대사공학적으로 설계된 코리네박테리움 글루타미쿰에서 유당이용 증대와 염색체에서의 유전자 발현을 통한 2'-푸코실락토오스의 안정적 생산

Abstract

The ingredient that differentiates human milk with mammalian milk is oligosaccharides. 50 ~ 80% of human milk oligosaccharides (HMOs) are fucosylated. 2'-Fucoyllactose (2'-FL) is the most abundant in fucosyl oligosaccharides. 2'-FL has prebiotic effects on promoting the growth of beneficial bacteria in the intestines, inhibits the growth of pathogenic bacteria, and improves immunity and brain development. Therefore, 2'-FL is getting attention as a key material for infant formula, functional foods, medicines and cosmetics. Corynebacterium glutamicum, which was used in this study, is a Gram-positive bacterium approved as GRAS (Generally Recognized As Safe) and has already been widely used for amino acid production industrially.

Previous studies have produced 2'-FL using metabolically engineered C. glutamicum. 2'-FL is synthesized by α-1,2 fucosylation of lactose and GDP-L-fucose. The GDP-L-fucose biosynthetic pathway was introduced into C. glutamicum and lactose was transported into the cells by introducing the lacYA operon. α-1,2 Fucosylation was achieved by expressing codon optimized α-1,2 fucosyltransferase (COfucT2) derived from Helicobacter pylori. Batch fermentation and fed-batch fermentation were carried out, resulting in production of 2'-FL of 0.6 g/L in batch fermentation and 11.5 g/L in fed-batch fermentation.

In this study, C. glutamicum was further engineered to enhance the production of 2-FL. The following three strategies were employed. First, some genes were removed so that only necessary genes can be expressed for 2-FL production in order to minimize a metabolic burden on the cell. Among the genes used previously, phosphomannomutase (manB) and GTP-mannose-1-phosphate guanylyltransferase (manC) already exist on the chromosome of C. glutamicum. The two genes were removed from the expression vector and as a result, 0.62 g/L of 2-FL was produced in batch fermentation. Second, in the lacYA operon, only lacY was expressed to improve the utilization of lactose, and 0.93 g/L of 2-FL was produced in batch fermentation. Third, the lactose permease gene (lacY) was expressed with the RBS (Ribosome Binding Site) and tac promoter, resulting in more lactose transport into the cell. As a result, 2'-FL was able to be produced at 1.94 g/L in batch fermentation and this is 3.3 times higher compared with the control strain which is developed in previous studies. Furthermore, the constructed strain was grown in fed-batch fermentation to improve the performance of 2-FL production. 25.5 g/L of 2-FL is produced in fed-batch fermentation, a 2.2 fold enhancement relative to the control strain.

Next, the use of antibiotics should be avoided for industrial fermentations. Since 2'-FL is used for foods and medicines, antibiotics-free production of 2-FL could improve consumers' perception and can save the cost of separation and purification of antibiotics. Therefore, to construct a fermentation system that stably produces 2-FL without using antibiotics, the genes necessary for producing 2'-FL were inserted into the chromosome of C. glutamicum. For the chromosomal integration, a double crossover method using the pK19mobsacB vector was used and the IS (Insertion Sequence) element was selected as a site for insertion. It was possible to introduce the genes on the chromosome of C. glutamicum by inserting COfucT2 into the site where ISCg2b is deleted. Then, ISCg2f and ISCg1a were further disrupted to provide a site for the insertion of the 2'-FL biosynthesis related genes, such as gmd and wcaG. As a result, 0.84 g/L of 2'-FL was produced in the batch fermentation without using kanamycin. When the genes (COfucT2, gmd, wcaG) are expressed simultaneously on plasmids and chromosomes, 3.01 g/L of 2-FL could be produced in flask fermentation without using antibiotics.

A fermentation system for producing high concentration of 2'-FL without using antibiotics was constructed. This study is expected to provide a technical basis for industrial production of 2'-FL by engineered C. glutamicum.