Scleric acid biosynthesis studies and semisynthetic derivatives Jingfan Zhang 1 , Christophe Corre 1,2 , Fabrizio Alberti 1,2 1 School of Life Sciences, University of Warwick, UK, 2 Department of Chemistry, University of Warwick, UK Microbial metabolites and synthetic derivatives are widely used for emerging health problems and agrochemicals. Using a combination of microbial genomics and genetic engineering of microorganisms, recent work by Drs Alberti and Corre resulted in the discovery of scleric acid, a new microbial metabolite. 1 Scleric acid was discovered by rational derepression of a silent gene cluster called scl biosynthetic gene cluster (BGC). 1 Scleric acid acts as an inhibitor of nicotinamide N -methyltransferase (NNMT), the overexpression of which is associated with tumorigenesis. 2 The overall aim of this project is to make analogues of scleric acid that have stronger binding to NNMT and can work as better inhibitors. To achieve this, precursor analogues are fed to selected knock-out strains to make new scleric acid derivatives. In order to optimize the production of biosynthetic analogues of scleric acid, it is also important to understand the assembly pathway of scleric acid, which is how every gene in the whole biosynthetic cluster works. In the scl gene cluster, two genes, SclG and SclL , are of great interest to us due to their potential roles in the biosynthesis. SclG has a putative function of being an ATP-grasp family enzyme. SclL has a predicted function of being LysR transcriptional regulator. SclG protein will be expressed (in E.coli BL21, E.coli Bap1 and Streptomyces albus J1074) to test its bioactivity in vivo . SclG was knocked out and complementation strains was made, to also serve this purpose. SclL was knocked out and overexpressed, in order to characterize it and learn if it is an activator or repressor, and to further improve the biosynthesis. At the same time, whole genome sequencing of Streptomyces sclerotialus was performed on nanopore to complement the sequence from Illumina data available on NCBI as well as checking whether the scl gene cluster extends beyond the transporter gene. References 1. Alberti, F.; Leng, D. J.; Wilkening, I.; Song, L. J.; Tosin, M.; Corre, C., Triggering the expression of a silent gene cluster from genetically intractable bacteria results in scleric acid discovery. Chemical Science 2019, 10 (2), 453-463. 2. van Haren, M. J.; Taig, R.; Kuppens, J.; Sastre Toraño, J.; Moret, E. E.; Parsons, R. B.; Sartini, D.;Emanuelli, M.; Martin, N. I., Inhibitors of nicotinamide N-methyltransferase designed to mimic the methylation reaction transition state. Organic & Biomolecular Chemistry 2017, 15 (31), 6656-6667.
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