Construction of recombinant Escherichia coli expressing xylitol-4-dehydrogenase and optimization for enhanced L-xylulose biotransformation from xylitol.

L-Xylulose is a rare ketopentose which inhibits α-glucosidase and is an indicator of hepatitis or liver cirrhosis. This pentose is also a precursor of other rare sugars such as L-xylose, L-ribose or L-lyxose. Recombinant E. coli expressing xylitol-4-dehydrogenase gene of Pantoea ananatis was constructed. A cost-effective culture media were used for L-xylulose production using the recombinant E. coli strain constructed. Response surface methodology was used to optimize these media components for L-xylulose production. A high conversion rate of 96.5% was achieved under an optimized pH and temperature using 20 g/L xylitol, which is the highest among the reports. The recombinant E. coli cells expressing the xdh gene were immobilized in calcium alginate to improve recycling of cells. Effective immobilization was achieved with 2% (w/v) sodium alginate and 3% (w/v) calcium chloride. The immobilized E. coli cells retained good stability and enzyme activity for 9 batches with conversion between 53 and 92% which would be beneficial for economical production of L-xylulose.

Photosynthetic Reduction of Xylose to Xylitol Using Cyanobacteria.

Photosynthetic generation of reducing power makes cyanobacteria an attractive host for biochemical reduction compared to cell-free and heterotrophic systems, which require burning of additional resources for the supply of reducing equivalent. Here, using xylitol synthesis as an example, efficient uptake and reduction of xylose photoautotrophically in Synechococcus elongatus PCC7942 are demonstrated upon introduction of an effective xylose transporter from Escherichia coli (Ec-XylE) and the NADPH-dependent xylose reductase from Candida boidinii (Cb-XR). Simultaneous activation of xylose uptake and matching of cofactor specificity enabled an average xylitol yield of 0.9 g g-1 xylose and a maximum productivity of about 0.15 g L-1 day-1 OD-1 with increased level of xylose supply. While long-term cellular maintenance still appears challenging, high-density conversion of xylose to xylitol using concentrated resting cell further pushes the titer of xylitol formation to 33 g L-1 in six days with 85% of maximum theoretical yield. While the results show that the unknown dissipation of xylose can be minimized when coupled to a strong reaction outlet, it remains to be the major hurdle hampering the yield despite the reported inability of cyanobacteria to metabolize xylose.

Exploiting the NADPH pool for xylitol production using recombinant Saccharomyces cerevisiae.

Xylitol is a five-carbon sugar alcohol that has a variety of uses in the food and pharmaceutical industries. In xylose assimilating yeasts, NAD(P)H-dependent xylose reductase (XR) catalyzes the reduction of xylose to xylitol. In the present study, XR with varying cofactor specificities was overexpressed in Saccharomyces cerevisiae to screen for efficient xylitol production. Xylose consumption and xylitol yields were higher when NADPH-dependent enzymes (Candida tropicalis XR and S. cerevisiae Gre3p aldose reductase) were expressed, indicating that heterologous enzymes can utilize the intracellular NADPH pool more efficiently than the NADH pool, where they may face competition from native enzymes. This was confirmed by overexpression of a NADH-preferring C. tropicalis XR mutant, which led to decreased xylose consumption and lower xylitol yield. To increase intracellular NADPH availability for xylitol production, the promoter of the ZWF1 gene, coding for the first enzyme of the NADPH-generating pentose phosphate pathway, was replaced with the constitutive GPD promoter in a strain expressing C. tropicalis XR. This change led to a ~12% increase in xylitol yield. Deletion of XYL2 and SOR1, whose gene products can use xylitol as substrate, did not further increase xylitol yield. Using wheat stalk hydrolysate as source of xylose, the constructed strain efficiently produced xylitol, demonstrating practical relevance of this approach.

Overexpression of Endogenous Xylose Reductase Enhanced Xylitol Productivity at 40 °C by Thermotolerant Yeast Kluyveromyces marxianus.

Xylitol is a valuable substance utilized by food and biochemical industries. NAD(P)H-dependent xylose reductase (XR)-encoded by the yeast KmXYL1 gene-is the key enzyme which facilitates reduction of xylose to xylitol. Multi-copy integration of a mutant KmXYL1 (mKmXYL1) gene was carried out using thermotolerant yeast Kluyveromyces marxianus KCTC17555ΔURA3, in order to enhance xylitol production. After multi-copy integration, the highest xylitol producing strain was isolated and named K. marxianus 17555-JBP2. This strain exhibited 440% higher xylitol production than the parental strain at 30 °C. Due to a multi-copy integration of the mKmXYL1 gene, various additional differences between K. marxianus 17555-JBP2 and the parental strain were observed, including a 66% increase in NAD(P)H-dependent XR activity at high temperature (45 °C). Quantitative real-time PCR and transcriptome analysis demonstrated that, relative to the parent strain, K. marxianus 17555-JBP2 exhibited two more copies of mKmXY1 genes and a 9.63-fold elevation in transcription of NAD(P)H-dependent XR. After optimization of bioreactor fermentation conditions (agitation speed), high-temperature (40 °C) xylitol productivity of K. marxianus 17555-JBP2 exhibited an 81% improvement relative to the parental strain. In this study, we demonstrated that the overexpression of endogenous XR could enhance xylitol productivity at 40 °C by thermotolerant K. marxianus.

Simultaneous fermentation of glucose and xylose at elevated temperatures co-produces ethanol and xylitol through overexpression of a xylose-specific transporter in engineered Kluyveromyces marxianus.

Engineered Kluyveromyces marxianus strains were constructed through over-expression of various transporters for simultaneous co-fermentation of glucose and xylose. The glucose was converted into ethanol, whereas xylose was converted into xylitol which has higher value than ethanol. Over-expressing xylose-specific transporter ScGAL2-N376F mutant enabled yeast to co-ferment glucose and xylose and the co-fermentation ability was obviously improved through increasing ScGAL2-N376F expression. The production of glycerol was blocked and acetate production was reduced by disrupting gene KmGPD1. The obtained K. marxianus YZJ119 utilized 120g/L glucose and 60g/L xylose simultaneously and produced 50.10g/L ethanol and 55.88g/L xylitol at 42°C. The yield of xylitol from consumed xylose was over 98% (0.99g/g). Through simultaneous saccharification and co-fermentation at 42°C, YZJ119 produced a maximal concentration of 44.58g/L ethanol and 32.03g/L xylitol or 29.82g/L ethanol and 31.72g/L xylitol, respectively, from detoxified or non-detoxified diluted acid pretreated corncob.

Effect of heterologous xylose transporter expression in Candida tropicalis on xylitol production rate.

Xylose utilization is inhibited by glucose uptake in xylose-assimilating yeasts, including Candida tropicalis, resulting in limitation of xylose uptake during the fermentation of glucose/xylose mixtures. In this study, a heterologous xylose transporter gene (At5g17010) from Arabidopsis thaliana was selected because of its high affinity for xylose and was codon-optimized for functional expression in C. tropicalis. The codon-optimized gene was placed under the control of the GAPDH promoter and was integrated into the genome of C. tropicalis strain LXU1 which is xyl2-disrupted and NXRG (codon-optimized Neurospora crassa xylose reductase) introduced. The xylose uptake rate was increased by 37-73 % in the transporter expression-enhanced strains depending on the glucose/xylose mixture ratio. The recombinant strain LXT2 in 500-mL flask culture using glucose/xylose mixtures showed a xylose uptake rate that was 29 % higher and a xylitol volumetric productivity (1.14 g/L/h) that was 25 % higher than the corresponding rates for control strain LXU1. Membrane protein extraction and Western blot analysis confirmed the successful heterologous expression and membrane localization of the xylose transporter in C. tropicalis.

Analysis and prediction of the physiological effects of altered coenzyme specificity in xylose reductase and xylitol dehydrogenase during xylose fermentation by Saccharomyces cerevisiae.

An advanced strategy of Saccharomyces cerevisiae strain development for fermentation of xylose applies tailored enzymes in the process of metabolic engineering. The coenzyme specificities of the NADPH-preferring xylose reductase (XR) and the NAD⁺-dependent xylitol dehydrogenase (XDH) have been targeted in previous studies by protein design or evolution with the aim of improving the recycling of NADH or NADPH in their two-step pathway, converting xylose to xylulose. Yeast strains expressing variant pairs of XR and XDH that according to in vitro kinetic data were suggested to be much better matched in coenzyme usage than the corresponding pair of wild-type enzymes, exhibit widely varying capabilities for xylose fermentation. To achieve coherence between enzyme properties and the observed strain performance during fermentation, we explored the published kinetic parameters for wild-type and engineered forms of XR and XDH as possible predictors of xylitol by-product formation (Y(xylitol)) in yeast physiology. We found that the ratio of enzymatic reaction rates using NADP(H) and NAD(H) that was calculated by applying intracellular reactant concentrations to rate equations derived from bi-substrate kinetic analysis, succeeded in giving a statistically reliable forecast of the trend effect on Y(xylitol). Prediction based solely on catalytic efficiencies with or without binding affinities for NADP(H) and NAD(H) were not dependable, and we define a minimum demand on the enzyme kinetic characterization to be performed for this purpose. An immediate explanation is provided for the typically lower Y(xylitol) in the current strains harboring XR engineered for utilization of NADH as compared to strains harboring XDH engineered for utilization of NADP⁺. The known XDH enzymes all exhibit a relatively high K(m) for NADP⁺ so that physiological boundary conditions are somewhat unfavorable for xylitol oxidation by NADP⁺. A criterion of physiological fitness is developed for engineered XR working together with wild-type XDH.

Effects of NADH-preferring xylose reductase expression on ethanol production from xylose in xylose-metabolizing recombinant Saccharomyces cerevisiae.

Efficient conversion of xylose to ethanol is an essential factor for commercialization of lignocellulosic ethanol. To minimize production of xylitol, a major by-product in xylose metabolism and concomitantly improve ethanol production, Saccharomyces cerevisiae D452-2 was engineered to overexpress NADH-preferable xylose reductase mutant (XR(MUT)) and NAD⁺-dependent xylitol dehydrogenase (XDH) from Pichia stipitis and endogenous xylulokinase (XK). In vitro enzyme assay confirmed the functional expression of XR(MUT), XDH and XK in recombinant S. cerevisiae strains. The change of wild type XR to XR(MUT) along with XK overexpression led to reduction of xylitol accumulation in microaerobic culture. More modulation of the xylose metabolism including overexpression of XR(MUT) and transaldolase, and disruption of the chromosomal ALD6 gene encoding aldehyde dehydrogenase (SX6(MUT)) improved the performance of ethanol production from xylose remarkably. Finally, oxygen-limited fermentation of S. cerevisiae SX6(MUT) resulted in 0.64 g l⁻¹ h⁻¹ xylose consumption rate, 0.25 g l⁻¹ h⁻¹ ethanol productivity and 39% ethanol yield based on the xylose consumed, which were 1.8, 4.2 and 2.2 times higher than the corresponding values of recombinant S. cerevisiae expressing XR(MUT), XDH and XK only.

Bioconversion of d-xylose to d-xylonate with Kluyveromyces lactis.

d-Xylonate was produced from d-xylose using Kluyveromyces lactis strains which expressed the gene for NADP(+)-dependent d-xylose dehydrogenase from Trichoderma reesei (xyd1). Up to 19 ± 2g d-xylonatel(-1) was produced when K. lactis expressing xyd1 was grown on 10.5 gd-galactosel(-1) and 40 g d-xylosel(-1). Intracellular accumulation of d-xylonate (up to ∼70 mg [gbiomass](-1)) was observed. d-Xylose was metabolised to d-xylonate, xylitol and biomass. Oxygen could be reduced to 6mmolO(2)l(-1)h(-1) without loss in titre or production rate, but metabolism of d-xylose and xylitol were more efficient when 12 mmolO(2)l(-1)h(-1) were provided. d-Xylose uptake was not affected by deletion of either the d-xylose reductase (XYL1) or a putative xylitol dehydrogenase encoding gene (XYL2) in xyd1 expressing strains. K. lactis xyd1ΔXYL1 did not produce extracellular xylitol and produced more d-xylonate than the xyd1 strain containing the endogenous XYL1. K. lactis xyd1ΔXYL2 produced high concentrations of xylitol and significantly less d-xylonate than the xyd1 strain with the endogenous XYL2.