Production of zeaxanthin in Escherichia coli transformed with different carotenogenic plasmids

• Published in: Applied microbiology and biotechnology 1997-08, Vol.48 (2), p.162-167
• Main Authors: Ruther, A, Misawa, N, Boger, P, Sandmann, G
• Format: Article
• Language: English
• Subjects: Bacteria; beta Carotene - analogs & derivatives; beta Carotene - biosynthesis; Biological and medical sciences; Biological Sciences; Biotechnology; Carotenoids; Child; E coli; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Growth conditions; Humans; Isopropyl Thiogalactoside - metabolism; Methods. Procedures. Technologies; Microbial engineering. Fermentation and microbial culture technology; Microbiological chemistry; Mixed Function Oxygenases - metabolism; Physiological aspects; Plasmids; Transformation, Bacterial; Xanthophylls; Yeasts; Zeaxanthins
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s002530051032

Carotenoids are of great commercial interest and attempts are made to produce different carotenoids in transgenic bacteria and yeasts. Development of appropriate systems and optimization of carotenoid yield involves transformation with several new genes on suitable plasmids. Therefore, the non-carotenogenic bacterium Escherichia coli JM101 was transformed in our study with several genes that mediated the biosynthetic production of the carotenoid zeaxanthin in this host. Selection of plasmids for the introduction of five essential genes for zeaxanthin formation showed that a pACYC-derived plasmid was the best. Multiplasmid transformation generally decreased production of zeaxanthin. By cotransformation with different plasmids, limitations in the biosynthetic pathway were found at the level of geranylgeranyl-pyrophosphate synthase and beta-carotene hydroxylase. In our study a maximum zeaxanthin content of 289 micrograms/g dry weight was obtained. This involved the construction of a plasmid that mediated high-level expression of beta-carotene hydroxylase. The level of expression was demonstrated on protein gels and solubilization by the mild detergent Brij 78 revealed that a significant portion of the expressed enzyme is located in the E. coli membranes where it can exert its catalytic function. Based on the results obtained, new strategies for vector construction and strain selection were proposed which could increase the present concentrations drastically. Optimal growth conditions of the transformed E. coli strains for carotenoid formation were found at a temperature of 28 degrees C and a cultivation period of 2 days.