Superior Yeast Research Articles

Novel Saccharomyces uvarum x Saccharomyces kudriavzevii synthetic hybrid with enhanced 2-phenylethanol production

BackgroundOver the last two decades, hybridization has been a powerful tool used to construct superior yeast for brewing and winemaking. Novel hybrids were primarily constructed using at least one Saccharomyces cerevisiae parent. However, little is known about hybrids used for other purposes, such as targeted flavor production, for example, 2-phenylethanol (2-PE). 2-PE, an aromatic compound widely utilised in the food, cosmetic, and pharmaceutical industries, presents challenges in biotechnological production due to its toxic nature. Consequently, to enhance productivity and tolerance to 2-PE, various strategies such as mutagenesis and genetic engineering are extensively explored to improved yeast strains. While biotechnological efforts have predominantly focused on S. cerevisiae for 2-PE production, other Saccharomyces species and their hybrids remain insufficiently described.ResultsTo address this gap, in this study, we analysed a new interspecies yeast hybrid, II/6, derived from S. uvarum and S. kudriavzevii parents, in terms of 2-PE bioconversion and resistance to its high concentration, comparing it with the parental strains. Two known media for 2-PE biotransformation and three different temperatures were used during this study to determine optimal conditions. In 72 h batch cultures, the II/6 hybrid achieved a maximum of 2.36 ± 0.03 g/L 2-PE, which was 2–20 times higher than the productivity of the parental strains. Our interest lay not only in determining whether the hybrid improved in productivity but also in assessing whether its susceptibility to high 2-PE titers was also mitigated. The results showed that the hybrid exhibited significantly greater resistance to the toxic product than the original strains.ConclusionsThe conducted experiments have confirmed that hybridization is a promising method for modifying yeast strains. As a result, both 2-PE production yield and tolerance to its inhibitory effects can be increased. Furthermore, this strategy allows for the acquisition of non-GMO strains, alleviating concerns related to additional legislative requirements or consumer acceptance issues for producers. The findings obtained have the potential to contribute to the development of practical solutions in the future.

NTH2 1271_1272delTA Gene Disruption Results in Salt Tolerance in Saccharomyces cerevisiae

Trehalose is a common energy reservoir, and its accumulation results in osmotic protection. This sugar can accumulate through its synthesis or slow degradation of the reservoir by trehalase enzymes. Saccharomyces cerevisiae contains two neutral trehalases, NTH1 and NTH2, responsible for 75% and 25% of the enzymatic metabolism. We were interested in the loss-of-function of both enzymes with CRISPR/Cas9. The later NTH2 was of great importance since it is responsible for minor metabolic degradation of this sugar. It was believed that losing its functionality results in limited osmotic protection. We constructed an osmotolerant superior yeast capable of growing in 0.85 M NaCl after independent nth21271_1272delTA mutation by CRISPR/Cas9 technology, compared with nth1 893_894insT and wild type. We suggest that this yeast model could give clues to breeding commercial yeast resulting in non-GMO salinity-tolerant strains.

How adaptive laboratory evolution can boost yeast tolerance to lignocellulosic hydrolyses.

The yeast Saccharomyces cerevisiae is an excellent candidate for establishing cell factories to convert lignocellulosic biomass into chemicals and fuels. To enable this technology, yeast robustness must be improved to withstand the fermentation inhibitors (e.g., weak organic acids, phenols, and furan aldehydes) resulting from biomass pretreatment and hydrolysis. Here, we discuss how evolution experiments performed in the lab, a method commonly known as adaptive laboratory evolution (ALE), may contribute to lifting yeast tolerance against the inhibitors of lignocellulosic hydrolysates (LCHs). The key is that, through the combination of whole-genome sequencing and reverse engineering, ALE provides a robust platform for discovering and testing adaptive alleles, allowing to explore the genetic underpinnings of yeast responses to LCHs. We review the insights gained from past evolution experiments with S. cerevisiae in LCH inhibitors and propose experimental designs to optimise the discovery of genetic variants adaptive to biomass toxicity. The knowledge gathered through ALE projects is envisaged as a roadmap to engineer superior yeast strains for biomass-based bioprocesses.

Selection of Superior Yeast Strains for the Fermentation of Lignocellulosic Steam-Exploded Residues.

The production of lignocellulosic ethanol calls for a robust fermentative yeast able to tolerate a wide range of toxic molecules that occur in the pre-treated lignocellulose. The concentration of inhibitors varies according to the composition of the lignocellulosic material and the harshness of the pre-treatment used. It follows that the versatility of the yeast should be considered when selecting a robust strain. This work aimed at the validation of seven natural Saccharomyces cerevisiae strains, previously selected for their industrial fitness, for their application in the production of lignocellulosic bioethanol. Their inhibitor resistance and fermentative performances were compared to those of the benchmark industrial yeast S. cerevisiae Ethanol Red, currently utilized in the second-generation ethanol plants. The yeast strains were characterized for their tolerance using a synthetic inhibitor mixture formulated with increasing concentrations of weak acids and furans, as well as steam-exploded lignocellulosic pre-hydrolysates, generally containing the same inhibitors. The eight non-diluted liquors have been adopted to assess yeast ability to withstand bioethanol industrial conditions. The most tolerant S. cerevisiae Fm17 strain, together with the reference Ethanol Red, was evaluated for fermentative performances in two pre-hydrolysates obtained from cardoon and common reed, chosen for their large inhibitor concentrations. S. cerevisiae Fm17 outperformed the industrial strain Ethanol Red, producing up to 18 and 39 g/L ethanol from cardoon and common reed, respectively, with ethanol yields always higher than those of the benchmark strain. This natural strain exhibits great potential to be used as superior yeast in the lignocellulosic ethanol plants.

Indigenous Yeast for Bioethanol Production

The second generation of bioethanol made from lignocellulosic biomass which is considered a clean energy source and has high potential of alternative and renewable energy sources. Yeast play an essential role in bioethanol production as fermentation agent a wide range of sugars to ethanol. The great yeast biodiveristy isolated from plants, fruit, or its part could be an potential source of strain. Indigenous yeasts screened and isolated from tuak, an traditional beverages, durian fruit, coconut water fermented some monosacharides of lignocellulose. Yeast isolates migh have ‘superior yeast’ which has characteristics such as tolerant to high temperature, acids, inhibitors, high ethanol level. These isolates are important for the development of efficient ethanol production, therefore very attractive for the fuel elcohol industry.

Enhancing bioethanol productivity by a yeast-immobilized catalytically active membrane in a fermentation-pervaporation coupling process

Bioethanol is a promising substitute for fossil fuels and is mainly produced through yeast fermentation. However, in conventional batch fermentation process extracellular ethanol accumulation restricts yeast excretion of intracellular ethanol, and results in suppressed yeast viability and ethanol productivity. The present study reports a yeast-immobilized catalytically active membrane for efficient extracellular ethanol removal towards enhanced bioethanol productivity in pervaporation membrane reactor. The membrane was fabricated by immersion phase inversion through coating a porous yeast/polyethersulfone catalytic layer on a polydimethylsiloxane (PDMS) pervaporation membrane. Benefited from the high porosity of biocatalytic layer, the immobilized yeast exhibited superior fermentation capacity in high substrate concentration when compared with free yeasts. Most importantly, by comparing the fermentation performances in catalytically active membrane reactor (CAMR), inert membrane reactor and batch reactor, CAMR exhibited the highest ethanol productivity (3.05 g L−1 h−1) and yeast mass concentration (26.41 g L−1), indicating a superior yeast growth rate and viability in CAMR. We attribute this to a “genuine” in situ removal ability of the bio-catalytically active membrane: the extracellular ethanol was promptly removed from the vicinity of the yeast, promoting a more rapid excretion of the inhibitor (intracellular ethanol) and thus maximally eliminated undesirable inhibition of the intracellular ethanol.

Novel Yeast Strains for the Efficient Saccharification and Fermentation of Starchy By-Products to Bioethanol

The use of solid starchy waste streams to produce value-added products, such as fuel ethanol, is a priority for the global bio-based economy. Despite technological advances, bioethanol production from starch is still not economically competitive. Large cost-savings can be achieved through process integration (consolidated bioprocessing, CBP) and new amylolytic microbes that are able to directly convert starchy biomass into fuel in a single bioreactor. Firstly, CBP technology requires efficient fermenting yeast strains to be engineered for amylase(s) production. This study addressed the selection of superior yeast strains with high fermentative performances to be used as recipient for future CBP engineering of fungal amylases. Twenty-one newly isolated wild-type Saccharomyces cerevisiae strains were screened at 30 °C in a simultaneous saccharification and fermentation (SSF) set up using starchy substrates at high loading (20% w/v) and the commercial amylases cocktail STARGEN™ 002. The industrial yeast Ethanol Red™ was used as benchmark. A cluster of strains produced ethanol levels (up to 118 g/L) significantly higher than those of Ethanol Red™ (about 109 g/L). In particular, S. cerevisiae L20, selected for a scale-up process into a 1-L bioreactor, confirmed the outstanding performance over the industrial benchmark, producing nearly 101 g/L ethanol instead of 94 g/L. As a result, this strain can be a promising CBP host for heterologous expression of fungal amylases towards the design of novel and efficient starch-to-ethanol routes.

HAA1 and PRS3 overexpression boosts yeast tolerance towards acetic acid improving xylose or glucose consumption: unravelling the underlying mechanisms.

Acetic acid tolerance and xylose consumption are desirable traits for yeast strains used in industrial biotechnological processes. In this work, overexpression of a weak acid stress transcriptional activator encoded by the gene HAA1 and a phosphoribosyl pyrophosphate synthetase encoded by PRS3 in a recombinant industrial Saccharomyces cerevisiae strain containing a xylose metabolic pathway was evaluated in the presence of acetic acid in xylose- or glucose-containing media. HAA1 or PRS3 overexpression resulted in superior yeast growth and higher sugar consumption capacities in the presence of 4g/L acetic acid, and a positive synergistic effect resulted from the simultaneous overexpression of both genes. Overexpressing these genes also improved yeast adaptation to a non-detoxified hardwood hydrolysate with a high acetic acid content. Furthermore, the overexpression of HAA1 and/or PRS3 was found to increase the robustness of yeast cell wall when challenged with acetic acid stress, suggesting the involvement of the modulation of the cell wall integrity pathway. This study clearly shows HAA1 and/or, for the first time, PRS3 overexpression to play an important role in the improvement of industrial yeast tolerance towards acetic acid. The results expand the molecular toolbox and add to the current understanding of the mechanisms involved in higher acetic acid tolerance, paving the way for the further development of more efficient industrial processes.

Dry-grind processing using amylase corn and superior yeast to reduce the exogenous enzyme requirements in bioethanol production.

BackgroundConventional corn dry-grind ethanol production process requires exogenous alpha and glucoamylases enzymes to breakdown starch into glucose, which is fermented to ethanol by yeast. This study evaluates the potential use of new genetically engineered corn and yeast, which can eliminate or minimize the use of these external enzymes, improve the economics and process efficiencies, and simplify the process. An approach of in situ ethanol removal during fermentation was also investigated for its potential to improve the efficiency of high-solid fermentation, which can significantly reduce the downstream ethanol and co-product recovery cost.ResultsThe fermentation of amylase corn (producing endogenous α-amylase) using conventional yeast and no addition of exogenous α-amylase resulted in ethanol concentration of 4.1 % higher compared to control treatment (conventional corn using exogenous α-amylase). Conventional corn processed with exogenous α-amylase and superior yeast (producing glucoamylase or GA) with no exogenous glucoamylase addition resulted in ethanol concentration similar to control treatment (conventional yeast with exogenous glucoamylase addition). Combination of amylase corn and superior yeast required only 25 % of recommended glucoamylase dose to complete fermentation and achieve ethanol concentration and yield similar to control treatment (conventional corn with exogenous α-amylase, conventional yeast with exogenous glucoamylase). Use of superior yeast with 50 % GA addition resulted in similar increases in yield for conventional or amylase corn of approximately 7 % compared to that of control treatment. Combination of amylase corn, superior yeast, and in situ ethanol removal resulted in a process that allowed complete fermentation of 40 % slurry solids with only 50 % of exogenous GA enzyme requirements and 64.6 % higher ethanol yield compared to that of conventional process.ConclusionsUse of amylase corn and superior yeast in the dry-grind processing industry can reduce the total external enzyme usage by more than 80 %, and combining their use with in situ removal of ethanol during fermentation allows efficient high-solid fermentation.

Improved stress resistance and ethanol production by segmental haploidization of the diploid genome in Saccharomyces cerevisiae

Saccharomyces cerevisiae strains from industrial and natural geographical environments are reported to show great variation in copy number of chromosomal regions. Such variation contributes to the mechanisms underlying adaptation to different environments. Here, we created and phenotypically analyzed segmentally haploidized strains, each harboring a deletion of one copy of approximately 100-300 kb of the left or right terminal region of 16 chromosomes in a diploid strain by using a PCR-mediated chromosomal deletion method. No haploidized strain of the 158-kb deleted right terminal region of chromosome III or the 172-kb deleted right terminal region of chromosome VI was produced; however, segmentally haploidized strains of the remaining 30 terminal regions were obtained. Among these 30 strains, two exhibited higher lactic acid resistance and two displayed higher thermo-tolerance at 41°C versus the host diploid strain. By contrast, four and two segmentally haploidized strains showed sensitivity to 6% lactic acid and low temperature at 13°C, respectively. The effect of the decreased copy number of the chromosomal terminal regions on ethanol production was analyzed. As compared with the host diploid strain, a 3.8% and 4.3% improvement in ethanol production in 10% glucose medium was observed for two strains in which one of two copies of the 197-kb left terminal region of chromosome V and one of two copies of the 195-kb left terminal region of chromosome X was deleted, respectively. These results indicate that artificial segmental haploidization might contribute to improvement of industrially important phenotypes and provide a new approach to breeding superior yeast strains.

Antimicrobial activity of mannose-derived glycosides

A set of 14 synthetic mannolipid-mimicking O-mannosides, S-mannosides, and mannosylsulfones varying in aglycone length were evaluated for their antimicrobial activity towards yeast Candida albicans as well as Gram-positive and Gram-negative bacteria, Staphylococcus aureus and Escherichia coli, respectively. S. aureus was the most susceptible to dodecyl α-d-mannopyranoside showing MIC value of 78 μM, while dodecyl α-d-thiomannopyranoside was superior yeast inhibitor exhibiting twofold lower MIC value. On the other hand, E. coli was resistant to both dodecyl glycosides. Mannosides exposure on RAW 264.7 cell line murine macrophages revealed the tight structure—immunobiological activity pattern. No significant cytotoxic effect and suppression of proliferation as a consequence of cell injury following 24 h exposure with 1–100 μg/cm3 mannosides were observed.

(474) A Selected Yeast for Control of Aspergillus flavus in Pistachio Orchards

Aspergillus flavus produces aflatoxin, a cancer-causing contaminant of pistachio in many production areas. A superior yeast strain of Pichia anomala has been demonstrated to inhibit the growth of A. flavus in the laboratory. It was selected for further study and potential release based on tests of durability and ability to inhibit A. flavus growth. This strain has been tested in the field for the past two years to evaluate its ability to survive in a field environment and to inhibit A. flavus production. The yeast was evaluated in the field to determine if: 1) the biocontrol yeast can survive in pistachio orchards; 2) the yeast has no phytotoxic effects on the pistachio trees or nuts; and 3) the yeast can be demonstrated to control A. flavus in the field. Studies during 2003 were conducted using a replicated experiment with three yeast concentrations and a water control. Treatments applied later in the season were found to be most effective. Highest yeast concentrations were observed just prior to harvest. Three spray concentrations and a water control were applied to evaluate possible phytotoxic effects on pistachio during 2004. No differences in leaf or nut appearance, in nut percent splits, or dry weight were observed for any of the treatments when compared to the water control. Artificial wounding experiments were also conducted during 2003 and 2004 to simulate the occurrence of early split nuts, the primary repository for A. flavus contamination. A 5× reduction in A. flavus colonization was observed on treated wounded nuts vs. untreated wounded nuts. A 5× reduction in A. flavus sporulation was also observed on treated wounded nuts vs. untreated wounded nuts.

Tryptic Digests of Sorghum Malt Sprouts: An Assessment of Their Usefulness as Organic Nitrogen Sources for the Yeast Saccharomyces Cerevisiae

The tryptic digests of sprouts from malts of two sorghum cultivars differed significantly in their yeast nutrient composition. While both digests contained elevated levels of Ca2+, Mg2+, and K+, the individual concentrations of the minerals varied with the sprouts' source. At 423.9 μg/g, Mg2+ levels were markedly (P < 0.001) higher in the tryptic digest of Local Red sorghum sprouts than in the Local White counterpart (287.3 μg/g), while the converse was true for Ca2+ and K+. Wide variation also occurred with respect to the nitrogen constituents. For example, the Local White sorghum sprouts digest contained considerably more ammonium, total free amino nitrogen and yeast-essential amino acids than did the Local Red counterpart. In contrast, Local Red malt sprout digest displayed significantly higher urea levels (124.7 mg/g compared with 98.94 mg/g) and superior yeast growth-promoting activity, suggesting that factors other than amino nitrogen also influenced the ability of the digests to support yeast growth. The digests of sorghum malt sprouts compared favorably with seven commercial nitrogenous bases in their ability to promote yeast growth. Results suggest that tryptic digests of sorghum malt sprouts are good alternative sources of nitrogenous and mineral nutrients for microbial cultivation.