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.

To explore the key sites affecting the intracellular assembly of key components of cellulosomes and obtain DocA mutants independent of Ca²⁺, Swiss-model, GROMACS, PyMOL, and other molecular dynamics simulation software were used for modeling and static and dynamic combination analysis. Site-specific mutation technology was used to mutate DocA, and Biacore was used to test the dependence of Ca²⁺ on the binding ability of protein DocA mutants and protein Coh, and to analyze the interaction and binding effect of mutant proteins in vitro. Forward intracellular mutant screening was performed based on semi-rational design and high throughput screening techniques. The orientation of mutations suitable for intracellular assembly was determined, and three directional mutant proteins, DocA-S1, DocA-S2, and DocA-S3, were obtained. Ca²⁺ independent DocA mutants were obtained gradually and their potential interaction mechanisms were analyzed. In the present study, intracellular self-assembly of key components of cellulosomes independent of Ca²⁺ was achieved, and DocA-S3 was applied to the assembly of key enzymes of L-lysine biosynthesis, in which DapA and DapB intracellular assembly increased L-lysine accumulation by 29.8% when compared with the control strains, providing a new strategy for improving the intracellular self-assembly of cellulosomes and amino acid fermentation efficiency.

To enhance the L-threonine synthesis level in Escherichia coli, this study constructed screening markers rich in L-threonine rare codons. By replacing all the threonine codons in the protein sequences with a high proportion of threonine with L-threonine rare codons and linking them to the fluorescent proteins with the same replacement, high-throughput screening of L-threonine production mutant strains was achieved. To address the metabolic imbalance caused by overexpression of a single enzyme, an artificial multi-enzyme complex system was constructed based on the principle of cellulosome self-assembly. By co-locating ThrC-DocA and ThrB-CohA, the substrate transfer path was shortened, achieving a 31.7% increase in L-threonine production. Furthermore, combined with multi-copy chromosomal integration technology via CRISPR-associated transposase (MUCICAT) technology, the thrC-docA-thrB-cohA gene cluster was integrated into the genome of the high-yield strains obtained through screening, eliminating the plasmid-dependent metabolic burden and significantly enhancing genetic stability. The modular assembly of metabolic pathways by using cellulosome elements provides a new paradigm for the optimization of complex pathways and lays a theoretical and technical foundation for the efficient production of L-threonine.