NDLI: Impact of rare codons and the functional coproduction of rate-limiting tRNAs on recombinant protein production in Bacillus megaterium

Content Provider	Springer Nature Link
Author	Finger, Constanze Gamer, Martin Klunkelfuß, Saskia Bunk, Boyke Biedendieck, Rebekka
Copyright Year	2015
Abstract	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.
Starting Page	8999
Ending Page	9010
Page Count	12
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	99
Issue Number	21
e-ISSN	14320614
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2015-07-03
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Bacillus megaterium tRNA genes Rare codon cluster Recombinant protein production Microbiology Microbial Genetics and Genomics Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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