Systematic engineering to enhance 3-carene production in yeast

• The NPP orthogonal pathway was introduced, and the well-matched key enzyme of Ss CarTS was selected and truncated to increase the yield of 3-carene. • The appropriate subcellular localization into endoplasmic reticulum for the truncated Ss CarT improved the production efficiency of 3-carene. • The molecular chaperone of PDI1 was coexpressed with the Ss CarTS to further increase the yield of 3-carene. • Obtained a 3-carene yield of 506.7 mg/L in a 5-L fed-batch culture system, which is the highest titre reported in microbes. 3-Carene is a bicyclic monoterpene with a pine aroma. It is widely used as a flavoring agent, food additive, and insecticide in food and agricultural applications. Therefore, establishing an efficient microbial pathway for 3-carene is important. At present, the low activity of 3-carene synthase (CarTS) and the insufficient supply of precursors in Saccharomyces cerevisiae significantly constrain the synthesis of 3-carene. In this study, the NPP orthogonal pathway, source screening, CarTS modification, enzymatic subcellular localization, and molecular chaperone screening were used to promote 3-carene synthesis. By introducing the NPP orthogonal pathway and truncated CarTS from Salvia stenophylla ( Ss CarTS), the yield of 3-carene increased by 32.1-fold. Subsequently, through enzymatic subcellular localization and molecular chaperone screening, the yield of 3-carene was increased by 39.0%, reaching 50.6 mg/L in shake flask cultures. Eventually, the final titer of 3-carene reached 506.7 mg/L in a 5-L bioreactor by optimizing the culture medium containing metal ions and vitamins, which is the highest reported titer of 3-carene in microbial cell factories. In this study, we provide a cell factory for the synthesis of 3-carene as well as reference data for the biosynthesis of other bicyclic monoterpenes.