A high-throughput screening method for decarboxylase mutants with efficient carbon fixing based on Biomek i7 automated workstation

Abstract:

[Background] 2,3-dihydroxybenzoic acid decarboxylase (2,3-DHBD) can catalyze the carboxylation of CO₂ with phenolic compounds to generate aromatic carboxylic acids, providing a new strategy for the synthesis of value-added chemicals from fixed CO₂. However, its application is limited by the low conversion efficiency. [Objective] To improve the catalytic efficiency of 2,3-DHBD, we established a high-throughput screening method based on the Biomek i7 automated workstation to achieve efficient screening of benzoic acid decarboxylases with high carboxylation activity, providing a method for enzyme engineering. [Methods] First, 508 clones were obtained by targeted hemi-saturation mutagenesis of the 2,3-DHBD from Aspergillus oryzae (2,3-DHBD_Ao). Then, mutant library construction, clone selection, culture, and enzyme activity assay were performed based on Biomek i7 automated workstation to achieve two rounds of screening for the mutants. The accuracy of the high-throughput screening method was evaluated by high performance liquid chromatography (HPLC). [Results] The decarboxylation and carboxylation reaction system catalyzed by the crude enzyme and the Biomek i7 automated workstation screening process were determined. The two rounds of high-throughput screening from 508 clones yielded 13 positive mutants, and the HPLC confirmed 3 positive mutants. The positive mutation rate of the constructed library was 0.6%, and the mutant with the highest carboxylation activity showed the activity 120% of the wild type. [Conclusion] A high-throughput screening method for the decarboxylases with high carboxylation activity was successfully established based on the Biomek i7 automated workstation. For the first time, the automated high-throughput equipment was combined with mutant screening, which provided the idea of determining the evaluation indexes during the screening. It took 175 h to conduct two rounds of screening for 508 clones, which shortened the screening cycle compared with manual screening.