Team:Ulink-SIP/parts/contribution
Contribution
Microbial production of 2-FL was suggested to be influenced by a number of factors including fucosylation activity of α-1,2-fucosyltransferase. Due to the vast knowledge of its functionality and the characteristics, α-1,2-fucosyltransferase from H. pylori (FucT2:BBa_K4081998) has been widely used for enzymatic- and microbial production of 2-FL. However, the low level of soluble FucT2 expression as well as its activity in recombinant E. coli clearly hinder the production of 2-FL, consequently reduce the overall productivity. Based on the literature survey, we found that there are many solutions:
Contribution Solution I
FucT2 from H. pylori is designed to increase solubility and activity by connecting three aspartic acid molecules to the N-terminus of FucT2.
The effect of FucT2s fused with three to six aspartate tags (D3-D6 tags) on 2-FL production was investigated via batch fermentations of four engineered E. coli. The result as shown in Fig. 4, expression of D4-FucT2, D5-FucT2 and D6-FucT2 could not be confirmed. Most FucT2 was expressed as insoluble form in the E. coli expressing FucT2, which is consistent with the previous study. The totaland insoluble levels of FucT2 expression significantly decreased in the D3-FucT2 strain, however, the levels of soluble FucT2 and D3FucT2 were invisible in the SDS-PAGE analysis with crude extracts. To confirm the soluble level of expression, purification of FucT2 and D3-FucT2 with His-tag was performed. As a result, soluble expression of FucT2 and D3-FucT2 was confirmed and the level of D3-FucT2 was much higher than those of FucT2 (Fig. 4b). So the conclusion is the titer and yield of 2-FL were further improved by adding the three aspartate molecules at the N-terminal of FucT2.
Fig. SDS-PAGE analysis of crude extracts (a) and purified enzymes (b) to investigate the effects of aspartate tags on the expression patterns of FucT2 variants in engineered E. coli strains. The abbreviations were defined as follows: arrows; FucT2, T; total protein fraction, S; soluble protein fraction, I; insoluble protein fraction, M; molecular weight marker.
Contribution Solution II
Replace with wcfB. Indeed, previous studies was clearly verified the presence of 2-FL produced by engineered E.coli expressing WcfB. Interestingly, di-fucosyllactose, a obvious by-product in the production process was not detected in the culture of engineered E. coli expressing WcfB contrary to the case of engineered E. coli expressing FucT2 from H. pylori (Fig). Through a screening process, putative α-1,2-fucosyltransferase of B. fragilis, WcfB was selected to replace FucT2 from H. pylori. The wcfB gene which is one of the extracellular polysaccharide biosynthesis genes in B. fragilis has been predicted as a gene coding for glycosyltransferase, especially fucosyltransferase due to the identity and similarity to α-1,2-fucosyltransferase in Yersinia enterocolitica and Homo sapiens. By the expression of WcfB in replacement of FucT2, 2-FL concentration and yield were improved by 4.0- and 8.4-folds in fed-batch fermentation. It is obvious that the in vivo fucosylation activity of WcfB is much higher than that of FucT2 in the engineered E. coli. It is noteworthy that WcfB does not catalyze the biosynthesis of di-fucosyllactose contrary to FucT2. It might be due to the difference in enzyme specificity between FucT2 and WcfB. This feature is a great advantage in commercial production of 2-FL since purification is one of the most costly steps in microbial production of value-added materials.
Fig. Representative MS spectrum of 2-FL production from
Purified fucosyllactoses from engineered E. coli expressing WcfB (c) and expressing FucT2 (d). Symbols for the types of monosaccharaides (glucose, ●; galactose, ■; fucose, ▲)
Reference List
[1] Seo, Jin-Ho, Chin, Young-Wook, Kim, & Ji-Yeong, et al. (2017). Improved production of 2 '-fucosyllactose in engineered escherichia coli by expressing putative alpha-1,2-fucosyltransferase, wcf from bacteroides fragilis. Journal of Biotechnology.
[2] Yu, S. , Liu, J. J. , Yun, E. J. , Kwak, S. , Kim, K. H. , & Jin, Y. S. . (2018). Production of a human milk oligosaccharide 2′-fucosyllactose by metabolically engineered saccharomyces cerevisiae. Microbial Cell Factories, 17.
[3] Chin, Y.W., Kim, J.Y., Lee, W.H., Seo, J.H., 2015. Enhanced production of 2′fucosyllactose in engineered Escherichia coli BL21star (DE3) by modulation of lactose metabolism and fucosyltransferase. J. Biotechnol. 210, 107-115.