Effect of Clp protease from Corynebacterium glutamicum on heterologous protein expression

Highlights

• Using Clp protease overexpression and deletion strategies.

• The ΔclpS strain had the strongest fluorescence value in C. glutamicum ATCC 13032.

• •glutamicum 1.15647 with clpS deleted had a higher fluorescence value than 13032-ΔclpS.

• The VHH yield of 15647-ΔclpS increased by nearly 65% to approximately 530 mg/L.

Abstract

The protease present in a host may reduce the yield and biological activity of heterologous proteins. In this study, we used protease overexpression and deletion strategies to examine the effect of the Clp protease system in Corynebacterium glutamicum on the recombinant protein and to produce a highly efficient heterologous protein expression host. In this study, we identified seven genes in the Clp protease family in Corynebacterium glutamicum ATCC 13032 through bioinformatics analysis, and studied their effects on the enhanced green fluorescent protein (EGFP) reporter protein. The fluorescence intensity of the knockout strain was significantly higher, and the effect of the clpS deletion strain was the most obvious. To verify the universal effect of the lack of clpS, the excellent industrial strain C. glutamicum 1.15647 was transformed to form recombinant 15647-ΔclpS. Based on the results, 15647-ΔclpS had a more significant effect on improving protein expression. Furthermore, recombinant human teriparatide (rhPTH) and variable domain of heavy chain of heavy-chain antibody (VHH) were selected to verify the universal applicability of the knockout strain for expressing heterologous proteins. Accordingly, we found that protease deficiency could increase the production of heterologous proteins. Finally, through a large-scale fermentation, the 15647-ΔclpS strain was used to produce VHH. Its yield was approximately 530 mg/L, which was 65% higher than that of WT-15647. In this study, a host that could effectively increase heterologous protein expression was successfully obtained.

Introduction

The gram-positive bacterium, Corynebacterium glutamicum, as a cell factory has been used in synthetic biology to produce high-value-added compounds and recombinant pharmaceutical proteins, owing to its many advantages, such as endotoxin-free, high-density production, and an ideal protein secretion system [1]. However, there are still serious problems with the overexpression of many heterologous proteins, such as low expression levels and undesired target protein degradation. Most current studies on improving cell expression levels focus on host selection and engineering, including the discovery of efficient promoters [2] and expression elements, modifying related metabolic pathways [3], enhancing secretion ability, weakening the protease system activity, and other strategies [[4], [5], [6]].

Undesired proteolysis of heterologous proteins expressed in the host is a serious problem because it can decrease the product yield or biological activity and cause the intact product to be contaminated by partially degraded products. This is caused by the presence of proteases in the host itself, which can eliminate proteins that may be irreversibly damaged, misfolded, and harm themselves due to spontaneous mutations or stress reactions, including heterologous proteins. The use of engineered expression hosts to obtain protease-deficient strains can alleviate proteolytic problems. Therefore, genetic manipulation of host proteases is an essential strategy for reducing host-specific degradation.

Currently, the proteases of many strains have been studied, such as the Lon, OmpT, DegP and RseP proteases of Escherichia. coli [[7], [8], [9], [10]], the HtrA protease of Listeria, and the ClpXp protease of Chlamydia trachomatis [11], a protease similar to Fus [12]. However, only few studies have assessed the protease system of C. glutamicum, and only few proteases have been found, such as SprA protease [13], FtsH protease [14] and the Clp protease family [15,16]. Clp protease is an ATP-dependent serine protease that is composed of catalytic and regulatory subunits that are widely present in various organisms [17]. The catalytic subunit is the ClpP protein family, which is comprised of two ClpP that form a cylindrical structure containing 14 enzyme active sites [18]. In contrast, the regulatory subunit is composed of the Hsp100/Clp chaperone protein hexamer with ATPase activity, which is bound to the same or different sides of the catalytic subunit [19,20].

Different types of Clp proteins have been detected in bacteria, fungi, cyanobacteria, animals, and plants [21]. ClpP, ClpA, and ClpX proteins were originally identified in E. coli. ClpP is a highly conserved AAA protease in most bacteria species, such as Bacillus subtilis, Mycobacterium tuberculosis, Helicobacter pylori and Staphylococcus aureus [22,23]. In addition, other proteins such as ClpB, ClpC, ClpD, ClpE, and ClpY, exist and most are present in bacteria and fungi [[24], [25], [26], [27]].

As the Clp protease has not been systematically studied, the Clp protein family was selected as the research target for the present study. Research on the Clp protease in C. glutamicum is expected to aid in the reduction of heterologous proteins degradation.

In this study, the effect of the Clp protease on heterologous protein expression in C. glutamicum was investigated. By employing protease gene overexpression strains and knockout strains, the rhPTH and VHH heterologous proteins were used to examine the effect of each protease on protein expression. Accordingly, protease knockout was found to promote the expression of heterologous proteins. Such findings imply that weakening the protease system is very effective at increasing protein expression and can add value to many applications in the pharmaceutical industry.