Abstract

Terpenes have many applications and are widely found in nature, but recent progress in synthetic biology has enabled the use of microorganisms as chassis cells for the synthesis of these compounds. Candida glycerinogenes (C. glycerinogenes) is an industrial strain that may be developed as a chassis for the synthesis of terpenes since it has a tolerance to hyperosmolality and high sugar, and has a complete mevalonate (MVA) pathway. However, monoterpenes such as pinene are highly toxic, and the tolerance of C. glycerinogenes to pinene was investigated. We also measured the content of mevalonate and squalene to evaluate the strength of the MVA pathway. To determine terpene synthesis capacity, a pathway for the synthesis of pinene was constructed in C. glycerinogenes. Pinene production was improved by overexpression, gene knockdown and antisense RNA inhibition. Pinene production was mainly enhanced by strengthening the upstream MVA pathway and inhibiting the production of by-products from the downstream pathway. With these strategies, yield could be increased by almost 16 times, to 6.0 mg/L. Overall, we successfully constructed a pinene synthesis pathway in C. glycerinogenes and enhanced pinene production through metabolic modification.

• Keywords: Antarctic seal;NMR;Phocidae;gut microbiome;marine mammal