Biology subjects

Burkart, M.

Publications and source records attributed to Burkart, M..

6 recordsLinked to original sources

bioRxiv2026

Computational Redesign of an Antifreeze Protein Using Deep Learning

Antifreeze proteins (AFPs) found in various cold-adapted organisms inhibit ice growth and are of interest for applications in food products, cryopreservation, agriculture, and materials science. Although high-resolution structures are available for several AFPs, the amino acids required for full antifreeze activity remain incompletely defined, and the development of AFP variants with properties such as enhanced solubility, high expression yield, and improved thermostability may further facilitate applications. Here, we used the deep learning model ProteinMPNN to redesign the globular fish antifreeze protein AFPIII, keeping the previously reported ice-binding residues fixed. We readily obtained sequences confidently predicted to adopt AFPIIIs structure and we selected five designed variants for expression, all of which expressed efficiently in E. coli. Circular dichroism spectroscopy showed that two of these variants retained secondary structure elements consistent with AFPIII, whereas the other three exhibited structural differences. One design was predicted and experimentally confirmed to have increased thermostability. All five variants displayed measurable thermal hysteresis activity. However, none reached the activity of wild-type AFPIII, suggesting that maintaining the currently established set of ice-binding residues is not sufficient to fully preserve this AFPs function; other, unidentified residues can also impact its activity. Our findings highlight the value of deep learning-based protein design methods both for generating AFP variants with desirable properties and for uncovering gaps in existing knowledge of well-characterized AFPs.

biophysics↗

bioRxiv2025

De Novo Protocell Membrane Formation Fueled by Primitive Metabolites

Lipid membranes define cell boundaries, acting as gatekeepers for transport and signaling. A central principle in biology is that all cellular membranes descend from a common ancestral membrane, as they cannot be generated in the absence of preexisting lipid structures. It is thus unclear how the first protocell membranes originated from membrane-less precursors. Here we demonstrate the de novo generation of lipid bilayers in the absence of any preexisting membranes, membrane-bound proteins, or lipid nanostructure templates. Using acetate as a two-carbon precursor, lipid tails are constructed by soluble enzymes and spontaneously conjugate to cysteine backbones, forming diacyl lipids that assemble into vesicles. Pore-forming peptides facilitate precursor transport into vesicles, allowing the continuous generation of new lipids. Formation of glycolipid membranes creates compartments that can maintain proton gradients. The de novo formation of membrane bilayers using primitive chemical building blocks may have been an intermediary step between primitive prebiotic biochemistry and the emergence of cellular life.

synthetic biology↗

bioRxiv2025

Identification and Overexpression of Endogenous Transcription Factors to Enhance Lipid Accumulation in the Commercially Relevant Species Chlamydomonas pacifica

Sustainable low-carbon energy solutions are critical to mitigating global carbon emissions. Algae-based platforms offer potential by converting carbon dioxide into valuable products while aiding carbon sequestration. However, scaling algae cultivation faces challenges like contamination in outdoor systems. Previously, our lab evolved Chlamydomonas pacifica, an extremophile green alga, which tolerates high temperature, pH, salinity, and light, making it ideal for large-scale bioproduct production, including biodiesel. Here, we enhanced lipid accumulation in evolved C. pacifica by identifying and overexpressing key endogenous transcription factors through genome-wide in-silico analysis and in-vivo testing. These factors include Lipid Remodeling Regulator 1 (CpaLRL1), Nitrogen Response Regulator 1 (CpaNRR1), Compromised Hydrolysis of Triacylglycerols 7 (CpaCHT7), and Phosphorus Starvation Response 1 (CpaPSR1). Under nitrogen deprivation, CpaLRL1, CpaNRR1, and CpaCHT7 overexpression enhanced lipid accumulation compared to wildtype. However, CpaPSR1 increased lipid accumulation compared to wildtype in normal media despite causing no effect under nitrogen depravation, highlighting the difference in function based on media conditions. Notably, lipid analysis of CpaPSR1 under normal media conditions revealed a 2.4-fold increase in triglycerides (TAGs) compared to the wild type, highlighting its potential for biodiesel production. This approach provides a framework for transcription factor-focused metabolic engineering in algae, advancing bioenergy and biomaterial production. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/651737v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@8ab995org.highwire.dtl.DTLVardef@19dff1forg.highwire.dtl.DTLVardef@1d865aborg.highwire.dtl.DTLVardef@1fafd12_HPS_FORMAT_FIGEXP M_FIG C_FIG Created with BioRender.com.

systems biology↗

bioRxiv2024

Adaptation to high pressure; insights from the genome of an evolved Escherichia coli strain with increased piezotolerance

Pressure is a key environmental parameter that influences the activity and distribution of microbial life on our planet. Despite its key role there is still not a definitive list of essential genes for microbial adaptation to life under increasing pressure. In this study we used a previously characterized Escherichia coli strain (AN62) evolved to grow at pressure (60 MPa) non-permissive to the parental strain and performed comparative genomics in order to identify the genome-level adaptations that might allowed the observed pressure-adapted phenotype. We identified 18 mutations in total of which 3 mutations were present in both the parental and evolved strain, 3 mutations were only present in the parental strain, and 12 mutations were observed only in the evolved AN62 strain. Among the characterized mutations we observed a point mutation in the acyl carrier protein (acpPV43G). Complementation experiments revealed that the observed V43G mutation in AcpP is responsible for increased levels of cis-vaccenic acid but is not alone responsible for the pressure adapted phenotype. Further molecular dynamics and docking simulations suggested that the V43G mutation promoted stronger binding of the AcpP protein to partner enzymes of the fatty acid biosynthesis pathway involved in fatty acid unsaturation. Data SummaryEscherichia coli reads from the parental and evolved strain have been deposited in the Sequence Read Archive (SRA) under accession number RJNA600359. All software used in the bioinformatic analysis is publicly available. Impact StatementPressure is a key environmental parameter. Two-thirds of our planet is covered by oceans with an average depth of 3800m, which means that the majority of the marine life experiences deep sea conditions. Our results offer a list of gene mutations that could contribute to an improved pressure growth phenotype in Escherichia coli, offering a unique insight on the genome level adaptations that might contribute to high pressure adaptation.

microbiology↗

bioRxiv2021

Chemoenzymatic Generation of Phospholipid Membranes Mediated by Type I Fatty Acid Synthase

The de novo formation of lipid membranes from minimal reactive precursors is a major goal in synthetic cell research. In nature, the synthesis of membrane phospholipids is orchestrated by numerous enzymes, including fatty acid synthases and membrane-bound acyltransferases. However, these enzymatic pathways are difficult to fully reproduce in vitro. As such, the reconstitution of phospholipid membrane synthesis from simple metabolic building blocks remains a challenge. Here, we describe a chemoenzymatic strategy for lipid membrane generation that utilizes a soluble bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA in situ from acetyl-CoA and malonyl-CoA. The fatty acid derivative spontaneously reacts with a cysteine-modified lysophospholipid by native chemical ligation (NCL), affording a non-canonical amidophospholipid that self-assembles into micron-sized membrane-bound vesicles. To our knowledge, this is the first example of reconstituting phospholipid membrane formation directly from acetyl-CoA and malonyl-CoA precursors. Our results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a highly selective bioconjugation reaction provides a biomimetic route for the de novo formation of membrane-bound vesicles.

synthetic biology↗

bioRxiv2019

Gating Mechanism of β-Ketoacyl-ACP Synthases

Formation of carbon-carbon bonds via {beta}-ketoacyl-acyl carrier protein (ACP) synthases (KS), are key reactions during de novo fatty acid and polyketide biosynthesis. KSs recognize multiple ACPs and choreograph ping-pong mechanisms often in an iterative fashion. Therefore, KSs must limit non-productive protein-protein interactions (PPIs) to achieve high degrees of reaction fidelity. To better understand the stereochemical features governing substrate discrimination during ACP*KS PPIs, we determined x-ray crystal structures complemented by molecular dynamic simulations of E. coli AcpP in complex with the elongating KSs, FabF and FabB. Covalently trapped substrate analogs were used to interrogate critical catalytic events accompanying carbon-carbon bond formation revealing a previously unknown gating mechanism during the binding and delivery of acyl-AcpPs. Two active site loops undergo large conformational excursions during this dynamic gating mechanism and are likely evolutionarily conserved features generally in elongating KSs.

biochemistry↗