Biology subjects

Kulakowski, S.

Publications and source records attributed to Kulakowski, S..

2 recordsLinked to original sources

bioRxiv2025

Biosensor-Driven Strain Engineering Reveals Key Cellular Processes for Maximizing Isoprenol Production in Pseudomonas putida

Synthetic biology tools have accelerated the generation of simple mutants, but combinatorial testing remains challenging. High-throughput methods struggle translating from proof-of-principle molecules to advanced bioproducts. We address this challenge with a biosensor-driven strategy for enhanced isoprenol production in Pseudomonas putida, a key precursor for sustainable aviation fuel and platform chemicals. This biosensor leverages P. putidas native response to short-chain alcohols via a previously uncharacterized hybrid histidine kinase signaling cascade. Refactoring the biosensor for a conditional growth-based selection enabled identification of competing cellular processes with a [~]16,500-member CRISPRi-library. An iterative combinatorial strain engineering approach yielded an integrated P. putida strain producing [~]900 mg/L isoprenol in glucose minimal medium, a 36-fold increase. Ensemble -omics analysis revealed metabolic rewiring, including amino acid accumulation as key drivers of enhanced production. Techno-economic analysis elucidated the path to economic viability and confirmed the benefits of adding amino acids outweigh the additional costs. This study establishes a robust biosensor driven approach for optimizing other heterologous pathways, accelerating microbial cell factory development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/643695v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@533605org.highwire.dtl.DTLVardef@ff98fforg.highwire.dtl.DTLVardef@9592fdorg.highwire.dtl.DTLVardef@1e52993_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Development of Modular Expression and Genome Editing Across Phylogenetically Distinct Diazotrophs

Diazotrophic bacteria can reduce atmospheric nitrogen into ammonia enabling bioavailability of the essential element. Many diazotrophs associate closely with plant roots increasing nitrogen availability, acting as plant growth promoters. These associations have the potential to reduce the need for costly synthetic fertilizers if they could be engineered for agricultural applications. However, despite the importance of diazotrophic bacteria, genetic tools are poorly developed in a limited number of species, in turn narrowing the crops and root microbiomes that can be targeted. Here we report optimized protocols and plasmids to manipulate phylogenetically diverse diazotrophs with the goal of enabling synthetic biology and genetic engineering. Three broad-host-range plasmids can be used across multiple diazotrophs, with the identification of one specific plasmid (containing origin of replication RK2 and a kanamycin resistance marker) showing the highest degree of compatibility across bacteria tested. We then demonstrated modular expression testing seven promoters and eleven ribosomal binding sites using proxy fluorescent proteins. Finally, we tested four small molecule inducible systems to report expression in three diazotrophs and demonstrate genome editing in Klebsiella michiganensis M5al. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/595406v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@ea73a4org.highwire.dtl.DTLVardef@4e540eorg.highwire.dtl.DTLVardef@706557org.highwire.dtl.DTLVardef@10d4c9f_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗