Biosynthesis and Molecular Engineering of Templated Natural Products

National Science Review REVIEW 4: 553–575, 2017 doi: 10.1093/nsr/nww045 Advance access publication 11 August 2016

CHEMISTRY Biosynthesis and molecular engineering of templated natural products Ming Chen1, Jingyu Liu1, Panpan Duan1, Mulin Li1 and Wen Liu1,2,3,∗ Downloaded from https://academic.oup.com/nsr/article/4/4/553/2615234 by guest on 27 September 2021

ABSTRACT Bioactive small molecules that are produced by living organisms, often referred to as natural products (NPs), historically play a critical role in the context of both medicinal chemistry and chemical biology. How nature creates these chemical entities with stunning structural complexity and diversity using a limited range of simple substrates has not been fully understood. Focusing on two types of NPs that share a highly evolvable ‘template’-biosynthetic logic, we here provide specific examples to highlight the conceptual and technological leaps in NP biosynthesis and witness the area of progress since the beginning of the twenty-first century. The biosynthesis of polyketides, non-ribosomal peptides and their hybrids thatshare an assembly-line enzymology of modular multifunctional proteins exemplifies an extended ‘central dogma’ that correlates the genotype of catalysts with the chemotype of products; in parallel, post-translational modifications of ribosomally synthesized peptides involve a number of unusual biochemical mechanisms for molecular maturation. Understanding the biosynthetic processes of these templated NPs would largely 1 State Key Laboratory facilitate the design, development and utilization of compatible biosynthetic machineries to address the of Bioorganic and challenge that often arises from structural complexity to the accessibility and efficiency of current chemical Natural Products Chemistry, Shanghai synthesis. Institute of Organic Keywords: natural products, biosynthesis, template enzymology Chemistry, Chinese Academy of Sciences, Shanghai 200032, INTRODUCTION bio-macromolecules in cellular networks, includ- 2 China; State Key Nature utilizes simple substrates, such as short car- ing proteins and nucleic acids, thereby enabling Laboratory of boxylic acids, amino acids and sugars, to prepare var- strategies using chemical probes to exquisitely con- Microbial ious building blocks; enzymatic polymerization and trol and examine life phenomena in biological sys- Metabolism, School of Life Science & combination/permutation of these monomers, in tems [6]. The generation of NP-like libraries isa Biotechnology, many cases followed by various post-modifications, major area of current interest in diversity-oriented Shanghai Jiao Tong eventually lead to the generation of diverse natural synthesis, which has emerged in aid of searching bi- University, Shanghai products (NPs) such as polyketides, peptides, ter- ologically active small molecules based on the ex- 200240, China and penoids, alkaloids and their hybrids in living organ- pansion of both their chemical and functional spaces 3Huzhou Center of isms (Fig. 1)[1]. These chemical entities exhibit an [7–9]. However, advances in chemical synthesis Bio-Synthetic extremely wide range of biological activities, which may not always keep pace with the high-throughput Innovation, Huzhou underlie the critical roles of NPs in both the history screening (HTS) technique [10], primarily due to 313000, China and context of medicinal chemistry and chemical bi- the tremendous challenge posed by the structural ology as various drugs, biological tools and synthetic complexity of NPs. ∗ Corresponding targets [2–4]. Of the chemotherapeutical agents We should always keep in mind that NPs do not author: E-mail: that have been clinically approved, particularly those initially exist for the purposeful use of human beings. [email protected] with anti-infection, antitumor and immunosuppres- In fact, the living organisms produce them, usually sive activities, approximately 50% are NPs and their as secondary metabolites, to modulate or participate Received 31 March semi-synthetic derivatives or are chemically synthe- in numerous internal biochemical processes and to 2016; Revised 13 sized but inspired by the pharmaceutically impor- fight against external environmental concerns, such May 2016; Accepted tant moieties arising from NPs [5]. Many of these as signal transduction, competition and adaptation 13 May 2016 small molecules are capable of specifically targeting [11–13]. These processes/situations are often

C The Author(s) 2016. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd. All rights reserved. For permissions,please e-mail: [email protected] 554 Natl Sci Rev, 2017, Vol. 4, No. 4 REVIEW

O O O O O N N N O N O O R OH N OH N N O O O indole.C2N R O NH O NH O N O N H N H H N O 2 COOH OH OH C C N O HO O O 6 2 OH O NH O O NH O O Taxol C5 isoprene unit O HO N NH (Antitumor) O O OH 2 SCoA HOOC OH ...... O O O O O N N Substrates Dactinomycin O OH SCoA (Antitumor) OH O O OH OH NH2 HOOC NH

Building Blocks Downloaded from https://academic.oup.com/nsr/article/4/4/553/2615234 by guest on 27 September 2021 OH O C6C2 ...... HO N OH H2N NH2 O HO O C6C1 O O O COOH NH2 O O HO O X CH3 O O C1 (X=O, N, C) Gentamicin C1a O N NH O O OCH OH Rifamycin B 3 (Antiinfection) (Antiinfection) H O HO H2N O OH O Erythromycin A O O HN O O (Antibacteria) O H H OH O OH H2N OH O Mitomycin ...... Artemisinin (Antitumor) HO O OH OH OH OH O OH (Antimalaria) H O H2N Amphotericin B O O HO (Antifungi) Natural Products

CH3 O O OH O O HO OH OH NH2 O S HO HO N OH O O Doxorubicin COOH NH2 (Antitumor) Thienamycin (Antiinfection)

Figure 1. Selected substrates, building blocks and representative natural products (NPs) with a variety of biological activities.

dynamic, and could feed back into the related bio- are produced by non-templated pathways, the tar- synthetic machineries for the diversification, opti- get products described here typically include polyke- mization and, ultimately, selection of suitable active tides and peptides (either ribosomally synthesized small molecules to combat the associated biochem- or non-ribosomally synthesized), both of which ical or environmental changes. Consequently, the share a ‘template’-biosynthetic logic that proceeds interaction between the metabolites of living organ- through the entire process for molecular assembly isms and the macromolecular targets that mediate and modification [14]. It should be noted that we do theirbiologicalfunctionsisconsideredasanintrinsic not intend to provide a comprehensive review of the engine that drives the co-evolution of these metabo- biosynthesis of these NPs over the past 15 years; the lites and their targets in nature [3]. It now seems that incredible and rapidly increasing volume of research NP evolution occurs over a continuous spectrum that has been completed during this time frame pre- spanning millennia, and the imaginable permuta- cludes such an effort. Instead, we report on the exam- tions or mutations of prototypes (e.g. biosynthetic ples in which we are involved or with which we are pathways, biochemical reactions, catalytic enzymes most familiar, to highlight the practices of what we and encoding genes) are manifested to some extent have learned from nature, how we recognize the gen- to create NPs that possess a multitude of biological erality and specificity in NP biosynthesis and, follow- functions and display an unimaginable diversity ing the natural theme in the development of biolog- of molecular architectures. This evolution has a ically active small molecules, whether we are able to constant theme, with the generality that typically ac- accelerate the NP-diversification process to advance counts for the relevance of NPs in biosynthesis and the expansion of their molecular utility. structure and with the specificity for unique members that can be individually recognized in nature. With a focus on templated bacterial NPs, this TEMPLATED ASSEMBLY-LINE review examines the progress of biosynthesis by ENZYMOLOGY exemplifying the conceptual and technological Polyketides, non-ribosomal peptides and their leaps in the twenty-first century. In contrast to hybrids, many of which have been successfully oligosaccharides, isoprenoids and other NPs that exploited as clinically used pharmaceuticals, REVIEW Chen et al. 555

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Figure 2. Assembly-line enzymology. (a) Conversion of the thiolation (T) domain/protein from an inactive apo-form into an active holo-form through phosphopantetheinyl transfer. (b) Modular PKSs and NRPSs for the formation of the C–C and C–N bonds in the biosynthesis of polyketides, non-ribosomal peptides and their hybrids.

veterinary agents and agrochemicals, consti- NRPSs are often giant enzymes organized into tute a large family of NPs whose biosynthetic modules (Fig. 2), which each contains a com- pathways share a templated multifunctional mon thiolation (T) domain that tethers an enzymology for skeleton assembly [15]. Polyke- activated monomer or growing intermediate via a tide synthases (PKSs) usually catalyse C–C 20-A-long˚ phosphopantetheinyl group derived from bond formation using short carboxylic acids as coenzyme A (CoA) (Fig. 2a) [18]. A prototype monomers (in activated (alkyl)malonyl thioester of a PKS module consists of an acyltransferase forms) [16], whereas non-ribosomal peptide (AT) domain that is responsible for selection and synthetases (NRPSs) incorporate amino acids transfer of an (alkyl)malonyl extender unit, a T through the formation of C–N bonds [17]. Con- domain for extender-unit loading, and a β-ketoacyl sistently with their catalytic cycles, both PKSs and synthase (KS) domain for Claisen condensation 556 Natl Sci Rev, 2017, Vol. 4, No. 4 REVIEW

of aligned acyl thioesters to elongate the growing [26–28]. The growing evidence that variable new polyketide chain. A minimum NRPS module enzymology can be complemented into archetypal contains an adenylation (A) domain that is re- PKS or NRPS paradigms leads to a common view sponsible for amino acid activation, a T domain that the template role of modular PKSs and NRPSs for thioesterification of the activated amino acid has not been fully recognized [29], particularly and a condensation (C) domain for transpeptida- regarding the incorporation of unusual building tion between the aligned peptidyl and aminoacyl blocks and new reactions that occur on and off the thioesters to elongate the growing peptide chain. assembly lines during and after chain elongation. Given the similarity in acyl transfer chemistry and catalytic logic [19], it is perhaps not surprising that Unusual starter and extender modular PKSs and NRPSs can be highly compatible

units utilized in the biosynthesis of Downloaded from https://academic.oup.com/nsr/article/4/4/553/2615234 by guest on 27 September 2021 (Fig. 2b) and their recombination results in a number of hybrid modular systems (e.g. PKS/NRPSs azinomycin B for transpeptidation between acyl and aminoacyl Azinomycin B (Fig. 3a), which was isolated from thioesters or NRPS/PKSs for decarboxylative Streptomyces sahachiroi and S. griseofuscus, is a potent condensation between peptidyl and acyl thioesters) antitumor antibiotic containing the functionalities to incorporate both carboxylic acids and amino 3-methoxy-5-methyl naphthoate (NPA), 2-amino- acids for production of various polyketide–peptide 1,3-dicarbonyl and epoxide that are densely assem- hybrid molecules. During each elongation process, bled with 1-azabicyclo[3.1.0]hexane in the struc- additional ketoreductase (KR), dehydratase (DH) ture [30]—an extremely rare ring system that is and enoylreductase (ER) domains in PKS modules shared only by the natural-product ficellomycin (de- control the stereochemistry and reductive extent spite their differences in stereochemistry and func- of the β-functionality of polyketide intermediates tional substitution) [31]. As a consequence of these [20]; similarly, specialized domains in NRPS structural hallmarks, azinomycin B selectively binds modules are responsible for various modifications within the major groove of DNA and forms co- of aminoacyl substrates or peptidyl intermediates valent interstrand cross-links (ISCs) [32–34]. This (e.g. N-, C-orO-methylation, acylation, reduc- compound has been shown to be active against the tion, heterocyclization and epimerization) [21]. L5178Y leukemic cell line and can remarkably in- Typically, a thioesterase (TE) domain within the crease the lifespan of P388 leukemic mice compared terminal PKS or NRPS module terminates chain with the clinically used DNA ISC drug mitomycin extension, either via an intermolecular transfer to C, which exhibited a comparable activity in a higher release a full-length linear intermediate or via an dosage [35,36]. intramolecular cyclization to generate a macrocyclic Previous isotope-labeling experiments, which product [22]. were conducted either in fermentation culture or In general, a modular PKS or NRPS system in a developed cell-free S. sahachiroi system, estab- appears to morph into an assembly line [15], which lished that the linear skeleton of azinomycin B con- programs monomer polymerization/modification tains an NPA and three amino acid-derived build- and chain tailoring/termination following a ing blocks [37–40]. Consistently, the five NRPSs, non-iteratively ‘one domain, one function’-like AziA1–A5 (Fig. 3a), constitute an atypical assem- co-linearity rule. Logically, the organization of bly line to program the sequential incorporation of biosynthetic information into multifunctional these monomers, two of which are carboxylic acids proteins (or their encoding genes) in this manner that form a C–O bond linkage [41]. In fact, NRPSs establishes an extended ‘central dogma’ that is able have now been shown to utilize hundreds of differ- to link the genotype of catalysts to the chemotype ent monomers far more than the 20 proteinogenic of products, thus setting the stage for assembly- amino acids [17], including D-configured and β- line engineering and bioinformatics-guided genome amino acids; methylated, glycosylated and phospho- mining toward the creation and discovery of new rylated residues and heterocyclic elements; as well polyketides, non-ribosomal peptides and their hy- as the carboxylic acids exemplified here in azino- brids [23–25]. Over the past 15 years, the advances mycin B biosynthesis. AziA1 is an A–T di-domain in genome sequencing have evidenced numerous protein that is responsible for starter-unit prim- such proteins that result from the variation, combi- ing by activating and loading 3-methoxy-5-methyl- nation, permutation and evolution of the functional NPA [42]. Skeleton assembly could be initiated by PKS and NRPS units (e.g. modules, domains and AziA2, an NRPS with a C–T–C-domain organiza- proteins). The fact that certain functional units tion. AziA2 likely takes over the starter unit and can be bypassed or used more than once in assem- catalyses the condensation with α-hydroxyisovaleryl bly lines has already broken the co-linearity rule thioester channeled by AziA3, which contains the REVIEW Chen et al. 557

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Figure 3. Proposed biosynthetic pathway of azinomycin B. (a) Atypical NRPSs for skeleton assembly. (b) Formation of the starter unit 3-methoxy-5- methyl-NPA that has a polyketide origin. (c) Formation of the unusual extender unit and its functionalization to afford the 1-azabicyclo[3.1.0]hexane ring system.

A, KR and T domains that are required for the in- whose activity is necessary for release of the full- corporation of Val-derived α-ketoisovaleric acid and length product. 5-NPA is then subjected to regiospe- the subsequent on-line reduction of its α-keto group cific hydroxylation by AziB1 and O-methylation of [14]. AziA4, an NRPS module with a typical archi- the resulting hydroxyl group by AziB2 [42]. Phylo- tecture (C–A–T), is assumedly unusual in substrate genetically, AziB belongs to a growing bacterial PKS choice; it elongates an unknown extender unit to family, the members of which, distinct from typical produce the aziridino[1,2a]pyrrolidinyl moiety. The modular PKSs in catalytic logic, act iteratively to pro- last NRPS, AziA5, has a reductase (RE) domain, induce aromatic compounds [44]. It catalyses the as- stead of the more commonly observed TE domain, sembly of a linear hexaketide from one acetyl-CoA in addition to the C–A–T organization for Thr in- and five malonyl-CoAs by successive decarboxyla- corporation, consistent with the hypothesis that the tive condensations and, during the process, the KR linear product is off-loaded as an aldehyde prior to domain conducts selective reductions at the C3, C5 the conversion of its C-terminal Thr residue to a 2- and C9 positions prior to intramolecular cyclization amino-1,3-dicarbonyl group [14]. and aromatization. Other members of this family The starter unit 3-methoxy-5-methyl-naphthoyl share a similar domain organization but primarily has an unusual polyketide origin (Fig. 3b). AziB differ from AziB in their elongation cycles and/or [41], a PKS in which the domains are organized as reduction patterns, as shown by (homo)orsellinic KS–AT–DH–KR–T (Fig. 3b), synthesizes 5-NPA acid synthases (aviM and TiaB, three elongations with the assistance of the thioesterase AziG [43], and no reduction) [45,46], 6-methylsalicyclic acid 558 Natl Sci Rev, 2017, Vol. 4, No. 4 REVIEW

synthases (ChlB1, PactS, MdpB and PokM1, three synthetic efforts toward diverse functionalization of elongations and reduction at C5) [47–51], (R)- the 2,2 -BiPy core for various uses. A number of bi- mellein synthase (Pks8, four elongations and reduc- ologically active NPs share this core structure, in- tionatC5)[52] and 2-hydroxy-5-methyl-NPA syn- cluding the non-symmetrically modified 2,2 -BiPy thase (NcsB, five elongations and reduction atC5 antibiotics caerulomycins (CAEs) and collismycins and C9) [53]. (COLs) [58–61] and symmetrically functionalized The 1-azabicyclo[3.1.0]hexane ring system, toxins orellanine and diglucopyranoside [62], as which largely underlies the DNA cross-linking well as aminoquinone streptonigrinoids, monoter- ability and biological activities of the molecule, penoid indole and pyridoacridine alkaloids [63–65], represents the most intriguing moiety in the struc- all of which embed one or both pyridine unit(s) ture of azinomycin B. The formation of this ring in a further cyclized complex scaffold. How nature

Downloaded from https://academic.oup.com/nsr/article/4/4/553/2615234 by guest on 27 September 2021 system involves the activity of the NRPS AziA4 to creates the 2,2 -BiPy products remained poorly un- incorporate a cryptic extender unit, which should derstood. Until recently, insights have been gained at least fulfill the atomic requirement in the con- through investigations into the polyketide–peptide struction of the aziridino[1,2a]pyrrolidinyl skeleton hybrid origin of CAEs and COLs [66–68]. (Fig. 3c) [41]. The preparation of this extender unit CAEs and COLs (Fig. 4a), the two groups of an- conceivably starts with AziC2-catalysed N-acylation tibiotics that are structurally similar to each other, of the Glu substrate using a strategy similar to share the 2,2 -BiPy core structure in which a di- or Lys or Arg biosynthesis [54], as the protection of tri-substituted ring A conjugates an unmodified ring the α-amino group precedes the conversion of the B. Their major difference is present at C5 of ringA, terminal carboxylic group of Glu into an aldehyde by which has (for COLs) or lacks (for CAEs) sulfur the kinase AziC3 and the reductase AziC4 though decoration. Consistently with their high structural phosphorylation-coupled activation and reduction. similarity, biogenesis-based comparative analysis re- AziC5 and AziC6, which are homologous to the N- vealed a highly conserved gene cassette for 2,2 -BiPy and C-terminal subunits of transketolases, respec- formation [68]. This cassette encodes an unusual hy- tively, could constitute a protein complex with the brid NRPS/PKS/NRPS system, which is composed cofactor thiamine pyrophosphate (TPP) for two- of, in CAE biosynthesis for example, the enzymes carbon unit extension at C5; after TPP elimination, CaeA1, CaeA2, CaeA3 and CaeA4, and three asso- a pyridoxal-5-phosphate (PLP)-dependent protein ciated proteins (e.g. CaeP1 and CaeP2, for picoli- would be responsible for transamination to generate nate formation; and CaeB1, a FAD-dependent pro- an amine diol. The following functionalization tein of unknown function) (Figs 4b and c). CaeA1 steps may include multiple oxidoreductions and is an NRPS containing the T–A loading module azabicycle formation (Fig. 3c). The characterization for priming picolinyl as a starter unit. CaeA2 is an of these reactions, which could occur on or off unusual PKS/NRPS hybrid protein that possesses the assembly line (with or without N-acylation a typical PKS module, KS–AT–T and an atypical protection), would reveal the elusive source of the NRPS module, condensation/cyclization (Cy)–A– extender unit. In fact, the starter and extender units T–terminal C (Ct), for sequential incorporation of significantly contribute to the chemical complexity malonyl-CoA and L-Cys. The functions of the Cy and structural diversity of the templated NPs whose and Ct domains in the NRPS module of CaeA2 re- biosynthesis features an assembly-line enzymology main unclear, although the Cy domain has previ- [55]. A fundamental understanding of how these ously been found as a C-domain variant with a dual building blocks develop would certainly enrich activity in many NRPS modules, required not only the toolbox of synthetic biology for manufacturing for amide bond formation, but also for the cycliza- designed products. tion of a newly incorporated Cys or Ser residue to yield a five-membered azoline ring after2 H Oelim- ination [17]. The NRPS CaeA3 contains a C–A–T module for Leu extension, and CaeA4 is a discrete On-line cyclization and differentiation of TE protein that either mediates product releasing or 2,2 -bipyridines edits a misprimed NRPS. 2,2-Bipyridine (2,2-BiPy) is an attractive core Overall, the biosynthesis of COLs involves a structure of a large class of chelating compounds head-to-tail homologous NRPS/PKS/NRPS sys- that are able to form stable complexes with metal tem to incorporate the building blocks, namely the ions [56,57]. These heterocyclic ligands have been starter unit picolinyl and the extender units malonyl, widely applied in the areas of coordination chem- cysteinyl and threoninyl, as in the biosynthesis of istry, asymmetric chemistry, material chemistry and CAEs [66,68]. The only exception is that the coun- analytical chemistry, thus continuously motivating terparts of the T–A di-domain NRPS CaeA1 are REVIEW Chen et al. 559

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Figure 4. Non-symmetrically modified 2,2-bipyridine (2,2-BiPy) antibiotics, encoding genes and biosynthetic pathways. (a) Structures of caerulomycin-A (CAE-A) and collismycin-A (COL-A). (b) Conserved gene cassettes from the cae and col clusters coding for 2,2-BiPy biosynthesis. (c) Proposed biosynthetic routes toward desulfurated and sulfurated 2,2-BiPys in the biosynthesis of CAEs and COLs, respectively.

the discrete proteins ColA1a and ColA1b (Fig. 4c). the sulfhydryl group of the residue Cys. Off-line tai- Characterization of the structurally related Leu- loring of these intermediates, including hydrolysis of containing 2,2-BiPy intermediates [66,67], being the extended Leu residue, reduction of the nascent desulfurated for CAEs and sulfurated for COLs, in carboxylate group to an aldehyde, transamination both biosynthetic pathways supports the hypothe- and oxidation of the resulting terminal amino group, sis that the two hybrid templates assemble a similar would lead to an oxime functionality that is common polyketide–peptide skeleton but differ in processing to both CAEs and COLs (Fig. 4c) [69,70]. 560 Natl Sci Rev, 2017, Vol. 4, No. 4 REVIEW

Extensive isotope-labeling experiments have re- tivities. Many members in this family also bear a di- vealed that the functional association of Cae/ColA1 alkyldecalin system within a rigid pentacyclic agly- and Cae/ColA2 is sufficient to construct the 2,2- cone, which is linked to tetronate either by a car- BiPy backbone [68,71,72]: unit B arises from pi- boxylic ester, such as chlorothricin (CHL), or by colinic acid, the carboxylate of which serves as C2 a carbonyl group, such as tetrocarcin A (TC–A) of unit A; and unit A formation involves a PKS- and kijanimicin (KIJ) (Fig. 5a). In contrast, pyrroin- catalysed two-carbon extension for C3 and C4, fol- domycins (PYRs) (Fig. 5a), which were isolated lowed by an NRPS-catalysed Cys incorporation to from S. rugosporus during a screening for agents ac- provide N1, C5 and C6, as well as the exocyclic C7 tive against bacterial pathogens, are perhaps the only (Fig. 4c). These findings raise an interesting ques- natural spirotetramates that have been identified to tion regarding the role of Cae/ColA3-catalysed Leu date [76,77]. Structurally, PYRs are highly related to extension, which appears to be ‘auxiliary’; however, the aforementioned spirotetronates, although they Downloaded from https://academic.oup.com/nsr/article/4/4/553/2615234 by guest on 27 September 2021 it has been validated to be indispensable for the show differences in their aglycones, which vary in generation of 2,2-BiPy intermediates. According to size, substitution pattern and heterocyclic function- these findings, 2,2-BiPy formation most likely oc- ality. curs on the assembly line and involves an unprece- The biosynthetic machineries used to produce dented intramolecular cyclization/rearrangement of spirotetronates and spirotetramates employ similar the linear picolinyl-acetyl-cysteinyl portion (with or modular PKS systems to assemble the main car- without Leu extension) [68]. This process might bon skeletons of their aglycones. As shown in CHL be associated with the activities of the atypical biosynthesis, six PKSs, ChlA1–A6, which contain cis domains Cy and Ct in the NRPS module of the functional domains that are co-linearly arranged CaeA2 as well as certain enzymes that act in trans, as a loading module and 11 extension modules for such as the aforementioned FAD-dependent protein chain elongation and modification, catalyse the for- Cae/ColB1. Briefly, a cyclization reaction gives rise mation of a nascent tetracosanoyl thioester interme- to a sulfur-containing seven-membered heterocycle, diate via 11 decarboxylation steps (Fig. 5b) [78]. which then undergoes a rearrangement to release Similar assembly logic has been observed for the the sulfhydryl group and provides a six-membered, biosynthesis of the spirotetramates PYRs, where the common intermediate as the branching point, either eight modular PKSs that are composed of a load- for desulphurization that leads to the formation of a ing module and 10 extension modules complete 10 sulfur-lacking ring A of the CAE members (route A) elongations and associated modifications to gener- or for dehydrogenation that results in the formation ate a docosanoyl thioester intermediate [79]. In ei- of a sulfur-containing ring A of the COL members ther case, given the presence of at least five modules (route B). sharing the KS–AT–DH–KR–T domain organiza- Modifications of growing acyl or peptidyl inter- tion (Fig. 5b), the resulting thioester intermediate mediates has long been known to be able to occur could be a polyene polyketide that has a minimum on modular PKSs or NRPSs in both cis and trans offivedoublebonds. manners. The former depends on the activities of Carbon chain extension in spirotetronate or the domain(s) embedded in the module, whereas spirotetramate biosynthesis terminates in a unique the latter involves the actions of protein(s) that are way though the incorporation of a three-carbon independent of the assembly-line machinery [19]. unit, either a glyceroyl or an amino acid, to form a As proposed here in 2,2-Bipy biosynthesis, on-line characteristic tetronate or tetramate moiety [79,80]. enzymatic cyclization to achieve correct active con- Specifically, for CHL-represented spirotetronates, formation is no exception [73]. Such PKS or NRPS tetronate formation involves a set of discrete func- precedents involving new chemistry have increas- tionally coupled proteins (Fig. 5b). For example, ingly been characterized, highlighting the promising ChlD1, a bi-functional enzyme carrying the AT and recruitment ability of thioester assembly-line plat- phosphatase activities, is responsible for loading forms, particularly when multiple internal and exter- the 1,3-biphosphoglycerate-derived glyceroyl unit nal activities are necessary for complex conversions. onto the thiolation protein ChlD2 [81,82]; ChlM, a KS III-like protein, then catalyses the condensation between the PKS-channeled tetracosanoyl thioester intermediate and the ChlD2-tethered glyc- Template effect on off-line cross-bridging eroyl thioester to release a tetronate-containing lin- The spirotetronate antibiotics [74,75], character- ear polyketide from the assembly line through the ized by a tetronate unit that is exclusively spiro- formation of C–O and C–C bonds [80]. With re- conjugated to a cyclohexene ring, are a large class spect to tetramate formation in PYR biosynthe- of NPs that exhibit a wide variety of biological ac- sis, the assembly line includes a C–A–T NRPS REVIEW Chen et al. 561

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Figure 5. Spirotetronates/spirotetramates and associated biosynthetic pathway. (a) Structures of chlorothricin (CHL), tetrocarcin A (TC-A), kijanimicin (KIJ) and pyrroindomycins (PYRs). (b) PKS assembly-line-programmed formation of a linear tetronate-containing intermediate that contains two pairs of 1,3-diene and alkene groups in CHL biosynthesis. 562 Natl Sci Rev, 2017, Vol. 4, No. 4 REVIEW

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NH NH NH HO HO HO PyrE3 PyrI4 HO O O HO O O HO O O PYRs H H

H H OH OH OH Internal pair of 1,3-diene and alkene Dialkyldecalin system Pentacyclic core Downloaded from https://academic.oup.com/nsr/article/4/4/553/2615234 by guest on 27 September 2021

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Modular type I PKS system OH OH OH ...... H HO 17 O HO 17 O HO 17 O 21 25 21 25 21 25 (S) (R) Modular 5 O O O O H KS AT KR T O O O O O O OH OH OH ...... SH Modular 3 KS AT T O O O

SH ...... AveC

Modular 1 MeO KS AT KR T O O Y X O O O HO O HO 17 21 25 SH 17 21 25 (S) MeO (S) O O

O O O O OH OH

Avermectin B1a: X-Y = CH=CH Avermectin B2a: X-Y = CH -CH(OH) O 2 OH

Figure 6. Template effects of the assembly lines on post-PKS cyclization reactions. (a) Enzymatic cascade [4 + 2] cycloaddition reactions in PYR biosynthesis. (b) Enzymatic spiroketalization in the biosynthesis of avermectins (AVEs).

module for additional amino acid extension, fol- corresponding to the cyclohexene-containing di- lowed by an intramolecular Dieckmann cyclization alkyldecalinand spiro-conjugate portions of the pen- to construct the five-membered heterocycle and tacyclic cores (Fig. 6a). Indeed, two dedicated pro- off-load the full-length linear intermediate as well teins, PyrE3 and PyrI4, act in tandem to catalyse [79]. The subsequent modifications may involve an the formation of the two cyclohexene units through acylation-elimination reaction (Fig. 5b), leading to [4 + 2] cycloadditions in a regio- and stereoselec- the generation of an additional double bond that is tive manner [84]: PyrE3 is responsible for endo- exocyclic to tetronate or tetramate [83]. selective dialkyldecalin formation, and PyrI4 acts as As characterized in the PYR biosynthetic path- an exo-selective spiro-conjugate synthase (Fig. 6a). way, the linear intermediate features at least six Spirotetronate NPs that are structurally related to double bonds in the structure. Intriguingly, these PYRs, such as CHL and versipelostatin (VST) [85], double bonds constitute two independent pairs of appear to share this cascade cross-bridging strategy, 1,3-diene and alkene groups as the structural hall- as the formation of a similar pentacyclic scaffold re- marks of the internal and terminal intramolecu- lies on the two highly ordered [4 + 2] cycloadditions lar [4 + 2] cycloaddition reactions, respectively, that occur on a tetronate-containing linear polyene REVIEW Chen et al. 563

substrate to generate dialkyldecalin first and spiro- ribosomal peptides and their hybrids. An additional conjugate next. example is present in the biosynthesis of aver- The Diels-Alder [4 + 2] cycloaddition that pro- mectins (AVEs) (Fig. 6b), a class of 16-membered ceeds through a single pericyclic transition state macrolide agrochemicals with widespread appli- plays a central role in the theory and practice of or- cations in the treatment of parasitic diseases. Four ganic chemistry [86]. The development of relevant modular PKSs, AveA1–A4, constitute an assembly synthetic methodology, including the artificial de- line involving the activities of a central KS–AT–T sign of biological Diels-Alder catalysts, has long been module and two flanking KS–AT–KR–T modules the focus of extensive efforts. There are few known to produce a polyketide intermediate that contains enzymes in nature that perform [4 + 2] cycloaddi- a C17,C25-dihydroxy C21-ketone motif [91]. This tions, and most of them possess multiple functions, intermediate is extremely unstable and readily lack catalytic efficiency and/or participate in reac- undergoes spontaneous carbonyl ketalization-based Downloaded from https://academic.oup.com/nsr/article/4/4/553/2615234 by guest on 27 September 2021 tions that occur non-enzymatically [87]. Validation dehydrative cyclization to produce the character- of whether these enzymes are Diels-Alderases has istic 6,6-spiroketal functionality, which lacks the proven challenging; however, this situation could stereochemistry of the spiro carbon C21 (Fig. 6b) change due to the recent emergence of monofunc- [91–93]. In contrast, the presence of AveC, a spe- tional proteins that are capable of effectively per- cialized spirocyclase, leads to its conversion into an forming [4 + 2] cyclization reactions in a completely enantiomerically pure 21S-6,6-spiroketal (Fig. 6b), enzyme-dependent manner [88], such as PyrE3 and supporting the hypothesis that this enzyme func- PyrI4 in PYR biosynthesis. Interestingly, the activ- tions to control stereochemistry and improve the ity of the flavoenzyme PyrE3 does not depend on reaction rate during the reaction in a manner similar a change in the redox state of the cofactor, indicat- to the [4 + 2] cycloaddition enzymes described ing that the flavine cofactor may play a structural above [88]. role in maintaining the requisite geometry of the active site for cyclization [84], similarly to S-adenosyl- L-methionine for SpnF in spinosyn A biosynthesis Diversity-oriented biosynthesis of [89]. Very recently, the crystal structures of PyrI4 alone and in complex with its product have been elu- nine-membered dilactones cidated, revealing a unique trapping action of the en- Antimycins (ANTs), which share a nine-membered zyme in establishment of the pentacyclic scaffold of dilactone core conjugated with an unusual 3- the molecules [90]. PyrI4 possesses a β-barrel cat- formamidosalicyclic acid (FSA) moiety (Fig. 7), are alytic core to accommodate the 1,3-diene and alkene a class of structurally related polyketide–peptide groups of the substrate; however, the transforma- hybrid antibiotics [94]. They are widely used in tion depends on its N-terminally extended, ligand chemical biology as the potent tools, because of binding-inducible sequence. This sequence is ini- their activity against mitochondrial respiration by tially disordered but is essential for substrate recog- specifically targeting cytochrome c reductase in the nition and accommodation. Following its structural electron transport chain [95]. However, an ANT rearrangement to form a locking α-helix, the lid-like derivative, generated by methylating the hydroxyl action imposes conformational constraints on the group of the FSA moiety, lacks this mode of action β-barrel catalytic core, which enhances the proxim- but directly inhibits the function of Bcl-2-related ity and polarization effects of the reaction groups to proteins that confer cellular resistance to a wide drive cyclization in a regio- and stereo-specific man- range of anti-cancer drugs [96,97]. Thus, the FSA- ner. This finding represents an important step inour conjugated dilactone has attracted much attention understanding of how the enzyme interacts with its as a privileged scaffold with the potential to bind ligand to catalyse a Diels-Alder-like [4 + 2] cycload- multiple protein targets. dition. Comparative genome mining revealed a uniform Consequently, the biosynthesis of paradigm for ANT biosynthesis in various Strepto- spirotetronates and spirotetramates features a myces strains, showing that a hybrid NRPS/PKS sys- cryptic template effect of the assembly line at the tem programs the dilactone core formation in a lin- post-modification stage, as the formation of two ear way by (i) priming FSA as an unusual starter pairs of 1,3-diene and alkene groups is programmed unit, the formation of which shares certain steps with on-line for the cascade enzymatic [4 + 2] cycload- Trp degradation [98]; (ii) sequentially incorporat- ditions that proceed off-line73 [ ]. Recently, close ing a Thr and a pyruvate (the latter is converted attention has been paid to cyclization reactions, on-line into an α-hydroxy unit to form the first es- particularly those with regio- and stereo-specificity ter bond) by the NRPS AntC (C–A–T–C–A–KR– that provide rigid scaffolds of polyketides, non- T); and (iii) performing two-carbon extension and 564 Natl Sci Rev, 2017, Vol. 4, No. 4 REVIEW Downloaded from https://academic.oup.com/nsr/article/4/4/553/2615234 by guest on 27 September 2021

Figure 7. Starter-unit choice, extender-unit incorporation and post-NRPS/PKS modification in diversity-oriented biosynthesis of an antimycin (ANT)-like nine-membered dilactone library.

subsequent lactonization using the PKS AntD (KS– tion. C7 alkylation occurs via PKS-catalysed elonga- AT–T–TE)to furnish the secondester bond(Fig.7) tion that utilizes alkylmalonyl as the extender unit, [99–101]. ANT maturation requires the reduction in contrast to C8 acylation, which is the result of of the C8-keto group, which is catalysed by the oxi- post-NRPS/PKS modification of the dilactone core doreductase AntM in trans before off-loading the lin- (Fig. 7). ear intermediate, to generate a hydroxyl group for The variation in C7 alkylation relies on the acyl attachment. The natural ANT family now com- activity of AntE (Fig. 7), the most promiscu- prises up to 44 structurally distinct entities [100], all ous member of the family of crotonyl-CoA of which differ only in C7 alkylation and C8 acyla- reductase/carboxylases (CCRs) that have been REVIEW Chen et al. 565

characterized to date [101,102]. In the presence that were amenable for isolation, characterization of NADPH and CO2, this CCR is capable of and scale-up preparation. Next, the highly promiscu- converting various α,β-unsaturated acyl-CoAs ous acyltransferase AntB was used for ANT matura- (linear, branched, aromatic, halogenated, trifluo- tion in vitro by building acyl groups at C8. Eventu- romethylated or alkynylated) into corresponding ally, the permutation of dozens of acyl-CoA donors alkylmalonyl-CoAs through reductive carboxylation with 13 dilactone acceptors produced 380 ANT- [102,103]. The crystallization of AntE in complex like products, among which 356 were new; this is with NADP+ indeed revealed a large cavity to approximately nine times the number of natural accommodate variable E-2,3-ene precursors [104]. ANT members. Remarkably, the functionalities in- Consistently, the PKS AntD functionally associates troduced into the dilactone core at the C5 , C7 and with AntE and prefers a longer extender unit for C8 positions cover a number of chemically active, incorporation into the polyketide skeleton. On the pharmaceutically important or naturally unavailable Downloaded from https://academic.oup.com/nsr/article/4/4/553/2615234 by guest on 27 September 2021 other hand, the diversity in C8 acylation depends groups (Fig. 7), largely facilitating the future investi- on the activity of the highly promiscuous acyl- gation in medicinal chemistry and chemical biology. transferase AntB (Fig. 7)[102,105]. This enzyme NPs provide a number of privileged scaffolds tolerates not only a diverse range of acyl donors in for diversity-oriented synthesis in library design, for terms of both length and structural features (e.g. which synthetic accessibility and efficiency often they can be linear, branched, aromatic, saturated, face challenges associated with the structural com- unsaturated, halogenated, or heterocyclic), but also plexity. Biosynthesis is a promising approach for a wide variety of acyl acceptors that are different in producing complex molecules; however, there have C7 alkylation. In addition, the ANT NRPS/PKS been relatively few successes in achieving a high level system was found to tolerate the change of the ofdiversificationusingthistechnique,duetothelim- starter unit and to initiate the assembly of a mod- itations in our understanding, development and uti- ified FSA-conjugated dilactone100 [ ], as feeding lization of compatible biosynthetic machineries. The of 6-fluoro-L-Trp for FSA fluorination led tothe construction of an ANT-like NP library exemplifies production of a series of 5-F-ANT products. the core strategy of diversity-oriented biosynthesis, Consequently, the entire ANT biosynthetic ma- which depends on the incorporation of an idea of chinery is highly compatible and serves as an combinatorial chemistry into different biosynthetic ideal model system to test ‘multiplex combinato- stages for starter-unit choice, extender-unit incor- rial biosynthesis’—a new approach that combines poration and post-NRPS-PKS modification. Given the techniques including multiple mutasynthesis, the increasing depth of knowledge in the field of pathway engineering and chemoenzymatic trans- biosynthesis [106–109] and the significant progress formation to construct a dilactone scaffold-bearing in protein/pathway engineering [23,24], multiplex natural-product-like library [102]. First, the inac- combinatorial biosynthesis would be a useful ap- tivation of the acyltransferase gene antB in the proach for generating libraries of polyketides, non- ANT-producer S. sp. NRRL 2288 eliminated the ribosomal peptides and their hybrids that share a diversity in C8 acylation and led to the synthe- similar biosynthetic logic. sis of diverse FSA-conjugated dilactones in vivo. This synthesis relies on the highly compatible NRPS/PKS system, which programs the sequential PARADIGMS FOR POST-TRANSLATIONAL incorporation of variable starter and extender units. MODIFICATIONS OF RIBOSOMALLY Taking advantage of the endogenous biotransformations in cells (i.e. FSA formation for starter-unit SYNTHESIZED PEPTIDES generation and β-oxidation of fatty acids to prepare Since the beginning of the twenty-first century, the α,β-unsaturated acyl-CoAs and their reductive car- peptide NPs with a ribosomal origin have become boxylation by AntE for extender-unit preparation), the chemical inspiration for the discovery of new single- or double-feeding of the precursors into the biosynthetic mechanisms [110]. Post-translational mutant S. sp. NRRL 2288 permitted the preparation modifications (PTMs) of genetically encoded pre- of both natural and unnatural building blocks in situ, cursor peptides have been demonstrated to be com- including the modified FSA starter unit derived from parable to NRPS paradigms in creating diverse 6-fluoro-L-Trp and various alkylmalonyl extender molecules, such as the lantibiotics that are char- units from exogenous carboxylates (e.g. cinnamate, acterized by lantionine bridges, bacteriocins and cyclohexanepropanoate, chloropentanoate and 10- cyanobactins that contain certain aromatic hetero- undecynoate). The combination of these two varia- cycles, and lasso peptides and microviridins that tions produced 13 dilactone scaffolds with distinct share macrolactam and/or macrolactone function- C5 substitution and C7 alkylation patterns in yields alities. In general, a precursor peptide consists of 566 Natl Sci Rev, 2017, Vol. 4, No. 4 REVIEW

O O H H H O N O N O N NH NH COOH N 2 2 H O OH N N O N N H S S S H O O O N H S N H N N N S N N 2 N NH N NH O H O N N O N N O HOO HN H S S S HN O S N NH O N O S O HO HO NH NS NH O O O O O HN HN NH HN S O S S N H N HO S N H N N N N S N O O OH O O Downloaded from https://academic.oup.com/nsr/article/4/4/553/2615234 by guest on 27 September 2021 HO HO O OH OH Thiostrepton Nosiheptide GE37468A

O H O O N NH S O N 2 O H O N O N N N N H NH2 O N HN N O S H O O NH2 N H NH N O S S NH N N N NH HN O O N N H O O N O HN O N O O HO O HO H O HN HN NH S O N HN O S N NH O NH S HO NH NH S N O HO S HN S O O O N S S N N HO H H N N HN O N S N H S HN N H OH O S N O O N O HO H2N O OH Sch18640 Lactazole A GE2270A

H S O N S O N H HN NH N H O O HO N N N HN O N S OHH O O O O OCH OH O S HN O 3 N N O HO O N N N S H N N N NH N S O N H H O N O N NH S S O HN HN HN O O NH O O N O S NH N N OH NH O O S N O O N OH NH O O N O S O N H O HO O NH OH NH HN OH S S O H N HO O N N S OH O O Sch40832 TP- 1161 Thiocillin I

HO OH

Figure 8. Structures of representative thiopeptide antibiotics.

an N-terminal sequence, termed leader peptide, re- The biosynthesis of thiopeptide antibiotics serves sponsible for PTM enzyme recognition and a C- as a model system for appreciating how nature de- terminal sequence, termed core peptide, subjected velops PTM strategies with the both generality and to a myriad of different PTMs that occur in leader specificity to convert ribosomally synthesized pep- peptide-dependent or -independent manners dur- tides and achieve the structural complexity and di- ing the conversion into mature product(s) [111]. versityofthemolecules[112,113].Thesesulfur-rich, Although the ribosome-based systems only employ highly modified peptide antibiotics share a charac- substrates within the 20 proteinogenic amino acid teristic macrocyclic core and an extended tail, both building blocks instead of a much wider array of of which constitute a thiopeptide-specific frame- those used by NRPS systems, the sequences of the work featuring a nitrogen-containing domain cen- resulting peptide substrates and associated enzy- tral to multiple azol(in)es and dehydroamino acids matic processing strategies can be highly permutable (Fig. 8). They act on the bacterial ribosome toin- and variable. hibit protein synthesis through a mechanism that is REVIEW Chen et al. 567

distinct from clinically used antibiotics, either by oc- tually, through the activity of the multifunctional cupying the crevice between the L11 protein and protein TbtD, the resulting linear intermediate 23S RNA in the 50S large ribosomal subunit or then undergoes an intramolecular [4 + 2] hetero- by targeting the aminoacyl tRNA chaperone EF- cycloaddition either by a Diels-Alder reaction or Tu in an elongation cycle. Many members in this by polar stepwise additions for macrocyclization, family exhibit potent activity against various drug- dehydration and aromatization to remove the leader resistant pathogens, including methicillin-resistant peptide (carboxamide form) from the heterocycle Staphylococcus aureus, penicillin-resistant Streptococ- central domain. cus pneumonia and vancomycin-resistant Enterococ- During thiopeptide biosynthesis, it should be cus species, thus continuously motivating the efforts noted that Ser/Thr residues have two possible fates. of analogue development toward overcoming their They are modified either to oxazoline or todehy- physical drawbacks (e.g. poor pharmacokinetics and droamino acid; however, cyclodehydration and de- Downloaded from https://academic.oup.com/nsr/article/4/4/553/2615234 by guest on 27 September 2021 low water solubility) in clinical use [114,115]. Re- hydration appear to be site- or residue-specific, par- cently, the interest in this class of peptide antibiotics ticularly for the members that contain a combination has apparently been renewed, largely due to the in- of thiazol(in)es and oxazol(in)es. Typically, oxazo- creasing number of serious microbial infections and line formation is less efficient than thiazoline gen- resistance, the promising antineoplastic activity of eration, possibly because the hydroxyl group has certain members against human cancer cells and the a lower nucleophilicity than the sulfhydryl group. identification of new entities observed from system- Some biosynthetic pathways of thiopeptides (e.g. atic analysis of the human microbiome [116–118]. GE2270A and TP-1161) have been shown to employ two cyclodehydratases [129,130]. This concur- rence represents a functional differentiation, with one cyclodehydratase specific for Cys and another General strategies for constructing the one for Ser/Thr, and the coordination of the PTMs thiopeptide-characteristic framework for specific production of thiazoline and oxazoline as Thiopeptide antibiotics structurally appear to wellasthedehydroaminoacidisindispensableforaf- be the variants deriving a six-membered nitro- fording the thiopeptide framework and maintaining gen heterocycle from a combination of both the overall efficiency of the biosynthesis119 [ ]. azol(in)e-containing (e.g. linear microcins and Consistently with the mechanistic commonality N-to-C macrocyclic cyanobactins) peptides and in building a thiopeptide-characteristic framework, dehydroamino acid-containing peptides (e.g. lan- comparative analysis of all available biosynthetic tibiotics) [112,113]. Consistently, the construction gene clusters of thiopeptides revealed that each of a characteristic thiopeptide framework relies on precursor peptide-encoding gene is exclusively the logical assembly of relevant enzymatic activities clustered with a highly conserved, usually six to process a precursor peptide, whose core peptide gene-containing cassette that codes for the for- is Cys and Ser/Thr-rich, and to build the above mation of azol(in)es, dehydroamino acids and structural hallmarks in a highly ordered manner the central domain [119]. Indeed, several re- [119]. Recent advances in mechanistic under- lated biosynthetic gene clusters, such as those standing of the functions of cyclodehydratases in for thiocillins and lactazoles [131–133], were microcin and cyanobactin biosynthesis [120–122], mined from the strains that were previously not dehydratases in lantibiotic biosynthesis [123] and known to be thiopeptide producers according the enzymes for [4 + 2] cycloaddition reactions to this genetic feature. ThioFinder, a web-based [124–126] have made it possible to achieve the in tool, was thereby developed to query sequenced vitro biosynthesis of the thiopeptide framework of bacterial genomes for their potential to produce thiomuracin (Fig. 9)[127]. First, in the presence of new thiopeptide members [134]. This tool is fully the Ocin-ThiF-like protein TbtF that is responsible supported by an open-access database ThioBase for engaging the precursor peptide TbtA, the (http://db-mml.sjtu.edu.cn/ThioFinder/), which YcaO-like cyclodehydratase TbtG successively documents the substantial information on thiopep- performs phosphorylation-based cyclodehydration tides regarding their chemical structure, biological on the six Cys residues of core peptide to produce activity, producing organism and biosynthetic thiazolines, which are then converted to thiazoles gene (cluster) along with the associated genome by the dehydrogenase TbtE. Next, the dehydratase if available. On the other hand, uncovering the pair TbtB and TbtC catalyses the dehydration of ribosomal origin of thiopeptides sets the stage for the Ser residues to generate four dehydroalanines sequence engineering of the precursor peptide to (Dhas) through an unusual tRNAGlu-dependent, diversify the peptidyl skeleton, which has resulted glutamylation-elimination process [128]. Even- in hundreds of analogues of the thiopeptides 568 Natl Sci Rev, 2017, Vol. 4, No. 4 REVIEW Downloaded from https://academic.oup.com/nsr/article/4/4/553/2615234 by guest on 27 September 2021

Figure 9. Common post-translational modifications (PTMs) to construct a characteristic thiopeptide framework as exemplified by thiomuracin biosynthesis.

thiocillins [135–137], GE37468 [138], GE2270A Specificity in functionalization of the [139], thiostrepton [125,140–142] and nosiheptide thiopeptide-characteristic framework [143]. This diversification, which is challenging in The family of thiopeptide antibiotics now contains current chemical synthesis-based approaches due over 100 naturally occurring entities, which are clas- to the structural complexity of thiopeptides, is of sified into five sub-families according to the oxida- significant interest in the search for new analogues tion extent and substitution pattern of the central that are suited to overcome bacterial resistance domain (Fig. 8): tetrasubstituted piperidine for se- in the clinic and in increasing our understanding ries a (e.g. thiostrepton), dehydropiperidine for se- of thiopeptide biosynthesis by evaluating the ries b (e.g. Sch 18640), imidazopiperidine for series effect of precursor peptide variations on PTM c (e.g. Sch 40832), trisubstituted pyridine for series d capacity. (e.g. thiocillins) and hydroxylated pyridine for series REVIEW Chen et al. 569

e (e.g. nosiheptide) [112,134]. The diverse function- quires an unusual ring-expanding conversion of the alization likely branches at a shared six-membered same substrate L-Trp (Fig. 10a) [150], featuring heterocycle that results from the common PTMs, a methyl transfer onto and a rearrangement of intramolecular [4 + 2] cycloaddition and subse- the indole part to give a quinoline ketone inter- quent dehydration [119]. Heterocycle aromatiza- mediate. A number of specialized enzymes partici- tion would result in protease activity-independent pate in this process [145,151,152], including TsrT elimination of leader peptide to give a pyridine for SAM radical-mediated 2-methylation, TsrA for domain, and further hydroxylation could produce PLP-dependent deamination, TsrE and TsrD for a hydroxypyridine. Alternatively, reduction(s) can oxidation-coupled ring opening and recyclization, be conducted to generate a piperidine or dehy- and TsrU for stereospecific reduction, as well as un- dropiperidine and, during this process, an unchar- characterized enzymes for epoxidation and side-ring acterized protease activity could be involved in the closing. Notably, both nosiheptide and thiostrep- Downloaded from https://academic.oup.com/nsr/article/4/4/553/2615234 by guest on 27 September 2021 specific cleavage of leader peptide and the simulta- ton share a common mode of action on the bac- neous release of the N-terminal amino acid residue terial ribosome, where, for example, the quinaldic for side-ring closure in the biosynthesis of certain bi- moiety of thiostrepton approaches A1067, one of cyclic members. the key nucleobases of the 23S rRNA that con- In addition to central domain diversity, the struc- tributes to mutation-induced bacterial resistance tures of thiopeptide antibiotics can differ with regard [153,154]. Focusing on this biologically relevant to the decoration of the macrocyclic core system, but tunable moiety, two thiostrepton derivatives, installation of the side-ring system and C-terminal 5 -fluorinated or 12 -methylated, were designed by tailoring of the extended side chain. Consistently, a modeling the molecules into the ribosome com- number of pathway-specific biosynthetic genes have plex and biosynthesized by feeding correspond- been identified to be clustered with the conserved ing exogenous analogues to the mutant strain that thiopeptide framework-forming gene cassette, un- lacks the endogenous quinoline ketone intermedi- derlying the biogenesis for individualized treatment ate [155–157]. These thiopeptides displayed a se- of precursor peptides in addition to the common ries of anticipated and unanticipated pharmaceu- PTMs. In particular, comparative investigations into tical advantages compared with the parent com- the biosynthesis of nosiheptide and thiostrepton pound, such as improved activity against a num- [131,144,145], both of which are bicyclic thiopep- ber of drug-resistant pathogens and improved wa- tide members, revealed a series of intriguing specific ter solubility that has largely affected the clinical PTM paradigms that involve seemingly ‘divergent’ use, thus confirming the rationale of modifying activities on a same substrate to achieve completely quinaldic moiety to develop thiostrepton-derived distinct building blocks and ‘convergent’ activities to antibiotics. In particular, the treatment of the intra- reach a same functionality through different enzy- cellular pathogen Mycobacterium marinum revealed matic transformations. that these antibiotics are able to induce host au- In nosiheptide biosynthesis, side-ring formation tophagy, which serves as an indirect but primary in- focuses on the conversion of a precursor peptide- tracellular anti-infection mechanism by enhancing independent L-Trp residue into 3-methyl-2-indolic cell defense [158]. Thiostrepton and its derivatives acid (MIA) (Fig. 10a) [146]. This complex rear- are thus the only antibiotics possessing dual action rangement involves multiple chemically difficult re- on both the bacterial pathogens and the infected actions; however, it depends only on the activity cells, which may inspire the future changes in the of NosL [147], an S-adenosylmethionine (SAM)- treatment of intracellular pathogens by taking the dependent radical protein, which itself catalyses an host response into account. unprecedented fragmentation-recombination that is Recently, an unmacrocyclized shunt intermedi- likely initiated by radical-mediated hydrogen ab- ate of nosiheptide that bears a complete indolic side straction from the amino group of L-Trp [148,149] ring along with thiazole and dehydroamino acid to remove the C–N unit and shift the carboxylate residues has been characterized [125], showing that group onto the indole ring. Prior to 4-methylation, the incorporation of MIA is a specific PTM prior which is catalysed by the SAM-dependent radical to the common [4 + 2] cycloaddition to afford a methyltransferase NosN [144], MIA can be acti- thiopeptide framework. In fact, a number of specific vated and subsequently attached onto the unmod- pre-cycloaddition PTMs, such as methylation, oxi- ified Cys side chain of the precursor peptide. The doreduction, C-terminal decarboxylation and cyclic closure of the side ring may involve an oxidation- residue formation, have been found in the biosyn- coupled lactonization to form the ester bond. In thesis of the monocyclic thiopeptide members such contrast, the formation of the quinaldic acid moi- as thiocillins and GE2270A [124,139]. Thiostrepton ety that resides in the thiostrepton side ring re- biosynthesis shares this manner in quinaldic acid 570 Natl Sci Rev, 2017, Vol. 4, No. 4 REVIEW

(a) Downloaded from https://academic.oup.com/nsr/article/4/4/553/2615234 by guest on 27 September 2021

(b)

Figure 10. Selected specific PTMs in the biosynthesis of the bicyclic thiopeptides nosiheptide and thiostrepton. (a) Divergence in processing L-Trp to form the indolic and quinaldic moieties. (b) Convergence of different enzymatic mechanisms in the formation of a common C-terminal amide.

formation and incorporation [151], which are amide is formed endogenously and requires the ac- evidently necessary for thiopeptide framework tivity of NosA on an extended Ser residue of the construction; however, [4 + 2] cycloaddi- precursor peptide (Fig. 10b) [159]. NosA cataly- tion/macrocyclization does not depend on the ses an unusual dealkylation reaction for nosiheptide closure of the side ring, given that quinaldic acid- maturation, taking advantage of the enamide moi- containing but side-ring-uncyclized products have ety arising from dehydration of this extended Ser been identified, indicating that side-ring closure that occurs as a common PTM at the early biosyn- is a post-thiopeptide PTM [140]. These findings thetic stage. Analysis of the crystal structure of NosA demonstrate that the common and specific PTMs and associated mutagenesis-based assays supported are interdependent and can occur alternately in the idea that tautomerization of the resulting Dha thiopeptide biosynthesis to achieve molecular tail to the corresponding methyl imine facilitates complexity and diversity [119]. the nucleophilic attack of H2O to produce a ter- Many thiopeptides, including the bicyclic mem- minally hydrated intermediate, which is then sub- bers nosiheptide and thiostrepton and the mono- jected to a Cα–N bond cleavage to yield nosiheptide cyclic member GE2270A, share a common C- along with pyruvate [160]. GE2270A biosynthesis terminal amide moiety, which, however, is synthe- could share this chemistry for terminal amide forma- sized through completely different routes. As exem- tion, which may occur as a pre-cycloaddition PTM plified by nosiheptide biosynthesis, the C-terminal [139]. In contrast, C-terminal amide formation in REVIEW Chen et al. 571

thiostrepton biosynthesis is exogenous and involves of NPs can be attributed to variations of a few com- the activity of the asparagine synthetase-like protein mon biosynthetic machineries. Although the differ- TsrC (Fig. 10b) [161]. This protein may utilize γ – ences between these paradigms have been invalu- carboxamide of Gln to generate a nascent ammonia able in delineating the biosynthesis of various classes in situ, the attachment of which to the ATP-activated of NPs, it also has become very clear that the plas- terminal carboxylate results in an amide, with Glu, ticity of various biosynthetic machineries is much AMP and PPi as the co-products. Interestingly, the more profound than previously appreciated. These carboxylate substrate of TsrC is prepared by TsrB, a features of the NPbiosyntheticmachineries will con- carboxyl esterase. This enzyme catalyses the hydrol- tinue to illuminate the opportunities and synthetic ysis of a methyl ester intermediate, indicating that biology strategies for enhancing NP structural diver- the precursor peptide of thiostrepton is C-terminally sity and drug discovery. methylated at the early stage of the biosynthesis Downloaded from https://academic.oup.com/nsr/article/4/4/553/2615234 by guest on 27 September 2021 (probably to facilitate dehydration of the C-terminal Ser residue by reducing the pKa value of the α hy- FUNDING drogen). This deesterification-amidation paradigm This work was supported in part by grants from National Nat- is common in the maturation of thiostrepton-like ural Science Foundation of China (21520102004, 31430005, thiopeptides, and either deesterification or amida- 21472231 and 21621002), Science and Technology Commission tion can be omitted from the biosynthetic pathway of Shanghai Municipality (Shanghai, China) (14JC1407700 and to generate thiopeptide members varying in the C- 15JC1400400), Chinese Academy of Sciences (XDB20020200) terminus as methyl esters, carboxylates or amides. and Chang-Jiang Scholars Program of China.

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