Mutational analysis in Corynebacterium stationis MFS transporters for improving nucleotide bioproduction

Area of Science:

• Microbial biotechnology
• Molecular biology
• Biochemistry

Background:

• Product secretion from engineered microbial cell factories is crucial for biomanufacturing.
• Nucleotide manufacturing in Corynebacterium stationis utilizes random mutagenesis for extracellular transport, but mechanisms remain unclear.
• Understanding transporter engineering is vital for improving microbial cell factory efficiency.

Purpose of the Study:

• To identify and characterize the nucleotide-exporting major facilitator superfamily (MFS) transporter in C. stationis.
• To elucidate the mechanism behind a hyperactive mutation (G64) in this MFS transporter.
• To provide insights for rational design of efflux transporters in microbial systems.

Main Methods:

• Genome mining to identify the MFS transporter.
• Site-directed mutagenesis to create the G64 hyperactive mutant.
• Structural estimation and molecular dynamics simulations to analyze transporter function.

Main Results:

• The nucleotide-exporting MFS transporter from C. stationis was identified.
• A hyperactive mutation at residue G64 was discovered, enhancing transporter activity.
• Mechanisms of enhanced activity involve improved transmembrane helix interactions and substrate-binding cavity alterations.

Conclusions:

• The study reveals how MFS transporters transition between inward- and outward-facing states to facilitate substrate efflux.
• Findings contribute to the rational design of improved efflux systems for microbial cell factories.
• This research deepens the understanding of nucleotide transport mechanisms in bacteria.