Comparative genomics of biotechnologically important yeasts.

Ascomycete yeasts are metabolically diverse, with great potential for biotechnology. Here, we report the comparative genome analysis of 29 taxonomically and biotechnologically important yeasts, including 16 newly sequenced. We identify a genetic code change, CUG-Ala, in Pachysolen tannophilus in the clade sister to the known CUG-Ser clade. Our well-resolved yeast phylogeny shows that some traits, such as methylotrophy, are restricted to single clades, whereas others, such as l-rhamnose utilization, have patchy phylogenetic distributions. Gene clusters, with variable organization and distribution, encode many pathways of interest. Genomics can predict some biochemical traits precisely, but the genomic basis of others, such as xylose utilization, remains unresolved. Our data also provide insight into early evolution of ascomycetes. We document the loss of H3K9me2/3 heterochromatin, the origin of ascomycete mating-type switching, and panascomycete synteny at the MAT locus. These data and analyses will facilitate the engineering of efficient biosynthetic and degradative pathways and gateways for genomic manipulation.

Spathaspora passalidarum selected for resistance to AFEX hydrolysate shows decreased cell yield.

This study employed cell recycling, batch adaptation, cell mating and high-throughput screening to select adapted Spathaspora passalidarum strains with improved fermentative ability. The most promising candidate YK208-E11 (E11) showed a 3-fold increase in specific fermentation rate compared to the parental strain and an ethanol yield greater than 0.45 g/g substrate while co-utilizing cellobiose, glucose and xylose. Further characterization showed that strain E11 also makes 40% less biomass compared to the parental strain when cultivated in rich media under aerobic conditions. A tetrazolium agar overlay assay in the presence of respiration inhibitors, including rotenone, antimycin A, KCN and salicylhydroxamic acid elucidated the nature of the mutational events. Results indicated that E11 has a deficiency in its respiration system that could contribute to its low cell yield. Strain E11 was subjected to whole genome sequencing and an ∼11 kb deletion was identified; the open reading frames absent in strain E11 code for proteins with predicted functions in respiration, cell division and the actin cytoskeleton, and may contribute to the observed physiology of the adapted strain. Results of the tetrazolium overlay also suggest that cultivation on xylose affects the respiration capacity in the wild-type strain, which could account for its faster fermentation of xylose as compared to glucose. These results support our previous finding that S. passalidarum has highly unusual physiological responses to xylose under oxygen limitation.

Effects of aeration on growth, ethanol and polyol accumulation by Spathaspora passalidarum NRRL Y-27907 and Scheffersomyces stipitis NRRL Y-7124.

Spathaspora passalidarum NN245 (NRRL-Y27907) is an ascomycetous yeast that displays a higher specific fermentation rate with xylose than with glucose. Previous studies have shown that its capacity for xylose fermentation increases while cell yield decreases with decreasing aeration. Aeration optimization plays a crucial role in maximizing bioethanol production from lignocellulosic hydrolysates. Here, we compared the kinetics of S. passalidarum NN245 and Scheffersomyces (Pichia) stipitis NRRL Y-7124 fermenting 15% glucose, 15% xylose, or 12% xylose plus 3% glucose under four different aeration conditions. The maximum specific fermentation rate for S. passalidarum was 0.153 g ethanol/g CDW · h with a yield of 0.448 g/g from 150 g/L xylose at an oxygen transfer rate of 2.47 mmol O2 /L h. Increasing the OTR to 4.27 mmol O2 /L h. decreased the ethanol yield from 0.46 to 0.42 g/g xylose while increasing volumetric ethanol productivity from 0.52 to 0.8 g/L h. Both yeasts had lower cells yields and higher ethanol yields when growing on xylose than when growing on glucose. Acetic acid accretions of both strains correlated positively with increasing aeration. S. passalidarum secreted lower amounts of polyols compared to S. stipitis under most circumstances. In addition, the composition of polyols differed: S. passalidarum accumulated mostly xylitol and R,R-2,3-butanediol (BD) whereas S. stipitis accumulated mostly xylitol and ribitol when cultivated in xylose or a mixture of 12% xylose and 3% glucose. R,R-2,3-BD accumulation by S. passalidarum during xylose fermentation can be as much as four times of that by S. stipitis, and R,R-2,3-BD is also the most abundant byproduct after xylitol. The ratios of polyols accumulated by the two species under different aeration conditions and the implications of these observations for strain and process engineering are discussed.

Comprehensive evaluation of two genome-scale metabolic network models for Scheffersomyces stipitis.

Genome-scale metabolic network models represent the link between the genotype and phenotype of the organism, which are usually reconstructed based on the genome sequence annotation and relevant biochemical and physiological information. These models provide a holistic view of the organism's metabolism, and constraint-based metabolic flux analysis methods have been used extensively to study genome-scale cellular metabolic networks. It is clear that the quality of the metabolic network model determines the outcome of the application. Therefore, it is critically important to determine the accuracy of a genome-scale model in describing the cellular metabolism of the modeled strain. However, because of the model complexity, which results in a system with very high degree of freedom, a good agreement between measured and computed substrate uptake rates and product secretion rates is not sufficient to guarantee the predictive capability of the model. To address this challenge, in this work we present a novel system identification based framework to extract the qualitative biological knowledge embedded in the quantitative simulation results from the metabolic network models. The extracted knowledge can serve two purposes: model validation during model development phase, which is the focus of this work, and knowledge discovery once the model is validated. This framework bridges the gap between the large amount of numerical results generated from genome-scale models and the knowledge that can be easily understood by biologists. The effectiveness of the proposed framework is demonstrated by its application to the analysis of two recently published genome-scale models of Scheffersomyces stipitis.

An optimized transformation protocol for Lipomyces starkeyi.

We report the development of an efficient genetic transformation system for Lipomyces starkeyi based on a modified lithium acetate transformation protocol. L. starkeyi is a highly lipogenic yeast that grows on a wide range of substrates. The initial transformation rate for this species was extremely low, and required very high concentrations of DNA. A systematic approach for optimizing the protocol resulted in an increase in the transformation efficiency by four orders of magnitude. Important parameters included cell density, the duration of incubation and recovery periods, the heat shock temperature, and the concentration of lithium acetate and carrier DNA within the transformation mixture. We have achieved efficiencies in excess of 8,000 transformants/µg DNA, which now make it possible to screen libraries in the metabolic engineering of this yeast. Metabolic engineering based on this transformation system could improve lipogenesis and enable formation of higher value products.

Comparative genomics of xylose-fermenting fungi for enhanced biofuel production.

Cellulosic biomass is an abundant and underused substrate for biofuel production. The inability of many microbes to metabolize the pentose sugars abundant within hemicellulose creates specific challenges for microbial biofuel production from cellulosic material. Although engineered strains of Saccharomyces cerevisiae can use the pentose xylose, the fermentative capacity pales in comparison with glucose, limiting the economic feasibility of industrial fermentations. To better understand xylose utilization for subsequent microbial engineering, we sequenced the genomes of two xylose-fermenting, beetle-associated fungi, Spathaspora passalidarum and Candida tenuis. To identify genes involved in xylose metabolism, we applied a comparative genomic approach across 14 Ascomycete genomes, mapping phenotypes and genotypes onto the fungal phylogeny, and measured genomic expression across five Hemiascomycete species with different xylose-consumption phenotypes. This approach implicated many genes and processes involved in xylose assimilation. Several of these genes significantly improved xylose utilization when engineered into S. cerevisiae, demonstrating the power of comparative methods in rapidly identifying genes for biomass conversion while reflecting on fungal ecology.

Cofermentation of glucose, xylose, and cellobiose by the beetle-associated yeast Spathaspora passalidarum.

Fermentation of cellulosic and hemicellulosic sugars from biomass could resolve food-versus-fuel conflicts inherent in the bioconversion of grains. However, the inability to coferment glucose and xylose is a major challenge to the economical use of lignocellulose as a feedstock. Simultaneous cofermentation of glucose, xylose, and cellobiose is problematic for most microbes because glucose represses utilization of the other saccharides. Surprisingly, the ascomycetous, beetle-associated yeast Spathaspora passalidarum, which ferments xylose and cellobiose natively, can also coferment these two sugars in the presence of 30 g/liter glucose. S. passalidarum simultaneously assimilates glucose and xylose aerobically, it simultaneously coferments glucose, cellobiose, and xylose with an ethanol yield of 0.42 g/g, and it has a specific ethanol production rate on xylose more than 3 times that of the corresponding rate on glucose. Moreover, an adapted strain of S. passalidarum produced 39 g/liter ethanol with a yield of 0.37 g/g sugars from a hardwood hydrolysate. Metabolome analysis of S. passalidarum before onset and during the fermentations of glucose and xylose showed that the flux of glycolytic intermediates is significantly higher on xylose than on glucose. The high affinity of its xylose reductase activities for NADH and xylose combined with allosteric activation of glycolysis probably accounts in part for its unusual capacities. These features make S. passalidarum very attractive for studying regulatory mechanisms enabling bioconversion of lignocellulosic materials by yeasts.

Evolutionary engineering of Saccharomyces cerevisiae for efficient aerobic xylose consumption.

Industrial biotechnology aims to develop robust microbial cell factories, such as Saccharomyces cerevisiae, to produce an array of added value chemicals presently dominated by petrochemical processes. Xylose is the second most abundant monosaccharide after glucose and the most prevalent pentose sugar found in lignocelluloses. Significant research efforts have focused on the metabolic engineering of S. cerevisiae for fast and efficient xylose utilization. This study aims to metabolically engineer S. cerevisiae, such that it can consume xylose as the exclusive substrate while maximizing carbon flux to biomass production. Such a platform may then be enhanced with complementary metabolic engineering strategies that couple biomass production with high value-added chemical. Saccharomyces cerevisiae, expressing xylose reductase, xylitol dehydrogenase and xylulose kinase, from the native xylose-metabolizing yeast Pichia stipitis, was constructed, followed by a directed evolution strategy to improve xylose utilization rates. The resulting S. cerevisiae strain was capable of rapid growth and fast xylose consumption producing only biomass and negligible amount of byproducts. Transcriptional profiling of this strain was employed to further elucidate the observed physiology confirms a strongly up-regulated glyoxylate pathway enabling respiratory metabolism. The resulting strain is a desirable platform for the industrial production of biomass-related products using xylose as a sole carbon source.

Interactions of fungi from fermented sausage with regenerated cellulose casings.

This research examined cellulolytic effects of fungi and other microbes present in cured sausages on the strength and stability of regenerated cellulose casings (RCC) used in the sausage industry. Occasionally during the curing process, RCC would split or fail, thereby leading to loss of product. The fungus Penicillium sp. BT-F-1, which was isolated from fermented sausages, and other fungi, which were introduced to enable the curing process, produced small amounts of cellulases on RCC in both liquid and solid cultivations. During continued incubation for 15-60 days in solid substrate cultivation (SSC) on RCC support, the fungus Penicillium sp isolate BT-F-1 degraded the casings' dry weights by 15-50% and decreased their tensile strengths by ~75%. Similarly commercial cellulase(s) resulted in 20-50% degradation of RCC in 48 h. During incubation with Penicillium sp BT-F-1, the surface structure of RCC collapsed, resulting in loss of strength and stability of casings. The matrix of industrial RCC comprised 88-93% glucose polymer residues with 0.8-4% xylan impurities. Premature casing failure appeared to result from operating conditions in the manufacturing process that allowed xylan to build up in the extrusion bath. The sausage fungus Penicillium sp BT-F-1 produced xylanases to break down soft xylan pockets prior to slow cellulosic dissolution of RCC.

Xylitol production from DEO hydrolysate of corn stover by Pichia stipitis YS-30.

Corn stover that had been treated with vapor-phase diethyl oxalate released a mixture of mono- and oligosaccharides consisting mainly of xylose and glucose. Following overliming and neutralization, a D-xylulokinase mutant of Pichia stipitis, FPL-YS30 (xyl3-∆1), converted the stover hydrolysate into xylitol. This research examined the effects of phosphoric or gluconic acids used for neutralization and urea or ammonium sulfate used as nitrogen sources. Phosphoric acid improved color and removal of phenolic compounds. D-Gluconic acid enhanced cell growth. Ammonium sulfate increased cell yield and maximum specific cell growth rate independently of the acid used for neutralization. The highest xylitol yield (0.61 g(xylitol)/g(xylose)) and volumetric productivity (0.18 g(xylitol)/g(xylose )l) were obtained in hydrolysate neutralized with phosphoric acid. However, when urea was the nitrogen source the cell yield was less than half of that obtained with ammonium sulfate.

Genome sequence of the lignocellulose-bioconverting and xylose-fermenting yeast Pichia stipitis.

Xylose is a major constituent of plant lignocellulose, and its fermentation is important for the bioconversion of plant biomass to fuels and chemicals. Pichia stipitis is a well-studied, native xylose-fermenting yeast. The mechanism and regulation of xylose metabolism in P. stipitis have been characterized and genes from P. stipitis have been used to engineer xylose metabolism in Saccharomyces cerevisiae. We have sequenced and assembled the complete genome of P. stipitis. The sequence data have revealed unusual aspects of genome organization, numerous genes for bioconversion, a preliminary insight into regulation of central metabolic pathways and several examples of colocalized genes with related functions. The genome sequence provides insight into how P. stipitis regulates its redox balance while very efficiently fermenting xylose under microaerobic conditions.

Pichia stipitis genomics, transcriptomics, and gene clusters.

Genome sequencing and subsequent global gene expression studies have advanced our understanding of the lignocellulose-fermenting yeast Pichia stipitis. These studies have provided an insight into its central carbon metabolism, and analysis of its genome has revealed numerous functional gene clusters and tandem repeats. Specialized physiological traits are often the result of several gene products acting together. When coinheritance is necessary for the overall physiological function, recombination and selection favor colocation of these genes in a cluster. These are particularly evident in strongly conserved and idiomatic traits. In some cases, the functional clusters consist of multiple gene families. Phylogenetic analyses of the members in each family show that once formed, functional clusters undergo duplication and differentiation. Genome-wide expression analysis reveals that regulatory patterns of clusters are similar after they have duplicated and that the expression profiles evolve along with functional differentiation of the clusters. Orthologous gene families appear to arise through tandem gene duplication, followed by differentiation in the regulatory and coding regions of the gene. Genome-wide expression analysis combined with cross-species comparisons of functional gene clusters should reveal many more aspects of eukaryotic physiology.

Deleting the para-nitrophenyl phosphatase (pNPPase), PHO13, in recombinant Saccharomyces cerevisiae improves growth and ethanol production on D-xylose.

Overexpression of D-xylulokinase in Saccharomyces cerevisiae engineered for assimilation of xylose results in growth inhibition that is more pronounced at higher xylose concentrations. Mutants deficient in the para-nitrophenyl phosphatase, PHO13, resist growth inhibition on xylose. We studied this inhibition under aerobic growth conditions in well-controlled bioreactors using engineered S. cerevisiae CEN.PK. Growth on glucose was not significantly affected in pho13Delta mutants, but acetate production increased by 75%. Cell growth, ethanol production, and xylose consumption all increased markedly in pho13Delta mutants. The specific growth rate and rate of specific xylose uptake were approximately 1.5 times higher in the deletion strain than in the parental strain when growing on glucose-xylose mixtures and up to 10-fold higher when growing on xylose alone. In addition to showing higher acetate levels, pho13Delta mutants also produced less glycerol on xylose, suggesting that deletion of Pho13p could improve growth by altering redox levels when cells are grown on xylose.

Engineering yeasts for xylose metabolism.

Technologies for the production of alternative fuels are receiving increased attention owing to concerns over the rising cost of petrol and global warming. One such technology under development is the use of yeasts for the commercial fermentation of xylose to ethanol. Several approaches have been employed to engineer xylose metabolism. These involve modeling, flux analysis, and expression analysis followed by the targeted deletion or altered expression of key genes. Expression analysis is increasingly being used to target rate-limiting steps. Quantitative metabolic models have also proved extremely useful: they can be calculated from stoichiometric balances or inferred from the labeling of intermediate metabolites. The recent determination of the genome sequence for P. stipitis is important, as its genome characteristics and regulatory patterns could serve as guides for further development in this natural xylose-fermenting yeast or in engineered Saccharomyces cerevisiae. Lastly, strain selection through mutagenesis, adaptive evolution or from nature can also be employed to further improve activity.

Nitrogen limitation, oxygen limitation, and lipid accumulation in Lipomyces starkeyi.

Lipid production by oleaginous yeasts is optimal at high carbon-to-nitrogen ratios. In the current study, nitrogen and carbon consumption by Lipomyces starkeyi were directly measured in defined minimal media with nitrogen content and agitation rates as variables. Shake flask cultures with an initial C:N ratio of 72:1 cultivated at 200rpm resulted in a lipid output of 10g/L, content of 55%, yield of 0.170g/g, and productivity of 0.06g/L/h. All of these values decreased by ≈50-60% when the agitation rate was raised to 300rpm or when the C:N ratio was lowered to 24:1, demonstrating the importance of these parameters. Under all conditions, L. starkeyi cultures tolerated acidified media (pH≈2.6) without difficulty, and produced considerable amounts of alcohols; including ethanol, mannitol, arabitol, and 2,3-butanediol. L. starkeyi also produced lipids from a corn stover hydrolysate, showing its potential to produce biofuels from renewable agricultural feedstocks.

Genomics and the making of yeast biodiversity.

Yeasts are unicellular fungi that do not form fruiting bodies. Although the yeast lifestyle has evolved multiple times, most known species belong to the subphylum Saccharomycotina (syn. Hemiascomycota, hereafter yeasts). This diverse group includes the premier eukaryotic model system, Saccharomyces cerevisiae; the common human commensal and opportunistic pathogen, Candida albicans; and over 1000 other known species (with more continuing to be discovered). Yeasts are found in every biome and continent and are more genetically diverse than angiosperms or chordates. Ease of culture, simple life cycles, and small genomes (∼10-20Mbp) have made yeasts exceptional models for molecular genetics, biotechnology, and evolutionary genomics. Here we discuss recent developments in understanding the genomic underpinnings of the making of yeast biodiversity, comparing and contrasting natural and human-associated evolutionary processes. Only a tiny fraction of yeast biodiversity and metabolic capabilities has been tapped by industry and science. Expanding the taxonomic breadth of deep genomic investigations will further illuminate how genome function evolves to encode their diverse metabolisms and ecologies.

Changing flux of xylose metabolites by altering expression of xylose reductase and xylitol dehydrogenase in recombinant Saccharomyces cerevisiae.

We changed the fluxes of xylose metabolites in recombinant Saccharomyces cerevisiae by manipulating expression of Pichia stipitis genes (XYL1 and XYL2) coding for xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively. XYL1 copy number was kept constant by integrating it into the chromosome. Copy numbers of XYL2 were varied either by integrating XYL2 into the chromosome or by transforming cells with XYL2 in a multicopy vector. Genes in all three constructs were under control of the strong constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Enzymatic activity of XR and XDH in the recombinant strains increased with the copy number of XYL1 and XYL2. XR activity was not detected in the parent but was present at a nearly constant level in all of the transformants. XDH activity increased 12-fold when XYL2 was on a multicopy vector compared with when it was present in an integrated single copy. Product formation during xylose fermentation was affected by XDH activity and by aeration in recombinant S. cerevisiae. Higher XDH activity and more aeration resulted in less xylitol and more xylulose accumulation during xylose fermentation. Secretion of xylulose by strains with multicopy XYL2 and elevated XDH supports the hypothesis that D-xylulokinase limits metabolic flux in recombinant S. cerevisiae.

Molecular characterization of a gene for aldose reductase ( CbXYL1) from Candida boidinii and its expression in Saccharomyces cerevisiae.

Candida boidinii produces significant amounts of xylitol from xylose, and assays of crude homogenates for aldose (xylose) reductase (XYL1p) have been reported to show relatively high activity with NADH as a cofactor even though XYL1p purified from this yeast does not have such activity. A gene coding for XYL1p from C. boidinii (CbXYL1) was isolated by amplifying the central region using primers to conserved domains and by genome walking. CbXYL1 has an open reading frame of 966 bp encoding 321 amino acids. The C. boidinii XYL1p is highly similar to other known yeast aldose reductases and is most closely related to the NAD(P)H-linked XYL1p of Kluyveromyces lactis. Cell homogenates from C. boidinii and recombinant Saccharomyces cerevisiae were tested for XYL1p activity to confirm the previously reported high ratio of NADH:NADPH linked activity. C. boidinii grown under fully aerobic conditions showed an NADH:NADPH activity ratio of 0.76, which was similar to that observed with the XYL1p from Pichia stipitis XYL1, but which is much lower than what was previously reported. Cells grown under low aeration showed an NADH:NADPH activity ratio of 2.13. Recombinant S. cerevisiae expressing CbXYL1 showed only NADH-linked activity in cell homogenates. Southern hybridization did not reveal additional bands. These results imply that a second, unrelated gene for XYL1p is present in C. boidinii.