(+)-Ambrein is the primary component of ambergris, a rare product found in sperm whales (Physeter microcephalus). Microbial production using sustainable resources is a promising way to replace animal extraction and chemical synthesis. We constructed an engineered yeast strain to produce (+)-ambrein de novo. Squalene is a substrate for the biosynthesis of (+)-ambrein. Firstly, strain LQ2, with a squalene yield of 384.4 mg/L was obtained by optimizing the mevalonate pathway. Then we engineered a method for the de novo production of (+)-ambrein using glucose as a carbon source by overexpressing codon-optimized tetraprenyl-β-curcumene cyclase (BmeTC) and its double mutant enzyme (BmeTC^Y167A/D373C), evaluating different promoters, knocking out GAL80, and fusing the protein with BmeTC and squalene synthase (AtSQS2). Nevertheless, the synthesis of (+)-ambrein is still limited, causing low catalytic activity in BmeTC. We carried out a protein surface amino acid modification of BmeTC. The dominant mutant BmeTC^{K6A/Q9E/N454A} for the first step was obtained to improve its catalytic activity. The yield of (+)-ambrein increased from 35.2 to 59.0 mg/L in the shake flask and finally reached 457.4 mg/L in the 2 L fermenter, the highest titer currently available for yeast. Efficiently engineered strains and inexpensive fermentation conditions for the industrial production of (+)-ambrein. The metabolic engineering tools provide directions for optimizing the biosynthesis of other high-value triterpenes.

• PMID: 38884886
• DOI full text
• PubMed
• PubTator

Reference Type

Journal Article

Authors

Lin C, Zhang X, Ji Z, Fan B, Chen Y, Wu Y, Gan Y, Li Z, Shang Y, Duan L, ... Show all

Primary Lit For

Additional Lit For

Review For