Abstract: Microbial production of α-farnesene from renewable feedstock is a promising alternative to traditional petroleum-based processes. Although it has been reported that α-farnesene can be heterologously produced by conventional model strains such as Escherichia coli and Saccharomyces cerevisiae, its fermentation scale is not easy to expand. Pichia pastoris is a good platform for the production of isoprene and has the potential for large-scale production of α-farnesene at high density. tHmg1, IDI1, ERG19, and AFSLERG20 were confirmed as the rate-limiting enzymes of the mevalonate pathway and the α-farnesene synthesis pathway. Then, the metabolic flux to α-farnesene synthesis was increased by combining overexpression rate limiting enzyme genes and optimizing the copy number of genes to balance the metabolic pathway. Strain F16 was obtained, and its α-farnesene yield was (1.09±0.02) g/L. Furthermore, the secretion of α-farnesene into extracellular space was promoted by adding unsaturated fatty acids, when 20 mmol/L of oleic acid was added to the medium, the highest α-farnesene titer in the shake flask was about 1.40 g/L [0.32 g/g (dry cell weight,DCW)], which was 3.1 times of that in the starting strain F1. For the first time, P. pastoris was used as a chassis microbial cell to produce α-farnesene and the results provide a new idea for the heterologous biosynthesis of other sesquiterpenes.
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