Simultaneous production of 2′‐fucosyllactose and difucosyllactose by engineered Escherichia coli with high secretion efficiency

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 ΔLFAR‐YA/FF+P‐PLA2 strain expressing phospholipase A2 (PLA2) from Streptomyces violaceoruber, whose native signal peptide was replaced with the PelB signal peptide (P‐PLA2), 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 ΔLFAR‐YA/FF+P‐PLA2 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.

Elimination of Byproduct Generation and Enhancement of 2′-Fucosyllactose Synthesis by Expressing a Novel α1,2-Fucosyltransferase in Engineered Escherichia coli

“…Taking advantage of the extent of the FucT2 reaction, Lee et al engineered E. coli expressing FKP and FucT2 to produce 2′-FL and DFL simultaneously, resulting in the formation of 1.22 g/L DFL and 0.47 g/L 2′-FL . An alternative α1,2-fucosyltransferase derived from B.…”

“…However, FucT2 and WbgL have been reported to have an additional α1,3-fucosyltransferase activity that can produce DFL. ,− Lee et al constructed a metabolically engineered E. coli harboring FucT2, which simultaneously produced 2′-FL and DFL with titers of 1.22 and 0.47 g/L, respectively . This characteristic of these α1,2-fucosyltransferases introduces new problems to the downstream purification process .…”

“…11,13−15 Lee et al constructed a metabolically engineered E. coli harboring FucT2, which simultaneously produced 2′-FL and DFL with titers of 1.22 and 0.47 g/L, respectively. 16 This characteristic of these α1,2-fucosyltransferases introduces new problems to the downstream purification process. 17 WcfB, an α1,2-fucosyltransferase from Bacteroides fragilis, showed higher fucosylation activity than FucT2 and did not produce DFL.…”

Biosynthesis of Human Milk Oligosaccharides: Enzyme Cascade and Metabolic Engineering Approaches

“…H. pylori FucT2-based metabolically engineered Escherichia coli simultaneously produces 2′-fucosyllactose (2′-FL) and 2′,3-difucosyllactose (DFL) Photobacterium multocida SiaTs possess at least six different enzymatic functions, including donor hydrolysis, α2,3-SiaT, α2,6-SiaT, α2,3-sialidase, α2,3- trans -sialidase, and α2,6- trans -sialidase activities. ,, Therefore, when the biosynthesis routes of HMOs are designed, it is important for selection of the suitable GTs, which may be helpful for enhancing the synthetic efficiency and specificity.…”

Directed Evolution of Soluble α-1,2-Fucosyltransferase Using Kanamycin Resistance Protein as a Phenotypic Reporter for Efficient Production of 2'-Fucosyllactose

“…Since in vitro multienzyme catalysis requires purified reagents and enzymes that increase production costs, production of 2'-FL using engineered microbes has been proven to be cost effective [ 5 , 6 ]. In engineered Escherichia coli [ 7 , 8 ] and Saccharomyces cerevisiae [ 9 , 10 ] capable of producing 2'-FL, lactose is fucosylated by a heterologous α-1,2-fucosyltransferase using guanosine 5'‐diphosphate (GDP)-L-fucose as a fucose donor. However, despite several attempts to construct high-performance strains, the 2'-FL titers are often reported to be low because of the insoluble and unstable nature of α-1,2-fucosyltransferase under process conditions [ 11 , 12 ].…”