Biology subjects

Cho, Y. I.

Publications and source records attributed to Cho, Y. I..

4 recordsLinked to original sources

bioRxiv2026

Confinement considerations for CRISPR toxin-antidote gene drive systems

Gene drives are potentially powerful tools, able to spread throughout target populations. They could be used to modify or suppress disease vectors, invasive species, and agricultural pests. Yet, in many scenarios, confinement of the drive to only a target population is required. Several types of drives are capable of this, among the most promising of which are CRISPR toxin-antidote drives. Though showing good performance in simple models, such drives have not been thoroughly assessed in spatially structured populations. Here we evaluate three modification drive variants with varying levels of confinement. We find that Toxin-Antidote Recessive Embryo (TARE) drive and 2-locus TARE drives can usually spread in connected populations or from a single sufficiently large release. However, they can still be stopped by a migration corridor or by a sufficiently high population density gradient. 1-locus 2-drive TARE, on the other hand, will not be able to spread outside of release areas and indeed will often retreat in the face of wild-type alleles. When these drives arrive at a point source such as a port, only the standard TARE drive has a significant chance of establishing if releases occur at sufficiently high frequency and quantity. However, in models with parental care among a limited number of offspring, TARE drives become more invasive, with lower introduction thresholds. Overall, we find that spatial and other ecological factors can substantially affect the outcome of a confined CRISPR toxin-antidote drive release.

ecology↗

bioRxiv2025

A Phosphopantetheinyl Transferase from Dictyobacter vulcani sp. W12 Expands the Combinatorial Biosynthetic Toolkit

The value of microbial natural product pathways extends beyond the chemicals they produce, as the enzymes they encode can be harnessed as biocatalysts. Microbial type II polyketide synthases (PKSs) are particularly noteworthy, as these enzyme assemblies produce complex polyaromatic pharmacophores. Combinatorial biosynthesis with type II PKSs has been described as a promising route for accessing never-before-seen bioactive molecules, but this potential is stymied in part by the lack of functionally compatible non-cognate proteins across type II PKS systems. Acyl carrier proteins (ACPs) are central to this challenge, as they shuttle reactive intermediates and malonyl building blocks between the other type II PKS domains during biosynthesis. Activating ACPs to their holo state via the phosphopantetheinyl transferase (PPTase)-catalyzed installation of a coenzyme A (CoA)-derived phosphopantetheine (Ppant) arm is critical to effectively study and strategically engineer type II PKSs, but not all ACPs can be activated using conventional PPTases. Here, we report the discovery of a previously unexplored non-actinobacterial PPTase from Dictyobacter vulcani sp. nov. W12 (vulcPPT). We explored its compatibility with both native and non-native ACPs, observing that vulcPPT activated all ACPs tested in this study, including a non-cognate, non-actinobacterial ACP which cannot be activated by the prototypical broad substrate PPTases AcpS and Sfp. Strategic optimization of phosphopantetheinylation reaction conditions increased apo to holo conversion. In addition to identifying a promising new promiscuous PPTase, this work establishes a road map for further investigation of PPTase compatibility and increases access to functional synthase components for use in combinatorial biosynthesis.

biochemistry↗

bioRxiv2023

Strategic engineering unlocks in vitro type II polyketide biosynthesis

Microbial polyketides represent a structurally diverse class of secondary metabolites with medicinally relevant properties and are synthesized by polyketide synthases (PKS). While Type I PKSs are large biosynthetic machineries composed of a single polypeptide chain, type II PKSs are minimally composed of a ketosynthase-chain length factor (KS-CLF) and a phosphopantetheinylated acyl carrier protein (holo-ACP) expressed separately. Although type II PKSs are found throughout the bacterial kingdom, and despite their importance to strategic bioengineering, type II PKSs have not been well-studied in vitro: In cases where the KS-CLF can be accessed via E. coli heterologous expression, the cognate ACPs are not activatable by the broad specificity Bacillus subtilis surfactin-producing phosphopantetheinyl transferase (PPTase) Sfp and, conversely, in systems where the ACP can be activated by Sfp, the corresponding KS-CLF is typically not readily obtained. Here, we report the high-yield heterologous expression of both cyanobacterial Gloeocapsa sp. PCC 7428 minimal type II PKS (gloPKS) components in E. coli, which allowed us to functionally reconstitute this minimal type II PKS in vitro. Initially, neither the cognate PPTase nor Sfp converted gloACP to its active holo state. However, by examining sequence differences between Sfp-compatible and -incompatible ACPs, we identified two conserved residues in gloACP that, when mutated, enabled high-yield phosphopantetheinylation of gloACP by Sfp. Using analogous mutations, other previously Sfp-incompatible type II PKS ACPs from different bacterial phyla were also rendered activatable by Sfp. This demonstrates the generalizability of our approach and breaks down a longstanding barrier to type II PKS studies and the exploration of complex biosynthetic pathways.

biochemistry↗

bioRxiv2021

Engineered chimeras unveil swappable modular features of fatty acid and polyketide synthase acyl carrier proteins

The strategic redesign of microbial biosynthetic pathways is a compelling route to access molecules of diverse structure and function in a potentially environmentally sustainable fashion. The promise of this approach hinges on an improved understanding of acyl carrier proteins (ACPs), which serve as central hubs in biosynthetic pathways. These small, flexible proteins mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural products. Past combinatorial biosynthesis efforts have failed due to incompatible ACP-enzyme pairings. Herein we report the design of chimeric ACPs with features of the actinorhodin polyketide synthase ACP (ACT) and of the E. coli fatty acid synthase (FAS) ACP (AcpP). We evaluate the ability of the chimeric ACPs to interact with the E. coli FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP interacts with FabF but ACT does not, we sought to exchange modular features of ACT with AcpP to confer functionality with FabF. The interactions of chimeric ACPs with FabF were interrogated using sedimentation velocity experiments, surface plasmon resonance analyses, mechanism-based crosslinking assays, and molecular dynamics simulations. Results suggest that the residues guiding AcpP-FabF compatibility and ACT-FabF incompatibility may reside in the loop I, -helix II region. These findings can inform the development of strategic secondary element swaps that expand the enzyme compatibility of ACPs across systems and therefore represent a critical step towards the strategic engineering of unnatural natural products.

biochemistry↗