Journal
METABOLIC ENGINEERING
Volume 78, Issue -, Pages 11-25Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2023.04.012
Keywords
Amino acid overproducer; tRNA modification; Codon expansion; Amber stop codon; High-throughput screening
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In this study, a selection system using engineered tRNAs and marker genes was developed to screen overproducers of specific amino acids. Random mutation libraries of Escherichia coli and Corynebacterium glutamicum were screened using growth-based and/or fluorescence-activated cell sorting (FACS)-based screening to obtain overproducers of five amino acids. This study provides a universal strategy for screening overproducers of proteinogenic and non-proteinogenic amino acids in amber-stop-codon-recoded or non-recoded hosts.
Amino acids have a multi-billion-dollar market with rising demand, prompting the development of high-performance microbial factories. However, a general screening strategy applicable to all proteinogenic and non-proteinogenic amino acids is still lacking. Modification of the critical structure of tRNA could decrease the aminoacylation level of tRNA catalyzed by aminoacyl-tRNA synthetases. Involved in a two-substrate sequential reaction, amino acids with increased concentration could elevate the reduced aminoacylation rate caused by specific tRNA modification. Here, we developed a selection system for overproducers of specific amino acids using corresponding engineered tRNAs and marker genes. As a proof-of-concept, overproducers of five amino acids such as L-tryptophan were screened out by growth-based and/or fluorescence-activated cell sorting (FACS)-based screening from random mutation libraries of Escherichia coli and Corynebacterium glutamicum, respectively. This study provided a universal strategy that could be applied to screen overproducers of proteinogenic and non-proteinogenic amino acids in amber-stop-codon-recoded or non-recoded hosts.
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