A novel high-temperature Kluyveromyces marxianus as a microbial cell factory host for sesquiterpene production

Artemisinin, a vital antimalarial agent, is traditionally extracted from Artemisia annua L. by plant-based extraction. The microbial biosynthesis of the artemisinin precursor, amorpha-4,11-diene (AD), followed by its chemical conversion into artemisinin provides a sustainable solution to meet the demand for artemisinin while overcoming the limitations of plant-based extraction. Kluyveromyces marxianus is a promising host for microbial cell factories owing to its robust thermotolerance, rapid growth kinetics, and ability to utilize a wide range of carbon sources. Herein, genetic modification tools for K. marxianus, including transformation methods and expression vectors, were identified and optimized, while the sesquiterpene biosynthetic capacity of K. marxianus was preliminarily evaluated. To enhance the precursor metabolite supply, eight key genes from the endogenous mevalonate pathway were overexpressed within the K. marxianus genome. A dual co-expression strategy, involving both extrachromosomal and chromosomally integrated expression of amorpha-4,11-diene synthase (ADS) was employed, followed by high-temperature fermentation to optimize production. Finally, an engineered K. marxianus strain was developed for the first time, capable of stable, efficient, and cost-effective production of AD, with a titer of 66.78 mg/L, a 113-fold increase compared with the wild-type strain. This engineered strain serves as a robust chassis and a valuable reference for the production and study of other terpenoids, laying the groundwork for further exploration of K. marxianus characteristics and the biosynthesis of AD.