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.

A method for sensitively monitoring enzyme kinetics and activities by using dual-color f luorescence crosscorrelation spectroscopy is described. This universal method enables the development of highly sensitive and precise assays for real-time kinetic analyses of any catalyzed cleavage or addition reaction, where a chemical linkage is formed or cleaved through an enzyme's action between two f luorophores that can be discriminated spectrally. In this work, a homogeneous assay with restriction endonuclease EcoRI and a 66-bp double-stranded DNA containing the GAATTC recognition site and f luorophores at each 5 end is described. The enzyme activity can be quantified down to the low picomolar range (>1.6 pM) where the rate constants are linearly dependent on the enzyme concentrations over two orders of magnitude. Furthermore, the reactions were monitored online at various initial substrate concentrations in the nanomolar range, and the reaction rates were clearly represented by the Michaelis-Menten equation with a K M of 14 ؎ 1 nM and a k cat of 4.6 ؎ 0.2 min ؊1 . In addition to kinetic studies and activity determinations, it is proposed that enzyme assays based on the dual-color f luorescence cross-correlation spectroscopy will be very useful for high-throughput screening and evolutionary biotechnology.Kinetic studies on enzymes are among the most important tools for understanding biological interactions at the molecular level. In combination with new approaches in genetic engineering and structure determination, there have been major efforts in recent years to develop more sensitive and precise techniques for characterizing the kinetics of enzyme reactions. These techniques have made it possible to develop efficient assays for analyzing catalytic parameters such as turnover rates, substrate specificity, as well as regio-and stereospecificity; they constitute a major part of the biochemical and pharmaceutical research. Moreover, the alteration of catalytic properties, either by evolutionary biotechnology (1-4) or by rational approaches, is of special interest for medical and industrial applications. The desired features are as follows: accelerated reaction rates; higher, relaxed, or even new substrate specificities; tolerance to nonnatural environments; and novel types of reactions. Modern rational design is the interplay between computer modeling and experimental testing of designed molecules by sensitive and quantitative assays. On the other hand, evolutionary approaches make use of rapid and highly sensitive screening assays to detect minute quantities of better adapted individuals among a large excess of alternatives. For all these purposes, fluorescence correlation spectroscopy (FCS) (5-8), with its ability to quantify molecular interactions at nanomolar and lower concentrations, has been proposed as an ideal tool (2). Most contemporary applications of FCS that are performed in confocal microscope (9) are based on the analysis of the molecular dynamics and the reaction kinetics of fluorescently labeled ...

Dual-color f luorescence cross-correlation spectroscopy (dual-color FCS) has previously been shown to be a suitable tool not only for binding but also for catalytic rate studies. In this work, its application as a rapid method for high-throughput screening (HTS) and evolutionary biotechnology is described. This application is called RAPID FCS (rapid assay processing by integration of dual-color FCS) and does not depend on the characterization of diffusion parameters that is the prerequisite for conventional f luorescence correlation spectroscopy. Dual-color FCS parameters were optimized to achieve the shortest analysis times. A simulated HTS with homogeneous assays for different restriction endonucleases (EcoRI, BamHI, SspI, and HindIII) achieved precise yes-or-no decisions within analysis times of about 1 s per sample. RAPID FCS combines these short analysis times with the development of fast and f lexible assays resulting in sensitive, homogeneous f luorescence-based assays, where a chemical linkage between different f luorophores is either cleaved or formed, or where differently labeled molecules interact by noncovalent binding. In principle, assay volumes can be reduced to submicroliters without decreasing the signal strength, making RAPID FCS an ideal tool for ultra-HTS when combined with nanotechnology. RAPID FCS can accurately probe 10 4 to 10 5 samples per day, and possibly more. In addition, this method has the potential to be an efficient tool for selection strategies in evolutionary biotechnology, where rare and specific binding or catalytic properties have to be screened in large numbers of samples.

Fluorescence-based assay technologies play an increasing role in high-throughput screening. They can be classified into different categories: f luorescence polarization, time-resolved f luorescence, f luorescence resonance energy transfer, and f luorescence correlation spectroscopy. In this work we present an alternative analytical technique for high-throughput screening, which we call confocal f luorescence coincidence analysis. Confocal f luorescence coincidence analysis extracts f luorescence f luctuations that occur coincidently in two different spectral ranges from a tiny observation volume of below 1 f l. This procedure makes it possible to monitor whether an association between molecular fragments that are labeled with different f luorophores is established or broken. Therefore, it provides access to the characterization of a variety of cleavage and ligation reactions in biochemistry. Confocal f luorescence coincidence analysis is a very sensitive and ultrafast technique with readout times of 100 ms and below. This feature is demonstrated by means of a homogeneous assay for restriction endonuclease EcoRI. The presented achievements break ground for throughput rates as high as 10 6 samples per day with using only small amounts of sample substance and therefore constitute a solid base for screening applications in drug discovery and evolutionary biotechnology.