Adaptation yields a highly efficient xylose-fermenting Zymomonas mobilis strain

Zymomonas mobilis is a superb ethanol producer with productivity exceeding yeast strains by several fold. Although metabolic engineering was successfully applied to expand its substrate range to include xylose, xylose fermentation lagged far behind glucose. In addition, xylose fermentation was often incomplete when its initial concentration was higher than 5%. Improvement of xylose fermentation is therefore necessary. In this work, we applied adaptation to improve xylose fermentation in metabolically engineered strains. As a result of adaptation over 80 days and 30 serial transfers in a medium containing high concentration of xylose, a strain, referred as A3, with markedly improved xylose metabolism was obtained. The strain was able to grow on 10% (w/v) xylose and rapidly ferment xylose to ethanol within 2 days and retained high ethanol yield. Similarly, in mixed glucose-xylose fermentation, a total of 9% (w/v) ethanol was obtained from two doses of 5% glucose and 5% xylose (or a total of 10% glucose and 10% xylose). Further investigation reveals evidence for an altered xylitol metabolism in A3 with reduced xylitol formation. Additionally xylitol tolerance in A3 was increased. Furthermore, xylose isomerase activity was increased by several times in A3, allowing cells to channel more xylose to ethanol than to xylitol. Taken together, these results strongly suggest that altered xylitol metabolism is key to improved xylose metabolism in adapted A3 strain. This work further demonstrates that adaptation and metabolic engineering can be used synergistically for strain improvement.     

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.     

Localisation of the glucose-fructose oxidoreductase in wild type and overproducing strains of Zymomonas mobilis

Abstract The enzyme glucose-fructose oxidoreductase (GFOR) from the Gram-negative ethanologenic bacterium Zymomonas mobilis was purified to homogeneity and was shown to be a tetrameric protein with a subunit size of M _r 42 500. Using immunogold-labelling in combination with electron microscopy, ultrathin sections of Z. mobilis wild type cells showed that the enzyme GFOR is located in the periplasm off the bacterial cells. Z. mobilis strains which carried the cloned gfo gene on plasmid pSUP104, had 5–6-fold increased GFOR enzyme activities. Moreover, these cells accumulated large amounts of a presumable unprocessed pre-GFOR protein ( M _r 48 000).     

一株葡萄糖和木糖共发酵高效产乙醇重组运动发酵单胞菌的性能评价

木糖是木质纤维素原料水解液中的第二大组分,木糖和葡萄糖的充分利用是有经济性地生产纤维素乙醇的关键。通过基因克隆手段构建了一株可以高效利用木糖产乙醇的重组运动发酵单胞菌Zymomonas mobilis TSH01,并进行了利用单糖溶液、混合糖溶液及玉米秸秆水解液发酵产乙醇效率的研究。结果表明,利用单一葡萄糖或单一木糖溶液发酵时,当糖浓度为8%、发酵72 h后,糖利用率分别为100%和98.9%,乙醇代谢收率分别为87.8%和78.3%;利用8%葡萄糖和8%木糖的混合溶液发酵时,72 h后,葡萄糖和木糖的利用率分别为98.5%和97.4%,乙醇代谢收率为94.9%。利用含3.2%葡萄糖和3.5%木糖的玉米秸秆水解液发酵72 h后,葡萄糖和木糖的利用率分别为100%和92.3%,乙醇代谢收率为91.5%。此外,磷酸二氢钾对发酵过程中木糖利用率以及乙醇收率的提高有明显促进作用。     

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; Δ32–46) 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; Δ2–20) 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 Δ2–20 deletion led to the restoration of growth on media with 1 M sucrose, whereas the presence of the Δ32–46 deletion led to a growth behavior similar to that of strain ACM3963, with no sorbitol formation from sucrose. Received: 14 December 1995 / Accepted: 4 March 1996     

Determination of glucose-fructose oxidoreductase activity in whole cells of Zymomonas mobilis

A method for the determination of glucose-fructose oxidoreductase (GFOR) activity in whole cells of Zymomonas mobilis is described. The K _m and the theoretical V _max for GFOR were 192 g glucose.l^-1 and 17 g gluconic acid.g^-1 cell.h^-1, respectively. The changes in enthalpy (31.1 kJ.mol^-1), entropy (0.41 kJ.K^-1), and Gibbs free energy (-97.5 kJ.mol^-1) related to glucose to gluconic acid conversion were also determined.     

Kinetic modeling to optimize pentose fermentation in Zymomonas mobilis

Zymomonas mobilis engineered to express four heterologous enzymes required for xylose utilization ferments xylose along with glucose. A network of pentose phosphate (PP) pathway enzymatic reactions interacting with the native glycolytic Entner Doudoroff (ED) pathway has been hypothesized. We have investigated this putative reaction network by developing a kinetic model incorporating all of the enzymatic reactions of the PP and ED pathways, including those catalyzed by the heterologous enzymes. Starting with the experimental literature on in vitro characterization of each enzymatic reaction, we have developed a kinetic model to enable dynamic simulation of intracellular metabolite concentrations along the network of interacting PP and ED metabolic pathways. This kinetic model is useful for performing in silico simulations to predict how varying the different enzyme concentrations will affect intracellular metabolite concentrations and ethanol production rate during continuous fermentation of glucose and xylose mixtures. Among the five enzymes whose concentrations were varied as inputs to the model, ethanol production in the continuous fermentor was optimized when xylose isomerase (XI) was present at the highest level, followed by transaldolase (TAL). Predictions of the model that the interconnecting enzyme phosphoglucose isomerase (PGI) does not need to be overexpressed were recently confirmed through experimental investigations. Through such systematic analysis, we can develop efficient strategies for maximizing the fermentation of both glucose and xylose, while minimizing the expression of heterologous enzymes.     

Glucose-fructose oxidoreductase, a periplasmic enzyme of Zymomonas mobilis, is active in its precursor form

Abstract Glucose-fructose oxidoreductase (GFOR) is a periplasmic enzyme of the ethanologenic, Gram-negative bacterium Zymomonas mobilis . It contains tightly bound NADP⁺ as cofactor. In Z. mobilis GFOR-recombinant strains, a precursor form of GFOR was accumulated. To assay the preGFOR for its NADP(H) content and enzymatic activity, it was purified from an overproducing strain. Using SDS-PAGE, the precursor subunit size was determined to approximately 45 kDa (compared with a 40 kDa subunit size for the mature GFOR subunit). The N-terminal amino acid sequence of the precursor was determined. The N-terminal residues of the GFOR matched with the signal sequence from the DNA sequence of the gene gfo . The precursor form of GFOR was enzymatically active and contained the cofactor NADP(H).     

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     

Separation of membrane fractions of Zymomonas mobilis

Abstract Membranes of Zymomonas mobilis ZM4 were separated by centrifugation on discontinuous density sorbitol gradient using a 2-step purification procedure. Four bands of respective densities 1.17 (L1), 1.20 (L2), 1.22 (H1), and 1.32 (H2) were obtained. NADH oxidase activity was detected in L1 and L2 fractions, indicating that they were derived from cytoplasmic membrane. H1 and H2 fractions gave a positive Limulus polyphemus lysate test of outer membrane endotoxin. Proteins of 2 cytoplasmic membrane bands and of 2 outer membrane bands showed respectively similar patterns when separated by electrophoresis.     

Response of Zymomonas mobilis levansucrase activity to sodium chloride

An activation of levansucrase-catalysed levan formation by NaCl, KCl and Na2 SO4 (0.03–0.7 M) was observed using cell-free extract of Zymomonas mobilis. A sigmoidal response of the rate of levansucrase-catalysed reaction to the sucrose concentration was significantly reduced in the presence of salts the Hill coefficient 2.10 and 1.0–1.2 respectively), possibly, due to the heterotropic activation of levansucrase as an allosteric enzyme. © Rapid Science Ltd. 1998     

A new biosensor for specific determination of sucrose using an oxidoreductase of Zymomonas mobilis and invertase

A new biosensor for specific determination of sucrose was developed using an oxidoreductase of Zymomonas mobilis and invertase. Cells of Z. mobilis were permeabilized with toluene in order to utilize the enzymes of glucose-fructose oxidoreductase and gluconolactonase inside the intact cells. Permeabilized cells and invertase were coimmobilized in a gelatin membrane, and a whole cell enzyme electrode was constructed by fixing the membrane on a pH electrode. The production of hydrogen ion was detected using the biosensor-connected microcomputer, and the concentration of sucrose was determined by using both the initial rate and the steady-state methods. Optimum conditions for biosensor response were pH 6.2 and temperature 35 degrees C. The effect of interfering compounds on the electrode response was investigated, and the interference by various sugars was eliminated by determining sucrose concentration using the steady-state method. The biosensor developed is simple and reproducible, and the calibration curve for sucrose is linear up to 70 g/L.     

Batch fermentation kinetics of sugar beet molasses by Zymomonas mobilis

A new osmotolerant mutant strain of Zymomonas mobilis was successfully used for ethanol production from beet molasses. Addition of magnesium sulfate to hydrolyzed molasses allowed repeated growth without the need of yeast extract addition. The kinetics and yields parameters of fermentation on media with different molasses concentrations were calculated. The anabolic parameters (specific growth rate, mu, and biomass yield, Y(X/S)) were inhibited at elevated molasses concentrations while the catabolic parameters (specific ethanol productivity, q(p), and ethanol yield, Y(p/s)) were not significantly affected. In addition to ethanol and substrate inhibition, osmotic pressure effects can explain the observed results.     

A model system for a fluorometric biosensor using permeabilized Zymomonas mobilis or enzymes with protein confined dinucleotides

Using permeabilized Zymomonas mobilis or glucose-fructose oxidoreductase isolated from this microorganism a model system for biosensors with a protein confined NADP(H) cofactor for the determination of glucose, fructose, gluconolactone, and sorbitol was developed. Either permeabilized microorganisms containing the oxidoreductase or the pure enzyme were confined via membrane separation in a small measuring chamber, that was integrated into a flow injection analysis system (FIA). The measuring principle was the monitoring of the NAD(P)H fluorescence, excited at 360 nm and measured at 450 nm. NADP(H), which is confined in the protein complex, was oxidized or reduced during the enzymatic reactions and the changes in the fluorescence intensity were related to the substrate concentration. The sensitivity of the system covered a range from 0.001 to 100 g/L of the analyte depending on substrate and operating conditions. The applicability of this model system for bioprocess monitoring was proved using samples from a Pseudomonas pseudoflava cultivation. (c) 1993 John Wiley & Sons, Inc.     

半定量RT-PCR检测运动发酵单胞菌中外源基因转录水平的研究

本研究运用半定量RT-PCR法检测运动发酵单胞菌重组菌中外源基因xylB的转录水平。提取野生型运动发酵单胞菌CP4及其2个重组菌的总RNA, 检测无DNA污染后定量至同一浓度、并反转录为cDNA。观测目的基因xylB和内标基因16S rRNA的PCR扩增曲线、并确定合适的循环数, 选用相同量的cDNA为模板, PCR检测各样本中xylB相对16S rRNA的转录水平。结果表明野生型菌株CP4中xylB基因没有转录, 而两株重组菌中皆有xylB的转录本, 且转录丰度基本一致, 酶活测定也进一步证实该基因在重组菌中有效表达。该方法可用于鉴定运动发酵单胞菌中特定基因的转录水平, 是一种快速有效的检测方法。     

重组运动发酵单胞菌的构建及木糖利用特性研究

将大肠杆菌(Escherichia coli)木糖代谢的关键酶基因.引入到运动发酵单胞菌中,获得能利用木糖发酵生产乙醇的重组工程菌株PZM.混合糖发酵过程中,重组菌利用葡萄糖和木糖生成乙醇的效率分别达到理论值的81.2%和63.1%.     

High efficiency ethanol fermentation by entrapment of Zymomonas mobilis into LentiKats

AIMS: To examine the potential of Zymomonas mobilis entrapped into polyvinylalcohol (PVA) lens-shaped immobilizates in batch and continuous ethanol production. METHODS AND RESULTS: Cells, free or immobilized in PVA hydrogel-based lens-shaped immobilizates - LentiKats, were cultivated on glucose medium in a 1 l bioreactor. In comparison with free cell cultivation, volumetric productivity of immobilized batch culture was nine times higher (43.6 g l(-1) h(-1)). The continuously operated system did not improve the efficiency (volumetric productivity of the immobilized cells 30.7 g l(-1) h(-1)). CONCLUSIONS: We demonstrated Z. mobilis capability, entrapped into LentiKats, in the cost-efficient batch system of ethanol production. SIGNIFICANCE AND IMPACT OF THE STUDY: The results reported here emphasize the potential of bacteria in combination with suitable fermentation technology in industrial scale. The innovation compared with traditional systems is characterized by excellent long-term stability, high volumetric productivity and other technological advantages.     

D-cycloserine biases enrichment for auxotrophic mutants of Zymomonas mobilis

Abstract Contrary to its effect on rich medium, d-cycloserine showed no bactericidal effect on Zymomonas mobilis cells cultured on mineral medium. Addition of a mixture of glycine and glutamic acid to the mineral medium restored its bactericidal action. However, mutant enrichments run in these conditions were biased, with mostly methionine mutants isolated. A decrease of the d-cycloserine concentration only reduced the bias.