Metabolic engineering of the non-conventional yeast Yarrowia lipolytica for the monoterpene geraniol heterologous production

Abstract

Green chemistry is a field of study focused on metabolic and bioprocess engineering for the production of various molecules. Employing genetically modified microorganisms, it turns feasible the sustainable synthesis of high-value compounds. One of these compounds is geraniol (3,7-dimethyl-trans-2,6-octadien-1-ol), which is a monoterpene used in many products as a fragrance and which presents potential as antimicrobial, antitumor, anti-inflammatory and antioxidant drug, for example. Notwithstanding its growing market, its attainment is restricted to extraction from essential oils by fractional distillation and to chemical synthesis. However, while the first is dependent on the long growth period of plants and on climatic conditions, the second requires restrictive conditions and numerous steps. As an alternative, the genetic engineering of microorganisms and the development of processes for the production of geraniol in biorreactors is investigated. Nowadays, Escherichia coli and Saccharomyces cerevisiae are the main organisms studied for this, but Yarrowia lipolytica, a non-conventional yeast species, might be explored for the production of geraniol. It presents characteristics related to availability of acetil-CoA precursor and to tolerance to geraniol antimicrobial activity, which are the major hurdles encountered in E. coli and S. cerevisiae. The yeast Y. lipolytica has high levels of cytosolic acetil-CoA, related to its elevated lipid synthesis. Furthermore, we hypothesize that its known tolerance for organic compounds could mean it is less impaired by the toxic geraniol. The present study aims at process development for geraniol production by Y. lipolytica. Firstly, genetic modification and heterologous expression of the geraniol synthase enzyme (GES gene) will be achieved using CRISPR-Cas9. The strain used for this already bears modifications for monoterpene synthesis: in addition to Cas9 expression, it superexpresses ACL and expresses SeACS for improvement of acetil-CoA levels, it superexpresses HMG, ERG12 and IDI for augmentation of mevalonate pathway flux, it expresses a modified ERG20 to make the GPP precursor available and it had its pSQS1 promoter exchanged by the weak pERG11 promoter to downregulate the squalene synthase. Therefore, this strain is already partially optimized for monoterpene synthesis and requires only the GES gene to produce geraniol. Three GES (from Catharanthus roseus, Valeriana officinalis and Ocimum basilicum) will be tested under control of the hybrid hp4d promoter. The activity of this promoter is related to the onset of the stationary phase of microbial growth and, consequently, can be used to decouple the toxic geraniol production from cell proliferation. After yeast metabolic engineering, the geraniol producing strain obtained will be grown in biorreactors. Geraniol production in fed-batch system will be evaluated to control by-product synthesis and maximize geraniol yield. Aeration, carbon source, pH and feeding strategies will be optimized. Hence, a process for ecological and sustainable production of the geraniol compound will be developed using a non-conventional yeast species.