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.

Hairy root transformation of Brassica rapa with bacterial halogenase genes and regeneration to adult plants to modify production of indolic compounds.

During the last years halogenated compounds have drawn a lot of attention. Metabolites with one or more halogen atoms are often more active than their non-halogenated derivatives like indole-3-acetic acid (IAA) and 4-Cl-IAA. Within this work, bacterial flavin-dependent tryptophan halogenase genes were inserted into Brassica rapa ssp. pekinensis (Chinese cabbage) with the aim to produce novel halogenated indole compounds. It was investigated which tryptophan-derived indole metabolites, such as indole glucosinolates or potential degradation products can be synthesized by the transgenic root cultures. In vivo and in vitro activity of halogenases heterologously produced was shown and the production of chlorinated tryptophan in transgenic root lines was confirmed. Furthermore, chlorinated indole-3-acetonitrile (Cl-IAN) was detected. Other tryptophan-derived indole metabolites, such as IAA or indole glucosinolates were not found in the transgenic roots in a chlorinated form. The influence of altered growth conditions on the amount of produced chlorinated compounds was evaluated. We found an increase in Cl-IAN production at low temperatures (8 °C), but otherwise no significant changes were observed. Furthermore, we were able to regenerate the wild type and transgenic root cultures to adult plants, of which the latter still produced chlorinated metabolites. Therefore, we conclude that the genetic information had been stably integrated. The transgenic plants showed a slightly altered phenotype compared to plants grown from seeds since they also still expressed the rol genes. By this approach we were able to generate various stably transformed plant materials from which it was possible to isolate chlorinated tryptophan and Cl-IAN.

An Unusual Flavin-Dependent Halogenase from the Metagenome of the Marine Sponge Theonella swinhoei WA.

Uncultured bacteria from sponges have been demonstrated to be responsible for the generation of many potent, bioactive natural products including halogenated metabolites.1 The identification of gene clusters from the metagenomes of such bacterial communities enables the discovery of enzymes that mediate new and useful chemistries and allows insight to be gained into the biogenesis of potentially pharmacologically important natural products. Here we report a new pathway to the keramamides (krm); the first functional evidence for the existence of a distinct producer in the Theonella swinhoei WA chemotype is revealed, and a key enzyme on the pathway, a unique flavin-dependent halogenase with a broad substrate specificity, with potential as a useful new biocatalytic tool, is described.

Characterization of the Aminotransferase ThdN from Thienodolin Biosynthesis in Streptomyces albogriseolus.

In Streptomyces albogriseolus the indolethiophen alkaloid thienodolin is derived from tryptophan. The first step in thienodolin biosynthesis is the regioselective chlorination of tryptophan in the 6-position of the indole ring. The second step is catalyzed by the aminotransferase ThdN. ThdN shows sequence homology (up to 69 % similarity) with known pyridoxal 5'-phosphate-dependent aminotransferases of the aspartate aminotransferase family from Gram-positive bacteria. thdN was heterologously expressed in Pseudomonas fluorescens, and the enzyme was purified by nickel-affinity chromatography. ThdN is a homodimeric enzyme with a mass of 90 600 kDa and catalyzes the conversion of l-tryptophan and a number of chlorinated and brominated l-tryptophans. The lowest KM values were found for 6-bromo- and 6-chlorotryptophan (40 and 66 µm, respectively). For l-tryptophan it was 454 µm, which explains why thienodolin is the major product and dechlorothienodolin is only a minor component. The turnover number (kcat ) for 7-chlorotryptophan (128 min-1 ) was higher than that for the natural substrate 6-chlorotryptophan (88 min-1 ).

Specific Enzymatic Halogenation-From the Discovery of Halogenated Enzymes to Their Applications In†Vitro and In†Vivo.

During the last 20 years, focus has shifted from haloperoxidases to flavin-dependent and non-heme-iron halogenases because of their proven involvement in the biosynthesis of halogenated metabolites in different organisms and the regioselectivity of their reactions. During the first 10-12 years, the main research topics were the detection of halogenases as well as the elucidation of three-dimensional structures and reaction mechanisms. This Review mainly deals with studies on halogenating enzymes published between 2010 and 2015. It focusses on the elucidation of the involvement of halogenating enzymes in halometabolite biosynthesis, application of halogenases in in vivo and in vitro systems, in vivo modification of biosynthetic pathways in bacteria and plants, improvement of enzyme stability, broadening of substrate specificity, and the combination of biocatalysis with chemical synthesis to produce new compounds.

A tryptophan 6-halogenase and an amidotransferase are involved in thienodolin biosynthesis.

The biosynthetic gene cluster for the plant growth-regulating compound thienodolin was identified in and cloned from the producer organism Streptomyces albogriseolus MJ286-76F7. Sequence analysis of a 27 kb DNA region revealed the presence of 21 ORFs, 14 of which are involved in thienodolin biosynthesis. Three insertional inactivation mutants were generated in the sequenced region to analyze their involvement in thienodolin biosynthesis and to functionally characterize specific genes. The gene inactivation experiments together with enzyme assays with enzymes obtained by heterologous expression and feeding studies showed that the first step in thienodolin biosynthesis is catalyzed by a tryptophan 6-halogenase and that the last step is the formation of a carboxylic amide group catalyzed by an amidotransferase. The results led to a hypothetical model for thienodolin biosynthesis.