In this study, a novel alginate lyase-encoding gene, VaAly7, was identified from Vibrio alginolyticus and efficiently expressed in Pichia pastoris, with an enzyme activity of 413 U/mL. The recombinant enzyme (VaAly7) was purified to electrophoretic homogeneity using QSFF strong anion exchange column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the molecular mass of the purified enzyme was approximately 44 kDa. The optimal temperature and pH of the enzyme were 30 ℃ and 7.0, respectively. This enzyme was activated by salts, and its activity was increased by about eight folds in the presence of 400 mmol/L NaCl. VaAly7 was a bifunctional alginate lyase with a preference for poly-guluronic acid and the shortest chain substrate that could be degraded by it was pentasaccharide. VaAly7 was used to degrade 10% polyguluronic acid solution, producing guluronate oligosaccharides (AOSs) with a yield of 86%. The degrees of polymerization of the products were in the range of 1–5 with an average molecular mass of 2.1 kDa. The enzyme shows potential in the preparation of guluronate oligosaccharides.

In this study, the de novo synthesis pathway of 2’-FL was established in Escherichia coli BL21star(DE3). The β-galactosidase gene (lacZ) and the UDP-glucose lipid carrier transferase gene (wcaJ) were knocked out by the CRISPR/Cas9 system. The effects of various exogenous α-1,2-fucosyltransferases on 2’-FL synthesis were investigated. Subsequently, the transcriptional levels of genes involved in the synthesis pathway were regulated, and the fermentation conditions were optimized. The results showed that after fermentation for 72 h, a 2’-FL concentration of 0.34 g/L was obtained in shake flasks with overexpression of the de novo synthesis pathway genes in E. coli BL21star (DE3), and the concentration of 2’-FL was increased to 2.12 g/L by knockout of the lacZ and wcaJ genes. Expression of α-1,2-fucosyltransferase Wcfb from Bacteroides fragilis in shake flasks resulted in the highest concentration of 4.12 g/L. Under the optimized fermentation conditions: 28 ℃ and 0.2 mmol/L final isopropyl-β-D-thiogalactoside (IPTG), the 2’-FL concentration was increased to 5.01 and 31.2 g/L in a shake flask and in a 5 L fermenter under fed-batch condition, respectively.

3-Fucosyllactose (3-FL) is a typical human milk oligosaccharides (HMOs) and plays a significant physiological function in the growth and brain development of infants. In this study, efficient biosynthesis of 3-FL was achieved by metabolic engineering of Escherichia coli BL21star(DE3). The α-1,3-fucosyltransferase gene futA from Helicobacter pylori and the L-fucokinase/GDP-L-fucose pyrophosphorylase gene fkp from Bacteroides fragilis were co-introduced into the initial strain, E. coli BL21star(DE3)ΔlacZΔwcaJ, resulting in the production of 1.01 g/L of 3-FL in a shake flask. By using the CRISPR/Cas9 system to delete the genes related to the fucose metabolism pathway, including the L-fucose isomerase gene fucI, the L-fucose kinase gene fucK and the L-rhamnose isomerase gene rhaA, the yield of 3-FL increased to 1.36 g/L. The α-1,3-fucosyltransferase FT1 from H. pylori NCTC11637 was the most effective in 3-FL biosynthesis, and the recombinant strain BLWFA-T expressing FT1 produced 1.56 g/L of 3-FL. Subsequently, the expression level of the fucose metabolism pathway-related genes was optimized, and the recombinant strain BLWFR-T2 expressing fkp through plasmid pETDuet-1 and expressing FT1 through plasmid pRSFDuet-1 produced 2.58 g/L of 3-FL. Finally, the regeneration of the cofactor guanosine triphosphate (GTP) was enhanced, and the recombinant strain BLWFR-T7 overexpressing the guanylate kinase gene gmk and the inosine-guanosine kinase gene gsk produced the highest level of 3-FL (3.01 g/L). After fed-batch fermentation for 78 h, BLWFA-T7 produced the highest yield of 3-FL (27.82 g/L) in a 5 L fermentor. This study provides a research basis for efficient microbial synthesis of 3-FL and other HMOs.