Genome engineering of Kluyveromyces marxianus for high D-( -)-lactic acid production under low pH conditions.

Saccharomyces cerevisiae is the workhorse of fermentation industry. Upon engineering for D-lactate production by a series of gene deletions, this yeast had deficiencies in cell growth and D-lactate production at high substrate concentrations. Complex nutrients or high cell density were thus required to support growth and D-lactate production with a potential to increase medium and process cost of industrial-scale D-lactate production. As an alternative microbial biocatalyst, a Crabtree-negative and thermotolerant yeast Kluyveromyces marxianus was engineered in this study to produce high titer and yield of D-lactate at a lower pH without growth defects. Only pyruvate decarboxylase 1 (PDC1) gene was replaced by a codon-optimized bacterial D-lactate dehydrogenase (ldhA). Ethanol, glycerol, or acetic acid was not produced by the resulting strain, KMΔpdc1::ldhA. Aeration rate at 1.5 vvm and culture pH 5.0 at 30 °C provided the highest D-lactate titer of 42.97 ± 0.48 g/L from glucose. Yield and productivity of D-lactate, and glucose-consumption rate were 0.85 ± 0.01 g/g, 0.90 ± 0.01 g/(L·h), and 1.06 ± 0.00 g/(L·h), respectively. Surprisingly, D-lactate titer, productivity, and glucose-consumption rate of 52.29 ± 0.68 g/L, 1.38 ± 0.05 g/(L·h), and 1.22 ± 0.00 g/(L·h), respectively, were higher at 42 °C compared to 30 °C. Sugarcane molasses, a low-value carbon, led to the highest D-lactate titer and yield of 66.26 ± 0.81 g/L and 0.91 ± 0.01 g/g, respectively, in a medium without additional nutrients. This study is a pioneer work of engineering K. marxianus to produce D-lactate at the yield approaching theoretical maximum using simple batch process. Our results support the potential of an engineered K. marxianus for D-lactate production on an industrial scale. KEY POINTS: • K. marxianus was engineered by deleting PDC1 and expressing codon-optimized D-ldhA. • The strain allowed high D-lactate titer and yield under pH ranging from 3.5 to 5.0. • The strain produced 66 g/L D-lactate at 30 °C from molasses without any additional nutrients.

NAD+-Dependent Formate Dehydrogenase from Themotolerant Yeast Ogataea parapolymorpha: Properties and Protein Engineering of the N-Terminal Sequence.

Previously, the gene of formate dehydrogenase (FDH, EC 1.2.1.2) from the thermotolerant methylotrophic yeast Ogataea parapolymorpha DL 1 (OpaFDH) was cloned in our laboratory. Recombinant enzyme with additional glycine amino acid residue (OpaFDH_GK) was obtained in Escherichia coli cells in active and soluble form with a yield of more than 1 g per liter of the medium. In the present work, a detailed comparison of this enzyme with FDHs from other sources was carried out. Among eukaryotic formate dehydrogenases, OpaFDH has the highest thermal stability. To elucidate effect of N-terminal residue on the properties of the enzyme, OpaFDH_K (identical to natural) and OpaFDH_AK variants containing an additional Ala residue at the N-terminus were also obtained. It was shown that addition of an Ala residue to the N-terminus reduces four-fold the rate constant of thermal inactivation compared with the addition of a Gly residue. Addition of six more histidine residues to the N-terminus of OpaFDH_AK leads to acceleration of purification, practically does not affect kinetic parameters, but somewhat reduces thermal stability, which, however, can be restored to the level of OpaFDH_AK stability by adding 0.5 M NaCl.

Distinct Metabolic Flow in Response to Temperature in Thermotolerant Kluyveromyces marxianus.

The intrinsic mechanism of the thermotolerance of Kluyveromyces marxianus was investigated by comparison of its physiological and metabolic properties at high and low temperatures. After glucose consumption, the conversion of ethanol to acetic acid became gradually prominent only at a high temperature (45°C) and eventually caused a decline in viability, which was prevented by exogenous glutathione. Distinct levels of reactive oxygen species (ROS), glutathione, and NADPH suggest a greater accumulation of ROS and enhanced ROS-scavenging activity at a high temperature. Fusion and fission forms of mitochondria were dominantly observed at 30°C and 45°C, respectively. Consistent results were obtained by temperature upshift experiments, including transcriptomic and enzymatic analyses, suggesting a change of metabolic flow from glycolysis to the pentose phosphate pathway. The results of this study suggest that K. marxianus survives at a high temperature by scavenging ROS via metabolic change for a period until a critical concentration of acetate is reached. IMPORTANCE Kluyveromyces marxianus, a thermotolerant yeast, can grow well at temperatures over 45°C, unlike Kluyveromyces lactis, which belongs to the same genus, or Saccharomyces cerevisiae, which is a closely related yeast. K. marxianus may thus bear an intrinsic mechanism to survive at high temperatures. This study revealed the thermotolerant mechanism of the yeast, including ROS scavenging with NADPH, which is generated by changes in metabolic flow.

Reconstruction and analysis of a Kluyveromyces marxianus genome-scale metabolic model.

BACKGROUND: Kluyveromyces marxianus is a thermotolerant yeast with multiple biotechnological potentials for industrial applications, which can metabolize a broad range of carbon sources, including less conventional sugars like lactose, xylose, arabinose and inulin. These phenotypic traits are sustained even up to 45 °C, what makes it a relevant candidate for industrial biotechnology applications, such as ethanol production. It is therefore of much interest to get more insight into the metabolism of this yeast. Recent studies suggested, that thermotolerance is achieved by reducing the number of growth-determining proteins or suppressing oxidative phosphorylation. Here we aimed to find related factors contributing to the thermotolerance of K. marxianus. RESULTS: Here, we reported the first genome-scale metabolic model of Kluyveromyces marxianus, iSM996, using a publicly available Kluyveromyces lactis model as template. The model was manually curated and refined to include the missing species-specific metabolic capabilities. The iSM996 model includes 1913 reactions, associated with 996 genes and 1531 metabolites. It performed well to predict the carbon source utilization and growth rates under different growth conditions. Moreover, the model was coupled with transcriptomics data and used to perform simulations at various growth temperatures. CONCLUSIONS: K. marxianus iSM996 represents a well-annotated metabolic model of thermotolerant yeast, which provides a new insight into theoretical metabolic profiles at different temperatures of K. marxianus. This could accelerate the integrative analysis of multi-omics data, leading to model-driven strain design and improvement.

Selection of thermotolerant Saccharomyces cerevisiae for high temperature ethanol production from molasses and increasing ethanol production by strain improvement.

A thermotolerant ethanol fermenting yeast strain is a key requirement for effective ethanol production at high temperature. This work aimed to select a thermotolerant yeast producing a high ethanol concentration from molasses and increasing its ethanol production by mutagenesis. Saccharomyces cerevisiae DMKU 3-S087 was selected from 168 ethanol producing strains because it produced the highest ethanol concentration from molasses at 40 °C. Optimization of molasses broth composition was performed by the response surface method using Box-Behnken design. In molasses broth containing optimal total fermentable sugars (TFS) of 200 g/L and optimal (NH4)2SO4 of 1 g/L, with an initial pH of 5.5 by shaking flask cultivation at 40 °C ethanol, productivity and yield were 58.4 ± 0.24 g/L, 1.39 g/L/h and 0.29 g/g, respectively. Batch fermentation in a 5 L stirred-tank fermenter with 3 L optimized molasses broth adjusted to an initial pH of 5.5 and fermentation controlled at 40 °C and 300 rpm agitation resulted in 72.4 g/L ethanol, 1.21 g/L/h productivity and 0.36 g/g yield at 60 h. Strain DMKU 3-S087 improvement was performed by mutagenesis using ultraviolet radiation and ethyl methane sulfonate (EMS). Six EMS mutants produced higher ethanol (65.2 ± 0.48-73.0 ± 0.54 g/L) in molasses broth containing 200 g/L TFS and 1 g/L (NH4)2SO4 by shake flask fermentation at 37 °C than the wild type (59.8 ± 0.25 g/L). Among these mutants, only mutant S087E100-265 produced higher ethanol (62.5 ± 0.26 g/L) than the wild type (59.5 ± 0.02 g/L) at 40 °C. In addition, mutant S087E100-265 showed better tolerance to high sugar concentration, furfural, hydroxymethylfurfural and acetic acid than the wild type.

Highly efficient conversion of xylose to ethanol without glucose repression by newly isolated thermotolerant Spathaspora passalidarum CMUWF1-2.

BACKGROUND: Efficient bioconversion of lignocellulosic biomass to bioethanol is one of key challenges in the situation of increasing bioethanol demand. The ethanologenic microbes for such conversion are required to possess abilities of utilization of various sugars including xylose and arabinose in lignocellulosic biomass. As required additional characteristics, there are a weak or no glucose repression that allows cells to simultaneously utilize various sugars together with glucose and thermotolerance for fermentation at high temperatures, which has several advantages including reduction of cooling cost. Spathaspora passalidarum ATCC MYA-4345, a type strains, isolated previously have mainly of these abilities or characteristics but its thermotolerance is not so strong and its glucose repression on xylose utilization is revealed. RESULTS: Newly isolated S. passalidarum CMUWF1-2 was found to have a high ability to produce ethanol from various sugars included in lignocellulosic biomass at high temperatures. The strain achieved ethanol yields of 0.43 g, 0.40 g and 0.20 g ethanol/g xylose at 30 °C, 37 °C and 40 °C, respectively. Interestingly, no significant glucose repression was observed in experiments with mixed sugars, being consistent with the strong resistance to 2-deoxyglucose, and antimycin A showed no effect on its growth in xylose medium. Moreover, the strain was tolerant to glucose and ethanol at concentrations up to 35.0% (w/v) and 8.0% (v/v), respectively. CONCLUSIONS: S. passalidarum CMUWF1-2 was shown to achieve efficient production of ethanol from various sugars and a high ethanol yield from xylose with little accumulation of xylitol. The strain also exhibited stress-resistance including thermotolerance and no detectable glucose repression as beneficial characteristics. Therefore, S. passalidarum CMUWF1-2 has remarkable potential for conversion of lignocellulosic biomass to bioethanol.

Electron transport chain in a thermotolerant yeast.

Yeasts capable of growing and surviving at high temperatures are regarded as thermotolerant. For appropriate functioning of cellular processes and cell survival, the maintenance of an optimal redox state is critical of reducing and oxidizing species. We studied mitochondrial functions of the thermotolerant Kluyveromyces marxianus SLP1 and the mesophilic OFF1 yeasts, through the evaluation of its mitochondrial membrane potential (ΔΨm), ATPase activity, electron transport chain (ETC) activities, alternative oxidase activity, lipid peroxidation. Mitochondrial membrane potential and the cytoplasmic free Ca2+ ions (Ca2+ cyt) increased in the SLP1 yeast when exposed to high temperature, compared with the mesophilic yeast OFF1. ATPase activity in the mesophilic yeast diminished 80% when exposed to 40° while the thermotolerant SLP1 showed no change, despite an increase in the mitochondrial lipid peroxidation. The SLP1 thermotolerant yeast exposed to high temperature showed a diminution of 33% of the oxygen consumption in state 4. The uncoupled state 3 of oxygen consumption did not change in the mesophilic yeast when it had an increase of temperature, whereas in the thermotolerant SLP1 yeast resulted in an increase of 2.5 times when yeast were grown at 30o, while a decrease of 51% was observed when it was exposed to high temperature. The activities of the ETC complexes were diminished in the SLP1 when exposed to high temperature, but also it was distinguished an alternative oxidase activity. Our results suggest that the mitochondria state, particularly ETC state, is an important characteristic of the thermotolerance of the SLP1 yeast strain.

Kluyveromyces marxianus as a host for heterologous protein synthesis.

The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K . marxianus can grow at temperatures 10 °C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced contamination risk; has ability to metabolize a wider variety of sugars, such as lactose and xylose; is the fastest growing eukaryote described so far; and does not require special cultivation techniques (such as fed-batch) to avoid fermentative metabolism. All these advantages exist together with a high secretory capacity, performance of eukaryotic post-translational modifications, and with a generally regarded as safe (GRAS) status. In the last years, replication origins from several Kluyveromyces spp. have been used for the construction of episomal vectors, and also integrative strategies have been developed based on the tendency for non-homologous recombination displayed by K. marxianus. The recessive URA3 auxotrophic marker and the dominant Kan(R) are mostly used for selection of transformed cells, but other markers have been made available. Homologous and heterologous promoters and secretion signals have been characterized, with the K. marxianus INU1 expression and secretion system being of remarkable functionality. The efficient synthesis of roughly 50 heterologous proteins has been demonstrated, including one thermophilic enzyme. In this mini-review, we summarize the physiological characteristics of K. marxianus relevant for its use in the efficient synthesis of heterologous proteins, the efforts performed hitherto in the development of a molecular toolbox for this purpose, and some successful examples.

High temperature alcoholic fermentation of orange peel by the newly isolated thermotolerant Pichia kudriavzevii KVMP10.

UNLABELLED: This work explores the potential for the development of orange peel based ethanol bioprocesses through isolation of the thermotolerant Pichia kudriavzevii KVMP10. A model solution of hydrolysed Valencia orange peel was employed to determine the ethanologenic potential of the yeast, which was maximized at 42°C producing 54 g l(-1) of ethanol. The effect of orange peel oil on bioethanol formation was investigated at 30 and 42°C confirming that the minimum inhibitory peel oil content was 0·01% (v/v). Pichia kudriavzevii KVMP10 demonstrated significant technological advantages for the production of sustainable bioenergy, such as utilization of both hexoses (glucose, sucrose, fructose and galactose) and pentoses (xylose) at high temperatures, exemplifying its great potential for application in orange peel based biorefineries for ethanol production. SIGNIFICANCE AND IMPACT OF THE STUDY: Citrus peel waste is one of the most underutilized and geographically diverse residues in the planet. In attempt to develop a citrus peel based biorefinery we report here the isolation of a yeast which exhibited favourable technological characteristics for the production of ethanol through utilization of the specific food waste. Pichia kudriavzevii KVMP10 was highly thermotolerant and utilized both hexoses and pentoses for ethanol production, which was achieved at elevated rates, highlighting its great potential for application in ethanol production processes from citrus peel.

Physiological responses contributing to multiple stress tolerance in Pichia kudriavzevii with potential enhancement for ethanol fermentation.

Economically feasible ethanol production requires efficient hydrolysis of lignocellulosic biomass and high-temperature processing to enable simultaneous saccharification and fermentation. During the lignocellulolysic hydrolysate, the yeast must encounter with a multiple of inhibitors such as heat and furfural. To solve this problem, a potential fermentative yeast strain that tolerated simultaneous multistress and enhance ethanol concentration was investigated. Twenty yeast isolates were classified into two major yeast species, namely Pichia kudriavzevii (twelve isolates) and Candida tropicalis (eight isolates). All P. kudriavzevii isolates were able to grow at high temperature (45 °C) and exhibited stress tolerance toward furfural. Among P. kudriavzevii isolates, NUCG-S3 presented the highest specific growth rate under each stress condition of heat and furfural, and multistress. Morphological changes in P. kudriavzevii isolates (NUCG-S2, NUCG-S3, NUKL-P1, NUKL-P3, and NUOR-J1) showed alteration in mean cell length and width compared to the non-stress condition. Ethanol production by glucose was also determined. The yeast strain, NUCG-S3, gave the highest ethanol concentrations at 99.46 ± 0.82, 62.23 ± 0.96, and 65.80 ± 0.62 g/l (P < 0.05) under temperature of 30 °C, 40 °C, and 42 °C, respectively. The tolerant isolated yeast NUCG-S3 achieved ethanol production of 53.58 ± 3.36 and 48.06 ± 3.31 g/l (P < 0.05) in the presence of 15 mM furfural and multistress (42 °C with 15 mM furfural), respectively. Based on the results of the present study, the novel thermos and furfural-tolerant yeast strain P. kudriavzevii NUCG-S3 showed promise as a highly proficient yeast for high-temperature ethanol fermentation.

A Thermotolerant Yeast Cyberlindnera rhodanensis DK Isolated from Laphet-so Capable of Extracellular Thermostable ß-Glucosidase Production.

This study aims to utilize the microbial resources found within Laphet-so, a traditional fermented tea in Myanmar. A total of 18 isolates of thermotolerant yeasts were obtained from eight samples of Laphet-so collected from southern Shan state, Myanmar. All isolates demonstrated the tannin tolerance, and six isolates were resistant to 5% (w/v) tannin concentration. All 18 isolates were capable of carboxy-methyl cellulose (CMC) degrading, but only the isolate DK showed ethanol production at 45 °C noticed by gas formation. This ethanol producing yeast was identified to be Cyberlindnera rhodanensis based on the sequence analysis of the D1/D2 domain on rRNA gene. C. rhodanensis DK produced 1.70 ± 0.01 U of thermostable extracellular ß-glucosidase when cultured at 37 °C for 24 h using 0.5% (w/v) CMC as a carbon source. The best two carbon sources for extracellular ß-glucosidase production were found to be either xylose or xylan, with ß-glucosidase activity of 3.07-3.08 U/mL when the yeast was cultivated in the yeast malt extract (YM) broth containing either 1% (w/v) xylose or xylan as a sole carbon source at 37 °C for 48 h. The optimal medium compositions for enzyme production predicted by Plackett-Burman design and central composite design (CCD) was composed of yeast extract 5.83 g/L, peptone 10.81 g/L and xylose 20.20 g/L, resulting in a production of 7.96 U/mL, while the medium composed (g/L) of yeast extract 5.79, peptone 13.68 and xylan 20.16 gave 9.45 ± 0.03 U/mL for 48 h cultivation at 37 °C. Crude ß-glucosidase exhibited a remarkable stability of 100%, 88% and 75% stable for 3 h at 35, 45 and 55 °C, respectively.

Metabolomics Study Reveals Biomarker L-Proline as Potential Stress-Protectant Compound for High-Temperature Bioethanol Fermentation by Yeast Pichia kudriavzevii 1P4.

High-temperature ethanol fermentation (> 40 °C) can be applied as effective bioprocess technology to increase ethanol production. Thermotolerant yeast Pichia kudriavzevii 1P4 showed the ability to produce ethanol at optimum 37 °C. Thus, this study evaluated the ethanol productivity of isolate 1P4 at high-temperature ethanol fermentation (42 and 45 °C) and the identification of metabolite biomarkers using untargeted metabolomics with liquid chromatography-tandem mass spectrometry (LC-MS/MS). 1P4 showed tolerance to temperature stress up to 45 °C and thus relevant for high-temperature fermentation. As measured by gas chromatography (GC), bioethanol production of 1P4 at 30, 37, 42, and 45 °C was 5.8 g/l, 7.1 g/l, 5.1 g/l, and 2.8 g/l, respectively. The classification of biomarker compounds was based on orthogonal projection analysis to latent structure discriminant analysis (OPLS-DA), resulting in L-proline being a suspected biomarker compound for isolate 1P4 tolerance against high-temperature stress. Indeed, supplementation of L-proline on fermentation medium supported the growth of 1P4 at high temperatures (> 40 °C) than without L-proline. The bioethanol production with the addition of the L-proline resulted in the highest ethanol concentration (7.15 g/l) at 42 °C. Supplementation of L-proline as a stress-protective compound increased ethanol productivity at high-temperature fermentation of 42 and 45 °C by 36.35% and 83.33%, respectively, compared without the addition of L-proline. Preliminary interpretation of these results indicates that bioprocess engineering through supplementation of stress-protective compounds L-proline increases the fermentation efficiency of isolate 1P4 at higher temperatures (42 °C and 45 °C).

Mitochondrial Irc3 helicase of the thermotolerant yeast Ogataea polymorpha displays dual DNA- and RNA-stimulated ATPase activity.

Irc3 is one of the six mitochondrial helicases described in Saccharomyces cerevisiae. Physiological functions of Irc3 are not completely understood as both DNA metabolic processes and mRNA translation have been suggested to be direct targets of the helicase. In vitro analysis of Irc3 has been hampered by the modest thermostability of the S. cerevisiae protein. Here, we purified a homologous helicase (Irc3op) of the thermotolerant yeast Ogataea polymorpha that retains structural integrity and catalytic activity at temperatures above 40 °C. Irc3op can complement the respiratory deficiency phenotype of a S. cerevisiae irc3Δ mutant, indicating conservation of biochemical functions. The ATPase activity of Irc3op is best stimulated by branched and double- stranded DNA cofactors. Single-stranded DNA binds Irc3op tightly but is a weak activator of the ATPase activity. We could also detect a lower level stimulation with RNA, especially with molecules possessing a compact three-dimensional structure. These results support the idea that that Irc3 might have dual specificity and remodel both DNA and RNA molecules in vivo. Furthermore, our analysis of kinetic parameters predicts that Irc3 could have a regulatory function via sensing changes of the mitochondrial ATP pool or respond to the accumulation of single-stranded DNA.

RNA polymerase II-driven CRISPR-Cas9 system for efficient non-growth-biased metabolic engineering of Kluyveromyces marxianus.

The thermotolerant yeast Kluyveromyces marxianus has gained significant attention in recent years as a promising microbial candidate for industrial biomanufacturing. Despite several contributions to the expanding molecular toolbox for gene expression and metabolic engineering of K. marxianus, there remains a need for a more efficient and versatile genome editing platform. To address this, we developed a CRISPR-based editing system that enables high efficiency marker-less gene disruptions and integrations using only 40 bp homology arms in NHEJ functional and non-functional K. marxianus strains. The use of a strong RNA polymerase II promoter allows efficient expression of gRNAs flanked by the self-cleaving RNA structures, tRNA and HDV ribozyme, from a single plasmid co-expressing a codon optimized Cas9. Implementing this system resulted in nearly 100% efficiency of gene disruptions in both NHEJ-functional and NHEJ-deficient K. marxianus strains, with donor integration efficiencies reaching 50% and 100% in the two strains, respectively. The high gRNA targeting performance also proved instrumental for selection of engineered strains with lower growth rate but improved polyketide biosynthesis by avoiding an extended outgrowth period, a common method used to enrich for edited cells but that fails to recover advantageous mutants with even slightly impaired fitness. Finally, we provide the first demonstration of simultaneous, markerless integrations at multiple loci in K. marxianus using a 2.6 kb and a 7.6 kb donor, achieving a dual integration efficiency of 25.5% in a NHEJ-deficient strain. These results highlight both the ease of use and general robustness of this system for rapid and flexible metabolic engineering in this non-conventional yeast.

Bioethanol Production from Cellulose-Rich Corncob Residue by the Thermotolerant Saccharomyces cerevisiae TC-5.

This study aimed to select thermotolerant yeast for bioethanol production from cellulose-rich corncob (CRC) residue. An effective yeast strain was identified as Saccharomyces cerevisiae TC-5. Bioethanol production from CRC residue via separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), and prehydrolysis-SSF (pre-SSF) using this strain were examined at 35-42 °C compared with the use of commercial S. cerevisiae. Temperatures up to 40 °C did not affect ethanol production by TC-5. The ethanol concentration obtained via the commercial S. cerevisiae decreased with increasing temperatures. The highest bioethanol concentrations obtained via SHF, SSF, and pre-SSF at 35-40 °C of strain TC-5 were not significantly different (20.13-21.64 g/L). The SSF process, with the highest ethanol productivity (0.291 g/L/h), was chosen to study the effect of solid loading at 40 °C. A CRC level of 12.5% (w/v) via fed-batch SSF resulted in the highest ethanol concentrations of 38.23 g/L. Thereafter, bioethanol production via fed-batch SSF with 12.5% (w/v) CRC was performed in 5-L bioreactor. The maximum ethanol concentration and ethanol productivity values were 31.96 g/L and 0.222 g/L/h, respectively. The thermotolerant S. cerevisiae TC-5 is promising yeast for bioethanol production under elevated temperatures via SSF and the use of second-generation substrates.

Physiological characterization of a new thermotolerant yeast strain isolated during Brazilian ethanol production, and its application in high-temperature fermentation.

BACKGROUND: The use of thermotolerant yeast strains can improve the efficiency of ethanol fermentation, allowing fermentation to occur at temperatures higher than 40 °C. This characteristic could benefit traditional bio-ethanol production and allow simultaneous saccharification and fermentation (SSF) of starch or lignocellulosic biomass. RESULTS: We identified and characterized the physiology of a new thermotolerant strain (LBGA-01) able to ferment at 40 °C, which is more resistant to stressors as sucrose, furfural and ethanol than CAT-1 industrial strain. Furthermore, this strain showed similar CAT-1 resistance to acetic acid and lactic acid, and it was also able to change the pattern of genes involved in sucrose assimilation (SUC2 and AGT1). Genes related to the production of proteins involved in secondary products of fermentation were also differentially regulated at 40 °C, with reduced expression of genes involved in the formation of glycerol (GPD2), acetate (ALD6 and ALD4), and acetyl-coenzyme A synthetase 2 (ACS2). Fermentation tests using chemostats showed that LBGA-01 had an excellent performance in ethanol production in high temperature. CONCLUSION: The thermotolerant LBGA-01 strain modulates the production of key genes, changing metabolic pathways during high-temperature fermentation, and increasing its resistance to high concentration of ethanol, sugar, lactic acid, acetic acid, and furfural. Results indicate that this strain can be used to improve first- and second-generation ethanol production in Brazil.

Survival of Kluyveromyces marxianus with stigmasterol as subjected to freezing stress.

Kluyveromyces marxianus is an aerobic yeast and is interested to be applied in many industries. This research was aimed to study the effect of the sterol alternative to ergosterol on the freezing stress of K. marxianus UBU1-11, a thermotolerant yeast. The 0-9 mgL-1 stigmasterol were added to the YM broth and applied for culturing. The growth of all conditions were not interfered by the addition of stigmasterol. The intra-cellular sterol content was detected in the medium with 5 mgL-1 stigmasterol and higher, where the maximum content was 0.32 mg g-1 cell dry weight. After frozen and thawed, the cultures contained stigmasterol had significantly higher viability than those without. It was found that the amount of stigmasterol contained in cells did not affect the number of survival. The stigmasterol provided a significant protection to the yeast cell when subjected to slow freezing. It also increased the survival rate of the culture subjected to subzero temperature storage.

Alleviation of catabolite repression in Kluyveromyces marxianus: the thermotolerant SBK1 mutant simultaneously coferments glucose and xylose.

BACKGROUND: Simultaneous cofermentation of glucose and xylose mixtures would be a cost-effective solution for the conversion of cellulosic biomass to high-value products. However, most yeasts ferment glucose and xylose sequentially due to glucose catabolite repression. A well known thermotolerant yeast, Kluyveromyces marxianus, was selected for this work because it possesses cost-effective advantages over Saccharomyces cerevisiae for biofuel production from cellulosic biomass. RESULTS: In the present study, we employed a directed evolutionary approach using 2-deoxyglucose to develop a thermotolerant mutant capable of simultaneous cofermentation of glucose and xylose by alleviating catabolite repression. The selected mutant, K. marxianus SBK1, simultaneously cofermented 40 g/L glucose and 28 g/L xylose to produce 23.82 g/L ethanol at 40 °C. This outcome corresponded to a yield of 0.35 g/g and productivity of 0.33 g/L h, representing an 84% and 129% improvement, respectively, over the parental strain. Interestingly, following mutagenesis the overall transcriptome of the glycolysis pathway was highly downregulated in K. marxianus SBK1, except for glucokinase-1 (GLK1) which was 21-fold upregulated. Amino acid sequence of GLK1 from K. marxianus SBK1 revealed three amino acid mutations which led to more than 22-fold lower enzymatic activity compared to the parental strain. CONCLUSIONS: We herein successfully demonstrated that the cofermentation of a sugar mixture is a promising strategy for the efficient utilization of cellulosic biomass by K. marxianus SBK1. Through introduction of additional biosynthetic pathways, K. marxianus SBK1 could become a chassis-type strain for the production of fuels and chemicals from cellulosic biomass.

Improvement of Brazilian bioethanol production - Challenges and perspectives on the identification and genetic modification of new strains of Saccharomyces cerevisiae yeasts isolated during ethanol process.

In Brazil, bioethanol is produced by sucrose fermentation from sugarcane by Saccharomyces cerevisiae in a fed-batch process that uses high density of yeast cells (15-25 % of wet weight/v) and high sugar concentration (18-22 % of total sugars). Several research efforts have been employed to improve the efficiency of this process through the isolation of yeasts better adapted to the Brazilian fermentation conditions. Two important wild strains named CAT-1 and PE-2 were isolated during the fermentation process and were responsible for almost 60 % of the total ethanol production in Brazil. However, in the last decade the fermentative substrate composition was much modified, since new sugar cane crops were developed, the use of molasses instead of sugar cane juice increase and with the prohibition of burning of sugarcane prior harvest. As consequence, these previously isolated strains are being replaced by new wild yeasts in most of ethanol plants. In this new scenario the isolation of novel better adapted yeasts with improved fermentative characteristics is still a big challenge. Here, we discuss the main aspects of Brazilian ethanol production and the efforts for the selection, characterization and genetic modifications of new strains with important phenotypic traits such as thermotolerance.

An integrated process for xylooligosaccharide and bioethanol production from corncob.

An integrated process for xylooligosaccharides (XOs) and bioethanol production from corncob was investigated. XOs were produced by a consecutive process of KOH treatment and hydrolysis by an in-house thermostable endo-xylanase from Streptomyces thermovulgaris. XO yields of 0.15 g/gKOH-treated corncob (22.13 g/L) and 0.52 g/graw corncob of cellulose-rich corncob (CRC) were obtained. After 96 h of enzymatic hydrolysis, CRC hydrolysate contained 62.16, 51.21, 10.03 and 0.92 g/L of total sugar, glucose, xylose and arabinose, respectively. Bioethanol production by separate hydrolysis and fermentation (SHF) using CRC hydrolysate, and by simultaneous saccharification and fermentation (SSF) using CRC was studied at 40 °C for thermotolerant Candida glabrata. SHF showed an ethanol yield of 0.28 g/gCRC (21.92 g/L) and ethanol productivity of 0.304 g/L/h with 93% theoretical yield. Surprisingly, by SSF, those parameters were 0.27 g/gCRC (31.32 g/L), 0.33 g/L/h and 89%, respectively. This integrated process might be a new cost-effective approach for corncob valorization.