Metabolic and evolutionary engineering of non-conventional yeasts for bioactive terpenoid production

Abstract

Geraniol is a monoterpene molecule used as a fragrance in various products such as creams and cleaning products. Its potential as an antimicrobial and antitumor drug, for example, is also vastly researched. Despite its growing market, its production is restricted to the extraction from essential oils by distillation or chemical synthesis, which are inefficient and expensive processes. In this context, the application of metabolic engineering for the synthesis of this compound by microorganisms is a promising alternative. Since now, the heterologous expression of the geraniol synthase enzyme was tested mainly in the microorganisms Escherichia coli and Saccharomyces cerevisiae, in which the low availability of the acetyl-CoA precursor and the toxicity of geraniol are the main obstacles to its biosynthesis. Yarrowia lipolytica, an unconventional oleaginous yeast, has attributes that can be exploited for the production of geraniol, possibly making it a more suitable host for its heterologous production. These characteristics include tolerance to organic compounds and high levels of cytosolic acetyl-CoA, due to its intense carbon flux to lipid metabolism. Furthermore, this yeast has the ability to consume several carbon sources, including oils and acetate, which is remarkable from the point of view of establishing bioprocesses. Due to these and other characteristics, naturally this is a yeast that has been used in several works for the production of different terpenes. Furthermore, a partner research group from Denmark, which can be said to be one of the world leaders in Y. lipolytica research, developed a strain already available for use in the project, with seven genetic modifications aimed at metabolic engineering for the production of monoterpenes. Hence, the present Initial Project Proposal aims to utilize this previously modified strain to obtain geraniol-producing Y. lipolytica strains and test them in fed-batch and chemostat cultures. Initially, experiments for evolution in flasks will be carried out to obtain Y. lipolytica strains that are more resistant to geraniol in the medium. Then, CRISPR-Cas9 technology will be applied to the expression of the enzyme geraniol synthase (GES) under the control of the semi-constitutive hp4d promoter, which shows higher activity at the start of the stationary phase of microbial growth. Thus, it is expected that the effect of geraniol toxicity during cell growth will be partially avoided. In this step, geraniol synthases originating from three different plants will be tested, in order to evaluate which has the best production in the oleaginous yeast. After that, the expression of the GES enzyme fused to geranyl pyrophosphate (GPP) synthase, which synthesizes the precursor GPP, will be evaluated under a semi-constitutive hp4d promoter and a constitutive TEFin promoter. As observed in S. cerevisiae, expression of the fused enzymes is expected to increase the production of geraniol by channeling the GPP substrate and by adding one more copy of GES to the yeast genome. Subsequently, the evolved and engineered strains will be analyzed for their ability to produce geraniol in bioreactors. Thenceforth, production in a fed-batch system will be evaluated and the aeration conditions, carbon sources, pH and feeding regimes will be optimized. Finally, laboratory evolution experiments will be carried out through long-term continuous cultures in benchtop reactors (chemostats), in order to select variants of engineered yeast that are even more tolerant to geraniol. Therefore, the project will carry out a complete process, from the construction of strains to the optimization of cultivation conditions and characterization of engineered yeast physiology, integrating training of students and techniques currently relevant in the areas of metabolic engineering and bioprocesses in a topic of scientific and socioeconomic importance. (AU)