Enzyme scaffolding and cellular compartments for metabolic engineering endeavours



Metabolic engineering aims at the production of certain metabolites at high titers and with a high productivity. Interference and loss of substrate to by-products is a general obstacle to overcome. Spatial organization of enzymes is one way to address the problem of interference with other metabolic processes.

The simplest way to organize enzymes spatially is to fix them to scaffolds, which can be made of proteins or other biomolecules (Idan and Hess, 2013). A scaffold system can promote the proximity of metabolic enzymes and increase the local concentration of intermediates, resulting on more efficient metabolic fluxes. Exemplarily, Dueber et al. (2009) showed a significantly improved production of glutaric acid by organizing of the enzymes along synthetic scaffolds.
However, while the use of synthetic scaffolds improves metabolic fluxes it does not provide full protection from interference with other present metabolic pathways. This can only be achieved by confining the pathway to compartments. A conceivable means for confining pathways exclusively to a compartment, are bacterial micro-compartments (BMCs). Most BMCs have an icosahedral shape, consisting of different semi-permeable, single-layered protein structures (Yeates et al., 2008). Recombinant production of empty BMCs in E. coli has been shown and GFP has been successfully targeted into BMCs (Choudhary et al., 2012; Parsons et al., 2010). The tools are therefore present for expression and constitution of microcompartments in heterologous organisms and targeting of metabolic pathways into them. A first example for the successful use of a BMC for pathway engineering has recently been shown for ethanol production (Lawrence et al., 2014).

Aims and methods.

Objectives of this project are the establishment of tools for artificial structural organization of metabolic pathways, including scaffolds to express enzymes in an organized close proximity and metabolic compartments, encapsulating enzymes in proteinaceous structures.
A preferable scaffold system, which will be tested is the dockerin/cohesin/scaffoldin system derived from bacterial cellulosomes (Vazana et al., 2012). The natural (extracellular) complexes are very large and highly organized. They comprise various mechanisms of very specific protein interactions, based on known motifs. These motifs have been successfully transferred to heterologous proteins, which are then readily incorporated into the highly ordered structure. Based on this architecture a wide range of possibilities exists to bring metabolic enzymes into close and ordered proximity.

A preferable bacterial microcompartment, which will be tested, is the 1,2-propanediol utilization metabolosome from Citrobacter freundii. Also in this case the molecular parts are known and have been characterized (Lawrence et al., 2014). Successful localization of heterologous proteins within these compartments has been shown. Therefore, they are considered as an optimal starting point to get hold of this technology before an ACIB proprietary system will be developed.

The cloning and expression of the single parts and the heterologous system will be based on Golden Gate Cloning (Engler and Marillonnet, 2011). The first tests to analyze the proper structure development will include fluorescent proteins, such as GFP and dsRed, which allow precise microscopic analyses of the formed structures in all organisms. As metabolic engineering examples two target molecules are chosen: itaconic acid (Steiger et al., 2013) and 5-amino valeric acid (Park et al., 2013). Both pathways comprise two enzymatic steps and both pathways branch off from common metabolite precursors, which are present in all organisms (L-lysine or citric acid, respectively). This guarantees simplicity to serve as model which can be analyzed in detail, but at the same time it involves a chain of reactions, which has the potential to benefit from the spatial organization of the enzymes.

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