Coupling carboxylic acid reductase to inorganic pyrophosphatase enhances cell-free in vitro aldehyde biosynthesis
Name
2015-12-12 - Coupling Car and Pyrophosphatase - Text-REVISEDv2 - FINAL-clean copy.pdf
Description
Accepted version
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873.06 KB
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Author(s)
Kunjapur, Aditya M
Cervantes, Bernardo
Prather, Kristala LJ
Date Issued
2016
Journal
Biochemical Engineering Journal
Publisher
Elsevier BV
Citation
Kunjapur, Aditya M., Bernardo Cervantes, and Kristala L. J. Prather. "Coupling Carboxylic Acid Reductase to Inorganic Pyrophosphatase Enhances Cell-Free in Vitro Aldehyde Biosynthesis." Biochemical Engineering Journal 109 (2016): 19-27.
Version
Author's final manuscript
Abstract
© 2015 Elsevier B.V. Carboxylic acid reductases (CARs) have been harnessed in metabolic pathways to produce aldehydes in engineered organisms. However, desired aldehyde products inhibit cell growth and limit product titers currently achievable from fermentative processes. Aldehyde toxicity can be entirely circumvented by performing aldehyde biosynthesis in non-cellular systems. Use of purified CARs for preparative-scale aldehyde synthesis has been limited by in vitro turnover of model CARs, such as Car. Ni from Nocardia iowensis, despite robust conversion of substrates associated with expression in heterologous hosts such as E. coli and yeast. In this study, we report that in vitro activity of Car. Ni is inhibited by formation of the co-product pyrophosphate, and that pairing of an inorganic pyrophosphatase (Ppa. Ec) with Car. Ni substantially improves the rate and yield of aldehyde biosynthesis. We demonstrate that, in the presence of Ppa. Ec, Michaelis-Menten kinetic models based on initial rate measurements accurately predict Car. Ni kinetics within an in vitro pathway over longer timescales. We rationalize our novel observations for Car. Ni by examining previously posed arguments for pyrophosphate hydrolysis made in the context of other adenylate-forming enzymes. Overall, our findings may aid in increasing adoption of CARs for cell-free in vitro aldehyde biosynthetic processes.
MIT Department
Massachusetts Institute of Technology. Department of Chemical Engineering
MIT Synthetic Biology Engineering Research Center
Massachusetts Institute of Technology. Microbiology Graduate Program
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Creative Commons Attribution-NonCommercial-NoDerivs License
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DOI of Published Version
https://doi.org/10.1016/J.BEJ.2015.12.018
