Development of Novel Biological Chassis based on Vibrio sp. dhg for Efficient Microbial Bioconversion

Abstract

Biorefinery refers to the concept of sustainably producing fuel, power, and chemicals from renewable materials such as biomass. Concerns about environmental pollution caused by the use of fossil fuels are further increasing the importance of biorefineries. Biorefinery is primarily based on microbial bioconversion, which utilizes biomass-derived carbohydrates to produce desired chemicals in genetically engineered microorganisms. Therefore, the selection of biomass feedstocks and target value-added chemicals and screening and engineering of microorganisms capable of efficient conversion are critical in improving the efficiency of biorefinery. Metabolic engineering of microorganisms is a rapidly evolving field that combines synthetic biology and metabolic modeling approaches to ensure efficient tuning of metabolic pathways. This is currently leading to a flood of publications outlining approaches for the production of value-added chemicals. However, recent advances also point out some insurmountable obstacles in this field. Optimization of basic cell properties, such as tolerance to chemical and physical stresses or high growth rate and substrate consumption rates, remains challenging. This is mainly due to the complexity of cellular networks and the lack of fundamental biochemical knowledge. Therefore, the development of novel microbial platforms with various advantageous properties superior to conventional platforms can improve the efficiency of the entire biorefinery. In this regard, Vibrio sp. dhg is expected to be widely used as an appropriate platform in microbial bioconversion. The strain is a marine bacterium isolated from seaweed sludge. It is Gram-negative, facultatively anaerobic, and halophilic. It has the advantage of being easy to be used by researchers due to its non-pathogenicity and simple cultivation condition. High productivity is expected due to its rapid growth and sugar catabolism. As it can catabolize various sugars from biomass feedstock, it is expected to be widely used in various non-edible biomass-based bioprocesses. Furthermore, genetic manipulation methods and synthetic biology tools for the strain developed in the previous study allow efficient construction of the desired phenotype. In this study, Vibrio sp. dhg was developed as a new biological chassis for an efficient microbial bioconversion process. To this end, I have mainly conducted research on further improvement of strains in terms of improving feedstock flexibility. In chapter 1, the overall motivation and research objectives of this study were described. In addition, the characteristics and advantages of Vibrio sp. dhg were evaluated in detail via a description of the previous study. In chapter 2, the materials and methods used in this study were summarized. Since the novel strain was engineered by methods different from those for conventional microbial hosts, it was divided into a separate chapter. In chapter 3, Vibrio sp. dhg was engineered to efficiently catabolize xylose. First, a heterologous xylose catabolic pathway was constructed in Vibrio sp. dhg, which cannot catabolize xylose naturally. Xylose catabolism was optimized by adaptive laboratory evolution (ALE), which obtained the highest xylose consumption rate as far as reported. In addition, mutational mechanisms in evolved strains were investigated by genome-scale analysis and experimental validation. In chapter 4, CCR in Vibrio sp. dhg was removed for simultaneous utilization of biomass-derived sugars. After the identification of CCR according to sugar combination, the phosphotransferase system (PTS) in the strain was disrupted. As a result, the strain was able to rapidly and simultaneously utilize multiple sugars. In chapter 5, production strains were constructed based on previously engineered strains. The strain to produce ethanol from brown macroalgal sugars and the strain to produce lactate from lignocellulosic sugar were constructed and culture conditions were optimized for maximum production. Compared to previous studies based on E. coli, significantly higher productivity was demonstrated. As demonstrated in this study, Vibrio sp. dhg has high potential as a novel microbial chassis for the bio-based industry. The high metabolic efficiency of the strain can improve the productivity of any target compound, which is greatly helpful in increasing the economic feasibility of bioproduction. Therefore, further studies on the deployment of Vibrio sp. dhg as microbial platforms are highly promising.