RESUMEN
Rheostat positions, which can be substituted with various amino acids to tune protein function across a range of outcomes, are a developing area for advancing personalized medicine and bioengineering. Current methods cannot accurately predict which proteins contain rheostat positions or their substitution outcomes. To compare the prevalence of rheostat positions in homologs, we previously investigated their occurrence in two pyruvate kinase (PYK) isozymes. Human liver PYK contained numerous rheostat positions that tuned the apparent affinity for the substrate phosphoenolpyruvate (Kapp-PEP) across a wide range. In contrast, no functional rheostat positions were identified in Zymomonas mobilis PYK (ZmPYK). Further, the set of ZmPYK substitutions included an unusually large number that lacked measurable activity. We hypothesized that the inactive substitution variants had reduced protein stability, precluding detection of Kapp-PEP tuning. Using modified buffers, robust enzymatic activity was obtained for 19 previously-inactive ZmPYK substitution variants at three positions. Surprisingly, both previously-inactive and previously-active substitution variants all had Kapp-PEP values close to wild-type. Thus, none of the three positions were functional rheostat positions, and, unlike human liver PYK, ZmPYK's Kapp-PEP remained poorly tunable by single substitutions. To directly assess effects on stability, we performed thermal denaturation experiments for all ZmPYK substitution variants. Many diminished stability, two enhanced stability, and the three positions showed different thermal sensitivity to substitution, with one position acting as a "stability rheostat." The differences between the two PYK homologs raises interesting questions about the underlying mechanism(s) that permit functional tuning by single substitutions in some proteins but not in others.
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Piruvato Quinasa , Zymomonas , Humanos , Zymomonas/enzimología , Zymomonas/genética , Zymomonas/química , Zymomonas/metabolismo , Piruvato Quinasa/química , Piruvato Quinasa/metabolismo , Piruvato Quinasa/genética , Sustitución de Aminoácidos , Estabilidad Proteica , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Estabilidad de Enzimas , Hígado/enzimología , Hígado/metabolismo , Hígado/química , Fosfoenolpiruvato/metabolismo , Fosfoenolpiruvato/químicaRESUMEN
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.
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Biocombustibles , Etanol , Fermentación , Ingeniería Metabólica , Estrés Fisiológico , Zymomonas , Zymomonas/metabolismo , Zymomonas/genética , Etanol/metabolismo , Ingeniería Metabólica/métodosRESUMEN
Isoprenoids are a diverse family of compounds that are synthesized from two isomeric compounds, isopentenyl diphosphate and dimethylallyl diphosphate. In most bacteria, isoprenoids are produced from the essential methylerythritol phosphate (MEP) pathway. The terminal enzymes of the MEP pathway IspG and IspH are [4Fe-4S] cluster proteins, and in Zymomonas mobilis, the substrates of IspG and IspH accumulate in cells in response to O2, suggesting possible lability of their [4Fe-4S] clusters. Here, we show using complementation assays in Escherichia coli that even under anaerobic conditions, Z. mobilis IspG and IspH are not as functional as their E. coli counterparts, requiring higher levels of expression to rescue viability. A deficit of the sulfur utilization factor (SUF) Fe-S cluster biogenesis pathway did not explain the reduced function of Z. mobilis IspG and IspH since no improvement in viability was observed in E. coli expressing the Z. mobilis SUF pathway or having increased expression of the E. coli SUF pathway. Complementation of single and double mutants with various combinations of Z. mobilis and E. coli IspG and IspH indicated that optimal growth required the pairing of IspG and IspH from the same species. Furthermore, Z. mobilis IspH conferred an O2-sensitive growth defect to E. coli that could be partially rescued by co-expression of Z. mobilis IspG. In vitro analysis showed O2 sensitivity of the [4Fe-4S] cluster of both Z. mobilis IspG and IspH. Altogether, our data indicate an important role of the cognate protein IspG in Z. mobilis IspH function under both aerobic and anaerobic conditions. IMPORTANCE: Isoprenoids are one of the largest classes of natural products, exhibiting diversity in structure and function. They also include compounds that are essential for cellular life across the biological world. In bacteria, isoprenoids are derived from two precursors, isopentenyl diphosphate and dimethylallyl diphosphate, synthesized primarily by the methylerythritol phosphate pathway. The aerotolerant Z. mobilis has the potential for methylerythritol phosphate pathway engineering by diverting some of the glucose that is typically efficiently converted into ethanol to produce isoprenoid precursors to make bioproducts and biofuels. Our data revealed the surprising finding that Z. mobilis IspG and IspH need to be co-optimized to improve flux via the methyl erythritol phosphate pathway in part to evade the oxygen sensitivity of IspH.
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Proteínas Bacterianas , Eritritol , Escherichia coli , Zymomonas , Zymomonas/metabolismo , Zymomonas/enzimología , Zymomonas/genética , Eritritol/metabolismo , Eritritol/análogos & derivados , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/enzimología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas Hierro-Azufre/metabolismo , Proteínas Hierro-Azufre/genética , Terpenos/metabolismo , OxidorreductasasRESUMEN
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.
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Adaptación Fisiológica , Proteínas Bacterianas , Cromosomas Bacterianos , Regulación Bacteriana de la Expresión Génica , Factores de Transcripción , Adaptación Fisiológica/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Cromosomas Bacterianos/química , Cromosomas Bacterianos/genética , Regulación Bacteriana de la Expresión Génica/genética , Mutación , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Estrés Fisiológico/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Zymomonas/genética , Zymomonas/metabolismo , Conformación de Ácido NucleicoRESUMEN
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.
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Escherichia coli , Etanol , Ácido Láctico , Ingeniería Metabólica , Piruvato Descarboxilasa , Zymomonas , Zymomonas/metabolismo , Zymomonas/genética , Piruvato Descarboxilasa/metabolismo , Piruvato Descarboxilasa/genética , Ingeniería Metabólica/métodos , Etanol/metabolismo , Ácido Láctico/metabolismo , Ácido Láctico/biosíntesis , Escherichia coli/metabolismo , Escherichia coli/genética , L-Lactato Deshidrogenasa/metabolismo , L-Lactato Deshidrogenasa/genética , Alanina/metabolismo , Ácido Pirúvico/metabolismo , FermentaciónRESUMEN
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.
Asunto(s)
Saccharum , Zymomonas , Celulosa , Sorbitol , Melaza , Maltosa , SacarosaRESUMEN
Rapid adaptation of metabolic capabilities is crucial for bacterial survival in habitats with fluctuating nutrient availability. In such conditions, the bacterial stringent response is a central regulatory mechanism activated by nutrient starvation or other stressors. This response is primarily controlled by exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) enzymes. To gain further insight into these enzymes, the high-resolution crystal structure of PPX from Zymomonas mobilis (ZmPPX) was determined at 1.8 Å. The phosphatase activity of PPX was strictly dependent on the presence of divalent metal cations. Notably, the structure of ZmPPX revealed the presence of two magnesium ions in the active site center, which is atypical compared to other PPX structures where only one divalent ion is observed. ZmPPX exists as a dimer in solution and belongs to the "long" PPX group consisting of four domains. Remarkably, the dimer configuration exhibits a substantial and deep aqueduct with positive potential along its interface. This aqueduct appears to extend towards the active site region, suggesting that this positively charged aqueduct could potentially serve as a binding site for polyP.
Asunto(s)
Magnesio , Zymomonas , Zymomonas/metabolismo , Ácido Anhídrido Hidrolasas/química , Ácido Anhídrido Hidrolasas/metabolismo , Bacterias/metabolismo , IonesRESUMEN
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.
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Zymomonas , Zymomonas/genética , Zymomonas/metabolismo , Plásmidos , Vectores Genéticos , Expresión GénicaRESUMEN
The bacterium Zymomonas mobilis is best known for fermentatively producing more ethanol than yeast. However, Z. mobilis has also puzzled researchers for decades with the counterintuitive observation that disrupting aerobic respiration benefits aerobic growth, implying that fermentation remains favorable. Retention of detrimental respiration genes seemed to defy natural selection. New findings by Felczak et al. help clarify the importance of respiration for Z. mobilis and the factors that led to the confusing prior results (M. M. Felczak, M. P. Bernard, and M. A. TerAvest, 2023, mBio 14:e02043-23, https://doi.org/10.1128/mbio.02043-23). The team overcame redundancy from multiple genome copies to delete what turned out to be a key terminal oxidase. Unlike previous studies, wherein mutants exhibited low respiration rates and had improved aerobic growth, this mutant was incapable of respiration and had poor aerobic growth. Thus, respiration is important but surprisingly exceeds what is optimal under lab conditions. Respiration likely protects against toxic effects of oxygen, ensuring retention of respiration genes in the Z. mobilis genome.
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Zymomonas , Zymomonas/genética , Fermentación , Etanol , Bacterias , Respiración , Saccharomyces cerevisiaeRESUMEN
Zymomonas mobilis (Z. mobilis), a bacterium known for its ethanol production capabilities, can also generate electricity by transitioning from ethanol production to electron generation. The purpose of this study is to investigate the ability of Z. mobilis to produce bioelectricity when utilized as a biocatalyst in a single-chamber microbial fuel cell (MFC). Given the bacterium's strong inclination towards ethanol production, a metabolic engineering strategy was devised to identify key reactions responsible for redirecting electrons from ethanol towards electricity generation. To evaluate the electroactivity of cultured Z. mobilis and its ethanol production in the presence of regulators, the reduction of soluble Fe(III) was utilized. Among the regulators tested, CuCl2 demonstrated superior effectiveness. Consequently, the MFC was employed to analyze the electrochemical properties of Z. mobilis using both a minimal and modified medium. By modifying the bacterial medium, the maximum current and power density of the MFC fed with Z. mobilis increased by more than 5.8- and sixfold, respectively, compared to the minimal medium. These findings highlight the significant impact of metabolic redirection in enhancing the performance of MFCs. Furthermore, they establish Z. mobilis as an active electrogenesis microorganism capable of power generation in MFCs.
Asunto(s)
Fuentes de Energía Bioeléctrica , Zymomonas , Etanol/metabolismo , Zymomonas/genética , Zymomonas/metabolismo , Compuestos Férricos/metabolismo , FermentaciónRESUMEN
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.
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Zymomonas , Zymomonas/metabolismo , Plásmidos/genética , Edición Génica , Fenilalanina/metabolismoRESUMEN
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.
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Riboswitch , Zymomonas , Zymomonas/genética , Riboswitch/genética , Teofilina/metabolismo , Plásmidos/genética , Edición Génica/métodos , Sistemas CRISPR-CasRESUMEN
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.
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Piruvato Quinasa , Zymomonas , Humanos , Piruvato Quinasa/metabolismo , Zymomonas/metabolismo , Sitios de Unión , Metabolismo de los Hidratos de Carbono , Piruvatos , Regulación AlostéricaRESUMEN
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.
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Ácido Acético , Zymomonas , Ácido Acético/metabolismo , Zymomonas/genética , Furaldehído/metabolismo , Barajamiento de ADN , Fermentación , Etanol/metabolismoRESUMEN
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.
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Sorghum , Zymomonas , Saccharomyces cerevisiae , Etanol , Fermentación , AlmidónRESUMEN
Molasses with abundant sugars is widely used for bioethanol production. Although the ethanologenic bacterium Zymomonas mobilis can use glucose, fructose, and sucrose for ethanol production, levan production from sucrose reduces the ethanol yield of molasses fermentation. To increase ethanol production from sucrose-rich molasses, Z. mobilis was adapted in molasses, sucrose, and fructose in parallel. Adaptation in fructose is the most effective route to generate an evolved strain F74 with improved molasses utilization, which is majorly due to a G99S mutation in Glf for enhanced fructose import. Subsequent sacB deletion and sacC overexpression in F74 to divert sucrose metabolism from levan production to ethanol production further enhanced ethanol productivity 28.6% to 1.35 g/L/h. The efficient utilization of molasses by diverting sucrose metabolic flux through adaptation and genome engineering not only generated an excellent ethanol producer using molasses but also provided the strategy for developing microbial cell factories.
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Ingeniería Metabólica , Zymomonas , Zymomonas/genética , Melaza , Sacarosa/metabolismo , Fructosa/metabolismo , Etanol/metabolismo , Fructanos/metabolismo , FermentaciónRESUMEN
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.
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Oryza , Zymomonas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Oryza/microbiología , Hidróxido de Sodio , Zymomonas/genética , Zymomonas/metabolismo , Etanol , Fermentación , Celulosa/metabolismo , Carbohidratos , Azúcares , HidrólisisRESUMEN
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.
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Zymomonas , Zymomonas/genética , Zymomonas/metabolismo , Glucosa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Transporte Biológico , Etanol/metabolismoRESUMEN
Zymomonas mobilis is an industrially relevant aerotolerant anaerobic bacterium that can convert up to 96% of consumed glucose to ethanol. This highly catabolic metabolism could be leveraged to produce isoprenoid-based bioproducts via the methylerythritol 4-phosphate (MEP) pathway, but we currently have limited knowledge concerning the metabolic constraints of this pathway in Z. mobilis. Here, we performed an initial investigation of the metabolic bottlenecks within the MEP pathway of Z. mobilis using enzyme overexpression strains and quantitative metabolomics. Our analysis revealed that 1-deoxy-d-xylulose 5-phosphate synthase (DXS) represents the first enzymatic bottleneck in the Z. mobilis MEP pathway. DXS overexpression triggered large increases in the intracellular levels of the first five MEP pathway intermediates, of which the buildup in 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (MEcDP) was the most substantial. The combined overexpression of DXS, 4-hydroxy-3-methylbut-2-enyl diphosphate (HMBDP) synthase (IspG), and HMBDP reductase (IspH) mitigated the bottleneck at MEcDP and mobilized carbon to downstream MEP pathway intermediates, indicating that IspG and IspH activity become the primary pathway constraints during DXS overexpression. Finally, we overexpressed DXS with other native MEP enzymes and a heterologous isoprene synthase and showed that isoprene can be used as a carbon sink in the Z. mobilis MEP pathway. By revealing key bottlenecks within the MEP pathway of Z. mobilis, this study will aid future engineering efforts aimed at developing this bacterium for industrial isoprenoid production. IMPORTANCE Engineered microorganisms have the potential to convert renewable substrates into biofuels and valuable bioproducts, which offers an environmentally sustainable alternative to fossil-fuel-derived products. Isoprenoids are a diverse class of biologically derived compounds that have commercial applications as various commodity chemicals, including biofuels and biofuel precursor molecules. Thus, isoprenoids represent a desirable target for large-scale microbial generation. However, our ability to engineer microbes for the industrial production of isoprenoid-derived bioproducts is limited by an incomplete understanding of the bottlenecks in the biosynthetic pathway responsible for isoprenoid precursor generation. In this study, we combined genetic engineering with quantitative analyses of metabolism to examine the capabilities and constraints of the isoprenoid biosynthetic pathway in the industrially relevant microbe Zymomonas mobilis. Our integrated and systematic approach identified multiple enzymes whose overexpression in Z. mobilis results in an increased production of isoprenoid precursor molecules and mitigation of metabolic bottlenecks.
Asunto(s)
Zymomonas , Zymomonas/genética , Biocombustibles , Composición de Base , Análisis de Secuencia de ADN , Filogenia , ARN Ribosómico 16S/metabolismo , Terpenos/metabolismo , Fosfatos/metabolismoRESUMEN
Cell-free systems have become valuable investigating tools for metabolic engineering research due to their easy access to metabolism without the interference of the membrane. Therefore, we applied Zymomonas mobilis cell-free system to investigate whether ethanol production is controlled by the genes of the metabolic pathway or is limited by cofactors. Initially, different glucose concentrations were added to the extract to determine the crude extract's capability to produce ethanol. Then, we investigated the genes of the metabolic pathway to find the limiting step in the ethanol production pathway. Next, to identify the bottleneck gene, a systemic approach was applied based on the integration of gene expression data on a cell-free metabolic model. ZMO1696 was determined as the bottleneck gene and an activator for its enzyme was added to the extract to experimentally assess its effect on ethanol production. Then the effect of NAD+ addition at the high concentration of glucose (1 M) was evaluated, which indicates no improvement in efficiency. Finally, the imbalance ratio of ADP/ATP was found as the controlling factor by measuring ATP levels in the extract. Furthermore, sodium gluconate as a carbon source was utilized to investigate the expansion of substrate consumption by the extract. 100% of the maximum theoretical yield was obtained at 0.01 M of sodium gluconate while it cannot be consumed by Z. mobilis. This research demonstrated the challenges and advantages of using Z. mobilis crude extract for overproduction.