(337bu) Engineering Yarrowia Lipolytica for the Biosynthesis of Geraniol and Nepetalactol

The monoterpenoids geraniol and nepetalactol are two key intermediates in the strictosidine pathway. Strictosidine serves as the central intermediate for 3,000 monoterpene indole alkaloids with anticancer and other pharmacological properties. Traditionally, 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.

In this work, a geraniol producing strain was first metabolically engineered in Yarrowia lipolytica using random integration. Firstly, two plant-derived geraniol synthases (GES) from Catharanthus roseus (Cr) and Valeriana officinalis (Vo) were screened with and without signal peptide targeting chloroplast. It was found that the truncated CrGES produced the most geraniol. 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 and effects of increasing glucose concentration were also investigated. The final strain overexpressing 3 copies of truncated CrGES and ERG10, HMGS, tHMG1, IDI was able to produce around 1 g/L of geraniol in shake flask cultivation for the random integration strain.

The final geraniol strain (3 copies of truncated CrGES and ERG10, HMGS, tHMG1, IDI) was also obtained by directed integration of genes in the Yarrowia lipolytica genome which will be used to further integrate the downstream nepetalactol pathway. An important step in the nepetalactol pathway is the conversion of geraniol to 8-hydroxy geraniol (8-OH geraniol) by the action of the enzyme geraniol 8-hydroxylase (G8H) which is a P450 enzyme and often difficult to express in heterologous hosts. In a previous report of strictosidine production (Brown et al., 2015), G8H was identified as the major bottleneck in the strictosidine pathway expressed in S. cerevisiae. Hence, we are in the process of screening G8H from multiple plant variants. G8H, being a P450 enzyme, requires an additional reductase partner and in some cases cytochrome b5 (CYB5) in order to achieve full activity. These enzyme variants are also being tested with CPRs and CYB5 to achieve maximum activity. The best variant will be chosen on the basis of higher conversion of geraniol to 8-OH geraniol and will be integrated into the geraniol producing strain to obtain an 8-OH geraniol producing strain. Finally, enzymes downstream of geraniol 8-hydroxylase (GOR and ISY) will be integrated into the final strain to produce nepetalactol in Yarrowia lipolytica.