Engineering corynebacterium glutamicum for the utilization of agar from red macroalgal biomass

Abstract

To overcome petroleum-based fuel and chemical industries, various forms of biomass have been used as an alternative solution for environmental issues. Existing biomass using food and non-food biomass from agriculture and wood showed many limitations including ethical issue and lignin problem. Third generation of biomass from marine is expected for solution of sustainable technology. Macroalgae has higher carbohydrate content, lower amounts of inhibitor substrates, no ethical issue from food, and no climate change issue from fertilization. Among them, red algae have many advantages comparing the other macro-algae. It has many carbohydrates than the other macroalgae and agar is large partition of red algae

about ~52 wt % of dry Gelidium amansii. Its carbohydrate contents consist of only galactose and 3,6-anhydro-α-L-galactose. For utilization of agar with high efficient, saccharification of agar is important due to generation of 5-hydroxymethylfulfural, cell growth inhibitor during acidification of agar. To cope this problem, enzymatic process using agarase should be established. In this study, engineering of Corynebacterium glutamicum was performed to achieve agar utilization system for economical and sustainable production of various biochemical and recombinant protein. C. glutamicum has been used as an industrial workhorse, for production of amino acid including glutamic acid and lysine. There are various genetic tools and heterologous enzymes required to enable C. glutamicum to utilize agar. For high stable recombinant protein expression, stable gene expression system is required. There are several kinds and dosages of mobile genetic elements (MGEs) in bacteria. In a few cases, they had minimal effect on cells. However recently, there are reports that MGEs inserted into gene of interest and reduced production yield and quality. There are many MGEs in C. glutamicum. Highly active MGEs were elucidated by high throughput screening. High productivity of recombinant protein and biochemical were obtained by deletion of specific MGEs. Second, high gene expression system is required for production of target proteins or biochemical. Adaptive laboratory evolution was performed using fluorescence activated cell sorter as an artificial selection pressure. From this adaptive evolution, specific genetic mutation was identified in origin of plasmid increasing copy number. Therefore, high productivity of recombinant proteins was obtained. Third, endo type $\beta$-agarase secretion system was engineered by Tat-pathway dependent secretion. Endo type $\beta$-agarase is necessary for hydrolysis of agar and is hard to reach economical productivity. High productivity yield was obtained by high copy plasmid and co-expression of Tat-pathway transporter. Finally, engineered C. glutamicum produced 3,6-anhydro-$\alpha$-L-galactose using enzymatic saccharified agar as a carbon source. To perform this result, I screened exo-type $\beta$-agarase from marine bacteria resource. Novel exo-type $\beta$-agarases were identified and characterized. Co-expression of endo- and exo-type $\beta$-agarases was constructed and high productivity of both $\beta$-agarases was achieved by fed-batch fermentation. At the same time, galactose utilization was introduced into C. glutamicum which is unable to catalyze galactose. Galactose metabolic pathway was optimized, and expression of galactose transporter showed improved cell growth rate in galactose. By combining these result, high productivity yield of 3,6-anhydro-α-L-galactose was achieved. Consequently, high value-added products including neoagaro-oligosaccharide, 3,6-anhydro-$\alpha$-L-galactose, $\beta$-agarases will be produced in this platform. This is the first report of developing C. glutamicum strain capable of using agar as carbon source and contribute to economical bioprocess in bioindustry.