A biocatalytic approach to the synthesis of myo-inositol derivatives

Supervisors: Gordon Florence, Terry K. Smith

Project Description:
Myo-inositol is ubiquitous in nature and is found as the structural core of a diverse range of derivatives including phosphatidylinositols and glycosylphosphatidylinositol (GPI) anchors. Many of these compounds have fundamental biological functions and given essentiality in living systems they have been the focus of significant research in both academia and industry.

The stereocontrolled synthesis of myo-inositol derivatives present a number of obstacles and challenges. 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). Thus, a reliable method for the production of an enantioenriched inositol phosphate remains to be established 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.1 As part of this work, recombinant TbINO1 was expressed in E. coli and an assay for D-myo-inositol-3-phosphate (IP) production developed.2 We have recently shown TbINO1 can be immbolized on Ni2+-Sephadex (loading ~200 mg/5 mL column). to produce >400 mg of enantiopure IP under flow conditions.3 However, our current system is far from optimal and further investigation of immobilization methodologies and precise reaction and stability tuning.

Objectives:

  • Optimize immobilization of TbINO1 for production of IP
  • Design and implement multi-enzyme cascade reaction system to transform glucose to IP.
  • Demonstrate viability of IP as a building block for the synthesis of inositol derivatives

Research Plan:

i) Immobilization and Reaction Optimisation: The non-covalent bound Ni-His-Tag interaction has several limitations including leaching of both Ni2+ and protein. The initial phase screen attachment methods for recombinant TbINO1 and assess activity and stability. We will investigate protein modification to; i) aid immobilization; and ii) broaden substrate scope.  Promising systems will be further optimized for enzyme loading, efficiency and recyclability. The final phase will seek process scale up to enable multigram production of IP under recycling conditions or ideally in continuous flow.

ii) Multienzyme Cascade Systems: While G6P is a viable starting material for the initial study of INO1 optimisation, added value to the process could be further increased via production of G6P in situ as part of a multienzyme cascade starting from glucose. This will pave the way for the viable production of isotopically labelled myo-inositol derivatives on large scale.

ii) Use of IP as a starting material for inositol synthesis: The ability to access significant quantities of enantiopure PI from the biocatalytic transformation presents a new starting point for the synthesis of a variety of chiral myo-inositol building blocks for application in synthesis.

References:

1) Mol. Microbiol. 2006, 61, 89.

2) Mol. Biol. Int. 2011, 389364.

3) React Chem Eng  2017,2, 44

 

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