Abstract
BackgroundEscherichia coli is, to date, the most used microorganism for the production of recombinant proteins and biotechnologically relevant metabolites. High density cell cultures allow efficient biomass and protein yields. However, their main limitation is the accumulation of acetate as a by-product of unbalanced carbon metabolism. Increased concentrations of acetate can inhibit cellular growth and recombinant protein production, and many efforts have been made to overcome this problem. On the other hand, it is known that E. coli is able to grow on acetate as the sole carbon source, although this mechanism has never been employed for the production of recombinant proteins.ResultsBy optimization of the fermentation parameters, we have been able to develop a new acetate containing medium for the production of a recombinant protein in E. coli BL21(DE3). The medium is based on a buffering phosphate system supplemented with 0.5% yeast extract for essential nutrients and sodium acetate as additional carbon source, and it is compatible with lactose induction. We tested these culture conditions for the production of MNEI, a single chain derivative of the sweet plant protein monellin, with potential for food and beverage industries. We noticed that careful oxygenation and pH control were needed for efficient protein production. The expression method was also coupled to a faster and more efficient purification technique, which allowed us to obtain MNEI with a purity higher than 99%.ConclusionsThe method introduced represents a new strategy for the production of MNEI in E. coli BL21(DE3) with a simple and convenient process, and offers a new perspective on the capabilities of this microorganism as a biotechnological tool. The conditions employed are potentially scalable to industrial processes and require only low-priced reagents, thus dramatically lowering production costs on both laboratory and industrial scale. The yield of recombinant MNEI in these conditions was the highest to date from E. coli cultures, reaching on average ~180 mg/L of culture, versus typical LB/IPTG yields of about 30 mg/L.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0299-0) contains supplementary material, which is available to authorized users.
Highlights
Escherichia coli is, to date, the most used microorganism for the production of recombinant proteins and biotechnologically relevant metabolites
A recent paper demonstrated that the phenotypic differences between the two strains is due to the high expression of acetyl-CoA synthetase in glucose exponential phase in BL21, which allows the simultaneous consumption of acetate and glucose [25]
Optimization of the acetate based medium for the production of MNEI in shake flasks In order to define a more convenient growth medium with respect to the conditions previously used in the production of MNEI for structural and functional studies [35, 37, 40, 41], we first tested whether lactose could replace IPTG induction in the control (LB) condition
Summary
Escherichia coli is, to date, the most used microorganism for the production of recombinant proteins and biotechnologically relevant metabolites. High density cell cultures allow efficient biomass and protein yields Their main limitation is the accumulation of acetate as a by-product of unbalanced carbon metabolism. Several techniques have been devised to limit acetate accumulation These include modifications of the growth medium composition through the addition of amino acids or minerals [15, 16], the design of different process strategies (i.e. fed batch or dialysis culture) [17, 18] or gene engineering on the microorganisms to reduce acetate production and consequent accumulation [10]. A recent paper demonstrated that the phenotypic differences between the two strains is due to the high expression of acetyl-CoA synthetase (acs) in glucose exponential phase in BL21, which allows the simultaneous consumption of acetate and glucose [25]
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