Unusual chain initiation mechanism in gladiolin and gladiostatin biosynthesis Yu Zhang 1 , Marianne Costa 1 , Fang Pang 1 , Lona Alkhalaf 1 , and Gregory L. Challis 1,2,3 1 Department of Chemistry, University of Warwick, UK, 2 Warwick Integrative Synthetic Biology Centre, University of Warwick, UK, 3 Monash University, Australia Gladiolin is a polyketide antibiotic, produced by Burkholderia gladioli BCC0238, with promising activity against Mycobacterium tuberculosis 1 . Chain initiation in gladiolin biosynthesis is proposed to involve loading of succinyl- CoA onto a stand-alone acyl carrier protein (ACP) domain (GbnA) by an acyltransferase AT domain (GbnB), followed by amidation catalyzed by an asparagine synthetase homologue (GbnC). Interestingly, an amidase (GbnM) subsequently converts the amide back into a carboxylic acid to produce the final gladiolin structure 2 , suggesting the amidation reaction plays a protective role during biosynthesis. Homologues of GbnA and GbnC (GdsC and GdsD respectively) were also found to be encoded by the biosynthetic gene cluster for gladiostatin, also produced by B. gladioli BCC0238, where they are proposed to be involved in forming the glutaramide moiety of this novel anticancer agent 3 . However, malonyl-CoA, not succinyl-CoA, is required for gladiostatin chain initiation and a dedicated AT domain is not encoded by the gladiostatin biosynthetic gene cluster. Here we describe initial attempts to reconstitute the activity of these enzymes in vitro and determine their substrate selectivity. Understanding the biosynthesis of these unusual polyketide synthase starter units will provide new opportunities for biosynthetic engineering. References 1. Song, L.; Jenner, M.; Masschelein, J.; Jones, C.; Bull, M. J.; Harris, S. R.; Hartkoorn, R. C.; Vocat, A.; Romero-Canelon, I.; Coupland, P., Discovery and biosynthesis of gladiolin: a Burkholderia gladioli antibiotic with promising activity against Mycobacterium tuberculosis. Journal of the American Chemical Society 2017, 139 (23), 7974-7981. 2. Costa, M. Studies on the Biosynthesis of Gladiolin. University of Warwick, 2018. Nakou, I. T.; Jenner, M.; Dashti, Y.; Romero- Canelon, I.; Masschelein, J.; Mahenthiralingam, E.; Challis, G. L., Genomics-Driven Discovery of a Novel Glutarimide Antibiotic from Burkholderia gladioli Reveals an Unusual Polyketide Synthase Chain Release Mechanism. Angew Chem Int Ed Engl 2020, 59 (51), 23145-23153.
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