TY  - JOUR
A1  - Pöschel, Laura
A1  - Gehr, Elisabeth
A1  - Buchhaupt, Markus
T1  - A pBBR1-based vector with IncP group plasmid compatibility for Methylorubrum extorquens
T2  - MicrobiologyOpen
N2  - Plasmids are one of the most important genetic tools for basic research and biotechnology, as they enable rapid genetic manipulation. Here we present a novel pBBR1-based plasmid for Methylorubrum extorquens, a model methylotroph that is used for the development of C1-based microbial cell factories. To develop a vector with compatibility to the so far mainly used pCM plasmid system, we transferred the pBBR1-based plasmid pMiS1, which showed an extremely low transformation rate and caused a strong growth defect. Isolation of a suppressor mutant with improved growth led to the isolation of the variant pMis1_1B. Its higher transformation rate and less pronounced growth defect phenotype could be shown to be the result of a mutation in the promotor region of the rep gene. Moreover, cotransformation of pMis1_1B and pCM160 was possible, but the resulting transformants showed stronger growth defects in comparison with a single pMis1_1B transformant. Surprisingly, cotransformants carrying pCM160 and a pMis1_1B derivative containing a mCherry reporter construct showed higher fluorescence levels than strains containing only the pMis1_1B-based reporter plasmids or a corresponding pCM160 derivative. Relative plasmid copy number determination experiments confirmed our hypothesis of an increased copy number of pMis1_1B in the strain carrying both plasmids. Despite the slight metabolic burden caused by pMis1_1B, the plasmid strongly expands the genetic toolbox for M. extorquens.
KW  - cotransformation
KW  - expression system
KW  - Methylorubrum extorquens AM1
KW  - plasmid
KW  - plasmid copy number
KW  - Rep gene
Y1  - 2022
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/79556
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-795568
VL  - 11
IS  - 5, e1325
PB  - John Wiley & Sons Ltd.
CY  - Malden, Mass.
ER  -