Cytoplasmic-peroxisomal spatial combination engineering in Candida tropicalis for enhanced terpenoid production

Abstract

Metabolic engineering of microorganisms for terpenoid biosynthesis is a green and sustainable alternative to plant extraction and chemical synthesis methods for producing plant-derived natural products. The compartmentalization of pathways or enzymes within organelles is increasingly recognized for its potential to eliminate metabolic crosstalk and enhance pathway efficacy in terpenoid production. However, the physicochemical environment of a given organelle may not meet the requirements for multiple enzymes. Here, a cytoplasmic-peroxisomal spatial-combination strategy was developed in Candida tropicalis to enhance terpenoid production. This strategy involved compartmentalizing the pathway from acetyl-CoA to mevalonate (MVA) within the cytoplasm and the subsequent steps from MVA to terpenoids within the peroxisome. Furthermore, we demonstrated that the pathway developed in this study is more effective because it provides optimal conditions for enzymes, and the use of the peroxisome membrane as a barrier to concentrate farnesyl diphosphate and geranylgeranyl pyrophosphate within the organelle. Additionally, we observed that this strategy efficiently enhanced productivity and was applicable to sesquiterpenoid, diterpene, and tetraterpenoid biosynthesis. Finally, under 5-L fed-batch fermentation, the titers of β-carotene and miltiradiene reached 9.9 g L⁻¹ and 4.2 g L⁻¹, respectively. This study provides new insights into terpenoid biosynthesis in yeast.