Bacterium Zymomonas Mobilis Research Articles

The Substitution of a Single Amino Acid Residue (Ser-116 → Asp) Alters NADP-containing Glucose-Fructose Oxidoreductase ofZymomonas mobilis into a Glucose Dehydrogenase with Dual Coenzyme Specificity

Glucose-fructose oxidoreductase (GFOR, EC 1.1.1.99.-) from the Gram-negative bacterium Zymomonas mobilis contains the tightly bound cofactor NADP. Based on the revision of the gfo DNA sequence, the derived GFOR sequence was aligned with enzymes catalyzing reactions with similar substrates. A novel consensus motif (AGKHVXCEKP) for a class of dehydrogenases was detected. From secondary structure analysis the serine-116 residue of GFOR was predicted as part of a Rossmann-type dinucleotide binding fold. An engineered mutant protein (S116D) was purified and shown to have lost tight cofactor binding based on (a) altered tryptophan fluorescence; (b) lack of NADP liberation through perchloric acid treatment of the protein; and (c) lack of GFOR enzyme activity. The S116D mutant showed glucose dehydrogenase activity (3.6 +/- 0.1 units/mg of protein) with both NADP and NAD as coenzymes (Km for NADP, 153 +/- 9 microM; for NAD, 375 +/- 32 microM). The single site mutation therefore altered GFOR, which in the wild-type situation contains NADP as nondissociable redox cofactor reacting in a ping-pong type mechanism, to a dehydrogenase with dissociable NAD(P) as cosubstrate and a sequential reaction type. After prolonged preincubation of the S116D mutant protein with excess NADP (but not NAD), GFOR activity could be restored to 70 units/mg, one-third of wild-type activity, whereas glucose dehydrogenase activity decreased sharply. A second site mutant (S116D/K121A/K123Q/I124K) showed no GFOR activity even after preincubation with NADP, but it retained glucose dehydrogenase activity (4.2 +/- 0.2 units/mg of protein).

Corn steep liquor as a cost-effective nutrition adjunct in high-performanceZymomonas ethanol fermentations

The ethanologenic bacterium Zymomonas mobilis has been demonstrated to possess several fermentation performance characteristics that are superior to yeast. In a recent survey conducted by the National Renewable Energy Laboratory (NREL), Zymomonas was selected as the most promising host for improvement by genetic engineering directed to pentose metabolism for the production of ethanol from lignocellulosic biomass and wastes. Minimization of costs associated with nutritional supplements and seed production is essential for economic large-scale production of fuel ethanol. Corn steep liquor (CSL) is a byproduct of corn wet-milling and has been used as a fermentation nutrient supplement in several different fermentations. This study employed pH-controlled batch fermenters to compare the growth and fermentation performance of Z. mobilis in glucose media with whole and clarified corn steep liquor as sole nutrient source, and to determine minimal amounts of CSL required to sustain high-performance fermentation. It was concluded that CSL can be used as a cost-effective single-source nutrition adjunct for Zymomonas fermentations. Supplementation with inorganic nitrogen significantly reduced the requirement for CSL. Depending on the type of process and mode of operation, there can be a significant contribution of nutrients from the seed culture, and this would also reduce the requirement for CSL. Removal of the insolubles (40% of the total solids) from CSL did not detract significantly from its nutritional effectiveness. On an equal-volume basis, clarified CSL was 1.33 times more "effective" (in terms of cell mass yield and fermentation time) than whole CSL. For fermentations at sugar loading of >5% (w/v), the recommended level of supplementation with clarified CSL is 1.0% (v/v). Based on CSL at US $50/t, the cost associated with using clarified CSL at 1.0% (v/v) is 88 cents/1000 L of medium and 5.3 cents/gal of undenatured ethanol for fermentation of 10% (w/v) glucose. This cost compares favorably to estimates for using inorganic nutrients. The cost impact is reduced to 3.1 cents/gal if there is a byproduct credit for selling the insolubles as animal feed at a price of about US $100/t. Therefore, the disposition of the CSL insolubles can significantly impact the calculations of cost associated with the use of CSL as a nutritional adjunct in large-scale fermentations.

Expression of the Zymomonas mobilis gfo gene or NADP-containing glucose:fructose oxidoreductase (GFOR) in Escherichia coli. Formation of enzymatically active preGFOR but lack of processing into a stable periplasmic protein.

Glucose:fructose oxidoreductase (GFOR) of the gram-negative bacterium Zymomonas mobilis is a periplasmic enzyme with tightly bound cofactor NADP. The preprotein carries an unusually long N-terminal signal peptide of 52 amino acid residues. Expression of the gfo gene in cells of Escherichia coli K12, under the control of a tac promoter, led to immunologically detectable proteins in western blots, and to the formation of an enzymatically active precursor form (preGFOR), located in the cytosol. Processing of preGFOR to the mature form was not observed in E. coli. Replacement of the authentic GFOR signal peptide by the shorter signal peptides of PhoA or OmpA from E. coli led to processing of the respective GFOR precursor proteins. However, the processed proteins were unstable and rapidly degraded in the periplasm unless an E. coli mutant was used that carried a triple lesion for periplasmic and outer-membrane proteases. When fusion-protein export was inhibited by sodium azide or carboxylcyanide m-chlorophenylhydrazone, the cytoplasmic precursor forms of the respective preGFOR were not degraded. A major protease-resistant GFOR peptide from the OmpA-GFOR fusion was found within spheroplasts of E. coli to which NADP had been added externally. The formation of this peptide did not occur in the presence of NAD. It is concluded that NADP is required for GFOR to fold into its native conformation and that its absence from the E. coli periplasm is responsible for failure to form a stable periplasmic protein. The results strongly suggest that, in Z. mobilis, additional protein factors are required for the transport of NADP across the plasma membrane and/or incorporation of NADP into the GFOR apoenzyme.

Carbohydrate metabolism in Zymomonas mobilis: a catabolic highway with some scenic routes

Sucrose, glucose and fructose are degraded in the Gram-negative bacterium Zymomonas mobilis via an anaerobic version of the Entner-Doudoroff pathway, to an equimolar mixture of ethanol and carbon dioxide. Sucrose is split extracellularly into glucose and fructose (or levan). The two sugars are transported into the cell via facilitated diffusion (uniport). A periplasmic enzyme, glucose-fructose oxidoreductase, provides the novel compatible solute, sorbitol, to counteract detrimental osmotic stress. Carbon flux and its regulation, and branches into anabolic pathways are discussed together with recent approaches to broaden the substrate range of the bacterium.

Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering.

The substrate fermentation range of the ethanologenic bacterium Zymomonas mobilis was expanded to include the pentose sugar, L-arabinose, which is commonly found in agricultural residues and other lignocellulosic biomass. Five genes, encoding L-arabinose isomerase (araA), L-ribulokinase (araB), L-ribulose-5-phosphate-4-epimerase (araD), transaldolase (talB), and transketolase (tktA), were isolated from Escherichia coli and introduced into Z. mobilis under the control of constitutive promoters that permitted their expression even in the presence of glucose. The engineered strain grew on and produced ethanol from L-arabinose as a sole C source at 98% of the maximum theoretical ethanol yield, based on the amount of consumed sugar. This indicates that arabinose was metabolized almost exclusively to ethanol as the sole fermentation product, with little by-product formation. Although no diauxic growth pattern was evident, the microorganism preferentially utilized glucose before arabinose, apparently reflecting the specificity of the indigenous facilitated diffusion transport system. This microorganism may be useful, along with the previously developed xylose-fermenting Z. mobilis (M. Zhang, C. Eddy, K. Deanda, M. Finkelstein, and S. Picataggio, Science 267:240-243, 1995), in a mixed culture for efficient fermentation of the predominant hexose and pentose sugars in agricultural residues and other lignocellulosic feedstocks to ethanol.

Carbohydrate metabolism inZymomonas mobilis: a catabolic highway with some scenic routes

Sucrose, glucose and fructose are degraded in the Gram-negative bacterium Zymomonas mobilis via an anaerobic version of the Entner-Doudoroff pathway, to an equimolar mixture of ethanol and carbon dioxide. Sucrose is split extracellularly into glucose and fructose (or levan). The two sugars are transported into the cell via facilitated diffusion (uniport). A periplasmic enzyme, glucose-fructose oxidoreductase, provides the novel compatible solute, sorbitol, to counteract detrimental osmotic stress. Carbon flux and its regulation, and branches into anabolic pathways are discussed together with recent approaches to broaden the substrate range of the bacterium.

Export of the periplasmic NADP-containing glucose-fructose oxidoreductase of Zymomonas mobilis.

Glucose-fructose oxidoreductase (GFOR) of the gram-negative bacterium Zymomonas mobilis is a periplasmic enzyme with the tightly bound cofactor NADP. The preprotein carries an unusually long N-terminal signal sequence of 52 amino acid residues. A sorbitol-negative mutant strain (ACM3963) was found to be deficient in GFOR activity and was used for the expression of plasmid-borne copies of the wild-type gfo gene or of alleles encoding alterations in the signal sequence of the pre-GFOR protein. Z. mobilis cells with the wild-type gfo allele translocated pre-GFOR, at least partially, via the Sec pathway since CCCP (carboxylcyanide-m-chlorophenylhydrazone; uncoupler of proton motive force) or sodium azide (inhibitor of SecA) abolished the processing of GFOR. A gfo allele with the hydrophobic region of the signal sequence removed (residues 32-46; Delta32-46) led to a protein that was no longer processed, but showed full enzymatic activity (180 U/mg) and had the cofactor NADP firmly bound. A deletion in the n-region of the signal sequence (residues 2-20; Delta2-20) or exchange of the entire GFOR signal sequence with the signal sequence of gluconolactonase of Z. mobilis led to active and processed GFOR. Strain ACM3963 could not grow in the presence of high sugar concentrations (1 M sucrose) unless sorbitol was added. The presence of the plasmid-borne gfo wild-type allele or of the Delta2-20 deletion led to the restoration of growth on media with 1 M sucrose, whereas the presence of the Delta32-46 deletion led to a growth behavior similar to that of strain ACM3963, with no sorbitol formation from sucrose.

Modelling, Simulation and Measurement of Metabolic Pathways in Zymomonas Mobilis

The metabolic pathways of the ethanol producing bacterium Zymomonas mobilis are modelled, simulated and compared with measured data obtained by 13C NMR and FIA (flow injection analysis). The modelling and simulation of either wild type or genetically manipulated metabolic pathways in the anaerobic bacterium is achieved by numerical solution of a system of ordinary differential equations. These equations describe the biochemical reaction network controlled by enzyme kinetics. The dynamic behaviour caused by extracellular changes of the substrate level in the fermentation broth is simulated as well as steady state conditions. Statements and predictions about intracellular reactions on growth conditions and genetically engineered cultures become possible. The gained models are based on assumptions concerning enzyme kinetics and their parameters both of them mostly unknown or only determined in vitro yet. By 13C NMR spectroscopy, stationary fluxes and metabolite concentrations can be determined in vivo and without any use of kinetic principles. Quick dynamic changes triggered by the external glucose level are measured by flow injection analysis. Thus, validation of parameters combined with kinetics and simulated metabolite concentrations can be achieved.

Batch fermentation kinetics of ethanol production by Zymomonas mobilis on cellulose hydrolysate

Growth and ethanol production by three strains (MSN77, thermotolerant, SBE15, osmotolerant and wild type ZM4) of the bacterium Zymomonas mobilis were tested in a rich medium containing the hexose fraction from a cellulose hydrolysate (Aspen wood). The variations of yield and kinetic parameters with fermentation time revealed an inhibition of growth by the ethanol produced. This inhibition may result from the increase in medium osmolality due to ethanol formation from glucose.

Quantification of small enthalpic differences in anaerobic microbial metabolism—a calorimetry-supported approach

A calorimetry-supported approach to the measurement of small enthalpic differences in microbial metabolism is presented. Anaerobic fermentations with the bacterium Zymomonas mobilis and the yeast Saccharomyces cerevisiae were carried out in a fermenter-calorimeter at a constant dilution rate ( D = 0.15 h −1). The glucose concentration of the feed was increased stepwise from 10 to 80 g l −1. Salts, vitamins and trace elements were present in excess. Different steady state conditions were analysed. For Z. mobilis, specific production rates for ethanol, carbon dioxide and heat were three times higher than those measured for S. cerevisiae. If the glucose feed concentration exceeded 20 g l −1 during the S. cerevisiae fermentations, glucose could be measured in the fermenter. Further increasing glucose concentrations of the feed caused higher specific ethanol, carbon dioxide and heat production rates, whereas the biomass yield Y X S decreased remarkably. This result can be explained as the Crabtree effect under anaerobic conditions and interpreted as metabolic uncoupling of anabolic biomass formation and catabolic energy substrate consumption. A futile cycle contributing to the heat production is considered to provide S. cerevisiae with a regulatory mechanism in the control of the flux of metabolites through the glycolytic pathway.

Ethanol transport in Zymomonas mobilis measured by using in vivo nuclear magnetic resonance spin transfer.

For the first time, unidirectional rate constants of ethanol diffusion through the lipid membrane of a microorganism, the bacterium Zymomonas mobilis, were determined, thus replacing indirect inferences with direct kinetic data. The rate constants k1 (in to out) were 6.8 +/- 0.4s(-1) at 29 degrees C and 2.7 +/- 0.3s(-1) at 20 degrees C. They were determined by using 1H selective nuclear magnetic resonance spin magnetization transfer. The measurements were done on l-ml cell suspensions. No addition of radiotracers, withdrawing of aliquots, physical separation methods, or chemical manipulations were required. Until now, the rate constants of ethanol transport in microorganisms have been unknown because ethanol diffuses through the cytoplasmic membrane too quickly for radiolabel approaches. Net velocities of ethanol exchange were calculated from unidirectional rate constants and cytoplasmic volume, which was also determined with the same nuclear magnetic resonance experiments. The results (i) confirmed that ethanol would not be rate limiting during the conversion of glucose by Z. mobilis and (ii) indicated that ethanol can serve as an in vivo marker of cytoplasmic volume changes. This was verified by monitoring for the first time the changes of both cytoplasmic volume and extracytoplasmic and cytoplasmic concentrations of alpha and beta anomers of D-glucose in cell suspensions of a microorganism. These findings may open up new possibilities for kinetic studies of ethanol and sugar transport in Z. mobilis and other organisms.

Energy coupling sites in the electron transport chain of Zymomonas mobilis

Energy-coupling sites in the electron transport chain of the obligately fermentative aerotolerant bacterium Zymomonas mobilis were examined. The H + O stoichiometry of the electron transport chain in intact bacteria oxidizing ethanol was close to 3.3. Cytoplasmic membrane vesicles coupled NADH oxidation to ATP synthesis. With ascorbate/phenazine methosulfate they showed oxygen uptake which was sensitive to antimycin A, but no significant ATP synthesis could be detected. Cells with a defective coupling site I, prepared by cultivation on a sulfate-deficient medium, showed a decreased rotenone sensitivity of respiration, and they lacked almost all the respiration-driven proton translocation and ATP synthesis. We conclude that, despite the reported composition of the electron transport chain, only energy coupling site 1 was functional in Z. mobilis.

Energy coupling sites in the electron transport chain ofZymomonas mobilis

Energy-coupling sites in the electron transport chain of the obligately fermentative aerotolerant bacterium Zymomonas mobilis were examined. The H+O stoichiometry of the electron transport chain in intact bacteria oxidizing ethanol was close to 3.3. Cytoplasmic membrane vesicles coupled NADH oxidation to ATP synthesis. With ascorbate/phenazine methosulfate they showed oxygen uptake which was sensitive to antimycin A, but no significant ATP synthesis could be detected. Cells with a defective coupling site I, prepared by cultivation on a sulfate-deficient medium, showed a decreased rotenone sensitivity of respiration, and they lacked almost all the respiration-driven proton translocation and ATP synthesis. We conclude that, despite the reported composition of the electron transport chain, only energy coupling site 1 was functional in Z. mobilis.

Metabolic Engineering of a Pentose Metabolism Pathway in Ethanologenic Zymomonas mobilis

The ethanol-producing bacterium Zymomonas mobilis was metabolically engineered to broaden its range of fermentable substrates to include the pentose sugar xylose. Two operons encoding xylose assimilation and pentose phosphate pathway enzymes were constructed and transformed into Z. mobilis in order to generate a strain that grew on xylose and efficiently fermented it to ethanol. Thus, anaerobic fermentation of a pentose sugar to ethanol was achieved through a combination of the pentose phosphate and Entner-Doudoroff pathways. Furthermore, this strain efficiently fermented both glucose and xylose, which is essential for economical conversion of lignocellulosic biomass to ethanol.

Analysis of Intact Hopanoids and Other Lipids from the Bacterium Zymomonas Mobilis by High-Performance Liquid Chromatography

Hopanoids and other lipids were extracted from Zymomonas mobilis and quantitatively analyzed by highperformance liquid chromatography. Previous methods for hopanoid analysis required derivatization of the hopanoids via periodate oxidation or acetylation. The current method employs a normal-phase silica gel column, a ternary gradient of hexane-isopropanol-water-triethylamine, and detection with a flame ionization detector. Three major hopanoid classes were separated and quantified by this new method, and together they comprised 30 to 40% of the total lipid in these cells. Mass spectrometry confirmed that chemical structures of these hopanoids were consistent with those previously proposed. In addition, several other common lipid classes (free fatty acids and six classes of common phospholipids), comprising the remaining 60 to 70% of the total lipids, were also separated and quantified. This new chromatographic method represents the first technique for the analysis and purification of intact hopanoids. This method should be useful for the analysis and purification of hopanoids from other bacterial species.

Useful mutants of Zymomonas mobilis alcohol dehydrogenase-2 obtained by the use of polymerase chain reaction random mutagenesis.

The alcohol dehydrogenase-2 isozyme of the ethanologenic bacterium Zymomonas mobilis (ZADH-2) is in the class of iron-activated dehydrogenases (Scope, 1983; Neale, et al., 1986; Reid and Fewson, 1994). These enzymes have not yet been characterised structurally except by sequence. No certain sequence similarities exist which allow the identification of active sites or other structural features. ZADH-2 can be purified in large amounts, and the ferrous ion substituted with cobaltous, which stabilizes the product (Neale et al., 1986: Tse, et al., 1989). The gene can be expressed at a high level in Escherichia coli, up to 30% of the soluble protein (Neale, et al., 1988). We have crystallized the enzyme and anticipate that full structural determination may be possible.

Crystallization and preliminary X-ray analysis of glucose-fructose oxidoreductase from Zymomonas mobilis.

Glucose-fructose oxidoreductase (E.C. 1.1.99.-) from the ethanol-producing Gram-negative bacterium Zymomonas mobilis is a periplasmic, soluble enzyme that forms a homotetramer of 160 kDa with one NADP(H) cofactor per subunit that is tightly, but noncovalently, bound. The enzyme was crystallized by the hanging drop vapor diffusion method using sodium citrate as precipitant. The obtained crystals belong to the space group P2(1)2(1)2, with unit cell constants of 84.6 A, 94.1 A, and 117.0 A, consistent with two monomers in the asymmetric unit. They diffract to a resolution of about 2 A and are suitable for X-ray structure determination.

Alcoholic fermentation of cellulose hydrolysate by Zymomonas mobilis

Growth and ethanol production by three strains of the bacterium Zymomonas mobilis were tested, at 40°C, in a medium containing cellulose hydrolysate (hexose fraction) as carbon source. The thermotolerant mutant C107 exhibited the best growth compared to wild type ZM4 and to the osmotolerant mutant SBE15. When cultivated in media supplemented with various nutrients, growth was only observed in presence of yeast extract (10 g/l) which acted both as a vitamin supplier and pH stabilizer. Using calcium pantothenate instead of yeast extract and sodium acetate to control pH resulted in growth inhibition by the high medium osmolality. Batch fermentation with pH control (KOH addition) showed good growth and ethanol production with the mineral medium.

Oxidative phosphorylation in Zymomonas mobilis

The obligately fermentative aerotolerant bacterium Zymomonas mobilis was shown to possess oxidative phosphorylation activity. Increased intracellular ATP levels were observed in aerated starved cell suspension in the presence of ethanol or acetaldehyde. Ethanolconsuming Z. mobilis generated a transmembrane pH gradient. ATP synthesis in starved Z. mobilis cells could be induced by external medium acidification of 3.5–4.0 pH units. Membrane vesicles of Z. mobilis coupled ATP synthesis to NADH oxidation. ATP synthesis was sensitive to the protonophoric uncoupler CCCP both in starved cells and in membrane vesicles. The H+-ATPase inhibitor DCCD was shown to inhibit the NADH-coupled ATP synthesis in membrane vesicles. The physiological role of oxidative phosphorylation in this obligately fermentative bacterium is discussed.

Estimation of kinetic and yield parameters: ethanol fermentation of a glucose and fructose mixture using Zymomonas mobilis

A new method is presented to calculate the kinetic and yield parameters of a fermentation. This method is based on polynomial fitting of the variations of substrates and biomass concentrations with time and calculation of instantaneous and overall parameters. Application to a mixed substrate and mixed product fermentation by the bacterium Zymomonas mobilis is presented.