Course-based Undergraduate Research Module for Enzyme Discovery Using Protein Structure Prediction

A major challenge for science educators is teaching foundational concepts while introducing their students to current research. Here we describe an active learning module developed to teach protein structure fundamentals while supporting ongoing research in enzyme discovery. It can be readily implemented in both entry-level and upper-division college biochemistry or biophysics courses. Pre-activity lectures introduced fundamentals of protein secondary structure and provided context for the research projects, while a homework assignment familiarized students with 3D visualization of biomolecules using UCSF Chimera, a free protein structure viewer. The activity is an online survey in which students compare structure elements in papain, a well-characterized cysteine protease from Carica papaya, to novel homologous proteases identified from the genomes of an extremophilic microbe (Halanaerobium praevalens) and two carnivorous plants (Drosera capensis and Cephalotus follicularis). Students were then able to identify, with varying levels of accuracy, a number of structural features in cysteine proteases that could expedite the identification of novel or biochemically interesting cysteine proteases for experimental validation in a university laboratory. Student responses to a post-activity survey were largely positive and constructive, indicating that the activity helped them learn about protein structure and describing points in the activity that could be improved.

Polysaccharide utilization loci-driven enzyme discovery reveals BD-FAE: a bifunctional feruloyl and acetyl xylan esterase active on complex natural xylans

Background Nowadays there is a strong trend towards a circular economy using lignocellulosic biowaste for the production of biofuels and other bio-based products. The use of enzymes at several stages of the production process (e.g., saccharification) can offer a sustainable route due to avoidance of harsh chemicals and high temperatures. For novel enzyme discovery, physically linked gene clusters targeting carbohydrate degradation in bacteria, polysaccharide utilization loci (PULs), are recognized ‘treasure troves’ in the era of exponentially growing numbers of sequenced genomes. Results We determined the biochemical properties and structure of a protein of unknown function (PUF) encoded within PULs of metagenomes from beaver droppings and moose rumen enriched on poplar hydrolysate. The corresponding novel bifunctional carbohydrate esterase (CE), now named BD-FAE, displayed feruloyl esterase (FAE) and acetyl esterase activity on simple, synthetic substrates. Whereas acetyl xylan esterase (AcXE) activity was detected on acetylated glucuronoxylan from birchwood, only FAE activity was observed on acetylated and feruloylated xylooligosaccharides from corn fiber. The genomic contexts of 200 homologs of BD-FAE revealed that the 33 closest homologs appear in PULs likely involved in xylan breakdown, while the more distant homologs were found either in alginate-targeting PULs or else outside PUL contexts. Although the BD-FAE structure adopts a typical α/β-hydrolase fold with a catalytic triad (Ser-Asp-His), it is distinct from other biochemically characterized CEs. Conclusions The bifunctional CE, BD-FAE, represents a new candidate for biomass processing given its capacity to remove ferulic acid and acetic acid from natural corn and birchwood xylan substrates, respectively. Its detailed biochemical characterization and solved crystal structure add to the toolbox of enzymes for biomass valorization as well as structural information to inform the classification of new CEs.

A multi-omics approach to lignocellulolytic enzyme discovery reveals a new ligninase activity from Parascedosporium putredinis NO1

Lignocellulose, the structural component of plant cells, is a major agricultural byproduct and the most abundant terrestrial source of biopolymers on Earth. The complex and insoluble nature of lignocellulose limits its conversion into value-added commodities, and currently, efficient transformation requires expensive pretreatments and high loadings of enzymes. Here, we report on a fungus from the Parascedosporium genus, isolated from a wheat-straw composting community, that secretes a large and diverse array of carbohydrate-active enzymes (CAZymes) when grown on lignocellulosic substrates. We describe an oxidase activity that cleaves the major β-ether units in lignin, thereby releasing the flavonoid tricin from monocot lignin and enhancing the digestion of lignocellulose by polysaccharidase mixtures. We show that the enzyme, which holds potential for the biorefining industry, is widely distributed among lignocellulose-degrading fungi from the Sordariomycetes phylum.

Organizing Multi-Enzyme Systems into Programmable Materials for Biocatalysis

Significant advances in enzyme discovery, protein and reaction engineering have transformed biocatalysis into a viable technology for the industrial scale manufacturing of chemicals. Multi-enzyme catalysis has emerged as a new frontier for the synthesis of complex chemicals. However, the in vitro operation of multiple enzymes simultaneously in one vessel poses challenges that require new strategies for increasing the operational performance of enzymatic cascade reactions. Chief among those strategies is enzyme co-immobilization. This review will explore how advances in synthetic biology and protein engineering have led to bioinspired co-localization strategies for the scaffolding and compartmentalization of enzymes. Emphasis will be placed on genetically encoded co-localization mechanisms as platforms for future autonomously self-organizing biocatalytic systems. Such genetically programmable systems could be produced by cell factories or emerging cell-free systems. Challenges and opportunities towards self-assembling, multifunctional biocatalytic materials will be discussed.

From Data Mining of Chitinophaga sp. Genome to Enzyme Discovery of a Hyperthermophilic Metallocarboxypeptidase

For several centuries, microorganisms and enzymes have been used for many different applications. Although many enzymes with industrial applications have already been reported, different screening technologies, methods and approaches are constantly being developed in order to allow the identification of enzymes with even more interesting applications. In our work, we have performed data mining on the Chitinophaga sp. genome, a gram-negative bacterium isolated from a bacterial consortium of sugarcane bagasse isolated from an ethanol plant. The analysis of 8 Mb allowed the identification of the chtcp gene, previously annotated as putative Cht4039. The corresponding codified enzyme, denominated as ChtCP, showed the HEXXH conserved motif of family M32 from thermostable carboxypeptidases. After expression in E. coli, the recombinant enzyme was characterized biochemically. ChtCP showed the highest activity versus benziloxicarbonil Ala-Trp at pH 7.5, suggesting a preference for hydrophobic substrates. Surprisingly, the highest activity of ChtCP observed was between 55 °C and 75 °C, and 62% activity was still displayed at 100 °C. We observed that Ca2+, Ba2+, Mn2+ and Mg2+ ions had a positive effect on the activity of ChtCP, and an increase of 30 °C in the melting temperature was observed in the presence of Co2+. These features together with the structure of ChtCP at 1.2 Å highlight the relevance of ChtCP for further biotechnological applications.

Correction to: Combined whole cell wall analysis and streamlined in silico carbohydrate‑active enzyme discovery to improve biocatalytic conversion of agricultural crop residues

An amendment to this paper has been published and can be accessed via the original article.

Symbiont-Mediated Digestion of Plant Biomass in Fungus-Farming Insects

Feeding on living or dead plant material is widespread in insects. Seminal work on termites and aphids has provided profound insights into the critical nutritional role that microbes play in plant-feeding insects. Some ants, beetles, and termites, among others, have evolved the ability to use microbes to gain indirect access to plant substrate through the farming of a fungus on which they feed. Recent genomic studies, including studies of insect hosts and fungal and bacterial symbionts, as well as metagenomics and proteomics, have provided important insights into plant biomass digestion across insect–fungal mutualisms. Not only do advances in understanding of the divergent and complementary functions of complex symbionts reveal the mechanism of how these herbivorous insects catabolize plant biomass, but these symbionts also represent a promising reservoir for novel carbohydrate-active enzyme discovery, which is of considerable biotechnological interest.