Mining and Understanding of New Transcriptional Regulatory Elements from Licorice-Derived Endophyte

\nThere is an increasing need for precise regulation of complex metabolic pathways in the cell factory development, and thus the discovery of new cis-regulatory elements and the responding trans elements is especially attracting the attention of scientific researchers. Previously, an endophyte S. rubidaea W12-1 was isolated from the root of Glycyrrhiza uralensis Fisch. derived from the northwest of China. Here, it was found that prodigiosin, a natural red pigment belonging to the class of alkaloids with multiple pharmacological activities, was produced by S. rubidaea W12-1 under specific conditions and growth stages, indicating that there should be interesting inducible promoters and regulatory factors in S. rubidaea. Therefore, whole genome sequencing and analysis were operated on S. rubidaea W12-1 to map the prodigiosin biosynthesis gene cluster, called pig cluster, and its upstream promoter Ppig. After DNA pull-down assay and proteomics, 22 transcription factor candidates being expected to interact with Ppig were screened out. Among them, AstD, DinB, GlgP, GlmS, FimA, MreB, PchP, RplB, RpoB, RpsD, SucB, TopA, TorZ, TPA, YaaA, YeiP were confirmed to be transcriptional repressors to Ppig according to the in vitro experiments in which the gene expression vectors (pRS415-PgapA-gene of the transcription factor candidate-Ppig-eGFP) were constructed and individually transferred into Escherichia coli followed by the cultivation and fluorescence detection of the engineered strains. Meanwhile, it was found that L-proline, an upstream compound of prodigiosin metabolism, could significantly enhance the transcriptional repression of SucB and significantly alleviate the transcriptional repression of FimA, TopA and TorZ on Ppig-eGFP, while malonyl-CoA, a second upstream compound of prodigiosin metabolism, could significantly enhance the transcriptional repression of CgtA and significantly alleviate the transcriptional repression of TorZ and YeiP on Ppig-eGFP. In summary, this study improved the understanding of the regulation mechanism of prodigiosin metabolism in Serratia, provided potential transcriptional regulatory elements in metabolic engineering and high-throughput screening of target strains, and contributed to enriching the genetic components derived from microorganisms in synthetic biology.\n