Chlorovirus promoters have strong regulatory activities in Escherichia coli and Saccharomyces cerevisiae

Chloroviruses are a group of large dsDNA viruses that infect certain unicellular, eukaryotic green algae. Sequence analysis revealed that chloroviruses have genomes of 300Kb or larger which possess more than 400 protein encoding genes; 40% of them resemble known proteins. It has been demonstrated that promoters from the prototype chlorovirus PBCV-1 are strong promoters in several phytopathogenic bacteria and higher plants, so the viral promoters might be strong in industrially important bacterial strains, or unicellular eukaryotic organisms, such as yeast or green algae, and be useful for developing transformation vectors. In this project we selected strong viral promoters in E. coli from random genome fragments generated by pooled genomic DNAs of multiple chlorovirus strains and shotgun library construction with a promoter-probing plasmid pKK232-8. When chloramphenicol acetyltransferase (CAT) controlled by its native promoter Pcat or by the artificially strong bacterial promoter Ptac, the cassettes Pcat-CAT and Ptac-CAT only allow E. coli DH5α grows in media supplemented with 800µg/ml or lower concentrations of chloramphenicol; while a quite few of screened viral promoters make the same E. coli strain be resistant to as high as 1,200µg/ml of chloramphenicol. Some of these promoters showed strong regulatory activity in yeast by expressing Pcv-HIS3 cassette in his3 strain. The function of viral promoters is currently being tested in E. coli K-12 strain MG1655 by promoter-swap for metabolic engineering purpose, such as upregulating some key genes in aspartate biosynthetic pathway to accumulate amino acids efficiently.