Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>Metabolic engineering of <it>Saccharomyces cerevisiae </it>for xylose fermentation into fuel ethanol has oftentimes relied on insertion of a heterologous pathway that consists of xylose reductase (XR) and xylitol dehydrogenase (XDH) and brings about isomerization of xylose into xylulose via xylitol. Incomplete recycling of redox cosubstrates in the catalytic steps of the NADPH-preferring XR and the NAD⁺-dependent XDH results in formation of xylitol by-product and hence in lowering of the overall yield of ethanol on xylose. Structure-guided site-directed mutagenesis was previously employed to change the coenzyme preference of <it>Candida tenuis </it>XR about 170-fold from NADPH in the wild-type to NADH in a Lys²⁷⁴→Arg Asn²⁷⁶→Asp double mutant which in spite of the structural modifications introduced had retained the original catalytic efficiency for reduction of xylose by NADH. This work was carried out to assess physiological consequences in xylose-fermenting <it>S. cerevisiae </it>resulting from a well defined alteration of XR cosubstrate specificity.</p> <p>Results</p> <p>An isogenic pair of yeast strains was derived from <it>S. cerevisiae </it>Cen.PK 113-7D through chromosomal integration of a three-gene cassette that carried a single copy for <it>C. tenuis </it>XR in wild-type or double mutant form, XDH from <it>Galactocandida mastotermitis</it>, and the endogenous xylulose kinase (XK). Overexpression of each gene was under control of the constitutive TDH3 promoter. Measurement of intracellular levels of XR, XDH, and XK activities confirmed the expected phenotypes. The strain harboring the XR double mutant showed 42% enhanced ethanol yield (0.34 g/g) compared to the reference strain harboring wild-type XR during anaerobic bioreactor conversions of xylose (20 g/L). Likewise, the yields of xylitol (0.19 g/g) and glycerol (0.02 g/g) were decreased 52% and 57% respectively in the XR mutant strain. The xylose uptake rate per gram of cell dry weight was identical (0.07 ± 0.02 h^-1) in both strains.</p> <p>Conclusion</p> <p>Integration of enzyme and strain engineering to enhance utilization of NADH in the XR-catalyzed conversion of xylose results in notably improved fermentation capabilities of recombinant <it>S. cerevisiae</it>.</p

Fermentation of mixed glucose-xylose substrates by engineered strains of Saccharomyces cerevisiae: role of the coenzyme specificity of xylose reductase, and effect of glucose on xylose utilization

<p>Abstract</p> <p>Background</p> <p>In spite of the substantial metabolic engineering effort previously devoted to the development of <it>Saccharomyces cerevisiae </it>strains capable of fermenting both the hexose and pentose sugars present in lignocellulose hydrolysates, the productivity of reported strains for conversion of the naturally most abundant pentose, xylose, is still a major issue of process efficiency. Protein engineering for targeted alteration of the nicotinamide cofactor specificity of enzymes catalyzing the first steps in the metabolic pathway for xylose was a successful approach of reducing xylitol by-product formation and improving ethanol yield from xylose. The previously reported yeast strain BP10001, which expresses heterologous xylose reductase from <it>Candida tenuis </it>in mutated (NADH-preferring) form, stands for a series of other yeast strains designed with similar rational. Using 20 g/L xylose as sole source of carbon, BP10001 displayed a low specific uptake rate <it>q</it>_xylose(g xylose/g dry cell weight/h) of 0.08. The study presented herein was performed with the aim of analysing (external) factors that limit <it>q</it>_xyloseof BP10001 under xylose-only and mixed glucose-xylose substrate conditions. We also carried out a comprehensive investigation on the currently unclear role of coenzyme utilization, NADPH compared to NADH, for xylose reduction during co-fermentation of glucose and xylose.</p> <p>Results</p> <p>BP10001 and BP000, expressing <it>C. tenuis </it>xylose reductase in NADPH-preferring wild-type form, were used. Glucose and xylose (each at 10 g/L) were converted sequentially, the corresponding <it>q</it>_substratevalues being similar for each strain (glucose: 3.0; xylose: 0.05). The distribution of fermentation products from glucose was identical for both strains whereas when using xylose, BP10001 showed enhanced ethanol yield (BP10001 0.30 g/g; BP000 0.23 g/g) and decreased yields of xylitol (BP10001 0.26 g/g; BP000 0.36 g/g) and glycerol (BP10001 0.023 g/g; BP000 0.072 g/g) as compared to BP000. Increase in xylose concentration from 10 to 50 g/L resulted in acceleration of substrate uptake by BP10001 (0.05 - 0.14 g/g CDW/h) and reduction of the xylitol yield (0.28 g/g - 0.15 g/g). In mixed substrate batches, xylose was taken up at low glucose concentrations (< 4 g/L) and up to fivefold enhanced xylose uptake rate was found towards glucose depletion. A fed-batch process designed to maintain a "stimulating" level of glucose throughout the course of xylose conversion provided a <it>q</it>_xylosethat had an initial value of 0.30 ± 0.04 g/g CDW/h and decreased gradually with time. It gave product yields of 0.38 g ethanol/g total sugar and 0.19 g xylitol/g xylose. The effect of glucose on xylose utilization appears to result from the enhanced flux of carbon through glycolysis and the pentose phosphate pathway under low-glucose reaction conditions.</p> <p>Conclusions</p> <p>Relative improvements in the distribution of fermentation products from xylose that can be directly related to a change in the coenzyme preference of xylose reductase from NADPH in BP000 to NADH in BP10001 increase in response to an increase in the initial concentration of the pentose substrate from 10 to 50 g/L. An inverse relationship between xylose uptake rate and xylitol yield for BP10001 implies that xylitol by-product formation is controlled not only by coenzyme regeneration during two-step oxidoreductive conversion of xylose into xylulose. Although xylose is not detectably utilized at glucose concentrations greater than 4 g/L, the presence of a low residual glucose concentration (< 2 g/L) promotes the uptake of xylose and its conversion into ethanol with only moderate xylitol by-product formation. A fed-batch reaction that maintains glucose in the useful concentration range and provides a constant <it>q</it>_glucosemay be useful for optimizing <it>q</it>_xylosein processes designed for co-fermentation of glucose and xylose.</p

Dynamic and Static Assembly of Sulfated Cellulose Nanocrystals with Alkali Metal Counter Cations

Sulfate groups on cellulose particles such as cellulose nanocrystals (CNCs) provide colloidal stability credit to electrostatic repulsion between the like-charged particles. The introduction of sodium counter cations on the sulfate groups enables drying of the CNC suspensions without irreversible aggregation. Less is known about the effect of other counter cations than sodium on extending the properties of the CNC particles. Here, we introduce the alkali metal counter cations, Li+, Na+, K+, Rb+, and Cs+, on sulfated CNCs without an ion exchange resin, which, so far, has been a common practice. We demonstrate that the facile ion exchange is an efficient method to exchange to any alkali metal cation of sulfate half esters, with exchange rates between 76 and 89%. The ability to form liquid crystalline order in rest was observed by the presence of birefringence patterns and followed the Hofmeister series prediction of a decreasing ability to form anisotropy with an increasing element number. However, we observed the K-CNC rheology and birefringence as a stand-out case within the series of alkali metal modifications, with dynamic moduli and loss tangent indicating a network disruptive effect compared to the other counter cations, whereas observation of the development of birefringence patterns in flow showed the absence of self- or dynamically-assembled liquid crystalline order