Carnosic acid (CA) is a phenolic diterpene compound, which is widely present in rosemary, sage and other labiaceae plants. Since CA has various physiological and pharmacological activities, it can be applied in the area of daily chemical, food, and medicine. In the biosynthesis of terpenoids, Candida tropicalis is a highly efficient production platform, because of its strong robustness, high temperature resistance, oil production and other properties. In this study, according to the codon preference and specificity of C. tropicalis, the whole sequence codon optimization and gene synthesis of the ferruginol synthase genes CYP76AH24 and the CA synthase CYP76AK6 from sage and the cytochrome reductase ATR1(CPR) from Arabidopsis were carried out. These genes were integrated into the genome of C. tropicalis 1C2PM04, which produces miltiradiene, the precursor of CA, and CA was successfully synthesized. Using modular engineering and the spatial combination of different subcellular compartments of yeast, the above three genes were introduced into the cytoplasm and peroxisome, and the CA production was 10.25 and 4.64 mg/L, respectively. By down-regulating squalene synthase ERG9, the consumption of farnesyl pyrophosphate pool was reduced and the metabolic flux was changed to further improve the CA production, and the titer reached 26.3 mg/L. Through the strategy of fusion expression of cytochrome P450 enzyme and cytochrome reductase, the rigid linker (E3AK, E3AK×2, E3AK×3) and flexible linker (G4S, G4S×2, G4S×3) were used as linker peptides to express CYP76AH24 and ATR1. The yield of CA increased by 1.54 times. By increasing the copy number of CPR and increasing the electron donor, the CA expression level was further improved, and the yield reached 78.24 mg/L.
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