The Impact of Metagenomics on Biocatalysis

In the ever-growing demand for sustainable ways to produce high-value small molecules, biocatalysis has come to the forefront of greener routes to these chemicals. As such, the need to constantly find and optimise suitable biocatalysts for specific transformations has never been greater. Metagenome mining has been shown to rapidly expand the toolkit of promiscuous enzymes needed for new transformations, without requiring protein engineering steps. If protein engineering is needed, the metagenomic candidate can often provide a better starting point for engineering than a previously discovered enzyme on the open database or from literature, for instance. In this review, we highlight where metagenomics has made substantial impact on the area of biocatalysis in recent years. We review the discovery of enzymes in previously unexplored or ‘hidden’ sequence space, leading to the characterisation of enzymes with enhanced properties that originate from natural selection pressures in native environments.     

A High‐Throughput Fluorescence Assay to Determine the Activity of Tryptophan Halogenases

Biocatalytic halogenation with tryptophan halogenases is hampered by severe limitations such as low activity and stability. These drawbacks can be overcome by directed evolution, but for screening large mutant libraries, a facile high‐throughput method is required. Therefore, we developed a quantitative halogenase assay based on a Suzuki–Miyaura cross‐coupling towards the formation of a fluorescent aryltryptophan. The technique was optimized for application in crude E. coli lysate without intermediary purification steps, and was used for quantitatively monitoring the formation of halogenated tryptophans with high specificity by facile fluorescence screening in microtiter plates. This novel screening approach was exploited to engineer a thermostable tryptophan 6‐halogenase. Libraries were constructed by error‐prone PCR and selected for improved thermal resistance simply by fluorogenic cross‐coupling. Our method led to an enzyme variant with substantially increased thermal stability and 2.5‐fold improved activity.     

Novel Arylindigoids by Late-Stage Derivatization of Biocatalytically Synthesized Dibromoindigo

Indigoids represent natural product-based compounds applicable as organic semiconductors and photoresponsive materials. Yet modified indigo derivatives are difficult to access by chemical synthesis. A biocatalytic approach applying several consecutive selective C−H functionalizations was developed that selectively provides access to various indigoids: Enzymatic halogenation of l -tryptophan followed by indole generation with tryptophanase yields 5-, 6- and 7-bromoindoles. Subsequent hydroxylation using a flavin monooxygenase furnishes dibromoindigo that is derivatized by acylation. This four-step one-pot cascade gives dibromoindigo in good isolated yields. Moreover, the halogen substituent allows for late-stage diversification by cross-coupling directly performed in the crude mixture, thus enabling synthesis of a small set of 6,6’-diarylindigo derivatives. This chemoenzymatic approach provides a modular platform towards novel indigoids with attractive spectral properties.     

Ein neues programmierbares Schema zur Bestimmung der Molekülsymmetrie

The symmetry of molecules can be determined, when their structure is known, using schemes of questions (algorithms), by means of which the existence of certain elements of symmetry is checked. In this paper, a new algorithm is presented, which renders feasible the prompt determination of symmetry point groups and visualizes relations between different types of symmetry. The application of the algorithm is demonstrated by means of a graphic scheme and a Pascal computer version.