Metabolic engineering of Saccharomyces cerevisiae for conversion of D-glucose to xylitol and other five-carbon sugars and sugar alcohols

Recombinant Saccharomyces cerevisiae strains that produce the sugar alcohols xylitol and ribitol and the pentose sugar D-ribose from D-glucose in a single fermentation step are described. A transketolase-deficient S. cerevisiae strain accumulated D-xylulose 5-phosphate intracellularly and released ribitol and pentose sugars (D-ribose, D-ribulose, and D-xylulose) into the growth medium. Expression of the xylitol dehydrogenase-encoding gene XYL2 of Pichia stipitis in the transketolase-deficient strain resulted in an 8.5-fold enhancement of the total amount of the excreted sugar alcohols ribitol and xylitol. The additional introduction of the 2-deoxy-glucose 6-phosphate phosphatase-encoding gene DOG1 into the transketolase-deficient strain expressing the XYL2 gene resulted in a further 1.6-fold increase in ribitol production. Finally, deletion of the endogenous xylulokinase-encoding gene XKS1 was necessary to increase the amount of xylitol to 50% of the 5-carbon sugar alcohols excreted.     

l-Arabinose/d-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of l-arabinose to l-arabonate with Saccharomyces cerevisiae

Four potential dehydrogenases identified through literature and bioinformatic searches were tested for l-arabonate production from l-arabinose in the yeast Saccharomyces cerevisiae. The most efficient enzyme, annotated as a d-galactose 1-dehydrogenase from the pea root nodule bacterium Rhizobium leguminosarum bv. trifolii, was purified from S. cerevisiae as a homodimeric protein and characterised. We named the enzyme as a l-arabinose/d-galactose 1-dehydrogenase (EC 1.1.1.-), Rl AraDH. It belongs to the Gfo/Idh/MocA protein family, prefers NADP⁺ but uses also NAD⁺ as a cofactor, and showed highest catalytic efficiency (k _cat/K _m) towards l-arabinose, d-galactose and d-fucose. Based on nuclear magnetic resonance (NMR) and modelling studies, the enzyme prefers the α-pyranose form of l-arabinose, and the stable oxidation product detected is l-arabino-1,4-lactone which can, however, open slowly at neutral pH to a linear l-arabonate form. The pH optimum for the enzyme was pH 9, but use of a yeast-in-vivo-like buffer at pH 6.8 indicated that good catalytic efficiency could still be expected in vivo. Expression of the Rl AraDH dehydrogenase in S. cerevisiae, together with the galactose permease Gal2 for l-arabinose uptake, resulted in production of 18 g of l-arabonate per litre, at a rate of 248 mg of l-arabonate per litre per hour, with 86 % of the provided l-arabinose converted to l-arabonate. Expression of a lactonase-encoding gene from Caulobacter crescentus was not necessary for l-arabonate production in yeast.     

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.     

Metabolic engineering of Saccharomyces cerevisiae for bioconversion of D-xylose to D-xylonate

An NAD(+)-dependent D-xylose dehydrogenase, XylB, from Caulobacter crescentus was expressed in Saccharomyces cerevisiae, resulting in production of 17 ± 2 g D-xylonate l(-1) at 0.23 gl(-1)h(-1) from 23 g D-xylose l(-1) (with glucose and ethanol as co-substrates). D-Xylonate titre and production rate were increased and xylitol production decreased, compared to strains expressing genes encoding T. reesei or pig liver NADP(+)-dependent D-xylose dehydrogenases. D-Xylonate accumulated intracellularly to ～70 mgg(-1); xylitol to ～18 mgg(-1). The aldose reductase encoding gene GRE3 was deleted to reduce xylitol production. Cells expressing D-xylonolactone lactonase xylC from C. crescentus with xylB initially produced more extracellular D-xylonate than cells lacking xylC at both pH 5.5 and pH 3, and sustained higher production at pH 3. Cell vitality and viability decreased during D-xylonate production at pH 3.0. An industrial S. cerevisiae strain expressing xylB efficiently produced 43 g D-xylonate l(-1) from 49 g D-xylose l(-1).     

Production and applications of carbohydrate-derived sugar acids as generic biobased chemicals

This review considers the chemical and biotechnological synthesis of acids that are obtained by direct oxidation of mono- or oligosaccharide, referred to as sugar acids. It focuses on sugar acids which can be readily derived from plant biomass sources and their current and future applications. The three main classes of sugar acids are aldonic, aldaric and uronic acids. Interest in organic acids derived from sugars has recently increased, as part of the interest to develop biorefineries which produce not only biofuels, but also chemicals to replace those currently derived from petroleum. More than half of the most desirable biologically produced platform chemicals are organic acids. Currently, the only sugar acid with high commercial production is d-gluconic acid. However, other sugar acids such as d-glucaric and meso-galactaric acids are being produced at a lower scale. The sugar acids have application as sequestering agents and binders, corrosion inhibitors, biodegradable chelators for pharmaceuticals and pH regulators. There is also considerable interest in the use of these molecules in the production of synthetic polymers, including polyamides, polyesters and hydrogels. Further development of these sugar acids will lead to higher volume production of the appropriate sugar acids and will help support the next generation of biorefineries.     

Identification and quantitation of phosphorus metabolites in yeast neutral pH extracts by nuclear magnetic resonance spectroscopy.

(31)P NMR spectroscopy offers a possibility to obtain a survey of all low-molecular-weight phosphorylated compounds in yeast. The yeast cells have been extracted using chloroform into a neutral aqueous phase. The use of high fields and the neutral pH extracts, which are suitable for NMR analysis, results in well-resolved (31)P NMR spectra. Two-dimensional NMR experiments, such as proton-detected heteronuclear single quantum ((1)H-(31)P HSQC) and (31)P correlation spectroscopy ((31)P COSY), have been used to assign the resonances. In the phosphomonoester region many of the signals could be assigned to known metabolites in the glycolytic and pentose phosphate pathways, although some signals remain unidentified. Accumulation of ribulose 5-phosphate, xylulose 5-phosphate, and ribose 5-phosphate was observed in a strain lacking transketolase activity when grown in synthetic complete medium. No such accumulation occurred when the cells were grown in yeast-peptone-dextrose medium. Trimetaphosphate (intracellular concentration about 0.2 mM) was detected in both cold methanol-chloroform and perchloric acid extracts.