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

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.

Influence of the combination of last sense codon and stop codon on expression efficiency of green fluorescent protein gene inEscherichia coliwhen the expression vector pKK223-3

“…In addition, the sequence context motif surrounding the ATG initiation codon (ATG-context) is an important factor that increases protein production [26][27][28][29][30][31][32] . 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 [33] .…”

EngineeringBacillusfor efficient production of heterologous protein: current progress, challenge and prospect

“…In addition, co‐expression of the responsible rare codon tRNA (rc tRNA ) genes could significantly improve the yields of GFP, formate dehydrogenase from Mycobacterium vaccae and extracellular hydrolase (TFH) from Thermobifida fusca in B. megaterium (Finger et al . ).…”

“…In B. licheniformis, a novel antisense sRNA aprA was identified, which acted as the RNA-based repressor of aprE, the gene encoding for subtilisin protease, and aprE transcriptional level and subtilisin yield were significantly increased in the aprA mutant strain (Hertel et al 2017). In addition, co-expression of the responsible rare codon tRNA (rctRNA) genes could significantly improve the yields of GFP, formate dehydrogenase from Mycobacterium vaccae and extracellular hydrolase (TFH) from Thermobifida fusca in B. megaterium (Finger et al 2015).…”

Integrated analysis of individual codon contribution to protein biosynthesis reveals a new approach to improving the basis of rational gene design

“… 31–34 Nevertheless, the introduction of synonymous mutations has sometimes resulted in a decrease in protein production 35 or loss of protein function due to instability, misfolding or aggregation. 24 , 36 , 37 Contrary to earlier codon optimization studies, recent works have demonstrated enhanced protein production by insertion of rare codons, 38 and, indeed, by random codon substitutions at the 5′-end region of genes. 39 …”