Screening l-Lysine-Overproducing Escherichia coli Using Artificial Rare Codons and a Rare Codon-Rich Marker

Abstract

l-Lysine, an essential amino acid for humans and mammals, is widely used in the food, feed, medicine, and cosmetics industries. In this study, a lysine over-producing Escherichia coli mutant was isolated using a fluorescence-based screen and an E. coli strain lacking five of the six L-lysine tRNA-UUU genes. Firstly, an l-lysine codon-rich protein was fused with a green fluorescent protein (all AAG codons were replaced with AAA), yielding a rare codon-rich screening marker positively correlated with l-lysine content. After association and room temperature plasma (ARTP) mutagenesis and induced fluorescent protein expression culture, mutant strains with strong fluorescence were sorted using flow cytometry. The fermentation performance of the high-yielding l-lysine strains were evaluated, which resulted in 16 of the 29 mutant strains showing increased L-lysine yields compared with those of the wild-type strains and a screening efficiency of up to 55.2%. Following a 48 h fermentation, the production of l-lysine (14.8 g/L) and biomass by E. coli QD01ΔtRNA L2 were 12.1 and 4.5% higher than those of the wild-type strain. The screening strategy for high-yielding strains based on the artificial rare cryptosystem established in this study will provide an efficient, accurate, and simple method for screening other amino-acid-producing microorganisms.

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“…Subsequently, through a combination of constant temperature and atmospheric pressure plasma mutagenesis, along with the induction of fluorescent protein expression, mutant strains exhibiting robust fluorescence were isolated via flow cytometry. Following a 48 h fermentation process, a high-yielding L-Lys strain named E. coli QD01 ΔtRNA L2 was obtained, demonstrating a remarkable production of 14.8 g/L L-Lys, surpassing the wild-type strain’s production by 12.1% [ 72 ]. The screening strategy established in this study provides an efficient, accurate, and easy method for screening product-targeting microorganisms.…”