance and enhanced catalase activity by a factor of 2–5, while the overexpression of Ndh had an opposite effect. Strains with a catalase-negative background were unable to grow already at 1 mM hydrogen peroxide, and their H2O2 resistance did not depend on AdhB or Ndh expression levels. Hence, the improved resistance of the ndh and adhB mutants to H2O2 resulted from their elevated catalase activity. The interrelation between these mutations, the catabolic redox balance, catalase activity, and oxidative stress defense in Z. mobilis is discussed.

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

The excessive consumption of sugars can cause health issues. Different strategies have been developed to reduce sugars in the diets. However, sugars in fruits and commercial products may be difficult to reduce, limiting their usage among certain populations of people. Zymomonas mobilis is a generally recognized as safe (GRAS) probiotic bacterium with the capability to produce levan-type prebiotics, and thrives in high-sugar environments with unique characteristics to be developed for lignocellulosic biofuel and biochemical production. In this study, the sugar reduction capabilities of Z. mobilis ZM4 were examined using two fruits of pear and persimmon and three high-sugar-content commercial products of two pear pastes (PPs) and one Chinese traditional wine (CTW). Our results demonstrated that Z. mobilis ZM4 can utilize sugars in fruits with about 20 g/L ethanol and less than 5 g/L sorbitol produced within 22 h using pears, and about 45 g/L ethanol and 30 g/L sorbitol produced within 34 h using persimmons. When PPs made from pears were used, Z. mobilis can utilize nearly all glucose (ca. 60 g/L) and most fructose (110 g/L) within 100 h with 40 ~ 60 g/L ethanol and more than 20 g/L sorbitol produced resulting in a final sorbitol concentration above 80 g/L. In the high-sugar-content alcoholic Chinese traditional wine, which contains mostly glucose and ethanol, Z. mobilis can reduce nearly all sugars with about 30 g/L ethanol produced, resulting in a final ethanol above 90 g/L. The ethanol yield and percentage yield of Z. mobilis in 50 ~ 60% CTW were 0.44 ~ 0.50 g/g and 86 ~ 97%, respectively, which are close to its theoretical yields—especially in 60% CTW. Although the ethanol yield and percentage yield in PPs were lower than those in CTW, they were similar to those in fruits of pears and persimmons with an ethanol yield around 0.30 ~ 0.37 g/g and ethanol percentage yield around 60 ~ 72%, which could be due to the formation of sorbitol and/or levan in the presence of both glucose and fructose. Our study also compared the fermentation performance of the classical ethanologenic yeast Saccharomyces cerevisiae BY4743 to Z. mobilis, with results suggesting that Z. mobilis ZM4 had better performance than that of yeast S. cerevisiae BY4743 given a higher sugar conversion rate and ethanol yield for sugar reduction. This work thus laid a foundation for utilizing the advantages of Z. mobilis in the food industry to reduce sugar concentrations or potentially produce alcoholic prebiotic beverages.Graphical

Syntrophy of Crypthecodinium cohnii and immobilized Zymomonas mobilis for docosahexaenoic acid production from sucrose-containing substrates

Marine heterotrophic dinoflagellate Crypthecodinium cohnii is an aerobic oleaginous microorganism that accumulates intracellular lipid with high content of 4,7,10,13,16,19-docosahexaenoic acid (DHA), a polyunsaturated ω−3 (22:6) fatty acid with multiple health benefits. C. cohnii can grow on glucose and ethanol, but not on sucrose or fructose. For conversion of sucrose-containing renewables to C. cohnii DHA, we investigated a syntrophic process, involving immobilized cells of ethanologenic bacterium Zymomonas mobilis for fermenting sucrose to ethanol. The non-respiring, NADH dehydrogenase-deficient Z. mobilis strain Zm6-ndh, with high ethanol yield both under anaerobic and aerobic conditions, was taken as the genetic background for inactivation of levansucrase (sacB). SacB mutation eliminated the levan-forming activity on sucrose. The double mutant Zm6-ndh-sacB cells were immobilized in Ca alginate, and applied for syntrophic conversion of sucrose to DHA of C. cohnii, either taking the ethanol-containing fermentation medium from the immobilized Z. mobilis for feeding to the C. cohnii fed-batch culture, or directly coculturing the immobilized Zm6-ndh-sacB with C. cohnii on sucrose. Both modes of cultivation produced C. cohnii CCMP 316 biomass with DHA content around 2–3 % of cell dry weight, corresponding to previously reported results for this strain on glucose.

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 model ethanologenic bacterium Zymomonas mobilis has many advantages for diverse biochemical production. Although the impact of temperature especially high temperature on the growth and ethanol production of Z. mobilis has been reported, the transcriptional profiles of Z. mobilis grown at different temperatures have not been systematically investigated. In this study, Z. mobilis wild-type strain ZM4 was used to study the effect of a broad range of temperatures of 24, 30, 36, 40, and 45 °C on cell growth and morphology, glucose utilization and ethanol production, as well as the corresponding global gene expression profiles using RNA-Seq-based transcriptomics. In addition, a recombinant Z. mobilis strain expressing reporter gene EGFP (ZM4_EGFP) was constructed to study the effect of temperature on heterologous protein expression at different temperatures. Our result demonstrated that the effect of temperature on the growth and morphology of ZM4 and ZM4_EGFP were similar. The biomass of these two strains decreased along with the temperature increase, and an optimal temperature range is needed for efficient glucose utilization and ethanol production. Temperatures lower or higher than normal temperature investigated in this work was not favorable for the glucose utilization and ethanol production as well as the expression of exogenous protein EGFP based on the results of flow cytometry and Western blot. Temperature also affected the transcriptional profiles of Z. mobilis especially under high temperature. Compared with ZM4 cultured at 30 °C, 478 genes were up-regulated and 481 genes were down-regulated at 45 °C. The number of differentially expressed genes of ZM4 cultured at other temperatures (24, 36 or 40 °C) was relatively small though compared with those at 30 °C. Since temperature usually increases during the fermentation process, and heat tolerance is one of the important robustness traits of industrial strains, candidate genes related to heat resistance based on our RNA-Seq result and literature report were then selected for genetics study using the strategies of plasmid overexpression of candidate gene or replacement of the native promoter of candidate gene by an inducible Ptet promoter. The genetics studies indicated that ZMO0236, ZMO1335, ZMO0994, operon groESL, and cspL, which encodes Mrp family chromosome partitioning ATPase, flavoprotein WrbA, an uncharacterized protein, chaperonin Cpn10 and GroEL, and an exogenous cold shock protein, respectively, were associated with heat tolerance, and recombinant strains over-expressing these genes can improve their heat tolerance. Our work thus not only explored the effects of temperature on the expression of exogenous gene EGFP and endogenous genes, but also selected and confirmed several genes associated with heat tolerance in Z. mobilis, which provided a guidance on identifying candidate genes associated with phenotypic improvement through systems biology strategy and genetics studies for other microorganisms.

The Ethanologenic Bacterium Zymomonas mobilis Divides Asymmetrically and Exhibits Heterogeneity in DNA Content.

The alphaproteobacterium Zymomonas mobilis exhibits extreme ethanologenic physiology, making this species a promising biofuel producer. Numerous studies have investigated its biology relevant to industrial applications and mostly at the population level. However, the organization of single cells in this industrially important polyploid species has been largely uncharacterized. In the present study, we characterized basic cellular behavior of Z. mobilis strain Zm6 under anaerobic conditions at the single-cell level. We observed that growing Z. mobilis cells often divided at a nonmidcell position, which contributed to variant cell size at birth. However, the cell size variance was regulated by a modulation of cell cycle span, mediated by a correlation of bacterial tubulin homologue FtsZ ring accumulation with cell growth. The Z. mobilis culture also exhibited heterogeneous cellular DNA content among individual cells, which might have been caused by asynchronous replication of chromosome that was not coordinated with cell growth. Furthermore, slightly angled divisions might have resulted in temporary curvatures of attached Z. mobilis cells. Overall, the present study uncovers a novel bacterial cell organization in Z. mobilisIMPORTANCE With increasing environmental concerns about the use of fossil fuels, development of a sustainable biofuel production platform has been attracting significant public attention. Ethanologenic Z. mobilis species are endowed with an efficient ethanol fermentation capacity that surpasses, in several respects, that of baker's yeast (Saccharomyces cerevisiae), the most-used microorganism for ethanol production. For development of a Z. mobilis culture-based biorefinery, an investigation of its uncharacterized cell biology is important, because bacterial cellular organization and metabolism are closely associated with each other in a single cell compartment. In addition, the current work demonstrates that the polyploid bacterium Z. mobilis exhibits a distinctive mode of bacterial cell organization, likely reflecting its unique metabolism that does not prioritize incorporation of nutrients for cell growth. Thus, another significant result of this work is to advance our general understanding in the diversity of bacterial cell architecture.

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

BackgroundAcid 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.ResultsIn 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.ConclusionAn 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.

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

Respiratory chain plays a pivotal role in the energy and redox balance of aerobic bacteria. By engineering respiration, it is possible to alter the efficiency of energy generation and intracellular redox state, and thus affect the key bioprocess parameters: cell yield, productivity and stress resistance. Here we summarize the current metabolic engineering and synthetic biology approaches to bacterial respiratory metabolism, with a special focus on the respiratory chain of the ethanologenic bacterium Zymomonas mobilis. Electron transport in Z. mobilis can serve as a model system of bacterial respiration with low oxidative phosphorylation efficiency. Its application for redox balancing and relevance for improvement of stress tolerance are analyzed.

Application of next generation sequencing in rare disorders

Phenolic aldehydes from lignocellulose pretreatment harshly inhibit the viability and metabolism of ethanol fermenting strains. Direct conversion of phenolic aldehydes is usually incomplete due to their low water solubility and recalcitrance to bioconversion. Here we consolidated phenolic aldehydes bioconversion and ethanol fermentation in a typical ethanologenic bacterium Zymomonas mobilis by constructing an intracellular oxidative pathway. The gene PP_2680 encoding NAD+-dependent aldehyde dehydrogenase from Pseudomonas putida KT2440 was expressed in Z. mobilis ZM4. The expression significantly improved both aldehyde inhibitor conversion and ethanol fermentability in corn stover hydrolysate. The purified PP_2680 aldehyde dehydrogenase showed strong in vitro oxidative capacity on phenolic aldehydes and its in vivo expression significantly up-regulated the key genes in the ED pathway and the oxidative phosphorylation. This study provided an important concept of simultaneous biodetoxification and fermentation in ethanologenic strains for the improvement of ethanol fermentability.

Special aspects of silage making process: microbiota of the feed formation

Phenolic aldehydes from lignocellulose pretreatment harshly inhibit the viability and metabolism of ethanol fermenting strains. Direct conversion of phenolic aldehydes is usually incomplete due to their low water solubility and recalcitrance to bioconversion. Here we consolidated phenolic aldehydes bioconversion and ethanol fermentation in a typical ethanologenic bacterium Zymomonas mobilis by constructing an intracellular oxidative pathway. The gene PP_2680 encoding NAD+-dependent aldehyde dehydrogenase from Pseudomonas putida KT2440 was expressed in Z. mobilis ZM4. The expression significantly improved both aldehyde inhibitor conversion and ethanol fermentability in corn stover hydrolysate. The purified PP_2680 aldehyde dehydrogenase showed strong in vitro oxidative capacity on phenolic aldehydes and its in vivo expression significantly up-regulated the key genes in the ED pathway and the oxidative phosphorylation. This study provided an important concept of simultaneous biodetoxification and fermentation in ethanologenic strains for the improvement of ethanol fermentability.

Expressing an oxidative dehydrogenase gene in ethanologenic strain Zymomonas mobilis promotes the cellulosic ethanol fermentability

Phenolic aldehydes from lignocellulose pretreatment harshly inhibit the viability and metabolism of ethanol fermenting strains. Direct conversion of phenolic aldehydes is usually incomplete due to their low water solubility and recalcitrance to bioconversion. Here we consolidated phenolic aldehydes bioconversion and ethanol fermentation in a typical ethanologenic bacterium Zymomonas mobilis by constructing an intracellular oxidative pathway. The gene PP_2680 encoding NAD+-dependent aldehyde dehydrogenase from Pseudomonas putida KT2440 was expressed in Z. mobilis ZM4. The expression significantly improved both aldehyde inhibitor conversion and ethanol fermentability in corn stover hydrolysate. The purified PP_2680 aldehyde dehydrogenase showed strong in vitro oxidative capacity on phenolic aldehydes and its in vivo expression significantly up-regulated the key genes in the ED pathway and the oxidative phosphorylation. This study provided an important concept of simultaneous biodetoxification and fermentation in ethanologenic strains for the improvement of ethanol fermentability.

Aerobic catabolism and respiratory lactate bypass in Ndh-negative Zymomonas mobilis

Ability to ferment in the presence of oxygen increases the robustness of bioprocesses and opens opportunity for novel industrial setups. The ethanologenic bacterium Zymomonas mobilis performs rapid and efficient anaerobic ethanol fermentation, yet its respiratory NADH dehydrogenase (Ndh)-deficient strain (ndh-) is known to produce ethanol with high yield also under oxic conditions. Compared to the wild type, it has a lower rate of oxygen consumption, and an increased expression of the respiratory lactate dehydrogenase (Ldh). Here we present a quantitative study of the product spectrum and carbon balance for aerobically growing ndh-. Ldh-deficient and Ldh-overexpressing ndh- strains were constructed and used to examine the putative role of the respiratory lactate bypass for aerobic growth and production. We show that aerobically growing ndh- strains perform fermentative metabolism with a near-maximum ethanol yield, irrespective of their Ldh expression background. Yet, Ldh activity strongly affects the aerobic product spectrum in glucose-consuming non-growing cells. Also, Ldh-deficiency hampers growth at elevated temperature (42 °C) and delays the restart of growth after 10–15 h of aerobic starvation.

Improving a recombinant Zymomonas mobilis strain 8b through continuous adaptation on dilute acid pretreated corn stover hydrolysate.

BackgroundComplete conversion of the major sugars of biomass including both the C5 and C6 sugars is critical for biofuel production processes. Several inhibitory compounds like acetate, hydroxymethylfurfural (HMF), and furfural are produced from the biomass pretreatment process leading to ‘hydrolysate toxicity,’ a major problem for microorganisms to achieve complete sugar utilization. Therefore, development of more robust microorganisms to utilize the sugars released from biomass under toxic environment is critical. In this study, we use continuous culture methodologies to evolve and adapt the ethanologenic bacterium Zymomonas mobilis to improve its ethanol productivity using corn stover hydrolysate.ResultsA turbidostat was used to adapt the Z. mobilis strain 8b in the pretreated corn stover liquor. The adaptation was initiated using pure sugar (glucose and xylose) followed by feeding neutralized liquor at different dilution rates. Once the turbidostat reached 60% liquor content, the cells began washing out and the adaptation was stopped. Several ‘sub-strains’ were isolated, and one of them, SS3 (sub-strain 3), had 59% higher xylose utilization than the parent strain 8b when evaluated on 55% neutralized PCS (pretreated corn stover) liquor. Using saccharified PCS slurry generated by enzymatic hydrolysis from 25% solids loading, SS3 generated an ethanol yield of 75.5% compared to 64% for parent strain 8b. Furthermore, the total xylose utilization was 57.7% for SS3 versus 27.4% for strain 8b. To determine the underlying genotypes in these new sub-strains, we conducted genomic resequencing and identified numerous single-nucleotide mutations (SNPs) that had arisen in SS3. We further performed quantitative reverse transcription PCR (qRT-PCR) on genes potentially affected by these SNPs and identified significant down-regulation of two genes, ZMO0153 and ZMO0776, in SS3 suggesting potential genetic mechanisms behind SS3’s improved performance.ConclusionWe have adapted/evolved Z. mobilis strain 8b for enhanced tolerance to the toxic compounds present in corn stover hydrolysates. The adapted strain SS3 has higher xylose utilization rate and produce more ethanol than the parent strain. We have identified transcriptional changes which may be responsible for these phenotypes, providing foundations for future research directions in improving Z. mobilis as biocatalysts for the production of ethanol or other fuel precursors.

Transcriptional analysis of adaptation to high glucose concentrations in Zymomonas mobilis.

The ethanologenic bacterium Zymomonas mobilis is usually tolerant to high concentrations of glucose. The addition of sorbitol decreases the lag phase and increases ethanol yield and productivity of the bacteria in high glucose concentrations. The molecular mechanisms of adaptation to high glucose concentrations and the effect of sorbitol are still unclear. In this study, microarray analysis was used to study the global transcriptional adaptation responses of Z. mobilis to high glucose concentrations. A total of 235 genes were differentially expressed when 220g/L glucose was added with or without 10mM sorbitol. These genes are involved in diverse aspects of cell metabolism and regulation, including membrane transporters, nitrogen metabolism, and plasmid-encoded genes. However, most differentially expressed genes were downregulated when sorbitol was added. Notably, the transcription of almost all genes involved in the Entner-Doudoroff and ethanol production pathways was not significantly affected. In addition, a prophage and a nitrogen-fixation cluster were significantly induced. These results revealed that Z. mobilis cells responded to high glucose concentrations by regulating the transcriptional levels of genes related to membrane channels and transporters, stress response mechanisms, and metabolic pathways. These data provide insight into the intracellular adaptation responses to high glucose concentrations and reveal strategies to engineer efficient ethanol fermentation in Z. mobilis.

Biotechnological potential of respiring Zymomonas mobilis: A stoichiometric analysis of its central metabolism

The active, yet energetically inefficient electron transport chain of the ethanologenic bacterium Zymomonas mobilis could be used in metabolic engineering for redox-balancing purposes during synthesis of certain products. Although several reconstructions of Z. mobilis metabolism have been published, important aspects of redox balance and aerobic catabolism have not previously been considered. Here, annotated genome sequences and metabolic reconstructions have been combined with existing biochemical evidence to yield a medium-scale model of Z. mobilis central metabolism in the form of COBRA Toolbox model files for flux balance analysis (FBA). The stoichiometric analysis presented here suggests the feasibility of several metabolic engineering strategies for obtaining high-value products, such as glycerate, succinate, and glutamate that would use the electron transport chain to oxidize the excess NAD(P)H, generated during synthesis of these metabolites. Oxidation of the excess NAD(P)H would also be needed for synthesis of ethanol from glycerol. Maximum product yields and the byproduct spectra have been estimated for each product, with glucose, xylose, or glycerol as the carbon substrates. These novel pathways represent targets for future metabolic engineering, as they would exploit both the rapid Entner–Doudoroff glycolysis, and the energetically uncoupled electron transport of Z. mobilis.