Evidence for an iterative module in chain elongation on the azalomycin polyketide synthase

Evidence for an iterative module in chain elongation on the azalomycin polyketide synthase Hui Hong1, Yuhui Sun2, Yongjun Zhou1, Emily Stephens1, Markiyan Samborskyy1 and Peter F. Leadlay*1 Full Research Paper Address: 1Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA, United Kingdom and 2Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Wuhan University, Ministry of Education, and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, People’s Republic of China Open Access Beilstein J. Org. Chem. 2016, 12, 2164–2172. doi:10.3762/bjoc.12.206 Received: 16 June 2016 Accepted: 23 September 2016 Published: 11 October 2016 Associate Editor: J. S. Dickschat Email: Peter F. Leadlay* - © 2016 Hong et al.; licensee Beilstein-Institut. License and terms: see end of document. * Corresponding author Keywords: colinearity; ebelactone; enzyme catalysis; marginolactone; natural products; polyketide synthase Abstract The assembly-line synthases that produce bacterial polyketide natural products follow a modular paradigm in which each round of chain extension is catalysed by a different set or module of enzymes. Examples of deviation from this paradigm, in which a module catalyses either multiple extensions or none are of interest from both a mechanistic and an evolutionary viewpoint. We present evidence that in the biosynthesis of the 36-membered macrocyclic aminopolyol lactones (marginolactones) azalomycin and kanchanamycin, isolated respectively from Streptomyces malaysiensis DSM4137 and Streptomyces olivaceus Tü4018, the first extension module catalyses both the first and second cycles of polyketide chain extension. To confirm the integrity of the azl gene cluster, it was cloned intact on a bacterial artificial chromosome and transplanted into the heterologous host strain Streptomyces lividans, which does not possess the genes for marginolactone production. When furnished with 4-guanidinobutyramide, a specific precursor of the azalomycin starter unit, the recombinant S. lividans produced azalomycin, showing that the polyketide synthase genes in the sequenced cluster are sufficient to accomplish formation of the full-length polyketide chain. This provides strong support for module iteration in the azalomycin and kanchanamycin biosynthetic pathways. In contrast, re-sequencing of the gene cluster for biosynthesis of the polyketide β-lactone ebelactone in Streptomyces aburaviensis has shown that, contrary to a recentlypublished proposal, the ebelactone polyketide synthase faithfully follows the colinear modular paradigm. Introduction Bacterial modular Type I polyketide synthases (PKSs) are multienzymes that govern the biosynthesis of diverse complex polyketide natural products, including clinically useful antibiot- ics, immunosuppressants, and antitumor compounds. They follow a remarkable assembly-line paradigm, in which each cycle of polyketide chain extension is accomplished by a differ- 2164 Beilstein J. Org. Chem. 2016, 12, 2164–2172. ent set or module of vertebrate fatty acid synthase (FAS)-related enzyme domains [1-4]. The direct connection between the number and type of modules and the chemical structure of the eventual product is often referred to as colinearity. Each module contains a ketosynthase (KS) domain, which recruits the growing polyketide acyl chain from the previous module and catalyses its Claisen-like carbon–carbon bond condensation with the incoming (alkyl)malonyl extender unit, tethered to an acyl carrier protein (ACP) domain. The choice of extender unit installed onto the ACP is dictated by an acyltransferase (AT domain). In addition to these conserved domains, a module may contain ketoreductase (KR), dehydratase (DH) and enoyl reductase (ER) domains that determine the degree and outcome of reductive processing of the newly-formed β-ketoacyl thioester. Finally, the extended chain is passed on to the following module. This processive assembly-line operation, in which all intermediates remain covalently attached to the multienzyme, helps to explain the efficiency of the process. It also neatly explains how the diversity of naturally-occurring complex polyketides is generated by a common biosynthetic mechanism, and provides clues to the evolution of these multienzymes through duplication, capture, deletion, and rearrangement of modules or individual domains [5]. It has both prompted efforts to manipulate PKS domains and modules into novel combinations, as a route to obtaining novel non-natural polyketide products [6,7], and facilitated the discovery of new biosynthetic gene clusters using whole-genome sequence analysis [8,9]. A number of assembly-line PKSs do not exactly follow the modular colinear paradigm, and there is great interest in characterising such exceptions, both for the insights these examples can potentially provide into the catalytic mechanism and specificity of chain extension, and to further our understanding of how these molecular machines have evolved [10-12]. It is clear, for example, that a large number of so-called trans-AT PKSs, where attachment of extender units to ACP domains is effected by stand-alone AT enzymes rather than by an intramodular AT domain, have an evolutionary history different from that of canonical (cis-AT) modular PKSs [13]. In trans-AT PKSs, domains are often found in unconventional order, and modules may be split between different PKS multienzyme subunits. In both types of modular PKS, domains may be present but apparently not used, or expected domains may be missing [10,12]. Perhaps the most striking deviations from colinearity are those where the number of modules in the PKS does not correspond to the number of extension units found in the chemical product [10,12]. Strains subjected either to random mutagenesis or to a targetted block in post-PKS steps have been found to accumulate aberrant products of either PKS module omission ("skipping") or the iterative use of a module ("stuttering") as minor congeners of a product mixture [14-16]. Efficient skipping of an interpolated heterologous module has also been observed in an engineered PKS assembly-line [17,18]. Naturally programmed skipping of a PKS module to make an alternative product is rare, the best-characterised example being the production of both the 12-membered macrolide methymycin and the 14-membered macrolide pikromycin from the same PKS [19], the smaller ring arising from use of an alternative start codon leading to a significantly-truncated final module incapable of condensation. However, an increasing number of PKS systems are now known in which the main product apparently requires iterative use of a module to accomplish two or even three successive rounds of chain extension. First noted in the stigmatellin PKS from Stigmatella aurantiaca [20], further examples have been uncovered in the PKSs for aureothin [21,22], borrelidin [23,24], lankacidin [25,26], neoaureothin [27], (...truncated)