Dual cytoplasmic-mitochondrial engineering for improved patchoulol production in Komagataella phaffii

ABSTRACT Mitochondria are a potential sub-organelle for the synthesis of terpenoids due to its abundant acetyl-CoA, cofactors, and compact compartment for targeting metabolic pathways and enzymes. Hence, conducting systematic investigations and harnessing the subcellular compartment of mitochondria may offer a viable avenue for enhancing terpenoids biosynthesis in yeast and other eukaryotic cells. Previous endeavors to boost terpenoids production in Komagataella phaffii mainly focused on diverse metabolic engineering strategies in the cytoplasm. Numerous studies have demonstrated that reconstructing specific metabolic pathways within mitochondria can lead to higher production yields than engineering the same pathways in the cytoplasm. In our study, we initially identified 6 well-performed mitochondrial targeting sequences (MTSs) through colocalization observation of EGFP and a mitochondrial red fluorescent probe via fluorescence microscopy. These MTSs were selected for the assembly of complete patchoulol biosynthesis pathways in the mitochondria, which resulted in a 1.7-fold increase in patchoulol production compared to the original strain. Furthermore, dual regulation of the cytoplasm and mitochondria was implemented in the P6H4＋PM2 strain to further enhance patchoulol synthesis to 109   mg/L, representing a 1.0-fold and 10.7-fold greater yield than that of the strains engineered solely in the cytoplasm or mitochondria, respectively. This study offers valuable guidance for effectively utilizing mitochondria to achieve higher patchoulol and other sesquiterpenes through dual cytoplasmic-mitochondrial engineering in K. phaffii .