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

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.

Achievements and perspectives of synthetic biology in botanical insecticides.

Botanical insecticides are the origin of all insecticidal compounds. They have been widely used to control pests in crops for a long time. Currently, the commercial production of botanical insecticides extracted from plants is limited because of insufficient raw material supply. Synthetic biology is a promising and effective approach for addressing the current problems of the production of botanical insecticides. It is an emerging biological research hotspot in the field of botanical insecticides. However, the biosynthetic pathways of many botanical insecticides are not completely elucidated. On the other hand, the cytotoxicity of botanical pesticides and low efficiency of these biosynthetic enzymes in new hosts make it still challenging for their heterologous production. In the present review, we summarized the recent developments in the heterologous production of botanical insecticides, analyzed the current challenges, and discussed the feasible production strategies, focusing on elucidating biosynthetic pathways, enzyme engineering, host engineering, and cytotoxicity engineering. Looking to the future, synthetic biology promises to further advance heterologous production of more botanical pesticides.