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.

Fine-tuning of xylose metabolism in genetically engineered Saccharomyces cerevisiae by scattered integration of xylose assimilation genes.

Manipulation of multiple genes is a common experience in metabolic engineering and synthetic biology studies. Chromosome integration of multiple genes in one single position is always performed, however, there is so far no study on the integration of multiple genes separately in various positions (here in after referred to as "scattered integration") and its effect on fine-tuning of cellular metabolism. In this study, scattered integration of the xylose assimilation genes PsXR, PsXDH and ScXK was investigated in Saccharomyces cerevisiae, and transcription analysis of these genes as well as their enzyme activities were compared with those observed when the genes were integrated into one single site (defined as "tandem integration" here). Not only notable differences in transcription levels and enzyme activities were observed when the genes were integrated by the two strategies, but also change of the cofactor preference of PsXR gene was validated. Xylose fermentation was further studied with the strains developed with these strategies, and elevated xylose utilization rate was obtained in the scattered integration strain. These results proved that by positioning multiple genes on different chromosomes, fine-tuning of cellular metabolism could be achieved in recombinant S. cerevisiae.

High expression of XYL2 coding for xylitol dehydrogenase is necessary for efficient xylose fermentation by engineered Saccharomyces cerevisiae.

The traditional ethanologenic yeast Saccharomyces cerevisiae cannot metabolize xylose, which is an abundant sugar in non-crop plants. Engineering this yeast for a practicable fermentation of xylose will therefore improve the economics of bioconversion for the production of fuels and chemicals such as ethanol. One of the most widely employed strategies is to express XYL1, XYL2, and XYL3 genes derived from Scheffersomyces stipitis (formerly Pichia stiptis) in S. cerevisiae. However, the resulting engineered strains have been reported to exhibit large variations in xylitol accumulation and ethanol yields, generating many hypotheses and arguments for elucidating these phenomena. Here we demonstrate that low expression levels of the XYL2 gene, coding for xylitol dehydrogenase (XDH), is a major bottleneck in efficient xylose fermentation. Through an inverse metabolic engineering approach using a genomic library of S. cerevisiae, XYL2 was identified as an overexpression target for improving xylose metabolism. Specifically, we performed serial subculture experiments after transforming a genomic library of wild type S. cerevisiae into an engineered strain harboring integrated copies of XYL1, XYL2 and XYL3. Interestingly, the isolated plasmids from efficient xylose-fermenting transformants contained XYL2. This suggests that the integrated XYL2 migrated into a multi-copy plasmid through homologous recombination. It was also found that additional overexpression of XYL2 under the control of strong constitutive promoters in a xylose-fermenting strain not only reduced xylitol accumulation, but also increased ethanol yields. As the expression levels of XYL2 increased, the ethanol yields gradually improved from 0.1 to 0.3g ethanol/g xylose, while the xylitol yields significantly decreased from 0.4 to 0.1g xylitol/g xylose. These results suggest that strong expression of XYL2 is a necessary condition for developing efficient xylose-fermenting strains.

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.

Cloning and overexpression of the xylitol dehydrogenase gene from Bacillus pallidus and its application to L-xylulose production.

The xylitol dehydrogenase gene (xdh) of Bacillus pallidus was cloned and overexpressed in Escherichia coli using pQE60 vector, for the first time. The open reading frame of 759 bp encoded a 253 amino acid protein with a calculated molecular mass of 27,333 Da. The recombinant xylitol dehydrogenase (XDH) was purified to homogeneity by three-step column chromatography, producing a single SDS-PAGE band of 28 kDa apparent molecular mass. The enzyme exhibited maximal activity at 55 °C in glycine-NaOH buffer pH 11.0, with 66% of initial enzyme activity retained after incubation at 40 °C for 1 h. In further application of the recombinant bacterium to L-xylulose production from xylitol (initial concentration 5%) using a resting cell reaction, 35% L-xylulose was produced within 24 h. This result indicates that this recombinant XDH is applicable in the large-scale production of L-xylulose.

Co-expression of xylose reductase gene from Candida shehatae and endogenous xylitol dehydrogenase gene in Saccharomyces cerevisiae and the effect of metabolizing xylose to ethanol.

The inability of Saccharomyces cerevisiae to utilize xylose is attributed to its inability to convert xylose to xylulose. Low xylose reductase (XR) and xylitol dehydrogenase (XDH) activities in S. cerevisiae are regarded as the reason of blocking the pathway from xylose to xylulose. We had found that Candida shehatae could also be another source for XR gene except Pichia stipitis in the previous study. In this study, we tried to investigate if the expressed XR from C. shehatae could work with the over-expressed endogenous XDH together to achieve the same goal of converting xylose to ethanol in S. cerevisiae. The XR gene (XYL1) from C. shehatae and endogenous XDH gene (XYL2) were both cloned and over-expressed in host S. cerevisiae cell. The specific enzyme activities of XR and XDH were measured and the result of fermentation revealed that the new combination of two enzymes from different sources other than P. stipitis could also coordinate and work with each other and confer xylose utilization ability to S. cerevisiae.

Proteome analysis of the xylose-fermenting mutant yeast strain TMB 3400.

Xylose fermentation in yeast has been a target of research for years, yet not all the factors that may affect xylose fermentation performance of yeast strains are known. In this study, the mutant S. cerevisiae strain TMB 3400, which has good xylose fermentation properties, was compared with its parental strain to examine the factors behind the improved xylose utilization at protein level. The proteome of the parental and the mutant strains were characterized by difference in gel electrophoresis (DiGE) to quantitatively identify proteins that are expressed at altered levels in the mutant. The most significant changes detected by proteome analysis were the 6-10-fold increased levels of xylose reductase, xylitol dehydrogenase and transketolase (Tkl1) in the mutant, which is in accordance with previous knowledge about xylose metabolism in yeast. The level of acetaldehyde dehydrogenase (Ald6) was also significantly increased. In addition, several proteins homologous to proteins from yeast species other than S. cerevisiae were identified in both strains, demonstrating the genetic heterogeneity of industrial yeast strains. The results were also compared with a previously reported transcription analysis performed with identical experimental set-up; however, very little correlation between the two datasets was observed. The results of the proteome analysis were in good agreement with a parallel study in which rationally designed overexpression of XR, XDH and the non-oxidative pentose phosphate pathway resulted in similar improvement in xylose utilization, which demonstrates the usefulness of proteome analysis for the identification of target genes for further metabolic engineering strategies in industrial yeast strains.