Biology subjects

Carruthers, D. N.

Publications and source records attributed to Carruthers, D. N..

2 recordsLinked to original sources

bioRxiv2025

Predictive genome-wide CRISPR-mediated gene downregulation for enhanced bioproduction

CRISPR interference (CRISPRi) has emerged as a valuable tool for redirecting metabolic flux to enhance bioproduction. However, conventional approaches for identifying target genes for CRISPRi-mediated downregulation have largely relied on heuristic methods and trial and error, which are labor-intensive and time-consuming. Additionally, the ability to achieve multigene knockdowns is limited by the constraints of molecular cloning techniques required for building multiplexed CRISPRi systems. In this study, we describe two novel methodologies FluxRETAP (Flux-REaction TArget Prioritization, a Genome-Scale Modeling Technique) and VAMMPIRE (a Versatile Assembly Method for MultiPlexing CRISPRi-mediated downREgulation). FluxRETAP accurately identified gene targets whose knockdown led to substantial increase of isoprenol titers, outperforming traditional heuristic selection. The use of VAMMPIRE enabled accurate and position-independent assembly of CRISPRi constructs containing up to five sgRNA arrays. The integration of FluxRETAP and VAMMPIRE has the potential to advance metabolic engineering by rapidly identifying CRISPRi-mediated knockdowns and knockdown combinations that enhance bioproduction titers, with potential applicability to other microbial systems.

synthetic biology↗

bioRxiv2020

Optimized gene expression from bacterial chromosome by high-throughput integration and screening

Chromosomal integration of recombinant genes is desirable compared to expression from plasmids due to increased stability, reduced cell-to-cell variability, and the elimination of antibiotics for plasmid maintenance. Here, we present a new approach for tuning pathway gene expression levels via random integrations and high-throughput screening. We demonstrate multiplexed gene integration and expression-level optimization for isobutanol production in Escherichia coli. The integrated strains could, with significantly lower expression levels than plasmid-based expression, produce high titers (10.0 {+/-} 0.9 g/L isobutanol in 48 h) and yields (69 % of the theoretical maximum). Close examination of pathway expression in the top-performing, as well as other isolates, reveals the complexity of cellular metabolism and regulation, underscoring the need for precise optimization while integrating pathway genes into the chromosome. This new method for multiplexed pathway gene integration and expression optimization could be readily extended to a wide range of pathways and chassis to create robust and efficient production strains.

synthetic biology↗