Activation mechanism and activity of globupain, a thermostable C11 protease from the Arctic Mid-Ocean Ridge hydrothermal system

Deep-sea hydrothermal vents offer unique habitats for heat tolerant enzymes with potential new enzymatic properties. Here, we present the novel C11 protease globupain, which was prospected from a metagenome-assembled genome of uncultivated Archaeoglobales sampled from the Soria Moria hydrothermal vent system located on the Arctic Mid-Ocean Ridge. Sequence comparisons against the MEROPS-MPRO database showed that globupain has the highest sequence identity to C11-like proteases present in human gut and intestinal bacteria. Successful recombinant expression in Escherichia coli of the wild-type zymogen and 13 mutant substitution variants allowed assessment of residues involved in maturation and activity of the enzyme. For activation, globupain required the addition of DTT and Ca2+. When activated, the 52kDa proenzyme was processed at K137 and K144 into a 12kDa light- and 32kDa heavy chain heterodimer. A structurally conserved H132/C185 catalytic dyad was responsible for the proteolytic activity, and the enzyme demonstrated the ability to activate in-trans. Globupain exhibited caseinolytic activity and showed a strong preference for arginine in the P1 position, with Boc-QAR-aminomethylcoumarin (AMC) as the best substrate out of a total of 17 fluorogenic AMC substrates tested. Globupain was thermostable (Tm activated enzyme = 94.51°C ± 0.09°C) with optimal activity at 75°C and pH 7.1. Characterization of globupain has expanded our knowledge of the catalytic properties and activation mechanisms of temperature tolerant marine C11 proteases. The unique combination of features such as elevated thermostability, activity at relatively low pH values, and ability to operate under high reducing conditions makes globupain a potential intriguing candidate for use in diverse industrial and biotechnology sectors

Sequence-based enzyme discovery from marine microbial diversity : Multiple approaches for the heterologous expression of genes in Escherichia coli

The vast biodiversity of marine environments is increasingly being recognized as a source for new enzymes with value in both basic research and applications within biotechnology. In this project, the major aim was to identify, produce and perform biochemical and structural characterization on a selection of marine enzymes. These efforts were focused on bacterial proteases (Paper I, II) viral nucleases (Section 4.1, and 5.3.2), putative endolysins as well as a DNA-polymerase (Paper III). A versatile workflow for the cloning and heterologous expression of genes in Escherichia coli, focused on subtilisin-like proteases is described in Paper I. Fragment exchange cloning was used to insert genes into expression vectors featuring combinations of maltose binding protein (MBP), small ubiquitin-related modifier (SUMO) for improved solubility, and His-tags for protein purification with affinity chromatography. A casein-based assay was also featured to screen for proteolytic activity. All four Bacilli subtilisins tested were successfully produced in soluble and active forms. Constructs featuring N-terminal fusion proteins led to highest soluble yields and activity values for most, but not all genes tested, highlighting the value of including multiple vector configurations. Using this workflow, an intracellular subtilisin protease (ISP) from Planococcus sp. AW02J18, was produced and characterized (Paper II). Optimal activity was observed at pH 11 and 45 °C, with an active range from pH 7.0 to 11 and no activity above 60 °C. Sequence analyses of the ISP pro-peptide pointed at the presence of a conserved LIPY/F motif, understood to be centrally involved in the autocatalytic maturation of the enzyme via mutational analyses. The 3D structure of Planococcus ISP was solved at 1.3 Å resolution with X-ray crystallography, producing the second unique ISP structure to date, and the first with an intact native catalytic triad. The combined mutational study of the LIPY/F motif and the structure of the inhibitory pro-peptide contributed to better understanding of the maturation process of Planococcus ISP, and that of ISPs in general. As a part of the Virus-X project, heterologous production trials were carried out for 42 putative viral nucleases originating from arctic viromes (Aevarsson et al. under review, see Appendix I), with annotated similarity to T4-Endonuclease V, λ-Exonuclease, and Exonuclease III (Sections 4.1 and 5.3.2). Candidate genes were synthesized as codon optimized (CO) for expression in E. coli and pre-cloned into pET-family vectors encoding a His-tag for IMAC at the N- or C-terminal. Only twelve candidates were produced as soluble proteins, of which six were able to be purified. In turn, the results exemplify the challenges of using E. coli as an expression host for environmental viral genes and suggests that CO alone leads to limited success. In Paper III, the discovery of a novel prophage is reported from within the genome of Hypnocyclicus thermotrophus, a Gram-negative, thermophilic bacterium isolated from the Seven Sisters hydrothermal vent field. Designated Hypnocyclicus thermotrophus phage H1 (HTH1), the identified prophage genome was 41.6 kbp long and consisted of 46 protein-coding genes. Analysis and functional annotation of the HTH1 genome with multiple in silico approaches suggested closest taxonomic association to the viral family Siphoviridae. The lytic cassette of HTH1 showed closest similarity to viruses of Gram-positive bacteria. However, HTH1 was found encoding an N-acetylmuramoyl-L-alanine amidase not observed in other compared phages. Nine genes putatively related to lysis and nucleic acid processing were selected for heterologous production in E. coli. Besides CO genes, codon harmonized (CH) variants of each gene were also tested to be able to compare the two approaches’ effects on the soluble yield and thermostability of heterologous proteins. Five genes led to soluble protein from their CO variants, of which 4 were also soluble as CH variants. When compared, CO variants achieved higher soluble protein yields, but CH variants led to proteins with higher thermostability, as assessed by differential scanning fluorimetry. Taken together, this work presents results encompassing key steps of a sequence-based pipeline for the discovery of marine microbial enzymes. In addition, reported findings expand existing knowledge of ISPs and prophages of Fusobacteria from hydrothermal vent environments. The cases presented within are connected by a common theme of improving soluble production of heterologous proteins in E. coli, via various, compatible means. Coupled with the contemporary bioinformatics tools facilitating the functional annotation of a wider range of viral genes, complementary implementation of these approaches suggests a promising blueprint for future studies aiming the bioprospecting of marine microbial genetic resources