Construction of an ultra-strong PtacM promoter via engineering the core-element spacer and 5' untranslated region for versatile applications in .

As one of the most important synthetic biology elements in transcriptional regulation, promoters play irreplaceable roles in metabolic engineering. For the industrial microorganism , both the construction of a promoter library with gradient strength and the creation of ultra-strong promoters are essential for the production of target enzymes and compounds. In this work, the spacer sequence (both length and base) between the -35 and -10 regions, and the 5'-terminal untranslated region (5'UTR) were particularly highlighted to investigate their contributions to promoter strength. We constructed a series of artificially induced promoters based on the classical tac promoter using ATCC13032 as the host. Here, we explored the effect of sequence length between the -35 and -10 regions on the strength of the tac promoter, and found that the mutant with 15 nt spacer length (PtacL15) was transcriptionally stronger than the classic Ptac (16 nt); subsequently, based on PtacL15, we explored the effect of the nucleotide sequence in the spacer region on transcriptional strength, and screened the strongest PtacL15m-110 (GAACAGGCTTTATCT), and PtacL15m-87 (AGTCGCTAAGACTCA); finally, we investigated the effect of the length of the 5'-terminal untranslated region (5'UTR) and screened out the optimal PtacM4 mutant with a 5'UTR length of 32 nt. Based on our new findings on the optimal spacer length (15 nt), nucleotide sequence (AGTCGCTAAGACTCA), and 5'UTR (truncated 32 nt), an ultra-strong PtacM, whose transcriptional strength was about 3.25 times that of the original Ptac, was obtained. We anticipate that these promoters with gradient transcriptional strength and the ultra-strong PtacM will play an important role in the construction of recombinant strains and industrial production.