Knowledge-based design of bimodular and trimodular polyketide synthases based on domain and module swaps: a route to simple statin analogues

Ranganathan, Anand; Timoney, Máire C.; Bycroft, Mark; Cortés, Jesús M.; Thomas, Iain P; Wilkinson, Barrie; Kellenberger, Laurenz; Hanefeld, Ulf; Galloway, Ian S.; Staunton, James; Leadlay, Peter F.

doi:10.1016/s1074-5521(00)80020-4

Cited by 106 publications

(98 citation statements)

References 42 publications

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“…Genetic engineering studies allow assembly of novel polyketide chains following fusion, swapping or repositioning of catalytic domains, modules or whole peptides within PKS polypeptides (Hutchinson & Fujii, 1995 ;Ranganathan et al, 1999 ;Tang et al, 2000). In engineered PKSs, co-expression of TE IIs in addition to other PKS proteins might help in achieving elevated levels of the polyketide products.…”

Section: -5346 # 2002 Sgmmentioning

confidence: 99%

Type II thioesterase from Streptomyces coelicolor A3(2) The GenBank accession number for the sequence reported in this paper is AF109727.

et al. 2002

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Type I polyketide synthases (PKSs) are complexes of large, multimodular enzymes that catalyse biosynthesis of polyketide compounds via repetitive reaction sequences, during which each step is catalysed by a separate enzymic domain. Many type I PKSs, and also non-ribosomal peptide synthetase clusters, contain additional thioesterase genes located adjacent to PKS genes. These are discrete proteins called type II thioesterases (TE IIs) to distinguish them from chain-terminating thioesterase (TE I) domains that are usually fused to the terminal PKS module. A gene of a new TE II, scoT, associated with the cluster of putative type I PKS genes from Streptomyces coelicolor A3(2), was found. The deduced amino acid sequence of the gene product shows extensive similarity to other authentic thioesterase enzymes, including conservation of characteristic motifs and residues involved in catalysis. When expressed in the heterologous host Streptomyces fradiae, scoT successfully complemented the resident TE II gene (tylO), and, by restoring a significant level of macrolide production, proved to be catalytically equivalent to the TylO protein. S 1 nuclease mapping of scoT revealed a single potential transcription start point with expression being switched on for a short period of time during a transition phase of growth.

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Section: -5346 # 2002 Sgmmentioning

confidence: 99%

Type II thioesterase from Streptomyces coelicolor A3(2) The GenBank accession number for the sequence reported in this paper is AF109727.

et al. 2002

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“…Early studies revealed the importance of ACP-KS interactions (6,7), as well as the utility of intermodular linker interactions (8), in the design of productive chimeric assembly lines. At the same time, the relatively broad, but not unrestricted, substrate tolerance of typical KS domains was also recognized (9).…”

mentioning

confidence: 99%

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases

Klaus

Ostrowski

Austerjost

et al. 2016

Journal of Biological Chemistry

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The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzymesubstrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs.The assembly line architecture of multimodular polyketide synthases (PKSs) 5 represents a promising catalytic framework for combinatorial biosynthesis (1). A particularly well studied example of this family of multienzyme systems is the 6-deoxyerythronolide B synthase (DEBS), which produces 6-deoxyerythronolide B (Fig. 1), the parent aglycone of the macrolide antibiotic erythromycin (2). DEBS is comprised of three distinct homodimeric proteins: DEBS1, DEBS2, and DEBS3, each containing two PKS modules, with each module being responsible for a distinct round of polyketide elongation and modification. DEBS uses propionyl-CoA to prime the loading didomain (LDD) of module 1 and six methylmalonyl-CoA-derived extender units in catalysis of the six cycles of polyketide chain elongation, followed by terminal release of the mature polyketide chain by thioesterase (TE)-catalyzed macrolactone formation.Since the original genetic characterization of DEBS over two decades ago (3, 4), extensive in vivo and in vitro analysis has revealed that specific protein-protein interactions play a critical role in the proper vectorial channeling of biosynthetic intermediates from one PKS module to the next (5). A particularly effective mini-assembly line for mechanistic analysis has been a simple bimodular derivative of DEBS (Fig. 2) harboring modules 1 and 2 with a fused TE domain. This bimodular PKS has served as a prototype for many analogous constructs.The potential for engineering chimeric PKSs by recombining modules from paralogous polyketide biosynthetic pathways has been explored for nearly two decades. Early studies revealed the importance of ACP-KS interactions (6, 7), as well as the utility of intermodul...

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“…With the exception of C-terminal͞ N-terminal linker pairs (6) that serve as docking sites for successive but noncovalently interacting modules, the structure and function of linkers in modular PKSs have been largely unexplored. There is growing appreciation, however, that a better understanding of the role of linkers is of critical importance to the continued success of biosynthetic engineering (6)(7)(8).…”

mentioning

confidence: 99%

The 2.7-Å crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase

Tang

Kim

Mathews

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

256

410

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The x-ray crystal structure of a 194-kDa fragment from module 5 of the 6-deoxyerythronolide B synthase has been solved at 2.7 Å resolution. Each subunit of the homodimeric protein contains a full-length ketosynthase (KS) and acyl transferase (AT) domain as well as three flanking ''linkers.'' The linkers are structurally well defined and contribute extensively to intersubunit or interdomain interactions, frequently by means of multiple highly conserved residues. The crystal structure also reveals that the active site residue Cys-199 of the KS domain is separated from the active site residue Ser-642 of the AT domain by Ϸ80 Å. This distance is too large to be covered simply by alternative positioning of a statically anchored, fully extended phosphopantetheine arm of the acyl carrier protein domain from module 5. Thus, substantial domain reorganization appears necessary for the acyl carrier protein to interact successively with both the AT and the KS domains of this prototypical polyketide synthase module. The 2.7-Å KS-AT structure is fully consistent with a recently reported lower resolution, 4.5-Å model of fatty acid synthase stucture, and emphasizes the close biochemical and structural similarity between polyketide synthase and fatty acid synthase enzymology. modular megasynthase ͉ multienzyme assembly ͉ polyketide synthase

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Knowledge-based design of bimodular and trimodular polyketide synthases based on domain and module swaps: a route to simple statin analogues

Cited by 106 publications

References 42 publications

Type II thioesterase from Streptomyces coelicolor A3(2) The GenBank accession number for the sequence reported in this paper is AF109727.

Type II thioesterase from Streptomyces coelicolor A3(2) The GenBank accession number for the sequence reported in this paper is AF109727.

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases

The 2.7-Å crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase

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