Conversion of xylan to ethanol by ethanologenic strains of Escherichia coli and Klebsiella oxytoca.

A two-stage process was evaluated for the fermentation of polymeric feedstocks to ethanol by a single, genetically engineered microorganism. The truncated xylanase gene (xynZ) from the thermophilic bacterium Clostridium thermocellum was fused with the N terminus of lacZ to eliminate secretory signals. This hybrid gene was expressed at high levels in ethanologenic strains of Escherichia coli KO11 and Klebsiella oxytoca M5A1(pLOI555). Large amounts of xylanase (25 to 93 mU/mg of cell protein) accumulated as intracellular products during ethanol production. Cells containing xylanase were harvested at the end of fermentation and added to a xylan solution at 60 degrees C, thereby releasing xylanase for saccharification. After cooling, the hydrolysate was fermented to ethanol with the same organism (30 degrees C), thereby replenishing the supply of xylanase for a subsequent saccharification. Recombinant E. coli metabolized only xylose, while recombinant K. oxytoca M5A1 metabolized xylose, xylobiose, and xylotriose but not xylotetrose. Derivatives of this latter organism produced large amounts of intracellular xylosidase, and the organism is presumed to transport both xylobiose and xylotriose for intracellular hydrolysis. By using recombinant M5A1, approximately 34% of the maximal theoretical yield of ethanol was obtained from xylan by this two-stage process. The yield appeared to be limited by the digestibility of commercial xylan rather than by a lack of sufficient xylanase or by ethanol toxicity. In general form, this two-stage process, which uses a single, genetically engineered microorganism, should be applicable for the production of useful chemicals from a wide range of biomass polymers.     

Engineering endoglucanase-secreting strains of ethanologenic Klebsiella oxytoca P2

Klebsiella oxytoca P2 was developed as a biocatalyst for the simultaneous saccharification and fermentation (SSF) of cellulose by chromosomally integrating Zymomonas mobilis genes (pdc, adhB) encoding the ethanol pathway. This strain contains the native ability to transport and metabolize cellobiose, eliminating the need to supplement with β-glucosidase during SSF. To increase the utility of this biocatalyst, we have now chromosomally integrated the celZ gene encoding the primary endoglucanase from Erwinia chrysanthemi. This gene was expressed at high levels by replacing the native promoter with a surrogate promoter derived from Z. mobilis DNA. With the addition of out genes encoding the type II protein secretion system from E. chrysanthemi, over half of the active endoglucanase (EGZ) was secreted into the extracellular environment. The two most active strains, SZ2(pCPP2006) and SZ6(pCPP2006), produced approximately 24 000 IU L⁻¹ of CMCase activity, equivalent to 5% of total cellular protein. Recombinant EGZ partially depolymerized acid-swollen cellulose and allowed the production of small amounts of ethanol by SZ6(pCPP2006) without the addition of fungal cellulase. However, additional endoglucanase activities will be required to complete the depolymerization of cellulose into small soluble products which can be efficiently metabolized to ethanol. Received 14 December 1998/ Accepted in revised form 04 March 1999     

Isolation and molecular characterization of high-performance cellobiose-fermenting spontaneous mutants of ethanologenic Escherichia coli KO11 containing the Klebsiella oxytoca casAB operon.

Escherichia coli KO11 was previously constructed to produce ethanol from acid hydrolysates of hemicellulose (pentoses and hexoses) by the chromosomal integration of Zymomonas mobilis genes encoding pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB). Klebsiella oxytoca P2 was constructed in an analogous fashion for the simultaneous saccharification and fermentation of cellulose and contains PTS enzymes for cellobiose. In this study, KO11 was further engineered for the fermentation of cellulose by adding the K. oxytoca casAB genes encoding Enzyme IIcellobiose and phospho-beta-glucosidase. Although the two K. oxytoca genes were well expressed in cloning hosts such as DH5 alpha, both were expressed poorly in E. coli KO11, a derivative of E. coli B. Spontaneous mutants which exhibited more than 15-fold-higher specific activities for cellobiose metabolism were isolated. The mutations of these mutants resided in the plasmid rather than the host. Three mutants were characterized by sequence analysis. All contained similar internal deletions which eliminated the casAB promoter and operator regions and placed the lacZ Shine-Dalgarno region immediately upstream from the casA Shine-Dalgarno region. KO11 harboring mutant plasmids (pLOI1908, pLOI1909, or pLOI1910) rapidly fermented cellobiose to ethanol, and the yield was more than 90% of the theoretical yield. Two of these strains were used with commercial cellulase to ferment mixed-waste office paper to ethanol.     

Genetic changes to optimize carbon partitioning between ethanol and biosynthesis in ethanologenic Escherichia coli

The production of ethanol from xylose by ethanologenic Escherichia coli strain KO11 was improved by adding various medium supplements (acetate, pyruvate, and acetaldehyde) that prolonged the growth phase by increasing cell yield and volumetric productivity (approximately twofold). Although added pyruvate and acetaldehyde were rapidly metabolized, the benefit of these additives continued throughout fermentation. Both additives increased the levels of extracellular acetate through different mechanisms. Since acetate can be reversibly converted to acetyl coenzyme A (acetyl-CoA) by acetate kinase and phosphotransacetylase, the increase in cell yield caused by each of the three supplements is proposed to result from an increase in the pool of acetyl-CoA. A similar benefit was obtained by inactivation of acetate kinase (ackA), reducing the production of acetate (and ATP) and sparing acetyl-CoA for biosynthetic needs. Inactivation of native E. coli alcohol-aldehyde dehydrogenase (adhE), which uses acetyl-CoA as an electron acceptor, had no beneficial effect on growth, which was consistent with a minor role for this enzyme during ethanol production. Growth of KO11 on xylose appears to be limited by the partitioning of carbon skeletons into biosynthesis rather than the level of ATP. Changes in acetyl-CoA production and consumption provide a useful approach to modulate carbon partitioning. Together, these results demonstrate that xylose fermentation to ethanol can be improved in KO11 by redirecting small amounts of pyruvate away from fermentation products and into biosynthesis. Though negligible with respect to ethanol yield, these small changes in carbon partitioning reduced the time required to complete the fermentation of 9.1% xylose in 1% corn steep liquor medium from over 96 h to less than 72 h.     

Fermentation of L-arabinose,D-xylose and D-glucose by ethanologenic recombinant Klebsiella oxytoca strain P2

Summary Recombinant Klebsiella oxytoca strain P2 carrying genes for pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis was evaluated for its ability to ferment arabinose, xylose and glucose alone and in mixtures in pH-controlled batch fermentations. This organism produced 0.34–0.43 g ethanol/g sugar at pH 6.0 and 30°C on 8% sugar substrate and demonstrated a preference for glucose. Sugar utilization was glucose > arabinose > xylose and ethanol production was xylose > glucose > arabinose.     

Development of industrial-medium-required elimination of the 2,3-butanediol fermentation pathway to maintain ethanol yield in an ethanologenic strain of Klebsiella oxytoca

Fermentation efficiency and nutrient costs are both significant factors in process economics for the microbial conversion of cellulosic biomass to commodity chemicals such as ethanol. In this study, we have developed a more industrial medium (OUM1) composed of 0.5% corn steep liquor (dry weight basis) supplemented with mineral salts (0.2%), urea (0.06%), and glucose (9%). Although the growth of strain P2 was vigorous in this medium, approximately 14% of substrate carbon was diverted into 2,3-butanediol and acetoin under the low pH conditions needed for optimal cellulase activity during simultaneous saccharification. Deleting the central region of the budAB genes encoding alpha-acetolactate synthase and alpha-acetolactate decarboxylase eliminated the butanediol and acetoin coproducts and increased ethanol yields by 12%. In OUM1 medium at pH 5.2, strain BW21 produced over 4% ethanol in 48 h (0.47 g ethanol per g glucose). Average productivity (48 h), ethanol titer, and ethanol yield for BW21 in OUM1 medium (pH 5.2) exceeded that of the parent (strain P2) in rich laboratory medium (Luria broth).     

Fermentation of sweet whey by ethanologenic Escherichia coli

Whey, an abundant byproduct of the dairy industry, contains large amounts of protein and lactose which could be used for fuel ethanol production. We have investigated a new organism as a candidate for such fermentations: recombinant Escherichia coli containing the genes encoding the ethanol pathway from Zymomonas mobilis. The highest level of ethanol achieved, 68 g/L, was produced after 108 hours in Luria broth containing 140 g lactose/L. Fermentations of lower lactose concentrations were completed more rapidly with approximately 88% of theoretical yields. Reconstituted sweet whey (60 g lactose/L)was fermented more slowly than lactose in Luria broth requiring 144 hours to produce 26 g ethanol/L. Supplementing sweet whey with a trace metal mix and ammonium sulfate reduced the required fermentation time to 72 hours and increased final ethanol concentration (28 g ethanol/L). By adding proteinases during fermentation, the requirement for ammonia was completely eliminated, and the rate of fermentation further improved (30 g ethanol/L after 48 hours). This latter incresed in rate of ethanol production and ethanol yield are presumed to result from incorporation of amino acids released by hydrolysis of whey proteins. The fermentation of sweet whey by ethanologenic E. coil reduced the nonvolatile residue by approximately 70%. This should reduce biological oxygen demand and reduce the cost of waste treatment. Whey supplemented with trace metals and small amounts of proteinase may represent an economically attractive feedstock for the production of ethanol and other useful chemicals.     

Mannose fermentation by an ethanologenic recombinant Escherichia coli

Summary Recombinant E. coli B (pLOI297) grows in Luria broth with mannose at a rate that is only about one-half of the rate with xylose and about one-quarter of the rate with glucose as carbon source. For a sugar concentration of about 2 % (w/v), the corresponding specific ethanol productivities (q_p) are 0.22, 0.45 and 0.70 g ethanol/g cell/h for mannose, xylose and glucose. At higher sugar concentrations (8–11 %), the sp. productivities are 0.12, 0.33 and 0.35 g ethanol/g cell/h for mannose, xylose and glucose. Using a synthetic softwood prehydrolysate medium, in which the mass ratio of mannose:xylose:glucose was approx. 1.0:0.6:0.4 (total sugar conc'n 4.5 %), the sp. productivities associated with glucose and xylose metabolism were decreased by about 50 % and 75 % respectively, whereas mannose metabolism appeared unaffected by the presence of the other sugars. In all cases, the sugar-to-ethanol conversion efficiency was >90 % of theoretical maximum     

Microcin-mediated interactions between Klebsiella pneumoniae and Escherichia coli strains

Amensal indirect interactions between a Klebsiella pneumoniae microcin-producing strain and several Escherichia coli strains, all of intestinal origin, were studied. Mixed batch cultures of both microcin-producing and microcin-sensitive strains showed that microcin production and excretion into the medium allowed the producer strain to prevail over sensitive strains, even when initial competition conditions were highly unfavourable for the producer. Mixed cultures also showed the production of a microcin-antagonist by the same microcin-producing strain when the nutrients in the medium had been depleted. The antagonist apparently promoted the viability of sensitive cells already damaged by microcin. These results have likely ecological implications.     

Conversion of corn milling fibrous co-products into ethanol by recombinant Escherichia coli strains K011 and SL40

Corn hulls and corn germ meal were both evaluated as feedstocks for production of ethanol for biofuel. Currently, these fibrous co-products are combined with corn steep liquor and the fermentation bottoms (if available) and marketed as cattle feed. Samples were obtained from wet and dry corn mills. The corn hulls and germ meal were evaluated for starch and hemicellulose compositions. Starch contents were 12 to 32% w/w and hemicellulose (arabinoxylans) contents were 23 to 64% w/w. Corn fibrous samples were hydrolysed, using dilute sulphuric acid, into mixed sugar streams containing arabinose, glucose and xylose. Total sugar concentrations in the hydrolysate varied from 8.4 to 10.8% w/v. The hydrolysates were fermented to ethanol using recombinant E. coli strains K011 and SL40. Ethanol yields were 0.38 to 0.41g ethanol produced/g total sugars consumed and fermentations were completed in 60h or less. However, residual xylose was detected for each hydrolysate fermentation and was especially significant for fermentations using strain SL40. Strain K011 was a superior ethanologenic strain compared with strain SL40 in terms of both ethanol yield and maximum productivity.     

Specific ethanol production rate in ethanologenic Escherichia coli strain KO11 Is limited by pyruvate decarboxylase

Modification of ethanol productivity and yield, using mineral medium supplemented with glucose or xylose as carbon sources, was studied in ethanologenic Escherichia coli KO11 by increasing the activity of five key carbon metabolism enzymes. KO11 efficiently converted glucose or xylose to ethanol with a yield close to 100% of the theoretical maximum when growing in rich medium. However, when KO11 ferments glucose or xylose in mineral medium, the ethanol yields decreased to only 70 and 60%, respectively. An increase in GALP(Ec) (permease of galactose-glucose-xylose) or PGK(Ec) (phosphoglycerate kinase) activities did not change xylose or glucose and ethanol flux. However, when PDC(Zm) (pyruvate decarboxylase from Zymomonas mobilis) activity was increased 7-fold, the yields of ethanol from glucose or xylose were increased to 85 and 75%, respectively, and organic acid formation rates were reduced. Furthermore, as a response to a reduction in acetate and ATP yield, and a limited PDC(Zm) activity, an increase in PFK(Ec) (phosphofructokinase) or PYK(Bs) (pyruvate kinase from Bacillus stearothermophilus) activity drastically reduced glucose or xylose consumption and ethanol formation flux. This experimental metabolic control analysis showed that ethanol flux in KO11 is negatively controlled by phosphofructokinase and pyruvate kinase, and positively influenced by the PDC(Zm) activity level.     

Fermentation of newsprint prehydrolysate by an ethanologenic recombinant Escherichia coli

Summary Recombinant E. coli B (pLOI297) produced ethanol from a nutrient-supplemented, newsprint prehydrolysate medium, at about a 20% reduction in both yield and productivity compared to a synthetic softwood hemicellulose hydrolysate medium (lacking acetic acid). With pH controlled at 7, the sugar-to-ethanol conversion efficiency with the newsprint prehydrolysate was 74.5% of theoretical maximum. The final ethanol concentration was 14.6 g/L. Reduced ethanol yield was due to by-product formation, principally lactic acid. The specific rates of glucose, mannose and xylose utilization in the synthetic medium were 0.73, 0.42 and 0.22 g/g cell/h respectively. The ethanol yield from the pretreatment processing of newsprint is estimated at 85L per dry metric ton.     

Sensitivity to selected beta-lactam antibiotics of clinical strains of Escherichia coli and Klebsiella pneumoniae

The studies aimed at analysing the resistance to some beta-lactam antibiotics among E. coli and K. pneumoniae clinical isolates and at evaluating. The extended spectrum of beta-lactamases (ESBL) production in the isolates. The analysis included 137 E. coli strains and 52 K. pneumoniae strains, isolated from hospitalized patients and out-patients treated in the first trimester of 1998. The strains were identified using the ATB computer system. Antibiotic sensitivity of the isolates was determined by disc-diffusion tests. ESBL production capacity of E. coli and K. pneumoniae strains was estimated by double-disc and ATB BLSA tests. Most of the analysed E. coli strains were found to exhibit significant sensitivity to compound penicillin preparations containing beta-lactam inhibitor (Augmentin, Tazocin) and to the third generation cefalosporins, in contrast, K. pneumoniae strains much more frequently were resistant to the drugs. Among the obtained isolates, 3 (2.2%) E. coli strains and 21 (40.4%) K. pneumoniae strains produced ESBL but all the isolates proved sensitive to imipenem. In evaluation of ESBL production-detecting tests, the double-disc test was found to be more reliable than ATB BLSA test.     

Efficient fermentation of Pinus sp. acid hydrolysates by an ethanologenic strain of Escherichia coli.

Process conditions for the acid hydrolysis of pine hemicellulose and cellulose have been described which provide a biocompatible sugar solution. By using an improved strain of recombinant Escherichia coli, strain KO11, hydrolysates supplemented with yeast extract and tryptone nutrients were converted to ethanol with an efficiency of 85% to over 100% on the basis of monomer sugar content (approximately 72 g/liter) and with the production of 35 g of ethanol per liter in 48 h. In the process described, approximately 347 liters of ethanol could be produced per dry metric ton of lignocellulose.     

Efficient fermentation of Pinus sp. acid hydrolysates by an ethanologenic strain of Escherichia coli.

Process conditions for the acid hydrolysis of pine hemicellulose and cellulose have been described which provide a biocompatible sugar solution. By using an improved strain of recombinant Escherichia coli, strain KO11, hydrolysates supplemented with yeast extract and tryptone nutrients were converted to ethanol with an efficiency of 85% to over 100% on the basis of monomer sugar content (approximately 72 g/liter) and with the production of 35 g of ethanol per liter in 48 h. In the process described, approximately 347 liters of ethanol could be produced per dry metric ton of lignocellulose.     

Simultaneous saccharification and fermentation of amorphous cellulose to ethanol by recombinant Klebsiella oxytoca SZ21 without supplemental cellulase

A derivative of Klebsiella oxytoca M5A1 containing chromosomally integrated genes for ethanol production from Zymomonas mobilis (pdc, adhB) and endoglucanase genes from Erwinia chrysanthemi (celY, celZ) produced over 20 000 U endoglucanase l^–1 activity during fermentation. In combination with the native ability to metabolize cellobiose and cellotriose, this strain was able to ferment amorphous cellulose to ethanol (58–76% of theoretical yield) without the addition of cellulase enzymes from other organisms.     

Elucidating mechanisms of solvent toxicity in ethanologenic Escherichia coli

Ethanol toxicity and its effect on ethanol production by the recombinant ethanologenic Escherichia coli strain KO11 were investigated in batch and continuous fermentation. During batch growth, ethanol produced by KO11 reduced both the specific cell growth rate (µ) and the cell yield (Y_X/S). The extent of inhibition increased with the production of both acetate and lactate. Subsequent accumulation of these metabolites and ethanol resulted in cessation of cell growth, redirection of metabolism to reduce ethanol production, and increased requirements for cell maintenance. These effects were found to depend on both the glycolytic flux and the flux from pyruvate to ethanol. Pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) activities measured during the batch fermentation suggested that decreased ethanol production resulted from enzyme inhibition rather than down‐regulation of genes in the ethanol‐producing pathway. Ethanol was added in continuous fermentation to provide an ethanol concentration of either 17 or 27 g/L, triggering sustained oscillations in the cell growth rate. Cell concentrations oscillated in‐phase with ethanol and acetate concentrations. The amplitude of oscillations depended on the concentration of ethanol in the fermentor. Through multiple oscillatory cycles, the yield (Y_P/S) and concentration of ethanol decreased, while production of acetate increased. These results suggest that KO11 favorably adapted to improve growth by synthesizing more ATP though acetate production, and recycling NADH by producing more lactate and less ethanol. Implications of these results for strategies to improve ethanol production are described. Biotechnol. Bioeng. 2010;106: 721–730. © 2010 Wiley Periodicals, Inc.     

Direct conversion of xylan to ethanol by recombinant Saccharomyces cerevisiae strains displaying an engineered minihemicellulosome

Arabinoxylan is a heteropolymeric chain of a β-1,4-linked xylose backbone substituted with arabinose residues, representing a principal component of plant cell walls. Here we developed recombinant Saccharomyces cerevisiae strains as whole-cell biocatalysts capable of combining hemicellulase production, xylan hydrolysis, and hydrolysate fermentation into a single step. These strains displayed a series of uni-, bi-, and trifunctional minihemicellulosomes that consisted of a miniscaffoldin (CipA3/CipA1) and up to three chimeric enzymes. The miniscaffoldin derived from Clostridium thermocellum contained one or three cohesin modules and was tethered to the cell surface through the S. cerevisiae a-agglutinin adhesion receptor. Up to three types of hemicellulases, an endoxylanase (XynII), an arabinofuranosidase (AbfB), and a β-xylosidase (XlnD), each bearing a C-terminal dockerin, were assembled onto the miniscaffoldin by high-affinity cohesin-dockerin interactions. Compared to uni- and bifunctional minihemicellulosomes, the resulting quaternary trifunctional complexes exhibited an enhanced rate of hydrolysis of arabinoxylan. Furthermore, with an integrated d-xylose-utilizing pathway, the recombinant yeast displaying the bifunctional minihemicellulosome CipA3-XynII-XlnD could simultaneously hydrolyze and ferment birchwood xylan to ethanol with a yield of 0.31 g per g of sugar consumed.     

Susceptibility of Klebsiella oxytoca to selected antibiotics

Fifty two clinical isolates of K. oxytoca were included. All of analysed strains were isolated from wound swabs. The aim of this study was to evaluate MIC value of amoxicillin with clavulanic acid, tigecycline and ciprofloxacin. The susceptibility to amoxicillin with clavulanic acid and tigecycline was tested by the Etest. The susceptibility to ciprofloxacine was tested by the agar dilution method. Among of analysed K. oxytoca strains 44 (84.6%) were susceptible to tigecycline, 27 (51.9%) to amoxicilline with clavulanic acid and 21 (40.4%) to ciprofloxacine. These data suggest that tigecycline, may be an effective therapeutic option for the treatment infections caused by K. oxytoca strains.     

Enzymatic degradation of nitriles by Klebsiella oxytoca

Klebsiella oxytoca, isolated from cyanide-containing wastewater, was able to utilize many nitriles as sole source of nitrogen. The major objective of this study was to explore the ability of K. oxytoca to utilize some nitriles and then further evaluate the pathways of transformation of cyanide compounds by K. oxytoca. Results from this study indicate that succinonitrile and valeronitrile were the most optimal sources of nitrogen for the growth of K. oxytoca. The biodegradation of acetonitrile proceeded with the formation of acetamide followed by acetic acid. The production of ammonia was also detected in this biodegradation experiment. Similar results were observed in the propionitrile biodegradation experiments. Collectively, this study suggests that the breakdown of acetonitrile or propionitrile by this bacterium was via a two-step enzymatic hydrolysis with amides as the intermediates and organic acids plus with ammonia as the end products.