Two-step functional screen on multiple proteinaceous substrates reveals temperature-robust proteases with a broad-substrate range

Abstract

To support the bio-based industry in development of environment-friendly processes and products, an optimal toolbox of biocatalysts is key. Although functional screen of (meta)genomic libraries may potentially contribute to identifying new enzymes, the discovery of new enzymes meeting industry compliance demands is still challenging. This is particularly noticeable in the case of proteases, for which the reports of metagenome-derived proteases with industrial applicability are surprisingly limited. Indeed, proteolytic clones have been typically assessed by its sole activity on casein or skim milk and limited to mild screening conditions. Here, we demonstrate the use of six industry-relevant animal and plant by-products, namely bone, feather, blood meals, gelatin, gluten, and zein, as complementary substrates in functional screens and show the utility of temperature as a screening parameter to potentially discover new broad-substrate range and robust proteases for the biorefinery industry. By targeting 340,000 clones from two libraries of pooled isolates of mesophilic and thermophilic marine bacteria and two libraries of microbial communities inhabiting marine environments, we identified proteases in four of eleven selected clones that showed activity against all substrates herein tested after prolonged incubation at 55 °C. Following sequencing, in silico analysis and recombinant expression in Escherichia coli, one functional protease, 58% identical at sequence level to previously reported homologs, was found to readily hydrolyze highly insoluble zein at temperatures up to 50 °C and pH 9–11. It is derived from a bacterial group whose ability to degrade zein was unknown. This study reports a two-step screen resulting in identification of a new marine metagenome-derived protease with zein-hydrolytic properties at common biomass processing temperatures that could be useful for the modern biorefinery industry. Key points • A two-step multi-substrate strategy for discovery of robust proteases. • Feasible approach for shortening enzyme optimization to industrial demands. • A new temperature-tolerant protease efficiently hydrolyzes insoluble zein.

Microbial proteases: ubiquitous enzymes with innumerable uses

“…The enzyme was further purified with a yield of 77.27% and a specific activity of 8.5 U/µg. García-Moyano et al ( 2021 ) through a two-step functional screening approach, identified a new marine metagenome-derived protease that can hydrolyze insoluble zein at temperatures up to 50 °C and pH 9–11. The enzyme was derived from a bacterial group who’s potential to degrade zein was unknown.…”

Design and Characterization of In-One Protease-Esterase PluriZyme

“…Adding a site supporting proteolytic activity was targeted, as proteases are pivotal enzymes for the hydrolysis of peptide bonds in materials where proteins are abundant components and are also widely used in organic synthesis [ 21 ]. This is why they constitute 60–65% of the global industrial market, growing at an annual growth rate of 5.6% [ 22 ]. Through evolution, proteases have adapted to the wide range of conditions found in complex organisms (variations in pH, reductive environment, etc.)…”

“…Proteases specifically cleave protein substrates either from the N or C termini (aminopeptidases and carboxypeptidases, respectively) and/or in the middle of the molecule (endopeptidases). Proteases can be easily screened by functional screens or in silico predictions in microorganisms or microbial communities by applying genomic and metagenomic approaches [ 22 ]. Of course, it should be stressed that the novelty itself does not guarantee a better enzymatic performance and better opportunities for commercialization, whose analysis requires laborious wet lab work.…”

“…This is not a trivial exercise, given that not all genes in a genome or a metagenome can be successfully cloned and expressed. This is especially important in the cases of proteases that suffer major problems of expression, compared to other types of enzymes that are as easy to be screened as proteases but better to be produced at high levels [ 22 ]. Proteases are used in a broad range of applications, including biorefineries targeting a broad range of biomasses [ 22 ].…”

Isolation, identification, and biochemical characterization of a novel bifunctional phosphomannomutase/phosphoglucomutase from the metagenome of the brown alga Laminaria digitata

“…Metagenomic approaches are typically employed to exploit the unculturable fraction of different environmental ecosystems, (Vieites et al, 2009), where metagenomic DNA (mDNA) is extracted from the environmental sample and analyzed using high throughput sequencing technologies to identify potential targeted enzymes (Dinsdale et al, 2008), which can then be cloned into heterologous expression systems and screened for the desired phenotype (Kennedy et al, 2011;Coughlan et al, 2015;Garcia-Moyano et al, 2021;Newgas et al, 2021). Alternatively, mDNA can be cloned directly into a suitable vector in heterologous hosts such as Escherichia coli, Streptomyces lividans, Pseudomonas putida, and Bacillus subtilis (Lam et al, 2015;Zhang et al, 2017), thereby generating a metagenomic library which can then be screened using phenotypic based assay systems to detect the newly acquired phenotype conferred on the host by the metagenomic clone (Coughlan et al, 2015;Lam et al, 2015;Ngara 10.3389/fmicb.2022.1000634 Frontiers in Microbiology 03 frontiersin.org .…”