Ubiquinone-10 production using Agrobacterium tumefaciens dps gene in Escherichia coli by coexpression system.
Ubiquinone (Coenzyme Q; abbreviation, UQ) acts as a mobile component of the respiratory chain by playing an essential role in the electron transport system, and has been widely used in pharmaceuticals. The biosynthesis of UQ involves 10 sequential reactions brought about by various enzymes. In this study we have cloned, expressed the decaprenyl diphosphate synthase, designated dps gene, from Agrobacterium tumefaciens, and succeeded in detecting UQ-10 in addition to innate UQ-8 in Escherichia coli. Furthermore, the production of UQ-10 was higher than UQ-8. To establish an efficient expression system for UQ- 10 production, 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-10 production by five times than that by expressing single dps gene in the shake flask culture. To study for a large-scale production of UQ-10 in E. coli, fed-batch fermentations were implemented to achieve a high cell density culture. A cell concentration of 85.40 g/L and 94.58 g/L dry cell weight (DCW), and UQ-10 content of 50.29 mg/L and 45.86 mg/L was obtained after 32.5 h and 27.5 h of cultivation, subsequent to isopropyl-beta-D-thiogalactopyranoside and lactose induction, respectively. In addition, plasmid stability was maintained at high level throughout the fermentation.
Zhang
Dawei
D
Beijing Key Lab of Bioprocess, College of Biology Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China.
Shrestha
Binaya
B
Li
Zhaopeng
Z
Tan
Tianwei
T
D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics*; D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000378:metabolism*; D001431:Bacteriological Techniques; D001483:Base Sequence; D019149:Bioreactors; D001709:Biotechnology; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004122:Dimethylallyltranstransferase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D007700:Kinetics; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D007652:Oxo-Acid-Lyases / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014451:Ubiquinone / Q000096:biosynthesis* / Q000737:chemistry
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D014451:Ubiquinone; D008780:Methyltransferases; C019616:UbiG protein, E coli; D019883:Alkyl and Aryl Transferases; C039821:decaprenyl pyrophosphate synthetase; C509554:4-hydroxybenzoic acid oligoprenyltransferase, E coli; D004122:Dimethylallyltranstransferase; D007652:Oxo-Acid-Lyases; C075960:chorismate pyruvate lyase; C025204:Ubiquinone Q2; D005947:Glucose; D005990:Glycerol