Dissertation > Biological Sciences > Microbiology > Microbial biochemistry

Metabolic Engineering of the Coenzyme Q10 Pathway in Rhodobacter Sphaeroides and Its Application

Author LuWenQiang
Tutor YuHongWei
School Zhejiang University
Course Biochemical Engineering
Keywords CoQ10 metabolic engineering constitutive expression metabolicbalance self-regulation
CLC Q936
Type PhD thesis
Year 2014
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Coenzyme Q10(CoQ10) is an essential electron carrier in the aerobic respiratory electron transfer system. It generates ATP for the body and has been proven effective in treating cardiovascular disease and hypertension. Also, CoQ10is an important antioxidant. Currently, CoQ10is widely used in health care and disease prevention. For improving the production of CoQ10, metabolic engineering of CoQ10producers has widely drawn the attention from the researchers. In this study, Rhodobacter sphaeroides, a natural CoQ10producer, was metabolically engineered to improve its production of CoQ10.For metabolic engineering of Rhodobacter sphaeroides, a constitutive expression plasmid was constructed. This plasmid harbors a broad-host-range plasmid backbone and a constitutive promoter. Real-time PCR verified that the plasmid could constitutively express the gene cloned in the plasmid in the presence of glucose. Taking UbiG as an example, the expression level of UbiG in the recombinant with UbiG cloned in the plasmid was133-times as high as that in the wild type. In this study, different strategies were developed based on this plasmid to improve the production of CoQ10.Metabolic balance is a key issue in metabolic engineering. In this study, we tried to improve the CoQ10production by enhancing the flux of the MEP pathway with a self-regulation system. This system was constructed based on the mechanism of Lac operator and ribosomal binding site regulation. By introducing5different ribosomal binding sites, a pathway-balanced mutant with87%CoQ10production higher than the wildtype was obtained. Also, this system was corfirmed to be robust and flexible in tuning metabolic balance.The quinone modification pathway directly influences the CoQ10production by transfor ming the precursors to CoQ10. We screened the key enzymes in the pathway and idenfied UbiE, UbiH and UbiG as the candidates. To expand the ubiquninoe modification pathway, different coexpression strategies were developed. It was found that by fusing UbiG and UbiE to the pufX peptide, which dereases the loss of intermediates diffusion, the flux of the pathway could be maximized and the production of CoQ10could be subsequently improved.Systematic engineering of the metabolic pathway is a highlight in metabolic engineering. It can integrate the positive results of the sectional pathway optimizations. In this study, the optimized MEP pathway and ubiquinone modification pathway were intergrated. As a result, a higher level of CoQ10production was achieved.In this study, we systematically engineered the CoQ10pathway in Rhodobacter sphaeroides and successfully improved the production of CoQ10by about3times.

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