Conversion of pentoses to ethanol by yeasts and fungi

Fermentation of D-xylose is of interest in enhancing the yield of ethanol obtainable from lignocellulosic hydrolysates. Such hydrolysates can contain both pentoses and hexoses, and while technology to convert hexoses to ethanol is well established, the fermentation of pentoses had been problematical. To overcome the difficulty, yeasts and fungi have been sought and identified in recent years that can convert D-xylose into ethanol. However, operation of their cultures in the presence of the pentose to obtain rapid and efficient ethanol production is somewhat more complex than in the archetype alcoholic fermentation, Saccharomyces cerevisiae on D-glucose. The complexity stems, in part, from the association of ethanol accumulation in cultures where D-xylose is the sole carbon source with conditions that limit growth, by oxygen in particular, although limitation by other nutrients might also be implicated. Aspects of screening for appropriate organisms and of the parameters that play a role in determining culture variables, especially those associated with ethanol productivity, are reviewed. Performance with D-xylose as sole carbon source, in sugar mixtures, and in lignocellulosic hydrolysates is discussed. A model that involves biochemical considerations of D-xylose metabolism is presented that rationalizes the effects of oxygen on cultures where D-xylose is the sole carbon source, notably effects of the specific rate of oxygen use on the rate and extent of ethanol accumulation. Alternate methods to direct fermentation of D-xylose have been developed that depend on its prior isomerization to D-xylose, followed by fermentation of the pentulose by certain yeasts and fungi. Factors involved in the biochemistry, use, and performance of these methods, which with some organisms involves sensitivity to oxygen, are reviewed.     

Confirmation and elimination of xylose metabolism bottlenecks in glucose phosphoenolpyruvate-dependent phosphotransferase system-deficient Clostridium acetobutylicum for simultaneous utilization of glucose, xylose, and arabinose

Efficient cofermentation of D-glucose, D-xylose, and L-arabinose, three major sugars present in lignocellulose, is a fundamental requirement for cost-effective utilization of lignocellulosic biomass. The Gram-positive anaerobic bacterium Clostridium acetobutylicum, known for its excellent capability of producing ABE (acetone, butanol, and ethanol) solvent, is limited in using lignocellulose because of inefficient pentose consumption when fermenting sugar mixtures. To overcome this substrate utilization defect, a predicted glcG gene, encoding enzyme II of the D-glucose phosphoenolpyruvate-dependent phosphotransferase system (PTS), was first disrupted in the ABE-producing model strain Clostridium acetobutylicum ATCC 824, resulting in greatly improved D-xylose and L-arabinose consumption in the presence of D-glucose. Interestingly, despite the loss of GlcG, the resulting mutant strain 824glcG fermented D-glucose as efficiently as did the parent strain. This could be attributed to residual glucose PTS activity, although an increased activity of glucose kinase suggested that non-PTS glucose uptake might also be elevated as a result of glcG disruption. Furthermore, the inherent rate-limiting steps of the D-xylose metabolic pathway were observed prior to the pentose phosphate pathway (PPP) in strain ATCC 824 and then overcome by co-overexpression of the D-xylose proton-symporter (cac1345), D-xylose isomerase (cac2610), and xylulokinase (cac2612). As a result, an engineered strain (824glcG-TBA), obtained by integrating glcG disruption and genetic overexpression of the xylose pathway, was able to efficiently coferment mixtures of D-glucose, D-xylose, and L-arabinose, reaching a 24% higher ABE solvent titer (16.06 g/liter) and a 5% higher yield (0.28 g/g) compared to those of the wild-type strain. This strain will be a promising platform host toward commercial exploitation of lignocellulose to produce solvents and biofuels.     

Sequential utilization of mixed monosaccharides by yeasts

Four yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilus, and Rhodotorula toruloides) were tested for their ability to grow and consume D-glucose, D-xylose, D-xylulose, and D-xylitol. Sequential utilization of substrates was observed when D-glucose as mixed with D-xylulose as the carbon source. Catabolite inhibition was tentatively concluded to be responsible for this regulatory mechanism. D-Glucose was also found to inhibit the utilization of D-xylose and D-xylitol in C. utilus and R. toruloides. D-Xylose, D-xylitol, and D-xylulose were consumed simultaneously by R. toruloides and C. utilus.     

Sequential utilization of mixed monosaccharides by yeasts.

Four yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilus, and Rhodotorula toruloides) were tested for their ability to grow and consume D-glucose, D-xylose, D-xylulose, and D-xylitol. Sequential utilization of substrates was observed when D-glucose as mixed with D-xylulose as the carbon source. Catabolite inhibition was tentatively concluded to be responsible for this regulatory mechanism. D-Glucose was also found to inhibit the utilization of D-xylose and D-xylitol in C. utilus and R. toruloides. D-Xylose, D-xylitol, and D-xylulose were consumed simultaneously by R. toruloides and C. utilus.     

Quantitative production of xylitol from D-xylose by a high-xylitol producing yeast mutant Candida tropicalis HXP2

Summary Xylitol was produced as a metabolic by-product by a number of yeasts when grown on medium containing D-xylose as carbon and energy sources. Among the yeast strains tested, a mutant strain of Candida tropicalis (HXP2) was found to produce xylitol from D-xylose with a high yield (>90%). Ethanol was also produced by HXP2 when D-glucose, D-fructose, or sucrose were used as substrates. The high-xylitol-producing yeast mutant is a good organism for the production of xylitol from biomass that contains D-xylose.     

Engineered Pseudomonas putida simultaneously catabolizes five major components of corn stover lignocellulose: Glucose,xylose, arabinose,p-coumaric acid,and acetic acid

Valorization of all major lignocellulose components, including lignin, cellulose, and hemicellulose is critical for an economically viable bioeconomy. In most biochemical conversion approaches, the standard process separately upgrades sugar hydrolysates and lignin. Here, we present a new process concept based on an engineered microbe that could enable simultaneous upgrading of all lignocellulose streams, which has the ultimate potential to reduce capital cost and enable new metabolic engineering strategies. Pseudomonas putida is a robust microorganism capable of natively catabolizing aromatics, organic acids, and D-glucose. We engineered this strain to utilize D-xylose by tuning expression of a heterologous D-xylose transporter, catabolic genes xylAB, and pentose phosphate pathway (PPP) genes tal-tkt. We further engineered L-arabinose utilization via the PPP or an oxidative pathway. This resulted in a growth rate on xylose and arabinose of 0.32 h⁻¹ and 0.38 h⁻¹, respectively. Using the oxidative L-arabinose pathway with the PPP xylose pathway enabled D-glucose, D-xylose, and L-arabinose co-utilization in minimal medium using model compounds as well as real corn stover hydrolysate, with a maximum hydrolysate sugar consumption rate of 3.3 g/L/h. After modifying catabolite repression, our engineered P. putida simultaneously co-utilized five representative compounds from cellulose (D-glucose), hemicellulose (D-xylose, L-arabinose, and acetic acid), and lignin-related compounds (p-coumarate), demonstrating the feasibility of simultaneously upgrading total lignocellulosic biomass to value-added chemicals.     

Genome-scale consequences of cofactor balancing in engineered pentose utilization pathways in Saccharomyces cerevisiae

Biofuels derived from lignocellulosic biomass offer promising alternative renewable energy sources for transportation fuels. Significant effort has been made to engineer Saccharomyces cerevisiae to efficiently ferment pentose sugars such as D-xylose and L-arabinose into biofuels such as ethanol through heterologous expression of the fungal D-xylose and L-arabinose pathways. However, one of the major bottlenecks in these fungal pathways is that the cofactors are not balanced, which contributes to inefficient utilization of pentose sugars. We utilized a genome-scale model of S. cerevisiae to predict the maximal achievable growth rate for cofactor balanced and imbalanced D-xylose and L-arabinose utilization pathways. Dynamic flux balance analysis (DFBA) was used to simulate batch fermentation of glucose, D-xylose, and L-arabinose. The dynamic models and experimental results are in good agreement for the wild type and for the engineered D-xylose utilization pathway. Cofactor balancing the engineered D-xylose and L-arabinose utilization pathways simulated an increase in ethanol batch production of 24.7% while simultaneously reducing the predicted substrate utilization time by 70%. Furthermore, the effects of cofactor balancing the engineered pentose utilization pathways were evaluated throughout the genome-scale metabolic network. This work not only provides new insights to the global network effects of cofactor balancing but also provides useful guidelines for engineering a recombinant yeast strain with cofactor balanced engineered pathways that efficiently co-utilizes pentose and hexose sugars for biofuels production. Experimental switching of cofactor usage in enzymes has been demonstrated, but is a time-consuming effort. Therefore, systems biology models that can predict the likely outcome of such strain engineering efforts are highly useful for motivating which efforts are likely to be worth the significant time investment.     

Engineering of an endogenous hexose transporter into a specific D-xylose transporter facilitates glucose-xylose co-consumption in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background

Engineering of Saccharomyces cerevisiae for the simultaneous utilization of hexose and pentose sugars is vital for cost-efficient cellulosic bioethanol production. This yeast lacks specific pentose transporters and depends on endogenous hexose transporters for low affinity pentose uptake. Consequently, engineered xylose-fermenting yeast strains first utilize D-glucose before D-xylose can be transported and metabolized.

Results

We have used an evolutionary engineering approach that depends on a quadruple hexokinase deletion xylose-fermenting S. cerevisiae strain to select for growth on D-xylose in the presence of high D-glucose concentrations. This resulted in D-glucose-tolerant growth of the yeast of D-xylose. This could be attributed to mutations at N367 in the endogenous chimeric Hxt36 transporter, causing a defect in D-glucose transport while still allowing specific uptake of D-xylose. The Hxt36-N367A variant transports D-xylose with a high rate and improved affinity, enabling the efficient co-consumption of D-glucose and D-xylose.

Conclusions

Engineering of yeast endogenous hexose transporters provides an effective strategy to construct glucose-insensitive xylose transporters that are well integrated in the carbon metabolism regulatory network, and that can be used for efficient lignocellulosic bioethanol production.

     

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.     

广谱碳源产油酵母菌的筛选

对10株酵母菌利用不同单糖为碳源条件下菌体内积累油脂的能力进行了初步考察,并对菌油进行了分离和脂肪酸组成分析。实验发现,以葡萄糖为唯一碳源时有9株菌油脂含量超过自身细胞干重的20%,可以界定为产油微生物。其中6#菌(T.cutaneumAS2.571)利用葡萄糖发酵菌体油脂含量达到65%(W/W)。所有实验菌株都能同化多种单糖,其中1#菌(L.starkeyiAS2.1390)、4#菌(R.toruloidesAS2.1389)和11#菌(L.starkeyiAS2.1608)表现出对碳源利用的广谱性,能转化五碳糖木糖和阿拉伯糖并在菌体内积累油脂,油脂含量最高达到26%。脂肪酸组成分析结果表明,菌油富含饱和及低度不饱和长链脂肪酸,其中棕榈酸、油酸和亚油酸三者之和占总脂肪酸组成的90%以上,脂肪酸组成分布类似于常见的植物油。这些结果对利用产油微生物转化木质纤维素水解混合糖获取油脂资源的研究具有重要意义。     

Specific features of fermentation of D-xylose and D-glucose by xylose-assimilating yeasts

The ability to assimilate D-glucose and D-xylose was studied in 21 yeast species of the following genera: Candida, Kluyveromyces, Pachysolen, Pichia, and Torulopsis. All the cultures fermented D-glucose with the formation of ethanol. During the assimilation of D-xylose, ethanol was produced by P. stipitis and C. shehatae, whereas xylitol was produced by C. didensiae, C. intermediae, C. parapsilosis, C. silvanorum, C. tropicalis, K. fragilis, K. marxianus, P. guillermondii, and T. molishiama. The yeast P. tannophilus produced comparable amounts of both alcohols. The possible use of xylose-assimilating yeasts for the production of xylitol and ethanol is discussed.     

Comparative study of the fermentation of D-glucose/D-xylose mixtures with Pachysolen tannophilus and Candida shehatae

We have performed a comparative analysis of the fermentation of the solutions of the mixtures of D-glucose and D-xylose with the yeasts Pachysolen tannophilus (ATCC 32691) and Candida shehatae (ATCC 34887), with the aim of producing bioethanol. All the experiments were performed in a batch bioreactor, with a constant aeration level, temperature of 30v°C, and a culture medium with an initial pH of 4.5. For both yeasts, the comparison was established on the basis of the following parameters: maximum specific growth rate, biomass productivity, specific rate of substrate consumption (q_s) and of ethanol production (q_E), and overall ethanol and xylitol yields. For the calculation of the specific rates of substrate consumption and ethanol production, differential and integral methods were applied to the kinetic data. From the experimental results, it is deduced that both Candida and Pachysolen sequentially consume the two substrates, first D-glucose and then D-xylose. In both yeasts, the specific substrate-consumption rate diminished over each culture. The values q_s and q_E proved higher in Candida, although the higher ethanol yield was of the same order for both yeasts, close to 0.4 kg kg^у.     

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.     

Conversion of pentoses by yeasts

The utilization and conversion of D-xylose, D-xylulose, L-arabinose, and xylitol by yeast strains have been investigated with the following results: (1) The majority of yeasts tested utilize D-xylose and produce polyols, ethanol, and organic acids. The type and amount of products formed varies with the yeast strains used. The most commonly detected product is xylitol. (2)The majority of yeasts tested utilize D-xylulose aerobically and fermentatively to produce ethanol, xylitol, D-arabitol, and organic acids. The type and amount of products varies depending upon the yeast strains used. (3) Xylitol is a poor carbon and energy source for most yeasts tested. Some yeast strains produce small amounts of ethanol from xylitol. (4) Most yeast strains utilize L-arabinose, and L-arabitol is the common product. Small amounts of ethanol are also produced by some yeast strains. (5) Of the four substrates examined, D-xylulose was the perferred substrate, followed by D-xylose, L-arabinose, and xylitol. (6) Mutant yeast strains that exhibit different metabolic product patterns can be induced and isolated from Candida sp. Saccharomyces cerevisiae, and other yeasts. These mutant strains can be used for ethanol production from D-xylose as well as for the study of metabolic regulation of pentose utilization in yeasts.     

D-Glucose Isomerase: Constitutive and Catabolite Repression-Resistant Mutants of Streptomyces phaeochromogenes

As in other Streptomyces species, the enzymatic conversion of D-glucose to D-fructose is carried out in Streptomyces phaeochromogenes NRRL B-3559 by the inducible enzyme, D-xylose keto isomerase (EC 5.3.1.5). Mutants of this microorganism were selected for their ability to grow on D-lyxose (2-epimer of D-xlose). As a result of the mutational event, the microorganism constitutively produced D-xylose isomerase. As in the parent strain, the constitutive formation of the isomerase was repressed by D-glucose. The fact that this mutant was unable to grow in low D-xylose concentrations in the presence of the D-glucose analogue, 3-O-methylglucose, permitted the isolation of D-xylose isomerase constitutive mutants which were insensitive to D-glucose repression.     

D-xylose utilization by Saccharomyces cerevisiae

Although it is generally accepted that Saccharomyces cerevisiae is unable to assimilate D-xylose, four strains were found to utilize xylose aerobically at different efficiencies in the presence of a mixture of substrates. The degree of D-xylose utilization by S. cerevisiae ATCC 26602 depended upon the presence of other substrates or yeast extract. The greatest amount of xylose (up to 69% over 7 d) was utilized when sugar substrates such as D-ribose were co-metabolized. Much lower degrees of utilization occurred with co-metabolism of organic acids, polyols or ethanol. A mixture of D-glucose, D-ribose, D-raffinose, glycerol and D-xylose resulted in greater xylose utilization than the presence of a single substrate and xylose. The absence of growth on a co-substrate alone did not prevent the utilization of xylose in its presence. Xylose was co-metabolized with ribose under anaerobic conditions but at a much slower rate than under aerobic conditions. When [14C]xylose was utilized in the presence of ribose under anaerobic conditions, the radioactive label was detected mainly in xylitol and not in the small amounts of ethanol produced. Under aerobic conditions the radioactive label was distributed between xylitol (91.3 +/- 0.8%), CO2 (2.6 +/- 2.3%) and biomass (1.7 +/- 0.6%). No other metabolic products were detected. Whereas most xylose was dissimilated rather than assimilated by S. cerevisiae, the organism apparently possesses a pathway which completely oxidizes xylose in the presence of another substrate.     

Fusarium oxysporum: status in bioethanol production

Fermentation of lignocellulosic materials to ethanol and other solvents provides an alternative way of treating wastes and producing chemical feedstocks and fuel additives. Considerable efforts have been made in past 10 years to improve the process based on lignocellulosic biomass and hydrolysate that contains a complex mixture of sugars, decomposition products of sugars, and sometimes the inhibitory levels of soluble lignin. Despite the relative abundance of D-xylose in crop and forest residues it has not been found efficiently fermentable by most of the microorganisms. Recent research has revealed that D-xylose may be fermented to ethanol and organic acids. Recently, several strains of Fusarium oxysporum have been found to have potential for converting not only D-xylose, but also cellulose to ethanol in a one-step process. Distinguishing features of F. oxysporum for ethanol production in comparison to other organisms are identified. These include the advantage of in situ cellulase production and cellulose fermentation, pentose fermentation, and the tolerance of sugars and ethanol. The main disadvantage is the slow conversion rate when compared with yeast.     

Comparative kinetics of D-xylose and D-glucose isomerase activities of the D-xylose isomerase from Thermus aquaticus HB8

The D-xylose isomerase from T. aquaticus accepts, besides D-xylose, also D-glucose, and, with lower efficiency, D-ribose, and D-arabinose as alternative substrates. The activity of the enzyme is strictly dependent on divalent cations. Mn2+ is most effective in the D-xylose isomerase reaction and Co2+ in the D-glucose isomerization. Mg2+ is active in both reactions, Zn2+ only in the further one. The enzyme is strongly inhibited by Cu2+, and weakly by Ni2+, Fe2+, and Ca2+. A hyperbolic dependence of the reaction velocity of the D-xylose isomerase on the concentration of D-xylose xylose and of D-glucose was found, while biphasic saturation curves were obtained by variation of the metal ion concentrations. The D-glucose isomerization reaction shows normal behaviour with respect to the metal ions. A kinetic model was derived on the basis of the assumption of two binding sites for divalent cations, one cofactor site with higher affinity and a second, low affinity site, which modulates the activity of the enzyme.     

Fermentation of D-xylose, xylitol, and D-xylulose by yeasts

Fifteen yeasts which can assimilate D-xylose were examined for the ability to convert this pentose to ethanol. In six of the seven genera investigated the conversion was enhanced when air had access to the medium. Therefore, the ability to convert D-xylose to ethanol under these conditions is probably common among yeasts. Growth under the same conditions on xylitol, a putative catabolite of D-xylose, led to only traces of ethanol. The effects of growth on another putative catabolite, D-xylose, were complex, but some of the strains which were among the better producers of ethanol from D-xylose produced less from D-xylulose.     

Microbial conversion of xylose into useful bioproducts

Microorganisms can produce a number of different bioproducts from the sugars in plant biomass. One challenge is devising processes that utilize all of the sugars in lignocellulosic hydrolysates. D-xylose is the second most abundant sugar in these hydrolysates. The microbial conversion of D-xylose to ethanol has been studied extensively; only recently, however, has conversion to bioproducts other than ethanol been explored. Moreover, in the case of yeast, D-xylose may provide a better feedstock for the production of bioproducts other than ethanol, because the relevant pathways are not subject to glucose-dependent repression. In this review, we discuss how different microorganisms are being used to produce novel bioproducts from D-xylose. We also discuss how D-xylose could be potentially used instead of glucose for the production of value-added bioproducts.