Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains

Background  

Fermentation of lignocellulosic biomass is an attractive alternative for the production of bioethanol. Traditionally, the yeast Saccharomyces cerevisiae is used in industrial ethanol fermentations. However, S. cerevisiae is naturally not able to ferment the pentose sugars D-xylose and L-arabinose, which are present in high amounts in lignocellulosic raw materials.     

Combined inactivation of the <Emphasis Type="Italic">Clostridium cellulolyticum</Emphasis> lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

Background  

The model bacterium Clostridium cellulolyticum efficiently degrades crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H₂ and CO₂, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels production. Therefore genetic engineering will likely be required to improve the ethanol yield. Plasmid transformation, random mutagenesis and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism, hindering genetic engineering.     

Improved strains of recombinant Escherichia coli for ethanol production from sugar mixtures

Hemicellulose hydrolysates of agricultural residues often contain mixtures of hexose and pentose sugars. Ethanologenic Escherichia coli that have been previously investigated preferentially ferment hexose sugars. In some cases, xylose fermentation was slow or incomplete. The purpose of this study was to develop improved ethanologenic E. coli strains for the fermentation of pentoses in sugar mixtures. Using fosfomycin as a selective agent, glucose-negative mutants of E. coli KO11 (containing chromosomally integrated genes encoding the ethanol pathway from Zymomonas mobilis) were isolated that were unable to ferment sugars transported by the phosphoenolpyruvate-dependent phosphotransferase system. These strains (SL31 and SL142) retained the ability to ferment sugars with independent transport systems such as arabinose and xylose and were used to ferment pentose sugars to ethanol selectively in the presence of high concentrations of glucose. Additional fosfomycin-resistant mutants were isolated that were superior to strain KO11 for ethanol production from hexose and pentose sugars. These hyperproductive strains (SL28 and SL40) retained the ability to metabolize all sugars tested, completed fermentations more rapidly, and achieved higher ethanol yields than the parent. Both SL28 and SL40 produced 60 gl^–1 ethanol from 120 gl^–1 xylose in 60 h, 20% more ethanol than KO11 under identical conditions. Further studies illustrated the feasibility of sequential fermentation. A mixture of hexose and pentose sugars was fermented with near theoretical yield by SL40 in the first step followed by a second fermentation in which yeast and glucose were added. Such a two-step approach can combine the attributes of ethanologenic E. coli for pentoses with the high ethanol tolerance of conventional yeasts in a single vessel.     

Characterization of Candida sp. NY7122, a novel pentose-fermenting soil yeast

Yeasts that ferment both hexose and pentose are important for cost-effective ethanol production. We found that the soil yeast strain NY7122 isolated from a blueberry field in Tsukuba (East Japan) could ferment both hexose and pentose (d-xylose and l-arabinose). NY7122 was closely related to Candida subhashii on the basis of the results of molecular identification using the sequence in the D1/D2 domains of 26S rDNA and 5.8S-internal transcribed spacer region. NY7122 produced at least 7.40 and 3.86 g l⁻¹ ethanol from 20 g l⁻¹ d-xylose and l-arabinose within 24 h. NY7122 could produce ethanol from pentose and hexose sugars at 37°C. The highest ethanol productivity of NY7122 was achieved under a low pH condition (pH 3.5). Fermentation of mixed sugars (50 g l⁻¹ glucose, 20 g l⁻¹ d-xylose, and 10 g l⁻¹ l-arabinose) resulted in a maximum ethanol concentration of 27.3 g l⁻¹ for the NY7122 strain versus 25.1 g l⁻¹ for Scheffersomyces stipitis. This is the first study to report that Candida sp. NY7122 from a soil environment could produce ethanol from both d-xylose and l-arabinose.     

Fermentation of mixed glucose-xylose substrates by engineered strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: role of the coenzyme specificity of xylose reductase,and effect of glucose on xylose utilization

Background  

In 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 q _xylose (g xylose/g dry cell weight/h) of 0.08. The study presented herein was performed with the aim of analysing (external) factors that limit q _xylose 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.     

Quantifying the metabolic capabilities of engineered <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> using linear programming analysis

Background  

The need for discovery of alternative, renewable, environmentally friendly energy sources and the development of cost-efficient, "clean" methods for their conversion into higher fuels becomes imperative. Ethanol, whose significance as fuel has dramatically increased in the last decade, can be produced from hexoses and pentoses through microbial fermentation. Importantly, plant biomass, if appropriately and effectively decomposed, is a potential inexpensive and highly renewable source of the hexose and pentose mixture. Recently, the engineered (to also catabolize pentoses) anaerobic bacterium Zymomonas mobilis has been widely discussed among the most promising microorganisms for the microbial production of ethanol fuel. However, Z. mobilis genome having been fully sequenced in 2005, there is still a small number of published studies of its in vivo physiology and limited use of the metabolic engineering experimental and computational toolboxes to understand its metabolic pathway interconnectivity and regulation towards the optimization of its hexose and pentose fermentation into ethanol.     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

Continuous ethanol production from wheat straw hydrolysate by recombinant ethanologenic <Emphasis Type="BoldItalic">Escherichia coli</Emphasis> strain FBR5

Continuous production of ethanol from alkaline peroxide pretreated and enzymatically saccharified wheat straw hydrolysate by ethanologenic recombinant Escherichia coli strain FBR5 was investigated under various conditions at controlled pH 6.5 and 35°C. The strain FBR5 was chosen because of its ability to ferment both hexose and pentose sugars under semi-anaerobic conditions without using antibiotics. The average ethanol produced from the available sugars (21.9–47.8 g/L) ranged from 8.8 to 17.3 g/L (0.28–0.45 g/g available sugars, 0.31–0.48 g/g sugar consumed) with ethanol productivity of 0.27–0.78 g l⁻¹ h⁻¹ in a set of 14 continuous culture (CC) runs (16–105 days). During these CC runs, no loss of ethanol productivity was observed. This is the first report on the continuous production of ethanol by the recombinant bacterium from a lignocellulosic hydrolysate.     

Metabolic evolution of non-transgenic <Emphasis Type="Italic">Escherichia coli</Emphasis> SZ420 for enhanced homoethanol fermentation from xylose

Efficient utilization of pentose sugars (xylose and arabinose) is an essential requirement for economically viable ethanol production from cellulosic biomass. The desirable pentose-fermenting ethanologenic biocatalysts are the native microorganisms or the engineered derivatives without recruited exogenous gene(s). We have used a metabolic evolution (adaptive selection) approach to improve a non-transgenic homoethanol Escherichia coli SZ420 (ldhA pflB ackA frdBC pdhR::pflBp6-aceEF-lpd) for xylose fermentation. An improved mutant, E. coli KC01, was evolved through a 3 month metabolic evolution process. This evolved mutant increased pyruvate dehydrogenase activity by 100%, cell growth rate (h⁻¹) by 23%, volumetric ethanol productivity by 65% and ethanol tolerance by 200%. These improvements enabled KC01 to complete 50 g xylose l⁻¹ fermentations with an ethanol titer of 23 g l⁻¹ and a yield of 90%. The improved cell growth and ethanol production of KC01 are likely attributed to its three fold increased ethanol tolerance.     

Genome-wide identification of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> genes required for tolerance to acetic acid

Background  

Acetic acid is a byproduct of Saccharomyces cerevisiae alcoholic fermentation. Together with high concentrations of ethanol and other toxic metabolites, acetic acid may contribute to fermentation arrest and reduced ethanol productivity. This weak acid is also a present in lignocellulosic hydrolysates, a highly interesting non-feedstock substrate in industrial biotechnology. Therefore, the better understanding of the molecular mechanisms underlying S. cerevisiae tolerance to acetic acid is essential for the rational selection of optimal fermentation conditions and the engineering of more robust industrial strains to be used in processes in which yeast is explored as cell factory.     

Evaluation of the bioconversion of genetically modified switchgrass using simultaneous saccharification and fermentation and a consolidated bioprocessing approach

Background

In mixed sugar fermentations with recombinant Saccharomyces cerevisiae strains able to ferment D-xylose and L-arabinose the pentose sugars are normally only utilized after depletion of D-glucose. This has been attributed to competitive inhibition of pentose uptake by D-glucose as pentose sugars are taken up into yeast cells by individual members of the yeast hexose transporter family. We wanted to investigate whether D-glucose inhibits pentose utilization only by blocking its uptake or also by interfering with its further metabolism.

Results

To distinguish between inhibitory effects of D-glucose on pentose uptake and pentose catabolism, maltose was used as an alternative carbon source in maltose-pentose co-consumption experiments. Maltose is taken up by a specific maltose transport system and hydrolyzed only intracellularly into two D-glucose molecules. Pentose consumption decreased by about 20 - 30% during the simultaneous utilization of maltose indicating that hexose catabolism can impede pentose utilization. To test whether intracellular D-glucose might impair pentose utilization, hexo-/glucokinase deletion mutants were constructed. Those mutants are known to accumulate intracellular D-glucose when incubated with maltose. However, pentose utilization was not effected in the presence of maltose. Addition of increasing concentrations of D-glucose to the hexo-/glucokinase mutants finally completely blocked D-xylose as well as L-arabinose consumption, indicating a pronounced inhibitory effect of D-glucose on pentose uptake. Nevertheless, constitutive overexpression of pentose-transporting hexose transporters like Hxt7 and Gal2 could improve pentose consumption in the presence of D-glucose.

Conclusion

Our results confirm that D-glucose impairs the simultaneous utilization of pentoses mainly due to inhibition of pentose uptake. Whereas intracellular D-glucose does not seem to have an inhibitory effect on pentose utilization, further catabolism of D-glucose can also impede pentose utilization. Nevertheless, the results suggest that co-fermentation of pentoses in the presence of D-glucose can significantly be improved by the overexpression of pentose transporters, especially if they are not inhibited by D-glucose.     

Towards industrial pentose-fermenting yeast strains

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.     

Enhanced ethanol production from industrial lignocellulose hydrolysates by a hydrolysate-cofermenting Saccharomyces cerevisiae strain

Industrial production of lignocellulosic ethanol requires a microorganism utilizing both hexose and pentose, and tolerating inhibitors. In this study, a hydrolysate-cofermenting Saccharomyces cerevisiae strain was obtained through one step in vivo DNA assembly of pentose-metabolizing pathway genes, followed by consecutive adaptive evolution in pentose media containing acetic acid, and direct screening in biomass hydrolysate media. The strain was able to coferment glucose and xylose in synthetic media with the respective maximal specific rates of glucose and xylose consumption, and ethanol production of 3.47, 0.38 and 1.62 g/g DW/h, with an ethanol titre of 41.07 g/L and yield of 0.42 g/g. Industrial wheat straw hydrolysate fermentation resulted in maximal specific rates of glucose and xylose consumption, and ethanol production of 2.61, 0.54 and 1.38 g/g DW/h, respectively, with an ethanol titre of 54.11 g/L and yield of 0.44 g/g. These are among the best for wheat straw hydrolysate fermentation through separate hydrolysis and cofermentation.

     

Butanol production by Clostridium beijerinckii ATCC 55025 from wheat bran

Wheat bran, a by-product of the wheat milling industry, consists mainly of hemicellulose, starch and protein. In this study, the hydrolysate of wheat bran pretreated with dilute sulfuric acid was used as a substrate to produce ABE (acetone, butanol and ethanol) using Clostridium beijerinckii ATCC 55025. The wheat bran hydrolysate contained 53.1 g/l total reducing sugars, including 21.3 g/l of glucose, 17.4 g/l of xylose and 10.6 g/l of arabinose. C. beijerinckii ATCC 55025 can utilize hexose and pentose simultaneously in the hydrolysate to produce ABE. After 72 h of fermentation, the total ABE in the system was 11.8 g/l, of which acetone, butanol and ethanol were 2.2, 8.8 and 0.8 g/l, respectively. The fermentation resulted in an ABE yield of 0.32 and productivity of 0.16 g l⁻¹ h⁻¹. This study suggests that wheat bran can be a potential renewable resource for ABE fermentation.     

Ethanol and sugar tolerance of Candida shehatae

Summary Candida shehatae ATCC 22984 fermented solutions of up to 260 g/L sugars derived by hydrolysis of whole barley. These solutions contained hexose: pentose 7030, the hexose being mainly glucose from the barley starch and the pentose being mainly xylose. At sugar concentrations of 180 g/L, fermentation was complete in 72 h, yielding 84 g/L ethanol, 0.47 g ethanol/g sugar. At 260 g/L, fermentation ceased when ethanol concentration reached 100 g/L, but resumed when the ethanol was removed by vacuum distillation, to yield finally 0.50 g ethanol/g sugar.     

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.     

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

Background  

Simultaneous saccharification and fermentation (SSF) is a promising process option for ethanol production from lignocellulosic materials. However, both the overall ethanol yield and the final ethanol concentration in the fermentation broth must be high. Hence, almost complete conversion of both hexoses and pentoses must be achieved in SSF at a high solid content. A principal difficulty is to obtain an efficient pentose uptake in the presence of high glucose and inhibitor concentrations. Initial glucose present in pretreated spruce decreases the xylose utilization by yeast, due to competitive inhibition of sugar transport. In the current work, prefermentation was studied as a possible means to overcome the problem of competitive inhibition. The free hexoses, initially present in the slurry, were in these experiments fermented before adding the enzymes, thereby lowering the glucose concentration.     

Fermentation of corn fiber hydrolysate to lactic acid by the moderate thermophile <Emphasis Type="Italic">Bacillus coagulans</Emphasis>

A strain of Bacillus coagulans that converted mixed sugars of glucose, xylose, and arabinose to l-lactic acid with 85% yield at 50°C was isolated from composted dairy manure. The strain was tolerant to aldehyde growth inhibitors at 2.5 g furfural/l, 2.5 g 5-hydroxymethylfurfural/l, 2.5 g vanillin/l, and 1.2 g p-hydroxybenzaldehyde/l. In a simultaneous saccharification and fermentation process, the strain converted a dilute-acid hydrolysate of 100 g corn fiber/l to 39 g lactic acid/l in 72 h at 50°C. Because of its inhibitor tolerance and ability to fully utilize pentose sugars, this strain has potential to be developed as a biocatalyst for the conversion of agricultural residues into valuable chemicals.     

A genetic overhaul of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> 424A(LNH-ST) to improve xylose fermentation

Robust microorganisms are necessary for economical bioethanol production. However, such organisms must be able to effectively ferment both hexose and pentose sugars present in lignocellulosic hydrolysate to ethanol. Wild type Saccharomyces cerevisiae can rapidly ferment hexose, but cannot ferment pentose sugars. Considerable efforts were made to genetically engineer S. cerevisiae to ferment xylose. Our genetically engineered S cerevisiae yeast, 424A(LNH-ST), expresses NADPH/NADH xylose reductase (XR) that prefer NADPH and NAD⁺-dependent xylitol dehydrogenase (XD) from Pichia stipitis, and overexpresses endogenous xylulokinase (XK). This strain is able to ferment glucose and xylose, as well as other hexose sugars, to ethanol. However, the preference for different cofactors by XR and XD might lead to redox imbalance, xylitol excretion, and thus might reduce ethanol yield and productivity. In the present study, genes responsible for the conversion of xylose to xylulose with different cofactor specificity (1) XR from N. crassa (NADPH-dependent) and C. parapsilosis (NADH-dependent), and (2) mutant XD from P. stipitis (containing three mutations D207A/I208R/F209S) were overexpressed in wild type yeast. To increase the NADPH pool, the fungal GAPDH enzyme from Kluyveromyces lactis was overexpressed in the 424A(LNH-ST) strain. Four pentose phosphate pathway (PPP) genes, TKL1, TAL1, RKI1 and RPE1 from S. cerevisiae, were also overexpressed in 424A(LNH-ST). Overexpression of GAPDH lowered xylitol production by more than 40%. However, other strains carrying different combinations of XR and XD, as well as new strains containing the overexpressed PPP genes, did not yield any significant improvement in xylose fermentation.     

Production of ethanol and n-butanol from hexose/pentose mixtures using consecutive fermentations with Saccharomyces cerevisiae and Clostridium acetobutylicum

Summary Saccharomyces cerevisiae and Clostridium acetobutylicum have been used to consecutively ferment molasses (5% solids) containing added pentose sugars (30 g/l). Butanol concentrations of 6.6 g/l and 3.7 g/l have been achieved from L-arabinose and D-xylose, respectively, in the presence of 22 g/l of ethanol.