Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 107, Issue 2-3, Pages 663-676Publisher
SPRINGER
DOI: 10.1007/s00253-022-12330-1
Keywords
Malonate transporter; Directed evolution; 3-Hydroxypropionate; Escherichia coli
Categories
Ask authors/readers for more resources
This study improved the transport performance of the MatPQM importer by obtaining MatP variants through directed evolution and a novel enzyme-inhibition-based high throughput screening approach. The best mutant, E103G/S194G/Y218H/L235P/N272S, showed twofold increased transport efficiency compared to the wild-type. Improved binding affinity of the mutant to the ligand was found to be the reason for the enhanced uptake activity of MatPQM. The engineered strain produced 20.08 g/L 3-HP with a yield of 0.87 mol/mol malonate, demonstrating the broad applications of this transporter mutant in microbial biosynthesis.
Our previous study's introduction of the malonic acid assimilation pathway into Escherichia coli enabled biosynthesis of 3-Hydroxypropionate (3-HP) from malonate. However, the relatively low uptake activity of tripartite ATP-independent periplasmic (TRAP) malonic acid transporter (MatPQM) is considered rate-limiting in malonate utilization. Here, to improve the transport performance of this importer, MatP variants were obtained via directed evolution and a novel developed enzyme-inhibition-based high throughput screening approach. This plate chromogenic screening method is based on the fact that malonic acid inhibits both of succinate dehydrogenase activity and further the capability of the reduction of methylene-blue to methylene-white. The best mutant E103G/S194G/Y218H/L235P/N272S showed twofold increased transport efficiency compared to the wild-type. ITC assay and structural analysis revealed that increased binding affinity of the mutant to the ligand was the reason for improved uptake activity of MatPQM. Finally, the engineered strain harboring the evolved mutant produced 20.08 g/L 3-HP with the yield of 0.87 mol/mol malonate in a bioreactor. Therefore, the well-established directed evolution strategy can be regarded as the reference work for other TRAP-type transporters engineering. And, this transporter mutant with enhanced malonic acid uptake activity has broad applications in the microbial biosynthesis of malonyl-CoA-derived valuable compounds in bacteria.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available