Biology subjects

Petzold, C.

Publications and source records attributed to Petzold, C..

6 recordsLinked to original sources

bioRxiv2026

Generative Design of New-to-nature Biosynthetic Assembly Lines with Genomic Language Modeling

Reprogramming biosynthetic assembly lines can extend biosynthesis beyond the chemical space explored by nature. However, this remains difficult because assembly-line function depends on coordinated interactions across large multidomain enzymes. Here, we couple gLM2, a genomic language model trained on metagenomic sequences, with discrete diffusion and domain-level conditioning to enable generative design and optimization of biosynthetic gene clusters. We apply this approach to a chimeric type I polyketide synthase (PKS) engineered to produce {delta}-valerolactam, a molecule not naturally synthesized by PKSs. Through iterative redesign of two multi-domain regions in the context of the full PKS sequence, gLM2 progressively improved {delta}-valerolactam production, yielding variants with up to 9.4-fold higher titer than the starting enzyme. Together, these results demonstrate that evolutionary sequence information can be learned and applied to complex, multi-domain enzyme design problems, expanding biosynthetic assembly lines to produce molecules outside their natural biosynthetic repertoire.

bioengineering↗

bioRxiv2024

Functional plasticity of HCO3- uptake and CO2 fixation in Cupriavidus necator H16

Uptake and fixation of CO2 are central to strategies for CO2-based biomanufacturing. Cupriavidus necator H16 has emerged as a promising industrial host for this purpose. Despite its prominence, the ability to engineer C. necator inorganic carbon uptake and fixation is underexplored. Here, we test the role of endogenous and heterologous genes on C. necator inorganic carbon metabolism. Deletion of one of the four carbonic anhydrases in C. necator, {beta}-carbonic anhydrase can, had the most deleterious effect on C. necator autotrophic growth. Replacement of this native uptake system with several classes of dissolved inorganic carbon (DIC) transporters from Cyanobacteria and chemolithoautotrophic bacteria recovered autotrophic growth and supported higher cell densities compared to wild-type (WT) C. necator in saturating CO2 in batch culture. Several heterologous strains with Halothiobacillus neopolitanus DAB2 (hnDAB2) expressed from the chromosome in combination with diverse rubisco homologs grew in CO2 equally or better than the wild-type strain. Our experiments suggest that the primary role of Can carbonic anhydrase during autotrophic growth is for bicarbonate accumulation to support anaplerotic metabolism, and an array of DIC transporters can complement this function. This work demonstrates flexibility in HCO3- uptake and CO2 fixation in C. necator, providing new pathways for CO2-based biomanufacturing. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/593039v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@83c601org.highwire.dtl.DTLVardef@13ca34forg.highwire.dtl.DTLVardef@5b7f8org.highwire.dtl.DTLVardef@1710e48_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Sample preparation and data collection for Serial Block Face Scanning Electron Microscopy of Mammalian Cell Monolayers

Volume electron microscopy encompasses a set of electron microscopy techniques that can be used to examine the ultrastructure of biological tissues and cells in three dimensions. Two block face techniques, focussed ion beam scanning electron microscopy (FIB-SEM) and serial block face scanning electron microscopy (SBF-SEM) have often been used to study biological tissue samples. More recently, these techniques have been adapted to in vitro tissue culture samples. Here we describe detailed protocols for two sample embedding methods for in vitro tissue culture cells intended to be studied using SBF-SEM. The first protocol focuses on cell pellet embedding and the second on en face embedding. En face embedding can be combined with light microscopy, and this CLEM workflow can be used to identify specific biological events in a light microscope, which can then be imaged using SBF-SEM. We systematically outline the steps necessary to fix, stain, embed and image adherent tissue culture cell monolayers by SBF-SEM. In addition to sample preparation, we discuss optimization of parameters for data collection. We highlight the challenges and key steps of sample preparation, and the consideration of imaging variables that will facilitate the acquisition of high quality datasets. Users experienced with electron microscopy sample preparation methodology will be able to complete this protocol in 10-11 days from initial seeding of cells in tissue culture to image acquisition.

cell biology↗

bioRxiv2023

3D reconstructions of parasite development and the intracellular niche of the microsporidian pathogen E. intestinalis

Microsporidia are an early-diverging group of fungal pathogens that infect a wide range of hosts. Several microsporidian species infect humans, and infections can lead to fatal disease in immunocompromised individuals. As obligate intracellular parasites with highly reduced genomes, microsporidia are dependent on metabolites from their hosts for successful replication and development. Our knowledge of how microsporidian parasites develop inside the host remains rudimentary, and our understanding of the intracellular niche occupied by microsporidia has thus far relied largely on 2D TEM images and light microscopy. Here, we use serial block face scanning electron microscopy (SBF-SEM) to capture 3D snapshots of the human-infecting microsporidian, Encephalitozoon intestinalis, within host cells. We track the development of E. intestinalis through its life cycle, which allows us to propose a model for how its infection organelle, the polar tube, is assembled de novo in each developing spore. 3D reconstructions of parasite-infected cells provide insights into the physical interactions between host cell organelles and parasitophorous vacuoles, which contain the developing parasites. The host cell mitochondrial network is substantially remodeled during E. intestinalis infection, leading to mitochondrial fragmentation. SBF-SEM analysis shows changes in mitochondrial morphology in infected cells, and live-cell imaging provides insights into mitochondrial dynamics during infection. Together, our data provide insights into parasite development, polar tube assembly, and microsporidia-induced mitochondrial remodeling in the host cell.

microbiology↗

bioRxiv2019

Human antiviral protein MxA forms novel metastable membrane-less cytoplasmic condensates exhibiting rapid reversible \"crowding\"-driven phase transitions

Phase-separated biomolecular condensates of proteins and nucleic acids form functional membrane-less organelles in the mammalian cell cytoplasm and nucleus. We report that the interferon (IFN)-inducible human \"myxovirus resistance protein A\" (MxA) forms membrane-less metastable condensates in the cytoplasm. Light and electron microscopy studies revealed that transient expression of HA- or GFP-tagged MxA in Huh7, HEK293T or Cos7 cells, or exposure of Huh7 cells to IFN-2a led to the appearance of MxA in the cytoplasm in membrane-less variably-sized spherical or irregular bodies, in filaments and even a reticulum. 1,6-Hexanediol treatment led to rapid disassembly of these condensates; however, FRAP revealed a relative rigidity with a mobile fraction of only 0.24{+/-}0.02 within condensates. In vesicular stomatitis virus (VSV)-infected Huh7 cells, the nucleocapsid (N) protein, which participates in forming phase-separated viral structures, associated with GFP-MxA condensates. Remarkably, the cytoplasmic GFP-MxA condensates disassembled within 1-3 min of exposure of cells to hypotonic medium (40-50 milliosmolar) and reassembled within 0.5-2 min of re-exposure of cells to isotonic medium (310-325 milliosmolar) through multiple cycles. Mechanistically, the extent of cytoplasmic \"crowding\" regulated this phase-separation process. GFP-MxA condensates also included the DNA sensor protein cyclic GMP-AMP synthase (cGAS), another protein known to be associated with liquid-like condensates. Functionally, GFP-MxA expression inhibited DNA/cGAS-responsive ISG54-luciferase activity but enhanced relative inducibility of ISG54-luc by IFN-, revealing a physical separation between condensate- and cytosol-based signaling pathways in the cytoplasm. Taken together, the data reveal a new aspect of the cell biology of MxA in the cell cytoplasm.\n\nImportanceThe human interferon-inducible \"myxovirus resistance protein A\" (MxA), which displays antiviral activity against several RNA and DNA viruses, exists in the cytoplasm in phase-separated membrane-less metastable condensates of variably-sized spherical or irregular bodies, in filaments and even in a reticulum. MxA condensate formation appeared necessary but not sufficient for antiviral activity. Remarkably, MxA condensates showed the unique property of rapid (within 1-3 min) reversible disassembly and reassembly in intact cells exposed sequentially to hypotonic and isotonic conditions Mechanistically, these phase transitions were regulated by the extent of cytoplasmic \"crowding.\" Moreover, GFP-MxA condensates included the DNA sensor protein cyclic GMP-AMP synthase (cGAS). Functionally, GFP-MxA expression inhibited DNA/cGAS-responsive ISG54-luciferase activity but enhanced inducibility of ISG54-luc by IFN-, revealing a biological distinction between condensate- and cytosol-based signaling pathways. Since intracellular edema and ionic changes are hallmarks of cytopathic viral effects, the rapid hypotonicity-driven disassembly of MxA condensates may modulate MxA.function during virus infection.

cell biology↗

bioRxiv2018

Methyl ketone production by Pseudomonas putida is enhanced by plant-derived amino acids

Plant biomass is an attractive source of renewable carbon for conversion to biofuels and bio-based chemicals. Conversion strategies often use a fraction of the total biomass, focusing on sugars from cellulose and hemicellulose. Strategies that use plant components such as plant-derived aromatics and amino acids have the potential to improve the efficiency of overall biomass conversion. Pseudomonas putida is a promising host for biomass conversion for its ability to metabolize a wide variety of organic compounds, including aromatics derived from lignin. P. putida was engineered to produce medium chain methyl ketones, which are promising diesel blendstocks and potential platform chemicals, from glucose and lignin-related aromatics, 4-hydroxybenzoate (4-HB) and protocatechuate (PCA). Unexpectedly, P. putida methyl ketone production was enhanced 2-to 5-fold compared to sugar controls when Arabidopsis thaliana hydrolysates derived from engineered plants that overproduce 4-HB and PCA, while E. coli production was lowered in these hydrolysates. This enhancement was more pronounced (~7-fold increase) with hydrolysates derived from non-engineered switchgrass (Panicum virgatum L.) suggesting it did not arise from overproduction of 4-HB and PCA. Global proteomic analysis of the methyl ketone-producing P. putida suggested that plant-derived amino acids may be the source of this enhancement. Mass spectrometry-based measurements of plant-derived amino acids demonstrated a high correlation between methyl ketone production and amino acid concentration in plant hydrolysates. Amendment of glucose-containing minimal media with a defined mixture of amino acids similar to those found in the hydrolysates studied led to a 9-fold increase in methyl ketone titer (1.1 g/L).

bioengineering↗