Dual Regulation of Cytoplasm and Peroxisomes for Improved Ay-Farnesene Production in Recombinant Pichia pastoris

Microbial production of alpha-farnesene from renewable raw materials is a feasible alternative to traditional petroleum craft. Recently, the research on improving alpha-farnesene production in Pichia pastoris mainly focused on cytoplasmic engineering, while comprehensive engineering of multiple subcellular compartments is rarely reported. Here, we first sought to confirm that the isopentenol utilization pathway (IUP) could act as a two-step shortcut for IPP synthesis in P. pastoris peroxisomes. In addition, we proposed dual regulation of cytoplasm and peroxisomes to boost alpha-farnesene synthesis in P. pastoris X33, thus the resultant strain produced 2.18 +/- 0.04 g/L, which was 1.3 times and 2.1 times than that of the strain only with peroxisomal or cytoplasmic engineering, respectively. The alpha-farnesene production achieved 2.56 +/- 0.04 g/L in shake flasks after carbon source cofeeding, which was the highest reported production in worldwide literatures to the best of my knowledge. Therefore, we propose these strategies as efficient approaches to enhancing alpha-farnesene production in P. pastoris, which might bring new ideas for the biosynthesis of high-value compounds.