Activation of Natural Products Biosynthetic Pathways via a Protein Modification Level Regulation Benyin Zhang, †,‡,⊥ Wenya Tian, †,⊥ Shuwen Wang, † Xiaoli Yan, † Xinying Jia, ∥ Gregory K. Pierens, ∥ Wenqing Chen, † Hongmin Ma, † Zixin Deng, † and Xudong Qu* ,†,§ † Key Laboratory of Combinatorial Biosynthesis and Drug Discovery Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan, Hubei, China, 430072 ‡ State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, Xining, Qinghai, China § Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing, Jiangsu, China ∥ Centre for Advanced Imaging, The University of Queensland, Brisbane, Queensland, Australia * S Supporting Information ABSTRACT: Natural products are critical for drug discovery and development; however their discovery is challenged by the wide inactivation or silence of microbial biosynthetic pathways. Currently strategies targeting this problem are mainly concentrated on chromosome dissembling, transcription, and translation-stage regulations as well as chemical stimulation. In this study, we developed a novel approach to awake cryptic/ silenced microbial biosynthetic pathways through augmentation of the conserved protein modification step-phosphopantethei- nylation of carrier proteins. Overexpression of phosphopante- theinyl transferase (Pptase) genes into 33 Actinomycetes achieved a significantly high activation ratio at which 23 (70%) strains produced new metabolites. Genetic and biochemical studies on the mode-of-action revealed that exogenous PPtases triggered the activation of carrier proteins and subsequent production of metabolites. With this approach we successfully identified five oviedomycin and halichomycin-like compounds from two strains. This study provides a novel approach to efficiently activate cryptic/silenced biosynthetic pathways which will be useful for natural products discovery. N atural products have long been appreciated for their critical role in drug discovery and development due to their diverse bioactivities. 1 It is revealed that more than 50% of small molecule drugs (including pesticides) are from natural products or their derivatives. 1,2 However, the rate of discovery of useful compounds from natural sources has declined progressively since 1970; thus, how to effectively discover natural products nowadays has been becoming a central question in the field of pharmaceuticals and its related fields. 1,2 Recently, the increasing number of sequenced genomes has indicated that microorganisms indeed have a far greater potential to produce metabolites than those previously isolated. It is revealed that more than 90% of microbial metabolites have not yet been accessed due to the inactivation or silencing of their biosynthetic machineries under normal laboratory culturing conditions. 3 One of the major reasons for this inactivation is the absence of the essential regulatory signals to trigger these pathways. The biosynthesis of natural products from biosynthetic gene clusters depends on orchestrated regulations, including dissembling of chromosome to chromatin (eukaryotic microbe only), transcription of relevant genes to mRNAs, translation of mRNA to proteins, and modification of proteins to an active form which performs the biosynthesis. Currently, different strategies targeting chromosome dissembling, 4,5 transcription, 6 and translation stages 7,8 as well as others 2,9−12 have been developed to successfully activate cryptic or silent biosynthetic pathways. Protein modification is one of the major regulation stages; however thus far there are no methods that have been described for natural products activation. We noticed that in the biosynthesis of three large natural product families, termed polyketide (PK), nonribosomal peptide (NRP), and fatty acid (FA), there is a conserved protein modification step, in which the encoded carrier proteins (apo form) of their synthases are phosphopantetheinylated by phosphopantetheinyl transferase (PPtase) and converted into an active form (holo form). 13 Interestingly, PPtases are also able to efficiently catalyze acyl-phosphopantetheinylation of the carrier proteins by accepting acyl-CoAs instead of HSCoA. 13,14 This misacylated acyl-phosphopantetheinylation is detrimental for metabolite biosynthesis, and the acyl group is required to be further removed by additional editing enzymes. 15 The promiscuity of PPtase, accepting both free CoA and acyl- CoA, contributes to activating and deactivating metabolite Received: March 17, 2017 Accepted: May 31, 2017 Published: May 31, 2017 Letters pubs.acs.org/acschemicalbiology © 2017 American Chemical Society 1732 DOI: 10.1021/acschembio.7b00225 ACS Chem. Biol. 2017, 12, 1732−1736