Strain selection, taxonomy, and genetics of xylose-fermenting yeasts

Transcription

1 Strain selection, taxonomy, and genetics of xylose-fermenting yeasts T. W. Jeffries* and C. P. Kurtzman *Forest Products Laboratory, US Department of Agriculture, Forest Service, Madison, Wisconsin, USA National Center for Agricultural Utilization Research, US Department of Agriculture, Agricultural Research Service, Peoria, Illinois, USA Xylose utilization is essential for the effcient conversion of lignocellulose to ethanol. The objective of this review is to trace the development of xylose-fermenting yeast strains from their discovery in Following initial reports, screens of known yeasts identified five species of interest: Candida shehatae, Candida tenuis, Pachysolen tannophilus, Pichia segobiensis, and Pichia stipitis. Candida shehatae strains can be divided into three varieties. Pachysolen tannophilus and Pichia stipitis have been studied most extensively and have the best-understood genetic systems. Improved mutants of P. tannophilis have been obtained by selecting for an inability to oxidize ethanol (eth) and for rapid growth on xylitol and nitrate. Improved P. stipitis mutants have been obtained by selecting for flocculation, decreased utilization of glucose, and growth on noninductive carbon sources. Bacterial xylose isomerase has been cloned and expressed in S. cerevisiae and Schizosaccharomyces pombe, but the heterologous enzyme is inactive. Xylose reductase and xylitol dehydrogenase have been cloned from P. stipitis and expressed in Saccharomyces cerevisiae, giving rise to transformant S. cerevisiae that grow on xylose but that ferment it poorly. A transformation and expression system based on the URA3 marker has recently been developed for P. stipitis so that contemporary genetic methods may be brought to bear on this organism. Keywords: Xylose; strain selection; taxonomy; genetics; yeast; fermentation Introduction Ethanol is an excellent transportation fuel, and its use could help avoid accumulation of carbon dioxide in the atmosphere. 1 2 Ethanol production from grain presently exceeds per year in the United States. 3 The potential for producing ethanol from lignocellulose is much greater. Large quantities of ethanol could be made from waste papers 4 and wood residues. 5 Estimates of biomass available for energy production in the United States range from 18 to 58 kj year By comparison, current U.S. energy consumption for transportation is about 23 kj year - 1, about half of which is derived from imported petroleum. Ethanol production from lignocellulose is limited more by institutional, technical, and economic factors than by the magnitude of the resource. One important technical factor restricting economic exploitation is the fermentation of xylose. A practicable xylose fermentation with acceptable yields and ethanol concentrations could lower the overall cost of ethanol from wood by 25%. 7 Address reprint requests to Dr. Jeffries at the Forest Products Laboratory, US Department of Agriculture, Forest Service, Madison, WI 53705, USA Received 13 September 1993; accepted 25 March 1994 Xylose is a prevalent sugar in both woody and herbaceous plants. Fast-growing hardwoods, such as hybrid poplar and American sycamore, and abundant, poorly utilized species, such as southern red oak, make up the bulk of the biomass resource for ethanol production. Hardwood hemicelluloses (along with most agricultural residues) contain 17% to 26% xylan. 8 By comparison, the cellulose content ranges from about 31% to 49% 9,10 (Table 1). However, composition alone does not indicate the full importance of the xylose component. Dilute acid hydrolysis can recover sugars in 85% yield from hemicellulose, whereas it can recover glucose from cellulose at a yield of only 55%. Depending on the initial composition, after acid hydrolysis, 35% to 50% of the fermentable sugars are obtained from hemicellulose, and in the case of angiosperms, the principal hemicellulosic sugar is xylose. 11 The importance of xylose for ethanol production from wood and pulping wastes has been recognized for many decades. In 1945, researchers at the Forest Products Laboratory selected Douglas fir as a feedstock for acid hydrolysis rather than more abundant and faster-growing hardwood species, largely because technology for fermenting pentose sugars did not exist, and the hemicellulose in Douglas fir consists almost entirely of hexose sugars. 12 Xylose is also a 922 Enzyme Microb. Technol., 1994, vol. 16, November 1994 Butterworth-Heinemann

2 Taxonomy of xylose-fermenting yeasts: T W. Jeffries and C. P. Kurtzman component of spent sulfite waste liquors in the sulfite pulping of hardwoods. Plant operators have produced yeast from spent sulfite liquors for many years, 13 and these feedstocks also have been used for ethanol production. 14 The ability of yeasts to ferment xylose has been recognized only since the early 1980s. The fermentation of xylose to ethanol was first reported by Karczewska in 1959, 15 but this observation went unnoticed until A major review in 1976 recorded that roughly half the known yeast species would assimilate D-xylose, but none would ferment it. 16 In 1979, as interest in ethanol production from wood grew along with bioconversion research, several laboratories began to investigate routes for the utilization of xylose. An initial breakthrough was the discovery that Saccharomyces cerevisiae, Schizosaccharomyces pombe, and some other yeasts could ferment the keto isomer of xylose, xyhdose. 17 This was significant because the commercial enzyme, xylose isomerase ( = glucose isomerase), will produce xylulose from xylose. This approach served as the basis for fermenting xylulose with Saccharomyces cerevisiae, 18,19 Schizosaccharomyces pombe, 20,21 Candida tropicalis, 22 and other yeasts. 23 Although this approach is technically feasible, incompatible temperature and ph optima for enzymatic isomeration and fermentation and the expense of the xylose isomerase have limited its practicability. However, these investigations did lead to several other lines of research. One was the attempt to introduce xylose isomerase into yeast in order to alleviate the oxidation/ reduction steps in xylose assimilation (see following). The other was the investigation of direct xylose fermentations by yeasts under aerobic conditions. At equilibrium, xylose isomerase converts only 17% of xylose into xylulose. Separation of these two sugars can be achieved by different utilization and purification, 24 but this is somewhat difficult, so for practical purposes, the fermentation of xylulose is conducted in the presence of xylose. Researchers in four laboratories discovered the direct conversion of xylose to ethanol almost simultaneously. Two groups observed ethanol production directly from xylose following screens of yeasts for anaerobic xylose metaholism. 25,26 One observation came from mutation and selection studies on a yeast strain known to assimilate xylose, 27 and one came from chance observations of ethanol production from xylose/xylulose mixtures under aerobic conditions. 28 Screens for xylose-fermenting yeasts Following initial observations, scientists in several laboratories screened yeasts for direct conversion of xylose to ethanol. Screening criteria included the abilities to use xylose and ferment glucose, the ethanol formation rate, ethanol yield, and ethanol tolerance. Other researchers screened for the capacities of yeasts to ferment xylan, 29 cellobiose, 30 spent sulfite liquors, 31] or hemicellulose hydrolyzates. 32 Even though the screening criteria were different, many of the same organisms were examined and often the same species came out on top (Table 2). Early comparative studies showed that yeasts capable of metabolizing xylose to ethanol fell into at least two classes: those that were able to produce ethanol under strictly anaerobic conditions, and those that required some oxygen for ethanol production. Pachysolen tannophilus is capable of carrying out a strictly anaerobic fermentation. 25 Enzyme Microb. Technol., 1994, vol. 16, November 923

3 Reviews C. tropicalis, 33 Kluyveromyces marxianus, 34 and many other yeasts, on the other hand, require oxygen. To date, no xylose-fermenting yeast has been shown to be capable of growing under strictly anaerobic conditions. The basis for the oxygen requirement is not fully known. The ability of Saccharomyces cerevisiae to grow anaerobically was recently attributed to a genetically divergent form of dihydroorotate dehydrogenase (DHOdehase), an NAD-dependent enzyme involved in pyrimidine biosynthesis. 35 In Schizosaccharomyces pombe a fermentative, petite-negative yeast incapable of anaerobic growth DHOdehase is localized in the mitochondrion where its activity is necessarily coupled to an intact electron transport chain. In S. cerevisiae, DHOdehase is cytosolic and is coupled to the reduction of fumarate to succinate. It is possible that the corresponding enzymes in known xylose-fementingyeasts are similar to the DHOdehase of S. pombe. The oxygen requirement for ethanol production appears to be attributable to factors such as the cofactor specificity of xylose reductase. 36 The requirement varies greatly among species and even with different strains within species. Product formation rates and yields shift with aeration, but the best fermentative oiganisms typically exhibit low oxygen requirements. Oxygen requirements for ethanol production are discussed extensively elsewhere in this series. Pachysolen tannophilus was the model organism for process development, mutagenesis, and strain selection studies for several years, but broad screening of yeast isolates showed that other species were better fermenters. In 1983, du Preez and van der Walt 37 first described Candida shehatae as a xylose-fermenting yeast. This organism produces ethanol at a higher rate and in better yield than does P. tannophilus. As such, it represented a major step forward in strain selection studies. Toivola et al. 38 were the first to publish results from a wide-ranging screen for xylosefermenting yeasts. They investigated more than 200 type species capable of both xylose utilization and glucose fermentation by inoculating Durham tubes of 1% yeast extract, 2% xylose with aerobically grown cells. Greater gas accumulation was accompanied by more ethanol production, but in some cases, ethanol production was detected by chromatography even though no gas accumulated. Yeast species exhibiting significant ethanol production included C. shehatae, Pichia stipitis, Pichia segobiensis, P. tannophilus, Candida tenuis, and Bretannomyces naardenensis. Du Preez and Prior 39 screened 56 yeast isolates and identified. stipitis and C. shehatae as the best species for xylose fermentation. Baraniak 40 had similar findings in a subsequent, more limited screen, and van der Walt et al. 41 later identified a novel xylose-fermenting species, Candida lyxosophila, from woodland soil isolates. Xylose is not the only sugar of interest in biomass conversion; cellobiose is the principal sugar obtained from enzymatic hydrolyzates of cellulose. Fermentation of cellobiose can speed up enzymatic saccharification and improve the overall process, so direct fermentation of these substrates is desirable. 42 Morikawa et al. 30 screened 213 species of yeasts for their abilities to ferment both xylose and cellobiose and identified Kluyveromyces cellobiovorus, a single strain belonging to a new species, as capable of fermenting both sugars. Lee et al. 29 screened more than 250 strains of yeasts for xylanase activities and their abilities to ferment xylan. Only 19 possessed xylanase activity. These consist of species within the genus Cyptococcus plus strains of P. stipitis and C. shehatae. Of the xylanase-positive strains, three also possessed cellulase activity, and two P. stipitis strains fermented xylan into ethanol. Further development of naturally occurring xylan-fermenting yeasts has not been reported. Strains of P. stipitis and C. shehatae differ considerably in their relative capacities to ferment xylose. Slininiger et al. 43 measured the abilities of three strains each of P. stipitis and C. shehatae and one strain of P. tannophilus at four different xylose concentrations ranging up to 20%. Of these organisms, P. stipitis NRRL Y-7124 formed the highest ethanol concentration (4.5%) while consuming the most xylose, but strains of C. shehatae fermented more rapidly. Maximum ethanol yields (0.42 to 0.43 g ethanol g -1 xylose consumed) were about the same for both P. stipitis and C. shehatae, and they were considerably greater than the maximum observed yield for P. tannophilus (0.21 g g -1 ). Du Preez et al. 44 compared the performance of P. stipitis CSIR-Y633 and C. shehatae Y-492 under conditions approximating optima for each strain. For both strains, the optimum ph occurred 924 Enzyme Microb. Technol., 1994, vol. 16, November

4 between 4 and 5.5, and the optimum temperature was 30 C. Both strains showed maximum volumetric ethanol productivities of about 0.9 g (l h) -1, but the maximum specific productivity for C. shehatae (0.48 g (l h) -1 g -l biomass) was about a third higher than for P stipitis (0.3 g (l h) -1 g -1 biomass). While the ethanol yield of C. shehatae was about 75% of theoretical, the maximal yields for P. stipitis were between 85% and 90%. Detailed physiological, biochemical, 45 and bioprocess engineering 46 studies of these organisms are presented elsewhere in this series. Taxonomy of xylose-fermenting yeasts: T W. Jeffries and C. P. Kurtzman Taxonomy and life cycles of major D-xylose-fermenting yeasts The discussion in this section focuses on the five yeasts identified by Toivola et al. 38 that produce 2% or more ethanol from D-xylose (Table 2). Species producing less ethanol from D-xylose will receive no further comment other than to note that they represent a phylogenetically diverse group of taxa. Candida shehatae By virtue of their placement in Candida, species assigned to this anamorphic or imperfect genus are not known to have a sexual state. The genus Candida is composed of about 200 species and is restricted to taxa that have multilateral budding and are ascomycetes. Occasionally, sexual states are found among Candida species, but reassignment of these species to a variety of different teleomorphic or perfect genera serves to emphasize the phylogenetic diversity of species assigned to Candida. Candida shehatae shows considerable phenotypic similarity to P. stipitis, and it has been suggested that the two might represent anamorphic and teleomorphic states of the same species. 47 However, Vaughan Martini 48 showed that there was little DNA relatedness between type strains of the two taxa and that they represent separate biological species. Kurtzman 49 examined the extent of genetic divergence among strains of C. shehatae from measurements of nuclear DNA relatedness. Surprisingly, the five strains compared represented three genetically divergent groups, each of which showed about 50%. DNA relatedness with the other groups (Figure 1). The amount of divergence observed suggested that members of the three groups were either varieties of C. shehatae or perhaps even genetically distinct species. Because comparisons of interfertility could not be made to address this question, varietal designations were assigned. The three varieties can be separated from each other and from other D-xylose-fermenting yeasts by the characteristics given in Table 2. The species reproduces vegetatively by multilateral budding, i.e., by the same method as observed in S. cerevisiae. Additionally, pseudohyphae are often formed. Candida tenuis On the basis of the assimilation and fermentation tests used for characterization of yeasts, C. tenuis is indistinguishable from P. stipitis. Nevertheless, comparisons of DNA complementarily show that the two taxa are different species. 46 Separation of the two species can be made from the presence of ascospore formation by P. stipitis. As with P. stipitis, C. tenuis reproduces vegetatively by multilateral budding and the formation of pseudohyphae. Pachysolen tannophilus Pachysolen tannophilus is one of the most unusual yeasts because ascus formation begins with the growth of a refractive tube from a vegetative cell and terminates when a single ascus is produced on the tip of the tube. The tube can be considered an ascospore. At maturity, the ascus wall deliquesces, releasing two to four hat-shaped ascospores. Cultures derived from single ascospores are sporogenous, indicating the species to be homothallic. 50 The vegetative cells producing ascospores often show prior conjugation with another cell. Wickerham 50 suggested that unconjugated cells that produce ascospores are diploid, but that diploidy also occurs when two haploid cells fuse. Following ascospore development, the diploid nucleus proceeds directly to meiosis and the production of a four-spored ascus. Consequently, even though the species is homothallic, a mating system approximating heterothallism can be developed. James and Zahab 51,52 and Maleszka et al. 53 used this approach to develop a genetic system for P. tannophilus. Their work showed that certain polyploid and aneuploid selections gave a greater fermentation of D-xylose. Cultures of P. tannophilus are often mucoid and have a faint smell of esters. Vegetative reproduction is by multilateral budding and formation of pseudohyphae. Pichia segobiensis Pichia segobiensis was detected as a significant D-xylose fermenter by Toivola et al., 38 but it seems to have been ignored by biotechnologists. Its growth reactions on various carbohydrates are similar to those of P. stipitis. Ascosporulation is often preceded by conjugation between independent cells or between a parent cell and its bud. Asci deliquesced at maturity and usually contain two hat-shaped ascospores. The species is presumed to be homothallic because parentbud conjugation can lead to ascus formation. 54 Vegetative reproduction is by multilateral budding and formation of pseudohyphae. Enzyme Microb. Technol., 1994, vol. 16, November 925

5 Reviews Pichia stipitis As with P. segobiensis, ascosporulation in P. stpitis is usually preceded by conjugation between independent cells or between a parent cell and its bud. At maturity, the deliquescent asci usually contain two hat-shaped ascospores. The species is believed to be homothallic. 47 Vegetative reproduction is by multilateral budding. Occasionally, poorly differentiated pseudohyphae are formed. Passoth et al. 55 determined the electrophoretic banding patterns of the chromosomes of P. stipitis and C. shehatae. Both species exhibited six chromosomal bands, except for one strain of C. shehatae which had only five bands. Strains of the two species showed considerable chromosomal length polymorphisms, but despite this, P. stipitis could be discerned from C. shehatae on the basis of electrokazyotypes. Relationships among species Phenoypic characters The five D-xylose-fermenting yeasts described here are quite similar in their growth profiles on standard carbon and nitrogen assimilation tests. 56,57 All give an ethanolic fermentation of glucose and xylose, and with the exception of P. tannophilus, galactose, sucrose, maltose, trehalose, cellobiose, melezitose, and starch are also fermented by some of the taxa. Key physiological characters for species separation are listed in Table 3. The morphology of vegetative cells is similar for all five species. Each undergoes multilateral budding and each may also form pseudohyphae. In culture, P. tannophilus differs from the others by formation of an extracellular polysaccharide that causes the growth to be mucoid. Molecular relationships In addition to the phenotypic similarity already noted, the Candida and Pichia species have similar G + C contents of nuclear DNA, and all have coenzyme Q-9 (Table 2). This is in contrast to P. tannophilus, which has a lower G + C content and coenzyme Q-8. Ribosomal RNA (rrna) nucleotide sequence divergence was used to estimate genetic distances among some of the D-xylose-fermenting yeasts. 47 Candida shehatae and P. stipitis appear to be only recently diverged, whereas P. tannophilus is more distantly related (Figure 2). Candida tenuis and P. segobiensis have not yet been compared with the other three species by this methodology, but their phenotypic and genotypic characteristics suggest that they may be closely related to P. stipitis and C. shehatae. Mutation and selection studies Candida sp. XF217 and Pachysolen tannophilus Mutation and selection has been used from the outset in developing improved xylose-fermenting yeasts. Gong et al. 23 first reported the development of a xylose-fermenting yeast mutant that produced ethanol from xylose under aerobic and microaerobic conditions. The Candida sp. XF217 strain was not characterized in subsequently published work. In 1984, James and Zahab 51 developed a genetic system for P. tannophilus, a strongly homothallic organism in which the haplophase is predominant. They were able to produce heterozygous diploids by cultivating two different auxotrophic strains together on minimal medium. The frequency of heterozygote formation is about one in 10 6 cells. The resulting diploids exhibit segregation when subjected to tetrad analysis. James and Zahab 52 produced polyploid and aneuploid strains of P. tannophilus by crossing prototrophic strains and then transferring the diploid premiotic nucleus from sporulation medium to growth medium. This interrupted the miotic cycle. The resulting haploid, diploid, and aneuploid strains were then analyzed for ethanol production in order to determine the effects of gene dosage. Increasing the number of chromosomes above the haploid level greatly increased the yield of ethanol from xylose and glucose. The greatest ethanol yield was observed with a putative tetraploid strain. Pachysolen tannophilus produces and respires ethanol simultaneously in culture. To obtain mutants that would form ethanol in greater yields, Lee et al. 58 selected strains unable to use ethanol as a sole carbon source. Eleven independent mutant loci were identified that conferred the 926 Enzyme Microb. Technol., 1994, vol. 16, November

6 Taxonomy of xylose-fermenting yeasts: T. W. Jeffries and C. P. Kurtzman inhibitor antimycin A. Other mutant strains were obtained from xylitol/urea medium. Pachysolen tannophilus mutants from the nitrate/xylitol medium exhibited both rapid growth on xylitol agar and higher specific ethanol production rates. Relative to the parental strain, the specific activities of some key enzymes, such as xylose reductase, xylitol dehydrogenase, and xylulokinase, were elevated from 1.5 to nearly 15-fold 62 (Table 5). One strain (NO 3 -NO 3-4) showed a 50% increase in the specific rate of ethanol production under anaerobic conditions and almost twice the ethanol production rate of the parent Y-2460 strain aerobically. Clark et al. 63 took the genetic studies with P. tannophilus mutants one step further by hybridizing the eth 2-1 mutant with a NO 3 -NO 3-4 lys strain. Segregants from this initial cross showed wide diversity in ethanol production, suggesting that several mutations were responsible for the performance of the NO 3 -NO 3-4 strain. After several backcrosses, a new mutant strain was obtained (P727) that possessed a higher fermentative rate and had diminished ethanol respiratory capacity. One of the biggest problems associated with hydrolyzate utilization is the repression of xylose utilization by glucose. Wedlock and Thornton 64 have reported that a hexokinase is associated with catabolite repression in P. tannophilus in much the same manner as is observed with S. cerevisiae. Wedlock et al. 65 obtained hexokinase-deficient mutants of P. tannophilus by selecting for resistance to 2-deoxyglucose, and found that unlike the wild-type strains, a mutant deficient in hexokinase 2 and glucokinase 1 (hxk2, glul) used glucose and xylose simultaneously. ethanol-defective phenotype. Of these, three led to greater yield and volumetric rates of ethanol production (Table 4). Carbon assimilation studies showed that the best mutant (eth2) is unable to metabolize ethanol, acetate, α-ketoglutarate, succinate, fumarate, and malate, and is apparently deficient in malate dehydrogenase. Jeffries 59 sought strains of P. tannophilus derepressed for key pentose phosphate pathway and fermentative enzymes. Starting from the observations that P. tannophilus cells grown on nitrate would not metabolize xylose anaerobically and that xylitol would not serve as a sole carbon source for growth, he selected mutant strains of P. tannophilus that were able to grow on xylitol using nitrate as a nitrogen source. Cultivation on nitrate is known to induce elevated levels of pentose phosphate pathway enzymes, such as glucose 6-phosphate dehydrogenase, xylose reductase, and xylitol dehydrogenase, 60 but nitrate is also known to depress the levels of alcohol dehydrogenase. 61 Therefore, mutant strains capable of more rapid growth on nitrate should have higher levels of these enzymes. Xylitol is only slowly metabolized, so mutants better able to grow on this substrate should be better xylose fermenters. Fermentative mutants were selected by enriching and plating on nitrate-xylitol medium in the presence of the respiratory Strain selection with C. shehatae and P. stipitis In the production of ethanol from glucose/xylose mixtures by P. stipitis, sugar uptake appears to be a rate-limiting step because xylose utilization is competitively inhibited by glucose. 66 To overcome this problem, Grootjen et al. 67 selected flocculating strains of P. stipitis. Some naturally occurring strains of P. stipitis tend to form pseudomycelia, and this trait can be accentuated. It is possible to obtain substantially higher levels of biomass in a gas-loop reactor using flocculating pseudomycelial yeasts. In practice, hemicellulose hydrolyzates contain substantial amounts of glucose. Glucose is used before xylose, and because the fermentation of xylose is less vigorous, ethanol production from glucose/xylose mixtures is limited. In an attempt to overcome this problem, Laplace et al. 68 selected Enzyme Microb. Technol., 1994, vol. 16, November 927

7 Reviews mutants of P. stipitis with reduced glucose utilization. They used nystatin enrichment in glucose medium, followed by cultivation on xylose, then plating on galactose. Colonies growing on galactose were replica plated onto glucose agar. Those showing limited growth on glucose were screened for their ability to use xylose. Of 20,000 original colonies on galactose agar, about 120 showed diminished growth on glucose, and 10 of these retained parental growth rates on xylose. Six of these were stable and were examined further. Two of the six strains showed diminished specific glucose uptake rates; three of the six showed increased specific xylose uptake rates. One strain, P. stipitis M5, showed both mutant traits. The specific ethanol production rates of the mutants on xylose were slightly greater than those of the parental strain. Hagedorn and Ciriacy 69 selected P. stipitis mutants unable to grow on xylose (xyl). Such strains are deficient for either xylose reductase or xylitol dehydrogenase; the deficiencies could be attributed to the structural genes XYL1 and XYL2, respectively. These findings suggest that in P. stipitis, xylose reductase and xylitol dehydrogenase are each coded by only one gene. This lends support to biochemical studies showing the presence of a single xylose reductase protein having dual cofactor specificity. Analysis of revertants from two xyl mutants revealed the presence of another gene coding for xylose reductase. Xylose reductase activity in these revertants requires NADPH as a cofactor. It is possible that this gene is normally cryptic. Jeffries 70 used a number of indirect methods to select P. stipitis mutants derepressed for fermentation of D-xylose. Pichia stipitis will use xylitol as a carbon source, albeit while showing marked petite/grande colony morphologies. 71 Also, it does not use nitrate as a nitrogen source. If strains are selected for improved growth on nitrate or xylitol, as was used for P. tannophilus, strains showing poorer fermentation are obtained (Jeffries, unpublished studies). To obtain derepressed strains, the unnatural sugar L-xylose can be employed as a noninductive carbon source. Xylose reductase shows activity against L-xylose, and when reduced, it forms xylitol; therefore derepressed strains can grow on L-xylose. This approach has been used to obtain a number of P. stipitis mutants showing 15% to 20% more ethanol production on xylose than the parental CBS 6450 strain. Protoplast fusion Protoplasm fusion has been used in several attempts to obtain improved fermentative strains. So many different (and largely unknown) traits are required for efficient ethanol production that wholesale merging of genomes from disparate strains has been attempted as a means for obtaining improved performance. Protoplasm fusion between S. cerevisiae and P. tannophilus NRRL Y-2460 was reported to result in hybrids that will produce and tolerate up to 10% ethanol (v/v) from molasses, but they do not produce as much ethanol as Y-2460 will on D-xylose. 72 Heluane et al. 73 have also produced hybrids between P. tannophilus and S. cerevisiae. The hybrids resembled S. cerevisiae morphologically and exhibited sugar assimilation patterns intermediate to the two yeasts. The hybrids assimilated but did not ferment xylose. Polyploids have been obtained for both C. shehatae 74 and P. stipitis, 75 but only slight improvements were observed in ethanol production from xylose. A triauxotrophic strain of P. stipitis was hybridized with a diauxotrophic strain of C. shehatae to yield prototrophic clones that appear to be partial hybrids. 76 The fermentative characteristics of these clones have not been reported. More recently, strains of C. shehatae and P. stipitis were hybridized with ethanol-tolerant S. cerevisiae in an attempt to obtain hybrids that would grow on xylose at high ethanol levels. 77 Mononucleate fusants were obtained, but these dissociated into segregants resembling the parental strains. DNA binding experiments on selected fusants of C. shehatae and P. stipitis showed that the nucleus was composed predominantly of Pichia DNA. 78 The fusants showed only marginal increases in cell DNA, and contained four chromosomes, similar to those found in Pichia. The results suggest that fusion led to integration of Candida genes with the genome of P. stipitis. Cloning and transformation in xylose-fermenting yeasts Until recently, development of improved fermentative strains has been hampered by the absence of a useful transformation system for xylose-fermenting yeasts. Ho et al. 79 reported the transformation of P. stipitis using kanamycin resistance as a selectable marker. Transformation frequencies were on the order of one to two per microgram of DNA. Jeffries 80 recently reported the development of an integrating transformation system for P. stipitis based on the P. stipitis ura3 auxotroph TJ26 and homologous recombination with P. stipitis URA3 cloned from P. stipitis CBS Autonomous replication sequences (ARS) were also cloned from this strain. When incorporated into vectors, 928 Enzyme Microb. Technol., 1994, vol. 16, November

8 Taxonomy of xylose-fermenting yeasts: T. W. Jeffries and C. P. Kurtzman they imparted vector replication as an episomal plasmid. The transformation frequencies with integrative vectors ranged from about 1,000 to 5,000 per microgram of DNA; frequencies with ARS vectors ranged from about 2,000 to 12,000 per microgram of DNA. Genetically improved strains derived from these transformation systems have not yet been reported. Morosoli et al. 81 have developed a transformation system for P. stipitis based on a his3 auxotroph of P. stipitis and the HIS3 gene from S. cerevisiae. It makes use of the yeast/e. coli shuttle vector pjh-s which contains an ARSl S. cerevisiae origin of replication. They linked the promoter region from P. stipitis XYL1to the structural xylanase gene using PCR, and found that the resulting cells produced small amounts of ethanol directly from xylan. Cloning of genes from xylose-fermenting yeasts Another approach to obtaining improved xylose-fermenting yeasts is to clone and express key genes for xylose metabolism into other organisms. 82 Stevis et al. 83 and Ho and Chang 84 reported cloning D-xylulokinase from P. tannophilus and S. cerevisiae by complementing a xylulokinase (xy1b) mutant of Escherichia coli; however, no sequence or cross-hybridization data have been reported that would confirm the nature of this clone. Skrzypek et al. 85 cloned and sequenced the ornithine carbamoyltransferase (OCTase) gene from P. tannophilus. This gene is the key enzyme in the biosynthesis of arginine and represents an important metabolic branch point. The gene was expressed in S. cerevisiae, raising the possibility that genetic transfer of xylosemetabolizing genes from P. tannophilus to S. cerevisiae might be possible. Maleszka and Skrzypek 86 have begun to assign the location of OCTase and other cloned genes to electrophoretically separated chromosomes of P. tannophilus. it was not catalytically active. Sarthy et al. 92 expressed E. coli xylose isomerase in S. cerevisiae but found that the cloned protein had about 10-3 as much activity as the native protein from E. coli (Figure 3). Xylose reductase and xylitol dehydrogenase At least three groups cloned and expressed P. stipitis xylose reductase in S. cerevisiae. Takuma et al. 93 found that S. cerevisiae carrying the gene for P. stipitis xylose reductase could take up xylose and convert it to xylitol, but could not grow on this sugar. Amore et al. 94 also found that S. cerevisiae transformed with P. stipitis xylose reductase would not grow on xylose, but Kotter et al. 95 could demonstrate growth on xylose if both xylose reductase and xylitol dehydrogenase were expressed. In a recent publication, Kotter and Ciriacy 96 showed that S. cerevisiae transformants expressing xylose reductase and xylitol dehydrogenase of P. stipitis would convert xylose to equimolar amounts of xylitol and ethanol under nonrespirative conditions. They attributed xylitol production to the production of NADPH by the oxidative pentose phosphate pathway. The transformed S. cerevisiae accumulated sedoheptulose-7-phosphate, indicating limitations in the nonoxidative steps of the pentose phosphate pathway in this yeast. Tantirungkij et al. 97 have also overexpressed P. stipitis xylose reductase and xylitol dehydrogenase in S. cerevisiae. Transformants of S. cerevisiae harboring xylose reductase alone could not assimilate xylose. Transformants bearing both enzymes could grow on xylose as a sole carbon source. They produced little ethanol but significant amounts of xylitol. In practice, xylitol might be a more valuable product. Hallborn et al. 98 showed that S. cerevisiae bearing P. stipitis xylose reductase converts up to 95% of the xylose to xylitol. Genetic transformation of Saccharomyces for improved xylose utilization Xylose isomerase Several research laboratories have attempted to create a xylose-fermenting yeast by expressing bacterial xylose isomerase in S. cerevisiae and other hexose-fermenting organisms. If successful, this would bypass the cofactor requirements of xylose reductase and xylitol dehydrogenase. Ueng et al. 87 cloned the gene for xylose isomerase from E. coli, and Chan et al. 88 expressed it in Schizosaccharomyces pombe. The transformed yeast reportedly produced 3.7% ethanol from xylose at 80% of the theoretical yield; however, these studies were performed in yeast extract, malt extract, and peptone (YEP) medium, and the malt extract probably accounted for at least a portion of the ethanol production. In subsequent reports, 89,90 this group demonstrated the production of only 0.4% to 2.0% ethanol. Production increased with the amount of YMP added. After accounting for the contribution of maltose, they reported a yield of 0.42 g ethanol g -1 xylose consumed. Amore et al. 91 expressed xylose isomerase genes from Bacillus and Actinoplanes in S. cerevisiae. Approximately 5% of the cellular protein consisted of xylose isomerase, but Enzyme Microb. Technol., 1994, vol. 16, November 929

9 Reviews Conclusion The economical ferrnention of xylose to ethanol and other products depends on the cost of providing the hemicellulosic feedstream and the efficiency of the fermentation. Eight years ago, Jeffries 99 postulated that before attempting commercialization, one should achieve 5% to 6% ethanol with a yield greater than 0.4 g g -1 within 36 h. It now appears that various groups have attained these benchmarks with model substrates or enzymatic hydrolyzates, but that acid hydrolyzates and industrial feedstocks still present some challenges. For some commercial applications, the fermentation of hemicellulosic sugar streams is near at hand, and further strain selection will improve prospects. References 930 Enzyme Microb. Technol., 1994, vol. 16, November

10 Taxonomy of xylose-fermenting yeasts: T. W. Jeffries and C. P. Kurtzman Enzyme Microb. Technol, r 1994, vol. 16, November 931

11 Reviews 932 Enzyme Microb, Technol., 1994, vol. 16, November