Natural promoters and promoter engineering strategies for metabolic regulation in Saccharomyces cerevisiae.

Sharomyces cerevisiae is currently one of the most important foreign gene expression systems. S. cerevisiae is an excellent host for high-value metabolite cell factories due to its advantages of simplicity, safety, and nontoxicity. A promoter, as one of the basic elements of gene transcription, plays an important role in regulating gene expression and optimizing metabolic pathways. Promoters control the direction and intensity of transcription, and the application of promoters with different intensities and performances will largely determine the effect of gene expression and ultimately affect the experimental results. Due to its significant role, there have been many studies on promoters for decades. While some studies have explored and analyzed new promoters with different functions, more studies have focused on artificially modifying promoters to meet their own scientific needs. Thus, this article reviews current research on promoter engineering techniques and related natural promoters in S. cerevisiae. First, we introduce the basic structure of promoters and the classification of natural promoters. Then, the classification of various promoter strategies is reviewed. Finally, by grouping related articles together using various strategies, this review anticipates the future development direction of promoter engineering.

Biosynthesis of valerenic acid by engineered Saccharomyces cerevisiae.

OBJECTIVE: To produce valerenic acid (VA) in Saccharomyces cerevisiae by engineering a heterologous synthetic pathway. RESULT: Valerena-4,7(11)-diene synthase (VDS) derived from Valeriana officinalis (valerian) was expressed in S. cerevisiae to generate valerena-4,7(11)-diene as the precursor of VA. By overexpressing the key genes of the mevalonate pathway ERG8, ERG12 and ERG19, and integrating 4 copies of MBP (maltose-binding protein)-VDS-ERG20 gene expression caskets into the genome, the production of valerena-4,7(11)-diene was improved to 75 mg/L. On this basis, the cytochrome P450 monooxygenase LsGAO2 derived from Lactuca sativa was expressed to oxidize valerena-4,7(11)-diene to produce VA, and the most effective VA production strain was used for fermentation. The yield of VA reached 2.8 mg/L in the flask and 6.8 mg/L in a 5-L bioreactor fed glucose. CONCLUSIONS: An S. cerevisiae strain was constructed and optimized to produce VA, but the valerena-4,7(11)-diene oxidation by LsGAO2 is still the rate-limiting step for VA synthesis that needs to be further optimized in future studies.

A novel behavioral three-way decision model with application to the treatment of mild symptoms of COVID-19.

The Coronavirus Disease 2019 (COVID-19) has popularized since late December 2019. In present, it is still highly transmissible and has severe impact on the public health and global economy. Due to the lack of specific drug and the appearance of different variants, the selection of the antiviral therapy to treat the patients with mild symptom is of vital importance. Hence, in this paper, we propose a novel behavioral Three-Way Decision (3WD) model and apply it to the medicine selection decision. First, a new relative utility function is constructed by considering the risk-aversion behavior and regret-aversion behavior of human beings. Second, based on the relative utility function, some new rules are defined to calculate the thresholds and conditional probabilities in 3WD and some corresponding theorems are explored and proved. Next, a new information fusion mechanism in the framework of evidential reasoning algorithm is developed. Then, the decision results are obtained based on the Bayesian decision procedure and the principle of maximum utility. Finally, an example with large-scale data set and an example about medicine selection for COVID-19 are provided to show the implementation process and effectiveness of the proposed method. Comparative analysis and sensitivity analysis are also performed to illustrate the superiority and the robustness of the current proposal.

Construction and optimization of Saccharomyces cerevisiae for synthesizing forskolin.

Forskolin, one of the primary active metabolites of labdane-type diterpenoids, exhibits significant medicinal value, such as anticancer, antiasthmatic, and antihypertensive activities. In this study, we constructed a Saccharomyces cerevisiae cell factory that efficiently produced forskolin. First, a chassis strain that can accumulate 145.8 mg/L 13R-manoyl oxide (13R-MO), the critical precursor of forskolin, was constructed. Then, forskolin was produced by integrating CfCYP76AH15, CfCYP76AH11, CfCYP76AH16, ATR1, and CfACT1-8 into the 13R-MO chassis with a titer of 76.25 µg/L. We confirmed that cytochrome P450 enzymes (P450s) are the rate-limiting step by detecting intermediate metabolite accumulation. Forskolin production reached 759.42 µg/L by optimizing the adaptations between CfCYP76AHs, t66CfCPR, and t30AaCYB5. Moreover, multiple metabolic engineering strategies, including regulation of the target genes' copy numbers, amplification of the endoplasmic reticulum (ER) area, and cofactor metabolism enhancement, were implemented to enhance the metabolic flow to forskolin from 13R-MO, resulting in a final forskolin yield of 21.47 mg/L in shake flasks and 79.33 mg/L in a 5 L bioreactor. These promising results provide guidance for the synthesis of other natural terpenoids in S. cerevisiae, especially for those containing multiple P450s in their synthetic pathways. KEY POINTS: • The forskolin biosynthesis pathway was optimized from the perspective of system metabolism for the first time in S. cerevisiae. • The adaptation and optimization of CYP76AHs, t66CfCPR, and t30AaCYB5 promote forskolin accumulation, which can provide a reference for diterpenoids containing complex pathways, especially multiple P450s pathways. • The forskolin titer of 79.33 mg/L is the highest production currently reported and was achieved by fed-batch fermentation in a 5 L bioreactor.