A substrate-selective co-fermentation strategy with Escherichia coli produces lactate by simultaneously consuming xylose and glucose

We describe a new approach for the simultaneous conversion of xylose and glucose sugar mixtures which potentially could be used for lignocellulosic biomass hydrolysate. In this study we used this approach to demonstrate the production of lactic acid. This process uses two substrate-selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. In addition to knockouts in pflB encoding for pyruvate formate lyase, the xylose-selective (glucose deficient) strain E. coli ALS1073 has deletions of the glk, ptsG, and manZ genes while the glucose-selective (xylose deficient) strain E. coli ALS1074 has a xylA deletion. By combining these two strains in a single process the xylose and glucose in a mixed sugar solution are simultaneously converted to lactate. Furthermore, the biomass concentrations of each strain can readily be adjusted in order to optimize the overall product formation. This approach to the utilization of mixed sugars eliminates the problem of diauxic growth, and provides great operational flexibility.     

Systems Metabolic Engineering of Escherichia coli Improves Coconversion of Lignocellulose‐Derived Sugars

Currently, microbial conversion of lignocellulose‐derived glucose and xylose to biofuels is hindered by the fact that most microbes (including Escherichia coli [E. coli], Saccharomyces cerevisiae, and Zymomonas mobilis) preferentially consume glucose first and consume xylose slowly after glucose is depleted in lignocellulosic hydrolysates. In this study, E. coli strains are developed that simultaneously utilize glucose and xylose in lignocellulosic biomass hydrolysate using genome‐scale models and adaptive laboratory evolution. E. coli strains are designed and constructed that coutilize glucose and xylose and adaptively evolve them to improve glucose and xylose utilization. Whole‐genome resequencing of the evolved strains find relevant mutations in metabolic and regulatory genes and the mutations’ involvement in sugar coutilization is investigated. The developed strains show significantly improved coconversion of sugars in lignocellulosic biomass hydrolysates and provide a promising platform for producing next‐generation biofuels.     

Conversion of glucose‐xylose mixtures to pyruvate using a consortium of metabolically engineered Escherichia coli

Two strains of Escherichia coli were engineered to accumulate pyruvic acid from two sugars found in lignocellulosic hydrolysates by knockouts in the aceE, ppsA, poxB, and ldhA genes. Additionally, since glucose and xylose are typically consumed sequentially due to carbon catabolite repression in E. coli, one strain (MEC590) was engineered to grow only on glucose while a second strain (MEC589) grew only on xylose. On a single substrate, each strain generated pyruvate at a yield of about 0.60 g/g in both continuous culture and batch culture. In a glucose‐xylose mixture under continuous culture, a consortium of both strains maintained a pyruvate yield greater than 0.60 g/g when three different concentrations of glucose and xylose were sequentially fed into the system. In a fed‐batch process, both sugars in a glucose‐xylose mixture were consumed simultaneously to accumulate 39 g/L pyruvate in less than 24 h at a yield of 0.59 g/g.     

Succinate production from xylose‐glucose mixtures using a consortium of engineered Escherichia coli

The conversion of variable sugar mixtures into biochemicals poses a challenge for a single microorganism. For example, succinate has not been effectively generated from mixtures of glucose and xylose. In this work, a consortium of two Escherichia coli strains converted xylose and glucose to succinate in a dual phase aerobic/anaerobic process. First, the optimal pathway from xylose or glucose to succinate was determined by expressing either heterologous pyruvate carboxylase or heterologous adenosine triphosphate‐forming phosphoenol pyruvate (PEP) carboxykinase. Expression of PEP carboxykinase (pck) resulted in higher yield (0.86 g/g) and specific productivity (155 mg/gh) for xylose conversion, while expression of pyruvate carboxylase (pyc) resulted in higher productivity (76 mg/gh) for glucose conversion. Then, processes using consortia of the two optimal xylose‐selective and glucose‐selective strains were designed for two different feed ratios of glucose/xylose. In each case the consortia generated over 40 g/L succinate efficiently with yields greater than 0.90 g succinate/g total sugar. This study demonstrates two advantages of microbial consortia for the conversion of sugar mixtures: each sugar‐to‐product pathway can be optimized independently, and the volumetric consumption rate for each sugar can be controlled independently, for example, by altering the biomass concentration of each consortium member strain.     

Regulation of Arabinose and Xylose Metabolism in Escherichia coli

Bacteria such as Escherichia coli will often consume one sugar at a time when fed multiple sugars, in a process known as carbon catabolite repression. The classic example involves glucose and lactose, where E. coli will first consume glucose, and only when it has consumed all of the glucose will it begin to consume lactose. In addition to that of lactose, glucose also represses the consumption of many other sugars, including arabinose and xylose. In this work, we characterized a second hierarchy in E. coli, that between arabinose and xylose. We show that, when grown in a mixture of the two pentoses, E. coli will consume arabinose before it consumes xylose. Consistent with a mechanism involving catabolite repression, the expression of the xylose metabolic genes is repressed in the presence of arabinose. We found that this repression is AraC dependent and involves a mechanism where arabinose-bound AraC binds to the xylose promoters and represses gene expression. Collectively, these results demonstrate that sugar utilization in E. coli involves multiple layers of regulation, where cells will consume first glucose, then arabinose, and finally xylose. These results may be pertinent in the metabolic engineering of E. coli strains capable of producing chemical and biofuels from mixtures of hexose and pentose sugars derived from plant biomass.The transporters and enzymes in many sugar metabolic pathways are conditionally expressed in response to their cognate sugar or a downstream pathway intermediate. While the induction of these pathways in response to a single sugar has been studied extensively (28), far less is known about how these pathways are induced in response to multiple sugars. One notable exception is the phenomenon observed when bacteria are grown in the presence of glucose and another sugar (10, 15). In such mixtures, the bacteria will often consume glucose first before consuming the other sugar, a process known as carbon catabolite repression (27). The classic example of carbon catabolite repression is the diauxic shift seen in the growth of Escherichia coli on mixtures of glucose and lactose, where the cells first consume glucose before consuming lactose. When the cells are consuming glucose, the genes in the lactose metabolic pathway are not induced, thus preventing the sugar from being consumed. A number of molecules participate in this regulation, including the cyclic AMP receptor protein (CRP), adenylate cyclase, cyclic AMP (cAMP), and EIIA from the phosphoenolpyruvate:glucose phosphotransferase system (PTS) (33). In addition to lactose, the metabolic genes for many other sugars are subject to catabolite repression by glucose in E. coli (27). While the preferential utilization of glucose is well known, it is an open question whether additional hierarchies exist among other sugars.Recently, substantial effort has been directed toward developing microorganisms capable of producing chemicals and biofuels from plant biomass (1, 34, 42). After glucose, l-arabinose and d-xylose are the next most abundant sugars found in plant biomass. Therefore, a key step in producing various chemicals and fuels from plant biomass will be the engineering of strains capable of efficiently fermenting these three sugars. However, one challenge concerns catabolite repression, which prevents microorganisms from fermenting these three sugars simultaneously and, as a consequence, may decrease the efficiency of the fermentation process. E. coli cells will first consume glucose before consuming either arabinose or xylose. As in the case of lactose, the genes in the arabinose and xylose metabolic pathways are not expressed when glucose is being consumed. In addition to glucose catabolite repression, a second hierarchy, between arabinose and xylose, appears to exist. Kang and coworkers have observed that the genes in the xylose metabolic pathway were repressed when cells were grown in a mixture of arabinose and xylose (21). Hernandez-Montalvo and coworkers also observed that E. coli utilizes arabinose before xylose (19). While a number of strategies exist for breaking the glucose-mediated repression of arabinose and xylose metabolism (8, 16, 19, 31), none exist for breaking the arabinose-mediated repression of xylose metabolism. Moreover, little is known about this repression beyond the observations made by these researchers.In this work, we investigate how the arabinose and xylose metabolic pathways are jointly regulated. We demonstrate that E. coli will consume arabinose before consuming xylose when it is grown in a mixture of the two sugars. Consistent with a mechanism involving catabolite repression, the genes in the xylose metabolic pathway are repressed in the presence of arabinose. We found that this repression is AraC dependent and is most likely due to binding by arabinose-bound AraC to the xylose promoters, with consequent inhibition of gene expression.     

Phosphatases and phosphate affect the formation of glucose from pentoses in Escherichia coli

Metabolically engineered Escherichia coli MEC143 with deletions of the ptsG, manZ, glk, pfkA, and zwf genes converts pentoses such as arabinose and xylose into glucose, with the dephosphorylation of glucose‐6‐phosphate serving as the final step. To determine which phosphatase mediates this conversion, we examined glucose formation from pentoses in strains containing knockouts of six different phosphatases singly and in combination. Deletions of single phosphatases and combinations of multiple phosphatases did not eliminate the accumulation of glucose from xylose or arabinose. Overexpression of one phosphatase, haloacid dehalogenase‐like phosphatase 12 coded by the ybiV gene, increased glucose yield significantly from 0.26 to 0.30 g/g (xylose) and from 0.32 to 0.35 g/g (arabinose). Growing cells under phosphate‐limited steady‐state conditions increased the glucose yield to 0.39 g glucose/g xylose, but did not affect glucose yield from arabinose (0.31 g/g). No single phosphatase is exclusively responsible for the conversion of glucose‐6‐phosphate to glucose in E. coli MEC143. Phosphate‐limited conditions are indeed able to enhance glucose formation in some cases, with this effect likely influenced by the different phosphate demands when E. coli metabolizes different carbon sources.     

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.     

Engineered Escherichia coli capable of co-utilization of cellobiose and xylose

Natural ability to ferment the major sugars (glucose and xylose) of plant biomass is an advantageous feature of Escherichia coli in biofuel production. However, excess glucose completely inhibits xylose utilization in E. coli and decreases yield and productivity of fermentation due to sequential utilization of xylose after glucose. As an approach to overcome this drawback, E. coli MG1655 was engineered for simultaneous glucose (in the form of cellobiose) and xylose utilization by a combination of genetic and evolutionary engineering strategies. The recombinant E. coli was capable of utilizing approximately 6 g/L of cellobiose and 2 g/L of xylose in approximately 36 h, whereas wild-type E. coli was unable to utilize xylose completely in the presence of 6 g/L of glucose even after 75 hours. The engineered strain also co-utilized cellobiose with mannose or galactose; however, it was unable to metabolize cellobiose in the presence of arabinose and glucose. Successful cellobiose and xylose co-fermentation is a vital step for simultaneous saccharification and co-fermentation process and a promising step towards consolidated bioprocessing.     

Fermentation of sugar mixtures using Escherichia coli catabolite repression mutants engineered for production of L-lactic acid

Conversion of lignocellulose to lactic acid requires strains capable of fermenting sugar mixtures of glucose and xylose. Recombinant Escherichia coli strains were engineered to selectively produce L-lactic acid and then used to ferment sugar mixtures. Three of these strains were catabolite repression mutants (ptsG ⁻) that have the ability to simultaneously ferment glucose and xylose. The best results were obtained for ptsG ⁻ strain FBR19. FBR19 cultures had a yield of 0.77 (g lactic acid/g added sugar) when used to ferment a 100 g/l total equal mixture of glucose and xylose. The strain also consumed 75% of the xylose. In comparison, the ptsG ⁺ strains had yields of 0.47–0.48 g/g and consumed 18–22% of the xylose. FBR19 was subsequently used to ferment a variety of glucose (0–40 g/l) and xylose (40 g/l) mixtures. The lactic acid yields ranged from 0.74 to 1.00 g/g. Further experiments were conducted to discover the mechanism leading to the poor yields for ptsG ⁺ strains. Xylose isomerase (XI) activity, a marker for induction of xylose metabolism, was monitored for FBR19 and a ptsG ⁺ control during fermentations of a sugar mixture. Crude protein extracts prepared from FBR19 had 10–12 times the specific XI activity of comparable samples from ptsG ⁺ strains. Therefore, higher expression of xylose metabolic genes in the ptsG ⁻ strain may be responsible for superior conversion of xylose to product compared to the ptsG ⁺ fermentations. Received 14 December 2000/ Accepted in revised form 28 June 2002     

Novel two‐stage fermentation process for bioethanol production using Saccharomyces pastorianus

Bioethanol produced from lignocellulosic materials has the potential to be economically feasible, if both glucose and xylose released from cellulose and hemicellulose can be efficiently converted to ethanol. Saccharomyces spp. can efficiently convert glucose to ethanol; however, xylose conversion to ethanol is a major hurdle due to lack of xylose‐metabolizing pathways. In this study, a novel two‐stage fermentation process was investigated to improve bioethanol productivity. In this process, xylose is converted into biomass via non‐Saccharomyces microorganism and coupled to a glucose‐utilizing Saccharomyces fermentation. Escherichia coli was determined to efficiently convert xylose to biomass, which was then killed to produce E. coli extract. Since earlier studies with Saccharomyces pastorianus demonstrated that xylose isomerase increased ethanol productivities on pure sugars, the addition of both E. coli extract and xylose isomerase to S. pastorianus fermentations on pure sugars and corn stover hydrolysates were investigated. It was determined that the xylose isomerase addition increased ethanol productivities on pure sugars but was not as effective alone on the corn stover hydrolysates. It was observed that the E. coli extract addition increased ethanol productivities on both corn stover hydrolysates and pure sugars. The ethanol productivities observed on the corn stover hydrolysates with the E. coli extract addition was the same as observed on pure sugars with both E. coli extract and xylose isomerase additions. These results indicate that the two‐stage fermentation process has the capability to be a competitive alternative to recombinant Saccharomyces cerevisiae‐based fermentations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:300–310, 2014     

Fermentation of xylose to succinate by enhancement of ATP supply in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

In Escherichia coli K12, succinate was not the dominant fermentation product from xylose. To reduce by-product formation and increase succinate accumulation, pyruvate formate lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, these mutations eliminated cell growth and xylose utilization. During anaerobic growth of bacteria, organic intermediates, such as pyruvate, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate level phosphorylation. In E. coli K12, conversion of xylose to pyruvate only yielded 0.67 net ATP per xylose during anaerobic fermentation. However, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose, which could meet the ATP needed for xylose metabolism. A pflB deletion strain cannot convert pyruvate to acetyl coenzyme A, the precursor for acetate and ethanol production, and could not produce the additional ATP. Thus, the double mutations eliminated cell growth and xylose utilization. To supply the sufficient ATPs, overexpression of ATP-forming phosphoenolpyruvate-carboxykinase from Bacillus subtilis 168 in an ldhA, pflB, and ppc deletion strain resulted in a significant increase in cell mass and succinate production. In addition, fermentation of corn stalk hydrolysate containing a high percentage of xylose and glucose produced a final succinate concentration of 11.13 g l⁻¹ with a yield of 1.02 g g⁻¹ total sugars during anaerobic fermentation.     

Improvement of <Emphasis Type="Italic">Escherichia coli</Emphasis> production strains by modification of the phosphoenolpyruvate:sugar phosphotransferase system

The application of metabolic engineering in Escherichia coli has resulted in the generation of strains with the capacity to produce metabolites of commercial interest. Biotechnological processes with these engineered strains frequently employ culture media containing glucose as the carbon and energy source. In E. coli, the phosphoenolpyruvate:sugar phosphotransferase system (PTS) transports glucose when this sugar is present at concentrations like those used in production fermentations. This protein system is involved in phosphoenolpyruvate-dependent sugar transport, therefore, its activity has an important impact on carbon flux distribution in the phosphoenolpyruvate and pyruvate nodes. Furthermore, PTS has a very important role in carbon catabolite repression. The properties of PTS impose metabolic and regulatory constraints that can hinder strain productivity. For this reason, PTS has been a target for modification with the purpose of strain improvement. In this review, PTS characteristics most relevant to strain performance and the different strategies of PTS modification for strain improvement are discussed. Functional replacement of PTS by alternative phosphoenolpyruvate-independent uptake and phosphorylation activities has resulted in significant improvements in product yield from glucose and productivity for several classes of metabolites. In addition, inactivation of PTS components has been applied successfully as a strategy to abolish carbon catabolite repression, resulting in E. coli strains that use more efficiently sugar mixtures, such as those obtained from lignocellulosic hydrolysates.     

Simultaneous utilization of glucose, xylose and arabinose in the presence of acetate by a consortium of Escherichia coli strains

ABSTRACT: BACKGROUND: The efficient microbial utilization of lignocellulosic hydrolysates has remained challenging because this material is composed of multiple sugars and also contains growth inhibitors such as acetic acid (acetate). Using an engineered consortium of strains derived from Escherichia coli C and a synthetic medium containing acetate, glucose, xylose and arabinose, we report on both the microbial removal of acetate and the subsequent simultaneous utilization of the sugars. RESULTS: In a first stage, a strain unable to utilize glucose, xylose and arabinose (ALS1392, strain E. coli C ptsG manZ glk crr xylA araA) removed 3 g/L acetate within 30 hours. In a subsequent second stage, three E. coli strains (ALS1370, ALS1371, ALS1391), which are each engineered to utilize only one sugar, together simultaneously utilized glucose, xylose and arabinose. The effect of non-metabolizable sugars on the metabolism of the target sugar was minimal. Additionally the deletions necessary to prevent the consumption of one sugar only minimally affected the consumption of a desired sugar. For example, the crr deletion necessary to prevent glucose consumption reduced xylose and arabinose utilization by less than 15 % compared to the wild-type. Similarly, the araA deletion used to exclude arabinose consumption did not affect xylose- and glucose-consumption. CONCLUSIONS: Despite the modest reduction in the overall rate of sugar consumption due to the various deletions that were required to generate the consortium of strains, the approach constitutes a significant improvement in any single-organism approach to utilize sugars found in lignocellulosic hydrolysate in the presence of acetate.     

Microbial removal of acetate selectively from sugar mixtures

Acetic acid is an unavoidable constituent of the biomass hydrolysates generated from acetylated hemicellulose and lignin, and acetate affects the performance of microbes used to convert these hydrolysates into biofuels or other biochemicals. In this study, acetate was selectively removed from synthetic mixtures of glucose and xylose using metabolically engineered Escherichia coli strains having mutations in the glucose phosphotransferase system (PTS) genes (ptsG, manZ, crr), glucokinase (glk), and xylose (xylA). In batch culture, ALS1060 (ptsG manZ glk xylA) consumed exclusively acetate to depletion, and then consumed the two sugars only at a very slow rate (a growth rate of about 0.01 h⁻¹). We also examined the effects of an additional knockout of either malX, fruA, fruB, bglF, or crr, genes that are involved in other PTSs, and a batch process using KD840 (ptsG manZ glk crr xylA) demonstrated a further reduction in glucose or xylose consumption by E. coli. These results demonstrate the feasibility of using a substrate-selective approach for the pre-treatment of biomass hydrolysate for microbial processes.     

Production of free fatty acids from switchgrass using recombinant Escherichia coli

Switchgrass is a promising feedstock to generate fermentable sugars required for the sustainable operation of biorefineries because of their abundant availability, easy cropping system, and high cellulosic content. The objective of this study was to investigate the potentiality of switchgrass as an alternative sugar supplier for free fatty acid (FFA) production using engineered Escherichia coli strains. Recombinant E. coli strains successfully produced FFAs using switchgrass hydrolysates. A total of about 3 g/L FFAs were attained from switchgrass hydrolysates by engineered E. coli strains. Furthermore, overall yield assessments of our bioconversion process showed that 88 and 46% of the theoretical maximal yields of glucose and xylose were attained from raw switchgrass during sugar generation. Additionally, 72% of the theoretical maximum yield of FFAs were achieved from switchgrass hydrolysates by recombinant E. coli during fermentation. These shake‐flask results were successfully scaled up to a laboratory scale bioreactor with a 4 L working volume. This study demonstrated an efficient bioconversion process of switchgrass‐based FFAs using an engineered microbial system for targeting fatty acid production that are secreted into the fermentation broth with associated lower downstream processing costs, which is pertinent to develop an integrated bioconversion process using lignocellulosic biomass. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:91–98, 2018     

Simultaneous consumption of pentose and hexose sugars: an optimal microbial phenotype for efficient fermentation of lignocellulosic biomass

Lignocellulosic biomass is an attractive carbon source for bio-based fuel and chemical production; however, its compositional heterogeneity hinders its commercial use. Since most microbes possess carbon catabolite repression (CCR), mixed sugars derived from the lignocellulose are consumed sequentially, reducing the efficacy of the overall process. To overcome this barrier, microbes that exhibit the simultaneous consumption of mixed sugars have been isolated and/or developed and evaluated for the lignocellulosic biomass utilization. Specific strains of Escherichia coli, Saccharomyces cerevisiae, and Zymomonas mobilis have been engineered for simultaneous glucose and xylose utilization via mutagenesis or introduction of a xylose metabolic pathway. Other microbes, such as Lactobacillus brevis, Lactobacillus buchneri, and Candida shehatae possess a relaxed CCR mechanism, showing simultaneous consumption of glucose and xylose. By exploiting CCR-negative phenotypes, various integrated processes have been developed that incorporate both enzyme hydrolysis of lignocellulosic material and mixed sugar fermentation, thereby enabling greater productivity and fermentation efficacy.     

Anaerobic xylose fermentation by <Emphasis Type="Italic">Spathaspora passalidarum</Emphasis>

A cost-effective conversion of lignocellulosic biomass into bioethanol requires that the xylose released from the hemicellulose fraction (20–40% of biomass) can be fermented. Baker’s yeast, Saccharomyces cerevisiae, efficiently ferments glucose but it lacks the ability to ferment xylose. Xylose-fermenting yeast such as Pichia stipitis requires accurately controlled microaerophilic conditions during the xylose fermentation, rendering the process technically difficult and expensive. In this study, it is demonstrated that under anaerobic conditions Spathaspora passalidarum showed high ethanol production yield, fast cell growth, and rapid sugar consumption with xylose being consumed after glucose depletion, while P. stipitis was almost unable to utilize xylose under these conditions. It is further demonstrated that for S. passalidarum, the xylose conversion takes place by means of NADH-preferred xylose reductase (XR) and NAD⁺-dependent xylitol dehydrogenase (XDH). Thus, the capacity of S. passalidarum to utilize xylose under anaerobic conditions is possibly due to the balance between the cofactor’s supply and demand through this XR–XDH pathway. Only few XRs with NADH preference have been reported so far. 2-Deoxy glucose completely inhibited the conversion of xylose by S. passalidarum under anaerobic conditions, but only partially did that under aerobic conditions. Thus, xylose uptake by S. passalidarum may be carried out by different xylose transport systems under anaerobic and aerobic conditions. The presence of glucose also repressed the enzymatic activity of XR and XDH from S. passalidarum as well as the activities of those enzymes from P. stipitis.     

Production of d-ribose by metabolically engineered Escherichia coli

Escherichia coli was metabolically engineered for the production of d-ribose, a functional five-carbon sugar, from xylose. For the accumulation of d-ribose, two genes of transketolase catalyzing the conversion of d-ribose-5-phosphate to sedoheptulose-7-phosphate in pentose phosphate pathway were disrupted to create a transketolase-deficient E. coli SGK013. In batch fermentation, E. coli SGK013 grew by utilizing glucose and then started to produce d-ribose from xylose after glucose depletion. E. coli SGK013 produced 0.75 g/L of d-ribose, which was identical to the standard d-ribose as confirmed by HPLC and LC/MS analyses. To improve D-ribose production, the ptsG gene encoding the glucose-specific IICB component was disrupted additionally, resulting in the construction of E. coli SGK015. The carbon catabolite repression-negative E. coli SGK015 utilized xylose and glucose simultaneously and produced up to 3.75 g/L of d-ribose, which is a 5-fold improvement compared to that of E. coli SGK013.