Genetic engineering of <Emphasis Type="Italic">Enterobacter asburiae</Emphasis> strain JDR-1 for efficient <Emphasis Type="SmallCaps">d</Emphasis>(−) lactic acid production from hemicellulose hydrolysate

In the dilute acid pretreatment of lignocellulose, xylose substituted with α-1,2-methylglucuronate is released as methylglucuronoxylose (MeGAX), which cannot be fermented by biocatalysts currently used to produce biofuels and chemicals. Enterobacter asburiae JDR-1, isolated from colonized wood, efficiently fermented both MeGAX and xylose in acid hydrolysates of sweetgum xylan. Deletion of pflB and als genes in this bacterium modified the native mixed acid fermentation pathways to one for homolactate production. The resulting strain, Enterobacter asburiae L1, completely utilized both xylose and MeGAX in a dilute acid hydrolysate of sweetgum xylan and produced lactate approximating 100% of the theoretical maximum yield. Enterobacter asburiae JDR-1 offers a platform to develop efficient biocatalysts for production of fuels and chemicals from hemicellulose hydrolysates of hardwood and agricultural residues.     

Genetic Engineering of Enterobacter asburiae Strain JDR-1 for Efficient Production of Ethanol from Hemicellulose Hydrolysates

Dilute acid pretreatment is an established method for hydrolyzing the methylglucuronoxylans of hemicellulose to release fermentable xylose. In addition to xylose, this process releases the aldouronate methylglucuronoxylose, which cannot be metabolized by current ethanologenic biocatalysts. Enterobacter asburiae JDR-1, isolated from colonized wood, was found to efficiently ferment both methylglucuronoxylose and xylose in acid hydrolysates of sweet gum xylan, producing predominantly ethanol and acetate. Transformation of E. asburiae JDR-1 with pLOI555 or pLOI297, each containing the PET operon containing pyruvate decarboxylase (pdc) and alcohol dehydrogenase B (adhB) genes derived from Zymomonas mobilis, replaced mixed-acid fermentation with homoethanol fermentation. Deletion of the pyruvate formate lyase (pflB) gene further increased the ethanol yield, resulting in a stable E. asburiae E1(pLOI555) strain that efficiently utilized both xylose and methylglucuronoxylose in dilute acid hydrolysates of sweet gum xylan. Ethanol was produced from xylan hydrolysate by E. asburiae E1(pLOI555) with a yield that was 99% of the theoretical maximum yield and at a rate of 0.11 g ethanol/g (dry weight) cells/h, which was 1.57 times the yield and 1.48 times the rate obtained with the ethanologenic strain Escherichia coli KO11. This engineered derivative of E. asburiae JDR-1 that is able to ferment the predominant hexoses and pentoses derived from both hemicellulose and cellulose fractions is a promising subject for development as an ethanologenic biocatalyst for production of fuels and chemicals from agricultural residues and energy crops.Lignocellulosic resources, including forest and agricultural residues and evolving energy crops, offer benign alternatives to petroleum-based resources for production of fuels and chemicals. As renewable resources, these lignocellulosic materials are expected to decrease dependence on exhaustible supplies of petroleum and mitigate the net release of carbon dioxide into the atmosphere. The development of economically acceptable bioconversion processes requires pretreatments that release the maximal quantities of hexoses (predominantly glucose released from cellulose) and pentoses (arabinose and xylose) from hemicelluloses and also requires microbial biocatalysts that efficiently convert these compounds to a single targeted product.As one of three main components of lignocellulosics, hemicellulose contains polysaccharides comprised of pentoses, hexoses and sugar acids that account for 20 to 35% of the total biomass from different sources (21). Methylglucuronoxylans (MeGAX_n), consisting of long chains of as many as 70 β-xylopyranose residues linked by β-1,4-glycosidic bonds (25), are the predominant components in the hemicellulose fractions of agricultural residues and energy crops, including corn stover, sugarcane bagasse, poplar, and switchgrass (7, 18, 23, 24). In hardwood and softwood xylans, a 4-O-methylglucuronic acid is attached at the 2′ position of every sixth to eighth xylose residue (12, 15). Dilute acid hydrolysis is commonly used to make the monosaccharides comprising hemicellulose accessible for fermentation (7, 22). However, the α-1,2 glucuronosyl linkage in xylan is resistant to dilute acid hydrolysis, which results in the release of methylglucuronoxylose (MeGAX) along with xylose and other monosaccharides. MeGAX is not fermented by bacterial biocatalysts currently used to convert hemicellulose to ethanol, such as Escherichia coli KO11 (2, 6). In sweet gum xylan, as much as 27% of the carbohydrate may be in this unfermentable fraction after dilute acid pretreatment (2, 20). Complete utilization of all hemicellulosic sugars can improve the efficiency of conversion of lignocellulosic materials to fuel ethanol and other value-added products.Our previous research on the processing of hemicelluloses for fermentation led to isolation of Enterobacter asburiae strain JDR-1. This isolate performed mixed-acid fermentation of the principal hexoses and pentoses that can be derived from cellulose and hemicellulose fractions of lignocellulosic biomass and exhibited a novel metabolic potential based on its ability to ferment MeGAX and xylose to ethanol and acetate as major fermentation products from sweet gum MeGAX_n hydrolysates generated by dilute acid pretreatment (2). This strain has been genetically modified to produce d-(−)-lactate as the predominant product from acid hydrolysates of MeGAX_n (3).In this study, the PET operon containing the pdc, adhA, and adhB genes from Zymomonas mobilis (10, 11) was incorporated into a pflB E. asburiae JDR-1 isolate by plasmid transformation to construct homoethanologenic strains. The resulting recombinant strains were compared with E. asburiae wild-type strain JDR-1 and the ethanologenic strain E. coli KO11 to evaluate their efficiencies of production of ethanol from dilute acid hydrolysates of sweet gum MeGAX_n.     

Paenibacillus sp. Strain JDR-2 and XynA1: a Novel System for Methylglucuronoxylan Utilization

Environmental and economic factors predicate the need for efficient processing of renewable sources of fuels and chemicals. To fulfill this need, microbial biocatalysts must be developed to efficiently process the hemicellulose fraction of lignocellulosic biomass for fermentation of pentoses. The predominance of methylglucuronoxylan (MeGAX_n), a β-1,4 xylan in which 10% to 20% of the xylose residues are substituted with α-1,2-4-O-methylglucuronate residues, in hemicellulose fractions of hardwood and crop residues has made this a target for processing and fermentation. A Paenibacillus sp. (strain JDR-2) has been isolated and characterized for its ability to efficiently utilize MeGAX_n. A modular xylanase (XynA₁) of glycosyl hydrolase family 10 (GH 10) was identified through DNA sequence analysis that consists of a triplicate family 22 carbohydrate binding module followed by a GH 10 catalytic domain followed by a single family 9 carbohydrate binding module and concluding with C-terminal triplicate surface layer homology (SLH) domains. Immunodetection of the catalytic domain of XynA₁ (XynA₁ CD) indicates that the enzyme is associated with the cell wall fraction, supporting an anchoring role for the SLH modules. With MeGAX_n as substrate, XynA₁ CD generated xylobiose and aldotetrauronate (MeGAX₃) as predominant products. The inability to detect depolymerization products in medium during exponential growth of Paenibacillus sp. strain JDR-2 on MeGAX_n, as well as decreased growth rate and yield with XynA₁ CD-generated xylooligosaccharides and aldouronates as substrates, indicates that XynA₁ catalyzes a depolymerization process coupled to product assimilation. This depolymerization/assimilation system may be utilized for development of biocatalysts to efficiently convert MeGAX_n to alternative fuels and biobased products.     

Structure, function, and regulation of the aldouronate utilization gene cluster from Paenibacillus sp. strain JDR-2

Direct bacterial conversion of the hemicellulose fraction of hardwoods and crop residues to biobased products depends upon extracellular depolymerization of methylglucuronoxylan (MeGAX_n), followed by assimilation and intracellular conversion of aldouronates and xylooligosaccharides to fermentable xylose. Paenibacillus sp. strain JDR-2, an aggressively xylanolytic bacterium, secretes a multimodular cell-associated GH10 endoxylanase (XynA1) that catalyzes depolymerization of MeGAX_n and rapidly assimilates the principal products, β-1,4-xylobiose, β-1,4-xylotriose, and MeGAX₃, the aldotetrauronate 4-O-methylglucuronosyl-α-1,2-xylotriose. Genomic libraries derived from this bacterium have now allowed cloning and sequencing of a unique aldouronate utilization gene cluster comprised of genes encoding signal transduction regulatory proteins, ABC transporter proteins, and the enzymes AguA (GH67 α-glucuronidase), XynA2 (GH10 endoxylanase), and XynB (GH43 β-xylosidase/α-arabinofuranosidase). Expression of these genes, as well as xynA1 encoding the secreted GH10 endoxylanase, is induced by growth on MeGAX_n and repressed by glucose. Sequences in the yesN, lplA, and xynA2 genes within the cluster and in the distal xynA1 gene show significant similarity to catabolite responsive element (cre) defined in Bacillus subtilis for recognition of the catabolite control protein (CcpA) and consequential repression of catabolic regulons. The aldouronate utilization gene cluster in Paenibacillus sp. strain JDR-2 operates as a regulon, coregulated with the expression of xynA1, conferring the ability for efficient assimilation and catabolism of the aldouronate product generated by a multimodular cell surface-anchored GH10 endoxylanase. This cluster offers a desirable metabolic potential for bacterial conversion of hemicellulose fractions of hardwood and crop residues to biobased products.     

Paenibacillus sp. strain JDR-2 and XynA1: a novel system for methylglucuronoxylan utilization

Environmental and economic factors predicate the need for efficient processing of renewable sources of fuels and chemicals. To fulfill this need, microbial biocatalysts must be developed to efficiently process the hemicellulose fraction of lignocellulosic biomass for fermentation of pentoses. The predominance of methylglucuronoxylan (MeGAXn), a beta-1,4 xylan in which 10% to 20% of the xylose residues are substituted with alpha-1,2-4-O-methylglucuronate residues, in hemicellulose fractions of hardwood and crop residues has made this a target for processing and fermentation. A Paenibacillus sp. (strain JDR-2) has been isolated and characterized for its ability to efficiently utilize MeGAXn. A modular xylanase (XynA1) of glycosyl hydrolase family 10 (GH 10) was identified through DNA sequence analysis that consists of a triplicate family 22 carbohydrate binding module followed by a GH 10 catalytic domain followed by a single family 9 carbohydrate binding module and concluding with C-terminal triplicate surface layer homology (SLH) domains. Immunodetection of the catalytic domain of XynA1 (XynA1 CD) indicates that the enzyme is associated with the cell wall fraction, supporting an anchoring role for the SLH modules. With MeGAXn as substrate, XynA1 CD generated xylobiose and aldotetrauronate (MeGAX3) as predominant products. The inability to detect depolymerization products in medium during exponential growth of Paenibacillus sp. strain JDR-2 on MeGAXn, as well as decreased growth rate and yield with XynA1 CD-generated xylooligosaccharides and aldouronates as substrates, indicates that XynA1 catalyzes a depolymerization process coupled to product assimilation. This depolymerization/assimilation system may be utilized for development of biocatalysts to efficiently convert MeGAXn to alternative fuels and biobased products.     

Rational and Evolutionary Engineering Approaches Uncover a Small Set of Genetic Changes Efficient for Rapid Xylose Fermentation in Saccharomyces cerevisiae

Economic bioconversion of plant cell wall hydrolysates into fuels and chemicals has been hampered mainly due to the inability of microorganisms to efficiently co-ferment pentose and hexose sugars, especially glucose and xylose, which are the most abundant sugars in cellulosic hydrolysates. Saccharomyces cerevisiae cannot metabolize xylose due to a lack of xylose-metabolizing enzymes. We developed a rapid and efficient xylose-fermenting S. cerevisiae through rational and inverse metabolic engineering strategies, comprising the optimization of a heterologous xylose-assimilating pathway and evolutionary engineering. Strong and balanced expression levels of the XYL1, XYL2, and XYL3 genes constituting the xylose-assimilating pathway increased ethanol yields and the xylose consumption rates from a mixture of glucose and xylose with little xylitol accumulation. The engineered strain, however, still exhibited a long lag time when metabolizing xylose above 10 g/l as a sole carbon source, defined here as xylose toxicity. Through serial-subcultures on xylose, we isolated evolved strains which exhibited a shorter lag time and improved xylose-fermenting capabilities than the parental strain. Genome sequencing of the evolved strains revealed that mutations in PHO13 causing loss of the Pho13p function are associated with the improved phenotypes of the evolved strains. Crude extracts of a PHO13-overexpressing strain showed a higher phosphatase activity on xylulose-5-phosphate (X-5-P), suggesting that the dephosphorylation of X-5-P by Pho13p might generate a futile cycle with xylulokinase overexpression. While xylose consumption rates by the evolved strains improved substantially as compared to the parental strain, xylose metabolism was interrupted by accumulated acetate. Deletion of ALD6 coding for acetaldehyde dehydrogenase not only prevented acetate accumulation, but also enabled complete and efficient fermentation of xylose as well as a mixture of glucose and xylose by the evolved strain. These findings provide direct guidance for developing industrial strains to produce cellulosic fuels and chemicals.     

Engineered Pseudomonas putida simultaneously catabolizes five major components of corn stover lignocellulose: Glucose,xylose, arabinose,p-coumaric acid,and acetic acid

Valorization of all major lignocellulose components, including lignin, cellulose, and hemicellulose is critical for an economically viable bioeconomy. In most biochemical conversion approaches, the standard process separately upgrades sugar hydrolysates and lignin. Here, we present a new process concept based on an engineered microbe that could enable simultaneous upgrading of all lignocellulose streams, which has the ultimate potential to reduce capital cost and enable new metabolic engineering strategies. Pseudomonas putida is a robust microorganism capable of natively catabolizing aromatics, organic acids, and D-glucose. We engineered this strain to utilize D-xylose by tuning expression of a heterologous D-xylose transporter, catabolic genes xylAB, and pentose phosphate pathway (PPP) genes tal-tkt. We further engineered L-arabinose utilization via the PPP or an oxidative pathway. This resulted in a growth rate on xylose and arabinose of 0.32 h⁻¹ and 0.38 h⁻¹, respectively. Using the oxidative L-arabinose pathway with the PPP xylose pathway enabled D-glucose, D-xylose, and L-arabinose co-utilization in minimal medium using model compounds as well as real corn stover hydrolysate, with a maximum hydrolysate sugar consumption rate of 3.3 g/L/h. After modifying catabolite repression, our engineered P. putida simultaneously co-utilized five representative compounds from cellulose (D-glucose), hemicellulose (D-xylose, L-arabinose, and acetic acid), and lignin-related compounds (p-coumarate), demonstrating the feasibility of simultaneously upgrading total lignocellulosic biomass to value-added chemicals.     

Metabolic engineering applications to renewable resource utilization

Lignocellulosic materials containing cellulose, hemicellulose, and lignin are the most abundant renewable organic resource on earth. The utilization of renewable resources for energy and chemicals is expected to increase in the near future. The conversion of both cellulose (glucose) and hemicellulose (hexose and pentose) for the production of fuel ethanol is being studied intensively, with a view to developing a technically and economically viable bioprocess. Whereas the fermentation of glucose can be carried out efficiently, the bioconversion of the pentose fraction (xylose and arabinose, the main pentose sugars obtained on hydrolysis of hemicellulose), presents a challenge. A lot of attention has therefore been focused on genetically engineering strains that can efficiently utilize both glucose and pentoses, and convert them to useful compounds, such as ethanol. Metabolic strategies seek to generate efficient biocatalysts (bacteria and yeast) for the bioconversion of most hemicellulosic sugars to products that can be derived from the primary metabolism, such as ethanol. The metabolic engineering objectives so far have focused on higher yields, productivities and expanding the substrate and product spectra.     

Synthesis of aromatic amino acids from 2G lignocellulosic substrates

Pseudomonas putida is a highly solvent-resistant microorganism and useful chassis for the production of value-added compounds from lignocellulosic residues, in particular aromatic compounds that are made from phenylalanine. The use of these agricultural residues requires a two-step treatment to release the components of the polysaccharides of cellulose and hemicellulose as monomeric sugars, the most abundant monomers being glucose and xylose. Pan-genomic studies have shown that Pseudomonas putida metabolizes glucose through three convergent pathways to yield 6-phosphogluconate and subsequently metabolizes it through the Entner–Doudoroff pathway, but the strains do not degrade xylose. The valorization of both sugars is critical from the point of view of economic viability of the process. For this reason, a P. putida strain was endowed with the ability to metabolize xylose via the xylose isomerase pathway, by incorporating heterologous catabolic genes that convert this C5 sugar into intermediates of the pentose phosphate cycle. In addition, the open reading frame T1E_2822, encoding glucose dehydrogenase, was knocked-out to avoid the production of the dead-end product xylonate. We generated a set of DOT-T1E-derived strains that metabolized glucose and xylose simultaneously in culture medium and that reached high cell density with generation times of around 100 min with glucose and around 300 min with xylose. The strains grew in 2G hydrolysates from diluted acid and steam explosion pretreated corn stover and sugarcane straw. During growth, the strains metabolized > 98% of glucose, > 96% xylose and > 85% acetic acid. In 2G hydrolysates P. putida 5PL, a DOT-T1E derivative strain that carries up to five independent mutations to avoid phenylalanine metabolism, accumulated this amino acid in the medium. We constructed P. putida 5PLΔgcd (xylABE) that produced up to 250 mg l⁻¹ of phenylalanine when grown in 2G pretreated corn stover or sugarcane straw. These results support as a proof of concept the potential of P. putida as a chassis for 2G processes.     

Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae

Xylose is the main pentose and second most abundant sugar in lignocellulosic feedstocks. To improve xylose utilization, necessary for the cost-effective bioconversion of lignocellulose, several metabolic engineering approaches have been employed in the yeast Saccharomyces cerevisiae. In this study, we describe the rational metabolic engineering of a S. cerevisiae strain, including overexpression of the Piromyces xylose isomerase gene (XYLA), Pichia stipitis xylulose kinase (XYL3) and genes of the non-oxidative pentose phosphate pathway (PPP). This engineered strain (H131-A3) was used to initialize a three-stage process of evolutionary engineering, through first aerobic and anaerobic sequential batch cultivation followed by growth in a xylose-limited chemostat. The evolved strain H131-A3-AL^CS displayed significantly increased anaerobic growth rate (0.203±0.006 h^?1) and xylose consumption rate (1.866 g g^?1 h^?1) along with high ethanol conversion yield (0.41 g/g). These figures exceed by a significant margin any other performance metrics on xylose utilization and ethanol production by S. cerevisiae reported to-date. Further inverse metabolic engineering based on functional complementation suggested that efficient xylose assimilation is attributed, in part, to the elevated expression level of xylose isomerase, which was accomplished through the multiple-copy integration of XYLA in the chromosome of the evolved strain.     

Pentose transport by the ruminal bacterium Butyrivibrio fibrisolvens

Abstract Butyrivibrio fibrisolvens is a fibrolytic ruminal bacterium that degrades hemicellulose and ferments the resulting pentose sugars. Washed cells of strain D1 accumulated radiolabelled xylose ( K _m= 1.5 μ M) and arabinose ( K _m= 0.2 μ M) when the organism was grown on xylose, arabinose, or glucose, but cultures grown on sucrose or cellobiose had little capacity to transport pentose. Glucose and xylose inhibited transport of each other non-competitively. Both sugars were utilized preferentially over arabinose, but since they did not inhibit transport of arabinose, it appeared that the preference was related to an internal metabolic step. Although the protonmotive force was completely abolished by ionophores, cells retained some ability to transport pentose. In contrast, the metabolic inhibitors iodoacetate, arsenate, and fluoride had little effect on protonmotive force but caused a large decrease in intracellular ATP and xylose and arabinose uptake. These results suggested that high-affinity, ATP-dependent mechanisms were responsible for pentose transport and hexose sugars affected the utilization of xylose and arabinose.     

Fermentation of xylose and hemicellulose hydrolysates by an ethanol-adapted culture ofBacteroides polypragmatus

Bacteroides polypragmatus type strain GP4 was adapted to grow in the presence of 3.5% (w/v) ethanol by successive transfers into 1% (w/v)d-xylose media supplemented with increasing concentrations of ethanol. The maximum specific growth rate of the ethanol-adapted culture (=0.30 h^-1) was not affected by up to 2% (w/v) ethanol but that of the non-adapted strain declined by about 50%. The growth rate of both cultures was limited by nutrient(s) contained in yeast extract. The ethanol yield of the adapted culture (1.01 mol/mol xylose) was higher than that (0.80 mol/mol xylose) of the non-adapted strain. The adapted culture retained the ability to simultaneously ferment pentose and hexose sugars, and moreover it was not inhibited by xylose concentrations of 7–9% (w/v). This culture also readily fermented hemicellulose hydrolysates obtained by mild acid hydrolysis of either hydrogen fluoride treated or steam exploded Aspen wood. The ethanol yield from the fermentation of the hydrolysates was comparable to that obtained from xylose.This paper is issued as NRCC No. 26338     

GH115 α-glucuronidase and GH11 xylanase from Paenibacillus sp. JDR-2: potential roles in processing glucuronoxylans

Paenibacillus sp. JDR-2 (Pjdr2) has been studied as a model for development of bacterial biocatalysts for efficient processing of xylans, methylglucuronoxylan, and methylglucuronoarabinoxylan, the predominant hemicellulosic polysaccharides found in dicots and monocots, respectively. Pjdr2 produces a cell-associated GH10 endoxylanase (Xyn10A₁) that catalyzes depolymerization of xylans to xylobiose, xylotriose, and methylglucuronoxylotriose with methylglucuronate-linked α-1,2 to the nonreducing terminal xylose. A GH10/GH67 xylan utilization regulon includes genes encoding an extracellular cell-associated Xyn10A₁ endoxylanase and an intracellular GH67 α-glucuronidase active on methylglucuronoxylotriose generated by Xyn10A₁ but without activity on methylglucuronoxylotetraose generated by a GH11 endoxylanase. The sequenced genome of Pjdr2 contains three paralogous genes potentially encoding GH115 α-glucuronidases found in certain bacteria and fungi. One of these, Pjdr2_5977, shows enhanced expression during growth on xylans along with Pjdr2_4664 encoding a GH11 endoxylanase. Here, we show that Pjdr2_5977 encodes a GH115 α-glucuronidase, Agu115A, with maximal activity on the aldouronate methylglucuronoxylotetraose selectively generated by a GH11 endoxylanase Xyn11 encoded by Pjdr2_4664. Growth of Pjdr2 on this methylglucuronoxylotetraose supports a process for Xyn11-mediated extracellular depolymerization of methylglucuronoxylan and Agu115A-mediated intracellular deglycosylation as an alternative to the GH10/GH67 system previously defined in this bacterium. A recombinantly expressed enzyme encoded by the Pjdr2 agu115A gene catalyzes removal of 4-O-methylglucuronate residues α-1,2 linked to internal xylose residues in oligoxylosides generated by GH11 and GH30 xylanases and releases methylglucuronate from polymeric methylglucuronoxylan. The GH115 α-glucuronidase from Pjdr2 extends the discovery of this activity to members of the phylum Firmicutes and contributes to a novel system for bioprocessing hemicelluloses.

     

Reduced Oxidative Pentose Phosphate Pathway Flux in Recombinant Xylose-Utilizing Saccharomyces cerevisiae Strains Improves the Ethanol Yield from Xylose

In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD⁺. In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g⁻¹) and the lowest xylitol yield (0.05 g g⁻¹) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.     

Sugar transporters in efficient utilization of mixed sugar substrates: current knowledge and outlook

There is increasing interest in production of transportation fuels and commodity chemicals from lignocellulosic biomass, most desirably through biological fermentation. Considerable effort has been expended to develop efficient biocatalysts that convert sugars derived from lignocellulose directly to value-added products. Glucose, the building block of cellulose, is the most suitable fermentation substrate for industrial microorganisms such as Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae. Other sugars including xylose, arabinose, mannose, and galactose that comprise hemicellulose are generally less efficient substrates in terms of productivity and yield. Although metabolic engineering including introduction of functional pentose-metabolizing pathways into pentose-incompetent microorganisms has provided steady progress in pentose utilization, further improvements in sugar mixture utilization by microorganisms is necessary. Among a variety of issues on utilization of sugar mixtures by the microorganisms, recent studies have started to reveal the importance of sugar transporters in microbial fermentation performance. In this article, we review current knowledge on diversity and functions of sugar transporters, especially those associated with pentose uptake in microorganisms. Subsequently, we review and discuss recent studies on engineering of sugar transport as a driving force for efficient bioconversion of sugar mixtures derived from lignocellulose.     

Construction of an Escherichia coli K-12 Mutant for Homoethanologenic Fermentation of Glucose or Xylose without Foreign Genes

Conversion of lignocellulosic feedstocks to ethanol requires microorganisms that effectively ferment both hexose and pentose sugars. Towards this goal, recombinant organisms have been developed in which heterologous genes were added to platform organisms such as Saccharomyces cerevisiae, Zymomonas mobilis, and Escherichia coli. Using a novel approach that relies only on native enzymes, we have developed a homoethanologenic alternative, Escherichia coli strain SE2378. This mutant ferments glucose or xylose to ethanol with a yield of 82% under anaerobic conditions. An essential mutation in this mutant was mapped within the pdh operon (pdhR aceEF lpd), which encodes components of the pyruvate dehydrogenase complex. Anaerobic ethanol production by this mutant is apparently the result of a novel pathway that combines the activities of pyruvate dehydrogenase (typically active during aerobic, oxidative metabolism) with the fermentative alcohol dehydrogenase.