(164an) Engineering Y. Lipolytica for the Biosynthesis of Geraniol

Geraniol is a monoterpene with wide applications in food, cosmetics, and pharmaceutical industries. Moreover, geraniol is the primary precursor of strictosidine which can be produced by incorporating a 12-step pathway in a geraniol producing strain. Strictosidine serves as the central intermediate for 3,000 monoterpene indole alkaloids with anticancer and other pharmacological properties. Traditionally, the monoterpenes are produced in their native plant sources in minute amounts. Microbial production of monoterpenes provides an alternative to traditional plant-based methods which require laborious extraction and separation methods and result in overharvesting of medicinal plants. Hence, efforts have been focused on metabolic engineering of microbes like E. coli and S. cerevisiae for the biosynthesis of natural products. However, E. coli lacks inner organelles that are present in yeast and cannot perform post-translational modifications making it an unsuitable host. Additionally, S. cerevisiae has low natural flux towards natural product synthesis making it an undesirable host. Alternatively, Yarrowia lipolytica can serve as an ideal host for natural product biosynthesis because of the availability of genetic engineering and synthetic biology tools.

This project aims to produce geraniol in metabolically engineered Yarrowia lipolytica. First, two plant-derived geraniol synthases (GES) from Catharanthus roseus (Cr) and Valeriana officinalis (Vo) were screened based on literature. Both wild type and truncated mutants of GES (without signal peptide targeting chloroplast) were examined by transforming yeast. It was found that the truncated CrGES produced the most geraniol and thus was used for further experimentation. The starting strain was obtained by overexpression of the truncated HMG1, IDI and truncated CrGES. Enhancing the precursor pool of acetyl-CoA was performed by overexpression of the MVA pathway genes such as ERG10, HMGS or MVK, PMK. Effect of copy number of truncated CrGES was also investigated. The final strain overexpressing 3 copies of truncated CrGES and ERG10, HMGS, tHMG1, IDI was able to produce around 700 mg/L of geraniol in shake flask cultivation. The optimization of fermentation conditions (carbon to nitrogen ratio) led the titer to increase around 1 g/L in shake flask cultivation. The bioreactor experiment is underway to boost the geraniol titers. The future work includes integrating the downstream pathway genes from geraniol to strictosidine to engineer a strictosidine producing strain.