Promoters that play an essential role in the gene regulation are of particular interest to the synthetic biology communities. Recent advances in high-throughput DNA sequencing have greatly increased the breadth of new genetic parts. The development of promoters with broad host properties could enable rapid phenotyping of genetic constructs in different hosts. In this study, we discovered that the GAL1/10 bidirectional promoter from Saccharomyces cerevisiae could be used for shuttle expression in Escherichia coli. Further investigation revealed that the GAL1/10 bidirectional promoter is subjected to catabolite repression in E. coli. We next constructed a set of Golden-Gate assembly vectors for shuttle expression between S. cerevisiae and E. coli. The utility of shuttle vectors was demonstrated for rapid phenotyping of a multigene pathway for cinnamyl alcohol production. Taken together, our work opens a new avenue for the future development of broad host expression systems between prokaryotic and eukaryotic hosts.