Ulrich Kettling

Cell‐Free Metabolic Engineering: Production of Chemicals by Minimized Reaction Cascades

The limited supply of fossil resources demands the development of renewable alternatives to petroleum-based products. Here, biobased higher alcohols such as isobutanol are versatile platform molecules for the synthesis of chemical commodities and fuels. Currently, their fermentation-based production is limited by the low tolerance of microbial production systems to the end products and also by the low substrate flux into cell metabolism. We developed an innovative cell-free approach, utilizing an artificial minimized glycolytic reaction cascade that only requires one single coenzyme. Using this toolbox the cell-free production of ethanol and isobutanol from glucose was achieved. We also confirmed that these streamlined cascades functioned under conditions at which microbial production would have ceased. Our system can be extended to an array of industrially-relevant molecules. Application of solvent-tolerant biocatalysts potentially allows for high product yields, which significantly simplifies downstream product recovery.

Analyzing single protein molecules using optical methods

Studies on single protein molecules have advanced from mere proofs of principle to insightful investigations of otherwise inaccessible biological phenomena. Recent studies predict a tremendous number of possible future applications. The long-term vision of biologists to watch single molecular processes in real time by peering into a cell with three-dimensional resolution might finally be realized. Another fascinating perspective is the identification and selection of single favorable variants from complex libraries of diverse biomolecules.

PRINCIPLES AND METHODS OF EVOLUTIONARY BIOTECHNOLOGY

Evolutionary biotechnology applies the principles of molecular evolution to biotechnology, leading to novel techniques for the creation of biomolecules with a great variety of functions for technical and medical purposes. Several basic principles for the application of evolutionary strategies can be derived from a comprehensive theory of molecular evolution. Prerequisites for evolutionary biotechnology are summarized with respect to the different classes of biomolecules and a few, selected applications are described in detail. Concepts for the technical implementation of evolutionary strategies are presented which allow automatized, high throughput processes.

Evolutionary Biotechnology - Reflections and Perspectives

With the designing of biomolecules, a new era in biotechnology has been initiated. One of the most promising strategies for successfully designing complex biomolecular functions is to exploit nature’s principles of Darwinian evolution, i.e. variation and selection. The application of these principles to directed evolution of molecules is the underlying concept of evolutionary biotechnology. Important prerequisites are a comprehensive understanding of the mode of molecular evolution as well as the ability to apply its principles to experimental systems in order to create and optimize molecular functions with scientific or economic value.

Dual-Color Confocal Fluorescence Spectroscopy and its Application in Biotechnology

In recent years, fluorescence correlation spectroscopy (FCS) has become an attractive analytical tool for the investigation of biomolecular processes at the single molecular level. This method was invented in the early 1970s by groups at Cornell University, Ithaca, N.Y. [9.1,9.2], and at the Karolinska Institute, Stockholm [9.3,9.4]. During the 1990s, modern confocal optics, new dyes as efficient fluorescent probes, sensitive photon detectors, and fast data processing tools have been introduced, mainly by Rigler and Eigen [9.5,9.6]. Due to these improvements, FCS permits the observation of the dynamics of single molecules in real time while they pass an open volume element of less than a femtoliter, i.e. the size of a common bacterial cell. Nowadays, FCS has found its way into several laboratories and companies all over the world as a tool for basic research as well as for industrial applications such as drug screening.