Metabolic Engineering of Saccharomyces cerevisiae for Heterologous Carnosic Acid Production

Abstract

Carnosic acid (CA), a phenolic tricyclic diterpene, has many biological effects, including anti-inflammatory, anticancer, antiobesity, and antidiabetic activities. In this study, an efficient biosynthetic pathway was constructed to produce CA in Saccharomyces cerevisiae. First, the CA precursor miltiradiene was synthesized, after which the CA production strain was constructed by integrating the genes encoding cytochrome P450 enzymes (P450s) and cytochrome P450 reductase (CPR) SmCPR. The CA titer was further increased by the coexpression of CYP76AH1 and SmCPR ∼t28SpCytb5 fusion proteins and the overexpression of different catalases to detoxify the hydrogen peroxide (H₂O₂). Finally, engineering of the endoplasmic reticulum and cofactor supply increased the CA titer to 24.65 mg/L in shake flasks and 75.18 mg/L in 5 L fed-batch fermentation. This study demonstrates that the ability of engineered yeast cells to synthesize CA can be improved through metabolic engineering and synthetic biology strategies, providing a theoretical basis for microbial synthesis of other diterpenoids.

Keywords: Saccharomyces cerevisiae; carnosic acid; miltiradiene; synthetic biology; terpenoid.

FIGURE 1

Overall strategy of CA production in S. cerevisiae. HMG1/HMG2, HMG-CoA reductase; IDI, isopentenyl pyrophosphate isomerase; ERG20, FPP synthase; ERG9, squalene synthetase; BTS1, GGPP synthase; SmCPS/SmKSL, miltiradiene synthase; CYP76AH24, 11-hydroxy-ferruginol synthase; CYP76AK6, CA synthase; CYP76AH1, ferruginol synthase; HMG-CoA, hydroxymethylglutaryl-CoA; SmCPR, cytochrome P450 reductase; Cytb5, cytochrome B5; CTT1, catalase; ER, endoplasmic reticulum; INO2, transcription factor that promotes phospholipid synthesis; HAC1, transcription factor that promotes protein folding; HEM3, heme synthase; TCA, tricarboxylic acid cycle; POS5, NADH kinase. Three arrows represent multistep reactions.

FIGURE 2

Miltiradiene production in S. cerevisiae. (A) Chromatogram of miltiradiene produced by strain WM1; (B) Chromatogram of the original strain 3HP-F; (C) GC–MS spectra of miltiradiene published in the literature (Hu et al., 2020); (D) GC–MS spectra of the chromatographic peak at RT = 17.82 min.

FIGURE 3

Protein fusion and linker selection. WM2: Coexpression of tSmCPS and tSmKSL. WMa–e: Fusion of tSmCPS and tSmKSL using five flexible linkers: GSTSSGSSG, GSTSSG, GGG, GGGS, no linker. *: The product titer of strain WM3a was significantly higher than that of WM3b. *p < 0.05; **p < 0.01; ***p < 0.001. Histograms illustrating the production of corresponding strains. Experiments were performed in triplicate.

FIGURE 4

CA production in S. cerevisiae increased in a stepwise manner. (A) Production of CA and miltiradiene by strains WCA1a–c. *: The CA titer of strain WCA1c is significantly higher than that of WCA1b. *p < 0.05; **p < 0.01; ***p < 0.001 (B) Production of miltiradiene, ferruginol and CA by strain WCA2-3. The data are the averages of three separate experiments.

FIGURE 5

Influence of different fusion strategies on miltiradiene, ferruginol and CA production. (A) WCA4a: CYP76AH1 ∼ t60SmCPR, SpCytb5; (B) WCA4b: SmCPR, CYP76AH1 ∼ t28SpCytb5; (C) WCA4c: SmCPR ∼ t28SpCytb5, CYP76AH1; (D) WCA4d: CYP76AH1 ∼ t60SmCPR ∼ t28SpCytb5. (E) WCA3: CYP76AH1, SmCPR and SpCytb5 in the natural state. The data are the averages of three separate experiments.

FIGURE 6

CA production in S. cerevisiae increased in a stepwise manner. (A) Effect of the overexpression of ScCTA1 and ScCTT1 catalases on the product titer. (B) Fermentation was conducted in YPD medium. WCA8, INO2 gene overexpressed; WCA9, HEM3 gene overexpressed; WCA10, POS5 gene overexpressed; WCA11, HAC1 gene overexpressed. The data are the averages of three separate experiments.

FIGURE 7

Production of CA in batch and fed-batch fermentations in a 5 L bioreactor. (A) Batch fermentation in a 5 L bioreactor using strain WCA11. The fermentation was conducted in YPD medium with 40 g/L glucose. (B) WCA11 fed-batch fermentation in a 5 L bioreactor. The data are the averages of three separate experiments.