Metabolic Engineering of Corynebacterium glutamicum for Producing Different Types of Triterpenoids.

Abstract

Triterpenoids widely exist in nature with diverse structures and possess various functional properties and biological effects. However, research on triterpenoids biosynthesis in Corynebacterium glutamicum is still limited to squalene, which restricts the development of C. glutamicum to produce high-value triterpenoids. In this study, C. glutamicum was developed as an efficient and flexible platform for the biosynthesis of different types of triterpenoids. Squalene was synthesized and the titer was improved to 400.1 mg/L in flask combining strategies of metabolic engineering and fermentation optimization. Particularly, intracellular squalene accounted for more than 97%, addressing the problem of leaking squalene in C. glutamicum, which may restrict the subsequent synthesis of other triterpenoids derived from squalene. Furthermore, 201.9 mg/L (3S)-2,3-oxidosqualene (SQO) and 264.9 mg/L (3S,22S)-2,3,22,23-dioxidosqualene (SDO) were successfully synthesized in strains harboring heterogeneous squalene epoxidase from Arabidopsis thaliana with different expression strengths. Therefore, a platform for de novo triterpenoids synthesis based on SQO or SDO was constructed in C. glutamicum. For instance, biosynthesis of α-amyrin and α-onocerin was achieved for the first time by introducing oxidosqualene cyclases in SQO- and SDO-producing C. glutamicum strains, respectively. After optimization, the titer of α-amyrin and α-onocerin was improved to 65.3 and 136.85 mg/L, respectively. Furthermore, ursolic acid, derived from α-amyrin, was synthesized after expressing cytochrome P450 enzyme and its compatible cytochrome P450 reductases with a titer of 486 μg/L. For the first time, reactions of epoxidation, cyclization, and oxidation from squalene were achieved in C. glutamicum, leading to the production of different types of triterpenoids. Our study provides a new platform for the production of triterpenoids, which will be helpful for the large-scale production of triterpenoids employing C. glutamicum as a chassis strain.