Promoter Engineering Enables Precise Metabolic Regulation Towards Efficient β-elemene Production in

Precisely controlling gene expression is beneficial for optimizing biosynthetic pathways for improving the production. However, promoters in nonconventional yeasts such as are always limited, which results in incompatible gene modulation. Here, we expanded the promoter library in based on transcriptional data, among which 13 constitutive promoters had the strengths ranging from 0-55% of P, the commonly used strong constitutive promoter, and 2 were growth phase-dependent promoters. Subsequently, 2 hybrid growth phase-dependent promoters were constructed and characterized, which had 2-fold higher activities. Finally, promoter engineering was applied to precisely regulate cellular metabolism for efficient production of β-elemene. The glyceraldehyde-3-phosphate dehydrogenase gene was downregulated to drive more flux into pentose phosphate pathway (PPP) and then to enhance the supply of acetyl-CoA by using phosphoketolase-phosphotransacetylase (PK-PTA) pathway. Coupled with the phase-dependent expression of synthase module (∼ fusion), the highest titer of 5.24 g/L with a yield of 0.037 g/(g glucose) was achieved in strain YY150U under fed-batch fermentation in shake flasks. This work characterized and engineered a series of promoters, that can be used to fine-tune genes for constructing efficient yeast cell factories.