Saccharomyces cerevisiae Cells Lacking the Zinc Vacuolar Transporter Zrt3 Display Improved Ethanol Productivity in Lignocellulosic Hydrolysates

Yeast-based bioethanol production from lignocellulosic hydrolysates (LH) is an attractive and sustainable alternative for biofuel production. However, the presence of acetic acid (AA) in LH is still a major problem. Indeed, above certain concentrations, AA inhibits yeast fermentation and triggers a regulated cell death (RCD) process mediated by the mitochondria and vacuole. Understanding the mechanisms involved in AA-induced RCD (AA-RCD) may thus help select robust fermentative yeast strains, providing novel insights to improve lignocellulosic ethanol (LE) production. Herein, we hypothesized that zinc vacuolar transporters are involved in vacuole-mediated AA-RCD, since zinc enhances ethanol production and zinc-dependent catalase and superoxide dismutase protect from AA-RCD. In this work, zinc limitation sensitized wild-type cells to AA-RCD, while zinc supplementation resulted in a small protective effect. Cells lacking the vacuolar zinc transporter Zrt3 were highly resistant to AA-RCD, exhibiting reduced vacuolar dysfunction. Moreover, zrt3Δ cells displayed higher ethanol productivity than their wild-type counterparts, both when cultivated in rich medium with AA (0.29 g L−1 h−1 versus 0.11 g L−1 h−1) and in an LH (0.73 g L−1 h−1 versus 0.55 g L−1 h−1). Overall, the deletion of ZRT3 emerges as a promising strategy to increase strain robustness in LE industrial production.

In Vitro and Clinical Evaluation of Cannabigerol (CBG) Produced via Yeast Biosynthesis: A Cannabinoid with a Broad Range of Anti-Inflammatory and Skin Health-Boosting Properties

Cannabigerol (CBG) is a minor non-psychoactive cannabinoid present in Cannabis sativa L. (C. sativa) at low levels (<1% per dry weight) that serves as the direct precursor to both cannabidiol (CBD) and tetrahydrocannabinol (THC). Consequently, efforts to extract and purify CBG from C. sativa is both challenging and expensive. However, utilizing a novel yeast fermentation technology platform, minor cannabinoids such as CBG can be produced in a more sustainable, cost-effective, and timely process as compared to plant-based production. While CBD has been studied extensively, demonstrating several beneficial skin properties, there are a paucity of studies characterizing the activity of CBG in human skin. Therefore, our aim was to characterize and compare the in vitro activity profile of non-psychoactive CBG and CBD in skin and be the first group to test CBG clinically on human skin. Gene microarray analysis conducted using 3D human skin equivalents demonstrates that CBG regulates more genes than CBD, including several key skin targets. Human dermal fibroblasts (HDFs) and normal human epidermal keratinocytes (NHEKs) were exposed in culture to pro-inflammatory inducers to trigger cytokine production and oxidative stress. Results demonstrate that CBG and CBD reduce reactive oxygen species levels in HDFs better than vitamin C. Moreover, CBG inhibits pro-inflammatory cytokine (Interleukin-1β, -6, -8, tumor necrosis factor α) release from several inflammatory inducers, such as ultraviolet A (UVA), ultraviolet B (UVB), chemical, C. acnes, and in several instances does so more potently than CBD. A 20-subject vehicle-controlled clinical study was performed with 0.1% CBG serum and placebo applied topically for 2 weeks after sodium lauryl sulfate (SLS)-induced irritation. CBG serum showed statistically significant improvement above placebo for transepidermal water loss (TEWL) and reduction in the appearance of redness. Altogether, CBG’s broad range of in vitro and clinical skin health-promoting activities demonstrates its strong potential as a safe, effective ingredient for topical use and suggests there are areas where it may be more effective than CBD.

Effect of Berry Extracts on Saccharomyces cerevisiae Yeast

Introduction. Fruit and berry extracts contain biologically active components and acids that can inhibit or activate Saccharomyces cerevisiae. The research objective was to study the effect of berry extracts on the activity of baking yeast S. cerevisiae and the biochemical properties of wheat dough. Study objects and methods. The experiment featured baking yeast Extra and dry berry extracts of raspberries, aronia, sea buckthorn, and rosehip (LLC Wisterra, Altai Region). The study involved standard and industry-specific control methods of raw materials and semi-finished bakery products, as well as som e standard methods of microbiological analysis. Results and discussion. The raspberry extract (3–4%) suppressed the growth and reproduction of the yeast: after 1 h of exposure, the yeast cell count dropped by 1.5–2 times compared to the control sample. The stimulating effect of the sea buckthorn extract increased the growth rate of yeast cells (up to 40% compared to the control). The extracts of aronia and rosehip had practically no effect on the growth rate of yeast cells. However, 2–3% aronia extract increased the fermentation of the dough, as evidenced by a higher dough fermentation property, which was 2 min versus 3 min at the control after 150 min of exposure. Fruit and berry extracts caused a natural increase in the acidity of the dough, which affected the growth rate of yeast cells. Sea buckthorn extracts increased the acidity so much (up to 4.24 pH units) that it could be regarded as acid stress, which increased the growth rate of yeast cells (1.53×106–1.55×106 vs. 1.10×106 in 1 mL of control sample). The lowest growth rate was detected in the samples with the raspberry extract, which is known to have a strong fungistatic effect: the count of yeast cells decreased by 1.5–2 times after an hour of fermentation. Conclusion. Berry extracts can be of practical interest to bakery enterprises as they help to control yeast fermentation and dough maturation time.

Bioproduction of 2-Phenylethanol through Yeast Fermentation on Synthetic Media and on Agro-Industrial Waste and By-Products: A Review

Due to its pleasant rosy scent, the aromatic alcohol 2-phenylethanol (2-PE) has a huge market demand. Since this valuable compound is used in food, cosmetics and pharmaceuticals, consumers and safety regulations tend to prefer natural methods for its production rather than the synthetic ones. Natural 2-PE can be either produced through the extraction of essential oils from various flowers, including roses, hyacinths and jasmine, or through biotechnological routes. In fact, the rarity of natural 2-PE in flowers has led to the inability to satisfy the large market demand and to a high selling price. Hence, there is a need to develop a more efficient, economic, and environmentally friendly biotechnological approach as an alternative to the conventional industrial one. The most promising method is through microbial fermentation, particularly using yeasts. Numerous yeasts have the ability to produce 2-PE using l-Phe as precursor. Some agro-industrial waste and by-products have the particularity of a high nutritional value, making them suitable media for microbial growth, including the production of 2-PE through yeast fermentation. This review summarizes the biotechnological production of 2-PE through the fermentation of different yeasts on synthetic media and on various agro-industrial waste and by-products.

Interaction of Long-Chain Alcohol with Dry Yeast, Cholesterol, and Sea Firefly Luciferase

Background: A hand sanitizer containing alcohol, usually ethanol or isopropanol, is typically used for disinfection, but given that cholesterol is one of the main components of virus envelopes, long-chain alcohol may be more effective. To better understand the potential disinfection activity of long-chain alcohols, we studied their interactions with dry yeast, cholesterol, and sea firefly luciferase. Methods and Results: We measured, at 30oC and 39oC, the minimum inhibition concentration (MIC) of dry yeast fermentation and the stability of cholesterol and sea firefly luciferase with alcohols, diols, cetyltrimethylammonium chloride, and stearyltrimethylammonium chloride. The MIC decreased with the chain length at C≤12 for dry yeast and cholesterol with alcohol at 30oC. At C13 and higher, the cut-off region was observed. At 39oC, the cut-off region shifted to C15 and higher. The reduction of MIC was measured with the diol or sea firefly luciferase at C≤14. Conclusion: The presence of the cut-off region is suggested to be related to whether the alcohol is in the liquid state. For the liquid alcohol, the longer the chain length, the lower the MIC. This suggests a potential disinfection activity of long-chain alcohol.

Oleaginous yeasts- substrate preference and lipid productivity: a view on the performance of microbial lipid producers

Background Oleaginous yeasts are promising microbial platforms for sustainable, bio-based production of biofuels and oleochemical building blocks. Bio-based residues provide sustainable and cost-effective carbon sources for fermentative yeast oil production without land-use change. Considering the regional abundancy of different waste streams, we chose complex biomass residue streams of marine origin; macroalgae hydrolysate, and terrestrial origin; wheat straw hydrolysate in the presence, and absence of corn steep liquor as a complex nitrogen source. We investigated the biomass and lipid yields of an array of well-described oleaginous yeasts; R. glutinis, T. asahii, R. mucilaginosa, R. toruloides, C. oleaginosus growing on these hydrolysates. Furthermore, their sugar utilization, fatty acid profile, and inhibitory effect of the hydrolysates on yeast growth were compared. For correlative reference, we initially performed comparative growth experiments for the strains on individual monomeric sugars separately. Each of these monomeric sugars was a dominant carbon source in the complex biomass hydrolysates evaluated in this study. In addition, we evaluated N-acetylglucosamine, the monomeric building block of chitin, as a low-cost nitrogen and carbon source in yeast fermentation. Results C. oleaginosus provided the highest biomass and lipid yields. In the wheat straw and brown algae hydrolysates, this yeast strain gained 7.5 g/L and 3.8 g/L lipids, respectively. Cultivation in algae hydrolysate resulted in a higher level of unsaturated fatty acids in the lipids accumulated by all yeast strains. R. toruloides and C. oleaginosus were able to effectively co-utilize mannitol, glucose, and xylose. Growth rates on wheat straw hydrolysate were enhanced in presence of corn steep liquor. Conclusions Among the yeast strains investigated in this study, C. oleaginosus proved to be the most versatile strain in terms of substrate utilization, productivity, and tolerance in the complex media. Various fatty acid profiles obtained on each substrate encourage the manipulation of culture conditions to achieve the desired fatty acid composition for each application. This could be accomplished by combining the element of carbon source with other formerly studied factors such as temperature and oxygen. Moreover, corn steep liquor showed promise for enhancement of growth in the oleaginous strains provided that carbon substrate is available.

Yeast cell factories for sustainable whey-to-ethanol valorisation towards a circular economy

Cheese whey is the major by-product of the dairy industry, and its disposal constitutes an environmental concern. The production of cheese whey has been increasing, with 190 million tonnes per year being produced nowadays. Therefore, it is emergent to consider different routes for cheese whey utilization. The great nutritional value of cheese whey turns it into an attractive substrate for biotechnological applications. Currently, cheese whey processing includes a protein fractionating step that originates the permeate, a lactose-reach stream further used for valorisation. In the last decades, yeast fermentation has brought several advances to the search for biorefinery alternatives. From the plethora of value-added products that can be obtained from cheese whey, ethanol is the most extensively explored since it is the alternative biofuel most used worldwide. Thus, this review focuses on the different strategies for ethanol production from cheese whey using yeasts as promising biological systems, including its integration in lignocellulosic biorefineries. These valorisation routes encompass the improvement of the fermentation process as well as metabolic engineering techniques for the introduction of heterologous pathways, resorting mainly to Kluyveromyces sp. and Saccharomyces cerevisiae strains. The solutions and challenges of the several strategies will be unveiled and explored in this review.

Directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae

Background Xylose contained in lignocellulosic biomass is an attractive carbon substrate for economically viable conversion to bioethanol. Extensive research has been conducted on xylose fermentation using recombinant Saccharomyces cerevisiae expressing xylose isomerase (XI) and xylose reductase/xylitol dehydrogenase (XR/XDH) pathways along with the introduction of a xylose transporter and amplification of the downstream pathway. However, the low utilization of xylose in the presence of glucose, due to the varying preference for cellular uptake, is a lingering challenge. Studies so far have mainly focused on xylose utilization inside the cells, but there have been little trials on the conversion of xylose to xylulose by cell before uptake. We hypothesized that the extracellular conversion of xylose to xylulose before uptake would facilitate better utilization of xylose even in the presence of glucose. To verify this, XI from Piromyces sp. was engineered and hyper-secreted in S. cerevisiae for the extracellular conversion of xylose to xylulose. Results The optimal pH of XI was lowered from 7.0 to 5.0 by directed evolution to ensure its high activity under the acidic conditions used for yeast fermentation, and hyper-secretion of an engineered XI-76 mutant (E56A and I252M) was accomplished by employing target protein-specific translational fusion partners. The purified XI-76 showed twofold higher activity than that of the wild type at pH 5. The secretory expression of XI-76 in the previously developed xylose utilizing yeast strain, SR8 increased xylose consumption and ethanol production by approximately 7–20% and 15–20% in xylose fermentation and glucose and xylose co-fermentation, respectively. Conclusions Isomerisation of xylose to xylulose before uptake using extracellular XI was found to be effective in xylose fermentation or glucose/xylose co-fermentation. This suggested that glucose competed less with xylulose than with xylose for uptake by the cell. Consequently, the engineered XI secretion system constructed in this study can pave the way for simultaneous utilization of C5/C6 sugars from the sustainable lignocellulosic biomass.

Value-Added Products from Ethanol Fermentation—A Review

Global demand for renewable and sustainable energy is increasing, and one of the most common biofuels is ethanol. Most ethanol is produced by Saccharomyces cerevisiae (yeast) fermentation of either crops rich in sucrose (e.g., sugar cane and sugar beet) or starch-rich crops (e.g., corn and starchy grains). Ethanol produced from these sources is termed a first-generation biofuel. Yeast fermentation can yield a range of additional valuable co-products that accumulate during primary fermentation (e.g., protein concentrates, water soluble metabolites, fusel alcohols, and industrial enzymes). Distillers’ solubles is a liquid co-product that can be used in animal feed or as a resource for recovery of valuable materials. In some processes it is preferred that this fraction is modified by a second fermentation with another fermentation organism (e.g., lactic acid bacteria). Such two stage fermentations can produce valuable compounds, such as 1,3-propanediol, organic acids, and bacteriocins. The use of lactic acid bacteria can also lead to the aggregation of stillage proteins and enable protein aggregation into concentrates. Once concentrated, the protein has utility as a high-protein feed ingredient. After separation of protein concentrates the remaining solution is a potential source of several known small molecules. The purpose of this review is to provide policy makers, bioethanol producers, and researchers insight into additional added-value products that can be recovered from ethanol beers. Novel products may be isolated during or after distillation. The ability to isolate and purify these compounds can provide substantial additional revenue for biofuel manufacturers through the development of marketable co-products.