Efficient ß-carotene production in engineered Saccharomyces cerevisiae using simple sugars and agricultural waste-based carbon and nitrogen sources.

ß-carotene, a precursor to vitamin A, holds significant promise for health and nutrition applications. This study introduces an optimized approach for ß-carotene production in Saccharomyces cerevisiae, leveraging metabolic engineering and a novel use of agricultural waste. The GAL80 gene deletion facilitated efficient ß-carotene synthesis from sucrose, avoiding the costly galactose induction, and achieved titers up to 727.8 ±â€¯68.0 mg/L with content levels of 71.8 ±â€¯0.4 mg/g dry cell weight (DCW). Furthermore, the application of agricultural by-products, specifically molasses and fish meal as carbon and nitrogen sources, was investigated. This approach yielded a substantial ß-carotene titer of 354.9 ±â€¯8.2 mg/L and a content of 60.5 ±â€¯4.3 mg/g DCW, showcasing the potential of these sustainable substrates for industrial-scale production. This study sets a new benchmark for cost-effective, green manufacturing of vital nutrients, demonstrating a scalable, eco-friendly alternative for ß-carotene production.

Red yeasts and their carotenogenic enzymes for microbial carotenoid production.

Carotenoids are C40 isoprene-based compounds with significant commercial interests that harbor diverse bioactivities. Prominent examples of carotenoids are beta-carotene, a precursor to vitamin A essential for proper eye health, and lycopene and astaxanthin, powerful antioxidants implicated in preventing cancers and atherosclerosis. Due to their benefits to human health, the market value for carotenoids is rapidly increasing and is projected to reach USD 1.7 billion by 2025. However, their production now relies on chemical synthesis and extraction from plants that pose risks to food management and numerous biological safety issues. Thus, carotenoid production from microbes is considered a promising strategy for achieving a healthy society with more sustainability. Red yeast is a heterogeneous group of basidiomycetous fungi capable of producing carotenoids. It is a critical source of microbial carotenoids from low-cost substrates. Carotenogenic enzymes from red yeasts have also been highly efficient, invaluable biological resources for biotechnological applications. In this minireview, we focus on red yeast as a promising source for microbial carotenoids, strain engineering strategies for improving carotenoid production in red yeasts, and potential applications of carotenogenic enzymes from red yeasts in conventional and nonconventional yeasts.

D-Lactic Acid Production from Sugarcane Bagasse by Genetically Engineered Saccharomyces cerevisiae.

Lactic acid (LA) is a promising bio-based chemical that has broad applications in food, nutraceutical, and bioplastic industries. However, production of the D-form of LA (D-LA) from fermentative organisms is lacking. In this study, Saccharomyces cerevisiae harboring the D-lactate dehydrogenase (DLDH) gene from Leuconostoc mesenteroides was constructed (CEN.PK2_DLDH). To increase D-LA production, the CRISPR/Cas12a system was used for the deletion of gpd1, gpd2, and adh1 to minimize glycerol and ethanol production. Although an improved D-LA titer was observed for both CEN.PK2_DLDHΔgpd and CEN.PK2_DLDHΔgpdΔadh1, growth impairment was observed. To enhance the D-LA productivity, CEN.PK2_DLDHΔgpd was crossed with the weak acid-tolerant S. cerevisiae BCC39850. The isolated hybrid2 showed a maximum D-LA concentration of 23.41 ± 1.65 g/L, equivalent to the improvement in productivity and yield by 2.2 and 1.5 folds, respectively. The simultaneous saccharification and fermentation using alkaline pretreated sugarcane bagasse by the hybrid2 led to an improved D-LA conversion yield on both the washed solid and whole slurry (0.33 and 0.24 g/g glucan). Our findings show the exploitation of natural yeast diversity and the potential strategy of gene editing combined with conventional breeding on improving the performance of S. cerevisiae for the production of industrially potent products.

Novel carotenogenic gene combinations from red yeasts enhanced lycopene and beta-carotene production in Saccharomyces cerevisiae from the low-cost substrate sucrose.

Carotenoids (C40H56) including lycopene and beta-carotene are relatively strong antioxidants that provide benefits to human health. Here, we screened highly efficient crt variants from red yeasts to improve lycopene and beta-carotene production in Saccharomyces cerevisiae. We identified that crt variants from Sporidiobolus pararoseus TBRC-BCC 63403 isolated from rice leaf in Thailand exhibited the highest activity in term of lycopene and beta-carotene production in the context of yeast. Specifically, the phytoene desaturase SpCrtI possessed up to 4-fold higher in vivo activity based on lycopene content than the benchmark enzyme BtCrtI from Blakeslea trispora in our engineered WWY005 strain. Also, the geranylgeranyl pyrophosphate (GGPP) synthase SpCrtE, the bifunctional phytoene synthase-lycopene cyclase SpCrtYB, and SpCrtI when combined led to 7-fold improvement in beta-carotene content over the benchmark enzymes from Xanthophyllomyces dendrorhous in the laboratory strain CEN.PK2-1C. Sucrose as an alternative to glucose was found to enhance lycopene production in cells lacking GAL80. Lastly, we demonstrated a step-wise improvement in lycopene production from shake-flasks to a 5-L fermenter using the strain with GAL80 intact. Altogether, our study represents novel findings on more effective crt genes from Sp. pararoseus over the previously reported benchmark genes and their potential applications in scale-up lycopene production.

Systematic engineering of Saccharomyces cerevisiae for D-lactic acid production with near theoretical yield.

D-lactic acid is a chiral three-carbon organic acid that can improve the thermostability of polylactic acid. Here, we systematically engineered Saccharomyces cerevisiae to produce D-lactic acid from glucose, a renewable carbon source, at near theoretical yield. Specifically, we screened D-lactate dehydrogenase (DLDH) variants from lactic acid bacteria in three different genera and identified the Leuconostoc pseudomesenteroides variant (LpDLDH) as having the highest activity in yeast. We then screened single-gene deletions to minimize the production of the side products ethanol and glycerol as well as prevent the conversion of D-lactic acid back to pyruvate. Based on the results of the DLDH screening and the single-gene deletions, we created a strain called ASc-d789M which overexpresses LpDLDH and contains deletions in glycerol pathway genes GPD1 and GPD2 and lactate dehydrogenase gene DLD1, as well as downregulation of ethanol pathway gene ADH1 using the L-methionine repressible promoter to minimize impact on growth. ASc-d789M produces D-lactic acid at a titer of 17.09 g/L in shake-flasks (yield of 0.89 g/g glucose consumed or 89% of the theoretical yield). Fed-batch fermentation resulted in D-lactic acid titer of 40.03 g/L (yield of 0.81 g/g glucose consumed). Altogether, our work represents progress towards efficient microbial production of D-lactic acid.

Sodium Acetate Responses in Saccharomyces cerevisiae and the Ubiquitin Ligase Rsp5.

Recent studies have revealed the feasibility of sodium acetate as a potentially novel inhibitor/stressor relevant to the fermentation from neutralized lignocellulosic hydrolysates. This mini-review focuses on the toxicity of sodium acetate, which is composed of both sodium and acetate ions, and on the involved cellular responses that it elicits, particularly via the high-osmolarity glycerol (HOG) pathway, the Rim101 pathway, the P-type ATPase sodium pumps Ena1/2/5, and the ubiquitin ligase Rsp5 with its adaptors. Increased understanding of cellular responses to sodium acetate would improve our understanding of how cells respond not only to different stimuli but also to composite stresses induced by multiple components (e.g., sodium and acetate) simultaneously. Moreover, unraveling the characteristics of specific stresses under industrially related conditions and the cellular responses evoked by these stresses would be a key factor in the industrial yeast strain engineering toward the increased productivity of not only bioethanol but also advanced biofuels and valuable chemicals that will be in demand in the coming era of bio-based industry.

Enhanced sodium acetate tolerance in Saccharomyces cerevisiae by the Thr255Ala mutation of the ubiquitin ligase Rsp5.

Sodium and acetate inhibit cell growth and ethanol fermentation by different mechanisms in Saccharomyces cerevisiae. We identified the substitution of a conserved Thr255 to Ala (T255A) in the essential Nedd4-family ubiquitin ligase Rsp5, which enhances cellular sodium acetate tolerance. The T255A mutation selectively increased the resistance of cells against sodium acetate, suggesting that S. cerevisiae cells possess an Rsp5-mediated mechanism to cope with the composite stress of sodium and acetate. The sodium acetate tolerance was dependent on the extrusion of intracellular sodium ions by the plasma membrane-localized sodium pumps Ena1, Ena2, and Ena5 (Ena1/2/5) and two known upstream regulators: the Rim101 pH signaling pathway and the Hog1 mitogen-activated protein kinase. However, the T255A mutation affected neither the ubiquitination level of the Rsp5 adaptor protein Rim8 nor the phosphorylation level of Hog1. These data raised the possibility that Rsp5 enhances the function of Ena1/2/5 specifically in response to sodium acetate through an unknown mechanism other than ubiquitination of Rim8 and activation of Hog1-mediated signaling. Also, an industrial yeast strain that expresses the T255A variant exhibited increased initial fermentation rates in the presence of sodium acetate. Hence, this mutation has potential for the improvement of bioethanol production from lignocellulosic biomass.