First- and second-generation integrated process for bioethanol production: Fermentation of molasses diluted with hemicellulose hydrolysate by recombinant Saccharomyces cerevisiae.

The integration of first- (1G) and second-generation (2G) ethanol production by adding sugarcane juice or molasses to lignocellulosic hydrolysates offers the possibility to overcome the problem of inhibitors (acetic acid, furfural, hydroxymethylfurfural and phenolic compounds), and add nutrients (such as salts, sugars and nitrogen sources) to the fermentation medium, allowing the production of higher ethanol titers. In this work, an 1G2G production process was developed with hemicellulosic hydrolysate (HH) from a diluted sulfuric acid pretreatment of sugarcane bagasse and sugarcane molasses. The industrial Saccharomyces cerevisiae CAT-1 was genetically modified for xylose consumption and used for co-fermentation of sucrose, fructose, glucose, and xylose. The fed-batch fermentation with high cell density that mimics an industrial fermentation was performed at bench scale fermenter, achieved high volumetric ethanol productivity of 1.59 g L-1 h-1, 0.39 g g-1 of ethanol yield, and 44.5 g L-1 ethanol titer, and shown that the yeast was able to consume all the sugars present in must simultaneously. With the results, it was possible to establish a mass balance for the global process: from pretreatment to the co-fermentation of molasses and HH, and it was possible to establish an effective integrated process (1G2G) with sugarcane molasses and HH co-fermentation employing a recombinant yeast.

Improved Sugarcane-Based Fermentation Processes by an Industrial Fuel-Ethanol Yeast Strain.

In Brazil, sucrose-rich broths (cane juice and/or molasses) are used to produce billions of liters of both fuel ethanol and cachaça per year using selected Saccharomyces cerevisiae industrial strains. Considering the important role of feedstock (sugar) prices in the overall process economics, to improve sucrose fermentation the genetic characteristics of a group of eight fuel-ethanol and five cachaça industrial yeasts that tend to dominate the fermentors during the production season were determined by array comparative genomic hybridization. The widespread presence of genes encoding invertase at multiple telomeres has been shown to be a common feature of both baker's and distillers' yeast strains, and is postulated to be an adaptation to sucrose-rich broths. Our results show that only two strains (one fuel-ethanol and one cachaça yeast) have amplification of genes encoding invertase, with high specific activity. The other industrial yeast strains had a single locus (SUC2) in their genome, with different patterns of invertase activity. These results indicate that invertase activity probably does not limit sucrose fermentation during fuel-ethanol and cachaça production by these industrial strains. Using this knowledge, we changed the mode of sucrose metabolism of an industrial strain by avoiding extracellular invertase activity, overexpressing the intracellular invertase, and increasing its transport through the AGT1 permease. This approach allowed the direct consumption of the disaccharide by the cells, without releasing glucose or fructose into the medium, and a 11% higher ethanol production from sucrose by the modified industrial yeast, when compared to its parental strain.

Hydrothermal pretreatment for the production of prebiotic oligosaccharides from tobacco stem.

Tobacco stem is an abundant and inexpensive renewable source to produce prebiotics by circular economy. In this study, hydrothermal pretreatments were evaluated on the release of xylooligosaccharides (XOS) and cello-oligosaccharides (COS) from the tobacco stem by a central composite rotational design associated with response surface methodology to evaluate the effects of temperature (161.72 to 218.3 °C) and solid load (SL) (2.93 to 17.07%). XOS were the main compounds released to the liquor. Desirability function was performed to maximize the production of XOS and minimize the effects of release of monosaccharides and degradation compounds. The result indicated yield of 96% w[XOS]/w[xylan] for 190 °C-2.93% SL. The highest value for COS and total oligomers content (COS + XOS) was 6.42 g/L and 17.7 g/L, respectively, for 190 °C-17.07% SL. The mass balance for the best yield XOS condition predicted 132 kg of XOS (X2-X6) from 1000 kg of tobacco stem.

Strategies for Efficient Expression of Heterologous Monosaccharide Transporters in Saccharomyces cerevisiae.

In previous work, we developed a Saccharomyces cerevisiae strain (DLG-K1) lacking the main monosaccharide transporters (hxt-null) and displaying high xylose reductase, xylitol dehydrogenase and xylulokinase activities. This strain proved to be a useful chassis strain to study new glucose/xylose transporters, as SsXUT1 from Scheffersomyces stipitis. Proteins with high amino acid sequence similarity (78-80%) to SsXUT1 were identified from Spathaspora passalidarum and Spathaspora arborariae genomes. The characterization of these putative transporter genes (SpXUT1 and SaXUT1, respectively) was performed in the same chassis strain. Surprisingly, the cloned genes could not restore the ability to grow in several monosaccharides tested (including glucose and xylose), but after being grown in maltose, the uptake of 14C-glucose and 14C-xylose was detected. While SsXUT1 lacks lysine residues with high ubiquitinylation potential in its N-terminal domain and displays only one in its C-terminal domain, both SpXUT1 and SaXUT1 transporters have several such residues in their C-terminal domains. A truncated version of SpXUT1 gene, deprived of the respective 3'-end, was cloned in DLG-K1 and allowed growth and fermentation in glucose or xylose. In another approach, two arrestins known to be involved in the ubiquitinylation and endocytosis of sugar transporters (ROD1 and ROG3) were knocked out, but only the rog3 mutant allowed a significant improvement of growth and fermentation in glucose when either of the XUT permeases were expressed. Therefore, for the efficient heterologous expression of monosaccharide (e.g., glucose/xylose) transporters in S. cerevisiae, we propose either the removal of lysines involved in ubiquitinylation and endocytosis or the use of chassis strains hampered in the specific mechanism of membrane protein turnover.

Analysis of glucose and xylose metabolism in new indigenous Meyerozyma caribbica strains isolated from corn residues.

Aiming to broaden the base of knowledge about wild yeasts, four new indigenous strains were isolated from corn residues, and phylogenetic-tree assemblings on ITS and LSU regions indicated they belong to Meyerozyma caribbica. Yeasts were cultivated under full- and micro-aerobiosis, starting with low or high cell-density inoculum, in synthetic medium or corn hydrolysate containing glucose and/or xylose. Cells were able to assimilate both monosaccharides, albeit by different metabolic routes (fermentative or respiratory). They grew faster in glucose, with lag phases ~ 10 h shorter than in xylose. The hexose exhaustion occurred between 24 and 34 h, while xylose was entirely consumed in the last few hours of cultivation (44-48 h). In batch fermentation in synthetic medium with high cell density, under full-aerobiosis, 18-20 g glucose l-1 were exhausted in 4-6 h, with a production of 6.5-7.0 g ethanol l-1. In the xylose medium, cells needed > 12 h to consume the carbohydrate, and instead of ethanol, cells released 4.4-6.4 g l-1 xylitol. Under micro-aerobiosis, yeasts were unable to assimilate xylose, and glucose was more slowly consumed, although the ethanol yield was the theoretical maximum. When inoculated into the hydrolysate, cells needed 4-6 h to deplete glucose, and xylose had a maximum consumption of 57%. Considering that the hydrolysate contained ~ 3 g l-1 acetic acid, it probably has impaired sugar metabolism. Thus, this study increases the fund of knowledge regarding indigenous yeasts and reveals the biotechnological potential of these strains.

Comparison of Spathaspora passalidarum and recombinant Saccharomyces cerevisiae for integration of first- and second-generation ethanol production.

First-generation ethanol (E1G) is based on the fermentation of sugars released from saccharine or starch sources, while second-generation ethanol (E2G) is focused on the fermentation of sugars released from lignocellulosic feedstocks. During the fractionation process to release sugars from hemicelluloses (mainly xylose), some inhibitor compounds are released hindering fermentation. Thus, the biggest challenge of using hemicellulosic hydrolysate is selecting strains and processes able to efficiently ferment xylose and tolerate inhibitors. With the aim of diluting inhibitors, sugarcane molasses (80% of sucrose content) can be mixed to hemicellulosic hydrolysate in an integrated E1G-E2G process. Cofermentations of xylose and sucrose were evaluated for the native xylose consumer Spathaspora passalidarum and a recombinant Saccharomyces cerevisiae strain. The industrial S. cerevisiae strain CAT-1 was modified to overexpress the XYL1, XYL2 and XKS1 genes and a mutant ([4-59Δ]HXT1) version of the low-affinity HXT1 permease, generating strain MP-C5H1. Although S. passalidarum showed better results for xylose fermentation, this yeast showed intracellular sucrose hydrolysis and low sucrose consumption in microaerobic conditions. Recombinant S. cerevisiae showed the best performance for cofermentation, and a batch strategy at high cell density in bioreactor achieved unprecedented results of ethanol yield, titer and volumetric productivity in E1G-E2G production process.

Deconstruction of banana peel for carbohydrate fractionation.

The deconstruction of banana peel for carbohydrate recovery was performed by sequential treatment (acid, alkaline, and enzymatic). The pretreatment with citric acid promoted the extraction of pectin, resulting in a yield of 8%. In addition, xylose and XOS, 348.5 and 17.3 mg/g xylan, respectively, were also quantified in acidic liquor as a result of partial depolymerization of hemicellulose. The spent solid was pretreated with alkaline solution (NaOH or KOH) for delignification and release of residual carbohydrates from the hemicellulose. The yields of xylose and arabinose (225.2 and 174.0 mg/g hemicellulose) were approximately 40% higher in the pretreatment with KOH, while pretreatment with NaOH promoted higher delignification (67%), XOS yield (32.6 mg/g xylan), and preservation of cellulosic fraction. Finally, the spent alkaline solid, rich in cellulose (76%), was treated enzymatically to release glucose, reaching the final concentration of 28.2 g/L. The mass balance showed that through sequential treatment, 9.9 g of xylose, 0.5 g of XOS, and 8.2 g of glucose were obtained from 100 g of raw banana peels, representing 65.8% and 46.5% conversion of hemicellulose and cellulose, respectively. The study of the fractionation of carbohydrates in banana peel proved to be a useful tool for valorization, mainly of the hemicellulose fraction for the production of XOS and xylose with high value applications in the food industry.

Hydrothermal treatment on depolymerization of hemicellulose of mango seed shell for the production of xylooligosaccharides.

Hydrothermal processing is an interesting biorefinery technology for converting lignocellulosic biomass into biofuels and biocompounds. This process is based on the selective solubilization and depolymerization of hemicellulose fraction (xylan) and may be considered beneficial, due to the possibility of obtaining xylooligosaccharides (XOS) with a degree of polymerization (DP) suitable for prebiotic applications. This study evaluated the effect of pressure (2.5 and 10 MPa) in a kinetic study (30 min) of hydrothermal treatment (180 °C) to optimize the extraction of XOS from mango seed shell. Total reducing sugars (TRS) values were close to the maximum in 15 min showing a slower rate for both pressures after this time, but at 10 MPa the value was 20 % lower than at 2.5 MPa. Based on these results, a new extraction was performed at 2.5 MPa and 15 min, and the extracted XOS were quantified, yielding 393.44 mg XOS/g xylan. XOS with a degree of polymerization between X2-X6 corresponded to 82.24 mg/g and XOS with X > 6 (or soluble xylan) corresponded to 311.20 mg/g. A low amount of xylose (8.81 mg/g xylan) was released, resulting in a hemicellulose conversion of 40.2 %. In general, approximately 8.1 kg of total XOS was produced from 100 kg of dried mango seed shell (X2-X6-1.7 kg and X > 6-6.4 kg).

The secreted acid trehalase encoded by the CgATH1 gene is involved in Candida glabrata virulence.

BACKGROUND: Candida glabrata yeast is the second cause of candidiasis worldwide. Differs from other yeasts since assimilates only glucose and trehalose (a characteristic used in rapid identification tests for this pathogen) by secreting into the medium a highly active acid trehalase encoded by the CgATH1 gene. OBJECTIVE: This study aimed to characterise the function of the acid trehalase in the physiopathology of C. glabrata. METHODS: Gene deletion was performed to obtain a mutant ath1Δ strain, and the ability of the ath1Δ strain to grow in trehalase, or the presence of trehalase activity in the ath1Δ yeast cells, was verified. We also tested the virulence of the ath1Δ strain in a murine model of infection. FINDINGS: The ath1Δ mutant strain grows normally in the presence of glucose, but loses its ability to grow in trehalose. Due to the high acid trehalase activity present in wild-type cells, the cytoplasmic neutral trehalase activity is only detected in the ath1Δ strain. We also observed a significantly lower virulence of the ath1Δ strain in a murine model of infection with either normal or immunocompromised mice. MAIN CONCLUSIONS: The acid trehalase is involved in the hydrolysis of external trehalose by C. glabrata, and the enzyme also plays a major virulence role during infectivity.

Biochemical analysis of cellobiose catabolism in Candida pseudointermedia strains isolated from rotten wood.

We isolated two Candida pseudointermedia strains from the Atlantic rain forest in Brazil, and analyzed cellobiose metabolization in their cells. After growth in cellobiose medium, both strains had high intracellular ß-glucosidase activity [~ 200 U (g cells)-1 for 200 mM cellobiose and ~ 100 U (g cells)-1 for 2 mM pNPßG] and negligible periplasmic cellobiase activity. During batch fermentation, the strain with the best performance consumed all the available cellobiose in the first 18 h of the assay, producing 2.7 g L-1 of ethanol. Kinetics of its cellobiase activity demonstrated a high-affinity hydrolytic system inside cells, with Km of 12.4 mM. Our data suggest that, unlike other fungal species that hydrolyze cellobiose extracellularly, both analyzed strains transport it to the cytoplasm, where it is then hydrolyzed by high-affinity intracellular ß-glucosidases. We believe this study increases the fund of knowledge regarding yeasts from Brazilian microbiomes.

d-Xylose consumption by nonrecombinant Saccharomyces cerevisiae: A review.

Xylose is the second most abundant sugar in nature. Its efficient fermentation has been considered as a critical factor for a feasible conversion of renewable biomass resources into biofuels and other chemicals. The yeast Saccharomyces cerevisiae is of exceptional industrial importance due to its excellent capability to ferment sugars. However, although S. cerevisiae is able to ferment xylulose, it is considered unable to metabolize xylose, and thus, a lot of research has been directed to engineer this yeast with heterologous genes to allow xylose consumption and fermentation. The analysis of the natural genetic diversity of this yeast has also revealed some nonrecombinant S. cerevisiae strains that consume or even grow (modestly) on xylose. The genome of this yeast has all the genes required for xylose transport and metabolism through the xylose reductase, xylitol dehydrogenase, and xylulokinase pathway, but there seems to be problems in their kinetic properties and/or required expression. Self-cloning industrial S. cerevisiae strains overexpressing some of the endogenous genes have shown interesting results, and new strategies and approaches designed to improve these S. cerevisiae strains for ethanol production from xylose will also be presented in this review.

Second-generation ethanol from non-detoxified sugarcane hydrolysate by a rotting wood isolated yeast strain.

This work aims to evaluate the production of second-generation ethanol from sugarcane bagasse hydrolysate without acetic acid (inhibitor) detoxification. Three isolated yeast strains from lignocellulosic materials were evaluated, and one strain (UFFS-CE-3.1.2), identified using large subunit rDNA sequences as Wickerhamomyces sp., showed satisfactory results in terms of ethanol production without acetic acid removal. A Plackett-Burman design was used to evaluate the influence of hydrolysate composition and nutrients supplementation in the fermentation medium for the second-generation ethanol production. Two fermentation kinetics were performed, with controlled pH at 5.5, or keeping the initial pH at 4.88. The fermentation conducted without pH adjustment and supplementation of nutrients reported the best result in terms of second-generation ethanol production. Wickerhamomyces sp., isolated as UFFS-CE-3.1.2, was considered promising in the production of second-generation ethanol by using crude (non-detoxified) sugarcane hydrolysate.

Cloning novel sugar transporters from Scheffersomyces (Pichia) stipitis allowing D-xylose fermentation by recombinant Saccharomyces cerevisiae.

OBJECTIVES: Since uptake of xylose limits its fermentation, we aimed to identify novel sugar transporters from Scheffersomyces stipitis that allow xylose uptake and fermentation by engineered Saccharomyces cerevisiae. RESULTS: An hxt-null S. cerevisiae strain, lacking the major hexose transporters (hxt1Δ-hxt7Δ and gal2Δ) but having high xylose reductase, xylitol dehydrogenase and xylulokinase activities, was transformed with a genomic DNA library from S. stipitis. Four plasmids allowing growth on xylose contained three genes encoding sugar transporters: the previously characterized XUT1 permease, and two new genes (HXT2.6 and QUP2) not previously identified as xylose transporters. High cell density fermentations with the recombinant strains showed that the XUT1 gene allowed ethanol production from xylose or xylose plus glucose as carbon sources, while the HXT2.6 permease produced both ethanol and xylitol, and the strain expressing the QUP2 gene produced mainly xylitol during xylose consumption. CONCLUSIONS: Cloning novel sugar transporters not previously identified in the S. stipitis genome using an hxt-null S. cerevisiae strain with a high xylose-utilizing pathway provides novel promising target genes for improved lignocellulosic ethanol production by yeasts.

Xylose and xylose/glucose co-fermentation by recombinant Saccharomyces cerevisiae strains expressing individual hexose transporters.

Since the uptake of xylose is believed to be one of the rate-limiting steps for xylose ethanol fermentation by recombinant Saccharomyces cerevisiae strains, we transformed a hxt-null strain lacking the major hexose transporters (hxt1Δ-hxt7Δ and gal2Δ) with an integrative plasmid to overexpress the genes for xylose reductase (XYL1), xylitol dehydrogenase (XYL2) and xylulokinase (XKS1), and analyzed the impact that overexpression of the HXT1, HXT2, HXT5 or HXT7 permeases have in anaerobic batch fermentations using xylose, glucose, or xylose plus glucose as carbon sources. Our results revealed that the low-affinity HXT1 permease allowed the maximal consumption of sugars and ethanol production rates during xylose/glucose co-fermentations, but was incapable to allow xylose uptake when this sugar was the only carbon source. The moderately high-affinity HXT5 permease was a poor glucose transporter, and it also did not allow significant xylose uptake by the cells. The moderately high-affinity HXT2 permease allowed xylose uptake with the same rates as those observed during glucose consumption, even under co-fermentation conditions, but had the drawback of producing incomplete fermentations. Finally, the high-affinity HXT7 permease allowed efficient xylose fermentation, but during xylose/glucose co-fermentations this permease showed a clear preference for glucose. Thus, our results indicate that approaches to engineer S. cerevisiae HXT transporters to improve second generation bioethanol production need to consider the composition of the biomass sugar syrup, whereby the HXT1 transporter seems more suitable for hydrolysates containing xylose/glucose blends, whereas the HXT7 permease would be a better choice for xylose-enriched sugar streams.

Draft Genome Sequence of the D-Xylose-Fermenting Yeast Spathaspora arborariae UFMG-HM19.1AT.

The draft genome sequence of the yeast Spathaspora arborariae UFMG-HM19.1A(T) (CBS 11463 = NRRL Y-48658) is presented here. The sequenced genome size is 12.7 Mb, consisting of 41 scaffolds containing a total of 5,625 predicted open reading frames, including many genes encoding enzymes and transporters involved in d-xylose fermentation.

Spathaspora brasiliensis sp. nov., Spathaspora suhii sp. nov., Spathaspora roraimanensis sp. nov. and Spathaspora xylofermentans sp. nov., four novel (D)-xylose-fermenting yeast species from Brazilian Amazonian forest.

Four new D-xylose fermenting yeast species of the clade Spathaspora were recovered from rotting-wood samples in a region of Amazonian forest, Northern Brazil. Three species produced unconjugated asci with a single elongated ascospore with curved ends. These species are described as Spathaspora brasiliensis, Spathaspora suhii and Spathaspora roraimanensis. Two isolates of an asexually reproducing species belonging to the Spathaspora clade were also obtained and they are described as Spathaspora xylofermentans. All these species are able to ferment D-xylose during aerobic batch growth in rich YP (1 % yeast extract, 2 % peptone and 2 % D-xylose) medium, albeit with differing efficiencies. The type strains are Spathaspora brasiliensis sp. nov UFMG-HMD19.3 (=CBMAI 1425=CBS 12679), Spathaspora suhii sp. nov. UFMG-XMD16.2 (=CBMAI 1426=CBS 12680), Spathaspora roraimanensis sp. nov. UFMG-XMD23.2 (CBMAI 1427=CBS 12681) and Spathaspora xylofermentans sp. nov. UFMG-HMD23.3 (=CBMAI 1428=CBS 12682).

Whole-genome sequencing of the efficient industrial fuel-ethanol fermentative Saccharomyces cerevisiae strain CAT-1.

The Saccharomyces cerevisiae strains widely used for industrial fuel-ethanol production have been developed by selection, but their underlying beneficial genetic polymorphisms remain unknown. Here, we report the draft whole-genome sequence of the S. cerevisiae strain CAT-1, which is a dominant fuel-ethanol fermentative strain from the sugarcane industry in Brazil. Our results indicate that strain CAT-1 is a highly heterozygous diploid yeast strain, and the ~12-Mb genome of CAT-1, when compared with the reference S228c genome, contains ~36,000 homozygous and ~30,000 heterozygous single nucleotide polymorphisms, exhibiting an uneven distribution among chromosomes due to large genomic regions of loss of heterozygosity (LOH). In total, 58 % of the 6,652 predicted protein-coding genes of the CAT-1 genome constitute different alleles when compared with the genes present in the reference S288c genome. The CAT-1 genome contains a reduced number of transposable elements, as well as several gene deletions and duplications, especially at telomeric regions, some correlated with several of the physiological characteristics of this industrial fuel-ethanol strain. Phylogenetic analyses revealed that some genes were likely associated with traits important for bioethanol production. Identifying and characterizing the allelic variations controlling traits relevant to industrial fermentation should provide the basis for a forward genetics approach for developing better fermenting yeast strains.

Engineering topology and kinetics of sucrose metabolism in Saccharomyces cerevisiae for improved ethanol yield.

Sucrose is a major carbon source for industrial bioethanol production by Saccharomyces cerevisiae. In yeasts, two modes of sucrose metabolism occur: (i) extracellular hydrolysis by invertase, followed by uptake and metabolism of glucose and fructose, and (ii) uptake via sucrose-proton symport followed by intracellular hydrolysis and metabolism. Although alternative start codons in the SUC2 gene enable synthesis of extracellular and intracellular invertase isoforms, sucrose hydrolysis in S. cerevisiae predominantly occurs extracellularly. In anaerobic cultures, intracellular hydrolysis theoretically enables a 9% higher ethanol yield than extracellular hydrolysis, due to energy costs of sucrose-proton symport. This prediction was tested by engineering the promoter and 5' coding sequences of SUC2, resulting in predominant (94%) cytosolic localization of invertase. In anaerobic sucrose-limited chemostats, this iSUC2-strain showed an only 4% increased ethanol yield and high residual sucrose concentrations indicated suboptimal sucrose-transport kinetics. To improve sucrose-uptake affinity, it was subjected to 90 generations of laboratory evolution in anaerobic, sucrose-limited chemostat cultivation, resulting in a 20-fold decrease of residual sucrose concentrations and a 10-fold increase of the sucrose-transport capacity. A single-cell isolate showed an 11% higher ethanol yield on sucrose in chemostat cultures than an isogenic SUC2 reference strain, while transcriptome analysis revealed elevated expression of AGT1, encoding a disaccharide-proton symporter, and other maltose-related genes. After deletion of both copies of the duplicated AGT1, growth characteristics reverted to that of the unevolved SUC2 and iSUC2 strains. This study demonstrates that engineering the topology of sucrose metabolism is an attractive strategy to improve ethanol yields in industrial processes.

Candida queiroziae sp. nov., a cellobiose-fermenting yeast species isolated from rotting wood in Atlantic Rain Forest.

Eight strains of a novel yeast species were isolated from rotting wood and wood-boring insects in Atlantic Rain Forest ecosystems in Brazil. Sequences of the D1/D2 domains of the large subunit of the rRNA gene showed that the yeast belongs to the Scheffersomyces clade and that it is related to Candida lignicola and Candida coipomoensis. The new species was isolated from rotting wood of three different localities and a wood-boring insect suggesting that these substrates are its ecological niche. This new yeast species is able to assimilate cellobiose and other compounds related to rotting wood. Strong fermentation of cellobiose in Durham tubes was observed for the strains of this new yeast. The new species produced an intracellular ß-glucosidase responsible for cellobiose hydrolysis. The novel species, Candida queiroziae sp. nov., is proposed to accommodate these isolates. The type strain of C. queiroziae is UFMG-CLM 5.1(T) (=CBS 11853(T) = NRRL Y-48722(T)).

Industrial fuel ethanol yeasts contain adaptive copy number changes in genes involved in vitamin B1 and B6 biosynthesis.

Fuel ethanol is now a global energy commodity that is competitive with gasoline. Using microarray-based comparative genome hybridization (aCGH), we have determined gene copy number variations (CNVs) common to five industrially important fuel ethanol Saccharomyces cerevisiae strains responsible for the production of billions of gallons of fuel ethanol per year from sugarcane. These strains have significant amplifications of the telomeric SNO and SNZ genes, which are involved in the biosynthesis of vitamins B6 (pyridoxine) and B1 (thiamin). We show that increased copy number of these genes confers the ability to grow more efficiently under the repressing effects of thiamin, especially in medium lacking pyridoxine and with high sugar concentrations. These genetic changes have likely been adaptive and selected for in the industrial environment, and may be required for the efficient utilization of biomass-derived sugars from other renewable feedstocks.