A new Zymomonas mobilis platform strain for the efficient production of chemicals.

BACKGROUND: Zymomonas mobilis is well known for its outstanding ability to produce ethanol with both high specific productivity and with high yield close to the theoretical maximum. The key enzyme in the ethanol production pathway is the pyruvate decarboxylase (PDC) which is converting pyruvate to acetaldehyde. Since it is widely considered that its gene pdc is essential, metabolic engineering strategies aiming to produce other compounds derived from pyruvate need to find ways to reduce PDC activity. RESULTS: Here, we present a new platform strain (sGB027) of Z. mobilis in which the native promoter of pdc was replaced with the IPTG-inducible PT7A1, allowing for a controllable expression of pdc. Expression of lactate dehydrogenase from E. coli in sGB027 allowed the production of D-lactate with, to the best of our knowledge, the highest reported specific productivity of any microbial lactate producer as well as with the highest reported lactate yield for Z. mobilis so far. Additionally, by expressing the L-alanine dehydrogenase of Geobacillus stearothermophilus in sGB027 we produced L-alanine, further demonstrating the potential of sGB027 as a base for the production of compounds other than ethanol. CONCLUSION: We demonstrated that our new platform strain can be an excellent starting point for the efficient production of various compounds derived from pyruvate with Z. mobilis and can thus enhance the establishment of this organism as a workhorse for biotechnological production processes.

Structure of the exopolyphosphatase (PPX) from Zymomonas mobilis reveals a two-magnesium-ions PPX.

Rapid adaptation of metabolic capabilities is crucial for bacterial survival in habitats with fluctuating nutrient availability. In such conditions, the bacterial stringent response is a central regulatory mechanism activated by nutrient starvation or other stressors. This response is primarily controlled by exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) enzymes. To gain further insight into these enzymes, the high-resolution crystal structure of PPX from Zymomonas mobilis (ZmPPX) was determined at 1.8 Å. The phosphatase activity of PPX was strictly dependent on the presence of divalent metal cations. Notably, the structure of ZmPPX revealed the presence of two magnesium ions in the active site center, which is atypical compared to other PPX structures where only one divalent ion is observed. ZmPPX exists as a dimer in solution and belongs to the "long" PPX group consisting of four domains. Remarkably, the dimer configuration exhibits a substantial and deep aqueduct with positive potential along its interface. This aqueduct appears to extend towards the active site region, suggesting that this positively charged aqueduct could potentially serve as a binding site for polyP.

Construction and comparison of different vehicles for heterologous gene expression in Zymomonas mobilis.

Zymomonas mobilis has the potential to be an optimal chassis for the production of bulk chemicals derived from pyruvate. However, a lack of available standardized and characterized genetic tools hinders both efficient engineering of Z. mobilis and progress in basic research on this organism. In this study, a series of different shuttle vectors were constructed based on the replication mechanisms of the native Z. mobilis plasmids pZMO1, pZMOB04, pZMOB05, pZMOB06, pZMO7 and p29191_2 and on the broad host range replication origin of pBBR1. These plasmids as well as genomic integration sites were characterized for efficiency of heterologous gene expression, stability without selection and compatibility. We were able to show that a wide range of expression levels could be achieved by using different plasmid replicons. The expression levels of the constructs were consistent with the relative copy numbers, as determined by quantitative PCR. In addition, most plasmids are compatible and could be combined. To avoid plasmid loss, antibiotic selection is required for all plasmids except the pZMO7-based plasmid, which is stable also without selection pressure. Stable expression of reporter genes without the need for selection was also achieved by genomic integration. All modules were adapted to the modular cloning toolbox Zymo-Parts, allowing easy reuse and combination of elements. This work provides an overview of heterologous gene expression in Z. mobilis and adds a rich set of standardized genetic elements to an efficient cloning system, laying the foundation for future engineering and research in this area.

Metabolic regulation boosts bioelectricity generation in Zymomonas mobilis microbial fuel cell, surpassing ethanol production.

Zymomonas mobilis (Z. mobilis), a bacterium known for its ethanol production capabilities, can also generate electricity by transitioning from ethanol production to electron generation. The purpose of this study is to investigate the ability of Z. mobilis to produce bioelectricity when utilized as a biocatalyst in a single-chamber microbial fuel cell (MFC). Given the bacterium's strong inclination towards ethanol production, a metabolic engineering strategy was devised to identify key reactions responsible for redirecting electrons from ethanol towards electricity generation. To evaluate the electroactivity of cultured Z. mobilis and its ethanol production in the presence of regulators, the reduction of soluble Fe(III) was utilized. Among the regulators tested, CuCl2 demonstrated superior effectiveness. Consequently, the MFC was employed to analyze the electrochemical properties of Z. mobilis using both a minimal and modified medium. By modifying the bacterial medium, the maximum current and power density of the MFC fed with Z. mobilis increased by more than 5.8- and sixfold, respectively, compared to the minimal medium. These findings highlight the significant impact of metabolic redirection in enhancing the performance of MFCs. Furthermore, they establish Z. mobilis as an active electrogenesis microorganism capable of power generation in MFCs.

Development of a counterselectable system for rapid and efficient CRISPR-based genome engineering in Zymomonas mobilis.

BACKGROUND: Zymomonas mobilis is an important industrial bacterium ideal for biorefinery and synthetic biology studies. High-throughput CRISPR-based genome editing technologies have been developed to enable targeted engineering of genes and hence metabolic pathways in the model ZM4 strain, expediting the exploitation of this biofuel-producing strain as a cell factory for sustainable chemicals, proteins and biofuels production. As these technologies mainly take plasmid-based strategies, their applications would be impeded due to the fact that curing of the extremely stable plasmids is laborious and inefficient. Whilst counterselection markers have been proven to be efficient for plasmid curing, hitherto only very few counterselection markers have been available for Z. mobilis. RESULTS: We constructed a conditional lethal mutant of the pheS gene of Z. mobilis ZM4, clmPheS, containing T263A and A318G substitutions and coding for a mutated alpha-subunit of phenylalanyl-tRNA synthetase to allow for the incorporation of a toxic analog of phenylalanine, p-chloro-phenylalanine (4-CP), into proteins, and hence leading to inhibition of cell growth. We demonstrated that expression of clmPheS driven by a strong Pgap promoter from a plasmid could render the Z. mobilis ZM4 cells sufficient sensitivity to 4-CP. The clmPheS-expressing cells were assayed to be extremely sensitive to 0.2 mM 4-CP. Subsequently, the clmPheS-assisted counterselection endowed fast curing of genome engineering plasmids immediately after obtaining the desired mutants, shortening the time of every two rounds of multiplex chromosome editing by at least 9 days, and enabled the development of a strategy for scarless modification of the native Z. mobilis ZM4 plasmids. CONCLUSIONS: This study developed a strategy, coupling an endogenous CRISPR-based genome editing toolkit with a counterselection marker created here, for rapid and efficient multi-round multiplex editing of the chromosome, as well as scarless modification of the native plasmids, providing an improved genome engineering toolkit for Z. mobilis and an important reference to develope similar genetic manipulation systems in other non-model organisms.

The 2.4 Å structure of Zymomonas mobilis pyruvate kinase: Implications for stability and regulation.

Human liver pyruvate kinase (hlPYK) catalyzes the final step in glycolysis, the formation of pyruvate (PYR) and ATP from phosphoenolpyruvate (PEP) and ADP. Fructose 1,6-bisphosphate (FBP), a pathway intermediate of glycolysis, serves as an allosteric activator of hlPYK. Zymomonas mobilis pyruvate kinase (ZmPYK) performs the final step of the Entner-Doudoroff pathway, which is similar to glycolysis in that energy is harvested from glucose and pyruvate is generated. The Entner-Doudoroff pathway does not have FBP as a pathway intermediate, and ZmPYK is not allosterically activated. In this work, we solved the 2.4 Å X-ray crystallographic structure of ZmPYK. The protein is dimeric in solution as determined by gel filtration chromatography, but crystallizes as a tetramer. The buried surface area of the ZmPYK tetramerization interface is significantly smaller than that of hlPYK, and yet tetramerization using the standard interfaces from higher organisms provides an accessible low energy crystallization pathway. Interestingly, the ZmPYK structure showed a phosphate ion in the analogous location to the 6-phosphate binding site of FBP in hlPYK. Circular Dichroism (CD) was used to measure melting temperatures of hlPYK and ZmPYK in the absence and presence of substrates and effectors. The only significant difference was an additional phase of small amplitude for the ZmPYK melting curves. We conclude that the phosphate ion plays neither a structural or allosteric role in ZmPYK under the conditions tested. We hypothesize that ZmPYK does not have sufficient protein stability for activity to be tuned by allosteric effectors as described for rheostat positions in the allosteric homologues.

Characterization and Application of the Sugar Transporter Zmo0293 from Zymomonas mobilis.

Zymomonas mobilis is a natural ethanologen with many desirable characteristics, which makes it an ideal industrial microbial biocatalyst for the commercial production of desirable bioproducts. Sugar transporters are responsible for the import of substrate sugars and the conversion of ethanol and other products. Glucose-facilitated diffusion protein Glf is responsible for facilitating the diffusion of glucose uptake in Z. mobilis. However, another sugar transporter-encoded gene, ZMO0293, is poorly characterized. We employed gene deletion and heterologous expression mediated by the CRISPR/Cas method to investigate the role of ZMO0293. The results showed that deletion of the ZMO0293 gene slowed growth and reduced ethanol production and the activities of key enzymes involved in glucose metabolism in the presence of high concentrations of glucose. Moreover, ZMO0293 deletion caused different transcriptional changes in some genes of the Entner Doudoroff (ED) pathway in the ZM4-ΔZM0293 strain but not in ZM4 cells. The integrated expression of ZMO0293 restored the growth of the glucose uptake-defective strain Escherichia coli BL21(DE3)-ΔptsG. This study reveals the function of the ZMO0293 gene in Z. mobilis in response to high concentrations of glucose and provides a new biological part for synthetic biology.

Comparative study of ethanol production from sodium hydroxide pretreated rice straw residue using Saccharomyces cerevisiae and Zymomonas mobilis.

Rice straw is a suitable alternative to a cheaper carbohydrate source for the production of ethanol. For pretreatment efficiency, different sodium hydroxide concentrations (0.5-2.5% w/v) were tested. When compared to other concentrations, rice straw processed with 2% NaOH (w/v) yielded more sugar (8.17 ± 0.01 mg/ml). An alkali treatment induces effective delignification and swelling of biomass. The pretreatment of rice straw with 2% sodium hydroxide (w/v) is able to achieve 55.34% delignification with 53.30% cellulose enrichment. The current study shows the effectiveness of crude cellulolytic preparation from Aspergillus niger resulting in 80.51 ± 0.4% cellulose hydrolysis. Rice straw hydrolysate was fermented using ethanologenic Saccharomyces cerevisiae (yeast) and Zymomonas mobilis (bacteria). Overall, superior efficiency of sugar conversion to ethanol 70.34 ± 0.3% was obtained with the yeast compared to bacterial strain 39.18 ± 0.5%. The current study showed that pretreatment with sodium hydroxide is an effective method for producing ethanol from rice straw and yeast strain S. cerevisiae having greater fermentative potential for bioethanol production than bacterial strain Z. mobilis.

Investigating ethanol production using the Zymomonas mobilis crude extract.

Cell-free systems have become valuable investigating tools for metabolic engineering research due to their easy access to metabolism without the interference of the membrane. Therefore, we applied Zymomonas mobilis cell-free system to investigate whether ethanol production is controlled by the genes of the metabolic pathway or is limited by cofactors. Initially, different glucose concentrations were added to the extract to determine the crude extract's capability to produce ethanol. Then, we investigated the genes of the metabolic pathway to find the limiting step in the ethanol production pathway. Next, to identify the bottleneck gene, a systemic approach was applied based on the integration of gene expression data on a cell-free metabolic model. ZMO1696 was determined as the bottleneck gene and an activator for its enzyme was added to the extract to experimentally assess its effect on ethanol production. Then the effect of NAD+ addition at the high concentration of glucose (1 M) was evaluated, which indicates no improvement in efficiency. Finally, the imbalance ratio of ADP/ATP was found as the controlling factor by measuring ATP levels in the extract. Furthermore, sodium gluconate as a carbon source was utilized to investigate the expansion of substrate consumption by the extract. 100% of the maximum theoretical yield was obtained at 0.01 M of sodium gluconate while it cannot be consumed by Z. mobilis. This research demonstrated the challenges and advantages of using Z. mobilis crude extract for overproduction.

Zymo-Parts: A Golden Gate Modular Cloning Toolbox for Heterologous Gene Expression in Zymomonas mobilis.

Zymomonas mobilis is a microorganism with extremely high sugar consumption and ethanol production rates and is generally considered to hold great potential for biotechnological applications. However, its genetic engineering is still difficult, hampering the efficient construction of genetically modified strains. In this work, we present Zymo-Parts, a modular toolbox based on Golden-Gate cloning offering a collection of promoters (including native, inducible, and synthetic constitutive promoters of varying strength), an array of terminators and several synthetic ribosomal binding sites and reporter genes. All these parts can be combined in an efficient and flexible way to achieve a desired level of gene expression, either from plasmids or via genome integration. Use of the GoldenBraid-based system also enables an assembly of operons consisting of up to five genes. We present the basic structure of the Zymo-Parts cloning system, characterize several constitutive and inducible promoters, and exemplify the construction of an operon and of chromosomal integration of a reporter gene. Finally, we demonstrate the power and utility of the Zymo-Parts toolbox for metabolic engineering applications by overexpressing a heterologous gene encoding for the lactate dehydrogenase of Escherichia coli to achieve different levels of lactate production in Z. mobilis.

Kinase expression enhances phenolic aldehydes conversion and ethanol fermentability of Zymomonas mobilis.

Kinases modulate the various physiological activities of microbial fermenting strains including the conversion of lignocellulose-derived phenolic aldehydes (4-hydroxyaldehyde, vanillin, and syringaldehyde). Here, we comprehensively investigated the gene transcriptional profiling of the kinases under the stress of phenolic aldehydes for ethanologenic Zymomonas mobilis using DNA microarray. Among 47 kinase genes, three genes of ZMO0003 (adenylylsulfate kinase), ZMO1162 (histidine kinase), and ZMO1391 (diacylglycerol kinase), were differentially expressed against 4-hydroxybenzaldehyde and vanillin, in which the overexpression of ZMO1162 promoted the phenolic aldehydes conversion and ethanol fermentability. The perturbance originated from plasmid-based expression of ZMO1162 gene contributed to a unique expression profiling of genome-encoding genes under all three phenolic aldehydes stress. Differentially expressed ribosome genes were predicted as one of the main contributors to phenolic aldehydes conversion and thus finally enhanced ethanol fermentability for Z. mobilis ZM4. The results provided an insight into the kinases on regulation of phenolic aldehydes conversion and ethanol fermentability for Z. mobilis ZM4, as well as the target object for rational design of robust biorefinery strains.

High-sodium maltobionate production by immobilized Zymomonas mobilis cells in polyurethane.

The purpose of this study was the production of maltobionic acid, in the form of sodium maltobionate, by Z. mobilis cells immobilized in polyurethane. The in situ immobilized system (0.125-0.35 mm) was composed of 7 g polyol, 3.5 g isocyanate, 0.02 g silicone, and 7 g Z. mobilis cell, at the concentration of 210 g/L. The bioconversion of maltose to sodium maltobionate was performed with different cell concentrations (7.0-9.0 gimobilized/Lreaction_medium), temperature (30.54-47.46 °C), pH (5.55-7.25), and substrate concentration (0.7-1.3 mol/L). The stability of the immobilized system was evaluated for 24 h bioconversion cycles and storage of 6 months. The maximum concentration of sodium maltobionate was 648.61 mmol/L in 34.34 h process (8.5 gdry_cell/Lreaction_medium) at 39 °C and pH 6.30. The immobilized system showed stability for 19 successive operational cycles of 24 h bioconversion and 6 months of storage, at 4 °C or 22 °C.

[Effect of bio-electrochemical system on the metabolic changes of Zymomonas mobilis].

A bio-electrochemical system can promote the interaction between microorganism and electrode and consequently change cellular metabolism. To investigate the metabolic performance of Zymomonas mobilis in the bio-electrochemical system, we applied an H-type bio-electrochemical reactor to control Z. mobilis fermentation under 3 V. Compared with the control group without applied voltage, the glycerol in the anode chamber increased by 24%, while the glucose consumption in the cathode chamber increased by 16%, and the ethanol and succinic acid concentration increased by 13% and 8%, respectively. Transcriptomic analysis revealed that the pathways related to organic acid metabolism, redox balance, and electron transfer played roles in metabolic changes. Three significantly differentially expressed genes, ZMO1060 (superoxide dismutase), ZMO0401 (diguanylate cyclase), and ZMO1819 (nitrogen fixation protein), were selected to verify their functions in the bio-electrochemical system. Overexpression of ZMO1060 and ZMO1819 improved the electrochemical activity of Z. mobilis. This study provides insights into the microbial metabolism regulated by the bio-electrochemical system.

Determination of Nucleotide Sequences within Promoter Regions Affecting Promoter Compatibility between Zymomonas mobilis and Escherichia coli.

A promoter plays a crucial role in controlling the expression of the target gene in cells, thus being one of the key biological parts for synthetic biology practices. Although significant efforts have been made to identify and characterize promoters with different strengths in various microorganisms, the compatibility of promoters within different hosts still lacks investigation. In this study, we chose the native Pgap promoter of Zymomonas mobilis to investigate nucleotide sequences within promoter regions affecting promoter compatibility between Escherichia coli and Z. mobilis. Pgap is one of the strongest promotors in Z. mobilis that has many excellent characteristics to be developed as microbial cell factories. Using EGFP as a reporter, a Z. mobilis-derived Pgap mutant library was constructed and sorted in E. coli, with candidate promoters exhibiting high fluorescence intensity collected. A total of 53 variants were finally selected and sequenced by Sanger sequencing. The sequencing results grouped these variants into 12 different Pgap variant types, among which seven types presented higher promoter strength than native Pgap in E. coli. The next-generation sequencing technique was then employed to identify key mutations within the Pgap promoter region that affect the promoter compatibility. Finally, six important sites were identified and confirmed to help increase Pgap strength in E. coli while keeping similar strength of native Pgap in Z. mobilis. Compared to native Pgap, synthetic promoters combining these sites had enhanced strength; especially, Pgap-6M combining all six sites exhibited 20-fold greater strength than native Pgap in E. coli. This study thus not only determined six important sites affecting promoter compatibility but also confirmed a series of Pgap promoter variants with strong promoter activity in both E. coli and Z. mobilis. In addition, a strategy was established in this study to investigate and determine nucleotide sequences in promoter regions affecting promoter compatibility, which can be applied in other microorganisms to help reveal universal factors affecting promoter compatibility and design promoters with desired strengths among different microbial cell factories.

Odd one out? Functional tuning of Zymomonas mobilis pyruvate kinase is narrower than its allosteric, human counterpart.

Various protein properties are often illuminated using sequence comparisons of protein homologs. For example, in analyses of the pyruvate kinase multiple sequence alignment, the set of positions that changed during speciation ("phylogenetic" positions) were enriched for "rheostat" positions in human liver pyruvate kinase (hLPYK). (Rheostat positions are those which, when substituted with various amino acids, yield a range of functional outcomes). However, the correlation was moderate, which could result from multiple biophysical constraints acting on the same position during evolution and/or various sources of noise. To further examine this correlation, we here tested Zymomonas mobilis PYK (ZmPYK), which has <65% sequence identity to any other PYK sequence. Twenty-six ZmPYK positions were selected based on their phylogenetic scores, substituted with multiple amino acids, and assessed for changes in Kapp-PEP . Although we expected to identify multiple, strong rheostat positions, only one moderate rheostat position was detected. Instead, nearly half of the 271 ZmPYK variants were inactive and most others showed near wild-type function. Indeed, for the active ZmPYK variants, the total range of Kapp,PEP values ("tunability") was 40-fold less than that observed for hLPYK variants. The combined functional studies and sequence comparisons suggest that ZmPYK has evolved functional and/or structural attributes that differ from the rest of the family. We hypothesize that including such "orphan" sequences in MSA analyses obscures the correlations used to predict rheostat positions. Finally, results raise the intriguing biophysical question as to how the same protein fold can support rheostat positions in one homolog but not another.

Creation of Markerless Genome Modifications in a Nonmodel Bacterium by Fluorescence-Aided Recombineering.

Metabolic engineering of nonmodel bacteria is often challenging because of the paucity of genetic tools for iterative genome modification necessary to equip bacteria with pathways to produce high-value products. Here, we outline a homologous recombination-based method developed to delete or add genes to the genome of a nonmodel bacterium, Zymomonas mobilis, at the desired locus using a suicide plasmid that contains gfp as a fluorescence marker to track its presence in cells. The suicide plasmid is engineered to contain two 500 bp regions homologous to the DNA sequence immediately flanking the target locus. A single crossover event at one of the two homologous regions facilitates insertion of the plasmid into the genome and subsequent homologous recombination events excise the plasmid from the genome, leaving either the original genotype or the desired modified genotype. A key feature of this plasmid is that Green Fluorescent Protein (GFP) expressed from the suicide plasmid allows easy identification and sorting of cells that have lost the plasmid by use of a fluorescence activated cell sorter. Subsequent PCR amplification of genomic DNA from strains lacking GFP allows rapid identification of the desired genotype, which is confirmed by DNA sequencing. This method provides an efficient and flexible platform for improved genetic engineering of Z. mobilis, which can be easily adapted to other nonmodel bacteria.

Deciphering Molecular Mechanism Underlying Self-Flocculation of Zymomonas mobilis for Robust Production.

Zymomonas mobilis metabolizes sugar anaerobically through the Entner-Doudoroff pathway with less ATP generated for lower biomass accumulation to direct more sugar for product formation with improved yield, making it a suitable host to be engineered as microbial cell factories for producing bulk commodities with major costs from feedstock consumption. Self-flocculation of the bacterial cells presents many advantages, such as enhanced tolerance to environmental stresses, a prerequisite for achieving high product titers by using concentrated substrates. ZM401, a self-flocculating mutant developed from ZM4, the unicellular model strain of Z. mobilis, was employed in this work to explore the molecular mechanism underlying this self-flocculating phenotype. Comparative studies between ZM401 and ZM4 indicate that a frameshift caused by a single nucleotide deletion in the poly-T tract of ZMO1082 fused the putative gene with the open reading frame of ZMO1083, encoding the catalytic subunit BcsA of the bacterial cellulose synthase to catalyze cellulose biosynthesis. Furthermore, the single nucleotide polymorphism mutation in the open reading frame of ZMO1055, encoding a bifunctional GGDEF-EAL protein with apparent diguanylate cyclase/phosphodiesterase activities, resulted in the Ala526Val substitution, which consequently compromised in vivo specific phosphodiesterase activity for the degradation of cyclic diguanylic acid, leading to intracellular accumulation of the signaling molecule to activate cellulose biosynthesis. These discoveries are significant for engineering other unicellular strains from Z. mobilis with the self-flocculating phenotype for robust production. IMPORTANCE Stress tolerance is a prerequisite for microbial cell factories to be robust in production, particularly for biorefinery of lignocellulosic biomass to produce biofuels, bioenergy, and bio-based chemicals for sustainable socioeconomic development, since various inhibitors are released during the pretreatment to destroy the recalcitrant lignin-carbohydrate complex for sugar production through enzymatic hydrolysis of the cellulose component, and their detoxification is too costly for producing bulk commodities. Although tolerance to individual stress has been intensively studied, the progress seems less significant since microbial cells are inevitably suffering from multiple stresses simultaneously under production conditions. When self-flocculating, microbial cells are more tolerant to multiple stresses through the general stress response due to enhanced quorum sensing associated with the morphological change for physiological and metabolic advantages. Therefore, elucidation of the molecular mechanism underlying such a self-flocculating phenotype is significant for engineering microbial cells with the unique multicellular morphology through rational design to boost their production performance.

Expression of Escherichia coli malic enzyme gene in Zymomonas mobilis for production of malic acid.

Malic acid is one of the organic acids which is used in various industries including food and pharmaceuticals. Biotechnological production of malic acid by an efficient microorganism is highly desirable as the process will be eco-friendly and cost-effective. In this study, malic acid synthesis by Zymomonas mobilis was studied by expressing Escherichia coli malic enzyme gene under Pchap, Ptac and Ppdc promoters. The mae+ recombinants were obtained by recombineering-based genomic integration of Pchap-mae, Ptac-mae and Ppdc-mae sequences. The Ppdc promoter showed the highest expression of malic enzyme and the Pchap the lowest. However, cell growth was limited in mae+ recombinant containing Ppdc promoter. The metabolic analysis showed the highest level of malic acid in Ppdc-mae recombinant (2.84 g/L), which was about eight times higher than that in the wild type strain. The study showed that these three promoters can be used to produce organic acids in Z. mobilis.

Zymomonas mobilis ZM4 Utilizes an NADP+-Dependent Acetaldehyde Dehydrogenase To Produce Acetate.

Zymomonas mobilis is a promising bacterial host for biofuel production, but further improvement has been hindered because some aspects of its metabolism remain poorly understood. For example, one of the main by-products generated by Z. mobilis is acetate, but the pathway for acetate production is unknown. Acetaldehyde oxidation has been proposed as the major source of acetate, and an acetaldehyde dehydrogenase was previously isolated from Z. mobilis via activity guided fractionation, but the corresponding gene has never been identified. We determined that the locus ZMO1754 (also known as ZMO_RS07890) encodes an NADP+-dependent acetaldehyde dehydrogenase that is responsible for acetate production by Z. mobilis. Deletion of this gene from the chromosome resulted in a growth defect in oxic conditions, suggesting that acetaldehyde detoxification is an important role of acetaldehyde dehydrogenase. The deletion strain also exhibited a near complete abolition of acetate production, both in typical laboratory conditions and during lignocellulosic hydrolysate fermentation. Our results show that ZMO1754 encodes the major acetate-forming acetaldehyde dehydrogenase in Z. mobilis, and we therefore rename the gene aldB based on functional similarity to the Escherichia coli acetaldehyde dehydrogenase. IMPORTANCE Biofuel production from nonfood crops is an important strategy for reducing carbon emissions from the transportation industry, but it has not yet become commercially viable. An important avenue to improve biofuel production is to enhance the characteristics of fermentation organisms by decreasing by-product formation via genetic engineering. Here, we identified and deleted a metabolic pathway and associated gene that lead to acetate formation in Zymomonas mobilis. Acetate is one of the major by-products generated during ethanol production by Z. mobilis, so this information may be used in the future to develop better strains for commercial biofuel production.

Cysteine supplementation enhanced inhibitor tolerance of Zymomonas mobilis for economic lignocellulosic bioethanol production.

Inhibitors in lignocellulosic hydrolysates are toxic to Zymomonas mobilis and reduce its bioethanol production. This study revealed cysteine supplementation enhanced furfural tolerance in Z. mobilis with a 2-fold biomass increase. Transcriptomic study illustrated that cysteine biosynthesis pathway was down-regulated while cysteine catabolism was up-regulated with cysteine supplementation. Mutants for genes involved in cysteine metabolism were constructed, and metabolites in cysteine metabolic pathway including methionine, glutathione, NaHS, glutamate, and pyruvate were supplemented into media. Cysteine supplementation boosted glutathione synthesis or H2S release effectively in Z. mobilis leading to the reduced accumulation of reactive oxygen species (ROS) induced by furfural, while pyruvate and glutamate produced in the H2S generation pathway promoted cell growth by serving as the carbon or nitrogen source. Finally, cysteine supplementation was confirmed to enhance Z. mobilis tolerance against ethanol, acetate, and corncob hydrolysate with an enhanced ethanol productivity from 0.38 to 0.55 g-1∙L-1∙h-1.