Broad-Host-Range ProUSER Vectors Enable Fast Characterization of Inducible Promoters and Optimization of p‑Coumaric Acid Production in Pseudomonas putida KT2440

• Published in: ACS synthetic biology 2016-07, Vol.5 (7), p.741-753
• Main Authors: Calero, Patricia, Jensen, Sheila I, Nielsen, Alex T
• Format: Article
• Language: English
• Subjects: Coumaric Acids - metabolism; Gene Deletion; Gene Expression Regulation, Bacterial; Genetic Vectors; Metabolic Engineering - methods; Promoter Regions, Genetic; Propionates; Pseudomonas putida - genetics; Pseudomonas putida - metabolism; Single-Cell Analysis - methods; Synthetic Biology - methods; Tyrosine - metabolism
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.6b00081

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.