Genetic engineering of ethanol production in Escherichia coli

The genes encoding essential enzymes of the fermentative pathway for ethanol production in Zymomonas mobilis, an obligately ethanologenic bacterium, were inserted into Escherichia coli under the control of a common promoter. Alcohol dehydrogenase II and pyruvate decarboxylase from Z. mobilis were expressed at high levels in E. coli, resulting in increased cell growth and the production of ethanol as the principal fermentation product from glucose. These results demonstrate that it is possible to change the fermentation products of an organism, such as E. coli, by the addition of genes encoding appropriate enzymes which form an alternative system for the regeneration of NAD+.     

Genetic engineering of ethanol production in Escherichia coli.

The genes encoding essential enzymes of the fermentative pathway for ethanol production in Zymomonas mobilis, an obligately ethanologenic bacterium, were inserted into Escherichia coli under the control of a common promoter. Alcohol dehydrogenase II and pyruvate decarboxylase from Z. mobilis were expressed at high levels in E. coli, resulting in increased cell growth and the production of ethanol as the principal fermentation product from glucose. These results demonstrate that it is possible to change the fermentation products of an organism, such as E. coli, by the addition of genes encoding appropriate enzymes which form an alternative system for the regeneration of NAD+.     

Immunocytochemical localization of glycolytic and fermentative enzymes in Zymomonas mobilis.

Gold-labeled antibodies were used to examine the subcellular locations of 11 glycolytic and fermentative enzymes in Zymomonas mobilis. Glucose-fructose oxidoreductase was clearly localized in the periplasmic region. Phosphogluconate lactonase and alcohol dehydrogenase I were concentrated in the cytoplasm near the plasma membrane. The eight remaining enzymes were more evenly distributed within the cytoplasmic matrix. Selected enzyme pairs were labeled on opposite sides of the same thin section to examine the frequency of colocalization. Results from these experiments provide evidence that glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and alcohol dehydrogenase I form an enzyme complex.     

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     

利用木糖和葡萄糖合成乙醇的新型重组大肠杆菌的研究

利用PCR方法从运动发酵单孢菌染色体DNA扩增出乙醇合成途径的关键酶基因pdc、adhB,分别用tac启动子控制表达,构建了可以在Escherichia coli JM109中表达的重组质粒pKK-PA、pEtac-PA.初步的乙醇发酵结果表明,在E.coli中只引入adhB基因不能拓宽其中的产乙醇途径,引入pdc基因可以与宿主自身的ADH酶协同作用,使碳流有效导向产乙醇方向.同时引入pdc、adhB基因可以在宿主E.coli中成功建立产乙醇途径.     

Gel electrophoretic analysis of Zymomonas mobilis glycolytic and fermentative enzymes: identification of alcohol dehydrogenase II as a stress protein.

The 13 major enzymes which compose the glycolytic and fermentative pathways in Zymomonas mobilis are particularly abundant and represent one-half of the soluble protein in exponential-phase cells. One- and two-dimensional polyacrylamide gel electrophoresis maps were developed for 12 of these enzymes. Assignments were made by comigration with purified proteins, comparison with overexpressed genes in recombinant strains, and Western blots (immunoblots). Although most glycolytic enzymes appeared resistant to turnover and accumulated in stationary-phase cells, the protein levels of pyruvate kinase, alcohol dehydrogenase I, and glucokinase declined. Alcohol dehydrogenase II was identified as a major stress protein and was induced both by exposure to ethanol and by elevated temperature (45 degrees C). This enzyme, encoded by the adhB gene, is expressed from tandem promoters which share partial sequence identity with the Escherichia coli consensus sequence for heat shock proteins.     

Glycolytic flux in Zymomonas mobilis: enzyme and metabolite levels during batch fermentation.

The rate at which Z. mobilis (Entner-Doudoroff pathway) converts high concentrations of glucose (20%) into ethanol plus CO2 changes as ethanol accumulates in the surrounding broth. This decline in glycolytic activity (per milligram of cell protein) does not result from inhibitory effects of ethanol, which can be reversed immediately by ethanol removal. The peak of fermentative activity (58 mumol of CO2 evolved per mg of cell protein per h) occurred after the accumulation of 1.1% ethanol (18 h) and declined to one-half this rate after 30 h (6.2% accumulated ethanol), although the cell number continued to increase. These times corresponded to the end of exponential growth and to the onset of the stationary phase (on the basis of measurement of cell protein), respectively. An examination of many of the requirements for fermentation (nucleotides, magnesium, enzyme levels, intracellular pH, delta pH) revealed three possible reasons for this early decline in activity: decreased abundance of nucleotides, a decrease in internal pH from 6.3 to 5.3, and a decrease in the specific activities of two glycolytic enzymes (pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase). 31P nuclear magnetic resonance spectra of perchlorate extracts from cells fermenting in broth revealed very low levels of glycolytic intermediates (Entner-Doudoroff pathway) in cells examined at the peak of fermentative activity (18-h cells) in comparison with cells examined at a later stage (30-h cells), consistent with limitation of the fermentation rate by glycolytic enzymes near the end of the pathway. It is likely that cell death (loss of colony-forming ability) and the collapse of delta pH also contribute to the further decline in fermentative activity after 30 h.     

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.     

Modulation of alcohol dehydrogenase isoenzyme levels in Zymomonas mobilis by iron and zinc.

Zymomonas mobilis is an unusual microorganism which utilizes both iron-containing alcohol dehydrogenase (ADHII) and zinc-containing alcohol dehydrogenase (ADHI) isoenzymes during fermentative growth. This organism is obligately ethanologenic, and alcohol dehydrogenase activity is essential. The activities of ADHI and ADHII were altered by supplementing growth medium with iron or zinc salts and by iron starvation. Growth under iron-limiting conditions (chelators, minimal medium) reduced ADHII activity but did not prevent the synthesis of the ADHII protein. The inactive form of this enzyme appeared quite stable, was not renatured by iron addition, and persisted in the cell. The iron-induced increase in ADHII activity required de novo synthesis which was blocked by antibiotic additions. The ability of Z. mobilis to synthesize ADHII and ADHI may be advantageous in nature.     

Use of the tac promoter and lacIq for the controlled expression of Zymomonas mobilis fermentative genes in Escherichia coli and Zymomonas mobilis.

The Zymomonas mobilis genes encoding alcohol dehydrogenase I (adhA), alcohol dehydrogenase II (adhB), and pyruvate decarboxylase (pdc) were overexpressed in Escherichia coli and Z. mobilis by using a broad-host-range vector containing the tac promoter and the lacIq repressor gene. Maximal IPTG (isopropyl-beta-D-thiogalactopyranoside) induction of these plasmid-borne genes in Z. mobilis resulted in a 35-fold increase in alcohol dehydrogenase I activity, a 16.7-fold increase in alcohol dehydrogenase II activity, and a 6.3-fold increase in pyruvate decarboxylase activity. Small changes in the activities of these enzymes did not affect glycolytic flux in cells which are at maximal metabolic activity, indicating that flux under these conditions is controlled at some other point in metabolism. Expression of adhA, adhB, or pdc at high specific activities (above 8 IU/mg of cell protein) resulted in a decrease in glycolytic flux (negative flux control coefficients), which was most pronounced for pyruvate decarboxylase. Growth rate and flux are imperfectly coupled in this organism. Neither a twofold increase in flux nor a 50% decline from maximal flux caused any immediate change in growth rate. Thus, the rates of biosynthesis and growth in this organism are not limited by energy generation in rich medium.     

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).     

Ethanol production from wood hydrolysate using genetically engineered Zymomonas mobilis

An ethanologenic microorganism capable of fermenting all of the sugars released from lignocellulosic biomass through a saccharification process is essential for secondary bioethanol production. We therefore genetically engineered the ethanologenic bacterium Zymomonas mobilis such that it efficiently produced bioethanol from the hydrolysate of wood biomass containing glucose, mannose, and xylose as major sugar components. This was accomplished by introducing genes encoding mannose and xylose catabolic enzymes from Escherichia coli. Integration of E. coli manA into Z. mobilis chromosomal DNA conferred the ability to co-ferment mannose and glucose, producing 91 % of the theoretical yield of ethanol within 36 h. Then, by introducing a recombinant plasmid harboring the genes encoding E. coli xylA, xylB, tal, and tktA, we broadened the range of fermentable sugar substrates for Z. mobilis to include mannose and xylose as well as glucose. The resultant strain was able to ferment a mixture of 20 g/l glucose, 20 g/l mannose, and 20 g/l xylose as major sugar components of wood hydrolysate within 72 h, producing 89.8 % of the theoretical yield. The recombinant Z. mobilis also efficiently fermented actual acid hydrolysate prepared from cellulosic feedstock containing glucose, mannose, and xylose. Moreover, a reactor packed with the strain continuously produced ethanol from acid hydrolysate of wood biomass from coniferous trees for 10 days without accumulation of residual sugars. Ethanol productivity was at 10.27 g/l h at a dilution rate of 0.25 h(-1).     

Isolation and properties of the glycolytic enzymes from Zymomonas mobilis. The five enzymes from glyceraldehyde-3-phosphate dehydrogenase through to pyruvate kinase.

The five glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase were each purified from extracts of Zymomonas mobilis cells, by using dye-ligand chromatography as the principal step. Two procedures, producing three and two of the enzymes respectively, are described in detail. Z. mobilis glyceraldehyde-phosphate dehydrogenase was found to be similar in most respects to the enzyme from other sources, except for having a slightly larger subunit size. Phosphoglycerate kinase has properties typical for this enzyme; however, it did not show the sulphate activation effects characteristic of this enzyme from most other sources. Phosphoglycerate mutase is a dimer, partially independent of 2,3-bisphosphoglycerate, and has a high specific activity. Enolase was found to be octameric; otherwise its properties were very similar to those of the yeast enzyme. Pyruvate kinase is unusual in being dimeric, and not requiring K+ for activity. It is not allosterically activated by sugar phosphates, having a high activity in the absence of any effectors. Some quantitative differences in the relative amounts of these enzymes, compared with eukaryotic species, are ascribed to the fact that Z. mobilis utilizes the Entner-Doudoroff pathway rather than the more common Embden-Meyerhoff glycolytic route.