Broad-Host-Range ProUSER Vectors Enable Fast Characterization of Inducible Promoters and Optimization of p-Coumaric Acid Production in Pseudomonas putida KT2440.
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.
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kogle Allé 6, 2970 Hørsholm, Denmark.
Patricia
P
Calero
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kogle Allé 6, 2970 Hørsholm, Denmark.
Sheila I
SI
Jensen
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kogle Allé 6, 2970 Hørsholm, Denmark.
Alex T
AT
Nielsen
D003373:Coumaric Acids / Q000378:metabolism*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D060847:Metabolic Engineering / Q000379:methods; D011401:Promoter Regions, Genetic; D011422:Propionates; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism*; D059010:Single-Cell Analysis / Q000379:methods; D058615:Synthetic Biology / Q000379:methods; D014443:Tyrosine / Q000378:metabolism
D003373:Coumaric Acids; D011422:Propionates; D014443:Tyrosine; C495469:p-coumaric acid
Pseudomonas putida KT2440; USER cloning; broad-host-range vectors; building blocks; p-coumaric acid