A novel DMAPP-responding genetic circuit sensor for high-throughput screening and evolving isoprene synthase

• Published in: Applied microbiology and biotechnology 2018-02, Vol.102 (3), p.1381-1391
• Main Authors: Liu, Chun-Li, Cai, Jing-Yi, Bi, Hao-Ran, Tan, Tian-Wei
• Format: Article
• Language: English
• Subjects: Alkyl and Aryl Transferases - genetics; Applied Genetics and Molecular Biotechnology; Arabinose; Arabinose - genetics; Arabinose operon; Biological evolution; Biomedical and Life Sciences; Biotechnology; C5 hydrocarbons; Circuits; Collating; Deoxyribonucleic acid; DNA; E coli; Enhanced green fluorescent protein; Enzymes; Escherichia coli; Escherichia coli - genetics; Flow Cytometry; Fluorescence; Gene expression; Gene Library; Gene Regulatory Networks; Genetic research; Green Fluorescent Proteins - biosynthesis; Green Fluorescent Proteins - genetics; Hemiterpenes - chemistry; High-throughput screening; High-Throughput Screening Assays; Isoprene; Life Sciences; Microbial Genetics and Genomics; Microbiology; Mutation; Nucleotide sequence; Operon; Organophosphorus Compounds - chemistry; Plant Proteins - genetics; Promoter Regions, Genetic; Properties; Pyrophosphates; Screening; Sensors; Substrates
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-017-8676-8

High-throughput screening is a popular tool for collating biological data which would otherwise require the use of excessive resources. In this study, an artificial genetic circuit sensor responding to dimethylallyl diphosphate (DMAPP) was constructed based on a modified L-arabinose operon for high-throughput screening and isoprene synthase ( ispS ) evolution in Escherichia coli ( E. coli ). As a first step, the DNA sequence of the L-arabinose ligand-binding domain (LBD) was replaced with an ispS gene to enable the AraC operon responding to DMAPP, which is the substrate of the IspS enzyme. Then, an enhanced GFP (eGFP) was also introduced as a reporter for pBAD promoter. The expression level of the reporter was monitored using either of the two tools: flow cytometer (FCM) and microplate reader. Sequentially, we observed that a high DMAPP concentration led to low eGFP fluorescence, and the overexpression of ispS gene, which consumes DMAPP, resulted in a high eGFP expression. These results demonstrated that the artificial genetic circuit sensor responded directly to the intracellular concentration of DMAPP, and the expression of IspS enzyme could be positively correlated to the expression level of eGFP. Finally, we identified two IspS mutants with different activities from an ispS gene library and further validated the screening method.