Modelling and optimisation of the one-pot, multi-enzymatic synthesis of chiral amino-alcohols based on microscale kinetic parameter determination

 

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Modelling and optimisation of the one-pot, multi-enzymatic synthesis of chiral amino-alcohols based on microscale kinetic parameter determination

Corresponding Author: Prof. Gary James Lye, Ph.D.
Corresponding Author’s Institution: University College London
First Author: Leonardo Rios-Solis, PH.D.
Order of Authors: Leonardo Rios-Solis, PH.D.; Phattaraporn Morris, PhD ; Christopher Grant, PhD; Akin
Odeleye, PhD; Helen C hailes, Professor; John M Ward, Professor; Paul A Dalby, Professor; Frank
Baganz, PhD; Gary J Lye, Professor

Abstract:

Advances in synthetic biology are facilitating the de novo design of complex, multi-step
enzymatic conversions for industrial organic synthesis. This work describes the integration of multistep
enzymatic pathway engineering with enzyme kinetics and bioreactor modelling to optimize the
synthesis of chiral amino alcohols using engineered E. coli transketolases (TK) and the
Chromobacterium violaceum transaminase (TAm). The specific target products were (2S, 3S)-2-
aminopentane-1,3-diol (APD) and (2S, 3R)-2-amino-1,3,4-butanetriol (ABT). Kinetic models and
parameters for each of the enzymatic steps were first obtained using automated microwell
experiments. These identified the TK-catalysed conversions as being up to 25 times faster than the
subsequent TAm conversions and inhibition of TAm by the amino donor used, (S)-(-)-α-
methylbenzylamine, as limiting the overall conversion yields. In order to better ‘match’ the relative
rates of the two enzymes an E. coli expression system, based on two compatible plasmids, was
constructed to produce both enzymes in a single host. By control of induction time and temperature it
was possible to produce six times more recombinant TAm than TK to help balance the reaction rates.
To overcome MBA inhibition fed-batch addition of the amino donor was introduced as well as the use
of isopropylamine as an alternate amino donor. Adopting these strategies, and using the kinetic models
to optimise feeding strategies, the one pot syntheses of APD and ABT were successfully scaled-up to
preparative scales. Excellent agreement was found between the kinetic profiles and yields predicted
and those achieved experimentally at the larger scale. In this case the integration of these multidisciplinary
approaches enabled us to achieve up to a 6 fold greater yield using concentrations an
order of magnitude higher than in previous preparative scale batch bioconversions carried out
sequentially.

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Modelling and optimisation of the one-pot, multi-enzymatic synthesis of chiral amino-alcohols based on microscale kinetic parameter determination

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