基于Biomek i7自动化工作站筛选高效固碳脱羧酶突变体的方法

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

摘要:

【背景】2,3-二羟基苯甲酸脱羧酶(2,3-dihydroxybenzoic acid decarboxylase, 2,3-DHBD)能够催化CO₂与酚类化合物羧化生成芳香羧酸，为固定CO₂合成增值化学品提供一种新的策略。由于目前转化效率低，限制了其应用。【目的】提高酶的催化效率，建立基于Biomek i7自动化工作站的高通量筛选方法以期实现高效筛选高羧化活性的苯甲酸脱羧酶的突变体，并建立一个从突变体构建到其酶活筛选的标准方法。【方法】基于Biomek i7自动化工作站对来源于米曲霉(Aspergillus oryzae)的2,3-DHBD (2,3-DHBD_Ao)进行突变体定点半饱和突变、克隆挑选、培养及酶活检测，实现508个克隆的两轮筛选，并利用HPLC评估高通量筛选方法的准确性。【结果】确定了高通量筛选粗酶催化的脱羧及羧化反应体系和Biomek i7自动化工作站筛选流程，对508个克隆进行两轮筛选，得到13个正向突变，HPLC检测最终确定3个正向突变体，构建的文库阳性突变率为0.6%，获得羧化活性最高的突变体是野生型2,3-DHBD_Ao的120%。【结论】利用Biomek i7自动化工作站建立了高通量筛选高羧化活性脱羧酶的方法，首次将自动化高通量设备与突变体筛选结合，提供了筛选时评价指标的确定思路，经过175 h即可完成508个克隆的两轮筛选工作，相较于人工筛选，该方法缩短了筛选周期，具有快速、准确和高效的特点。

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.