Development of a system for heterologous expression of the neurotoxin anatoxin-a(s) gene cluster

• Grant number: 17/22945-8
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: March 01, 2018
• End date: February 28, 2019
• Field of knowledge: Health Sciences - Collective Health - Public Health
• Principal Investigator: Marli de Fátima Fiore
• Grantee: Stella de Lima Camargo
• Supervisor: Bradley S. Moore
• Host Institution: Centro de Energia Nuclear na Agricultura (CENA). Universidade de São Paulo (USP). Piracicaba , SP, Brazil
• Institution abroad: University of California, San Diego (UC San Diego), United States
• Associated to the scholarship: 17/06869-0 - Heterologous expression, purification and characterization of enzymes involved in the biosynthetic pathway of the cyanobacterial neurotoxin anatoxin-a(s), BP.DR

Abstract

Sphaerospermopsis torques-reginae is a planktonic cyanobacterial species with broad distribution in several Brazilian aquatic environments. Some strains of these species are known to produce the neurotoxic organophosphate anatoxin-a(s) (ANS), which is a powerful and irreversible inhibitor of acetylcholinesterase, an enzyme that acts on the peripheral nervous system of mammals. Although the ANS structure has been elucidated, its biosynthetic pathway is still unknown. In the attempt to find the gene cluster involved in the ANS biosynthetic pathway, the sequencing and assembly of the genome of S. torques-reginae ITEP-024 (Brazilian ANS-producing strain) were carried out by our research group, as part of the project "Heterologous expression, purification and characterization of the enzymes involved in the biosynthetic pathway of the cyanobacterial neurotoxin anatoxin-a(s)" (FAPESP n° 2017/06869-0). The genome sequence allowed the detection of a gene cluster potentially involved in ANS production. The ribosomal 10.9 kb ANS biosynthetic gene cluster containing eight genes is suitable to be expressed in a heterologous expression system, an essential strategy for improve our knowledge and pathway engineering of various potentially-valuable natural products. Thus, the aim of this research proposal is to develop a system using the transformation-associated recombination (TAR) cloning of ANS gene cluster. The TAR-based genetic platform allows to directly clone, refactor and heterologously express the whole biosynthetic pathway. Therefore, cells from S. torques-reginae ITEP-024 culture will be grown in liquid ASM-1 medium for 21 days and used for the preparation of genomic DNA fragments for TAR and the gene cluster capture vector. Thereafter, direct cloning of ANS gene cluster will be performed using TAR and, finally, heterologous expression of the ANS biosynthetic pathway will be carried out. These experiments have the goal to prove the proposed biosynthetic pathway. After the expression of ANS cluster in the developed heterologous system, the production of the ANS will be evaluated by chemical analyses using mass spectrometry methods. The use of TAR cloning to directly capture the gene cluster from genomic DNA offers a fast and efficient way for production and genetic manipulations, thus allowing rapid and detailed investigation of promising biosynthetic pathways. This research will contribute to the understanding of the biosynthetic pathway of the ANS cyanotoxin and the development of fast and sensitive methods for the detection and monitoring of this toxin in fresh waters used for public supply. (AU)