CRISPRi‐mediated programming essential gene can as a Direct Enzymatic Performance Evaluation & Determination (DEPEND) system

• Published in: Biotechnology and bioengineering Vol. 117; no. 9; pp. 2842 - 2851
• Main Authors: Tan, Shih‐I, Yu, Peng‐Jui, Ng, I‐Son
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.09.2020
• Subjects: 5-Aminolevulinate Synthetase - genetics; 5-Aminolevulinate Synthetase - metabolism; Aminolevulinic acid; aminolevulinic acid synthetase; Biosensing Techniques - methods; Biosensors; Carbon dioxide; Carbonic anhydrase; Carbonic anhydrases; Carbonic Anhydrases - genetics; Carbonic Anhydrases - metabolism; CRISPR-Cas Systems - genetics; CRISPRi; Enzymatic activity; Enzymes; Escherichia coli - genetics; essential gene; Genes, Essential - genetics; Glycine; Inspection; Performance evaluation; Protein engineering; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Ribonucleic acid; RNA; RNA, Guide, CRISPR-Cas Systems; Screening; whole‐cell biosensor; carbonic anhydrase; performance; CRISPRi; essential gene; whole-cell biosensor; aminolevulinic acid synthetase
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.27443

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. Protein quality and quantity was tremendously affected by the amino acid and DNA sequence, which make it difficult to select the appropriate protein candidate. DEPEND, Direct Enzymatic Performance Evaluation and Determination system, is a dependable platform to rapidly screen the positive protein candidate in a single‐step transformation of plasmids expressing a CRISPRi to confer a survival stress coupled with a gene to release the stress. This platform may accelerate protein engineering in the future.