Systems metabolic engineering of microbial cell factories for the synthesis of value-added chemicals.

다국어 초록 (Multilingual Abstract)

Microbial cell factories offer us an excellent opportunity for the conversion of many different cheaply available raw materials into valuable chemicals. Systems metabolic engineering aims at developing rational strategies for the engineering of microbial hosts by providing global level information of a cell. This dissertation focuses on metabolic engineering, bioprocess modeling and pathway analysis, to develop robust microbial cell factories for the synthesis of value-added chemicals. The following research tasks were completed in this regard. First, statistical models were developed for the prediction of product yields in engineered microbial cell factories - Saccharomyces cerevisiae and Escherichia coli (Chapter 2). A large space of experimental data for chemical production from recent references was collected and a statistics-based model was developed to calculate production yield. The input variables (numerical or categorical variables) for the model represented the number of enzymatic steps in the biosynthetic pathway of interest, metabolic modifications, cultivation modes, nutrition and oxygen availability. In addition, the use of 13C-isotopomer analysis method was proposed for the accurate determination of product yields in engineered microbes under complex cultivation conditions (Chapter 3). Second, metabolic engineering of the cyanobacterium, Synechocystis sp. PCC 6803 was performed for synthesizing isobutanol under phototrophic conditions (Chapter 4). With the expression of the heterologous genes from the Ehrlich Pathway, by incorporating an in situ isobutanol harvesting system, and also by employing mixotrophic conditions, the engineered Synechocystis 6803 strain accumulated a maximum of ~300 mg/L of isobutanol in a 21 day culture. In addition, Synechocystis 6803 was engineered for the synthesis of D-lactic acid (Chapter 5), via overexpression of a novel D-lactate dehydrogenase (encoded by gldA101). The production of D-lactate was further improved by employing three strategies: (i) cofactor balancing, (ii) codon optimization, and (iii) process optimization. The engineered Synechocystis 6803 produced 2.2 g/L D-lactate under photoautotrophic conditions with acetate, the highest reported lactate titer among all known cyanobacterial strains. Finally, an E. coli cell factory was engineered to study the fermentation kinetics for scaled-up isobutanol production (Chapter 6). Through kinetic modeling (to describe the dynamics of biomass, products and glucose concentration) and isotopomer analysis, we have also offered metabolic insights into the performance trade-off between two engineered isobutanol producing E. coli strains (a high performance and a low performance strain). The kinetic model can also predict isobutanol production under different fermentation conditions. I and my colleagues have also demonstrated that E. coli cell factory can also be used for converting waste acetate into free fatty acids through metabolic engineering. In conclusion, the opportunities and commercial limitations with current biotechnology as well as the role of systems metabolic engineering for the development of high performance microbial cell factories were discussed (Chapter 7).