Production Of Glycolysis Metabolites In Microfluidic Devices: D-1,3-Bisphosphoglycerate

1,3-Diphosphoglycerate (DPG) is a metabolite synthesised in the glycolysis and gluconeogenesis pathways and is involved in the control of cellular metabolism, oxygen affinity of red cells and other cellular functions. DPG can be produced by the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (DGA3P) by D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH,) in the presence of NAD and inorganic phosphate or by the phosphorylation of 3-phosphoglycerate (PPG) by phosphoglycerate kinase (PGK,) in the presence of ATP (Figure 1). The production of DPG by DG3P is preferred due to the unfavourable reaction thermodynamics of PGK. Nonetheless, the enzymatic synthesis of the DPG is particularly challenging due the high rate of decomposition (Negelein, 1965) and has been until now. To address this challenge, we want to exploit the advances made in microreactor technology, and in microfluidics for rapid molecule isolation. This project will highlight the potential of these novel technologies for industrial application. The research supports the development of novel therapies with minimal cost ("Healthcare Technologies"), and flexible and reconfigurable manufacturing systems and microsystems ("Manufacturing the future").

Objectives and Approach

Performing flow chemistry in structured microfluidic reactors offers an increased control over the reaction conditions and easier compartmentalisation of multi-enzyme reactions, in addition to the possibility to combine reaction and separation steps (O'Sullivan et al., 2012). Furthermore, these devices offer enhanced mass and heat transport due to the high surface-to-volume ratios, compared with traditional batch reactors (Marques & Fernandes, 2011, Wohlgemuth et al. 2015). These characteristics of microfluidic reactors are of particular interest when unstable compounds are produced and where there is a need to precipitate and isolate an unstable compound rapidly.

To demonstrate the value of this novel engineering approach, we propose to produce DPG continuously in an integrated microfluidic system, comprising reaction and separation unit operations (Figure 2), with the enzyme either in solution or immobilised. The entire train of unit operations would have analytical techniques incorporated, either on-line (Raman, NIR and UV spectroscopy), in-line or at-line (HPLC, GC), which will allow the establishment of the Process Analytical Tools approaches.

Project Outline

1. Development of analytical techniques, e.g. to analyse DPG
2. Development of pre-cursor reaction for (batch and microfluidic reactor) to produce glyceraldehyde cost-effectively
3. Structured microfluidic reactor optimisation
3.1. Evaluate impact of two-phase system on enzyme activity, including organic-aqueous and aqueous-aqueous
3.2. Determine product degradation rate and based on reactor modelling assess the type of structured microfluidic reactor and biocatalyst form to use (Van Daele et al., 2016)
3.3 Evaluating microfluidic reactor cascades, including chemo-enzyme and enzyme-enzyme
3.3. Study interaction of residence times of the reactor and the entire set of unit operations, versus stability to minimise product degradation
4. Precipitation Unit
4.1. Establishment of precipitation conditions using different precipitation agents
4.2. Assess suitability of in-house developed flocculation device to carry out precipitation processes
5. Integrated System
5.1. Incorporation of analytic technology to control reaction and precipitation conditions.
5.2. Evaluate industrial implementation