PRODUCTION OF ZEAXANTHIN IN ESCHERICHIA-COLI TRANSFORMED WITH DIFFERENT CAROTENOGENIC PLASMIDS

Carotenoids are of great commercial interest and attempts are made to produce different carotenoids in transgenic bacteria and yeasts. Devel opment of appropriate systems and optimization of carotenoid yield inv olves transformation with several new genes on suitable plasmids. Ther efore, the non-carotenogenic bacterium Escherichia coli JM101 was tran sformed in our study with several genes that mediated the biosynthetic production of the carotenoid zeaxanthin in this host, Selection of pl asmids for the introduction of five essential genes for zeaxanthin for mation showed ed that a pACYC-derived plasmid was the best. Multiplasm id transformation generally decreased production of zeaxanthin. By cot ransformation with different plasmids, limitations in the biosynthetic pathway were found at the level of geranylgeranyl-pyrophosphate synth ase and beta-carotene hydroxylase. In our study a maximum zeaxanthin c ontent of 289 mu g/g dry weight was obtained. This involved the constr uction of a plasmid that mediated high-level expression of beta-carote ne hydroxylase. The level of expression was demonstrated on protein ge ls and solubilization by the mild detergent Brij 78 revealed that a si gnificant portion of the expressed enzyme is located in the E. coli me mbranes where it can exert its catalytic function. Based on the result s obtained, new strategies for vector construction and strain selectio n were proposed which could increase the present concentrations drasti cally. Optimal growth conditions of the transfomed E. coli strains for carotenoid formation were found at a temperature of 28 degrees C and a cultivation period of 2 days.