Enzyme and Metabolic Engineering Strategies for Biosynthesis of α-Farnesene in Saccharomyces cerevisiae

Abstract

α-Farnesene, a type of acyclic sesquiterpene, is an important raw material in agriculture, aircraft fuel, and the chemical industry. In this study, we constructed an efficient α-farnesene-producing yeast cell factory by combining enzyme and metabolic engineering strategies. First, we screened different plants for α-farnesene synthase (AFS) with the best activity and found that AFS from Camellia sinensis (CsAFS) exhibited the most efficient α-farnesene production in Saccharomyces cerevisiae 4741. Second, the metabolic flux of the mevalonate pathway was increased to improve the supply of the precursor farnesyl pyrophosphate. Third, inducing site-directed mutagenesis in CsAFS, the CsAFSW281C variant was obtained, which considerably increased α-farnesene production. Fourth, the N-terminal serine–lysine–isoleucine–lysine (SKIK) tag was introduced to construct the SKIK∼CsAFSW281C variant, which further increased α-farnesene production to 2.8 g/L in shake-flask cultures. Finally, the α-farnesene titer of 28.3 g/L in S. cerevisiae was obtained by fed-batch fermentation in a 5 L bioreactor.

Integrated HS-GC–IMS and UPLC-Q-Orbitrap HRMS-based metabolomics revealed the characteristics and differential volatile and nonvolatile metabolites of different citrus peels

“…The relative concentration of farnesene was higher among the terpenoids in CLU and GP. This compound serves as a crucial raw material in agriculture, aviation fuel, and the chemical industry (S. Wang et al, 2023 , Wang et al, 2023 , Wang et al, 2023 ). Geranyl acetate was more elevated in QJ and CLU.…”

“…4-Coumaric acid was significantly upregulated in DHP and WG and can regulate the intestinal microflora to protect the gastrointestinal barrier (Z. Y. Wang et al, 2023 , Wang et al, 2023 , Wang et al, 2023 ). Caffeic acid and isoferulic acid can be used as characteristic metabolites to identify PK, and their supplementation can enhance intestinal integrity and barrier function by modulating the intestinal microbiota and its metabolites ( Jiang et al, 2020 ; Wen et al, 2023 ).…”

“…Zhang et al screened 30 potential volatile biomarkers to distinguish among loose-skin mandarins, sweet oranges, pomelos, and lemons and 17 effective biomarkers to distinguish clementine mandarins from the other loose-skin mandarins and sweet oranges ( Zhang et al, 2019 ). Wang et al used gas chromatography–mass spectrometry to analyze the volatile components of Zangju peel during the whole growth period, which provided a basis for comprehensively understanding its flavor characteristics in the post-ripening period and its applications in food, cosmetics, flavors, and fragrances (P. Wang et al, 2023 , Wang et al, 2023 , Wang et al, 2023 ). Liquid chromatography–mass spectrometry has also become a central platform in metabolomics, specifically in the study of natural products and the plant sciences ( Salem et al, 2020 ).…”

Combining Metabolic Engineering and Lipid Droplet Assembly to Achieve Campesterol Overproduction in Saccharomyces cerevisiae

“…40−42 The biggest advantage of high-density fermentation is that each parameter value can be finely controlled during the fermentation process to maintain optimal fermentation conditions for cell growth and the production of the target product. 43,44 On the one hand, the productivity of the final product can be maximized, and on the other hand, the volume of the fermentation tank can be fully utilized and separation costs can be reduced. 45,46 In the present study, due to the knockout of the ERG5 gene, the synthesis pathway of ergosterol in S. cerevisiae has been disrupted.…”

Catalytic Mechanism and Heterologous Biosynthesis Application of Sesquiterpene Synthases

“…The mutant library was screened based on the vanillin-sulfuric acid method. Wang et al performed site-directed mutagenesis of FS3 from Camellia sinensis based on multiple sequence alignment and found that α-farnesene production in the FS3 W281C variant was markedly increased and was 1.9-fold higher than that in the WT, reaching 28.3 g/L.…”