Background: Ginsenoside compound K (CK), one of the primary active metabolites of protopanaxadiol-type ginsenosides, is produced by the intestinal flora that degrade ginseng saponins and exhibits diverse biological properties such as anticancer, anti-inflammatory, and anti-allergic properties. However, it is less abundant in plants. Therefore, enabling its commercialization by construction of a Saccharomyces cerevisiae cell factory is of considerable significance. Results: We induced overexpression of PGM2, UGP1, and UGT1 genes in WLT-MVA5, and obtained a strain that produces ginsenoside CK. The production of CK at 96 h was 263.94 ± 2.36 mg/L, and the conversion rate from protopanaxadiol (PPD) to ginsenoside CK was 64.23 ± 0.41%. Additionally, it was observed that the addition of glycerol was beneficial to the synthesis of CK. When 20% glucose (C mol) in the YPD medium was replaced by the same C mol glycerol, CK production increased to 384.52 ± 15.23 mg/L, which was 45.68% higher than that in YPD medium, and the PPD conversion rate increased to 77.37 ± 3.37% as well. As we previously observed that ethanol is beneficial to the production of PPD, ethanol and glycerol were fed simultaneously in the 5-L bioreactor fed fermentation, and the CK levels reached 1.70 ± 0.16 g/L. Conclusions: In this study, we constructed an S. cerevisiae cell factory that efficiently produced ginsenoside CK. Glycerol effectively increased the glycosylation efficiency of PPD to ginsenoside CK, guiding higher carbon flow to the synthesis of ginsenosides and effectively improving CK production. CK production attained in a 5-L bioreactor was 1.7 g/L after simultaneous feeding of glycerol and ethanol.

Diterpenoids are a large class of natural products with complex structures and broad commercial applications as food additives, important medicines, and fragrances. However, their low abundance in plants and high structural complexity limit their applications. Therefore, it is important to create an efficient diterpenoid-producing yeast cell factory of the production of various high-value diterpenoid compounds in a cost-effective manner In this study, 13R-manoyl oxide (13R-MO; 2.31 mg/L) was produced by expressing CfTPS2 and CfTPS3 from Coleus forskohlii in Saccharomyces cerevisiae. The 13R-MO titer was increased by 142-fold to 328.15 mg/L via the stepwise metabolic engineering of the original strain, including the overexpression of the rate-limiting genes (tHMG1 and ERG20) of the mevalonate pathway, transcription and protein level regulation of ERG9, Bts1p and Erg20F96Cp fusion, and the overexpression of tCfTPS2 and tCfTPS3 (excision of the N-terminal plastid transit peptide sequences of CfTPS2 and CfTPS3). The final titer of 13R-MO reached up to 3 g/L by fed-batch fermentation in a 5 L bioreactor. In this study, an efficient 13R-MO yeast cell factory was constructed, which achieved the de novo production of 3 g/L of 13R-MO from glucose. To the best of our knowledge, this is the highest 13R-MO titer reported to date. Furthermore, the metabolic engineering strategies presented here could be used to produce other valuable diterpenoid compounds in yeast.

In this article, we report a method for preparing an immobilized bacterial agent of petroleum-degrading bacteria Gordonia alkanivorans W33 by combining high-density fermentation and bacterial immobilization technology and testing its bioremediation effect on petroleum-contaminated soil. After determining the optimal combination of MgCl₂, CaCl₂ concentration, and culture time in the fermentation conditions by conducting a response surface analysis, the cell concentration reached 7.48 × 10⁹ CFU/mL by 5 L fed-batch fermentation. The W33-vermiculite-powder-immobilized bacterial agent mixed with sophorolipids and rhamnolipids in a weight ratio of 9:10 was used for the bioremediation of petroleum-contaminated soil. After 45 days of microbial degradation, 56.3% of the petroleum in the soil with 20,000 mg/kg petroleum content was degraded, and the average degradation rate reached 250.2 mg/kg/d.