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.

The E3 ubiquitin ligase Hace1 is required for early embryonic development in Xenopus laevis.

BACKGROUND: HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1) regulates a wide variety of cellular processes. It has been shown that one of the targets of HACE1 is the GTP-bound form of the small GTPase Rac1. However, the role of HACE1 in early development remains unknown. RESULTS: In situ hybridization revealed that Xenopus laevis hace1 is specifically expressed in the ectoderm at the blastula and gastrula stages and in the epidermis, branchial arch, kidney, and central nervous system at the tailbud stage. Knockdown of hace1 in Xenopus laevis embryos via antisense morpholino oligonucleotides led to defects in body axis elongation, pigment formation, and eye formation at the tadpole stage. Experiments with Keller sandwich explants showed that hace1 knockdown inhibited convergent extension, a morphogenetic movement known to be crucial for body axis elongation. In addition, time lapse imaging of whole embryos during the neurula stage indicated that hace1 knockdown also delayed neural tube closure. The defects caused by hace1 knockdown were partly rescued by knockdown of rac1. Moreover, embryos expressing a constitutively active form of Rac1 displayed phenotypes similar to those of embryos with hace1 knocked down. CONCLUSIONS: Our results suggest that Xenopus laevis hace1 plays an important role in early embryonic development, possibly via regulation of Rac1 activity.

Anti-inflammatory Effects of Bacteroidota Strains Derived From Outstanding Donors of Fecal Microbiota Transplantation for the Treatment of Ulcerative Colitis.

BACKGROUND: The proportion of certain Bacteroidota species decreased in patients with ulcerative colitis, and the recovery of Bacteroidota is associated with the efficacy of fecal microbiota transplantation therapy. We hypothesized that certain Bacteroidota may advance ulcerative colitis treatment. Accordingly, we aimed to evaluate the anti-inflammatory effects of Bacteroidota strains isolated from donors. METHODS: Donors with proven efficacy of fecal microbiota transplantation for ulcerative colitis were selected, and Bacteroidota strains were isolated from their stools. The immune function of Bacteroidota isolates was evaluated through in vitro and in vivo studies. RESULTS: Twenty-four Bacteroidota strains were isolated and identified. Using an in vitro interleukin (IL)-10 induction assay, we identified 4 Bacteroidota strains with remarkable IL-10-induction activity. Of these, an Alistipes putredinis strain exhibited anti-inflammatory effects in a mouse model of colitis induced by sodium dextran sulfate and oxazolone. However, 16S rRNA gene-based sequencing analysis of A. putredinis cultures in the in vivo study revealed unexpected Veillonella strain contamination. A second in vitro study confirmed that the coculture exhibited an even more potent IL-10-inducing activity. Furthermore, the production of A. putredinis-induced IL-10 was likely mediated via toll-like receptor 2 signaling. CONCLUSIONS: This study demonstrated that A. putredinis, a representative Bacteroidota species, exhibits anti-inflammatory effects in vivo and in vitro; however, the effects of other Bacteroidota species remain unexplored. Our fecal microbiota transplantation-based reverse translation approach using promising bacterial species may represent a breakthrough in microbiome drug development for controlling dysbiosis during ulcerative colitis.

We isolated Bacteroidota species from the feces of donors who were effectively cured of UC with fecal microbiota transplantation and proved the anti-inflammatory effects of Bacteroidota species, especially Alistipes putredinis, through cell experiments and in vivo experiments.