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

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

BACKGROUND: Lignocellulose 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. RESULTS: With all strains, yield coefficients based on total sugar consumed were high for ethanol (0.39 to 0.40 g/g) and notably low for fermentation by-products (glycerol: ≤0.10 g/g; xylitol: ≤0.08 g/g; acetate: 0.04 g/g). In contrast to the specific glucose utilization rate that was similar for all strains (q(Glucose) ≈ 2.9 g/g(cell dry weight (CDW))/h), the xylose consumption rate was enhanced by a factor of 11.5 (IBB10A02; q(Xylose) = 0.23 g/g(CDW)/h) and 17.5 (IBB10B05; q(Xylose) = 0.35 g/g(CDW)/h) as compared to the q(Xylose) of the non-evolved strain BP10001. In xylose-supplemented (50 g/L) hydrolyzates prepared from 5% dry mass, strain IBB10B05 displayed a q(Xylose) of 0.71 g/g(CDW)/h and depleted xylose in 2 days with an ethanol yield of 0.30 g/g. Under the conditions used, IBB10B05 was also capable of slow anaerobic growth. CONCLUSIONS: Laboratory evolution of strain BP10001 resulted in effectively enhanced q(Xylose) 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

125th anniversary review: fuel alcohol: current production and future challenges

Global research and industrial development of liquid transportation biofuels are moving at a rapid pace. This is mainly due to the significant roles played by biofuels in decarbonising our future energy needs, since they act to mitigate the deleterious impacts of greenhouse gas emissions to the atmosphere that are contributors of climate change. Governmental obligations and international directives that mandate the blending of biofuels in petrol and diesel are also acting as great stimuli to this expanding industrial sector. Currently, the predominant liquid biofuel is bioethanol (fuel alcohol) and its worldwide production is dominated by maize-based and sugar cane-based processes in North and South America, respectively. In Europe, fuel alcohol production employs primarily wheat and sugar beet. Potable distilled spirit production and fuel alcohol processes share many similarities in terms of starch bioconversion, fermentation, distillation and co-product utilisation, but there are some key differences. For example, in certain bioethanol fermentations, it is now possible to yield consistently high ethanol concentrations of ~20% (v/v). Emerging fuel alcohol processes exploit lignocellulosic feedstocks and scientific and technological constraints involved in depolymerising these materials and efficiently fermenting the hydrolysate sugars are being overcome. These so-called secondgeneration fuel alcohol processes are much more environmentally and ethically acceptable compared with exploitation of starch and sugar resources, especially when considering utilisation of residual agricultural biomass and biowastes. This review covers both first and second-generation bioethanol processes with a focus on current challenges and future opportunities of lignocellulose-to-ethanol as this technology moves from demonstration pilot-plants to full-scale industrial facilities

Stepwise metabolic adaption from pure metabolization to balanced anaerobic growth on xylose explored for recombinant Saccharomyces cerevisiae

BACKGROUND: To effectively convert lignocellulosic feedstocks to bio-ethanol anaerobic growth on xylose constitutes an essential trait that Saccharomyces cerevisiae strains normally do not adopt through the selective integration of a xylose assimilation route as the rate of ATP-formation is below energy requirements for cell maintenance (m(ATP)). To enable cell growth extensive evolutionary and/or elaborate rational engineering is required. However the number of available strains meeting demands for process integration are limited. In this work evolutionary engineering in just two stages coupled to strain selection under strict anaerobic conditions was carried out with BP10001 as progenitor. BP10001 is an efficient (Y(ethanol) = 0.35 g/g) but slow (q(ethanol) = 0.05 ± 0.01 g/g(BM)/h) xylose-metabolizing recombinant strain of Saccharomyces cerevisiae that expresses an optimized yeast-type xylose assimilation pathway. RESULTS: BP10001 was adapted in 5 generations to anaerobic growth on xylose by prolonged incubation for 91 days in sealed flasks. Resultant strain IBB10A02 displayed a specific growth rate μ of 0.025 ± 0.002 h(-1) but produced large amounts of glycerol and xylitol. In addition growth was strongly impaired at pH below 6.0 and in the presence of weak acids. Using sequential batch selection and IBB10A02 as basis, IBB10B05 was evolved (56 generations). IBB10B05 was capable of fast (μ = 0.056 ± 0.003 h(-1); q(ethanol) = 0.28 ± 0.04 g/g(BM)/h), efficient (Y(ethanol) = 0.35 ± 0.02 g/g), robust and balanced fermentation of xylose. Importantly, IBB10A02 and IBB10B05 displayed a stable phenotype. Unlike BP10001 both strains displayed an unprecedented biphasic formation of glycerol and xylitol along the fermentation time. Transition from a glycerol- to a xylitol-dominated growth phase, probably controlled by CO(2)/HCO(3)(-), was accompanied by a 2.3-fold increase of m(ATP) while Y(ATP) (= 87 ± 7 mmol(ATP)/g(BM)) remained unaffected. As long as glycerol constituted the main by-product energetics of anaerobic growth on xylose and glucose were almost identical. CONCLUSIONS: In just 61 generation IBB10B05, displaying ~530% improved strain fitness, was evolved from BP10001. Its excellent xylose fermentation properties under industrial relevant conditions were proven and rendered it competitive. Based on detailed analysis of growth energetics we showed that m(ATP) was predominantly determined by the type of polyol formed rather than, as previously assumed, substrate-specific

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

From Beverages to Biofuels: The Journeys of Ethanol-Producing Microorganisms

Microbial fermentation for bio-based products is quickly becoming an integral component of the world infrastructure, as the processes encompassing the synthesis of these natural products becomes more efficient and cost effective to compete with existing commodities. Bioethanol is currently one of the most desired fermentation products, as this constituent can be applied to multiple uses in not only contributing to the more traditional routes of beer brewing and winemaking, but also in the foundation for green fuel sources. By optimizing yields, the innovative processes could be applied towards engineering more rapid and productive biomanufacturing. In order to achieve these goals, we as researchers must understand the underlying principles and intricate networks that play a role within the microenvironment and also on the cellular level in key fermentative microbes such as Saccharomyces cerevisiae and Zymomonas mobilis. In-depth pathway analysis could lead to the development of more favorable metabolic outcomes. This review focuses on the key metabolic networks and cellular frameworks in these model organisms, and how biosynthesis of ethanol yields can be optimized throughout the fermentation process

Genomic and transcriptomic analysis of Saccharomyces cerevisiae isolates with focus in succinic acid production

Succinic acid is a platform chemical that plays an important role as precursor for the synthesis of many valuable bio-based chemicals. Its microbial production from renewable resources has seen great developments, specially exploring the use of yeasts to overcome the limitations of using bacteria. The objective of the present work was to screen for succinate-producing isolates, using a yeast collection with different origins and characteristics. Four strains were chosen, two as promising succinic acid producers, in comparison with two low producers. Genome of these isolates was analysed, and differences were found mainly in genes SDH1, SDH3, MDH1 and the transcription factor HAP4, regarding the number of single nucleotide polymorphisms and the gene copy-number profile. Real-time PCR was used to study gene expression of 10 selected genes involved in the metabolic pathway of succinic acid production. Results show that for the non-producing strain, higher expression of genes SDH1, SDH2, ADH1, ADH3, IDH1 and HAP4 was detected, together with lower expression of ADR1 transcription factor, in comparison with the best producer strain. This is the first study showing the capacity of natural yeast isolates to produce high amounts of succinic acid, together with the understanding of the key factors associated, giving clues for strain improvement.This work was supported by the TRANSBIO project from the European Community’s Seventh Framework Programme (FP7/2007-2013, grant agreement No. 289603), by the EcoAgriFood project (NORTE-01-0145-FEDER-000009) via the North Portugal Regional Operational Programme (Norte 2020) under the Portugal 2020 Partnership Agreement, and by FCT I.P. through the strategic funding UID/BIA/04050/2013.info:eu-repo/semantics/publishedVersio

Prefermentation improves xylose utilization in simultaneous saccharification and co-fermentation of pretreated spruce

<p>Abstract</p> <p>Background</p> <p>Simultaneous saccharification and fermentation (SSF) is a promising process option for ethanol production from lignocellulosic materials. However, both the overall ethanol yield and the final ethanol concentration in the fermentation broth must be high. Hence, almost complete conversion of both hexoses and pentoses must be achieved in SSF at a high solid content. A principal difficulty is to obtain an efficient pentose uptake in the presence of high glucose and inhibitor concentrations. Initial glucose present in pretreated spruce decreases the xylose utilization by yeast, due to competitive inhibition of sugar transport. In the current work, prefermentation was studied as a possible means to overcome the problem of competitive inhibition. The free hexoses, initially present in the slurry, were in these experiments fermented before adding the enzymes, thereby lowering the glucose concentration.</p> <p>Results</p> <p>This work shows that a high degree of xylose conversion and high ethanol yields can be achieved in SSF of pretreated spruce with a xylose fermenting strain of <it>Saccharomyces cerevisiae </it>(TMB3400) at 7% and 10% water insoluble solids (WIS). Prefermentation and fed-batch operation, both separately and in combination, improved xylose utilization. Up to 77% xylose utilization and 85% of theoretical ethanol yield (based on total sugars), giving a final ethanol concentration of 45 g L^-1, were obtained in fed-batch SSF at 10% WIS when prefermentation was applied.</p> <p>Conclusion</p> <p>Clearly, the mode of fermentation has a high impact on the xylose conversion by yeast in SSF. Prefermentation enhances xylose uptake most likely because of the reduced transport inhibition, in both batch and fed-batch operation. The process significance of this will be even greater for xylose-rich feedstocks.</p