Production of extracellular PETase from Ideonella sakaiensis using sec-dependent signal peptides in E. coli

https://doi.org/10.1016/j.bbrc.2018.11.087Get rights and content

Highlights

  • PETase from Ideonella sakaiensis (IsPETase) was successfully produced using the protein secretory expression system.

  • Extracellular production of IsPETase is achieved by sec-dependent secretion system.

  • The extracellularly produced IsPETase shows a PET degradation activity.

Abstract

Poly(ethylene terephthalate) (PET) is the most commonly used polyester polymer resin in fabrics and storage materials, and its accumulation in the environment is a global problem. The ability of PET hydrolase from Ideonella sakaiensis 201-F6 (IsPETase) to degrade PET at moderate temperatures has been studied extensively. However, due to its low structural stability and solubility, it is difficult to apply standard laboratory-level IsPETase expression and purification procedures in industry. To overcome this difficulty, the expression of IsPETase can be improved by using a secretion system. This is the first report on the production of an extracellular IsPETase, active against PET film, using Sec-dependent translocation signal peptides from E. coli. In this work, we tested the effects of fusions of the Sec-dependent and SRP-dependent signal peptides from E. coli secretory proteins into IsPETase, and successfully produced the extracellular enzyme using pET22b-SPMalE:IsPETase and pET22b-SPLamB:IsPETase expression systems. We also confirmed that the secreted IsPETase has PET-degradation activity. The work will be used for development of a new E. coli strain capable of degrading and assimilating PET in its culture medium.

Introduction

Poly (ethylene terephthalate) (PET) is one of the most common consumer plastics, and is in tremendous demand for use in particular containers and fibers [1]. For decades, use of PET plastics has dramatically increased due to its low price, lightness, processability, and bio-inertness. However, its non-degradability is a mixed blessing, and environmental groups, governments, and the public are greatly concerned that its extreme resistance to decomposition has allowed it to accumulate in large amounts of PET waste in the oceans and other ecosystems. Much effort has therefore been exerted towards developing eco-friendly degradation of PET using microorganisms. Recently, a gram-negative bacterium, Ideonella sakaiensis 201-F6, that is able to use PET as an energy and carbon source was isolated [[2], [3], [4]]. The bacterium utilizes two important hydrolases to degrade PET: PET hydrolase (IsPETase) and mono (2-hydroxyethyl) terephthalate hydrolase (IsMHETase) [2]. The PETase has been shown to hydrolyze PET into MHET, a monomer, at a moderate temperature (30 °C), while the MHETase further degrades MHET into ethylene glycol and terephthalate [2]. Structural investigations of IsPETase have revealed that the enzyme's mechanism of action is unique, having higher activity on PET films than other hydrolases/esterases, and that it is able to utilize extremely large and hydrophobic polymers [[5], [6], [7], [8], [9]]. Therefore, enzymatic hydrolysis of PET using this enzyme is expected to provide a solution to environmental contamination by plastics.

Recent studies on IsPETase have primarily employed recombinantly expressed and purified enzymes [[5], [6], [7],9]. However, recombinant expression systems have serious disadvantages when applied to microbial degradation of PET, including a high cost of purification, and low stability, solubility, and yield of the recombinant enzymes. A potential means of solving these problems is fusion of the enzyme to an N-terminus signal peptide, which allows for translocation of the protein precursors from the cytoplasm to the periplasmic and/or the extracellular space [10]. I. sakaiensis naturally secretes these enzymes into the extracellular space because PET polymer cannot cross the lipid bilayer membranes of gram-negative microbes. Continuous secretory production of IsPETase by host cells in batch/fed-batch/continuous culture medium may overcome the enzyme's loss of activity over time, resulting from its relatively low structural stability, and its melting temperature of 46.8 °C [6] (Fig. 1). In addition, it is generally known that the oxidative environment of the periplasm helps in the formation of disulfide bonds [11]. The unique existence of two disulfide bridges in IsPETase and their importance in enzyme activity [6] implies that periplasmic expression of IsPETase can aid its proper folding.

A strongly preferred secretory expression system in gram-negative Escherichia coli K12 strain is the type II secretion mechanism, which is a two-step process and involves periplasmic and extracellular translocation [12]. Three known pathways (the Sec-dependent pathway, signal recognition particle (SRP) pathway, and twin-arginine translocation (TAT) pathway) are involved in the periplasmic translocation within this system [13]. The Sec-dependent and SRP pathways, which are difficult to discriminate in vivo, are related to post-/co-translational translocation of prefolded polypeptides through the inner membrane, while the TAT pathway exports the folded protein [14]. In this study, we tested the effects of fusing Sec-dependent signal peptides from E. coli secretory proteins into IsPETase, and succeeded in producing the extracellular enzyme, which is active toward PET film, using E. coli. This work may contribute to the development of a recombinant PET-degrading microbe for use in environmental remediation.

Section snippets

Construction of expression vectors

The codon-optimized gene for IsPETase was synthesized [6], and subcloned into a pET22b(+) vector (Novagen/Merck) without its leader sequence. Due to the existence of an Nco I restriction site (CCATGG), we added two nucleotides (CG) in front of the inserted gene to prevent a frame shift, resulting in the addition of Met and Ala at the N-terminus of the secreted proteins. The PelB leader sequence of the cloning product (pET22b:IsPETase) was replaced using Nde I and NcoI restriction enzymes with

Selection of signal peptides

We previously reported expression and purification of IsPETase without its leader peptide using pET15b, which contains a T7 promoter for overexpressing the enzyme in E. coli Rosetta Gami-B cytoplasm [6]. For production of extracellular IsPETase, a signal peptide compatible with E. coli secretion systems needed to be introduced. Because of the importance of selecting an optimal signal peptide, we predicted by which secretory pathway, the Sec-dependent pathway or TAT pathway, the original Is

Acknowledgements

This work was carried out with the support the Next Generation BioGreen 21 Program (Code No. PJ01326503), Rural Development Administration, Republic of Korea, and also supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning through the National Research Foundation of Korea (NRF-2017M1A2A2087631). H. Seo, H.F Son and Hsingle bondY Sagong were supported by the Global PhD Fellowship

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