Microscale evaluation of de novo engineered whole cell biocatalysts

Biocatalysis has emerged as a powerful tool for the synthesis of high value optically pure compounds. With advances in synthetic biology, it is now possible to design de novo non-native pathways to perform non-natural chiral bioconversions. However these systems are difficult to assemble and operate productively, severely hampering their industrial application. The purpose of this study was to develop a microscale toolbox for the rapid design and evaluation of synthetic pathways, in order to increase their operational productivities and speed-up their process development. The first aim of this work was to establish a microscale platform to accelerate the evaluation of different variants of transketolase (TK) and transaminase (TAm), in order to design and construct a de novo pathway for the one-pot synthesis of chiral amino alcohols. The second aim was to develop a microscale methodology to rapidly establish the complete kinetic models of the selected TKs and TAms, which would allow efficient operation of the one-pot synthesis. The third aim was to scale-up the production of the biocatalyst to pilot plant, while controlling and maintaining the desired level of expression of each enzyme. Finally the fourth aim of this project was to scale-up and simulate the complete one-pot syntheses to preparative scale, while predicting and applying the best reaction strategies and reactor configurations. The experimental microscale toolbox was based on 96 microwell plates with automation capacities, where the one-pot syntheses of the diastereoisomers (2S,3S)-2 aminopentane-1,3-diol (APD) and (2S,3R)-2-amino-1,3,4-butanetriol (ABT) were designed and performed with final product yields of 90% and 87% mol/mol respectively. For the synthesis of ABT and APD, the wild type E. coli TK and the engineered D469E TK were identified as the best candidates respectively, and both enzymes were paired with the TAm from Chromobacterium violaceum. A microscale methodology for kinetic model establishment was developed based on programmable non linear methods. The TAm step was found to be the bottleneck of the multi-step syntheses, due to the high a Michaelis constant of intermediate substrate erythrulose for the synthesis of ABT, and the low catalytic constants for the synthesis of APD. Also the amino donor substrate was discovered to be toxic for the TAm, as well as causing side reactions, thus affecting the overall performance of the de novo pathway. The production of the E. coli whole cells containing the de novo pathway were successfully scaled-up to pilot plant without losing catalytic activity. By manipulating the fermentation temperature and induction time of TAm, it was found the desired level of expression of each enzyme could be achieved. Finally, the complete one-pot syntheses were simulated using the previously established microscale kinetic models, which were found to be predictive of preparative scale bioconversions. A reactor with fed-batch addition of the amino donor was predicted as the best operating strategy in each case. Using this strategy, the one-pot syntheses allowed up to a 6-fold increase in product yield (% mol/mol), while using concentrations one order of magnitude higher than previously published preparative scale data. As a conclusion, this work is the first of its kind to develop such a microscale modelling toolbox, which is designed to exploit the synthetic potential of engineered and recombinant enzymes, in order to design, simulate and optimize de novo engineered pathways. This makes the results of this work an original contribution for the process development of synthetic pathways.