Abstract
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.
Key points
• We reported directed evolution of a TRAP-type malonic acid transporter.
• We found the enhanced malonate uptake activity of mutant lies in improved affinity.
• We enhanced 3-HP bioproduction with high yield by employing the best mutant.
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Funding
This work was supported by grants from the National Natural Science Foundation of China (22278233,31860011), the Natural Science Foundation of Shandong Province, China (ZR2020MC009), the “First class grassland science discipline” program in Shandong Province. The authors would also like to thank the “Laboratory for Agricultural Molecular Biology of Qingdao Agricultural University” for providing laboratory apparatus.
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BL and JY designed the study. BL, XZ, CM, and LW performed the study. BL and XZ analyzed the data. BL, XZ, and JY wrote the paper. All authors read and approved the final manuscript.
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Liang, B., Zhang, X., Meng, C. et al. Directed evolution of tripartite ATP-independent periplasmic transporter for 3-Hydroxypropionate biosynthesis. Appl Microbiol Biotechnol 107, 663–676 (2023). https://doi.org/10.1007/s00253-022-12330-1
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DOI: https://doi.org/10.1007/s00253-022-12330-1