Broad host range ProUSER vectors enable fast characterization of inducible promoters and optimization of p-coumaric acid production in Pseudomonas putida KT2440

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Abstract

Pseudomonas putida 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 PrhaB and Pm promoters were orthogonal and showed titratable, high, and homogeneous expression. To optimize the production of p-coumaric acid, P. putida was engineered to prevent degradation of tyrosine and p-coumaric acid. Pm and PrhaB 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 p-coumaric acid production of 1.2 mM, the highest achieved in Pseudomonads 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.
Original languageEnglish
JournalA C S Synthetic Biology
Volume5
Issue number7
Pages (from-to)741-753
Number of pages13
ISSN2161-5063
DOIs
Publication statusPublished - 2016

Keywords

  • Building blocks
  • Broad-host-range vectors
  • USER cloning
  • P-coumaric acid
  • Pseudomonas putida KT2440

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