International collaboration in the chemistry of alkaloid natural product biosynthesis

• Grant number: 12/50026-3
• Support Opportunities: Regular Research Grants
• Duration: September 01, 2012 - August 31, 2015
• Field of knowledge: Physical Sciences and Mathematics - Chemistry - Organic Chemistry
• Convênio/Acordo: NSF - ICC
• Principal Investigator: Roberto Gomes de Souza Berlinck
• Grantee: Roberto Gomes de Souza Berlinck
• Principal researcher abroad: David H. Shernan
• Institution abroad: University of Michigan, United States
• Host Institution: Instituto de Química de São Carlos (IQSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil

Abstract

One of the most underexplored classes of natural products from a metabolic pathway perspective includes complex alkaloids derived from terrestrial and marine sources. In addition to the fascinating genetic and biochemical mechanisms involved in construction of these secondary metabolites, the introduction of stereochemistry and intriguing examples of pathways that lead to antipodal products indicates a rich opportunity to gain new insights into the life processes that generate these important compounds. In this proposal, the Sherman and Berlinck laboratories plan to combine research expertise and interests to explore the molecular genetic and biochemical basis for assembly and tailoring of two marine derived Penicillium sp. metabolites and compare them with two pairs of structurally related antipodal metabolites derived from a marine and terrestrial Aspergillus sp. A combination of genome sequencing, bioinformatic assembly, gene disruption, strain optimization and metabolite analysis, as well as biochemical and enzymatic studies will be pursued to dissect the key enzymes that control formation of the core alkaloid structures and modify them into structurally complex natural products. Objective for the current proposal include: 1. Conduct total genome sequencing, contig assembly, pathway mining and bioinformatic analysis of Penicillium oxalicum and Penicillium citrinum that produce the maleagrine/oxaline and citrinalin A/B metabolites, respectively. Perform a deep annotation of the gene cluster open reading frames, and conduct a comparative analysis with the previously characterized antipodal stephacidin/notoamide biosynthetic pathways derived from marine and terrestrial Aspergillus sp. Pursue biochemical studies following cloning and overexpression of non-ribosomal peptide synthetase (NRPS), and select tailoring enzymes identified through deep annotation. 2. Develop a genetic transformation/conjugation system for Penicillium oxalicum and Penicillium citrinum and select key genes for disruption studies. We will focus on spiro-oxindole core formation, and post-core ring system tailoring steps including prenylation, oxidative pinacol rearrangement, intramolecular Diels Alder reaction, and nitro group formation. 3. Conduct strain improvement to maximize levels of accumulated products generated by gene-disruption mutant strains in the alkaloid biosynthetic pathways. Metabolites will be purified and complete structure elucidation will be performed to establish the identity of select substrates for analysis of individual pathway enzymes. 4. Employ in vivo and in vitro pathway engineering approaches using heterologous genes and enzymes for halogenation, amide hydrolysis, amine oxidation, pyran ring expansion and other functional groups to increase chemical diversity of alkaloid natural products. (AU)

Scientific publications

(5)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

DE CASTRO, MARCOS V.; IOCA, LAURA P.; WILLIAMS, DAVID E.; COSTA, BRUNA Z.; MIZUNO, CAROLINA M.; SANTOS, MARIO F. C.; DE JESUS, KAREN; FERREIRA, EVERTON L. F.; SELEGHIM, MIRNA H. R.; SETTE, LARA D.; et al. Condensation of Macrocyclic Polyketides Produced by Penicillium sp DRF2 with Mercaptopyruvate Represents a New Fungal Detoxification Pathway. Journal of Natural Products, v. 79, n. 6, p. 1668-1678, JUN 2016. (05/60175-2, 10/50190-2, 12/50026-3, 13/50228-8)

FERREIRA, EVERTON L. F.; WILLIAMS, DAVID E.; IOCA, LAURA P.; MORAIS-URANO, RAQUEL P.; SANTOS, MARIO F. C.; PATRICK, BRIAN O.; ELIAS, LUCIANA M.; LIRA, SIMONE P.; FERREIRA, ANTONIO G.; PASSARINI, MICHEL R. Z.; et al. Structure and Biogenesis of Roussoellatide, a Dichlorinated Polyketide from the Marine-Derived Fungus Roussoella sp DLM33. ORGANIC LETTERS, v. 17, n. 21, p. 5152-5155, NOV 6 2015. (12/50026-3, 13/50228-8)

NEWMISTER, SEAN A.; GOBER, CLAIRE M.; ROMMINGER, STELAMAR; YU, FENGAN; TRIPATHI, ASHOOTOSH; PARRA, LIZBETH LORENA L.; WILLIAMS, ROBERT M.; BERLINCK, ROBERTO G. S.; JOULLIE, MADELEINE M.; SHERMAN, DAVID H.. OxaD: A Versatile Indolic Nitrone Synthase from the Marine-Derived Fungus Penicillium oxalicum F30. Journal of the American Chemical Society, v. 138, n. 35, p. 11176-11184, SEP 7 2016. (12/50026-3, 14/05670-7, 13/50228-8)

NEWMISTER, SEAN A.; ROMMINGER, STELAMAR; SCHMIDT, JENNIFER J.; WILLIAMS, ROBERT M.; SMITH, JANET L.; BERLINCK, ROBERTO G. S.; SHERMAN, DAVID H.. Unveiling sequential late-stage methyltransferase reactions in the meleagrin/oxaline biosynthetic pathway. ORGANIC & BIOMOLECULAR CHEMISTRY, v. 16, n. 35, p. 6450-6459, SEP 21 2018. (13/50228-8, 14/05670-7, 12/50026-3)

MERCADO-MARIN, EDUARDO V.; GARCIA-REYNAGA, PABLO; ROMMINGER, STELAMAR; PIMENTA, ELI F.; ROMNEY, DAVID K.; LODEWYK, MICHAEL W.; WILLIAMS, DAVID E.; ANDERSEN, RAYMOND J.; MILLER, SCOTT J.; TANTILLO, DEAN J.; et al. Total synthesis and isolation of citrinalin and cyclopiamine congeners. Nature, v. 509, n. 7500, p. 318-324, MAY 15 2014. (12/50026-3)