Hyperproduction of phenylalanine by Escherichia coli: application of a temperature-controllable expression vector carrying the repressor-promoter system of bacteriophage lambda |
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| Affiliation: | 1. College of Landscape Architecture, Sichuan Agricultural University, Chengdu 611130, China;2. College of Biology and Pharmacy, Yulin Normal University, Yulin 537000, China;3. Department of Electrical and Biomedical Engineering, University of Nevada, Reno 89503, USA;1. The Dept. of Bio and Fermentation Convergence Technology, BK21 PLUS project, Kookmin University, Seoul, Republic of Korea;2. The Dept. of Biotechnology, Graduate School, Korea University, Seoul, Republic of Korea;1. Genetics and Physiology of Microalgae, Institute of Botany, B22, University of Liège, Belgium;2. Centre of Analytical Research and Technology (CART) – LSM/Inorganic Analytical Chemistry, Department of Chemistry, B6C, University of Liège, Belgium;3. The Fritz Haber Research Center for Molecular Dynamics, Institute of Chemistry, Hebrew University of Jerusalem, Jerusalem 91904, Israel;4. Theoretical Physical Chemistry, Department of Chemistry, B6C, University of Liège, Belgium;5. Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA |
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| Abstract: | For the high production of phenylalanine by Escherichia coli, we cloned the pheAFR and aroFFR genes (FR = feedback resistant), which encoded chorismate mutase P-prephenate dehydratase and 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase that are feedback inhibition-free as to the endproducts, into a temperature-controllable expression vector composed of the PR and PL promoter and a temperature sensitive repressor, cI857, of bacteriophage lambda. The plasmid obtained was designated as pSY130-14, and the temperature dependency of expression of the cloned genes and of phenylalanine production was investigated at different temperatures between 30 and 42°C using the strain AT2471 harbouring the plasmid. Above 35°C, the pheAFR gene and aroFFR gene expressions, and activities of both enzymes continued to increase up to 42°C. The cell concentration remained constant up to 38.5°C, but started to decrease sharply above 40°C, while the cell concentration of the host strain, AT2471, remained constant at all temperatures tested. The concentration of phenylalanine also depended on the temperature, and the highest production of phenylalanine, 18.6 g l−1, was obtained from glucose at 38.5°C in a 2.5 1 reactor. |
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