Sonja Billerbeck

Exploiting cell-free systems: Implementation and debugging of a system of biotransformations

The orchestration of a multitude of enzyme catalysts allows cells to carry out complex and thermodynamically unfavorable chemical conversions. In an effort to recruit these advantages for in vitro biotransformations, we have assembled a 10‐step catalytic system—a system of biotransformations (SBT)—for the synthesis of unnatural monosaccharides based on the versatile building block dihydroxyacetone phosphate (DHAP). To facilitate the assembly of such a network, we have insulated a production pathway from Escherichia colis central carbon metabolism. This pathway consists of the endogenous glycolysis without triose‐phosphate isomerase to enable accumulation of DHAP and was completed with lactate dehydrogenase to regenerate NAD+. It could be readily extended for the synthesis of unnatural sugar molecules, such as the unnatural monosaccharide phosphate 5,6,7‐trideoxy‐D‐threo‐heptulose‐1‐phosphate from DHAP and butanal. Insulation required in particular inactivation of the amn gene encoding the AMP nucleosidase, which otherwise led to glucose‐independent DHAP production from adenosine phosphates. The work demonstrates that a sufficiently insulated in vitro multi‐step enzymatic system can be readily assembled from central carbon metabolism pathways. Biotechnol. Bioeng. 2010; 106: 376–389.

The good of two worlds: increasing complexity in cell-free systems

In vitro biocatalytic systems have moved far beyond established uses in food, diagnostic, and chemical applications. As new strategies to construct and manage multiple enzymes in ever more complex systems are developed, novel applications emerge. In the field of chemistry, complex protein networks are applied to enable the production of fine chemicals, such as dihydroxyacetone phosphate, and even bulk chemicals, such as biofuels, from cheap sugars. Cell-free protein synthesis is applied to expanding protein and nucleic acid biochemistry and enabling novel assay formats, while programmable DNA-circuits can be exploited to engineer sensitive detection methods. Novel developments in chemical analytics such as real-time mass spectrometry to follow the metabolism online, directed physical assembly of network members facilitating substrate channeling, and encapsulation forming biofunctional subunits enable a better control and potential for optimization.

Forward design of a complex enzyme cascade reaction

Enzymatic reaction networks are unique in that one can operate a large number of reactions under the same set of conditions concomitantly in one pot, but the nonlinear kinetics of the enzymes and the resulting system complexity have so far defeated rational design processes for the construction of such complex cascade reactions. Here we demonstrate the forward design of an in vitro 10-membered system using enzymes from highly regulated biological processes such as glycolysis. For this, we adapt the characterization of the biochemical system to the needs of classical engineering systems theory: we combine online mass spectrometry and continuous system operation to apply standard system theory input functions and to use the detailed dynamic system responses to parameterize a model of sufficient quality for forward design. This allows the facile optimization of a 10-enzyme cascade reaction for fine chemical production purposes.

Towards functional orthogonalisation of protein complexes: individualisation of GroEL monomers leads to distinct quasihomogeneous single rings.

The essential molecular chaperonin GroEL is an example of a functionally highly versatile cellular machine with a wide variety of in vitro applications ranging from protein folding to drug release. Directed evolution of new functions for GroEL is considered difficult, due to its structure as a complex homomultimeric double ring and the absence of obvious molecular engineering strategies. In order to investigate the potential to establish an orthogonal GroEL system in Escherichia coli, which might serve as a basis for GroEL evolution, we first successfully individualised groEL genes by inserting different functional peptide tags into a robustly permissive site identified by transposon mutagenesis. These peptides allowed fundamental aspects of the intracellular GroEL complex stoichiometry to be studied and revealed that GroEL single‐ring complexes, which assembled in the presence of several functionally equivalent but biochemically distinct monomers, each consist almost exclusively of only one type of monomer. At least in the case of GroEL, individualisation of monomers thus leads to individualisation of homomultimeric protein complexes, effectively providing the prerequisites for evolving an orthogonal intracellular GroEL folding machine.

A genetic replacement system for selection-based engineering of essential proteins

BackgroundEssential genes represent the core of biological functions required for viability. Molecular understanding of essentiality as well as design of synthetic cellular systems includes the engineering of essential proteins. An impediment to this effort is the lack of growth-based selection systems suitable for directed evolution approaches.ResultsWe established a simple strategy for genetic replacement of an essential gene by a (library of) variant(s) during a transformation.The system was validated using three different essential genes and plasmid combinations and it reproducibly shows transformation efficiencies on the order of 107 transformants per microgram of DNA without any identifiable false positives. This allowed for reliable recovery of functional variants out of at least a 105-fold excess of non-functional variants. This outperformed selection in conventional bleach-out strains by at least two orders of magnitude, where recombination between functional and non-functional variants interfered with reliable recovery even in recA negative strains.ConclusionsWe propose that this selection system is extremely suitable for evaluating large libraries of engineered essential proteins resulting in the reliable isolation of functional variants in a clean strain background which can readily be used for in vivo applications as well as expression and purification for use in in vitro studies.

A modular yeast biosensor for low-cost point-of-care pathogen detection

A yeast-based dipstick biosensor enables low-cost, on-site surveillance of pathogens. The availability of simple, specific, and inexpensive on-site detection methods is of key importance for deployment of pathogen surveillance networks. We developed a nontechnical and highly specific colorimetric assay for detection of pathogen-derived peptides based on Saccharomyces cerevisiae—a genetically tractable model organism and household product. Integrating G protein–coupled receptors with a visible, reagent-free lycopene readout, we demonstrate differential detection of major human, plant, and food fungal pathogens with nanomolar sensitivity. We further optimized a one-step rapid dipstick prototype that can be used in complex samples, including blood, urine, and soil. This modular biosensor can be economically produced at large scale, is not reliant on cold-chain storage, can be detected without additional equipment, and is thus a compelling platform scalable to global surveillance of pathogens.

Technologies for Biosystems Engineering

The rising knowledge for a variety of model organisms about chromosome compositions, gene regulation and molecular interactions which influence cell development and biological systems behavior drives an engineering effort to design and construct ever more complex novel molecular or cellular functions and behaviors. As molecular functions are encoded on DNA level, engineering of new and complex systems starts with the engineering of its encoding DNA. Although methods for genetic engineering are available since decades, their focus was the modification of rather small systems, such as cloning or modifying single genes. Therefore, the engineering of complex biological systems on DNA level, involving multiple genes up to multiple pathways or even entire genomes, requires revisiting the general usefulness of available methods and their potential for scalability. Here we review available methods and their applicability for biosystems engineering approaches as well as recent technological advances which expand the toolbox for understanding and engineering complex biological systems with novel traits.

Multienzymnetzwerke für die Feinchemie

ZusammenfassungDie Produktion von Feinchemikalien beruht auf Prozessen mit mehreren Reaktionsschritten. Wir arbeiten an Konzepten für die Rekrutierung, Isolierung und Optimierung von in vitro-Enzymkaskaden aus Zellextrakten für die Synthese von komplexen Zuckern.AbstractThe production of fine chemicals relies on complex multi-step synthesis. We work on the scientific and technical challenges to recruit, insulate and optimize multi-enzyme in vitro networks from cell free extracts for the synthesis of complex sugars.