Rios-Solis, L;
Halim, M;
Cazares, A;
Morris, P;
Ward, JM;
Hailes, HC;
Dalby, PA;
... Lye, GJ; + view all
(2011)
A toolbox approach for the rapid evaluation of multi-step enzymatic syntheses comprising a 'mix and match' E. coli expression system with microscale experimentation.
Biocatalysis and Biotransformation
, 29
(5)
pp. 192-203.
10.3109/10242422.2011.609589.
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Abstract
This work describes an experimental 'toolbox' for the rapid evaluation and optimisation of multi-step enzymatic syntheses comprising a 'mix and match' E. coli-based expression system and automated microwell scale experimentation. The approach is illustrated with a de novo designed pathway for the synthesis of optically pure amino alcohols using the enzymes transketolase (TK) and transaminase (TAm) to catalyze asymmetric carbon-carbon bond formation and selective chiral amine group addition respectively. The E. coli expression system, based on two compatible plasmids, enables pairs of enzymes from previously engineered and cloned TK and TAm libraries to be evaluated for the sequential conversion of different initial substrates. This is complemented by the microwell experimentation which enables efficient investigation of different biocatalyst forms, use of different amine donors and substrate feeding strategies. Using this experimental 'toolbox', 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, which gave final product yields of 90% mol/mol for APD and 87% mol/mol for ABT (relative to the initial TK substrates) within 25 hours. For the synthesis of APD, the E coli TK mutant D469E was paired with the TAm from Chromobacterium violaceum 2025 while for ABT synthesis the wild-type E. coli TK exhibited the highest specific activity and ee( enantiomeric excess) of >95%. For both reactions, whole-cell forms of the TK-TAm biocatalyst performed better than cell lysates while isopropylamine (IPA) was a preferable amine donor than methylbenzylamine (MBA) since side reactions with the initial TK substrates were avoided. The available libraries of TK and TAm enzymes and scalable nature of the microwell data suggest this 'toolbox' provides an efficient approach to early stage bioconversion process design in the chemical and pharmaceutical sectors. © 2011 Informa UK, Ltd.
| Type: | Article |
|---|---|
| Title: | A toolbox approach for the rapid evaluation of multi-step enzymatic syntheses comprising a 'mix and match' E. coli expression system with microscale experimentation |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.3109/10242422.2011.609589 |
| Publisher version: | http://dx.doi.org/10.3109/10242422.2011.609589 |
| Language: | English |
| Additional information: | This version is the author accepted manuscript. For information on re-use, please refer to the publisher's terms and conditions. |
| Keywords: | transketolase, transaminase, biocatalyst, multi-step enzymatic synthesis, chiral amino alcohols, de novo pathway |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Chemistry |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/10194394 |
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