Genome-scale modeling and in silico analysis of ethanologenic bacteria Zymomonas mobilis

Bioethanol has been recognized as a potential alternative energy source. Among various ethanol-producing microbes, Zymomonas mobilis has acquired special attention due to its higher ethanol yield and tolerance. However, cellular metabolism in Z. mobilis remains unclear, hindering its practical application for bioethanol production. To elucidate such physiological characteristics, we reconstructed and validated a genome-scale metabolic network (iZM363) of Z. mobilis ATCC31821 (ZM4) based on its annotated genome and biochemical information. The phenotypic behaviors and metabolic states predicted by our genome-scale model were highly consistent with the experimental observations of Z. mobilis ZM4 strain growing on glucose as well as NMR-measured intracellular fluxes of an engineered strain utilizing glucose, fructose, and xylose. Subsequent comparative analysis with Escherichia coli and Saccharomyces cerevisiae as well as gene essentiality and flux coupling analyses have also confirmed the functional role of pdc and adh genes in the ethanologenic activity of Z. mobilis, thus leading to better understanding of this natural ethanol producer. In future, the current model could be employed to identify potential cell engineering targets, thereby enhancing the productivity of ethanol in Z. mobilis.     

Over-expression of xylulokinase in a xylose-metabolising recombinant strain of Zymomonas mobilis

The broad host range vector pBBR1MCS-2 has been evaluated as an expression vector for Zymomonas mobilis. The transformation efficiency of this vector was 2 x 10(3) CFU per mug of DNA in a recombinant strain of Z. mobilis ZM4/AcR containing the plasmid pZB5. Stable replication for this expression vector was demonstrated for 50 generations. This vector was used to study xylose metabolism in acetate resistant Z. mobilis ZM4/AcR (pZB5) by over-expression of xylulokinase (XK), as previous studies had suggested that XK could be the rate-limiting enzyme for such strains. Based on the above vector, a recombinant plasmid pJX1 harboring xylB (expressing XK) under control of a native Z. mobilis promotor Ppdc was constructed. When this plasmid was introduced into ZM4/AcR (pZB5) a 3-fold higher XK expression was found compared to the control strain. However, fermentation studies with ZM4/AcR (pZB5, pJX1) on xylose medium did not result in any increase in rate of growth or xylose metabolism, suggesting that XK expression was not rate-limiting for ZM4/AcR (pZB5) and related strains.     

Kinetic and nuclear magnetic resonance studies of xylose metabolism by recombinant Zymomonas mobilis ZM4(pZB5)

The specific rates of growth, substrate utilization, and ethanol production as well as yields of biomass and ethanol production on xylose for the recombinant Zymomonas mobilis ZM4(pZB5) were shown to be much less than those on glucose or glucose-xylose mixtures. Typical fermentations with ZM4(pZB5) growing on glucose-xylose mixtures followed two-phase growth kinetics with the initial uptakes of glucose and xylose being followed by slower growth and metabolic uncoupling on xylose after glucose depletion. The reductions in rates and yields from xylose metabolism were considered in the present investigation and may be due to a number of factors, including the following: (i) the increased metabolic burden from maintenance of plasmid-related functions, (ii) the production of by-products identified as xylitol, acetate, lactate, acetoin, and dihydroxyacetone by (13)C-nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography, (iii) growth inhibition due to xylitol by the putative inhibitory compound xylitol phosphate, and (iv) the less energized state of ZM4(pZB5). In vivo (31)P-NMR studies have established that the levels of NTP and UDP sugars on xylose were less than those on glucose, and this energy limitation is likely to restrict the growth of the recombinant strain on xylose media.     

Genome sequence of the ethanol-producing Zymomonas mobilis subsp. pomaceae lectotype strain ATCC 29192

Zymomonas mobilis is an alphaproteobacterium studied for bioethanol production. Different strains of this organism have been hitherto sequenced; they all belong to the Z. mobilis subsp. mobilis taxon. Here we report the finished and annotated genome sequence of strain ATCC 29192, a cider-spoiling agent isolated in the United Kingdom. ATCC 29192 is the lectotype of the second-best-characterized subspecies of Z. mobilis, Z. mobilis subsp. pomaceae. The nucleotide sequence of ATCC 29192 deviates from that of Z. mobilis subsp. mobilis representatives, which justifies its distinct taxonomic positioning and proves particularly useful for comparative and functional genomic analyses.     

Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli.

Lactose and all of the major sugars (glucose, xylose, arabinose, galactose, and mannose) present in cellulose and hemicellulose were converted to ethanol by recombinant Escherichia coli containing plasmid-borne genes encoding the enzymes for the ethanol pathway from Zymomonas mobilis. Environmental tolerances, plasmid stability, expression of Z. mobilis pyruvate decarboxylase, substrate range, and ethanol production (from glucose, lactose, and xylose) were compared among eight American Type Culture Collection strains. E. coli ATCC 9637(pLO1297), ATCC 11303(pLO1297), and ATCC 15224(pLO1297) were selected for further development on the basis of environmental hardiness and ethanol production. Volumetric ethanol productivities per hour in batch culture were 1.4 g/liter for glucose (12%), 1.3 g/liter for lactose (12%), and 0.64 g/liter for xylose (8%). Ethanol productivities per hour ranged from 2.1 g/g of cell dry weight with 12% glucose to 1.3 g/g of cell dry weight with 8% xylose. The ethanol yield per gram of xylose was higher for recombinant E. coli than commonly reported for Saccharomyces cerevisiae with glucose. Glucose (12%), lactose (12%), and xylose (8%) were converted to (by volume) 7.2% ethanol, 6.5% ethanol, and 5.2% ethanol, respectively.     

Efficient ethanol production from glucose, lactose, and xylose by recombinant Escherichia coli

Lactose and all of the major sugars (glucose, xylose, arabinose, galactose, and mannose) present in cellulose and hemicellulose were converted to ethanol by recombinant Escherichia coli containing plasmid-borne genes encoding the enzymes for the ethanol pathway from Zymomonas mobilis. Environmental tolerances, plasmid stability, expression of Z. mobilis pyruvate decarboxylase, substrate range, and ethanol production (from glucose, lactose, and xylose) were compared among eight American Type Culture Collection strains. E. coli ATCC 9637(pLO1297), ATCC 11303(pLO1297), and ATCC 15224(pLO1297) were selected for further development on the basis of environmental hardiness and ethanol production. Volumetric ethanol productivities per hour in batch culture were 1.4 g/liter for glucose (12%), 1.3 g/liter for lactose (12%), and 0.64 g/liter for xylose (8%). Ethanol productivities per hour ranged from 2.1 g/g of cell dry weight with 12% glucose to 1.3 g/g of cell dry weight with 8% xylose. The ethanol yield per gram of xylose was higher for recombinant E. coli than commonly reported for Saccharomyces cerevisiae with glucose. Glucose (12%), lactose (12%), and xylose (8%) were converted to (by volume) 7.2% ethanol, 6.5% ethanol, and 5.2% ethanol, respectively.     

Genome sequence of the ethanol-producing Zymomonas mobilis subsp. mobilis lectotype strain ATCC 10988

Zymomonas mobilis ATCC 10988 is the type strain of the Z. mobilis subsp. mobilis taxon, members of which are some of the most rigorous ethanol-producing bacteria. Isolated from Agave cactus fermentations in Mexico, ATCC 10988 is one of the first Z. mobilis strains to be described and studied. Its robustness in sucrose-substrate fermentations, physiological characteristics, large number of plasmids, and overall genomic plasticity render this strain important to the study of the species. Here we report the finishing and annotation of the ATCC 10988 chromosomal and plasmid genome.     

High productivity ethanol fermentation on a mineral medium using a flocculent strain ofZymomonas mobilis

Summary A flocculent strain ofZymomonas mobilis (ZM4F JM1) was isolated in continuous culture. The parent strain, ZM4F, had lost its flocculating properties. The isolation was done in a conical fermentor at high dilution rate. Ethanol production by the new strain was then compared on a rich and mineral medium. The mineral medium showed high performance and could be used for industrial production of ethanol since it reduced one hundred fold the vitamin cost of the fermentation.     

Disruption of the Zymomonas mobilis extracellular sucrase gene (sacC) improves levan production

AIMS: Disruption of the extracellular Zymomonas mobilis sucrase gene (sacC) to improve levan production. METHODS AND RESULTS: A PCR-amplified tetracycline resistance cassette was inserted within the cloned sacC gene in pZS2811. The recombinant construct was transferred to Z. mobilis by electroporation. The Z. mobilis sacC gene, encoding an efficient extracellular sucrase, was inactivated. A sacC defective mutant of Z. mobilis, which resulted from homologous recombination, was selected and the sacC gene disruption was confirmed by PCR. Fermentation trials with this mutant were conducted, and levansucrase activity and levan production were measured. In sucrose medium, the sacC mutant strain produced threefold higher levansucrase (SacB) than the parent strain. This resulted in higher levels of levan production, whilst ethanol production was considerably decreased. CONCLUSIONS: Zymomonas mobilis sacC gene encoding an extracellular sucrase was inactivated by gene disruption. This sacC mutant strain produced higher level of levan in sucrose medium because of the improved levansucrase (SacB) than the parent strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The Z. mobilis CT2, sacC mutant produces high level of levansucrase (SacB) and can be used for the production of levan.     

Ethanol production from fructose in continuous culture by free and flocculent cells of Zymomonas mobilis

Summary Zymomonas mobilis strain ZM4 was used for ethanol production from fructose (100 g/l) in continuous culture with a mineral (containing Ca pantothenate) or a rich (containing yeast extract) mediium. With both media high conversion yields were observed but the ethanol productivity was limited by the low biomass content of the fermentor. A new flocculent strain of Z.mobilis (ZM4F) was cultivated in a CSTR with an internal settler and showed a maximal productivity of 93 g/l.h (fructose conversion of 80%). When the fructose conversion was 96% an ethanol productivity of 85.6 g/l.h with an ethanol yield of 0.49 g/g (96% of theoretical) was observed.     

Effects of potassium chloride on ethanol production by an osmotolerant mutant of Zymomonas mobilis

The effect of increasing the KCl concentration in the culture medium of an alcoholic fermentation of glucose using the bacterium Zymomonas mobilis was investigated. Data obtained with the wild-type strain (ZM4, ATCC 31821) and with a newly isolated osmotolerant mutant (SBE15) were compared. It was observed that, at high salt concentration, inhibition of growth occured (specific growth rate and biomass yield) while ethanol production (specific ethanol productivity and ethanol yield) was unaffected. In contrast, the specific rate of in-vitro ethanol production, using either cell-free extract or washed cells, was strongly inhibited by increasing the KCl concentration in the incubation mixture. Therefore, it was concluded that the intracellular concentration of KCl was maintained below the inhibitory concentration by an active transport system. In addition, the fermentation performances of the osmotolerant mutant strain were higher than those of the parent strain at all the KCl concentrations tested, suggesting the utility of the former to run ethanolic fermentations in crude industrial media with a high salt content. Furthermore, the fermentation data on media containing added KCl agreed well with those obtained on molasses media, suggesting that the inhibition observed on these media was due to their high osmolality. Correspondence to: J. Baratti     

Zymomonas mobilis CP4: a clarification of strains via plasmid profiles

There has been confusion regarding the nomenclature of cultures of Zymomonas mobilis CP4 (Gonçalves de Lima et al., 1970). Thus five cultures from different laboratories but originally received from a single source (Recife, Brazil) were compared by analysis of their plasmid profiles and the restriction digest patterns of the plasmids. Four cultures of Z. mobilis CP4 showed identical plasmid profiles and restriction digest patterns. One culture of CP4 (ZM4, ATCC 31821), cultured in P.L. Rogers' laboratory in Australia, and cultures derived from it and deposited in connection with recent patents, strain ZM401 (ATCC 31822) and strain ZM481 (ATCC 31823), differed from the first four cultures with respect to both plasmid profiles and restriction digest pattern. The reason for the plasmid differences between the two groups of cultures is unclear, but may have arisen in the original distribution, by natural selection while in continued culture or perhaps by selection of a minor strain in the original culture. In order to clarify the status of the two variant groups, the cultures that possess the same plasmid profile as the CP4 culture currently being distributed from the Recife culture collection are now designated CP4 (and derivatives). The cultures that possess the plasmid profile of the Rogers' CP4 (ZM4) are now designated by the Rogers' laboratory notation as ZM4 (and derivatives) and prior citation to CP4 removed from their nomenclature.     

Use of wheat bran as a nutritive supplement for the production of ethanol by Zymomonas mobilis

A strain of Zymomonas mobilis (ZYM-TS 1) was isolated from fermenting palm wine (toddy). In addition to glucose, sucrose, and fructose, the organism utilized hydrolysates of corn ( Zea mays ) flour, corn starch and ragi ( Eleusine coracana ) flour. Amounts of ethanol produced in media (adjusted to 10% (w/v) total carbohydrates) fortified with wheat bran extract only, were comparable with those obtained from the defined media containing yeast extract, (NH₄)₂SO₄, KH₂PO₄, and MgSO₄.7H₂O. The results indicate that wheat bran extract can supply all the necessary nutrients required for ethanol production by Zymomonas mobilis .     

Cell immobilization in kappa-carrageenan for ethanol production

A combination of extended Monod kinetics and the diffusional equation was used for evaluating the effectiveness factor of entrapped immobilized cells. Based on the kinetics of Zymomonas mobilis reported in the literature, the numerical results have revealed that the problem of mass transfer diffusional restrictions can be neglected by using small beads (1 mm in diameter) with a corresponding cell loading up to 276 g/L gel. On the basis of the numerical results obtained, the application of immobilized cells for continuous ethanol production was investigated. The kappa-carrageenan method was utilized to entrap Z. mobilis CP4, a potential ethanol producer. A two stage fermentation process has also been developed for ethanol production by the Z. mobilis carrageenan-bound cells. About 90 g/L ethanol was produced by immobilized cells at a total residence time of 1.56 h. The ethanol yield was estimated to be 93% of theoretical. The results obtained in this study also indicated that the control of optimum pH in an immobilized cell column is necessary to enhance the rate of ethanol production.     

Xanthan gum and ethanol production by Xanthomonas campestris and Zymomonas mobilis from peach pulp

The wild type of Xanthomonas campestris and a mutant strain of Zymomonas mobilis CP4, tolerant to sucrose up to 40% (w/v), were used to produce either xanthan gum or ethanol, respectively, from peach pulp supplemented with different salts. Both bacteria grew well (2.7 mg/ml for X. campestris and 1.45 mg/ml for Z. mobilis) in fine peach pulp and the production of xanthan gum or ethanol was 0.1–0.2 g/l or 110 g/l, respectively.     

A structured kinetic model for Zymomonas mobilis ATCC10988

The inhibitory effects of glucose and ethanol on Zymomonas mobilis ATCC10988 were isolated through kinetic analysis of transient batch fermentation data. Growth of Z. mobilis was inhibited above a glucose concentration of 80 g/L. Growth was mildly inhibited by ethanol to 50 g/L, and severely inhibited above this concentration. Specific rates of ethanol production and glucose uptake were essentially invariant during batch fermentation. A structured kinetic model was developed, by way of augmentation of the Extended Bottleneck model, to quantify the kinetics of the growth and product formation processes. The model successfully describes the transient batch fermentation of Z. mobilis over a wide range of initial glucose concentration in a semidefined medium.     

Flocculating Zymomonas mobilis is a promising host to be engineered for fuel ethanol production from lignocellulosic biomass

Whereas Saccharomyces cerevisiae uses the Embden‐Meyerhof‐Parnas pathway to metabolize glucose, Zymomonas mobilis uses the Entner‐Doudoroff (ED) pathway. Employing the ED pathway, 50% less ATP is produced, which could lead to less biomass being accumulated during fermentation and an improved yield of ethanol. Moreover, Z. mobilis cells, which have a high specific surface area, consume glucose faster than S. cerevisiae, which could improve ethanol productivity. We performed ethanol fermentations using these two species under comparable conditions to validate these speculations. Increases of 3.5 and 3.3% in ethanol yield, and 58.1 and 77.8% in ethanol productivity, were observed in ethanol fermentations using Z. mobilis ZM4 in media containing ～100 and 200 g/L glucose, respectively. Furthermore, ethanol fermentation bythe flocculating Z. mobilis ZM401 was explored. Although no significant difference was observed in ethanol yield and productivity, the flocculation of the bacterial species enabled biomass recovery by cost‐effective sedimentation, instead of centrifugation with intensive capital investment and energy consumption. In addition, tolerance to inhibitory byproducts released during biomass pretreatment, particularly acetic acid and vanillin, was improved. These experimental results indicate that Z. mobilis, particularly its flocculating strain, is superior to S. cerevisiae as a host to be engineered for fuel ethanol production from lignocellulosic biomass.     

产乙醇运动发酵单胞菌的研究进展

运动发酵单胞菌作为天然生产乙醇的主要微生物之一,具有特殊的Entner Doudoroff途径和其他一些特殊的糖代谢和能量代谢方式,因此具有乙醇产率高和乙醇耐受力强的显著特点。通过简述运动发酵单胞菌的糖代谢和能量代谢、乙醇和高渗透压等耐性及其遗传改造三方面的研究进展,阐明其应用于燃料乙醇生产的巨大潜力     

Interaction of liposomes with Zymomonas mobilis cells

Abstract Zymomonas mobilis (strain ZM4), an ethanoltolerant bacterium, interacts efficiently with liposomes consisting of Escherichia coli phospholipids. The parameters of the cell-liposome interaction have been determined. The process depends on temperature, presence of divalent cation, pH, liposome/cell ratio and is insensitive to pre-treatment of ZM4 cells with EDTA. Optimal transfer of exogenous phospholipids into cells was achieved at 37°C and pH 6.6, when the interaction medium contained 20 mM CaCl₂.