Simultaneous production of 2′‐fucosyllactose and difucosyllactose by engineered <i>Escherichia coli</i> with high secretion efficiency
Hyun‐Jae Lee, Dong Joo Shin, Kanghee Han, Young‐Wook Chin, Jong Pil Park, Kyeongsoon Park, Chang‐Hyung Choi, Bo‐Ram Park, Soo‐Jung Kim, Sun‐Ki Kim
Abstract
Abstract Background and aim Difucosyllactose (Di‐FL) has strong antimicrobial activity against various pathogens, including group B Streptococcus , identified as the leading cause of neonatal sepsis. In this study, we sought to develop Escherichia coli as a microbial cell factory for efficiently producing Di‐FL as well as 2′‐fucosyllactose (2′‐FL), the most abundant fucosylated oligosaccharide in human milk, by utilizing the salvage guanosine 5′‐diphosphate (GDP)‐ l ‐fucose biosynthetic pathway. Main methods and major results The biosynthetic pathway for producing fucosylated oligosaccharides via the salvage pathway requires two enzymes, l ‐fucokinase/GDP‐ l ‐fucose phosphorylase (FKP) from Bacteroides fragilis and α‐1,2‐fucosyltransferase (FucT2) from Helicobacter pylori . To decrease the intracellular accumulation of 2′‐FL while increasing substrate accessibility to FKP and FucT2, we evaluated whether extracellular secretion of FKP and FucT2 would enhance the production of fucosylated oligosaccharides. Among various engineered strains constructed in this study, the Δ LFAR‐YA /FF+P‐PLA 2 strain expressing phospholipase A 2 (PLA 2 ) from Streptomyces violaceoruber , whose native signal peptide was replaced with the PelB signal peptide (P‐PLA 2 ), could secrete both FKP and FucT2 into the culture medium. Notably, it was observed that FKP and FucT2 present in the extracellular fraction could catalyze the synthesis of Di‐FL from lactose and fucose. As a result, a batch fermentation with the Δ LFAR‐YA /FF+P‐PLA 2 strain resulted in the production of 1.22 ± 0.01 g L −1 Di‐FL and 0.47 ± 0.01 g L −1 2′‐FL, whereas the control strain could only produce 0.65 ± 0.01 g L −1 2′‐FL. Conclusions and implications This study highlights the benefits of extracellular secretion of enzymes to improve biotransformation efficiency, as the transport of substrates and/or products across the cell membrane is limited.