Transcription factor shapes chromosomal conformation and regulates gene expression in bacterial adaptation.

Genomic mutations allow bacteria to adapt rapidly to adverse stress environments. The three-dimensional conformation of the genome may also play an important role in transcriptional regulation and environmental adaptation. Here, using chromosome conformation capture, we investigate the high-order architecture of the Zymomonas mobilis chromosome in response to genomic mutation and ambient stimuli (acetic acid and furfural, derived from lignocellulosic hydrolysate). We find that genomic mutation only influences the local chromosome contacts, whereas stress of acetic acid and furfural restrict the long-range contacts and significantly change the chromosome organization at domain scales. Further deciphering the domain feature unveils the important transcription factors, Ferric uptake regulator (Fur) proteins, which act as nucleoid-associated proteins to promote long-range (>200 kb) chromosomal communications and regulate the expression of genes involved in stress response. Our work suggests that ubiquitous transcription factors in prokaryotes mediate chromosome organization and regulate stress-resistance genes in bacterial adaptation.

Comprehensive network of stress-induced responses in Zymomonas mobilis during bioethanol production: from physiological and molecular responses to the effects of system metabolic engineering.

Nowadays, biofuels, especially bioethanol, are becoming increasingly popular as an alternative to fossil fuels. Zymomonas mobilis is a desirable species for bioethanol production due to its unique characteristics, such as low biomass production and high-rate glucose metabolism. However, several factors can interfere with the fermentation process and hinder microbial activity, including lignocellulosic hydrolysate inhibitors, high temperatures, an osmotic environment, and high ethanol concentration. Overcoming these limitations is critical for effective bioethanol production. In this review, the stress response mechanisms of Z. mobilis are discussed in comparison to other ethanol-producing microbes. The mechanism of stress response is divided into physiological (changes in growth, metabolism, intracellular components, and cell membrane structures) and molecular (up and down-regulation of specific genes and elements of the regulatory system and their role in expression of specific proteins and control of metabolic fluxes) changes. Systemic metabolic engineering approaches, such as gene manipulation, overexpression, and silencing, are successful methods for building new metabolic pathways. Therefore, this review discusses systems metabolic engineering in conjunction with systems biology and synthetic biology as an important method for developing new strains with an effective response mechanism to fermentation stresses during bioethanol production. Overall, understanding the stress response mechanisms of Z. mobilis can lead to more efficient and effective bioethanol production.

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.

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.

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.

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.

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.

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.

Molecular insight into regioselectivity of transfructosylation catalyzed by GH68 levansucrase and ß-fructofuranosidase.

Glycoside hydrolase family 68 (GH68) enzymes catalyze ß-fructosyltransfer from sucrose to another sucrose, the so-called transfructosylation. Although regioselectivity of transfructosylation is divergent in GH68 enzymes, there is insufficient information available on the structural factor(s) involved in the selectivity. Here, we found two GH68 enzymes, ß-fructofuranosidase (FFZm) and levansucrase (LSZm), encoded tandemly in the genome of Zymomonas mobilis, displayed different selectivity: FFZm catalyzed the ß-(2→1)-transfructosylation (1-TF), whereas LSZm did both of 1-TF and ß-(2→6)-transfructosylation (6-TF). We identified His79FFZm and Ala343FFZm and their corresponding Asn84LSZm and Ser345LSZm respectively as the structural factors for those regioselectivities. LSZm with the respective substitution of FFZm-type His and Ala for its Asn84LSZm and Ser345LSZm (N84H/S345A-LSZm) lost 6-TF and enhanced 1-TF. Conversely, the LSZm-type replacement of His79FFZm and Ala343FFZm in FFZm (H79N/A343S-FFZm) almost lost 1-TF and acquired 6-TF. H79N/A343S-FFZm exhibited the selectivity like LSZm but did not produce the ß-(2→6)-fructoside-linked levan and/or long levanooligosaccharides that LSZm did. We assumed Phe189LSZm to be a responsible residue for the elongation of levan chain in LSZm and mutated the corresponding Leu187FFZm in FFZm to Phe. An H79N/L187F/A343S-FFZm produced a higher quantity of long levanooligosaccharides than H79N/A343S-FFZm (or H79N-FFZm), although without levan formation, suggesting that LSZm has another structural factor for levan production. We also found that FFZm generated a sucrose analog, ß-D-fructofuranosyl α-D-mannopyranoside, by ß-fructosyltransfer to d-mannose and regarded His79FFZm and Ala343FFZm as key residues for this acceptor specificity. In summary, this study provides insight into the structural factors of regioselectivity and acceptor specificity in transfructosylation of GH68 enzymes.

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.

Enhancing Secretion of Endoglucanase in Zymomonas mobilis by Disturbing Peptidoglycan Synthesis.

Zymomonas mobilis (Z. mobilis) is a potential candidate strain for consolidated bioprocessing (CBP) in lignocellulosic biorefinery. However, the low-level secretion of cellulases limits this CBP process, and the mechanism of protein secretion that is affected by cell wall peptidoglycan is also not well understood. Here, we constructed several penicillin-binding protein (PBP)-deficient strains derived from Z. mobilis S192 to perturb the cell wall peptidoglycan network and then investigated the effects of peptidoglycan on the endoglucanase secretion. The results showed that extracellular recombinant endoglucanase production was significantly enhanced in PBP mutant strains, notably, Δ1089/0959 (4.09-fold) and Δ0959 (5.76-fold) in comparison to parent strains. For PBP-deficient strains, the growth performance was not significantly inhibited, but cell morphology was altered. In addition, enhanced antibiotic sensitivity and reduced inhibitor tolerance were also detected in our study. The concentration of intracellular soluble peptidoglycan was increased, especially for single-gene deletion. Outer membrane permeability of PBP-deficient strains was also improved, notably, Δ1089/0959 (1.14-fold) and Δ0959 (1.07-fold), which might explain the increased endoglucanase extracellular secretion. Our findings indicated that PBP-deficient Z. mobilis was capable of increasing endoglucanase extracellular secretion via cell wall peptidoglycan disturbance, and it will provide a foundation for the development of CBP technology in Z. mobilis in the future. IMPORTANCE Cell wall peptidoglycan has the function to maintain cell robustness and acts as the barrier to secret recombinant proteins from the cytoplasm to extracellular space in Z. mobilis and other bacteria. Herein, we perturbed the peptidoglycan synthesis network via knocking out PBPs (ZMO0197, ZMO0959, ZMO1089) to enhance recombinant endoglycanase extracellular secretion in Z. mobilis S912. This study could lay the foundation for understanding the regulatory network of cell wall synthesis and guide the construction of CBP strains in Z. mobilis.

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.

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.

Biochemical and Mutational Analysis of Radical S-Adenosyl-L-Methionine Adenosylhopane Synthase HpnH from Zymomonas mobilis Reveals that the Conserved Residue Cysteine-106 Reduces a Radical Intermediate and Determines the Stereochemistry.

Adenosylhopane is a crucial precursor of C35 hopanoids, which are believed to modulate the fluidity and permeability of bacterial cell membranes. Adenosylhopane is formed by a crosslinking reaction between diploptene and a 5'-deoxyadenosyl radical that is generated by the radical S-adenosyl-L-methionine (SAM) enzyme HpnH. We previously showed that HpnH from Streptomyces coelicolor A3(2) (ScHpnH) converts diploptene to (22R)-adenosylhopane. However, the mechanism of the stereoselective C-C bond formation was unclear. Thus, here, we performed biochemical and mutational analysis of another HpnH, from the ethanol-producing bacterium Zymomonas mobilis (ZmHpnH). Similar to ScHpnH, wild-type ZmHpnH afforded (22R)-adenosylhopane. Conserved cysteine and tyrosine residues were suggested as possible hydrogen sources to quench the putative radical reaction intermediate. A Cys106Ala mutant of ZmHpnH had one-fortieth the activity of the wild-type enzyme and yielded both (22R)- and (22S)-adenosylhopane along with some related byproducts. Radical trapping experiments with a spin-trapping agent supported the generation of a radical intermediate in the ZmHpnH-catalyzed reaction. We propose that the thiol of Cys106 stereoselectively reduces the radical intermediate generated at the C22 position by the addition of the 5'-deoxadenosyl radical to diploptene, to complete the reaction.

Model-driven analysis of mutant fitness experiments improves genome-scale metabolic models of Zymomonas mobilis ZM4.

Genome-scale metabolic models have been utilized extensively in the study and engineering of the organisms they describe. Here we present the analysis of a published dataset from pooled transposon mutant fitness experiments as an approach for improving the accuracy and gene-reaction associations of a metabolic model for Zymomonas mobilis ZM4, an industrially relevant ethanologenic organism with extremely high glycolytic flux and low biomass yield. Gene essentiality predictions made by the draft model were compared to data from individual pooled mutant experiments to identify areas of the model requiring deeper validation. Subsequent experiments showed that some of the discrepancies between the model and dataset were caused by polar effects, mis-mapped barcodes, or mutants carrying both wild-type and transposon disrupted gene copies-highlighting potential limitations inherent to data from individual mutants in these high-throughput datasets. Therefore, we analyzed correlations in fitness scores across all 492 experiments in the dataset in the context of functionally related metabolic reaction modules identified within the model via flux coupling analysis. These correlations were used to identify candidate genes for a reaction in histidine biosynthesis lacking an annotated gene and highlight metabolic modules with poorly correlated gene fitness scores. Additional genes for reactions involved in biotin, ubiquinone, and pyridoxine biosynthesis in Z. mobilis were identified and confirmed using mutant complementation experiments. These discovered genes, were incorporated into the final model, iZM4_478, which contains 747 metabolic and transport reactions (of which 612 have gene-protein-reaction associations), 478 genes, and 616 unique metabolites, making it one of the most complete models of Z. mobilis ZM4 to date. The methods of analysis that we applied here with the Z. mobilis transposon mutant dataset, could easily be utilized to improve future genome-scale metabolic reconstructions for organisms where these, or similar, high-throughput datasets are available.

Peculiar substrate specificity of δ1-pyrroline-5-carboxylate reductase in the obligately fermentative bacterium Zymomonas mobilis.

BACKGROUND: The enzyme that catalyzes the last step in proline synthesis, δ1-pyrroline-5-carboxylate reductase, showed in most cases a distinct preference in vitro for NADPH as the electron donor. METHODS AND RESULTS: A Zymomonas mobilis gene coding for a δ1-pyrroline-5-carboxylate reductase was cloned and heterologously expressed, and the recombinant protein was purified and characterized. The enzyme showed higher affinity to, and higher catalytic rate with NADH, with a specific activity of about 600 nkat (mg protein)-1. The molecular basis of this feature was investigated by analysis of the dinucleotide binding domain in silico. CONCLUSIONS: We postulate that the main determinants of coenzyme preference for P5C reductases are the length and the sequence of the motif A, whereas the overall sequence identity is insufficient to predict it a priori. Results are discussed in view of the obligately fermentative metabolism of this bacterium.

Characterization and repurposing of the endogenous Type I-F CRISPR-Cas system of Zymomonas mobilis for genome engineering.

Application of CRISPR-based technologies in non-model microorganisms is currently very limited. Here, we reported efficient genome engineering of an important industrial microorganism, Zymomonas mobilis, by repurposing the endogenous Type I-F CRISPR-Cas system upon its functional characterization. This toolkit included a series of genome engineering plasmids, each carrying an artificial self-targeting CRISPR and a donor DNA for the recovery of recombinants. Through this toolkit, various genome engineering purposes were efficiently achieved, including knockout of ZMO0038 (100% efficiency), cas2/3 (100%), and a genomic fragment of >10 kb (50%), replacement of cas2/3 with mCherry gene (100%), in situ nucleotide substitution (100%) and His-tagging of ZMO0038 (100%), and multiplex gene deletion (18.75%) upon optimal donor size determination. Additionally, the Type I-F system was further applied for CRISPRi upon Cas2/3 depletion, which has been demonstrated to successfully silence the chromosomally integrated mCherry gene with its fluorescence intensity reduced by up to 88%. Moreover, we demonstrated that genome engineering efficiency could be improved under a restriction-modification (R-M) deficient background, suggesting the perturbance of genome editing by other co-existing DNA targeting modules such as the R-M system. This study might shed light on exploiting and improving CRISPR-Cas systems in other microorganisms for genome editing and metabolic engineering practices.

Transporter proteins in Zymomonas mobilis contribute to the tolerance of lignocellulose-derived phenolic aldehyde inhibitors.

Transporter proteins are of great importance for improving the tolerance of fermentation strains to lignocellulose-derived furans and phenolic inhibitors. Different from the documented transporter proteins responsible for the tolerance of furfural and 5-hydroxymethyl-furfural (HMF), transporters responsible for that of varied phenolic aldehyde inhibitors were less investigated and elucidated. Here, an interesting phenomenon was found that no phenolic alcohols were accumulated from phenolic aldehydes degradation in Zymomonas mobilis. A transcriptional profiling of transporter genes was established in Z. mobilis ZM4 under phenolic aldehydes stress using DNA microarray. Six transporter proteins were identified as the potential candidates responsible for the tolerance of phenolic aldehydes including ABC transporter (ZMO0799 and ZMO0800), MFS transporter (ZMO1288 and ZMO1856), and RND transporter (ZMO0282 and ZMO0798). Furthermore, the analysis showed that the key transporters were significantly correlated with oxidoreductases and transcriptional regulators. This work would provide several important transporter genes serving as synthetic biology tools for improving the robustness of biorefinery strains.

The Quorum Sensing Auto-Inducer 2 (AI-2) Stimulates Nitrogen Fixation and Favors Ethanol Production over Biomass Accumulation in Zymomonas mobilis.

Autoinducer 2 (or AI-2) is one of the molecules used by bacteria to trigger the Quorum Sensing (QS) response, which activates expression of genes involved in a series of alternative mechanisms, when cells reach high population densities (including bioluminescence, motility, biofilm formation, stress resistance, and production of public goods, or pathogenicity factors, among others). Contrary to most autoinducers, AI-2 can induce QS responses in both Gram-negative and Gram-positive bacteria, and has been suggested to constitute a trans-specific system of bacterial communication, capable of affecting even bacteria that cannot produce this autoinducer. In this work, we demonstrate that the ethanologenic Gram-negative bacterium Zymomonas mobilis (a non-AI-2 producer) responds to exogenous AI-2 by modulating expression of genes involved in mechanisms typically associated with QS in other bacteria, such as motility, DNA repair, and nitrogen fixation. Interestingly, the metabolism of AI-2-induced Z. mobilis cells seems to favor ethanol production over biomass accumulation, probably as an adaptation to the high-energy demand of N2 fixation. This opens the possibility of employing AI-2 during the industrial production of second-generation ethanol, as a way to boost N2 fixation by these bacteria, which could reduce costs associated with the use of nitrogen-based fertilizers, without compromising ethanol production in industrial plants.

Regulated redirection of central carbon flux enhances anaerobic production of bioproducts in Zymomonas mobilis.

The microbial production of chemicals and fuels from plant biomass offers a sustainable alternative to fossilized carbon but requires high rates and yields of bioproduct synthesis. Z. mobilis is a promising chassis microbe due to its high glycolytic rate in anaerobic conditions that are favorable for large-scale production. However, diverting flux from its robust ethanol fermentation pathway to nonnative pathways remains a major engineering hurdle. To enable controlled, high-yield synthesis of bioproducts, we implemented a central-carbon metabolism control-valve strategy using regulated, ectopic expression of pyruvate decarboxylase (Pdc) and deletion of chromosomal pdc. Metabolomic and genetic analyses revealed that glycolytic intermediates and NADH accumulate when Pdc is depleted and that Pdc is essential for anaerobic growth of Z. mobilis. Aerobically, all flux can be redirected to a 2,3-butanediol pathway for which respiration maintains redox balance. Anaerobically, flux can be redirected to redox-balanced lactate or isobutanol pathways with ≥65% overall yield from glucose. This strategy provides a promising path for future metabolic engineering of Z. mobilis.