Selective microbial production of lacto-N-fucopentaose I in Escherichia coli using engineered α-1,2-fucosyltransferases.

Lacto-N-fucopentaose I (LNFP I) is the second most abundant fucosylated human milk oligosaccharide (HMO) in breast milk after 2'-fucosyllactose (2'-FL). Studies have reported that LNFP I exhibits antimicrobial activity against group B Streptococcus and antiviral effects against Enterovirus and Norovirus. Microbial production of HMOs by engineered Escherichia coli is an attractive, low-cost process, but few studies have investigated production of long-chain HMOs, including the pentasaccharide LNFP I. LNFP I is synthesized by α1,2-fucosyltransfer reaction to the N-acetylglucosamine moiety of the lacto-N-tetraose skeleton, which is catalyzed by α1,2-fucosyltransferase (α1,2-FucT). However, α1,2-FucTs competitively transfer fucose to lactose, resulting in formation of the byproduct 2'-FL. In this study, we constructed LNFP I-producing strains of E. coli with various α1,2-fucTs, and observed undesired 2'-FL accumulation during fed-batch fermentation, although, in test tube assays, some strains produced LNFP I without 2'-FL. We hypothesized that promiscuous substrate selectivity of α1,2-FucT was responsible for 2'-FL production. Therefore, to decrease the formation of byproduct 2'-FL, we designed 15 variants of FsFucT from Francisella sp. FSC1006 by rational and semi-rational design approaches. Five of these variants of FsFucT surpassed a twofold reduction in 2'-FL production compared with wild-type FsFucT while maintaining comparable levels of LNFP I production. These designs encompassed substitutions in either a loop region of the enzyme (residues 154-171), or in specific residues (Q7, H162, and L164) that influence substrate binding either directly or indirectly. In particular, the E. coli strain that expressed FsFucT_S3 variants, with a substituted loop region (residues 154-171) forming an α-helix structure, achieved an accumulation of 19.6 g/L of LNFP I and 0.04 g/L of 2'-FL, while the E. coli strain expressing the wild-type FsFucT accumulated 12.2 g/L of LNFP I and 5.85 g/L of 2'-FL during Fed-bach fermentation. Therefore, we have successfully demonstrated the selective and efficient production of the pentasaccharide LNFP I without the byproduct 2'-FL by combining protein engineering of α1,2-FucT designed through in silico structural modeling of an α1,2-FucT and docking simulation with various ligands, with metabolic engineering of the host cell.

Efficient production of lacto-N-fucopentaose III in engineered Escherichia coli using α1,3-fucosyltransferase from Parabacteroides goldsteinii.

Lacto-N-fucopentaose III (LNFP III) is a human milk oligosaccharide (HMO) with potential health benefits in infants, including in immune development and modulation of the intestinal environment. Low-cost fermentative production of various HMOs from lactose by engineered Escherichia coli has attracted attention, but few reports have investigated long-chain HMO production, such as of the pentasaccharide LNFP III. LNFP III is synthesized by α1,3-fucosyltransfer reaction to the glucosamine (GlcNAc) moiety in the lacto-N-neotetraose (LNnT) skeleton by α1,3-fucosyltransferase (α1,3-FucT). However, the known α1,3-FucTs also transfer fucose to the reducing terminal glucose moiety of LNnT or the starting material lactose, resulting in various byproducts. Here, we found a useful α1,3-FucT from Parabacteroides goldsteinii (PgsFucT), which is only reactive for GlcNAc in the N-acetyllactosamine (LacNAc) skeleton in vivo. On the basis of sequence alignment with a FucT of known structure, we also generated α1,3-FucT variants with altered reactivity for LacNAc or lactose. An E. coli strain heterologously expressing PgsFucT accumulated 3.84 g/L of LNFP III after 48 h of culture in a 3-L jar-fermenter. The amounts of various byproduct sugars were remarkably decreased compared with a strain expressing the previously characterized α1,3-fucT from Bacteroides fragilis.

Transporter Engineering Enables the Efficient Production of Lacto-N-triose II and Lacto-N-tetraose in Escherichia coli.

Lacto-N-triose (LNT II) and lacto-N-tetraose (LNT) are human milk oligosaccharides (HMOs) with various potential functions for infants. HMO production by Escherichia coli fermentation has attracted attention in recent years. However, little is known about the cellular export of HMOs. In this study, we identified four endogenous E. coli transporter genes (setA, setB, ydeA, and mdfA), overexpression of which significantly increased the efficiency of LNT II production. The setA-enhanced strain accumulated 34.2 g/L LNT II in a 3 L bioreactor. In the production of LNT, which uses LNT II as an intermediate, disruption of setA remarkably decreased the LNT II accumulation and enhanced the titer of LNT. Furthermore, by heterologous expression of extracellular ß-1,3-N-acetylglucosaminidase from Bifidobacterium bifidum, which degrades LNT II, we eliminated LNT II completely. This study shows that regulation of sugar efflux transporters in E. coli can increase the production of HMOs and decrease the amounts of undesired byproducts.

A Tryptophan Prenyltransferase with Broad Substrate Tolerance from Bacillus subtilis subsp. natto.

Bacillus subtilis subsp. natto secretes the ComXnatto pheromone as a quorum-sensing pheromone to produce poly-γ-glutamate for biofilm formation. The amino-acid sequence of the pheromone is Lys-Trp-Pro-Pro-Ile-Glu, and the tryptophan residue is post-translationally modified with a farnesyl group to form a tricyclic scaffold. Unlike other Bacillus ComX pheromones, the tryptophan residue is distant from the C-terminal end of the precursor peptide ComXnatto . Here, we report the functional analysis of ComQnatto , which catalyzes a unique farnesyl-transfer reaction. ComQnatto recognizes not only full-length ComXnatto but also N- and/or C-terminal truncated ComXnatto analogues and even a single tryptophan for modification with a farnesyl group in vitro. These results, together with the calculated kinetic parameters, suggest that ComQnatto does not require a leader sequence for substrate recognition and is a promising enzyme with broad substrate tolerance for the synthesis of various prenylated tryptophan derivatives.

Posttranslational isoprenylation of tryptophan in bacteria.

Posttranslational isoprenylation is generally recognized as a universal modification of the cysteine residues in peptides and the thiol groups of proteins in eukaryotes. In contrast, the Bacillus quorum sensing peptide pheromone, the ComX pheromone, possesses a posttranslationally modified tryptophan residue, and the tryptophan residue is isoprenylated with either a geranyl or farnesyl group at the gamma position to form a tricyclic skeleton that bears a newly formed pyrrolidine, similar to proline. The post-translational dimethylallylation of two tryptophan residues of a cyclic peptide, kawaguchipeptin A, from cyanobacteria has also been reported. Interestingly, the modified tryptophan residues of kawaguchipeptin A have the same scaffold as that of the ComX pheromones, but with the opposite stereochemistry. This review highlights the biosynthetic pathways and posttranslational isoprenylation of tryptophan. In particular, recent studies on peptide modifying enzymes are discussed.

Identification of Pyridinium with Three Indole Moieties as an Antimicrobial Agent.

A novel pyridinium with three indole moieties, tricepyridinium, was obtained from the culture of an Escherichia coli clone incorporating metagenomic libraries from the marine sponge Discodermia calyx. For the important structural elements of tricepyridinium to be investigated for antibacterial activity, tricepyridinium and its analogues were chemically synthesized. Tricepyridinium had antimicrobial activity, but not against E. coli, and cytotoxicity against P388 cells. Additional bioassays with its synthetic analogues revealed that the intriguing combination of the indole moieties, most likely derived from three tryptamines, as well as the pyridinium moiety were chiefly responsible for its potent biological activities.

Stereospecific prenylation of tryptophan by a cyanobacterial post-translational modification enzyme.

Prenylation is a key post-translational reaction to increase the structural diversity and bioactivity of peptides and proteins. Until now, only one post-translational modification enzyme, ComQ, has been identified to mediate the prenylation of a tryptophan residue in ribosomally synthesized peptides. Here, we report the in vitro characterization of KgpF, a novel prenyltransferase which transfers dimethylallyl moieties to tryptophan residues during kawaguchipeptin A biosynthesis. The stereospecific prenylation by KgpF was determined by a combination of in vitro dimethylallylation of Fmoc-tryptophan by KgpF and chemical synthesis of dimethylallylated Fmoc-tryptophan diastereomers. KgpF modified the tryptophan derivative with a dimethylallyl group at the 3 position of its indole ring, resulting in the formation of a tricyclic structure with the same scaffold as prenylation by ComQ, but with the opposite stereochemistry.