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.

Interfacial plasticity facilitates high reaction rate of E. coli FAS malonyl-CoA:ACP transacylase, FabD

Fatty 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 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.

Comparative Genomics Underlines Multiple Roles of Profftella, an Obligate Symbiont of Psyllids: Providing Toxins, Vitamins, and Carotenoids

The Asian citrus psyllid Diaphorina citri (Insecta: Hemiptera: Psylloidea), a serious pest of citrus species worldwide, harbors vertically transmitted intracellular mutualists, Candidatus Profftella armatura (Profftella_DC, Gammaproteobacteria: Burkholderiales) and Candidatus Carsonella ruddii (Carsonella_DC, Gammaproteobacteria: Oceanospirillales). Whereas Carsonella_DC is a typical nutritional symbiont, Profftella_DC is a unique defensive symbiont with organelle-like features, including intracellular localization within the host, perfect infection in host populations, vertical transmission over evolutionary time, and drastic genome reduction down to much less than 1 Mb. Large parts of the 460-kb genome of Profftella_DC are devoted to genes for synthesizing a polyketide toxin; diaphorin. To better understand the evolution of this unusual symbiont, the present study analyzed the genome of Profftella_Dco, a sister lineage to Profftella_DC, using Diaphorina cf. continua, a host psyllid congeneric with D. citri. The genome of coresiding Carsonella (Carsonella_Dco) was also analyzed. The analysis revealed nearly perfect synteny conservation in these genomes with their counterparts from D. citri. The substitution rate analysis further demonstrated genomic stability of Profftella which is comparable to that of Carsonella. Profftella_Dco and Profftella_DC shared all genes for the biosynthesis of diaphorin, hemolysin, riboflavin, biotin, and carotenoids, underlining multiple roles of Profftella, which may contribute to stabilizing symbiotic relationships with the host. However, acyl carrier proteins were extensively amplified in polyketide synthases DipP and DipT for diaphorin synthesis in Profftella_Dco. This level of acyl carrier protein augmentation, unprecedented in modular polyketide synthases of any known organism, is not thought to influence the polyketide structure but may improve the synthesis efficiency.

Acyltransferase-mediated selection of the length of the fatty acyl chain and of the acylation site governs activation of bacterial RTX toxins

In a wide range of organisms, from bacteria to humans, numerous proteins have to be posttranslationally acylated to become biologically active. Bacterial repeats in toxin (RTX) cytolysins form a prominent group of proteins that are synthesized as inactive protoxins and undergo posttranslational acylation on ε-amino groups of two internal conserved lysine residues by co-expressed toxin-activating acyltransferases. Here, we investigated how the chemical nature, position, and number of bound acyl chains govern the activities of Bordetella pertussis adenylate cyclase toxin (CyaA), Escherichia coli α-hemolysin (HlyA), and Kingella kingae cytotoxin (RtxA). We found that the three protoxins are acylated in the same E. coli cell background by each of the CyaC, HlyC, and RtxC acyltransferases. We also noted that the acyltransferase selects from the bacterial pool of acyl–acyl carrier proteins (ACPs) an acyl chain of a specific length for covalent linkage to the protoxin. The acyltransferase also selects whether both or only one of two conserved lysine residues of the protoxin will be posttranslationally acylated. Functional assays revealed that RtxA has to be modified by 14-carbon fatty acyl chains to be biologically active, that HlyA remains active also when modified by 16-carbon acyl chains, and that CyaA is activated exclusively by 16-carbon acyl chains. These results suggest that the RTX toxin molecules are structurally adapted to the length of the acyl chains used for modification of their acylated lysine residue in the second, more conserved acylation site.

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

Fatty 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.