Benjamin Busch

Functionally Distinct Modules Operate Two Consecutive α,β→β,γ Double-Bond Shifts in the Rhizoxin Polyketide Assembly Line†

Shift work: Biochemical analysis of the rhizoxin pathway revealed that the diene moiety is not shifted all at once, but through distinct enzymatic operations. The first shift occurs by a formal β,γ-dehydration in module 7, while the second double bond is first generated by module 8 and then shifted by an unprecedented “shift module” with a novel type of DH* domain (see scheme). ACP=acyl carrier protein, DH*=dehydratase-like shift domain.

Vinylogous chain branching catalysed by a dedicated polyketide synthase module

Bacteria use modular polyketide synthases (PKSs) to assemble complex polyketides, many of which are leads for the development of clinical drugs, in particular anti-infectives and anti-tumoral agents. Because these multifarious compounds are notoriously difficult to synthesize, they are usually produced by microbial fermentation. During the past two decades, an impressive body of knowledge on modular PKSs has been gathered that not only provides detailed insight into the biosynthetic pathways but also allows the rational engineering of enzymatic processing lines to yield structural analogues. Notably, a hallmark of all PKS modules studied so far is the head-to-tail fusion of acyl and malonyl building blocks, which leads to linear backbones. Yet, structural diversity is limited by this uniform assembly mode. Here we demonstrate a new type of PKS module from the endofungal bacterium Burkholderia rhizoxinica that catalyses a Michael-type acetyl addition to generate a branch in the carbon chain. In vitro reconstitution of the entire PKS module, X-ray structures of a ketosynthase-branching didomain and mutagenesis experiments revealed a crucial role of the ketosynthase domain in branching the carbon chain. We present a trapped intermediary state in which acyl carrier protein and ketosynthase are covalently linked by the branched polyketide and suggest a new mechanism for chain alkylation, which is functionally distinct from terpenoid-like β-branching. For the rice seedling blight toxin rhizoxin, one of the strongest known anti-mitotic agents, the non-canonical polyketide modification is indispensable for phytotoxic and anti-tumoral activities. We propose that the formation of related pharmacophoric groups follows the same general scheme and infer a unifying vinylogous branching reaction for PKS modules with a ketosynthase-branching–acyl-carrier-protein architecture. This study unveils the structure and function of a new PKS module that broadens the biosynthetic scope of polyketide biosynthesis and sets the stage for rationally creating structural diversity.

Symbiotic Cooperation in the Biosynthesis of a Phytotoxin

Natural products play a key role in symbiotic interactions between microorganisms and higher organisms, covering all kingdoms of life. The function of these secondary metabolites may range from signaling compounds in mutualism to virulence factors and antibiotics in parasitic relationships. In many cases the interactions involve multiple partners and thus the biogenetic basis of chemical mediators can be quite complex. This complexity is well exemplified by the unparalleled tripartite relationship among the rice-seedling-blight fungus Rhizopus microsporus, its host plant Oryza sativa and endosymbiotic bacteria that reside in the fungal cytosol. The bacterial symbionts (Burkholderia species) produce a phytotoxin complex to assist the phytopathogenic fungus in colonizing rice seedlings. In turn the bacteria profit from a safe niche and access to nutrients released from the decaying plant. Initially, the macrolide rhizoxin (1, Figure 1) and various congeners such as WF-1360F (2) were isolated from cultures of R. microsporus van Tieghem var. chinensis and identified as the causative agent of rice seedling blight. Rhizoxin efficiently inhibits eukaryotic cell proliferation by binding to b-tubulin and thus blocking the formation of the mitotic spindle. Notably, the pure compound alone evokes the typical symptoms of seedling root swelling. Only recently, through detection, isolation, and cultivation of the endosymbionts we could unequivocally prove that actually associated bacteria are the true producers of the toxin complex. The importance of this metabolic capability has been underlined by the finding that fungal reproduction depends entirely on the presence of the bacterial symbionts. Survival of the toxinogenic symbiosis is warranted by the strict sporulation control and exclusive dispersal of spores harboring endosymbionts. Moreover, the unusual mutualism has been fine-tuned through symbiosis factors such as a type-III secretion system and a novel lipopolysaccharide O-antigen that sets the symbionts into a “stealth mode” by decorating the outer membrane of the endosymbionts. The host, on the other hand, acquired resistance towards rhizoxin by mutation of the b-tubulin. Because of its ecological and medicinal relevance as an antimitotic agent, the biosynthesis of rhizoxin has been studied. Cloning, sequencing, and molecular analyses of the rhizoxin (rhi) biosynthetic gene cluster in the genome of Burkholderia rhizoxinica revealed the molecular basis for a complex polyketide assembly line required for the biosynthesis of the virulence factor. Whereas the biosynthesis of the macrolide backbone has been decoded by mutational analyses, polyketide tailoring mechanisms and the biological role of the bis(epoxidation) have remained elusive. Herein we elucidate the dual epoxidation of rhizoxin and its impact on rice seedling blight and report an unprecedented case for symbiotic cooperation in the biosynthesis of an ecologically relevant natural product. In a broader survey on rhizoxin-positive Rhizopus species we discovered that the unusual bacterial–fungal association is not restricted to a single isolate but has spread worldwide. We have identified eight related Burkholderia–Rhizopus associations from geographically highly different regions on five continents; these findings underlign the ecological imporFigure 1. A) Structures of rhizoxin and congeners. B) Phylogenetic relationship of Rhizopus microsporus strains; structures of the corresponding metabolites indicate which strains can produce bisepoxides. The numbers on top of the branches indicate the clade probability values; the scale on the left site relates the length of a branch to the distance (number of changes that have taken place along a branch).

Exploiting Enzymatic Promiscuity to Engineer a Focused Library of Highly Selective Antifungal and Antiproliferative Aureothin Analogues

Aureothin is a shikimate-polyketide hybrid metabolite from Streptomyces thioluteus with a rare nitroaryl moiety, a chiral tetrahydrofuran ring, and an O-methylated pyrone ring. The antimicrobial and antitumor activities of aureothin have caught our interest in modulating its structure as well as its bioactivity profile. In an integrated approach using mutasynthesis, biotransformation, and combinatorial biosynthesis, a defined library of aureothin analogues was generated. The promiscuity of the polyketide synthase assembly line toward different starter units and the plasticity of the pyrone and tetrahydrofuran ring formation were exploited. A selection of 15 new aureothin analogues with modifications at the aryl residue, the pyrone ring, and the oxygenated backbone was produced on a preparative scale and fully characterized. Remarkably, various new aureothin derivatives are less cytotoxic than aureothin but have improved antiproliferative activities. Furthermore, we found that the THF ring is crucial for the remarkably selective activity of aureothin analogues against certain pathogenic fungi.

Evolution of metabolic diversity in polyketide-derived pyrones: using the non-colinear aureothin assembly line as a model system.

Polyketide-derived pyrones are structurally diverse secondary metabolites that are represented in all three kingdoms of life and are endowed with various biological functions. The aureothin family of Streptomyces metabolites was chosen as a model to study the factors governing structural diversity and the evolutionary processes involved. This review highlights recent insights into the non-colinear aureothin and neoaureothin modular type I polyketide synthase (PKS), aromatic starter unit biosynthesis, polyketide tailoring reactions, and a non-enzymatic polyene splicing cascade. Pyrone biosynthesis in bacteria, fungi, and plants is compared. Finally, various strategies to increase metabolic diversity of aureothin derivatives through mutasynthesis, pathway engineering, and biotransformation are presented. The unusual aureothin and neoaureothin assembly lines thus not only represent a model for PKS evolution, but provided important insights into non-canonical enzymatic processes that could be employed for the production of antitumor and antifungal agents.

Multifactorial Control of Iteration Events in a Modular Polyketide Assembly Line

The poly-ketide synthases (PKSs) in charge of selecting, fusing, andprocessing the building blocks are organized into modulesthat consist of individual catalytic domains. Typically, there isa unidirectional progress of chain elongation, where eachdomainisonlyusedonceinthebiosynthesisofonepolyketidemolecule. Thus, for most synthases there is a strict colinearitybetween the number and architecture of the modules and thenumberofelongationsanddegreeofb-ketoprocessing.

Freedom and Constraint in Engineered Noncolinear Polyketide Assembly Lines

Many pharmacologically important natural products are assembled by modular type I polyketide synthases (PKS), which typically act in a unidirectional fashion. The synthases producing the unusual nitro-substituted polyketides neoaureothin (nor, also called spectinabilin) and aureothin (aur) are exceptional, as they employ individual modules iteratively. Here, we investigate the plasticity of the nor PKS and the factors governing the number of elongations catalyzed by the noncanonical module. Surprisingly, we observe that the nor PKS can mediate an additional chain elongation to yield the higher homolog homoneoaureothin. Furthermore, we design several truncated variants of the nor PKS to use them in the context of artificial assembly lines for aureothin and homoaureothin. The resulting polypropionate derivatives provide valuable insights into chain length control and reveal structure-activity relationships relating to the size of the polypropionate backbones. Overall, we show that iterative modules are remarkably adaptable while downstream modules are gatekeepers that select for correct polyketide chain length.

Interchenar Retrotransfer of Aureothin Intermediates in an Iterative Polyketide Synthase Module

The course of the enigmatic iterative use of a polyketide synthase module was deduced from targeted domain inactivation in the aureothin assembly line. Mutational analyses revealed that the N-terminus of AurA is not involved in the iteration process, ruling out an ACP-ACP shuttle. Furthermore, an AurA(KS°, ACP°)-AurA(AT(0)) heterodimer proved to be nonfunctional, whereas aureothin production was restored in a ΔaurA mutant complemented with AurA(KS°)-AurA(ACP°). This finding supports a model according to which the ACP-bound polyketide intermediate is transferred back to the KS domain on the opposite PKS strand.

Structural characterization of two lipopolysaccharide O-antigens produced by the endofungal bacterium Burkholderia sp. HKI-402 (B4)

Two different polysaccharides were isolated and identified from the lipopolysaccharide fraction of endofungal bacterium Burkholderia sp. HKI-402 (B4). The complete structure was elucidated by chemical analysis and 2D NMR spectroscopy as the following:

Pyran formation by an atypical CYP-mediated four-electron oxygenation-cyclization cascade in an engineered aureothin pathway.

Small changes, big effect: A new aureothin derivative, aureopyran, which features an unusual pyran backbone, was generated by simply altering the enzymatic methylation topology. The α-pyrone ring hampers the correct placement of the polyketide backbone in the multifunctional cytochrome P450 monooxygenase AurH. Instead of a tetrahydrofuran ring, an oxo intermediate is formed that readily undergoes a rare electrocyclization reaction.