Metabolic engineering Corynebacterium glutamicum ATCC13032 for 2′-fucosyllactose production

Abstract

2′-Fucosyllactose is the most abundant human milk oligosaccharides and one of the three essential nutrients for infant growth. Corynebacterium glutamicum is one of the most common industrial fermentation bacteria but cannot synthesize 2′-fucosyllactose. In this study, C. glutamicum ATCC13032 was engineered for 2′-fucosyllactose production from fucose and lactose. The gene futC from Helicobacter pylori encoding α-1,2-fucosyltransferase was codon optimized and mutated at four amino acids (F40S/Q150H/C151R/Q239S). The modified gene futC and the gene fkp from Bacteroides thetaiotaomicron encoding fucokinase/GDP-fucose pyrophosphorylase were overexpressed in plasmid pEC and transformed into C. glutamicum, resulting in CW002. CW002 did not synthesize 2′-fucosyllactose possibly because the substrates fucose and/or lactose did not pass through the cell membrane. Therefore, the gene lacY encoding lactose permease and the gene fucP encoding fucose permease from Escherichia coli were overexpressed in plasmid pXTuf and transformed into CW002, resulting in CW006. CW006 did synthesize 2′-fucosyllactose as expected. It is interesting that the production of 2′-fucosyllactose was decreased or stopped when the expression combination of these four genes was changed, suggesting that the expression levels of the four genes in CW006 might have to well balanced. C. glutamicum CW006 produced 2.07 g/L 2′-fucosyllactose in a 2.4 L bioreactor.

Keywords

Corynebacterium glutamicum / Human milk oligosaccharide / 2′-fucosyllactose / fkp / futC

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Jiangnan University