A new physical and biological strategy to reduce the content of zearalenone in infected wheat kernels: the effect of cold needle perforation, microorganisms, and purified enzyme.

With the aim of reintroducing wheat grains naturally contaminated with mycotoxins into the food value chain, a decontamination strategy was developed in this study. For this purpose, in a first step, the whole wheat kernels were pre-treated using cold needle perforation. The pore size was evaluated by scanning electron microscopy and the accessibility of enzymes and microorganisms determined using fluorescent markers in the size range of enzymes (5 nm) and microorganisms (10 µm), and fluorescent microscopy. The perforated wheat grains, as well as non-perforated grains as controls, were then incubated with selected microorganisms (Bacillus megaterium Myk145 and B. licheniformis MA572) or with the enzyme ZHD518. The two bacilli strains were not able to significantly reduce the amount of zearalenone (ZEA), neither in the perforated nor in the non-perforated wheat kernels in comparison with the controls. In contrast, the enzyme ZHD518 significantly reduced the initial concentration of ZEA in the perforated and non-perforated wheat kernels in comparison with controls. Moreover, in vitro incubation of ZHD518 with ZEA showed the presence of two non-estrogenic degradation products of ZEA: hydrolysed zearalenone (HZEA) and decarboxylated hydrolysed ZEA (DHZEA). In addition, the physical pre-treatment led to a reduction in detectable mycotoxin contents in a subset of samples. Overall, this study emphasizes the promising potential of combining physical pre-treatment approaches with biological decontamination solutions in order to address the associated problem of mycotoxin contamination and food waste reduction.

Excelzyme: A Swiss University-Industry Collaboration for Accelerated Biocatalyst Development.

Excelzyme, an enzyme engineering platform located at the Zurich University of Applied Sciences, is dedicated to accelerating the development of tailored biocatalysts for large-scale industrial applications. Leveraging automation and advanced computational techniques, including machine learning, efficient biocatalysts can be generated in short timeframes. Toward this goal, Excelzyme systematically selects suitable protein scaffolds as the foundation for constructing complex enzyme libraries, thereby enhancing sequence and structural biocatalyst diversity. Here, we describe applied workflows and technologies as well as an industrial case study that exemplifies the successful application of the workflow.

Effective engineering of a ketoreductase for the biocatalytic synthesis of an ipatasertib precursor.

Semi-rational enzyme engineering is a powerful method to develop industrial biocatalysts. Profiting from advances in molecular biology and bioinformatics, semi-rational approaches can effectively accelerate enzyme engineering campaigns. Here, we present the optimization of a ketoreductase from Sporidiobolus salmonicolor for the chemo-enzymatic synthesis of ipatasertib, a potent protein kinase B inhibitor. Harnessing the power of mutational scanning and structure-guided rational design, we created a 10-amino acid substituted variant exhibiting a 64-fold higher apparent kcat and improved robustness under process conditions compared to the wild-type enzyme. In addition, the benefit of algorithm-aided enzyme engineering was studied to derive correlations in protein sequence-function data, and it was found that the applied Gaussian processes allowed us to reduce enzyme library size. The final scalable and high performing biocatalytic process yielded the alcohol intermediate with ≥ 98% conversion and a diastereomeric excess of 99.7% (R,R-trans) from 100 g L-1 ketone after 30 h. Modelling and kinetic studies shed light on the mechanistic factors governing the improved reaction outcome, with mutations T134V, A238K, M242W and Q245S exerting the most beneficial effect on reduction activity towards the target ketone.

Advances in Noncanonical Amino Acid Incorporation for Enzyme Engineering Applications.

Incorporation of noncanonical amino acids (ncAAs) via genetic code expansion (GCE) opens up new possibilities for chemical biology. The technology has led to the development of novel xenobiotic enzymes with tailored properties which can serve as entry points into a multitude of applications, including protein conjugation, immobilization, or labeling. In this review, we discuss recent progress in the use of GCE to create biocatalysts possessing reaction repertoires that lie beyond what is achievable with canonical amino acids (cAAs). Furthermore, we highlight how GCE enables to gain mechanistic insights into protein function by the incorporation of judiciously selected ncAAs. As the amino acid alphabet continues to grow and improved tools for ncAA incorporation are being developed, we anticipate the creation of additional powerful biological catalysts for synthetic application which merge the chemical versatility of anthropogenic building blocks with the exquisite selectivities of enzymes.

Improving Enzyme Fitness with Machine Learning.

The combinatorial composition of proteins has triggered the application of machine learning in enzyme engineering. By predicting how protein sequence encodes function, researchers aim to leverage machine learning models to select a reduced number of optimized sequences for laboratory measurement with the aim to lower costs and shorten timelines of enzyme engineering campaigns. In this review, we will highlight successful algorithm-aided protein engineering examples, including work carried out within the NCCR Catalysis. In this context, we will discuss the underlying computational methods developed to improve enzyme properties such as enantioselectivity, regioselectivity, activity, and stability. Considering the rapid maturing of computational techniques, we expect that their continued application in enzyme engineering campaigns will be key to deliver additional powerful biocatalysts for sustainable chemical synthesis.

The catalytic role of glutathione transferases in heterologous anthocyanin biosynthesis.

Anthocyanins are ubiquitous plant pigments used in a variety of technological applications. Yet, after over a century of research, the penultimate biosynthetic step to anthocyanidins attributed to the action of leucoanthocyanidin dioxygenase has never been efficiently reconstituted outside plants, preventing the construction of heterologous cell factories. Through biochemical and structural analysis, here we show that anthocyanin-related glutathione transferases, currently implicated only in anthocyanin transport, catalyse an essential dehydration of the leucoanthocyanidin dioxygenase product, flavan-3,3,4-triol, to generate cyanidin. Building on this knowledge, introduction of anthocyanin-related glutathione transferases into a heterologous biosynthetic pathway in baker's yeast results in >35-fold increased anthocyanin production. In addition to unravelling the long-elusive anthocyanin biosynthesis, our findings pave the way for the colourants' heterologous microbial production and could impact the breeding of industrial and ornamental plants.

LibGENiE - A bioinformatic pipeline for the design of information-enriched enzyme libraries.

Enzymes are potent catalysts with high specificity and selectivity. To leverage nature's synthetic potential for industrial applications, various protein engineering techniques have emerged which allow to tailor the catalytic, biophysical, and molecular recognition properties of enzymes. However, the many possible ways a protein can be altered forces researchers to carefully balance between the exhaustiveness of an enzyme screening campaign and the required resources. Consequently, the optimal engineering strategy is often defined on a case-by-case basis. Strikingly, while predicting mutations that lead to an improved target function is challenging, here we show that the prediction and exclusion of deleterious mutations is a much more straightforward task as analyzed for an engineered carbonic acid anhydrase, a transaminase, a squalene-hopene cyclase and a Kemp eliminase. Combining such a pre-selection of allowed residues with advanced gene synthesis methods opens a path toward an efficient and generalizable library construction approach for protein engineering. To give researchers easy access to this methodology, we provide the website LibGENiE containing the bioinformatic tools for the library design workflow.

Engineering Fe(II)/α-Ketoglutarate-Dependent Halogenases and Desaturases.

Fe(II)/α-ketoglutarate-dependent dioxygenases (α-KGDs) are widespread enzymes in aerobic biology and serve a remarkable array of biological functions, including roles in collagen biosynthesis, plant and animal development, transcriptional regulation, nucleic acid modification, and secondary metabolite biosynthesis. This functional diversity is reflected in the enzymes' catalytic flexibility as α-KGDs can catalyze an intriguing set of synthetically valuable reactions, such as hydroxylations, halogenations, and desaturations, capturing the interest of scientists across disciplines. Mechanistically, all α-KGDs are understood to follow a similar activation pathway to generate a substrate radical, yet how individual members of the enzyme family direct this key intermediate toward the different reaction outcomes remains elusive, triggering structural, computational, spectroscopic, kinetic, and enzyme engineering studies. In this Perspective, we will highlight how first enzyme and substrate engineering examples suggest that the chemical reaction pathway within α-KGDs can be intentionally tailored using rational design principles. We will delineate the structural and mechanistic investigations of the reprogrammed enzymes and how they begin to inform about the enzymes' structure-function relationships that determine chemoselectivity. Application of this knowledge in future enzyme and substrate engineering campaigns will lead to the development of powerful C-H activation catalysts for chemical synthesis.

Algorithm-aided engineering of aliphatic halogenase WelO5* for the asymmetric late-stage functionalization of soraphens.

Late-stage functionalization of natural products offers an elegant route to create novel entities in a relevant biological target space. In this context, enzymes capable of halogenating sp3 carbons with high stereo- and regiocontrol under benign conditions have attracted particular attention. Enabled by a combination of smart library design and machine learning, we engineer the iron/α-ketoglutarate dependent halogenase WelO5* for the late-stage functionalization of the complex and chemically difficult to derivatize macrolides soraphen A and C, potent anti-fungal agents. While the wild type enzyme WelO5* does not accept the macrolide substrates, our engineering strategy leads to active halogenase variants and improves upon their apparent kcat and total turnover number by more than 90-fold and 300-fold, respectively. Notably, our machine-learning guided engineering approach is capable of predicting more active variants and allows us to switch the regio-selectivity of the halogenases facilitating the targeted analysis of the derivatized macrolides' structure-function activity in biological assays.

Swiss Women in Chemistry - Two Years Later ...

The SCS Swiss Women in Chemistry network was launched in September 2019. Under the umbrella of the Swiss Chemical Society, its aim is to create visibility, facilitate networking and provide a supportive community for female chemists in Switzerland across all career stages both in industry and academia. The current article provides an overview on the platform's activities over the past two years.

Asymmetric Cation-Olefin Monocyclization by Engineered Squalene-Hopene Cyclases.

Squalene-hopene cyclases (SHCs) have great potential for the industrial synthesis of enantiopure cyclic terpenoids. A limitation of SHC catalysis has been the enzymes' strict (S)-enantioselectivity at the stereocenter formed after the first cyclization step. To gain enantio-complementary access to valuable monocyclic terpenoids, an SHC-wild-type library including 18 novel homologs was set up. A previously not described SHC (AciSHC) was found to synthesize small amounts of monocyclic (R)-γ-dihydroionone from (E/Z)-geranylacetone. Using enzyme and process optimization, the conversion to the desired product was increased to 79 %. Notably, analyzed AciSHC variants could finely differentiate between the geometric geranylacetone isomers: While the (Z)-isomer yielded the desired monocyclic (R)-γ-dihydroionone (>99 % ee), the (E)-isomer was converted to the (S,S)-bicyclic ether (>95 % ee). Applying the knowledge gained from the observed stereodivergent and enantioselective transformations to an additional SHC-substrate pair, access to the complementary (S)-γ-dihydroionone (>99.9 % ee) could be obtained.

Exploring the Biocatalytic Potential of Fe/α-Ketoglutarate-Dependent Halogenases.

Freestanding Fe/α-ketoglutarate-dependent halogenases are oxidoreductases that catalyze the installation of halogen atoms into unactivated sp3 -hybridized carbon centers with high stereo- and regioselectivity. Since their discovery in 2014, a small number of indole alkaloid and amino acid halogenases have been identified and characterized. First enzyme engineering examples suggest that the accessible substrate range of these enzymes may be expanded through the use of rational enzyme design and directed evolution. Structural investigations of non-heme iron halogenases acting on freestanding as well as tethered substrates are beginning to inform about the principles of the underlying halogenation mechanism.

Evolved Aliphatic Halogenases Enable Regiocomplementary C-H Functionalization of a Pharmaceutically Relevant Compound.

Non-heme iron halogenases are synthetically valuable biocatalysts that are capable of halogenating unactivated sp3 -hybridized carbon centers with high stereo- and regioselectivity. The reported substrate scope of these enzymes, however, is limited primarily to the natural substrates and their analogues. We engineered the halogenase WelO5* for chlorination of a martinelline-derived fragment. Using structure-guided evolution, a halogenase variant with a more than 290-fold higher total turnover number and a 400-fold higher apparent kcat compared to the wildtype enzyme was generated. Moreover, we identified key positions in the active site that allow direction of the halogen to different positions in the target substrate. This is the first example of enzyme engineering to expand the substrate scope of a non-heme iron halogenase beyond the native indole-alkaloid-type substrates. The highly evolvable nature of WelO5* underscores the usefulness of this enzyme family for late-stage halogenation.

Enzymatic PET Degradation.

Plastic, in the form of packaging material, disposables, clothing and other articles with a short lifespan, has become an indispensable part of our everyday life. The increased production and use of plastic, however, accelerates the accumulation of plastic waste and poses an increasing burden on the environment with negative effects on biodiversity and human health. PET, a common thermoplastic, is recycled in many countries via thermal, mechanical and chemical means. Recently, several enzymes have been identified capable of degrading this recalcitrant plastic, opening possibilities for the biological recycling of the omnipresent material. In this review, we analyze the current knowledge of enzymatic PET degradation and discuss advances in improving the involved enzymes via protein engineering. Looking forward, the use of plastic degrading enzymes may facilitate sustainable plastic waste management and become an important tool for the realization of a circular plastic economy.

Novel Old Yellow Enzyme Subclasses.

Many drug candidate molecules contain at least one chiral centre, and consequently, the development of biocatalytic strategies to complement existing metal- and organocatalytic approaches is of high interest. However, time is a critical factor in chemical process development, and thus, the introduction of biocatalytic steps, even if more suitable, is often prevented by the limited availability of off-the-shelf enzyme libraries. To expand the biocatalytic toolbox with additional ene reductases, we screened 19 bacterial strains for double bond reduction activity by using the model substrates cyclohexanone and carvone. Overall, we identified 47 genes coding for putative ene reductases. Remarkably, bioinformatic analysis of all genes and the biochemical characterization of four representative novel ene reductases led us to propose the existence of two new Old Yellow Enzyme subclasses, which we named OYE class III and class IV. Our results demonstrate that although, on a DNA level, each new OYE subclass features a distinct combination of sequence motifs previously known from the classical and the thermophilic-like group, their substrate scope more closely resembles the latter subclass.

Linear enzyme cascade for the production of (-)-iso-isopulegol.

Biocatalysis has developed enormously in the last decade and now offers solutions for the sustainable production of chiral and highly functionalised asset molecules. Products generated by enzymatic transformations are already being used in the food, feed, chemical, pharmaceutical and cosmetic industry, and the accessible compound panoply is expected to expand even further. In particular, the combination of stereo-selective enzymes in linear cascade reactions is an elegant strategy toward enantiomeric pure compounds, as it reduces the number of isolation and purification steps and avoids accumulation of potentially unstable intermediates. Here, we present the set-up of an enzyme cascade to selectively convert citral to (-)-iso-isopulegol by combining an ene reductase and a squalene hopene cyclase. In the initial reaction step, the ene reductase YqjM from Bacillus subtilis selectively transforms citral to (S)-citronellal, which is subsequently cyclised exclusively to (-)-iso-isopulegol by a mutant of the squalene hopene cyclase from Alicyclobacillus acidocaldarius (AacSHC). With this approach, we can convert citral to an enantiopure precursor for isomenthol derivatives.

Directed evolution of carbon-hydrogen bond activating enzymes.

As industrial biocatalysis is maturing, access to enzymatic activities beyond chiral resolutions, asymmetric ketone reductions and reductive aminations is gradually becoming reality. Especially the utilization of carbon-hydrogen bond (C-H) activating enzymes is very attractive as they catalyze a variety of chemically extremely challenging transformations. Because of their intrinsic complexity, the use of these enzymes in manufacturing has been limited. However, recent advances in enzyme engineering and bioinformatics have led to activity improvements for native and non-native substrates, the introduction of new-to-nature chemistries and the identification of promising novel enzyme families. Looking forward, the use of automation and advanced computer algorithms will help to streamline the evolution process of C-H activating enzymes leading to more robust and active biocatalysts.