Improvement of xylose fermentation in respiratory-deficient xylose-fermenting Saccharomyces cerevisiae

Effective conversion of xylose in lignocelluloses is expected to reduce the production cost of second-generation biofuels significantly. The factors affecting xylose fermentation in Saccharomyces cerevisiae that express xylose reductase-xylitol dehydrogenase (XR-XDH) are studied. Although overproduction of non-oxidative pentose phosphate pathway significantly increased the aerobic-specific growth rate on xylose and slightly improved conversion of xylose to ethanol under oxygen-limited conditions, the elimination of respiration by deleting cytochrome C oxidase subunit IV gene impeded aerobic growth on xylose. However, the adaptive evolution of the respiratory-deficient strain with an NADP(+)-preferring XDH mutant in xylose media dramatically improved its xylose-fermenting ability. The specific growth rate, ethanol yield, and xylitol yield of the evolved strain on xylose were 0.06h(-1), 0.39gg(-1), and 0.13gg(-1) consumed xylose, respectively. Similar to anaerobic fermentation, the evolved strain exhibited accumulated ethanol rather than recycled it under aerobic conditions.     

Comparative study on a series of recombinant flocculent Saccharomyces cerevisiae strains with different expression levels of xylose reductase and xylulokinase

Ethanol production from xylose is important for the utilization of lignocellulosic biomass as raw materials. Recently, we reported the development of an industrial xylose-fermenting Saccharomyces cerevisiae strain, MA-R4, which was engineered by chromosomal integration to express the genes encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis along with S. cerevisiae xylulokinase gene constitutively using the alcohol-fermenting flocculent yeast strain, IR-2. IR-2 has the highest xylulose-fermenting ability of the industrial diploid strains, making it a useful host strain for genetically engineering xylose-utilizing S. cerevisiae. To optimize the activities of xylose metabolizing enzymes in the metabolic engineering of IR-2 for further improvement of ethanol production from xylose, we constructed a set of recombinant isogenic strains harboring different combinations of genetic modifications present in MA-R4, and investigated the effect of constitutive expression of xylulokinase and of different levels of xylulokinase and xylose reductase activity on xylose fermentation. This strain comparison showed that constitutive expression of xylulokinase increased ethanol production from xylose at the expense of xylitol excretion, and that high activity of xylose reductase resulted in an increased rate of xylose consumption and an increased glycerol yield. Moreover, strain MA-R6, which has moderate xylulokinase activity, grew slightly better but accumulated more xylitol than strain MA-R4. These results suggest that fine-tuning of introduced enzyme activity in S. cerevisiae is important for improving xylose fermentation to ethanol.     

Improvements in ethanol production from xylose by mating recombinant xylose-fermenting Saccharomyces cerevisiae strains

To improve the ability of recombinant Saccharomyces cerevisiae strains to utilize the hemicellulose components of lignocellulosic feedstocks, the efficiency of xylose conversion to ethanol needs to be increased. In the present study, xylose-fermenting, haploid, yeast cells of the opposite mating type were hybridized to produce a diploid strain harboring two sets of xylose-assimilating genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase. The hybrid strain MN8140XX showed a 1.3- and 1.9-fold improvement in ethanol production compared to its parent strains MT8-1X405 and NBRC1440X, respectively. The rate of xylose consumption and ethanol production was also improved by the hybridization. This study revealed that the resulting improvements in fermentation ability arose due to chromosome doubling as well as the increase in the copy number of xylose assimilation genes. Moreover, compared to the parent strain, the MN8140XX strain exhibited higher ethanol production under elevated temperatures (38 °C) and acidic conditions (pH 3.8). Thus, the simple hybridization technique facilitated an increase in the xylose fermentation activity.     

APJ1 and GRE3 homologs work in concert to allow growth in xylose in a natural Saccharomyces sensu stricto hybrid yeast

Creating Saccharomyces yeasts capable of efficient fermentation of pentoses such as xylose remains a key challenge in the production of ethanol from lignocellulosic biomass. Metabolic engineering of industrial Saccharomyces cerevisiae strains has yielded xylose-fermenting strains, but these strains have not yet achieved industrial viability due largely to xylose fermentation being prohibitively slower than that of glucose. Recently, it has been shown that naturally occurring xylose-utilizing Saccharomyces species exist. Uncovering the genetic architecture of such strains will shed further light on xylose metabolism, suggesting additional engineering approaches or possibly even enabling the development of xylose-fermenting yeasts that are not genetically modified. We previously identified a hybrid yeast strain, the genome of which is largely Saccharomyces uvarum, which has the ability to grow on xylose as the sole carbon source. To circumvent the sterility of this hybrid strain, we developed a novel method to genetically characterize its xylose-utilization phenotype, using a tetraploid intermediate, followed by bulk segregant analysis in conjunction with high-throughput sequencing. We found that this strain's growth in xylose is governed by at least two genetic loci, within which we identified the responsible genes: one locus contains a known xylose-pathway gene, a novel homolog of the aldo-keto reductase gene GRE3, while a second locus contains a homolog of APJ1, which encodes a putative chaperone not previously connected to xylose metabolism. Our work demonstrates that the power of sequencing combined with bulk segregant analysis can also be applied to a nongenetically tractable hybrid strain that contains a complex, polygenic trait, and identifies new avenues for metabolic engineering as well as for construction of nongenetically modified xylose-fermenting strains.     

Expression of a heterologous xylose transporter in a <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain engineered to utilize xylose improves aerobic xylose consumption

The goal of this investigation was to determine the effect of a xylose transport system on glucose and xylose co-consumption as well as total xylose consumption in Saccharomyces cerevisiae. We expressed two heterologous transporters from Arabidopsis thaliana in recombinant xylose-utilizing S. cerevisiae cells. Strains expressing the heterologous transporters were grown on glucose and xylose mixtures. Sugar consumption rates and ethanol concentrations were determined and compared to an isogenic control strain lacking the A. thaliana transporters. Expression of the transporters increased xylose uptake and xylose consumption up to 46% and 40%, respectively. Xylose co-consumption rates (prior to glucose depletion) were also increased by up to 2.5-fold compared to the control strain. Increased xylose consumption correlated with increased ethanol concentration and productivity. During the xylose/glucose co-consumption phase, strains expressing the transporters had up to a 70% increase in ethanol production rate. It was concluded that in these strains, xylose transport was a limiting factor for xylose utilization and that increasing xylose/glucose co-consumption is a viable strategy for improving xylose fermentation.     

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

Background

Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose.

Results

The specific aerobic arabinose growth rate was identical, 0.03 h^-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h^-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)^-1 h^-1 than the xylose isomerase strain, which only reached 0.03 h^-1 and 0.02 g (g cells)^-1h^-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g^-1 compared with 0.18 g g^-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g^-1 compared with 0.32 g g^-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l^-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l^-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)^-1 h^-1 compared with 0.01 g (g cells)^-1 h^-1 for the xylose reductase/xylitol dehydrogenase strain and the xylose isomerase strain, respectively.

Conclusion

The combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme.     

Reduced oxidative pentose phosphate pathway flux in recombinant xylose-utilizing Saccharomyces cerevisiae strains improves the ethanol yield from xylose

In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD(+). In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g(-1)) and the lowest xylitol yield (0.05 g g(-1)) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.     

Directed evolution of xylose isomerase for improved xylose catabolism and fermentation in the yeast Saccharomyces cerevisiae

The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tal1 and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.     

Endogenous xylose pathway in Saccharomyces cerevisiae

The baker's yeast Saccharomyces cerevisiae is generally classified as a non-xylose-utilizing organism. We found that S. cerevisiae can grow on D-xylose when only the endogenous genes GRE3 (YHR104w), coding for a nonspecific aldose reductase, and XYL2 (YLR070c, ScXYL2), coding for a xylitol dehydrogenase (XDH), are overexpressed under endogenous promoters. In nontransformed S. cerevisiae strains, XDH activity was significantly higher in the presence of xylose, but xylose reductase (XR) activity was not affected by the choice of carbon source. The expression of SOR1, encoding a sorbitol dehydrogenase, was elevated in the presence of xylose as were the genes encoding transketolase and transaldolase. An S. cerevisiae strain carrying the XR and XDH enzymes from the xylose-utilizing yeast Pichia stipitis grew more quickly and accumulated less xylitol than did the strain overexpressing the endogenous enzymes. Overexpression of the GRE3 and ScXYL2 genes in the S. cerevisiae CEN.PK2 strain resulted in a growth rate of 0.01 g of cell dry mass liter(-1) h(-1) and a xylitol yield of 55% when xylose was the main carbon source.     

Xylulokinase overexpression in two strains of Saccharomyces cerevisiae also expressing xylose reductase and xylitol dehydrogenase and its effect on fermentation of xylose and lignocellulosic hydrolysate

Fermentation of the pentose sugar xylose to ethanol in lignocellulosic biomass would make bioethanol production economically more competitive. Saccharomyces cerevisiae, an efficient ethanol producer, can utilize xylose only when expressing the heterologous genes XYL1 (xylose reductase) and XYL2 (xylitol dehydrogenase). Xylose reductase and xylitol dehydrogenase convert xylose to its isomer xylulose. The gene XKS1 encodes the xylulose-phosphorylating enzyme xylulokinase. In this study, we determined the effect of XKS1 overexpression on two different S. cerevisiae host strains, H158 and CEN.PK, also expressing XYL1 and XYL2. H158 has been previously used as a host strain for the construction of recombinant xylose-utilizing S. cerevisiae strains. CEN.PK is a new strain specifically developed to serve as a host strain for the development of metabolic engineering strategies. Fermentation was carried out in defined and complex media containing a hexose and pentose sugar mixture or a birch wood lignocellulosic hydrolysate. XKS1 overexpression increased the ethanol yield by a factor of 2 and reduced the xylitol yield by 70 to 100% and the final acetate concentrations by 50 to 100%. However, XKS1 overexpression reduced the total xylose consumption by half for CEN.PK and to as little as one-fifth for H158. Yeast extract and peptone partly restored sugar consumption in hydrolysate medium. CEN.PK consumed more xylose but produced more xylitol than H158 and thus gave lower ethanol yields on consumed xylose. The results demonstrate that strain background and modulation of XKS1 expression are important for generating an efficient xylose-fermenting recombinant strain of S. cerevisiae.     

Alcoholic glucose and xylose fermentations by the coculture process: compatibility and typing of associated strains.

As part of the simultaneous fermentation of both glucose and xylose to ethanol by a coculture process, compatibilities between xylose-fermenting yeasts and glucose-fermenting species were investigated. Among the Saccharomyces species tested, none inhibited growth of the xylose-fermenting yeasts. By contrast, many xylose-fermenting yeasts, among the 11 tested, exerted an inhibitory effect on growth of the selected Saccharomyces species. Killer character was demonstrated in three strains of Pichia stipitis. Such strains, despite their high fermentative performances, cannot be used to ferment D-xylose in association with the selected Saccharomyces species. From compatibility tests between xylose-fermenting yeasts and Saccharomyces species, pairs of microorganisms suitable for simultaneous xylose and glucose fermentations by coculture are proposed. Strains associated in the coculture process are distinguished by their resistance to mitochondrial inhibitors. The xylose-fermenting yeasts are able to grow on media containing erythromycin (1 g/L) or diuron (50 mg/L), whereas the Saccharomyces species are inhibited by these mitochondrial inhibitors.     

Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae

Saccharomyces cerevisiae TMB3001 has previously been engineered to utilize xylose by integrating the genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) and overexpressing the native xylulokinase (XK) gene. The resulting strain is able to metabolize xylose, but its xylose utilization rate is low compared to that of natural xylose utilizing yeasts, like Pichia stipitis or Candida shehatae. One difference between S. cerevisiae and the latter species is that these possess specific xylose transporters, while S. cerevisiae takes up xylose via the high-affinity hexose transporters. For this reason, in part, it has been suggested that xylose transport in S. cerevisiae may limit the xylose utilization.We investigated the control exercised by the transport over the specific xylose utilization rate in two recombinant S. cerevisiae strains, one with low XR activity, TMB3001, and one with high XR activity, TMB3260. The strains were grown in aerobic sugar-limited chemostat and the specific xylose uptake rate was modulated by changing the xylose concentration in the feed, which allowed determination of the flux response coefficients. Separate measurements of xylose transport kinetics allowed determination of the elasticity coefficients of transport with respect to extracellular xylose concentration. The flux control coefficient, C(J) (transp), for the xylose transport was calculated from the response and elasticity coefficients. The value of C(J) (transp) for both strains was found to be < 0.1 at extracellular xylose concentrations > 7.5 g L(-1). However, for strain TMB3260 the flux control coefficient was higher than 0.5 at xylose concentrations < 0.6 g L(-1), while C(J) (transp) stayed below 0.2 for strain TMB3001 irrespective of xylose concentration.     

Enhancement in xylose utilization using <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> NIRE-K1 through evolutionary adaptation approach

The evolutionary adaptation was carried out on the thermotolerant yeast Kluyveromyces marxianus NIRE-K1 at 45 °C up to 60 batches to enhance its xylose utilization capability. The adapted strain showed higher specific growth rate and 3-fold xylose uptake rate and short lag phase as compared to the native strain. During aerobic growth adapted yeast showed 2.81-fold higher xylose utilization than that of native. In anaerobic batch fermentation, adapted yeast utilized about 91 % of xylose in 72 h and produced 2.88 and 18.75 g l^?1 of ethanol and xylitol, respectively, which were 5.11 and 5.71-fold higher than that of native. Ethanol yield, xylitol yield and specific sugar consumption rate obtained by the adapted cells were found to be 1.57, 1.65 and 4.84-fold higher than that of native yeast, respectively. Aforesaid results suggested that the evolutionary adaptation will be a very effective strategy in the near future for economic lignocellulosic ethanol production.     

Engineering Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: reflections and perspectives

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.     

High expression of XYL2 coding for xylitol dehydrogenase is necessary for efficient xylose fermentation by engineered Saccharomyces cerevisiae

The traditional ethanologenic yeast Saccharomyces cerevisiae cannot metabolize xylose, which is an abundant sugar in non-crop plants. Engineering this yeast for a practicable fermentation of xylose will therefore improve the economics of bioconversion for the production of fuels and chemicals such as ethanol. One of the most widely employed strategies is to express XYL1, XYL2, and XYL3 genes derived from Scheffersomyces stipitis (formerly Pichia stiptis) in S. cerevisiae. However, the resulting engineered strains have been reported to exhibit large variations in xylitol accumulation and ethanol yields, generating many hypotheses and arguments for elucidating these phenomena. Here we demonstrate that low expression levels of the XYL2 gene, coding for xylitol dehydrogenase (XDH), is a major bottleneck in efficient xylose fermentation. Through an inverse metabolic engineering approach using a genomic library of S. cerevisiae, XYL2 was identified as an overexpression target for improving xylose metabolism. Specifically, we performed serial subculture experiments after transforming a genomic library of wild type S. cerevisiae into an engineered strain harboring integrated copies of XYL1, XYL2 and XYL3. Interestingly, the isolated plasmids from efficient xylose-fermenting transformants contained XYL2. This suggests that the integrated XYL2 migrated into a multi-copy plasmid through homologous recombination. It was also found that additional overexpression of XYL2 under the control of strong constitutive promoters in a xylose-fermenting strain not only reduced xylitol accumulation, but also increased ethanol yields. As the expression levels of XYL2 increased, the ethanol yields gradually improved from 0.1 to 0.3g ethanol/g xylose, while the xylitol yields significantly decreased from 0.4 to 0.1g xylitol/g xylose. These results suggest that strong expression of XYL2 is a necessary condition for developing efficient xylose-fermenting strains.     

Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Two 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.     

转醛醇酶基因差异表达影响酵母发酵木糖及耐受乙酸性能

【目的】木糖发酵是纤维素燃料乙醇生产的一个关键瓶颈,同时木质纤维素水解液中的乙酸严重抑制酿酒酵母的木糖发酵过程,因此通过基因工程手段提高菌株对木糖的利用以及对乙酸的耐受性具有重要意义。本研究以非氧化磷酸戊糖途径(PPP途径)中关键基因转醛醇酶基因(TAL1)为研究对象,探讨了3种不同启动子PTDH3、PAHP1和PUBI4,控制其表达对菌株利用木糖和耐受乙酸的影响。【方法】通过同源重组用3种启动子替换酿酒酵母基因工程菌NAPX37的TAL1基因的启动子PTAL1,再通过孢子分离和单倍体交配构建了纯合子,利用批次发酵比较了在以木糖为唯一碳源和混合糖(葡萄糖和木糖)为碳源条件下,3种启动子控制TAL1基因表达导致的发酵和乙酸耐受能力的差异。【结果】启动子PTDH3、PAHP1和PUBI4在不同程度上提高了TAL1基因的转录水平,提高了菌株对木糖的利用速率及乙酸耐受能力,提高了菌株在60 mmol/L乙酸条件下的葡萄糖利用速率。在以木糖为唯一碳源且无乙酸存在、以及混合糖为碳源的条件下,PAHP1启动子控制TAL1表达菌株的发酵结果优于PTDH3和PUBI4启动子的菌株,PAHP1启动子控制的TAL1基因的转录水平比较合适。在木糖为唯一碳源且乙酸为30 mmol/L时,PUBI4启动子控制TAL1基因表达的菌株发酵结果则优于PAHP1和PTDH3启动子菌株,此时PUBI4启动子控制的TAL1的转录水平比较合适。【结论】启动子PTDH3、PAHP1和PUBI4不同程度地提高TAL1基因的表达,在不同程度上改善了酵母菌株的木糖发酵速率和耐受乙酸性能,改善程度受发酵条件的影响。