Protein representations: Encoding biological information for machine learning in biocatalysis.

Enzymes offer a more environmentally friendly and low-impact solution to conventional chemistry, but they often require additional engineering for their application in industrial settings, an endeavour that is challenging and laborious. To address this issue, the power of machine learning can be harnessed to produce predictive models that enable the in silico study and engineering of improved enzymatic properties. Such machine learning models, however, require the conversion of the complex biological information to a numerical input, also called protein representations.

Action and cooperation in alginate degradation by three enzymes from the human gut bacterium Bacteroides eggerthii DSM 20697.

Alginate is a polysaccharide consumed by humans in edible seaweed and different foods where it is applied as a texturizing hydrocolloid or in encapsulations of drugs and probiotics. While gut bacteria are found to utilize and ferment alginate to health-beneficial short-chain fatty acids, knowledge on the details of the molecular reactions is sparse. Alginates are composed of mannuronic acid (M) and its C-5 epimer guluronic acid (G).

Enzymatic glycosylation of aloesone performed by plant UDP-dependent glycosyltransferases.

Aloesone is a bioactive natural product and biosynthetic precursor of rare glucosides found in rhubarb and some aloe plants including Aloe vera. This study aimed to investigate biocatalytic aloesone glycosylation and more than 400 uridine diphosphate-dependent glycosyltransferase (UGT) candidates, including multifunctional and promiscuous enzymes from a variety of plant species were assayed. As a result, 137 selective aloesone UGTs were discovered, including four from the natural producer rhubarb.

GASP: A Pan-Specific Predictor of Family 1 Glycosyltransferase Acceptor Specificity Enabled by a Pipeline for Substrate Feature Generation and Large-Scale Experimental Screening.

Glycosylation represents a major chemical challenge; while it is one of the most common reactions in Nature, conventional chemistry struggles with stereochemistry, regioselectivity, and solubility issues. In contrast, family 1 glycosyltransferase (GT1) enzymes can glycosylate virtually any given nucleophilic group with perfect control over stereochemistry and regioselectivity. However, the appropriate catalyst for a given reaction needs to be identified among the tens of thousands of available sequences.

A growth selection system for sucrose synthases (SuSy): design and test.

High throughput screening (HTS) methods of enzyme variants are essential for the development of robust biocatalysts suited for low impact, industrial scale, biobased synthesis of a myriad of compounds. However, for the majority of enzyme classes, current screening methods have limited throughput, or need expensive substrates in combination with sophisticated setups. Here, we present a straightforward, high throughput selection system that couples sucrose synthase activity to growth.

The sugar donor specificity of plant family 1 glycosyltransferases.

Plant family 1 glycosyltransferases (UGTs) represent a formidable tool to produce valuable natural and novel glycosides. Their regio- and stereo-specific one-step glycosylation mechanism along with their inherent wide acceptor scope are desirable traits in biotechnology. However, their donor scope and specificity are not well understood.

A life cycle assessment of early-stage enzyme manufacturing simulations from sustainable feedstocks.

Enzyme-catalyzed reactions have relatively small environmental footprints. However, enzyme manufacturing significantly impacts the environment through dependence on traditional feedstocks. With the objective of determining the environmental impacts of enzyme production, the sustainability potential of six cradle-to-gate enzyme manufacturing systems focusing on glucose, sea lettuce, acetate, straw, and phototrophic growth, was thoroughly evaluated.

Chemoenzymatic indican for light-driven denim dyeing.

Blue denim, a billion-dollar industry, is currently dyed with indigo in an unsustainable process requiring harsh reducing and alkaline chemicals. Forming indigo directly in the yarn through indican (indoxyl-β-glucoside) is a promising alternative route with mild conditions. Indican eliminates the requirement for reducing agent while still ending as indigo, the only known molecule yielding the unique hue of blue denim.

The function of UDP-glycosyltransferases in plants and their possible use in crop protection.

Glycosyltransferases catalyse the transfer of a glycosyl moiety from a donor to an acceptor. Members of this enzyme class are ubiquitous throughout all kingdoms of life and are involved in the biosynthesis of countless types of glycosides. Family 1 glycosyltransferases, also referred to as uridine diphosphate-dependent glycosyltransferases (UGTs), glycosylate small molecules such as secondary metabolites and xenobiotics.

An improved integrative GFP-based vector for genetic engineering of Parageobacillus thermoglucosidasius facilitates the identification of a key sporulation regulator.

Parageobacillus thermoglucosidasius is a thermophilic Gram-positive bacterium, which is a promising host organism for sustainable bio-based production processes. However, to take full advantage of the potential of P. thermoglucosidasius, more efficient tools for genetic engineering are required.

Functional characterization of the phosphotransferase system in Parageobacillus thermoglucosidasius.

Parageobacillus thermoglucosidasius is a thermophilic bacterium characterized by rapid growth, low nutrient requirements, and amenability to genetic manipulation. These characteristics along with its ability to ferment a broad range of carbohydrates make P. thermoglucosidasius a potential workhorse in whole-cell biocatalysis.

Sequence mining yields 18 phloretin C-glycosyltransferases from plants for the efficient biocatalytic synthesis of nothofagin and phloretin-di-C-glycoside.

C-glycosyltransferases (C-GTs) offer selective and efficient synthesis of natural product C-glycosides under mild reaction conditions. In contrast, the chemical synthesis of these C-glycosides is challenging and environmentally harmful. The rare occurrence of C-glycosylated compounds in Nature, despite their stability, suggests that their biosynthetic enzymes, C-GTs, might be scarce.

Family 1 glycosyltransferases (GT1, UGTs) are subject to dilution-induced inactivation and low chemo stability toward their own acceptor substrates.

Glycosylation reactions are essential but challenging from a conventional chemistry standpoint. Conversely, they are biotechnologically feasible as glycosyltransferases can transfer sugar to an acceptor with perfect regio- and stereo-selectivity, quantitative yields, in a single reaction and under mild conditions. Low stability is often alleged to be a limitation to the biotechnological application of glycosyltransferases.

Structure-guided engineering of key amino acids in UGT85B1 controlling substrate and stereo-specificity in aromatic cyanogenic glucoside biosynthesis.

Cyanogenic glucosides are important defense molecules in plants with useful biological activities in animals. Their last biosynthetic step consists of a glycosylation reaction that confers stability and increases structural diversity and is catalyzed by the UDP-dependent glycosyltransferases (UGTs) of glycosyltransferase family 1. These versatile enzymes have large and varied substrate scopes, and the structure-function relationships controlling scope and specificity remain poorly understood.

Discovery of fungal oligosaccharide-oxidising flavo-enzymes with previously unknown substrates, redox-activity profiles and interplay with LPMOs.

Oxidative plant cell-wall processing enzymes are of great importance in biology and biotechnology. Yet, our insight into the functional interplay amongst such oxidative enzymes remains limited. Here, a phylogenetic analysis of the auxiliary activity 7 family (AA7), currently harbouring oligosaccharide flavo-oxidases, reveals a striking abundance of AA7-genes in phytopathogenic fungi and Oomycetes.

Natural product C-glycosyltransferases - a scarcely characterised enzymatic activity with biotechnological potential.

Covering: up to 2020C-Glycosyltransferases are enzymes that catalyse the transfer of sugar molecules to carbon atoms in substituted aromatic rings of a variety of natural products. The resulting β-C-glycosidic bond is more stable in vivo than most O-glycosidic bonds, hence offering an attractive modulation of a variety of compounds with multiple biological activities. While C-glycosylated natural products have been known for centuries, our knowledge of corresponding C-glycosyltransferases is scarce.

Molecular characteristics of plant UDP-arabinopyranose mutases.

l-arabinofuranose is a ubiquitous component of the cell wall and various natural products in plants, where it is synthesized from cytosolic UDP-arabinopyranose (UDP-Arap). The biosynthetic machinery long remained enigmatic in terms of responsible enzymes and subcellular localization. With the discovery of UDP-Arap mutase in plant cytosol, the demonstration of its role in cell-wall arabinose incorporation and the identification of UDP-arabinofuranose transporters in the Golgi membrane, it is clear that the cytosolic UDP-Arap mutases are the key enzymes converting UDP-Arap to UDP-arabinofuranose for cell wall and natural product biosynthesis.

Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes.

Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, co-expression partners, purification methods, and optimization of protein solubility and stability.

X-ray diffraction analysis and in vitro characterization of the UAM2 protein from Oryza sativa.

The role of seemingly non-enzymatic proteins in complexes interconverting UDP-arabinopyranose and UDP-arabinofuranose (UDP-arabinosemutases; UAMs) in the plant cytosol remains unknown. To shed light on their function, crystallographic and functional studies of the seemingly non-enzymatic UAM2 protein from Oryza sativa (OsUAM2) were undertaken. Here, X-ray diffraction data are reported, as well as analysis of the oligomeric state in the crystal and in solution.

A Novel Fic (Filamentation Induced by cAMP) Protein from Clostridium difficile Reveals an Inhibitory Motif-independent Adenylylation/AMPylation Mechanism.

Filamentation induced by cAMP (Fic) domain proteins have been shown to catalyze the transfer of the AMP moiety from ATP onto a protein target. This type of post-translational modification was recently shown to play a crucial role in pathogenicity mediated by two bacterial virulence factors. Herein we characterize a novel Fic domain protein that we identified from the human pathogen Clostridium difficile The crystal structure shows that the protein adopts a classical all-helical Fic fold, which belongs to class II of Fic domain proteins characterized by an intrinsic N-terminal autoinhibitory α-helix.