Bayir, N; (2017) Modelling of integrated de-novo pathways for amino alcohol synthesis. Doctoral thesis , UCL (University College London).

Abstract

Chiral amino alcohols are industrially valuable intermediates used in the synthesis of many pharmaceuticals and fine chemicals. Transamination (TAm) is not only one of the most interesting industrial enzymatic reactions on its own nowadays but also the second enzymatic step of the de novo pathway studied by our research group (Bioconversion-Chemistry and Engineering Interface Programme-BiCE) for the synthesis of chiral amino alcohols. In this thesis, firstly a study on the kinetic modelling of TAm reactions was presented. Kinetic behaviour of two ω-TAm reactions employing different amino acceptors but the same amino donor was investigated. Using a Genetic Algorithm, kinetic models could be determined with only 10-12 progress curves for both reaction schemes without having any experimental data for the reverse reaction. Also, a sensitivity analysis on the kinetic parameters of both reactions was carried out. Having gained enough insight into the kinetic modelling of TAm reactions, two different sequential enzyme reactions leading to the synthesis of 2-amino-1,3,4- butanetriol (ABT) were studied. The first one involved three different enzymes, namely TAm (pQR977), Transketolase (TK) and TAm (pQR801). 100% conversion was proven possible with both experimental and simulation results in a 28 hour-long fed-batch reaction. Although, the concentrations tested in the experiments were low, this three-step sequential enzyme reaction showed great promise for further being optimized for industrial applications. The second one was a recycling sequential enzyme reaction involving three different enzymatic reactions taking place simultaneously. Two of the reactions were catalyzed by TAm (pQR1021) and one of them was catalyzed by TK. With the help of second TAm reaction not only desired product was synthesized but also one of the intermediary precursors was recycled within the system. The long term goal of this project was the integration of de novo pathways leading to the synthesis of chiral amino alcohols into host cell metabolism. Thus, the recycling sequential enzyme reaction employing only two different enzymes and two different substrates was found promising for achieving this goal. With both experimental and simulation results the favorability of this reaction was demonstrated and low amounts of ABT (~6 mM) was synthesized in a 8-hour long reaction using purified enzymes. One of the precursors in both sequential enzyme reactions was serine. In the final results chapter, a metabolic modelling methodology was used for identifying optimal metabolic engineering strategies, namely enzyme knock-outs and enzyme expression level modulations, for the overproduction of serine by employing a partial mechanistic model of central carbon metabolism of Escherichia coli. Simulation results showed 89% increase in serine synthesis in the case of doubling the expression level of SerSynth, a hypothetical enzyme for the synthesis of serine from 3-phosphoglycerate.

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