Biology subjects

Burkart, M. D.

Publications and source records attributed to Burkart, M. D..

5 recordsLinked to original sources

bioRxiv2025

Microplastic pollution induces algae blooms in experimental ponds but bioplastics are less harmful

An ever-growing sea of plastic waste permeates even the most remote ecosystems; however, its ecological impact is unclear. Less persistent bioplastic alternatives are available but also have unknown environmental effects. We conducted a three-month experiment exposing plankton in experimental ponds to 10 concentrations of three different thermoplastic polyurethane microplastics, including two biodegradable bioplastics. Algal blooms with dense chlorophyll occurred consistently at high concentrations of the petroleum-derived thermoplastic polyurethane, but only occasionally with the two bioplastics. Herbivorous zooplankton density was strongly reduced by typical thermoplastic polyurethane and only weakly by bioplastics, therefore the effect on algae is at least partly due to reductions in top-down grazing pressure. Microbial communities exhibited compositional shifts in response to all three plastic types, with petroleum-derived plastic associated with the most pronounced differences across both prokaryotic and eukaryotic domains. Our results show that plastic pollution may contribute to the growing global problems of eutrophication, coastal hypoxia and harmful algae blooms, and that biodegradable plastics may have smaller environmental footprints.

ecology↗

bioRxiv2025

Sustainable production of plastic-degrading enzymes in Chlamydomonas pacifica

The discovery of a new extremophile alga, Chlamydomonas pacifica, provides an opportunity to expand on heterologous protein expression beyond the traditional Chlamydomonas reinhardtii. C. pacifica is a unicellular extremophile capable of surviving at high pH, high temperatures, and high salinity. These various growth conditions allow C. pacifica to outcompete any invading contaminants in open-air environments. Developing this novel species as a platform for recombinant protein production could significantly advance commercial microalgal recombinant protein production. We have previously shown that C. reinhardtii can secrete a plastic-degrading enzyme: a PETase known as PHL7. This PETase is capable of cleaving ester bonds and has been used commercially for the degradation of PET plastics. However, the expression of such an enzyme has yet to be done in open raceway ponds and on a large scale. Here, we describe the culturing of PHL7 transgenic C. pacifica strain in three 80L raceway ponds and the measurements of recombinant enzymatic expression and activity found in the culture media. Our work provides proof of concept that this new organism can produce functional PHL7 enzymes in addition to producing the valuable components that inherently exist in the C. pacifica algae biomass. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/654053v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@10065a3org.highwire.dtl.DTLVardef@110c456org.highwire.dtl.DTLVardef@1c9efc6org.highwire.dtl.DTLVardef@9ceea1_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2022

Mechanism-based crosslinking probes capture E. coli ketosynthase FabB in conformationally-distinct catalytic states.

Ketosynthases (KS) catalyse essential carbon-carbon bond forming reactions in fatty acid biosynthesis using a two-step, ping-pong reaction mechanism. In E. coli, there are two homodimeric elongating KSs, FabB and FabF, both of which possess overlapping substrate selectivity. However, FabB is essential for the biosynthesis of unsaturated fatty acids (UFAs) required for cell survival in the absence of exogenous UFAs. Additionally, FabB has reduced activity towards substrates longer than 14 carbons, whereas FabF efficiently catalyses the elongation of saturated C14 and unsaturated C16:1 acyl-acyl carrier protein (ACP) complexes. In this study, we solved two crosslinked crystal structures of FabB in complex with ACPs functionalized with long-chain fatty acid crosslinking probes that approximate catalytic steps. Both homodimeric structures possess asymmetric substrate binding pockets, suggestive of cooperative relationships between the two FabB monomers when engaged with C14 and C16 acyl chains. In addition, these structures capture an unusual rotamer of the active site gating residue, F392, potentially representative of the catalytic state prior to substrate release. These structures demonstrate the utility of mechanism-based crosslinking methods to capture and elucidate at near atomic resolution conformational transitions accompanying KS-mediated catalysis. SynopsisCrystal structures of KS-ACP crosslinked complex elucidate chain length preference and substrate processing mechanism of E. coli FabB.

biochemistry↗

bioRxiv2021

Structure and mechanistic analyses of the gating mechanism of elongating ketosynthases

Ketosynthases (KSs) catalyze carbon-carbon bond forming reactions in fatty acid synthases (FASs) and polyketide synthases (PKSs). KSs utilize a two-step ping pong kinetic mechanism to carry out an overall decarboxylative thio-Claisen condensation that can be separated into the transacylation and condensation reactions. In both steps, an acyl carrier protein (ACP) delivers thioester tethered substrates to the active sites of KSs. Therefore, protein-protein interactions (PPIs) and KS-mediated substrate recognition events are required for catalysis. Recently, crystal structures of Escherichia coli elongating type II FAS KSs, FabF and FabB, in complex with E. coli ACP, AcpP, revealed distinct conformational states of two active site KS loops. These loops were proposed to operate via a gating mechanism to coordinate substrate recognition and delivery followed by catalysis. Here we interrogate this proposed gating mechanism by solving two additional high-resolution structures of substrate engaged AcpP-FabF complexes, one of which provides the missing AcpP-FabF gate-closed conformation. Clearly defined interactions of one of these active site loops with AcpP are present in both the open and closed conformations, suggesting AcpP binding triggers or stabilizes gating transitions, further implicating PPIs in carrier protein-dependent catalysis. We functionally demonstrate the importance of gating in the overall KS condensation reaction and provide experimental evidence for its role in the transacylation reaction. Furthermore, we evaluate the catalytic importance of these loops using alanine scanning mutagenesis and also investigate chimeric FabF constructs carrying elements found in type I PKS KS domains. These findings broaden our understanding of the KS mechanism which advances future engineering efforts in both FASs and evolutionarily related PKSs.

biochemistry↗

bioRxiv2020

Structural 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.

biochemistry↗