Rational Design of an α-1,3-Fucosyltransferase for the Biosynthesis of 3-Fucosyllactose in Bacillus subtilis ATCC 6051a via De Novo GDP-l-Fucose Pathway.

3-Fucosyllactose (3-FL) is an important oligosaccharide and nutrient in breast milk that can be synthesized in microbial cells by α-1,3-fucosyltransferase (α-1,3-FucT) using guanosine 5'-diphosphate (GDP)-l-fucose and lactose as substrates. However, the catalytic efficiency of known α-1,3-FucTs from various sources was limited due to their low solubility. To enhance the microbial production of 3-FL, the efficiencies of α-1,3-FucTs were evaluated and in Bacillus subtilis (B. subtilis) chassis cells that had been endowed with a heterologous synthetic pathway for GDP-l-fucose, revealing that the activity of FucTa from Helicobacter pylori (H. pylori) was higher than that of any of other reported homologues. To further improve the catalytic performance of FucTa, a rational design approach was employed, involving intracellular evaluation of the mutational sites of M32 obtained through directed evolution, analysis of the ligand binding site diversity, and protein structure simulation. Among the obtained variants, the FucTa-Y218 K variant exhibited the highest 3-FL yield, reaching 7.55 g/L in the shake flask growth experiment, which was 3.48-fold higher than that achieved by the wild-type enzyme. Subsequent fermentation optimization in a 5 L bioreactor resulted in a remarkable 3-FL production of 36.98 g/L, highlighting the great prospects of the designed enzyme and the strains for industrial applications.

Rational design of GDP­D­mannose mannosyl hydrolase for microbial L­fucose production.

BACKGROUND: L­Fucose is a rare sugar that has beneficial biological activities, and its industrial production is mainly achieved with brown algae through acidic/enzymatic fucoidan hydrolysis and a cumbersome purification process. Fucoidan is synthesized through the condensation of a key substance, guanosine 5'­diphosphate (GDP)­L­fucose. Therefore, a more direct approach for biomanufacturing L­fucose could be the enzymatic degradation of GDP­L­fucose. However, no native enzyme is known to efficiently catalyze this reaction. Therefore, it would be a feasible solution to engineering an enzyme with similar function to hydrolyze GDP­L­fucose. RESULTS: Herein, we constructed a de novo L­fucose synthetic route in Bacillus subtilis by introducing heterologous GDP­L­fucose synthesis pathway and engineering GDP­mannose mannosyl hydrolase (WcaH). WcaH displays a high binding affinity but low catalytic activity for GDP­L­fucose, therefore, a substrate simulation­based structural analysis of the catalytic center was employed for the rational design and mutagenesis of selected positions on WcaH to enhance its GDP­L­fucose­splitting efficiency. Enzyme mutants were evaluated in vivo by inserting them into an artificial metabolic pathway that enabled B. subtilis to yield L­fucose. WcaHR36Y/N38R was found to produce 1.6 g/L L­fucose during shake­flask growth, which was 67.3% higher than that achieved by wild­type WcaH. The accumulated L­fucose concentration in a 5 L bioreactor reached 6.4 g/L. CONCLUSIONS: In this study, we established a novel microbial engineering platform for the fermentation production of L­fucose. Additionally, we found an efficient GDP­mannose mannosyl hydrolase mutant for L­fucose biosynthesis that directly hydrolyzes GDP­L­fucose. The engineered strain system established in this study is expected to provide new solutions for L­fucose or its high value­added derivatives production.

A wheat bran inducible expression system for the efficient production of α-L-arabinofuranosidase in Bacillus subtilis.

α-l-arabinofuranosidases (EC 3.2.1.55; AFs) cause the release of arabinosyl residues from hemicellulose polymers such as xylans, and are receiving increased levels of research attention as they could be applied in a range of processes that involve the enzymatic degradation of xylans. The secretory production of bacterial AFs has not been attempted previously. In this study, we designed a unique induction system for the production of a recombinant AF in Bacillus subtilis in order to exploit its enzymic degradation of wheat bran. We found that non-starch phytochemicals were more efficient than d-xylose when inducing the expression of T7 RNA polymerase and driving the transcription of AF by the T7 promoter. The host cell, B. subtilis (ATCC 6051a-derived strain 164T7P) was engineered to incorporate a DNA cassette that expressed T7 RNA polymerase under the control of a d-xylose inducible promoter (PxylA). The T7 promoter engineered into 164T7P was initially tested and compared with P43 in terms of GFP expression; we found that the expression level of GFP by the T7 promoter was ten-fold higher than that achieved by P43. When cultured in a flask with gentle shaking, and with d-xylose as an inducer, the recombinant strain successfully expressed arbf, a family 51 (GH 51) glycoside hydrolase from Bacillus licheniformis, and secreted 141.4 ±â€¯4.8 U/mL of enzyme, with a Km of 1.4 ±â€¯0.1 mM and a kcat of 139.4 s-1. However, the protein was devoid of a secretary signal peptide. When cultures were supplemented with wheat bran, the maximal yield of the secreted AF reached 194.8 ±â€¯4.1 U/mL. The results provide a foundation for the high level production of heterologous proteins using wheat bran as the inducer in B. subtilis.