Combinatorial Tuning of 5′UTR and N-Terminal Coding Sequences for Enhanced Recombinant Protein Expression in Corynebacterium glutamicum

The 5'UTR sequence and N-terminal coding sequence (NCS) have been used to regulate gene expression in Corynebacterium glutamicum (C. glutamicum) microbial cell factories. However, there is currently insufficient research on the relationship between these expression element sequences and the protein expression rate in C. glutamicum. This study established a pattern between 5'UTR and NCS feature sequences and protein expression and validated their effects on protein expression. First, a 5'UTR library and a NCS library containing base N were constructed separately, and a continuous regulatory range across 5 orders of magnitude for the enhanced green fluorescent protein (eGFP) expression was achieved in both libraries by fluorescence activated cell sorting (FACS) and high-throughput sequencing. Next, the relationship between sequence information and protein expression was established based on the 5'UTR sequence and NCS sequence characteristics analysis in terms of CG content, minimum free energy (MFE), tRNA adaptability index, and deep learning. Moreover, four 5'UTR characteristic sequences and four NCS characteristic sequences were finally screened, which showed strong compatibility with different exogenous proteins. Furthermore, dynamic adjustment of eGFP fluorescence intensity from 45% to 511% was achieved through 16 different combinations of the screened four 5'UTR and four NCS sequences, confirming the synergistic effect of these two components. At the same time, these combinations also have a wide range of dynamic regulation of protein expression levels of other recombinant proteins such as mCherry and heavy chain antibody. This study provided a potential tool for finely regulating gene expression or protein production in C. glutamicum.