Metabolic engineering of the yeast Yarrowia lipolytica for the monoterpene geraniol heterologous production.

Author(s)

Enzo Bento Queiroz

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Escola Politécnica (EP/BC)
• Defense date: 2023-06-28

Examining board members

Thiago Olitta Basso; Andreas Karoly Gombert; Luiziana Ferreira da Silva

• Advisor: Thiago Olitta Basso

Abstract

Geraniol is a monoterpene, a class of secondary metabolites of plants, used as a fragrance in various hygiene and cosmetic products. In addition, this molecule also has potential, for example, as an antimicrobial and antitumor drug and as a chemical precursor of other compounds relevant to industry and human health. Despite its growing market, obtainment of geraniol is restricted to the extraction of plant essential oils or chemical synthesis, which are processes with inefficient, expensive or polluting steps. In this context, the use of metabolic engineering for the production of terpenes by microorganisms is a promising alternative that has been explored by the scientific community. Yarrowia lipolytica, a non-conventional oleaginous yeast, has attributes that can be exploited for the sustainable and efficient production of geraniol. These characteristics include tolerance to organic compounds and high levels of cytosolic acetyl-CoA, the molecule that initiates the monoterpene biosynthesis pathway. Thus, the present project aimed to develop a Y. lipolytica strain engineered for the biosynthesis of geraniol. For this, the Y. lipolytica strain to be transformed was analyzed for its growth and cellular morphology and its growth physiology was studied. Then, the plasmids required for geraniol synthase (GES) expression and its integration into the Y. lipolytica genome were replicated and confirmed. Finally, the CRISPRCas9 genome editing system was applied for the expression of the GES enzyme from Catharanthus roseus in the Y. lipolytica strain with modifications for the production of monoterpenes. Integration of expression cassette at the target locus was confirmed with diganosis PCR with genomic DNA as template. Production of 35 ± 10 mg L-1 geraniol (which represents a yield of 0.08 % C-mol geraniol C-mol-1 glucose) by the genetically modified BYa3105 strain was verified via GC-MS. Therefore, the basis for a bioprocess of geraniol production have been established and may be further explored in the next stages of the project. This is the first known report of de novo geraniol biosynthesis by Y. lipolytica. (AU)

• FAPESP's process: 21/02155-8 - Metabolic engineering of the non-conventional yeast Yarrowia lipolytica for the monoterpene geraniol heterologous production
• Grantee: Enzo Bento Queiroz
• Support Opportunities: Scholarships in Brazil - Master