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The bacterium Zymomonas mobilis is investigated as a model organism for the cultivation and separation of ethanol as a product by in situ extraction in continuous flow microreactors. The considered microreactor is the Coiled Flow Inverter (CFI), which consists of a capillary coiled onto a support structure. Like other microreactors, the CFI benefits from a high surface-to-volume ratio, which enhances mass and heat transfer. Compared to many other microreactors, the CFI offers the advantage of operating without internal structures, which are often used to ensure good mixing. The simplicity of the design makes the CFI particularly suitable for biochemical applications as cells do not get stuck or damaged by internal structures. Despite this simplicity, good mixing is achieved through flow vortices caused by Taylor and Dean vortices. The reaction system consists of two phases, in which the aqueous phase carries the bacterium and an oleyl alcohol phase is used to extract the ethanol produced. Key parameters for evaluation are bacteria growth and the amount of ethanol produced by the microorganism. The results show the suitability of the CFI for microbial production of valuable compounds. A maximum ethanol concentration of 1.26 g L−1 was achieved for the experiment in the CFI. Overall, the cultivation in the CFI led to faster growth of Z. mobilis, resulting in 25% higher ethanol production than in conducted batch experiments. [ABSTRACT FROM AUTHOR]

Forestry residues (FR) and medicinal-aromatic plant waste (MAPW) are considered potential resources for energy recovery. In this context, we explore the bioethanol production potential of three microbial strains Aspergillus niger, Zymomonas mobilis, and Trichoderma longibrachiatum using this lignocellulosic hydrolysate as a substrate. The blend of FR and MAPW was pretreated by different methods like acid sulfuric (AS), steam explosion (SE), and enzymatic (E). The ethanol yield was measured by gas chromatography (GC). Zymomonas mobilis demonstrated the highest ethanol yield of 5.95% on untreated substrate. Conversely, Aspergillus niger exhibited peak performance with an ethanol yield of 10.78% following AS, SE and E combined pretreatment. Trichoderma longibrachiatum, yielded ethanol ranging from 1.27% to 2.47%. Furthermore, the use of immobilized d' Aspergillus niger strains revealed a small decrease in ethanol yield from 11.34% in the first cycle to only 5.02% in the sixth cycle. In conclusion, Aspergillus niger emerges as a promising candidate due to its dual functionality in pretreatment and ethanol fermentation, offering pathways for advancing sustainable biofuel technologies. [ABSTRACT FROM AUTHOR]

Forestry residues (FR) and medicinal-aromatic plant waste (MAPW) are considered potential resources for energy recovery. In this context, we explore the bioethanol production potential of three microbial strains Aspergillus niger, Zymomonas mobilis, and Trichoderma longibrachiatum using this lignocellulosic hydrolysate as a substrate. The blend of FR and MAPW was pretreated by different methods like acid sulfuric (AS), steam explosion (SE), and enzymatic (E). The ethanol yield was measured by gas chromatography (GC). Zymomonas mobilis demonstrated the highest ethanol yield of 5.95% on untreated substrate. Conversely, Aspergillus niger exhibited peak performance with an ethanol yield of 10.78% following AS, SE and E combined pretreatment. Trichoderma longibrachiatum, yielded ethanol ranging from 1.27% to 2.47%. Furthermore, the use of immobilized d’ Aspergillus niger strains revealed a small decrease in ethanol yield from 11.34% in the first cycle to only 5.02% in the sixth cycle. In conclusion, Aspergillus niger emerges as a promising candidate due to its dual functionality in pretreatment and ethanol fermentation, offering pathways for advancing sustainable biofuel technologies.

Abstract 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. Graphical Abstract

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.

Lactate is an important monomer for the synthesis of poly-lactate (PLA), which is a substitute for the petrochemical plastics. To achieve the goal of high lactate titer, rate, and yield for commercial production, efficient lactate production pathway is needed as well as genetic targets that affect high lactate production and tolerance. In this study, an LldR-based d-lactate biosensor with a broad dynamic range was first applied into Zymomonas mobilis to select mutant strains with strong GFP fluorescence, which could be the mutant strains with increased d-lactate production. Then, LldR-based d-lactate biosensor was combined with a genome-wide CRISPR interference (CRISPRi) library targeting the entire genome to generate thousands of mutants with gRNA targeting different genetic targets across the whole genome. Specifically, two mutant libraries were selected containing 105 and 104 mutants with different interference sites from two rounds of fluorescence-activated cell sorting (FACS), respectively. Two genetic targets of ZMO1323 and ZMO1530 were characterized and confirmed to be associated with the increased d-lactate production, further knockout of ZMO1323 and ZMO1530 resulted in a 15% and 21% increase of d-lactate production, respectively. This work thus not only established a high-throughput approach that combines genome-scale CRISPRi and biosensor-assisted screening to identify genetic targets associated with d-lactate production in Z. mobilis, but also provided a feasible high-throughput screening approach for rapid identification of genetic targets associated with strain performance for other industrial microorganisms.

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Zymomonas mobilis is a gram-negative facultative anaerobic spore, which is generally recognized as a safe. As a promising ethanologenic organism for large-scale bio-ethanol production, Z. mobilis has also shown a good application prospect in food processing and food additive synthesis for its unique physiological characteristics and excellent industrial characteristics. It not only has obvious advantages in food processing and becomes the biorefinery chassis cell for food additives, but also has a certain healthcare effect on human health. Until to now, most of the research is still in theory and laboratory scale, and further research is also needed to achieve industrial production. This review summarized the physiological characteristics and advantages of Z. mobilis in food industry for the first time and further expounds its research status in food industry from three aspects of food additive synthesis, fermentation applications, and prebiotic efficacy, it will provide a theoretical basis for its development and applications in food industry. This review also discussed the shortcomings of its practical applications in the current food industry, and explored other ways to broaden the applications of Z. mobilis in the food industry, to promote its applications in food processing. Potential applications of Zymomonas mobilis in food industry summarized for the first time. Research status of Z. mobilis in food additive synthesis, fermentation applications, and probiotics are discussed in details. Future research perspectives of Z. mobilis in food industry further proposed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Due to the electrical nature of the cell, it is possible to modulate its behavior through the application of non-lethal external electric fields to improve fermentation processes. In this work, a microbial cell system with a chamber and two electrodes inside and connected to a voltage source was used. One of the electrodes was kept isolated to create an electric field without the flow of current. Cultures with two ethanol-producing microbial strains (Saccharomyces cerevisiae and Zymomonas mobilis) were conducted in this device. The application of voltages between 0 and 18 V was evaluated to determine the impact of the generated electric field on ethanol production. To analyze the possible effect of the field on the central carbon metabolism in each strain, biochemical-based kinetic models were formulated to describe the experimental fermentation kinetics obtained.It was found that low applied voltages did not have significant effects on growth rate in either strain, but all voltages evaluated increased substrate consumption and ethanol production rate in Z. mobilis, while only 18 V affected these rates in S. cerevisiae, indicating that Z. mobilis was the most sensitive to the electric field. At the end of the fermentation, significant increases in ethanol yields of 10.7% and 19.5% were detected for S. cerevisiae and Z. mobilis, respectively. The proposed mathematical models showed that substrate transport through the membrane catalyzed by the phosphotransferase system (PTS) for Z. mobilis and hexose transport proteins mechanism and hexokinase (HK) activity for S. cerevisiae and the transformation of pyruvate to ethanol, catalyzed by the decarboxylase (PDC) and alcohol dehydrogenase (ADH) enzymes, were the reactions most affected by the application of the external field.

Abstract Byproducts from the sugarcane manufacturing process, specifically sugarcane molasses (SM) and sugarcane bagasse (SB), can be used as alternative raw materials for sorbitol production via the biological fermentation process. This study investigated the production of sorbitol from SM and sugarcane bagasse hydrolysate (SBH) using a thermally adapted Zymomonas mobilis ZM AD41. Various combinations of SM and SBH on sorbitol production using batch fermentation process were tested. The results revealed that SM alone (FM1) or a mixture of SM and SBH at a ratio of 3:1 (FM2) based on the sugar mass in the raw material proved to be the best condition for sorbitol production by ZM AD41 at 37 °C. Further optimization conditions for sorbitol production revealed that a sugar concentration of 200 g/L and a CaCl2 concentration of 5.0 g/L yielded the highest sorbitol content. The maximum sorbitol concentrations produced by ZM AD41 in the fermentation medium containing SM (FM1) or a mixture of SM and SBH (FM2) were 31.23 and 30.45 g/L, respectively, comparable to those reported in the literature using sucrose or a mixture of sucrose and maltose as feedstock. These results suggested that SBH could be used as an alternative feedstock to supplement or blend with SM for sustainable sorbitol production. In addition, the fermentation conditions established in this study could also be applied to large-scale sorbitol production. Moreover, the thermally adapted Z. mobilis ZM AD41 is also a promising sorbitol-producing bacterium for large-scale production at a relatively high fermentation temperature using agricultural byproducts, specifically SM and SB, as feedstock, which could reduce the operating cost due to minimizing the energy required for the cooling system.

Byproducts from the sugarcane manufacturing process, specifically sugarcane molasses (SM) and sugarcane bagasse (SB), can be used as alternative raw materials for sorbitol production via the biological fermentation process. This study investigated the production of sorbitol from SM and sugarcane bagasse hydrolysate (SBH) using a thermally adapted Zymomonas mobilis ZM AD41. Various combinations of SM and SBH on sorbitol production using batch fermentation process were tested. The results revealed that SM alone (FM1) or a mixture of SM and SBH at a ratio of 3:1 (FM2) based on the sugar mass in the raw material proved to be the best condition for sorbitol production by ZM AD41 at 37 °C. Further optimization conditions for sorbitol production revealed that a sugar concentration of 200 g/L and a CaCl2 concentration of 5.0 g/L yielded the highest sorbitol content. The maximum sorbitol concentrations produced by ZM AD41 in the fermentation medium containing SM (FM1) or a mixture of SM and SBH (FM2) were 31.23 and 30.45 g/L, respectively, comparable to those reported in the literature using sucrose or a mixture of sucrose and maltose as feedstock. These results suggested that SBH could be used as an alternative feedstock to supplement or blend with SM for sustainable sorbitol production. In addition, the fermentation conditions established in this study could also be applied to large-scale sorbitol production. Moreover, the thermally adapted Z. mobilis ZM AD41 is also a promising sorbitol-producing bacterium for large-scale production at a relatively high fermentation temperature using agricultural byproducts, specifically SM and SB, as feedstock, which could reduce the operating cost due to minimizing the energy required for the cooling system. [ABSTRACT FROM AUTHOR]

In this study, bioethanol production was compared via a novel two-stage fermentation using Zymomonas mobilis and/or Pichia stipitis with low inoculum size (5%). A synthetic glucose (30 g/l)/xylose (20 g/l) medium simulated the real hydrolyzed lignocellulose, and the inhibitory effects of different compounds and high sugar and bioethanol concentrations were restricted to find strains synergistic interactions. Higher ethanol yield and sugar conversion alongside lower time and cost were also considered. Xylose was entirely consumed by single P. stipitis only, but glucose was completely fermented in all strategies. Maximum glucose and xylose conversion efficiencies were obtained by single Z. mobilis and P. stipitis cultures, respectively. Sequential culture provided the same glucose conversion and consumption rate (100% to 1.01 gg/l.h) and yield (0.50 ge/gg) as single Z. mobilis and the nearest xylose conversion efficiency (30% to 0.14 gx/l.h to 0.16 ge/gx) to single P. stipitis (47% to 0.34 gx/l.h to 0.27 ge/gx). However, this culture was inefficient since Z. mobilis was removed. By individually immobilizing the strains on stable calcium alginate beads, the sequential-co-immobilized strategy resulted in higher xylose conversion, yield, and overall productivity of 83%, 0.45 ge/gx, and 0.30 ge/l.h than the sequential-co culture (16%, 0.31 ge/gx, and 0.14 ge/l.h), respectively. This strategy provides synergistic interactions by allocating a part of glucose to rapid growth of P. stipitis and utilizing intermediate metabolites of xylose fermentation by Z. mobilis to enhance overall efficiency. The sequential-co-immobilized strategy was also examined in 2 L bioreactor where xylose uptake rate, xylose conversion, and ethanol productivity increased 0.04 gx/l.h, 11%, and 0.27 ge/l.h compared to the sequential-co, respectively. The sequential-co-immobilized culture was found as an efficient strategy in production of second-generation bioethanol from lignocellulose. [ABSTRACT FROM AUTHOR]

Palm wine (tuak) is a traditional drink recognized worldwide as the traditional drink of Africa, Southeast Asia, and Central America. The history of alcoholic beverages in Indonesia is from the prehistoric era and is recorded in Borobudur, the largest Buddha temple in Indonesia, built in the eighth century. Although Indonesian society has experienced many social and cultural changes, the tradition of tuak production for daily drinks and ceremonies is strong and widespread and has been introduced as a tourism commodity in regions of Indonesia, especially in Bali. However, there are significant challenges in improving this traditional drink's quality and economic value. In this review, we conducted a desktop study and field survey to learn how tuak is part of Indonesian fermented beverage history, especially in Bali, linked to daily life in most traditional communities and supported by local regulators for the existence of this traditional heritage in society and economy. The nutritional composition of tuak comprises monosaccharides such as glucose and fructose, disaccharides including sucrose and maltose, organic acids, and bioactive compounds encompassing saponins, phenols, terpenoids, alkaloids, and flavonoids. Additionally, tuak contains minerals, alcohols, and essential aromatic compounds. The microorganisms engaged in the fermentation process predominantly comprise ambient air microbes and plant-associated microbes, with a significant prevalence of Saccharomyces cerevisiae, Saccharomyces chevalieri, and Zymomonas mobilis. Additionally, Candida tropicalis yeast is present in the microbial consortium involved in the fermentation. This understanding will provide comprehensive information from a socio-economic perspective to preserve production, celebrate culture and to further develop the value of traditional beverages. [ABSTRACT FROM AUTHOR]

Zymomonas mobilis is an ethanologenic bacterium that can produce hopanoids using farnesyl pyrophosphate (FPP), which can be used as the precursor by β-farnesene synthase for β-farnesene production. To explore the possibility and bottlenecks of developing Z. mobilis for β-farnesene production, five heterologous β-farnesene synthases were selected and screened, and AaBFS from Artemisia annua had the highest β-farnesene titer. Recombinant strains with AaBFS driven by the strong constitutive promoter Pgap (Pgap–AaBFS) doubled its β-farnesene production to 25.73 ± 0.31 mg/L compared to the recombinant strain with AaBFS driven by Ptet (Ptet–AaBFS), which can be further improved by overexpressing the Pgap–AaBFS construct using the strategies of multiple plasmids (41.00 ± 0.40 mg/L) or genomic multi-locus integration (48.33 ± 3.40 mg/L). The effect of cofactor NADPH balancing on β-farnesene production was also investigated, which can be improved only in zwf-overexpressing strains but not in ppnK-overexpressing strains, indicating that cofactor balancing is important and sophisticated. Furthermore, the β-farnesene titer was improved to 73.30 ± 0.71 mg/L by overexpressing dxs, ispG, and ispH. Finally, the β-farnesene production was increased to 159.70 ± 7.21 mg/L by fermentation optimization, including the C/N ratio, flask working volume, and medium/dodecane ratio, which was nearly 13-fold improved from the parental strain. This work thus not only generated a recombinant β-farnesene production Z. mobilis strain but also unraveled the bottlenecks to engineer Z. mobilis for farnesene production, which will help guide the future rational design and construction of cell factories for terpenoid production in non-model industrial microorganisms.

Abstract Background Recent 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. Results We 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. Conclusions This 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.

The bacterium Zymomonas mobilis is investigated as a model organism for the cultivation and separation of ethanol as a product by in situ extraction in continuous flow microreactors. The considered microreactor is the Coiled Flow Inverter (CFI), which consists of a capillary coiled onto a support structure. Like other microreactors, the CFI benefits from a high surface-to-volume ratio, which enhances mass and heat transfer. Compared to many other microreactors, the CFI offers the advantage of operating without internal structures, which are often used to ensure good mixing. The simplicity of the design makes the CFI particularly suitable for biochemical applications as cells do not get stuck or damaged by internal structures. Despite this simplicity, good mixing is achieved through flow vortices caused by Taylor and Dean vortices. The reaction system consists of two phases, in which the aqueous phase carries the bacterium and an oleyl alcohol phase is used to extract the ethanol produced. Key parameters for evaluation are bacteria growth and the amount of ethanol produced by the microorganism. The results show the suitability of the CFI for microbial production of valuable compounds. A maximum ethanol concentration of 1.26 g L−1 was achieved for the experiment in the CFI. Overall, the cultivation in the CFI led to faster growth of Z. mobilis, resulting in 25% higher ethanol production than in conducted batch experiments.

Background: Recent 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. Results: We 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. Conclusions: This 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. [ABSTRACT FROM AUTHOR]

Integration of methanogenic archaea with photocatalysts presents a sustainable solution for solar-driven methanogenesis. However, maximizing CH4 conversion efficiency remains challenging due to the intrinsic energy conservation and strictly restricted substrates of methanogenic archaea. Here, we report a solar-driven biotic--abiotic hybrid (biohybrid) system by incorporating cadmium sulfide (CdS) nanoparticles with a rationally designed methanogenic archaeon Methanosarcina acetivorans C2A, in which the glucose synergist protein and glucose kinase, an energy-efficient route for glucose transport and phosphorylation from Zymomonas mobilis, were implemented to facilitate nonnative substrate glucose for methanogenesis. We demonstrate that the photo-excited electrons facilitate membrane-bound electron transport chain, thereby augmenting the Na+ and H+ ion gradients across membrane to enhance adenosine triphosphate (ATP) synthesis. Additionally, this biohybrid system promotes the metabolism of pyruvate to acetyl coenzyme A (AcCoA) and inhibits the flow of AcCoA to the tricarboxylic acid (TCA) cycle, resulting in a 1.26-fold augmentation in CH4 production from glucose-derived carbon. Our results provide a unique strategy for enhancing methanogenesis through rational biohybrid design and reprogramming, which gives a promising avenue for sustainably manufacturing value-added chemicals. [ABSTRACT FROM AUTHOR]

The objective of this study was to purify sodium maltobionate using Zymomonas mobilis cells immobilized in situ on flexible polyurethane (PU) and convert it into maltobionic acid for further evaluation of bioactivity (iron chelating ability, antibacterial potential and cytoprotection) and incorporation into films based on cassava starch, chitosan, and cellulose acetate. Sodium maltobionate exhibited a purity of 98.1% and demonstrated an iron chelating ability of approximately 50% at concentrations ranging from 15 to 20 mg mL−1. Maltobionic acid displayed minimal inhibitory concentrations (MIC) of 8.5, 10.5, 8.0, and 8.0 mg mL−1 for Salmonella enterica serovar Choleraesuis, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes, respectively. Maltobionic acid did not exhibit cytotoxicity in HEK-293 cells at concentrations up to 500 µg mL−1. Films incorporating 7.5% maltobionic acid into cassava starch and chitosan demonstrated inhibition of microbial growth, with halo sizes ranging from 15.67 to 22.33 mm. These films had a thickness of 0.17 and 0.13 mm, water solubility of 62.68% and 78.85%, and oil solubility of 6.23% and 11.91%, respectively. The cellulose acetate film exhibited a non-uniform visual appearance due to the low solubility of maltobionic acid in acetone. Mechanical and optical properties were enhanced with the addition of maltobionic acid to chitosan and cassava films. The chitosan film with 7.5% maltobionic acid demonstrated higher tensile strength (30.3 MPa) and elongation at break (9.0%). In contrast, the cassava starch film exhibited a high elastic modulus (1.7). Overall, maltobionic acid, with its antibacterial activity, holds promise for applications in active films suitable for food packaging. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Zymomonas mobilis is regarded as a potential chassis for the production of platform chemicals. Genome editing using the CRISPR-Cas system could meet the need for gene modification in metabolic engineering. However, the low curing efficiency of CRISPR editing plasmid is a common bottleneck in Z. mobilis. In this study, we utilized a theophylline-dependent riboswitch to regulate the expression of the replicase gene of the editing plasmid, thereby promoting the elimination of exogeneous plasmid. The riboswitch D (RSD) with rigorous regulatory ability was identified as the optimal candidate by comparing the transformation efficiency of four theophylline riboswitch-based backbone editing plasmids, and the optimal theophylline concentration for inducing RSD was determined to be 2 mM. A highly effective method for eliminating the editing plasmid, cells with RSD-based editing plasmid which were cultured in liquid and solid RM media in alternating passages at 37 °C without shaking, was established by testing the curing efficiency of backbone editing plasmids pMini and pMini-RSD in RM medium with or without theophylline at 30 °C or 37 °C. Finally, the RSD-based editing plasmid was applied to genome editing, resulting in an increase of more than 10% in plasmid elimination efficiency compared to that of pMini-based editing plasmid. Key points: • An effective strategy for curing CRISPR editing plasmid has been established in Z. mobilis. • Elimination efficiency of the CRISPR editing plasmid was enhanced by 10% to 20% under the regulation of theophylline-dependent riboswitch RSD. [ABSTRACT FROM AUTHOR]

Stable isotope tracers are a powerful tool for the quantitative analysis of microbial metabolism, enabling pathway elucidation, metabolic flux quantification, and assessment of reaction and pathway thermodynamics. 13C and 2H metabolic flux analysis commonly relies on isotopically labeled carbon substrates, such as glucose. However, the use of 2H-labeled nutrient substrates faces limitations due to their high cost and limited availability in comparison to 13C-tracers. Furthermore, isotope tracer studies in industrially relevant bacteria that metabolize complex substrates such as cellulose, hemicellulose, or lignocellulosic biomass, are challenging given the difficulty in obtaining these as isotopically labeled substrates. In this study, we examine the potential of deuterated water (2H 2 O) as an affordable, substrate-neutral isotope tracer for studying central carbon metabolism. We apply 2H 2 O labeling to investigate the reversibility of glycolytic reactions across three industrially relevant bacterial species - C. thermocellum, Z. mobilis, and E. coli -harboring distinct glycolytic pathways with unique thermodynamics. We demonstrate that 2H 2 O labeling recapitulates previous reversibility and thermodynamic findings obtained with established 13C and 2H labeled nutrient substrates. Furthermore, we exemplify the utility of this 2H 2 O labeling approach by applying it to high-substrate C. thermocellum fermentations -a setting in which the use of conventional tracers is impractical-thereby identifying the glycolytic enzyme phosphofructokinase as a major bottleneck during high-substrate fermentations and unveiling critical insights that will steer future engineering efforts to enhance ethanol production in this cellulolytic organism. This study demonstrates the utility of deuterated water as a substrate-agnostic isotope tracer for examining flux and reversibility of central carbon metabolic reactions, which yields biological insights comparable to those obtained using costly 2H-labeled nutrient substrates. • Deuterated water (2H 2 O) is a useful isotope tracer for examining flux and reversibility of metabolic reactions. • 2H 2 O labeling reveals the reversibility of glycolytic reactions across industrially relevant bacterial species. • 2H 2 O labeling generates biological insights consistent with established isotopically labeled nutrient substrates. • The glycolytic enzyme phosphofructokinase as a major bottleneck during C. thermocellum high-substrate fermentations. [ABSTRACT FROM AUTHOR]

Zymomonas mobilis, a Gram-negative bacteria observed in some popular beverages, is considered safe and has been studied for its potential therapeutic benefits. In this study, we explored its effects on the inflammatory process, tissue integrity, differential gene expression, and microbiota composition in an experimental dextran sulfate sodium (DSS)-induced colitis model in mice. As a result, Z. mobilis alleviated the symptoms caused by DSS administration, as indicated by reduced weight loss, disease activity index, a significant reduction in the colon weight/length ratio, and histopathological improvement. Also, Z. mobilis could restore the mucosal barrier as well as increase the expression of Muc3 and Ocln genes. An analysis of 16S rRNA sequences showed that Z. mobilis alters gut microbiota, increasing Akkermansia muciniphila abundance and decreasing Escherichia coli. Furthermore, Z. mobilis seems to be involved in potentiating a regulatory phenotype by inducing immunomodulatory genes like Tgfb, Il5, Il10, and Foxp3 and reducing the relative mRNA expression of proinflammatory cytokines TNF, Il6, and Il17. Our data suggest that Z. mobilis could alleviate disease progression and be considered a possible probiotic adjuvant for pathologies of the bowel. [ABSTRACT FROM AUTHOR]

Understanding the energy metabolism and its regulation is one of the clues to metabolic engineering of stress-resistant lignocellulose-converting microbial strains, also including the promising ethanologen Zymomonas mobilis. Z. mobilis is an obligately fermentative, facultatively anaerobic bacterium, carrying an active respiratory chain with low energy-coupling efficiency. Its respiration does not supply energy to aerobically growing cultures on sugary media, yet oxidative phosphorylation has been demonstrated in non-growing cells with ethanol. Here, we show, for the first time, that in respiring, non-growing Z. mobilis cells receiving regular small amounts of ethanol, oxidative phosphorylation significantly contributes to the maintenance of their viability. No improvement of viability is seen in the NADH dehydrogenase (ndh)-deficient respiratory mutant, which is unable to oxidize ethanol. The ethanol effect is also hampered by the protonophoric uncoupler CCCP, or the inhibitor of ATP synthase, DCCD. At higher concentrations (6% v/v), ethanol causes stress that slows down culture growth. By monitoring the activity of several respiratory gene promoters under ethanol stress with the green fluorescent protein reporter system, we demonstrate downregulation of these promoters, in particular the ndh promoter. We speculate that the decrease in respiratory chain activity in response to stress conditions mitigates the production of reactive oxygen species. [ABSTRACT FROM AUTHOR]

Zymomonas mobilis is an emerging chassis for being engineered to produce bulk products due to its unique glycolysis through the Entner–Doudoroff pathway with less ATP produced for lower biomass accumulation and higher product yield. When self‐flocculated, the bacterial cells are more productive, since they can self‐immobilize within bioreactors for high density, and are more tolerant to stresses for higher product titers, but this morphology needs to be controlled properly to avoid internal mass transfer limitation associated with their strong self‐flocculation. Herewith we explored the regulation of cyclic diguanosine monophosphate (c‐di‐GMP) on self‐flocculation of the bacterial cells through activating cellulose biosynthesis. While ZMO1365 and ZMO0919 with GGDEF domains for diguanylate cyclase activity catalyze c‐di‐GMP biosynthesis, ZMO1487 with an EAL domain for phosphodiesterase activity catalyzes c‐di‐GMP degradation, but ZMO1055 and ZMO0401 contain the dual domains with phosphodiesterase activity predominated. Since c‐di‐GMP is synthesized from GTP, the intracellular accumulation of this signal molecule through deactivating phosphodiesterase activity is preferred for activating cellulose biosynthesis to flocculate the bacterial cells, because such a strategy exerts less perturbance on intracellular processes regulated by GTP. These discoveries are significant for not only engineering unicellular Z. mobilis strains with the self‐flocculating morphology to boost production but also understanding mechanism underlying c‐di‐GMP biosynthesis and degradation in the bacterium. [ABSTRACT FROM AUTHOR]

TetR-family transcriptional regulators are widely distributed among bacteria and involved in various cellular processes such as multidrug and inhibitor resistance. Zymomonas mobilis is a industrial bacterium for lignocellulosic ethanol production. Although TetR-family regulators and their associated RND-family efflux pumps in Z. mobilis have been identified to be differentially expressed under various inhibitors and stressful conditions, there are no systematic investigation yet. In this study, bioinformatic analyses indicated that there are three TetR-family transcriptional regulators (ZMO0281, ZMO0963, ZMO1547) and two RND-family efflux pumps (ZMO0282-0285, ZMO0964-0966) adjacent to corresponding TetR-family regulators of ZMO0281 and ZMO0963 in Z. mobilis. Genetics studies were then carried out with various mutants of TetR-family regulators constructed, and ZMO0281 was characterized to be related to acetate tolerance. Combining transcriptomics and dual-reporter gene system, this study demonstrated that three TetR-family regulators repressed their adjacent genes specifically. Moreover, TetR-family regulator ZMO0281 might also be involved in other cellular processes in the presence of acetate. In addition, the upregulation of RND-family efflux pumps due to ZMO0281 deletion might lead to an energy imbalance and decreased cell growth in Z. mobilis under acetate stress. The systematic investigation of all three TetR-family regulators and their roles on a major lignocellulosic inhibitor acetate tolerance in Z. mobilis thus not only unravels the molecular mechanisms of TetR-family regulators and their potential cross-talks on regulating RND-family efflux pumps and other genes in Z. mobilis, but also provides guidance on understanding the roles of multiple regulators of same family in Z. mobilis and other microorganisms for efficient lignocellulosic biochemical production.

Abstract 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 P gap 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.

High-quality genome-scale metabolic models (GEMs) could play critical roles on rational design of microbial cell factories in the classical Design-Build-Test-Learn cycle of synthetic biology studies. Despite of the constant establishment and update of GEMs for model microorganisms such as Escherichia coli and Saccharomyces cerevisiae, high-quality GEMs for non-model industrial microorganisms are still scarce. Zymomonas mobilis subsp. mobilis ZM4 is a non-model ethanologenic microorganism with many excellent industrial characteristics that has been developing as microbial cell factories for biochemical production. Although five GEMs of Z. mobilis have been constructed, these models are either generating ATP incorrectly, or lacking information of plasmid genes, or not providing standard format file. In this study, a high-quality GEM iZM516 of Z. mobilis ZM4 was constructed. The information from the improved genome annotation, literature, datasets of Biolog Phenotype Microarray studies, and recently updated Gene-Protein-Reaction information was combined for the curation of iZM516. Finally, 516 genes, 1389 reactions, 1437 metabolites, and 3 cell compartments are included in iZM516, which also had the highest MEMOTE score of 91% among all published GEMs of Z. mobilis. Cell growth was then predicted by iZM516, which had 79.4% agreement with the experimental results of the substrate utilization. In addition, the potential endogenous succinate synthesis pathway of Z. mobilis ZM4 was proposed through simulation and analysis using iZM516. Furthermore, metabolic engineering strategies to produce succinate and 1,4-butanediol (1,4-BDO) were designed and then simulated under anaerobic condition using iZM516. The results indicated that 1.68 mol/mol succinate and 1.07 mol/mol 1,4-BDO can be achieved through combinational metabolic engineering strategies, which was comparable to that of the model species E. coli. Our study thus not only established a high-quality GEM iZM516 to help understand and design microbial cell factories for economic biochemical production using Z. mobilis as the chassis, but also provided guidance on building accurate GEMs for other non-model industrial microorganisms.

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.

Abstract Background Lignocellulose-derived aldehyde inhibitors seriously blocked the biorefinery of biofuels and biochemicals. To date, the economic production of lignocellulose-based products heavily relied on high productivities of fermenting strains. However, it was expensive and time-consuming for the achievable rational modification to strengthen stress tolerance robustness of aldehyde inhibitors. Here, it aimed to improve aldehyde inhibitors tolerance and cellulosic bioethanol fermentability for the chassis Zymomonas mobilis ZM4 pretreated using energy-efficient and eco-friendly cold plasma. Results It was found that bioethanol fermentability was weaker in CSH (corn stover hydrolysates) than that in synthetic medium for Z. mobilis, and thus was attributed to the inhibition of the lignocellulose-derived aldehyde inhibitors in CSH. Convincingly, it further confirmed that the mixed aldehydes severely decreased bioethanol accumulation through additional aldehydes supplementary assays in synthetic medium. After assayed under different processing time (10–30 s), discharge power (80–160 W), and working pressure (120–180 Pa) using cold atmosphere plasma (CAP), it achieved the increased bioethanol fermentability for Z. mobilis after pretreated at the optimized parameters (20 s, 140 W and 165 Pa). It showed that cold plasma brought about three mutation sites including ZMO0694 (E220V), ZMO0843 (L471L) and ZMO0843 (P505H) via Genome resequencing-based SNPs (single nucleotide polymorphisms). A serial of differentially expressed genes (DEGs) were further identified as the potential contributors for stress tolerance via RNA-Seq sequencing, including ZMO0253 and ZMO_RS09265 (type I secretion outer membrane protein), ZMO1941 (Type IV secretory pathway protease TraF-like protein), ZMOr003 and ZMOr006 (16S ribosomal RNA), ZMO0375 and ZMO0374 (levansucrase) and ZMO1705 (thioredoxins). It enriched cellular process, followed by metabolic process and single-organism process for biological process. For KEGG analysis, the mutant was also referred to starch and sucrose metabolism, galactose metabolism and two-component system. Finally, but interestingly, it simultaneously achieved the enhanced stress tolerance capacity of aldehyde inhibitors and bioethanol fermentability in CSH for the mutant Z. mobilis. Conclusions Of several candidate genetic changes, the mutant Z. mobilis treated with cold plasma was conferred upon the facilitated aldehyde inhibitors tolerance and bioethanol production. This work would provide a strain biocatalyst for the efficient production of lignocellulosic biofuels and biochemicals.

Abstract Acetic acid and furfural (AF) are two major inhibitors of microorganisms during lignocellulosic ethanol production. In our previous study, we successfully engineered Zymomonas mobilis 532 (ZM532) strain by genome shuffling, but the molecular mechanisms of tolerance to inhibitors were still unknown. Therefore, this study investigated the responses of ZM532 and its wild-type Z. mobilis (ZM4) to AF using multi-omics approaches (transcriptomics, genomics, and label free quantitative proteomics). Based on RNA-Seq data, two differentially expressed genes, ZMO_RS02740 (up-regulated) and ZMO_RS06525 (down-regulated) were knocked out and over-expressed through CRISPR-Cas technology to investigate their roles in AF tolerance. Overall, we identified 1865 and 14 novel DEGs in ZM532 and wild-type ZM4. In contrast, 1532 proteins were identified in ZM532 and wild-type ZM4. Among these, we found 96 important genes in ZM532 involving acid resistance mechanisms and survival rates against stressors. Furthermore, our knockout results demonstrated that growth activity and glucose consumption of mutant strains ZM532∆ZMO_RS02740 and ZM4∆ZMO_RS02740 decreased with increased fermentation time from 42 to 55 h and ethanol production up to 58% in ZM532 than that in ZM532∆ZMO_RS02740. Hence, these findings suggest ZMO_RS02740 as a protective strategy for ZM ethanol production under stressful conditions.

Advances in synthetic biology have enabled the incorporation of novel biochemical pathways for the production of high-value products into industrially important bacterial hosts. However, attempts to redirect metabolic fluxes towards desired products often lead to the buildup of toxic or undesirable intermediates or, more generally, unwanted metabolic cross-talk. The use of shells derived from self-assembling protein-based prokaryotic organelles, referred to as bacterial microcompartments (BMCs), as a scaffold for metabolic enzymes represents a sophisticated approach that can both insulate and integrate the incorporation of challenging metabolic pathways into industrially important bacterial hosts. Here we took a synthetic biology approach and introduced the model shell system derived from the myxobacterium Haliangium ochraceum (HO shell) into the industrially relevant organism Zymomonas mobilis with the aim of constructing a BMC-based spatial scaffolding platform. SDS-PAGE, transmission electron microscopy, and dynamic light scattering analyses collectively demonstrated the ability to express and purify empty capped and uncapped HO shells from Z. mobilis. As a proof of concept to internally load or externally decorate the shell surface with enzyme cargo, we have successfully targeted fluorophores to the surfaces of the BMC shells. Overall, our results provide the foundation for incorporating enzymes and constructing BMCs with synthetic biochemical pathways for the future production of high-value products in Z. 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. [ABSTRACT FROM AUTHOR]

The microbe, Zymomonas mobilis , can efficiently convert sugars to 2,3 butanediol (BDO), an important intermediate for downstream chemical products, only in a well-controlled microaerated environment. However, controlling oxygen distribution in industrial-scale bioreactors is challenging, and at-scale BDO production is hard to optimize using traditional engineering methods. This study takes a step towards addressing this problem through a computational model for reacting multiphase flows in large-scale bioreactors. A phenomenological metabolic model was first developed and validated against experiments, and then it was coupled to a multiphase computational fluid dynamics (CFD) solver using a subcycling algorithm for simulating long conversion times (∼ 30 h). Large-scale (∼ 500 m3) bubble column simulations using this coupled approach demonstrated a 25% improvement in BDO yield with low sparging rates (0.002 m/s) and low height-to-diameter ratio (0.875) compared to a baseline bubble column (35 m height, 5 m diameter) with a superficial gas velocity of 0.06 m/s. [Display omitted] • A computational model for butanediol production in bubble columns is presented. • A validated bioreaction kinetics model is coupled to multiphase flow equations. • Large time scale disparity between transport and chemical kinetics is overcome through a subcycling algorithm. • Lower aspect ratio bubble columns and homogenous microaerated environments improve BDO yields by 25%. [ABSTRACT FROM AUTHOR]

BACKGROUND: The present study aimed to demonstrate the superiority of bioethanol yield and its quality from sorghum using the granular starch degrading enzyme Stargen™ 002 over simultaneous saccharification and fermentation, and separate hydrolysis and fermentation using Zymomonas mobilis CCM 3881 and Ethanol Red® yeast. RESULTS: Bacteria were found to produce ethanol at higher yield than the yeast in all fermentations. The highest ethanol yield was obtained with Z. mobilis during 48 h of simultaneous saccharification and fermentation (83.85% theoretical yield) and fermentation with Stargen™ 002 (81.27% theoretical yield). Pre‐liquefaction in fermentation with Stargen™ 002 did not improve ethanol yields for both Z. mobilis and Saccharomyces cerevisiae. Chromatographic analysis showed twice less total volatile compounds in distillates obtained after bacterial (3.29–5.54 g L−1) than after yeast (7.84–9.75 g L−1) fermentations. Distillates obtained after bacterial fermentation were characterized by high level of aldehydes (up to 65% of total volatiles) and distillates obtained after yeast fermentation of higher alcohols (up to 95% of total volatiles). The process of fermentation using granular starch hydrolyzing enzyme cocktail Stargen™ 002 resulted in low amounts of all volatile compounds in distillates obtained after bacterial fermentation, but the highest amounts in distillates obtained after yeast fermentation. CONCLUSION: The present study emphasizes the great potential of bioethanol production from sorghum with Z. mobilis using granular starch hydrolyzing enzyme Stargen™ 002, which leads to reduced water and energy consumption, especially when energy sources are strongly related to global climate change. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Genomic DNA has high phosphate content; therefore, monoploid prokaryotes need an external phosphate source or an internal phosphate storage polymer for replication and cell division. For two polyploid prokaryotic species, the halophilic archaeon Haloferax volcanii and the cyanobacterium Synechocystis PCC 6803, it has been reported that they can grow in the absence of an external phosphate source by reducing the genome copy number per cell. To unravel whether this feature might be widespread in and typical for polyploid prokaryotes, three additional polyploid prokaryotic species were analyzed in the present study, i.e., the alphaproteobacterium Zymomonas mobilis, the gammaproteobacterium Azotobacter vinelandii, and the haloarchaeon Halobacterium salinarum. Polyploid cultures were incubated in the presence and in the absence of external phosphate, growth was recorded, and genome copy numbers per cell were quantified. Limited growth in the absence of phosphate was observed for all three species. Phosphate was added to phosphate-starved cultures to verify that the cells were still viable and growth-competent. Remarkably, stationary-phase cells grown in the absence or presence of phosphate did not become monoploid but stayed oligoploid with about five genome copies per cell. As a negative control, it was shown that monoploid Escherichia coli cultures did not exhibit any growth in the absence of phosphate. Taken together, all five polyploid prokaryotic species that have been characterized until now can grow in the absence of environmental phosphate by reducing their genome copy numbers, indicating that cell proliferation outperforms other evolutionary advantages of polyploidy. [ABSTRACT FROM AUTHOR]

In the current investigation, the woody stem of Moringa oleifera was processed by chipping and milling and was subsequently exposed to a combination of pretreatments involving a 3% v/v solution of nitric acid and autohydrolysis. The simultaneous saccharification and fermentation (SSF) of the pretreated hydrolysate of M. oleifera was conducted using Zymomonas mobilis and Kluyveromyces marxianus in occurrence of commercial cellulase enzyme, Tween 80, and sodium azide. The fermentation process parameters for Z. mobilis were optimized individually, including a substrate concentration of 5% (w/v), concentration of inoculum 5% (v/v), pH 5.4, and temperature 34 °C. Similarly, for K. marxianus, the process parameters were optimized individually, with a substrate concentration of 5% (w/v), an inoculum concentration of 3% (v/v), a pH of 5.1, and a temperature of 41 °C. The highest cellulosic ethanol concentration was achieved by the micro-organism K. marxianus after a fermentation period of 96 h. [ABSTRACT FROM AUTHOR]

High-temperature ethanol fermentation (HTEF) using high-potential thermotolerant ethanologenic microorganisms is a promising platform for ethanol production in tropical or subtropical areas. This study aims to evaluate the ethanol production potential of recombinant Zymomonas mobilis R301 overexpressing groESL genes under normal and high-temperature conditions and the expression of genes involved in the heat shock response and ethanol production pathway during ethanol fermentation using sweet sorghum juice (SSJ) as feedstock. Growth characterization analysis revealed that the recombinant Z. mobilis R301 exhibited multi-stress tolerance toward heat, acetic acid, and furfural. Based on the statistical experimental design, the optimum conditions for ethanol production from SSJ by the recombinant R301 at 30 °C were a sugar concentration of 171.67 g/L, cell concentration of 9.42% (v/v), and yeast extract concentration of 10.89 g/L, while those at 40 °C were a sugar concentration of 199.48 g/L, yeast extract concentration of 10.88 g/L, MgSO4 concentration of 1.05 g/L, and initial pH of 6.8. The maximum ethanol concentrations and productivities achieved in this study were 63.26 g/L and 1.17 g/L.h at 30 °C and 58.62 g/L and 1.22 g/L.h at 40 °C. The overexpression of the groES and groEL genes and upregulation of other heat shock-responsive genes at 40 °C enhanced cell growth, viability, and fermentation capacity of recombinant Z. mobilis R301 under heat stress. The current study demonstrated that recombinant Z. mobilis R301 exhibited high potential for ethanol production from SSJ or other sugar-based raw materials under high-temperature conditions. [ABSTRACT FROM AUTHOR]

Background: Lignocellulose-derived aldehyde inhibitors seriously blocked the biorefinery of biofuels and biochemicals. To date, the economic production of lignocellulose-based products heavily relied on high productivities of fermenting strains. However, it was expensive and time-consuming for the achievable rational modification to strengthen stress tolerance robustness of aldehyde inhibitors. Here, it aimed to improve aldehyde inhibitors tolerance and cellulosic bioethanol fermentability for the chassis Zymomonas mobilis ZM4 pretreated using energy-efficient and eco-friendly cold plasma. Results: It was found that bioethanol fermentability was weaker in CSH (corn stover hydrolysates) than that in synthetic medium for Z. mobilis, and thus was attributed to the inhibition of the lignocellulose-derived aldehyde inhibitors in CSH. Convincingly, it further confirmed that the mixed aldehydes severely decreased bioethanol accumulation through additional aldehydes supplementary assays in synthetic medium. After assayed under different processing time (10–30 s), discharge power (80–160 W), and working pressure (120–180 Pa) using cold atmosphere plasma (CAP), it achieved the increased bioethanol fermentability for Z. mobilis after pretreated at the optimized parameters (20 s, 140 W and 165 Pa). It showed that cold plasma brought about three mutation sites including ZMO0694 (E220V), ZMO0843 (L471L) and ZMO0843 (P505H) via Genome resequencing-based SNPs (single nucleotide polymorphisms). A serial of differentially expressed genes (DEGs) were further identified as the potential contributors for stress tolerance via RNA-Seq sequencing, including ZMO0253 and ZMO_RS09265 (type I secretion outer membrane protein), ZMO1941 (Type IV secretory pathway protease TraF-like protein), ZMOr003 and ZMOr006 (16S ribosomal RNA), ZMO0375 and ZMO0374 (levansucrase) and ZMO1705 (thioredoxins). It enriched cellular process, followed by metabolic process and single-organism process for biological process. For KEGG analysis, the mutant was also referred to starch and sucrose metabolism, galactose metabolism and two-component system. Finally, but interestingly, it simultaneously achieved the enhanced stress tolerance capacity of aldehyde inhibitors and bioethanol fermentability in CSH for the mutant Z. mobilis. Conclusions: Of several candidate genetic changes, the mutant Z. mobilis treated with cold plasma was conferred upon the facilitated aldehyde inhibitors tolerance and bioethanol production. This work would provide a strain biocatalyst for the efficient production of lignocellulosic biofuels and biochemicals. [ABSTRACT FROM AUTHOR]

Xylose fermentation is an essential issue for lignocellulosic ethanol production, which is often inhibited by degradation products generated during biomass pretreatment. In this study, enzymatic hydrolysate of dilute acid pretreated corn stover was fermented using Zymomonas mobilis 8b. With the solid loading of corn stover increased from 5 to 24 wt.%, the xylose consumption volume decreased from 97.0 to 21.5%. The ethanol produced ranged from 7.8 to 28.6 g/L. In order to improve xylose consumption, corn slurry was integrated into the corn stover ethanol process. Integration strategies, glucoamylase dosage, and corn–corn stover mixing ratio were investigated. Results showed that introducing corn slurry into corn stover hydrolysate was an effective way to improve xylose fermentation as well as enhance ethanol titer. Specifically, when liquefied corn slurry was introduced into corn stover hydrolysate at the ratio of 1:1 and total solid loading of 24%, the xylose consumption volume exceeded 88% with the final ethanol titer reached 68.6 g/L. This study indicates that integration of the first- and the second-generation ethanol processes is beneficial to xylose fermentation. [ABSTRACT FROM AUTHOR]

The microbial production of fuel ethanol is an attractive and sustainable biotechnological approach. This study presents a metabolic engineering strategy of Zymomonas mobilis aimed at coproducing bioethanol and fatty acids. The increased flux of fatty acids stabilizes the cell membrane and thus counteracts the progressively higher ethanol toxicity. In a glucose medium, the highest ethanol titer achieved was 146.7 g/kg of broth, surpassing the wild-type Z. mobilis CP4 and angel yeast by 30% and 45%, respectively. The recombinant strain exhibited a total fatty acid titer of 0.4 g/L from 230 g/L total sugar solution (5 L bioreactor), representing a 12-fold increase compared to the wild-type Z. mobilis CP4. Furthermore, when using a 4:2:1 mixture of glucose: xylose: mannose (w/v), an ethanol concentration of 142.8 g/kg of broth was attained, only 2.66% lower than that of the glucose-only medium. These findings highlight the enormous potential of this genetically engineered strain for the sustainable production of ethanol and fatty acids from lignocellulosic renewable carbon sources. [ABSTRACT FROM AUTHOR]

Acetic acid and furfural (AF) are two major inhibitors of microorganisms during lignocellulosic ethanol production. In our previous study, we successfully engineered Zymomonas mobilis 532 (ZM532) strain by genome shuffling, but the molecular mechanisms of tolerance to inhibitors were still unknown. Therefore, this study investigated the responses of ZM532 and its wild-type Z. mobilis (ZM4) to AF using multi-omics approaches (transcriptomics, genomics, and label free quantitative proteomics). Based on RNA-Seq data, two differentially expressed genes, ZMO_RS02740 (up-regulated) and ZMO_RS06525 (down-regulated) were knocked out and over-expressed through CRISPR-Cas technology to investigate their roles in AF tolerance. Overall, we identified 1865 and 14 novel DEGs in ZM532 and wild-type ZM4. In contrast, 1532 proteins were identified in ZM532 and wild-type ZM4. Among these, we found 96 important genes in ZM532 involving acid resistance mechanisms and survival rates against stressors. Furthermore, our knockout results demonstrated that growth activity and glucose consumption of mutant strains ZM532∆ZMO_RS02740 and ZM4∆ZMO_RS02740 decreased with increased fermentation time from 42 to 55 h and ethanol production up to 58% in ZM532 than that in ZM532∆ZMO_RS02740. Hence, these findings suggest ZMO_RS02740 as a protective strategy for ZM ethanol production under stressful conditions. [ABSTRACT FROM AUTHOR]

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Cassava is one of the most widely produced agricultural products in Indonesia with cassava peel waste of 20%. Cassava peel contains carbohydrates and stores a high cellulose content so that it has a potential to be a bioethanol. This study aims to utilize cassava peel waste for bioethanol production with an alkaline pretreatment process, cellulase enzyme hydrolysis and fermentation by using Zymomonas mobilis bacteria. Alkaline pretreatment with 14% NaOH is used to hydrolyze lignocellulose. The hydrolysis optimization process enzymatically applies the Surface Response Method (RSM) to determine the optimum conditions at hydrolysis pH in the range of 2-10 and hydrolysis temperature in the range of 30-70 °C by analyzing glucose levels using the Dinitrosalicylic Acid (DNS) method and UV-vis spectrophotometry instruments. Surface Response Method (RSM) is likewise implemented to decide the greatest conditions of the fermentation process. The pH measurement ranges 2-10, and fermentation time takes 6 to 168 hours. Based on the results of research, it results a lignin content of 6.68% (b/b), cellulose content of 58.4% (b/b), and hemicellulose content of 27.3% (b/b). The optimum conditions of the hydrolysis process obtained an optimum glucose level of 9.22mg/mL at pH 2 and a hydrolysis temperature of 50°C. The optimum conditions of the fermentation process use Zymomonas mobilis at pH 6 while fermentation time takes 168 hours analyzed using a refractometer produced a bioethanol content of 37.75% (v/v) and a gas chromatography produced a bioethanol content of 54.94% (v/v). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The second generation (2 G) biofuels were introduced to solve the issues associated with first-generation biofuel (dependency on food materials) and fossil fuels, such as reservoirs diminution, high demand, price fluctuation, and lethal greenhouse gases emission. Butanol and ethanol are the main 2 G biofuels. They are used as a disinfectant, antiseptic, and chemical solvent in the pharmaceutical, plastic, textiles, cosmetics, and fuel industries. Currently, their bacterial biological production from lignocellulosic material at the industrial level with primitive microorganisms is under development and not economical and qualitative compatible as compared to that of fossil origin, due to the slow growth rate, low titer, recalcitrant nature of lignocellulose, strain intolerance to a higher amount of butanol and ethanol, and strain inability to tolerate inhibitors accumulated during pretreatment of lignocellulosic materials. Therefore, metabolic engineering strategies such as redirection of carbon flux, knocking out competing pathways, enhancing strain robustness and wide range of substrate utilization ability, and overexpression of enzymes involved in their biological synthesis have been applied to bacteria for enhancing their ability for 2 G ethanol and butanol production in a highly cost-effective amount from lignocellulosic materials. Herein, we summarized and reviewed the progress in metabolic engineering of bacterial species such as Clostridium spp, Escherichia coli , and Zymomonas mobilis for the synthesis of 2 G butanol and ethanol, especially from lignocellulosic materials. [ABSTRACT FROM AUTHOR]