New constitutive promoters screened from metagenomic library of rumen microbes

Abstract:

[Objective] Screening of novel promoter elements from the genome of microorganisms of extreme environmental origin for the design of synthetic biological chassis cells.[Methods] We used a promoter-probe plasmid pUC18-GFP containing a green fluorescent protein structural gene and a ribosome bind site to construct a rumen metagenomic library. This method allows us to obtain the DNA fragments with constitutive promoter function rapidly and efficiently from this library. We obtained possible promoter regions through the neural network-based promoter prediction analysis. Then, we verified the function of the promoter initiation by using GFP and maltotetraose amylase from Pseudomonas stutzeri as the reporter.[Results] We obtained twenty-two DNA fragments functioning as constitutive promoters from about 3750 transformants. These fragments share very low sequence identities with the reported gene sequences in the NCBI database, and present different starting efficiencies. In addition, we obtained two new promoter fragments RFa1p2 (76 bp) and RFb4p (547 bp) by promoter prediction and sub-cloning. These new constitutive promoters are able to express heterologous proteins efficiently in the absence of any inductor in the genetically engineered E. coli cells.