Saccharomyces cerevisiae strain comparison in glucose–xylose fermentations on defined substrates and in high-gravity SSCF: convergence in strain performance despite differences in genetic and evolutionary engineering history

BackgroundThe most advanced strains of xylose-fermenting Saccharomyces cerevisiae still utilize xylose far less efficiently than glucose, despite the extensive metabolic and evolutionary engineering applied in their development. Systematic comparison of strains across literature is difficult due to widely varying conditions used for determining key physiological parameters. Here, we evaluate an industrial and a laboratory S. cerevisiae strain, which has the assimilation of xylose via xylitol in common, but differ fundamentally in the history of their adaptive laboratory evolution development, and in the cofactor specificity of the xylose reductase (XR) and xylitol dehydrogenase (XDH).ResultsIn xylose and mixed glucose–xylose shaken bottle fermentations, with and without addition of inhibitor-rich wheat straw hydrolyzate, the specific xylose uptake rate of KE6-12.A (0.27–1.08 g gCDW−1 h−1) was 1.1 to twofold higher than that of IBB10B05 (0.10–0.82 g gCDW−1 h−1). KE6-12.A further showed a 1.1 to ninefold higher glycerol yield (0.08–0.15 g g−1) than IBB10B05 (0.01–0.09 g g−1). However, the ethanol yield (0.30–0.40 g g−1), xylitol yield (0.08–0.26 g g−1), and maximum specific growth rate (0.04–0.27 h−1) were in close range for both strains. The robustness of flocculating variants of KE6-12.A (KE-Flow) and IBB10B05 (B-Flow) was analyzed in high-gravity simultaneous saccharification and co-fermentation. As in shaken bottles, KE-Flow showed faster xylose conversion and higher glycerol formation than B-Flow, but final ethanol titres (61 g L−1) and cell viability were again comparable for both strains.ConclusionsIndividual specific traits, elicited by the engineering strategy, can affect global physiological parameters of S. cerevisiae in different and, sometimes, unpredictable ways. The industrial strain background and prolonged evolution history in KE6-12.A improved the specific xylose uptake rate more substantially than the superior XR, XDH, and xylulokinase activities were able to elicit in IBB10B05. Use of an engineered XR/XDH pathway in IBB10B05 resulted in a lower glycerol rather than a lower xylitol yield. However, the strain development programs were remarkably convergent in terms of the achieved overall strain performance. This highlights the importance of comparative strain evaluation to advance the engineering strategies for next-generation S. cerevisiae strain development.

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title: Development of efficient xylose fermentation in Saccharomyces cerevisiae: xylose isomerase as a key component.
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title: Development of a D-xylose fermenting and inhibitor tolerant industrial Saccharomyces cerevisiae strain with high performance in lignocellulose hydrolysates using metabolic and evolutionary engineering
abstract: BackgroundThe production of bioethanol from lignocellulose hydrolysates requires a robust, D-xylose-fermenting and inhibitor-tolerant microorganism as catalyst. The purpose of the present work was to develop such a strain from a prime industrial yeast strain, Ethanol Red, used for bioethanol production.ResultsAn expression cassette containing 13 genes including Clostridium phytofermentans XylA, encoding D-xylose isomerase (XI), and enzymes of the pentose phosphate pathway was inserted in two copies in the genome of Ethanol Red. Subsequent EMS mutagenesis, genome shuffling and selection in D-xylose-enriched lignocellulose hydrolysate, followed by multiple rounds of evolutionary engineering in complex medium with D-xylose, gradually established efficient D-xylose fermentation. The best-performing strain, GS1.11-26, showed a maximum specific D-xylose consumption rate of 1.1 g/g DW/h in synthetic medium, with complete attenuation of 35 g/L D-xylose in about 17 h. In separate hydrolysis and fermentation of lignocellulose hydrolysates of Arundo donax (giant reed), spruce and a wheat straw/hay mixture, the maximum specific D-xylose consumption rate was 0.36, 0.23 and 1.1 g/g DW inoculum/h, and the final ethanol titer was 4.2, 3.9 and 5.8% (v/v), respectively. In simultaneous saccharification and fermentation of Arundo hydrolysate, GS1.11-26 produced 32% more ethanol than the parent strain Ethanol Red, due to efficient D-xylose utilization. The high D-xylose fermentation capacity was stable after extended growth in glucose. Cell extracts of strain GS1.11-26 displayed 17-fold higher XI activity compared to the parent strain, but overexpression of XI alone was not enough to establish D-xylose fermentation. The high D-xylose consumption rate was due to synergistic interaction between the high XI activity and one or more mutations in the genome. The GS1.11-26 had a partial respiratory defect causing a reduced aerobic growth rate.ConclusionsAn industrial yeast strain for bioethanol production with lignocellulose hydrolysates has been developed in the genetic background of a strain widely used for commercial bioethanol production. The strain uses glucose and D-xylose with high consumption rates and partial cofermentation in various lignocellulose hydrolysates with very high ethanol yield. The GS1.11-26 strain shows highly promising potential for further development of an all-round robust yeast strain for efficient fermentation of various lignocellulose hydrolysates.

title: Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain.
abstract: We have recently reported about a Saccharomyces cerevisiae strain that, in addition to the Piromyces XylA xylose isomerase gene, overexpresses the native genes for the conversion of xylulose to glycolytic intermediates. This engineered strain (RWB 217) exhibited unprecedentedly high specific growth rates and ethanol production rates under anaerobic conditions with xylose as the sole carbon source. However, when RWB 217 was grown on glucose-xylose mixtures, a diauxic growth pattern was observed with a relatively slow consumption of xylose in the second growth phase. After prolonged cultivation in an anaerobic, xylose-limited chemostat, a culture with improved xylose uptake kinetics was obtained. This culture also exhibited improved xylose consumption in glucose-xylose mixtures. A further improvement in mixed-sugar utilization was obtained by prolonged anaerobic cultivation in automated sequencing-batch reactors on glucose-xylose mixtures. A final single-strain isolate (RWB 218) rapidly consumed glucose-xylose mixtures anaerobically, in synthetic medium, with a specific rate of xylose consumption exceeding 0.9 gg(-1)h(-1). When the kinetics of zero trans-influx of glucose and xylose of RWB 218 were compared to that of the initial strain, a twofold higher capacity (V(max)) as well as an improved K(m) for xylose was apparent in the selected strain. It is concluded that the kinetics of xylose fermentation are no longer a bottleneck in the industrial production of bioethanol with yeast.

title: Functional Expression of a Bacterial Xylose Isomerase in Saccharomyces cerevisiae
abstract: In industrial fermentation processes, the yeast Saccharomyces cerevisiae is commonly used for ethanol production. However, it lacks the ability to ferment pentose sugars like d-xylose and l-arabinose. Heterologous expression of a xylose isomerase (XI) would enable yeast cells to metabolize xylose. However, many attempts to express a prokaryotic XI with high activity in S. cerevisiae have failed so far. We have screened nucleic acid databases for sequences encoding putative XIs and finally were able to clone and successfully express a highly active new kind of XI from the anaerobic bacterium Clostridium phytofermentans in S. cerevisiae. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize d-xylose and to use it as the sole carbon and energy source. The new enzyme has low sequence similarities to the XIs from Piromyces sp. strain E2 and Thermus thermophilus, which were the only two XIs previously functionally expressed in S. cerevisiae. The activity and kinetic parameters of the new enzyme are comparable to those of the Piromyces XI. Importantly, the new enzyme is far less inhibited by xylitol, which accrues as a side product during xylose fermentation. Furthermore, expression of the gene could be improved by adapting its codon usage to that of the highly expressed glycolytic genes of S. cerevisiae. Expression of the bacterial XI in an industrially employed yeast strain enabled it to grow on xylose and to ferment xylose to ethanol. Thus, our findings provide an excellent starting point for further improvement of xylose fermentation in industrial yeast strains.

title: Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae
abstract: BackgroundTwo heterologous pathways have been used to construct recombinant xylose-fermenting Saccharomyces cerevisiae strains: i) the xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway and ii) the xylose isomerase (XI) pathway. In the present study, the Pichia stipitis XR-XDH pathway and the Piromyces XI pathway were compared in an isogenic strain background, using a laboratory host strain with genetic modifications known to improve xylose fermentation (overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deletion of the aldose reductase gene GRE3). The two isogenic strains and the industrial xylose-fermenting strain TMB 3400 were studied regarding their xylose fermentation capacity in defined mineral medium and in undetoxified lignocellulosic hydrolysate.ResultsIn defined mineral medium, the xylose consumption rate, the specific ethanol productivity, and the final ethanol concentration were significantly higher in the XR- and XDH-carrying strain, whereas the highest ethanol yield was achieved with the strain carrying XI. While the laboratory strains only fermented a minor fraction of glucose in the undetoxified lignocellulose hydrolysate, the industrial strain TMB 3400 fermented nearly all the sugar available. Xylitol was formed by the XR-XDH-carrying strains only in mineral medium, whereas in lignocellulose hydrolysate no xylitol formation was detected.ConclusionDespite by-product formation, the XR-XDH xylose utilization pathway resulted in faster ethanol production than using the best presently reported XI pathway in the strain background investigated. The need for robust industrial yeast strains for fermentation of undetoxified spruce hydrolysates was also confirmed.

title: Evolutionary engineering of industrially important microbial phenotypes.
abstract: The tremendous complexity of dynamic interactions in cellular systems often impedes practical applications of metabolic engineering that are largely based on available molecular or functional knowledge. In contrast, evolutionary engineering follows nature's 'engineering' principle by variation and selection. Thus, it is a complementary strategy that offers compelling scientific and applied advantages for strain development and process optimization, provided a desired phenotype is amenable to direct or indirect selection. In addition to simple empirical strain development by random mutation and direct selection on plates, evolutionary engineering also encompasses recombination and continuous evolution of large populations over many generations. Two distinct evolutionary engineering applications are likely to gain more relevance in the future: first, as an integral component in metabolic engineering of strains with improved phenotypes, and second, to elucidate the molecular basis of desired phenotypes for subsequent transfer to other hosts. The latter will profit from the broader availability of recently developed methodologies for global response analysis at the genetic and metabolic level. These methodologies facilitate identification of the molecular basis of evolved phenotypes. It is anticipated that, together with novel analytical techniques, bioinformatics, and computer modeling of cellular functions and activities, evolutionary engineering is likely to find its place in the metabolic engineer's toolbox for research and strain development. This review presents evolutionary engineering of whole cells as an emerging methodology that draws on the latest advances from a wide range of scientific and technical disciplines.

title: Stepwise metabolic adaption from pure metabolization to balanced anaerobic growth on xylose explored for recombinant Saccharomyces cerevisiae
abstract: BackgroundTo 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 (mATP). 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 (Yethanol = 0.35 g/g) but slow (qethanol = 0.05 ± 0.01 g/gBM/h) xylose-metabolizing recombinant strain of Saccharomyces cerevisiae that expresses an optimized yeast-type xylose assimilation pathway.ResultsBP10001 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; qethanol = 0.28 ± 0.04 g/gBM/h), efficient (Yethanol = 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 CO2/HCO3-, was accompanied by a 2.3-fold increase of mATP while YATP (= 87 ± 7 mmolATP/gBM) remained unaffected. As long as glycerol constituted the main by-product energetics of anaerobic growth on xylose and glucose were almost identical.ConclusionsIn 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 mATP was predominantly determined by the type of polyol formed rather than, as previously assumed, substrate-specific.

title: Generation of the improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3400 by random mutagenesis and physiological comparison with Pichia stipitis CBS 6054.
abstract: The recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3399 was constructed by chromosomal integration of the genes encoding D-xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK). S. cerevisiae TMB 3399 was subjected to chemical mutagenesis with ethyl methanesulfonate and, after enrichment, 33 mutants were selected for improved growth on D-xylose and carbon dioxide formation in Durham tubes. The best-performing mutant was called S. cerevisiae TMB 3400. The novel, recombinant S. cerevisiae strains were compared with Pichia stipitis CBS 6054 through cultivation under aerobic, oxygen-limited, and anaerobic conditions in a defined mineral medium using only D-xylose as carbon and energy source. The mutation led to a more than five-fold increase in maximum specific growth rate, from 0.0255 h(-1) for S. cerevisiae TMB 3399 to 0.14 h(-1) for S. cerevisiae TMB 3400, whereas P. stipitis grew at a maximum specific growth rate of 0.44 h(-1). All yeast strains formed ethanol only under oxygen-limited and anaerobic conditions. The ethanol yields and maximum specific ethanol productivities during oxygen limitation were 0.21, 0.25, and 0.30 g ethanol g xylose(-1) and 0.001, 0.10, and 0.16 g ethanol g biomass(-1) h(-1) for S. cerevisiae TMB 3399, TMB 3400, and P. stipitis CBS 6054, respectively. The xylitol yield under oxygen-limited and anaerobic conditions was two-fold higher for S. cerevisiae TMB 3399 than for TMB 3400, but the glycerol yield was higher for TMB 3400. The specific activity, in U mg protein(-1), was higher for XDH than for XR in both S. cerevisiae TMB 3399 and TMB 3400, while P. stipitis CBS 6054 showed the opposite relation. S. cerevisiae TMB 3400 displayed higher specific XR, XDH and XK activities than TMB 3399. Hence, we have demonstrated that a combination of metabolic engineering and random mutagenesis was successful to generate a superior, xylose-utilizing S. cerevisiae, and uncovered distinctive physiological properties of the mutant.

title: Sequence-dependent prediction of recombination hotspots in Saccharomyces cerevisiae.
abstract: Meiotic recombination does not occur randomly across the genome, but instead occurs at relatively high frequencies in some genomic regions (hotspots) and relatively low frequencies in others (coldspots). Hotspots and coldspots would shed light on the mechanism of recombination, but the accurate prediction of hot/cold spots is still an open question. In this study, we presented a model to predict hot/cold spots in yeast using increment of diversity combined with quadratic discriminant analysis (IDQD) based on sequence k-mer frequencies. 5-fold cross validation showed a total prediction accuracy of 80.3%. Compared with other machine-learning algorithms, IDQD approach is as powerful as random forest (RF) and outperforms support vector machine (SVM) in identifying hotspots and coldspots. We also predicted increased recombination rates in the upstream regions of transcription start sites and in the downstream regions of transcription termination sites. Additionally, genome-wide recombination map in yeast obtained by IDQD model is in close agreement with the experimentally generated map, especially for the Peak locations, although some fine-scale differences exist. Our results highlight the sequence dependency of recombination.

title: Evolutionary Engineering of Saccharomyces cerevisiae for Anaerobic Growth on Xylose
abstract: Xylose utilization is of commercial interest for efficient conversion of abundant plant material to ethanol. Perhaps the most important ethanol-producing organism, Saccharomyces cerevisiae, however, is incapable of xylose utilization. While S. cerevisiae strains have been metabolically engineered to utilize xylose, none of the recombinant strains or any other naturally occurring yeast has been able to grow anaerobically on xylose. Starting with the recombinant S. cerevisiae strain TMB3001 that overexpresses the xylose utilization pathway from Pichia stipitis, in this study we developed a selection procedure for the evolution of strains that are capable of anaerobic growth on xylose alone. Selection was successful only when organisms were first selected for efficient aerobic growth on xylose alone and then slowly adapted to microaerobic conditions and finally anaerobic conditions, which indicated that multiple mutations were necessary. After a total of 460 generations or 266 days of selection, the culture reproduced stably under anaerobic conditions on xylose and consisted primarily of two subpopulations with distinct phenotypes. Clones in the larger subpopulation grew anaerobically on xylose and utilized both xylose and glucose simultaneously in batch culture, but they exhibited impaired growth on glucose. Surprisingly, clones in the smaller subpopulation were incapable of anaerobic growth on xylose. However, as a consequence of their improved xylose catabolism, these clones produced up to 19% more ethanol than the parental TMB3001 strain produced under process-like conditions from a mixture of glucose and xylose.

title: Evolutionary engineering strategies to enhance tolerance of xylose utilizing recombinant yeast to inhibitors derived from spruce biomass
abstract: BackgroundOne of the crucial factors for a sustainable and economical production of lignocellulosic based bioethanol is the availability of a robust fermenting microorganism with high tolerance to inhibitors generated during the pretreatment of lignocellulosic raw materials, since these inhibitors are known to severely hinder growth and fermentation. long-term adaptation in repetitive batch cultures in shake flasks using a cocktail of 12 different inhibitors and a long-term chemostat adaptation using spruce hydrolysate were used as evolutionary engineering strategies to improve the inhibitor tolerance in the metabolically engineered xylose utilizing Saccharomyces cerevisiae strain, TMB3400. The yeast was evolved for a period of 429 and 97 generations in repetitive batch cultures and chemostat cultivation, respectively. During the evolutionary engineering in repetitive batch cultures the maximum specific growth rate increased from 0.18 h-1 to 0.33 h-1 and the time of lag phase was decreased from 48 h to 24 h. In the chemostat adaptation, after 97 generations, the specific conversion rates of HMF and furfural were found to be 3.5 and 4 folds higher respectively, compared to rates after three generations. Two evolved strains (RK60-5, RKU90-3) and one evolved strain (KE1-17) were isolated from evolutionary engineering in repetitive batches and chemostat cultivation, respectively. The strains displayed significantly improved growth performance over TMB3400 when cultivated in spruce hydrolysate under anaerobic conditions, the evolved strains exhibited 25 to 38% increase in specific consumption rate of sugars and 32 to 50% increased specific ethanol productivity compared to TMB3400. The evolved strains RK60-5 and RKU90-3 were unable to consume xylose under anaerobic conditions, whereas, KE1-17 was found to consume xylose at similar rates as TMB3400.ConclusionUsing evolutionary engineering strategies in batch and chemostat cultivations we have generated three evolved strains that show significantly better tolerance to inhibitors in spruce hydrolysate and displayed a shorter time for overall fermentation of sugars compared to the parental strain.

title: Industrial yeasts strains for biorefinery solutions: Constructing and selecting efficient barcoded xylose fermenting strains for ethanol
abstract: There have been intense efforts in the scientific community and industry to engineer S. cerevisiae strains to co-consume xylose and glucose. In this context, this paper describes a process for introducing a barcode into an industrial xylose fermenting yeast strains to ensure quick and reliable identification of patented industrial strains. Furthermore, this work presents a novel procedure for screening and selecting the clones that reflects the characteristics of the industrial process. The key points of efficient selection of high-performing clones are elaborated and discussed. In spite of the unforeseen effects on yeast physiology after the introduction of a new sequence into the genome, we obtained barcoded clones with the same xylose consumption rates and ethanol yields as the cells without the barcode.

title: Evolutionary engineering of Saccharomyces cerevisiae for improved industrially important properties.
abstract: This article reviews evolutionary engineering of Saccharomyces cerevisiae. Following a brief introduction to the 'rational' metabolic engineering approach and its limitations such as extensive genetic and metabolic information requirement on the organism of interest, complexity of cellular physiological responses, and difficulties of cloning in industrial strains, evolutionary engineering is discussed as an alternative, inverse metabolic engineering strategy. Major evolutionary engineering applications with S. cerevisiae are then discussed in two general categories: (1) evolutionary engineering of substrate utilization and product formation and (2) evolutionary engineering of stress resistance. Recent developments in functional genomics methods allow rapid identification of the molecular basis of the desired phenotypes obtained by evolutionary engineering. To conclude, when used alone or in combination with rational metabolic engineering and/or computational methods to study and analyze processes of adaptive evolution, evolutionary engineering is a powerful strategy for improvement in industrially important, complex properties of S. cerevisiae.

title: Engineering Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: Reflections and perspectives
abstract: Conversion of the abundant lignocellulosic biomass into ethanol is an environmentally sustainable solution to the energy crisis. Fermentation of lignocellulosic hydrolysates by Saccharomyces cerevisiae is not cost‐effective yet as substantial amounts of xylose in the hydrolysates cannot be utilized by native S. cerevisiae strains. Extensive studies including both metabolic and evolutionary engineering have been carried out to develop an efficient xylose‐fermenting S. cerevisiae strain, yet the ethanol yield and productivity from xylose fermentation of the best one are still far below expectation. This review compares the engineering approaches and resulted anaerobic xylose fermentation performance of recently reported xylose‐utilizing S. cerevisiae strains, with the aim to understand the intrinsic reason for their low xylose fermentation capabilities. These comparative analyses revealed that some of the current engineering targets and the so‐called “hot issues” might be overrated. Our opinions on the underrated parts and future efforts in this field are also presented. Overall, this review serves as a comprehensive reference to understanding xylose fermentation by S. cerevisiae.

title: Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae
abstract: During hydrolysis of lignocellulosic biomass, monomeric sugars and a broad range of inhibitory compounds are formed and released. These inhibitors, which can be organized around three main groups, furans, weak acids and phenolics, reduce ethanol yield and productivity by affecting the microorganism performance during the fermentation step. Among the microorganisms that have been evaluated for lignocellulosic hydrolysate ethanol fermentation, the yeast Saccharomyces cerevisiae appears to be the least sensitive. In order to overcome the effect of inhibitors, strategies that include improvement of natural tolerance of microorganism and use of fermentation control strategies have been developed. An overview of the origin, effects and mechanisms of action of known inhibitors on S. cerevisiae is given. Fermentation control strategies as well as metabolic, genetic and evolutionary engineering strategies to obtain S. cerevisiae strains with improved tolerance are discussed.

title: Model-Based Fed-Batch for High-Solids Enzymatic Cellulose Hydrolysis
abstract: While many kinetic models have been developed for the enzymatic hydrolysis of cellulose, few have been extensively applied for process design, optimization, or control. High-solids operation of the enzymatic hydrolysis of lignocellulose is motivated by both its operation decreasing capital costs and increasing product concentration and hence separation costs. This work utilizes both insights obtained from experimental work and kinetic modeling to develop an optimization strategy for cellulose saccharification at insoluble solids levels greater than 15% (w/w), where mixing in stirred tank reactors (STRs) becomes problematic. A previously developed model for batch enzymatic hydrolysis of cellulose was modified to consider the effects of feeding in the context of fed-batch operation. By solving the set of model differential equations, a feeding profile was developed to maintain the insoluble solids concentration at a constant or manageable level throughout the course of the reaction. Using this approach, a stream of relatively concentrated solids (and cellulase enzymes) can be used to increase the final sugar concentration within the reactor without requiring the high initial levels of insoluble solids that would be required if the operation were performed in batch mode. Experimental application in bench-scale STRs using a feed stream of dilute acid-pretreated corn stover solids and cellulase enzymes resulted in similar cellulose conversion profiles to those achieved in batch shake-flask reactors where temperature control issues are mitigated. Final cellulose conversions reached approximately 80% of theoretical for fed-batch STRs fed to reach a cumulative solids level of 25% (w/w) initial insoluble solids.

title: Improving simultaneous saccharification and co-fermentation of pretreated wheat straw using both enzyme and substrate feeding
abstract: BackgroundSimultaneous saccharification and co-fermentation (SSCF) has been recognized as a feasible option for ethanol production from xylose-rich lignocellulosic materials. To reach high ethanol concentration in the broth, a high content of water-insoluble solids (WIS) is needed, which creates mixing problems and, furthermore, may decrease xylose uptake. Feeding of substrate has already been proven to give a higher xylose conversion than a batch SSCF. In the current work, enzyme feeding, in addition to substrate feeding, was investigated as a means of enabling a higher WIS content with a high xylose conversion in SSCF of a xylose-rich material. A recombinant xylose-fermenting strain of Saccharomyces cerevisiae (TMB3400) was used for this purpose in fed-batch SSCF experiments of steam-pretreated wheat straw.ResultsBy using both enzyme and substrate feeding, the xylose conversion in SSCF could be increased from 40% to 50% in comparison to substrate feeding only. In addition, by this design of the feeding strategy, it was possible to process a WIS content corresponding to 11% in SSCF and obtain an ethanol yield on fermentable sugars of 0.35 g g-1.ConclusionA combination of enzyme and substrate feeding was shown to enhance xylose uptake by yeast and increase overall ethanol yield in SSCF. This is conceptually important for the design of novel SSCF processes aiming at high-ethanol titers. Substrate feeding prevents viscosity from becoming too high and thereby allows a higher total amount of WIS to be added in the process. The enzyme feeding, furthermore, enables keeping the glucose concentration low, which kinetically favors xylose uptake and results in a higher xylose conversion.

title: Designing simultaneous saccharification and fermentation for improved xylose conversion by a recombinant strain of Saccharomyces cerevisiae.
abstract: Wheat straw is an abundant agricultural residue which can be used as a raw material for bioethanol production. Due to the high xylan content in wheat straw, fermentation of both xylose and glucose is crucial to meet desired overall yields of ethanol. In the present work a recombinant xylose fermenting strain of Saccharomyces cerevisiae, TMB3400, cultivated aerobically on wheat straw hydrolysate, was used in simultaneous saccharification and fermentation (SSF) of steam pretreated wheat straw. The influence of fermentation strategy and temperature was studied in relation to xylose consumption, ethanol formation and by-product formation. In addition, model SSF experiments were made to further investigate the influence of temperature on xylose fermentation and by-product formation. In particular for SSF at the highest value of fibre content tested (9% water insoluble substance, WIS), it was found that a fed-batch strategy was clearly superior to the batch process in terms of ethanol yield, where the fed-batch gave 71% of the theoretical yield (based on all available sugars) in comparison to merely 59% for the batch. Higher ethanol yields, close to 80%, were obtained at a WIS-content of 7%. Xylose fermentation significantly contributed to the overall ethanol yields. The choice of temperature in the range 30-37 degrees C was found to be important, especially at higher contents of water insoluble solids (WIS). The optimum temperature was found to be 34 degrees C for the raw material and yeast strain studied. Model SSF experiments with defined medium showed strong temperature effects on the xylose uptake rate and xylitol yield.

title: 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
abstract: BackgroundMetabolic engineering of Saccharomyces cerevisiae 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 Candida tenuis 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 S. cerevisiae resulting from a well defined alteration of XR cosubstrate specificity.ResultsAn isogenic pair of yeast strains was derived from S. cerevisiae Cen.PK 113-7D through chromosomal integration of a three-gene cassette that carried a single copy for C. tenuis XR in wild-type or double mutant form, XDH from Galactocandida mastotermitis, 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.ConclusionIntegration 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 S. cerevisiae.

title: Statistical reconciliation of the elemental and molecular biomass composition of Saccharomyces cerevisiae.
abstract: A systematic mathematical procedure capable of detecting the presence of a gross error in the measurements and of reconciling connected data sets by using the maximum likelihood principle is applied to the biomass composition data of yeast. The biomass composition of Saccharomyces cerevisiae grown in a chemostat under glucose limitation was analyzed for its elemental and for its molecular composition. Both descriptions initially resulted in conflicting results concerning the elemental composition, molecular weight, and degrees of reduction. The application of the statistical reconciliation method, based on elemental balances and equality relations, is used to obtain a consistent biomass composition. Simultaneously, the error margins of the data sets are significantly reduced in the reconciliation process. On the basis of statistical analysis it was found that inclusion of about 4% water in the list of biomass constituents is essential to adequately describe the dry biomass and match both set of measurements. The reconciled carbon content of the biomass varied 4% from the ones obtained from the molecular analysis. The proposed method increases the accuracy of biomass composition data of its elements and its molecules by providing a best estimate based on all available data and thus provides an improved and consistent basis for metabolic flux analysis as well as black box modeling approaches.

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

title: Process intensification through microbial strain evolution: mixed glucose-xylose fermentation in wheat straw hydrolyzates by three generations of recombinant Saccharomyces cerevisiae
abstract: 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 0.40 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 (qGlucose ≈ 2.9 g/gcell dry weight (CDW)/h), the xylose consumption rate was enhanced by a factor of 11.5 (IBB10A02; qXylose = 0.23 g/gCDW/h) and 17.5 (IBB10B05; qXylose = 0.35 g/gCDW/h) as compared to the qXylose of the non-evolved strain BP10001. In xylose-supplemented (50 g/L) hydrolyzates prepared from 5% dry mass, strain IBB10B05 displayed a qXylose of 0.71 g/gCDW/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.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.

title: Kinetic modeling of multi-feed simultaneous saccharification and co-fermentation of pretreated birch to ethanol.
abstract: Fed-batch simultaneous saccharification and fermentation (SSF) is a feasible option for bioethanol production from lignocellulosic raw materials at high substrate concentrations. In this work, a segregated kinetic model was developed for simulation of fed-batch simultaneous saccharification and co-fermentation (SSCF) of steam-pretreated birch, using substrate, enzymes and cell feeds. The model takes into account the dynamics of the cellulase-cellulose system and the cell population during SSCF, and the effects of pre-cultivation of yeast cells on fermentation performance. The model was cross-validated against experiments using different feed schemes. It could predict fermentation performance and explain observed differences between measured total yeast cells and dividing cells very well. The reproducibility of the experiments and the cell viability were significantly better in fed-batch than in batch SSCF at 15% and 20% total WIS contents. The model can be used for simulation of fed-batch SSCF and optimization of feed profiles.

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

title: Engineering of a matched pair of xylose reductase and xylitol dehydrogenase for xylose fermentation by Saccharomyces cerevisiae.
abstract: 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.

title: Stress‐related challenges in pentose fermentation to ethanol by the yeast Saccharomyces cerevisiae
abstract: Conversion of agricultural residues, energy crops and forest residues into bioethanol requires hydrolysis of the biomass and fermentation of the released sugars. During the hydrolysis of the hemicellulose fraction, substantial amounts of pentose sugars, in particular xylose, are released. Fermentation of these pentose sugars to ethanol by engineered Saccharomyces cerevisiae under industrial process conditions is the subject of this review. First, fermentation challenges originating from the main steps of ethanol production from lignocellulosic feedstocks are discussed, followed by genetic modifications that have been implemented in S. cerevisiae to obtain xylose and arabinose fermenting capacity per se. Finally, the fermentation of a real lignocellulosic medium is discussed in terms of inhibitory effects of furaldehydes, phenolics and weak acids and the presence of contaminating microbiota.

title: Effect of acetic acid and pH on the cofermentation of glucose and xylose to ethanol by a genetically engineered strain of Saccharomyces cerevisiae.
abstract: A current challenge of the cellulosic ethanol industry is the effect of inhibitors present in biomass hydrolysates. Acetic acid is an example of one such inhibitor that is released during the pretreatment of hemicellulose. This study examined the effect of acetic acid on the cofermentation of glucose and xylose under controlled pH conditions by Saccharomyces cerevisiae 424A(LNH-ST), a genetically engineered industrial yeast strain. Acetic acid concentrations of 7.5 and 15 g L(-1), representing the range of concentrations expected in actual biomass hydrolysates, were tested under controlled pH conditions of 5, 5.5, and 6. The presence of acetic acid in the fermentation media led to a significant decrease in the observed maximum cell biomass concentration. Glucose- and xylose-specific consumption rates decreased as the acetic acid concentration increased, with the inhibitory effect being more severe for xylose consumption. The ethanol production rates also decreased when acetic acid was present, but ethanol metabolic yields increased under the same conditions. The results also revealed that the inhibitory effect of acetic acid could be reduced by increasing media pH, thus confirming that the undissociated form of acetic acid is the inhibitory form of the molecule.

title: Bioethanol production performance of five recombinant strains of laboratory and industrial xylose-fermenting Saccharomyces cerevisiae.
abstract: In this study, five recombinant Saccharomyces cerevisiae strains were compared for their xylose-fermenting ability. The most efficient xylose-to-ethanol fermentation was found by using the industrial strain MA-R4, in which the genes for xylose reductase and xylitol dehydrogenase from Pichia stipitis along with an endogenous xylulokinase gene were expressed by chromosomal integration of the flocculent yeast strain IR-2. The MA-R4 strain rapidly converted xylose to ethanol with a low xylitol yield. Furthermore, the MA-R4 strain had the highest ethanol production when fermenting not only a mixture of glucose and xylose, but also mixed sugars in the detoxified hydrolysate of wood chips. These results collectively suggest that MA-R4 may be a suitable recombinant strain for further study into large-scale ethanol production from mixed sugars present in lignocellulosic hydrolysates.

title: Fermentation performance of engineered and evolved xylose‐fermenting Saccharomyces cerevisiae strains
abstract: Lignocellulose hydrolysate is an abundant substrate for bioethanol production. The ideal microorganism for such a fermentation process should combine rapid and efficient conversion of the available carbon sources to ethanol with high tolerance to ethanol and to inhibitory components in the hydrolysate. A particular biological problem are the pentoses, which are not naturally metabolized by the main industrial ethanol producer Saccharomyces cerevisiae. Several recombinant, mutated, and evolved xylose fermenting S. cerevisiae strains have been developed recently. We compare here the fermentation performance and robustness of eight recombinant strains and two evolved populations on glucose/xylose mixtures in defined and lignocellulose hydrolysate‐containing medium. Generally, the polyploid industrial strains depleted xylose faster and were more resistant to the hydrolysate than the laboratory strains. The industrial strains accumulated, however, up to 30% more xylitol and therefore produced less ethanol than the haploid strains. The three most attractive strains were the mutated and selected, extremely rapid xylose consumer TMB3400, the evolved C5 strain with the highest achieved ethanol titer, and the engineered industrial F12 strain with by far the highest robustness to the lignocellulosic hydrolysate.

title: Towards industrial pentose-fermenting yeast strains
abstract: Production of bioethanol from forest and agricultural products requires a fermenting organism that converts all types of sugars in the raw material to ethanol in high yield and with a high rate. This review summarizes recent research aiming at developing industrial strains of Saccharomyces cerevisiae with the ability to ferment all lignocellulose-derived sugars. The properties required from the industrial yeast strains are discussed in relation to four benchmarks: (1) process water economy, (2) inhibitor tolerance, (3) ethanol yield, and (4) specific ethanol productivity. Of particular importance is the tolerance of the fermenting organism to fermentation inhibitors formed during fractionation/pretreatment and hydrolysis of the raw material, which necessitates the use of robust industrial strain background. While numerous metabolic engineering strategies have been developed in laboratory yeast strains, only a few approaches have been realized in industrial strains. The fermentation performance of the existing industrial pentose-fermenting S. cerevisiae strains in lignocellulose hydrolysate is reviewed. Ethanol yields of more than 0.4 g ethanol/g sugar have been achieved with several xylose-fermenting industrial strains such as TMB 3400, TMB 3006, and 424A(LNF-ST), carrying the heterologous xylose utilization pathway consisting of xylose reductase and xylitol dehydrogenase, which demonstrates the potential of pentose fermentation in improving lignocellulosic ethanol production.

title: Metabolic Engineering: Principles And Methodologies
abstract: The Essence of Metabolic Engineering. Review of Cellular Metabolism. Comprehensive Models for Cellular Reactions.  Regulation of Metabolic Pathways. Examples of Pathway Manipulations: Metabolic Engineering in Practice. Metabolic Pathway Synthesis. Metabolic Flux Analysis. Methods for the Experimental Determination of Metabolic Fluxes by Isotope Labeling. Applications of Metabolic Flux Analysis.  Analysis of Structure of Metabolic Networks. Flux Analysis of Metabolic Networks. Thermodynamics of Cellular Processes.

title: The xylose reductase/xylitol dehydrogenase/xylulokinase ratio affects product formation in recombinant xylose-utilising Saccharomyces cerevisiae
abstract: Data simulations based on a kinetic model implied that under simplified simulation conditions a 1:≥10:≥4 relation of the xylose reductase (XR)/xylitol dehydrogenase (XDH)/xylulokinase (XK) ratio was optimal in minimising xylitol formation during xylose utilisation in yeast. The steady-state level of the intermediary xylitol depended also, to a great extent, on the NADH and NAD+ concentrations. Anaerobic xylose utilisation was investigated for three different recombinant, XR-, XDH- and XK-expressing Saccharomyces cerevisiae strains, TMB 3002, TMB 3003 and TMB 3004, to verify the model predictions. Overexpression of XK was found to be necessary for ethanol formation from xylose. Furthermore, the xylitol formation decreased with decreasing XR/XDH ratio, while the ethanol formation increased. Of the three strains, TMB 3004, which was the strain with a XR/XDH/XK ratio corresponding to the theoretical optimal ratio, fermented xylose to ethanol most efficiently.

title: Molecular Basis for Anaerobic Growth of Saccharomyces cerevisiae on Xylose, Investigated by Global Gene Expression and Metabolic Flux Analysis
abstract: Yeast xylose metabolism is generally considered to be restricted to respirative conditions because the two-step oxidoreductase reactions from xylose to xylulose impose an anaerobic redox imbalance. We have recently developed, however, a Saccharomyces cerevisiae strain that is at present the only known yeast capable of anaerobic growth on xylose alone. Using transcriptome analysis of aerobic chemostat cultures grown on xylose-glucose mixtures and xylose alone, as well as a combination of global gene expression and metabolic flux analysis of anaerobic chemostat cultures grown on xylose-glucose mixtures, we identified the distinguishing characteristics of this unique phenotype. First, the transcript levels and metabolic fluxes throughout central carbon metabolism were significantly higher than those in the parent strain, and they were most pronounced in the xylose-specific, pentose phosphate, and glycerol pathways. Second, differential expression of many genes involved in redox metabolism indicates that increased cytosolic NADPH formation and NADH consumption enable a higher flux through the two-step oxidoreductase reaction of xylose to xylulose in the mutant. Redox balancing is apparently still a problem in this strain, since anaerobic growth on xylose could be improved further by providing acetoin as an external NADH sink. This improved growth was accompanied by an increased ATP production rate and was not accompanied by higher rates of xylose uptake or cytosolic NADPH production. We concluded that anaerobic growth of the yeast on xylose is ultimately limited by the rate of ATP production and not by the redox balance per se, although the redox imbalance, in turn, limits ATP production.

title: Tolerance to furfural-induced stress is associated with pentose phosphate pathway genes ZWF1, GND1, RPE1, and TKL1 in Saccharomyces cerevisiae
abstract: Engineering yeast to be more tolerant to fermentation inhibitors, furfural and 5-hydroxymethylfurfural (HMF), will lead to more efficient lignocellulose to ethanol bioconversion. To identify target genes involved in furfural tolerance, a Saccharomyces cerevisiae gene disruption library was screened for mutants with growth deficiencies in the presence of furfural. It was hypothesized that overexpression of these genes would provide a growth benefit in the presence of furfural. Sixty two mutants were identified whose corresponding genes function in a wide spectrum of physiological pathways, suggesting that furfural tolerance is a complex process. We focused on four mutants, zwf1, gnd1, rpe1, and tkl1, which represent genes encoding pentose phosphate pathway (PPP) enzymes. At various concentrations of furfural and HMF, a clear association with higher sensitivity to these inhibitors was demonstrated in these mutants. PPP mutants were inefficient at reducing furfural to the less toxic furfuryl alcohol, which we propose is a result of an overall decreased abundance of reducing equivalents or to NADPH's role in stress tolerance. Overexpression of ZWF1 in S. cerevisiae allowed growth at furfural concentrations that are normally toxic. These results demonstrate a strong relationship between PPP genes and furfural tolerance and provide additional putative target genes involved in furfural tolerance.

title: Role of pretreatment and conditioning processes on toxicity of lignocellulosic biomass hydrolysates
abstract: The Department of Energy’s Office of the Biomass Program has set goals of making ethanol cost competitive by 2012 and replacing 30% of 2004 transportation supply with biofuels by 2030. Both goals require improvements in conversions of cellulosic biomass to sugars as well as improvements in fermentation rates and yields. Current best pretreatment processes are reasonably efficient at making the cellulose/hemicellulose/lignin matrix amenable to enzymatic hydrolysis and fermentation, but they release a number of toxic compounds into the hydrolysate which inhibit the growth and ethanol productivity of fermentation organisms. Conditioning methods designed to reduce the toxicity of hydrolysates are effective, but add to process costs and tend to reduce sugar yields, thus adding significantly to the final cost of production. Reducing the cost of cellulosic ethanol production will likely require enhanced understanding of the source and mode of action of hydrolysate toxic compounds, the means by which some organisms resist the actions of these compounds, and the methodology and mechanisms for conditioning hydrolysate to reduce toxicity. This review will provide an update on the state of knowledge in these areas and can provide insights useful for the crafting of hypotheses for improvements in pretreatment, conditioning, and fermentation organisms.

title: Effects of acetic acid on the kinetics of xylose fermentation by an engineered, xylose-isomerase-based Saccharomyces cerevisiae strain.
abstract: Acetic acid, an inhibitor released during hydrolysis of lignocellulosic feedstocks, has previously been shown to negatively affect the kinetics and stoichiometry of sugar fermentation by (engineered) Saccharomyces cerevisiae strains. This study investigates the effects of acetic acid on S. cerevisiae RWB 218, an engineered xylose-fermenting strain based on the Piromyces XylA (xylose isomerase) gene. Anaerobic batch cultures on synthetic medium supplemented with glucose-xylose mixtures were grown at pH 5 and 3.5, with and without addition of 3 g L(-1) acetic acid. In these cultures, consumption of the sugar mixtures followed a diauxic pattern. At pH 5, acetic acid addition caused increased glucose consumption rates, whereas specific xylose consumption rates were not significantly affected. In contrast, at pH 3.5 acetic acid had a strong and specific negative impact on xylose consumption rates, which, after glucose depletion, slowed down dramatically, leaving 50% of the xylose unused after 48 h of fermentation. Xylitol production was absent (<0.10 g L(-1)) in all cultures. Xylose fermentation in acetic -acid-stressed cultures at pH 3.5 could be restored by applying a continuous, limiting glucose feed, consistent with a key role of ATP regeneration in acetic acid tolerance.

title: Physiological response of Saccharomyces cerevisiae to weak acids present in lignocellulosic hydrolysate.
abstract: Weak acids are present in lignocellulosic hydrolysate as potential inhibitors that can hamper the use of this renewable resource for fuel and chemical production. To study the effects of weak acids on yeast growth, physiological investigations were carried out in batch cultures using glucose as carbon source in the presence of acetic, formic, levulinic, and vanillic acid at three different concentrations at pH 5.0. The results showed that acids at moderate concentrations can stimulate the glycolytic flux, while higher levels of acid slow down the glycolytic flux for both aerobically and anaerobically grown yeast cells. In particular, the flux distribution between respiratory and fermentative growth was adjusted to achieve an optimal ATP generation to allow a maintained energy level as high as it is in nonstressed cells grown exponentially on glucose under aerobic conditions. In addition, yeast cells exposed to acids suffered from severe reactive oxygen species stress and depletion of reduced glutathione commensurate with exhaustion of the total glutathione pool. Furthermore, a higher cellular trehalose content was observed as compared to control cultivations, and this trehalose probably acts to enhance a number of stress tolerances of the yeast.

title: Energy flux and osmoregulation of Saccharomyces cerevisiae grown in chemostats under NaCl stress
abstract: The energetics and accumulation of solutes in Saccharomyces cerevisiae were investigated for cells grown aerobically in a chemostat under NaCl stress and glucose limitation. Changed energy requirements in relation to external salinity were examined by energy balance determinations performed by substrate and product analyses, with the latter including heat measurements by microcalorimetry. In both 0 and 0.9 M NaCl cultures, the catabolism was entirely respiratory at the lowest dilution rates tested but shifted to a mixed respiratory-fermentative metabolism at higher dilution rates. This shift occurred at a considerably lower dilution rate for salt-grown cells. The intracellular solute concentrations, as calculated on the basis of intracellular soluble space determinations, showed that the internal Na+ concentration increased from about 0.02 molal in basal medium to about 0.18 molal in 0.9 M NaCl medium, while intracellular K+ was maintained around 0.29 molal despite the variation in external salinity. The intracellular glycerol concentration increased from below 0.05 molal at low salinity to about 1.2 molal at 0.9 M NaCl. The concentrations of the internal solutes, however, changed insignificantly with growth rate and energy metabolism. The additional maintenance energy expenditure for growth at 0.9 M NaCl was, depending on the growth rate, 14 to 31% of the total energy requirement for growth at 0 M NaCl. Including the energy conserved in glycerol, the total additional energy demand for growth at 0.9 M NaCl corresponded to 28 to 51% of the energy required for growth at 0 M NaCl.

title: Furfural, 5-hydroxymethyl furfural, and acetoin act as external electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae.
abstract: The electron acceptors acetoin, acetaldehyde, furfural, and 5-hydroxymethylfurfural (HMF) were added to anaerobic batch fermentation of xylose by recombinant, xylose utilising Saccharomyces cerevisiae TMB 3001. The intracellular fluxes during xylose fermentation before and after acetoin addition were calculated with metabolic flux analysis. Acetoin halted xylitol excretion and decreased the flux through the oxidative pentose phosphate pathway. The yield of ethanol increased from 0.62 mol ethanol/mol xylose to 1.35 mol ethanol/mol xylose, and the cell more than doubled its specific ATP production after acetoin addition compared to fermentation of xylose only. This did, however, not result in biomass growth. The xylitol excretion was also decreased by furfural and acetaldehyde but was unchanged by HMF. Thus, furfural present in lignocellulosic hydrolysate can be beneficial for ethanolic fermentation of xylose. Enzymatic analyses showed that the reduction of acetoin and furfural required NADH, whereas the reduction of HMF required NADPH. The enzymatic activity responsible for furfural reduction was considerably higher than for HMF reduction and also in situ furfural conversion was higher than HMF conversion.

title: Metabolic effects of furaldehydes and impacts on biotechnological processes
abstract: There is a growing awareness that lignocellulose will be a major raw material for production of both fuel and chemicals in the coming decades—most likely through various fermentation routes. Considerable attention has been given to the problem of finding efficient means of separating the major constituents in lignocellulose (i.e., lignin, hemicellulose, and cellulose) and to efficiently hydrolyze the carbohydrate parts into sugars. In these processes, by-products will inevitably form to some extent, and these will have to be dealt with in the ensuing microbial processes. One group of compounds in this category is the furaldehydes. 2-Furaldehyde (furfural) and substituted 2-furaldehydes—most importantly 5-hydroxymethyl-2-furaldehyde—are the dominant inhibitory compounds found in lignocellulosic hydrolyzates. The furaldehydes are known to have biological effects and act as inhibitors in fermentation processes. The effects of these compounds will therefore have to be considered in the design of biotechnological processes using lignocellulose. In this short review, we take a look at known metabolic effects, as well as strategies to overcome problems in biotechnological applications caused by furaldehydes.

title: Pichia stipitis xylose reductase helps detoxifying lignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfural (HMF)
abstract: BackgroundPichia stipitis xylose reductase (Ps-XR) has been used to design Saccharomyces cerevisiae strains that are able to ferment xylose. One example is the industrial S. cerevisiae xylose-consuming strain TMB3400, which was constructed by expression of P. stipitis xylose reductase and xylitol dehydrogenase and overexpression of endogenous xylulose kinase in the industrial S. cerevisiae strain USM21.ResultsIn this study, we demonstrate that strain TMB3400 not only converts xylose, but also displays higher tolerance to lignocellulosic hydrolysate during anaerobic batch fermentation as well as 3 times higher in vitro HMF and furfural reduction activity than the control strain USM21. Using laboratory strains producing various levels of Ps-XR, we confirm that Ps-XR is able to reduce HMF both in vitro and in vivo. Ps-XR overexpression increases the in vivo HMF conversion rate by approximately 20%, thereby improving yeast tolerance towards HMF. Further purification of Ps-XR shows that HMF is a substrate inhibitor of the enzyme.ConclusionWe demonstrate for the first time that xylose reductase is also able to reduce the furaldehyde compounds that are present in undetoxified lignocellulosic hydrolysates. Possible implications of this newly characterized activity of Ps-XR on lignocellulosic hydrolysate fermentation are discussed.

title: Aldo-keto reductases as modulators of stress response.
abstract: Human aldose reductase (AKR1B1) has been implicated as a factor in the pathogenesis of diabetic complications. However, little is known about the physiological role of this enzyme or of related aldo-keto reductases in human tissues. In mammalian systems, a gene knock out approach is often employed as an experimental strategy to probe for gene function. However, in the murine system, phenotypic characterization of an aldose reductase (AKR1B3) knock out is likely to be complicated due to functional compensation by redundant AKRs including AKRs 1A (aldehyde reductase), 1B7 (FR-1) and 1B8 (MVDP). As an alternate strategy, we are examining the budding yeast Saccharomyces cerevisiae as a model system for a functional genomics study of AKRs. A distinct advantage of this system centers on the ability to readily ablate multiple targeted genes in a single strain. In addition to providing insights into functional redundancy, this system allows us to use a genetic approach to study possible effector pathways associated with one or more individual genes. Yeast open reading frames (ORFs) encoding AKRs with functional similarity to human aldose reductase (AKR1B1) were identified by BLAST analysis and were functionally validated by studies of recombinant proteins. By ablating three of the yeast AKR genes most functionally similar to AKR1B1, we have created a unique strain of S. cerevisiae that shows enhanced sensitivity to stress. Ongoing studies with oligonucleotide arrays show that the triple null strain has an altered transcription profile consistent with an enhanced stress response in comparison with the parental strain. These data indicate that AKR-null strains may provide new insights into signaling mechanisms involving this family of proteins.

title: Expression of protein engineered NADP+-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant Saccharomyces cerevisiae
abstract: A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD+-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP+. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP+-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP+-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.

title: 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.

title: Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae.
abstract: In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.

title: Alcoholic fermentation of xylose and mixed sugars using recombinant Saccharomyces cerevisiae engineered for xylose utilization
abstract: Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h−1, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h−1. The adapted strain could ferment 20 g l−1 of xylose to ethanol with a yield of 0.37 g g−1 and production rate of 0.026 g l−1 h−1. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g−1) and production rate (0.07 g l−1 h−1) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g−1.

title: Fermentation of xylose/glucose mixtures by metabolically engineered Saccharomyces cerevisiae strains expressing XYL1 and XYL2 from Pichia stipitis with and without overexpression of TAL1.
abstract: Anaerobic xylose conversion by two metabolically engineered Saccharomyces cerevisiae strains in the presence and absence of simultaneous glucose metabolism was investigated. One strain expressed XYL1 encoding xylose reductase (XR) and XYL2 encoding xylitol dehydrogenase (XDH) from Pichia stipitis, whereas the other additionally overexpressed TAL1 encoding transaldolase (TAL). Both strains formed xylitol as the main product of xylose metabolism. The TAL1-overexpressing strain gave a higher biomass yield and produced less carbon dioxide and somewhat less xylitol compared with the XYL1 + XYL2 strain, indicating that TAL limited xylose metabolism in the latter. The ethanol yield was similar with both strains. The simultaneous metabolism of glucose enhanced xylose metabolism by causing a higher rate of xylose consumption and less xylitol and xylulose excretion, compared with xylose metabolism alone. Simultaneous xylose and glucose metabolism affected the growth rate negatively compared with growth on glucose alone. Additionally, comparison of the specific growth rate of the host strain, a reference strain with a plasmid without XYL1, XYL2 or TAL1, the XYL1 + XYL2 strain and the XYL1 + XYL2 + TAL1 strain on glucose, showed that the presence of plasmids and expression of genes on the plasmids caused a decrease in specific growth rates related to the number of plasmids present and the number of structural genes on the plasmids. Both strains exhibited high XR and XDH activities in batch cultivation, but rapidly lost the activities in chemostat cultivation. Limitations in the xylose-metabolising pathway and further improvement of recombinant xylose-metabolising S. cerevisiae are discussed.

title: 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
abstract: 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.

title: Characterization of the effectiveness of hexose transporters for transporting xylose during glucose and xylose co‐fermentation by a recombinant Saccharomyces yeast
abstract: We have developed recombinant Saccharomyces yeasts that can effectively co‐ferment glucose and xylose to ethanol. However, these yeasts still ferment glucose more efficiently than xylose. The transport of xylose could be one of the steps limiting the fermentation of xylose. In this study, we characterized the changes in the expression pattern of the hexose transporter and related genes during co‐fermentation of glucose and xylose using one of our recombinant yeasts, Saccharomyces cerevisiae 424A(LNH‐ST). The transcription of the hexose transporter and related genes was strongly influenced by the presence of glucose; HXT1, HXT2 and HXT3 were greatly activated by glucose and HXT5, HXT7 and AGT1 were significantly repressed by glucose. We also examined the effectiveness of individual transporters encoded by HXT1, HXT2, HXT4, HXT5, HXT7 and GAL2 genes for transporting xylose during co‐fermentation of glucose and xylose in a Saccharomyces hxt° mutant (RE700A). We compared these hxt° derivatives to RE700A wild‐type strain (S. cerevisiae MC996A) where all of them contained the same xylose metabolizing genes present in our xylose‐fermenting yeasts such as 424A(LNH‐ST). Our results showed that recombinant RE700A containing the cloned HXT7 or HXT5 were substantially more effective for fermenting xylose to ethanol. In addition, we found that the efficiency of transporters for intracellular accumulation of xylose was as follows: HXT7 > HXT5 > GAL2 > WT > HXT1 > HXT4 > > > RE700A. Furthermore, we provided evidence that the Saccharomyces galactose transporter system could be a highly effective xylose transporter. The information reported here should be of great importance for improving the Saccharomyces yeast transport of xylose.

title: Genetically Engineered SaccharomycesYeast Capable of Effective Cofermentation of Glucose and Xylose
abstract: Xylose is one of the major fermentable sugars present in cellulosic biomass, second only to glucose. However, Saccharomycesspp., the best sugar-fermenting microorganisms, are not able to metabolize xylose. We developed recombinant plasmids that can transformSaccharomyces spp. into xylose-fermenting yeasts. These plasmids, designated pLNH31, -32, -33, and -34, are 2μm-based high-copy-number yeast-E. coli shuttle plasmids. In addition to the geneticin resistance and ampicillin resistance genes that serve as dominant selectable markers, these plasmids also contain three xylose-metabolizing genes, a xylose reductase gene, a xylitol dehydrogenase gene (both from Pichia stipitis), and a xylulokinase gene (from Saccharomyces cerevisiae). These xylose-metabolizing genes were also fused to signals controlling gene expression from S. cerevisiae glycolytic genes. Transformation of Saccharomyces sp. strain 1400 with each of these plasmids resulted in the conversion of strain 1400 from a non-xylose-metabolizing yeast to a xylose-metabolizing yeast that can effectively ferment xylose to ethanol and also effectively utilizes xylose for aerobic growth. Furthermore, the resulting recombinant yeasts also have additional extraordinary properties. For example, the synthesis of the xylose-metabolizing enzymes directed by the cloned genes in these recombinant yeasts does not require the presence of xylose for induction, nor is the synthesis repressed by the presence of glucose in the medium. These properties make the recombinant yeasts able to efficiently ferment xylose to ethanol and also able to efficiently coferment glucose and xylose present in the same medium to ethanol simultaneously.

title: Influence of the propagation strategy for obtaining robust Saccharomyces cerevisiae cells that efficiently co-ferment xylose and glucose in lignocellulosic hydrolysates
abstract: Development of xylose‐fermenting yeast strains that are tolerant to the inhibitors present in lignocellulosic hydrolysates is crucial to achieve efficient bioethanol production processes. In this study, the importance of the propagation strategy for obtaining robust cells was studied. Addition of hydrolysate during propagation of the cells adapted them to the inhibitors, resulting in more tolerant cells with shorter lag phases and higher specific growth rates in minimal medium containing acetic acid and vanillin than unadapted cells. Addition of hydrolysate during propagation also resulted in cells with better fermentation capabilities. Cells propagated without hydrolysate were unable to consume xylose in wheat straw hydrolysate fermentations, whereas 40.3% and 97.7% of the xylose was consumed when 12% and 23% (v/v) hydrolysate, respectively, was added during propagation. Quantitative polymerase chain reaction revealed changes in gene expression, depending on the concentration of hydrolysate added during propagation. This study highlights the importance of using an appropriate propagation strategy for the optimum performance of yeast in fermentation of lignocellulosic hydrolysates.