Biotechnology and Bioprocess Engineering 2021; 26(4): 621-629  
Systems Biology Engineering of the Pantothenate Pathway to Enhance 3HB Productivity in Escherichia coli
Samer Younes, Dania Awad, Elias Kassab, Martina Haack, Claudia Schuler, Norbert Mehlmer, and Thomas Brueck
Samer Younes, Dania Awad, Elias Kassab, Martina Haack, Claudia Schuler, Norbert Mehlmer, Thomas Brueck*
Werner Siemens-Chair of Synthetic Biotechnology, Department of Chemistry, Technical University of Munich (TUM), Garching 85748, Germany
Tel: +49-89-289-13250; Fax: +49-89-289-13255
E-mail: brueck@tum.de
Both authors contributed equally to this work
Received: February 23, 2021; Revised: June 6, 2021; Accepted: June 7, 2021; Published online: August 30, 2021.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
The monomer, 3-hydroxybutyrate (3HB), plays an interesting role as a precursor for antibiotics, vitamins, and bioplastics such as polyhydroxy butyrates (PHB). The biotechnological production of both compounds in Escherichia coli has seen increased interest in the last decade. The central metabolite in the 3HB production pathway is acetyl-CoA, the derivative of coenzyme A (CoA). Enriching the intracellular pool of these cofactors could improve 3HB titers. In our study, we opted to increase CoA titers by upregulating pantothenate kinase (PanK), the rate-limiting step in CoA biosynthetic pathway. To this end, 4 PanKs genes of different taxonomic origins (mammalian, fungal, and bacterial) were individually expressed and evaluated in 3HB producing E. coli cells. In shake flask studies, strains expressing Aspergillus nidulans PankII and Mus musculus PanK1β achieved the highest 3HB titers. In a bioreactor fermentation, the strain harboring murine PanK1β resulted in 7.6 g/L compared to 5.4 g/L of 3HB in the control strain. Although structurally different from the bacterial PankI, our study showed that eukaryotic Panks can supplement the kinase activity in prokaryotes. Expressing the exogenous PanKs offer several advantages over the host’s enzyme; PanKII is only inhibited by acetyl-CoA, for which the 3HB-production system would provide a constant metabolic sink. Additionally, PanK1β is weakly regulated by acetyl-CoA, and its activity is stimulated by free CoA. Overexpressing eukaryotic PanKs constitutes a suitable strategy for improving 3HB titers in E. coli.
Keywords: 3-hydroxybutyrate, pantothenate kinase, Escherichia coli, bioplastics, CoA, acetyl-CoA


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