Mario Klimacek

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

BackgroundIn spite of the substantial metabolic engineering effort previously devoted to the development of Saccharomyces cerevisiae 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 Candida tenuis 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 qxylose (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 qxylose of 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.ResultsBP10001 and BP000, expressing C. tenuis xylose reductase in NADPH-preferring wild-type form, were used. Glucose and xylose (each at 10 g/L) were converted sequentially, the corresponding qsubstrate values 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 qxylose that 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.ConclusionsRelative 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 qglucose may be useful for optimizing qxylose in processes designed for co-fermentation of glucose and xylose.

Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases.

The co-ordinates reported have been submitted to the Protein Data Bank under accession number 1MI3. Xylose reductase (XR; AKR2B5) is an unusual member of aldo-keto reductase superfamily, because it is one of the few able to efficiently utilize both NADPH and NADH as co-substrates in converting xylose into xylitol. In order to better understand the basis for this dual specificity, we have determined the crystal structure of XR from the yeast Candida tenuis in complex with NAD(+) to 1.80 A resolution (where 1 A=0.1 nm) with a crystallographic R -factor of 18.3%. A comparison of the NAD(+)- and the previously determined NADP(+)-bound forms of XR reveals that XR has the ability to change the conformation of two loops. To accommodate both the presence and absence of the 2-phosphate, the enzyme is able to adopt different conformations for several different side chains on these loops, including Asn(276), which makes alternative hydrogen-bonding interactions with the adenosine ribose. Also critical is the presence of Glu(227) on a short rigid helix, which makes hydrogen bonds to both the 2- and 3-hydroxy groups of the adenosine ribose. In addition to changes in hydrogen-bonding of the adenosine, the ribose unmistakably adopts a 3- endo conformation rather than the 2- endo conformation seen in the NADP(+)-bound form. These results underscore the importance of tight adenosine binding for efficient use of either NADH or NADPH as a co-substrate in aldo-keto reductases. The dual specificity found in XR is also an important consideration in designing a high-flux xylose metabolic pathway, which may be improved with an enzyme specific for NADH.

Limitations in xylose-fermenting Saccharomyces cerevisiae, made evident through comprehensive metabolite profiling and thermodynamic analysis.

ABSTRACT Little is known about how the general lack of efficiency with which recombinant Saccharomyces cerevisiae strains utilize xylose affects the yeast metabolome. Quantitative metabolomics was therefore performed for two xylose-fermenting S. cerevisiae strains, BP000 and BP10001, both engineered to produce xylose reductase (XR), NAD+-dependent xylitol dehydrogenase and xylulose kinase, and the corresponding wild-type strain CEN.PK 113-7D, which is not able to metabolize xylose. Contrary to BP000 expressing an NADPH-preferring XR, BP10001 expresses an NADH-preferring XR. An updated protocol of liquid chromatography/tandem mass spectrometry that was validated by applying internal 13C-labeled metabolite standards was used to quantitatively determine intracellular pools of metabolites from the central carbon, energy, and redox metabolism and of eight amino acids. Metabolomic responses to different substrates, glucose (growth) or xylose (no growth), were analyzed for each strain. In BP000 and BP10001, flux through glycolysis was similarly reduced (∼27-fold) when xylose instead of glucose was metabolized. As a consequence, (i) most glycolytic metabolites were dramatically (≤120-fold) diluted and (ii) energy and anabolic reduction charges were affected due to decreased ATP/AMP ratios (3- to 4-fold) and reduced NADP+ levels (∼3-fold), respectively. Contrary to that in BP000, the catabolic reduction charge was not altered in BP10001. This was due mainly to different utilization of NADH by XRs in BP000 (44%) and BP10001 (97%). Thermodynamic analysis complemented by enzyme kinetic considerations suggested that activities of pentose phosphate pathway enzymes and the pool of fructose-6-phosphate are potential factors limiting xylose utilization. Coenzyme and ATP pools did not rate limit flux through xylose pathway enzymes.

Engineering of a matched pair of xylose reductase and xylitol dehydrogenase for xylose fermentation by Saccharomyces cerevisiae

Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation has often relied on insertion of a heterologous pathway consisting of nicotinamide adenine dinucleotide (phosphate) NAD(P)H-dependent xylose reductase (XR) and NAD(+)-dependent xylitol dehydrogenase (XDH). Low ethanol yield, formation of xylitol and other fermentation by-products are seen for many of the S. cerevisiae strains constructed in this way. This has been ascribed to incomplete coenzyme recycling in the steps catalyzed by XR and XDH. Despite various protein-engineering efforts to alter the coenzyme specificity of XR and XDH individually, a pair of enzymes displaying matched utilization of NAD(H) and NADP(H) was not previously reported. We have introduced multiple site-directed mutations in the coenzyme-binding pocket of Galactocandida mastotermitis XDH to enable activity with NADP(+), which is lacking in the wild-type enzyme. We describe four enzyme variants showing activity for xylitol oxidation by NADP(+) and NAD(+). One of the XDH variants utilized NADP(+) about 4 times more efficiently than NAD(+). This is close to the preference for NADPH compared with NADH in mutants of Candida tenuis XR. Compared to an S. cerevisiae-reference strain expressing the genes for the wild-type enzymes, the strains comprising the gene encoding the mutated XDH in combination a matched XR mutant gene showed up to 50% decreased glycerol yield without increase in ethanol during xylose fermentation.

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

Highlights ► Fermentation of xylose into ethanol is important in “biomass-to-biofuel” processes. ► Protein engineering has been extensively used to improve xylose fermentation. ► Published data on variants of XR and XDH are explored as predictors of strain performance. ► Applying internal reactant concentrations to bi-substrate kinetic equations give a reliable forecast on xylitol formation. ► A criterion of physiological fitness of engineered forms of XR is developed.

Process intensification through microbial strain evolution: mixed glucose-xylose fermentation in wheat straw hydrolyzates by three generations of recombinant Saccharomyces cerevisiae

BackgroundLignocellulose hydrolyzates present difficult substrates for ethanol production by the most commonly applied microorganism in the fermentation industries, Saccharomyces cerevisiae. High resistance towards inhibitors released during pretreatment and hydrolysis of the feedstock as well as efficient utilization of hexose and pentose sugars constitute major challenges in the development of S. cerevisiae strains for biomass-to-ethanol processes. Metabolic engineering and laboratory evolution are applied, alone and in combination, to adduce desired strain properties. However, physiological requirements for robust performance of S. cerevisiae in the conversion of lignocellulose hydrolyzates are not well understood. The herein presented S. cerevisiae strains IBB10A02 and IBB10B05 are descendants of strain BP10001, which was previously derived from the widely used strain CEN.PK 113-5D through introduction of a largely redox-neutral oxidoreductive xylose assimilation pathway. The IBB strains were obtained by a two-step laboratory evolution that selected for fast xylose fermentation in combination with anaerobic growth before (IBB10A02) and after adaption in repeated xylose fermentations (IBB10B05). Enzymatic hydrolyzates were prepared from up to 15% dry mass pretreated (steam explosion) wheat straw and contained glucose and xylose in a mass ratio of approximately 2.ResultsWith all strains, yield coefficients based on total sugar consumed were high for ethanol (0.39 to 0.40xa0g/g) and notably low for fermentation by-products (glycerol: ≤0.10xa0g/g; xylitol: ≤0.08xa0g/g; acetate: 0.04xa0g/g). In contrast to the specific glucose utilization rate that was similar for all strains (qGlucoseu2009≈u20092.9xa0g/gcell dry weight (CDW)/h), the xylose consumption rate was enhanced by a factor of 11.5 (IBB10A02; qXyloseu2009=u20090.23xa0g/gCDW/h) and 17.5 (IBB10B05; qXyloseu2009=u20090.35xa0g/gCDW/h) as compared to the qXylose of the non-evolved strain BP10001. In xylose-supplemented (50xa0g/L) hydrolyzates prepared from 5% dry mass, strain IBB10B05 displayed a qXylose of 0.71xa0g/gCDW/h and depleted xylose in 2xa0days with an ethanol yield of 0.30xa0g/g. Under the conditions used, IBB10B05 was also capable of slow anaerobic growth.ConclusionsLaboratory evolution of strain BP10001 resulted in effectively enhanced qXylose at almost complete retention of the fermentation capabilities previously acquired by metabolic engineering. Strain IBB10B05 is a sturdy candidate for intensification of lignocellulose-to-bioethanol processes.

A catalytic consensus motif for D-mannitol 2-dehydrogenase, a member of a polyol-specific long-chain dehydrogenase family, revealed by kinetic characterization of site-directed mutants of the enzyme from Pseudomonas fluorescens.

Lys-295, Asn-300 and His-303 of D-mannitol 2-dehydrogenase from Pseudomonas fluorescens were mutated individually into alanine (K295A, N300A and H303A respectively). Purified mutants displayed catalytic efficiencies for NAD(+)-dependent oxidation of D-mannitol 300-fold (H303A), 1000-fold (N300A) and approx. 400000-fold (K295A) below the wild-type level. Comparison of primary kinetic isotope effects on kinetic parameters for D-fructose reduction by wild-type and mutants at pH 10.0 demonstrate that Asn-300 has an auxiliary role in stabilization of the transition state of hydride transfer, and His-303 contributes to substrate positioning. The large solvent isotope effect of 11+/-1 on k (cat) for mannitol oxidation by K295A at pH((2)H) 10.5 suggests a role for Lys-295 in general base enzymic catalysis. Positional conservation of Lys-295, Asn-300 and His-303 across a family of polyol-specific long-chain dehydrogenases suggests a unique catalytic signature: Lys-Xaa(4)-Asn-Xaa(2)-His (where Xaa denotes any amino acid).

Characterization of recombinant xylitol dehydrogenase from Galactocandida mastotermitis expressed in Escherichia coli.

The plasmid-encoded gene of xylitol dehydrogenase from the yeast Galactocandida mastotermitis was expressed in Escherichia coli at 25 degrees C. Recombinant enzyme was isolated in 70% yield using two steps of biomimetic affinity chromatography with the dye ligand Procion Red HE3B immobilized onto Sepharose 4B-CL. Similar to natural enzyme, recombinant xylitol dehydrogenase is a functional homotetramer with a stoichiometric content of catalytic zinc in each 37-kDa subunit. Though lacking bound Mg(2+) found in xylitol dehydrogenase isolated from yeast cell extracts, the recombinant enzyme is as active and stable as the native enzyme. Stereospecificity of enzymic hydrogen transfer from NADH has been determined by 1H-NMR and is 4-pro-R. A detailed steady-state kinetic analysis has been carried out for the enzymic reaction, polyol+NAD(+)<-->ketose+NADH+H(+), at pH 7.5 and 25 degrees C using xylitol and D-xylulose, the physiological polyol-ketose pair, as well as D-sorbitol and D-fructose. Primary deuterium kinetic isotope effects on steady-state kinetic parameters for oxidation of D-sorbitol and reduction of D-fructose have been measured at pH 7.5. Combined results of initial-rate analysis and isotope effect studies suggest that the enzyme utilizes a preferentially ordered kinetic mechanism in which NAD(+) binds before D-sorbitol and D-fructose is released before NADH. Dissociation of NADH appears to be the main rate-limiting step for D-sorbitol oxidation under substrate-saturated reaction conditions.

Exploring the active site of yeast xylose reductase by site‐directed mutagenesis of sequence motifs characteristic of two dehydrogenase/reductase family types

Starting from a common tyrosine, yeast xylose reductases (XRs) contain two conserved sequence motifs corresponding to the catalytic signatures of single‐domain reductases/epimerases/dehydrogenases (Tyr n ‐(X)3‐Lys n+4) and aldo/keto reductases (AKRs) (Tyr n ‐(X)28‐Lys n+29). Tyr51, Lys55 and Lys80 of XR from Candida tenuis were replaced by site‐directed mutagenesis. The purified Tyr51→ Phe and Lys80→Ala mutants showed turnover numbers and catalytic efficiencies for NADH‐dependent reduction of D‐xylose between 2500‐ and 5000‐fold below wild‐type levels, suggesting a catalytic role of both residues. Replacing Lys55 by Asn, a substitution found in other AKRs, did not detectably affect binding of coenzymes, and enzymatic catalysis to carbonyl/alcohol interconversion. The contribution of Tyr51 to rate enhancement of aldehyde reduction conforms with expectations for the general acid catalyst of the enzymatic reaction.

Co-fermentation of hexose and pentose sugars in a spent sulfite liquor matrix with genetically modified Saccharomyces cerevisiae

Spent sulfite liquor (SSL) is a by-product of pulp and paper manufacturing and is a promising substrate for second-generation bioethanol production. The Saccharomyces cerevisiae strain IBB10B05 presented herein for SSL fermentation was enabled to xylose utilization by metabolic pathway engineering and laboratory evolution. Two SSLs from different process stages and with variable dry matter content were analyzed; SSL-Thin (14%) and SSL-S2 (30%). Hexose and pentose fermentation by strain IBB10B05 was efficient in 70% SSL matrix without any pretreatment. Ethanol yields varied between 0.31 and 0.44g/g total sugar, depending on substrate and process conditions used. Control of pH at 7.0 effectively reduced the inhibition by the acetic acid contained in the SSLs (up to 9g/L), thus enhancing specific xylose uptake rates (q(Xylose)) as well as ethanol yields. The total molar yield of fermentation by-products (glycerol, xylitol) was constant (0.36±0.03mol/mol xylose) at different q(Xylose). Compound distribution changed with glycerol and xylitol being chiefly formed at low and high q(Xylose), respectively.