Building terpene production platforms in yeast.

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• Terpenes and terpenoids are valuable compounds with complex chemical synthesis.
• Biological production platforms offer a sustainable alternative for terpene synthesis.
• Current yeast strains require optimization for efficient terpene precursor accumulation.

Purpose of the Study:

• To develop a robust yeast production platform for terpenes.
• To enhance the accumulation of farnesyl pyrophosphate (FPP), a key terpene precursor.
• To engineer yeast for the production of specific sesquiterpenes and triterpenes.

Main Methods:

• Employing unbiased genetic selection (EMS mutagenesis) to identify yeast mutants with enhanced sterol uptake (SUE mutations).
• Screening for high farnesol accumulation as an indicator of increased FPP levels.
• Introducing insertional mutations in ERG9 (squalene synthase) and ERG1 (squalene epoxidase) genes.
• Transforming engineered yeast with terpene synthase genes (HPS for sesquiterpenes, heterologous genes for triterpenes).

Main Results:

• Identified SUE mutations enabling dispensable mevalonate pathways and exogenous sterol uptake.
• Developed yeast lines accumulating over 70 mg/L farnesol.
• Engineered strain ZX178-08 produced 116 mg/L premnaspirodiene (sesquiterpene), increased to 170 mg/L with tHMGR co-expression.
• Achieved over 60 mg/L botryococcene (triterpene) and >270 mg/L squalene production with optimized ERG1 mutants.

Conclusions:

• The developed yeast platform demonstrates significant potential for microbial terpene and terpenoid production.
• Metabolic engineering strategies, including SUE mutations and pathway optimization, are effective for enhancing precursor pools.
• This platform provides a versatile and efficient system for producing diverse terpenes, outperforming parental strains significantly.