TY  - JOUR
AU  - Weihmann, Robin
AU  - Domröse, Andreas
AU  - Drepper, Thomas
AU  - Jaeger, Karl‐Erich
AU  - Loeschcke, Anita
TI  - Protocols for yTREX /Tn5‐based gene cluster expression in Pseudomonas putida
JO  - Microbial biotechnology
VL  - 13
IS  - 1
SN  - 1751-7915
CY  - Oxford
PB  - Wiley-Blackwell
M1  - FZJ-2019-05374
SP  - 250-262
PY  - 2020
AB  - Bacterial gene clusters, which represent a genetic treasure trove for secondary metabolite pathways, often need to be activated in a heterologous host to access the valuable biosynthetic products. We provide here a detailed protocol for the application of the yTREX ‘gene cluster transplantation tool’: Via yeast recombinational cloning, a gene cluster of interest can be cloned in the yTREX vector, which enables the robust conjugational transfer of the gene cluster to bacteria like Pseudomonas putida, and their subsequent transposon Tn5‐based insertion into the host chromosome. Depending on the gene cluster architecture and chromosomal insertion site, the respective pathway genes can be transcribed effectively from a chromosomal promoter, thereby enabling the biosynthesis of a natural product. We describe workflows for the design of a gene cluster expression cassette, cloning of the cassette in the yTREX vector by yeast recombineering, and subsequent transfer and expression in P. putida. As an example for yTREX‐based transplantation of a natural product biosynthesis, we provide details on the cloning and activation of the phenazine‐1‐carboxylic acid biosynthetic genes from Pseudomonas aeruginosa in P. putidaKT2440 as well as the use of β‐galactosidase‐encoding lacZ as a reporter of production levels.
LB  - PUB:(DE-HGF)16
C6  - pmid:31162833
UR  - <Go to ISI:>//WOS:000596580200022
DO  - DOI:10.1111/1751-7915.13402
UR  - https://juser.fz-juelich.de/record/866203
ER  -