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

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.

The biosynthesis of macrocyclic polyketides, and especially that of macrolactones, has been extensively studied [12][13][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 methylmalonyl-CoA, 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 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.
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 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 polyketide synthase (PKS) comprises four typical type I PKSs, VinP1, VinP2, VinP3, and VinP4, and is likely responsible for vicenilactam formation (Fig. 5) [24].The PKS domain structure corresponds well to the  14), including specificity for the extender unit and the degree of reduction at the β-position.However, the mechanism of the C9-C10 double-bond formation remains unclear.The DH domain in module 5 of VinP5 is presumably involved in a unique dehydration process that generates the corresponding trisubstituted olefin.Ultimately, the C-terminal thioesterase domain of the VinP4 PKS is responsible for macrolactamization, resulting in the formation of vicenilactam (14) [26,27].
Prior to macrolactamization, an amidohydrolase, VinJ, removes the terminal alanyl moiety from the elongated polyketide chain (Fig. 4) [25,28].VinJ appears to recognize the elongated polyketide chain with L-Ala at its terminus, although the exact substrate of recognition and timing of which VinJ recognizes, remain unclear.We propose that the attachment of L-Ala to form L-Ala-3AIB-VinL (18) is likely a protective step, blocking the nucleophilic amino group in 3AIB during polyketide elongation (the thermodynamically preferable six-membered lactam can form after the first polyketide extension step if the β-amino group is free; however, the terminal amino group of L-Ala in the dipeptide intermediate cannot access the thioester moiety on the ACP domain of PKS due to the rigid conformation of the amide bond).Thus, the removal of L-Ala from the elongated polyketide intermediate appears to be a deprotection step, generating the nucleophilic amino group derived from 3AIB.This protection-deprotection logic resembles the methodology employed in organic synthesis.Nature seems to favor this type of methodology for the efficient biosynthesis of natural products; for example, amidohydrolasemediated cleavage reactions are employed in the selective biosynthesis of other dead-end natural products, such as desertomycin [29] and butirosin [30].

Incorporation of β-amino acids into the polyketide synthase: a common mechanism
A series of biosynthetic studies on vicenistatin revealed a common logic regarding the mechanism of β-amino acid starter unit incorporation into the polyketide pathway (Fig. 4) [31].First, a β-amino acid-selective adenylation enzyme activates a pathway-specific β-amino acid and ligates it to a standalone ACP, yielding β-aminoacyl-ACP.Second, another adenylation enzyme activates L-Ala, L-Ser, or Gly, and catalyzes an amide bond-forming reaction with β-aminoacyl-ACP to give dipeptidyl-ACP, presumably for protection of the β-amino group.Third, dipeptidyl-ACP is recognized by a dipeptidyltransferase and transferred to the loading ACP domain at the start of the initial PKS module.Finally, the terminal aminoacyl group is removed by an amidohydrolase prior to macrolactam formation.We have identified the BGCs for incednine (2) [32], cremimycin (3) [33], hitachimycin (4) [34], and fluvircin B 2 (5) [35]; these studies indicated that homologous enzymes are encoded in all BGCs.Thus, this biosynthetic logic is commonly utilized in the biosynthesis of macrolactam antibiotics with β-amino acid starter units.The existence of five homologous enzymes, including a standalone ACP, highlights the potential for BGC identification corresponding to other macrolactam antibiotics that incorporate β-amino acid starter units [9,10].An important detail in the biosynthesis of vicenistatin and fluvircin B 2 is that a decarboxylation reaction (removal of the β-carboxy group from β-aminoacyl-ACP) must occur before the second aminoacylation to give dipeptidyl-ACP.
It is also intriguing how these β-amino acid-selective adenylation enzymes recognize their cognate standalone ACPs (Fig. 8).To elucidate the mechanism by which βamino acid-selective adenylation enzymes recognize standalone ACPs, we constructed a cross-linked complex using HitB and HitD in hitachimycin biosynthesis [47].In this system, the original cysteamine moiety in pantetheine is replaced with 1,2-ethylenediamine and further acylated with α-bromoacetic acid, to yield the α-bromoacetamidecontaining pantetheine mimic C2Br (25).This analog provides an electrophilic functional group for the cross-linking reaction.C2Br (25) is enzymatically converted to the corresponding acylated CoA mimic by the CoA biosynthetic enzymes CoaA, CoaD, and CoaE [48].A promiscuous  [44,45] 3-aminonon-5,7-dienoic acid (23 [46] 3-aminoundeca-5,7-dienoic acid (24 Fig. 7 Non-ribosomal (amino acid specificity-conferring) codes for representative β-amino acid selective adenylation enzymes.Unique amino acid residues conserved in each family of enzymes are highlighted in color Fig. 8 Cross-linking reaction of adenylation enzyme (A domain) and crypto-ACP Biosynthesis of macrolactam antibiotics with β-amino acid polyketide starter units phosphopantetheinyl transferase, Sfp [49], is then used to transfer the C2Br mimic to the apo form of a standalone ACP to give C2Br-ACP (26) as crypto-ACP.Next, we mutated an aspartate residue, which is conserved among adenylation enzymes (shown here as A domain), to cysteine.In the wild-type enzyme, the aspartate residue interacts with the amino group of the β-amino acid substrate.When the ACP is correctly recognized by the adenylation enzyme, the thiol group of the cysteine residue functions as a nucleophile, reacting with the α-bromoacetyl group of crypto-ACP to form a cross-linked complex.The crystal structure of the HitB-HitD cross-linked complex revealed the interface between these proteins, illustrating their favorable interaction.This unique protein-protein interaction (PPI) appears to be a key factor for the selective transfer of the β-amino acid to the cognate ACP to produce β-aminoacyl-ACP in selective biosynthesis.It is also intriguing how VinM selectively recognizes 3AIB-VinL (17) by distinguishing it from the holo form of VinL (15) and 3-MeAsp-VinL (16).To address the importance of the length of the β-aminoacyl moiety in 3AIB-VinL (17), we prepared longer versions of the αbromoacetamide pantetheine mimic by replacing the 1,2ethylenediamine moiety with 1,3-propanediamine, 1,4-propanediamine, or 1,6-hexanediamine, yielding C3Br (27), C4Br (28), and C6Br (29) (Fig. 8) [50].When we used C6Br-crypto-VinL for the cross-linking reaction with the VinM cysteine mutant, the cross-linked complex was efficiently produced.The crystal structure of the VinM-VinL complex with C6Br (29) illustrated the PPI between VinM and VinL.Interestingly, the pantoate moiety of C6Br is bent, and the dimethyl group interacts with a tyrosine residue that is conserved among the VinM family of adenylation enzymes.Therefore, VinM appears to recognize the pantoate moiety in 3AIB-VinL (17) and adjusts the location of the nucleophilic β-amino group for the amide bond-forming reaction to give L-Ala-3AIB-VinL (18).This result indicates that the phosphopantetheinyl group is not only a linker that connects the ACP and the acyl group, but is also selectively recognized by enzymes that transfer acyl groups.
Overall, unique β-amino acids are biosynthesized by pathway-specific enzymes in each macrolactam producer strain to supply the appropriate starter units.
In incednine biosynthesis, the aglycone incednam (33) appears to undergo glycosylation with two unique sugars, N-methylxylosamine and N-demethylforosamine, presumably by glycosyltransferases IdnS4 and IdnS14, and possibly with the assistance of an auxiliary cytochrome P450-type protein, IdnO2 (Fig. 13) [32].UDP-N-methylxylosamine (34) is likely biosynthesized from UDP-Nacetyl-D-glucosamine (GlcNAc, 35) through the action of UDP-GlcNAc 6'-dehydrogenase IdnS2, UDP-N-   12).Incednam ( 33) is unlikely to be the macrolactam that is initially formed by PKSs IdnP1, IdnP2, IdnP3, IdnP4, and IdnP5, as it contains a hydroxy group at the C-10 position.This hydroxy group is presumably introduced by a cytochrome P450 monooxygenase, IdnO1, during post-PKS modification, although the exact point at which it does in the enzymatic pathway remains unclear.
In hitachimycin biosynthesis, the C10-OH group is anticipated to be O-methylated by the methyltransferase HitM6 in the final step of post-PKS modification.

Future perspectives
The next challenge for the field is to build upon the accumulated knowledge of the biosynthetic mechanisms of βamino acid macrolactam antibiotics to engineer designer macrolactam compounds.As mentioned earlier, we have successfully produced hitachimycin analogs through mutasynthesis with β-Phe derivatives.However, the scope of the derivatives was limited because the β-amino acid-selective adenylation enzyme HitB did not accept most o-and psubstituted β-Phe derivatives, with the exception of small fluorine-substituted analogs.To address this limitation, the active site of HitB must be engineered to accommodate oand p-substituted β-Phe derivatives, which would enable the production of a diverse range of hitachimycin analogs.Consequently, the engineering of adenylation enzymes to broaden their substrate scope is a crucial next step.
Moreover, β-amino acid-selective-adenylation enzymes can be exchanged to alter the β-amino acid starter units, leading to the production of new macrolactam compounds.Given the unique PPI revealed by the HitB-HitD crosslinked complex, it is essential to confirm whether the exchanged β-amino acid-selective adenylation enzyme can interact with different ACPs to ligate non-native β-amino acids.To overcome potential challenges in PPIs, designing specific interactions between each adenylation enzyme and the ACP may be necessary to introduce unnatural β-amino acids into the pathway, facilitating the production of designer macrolactams.The engineering of type I PKSs, including the exchange, introduction, and deletion of domains, can also be applied to the biosynthetic machinery of macrolactam antibiotics.
This biosynthetic journey, starting from the discovery of vicenistatin, has spanned three decades.Challenges still lie ahead as we strive to create novel macrolactam compounds with improved biological activities.

Fig. 3
Fig. 3 Incorporation studies to investigate the origins of vicenistatin