A particular silent codon exchange in a recombinant gene greatly influences host cell metabolic activity

Abstract

BackgroundRecombinant protein production using Escherichia coli as expression host is highly efficient, however, it also induces strong host cell metabolic burden. Energy and biomass precursors are withdrawn from the host’s metabolism as they are required for plasmid replication, heterologous gene expression and protein production. Rare codons in a heterologous gene may be a further drawback. This study aims to investigate the influence of particular silent codon exchanges within a heterologous gene on host cell metabolic activity. Silent mutations were introduced into the coding sequence of a model protein to introduce all synonymous arginine or leucine codons at two randomly defined positions, as well as substitutions leading to identical amino acid exchanges with different synonymous codons. The respective E. coli clones were compared during cultivation in a mineral autoinduction medium using specialized online and offline measuring techniques to quantitatively analyze effects on respiration, biomass and protein production, as well as on carbon source consumption, plasmid copy number, intracellular nucleobases and mRNA content of each clone.ResultsHost stain metabolic burden correlates with recombinant protein production. Upon heterologous gene expression, tremendous differences in respiration, biomass and protein production were observed. According to their different respiration activity the E. coli clones could be classified into two groups, Type A and Type B. Type A clones tended to higher product formation, Type B clones showed stronger biomass formation. Whereas codon usage and intracellular nucleobases had no influence on the Type-A–Type-B-behavior, plasmid copy number, mRNA content and carbon source consumption strongly differed between the two groups.ConclusionsParticular silent codon exchanges in a heterologous gene sequence led to differences in initial growth of Type A and Type B clones. Thus, the biomass concentration at the time point of induction varied. In consequence, not only plasmid copy number and expression levels differed between the two groups, but also the kinetics of lactose and glycerol consumption. Even though the underlying molecular mechanisms are not yet identified we observed the astonishing phenomenon that particular silent codon exchanges within a heterologous gene tremendously affect host cell metabolism and recombinant protein production. This could have great impact on codon optimization of heterologous genes, screening procedures for improved variants, and biotechnological protein production processes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0348-8) contains supplementary material, which is available to authorized users.

Online measurement of the respiratory activity in shake flasks enables the identification of cultivation phases and patterns indicating recombinant protein production in various Escherichia coli host strains

“…After an initial exponential increase, the OTR of BL21(DE3), Rosetta(DE3), and HMS174(DE3) decreases slightly for several hours (Figure A, B, and C). This phase is typically associated with recombinant protein production . Afterward, the OTR increases again, which is indicative for a second noninduced growth phase .…”

“…In their work, a correlation between OTR reflecting the metabolic state of the culture and recombinant protein production was observed. In addition, Rahmen et al confirmed these findings for E. coli BL21(DE3) clones expressing identical proteins only differing in a single amino acid or a single codon of the heterologous protein …”

Scale‐up of a Type I secretion system in E. coli using a defined mineral medium

“…The phenomenon of a more or less constant OTR after induction is well known from auto‐induction media containing glucose, lactose, and glycerol. Several studies identified a phase of rather constant OTR after induction with IPTG or its analogue lactose as production phase and indication of the strength of metabolic burden . An equivalent behavior is shown here for the first time in a mineral medium using arabinose as inducer.…”

Respiration activity monitoring system for any individual well of a 48-well microtiter plate

“…But considering different well geometries and shaking parameters, the resulting increased maximum oxygen transfer capacities can be comparable to stirred tank systems [30–32]. Based on the literature [16, 28, 33, 34], all cultivations of this section are expected to be oxygen unlimited.…”

“…The OTR provides important information about growth behavior and metabolism, it enables mass balancing and evaluation of stoichiometric relationships for aerobic cultivations [12–15]. Even estimations about the product formation are possible on the basis of the OTR [16, 17]. According to the state of the art, the OTR is determined via off gas analytics in fermenter scale [18].…”