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Improved protein production and codon optimization analyses in Escherichia coli by bicistronic design.

ABSTRACT: Different codon optimization algorithms are available that aim at improving protein production by optimizing translation elongation. In these algorithms, it is generally not considered how the altered protein coding sequence will affect the secondary structure of the corresponding RNA transcript, particularly not the effect on the 5'-UTR structure and related ribosome binding site availability. This is a serious drawback, because the influence of codon usage on mRNA secondary structures, especially near the start of a gene, may strongly influence translation initiation. In this study, we aim to reduce the effect of codon usage on translation initiation by applying a bicistronic design (BCD) element. Protein production of several codon-optimized gene variants is tested in parallel for a BCD and a standard monocistronic design (MCD). We demonstrate that these distinct architectures can drastically change the relative performance of different codon optimization algorithms. We conclude that a BCD is indispensable in future studies that aim to reveal the impact of codon optimization and codon usage correlations. Furthermore, irrespective of the algorithm used, using a BCD does improve protein production compared with an MCD. The overall highest expression from BCDs for both GFP and RFP is at least twofold higher than the highest levels found for the MCDs, while for codon variants having very low expression from the MCD, even 10-fold to 100-fold increases in expression were achieved by the BCD. This shows the great potential of the BCD element for recombinant protein production.

SUBMITTER: Nieuwkoop T

PROVIDER: S-EPMC6302717 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Improved protein production and codon optimization analyses in Escherichia coli by bicistronic design.

Nieuwkoop Thijs T Claassens Nico J NJ van der Oost John J

Microbial biotechnology 20181128 1

Different codon optimization algorithms are available that aim at improving protein production by optimizing translation elongation. In these algorithms, it is generally not considered how the altered protein coding sequence will affect the secondary structure of the corresponding RNA transcript, particularly not the effect on the 5'-UTR structure and related ribosome binding site availability. This is a serious drawback, because the influence of codon usage on mRNA secondary structures, especia ...[more]

PMID: 30484964

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Project description:The ability to control the synthesis of products in the absence of cell division remains an attractive alternative for the optimization of microbial processes. If this could be achieved, it would dramatically expand the productivity of many microbial processes by increasing product synthesis in the absence of an increase in cell mass. In the present work we have looked at the factors involved in the design of a host where the fluxes would be channeled towards product formation rather than biomass synthesis. To identify the genes responsible for diverting the metabolic flux specifically towards product formation we have used for this study is quiescent-cell (Q-cell) expression system, in which a plasmid-encoded protein is expressed in nongrowing but metabolically active cells. These cells channel the metabolic flux towards recombinant protein production and therefore the specific product yield per unit biomass is significantly higher. Indole which is previously known to be a signalling molecule is here used as an inducer of Quiescence. E.coli L-Asparaginase is used as a model protein in this work. Fed batch cultures were carried out in Sartorius BIOSTAT B plus 5liter bioreactor. The data acquisition was done with the MFCS Shell software (version) provided with the bioreactor. The Sartorius BIOSTAT B plus had proportional integral derivative-based control loops for temperature, pH, antifoam, and dissolved-oxygen (DO) regulation. The culture was grown in a batch mode till 10-12 OD (µ=0.5) and then the feed was attached. In the next hour the culture was induced for product formation with 1M IPTG. In case of Q culture quiescence was induced with 0.5M Indole just 5min after induction with IPTG. Post induction every hour samples were collected and frozen with liquid nitrogen and stored at -80ºC for further analytical use. Total RNA extraction, RNA quality control (Agilent BioAnalyser), total RNA labeling, microarray hybridization and scanning were performed according to the Affymetrix GeneChip Expression analysis .

Dataset Information

Improved protein production and codon optimization analyses in Escherichia coli by bicistronic design.

Publications

Improved protein production and codon optimization analyses in Escherichia coli by bicistronic design.

Similar Datasets

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