| Ubiquinone (Coenzyme Q; abbreviation, UQ) is an essential electron carrier in the mitochondrial respiratory chain, and widely used as an essential component of ATP generation in the oxidative phosphorylation process and as an lipid-soluble antioxidant preventing lipid peroxidation. Therefore it has been widely used in pharmaceuticals and cosmetics.In this study, ubiquinone-10production was performed in Escherichia coli Schizosaccharomyces pombe and Agrobacterium tumefacients, respectively. In Escherichia coli, we have cloned, expressed the decaprenyl diphosphate synthase, designated dps, gene from Agrobacterium tumefaciens, and succeeded in detecting UQ-10in addition to innate UQ-8in Escherichia coli. Furthermore, the production of UQ-10was higher than UQ-8. To establish an efficient expression system for UQ-10production, we used genes, including ubiC, ubiA and ubiG involved in UQ biosynthesis in E. coli, to construct a better co-expression system. The expression coupled by dps and ubiCA was effective for increasing UQ-10production by five times than that by expressing single dps gene in the shake flask culture. To study for a large scale production of UQ-10in E. coli, fed-batch fermentations were implemented to achieve a high cell density culture. A cell concentration of84.50g/L and94.58g/L dry cell weight (DCW), and UQ-10content of50.29mg/L and45.86mg/L was obtained after32.5h and27.5h cultivation subsequent to IPTG and lactose induction, respectively. In addition, plasmid stability was maintained at high level throughout the fermentation.we cloned and overexpressed the full length pptl (MTpptl) gene which encodes p-hydroxybenzoate:polyprenyltransferase and ERpptl gene which was modified to be localized on endoplasmic reticulum in fission yeast. The yeast MTpptl and ERpptl transgenic lines showed about3.7and5.1times increment in UQ content and the recombinant yeasts with a higher UQ level are more resistant to H2O2, Cu2+and NaCl, and interestingly their growth was also faster than the wild type at lower temperature. For large scale cultivation, the direct feedback control of glucose using an on-line ethanol concentration monitor for ubiquinone production of yeast ERppt1by high-cell-density fermentation was investigated and the fermentation parameters(e.g., dissolved oxygen, pH, ethanol concentration, oxygen uptake rate, carbon dioxide evolution rate and respiration quotient) were also discussed. After90h cultures, the yeast dry cell weight reached57g/L and the ubiquinone yield reached24mg/L. In addition, plasmid stability was maintained at high level throughout the fermentation.Dps gene, which encodes polyprenyl diphosphate (PPP) synthases, involved in ubiquinone biosynthesis from Agrobacterium tumefaciens, and ubiA gene in Escherichia coli, coq2gene in Saccharomyces cerevisiae and pptl gene in Schizosaccahromyces pombe which encodes4-hydroxybenzoate:polyprenyl diphosphate (4-HB:PPP) transferase, were reconfigured into an openron under the control of a single promter to yield various plasmids including pBIV-dps, pBIV-dpsq, pBIV-dpsp and pBIV-dpsca. The recombinant A. tumefaciens containing dps-ubiC-ubiA gene showed the highest level ubiquinone production (2.18mg/g) than that of the other recombinants and the original bacterium. In an aerobic fed-batch fermentation, A. tumefaciens containing the pBIV-dpsca plasmid produced33mg of ubiquinone-10per liter which was1.52times higher than that of original type. while in microaerobic fed-batch fermentation, recombinant cell pBIV-dpsca produced66.8mg/L of ubiquinone-10. Compared to the original A. tumefaciens, the ubiquinone-10yield and productivities of the recombinant bacterium pBIV-dpsca increased2.19and1.85times, respectively, under microaerobic fed-batch conditions. |
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