caa a22 4500 605505624 CHVBK 20210128100623.0 cr unu---uuuuu 210128e20151101xx s 000 0 eng 10.1007/s00253-015-6744-5 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6744-5 Impact of rare codons and the functional coproduction of rate-limiting tRNAs on recombinant protein production in Bacillus megaterium [Elektronische Daten] [Constanze Finger, Martin Gamer, Saskia Klunkelfuß, Boyke Bunk, Rebekka Biedendieck] The Gram-positive bacterium Bacillus megaterium was systematically developed for the plasmid-based production of recombinant proteins at the gram-per-liter scale. The amount of protein produced per cell was found strongly correlated to the codon usage of the heterologous gene of interest in comparison to the codon usage of B. megaterium. For analyzing the influence of rare codons on the translational efficiency and protein production in B. megaterium, a test system using the gene for the green fluorescent protein (GFP) as reporter was established. For this purpose, four consecutive identical codons were introduced into the 5′ end of gfp and the resulting variations in GFP formation were quantified. Introduction of the rare codons GCC, CGG, and ACC for alanine, arginine, and threonine reduced GFP production 2.1-, 3.3-, and 1.7-fold in comparison to the favored codons GCU, CGU, and ACA, respectively. Coexpression of the corresponding rare codon tRNA (rctRNA) genes improved GFP production 4.2-, 2.7-, and 1.7-fold, respectively. The system was applied to the production of a formate dehydrogenase (FDH) from Mycobacterium vaccae and an extracellular hydrolase (TFH) from Thermobifida fusca. Coexpression of one to three different rctRNA genes resulted in an up to 18-fold increased protein production. Interestingly, rctRNA gene coexpression also elevated the production of M. vaccae FDH and T. fusca TFH from codon optimized genes, indicating a general positive effect by rctRNA gene overexpression on the protein production in B. megaterium. Thus, the basis for a B. megaterium enhanced production strain coexpressing rctRNA genes was laid. Springer-Verlag Berlin Heidelberg, 2015 Bacillus megaterium nationallicence tRNA genes nationallicence Rare codon cluster nationallicence Recombinant protein production nationallicence Finger Constanze Institute of Microbiology, Technische Universität Braunschweig, Spielmannstraße 7, 38106, Braunschweig, Germany aut Gamer Martin Institute of Microbiology, Technische Universität Braunschweig, Spielmannstraße 7, 38106, Braunschweig, Germany aut Klunkelfuß Saskia Institute of Microbiology, Technische Universität Braunschweig, Spielmannstraße 7, 38106, Braunschweig, Germany aut Bunk Boyke German Collection for Microorganisms and Cell Cultures (DSMZ), Inhoffenstraße 7, 38124, Braunschweig, Germany aut Biedendieck Rebekka Institute of Microbiology, Technische Universität Braunschweig, Spielmannstraße 7, 38106, Braunschweig, Germany aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/21(2015-11-01), 8999-9010 0175-7598 99:21<8999 2015 99 253 https://doi.org/10.1007/s00253-015-6744-5 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6744-5 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Finger Constanze Institute of Microbiology, Technische Universität Braunschweig, Spielmannstraße 7, 38106, Braunschweig, Germany aut NATIONALLICENCE 700 1- Gamer Martin Institute of Microbiology, Technische Universität Braunschweig, Spielmannstraße 7, 38106, Braunschweig, Germany aut NATIONALLICENCE 700 1- Klunkelfuß Saskia Institute of Microbiology, Technische Universität Braunschweig, Spielmannstraße 7, 38106, Braunschweig, Germany aut NATIONALLICENCE 700 1- Bunk Boyke German Collection for Microorganisms and Cell Cultures (DSMZ), Inhoffenstraße 7, 38124, Braunschweig, Germany aut NATIONALLICENCE 700 1- Biedendieck Rebekka Institute of Microbiology, Technische Universität Braunschweig, Spielmannstraße 7, 38106, Braunschweig, Germany aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/21(2015-11-01), 8999-9010 0175-7598 99:21<8999 2015 99 253