| Congresso Brasileiro de Microbiologia 2023 | Resumo: 209-1 | ||||
Resumo:Abstract: Geraniol is a monoterpene found in essential oils from a plethora of plant species. It is used as a fragrance in cleaning and personal hygiene products, but also has potential pharmaceutical applicability as an antibiotic and anti-inflammatory drug. Geraniol industrial production may be conducted by extraction from plant tissues. This method, however, requires great quantities of plant material and is influenced by weather conditions during the time-consuming growth of these organisms. Chemical synthesis is most often applied nowadays, but require unsustainable fossil feedstock and harsh reaction conditions. A more sustainable and cheap alternative in development is the heterologous biosynthesis of geraniol by microorganisms using renewable materials. One possible host for this task is Yarrowia lipolytica, a Saccharomycetales fungi which metabolizes different sugars, lipids and organic compounds as carbon and energy sources and presents high pool of cytosolic Acetyl-CoA, the precursor for the geraniol synthesis metabolic pathway. In this work, integration of the Catharanthus roseus geraniol synthase (crGES) gene into the genome of Y. lipolytica was conducted, with the aim of developing a geraniol producing strain. For this, the hp4d quasi-constitutive synthetic promoter, the codon-optimized crGES gene and the TLip2 terminator sequences, flanked by sequences homologous to a specific genomic region devoid of genes, were chosen to compose a donor DNA expression cassette and then synthesized by a third party. A Y. lipolytica strain previously modified for high-titre monoterpene biosynthesis and for genomic editing via an integrated Cas9 was transformed with the obtained donor DNA and with an epissomal plasmid with the required sequence for expression of a gRNA. Transformant colonies whose genome was properly repaired with the donor DNA after the double-strand break caused by the Cas9-gRNA system were obtained after growth in selective medium. Genomic DNA was extracted using a commercial kit and used for confirmation of the cassette integration by PCR. Thus, a novel Y. lipolytica strain with the crGES gene integrated in its genome was obtained. Further experiments require the confirmation of the gene expression and of geraniol biosynthesis by the genetically modified Y. lipolytica.
Development agencies: This work was funded by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo), grant numbers 2021/02155-8 and 2022/03052-0. Palavras-chave: CRISPR, Metabolic engineering, Yarrowia lipolytica, Terpene Agência de fomento:FAPESP | |||||