Zhenqiu Li,Ruiping Gao,Qinggang Hao,Huifang Zhao,Longbin Cheng,Fang He,Li Liu,Xiuhua Liu,Wayne K. W. Chou,Huajie Zhu,David E. Cane
The T296V mutant of amorpha-4,11-diene synthase catalyzes the abortive conversion of the natural substrate (E,E)-farnesyl diphosphate mainly into the acyclic product (E)-β-farnesene (88%) instead of the natural bicyclic sesquiterpene amorphadiene (7%). Incubation of the T296V mutant with (3R,6E)-nerolidyl diphosphate resulted in cyclization to amorphadiene. Analysis of additional mutants of amino acid residue 296 and in vitro assays with the intermediate analogue (2Z,6E)-farnesyl diphosphate as well as (3S,6E)-nerolidyl diphosphate demonstrated that the T296V mutant can no longer catalyze the allylic rearrangement of farnesyl diphosphate to the normal intermediate (3R,6E)-nerolidyl diphosphate, while retaining the ability to cyclize (3R,6E)-nerolidyl diphosphate to amorphadiene. The T296A mutant predominantly retained amorphadiene synthase activity, indicating that neither the hydroxyl nor the methyl group of the Thr296 side chain is required for cyclase activity.
Min Zhang,Yongfu Ruan,Yi Hu,Yiying Huo,Meng Wang,Zhiwei Zhu,Yunming Fang
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.
Due to complex chemical mechanism of sesquiterpene synthases, amorpha-4, 11-diene (AD) from microbial fermentation contained isomeride β-farnesene (FA) with similar boiling point. For purifying AD, FA was converted to biofuel-valued squalene (SQ) with high boiling point using solid Pd catalysts. (NHT)OS-Pd catalysts resulted in FA conversion of >99.9% and SQ selectivity of >99.0% at 80 °C for 5 h. Moreover, this catalyst exhibited great stability and recycling performance. When (NHT)OS-Pd was applied to AD mixture, almost FA was converted to SQ and AD remained in the pre-reaction state. As SQ had a higher boiling point, AD was easily distilled out.
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