Obtaining a series of native gradient promoter-5'-UTR sequences in Corynebacterium glutamicum ATCC 13032

Corynebacterium glutamicum is an important industrial microorganism used for the production of many valuable compounds, especially amino acids and their derivatives. For fine-tuning of metabolic pathways, synthetic biological tools are largely based on the rational application of promoters. However, the limited number of promoters make it difficult.

In this study, according to the analysis of RNA-Seq data, 90 DNA fragments with lengths of 200-500 bp that may contain promoter-5'-UTR (PUTR) sequences were amplified and linked to a fluorescent protein gene. When compared with the common strong PUTR P_sodUTR, 17 strong PUTRs were obtained, which maintained stable expression strengths from the early to post stationary phase. Among them, P_NCgl1676UTR was the strongest and its fluorescent protein expression level was more than five times higher than that of P_sodUTR. Furthermore, nine typical chemicals related to the biosynthesis of sulfur-containing amino acids (such as L-methionine, L-cysteine) were selected as stress substances to preliminarily explore the stress on these PUTRs. The results showed that the expression of P_brnFUTR was activated by L-methionine, while that of P_NCgl1202UTR was severely inhibited by L-lysine.

These findings demonstrated that the selected PUTRs can stably express different genes, such as the red fluorescence protein gene, and can be useful for fine-tuning regulation of metabolic networks in C. glutamicum or for establishing high-throughput screening strategies through biosensor for the production of useful compounds.