Industrial Saccharomyces Cerevisiae Strains Research Articles

Genomic and metabolic instability during long-term fermentation of an industrial Saccharomyces cerevisiae strain engineered for C5 sugar utilization.

The genetic stability and metabolic robustness of production strains is one of the key criteria for the production of bio-based products by microbial fermentation on an industrial scale. These criteria were here explored in an industrial ethanol-producer strain of Saccharomyces cerevisiae able to co-ferment D-xylose and L-arabinose with glucose through the chromosomal integration of several copies of pivotal genes for the use of these pentose (C5) sugars. Using batch sequential cultures in a controlled bioreactor that mimics long-term fermentation in an industrial setting, this strain was found to exhibit significant fluctuations in D-xylose and L-arabinose consumption as early as the 50th generation and beyond. These fluctuations seem not related to the few low-consumption C5 sugar clones that appeared throughout the sequential batch cultures at a frequency lower than 1.5% and that were due to the reduction in the number of copies of transgenes coding for C5 sugar assimilation enzymes. Also, subpopulations enriched with low or high RAD52 expression, whose expression level was reported to be proportional to homologous recombination rate did not exhibit defect in C5-sugar assimilation, arguing that other mechanisms may be responsible for copy number variation of transgenes. Overall, this work highlighted the existence of genetic and metabolic instabilities in an industrial yeast which, although modest in our conditions, could be more deleterious in harsher industrial conditions, leading to reduced production performance.

Modification of the second PEP4-allele facilitates an industrial Saccharomyces cerevisiae to tolerate tartaric acid stress

The practical significance of constructing robust industrial production strains against organic acid stress lies not only in improving fermentation efficiency but also in reducing manufacturing costs. In a previous study, we constructed an industrial Saccharomyces cerevisiae strain by modifying another PEP4-allele of a mutant that already had one PEP4-allele disrupted. This modification enhanced cellular tolerance to citric acid stress during growth. Unlike citric acid, which S. cerevisiae can consume, tartaric acid is often added to grape must during winemaking to increase total acidity and is not metabolizable. The results of the present study indicate that the modification of the second PEP4-allele improves the cellular tolerance of the strain with one PEP4-allele disrupted against tartaric acid stress during growth and contributes to maintaining intracellular pH homeostasis in cells subjected to tartaric acid stress. Moreover, under tartaric acid stress, a significant improvement in glucose-ethanol conversion performance, conferred by the modification of the second PEP4-allele, was observed. This study not only broadens our understanding of the role of the PEP4-allele in cellular regulation but also provides a prospective approach to reducing the concentration of sulfur dioxide used in winemaking.

Multi-Response Optimization of Thermochemical Pretreatment of Soybean Hulls for 2G-Bioethanol Production

Soybean is a major crop used in the production of human food. The soybean hull (SH) is also known as the seed coat and it constitutes about 5–8% of the total seed on a dry weight basis, depending on the variety and the seed size. Dilute sulfuric acid was employed for the thermochemical pretreatment of SH prior to enzymatic saccharification and alcoholic fermentation. Empirical modeling of response surface, severity factor and multi-response desirability function methodology, were used to perform the process optimization. Temperature, acid concentration and reaction time were defined as operational variables, while furfural, 5-hydroxymethylfurfural and solubilized hemicellulose and cellulose were defined as response variables. Mathematical models satisfactorily described the process and optimal conditions were found at 121 °C; 2.5% w/v H2SO4 and 60 min. More than 80% and 90% of hemicelluloses and celluloses, respectively, were able to solubilize at this point. The fermentation performance of an industrial Saccharomyces cerevisiae strain was also evaluated. The glucose available in the hydrolysates was completely consumed in less than 12 h, with an average ethanol yield of 0.45 gethanol/gglucose. Thus, the thermochemical conditioning of SH with dilute sulfuric acid is a suitable operation for 2G-bioethanol production.

Improving Saccharomyces cerevisiae acid and oxidative stress resistance using a prokaryotic gene identified by functional metagenomics

Innovations in obtaining products from lignocellulosic biomass have been largely based on the improvement of microorganisms and enzymes capable of degrading these materials. To complete the whole process, microorganisms must be able to ferment the resulting sugars and tolerate high concentrations of product, osmotic pressure, ion toxicity, temperature, toxic compounds from lignocellulose pretreatment, low pH, and oxidative stress. In this work, we engineered laboratory and industrial Saccharomyces cerevisiae strains by combining a gene (hu) recovered from a metagenomic approach with different native and synthetic promoters to obtain improved acid and oxidative stress resistance. Laboratorial strains harboring hu gene under the control of the synthetic stress responsive PCCW14v5 showed increased survival rates after 2 h exposure to pH 1.5. The hu gene was also able to significantly enhance the tolerance of the industrial strain to high concentrations of H2O2 when combined with PTEF1, PYGP1 or PYGP1v7 after 3 h exposure.

Microcystin-Detoxifying Recombinant Saccharomyces cerevisiae Expressing the mlrA Gene from Sphingosinicella microcystinivorans B9.

Contamination of water by microcystins is a global problem. These potent hepatotoxins demand constant monitoring and control methods in potable water. Promising approaches to reduce contamination risks have focused on natural microcystin biodegradation led by enzymes encoded by the mlrABCD genes. The first enzyme of this system (mlrA) linearizes microcystin structure, reducing toxicity and stability. Heterologous expression of mlrA in different microorganisms may enhance its production and activity, promote additional knowledge on the enzyme, and support feasible applications. In this context, we intended to express the mlrA gene from Sphingosinicella microcystinivorans B9 in an industrial Saccharomyces cerevisiae strain as an innovative biological alternative to degrade microcystins. The mlrA gene was codon-optimized for expression in yeast, and either expressed from a plasmid or through chromosomal integration at the URA3 locus. Recombinant and wild yeasts were cultivated in medium contaminated with microcystins, and the toxin content was analyzed during growth. Whereas no difference in microcystins content was observed in cultivation with the chromosomally integrated strain, the yeast strain hosting the mlrA expression plasmid reduced 83% of toxins within 120 h of cultivation. Our results show microcystinase A expressed by industrial yeast strains as a viable option for practical applications in water treatment.

Renewing Lost Genetic Variability with a Classical Yeast Genetics Approach.

Due to their long domestication time course, many industrial Saccharomyces cerevisiae strains are adopted in numerous processes mostly for historical reasons instead of scientific and technological needs. As such, there is still significant room for improvement for industrial yeast strains relying on yeast biodiversity. This paper strives to regenerate biodiversity with the innovative application of classic genetic methods to already available yeast strains. Extensive sporulation was indeed applied to three different yeast strains, specifically selected for their different origins as well as backgrounds, with the aim of clarifying how new variability was generated. A novel and easy method to obtain mono-spore colonies was specifically developed, and, to reveal the extent of the generated variability, no selection after sporulation was introduced. The obtained progenies were then tested for their growth in defined mediums with high stressor levels. A considerable and strain-specific increase in both phenotypic and metabolomic variability was assessed, and a few mono-spore colonies were found to be of great interest for their future exploitation in selected industrial processes.

Lipidome variation of industrial Saccharomyces cerevisiae strains analyzed by LC-QTOF/MS-based untargeted lipidomics

Although various yeast strains used in the food industry have been characterized by multilayer analysis, knowledge of the variation of lipid profiles involved in fermentation characteristics and stress tolerance remains in its infancy. In this study, untargeted lipidomics was applied to 10 yeast strains, including laboratory, baker's, wine, and sake yeasts, which exhibit distinct fermentation phenotypes, to obtain a comprehensive overview of the yeast lipidome. The relative standard deviation (RSD) in the abundance of the 352 identified lipid molecular species was investigated to reveal the specific and common lipids. Lipids containing very long-chain fatty acids and hydroxy long-chain fatty acids showed relatively large RSD, whereas lipids containing acyl chains, which are commonly found in yeast, such as C16-C18, showed less RSD among the 10 strains. Furthermore, principal component analysis of lipid profiles showed similar trends among industrial yeast strains. As lipids are involved in yeast phenotypes, including stress tolerance and fermentation characteristics, correlation analysis was performed with lipid abundance and phenotypes. The results revealed that molecular species with a high RSD in abundance among the 10 strains were correlated with specific stress tolerance and fermentation phenotypes.

Prospecting and engineering yeasts for ethanol production under inhibitory conditions: an experimental design analysis.

The recently discovered wild yeast Wickerhamomyces sp. UFFS-CE-3.1.2 was analyzed through a high-throughput experimental design to improve ethanol yields in synthetic media with glucose, xylose, and cellobiose as carbon sources and acetic acid, furfural, formic acid, and NaCl as fermentation inhibitors. After Plackett-Burman (PB) and central composite design (CCD), the optimized condition was used in a fermentation kinetic analysis to compare this yeast's performance with an industrial Saccharomyces cerevisiae strain (JDY-01) genetically engineered to achieve a higher xylose fermentation capacity and fermentation inhibitors tolerance by overexpressing the genes XYL1, XYL2, XKS1, and TAL1. Our results show that furfural and NaCl had no significant effect on sugar consumption by UFFS-CE-3.1.2. Surprisingly, acetic acid negatively affected glucose but not xylose and cellobiose consumption. In contrast, the pH positively affected all the analyzed responses, indicating a cell's preference for alkaline environments. In the CCD, sugar concentration negatively affected the yields of ethanol, xylitol, and cellular biomass. Therefore, fermentation kinetics were carried out with the average concentrations of sugars and fermentation inhibitors and the highest tested pH value (8.0). Although UFFS-CE-3.1.2 fermented glucose efficiently, xylose and cellobiose were mainly used for cellular growth. Interestingly, the genetically engineered strain JDY-01 consumed ~ 30% more xylose and produced ~ 20% more ethanol. Also, while UFFS-CE-3.1.2 only consumed 32% of the acetic acid of the medium, JDY-01 consumed > 60% of it, reducing its toxic effects. Thus, the overexpressed genes played an essential role in the inhibitors' tolerance, and the applied engineering strategy may help improve 2G ethanol production.

Saccharomyces cerevisiae employs complex regulation strategies to tolerate low pH stress during ethanol production

BackgroundIndustrial bioethanol production may involve a low pH environment caused by inorganic acids, improving the tolerance of Saccharomyces cerevisiae to a low pH environment is of industrial importance to increase ethanol yield, control bacterial contamination, and reduce production cost. In our previous study, acid tolerance of a diploid industrial Saccharomyces cerevisiae strain KF-7 was chronically acclimatized by continuous ethanol fermentation under gradually increasing low-pH stress conditions. Two haploid strains B3 and C3 having excellent low pH tolerance were derived through the sporulation of an isolated mutant. Diploid strain BC3 was obtained by mating these two haploids. In this study, B3, C3, BC3, and the original strain KF-7 were subjected to comparison transcriptome analysis to investigate the molecular mechanism of the enhanced phenotype.ResultThe comparison transcriptome analysis results suggested that the upregulated vitamin B1 and B6 biosynthesis contributed to the low pH tolerance. Amino acid metabolism, DNA repairment, and general stress response might also alleviate low pH stress.ConclusionSaccharomyces cerevisiae seems to employ complex regulation strategies to tolerate low pH during ethanol production. The findings provide guides for the construction of low pH-tolerant industrial strains that can be used in industrial fermentation processes.

A Hierarchical Transcriptional Regulatory Network Required for Long-Term Thermal Stress Tolerance in an Industrial Saccharomyces cerevisiae Strain.

Yeast cells suffer from continuous and long-term thermal stress during high-temperature ethanol fermentation. Understanding the mechanism of yeast thermotolerance is important not only for studying microbial stress biology in basic research but also for developing thermotolerant strains for industrial application. Here, we compared the effects of 23 transcription factor (TF) deletions on high-temperature ethanol fermentation and cell survival after heat shock treatment and identified three core TFs, Sin3p, Srb2p and Mig1p, that are involved in regulating the response to long-term thermotolerance. Further analyses of comparative transcriptome profiling of the core TF deletions and transcription regulatory associations revealed a hierarchical transcriptional regulatory network centered on these three TFs. This global transcriptional regulatory network provided a better understanding of the regulatory mechanism behind long-term thermal stress tolerance as well as potential targets for transcriptome engineering to improve the performance of high-temperature ethanol fermentation by an industrial Saccharomyces cerevisiae strain.

CRISPR/Cas9-mediated Inactivation of arginase in a yeast strain isolated from Nuruk and its impact on the whole genome

Despite the advantages of CRISPR/Cas9 technology in the food industry, controversy over its off-target effects exists. We engineered an industrial Saccharomyces cerevisiae strain isolated from a Korean rice wine starter, Nuruk, using CRISPR/Cas9 to decrease ethyl carbamate (EC) formation. We disrupted the CAR1 gene encoding arginase, which plays a key role in EC formation. Subsequently, we compared the whole genome of the engineered strain to that of the wild type by analyzing heterozygous and homozygous mutations through variant calling. Homozygous mutations in the genome of the engineered strains were identified as the target mutations in CAR1 induced by CRISPR/Cas9, and no other off-target effects were observed. Our findings have critical implications for the use of CRISRP/Cas9 technology in yeasts in the food industry.

Modification of the second PEP4-allele enhances citric acid stress tolerance during cultivation of an industrial rice wine yeast strain with one PEP4-allele disrupted

To investigate the effect of PEP4 alleles on cellular tolerance of rice wine Saccharomyces cerevisiae against citric acid stress, one PEP4-allele disrupted strain and one PEP4-allele modified along with one PEP4-allele disrupted rice wine Saccharomyces cerevisiae strains were constructed. The PEP4-allele disruption and modification significantly decreased intracellular PrA activity, and a dose-response relationship was found between intracellular Proteinase A activity and number of intact PEP4-alleles. Under cultivation conditions whether with sufficiency or starvation of amino acid nitrogen source, the second PEP4-allele modification was all found beneficial for cellular citric acid stress tolerance of the industrial Saccharomyces cerevisiae strain SWY85. These results allude to PEP4-allele having a physiological role in the metabolic regulations correlated to citric acid tolerance in yeast cell.

Deletion of the MBP1 Gene, Involved in the Cell Cycle, Affects Respiration and Pseudohyphal Differentiation in Saccharomyces cerevisiae.

ABSTRACTMbp1p is a component of MBF (MluI cell cycle box binding factor, Mbp1p-Swi6p) and is well known to regulate the G1–S transition of the cell cycle. However, few studies have provided clues regarding its role in fermentation. This work aimed to recognize the function of the MBP1 gene in ethanol fermentation in a wild-type industrial Saccharomyces cerevisiae strain. MBP1 deletion caused an obvious decrease in the final ethanol concentration under oxygen-limited (without agitation), but not under aerobic, conditions (130 rpm). Furthermore, the mbp1Δ strain showed 84% and 35% decreases in respiration intensity under aerobic and oxygen-limited conditions, respectively. These findings indicate that MBP1 plays an important role in responding to variations in oxygen content and is involved in the regulation of respiration and fermentation. Unexpectedly, mbp1Δ also showed pseudohyphal growth, in which cells elongated and remained connected in a multicellular arrangement on yeast extract-peptone-dextrose (YPD) plates. In addition, mbp1Δ showed an increase in cell volume, associated with a decrease in the fraction of budded cells. These results provide more detailed information about the function of MBP1 and suggest some clues to efficiently improve ethanol production by industrially engineered yeast strains.IMPORTANCE Saccharomyces cerevisiae is an especially favorable organism used for ethanol production. However, inhibitors and high osmolarity conferred by fermentation broth, and high concentrations of ethanol as fermentation runs to completion, affect cell growth and ethanol production. Therefore, yeast strains with high performance, such as rapid growth, high tolerance, and high ethanol productivity, are highly desirable. Great efforts have been made to improve their performance by evolutionary engineering, and industrial strains may be a better start than laboratory ones for industrial-scale ethanol production. The significance of our research is uncovering the function of MBP1 in ethanol fermentation in a wild-type industrial S. cerevisiae strain, which may provide clues to engineer better-performance yeast in producing ethanol. Furthermore, the results that lacking MBP1 caused pseudohyphal growth on YPD plates could shed light on the development of xylose-fermenting S. cerevisiae, as using xylose as the sole carbon source also caused pseudohyphal growth.

Resveratrol Production from Hydrothermally Pretreated Eucalyptus Wood Using Recombinant Industrial Saccharomyces cerevisiae Strains.

Resveratrol is a phenolic compound with strong antioxidant activity, being promising for several applications in health, food, and cosmetics. It is generally extracted from plants or chemically synthesized, in both complex and not sustainable processes, but microbial biosynthesis of resveratrol can counter these drawbacks. In this work, resveratrol production by microbial biosynthesis from lignocellulosic materials was assessed. Three robust industrial Saccharomyces cerevisiae strains known for their thermotolerance and/or resistance to inhibitory compounds were identified as suitable hosts for de novo resveratrol production from glucose and ethanol. Through the CRISPR/Cas9 system, all industrial strains, and a laboratory one, were successfully engineered with the resveratrol biosynthetic pathway via the phenylalanine intermediate. All strains were further screened at 30 °C and 39 °C to evaluate thermotolerance, which is a key feature for Simultaneous Saccharification and Fermentation processes. Ethanol Red RBP showed the best performance at 39 °C, with more than 2.6-fold of resveratrol production in comparison with the other strains. This strain was then used to assess resveratrol production from glucose and ethanol. A maximum resveratrol titer of 187.07 ± 19.88 mg/L was attained from a medium with 2% glucose and 5% ethanol (w/v). Lastly, Ethanol Red RBP produced 151.65 ± 3.84 mg/L resveratrol from 2.95% of cellulose from hydrothermally pretreated Eucalyptus globulus wood, at 39 °C, in a Simultaneous Saccharification and Fermentation process. To the best of our knowledge, this is the first report of lignocellulosic resveratrol production, establishing grounds for the implementation of an integrated lignocellulose-to-resveratrol process in an industrial context.

Comparative 13 C-metabolic flux analysis indicates elevation of ATP regeneration, carbon dioxide, and heat production in industrial Saccharomyces cerevisiae strains.

Various industrial Saccharomyces cerevisiae strains are used for specific processes, such as sake, wine brewing and bread making. Understanding mechanisms underlying the fermentation performance of these strains would be useful for further engineering of the S. cerevisiae metabolism. However, the relationship between the fermentation performance, intra-cellular metabolic states, and other phenotypic characteristics of industrial yeasts is still unclear. In this study, 13 C-metabolic flux analysis of four diploid yeast strains-laboratory, sake, bread, and wine yeasts-was conducted. While the Crabtree effect was observed for all strains, the metabolic flux level of glycolysis was elevated in bread and sake yeast. Furthermore, increased flux levels of the TCA cycle were commonly observed in the three industrial strains. The specific rates of CO2 production, net ATP regeneration, and metabolic heat generation estimated from the metabolic flux distribution were two to three times greater than those of the laboratory strain. The elevation in metabolic heat generation was correlated with the tolerance to low-temperature stress. These results indicate that the metabolic flux distribution of sake and bread yeast strains contributes to faster production of ethanol and CO2 . It is also suggested that the generation of metabolic heat is preferable under the actual industrial fermentation conditions.

Marker Assisted Selection of Malic-Consuming Saccharomyces cerevisiae Strains for Winemaking. Efficiency and Limits of a QTL's Driven Breeding Program.

Natural Saccharomyces cerevisiae yeast strains exhibit very large genotypic and phenotypic diversity. Breeding programs that take advantage of this characteristic are widely used for selecting starters for wine industry, especially in the recent years when winemakers need to adapt their production to climate change. The aim of this work was to evaluate a marker assisted selection (MAS) program to improve malic acid consumption capacity of Saccharomyces cerevisiae in grape juice. Optimal individuals of two unrelated F1-hybrids were crossed to get a new genetic background carrying many “malic consumer” loci. Then, eleven quantitative trait loci (QTLs) already identified were used for implementing the MAS breeding program. By this method, extreme individuals able to consume more than 70% of malic acid in grape juice were selected. These individuals were tested in different enological matrixes and compared to their original parental strains. They greatly reduced the malic acid content at the end of alcoholic fermentation, they appeared to be robust to the environment, and they accelerated the ongoing of malolactic fermentations by Oenococcus oeni. This study illustrates how MAS can be efficiently used for selecting industrial Saccharomyces cerevisiae strains with outlier properties for winemaking.

Reducing glucoamylase usage for commercial-scale ethanol production from starch using glucoamylase expressing Saccharomyces cerevisiae

The development of yeast that converts raw corn or cassava starch to ethanol without adding the exogenous α-amylase and/or glucoamylase would reduce the overall ethanol production cost. In this study, two copies of codon-optimized Saccharomycopsis fibuligera glucoamylase genes were integrated into the genome of the industrial Saccharomyces cerevisiae strain CCTCC M94055, and the resulting strain CIBTS1522 showed comparable basic growth characters with the parental strain. We systemically evaluated the fermentation performance of the CIBTS1522 strain using the raw corn or cassava starch at small and commercial-scale, and observed that a reduction of at least 40% of the dose of glucoamylase was possible when using the CIBTS1522 yeast under real ethanol production condition. Next, we measured the effect of the nitrogen source, the phosphorous source, metal ions, and industrial microbial enzymes on the strain’s cell wet weight and ethanol content, the nitrogen source and acid protease showed a positive effect on these parameters. Finally, orthogonal tests for some other factors including urea, acid protease, inoculum size, and glucoamylase addition were conducted to further optimize the ethanol production. Taken together, the CIBTS1522 strain was identified as an ideal candidate for the bioethanol industry and a better fermentation performance could be achieved by modifying the industrial culture media and condition.

Improving multiple stress-tolerance of a flocculating industrial Saccharomyces cerevisiae strain by random mutagenesis and hybridization

Breeding stress-tolerant industrial Saccharomyces cerevisiae strains is crucial to reduce the cost of bioethanol production. In this study, a novel strategy, atmospheric and room temperature plasma (ARTP) mutagenesis combined with genome shuffling and hybridization, was successfully applied to obtain a multiple stress-tolerant flocculating industrial S. cerevisiae strain E-158. The enhanced strain E-158 grew better under various stress conditions compared with the original strain KF-7. The concentrations of ethanol produced by strain E-158 during batch fermentation were 10.14 %–81.02 % higher than those produced by strain KF-7 under high ethanol, high temperature, and high osmosis stress conditions. The stress tolerance of E-158 kept stable even after 30 times of sub-cultivation. The lower reactive oxygen species (ROS) level and higher superoxide dismutase (SOD) and catalase (CAT) activities in strain E-158 manifested that the antioxidant defense ability of E-158 was improved and contributed to its multiple stress-tolerance. This study provided a stress-tolerant strain for industrial ethanol production and a feasible strategy for breeding multiple stress-tolerant industrial S. cerevisiae strain.

Proteomics Answers Which Yeast Genes Are Specific for Baking, Brewing, and Ethanol Production.

Yeast strains are convenient models for studying domestication processes. The ability of yeast to ferment carbon sources from various substrates and to produce ethanol and carbon dioxide is the core of brewing, winemaking, and ethanol production technologies. The present study reveals the differences among yeast strains used in various industries. To understand this, we performed a proteomic study of industrial Saccharomyces cerevisiae strains followed by a comparative analysis of available yeast genetic data. Individual protein expression levels in domesticated strains from different industries indicated modulation resulting from response to technological environments. The innovative nature of this research was the discovery of genes overexpressed in yeast strains adapted to brewing, baking, and ethanol production, typical genes for specific domestication were found. We discovered a gene set typical for brewer’s yeast strains. Baker’s yeast had a specific gene adapted to osmotic stress. Toxic stress was typical for yeast used for ethanol production. The data obtained can be applied for targeted improvement of industrial strains.

Genome sequence of Acremonium strictum AAJ6 strain isolated from the Cerrado biome in Brazil and CAZymes expression in thermotolerant industrial yeast for ethanol production

Increased demand for biofuels promotes the search for new biomass-degrading fungi. Acremonium strictum is an environmentally widespread filamentous fungi found on plant debris; that secretes lignocellulose-degrading enzymes. A recently isolated A. strictum strain, AAJ6; native to the Brazilian Cerrado biome was evaluated for its capacity to degrade lignocellulosic substrates. In this study, whole-genome sequencing of AAJ6 was performed and 775 CAZy domains were identified which correlated to those of A. strictum strain DS1bioAY4a and other lignocellulolytic fungi; suggesting AAJ6 is a high CAZyme producer. We expressed the glycoside hydrolase families GH74 and GH3 from plasmid or genome-integrated to evaluate the ethanol production from cellulosic substrates in Brazilian industrial Saccharomyces cerevisiae strains (PE-2 and SA-1) evolved for thermotolerance (AMY12 and AMY35). Those expressing the genome-integrated enzymes showed the highest β-glucosidase activity and growth in medium with cellobiose at 40°C. The strain AGY005 (integrated cassettes) showed 19, 23 and 46% higher ethanol production in SHF, pSSF (partial hydrolysis SSF) and SSF processes, respectively, using Avicel, and ∼50% more ethanol using pre-treated sugarcane bagasse, compared to the strain with a plasmid-based expression. These results indicate the improved performance of thermotolerant industrial strains with genome-integrated CAZymes in the SSF process for 2G ethanol.