RESUMO
The fortieth anniversary of biocatalysis started at Ciba-Geigy and later at Novartis is a great time to pause and reflect on development of science and technology in this field. Enzyme-based synthesis became a highly valued enabling tool for pharmaceutical research and development over the last decades. In this perspective we aim to discuss how the scientific approaches and trends evolved over the time and present future challenges and opportunities.
Assuntos
BiocatáliseRESUMO
The display of complex proteins on the surface of cells is of great importance for protein engineering and other fields of biotechnology. Herein, we describe a modular approach, in which the membrane anchor protein Lpp-OmpA and a protein of interest (passenger) are expressed independently as genetically fused SpyCatcher and SpyTag units and assembled inâ situ by post-translational coupling. Using fluorescent proteins, we first demonstrate that this strategy allows the construct to be installed on the surface of E. coli cells. The scope of our approach was then demonstrated by using three different functional enzymes, the stereoselective ketoreductase Gre2p, the homotetrameric glucose 1-dehydrogenase GDH, and the bulky heme- and diflavin-containing cytochrome P450 BM3 (BM3). In all cases, the SpyCatcher-SpyTag method enabled the generation of functional whole-cell biocatalysts, even for the bulky BM3, which could not be displayed by conventional fusion with Lpp-OmpA. Furthermore, by using a GDH variant carrying an internal SpyTag, the system could be used to display an enzyme with unmodified N- and C-termini.
Assuntos
Técnicas de Visualização da Superfície Celular/métodos , Enzimas/metabolismo , Membrana Celular/metabolismo , Enzimas/genética , Escherichia coli/citologia , Escherichia coli/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Biofilms are the natural form of life of the majority of microorganisms. These multispecies consortia are intensively studied not only for their effects on health and environment but also because they have an enormous potential as tools for biotechnological processes. Further exploration and exploitation of these complex systems will benefit from technical solutions that enable integrated, machine-assisted cultivation and analysis. We here introduce a microfluidic platform, where readily available microfluidic chips are connected by automated liquid handling with analysis instrumentation, such as fluorescence detection, microscopy, chromatography and optical coherence tomography. The system is operable under oxic and anoxic conditions, allowing for different gases and nutrients as feeding sources and it offers high spatiotemporal resolution in the analysis of metabolites and biofilm composition. We demonstrate the platform's performance by monitoring the productivity of biofilms as well as the spatial organization of two bacterial species in a co-culture, which is driven by chemical gradients along the microfluidic channel.
Assuntos
Biofilmes , Dispositivos Lab-On-A-Chip , Bacillus subtilis/crescimento & desenvolvimento , Cromatografia/métodos , Escherichia coli/crescimento & desenvolvimento , Hibridização in Situ Fluorescente , Microscopia/métodos , Espectrometria de Fluorescência/métodos , Tomografia de Coerência Óptica/métodosRESUMO
All-enzyme hydrogels are efficient reagents for continuous flow biocatalysis. These materials can be obtained by self-assembly of two oligomeric enzymes, modified with the complementary SpyTag and SpyCatcher units. To facilitate access to the large proportion of biocatalytically relevant monomeric enzymes, we demonstrate that the tagging valency of the monomeric (S)-stereoselective ketoreductase Gre2p from Saccharomyces cerevisiae can be designed to assemble stable, active hydrogels with the cofactor-regenerating glucose 1-dehydrogenase GDH from Bacillus subtilis. Mounted in microfluidic reactors, these gels revealed high conversion rates and stereoselectivity in the reduction of prochiral methylketones under continuous flow for more than 8 days. The sequential use as well as parallelization by 'numbering up' of the flow reactor modules demonstrate that this approach is suitable for syntheses on the semipreparative scale.
RESUMO
Continuous flow biocatalysis is an emerging field of industrial biotechnology that uses enzymes immobilized in flow channels for the production of value-added chemicals. We describe the construction of self-assembling all-enzyme hydrogels that are comprised of two tetrameric enzymes. The stereoselective dehydrogenase LbADH and the cofactor-regenerating glucose 1-dehydrogenase GDH were genetically fused with a SpyTag or SpyCatcher domain, respectively, to generate two complementary homo-tetrameric building blocks that polymerize under physiological conditions into porous hydrogels. Mounted in microfluidic reactors, the gels show excellent stereoselectivity with near quantitative conversion in the reduction of prochiral ketones along with high robustness under process and storage conditions. The gels function as compartment that retains intermediates thus enabling high total turnover numbers of the expensive cofactor NADP(H).
Assuntos
Hidrogéis/metabolismo , Oxirredutases/metabolismo , Biocatálise , Hidrogéis/química , Estrutura Molecular , Oxirredutases/química , EstereoisomerismoRESUMO
We herein describe the engineering of E.â coli strains that display orthogonal tags for immobilization on their surface and overexpress a functional heterologous "protein content" in their cytosol at the same time. Using the outer membrane protein Lpp-ompA, cell-surface display of the streptavidin-binding peptide, the SpyTag/SpyCatcher system, or a HaloTag variant allowed us to generate bacterial strains that can selectively bind to solid substrates, as demonstrated with magnetic microbeads. The simultaneous cytosolic expression of functional content was demonstrated for fluorescent proteins or stereoselective ketoreductase enzymes. The latter strains gave high selectivities for specific immobilization onto complementary surfaces and also in the whole-cell stereospecific transformation of a prochiral CS -symmetric nitrodiketone.