Characterization of the Positive Transcription Regulator PfaR for Improving Eicosapentaenoic Acid Production in Shewanella putrefaciens W3-18-1.

The biosynthetic pathway of eicosapentaenoic acid (EPA) has previously been reported in marine bacteria, while the regulatory mechanism remains poorly understood. In this study, a putative transcriptional regulator PfaR encoded adjacent to the PFA biosynthesis gene cluster (pfaEABCD) was computationally and experimentally characterized. Comparative analyses on the wild type (WT) strain, in-frame deletion, and overexpression mutants revealed that PfaR positively regulated EPA synthesis at low temperature. RNA-Seq and real-time quantitative PCR analyses demonstrated that PfaR stimulated the transcription of pfaABCD. The transcription start site of pfaR was mapped by using primer extension and highly conserved promoter motifs bound by the housekeeping Sigma 70 factor that were identified in the upstream of pfaR. Moreover, overexpression of PfaR in WT strain W3-18-1 at low temperature could improve EPA productivity from 0.07% to 0.13% (percentage of EPA to dry weight, mg/mg) of dry weight. Taken together, these findings could provide important implications into the transcriptional control and metabolic engineering in terms of EPA productivity for industrial strains. IMPORTANCE We have experimentally confirmed that PfaR is a positive transcription regulator that promotes EPA synthesis at low temperature in Shewanella putrefaciens W3-18-1. Overexpression of PfaR in WT strain W3-18-1 could lead to a 1.8-fold increase in EPA productivity at low temperature. It is further shown that PfaR may be regulated by housekeeping Sigma 70 factor at low temperature.