Abstract
BackgroundEscherichia coli (E. coli) is a bacteria that is widely employed in many industries for the production of high interest bio-products such as recombinant proteins. Nevertheless, the use of E. coli for recombinant protein production may entail some disadvantages such as acetate overflow. Acetate is accumulated under some culture conditions, involves a decrease in biomass and recombinant protein production, and its metabolism is related to protein lysine acetylation. Thereby, the carbon and nitrogen sources employed are relevant factors in cell host metabolism, and the study of the central metabolism of E. coli and its regulation is essential for optimizing the production of biomass and recombinant proteins. In this study, our aim was to find the most favourable conditions for carrying out recombinant protein production in E. coli BL21 using two different approaches, namely, manipulation of the culture media composition and the deletion of genes involved in acetate metabolism and Nε-lysine acetylation.ResultsWe evaluated protein overexpression in E. coli BL21 wt and five mutant strains involved in acetate metabolism (Δacs, ΔackA and Δpta) and lysine acetylation (ΔpatZ and ΔcobB) grown in minimal medium M9 (inorganic ammonium nitrogen source) and in complex TB7 medium (peptide-based nitrogen source) supplemented with glucose (PTS carbon source) or glycerol (non-PTS carbon source). We observed a dependence of recombinant protein production on acetate metabolism and the carbon and nitrogen source employed. The use of complex medium supplemented with glycerol as a carbon source entails an increase in protein production and an efficient use of resources, since is a sub-product of biodiesel synthesis. Furthermore, the deletion of the ackA gene results in a fivefold increase in protein production with respect to the wt strain and a reduction in acetate accumulation.ConclusionThe results showed that the use of diverse carbon and nitrogen sources and acetate metabolism knockout strains can redirect E. coli carbon fluxes to different pathways and affect the final yield of the recombinant protein bioprocess. Thereby, we obtained a fivefold increase in protein production and an efficient use of the resources employing the most suitable strain and culture conditions.
Highlights
Escherichia coli (E. coli) is a bacteria that is widely employed in many industries for the production of high interest bio-products such as recombinant proteins
Physiological characterization of the strains overexpressing pRSETA‐Green Fluorescent Protein expression levels (GFP) To determine the specific growth rates, biomass yields (YX/S) and specific carbon consumption rates (qs) for E. coli BL21 wt and deficient strains (ΔpatZ, ΔcobB, Δacs, ΔackA and Δpta) overexpressing pRSETA-GFP, all strains were grown in minimal medium M9 or in complex TB7 medium supplemented with 20 mM glucose or 40 mM glycerol
As regards to glyoxylate shunt we studied the relative gene transcription value of aceA, whose expression was downregulated in E. coli BL21 grown in glycerol. aceA expression is activated by catabolite repressor activator (Cra) and repressed by aerobic respiration control protein (ArcA) and cAMP-cAMP receptor protein (Crp) [38, 39]
Summary
Escherichia coli (E. coli) is a bacteria that is widely employed in many industries for the production of high interest bio-products such as recombinant proteins. In spite of all the advantages that this bacteria offers, the use of E. coli may entail production of by-products such as acetate, which involves a decrease in biomass and recombinant protein production [4, 5] For this reason, several strategies have been developed to limit acetate accumulation, such as the use of different media and culture conditions [6,7,8], the employment of genetic engineering to limit the formation and accumulation of this compound, the expression of sRNA or the enhancement of the respiratory activity [9,10,11,12,13,14,15]. K12 is the most studied E. coli strain (K strain), E. coli BL21 (B strain) is the most used for recombinant protein production because B strains lack some proteases, achieve higher biomass yields and produces much less acetate than E. coli K12, even in the presence of excess glucose [13, 16]
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