Harnessing enzyme expression for production of target chemicals is a critical and multifarious process, where screening of different genes by inspection of enzymatic activity plays an imperative role. Here, we conceived an idea to improve the time‐consuming and labor‐intensive process of enzyme screening. Controlling cell growth was achieved by the Cluster Regularly Interspaced Short Palindromic Repeat (CRISPRi) system with different single guide RNA targeting the essential gene can (CRISPRi::CA) that encodes a carbonic anhydrase for CO₂ uptake. CRISPRi::CA comprises a whole‐cell biosensor to monitor CO₂ concentration, ranging from 1% to 5%. On the basis of CRISPRi::CA, an effective and simple Direct Enzymatic Performance Evaluation & Determination (DEPEND) system was developed by a single step of plasmid transformation for targeted enzymes. As a result, the activity of different carbonic anhydrases corresponded to the colony‐forming units. Furthermore, the enzymatic performance of 5‐aminolevulinic acid synthetase (ALAS), which converts glycine and succinate‐CoA to release a molecule of CO₂, has also been distinguished, and the effect of the chaperone GroELS on ALAS enzyme folding was successfully identified in the DEPEND system. We provide a highly feasible, time‐saving, and flexible technology for the screening and inspection of high‐performance enzymes, which may accelerate protein engineering in the future.

中文翻译：

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CRISPRi介导的编程必需基因可以作为直接酶性能评估和测定（DEPEND）系统。


利用酶表达来生产目标化学品是一个关键且复杂的过程，其中通过检查酶活性筛选不同基因至关重要。在这里，我们想到了一个想法来改进酶筛选的耗时和劳动密集型过程。控制细胞生长是通过簇规则间隔短回文重复序列 (CRISPRi) 系统实现的，该系统具有针对编码用于 CO ₂吸收的碳酸酐酶的必需基因can (CRISPRi::CA) 的不同单引导 RNA。 CRISPRi::CA 包含一个全细胞生物传感器，用于监测 CO ₂浓度（范围从 1% 到 5%）。在CRISPRi::CA的基础上，通过对目标酶进行一步质粒转化，开发了一种有效、简单的直接酶学性能评估和测定（DEPEND）系统。结果，不同碳酸酐酶的活性对应于菌落形成单位。此外，5-氨基乙酰丙酸合成酶（ALAS）可转化甘氨酸和琥珀酸辅酶A以释放CO ₂分子，其酶促性能也已得到区分，并且分子伴侣GroELS对ALAS酶折叠的影响已在依赖系统。我们为高性能酶的筛选和检测提供高度可行、省时且灵活的技术，这可能会加速未来的蛋白质工程。