Biosynthesis of macrolactam antibiotics with β-amino acid polyketide starter units

The Journal of Antibiotics https://doi.org/10.1038/s41429-024-00742-2 REVIEW ARTICLE Biosynthesis of macrolactam antibiotics with β-amino acid polyketide starter units Fumitaka Kudo 1 1234567890();,: 1234567890();,: Received: 31 January 2024 / Revised: 4 April 2024 / Accepted: 6 May 2024 © The Author(s) 2024. This article is published with open access Abstract Macrolactam antibiotics incorporating β-amino acid polyketide starter units, isolated primarily from Actinomycetes species, show significant biological activities. This review provides a detailed analysis into the biosynthetic studies of vicenistatin, a macrolactam antibiotic with a 3-aminoisobutyrate starter unit, as well as biosynthetic research on related macrolactam compounds. Firstly, the elucidation of a common mechanism for the incorporation of β-amino acid starter units into the polyketide synthase (PKS) is described. Secondly, the unique biosynthetic mechanisms of the β-amino acids that are used to supply the main macrolactam biosynthetic pathways with starter units are discussed. Thirdly, some distinctive post-PKS modification mechanisms that complete macrolactam antibiotic biosynthesis are summarized. Finally, future directions for creating new macrolactam compounds through engineered biosynthesis pathways are described. Introduction Macrolactam antibiotics that incorporate β-amino acids as polyketide starter units have been isolated mostly from the Actinomycetes species. These compounds exhibit significant biological activities, including antibacterial, antifungal, and antitumor effects [1]. Vicenistatin (1) [2], incednine (2) [3], cremimycin (3) [4], hitachimycin (stubomycin) (4) [5, 6], and fluvirucin B2 (Sch 38518, 5) [7, 8] are examples of such macrolactam antibiotics that have been selected as research targets in our laboratory (Fig. 1); however, many other related macrolactam compounds have also been discovered [9–11]. Vicenistatin (1) features 3-aminoisobutylate (3AIB, 6) as the polyketide starter unit. Similarly, incednine (2) has 3-aminobutyrate (3ABA, 7), cremimycin (3) has 3-aminononanoate (3ANA, 8), hitachimycin (4) has β-phenylalanine (β-Phe, 9), and fluvirucin B2 (5) has Fumitaka Kudo was awarded the Sumiki-Umezawa Memorial Award from the Japan Antibiotic Research Association in 2023. This review article is partly based on his award-winning research. * Fumitaka Kudo 1 Department of Chemistry, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro-ku, Tokyo 152-8551, Japan β-alanine (β-Ala, 10). The polyketide chain elongates from the β-amino acid starter unit and cyclizes between the βamino group of the starter unit and the carboxylate moiety of the final extension product, yielding the corresponding macrolactam. As such, this class of macrolactams are biosynthesized via the typical polyketide pathway but with the incorporation of the unique β-amino acid starter units. The biosynthesis of macrocyclic polyketides, and especially that of macrolactones, has been extensively studied [12–14] (Fig. 2). In general, acetate or propionate serves as the starter unit, and malonate and/or methylmalonate act as the extender units to construct the macrocyclic polyketide skeleton. The extender units typically exist as coenzyme A (CoA) thioesters, such as malonyl-CoA and methylmalonylCoA, which are transferred to the acyl carrier protein (ACP) domain of the polyketide synthase (PKS) by an acyltransferase (AT) domain, yielding malonyl-ACP/methylmalonyl-ACP [15]. Similarly, starter units are ligated to the ACP, forming acyl-ACP, and there are several methods to achieve this ligation [16]. The starter acyl-ACP is recognized by the β-ketosynthase (KS) domain. The acyl group is transferred to a cysteine residue of the active site of the KS domain and condensed with the extender malonyl-ACP/ methylmalonyl-ACP to produce β-ketoacyl-ACP with the release of carbon dioxide. The β-carbonyl group of β-ketoacyl-ACP is subsequently reduced by the β-ketoreductase (KR) domain to yield β-hydroxyacyl-ACP, which is further processed by the dehydratase (DH) domain F. Kudo Fig. 1 Macrolactam antibiotics studied in our laboratory Fig. 2 General mechanism of type I polyketide synthase (PKS) for macrocyclic polyketides in bacteria to generate α,β-unsaturated acyl-ACP. Finally, the enoyl reductase (ER) domain reduces α,β-unsaturated acyl-ACP to provide a fully reduced acyl-ACP. The second round of polyketide chain elongation is catalyzed by a different set of catalytic domains to extend the chain by one acetate unit. The number of rounds of extension determine the length of the corresponding polyketide and each round is catalyzed by a PKS module consisting of the essential catalytic domains: AT, ACP and KS. The degree of reduction of the polyketide chain is determined by the particular combination of additional tailoring domains (KR, DH, ER), within the module. For example, if the KR domain is absent, the β-carbonyl group remains in the polyketide chain; if the DH domain is absent, the βhydroxy group remains; and if the ER domain is absent, an olefinic moiety remains. Finally, the thioesterase (TE) domain, located in the terminal PKS module, catalyzes an acyl transfer from the final thioester of the ACP-bound polyketide chain to form an acyl-TE complex, subsequently facilitating lactonization with a hydroxyl group on the elongated polyketide yielding a macrolactone. Post-PKS modifications, including polyketide skeletal modification, oxidation (hydroxylation, epoxidation), glycosylation, methylation, and acylation, are required to complete the biosynthesis of the dead-end polyketide compound [17]. In the biosynthesis of macrolactam antibiotics, the aforementioned PKS reaction is used to construct the polyketide skeleton; however, unique nitrogen-containing starter units are employed. There are several key points of interest regarding β-amino acid starter units. Firstly, their mechanism of incorporation into the PKS machinery. Secondly, as most β-amino acids are non-proteinogenic, their biosynthetic mechanisms are presumably unique. Lastly, the post-PKS modification of macrolactams appears crucial for their biological activities. This review provides a detailed summary of the biosynthetic studies of vicenistatin, covering the entire biosynthetic pathway. A common mechanism for the Biosynthesis of macrolactam antibiotics with β-amino acid polyketide starter units Fig. 3 Incorporation studies to investigate the origins of vicenistatin incorporation of β-amino acids into the PKS machinery is outlined, emphasizing that adenylation enzymes, selective for β-amino acids, act as gatekeepers, thereby determining incorporation of the unique β-amino acid starter units. Next, the unique mechanisms of β-amino acid biosynthesis and post-PKS modification are described. Finally, future perspectives for creating new molecules based on these biosynthetic studies are discussed. Vicenistatin: a macrolactam antibiotic with a β-amino acid (...truncated)