Topology engineering via protein catenane construction to strengthen an industrial biocatalyst

Highlights

• Two different protein catenanes were constructed for an industrial biocatalyst.

• Protein catenation enhanced both the thermo- and proteolytic stabilities of gamma lactamase.

• Catenated enzymes displayed higher enzyme efficiencies (Kcat/Km).

Abstract

Protein topology engineering has emerged as a new dimension to alter protein stability and function. Inspired by the art of nature, where backbone cyclization is frequently adopted to enhance the stability of natural peptide products and thermostable enzymes; herein, we report protein topology engineering of an industrial thermolabile gamma lactamase via catenation. Two different protein catenanes were successfully constructed via SpyTag/SpyCatcher modules and two different peptide dimer domains. The designed protein catenanes were functionally synthesized in Escherichia coli. A comparison of their biochemical properties revealed that protein topology played a key role in the stability of gamma lactamase. Protein catenation enhanced both the thermo- and proteolytic stabilities of gamma lactamase. Gamma lactamase was stabilized by ∼8 °C in one of the catenated forms. Moreover, Cat1-MhIHL-V54L and Cat2-MhIHL-V54L displayed 1.8- and 2.4-fold higher enzyme efficiencies (Kcat/Km), respectively, than the unattenuated enzyme. Therefore, our results proved that protein catenane construction could be a general strategy to strengthen industrial biocatalysts by mechanisms distinct from those of the conventional direct evolution schemes, whereby our results offer wide applications in the fine chemical industry.

Introduction

Polypeptides or proteins are among the most complex, diverse and functional macromolecule polymers generated in nature, which is the source of all living matter. Unlike organic polymers, polypeptides or proteins are initially synthesized as linear chains by sophisticated cellular translational machineries called ribosomes (Brar and Weissman, 2015). One challenge these polypeptides or proteins face in a complex cellular environment is the presence of a large number of peptidases and proteases, which impair protein stability. To resist protease degradation, small polypeptides have evolved diverse strategies to change their properties, including topological properties and post-translational modifications (Arnison et al., 2013). For example, ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly growing family of natural products initially synthesized from 20 proteinogenic amino acids (Arnison et al., 2013). However, extensive post-translational modification strategies have emerged during evolution, resulting in highly stable compounds. Cyclization is one of the most frequent modifications among these strategies (Arnison et al., 2013). For instance, sactipeptides are medium-sized peptides with several intramolecular thioether bonds that crosslink the α-carbon of an acceptor amino acid and the sulfur atom of a cysteine residue (Fluhe and Marahiel, 2013). Lasso peptides, on the other hand, have evolved a defining lasso structural feature by an isopeptide bond formed between the N-terminal α-amino group and the carboxylic acid side chain of an aspartate or glutamate at positions 7, 8, or 9 of the amino acid sequence (Hegemann et al., 2015; Maksimov et al., 2012; Zhu et al., 2019). Other peptides, such as lanthipeptides, cyanobactins, and thiopeptides have also evolved diverse macrocyclization strategies to decrease conformational flexibility and increase metabolic stability (Arnison et al., 2013).

In contrast to small peptides, proteins are translated as linear chains and directly folded into defined 3D structures. Few post-translational modifications have been observed for most known enzymes, and these modifications include phosphorylation, acetylation, glycosylation, methylation, and ubiquitylation (Witze et al., 2007). Topological heterogeneity including cyclization is not common strategy adopted by proteins due to their folded structures except for disulfide bond formation (Appleby et al., 2001; Liu et al., 2016). Nevertheless, nature does evolve such strategies under extraordinary conditions for protein stability as well. One special case is the bacterial pili and adhesins that are formed by the assembly of multi-subunit proteins (Kang and Baker, 2012). Recently, structural characterization has shown that the pili of gram-positive pathogens are covalent polymers. Further studies have revealed that autocatalytically generated isopeptide or thioester bond crosslinks are common modifications formed to stabilize each subdomain (Pointon et al., 2010; Zakeri et al., 2012). This unusual cyclization strategy can remarkably increase the robustness of the protein, conferring an organic-polymer–like property on the protein. These observations also inspire new perspectives in biotechnological applications. For instance, SpyTag/SpyCatcher is a peptide/protein partner developed from the adhesin domain (CnaB2) of Streptococcus pyogenes fibronectin-binding protein FbaB (Zakeri et al., 2012). To develop this system, a 13-amino-acid-long peptide (SpyTag) and modified partner protein (138 amino acids, 15 kDa, SpyCatcher) are generated by splitting CnaB2. When these two parts encounter each other, isopeptide bonds spontaneously form between them. After the invention of this technology, SpyTag/SpyCatcher has quickly been adopted as one of the most useful tools for topology engineering and widely applied in synthetic biology, nanobiotechnology, and biomaterials (Sutherland et al., 2019).

Besides the isopeptide bond cyclization, bioengineers have been developing other tools for topology engineering of target proteins by either chemical or biological approaches. For example, Link et al. have recently reported a residue-specific incorporation technique to introduce azidohomoalanine and p-ethynylphenylalanine into a model leucine zipper protein and the globular protein G (Abdeljabbar et al., 2014). These two noncanonical amino acids can autocatalytically be crosslinked via the azide-alkyne ligation chemistry, resulting in more stable proteins. Another interesting study conducted by the same group has demonstrated the construction of a lasso peptide fusion protein by the manipulation of the lasso peptide biosynthesis machinery (Zong et al., 2016). The lasso structure introduced at the N-terminus presumably confers more stability on the protein against proteolytic degradation. Lately, Cornelissen et al. have used a mEETI-II knottin mini protein from the cysteine-stabilized knot class to functionalize a Thermotoga maritima encapsulin nanoparticle (Klem et al., 2018). The generated cyclotide can protect the protein cage from trypsin degradation, and thus this study provides another successful case of manipulating a folded peptide for topology engineering. We have recently shown that an artificial protein dodecahedron can also be used as a scaffold platform for enzyme protection (Li et al., 2018). All these examples have proven that topology engineering is a very promising strategy to strengthen target enzymes and a new direction for protein engineering in the future.

Molecular catenanes featuring rings interlocked with mechanical bonds are unusual forms seldom observed in proteins and have been considered an important topology for protein stability (Wang and Zhang, 2018). It has been shown that protein catenation can benefit folded structural domains (Wang and Zhang, 2016, 2017). Biocatalysts frequently show matchless regio- or stereo-selectivity in comparison with chemical catalysts but suffer from instability (Bommarius and Paye, 2013). Therefore, protein catenation can have wide applications in industrial biotechnology. Herein, we constructed two different protein catenanes for the gamma lactamase (γ-lactamase) MhIHL-V54L. In vitro studies have shown that different topologies can significantly change the properties of target enzymes. Our data prove that topology plays a key role in the stability of biocatalysts, and protein catenane construction can be adopted as a general strategy to strengthen industrial biocatalysts.