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

被引：80

作者：

Calero, Patricia ^{[1
]}

Jensen, Sheila I. ^{[1
]}

Nielsen, Alex T. ^{[1
]}

机构：

[1] Tech Univ Denmark, Novo Nordisk Fdn, Ctr Biosustainabil, Kogle Alle 6, DK-2970 Horsholm, Denmark

来源：

ACS SYNTHETIC BIOLOGY | 2016年 / 5卷 / 07期

关键词：

building blocks; broad-host-range vectors; USER cloning; p-coumaric acid; Pseudomonas putida KT2440; HIGH-LEVEL EXPRESSION; ESCHERICHIA-COLI; GENE-EXPRESSION; RNA-POLYMERASE; L-TYROSINE; PLASMID; ARABINOSE; PATHWAY; CONSTRUCTION; CLONING;

D O I：

10.1021/acssynbio.6b00081

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：741 / 753

页数：13

共 86 条

[1] REGULATOR AND ENZYME SPECIFICITIES OF THE TOL PLASMID-ENCODED UPPER PATHWAY FOR DEGRADATION OF AROMATIC-HYDROCARBONS AND EXPANSION OF THE SUBSTRATE RANGE OF THE PATHWAY [J].