Broad Substrate Specificity of the Loading Didomain of the Lipomycin Polyketide Synthase

LipPks1, a polyketide synthase subunit of the lipomycin synthase, is believed to catalyze the polyketide chain initiation reaction using isobutyryl-CoA as a substrate, followed by an elongation reaction with methylmalonyl-CoA to start the biosynthesis of antibiotic α-lipomycin in <i>Streptomyces aureofaciens</i> Tü117. Recombinant LipPks1, containing the thioesterase domain from the 6-deoxyerythronolide B synthase, was produced in <i>Escherichia coli</i>, and its substrate specificity was investigated <i>in vitro</i>. Surprisingly, several different acyl-CoAs, including isobutyryl-CoA, were accepted as the starter substrates, while no product was observed with acetyl-CoA. These results demonstrate the broad substrate specificity of LipPks1 and may be applied to producing new antibiotics

Acylation and transacylation activities of WT, H640A, S641A, and H640A+S641A.

<p>A) Transacylation activity as observed by high-resolution LC/QTOFMS. Data for the variants (WT, H640A, S641A, H640A+S641A) incubated with malonyl-CoA and a wildtype negative control without malonyl-CoA are shown (WT – Mal-CoA). B) Formation of malonyl-AT complex for wildtype (monoisotopic peptide m/z  =  1000.9903, z  =  4) and H640A (monoisotopic peptide m/z  =  984.4849, z  =  4) as observed by high-resolution LC/QTOFMS. Data for wildtype and H640A (WT, H640A) incubated with malonyl-CoA as well as a wildtype negative control without malonyl-CoA (WT – Mal-CoA) are shown. The mass for each chromatogram is shown in parenthesis to the right. Additional details on chromatogram preparation in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109421#pone.0109421.s001" target="_blank">Methods S1</a>.</p

Understanding the Role of Histidine in the GHSxG Acyltransferase Active Site Motif: Evidence for Histidine Stabilization of the Malonyl-Enzyme Intermediate

<div><p>Acyltransferases determine which extender units are incorporated into polyketide and fatty acid products. The ping-pong acyltransferase mechanism utilizes a serine in a conserved GHSxG motif. However, the role of the conserved histidine in this motif is poorly understood. We observed that a histidine to alanine mutation (H640A) in the GHSxG motif of the malonyl-CoA specific yersiniabactin acyltransferase results in an approximately seven-fold higher hydrolysis rate over the wildtype enzyme, while retaining transacylation activity. We propose two possibilities for the reduction in hydrolysis rate: either H640 structurally stabilizes the protein by hydrogen bonding with a conserved asparagine in the ferredoxin-like subdomain of the protein, or a water-mediated hydrogen bond between H640 and the malonyl moiety stabilizes the malonyl-O-AT ester intermediate.</p></div

Acyltransferase reaction mechanism and sequence logos.

<p>A) AT reaction mechanism: ATs utilize a ping-pong mechanism to transfer acyl groups from CoA to ACP (for most ATs, R₁ = H or CH₃). Hydrolysis, shown in the bottom branch is a competing, unproductive reaction. B) Sequence logo for the motif containing the active site serine of pfam PF00698, which encompasses ATs from fatty acid and polyketide biosynthesis. C) Sequence logo for pfam PF01734, which are evolutionarily related phospholipases. Logos created using Skyline <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109421#pone.0109421-Wheeler1" target="_blank">[12]</a>.</p

Crystal structure with malonate of the acyltransfserase from DynE8, an iterative type I PKS.

<p>Residues involved in catalysis are labeled and shown as sticks. The hydrogen bonding water highlighted in the text is shown as a sphere. Figure 3 was prepared using Pymol from the PDB entry 4AMP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109421#pone.0109421-Liew1" target="_blank">[11]</a>.</p

Hydrolysis rates for yersiniabactin AT mutants at a concentration of 35 µM malonyl-CoA.

<p>n.d.  =  below limit of detection; error bars are the standard deviation of 3 replicates.</p><p>Hydrolysis rates for yersiniabactin AT mutants at a concentration of 35 µM malonyl-CoA.</p

Alteration of Polyketide Stereochemistry from <i>anti</i> to <i>syn</i> by a Ketoreductase Domain Exchange in a Type I Modular Polyketide Synthase Subunit

Polyketide natural products have broad applications in medicine. Exploiting the modular nature of polyketide synthases to alter stereospecificity is an attractive strategy for obtaining natural product analogues with altered pharmaceutical properties. We demonstrate that by retaining a dimerization element present in LipPks1+TE, we are able to use a ketoreductase domain exchange to alter α-methyl group stereochemistry with unprecedented retention of activity and simultaneously achieve a novel alteration of polyketide product stereochemistry from <i>anti</i> to <i>syn</i>. The substrate promiscuity of LipPks1+TE further provided a unique opportunity to investigate the substrate dependence of ketoreductase activity in a polyketide synthase module context

Engineering a Polyketide Synthase for <i>In Vitro</i> Production of Adipic Acid

Polyketides have enormous structural diversity, yet polyketide synthases (PKSs) have thus far been engineered to produce only drug candidates or derivatives thereof. Thousands of other molecules, including commodity and specialty chemicals, could be synthesized using PKSs if composing hybrid PKSs from well-characterized parts derived from natural PKSs was more efficient. Here, using modern mass spectrometry techniques as an essential part of the design–build–test cycle, we engineered a chimeric PKS to enable production one of the most widely used commodity chemicals, adipic acid. To accomplish this, we introduced heterologous reductive domains from various PKS clusters into the borrelidin PKS’ first extension module, which we previously showed produces a 3-hydroxy-adipoyl intermediate when coincubated with the loading module and a succinyl-CoA starter unit. Acyl-ACP intermediate analysis revealed an unexpected bottleneck at the dehydration step, which was overcome by introduction of a carboxyacyl-processing dehydratase domain. Appending a thioesterase to the hybrid PKS enabled the production of free adipic acid. Using acyl-intermediate based techniques to “debug” PKSs as described here, it should one day be possible to engineer chimeric PKSs to produce a variety of existing commodity and specialty chemicals, as well as thousands of chemicals that are difficult to produce from petroleum feedstocks using traditional synthetic chemistry

<i>In Vitro</i> Analysis of Carboxyacyl Substrate Tolerance in the Loading and First Extension Modules of Borrelidin Polyketide Synthase

The borrelidin polyketide synthase (PKS) begins with a carboxylated substrate and, unlike typical decarboxylative loading PKSs, retains the carboxy group in the final product. The specificity and tolerance of incorporation of carboxyacyl substrate into type I PKSs have not been explored. Here, we show that the first extension module is promiscuous in its ability to extend both carboxyacyl and non-carboxyacyl substrates. However, the loading module has a requirement for substrates containing a carboxy moiety, which are not decarboxylated <i>in situ</i>. Thus, the loading module is the basis for the observed specific incorporation of carboxylated starter units by the borelidin PKS

Comprehensive <i>in Vitro</i> Analysis of Acyltransferase Domain Exchanges in Modular Polyketide Synthases and Its Application for Short-Chain Ketone Production

Type I modular polyketide synthases (PKSs) are polymerases that utilize acyl-CoAs as substrates. Each polyketide elongation reaction is catalyzed by a set of protein domains called a module. Each module usually contains an acyltransferase (AT) domain, which determines the specific acyl-CoA incorporated into each condensation reaction. Although a successful exchange of individual AT domains can lead to the biosynthesis of a large variety of novel compounds, hybrid PKS modules often show significantly decreased activities. Using monomodular PKSs as models, we have systematically analyzed the segments of AT domains and associated linkers in AT exchanges <i>in vitro</i> and have identified the boundaries within a module that can be used to exchange AT domains while maintaining protein stability and enzyme activity. Importantly, the optimized domain boundary is highly conserved, which facilitates AT domain replacements in most type I PKS modules. To further demonstrate the utility of the optimized AT domain boundary, we have constructed hybrid PKSs to produce industrially important short-chain ketones. Our <i>in vitro</i> and <i>in vivo</i> analysis demonstrated production of predicted ketones without significant loss of activities of the hybrid enzymes. These results greatly enhance the mechanistic understanding of PKS modules and prove the benefit of using engineered PKSs as a synthetic biology tool for chemical production