Systematic metabolic engineering of s for β-farnesene production.

is an ethanologenic bacterium that can produce hopanoids using farnesyl pyrophosphate (FPP), which can be used as the precursor by β-farnesene synthase for β-farnesene production. To explore the possibility and bottlenecks of developing for β-farnesene production, five heterologous β-farnesene synthases were selected and screened, and from had the highest β-farnesene titer. Recombinant strains with driven by the strong constitutive promoter P (P-) doubled its β-farnesene production to 25.73 ± 0.31 mg/L compared to the recombinant strain with driven by P (P-), which can be further improved by overexpressing the P- construct using the strategies of multiple plasmids (41.00 ± 0.40 mg/L) or genomic multi-locus integration (48.33 ± 3.40 mg/L). The effect of cofactor NADPH balancing on β-farnesene production was also investigated, which can be improved only in -overexpressing strains but not in -overexpressing strains, indicating that cofactor balancing is important and sophisticated. Furthermore, the β-farnesene titer was improved to 73.30 ± 0.71 mg/L by overexpressing , , and . Finally, the β-farnesene production was increased to 159.70 ± 7.21 mg/L by fermentation optimization, including the C/N ratio, flask working volume, and medium/dodecane ratio, which was nearly 13-fold improved from the parental strain. This work thus not only generated a recombinant β-farnesene production strain but also unraveled the bottlenecks to engineer for farnesene production, which will help guide the future rational design and construction of cell factories for terpenoid production in non-model industrial microorganisms.