| dc.description.abstract | The negative impact of burning fossil fuels and oil supply concerns have prompted researchers to look for alternative renewable fuel sources. Sustainable lignocellulosic and photosynthetic biofuels are promising renewable energy resources that reduce greenhouse gas emission and pose least risks to the food supply. However, the metabolic pathways involved in lignocellulosic biomass utilization and subsequent biofuel or bioproduct production in microorganisms are still unclear, which hinders high yield biofuel production through genetic engineering. In the present study, systems biology guided genetic engineering technologies and strategies have been systematically investigated to pave a biological route to produce renewable fuels and bioproducts. Two effective cell factories, photosynthetic Synechococcus elongatus and ligninolytic Pseudomonas putida, have been designed and evaluated to produce the sustainable limonene and polyhydroxyalkanoates (PHAs), respectively. First, proteomics, transcriptomics and metabolomics technologies have been employed to find the key limiting steps in the limonene biosynthesis pathway (MEP). The comprehensive proteomics analysis and subsequent genetic engineering results highlighted that regulations in the MEP were not the rate limiting factors for limonene productivity in cyanobacteria. Further metabolomics analysis suggested that the carbon partitions between the primary and secondary metabolites could have a strong impact on limonene productivity. By mutating the sucrose biosynthesis pathway in S. elongatus PCC7942, the limonene productivity was significantly increased. Interestingly, the increased glycogen content in sucrose phosphate synthase mutated strain led to the hypothesis of redirecting the carbon flux to the MEP pathway by mutating the glycogen biosynthesis pathway. This hypothesis was supported by an increased yield of 14.9 mg/L by the glucose-1-phosphate adenylyltransferase(glgC) mutated strain under continuous growing conditions. Second, to produce high yields of PHAs in heterotrophic Pseudomonas putida from lignin derivatives, metabolic engineering strategies with substrate optimization were designed. The lignin with high biological reactivity was prepared with an innovative fractionation strategy. Comparative proteomics between several lignin substrate was conducted for a better understanding of lignin degradation mechanism and PHA biosynthesis in Pseudomonas putida. Further genetic engineering strategies based on the pathway findings from proteomics promoted the cell growth, lignin consumption and PHA accumulations. In conclusion, with deeper understanding of metabolic pathways regulations from multidenominational omics study, we successfully designed new metabolic engineering strategies to get most effective strains and hence improve the production of the limonene and PHAs in microorganisms. | |
