Squalene (C30H50) is an acyclic triterpenoid compound renowned for its myriad physiological functions, such as anticancer and antioxidative properties, rendering it invaluable in both the food and pharmaceutical sectors. Owing to the constraints on natural resources, microbial fermentation has emerged as a prominent trend. Schizochytrium sp. known to harbor the intact Mevalonate (MVA) pathway, possesses the inherent capability to biosynthesize squalene. However, there is a dearth of reported key genes in both the MVA pathway and the squalene synthesis pathway, along with the associated promoter elements for their modification. This study commenced by cloning and characterizing 13 endogenous promoters derived from transcriptome sequencing data. Subsequently, five promoters exhibiting varying expression intensities were chosen from the aforementioned pool to facilitate the overexpression of the squalene synthase gene SQS, pivotal in the MVA pathway. Ultimately, a transformed strain designated as SQS-3626, exhibiting squalene production 2.8 times greater than that of the wild-type strain, was identified. Lastly, the optimization of nitrogen source concentrations and trace element contents in the fermentation medium was conducted. Following 120 hours of fed-batch fermentation, the accumulated final squalene yield in the transformed strain SQS-3626 reached 2.2 g/L.

The isopentenol utilization pathway (IUP) is potential in terpenoids synthesis. This study aimed to construct IUP-employed E. coli chassis for stably synthesizing terpenoids. As to effectiveness, promotor engineering strategy was employed to regulate IUP expression level, while ribosome-binding site (RBS) library of the key enzyme was constructed for screening the optimal RBS, followed by optimization of concentration of inducer and substrates, the titer of reporting production, lycopene, from 0.087 to 8.67 mg/OD600. As about stability, the IUP expression cassette was integrated into the genome through transposition tool based on CRISPR-associated transposases. Results showed that the strain with 13 copies produced 1.78-fold lycopene titer that of the controlled strain with IUP-harbored plasmid, and it exhibited stable expression after ten successions while the plasmid loss was observed in the controlled strain in the 3rd succession. This strategy provides valuable information for rapid construction of highly effective and stable chassis employing IUP for terpenoids production.

Yarrowia lipolytica, an oleaginous yeast with the GRAS status, has been developed as a platform for the chemical production. Specifically, promoter engineering is an important approach to regulating gene expression at the transcriptional level, which is of great significance in constructing microbial cell factories. Until now, some work on the promoter study has been carried out in Y. lipolytica. However, compared with other microorganisms, such as Saccharomyces cerevisiae, Bacillus subtilis, and etc., obtained achievements still need further replenishment and development for Y. lipolytica. For this, we screened 81 endogenous promoters in Y. lipolytica, mainly involved in carbon metabolism, amino acid metabolism, and lipid metabolism. As a result, we obtained 15 strong promoters, 41 medium strength promoters, and 25 weak promoters, with the strength spanning from 0.06% to 1.60-fold of PTEF promoter. In general, our study provides a unique and available endogenous promoter library for Y. lipolytica.