Influence of specific growth rate over the secretory expression of recombinant potato carboxypeptidase inhibitor in fed-batch cultures of Escherichia coli

Abstract

A high cell density cultivation protocol was developed for the secretory production of potato carboxypeptidase inhibitor (PCI) in Escherichia coli. The strain BW25113 (pIMAM3) was cultured in fed-batch mode employing minimal media and an exponential feed profile where the specific growth rate was fixed by limitation of the fed carbon source (glycerol). Plasmid loss rates were found to be proportional to the specific growth rate. Distribution of PCI along the cell compartments and the culture media was also dependent on the fixed growth rate. When specific growth rate was kept at μ = 0.10 h⁻¹, 1.4 g PCI L⁻¹ were obtained when adding the product present in periplasmic extracts and supernatant fractions, with a 50% of the total expressed protein recovered from the extracellular medium. This constituted a 1.2-fold increase compared to growth at μ = 0.15 h⁻¹, and 2.0-fold compared to μ = 0.25 h⁻¹. Last, a cell permeabilization treatment with Triton X-100 and glycine was employed to direct most of the product to the culture media, achieving over 81% of extracellular PCI. Overall, our results point out that production yields of secretory proteins in fed-batch cultures of E. coli can be improved by means of process variables, with applications to the production of small disulfide-bridged proteins. Overall, our results point out that control of the specific growth rate is a successful strategy to improve the production yields of secretory expression in fed-batch cultures of E. coli, with applications to the production of small disulfide-bridged proteins.

Introduction

Secretory expression of heterologous proteins in Escherichia coli has a number of advantages over more common cytosolic expression. First, secretion of the recombinant product is attractive form a downstream processing stand-point, since no cell-disruption steps are needed and contamination with other proteins is reduced both in the periplasm and culture media [1], [2]. Secondly, the formation of disulfide bridges is actively catalyzed in the periplasmic space [3], [4]. Also, for proteins that are toxic to the host, secretion may palliate their detrimental effect over culture growth [2].

Several proteins have been successfully produced in the periplasmic space and culture supernatants of high cell density cultures of E. coli [5], [6], but since the capacity of the bacterial secretion machinery is limited and there are several factors that affect protein expression and translocation [7], [8], achieving high protein yields of protein exported through the inner membrane can be a complex task. In this sense, it has been proven that translational and translocation levels have to be properly coupled to reach a state where most of the expressed heterologous protein is secreted [9], [10]. This can be achieved by manipulations of genetic parameters like the promoter strength [11], the nature of the signal sequence [12], [13] or the plasmid copy number [14], but optimization of the culture protocols is also necessary. Previous studies show the influence of culture media composition, growth kinetics, induction moment and temperature over secretory protein yields [1], [2], [5].

Potato carboxypeptidase inhibitor (PCI) is a small protein naturally occurring in leafs and stems of Solanum tubesorum [15]. Composed by 39 residues and three disulfide bridges, it has potential biomedical applications given its proven antitumoral properties [16], [17]. PCI had previously been produced in E. coli using the pIN-III-ompA-derived plasmid pIMAM3, which allows for the translocation of the protein to the periplasmic space where formation of its disulfide bonds was successfully achieved and the active form could be recovered from culture supernatants [18], [19]. Excretion of PCI out of the cell envelope is probably favored by its small size and compact structure. A fed-batch procedure had previously been designed for the overexpression of PCI in high cell density cultures in semi-complex media, but relatively low levels of biomass (15 g DCW L⁻¹) were achieved, and the process was not automated, with feedstock additions not responding to any monitored variable. The aim of this work was to design a robust, automated and repeatable fed-batch process at bench-top level in order to increase the production of biologically active PCI by maximizing both the biomass concentrations and the expression-secretion of the inhibitor. Since it was observed that the specific growth rate (μ) had a major influence in the amounts of excreted PCI, a series of fermentations at different fixed growth rates were carried out. The dynamics of the PCI concentration profiles in the cytosol, periplasmic space and culture media was analyzed in order to identify and overcome the bottlenecks in the secretory production of this protein.