Ganodone, a Bioactive Benzofuran from the Fruiting Bodies of <i>Ganoderma tsugae</i>

Extracts of <i>Ganoderma tsugae</i>, also known as the Hemlock varnish shelf mushroom, and related Reishi mushrooms are well documented in traditional Chinese medicine. Several <i>Ganoderma</i> sp. are currently cultivated for use in coffee, teas, and dietary supplements. We now report on the isolation and characterization of an unprecedented benzofuran, ganodone (<b>1</b>), from the fruiting bodies of mature growth <i>G. tsugae</i>. This discovery provides a key next step in evaluating the active components in their associated herbal supplements

Ganodone, a Bioactive Benzofuran from the Fruiting Bodies of <i>Ganoderma tsugae</i>

Extracts of <i>Ganoderma tsugae</i>, also known as the Hemlock varnish shelf mushroom, and related Reishi mushrooms are well documented in traditional Chinese medicine. Several <i>Ganoderma</i> sp. are currently cultivated for use in coffee, teas, and dietary supplements. We now report on the isolation and characterization of an unprecedented benzofuran, ganodone (<b>1</b>), from the fruiting bodies of mature growth <i>G. tsugae</i>. This discovery provides a key next step in evaluating the active components in their associated herbal supplements

Structure of FD-895 Revealed through Total Synthesis

The total synthesis of FD-895 was completed through a strategy that featured the use of a tandem esterification ring-closing metathesis (RCM) process to construct the 12-membered macrolide and a modified Stille coupling to append the side chain. These studies combined with detailed analysis of all four possible C16–C17 stereoisomers were used to confirm the structure of FD-895 and identify an analog with an enhanced subnanomolar bioactivity

Matching Protein Interfaces for Improved Medium-Chain Fatty Acid Production

Medium-chain fatty acids (MCFAs) are key intermediates in the synthesis of medium-chain chemicals including α-olefins and dicarboxylic acids. In bacteria, microbial production of MCFAs is limited by the activity and product profile of fatty acyl-ACP thioesterases. Here, we engineer a heterologous bacterial medium-chain fatty acyl-ACP thioesterase for improved MCFA production in <i>Escherichia coli</i>. Electrostatically matching the interface between the heterologous medium-chain <i>Acinetobacter baylyi</i> fatty acyl-ACP thioesterase (AbTE) and the endogenous <i>E. coli</i> fatty acid ACP (<i>E. coli</i> AcpP) by replacing small nonpolar amino acids on the AbTE surface for positively charged ones increased secreted MCFA titers more than 3-fold. Nuclear magnetic resonance titration of <i>E. coli</i> ¹⁵N-octanoyl-AcpP with a single AbTE point mutant and the best double mutant showed a progressive and significant increase in the number of interactions when compared to AbTE wildtype. The best AbTE mutant produced 131 mg/L of MCFAs, with MCFAs being 80% of all secreted fatty acid chain lengths after 72 h. To enable the future screening of larger numbers of AbTE variants to further improve MCFA titers, we show that a previously developed G-protein coupled receptor (GPCR)-based MCFA sensor differentially detects MCFAs secreted by <i>E. coli</i> expressing different AbTE variants. This work demonstrates that engineering the interface of heterologous enzymes to better couple with endogenous host proteins is a useful strategy to increase the titers of microbially produced chemicals. Further, this work shows that GPCR-based sensors are producer microbe agnostic and can detect chemicals directly in the producer microbe supernatant, setting the stage for the sensor-guided engineering of MCFA producing microbes

Sulfonyl 3‑Alkynyl Pantetheinamides as Mechanism-Based Cross-Linkers of Acyl Carrier Protein Dehydratase

Acyl carrier proteins (ACPs) play a central role in acetate biosynthetic pathways, serving as tethers for substrates and growing intermediates. Activity and structural studies have highlighted the complexities of this role, and the protein–protein interactions of ACPs have recently come under scrutiny as a regulator of catalysis. As existing methods to interrogate these interactions have fallen short, we have sought to develop new tools to aid their study. Here we describe the design, synthesis, and application of pantetheinamides that can cross-link ACPs with catalytic β-hydroxy-ACP dehydratase (DH) domains by means of a 3-alkynyl sulfone warhead. We demonstrate this process by application to the Escherichia coli fatty acid synthase and apply it to probe protein–protein interactions with noncognate carrier proteins. Finally, we use solution-phase protein NMR spectroscopy to demonstrate that sulfonyl 3-alkynyl pantetheinamide is fully sequestered by the ACP, indicating that the <i>crypto</i>-ACP closely mimics the natural DH substrate. This cross-linking technology offers immediate potential to lock these biosynthetic enzymes in their native binding states by providing access to mechanistically cross-linked enzyme complexes, presenting a solution to ongoing structural challenges

Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions

<div><p>Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga <em>Chlamydomonas reinhardtii</em> (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar <em>in silico</em> binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate <em>in vitro</em> that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated <em>in vivo</em>, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the <em>C. reinhardtii</em> chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes.</p> </div

Thioesterase activity assay.

<p>Activity of plant and algal thioesterases and porcine pancreas type II lipase were determined by monitoring the hydrolysis of para-nitrophenylhexanoate for 16 hours at 30°C. (<i>A</i>) pH 7, TEs expressed in <i>E. coli</i>; (<i>B</i>) pH 8, TEs expressed in <i>E. coli</i>; (<i>C</i>) pH 7, TEs expressed in <i>C. reinhardtii</i>; (<i>D</i>) pH 8, TEs expressed in <i>C. reinhardtii</i>.</p

Fatty acid analysis of <i>C. reinhardtii</i> strains expressing thioesterases.

<p>Fatty acid composition of Cr strains was determined by GC/MS analysis and comparison to authentic standards. Peak areas were integrated and compared to an external standard for quantification. Bar graphs denote abundances of (<i>A</i>) Myristic acid (14:0), (<i>B</i>) Palmitic acid (16:0), and (<i>C</i>) Oleic acid (18:1), and labels on the Y-axis correspond to the percentages of these fatty acids of the total fatty acid content. (<i>D</i>) Full GC/MS chromatograms of Cr strains expressing CrTE (red), UcTE (Blue) and wildtype CrTE (black). Three separate cultures of each strain were analyzed for fatty acid content and composition, and data were recorded and averaged with a mean deviation of 7% in each experiment. Statistical analyses were performed using SPSS (v13.0), and for all data analysis, a <i>p</i>-value<0.5 was considered statistically significant.</p

Thioesterase modeling, docking of ACP-TE protein-protein interactions, and blind substrate docking of fatty acid substrate to <i>C. reinhardtii</i> TE.

<p>(<i>A</i>) Docking of CrTE (grey) with Cr-cACP (blue) showing a <10 Å distance between Cr-cACP Ser₄₃ (orange) and the active site Cys₃₀₆His₂₇₀Asn₂₆₈ triad (magenta) of CrTE. (<i>B</i>) Docked complex of CrTE (grey) and ChTE (yellow) showing similar binding modes of Cr-cACP to both plant and algal thioesterases. (<i>C</i>) Surface representation of blind docking of stearyl-4′-phosphopantetheine to CrTE showing the thioester bond of the substrate in close proximity to the TE active site and stearate in the binding tunnel of CrTE.</p

Schematic of activity-based crosslinking between CrACP and TEs.

<p>(<i>A</i>) <i>Apo</i>-CrACP is formed by treating <i>holo</i>-CrACP with ACP hydrolase from <i>P. aeruginosa </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042949#pone.0042949-Murugan1" target="_blank">[36]</a>, removing the pantetheine moiety from the conserved serine of CrACP. Presence of <i>apo</i>-CrACP is confirmed using a one-pot fluorescent labeling method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042949#pone.0042949-Worthington3" target="_blank">[37]</a>, detected by visualization of a resulting SDS-PAGE gel at 365 nm. (+) formation of fluorescent <i>crypto</i>-CrACP; (−) control reaction in which fluorescent pantetheine analogue <b>1</b> was omitted. (<i>B</i>) Activity-based crosslinking scheme. <i>Apo</i>-CrACP is incubated with <b>2</b> or <b>3</b>, Sfp, ATP, CoA-A, CoA-D, and CoA-E to generate the corresponding <i>crypto</i>-CrACPs <b>4</b> and <b>5</b>. Upon incubation of <i>crypto</i>-CrACP with TE, protein-protein interactions trigger a site-specific covalent crosslinking reaction with the chloroacrylamide in <b>4</b> or the α-bromoamide in <b>5</b>, forming an ACP-TE crosslinked complex. (<i>C</i>) Predicted enzymatic mechanism of the hydrolytic release of a fatty acid by CrTE using a Cys-Asn-His catalytic triad. (<i>D</i>) Mechanism of irreversible crosslink between TE and <i>crypto</i>-CrACP containing a reactive bromide on the carbon α to the site of nucleophilic attack by the TE.</p