Engineering of microbial cell factories for the sustainable production of fuels and chemicals using a novel carbon elongation pathway
Doctor of Philosophy
Concerns over sustained availability of fossil resources along with environmental impact of their use have stimulated the development of alternative methods for fuel and chemical production from renewable resources. Feedstocks from renewables are commonly C5~C6 sugars and C3 glycerol obtained from hydrolytic degradation of biomass and lipids respectively, and some successful results in production of chemicals in the C1-C6 range have been reported largely due to fact that those chemicals can be produced directly or be readily synthesized from common compounds within central metabolism. The biomanufacturing of chemicals of which the number of carbon exceed 6, on the other hand, requires elaborate intracellular carbon-carbon bond forming reactions. Although numerous studies have employed nature designed carbon elongation pathway such as fatty acid biosynthesis and isoprenoid pathway in attempt to produce those chemicals, most metabolic platforms suffer from low titer and yield of products largely because of both cellular regulation and high expenditure of cellular energy on the biosynthesis of starter and extender units for carbon elongation. These obstacles motivated us to an investigation of new approaches to develop cell factories to enable sustainable production of chemicals. To this end, we selected the reversed β-oxidation (r-BOX) as carbon elongation module due to its orthogonality and superiority in energy consumption and functionality compared with that of other carbon elongation modules. The construction of a functional r-BOX, however, was only achieved by manipulating multiple global regulators which rendered it less applicable to regulate carbon elongation and fine-tuning the addition of functional groups. To address these difficulties, we adopted a bottom-up synthetic biology approach to facilitate r-BOX in E. coli which consist of four individual enzymatic reactions: thiolase, 3-hydroxy acyl-CoA dehydrogenase, trans-enoyl-CoA dehydratase and 2-enoyl-CoA reductase. In addition, further assessment on the efficiency of newly developed cell factories was carried out based on the three criteria: i) carbon elongation where the biosynthesis of decanoyl-CoA from acetyl-CoA was confirmed by using corresponding n-acids and n-alcohols as proxy products, ii) functionality on the carbon back bone where the production of α,β-unsaturated carboxylic acids were demonstrated and iii) selectivity in carbon chain length using decanoic acid as a proxy product. In these respects, the cell factories implemented with r-BOX along with various acyl-CoA hydrolysis module exhibited superiority in titer, yield and ratio. Thus, we demonstrated that metabolic engineering strategies covered in the present study to develop cell-factories were useful as a tool for the production of various chain length of chemicals with both different functional groups and high yield.