초록
<P><I>Pseudomonas putida</I> KT2440 has gained increasing interest as a host for the production of biochemicals. Because of the lack of a systematic characterization of inducible promoters in this strain, we generated ProUSER broad-host-expression plasmids that facilitate fast uracil-based cloning. A set of ProUSER-reporter vectors was further created to characterize different inducible promoters. The <I>PrhaB</I> and <I>Pm</I> promoters were orthogonal and showed titratable, high, and homogeneous expression. To optimize the production of <I>p</I>-coumaric acid, <I>P. putida</I> was engineered to prevent degradation of tyrosine and <I>p</I>-coumaric acid. <I>Pm</I> and <I>PrhaB</I> were used to control the expression of a tyrosine ammonia lyase or AroG* and TyrA* involved in tyrosine production, respectively. Pathway expression was optimized by modulating inductions, resulting in small-scale <I>p</I>-coumaric acid production of 1.2 mM, the highest achieved in <I>Pseudomonads</I> under comparable conditions. With broad-host-range compatibility, the ProUSER vectors will serve as useful tools for optimizing gene expression in a variety of bacteria.</P><P><B>Graphic Abstract</B><BR><IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/asbcd6/2016/asbcd6.2016.5.issue-7/acssynbio.6b00081/production/images/medium/sb-2016-000817_0006.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/sb6b00081'>ACS Electronic Supporting Info</A></P>