Improved strains of recombinant Escherichia coli for ethanol production from sugar mixtures

Hemicellulose hydrolysates of agricultural residues often contain mixtures of hexose and pentose sugars. Ethanologenic Escherichia coli that have been previously investigated preferentially ferment hexose sugars. In some cases, xylose fermentation was slow or incomplete. The purpose of this study was to develop improved ethanologenic E. coli strains for the fermentation of pentoses in sugar mixtures. Using fosfomycin as a selective agent, glucose-negative mutants of E. coli KO11 (containing chromosomally integrated genes encoding the ethanol pathway from Zymomonas mobilis) were isolated that were unable to ferment sugars transported by the phosphoenolpyruvate-dependent phosphotransferase system. These strains (SL31 and SL142) retained the ability to ferment sugars with independent transport systems such as arabinose and xylose and were used to ferment pentose sugars to ethanol selectively in the presence of high concentrations of glucose. Additional fosfomycin-resistant mutants were isolated that were superior to strain KO11 for ethanol production from hexose and pentose sugars. These hyperproductive strains (SL28 and SL40) retained the ability to metabolize all sugars tested, completed fermentations more rapidly, and achieved higher ethanol yields than the parent. Both SL28 and SL40 produced 60 gl^–1 ethanol from 120 gl^–1 xylose in 60 h, 20% more ethanol than KO11 under identical conditions. Further studies illustrated the feasibility of sequential fermentation. A mixture of hexose and pentose sugars was fermented with near theoretical yield by SL40 in the first step followed by a second fermentation in which yeast and glucose were added. Such a two-step approach can combine the attributes of ethanologenic E. coli for pentoses with the high ethanol tolerance of conventional yeasts in a single vessel.     

Use of catabolite repression mutants for fermentation of sugar mixtures to ethanol

Use of agricultural biomass, other than corn-starch, to produce fuel ethanol requires a microorganism that can ferment the mixture of sugars derived from hemicellulose. Escherichia coli metabolizes a wide range of substrates and has been engineered to produce ethanol in high yield from sugar mixtures. E. coli metabolizes glucose in preference to other sugars and, as a result, utilization of the pentoses in hemicellulose-derived sugar mixtures is delayed and may be incomplete. Residual sugar lowers the ethanol yield and is problematic for downstream processing of fermentation products. Therefore, a catabolite repression mutant that simultaneously utilizes glucose and pentoses would be useful for fermentation of complex substrate mixtures. We constructed ethanologenic E. coli strains with a glucose phosphotransferase (ptsG) mutation and used the mutants to ferment glucose, arabinose, and xylose, singly and in mixtures, to ethanol. Yields were 87-94% of theoretical for both the wild type and mutants, but the mutants had an altered pattern of mixed sugar utilization. Phosphotransferase mutants metabolized the pentoses simultaneously with glucose, rather than sequentially. Based upon fermentations of sugar mixtures, a catabolite-repression mutant of ethanologenic E. coli is expected to provide more efficient fermentation of hemicellulose hydrolysates by allowing direct utilization of pentoses.     

Ethanol production from rice hull using Pichia stipitis and optimization of acid pretreatment and detoxification processes

The goal of this study was to produce ethanol from rice hull hydrolysates (RHHs) using Pichia stipitis strains and to optimize dilute acid hydrolysis and detoxification processes by response surface methodology (RSM). The optimized conditions were found as 127.14°C, solid:liquid ratio of 1:10.44 (w/v), acid ratio of 2.52% (w/v), and hydrolysis time of 22.01 min. At these conditions, the fermentable sugar concentration was 21.87 g/L. Additionally, the nondetoxified RHH at optimized conditions contained 865.2 mg/L phenolics, 24.06 g/L fermentable sugar, no hydroxymethylfurfural (HMF), 1.62 g/L acetate, 0.36 g/L lactate, 1.89 g/L glucose, and 13.49 g/L fructose + xylose. Furthermore, RHH was detoxified with various methods and the best procedures were found to be neutralization with CaO or charcoal treatment in terms of the reduction of inhibitory compounds as compared to nondetoxified RHH. After detoxification procedures, the content of hydrolysates consisted of 557.2 and 203.1 mg/L phenolics, 19.7 and 21.60 g/L fermentable sugar, no HMF, 0.98 and 1.39 g/L acetate, 0 and 0.04 g/L lactate, 1.13 and 1.03 g/L glucose, and 8.46 and 12.09 g/L fructose + xylose, respectively. Moreover, the base‐line mediums (control), and nondetoxified and detoxified hydrolysates were used to produce ethanol by using P. stipitis strains. The highest yields except that of base‐line mediums were achieved using neutralization (35.69 and 38.33% by P. stipitis ATCC 58784 and ATCC 58785, respectively) and charcoal (37.55% by P. stipitis ATCC 58785) detoxification methods. Results showed that the rice hull can be utilized as a good feedstock for ethanol production using P. stipitis. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:872–882, 2016     

Ethanolic fermentation of hydrolysates from ammonia fiber expansion (AFEX) treated corn stover and distillers grain without detoxification and external nutrient supplementation

External nutrient supplementation and detoxification of hydrolysate significantly increase the production cost of cellulosic ethanol. In this study, we investigated the feasibility of fermenting cellulosic hydrolysates without washing, detoxification or external nutrient supplementation using ethanologens Escherichia coli KO11 and the adapted strain ML01 at low initial cell density (16 mg dry weight/L). The cellulosic hydrolysates were derived from enzymatically digested ammonia fiber expansion (AFEX)-treated corn stover and dry distiller's grain and solubles (DDGS) at high solids loading (18% by weight). The adaptation was achieved through selective evolution of KO11 on hydrolysate from AFEX-treated corn stover. All cellulosic hydrolysates tested (36-52 g/L glucose) were fermentable. Regardless of strains, metabolic ethanol yields were near the theoretical limit (0.51 g ethanol/g consumed sugar). Volumetric ethanol productivity of 1.2 g/h/L was achieved in fermentation on DDGS hydrolysate and DDGS improved the fermentability of hydrolysate from corn stover. However, enzymatic hydrolysis and xylose utilization during fermentation were the bottlenecks for ethanol production from corn stover at these experimental conditions. In conclusion, fermentation under the baseline conditions was feasible. Utilization of nutrient-rich feedstocks such as DDGS in fermentation can replace expensive media supplementation.     

Fermentation to ethanol of pentose-containing spent sulphite liquor

Ethanolic fermentation of spent sulphite liquor with ordinary bakers' yeast is incomplete because this yeast cannot ferment the pentose sugars in the liquor. This results in poor alcohol yields, and a residual effluent problem By using the yeast Candida shehatae (R) for fermentation of the spent sulphite liquor from a large Canadian alcohol-producing sulphite pulp and paper mill, pentoses as well as hexoses were fermented nearly completely, alcohol yields were raised by 33%, and sugar removal increased by 46%. Inhibitors were removed prior to fermentation by steam stripping. Major benefits were obtained by careful recycling of this yeast, which was shown to be tolerant both of high sugar concentrations and high alcohol concentrations. When sugar concentrations over 250 g/L (glucose: xylose 70:30) were fermented, ethanol became an inhibitor when its concentration reached 90 g/L. However, when the ethanol was removed by low-temperature vacuum distillation, fermentation continued and resulted in a yield of 0.50 g ethanol/g sugar consumed. Further improvement was achieved by combining enzyme saccharification of sugar oligomers with fermentation. This yeast is able to ferment both hexoses and pentoses simultaneously, efficiently, and rapidly. Present indications are that it is well suited to industrial operations wherever hexoses and pentoses are both to be fermented to ethanol, for example, in wood hydrolysates.     

Ethanol production from corn cob hydrolysates by <Emphasis Type="Italic">Escherichia coli</Emphasis> KO11

Corn cob hydrolysates, with xylose as the dominant sugar, were fermented to ethanol by recombinant Escherichia coli KO11. When inoculum was grown on LB medium containing glucose, fermentation of the hydrolysate was completed in 163 h and ethanol yield was 0.50 g ethanol/g sugar. When inoculum was grown on xylose, ethanol yield dropped, but fermentation was faster (113 h). Hydrolysate containing 72.0 g/l xylose and supplemented with 20.0 g/l rice bran was readily fermented, producing 36.0 g/l ethanol within 70 h. Maximum ethanol concentrations were not higher for fermentations using higher cellular concentration inocula. A simulation of an industrial process integrating pentose fermentation by E. coli and hexose fermentation by yeast was carried out. At the first step, E. coli fermented the hydrolysate containing 85.0 g/l xylose, producing 40.0 g/l ethanol in 94 h. Baker's yeast and sucrose (150.0 g/l) were then added to the spent fermentation broth. After 8 h of yeast fermentation, the ethanol concentration reached 104.0 g/l. This two-stage fermentation can render the bioconversion of lignocellulose to ethanol more attractive due to increased final alcohol concentration. Journal of Industrial Microbiology & Biotechnology (2002) 29, 124–128 doi:10.1038/sj.jim.7000287 Received 20 February 2002/ Accepted in revised form 04 June 2002     

Fermentation of sugar cane bagasse hemicellulosic hydrolysate and sugar mixtures to ethanol by recombinant Escherichia coli KO11

Escherichia coli KO11, carrying the ethanol pathway genes pdc (pyruvate decarboxylase) and adh (alcohol dehydrogenase) from Zymomonas mobilis integrated into its chromosome, has the ability to metabolize pentoses and hexoses to ethanol, both in synthetic medium and in hemicellulosic hydrolysates. In the fermentation of sugar mixtures simulating hemicellulose hydrolysate sugar composition (10.0 g of glucose/l and 40.0 g of xylose/l) and supplemented with tryptone and yeast extract, recombinant bacteria produced 24.58 g of ethanol/l, equivalent to 96.4% of the maximum theoretical yield. Corn steep powder (CSP), a byproduct of the corn starch-processing industry, was used to replace tryptone and yeast extract. At a concentration of 12.5 g/l, it was able to support the fermentation of glucose (80.0 g/l) to ethanol, with both ethanol yield and volumetric productivity comparable to those obtained with fermentation media containing tryptone and yeast extract. Hemicellulose hydrolysate of sugar cane bagasse supplemented with tryptone and yeast extract was also readily fermented to ethanol within 48 h, and ethanol yield achieved 91.5% of the theoretical maximum conversion efficiency. However, fermentation of bagasse hydrolysate supplemented with 12.5 g of CSP/l took twice as long to complete. This revised version was published online in November 2006 with corrections to the Cover Date.     

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.     

Prefermentation improves xylose utilization in simultaneous saccharification and co-fermentation of pretreated spruce

Background  

Simultaneous saccharification and fermentation (SSF) is a promising process option for ethanol production from lignocellulosic materials. However, both the overall ethanol yield and the final ethanol concentration in the fermentation broth must be high. Hence, almost complete conversion of both hexoses and pentoses must be achieved in SSF at a high solid content. A principal difficulty is to obtain an efficient pentose uptake in the presence of high glucose and inhibitor concentrations. Initial glucose present in pretreated spruce decreases the xylose utilization by yeast, due to competitive inhibition of sugar transport. In the current work, prefermentation was studied as a possible means to overcome the problem of competitive inhibition. The free hexoses, initially present in the slurry, were in these experiments fermented before adding the enzymes, thereby lowering the glucose concentration.     

A strain of <Emphasis Type="Italic">Meyerozyma guilliermondii</Emphasis> isolated from sugarcane juice is able to grow and ferment pentoses in synthetic and bagasse hydrolysate media

The search for new microbial strains that are able to withstand inhibitors released from hemicellulosic hydrolysis and are also still able to convert sugars in ethanol/xylitol is highly desirable. A yeast strain isolated from sugarcane juice and identified as Meyerozyma guilliermondii was evaluated for the ability to grow and ferment pentoses in synthetic media and in sugarcane bagasse hydrolysate. The yeast grew in xylose, arabinose and glucose at the same rate at an initial medium pH of 5.5. At pH 4.5, the yeast grew more slowly in arabinose. There was no sugar exhaustion within 60 h. At higher xylose concentrations with a higher initial cell concentration, sugar was exhausted within 96 h at pH 4.5. An increase of 350 % in biomass was obtained in detoxified hydrolysates, whereas supplementation with 3 g/L yeast extract increased biomass production by approximately 40 %. Ethanol and xylitol were produced more significantly in supplemented hydrolysates regardless of detoxification. Xylose consumption was enhanced in supplemented hydrolysates and arabinose was consumed only when xylose and glucose were no longer available. Supplementation had a greater impact on ethanol yield and productivity than detoxification; however, the product yields obtained in the present study are still much lower when compared to other yeast species in bagasse hydrolysate. By the other hand, the fermentation of both xylose and arabinose and capability of withstanding inhibitors are important characteristics of the strain assayed.     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

An interlaboratory comparison of the performance of ethanol-producing micro-organisms in a xylose-rich acid hydroysate

A xylose-rich, dilute-acid-pretreated corncob hydrolase was fermented by Escherichia coli ATCC 11303, recombinant (rec) E. coli (pLOI 297 and KO11), Pichia stipitis (CBS 5773, 6054 adn R), Saccharomyces cerevisiae siolate 3 in combination with xylose isomerase, rec S. cerevisiae (TJ1, H550 and H477), and Fusraium oxysporum VIT-D-80134 in an interlaboratory comparison. The micro-organisms were studied according to three different options: (A) fermentation under consistent conditions, (B) fermentation under optimal conditions for the organisms, and (C) fermentation under optimal conditions for the organism with detoxification if the hydrolysate. The highest yields of tehanol, 0.24 g/g (A), 0.36 g/g (B) and 0.54 g/g (C), were obtained from rec E. coli B, KO11. P. stipitis and F. oxysporum were sensitive to the inhibitors present in the hydrolysate and produced a minimum yields of 0.34 g/g (C) and 0.04 g/g (B), respectively. The analysis of the corn-cob hydrolysate and aspects of process economy of the different fermentation options (pH, sterilization, nutrient supplementation, adaptation, detoxification) are discussed.Correspondece to: B. Hahn-Hägerdal     

The acetone butanol fermentation on glucose and xylose. I. Regulation and kinetics in batch cultures

The kinetics in batch culture of the acetone butanol fermentation by Clostridium acetobutylicum is compared on glucose, xylose, and mixtures of both sugars. The fastest initial growth and transition from an acid to a solvent metabolism occurs on glucose, with a final 62 g/L glucose conversion. On xylose, an initial slower growth rate and a longer metabolic transition result in higher cellular and acids concentration, thus in a level of fermented sugar limited to 47 g/L. Batch fermentations on mixtures of glucose and xylose show that both sugars can be fermented, with a higher rate for glucose. However, xylose fermentation is inducible and inhibited at glucose level above 15 g/L. Mixtures of glucose and xylose yield the highest amount of fermented sugars, up to 68 g/L, as a result of both a fast metabolic transition on glucose and a strong acid reconsumption on xylose. In all cases, solvent production is triggered at a total acid concentration between 4 and 5 g/L, whereas the final inhibition of the fermentation takes place at a total butanol and acid concentration between 18 and 20 g/L.     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

Loss of ethanologenicity in Escherichia coli B recombinants pLOI297 and KO11 during growth in the absence of antibiotics

Summary The two cultures under investigation were genetically engineered for the purpose of producing fuel ethanol from biomass and wastes. In this study, stability was viewed in pragmatic terms whereby the fermentation performance of the different genetic constructs was assessed solely with respect to their capacity to maintain a high efficiency of sugar-to-ethanol conversion. Two serial transfers of test tube batch cultures accounted for about 12 generations of semi-continuous growth in LB medium containing one of 4 different sugars - glucose, galactose, mannose or xylose. Both the plasmid-bearing recombinant E. coli B (ATCC 11303) pLOI297 and the chromosomally-integrated recombinant KO11 exhibited dramatic loss of ethanologenicity during growth in a selective (antibiotic-supplemented) medium with mannose as fermentation substrate. In the absence of antibiotics, both recombinants exhibited instability, with the exception of KO11 with xylose as substrate. These observations with short term cultures call into question previous claims regarding the stability of these genetic constructs.     

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。     

The role of peroxisomes in xylose alcoholic fermentation in the engineered Saccharomyces cerevisiae

Xylose is a second‐most abounded sugar after glucose in lignocellulosic hydrolysates and should be efficiently fermented for economically viable second‐generation ethanol production. Despite significant progress in metabolic and evolutionary engineering, xylose fermentation rate of recombinant Saccharomyces cerevisiae remains lower than that for glucose. Our recent study demonstrated that peroxisome‐deficient cells of yeast Ogataea polymorpha showed a decrease in ethanol production from xylose. In this work, we have studied the role of peroxisomes in xylose alcoholic fermentation in the engineered xylose‐utilizing strain of S. cerevisiae. It was shown that peroxisome‐less pex3Δ mutant possessed 1.5‐fold decrease of ethanol production from xylose. We hypothesized that peroxisomal catalase Cta1 may have importance for hydrogen peroxide, the important component of reactive oxygen species, detoxification during xylose alcoholic fermentation. It was clearly shown that CTA1 deletion impaired ethanol production from xylose. It was found that enhancing the peroxisome population by modulation the peroxisomal biogenesis by overexpression of PEX34 activates xylose alcoholic fermentation.     

Engineering a homo-ethanol pathway in Escherichia coli: increased glycolytic flux and levels of expression of glycolytic genes during xylose fermentation

Replacement of the native fermentation pathway in Escherichia coli B with a homo-ethanol pathway from Zymomonas mobilis (pdc and adhB genes) resulted in a 30 to 50% increase in growth rate and glycolytic flux during the anaerobic fermentation of xylose. Gene array analysis was used as a tool to investigate differences in expression levels for the 30 genes involved in xylose catabolism in the parent (strain B) and the engineered strain (KO11). Of the 4,290 total open reading frames, only 8% were expressed at a significantly higher level in KO11 (P < 0.05). In contrast, over half of the 30 genes involved in the catabolism of xylose to pyruvate were expressed at 1.5-fold- to 8-fold-higher levels in KO11. For 14 of the 30 genes, higher expression was statistically significant at the 95% confidence level (xylAB, xylE, xylFG, xylR, rpiA, rpiB, pfkA, fbaA, tpiA, gapA, pgk, and pykA) during active fermentation (6, 12, and 24 h). Values at single time points for only four of these genes (eno, fbaA, fbaB, and talA) were higher in strain B than in KO11. The relationship between changes in mRNA (cDNA) levels and changes in specific activities was verified for two genes (xylA and xylB) with good agreement. In KO11, expression levels and activities were threefold higher than in strain B for xylose isomerase (xylA) and twofold higher for xylulokinase (xylB). Increased expression of genes involved in xylose catabolism is proposed as the basis for the increase in growth rate and glycolytic flux in ethanologenic KO11.     

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.     

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.