期刊
BIOTECHNOLOGY AND BIOENGINEERING
卷 117, 期 9, 页码 2842-2851出版社
WILEY
DOI: 10.1002/bit.27443
关键词
aminolevulinic acid synthetase; carbonic anhydrase; CRISPRi; essential gene; performance; whole-cell biosensor
资金
- Ministry of Science and Technology [MOST 108-2621-M-006-015, MOST 108-2221-E-006-004-MY3, MOST 108-2218-E-006-006]
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 genecan(CRISPRi::CA) that encodes a carbonic anhydrase for CO(2)uptake. CRISPRi::CA comprises a whole-cell biosensor to monitor CO(2)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.
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