A biocatalytic approach to the synthesis of myo-inositol derivatives

Myo-inositol is ubiquitous in nature and is found as the structural core of a diverse range of derivatives including phosphatidylinositols, phosphatidylinositol phosphates, glycosylphosphatidylinositol (GPI) anchors and mycothiol. Many of these compounds have important and fundamental biological functions, including cellular signalling, protein anchoring and pathogen virulence. Given their essential function in living systems they have been the focus of significant research in both academia and industry.

The stereocontrolled synthesis of protected myo-inositol derivatives present a number of obstacles and challenges requiring often lengthy synthetic sequences. An alternative to classical synthetic routes would be to adopt a chemoenzymatic approach in which biocatalysis introduces chirality. To date the concept of using a chrial inositol phosphate derivative as a starting point has not been realized, primarily due to the lack of availability and prohibitive cost (e.g. D-myo-inositol-3-phosphate = €196/mg; D-myo-inositol-1-phosphate = €69/mg). Thus, a reliable method for the production of an enantioenriched inositol phosphate from a cheap and readily available starting material is essential to enable a thorough investigation of such an approach and has the potential to greatly simplify access to complex inositol derivatives.

The isomerisation of glucose 6-phosphate to D-myo-inositol-3-phosphate (IP) mediated by inositol phosphate synthase (INO1) is the first biosynthetic step in the production of all inositol-containing metabolites and the enzyme is present in all eukaryotes and prokaryotes. In 2006 Smith and Martin detailed the role of the INO1 in bloodstream T. brucei (TbINO1) in production of the parasite’s variable surface GPI-anchored protein coat. As part of this work, recombinant TbINO1 was expressed in E. coli and an assay for D-myo-inositol-3-phosphate (IP) production was developed. In preliminary studies recombinant T. brucei INO1 was expressed (~100 mgL-1) and proved effective at transforming up to 10 mg of glucose 6-phosphate (€0.10/mg) in batch reactions. These batch reactions were not amenable to scale-up due to protein precipitation at increased concentrations. In order to resolve this limitation, TbINO1 was immobilized onto a Ni2+-Sephadex column via its His-Tag (loading ~200 mg/5 mL column). Despite the immobilization method being unoptimized, we were able to produce >400 mg of enantiopure IP in flow at 37 °C. However, our current system is far from optimal and suffers significant loss of catalytic activity after 36 h, requiring further investigation of immobilization methodologies and precise reaction and stability tuning.

Objectives:
i) Optimize immobilization of TbINO1 for production of D-myo-inositol 3-phosphate.
ii) Design and implement multi-enzyme cascade reaction system to transform glucose to D-myo-inositol 3-phosphate.
iii) Demonstrate viability of D-myo-inositol-3-phosphate as a starting point for the synthesis of enantiopure inositol derivatives

For further information on these opportunities and informal enquiries please contact Dr Gordon Florence ([Email Address Removed]).

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