Exploration and expanding the terpenome of Streptomyces sp. CBMAI 2042 via genome mining

• Grant number: 22/03936-6
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: September 01, 2022
• End date: August 31, 2023
• Field of knowledge: Physical Sciences and Mathematics - Chemistry - Organic Chemistry
• Principal Investigator: Ljubica Tasic
• Grantee: Douglas Cunha Sachito
• Supervisor: Rudolf Konrad Allemann
• Host Institution: Instituto de Química (IQ). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Institution abroad: Cardiff University, Wales
• Associated to the scholarship: 19/04855-7 - Exploration and expanding the terpenome of Streptomyces sp. CBMAI 2042 via genome mining, BP.DD

Abstract

Terpenes are the most abundant class of natural products in nature. These metabolites have a variety of applications, including pharmaceuticals, perfumes and flavors, bulk chemicals and fuels.This class of natural products is structurally diverse and derived from C5 precursor molecules, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). These precursor molecules are joined by enzymes known as prenyltransferases (PTs) to produce elongated polyisoprenes. Another group of enzymes fundamental to the structural diversification of terpenoids are terpene cyclases (TCs), which catalyse the conversion of linear oligoprenyl pyrophosphate via cationic intermediates into a variety of (poly)cyclic hydrocarbon molecules. Nevertheless, studies with bacterial terpene cyclases (BTCs) have shown that the overall amino acid sequences of these enzymes have no significant similarity to terpene cyclases of plant or fungal origin and only low amino acid sequence similarity to other BTCs. Therefore, studies in this area present an even greater challenge for the biochemical characterization of BTCs and assignment of the (poly)cyclic product.To overcome this challenge in searching for cryptic bacterial TCs, Yamada Y. and co-workers applied an alternative genome mining technique known as HMMs (Hidden Markov Models), which, in addition to aligning conserved regions in proteins, also performs a search of protein family databases and then correlates the tertiary structure with known bacterial cyclases. This new approach made it possible to expand the amount of information about enzymes of this class that was hidden in the Streptomyces genome. This method expanded the number of previously known 140 to 262 TCs.Previous studies of the volatile organic compounds of Streptomyces sp. CBMAI 2042, an endophyte isolated from Citrus sinensis, unveiled at least 28 cyclic sesquiterpenes. In addition, genome mining of S. sp. CBMAI 2042 revealed three genes encoding sesquiterpene cyclases. These genes are hereafter referred to as ts-1, ts-2 and ts-3.In this genome-guided study, three recombinant bacterial cyclases were produced in E. coli Rosetta (DE3), purified and incubated with farnesyl pyrophosphate (FPP). Enzyme substrate assays showed that all three recombinant enzymes were active and their incubationproducts were cyclic sesquiterpenes. In addition, one of the terpene cyclase studied in this project showed high promiscuity for the formation of cyclic products, a suitable and promising target enzyme for biotechnological applications. To increase the knowledge of bacterial cyclases, in this BEPE project we propose to explore the machinery of these enzymes by performing incubation experiments with unnatural precursors and enzyme. (AU)