CRISPR/Cas9 has been widely used in engineering Saccharomyces cerevisiae for gene insertion, replacement and deletion due to its simplicity and high efficiency. The selectable markers of CRISPR/Cas9 systems are particularly useful for genome editing and Cas9-plasmids removing in yeast. In our previous research, GAL80 gene has been deleted by the plasmid pML104-mediated CRISPR/Cas9 system in an engineered yeast, in order to eliminate the requirement of galactose supplementation for induction. The maximum artemisinic acid production by engineered S. cerevisiae 1211-2 (740 mg/L) was comparable to that of the parental strain 1211 without galactose induction. Unfortunately, S. cerevisiae 1211-2 was inefficient in the utilization of the carbon source ethanol in the subsequent 50 L pilot fermentation experiment. The artemisinic acid yield in the engineered S. cerevisiae 1211-2 was only 20%-25% compared with that of S. cerevisiae 1211. The mutation of the selection marker URA3 was supposed to affect the growth and artemisinic acid production. A ura3 mutant was successfully restored by a recombinant plasmid pML104-KanMx4-u along with a 90 bp donor DNA, resulting in S. cerevisiae 1211-3. This mutant could grow normally in a fed-batch fermentor with mixed glucose and ethanol feeding, and the final artemisinic acid yield (> 20 g/L) was comparable to that of the parental strain S. cerevisiae 1211. In this study, an engineered yeast strain producing artemisinic acid without galactose induction was obtained. More importantly, it was the first report showing that the auxotrophic marker URA3 significantly affected artemisinic acid production in a pilot-scale fermentation with ethanol feeding, which provides a reference for the production of other natural products in yeast chassis.
Artemisinins ; Fermentation ; Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism*

Lager yeast is the most popular yeast strain used for beer production in China. The flocculation of yeast plays an important role in cell separation at the end of fermentation. Therefore, appropriately enhancing the flocculation capability of the lager yeast without affecting its fermentation performance would be desirable for beer industry. Our previous study showed that the defect of gene RIM21 might contribute to the enhanced flocculation capability of a lager yeast G03. To further investigate the role of the RIM21 gene in flocculation of strain G03, this study constructed a RIM21-deleted mutant strain G03-RIM21Δ through homologous recombination. Deletion of RIM21 improved the flocculation capability of strain G03 during wort fermentation at 11 °C without changing its fermentation performance significantly. The expression of FLO5, Lg-FLO1 and some other genes involved in cell wall integrity pathway were up-regulated in strain G03-RIM21Δ. In addition, the disruption of RIM21 enhanced resistance of yeast cells to cell wall inhibitors. These results provide a basis for elucidating the flocculation mechanism of lager yeast under low-temperature fermentation conditions.
Beer ; Fermentation ; Flocculation ; Receptors, Cell Surface ; Saccharomyces/metabolism* ; Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism*

Naringenin is a natural flavonoid compound with anti-inflammatory, anti-oxidation, anti-viral, anti-atherosclerosis and other pharmacological activities. It is also an important precursor of other flavonoid synthesis and with great value of application. At present, the production of flavonoids such as naringenin by microbial methods has a low yield due to imbalance of metabolic pathways, which greatly limits its industrial application. In this study, a naringenin-producing strain of Saccharomyces cerevisiae Y-01 was used in the research object. The expression levels of 4-coumaric acid: CoA ligase (4CL), chalcone synthase (CHS) and chalcone isomerase (CHI) were controlled by promoter and copy numbers to investigate the quantitative effect of key enzyme expression level on the accumulation level of target products. The results showed that the correlation between naringenin production and 4CL or CHI expression was not significant while there was a positive correlation with the expression level of CHS. Strain Y-04 with high yield of naringenin was obtained by regulating the expression level of chs gene, and the yield was increased by 4.1-folds compared with the original strain Y-01. This study indicated that CHS is a key regulatory target of naringenin synthesis. Rational regulation of CHS expression can significantly promote the accumulation of naringenin. The related results provide an important theoretical reference for the use of metabolic engineering to strengthen microbial synthesis of important flavonoids such as naringenin.
Flavanones ; metabolism ; Metabolic Engineering ; Saccharomyces cerevisiae

As a generally-recognized-as-safe microorganism, Saccharomyces cerevisiae is a widely studied chassis cell for the production of high-value or bulk chemicals in the field of synthetic biology. In recent years, a large number of synthesis pathways of chemicals have been established and optimized in S. cerevisiae by various metabolic engineering strategies, and the production of some chemicals have shown the potential of commercialization. As a eukaryote, S. cerevisiae has a complete inner membrane system and complex organelle compartments, and these compartments generally have higher concentrations of the precursor substrates (such as acetyl-CoA in mitochondria), or have sufficient enzymes, cofactors and energy which are required for the synthesis of some chemicals. These features may provide a more suitable physical and chemical environment for the biosynthesis of the targeted chemicals. However, the structural features of different organelles hinder the synthesis of specific chemicals. In order to ameliorate the efficiency of product biosynthesis, researchers have carried out a number of targeted modifications to the organelles grounded on an in-depth analysis of the characteristics of different organelles and the suitability of the production of target chemicals biosynthesis pathway to the organelles. In this review, the reconstruction and optimization of the biosynthesis pathways for production of chemicals by organelle mitochondria, peroxisome, golgi apparatus, endoplasmic reticulum, lipid droplets and vacuole compartmentalization in S. cerevisiae are reviewed in-depth. Current difficulties, challenges and future perspectives are highlighted.
Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism* ; Golgi Apparatus/metabolism* ; Metabolic Engineering ; Vacuoles/metabolism*

Monoterpenes are widely used in cosmetics, food, medicine, agriculture and other fields. With the development of synthetic biology, it is considered as a potential way to create microbial cell factories to produce monoterpenes. Engineering Saccharomyces cerevisiae to produce monoterpenes has been a research hotspot in synthetic biology. In S. cerevisiae, the production of geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) is catalyzed by a bifunctional enzyme farnesyl pyrophosphate synthetase(encoded by ERG20 gene) which is inclined to synthesize FPP essential for yeast growth. Therefore, reasonable control of FPP synthesis is the basis for efficient monoterpene synthesis in yeast cell factories. In order to achieve dynamic control from GPP to FPP biosynthesis in S. cerevisiae, we obtained a novel chassis strain HP001-pERG1-ERG20 by replacing the ERG20 promoter of the chassis strain HP001 with the promoter of cyclosqualene cyclase(ERG1) gene. Further, we reconstructed the metabolic pathway by using GPP and neryl diphosphate(NPP), cis-GPP as substrates in HP001-pERG1-ERG20. The yield of GPP-derived linalool increased by 42.5% to 7.6 mg·L~(-1), and that of NPP-derived nerol increased by 1 436.4% to 8.3 mg·L~(-1). This study provides a basis for the production of monoterpenes by microbial fermentation.
Fermentation ; Geranyltranstransferase/genetics* ; Monoterpenes/metabolism* ; Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism*

Higher alcohols are one of the main by-products of Saccharomyces cerevisiae in brewing. High concentration of higher alcohols in alcoholic beverages easily causes headache, thirst and other symptoms after drinking. It is also the main reason for chronic drunkenness and difficulty in sobering up after intoxication. The main objective of this review is to present an overview of the flavor characteristics and metabolic pathways of higher alcohols as well as the application of mutagenesis breeding techniques in the regulation of higher alcohol metabolism in S. cerevisiae. In particular, we review the application of metabolic engineering technology in genetic modification of amino transferase, α-keto acid metabolism, acetate metabolism and carbon-nitrogen metabolism. Moreover, key challenges and future perspectives of realizing optimization of higher alcohols metabolism are discussed. This review is intended to provide a comprehensive understanding of metabolic regulation system of higher alcohols in S. cerevisiae and to provide insights into the rational development of the excellent industrial S. cerevisiae strains producing higher alcohols.
Alcoholic Beverages ; Alcohols/analysis* ; Fermentation ; Saccharomyces cerevisiae/metabolism* ; Saccharomyces cerevisiae Proteins/metabolism*

Patchoulol is an important sesquiterpenoid in the volatile oil of Pogostemon cablin, and is also considered to be the main contributing component to the pharmacological efficacy and fragrance of P. cablin oil, which has antibacterial, antitumor, antioxidant, and other biological activities. Currently, patchoulol and its essential oil blends are in high demand worldwide, but the traditional plant extraction method has many problems such as wasting land and polluting the environment. Therefore, there is an urgent need for a new method to produce patchoulol efficiently and at low cost. To broaden the production method of patchouli and achieve the heterologous production of patchoulol in Saccharomyces cerevisiae, the patchoulol synthase(PS) gene from P. cablin was codon optimized and placed under the inducible strong promoter GAL1 to transfer into the yeast platform strain YTT-T5, thereby obtaining strain PS00 with the production of(4.0±0.3) mg·L~(-1) patchoulol. To improve the conversion rate, this study used protein fusion method to fuse SmFPS gene from Salvia miltiorrhiza with PS gene, leading to increase the yield of patchoulol to(100.9±7.4) mg·L~(-1) by 25-folds. By further optimizing the copy number of the fusion gene, the yield of patchoulol was increased by 90% to(191.1±32.7) mg·L~(-1). By optimizing the fermentation process, the strain was able to achieve a patchouli yield of 2.1 g·L~(-1) in a high-density fermentation system, which was the highest yield so far. This study provides an important basis for the green production of patchoulol.
Saccharomyces cerevisiae/metabolism* ; Sesquiterpenes/metabolism* ; Pogostemon ; Oils, Volatile/metabolism*

Limonene and its derivative perillic acid are widely used in food, cosmetics, health products, medicine and other industries as important bioactive natural products. However, inefficient plant extraction and high energy-consuming chemical synthesis hamper the industrial production of limonene and perillic acid. In this study, limonene synthase from Mentha spicata was expressed in Saccharomyces cerevisiae by peroxisome compartmentalization, and the yield of limonene was 0.038 mg/L. The genes involved in limonene synthesis, ERG10, ERG13, tHMGR, ERG12, ERG8, IDI1, MVD1, ERG20ww and tLS, were step-wise expressed via modular engineering to study their effects on limonene yield. The yield of limonene increased to 1.14 mg/L by increasing the precursor module. Using the plasmid with high copy number to express the above key genes, the yield of limonene significantly increased up to 86.74 mg/L, which was 4 337 times higher than that of the original strain. Using the limonene-producing strain as the starting strain, the production of perillic acid was successfully achieved by expressing cytochrome P450 enzyme gene from Salvia miltiorrhiza, and the yield reached 4.42 mg/L. The results may facilitate the construction of cell factory with high yield of monoterpene products by S. cerevisiae.
Saccharomyces cerevisiae/metabolism* ; Limonene/metabolism* ; Metabolic Engineering ; Monoterpenes/metabolism*

Prior research reported the oscillatory behavior characterized by long period and high amplitude during high gravity continuous ethanol fermentations at the dilution rate of 0.027 h(-1). In this paper, high gravity continuous ethanol fermentations using Saccharomyces cerevisia at different dilution rates were carried out. Similar oscillations were observed when the dilution rate was switched to 0.04 h(-1). Both oscillatory and steady processes can be achieved at dilution rates of 0.027 or 0.04 h(-1), which depends on the initial status of the fermentation system. However, compared to steady process at the same dilution rate of 0.04 h(-1), the average residual sugar concentration was lowered by 14.8% for the oscillatory process, while the average ethanol concentration and productivity were increased by 12.6% and 12.3%, respectively. Further investigation revealed that besides the lag time, oscillatory processes were different from steady ones in kinetics because a higher specific growth rate can be achieved at the same residual sugar and ethanol concentrations (increased by 53.8% in average).
Bioreactors ; microbiology ; Carbohydrates ; Ethanol ; metabolism ; Fermentation ; Hypergravity ; Saccharomyces cerevisiae ; metabolism

OBJECTIVETo investigate the characteristics of Zn2+ biosorption and the release of cations during the process of Zn2+ biosorption by intact cells of Saccharomyces cerevisiae.

METHODSThe batch adsorption test was used to study the biosorption equilibrium and isotherm. Zn2+ concentration was measured with atomic adsorption spectrophotometer (AAS) AAS 6 Vario.

RESULTSWhen the initial concentration of Zn2+ ranged between 0.08 and 0.8 mmol/L, the initial pH was natural (about 5.65), the sorbent concentration was about 1 g/L and the capacity of Zn2+ biosorption was from 74.8 to 654.8 micromol/g. The pH value increased by 0.55-1.28 and the intracellular cations (K+, Mg2+, Na+, Ca2+) of the cells were released during the process of Zn2+ biosorption.

CONCLUSIONIon exchange was one of the mechanisms for Zn2+ biosorption. The biomass of Saccharomyces cerevisiae is a potential biosorbent for the removal of Zn2+ from aqueous solution. More work needs to be done before putting it into practical application.