Engineered chimeras unveil swappable modular features of fatty acid and polyketide synthase acyl carrier proteins

The strategic redesign of microbial biosynthetic pathways is a compelling route to access molecules of diverse structure and function in a potentially environmentally sustainable fashion. The promise of this approach hinges on an improved understanding of acyl carrier proteins (ACPs), which serve as central hubs in biosynthetic pathways. These small, flexible proteins mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural products. Past combinatorial biosynthesis efforts have failed due to incompatible ACP-enzyme pairings. Herein we report the design of chimeric ACPs with features of the actinorhodin polyketide synthase ACP (ACT) and of the E. coli fatty acid synthase (FAS) ACP (AcpP). We evaluate the ability of the chimeric ACPs to interact with the E. coli FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP interacts with FabF but ACT does not, we sought to exchange modular features of ACT with AcpP to confer functionality with FabF. The interactions of chimeric ACPs with FabF were interrogated using sedimentation velocity experiments, surface plasmon resonance analyses, mechanism-based crosslinking assays, and molecular dynamics simulations. Results suggest that the residues guiding AcpP-FabF compatibility and ACT-FabF incompatibility may reside in the loop I, α-helix II region. These findings can inform the development of strategic secondary element swaps that expand the enzyme compatibility of ACPs across systems and therefore represent a critical step towards the strategic engineering of unnatural natural products.

Download Full-text

Structure and Dynamic Basis of Molecular Recognition Between Acyltransferase and Carrier Protein in E. coli Fatty Acid Synthesis

10.1101/2020.05.15.098798 ◽

2020 ◽

Cited By ~ 1

Author(s):

Laetitia E. Misson ◽

Jeffrey T. Mindrebo ◽

Tony D. Davis ◽

Ashay Patel ◽

J. Andrew McCammon ◽

...

Keyword(s):

Molecular Dynamics ◽

Fatty Acid ◽

Fatty Acid Synthase ◽

Polyketide Synthase ◽

De Novo ◽

Md Simulations ◽

Type Ii ◽

Carrier Proteins ◽

Acyl Carrier Proteins ◽

Binding Interface

AbstractFatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP-AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the Escherichia coli FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP-AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the first x-ray crystal structure of a type II ACP-AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long timescale molecular dynamics (MD) simulations. Together, these complementary approaches reveal key catalytic features of FAS ACP-AT interactions. These mechanistic inferences suggest that AcpP adopts multiple, productive conformations at the AT binding interface, allowing the complex to sustain high transacylation rates. Furthermore, MD simulations support rigid body subdomain motions within the FabD structure that may play a key role in AT activity and substrate selectivity.Significance StatementThe essential role of acyltransferases (ATs) in fatty acid synthase (FAS) and polyketide synthase (PKS) pathways, namely the selection and loading of starter and extender units onto acyl carrier proteins (ACPs), relies on catalytically productive ACP-AT interactions. Here, we describe and interrogate the first structure of a type II FAS malonyl-CoA:ACP-transacylase (MAT) in covalent complex with its cognate ACP. We combine structural, mutational, crosslinking and kinetic data with molecular dynamics simulations to describe a highly flexible and robust protein-protein interface, substrate-induced active site reorganization, and key subdomain motions that likely govern FAS function. These findings strengthen a mechanistic understanding of molecular recognitions between ACPs and partner enzymes and provide new insights for engineering AT-dependent biosynthetic pathways.

Download Full-text

Acyltransferases as Tools for Polyketide Synthase Engineering

Antibiotics ◽

10.3390/antibiotics7030062 ◽

2018 ◽

Vol 7 (3) ◽

pp. 62 ◽

Cited By ~ 14

Author(s):

Ewa Musiol-Kroll ◽

Wolfgang Wohlleben

Keyword(s):

Natural Products ◽

Rational Design ◽

Polyketide Synthase ◽

Antifungal Agents ◽

Relevant Literature ◽

Structural Diversity ◽

Building Blocks ◽

Assembly Lines ◽

Acyl Carrier Proteins ◽

Polyketide Biosynthesis

Polyketides belong to the most valuable natural products, including diverse bioactive compounds, such as antibiotics, anticancer drugs, antifungal agents, immunosuppressants and others. Their structures are assembled by polyketide synthases (PKSs). Modular PKSs are composed of modules, which involve sets of domains catalysing the stepwise polyketide biosynthesis. The acyltransferase (AT) domains and their “partners”, the acyl carrier proteins (ACPs), thereby play an essential role. The AT loads the building blocks onto the “substrate acceptor”, the ACP. Thus, the AT dictates which building blocks are incorporated into the polyketide structure. The precursor- and occasionally the ACP-specificity of the ATs differ across the polyketide pathways and therefore, the ATs contribute to the structural diversity within this group of complex natural products. Those features make the AT enzymes one of the most promising tools for manipulation of polyketide assembly lines and generation of new polyketide compounds. However, the AT-based PKS engineering is still not straightforward and thus, rational design of functional PKSs requires detailed understanding of the complex machineries. This review summarizes the attempts of PKS engineering by exploiting the AT attributes for the modification of polyketide structures. The article includes 253 references and covers the most relevant literature published until May 2018.

Download Full-text

Novel Polyketide Synthase from Nectria haematococca

Applied and Environmental Microbiology ◽

10.1128/aem.70.5.2984-2988.2004 ◽

2004 ◽

Vol 70 (5) ◽

pp. 2984-2988 ◽

Cited By ~ 48

Author(s):

Stephane Graziani ◽

Christelle Vasnier ◽

Marie-Josee Daboussi

Keyword(s):

Polyketide Synthase ◽

Fungal Growth ◽

Type I ◽

Biosynthetic Pathways ◽

Carrier Proteins ◽

Nectria Haematococca ◽

Acyl Carrier Proteins ◽

Asexual Sporulation ◽

Ketoacyl Synthase ◽

Pigment Biosynthesis

ABSTRACT We identified a polyketide synthase (PKS) gene, pksN, from a strain of Nectria haematococca by complementing a mutant unable to synthesize a red perithecial pigment. pksN encodes a 2,106-amino-acid polypeptide with conserved motifs characteristic of type I PKS enzymatic domains: β-ketoacyl synthase, acyltransferase, duplicated acyl carrier proteins, and thioesterase. The pksN product groups with the Aspergillus nidulans WA-type PKSs involved in conidial pigmentation and melanin, bikaverin, and aflatoxin biosynthetic pathways. Inactivation of pksN did not cause any visible change in fungal growth, asexual sporulation, or ascospore formation, suggesting that it is involved in a specific developmental function. We propose that pksN encodes a novel PKS required for the perithecial red pigment biosynthesis.

Download Full-text

Streptomyces coelicolor phosphopantetheinyl transferase: a promiscuous activator of polyketide and fatty acid synthase acyl carrier proteins

Journal of the Chemical Society Perkin Transactions 1 ◽

10.1039/b204633b ◽

2002 ◽

pp. 1644-1649 ◽

Cited By ~ 25

Author(s):

Russell J. Cox ◽

John Crosby ◽

Oliver Daltrop ◽

Frank Glod ◽

Malgorzata E. Jarzabek ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthase ◽

Streptomyces Coelicolor ◽

Phosphopantetheinyl Transferase ◽

Carrier Proteins ◽

Acyl Carrier Proteins

Download Full-text

Reconstitution of the FK228 Biosynthetic Pathway Reveals Cross Talk between Modular Polyketide Synthases and Fatty Acid Synthase

Applied and Environmental Microbiology ◽

10.1128/aem.01513-10 ◽

2010 ◽

Vol 77 (4) ◽

pp. 1501-1507 ◽

Cited By ~ 23

Author(s):

Shane R. Wesener ◽

Vishwakanth Y. Potharla ◽

Yi-Qiang Cheng

Keyword(s):

Fatty Acid ◽

Cross Talk ◽

Biosynthetic Pathway ◽

Fatty Acid Synthase ◽

Polyketide Synthase ◽

Fatty Acid Biosynthesis ◽

Chain Elongation ◽

E Coli ◽

Genetic Components ◽

Polyketide Chain

ABSTRACTFunctional cross talk between fatty acid biosynthesis and secondary metabolism has been discovered in several cases in microorganisms; none of them, however, involves a modular biosynthetic enzyme. Previously, we reported a hybrid modular nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) pathway for the biosynthesis of FK228 anticancer depsipeptide inChromobacterium violaceumstrain 968. This pathway contains two PKS modules on the DepBC enzymes that lack a functional acyltransferase (AT) domain, and no apparent AT-encoding gene exists within the gene cluster or its vicinity. We report here that, through reconstitution of the FK228 biosynthetic pathway inEscherichia colicells, two essential genes,fabD1andfabD2, both encoding a putative malonyl coenzyme A (CoA) acyltransferase component of the fatty acid synthase complex, are positively identified to be involved in FK228 biosynthesis. Either gene product appears sufficient to complement the AT-less PKS modules on DepBC for polyketide chain elongation. Concurrently, a gene (sfp) encoding a putative Sfp-type phosphopantetheinyltransferase was identified to be necessary for FK228 biosynthesis as well. Most interestingly, engineeredE. colistrains carrying variable genetic components produced significant levels of FK228 under both aerobic and anaerobic cultivation conditions. Discovery of thetranscomplementation of modular PKSs by housekeeping ATs reveals natural product biosynthesis diversity. Moreover, demonstration of anaerobic production of FK228 by an engineered facultative bacterial strain validates our effort toward the engineering of novel tumor-targeting bioagents.

Download Full-text

Comparative structure, dynamics and evolution of acyl-carrier proteins from Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii

Biochemical Journal ◽

10.1042/bcj20190797 ◽

2020 ◽

Vol 477 (2) ◽

pp. 491-508 ◽

Cited By ~ 1

Author(s):

Ravi P. Barnwal ◽

Mandeep Kaur ◽

Alec Heckert ◽

Janeka Gartia ◽

Gabriele Varani

Keyword(s):

Fatty Acid ◽

Borrelia Burgdorferi ◽

Active Sites ◽

Pathogenic Bacteria ◽

Fatty Acid Synthase ◽

Brucella Melitensis ◽

Carrier Proteins ◽

Acyl Carrier Proteins ◽

Rickettsia Prowazekii ◽

Structure Dynamics

Acyl carrier proteins (ACPs) are small helical proteins found in all kingdoms of life, primarily involved in fatty acid and polyketide biosynthesis. In eukaryotes, ACPs are part of the fatty acid synthase (FAS) complex, where they act as flexible tethers for the growing lipid chain, enabling access to the distinct active sites in FAS. In the type II synthesis systems found in bacteria and plastids, these proteins exist as monomers and perform various processes, from being a donor for synthesis of various products such as endotoxins, to supplying acyl chains for lipid A and lipoic acid FAS (quorum sensing), but also as signaling molecules, in bioluminescence and activation of toxins. The essential and diverse nature of their functions makes ACP an attractive target for antimicrobial drug discovery. Here, we report the structure, dynamics and evolution of ACPs from three human pathogens: Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii, which could facilitate the discovery of new inhibitors of ACP function in pathogenic bacteria.

Download Full-text