Ribosome binding site (RBS) is an important biological control element for fine-regulation of genes expression, which has been widely applied in microbial metabolic engineering and synthetic biology. Corynebacterium glutamicum is an important industrial microorganism for industrial fermentation of several amino acids and organic acids. However, there were few researches on the RBS element for C. glutamicum. In this study, a random RBS library was designed and constructed based on the common RBS characteristics in C. glutamicum. Then, a high-throughput screening technology was developed by coupling flow cytometry and 96-well plate screening technology for screening of the artificial RBS library. After two-step screening, an artificial RBS element library with wide range of regulation and uniform coverage was constructed, and the regulation strength range of the library was 29.04-fold. Next, the artificial RBS library was further characterized by α-amylase expression system, and the results showed that the RBS library had high stability and versatility. Subsequently, the artificial library was applied for the regulation and optimization of the L-homoserine biosynthetic pathway. The expressions of LysCr and Homr, the key enzymes in L-homoserine synthesis pathway, were fine-regulated using RBS elements with different intensities. Finally, the best combination of LysCr and Homr for L-homoserine synthesis was obtained, and the resulted strain accumulated 12.7 g/L of L-homoserine, which was 3.6 times higher than that of the wild-type strain. Therefore, an artificial RBS library was constructed and exhibited wide regulation range and high stability in this study, which provided the effective control elements for metabolic engineering and gene expression regulation in C. glutamicum.
ZHANG Yue
,
ZHANG Jiwei
,
WU Shuo
,
XU Ning
,
LIU Jun
,
WEI Liang
. Construction and application of an artificial ribosomal binding site (RBS) library in Corynebacterium glutamicum[J]. Food and Fermentation Industries, 2023
, 49(23)
: 25
-32
.
DOI: 10.13995/j.cnki.11-1802/ts.035178
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