Human milk oligosaccharides (HMOs) have been proven to be beneficial to infants’ intestinal health and immune systems. 2′-Fucosyllactose (2′-FL) is the most abundant and thoroughly studied HMO and has been approved to be an additive of infant formula. How to construct efficient and safe microbial cell factories for the production of 2′-FL attracts increasing attention. In this work, we engineered the Bacillus subtilis as an efficient 2′-FL producer by engineering the substrate transport and cofactor guanosine 5′-triphosphate (GTP) regeneration systems. First, we constructed a synthesis pathway for the 2′-FL precursor guanosine 5′-diphosphate-l-fucose (GDP-l-fucose) by introducing the salvage pathway gene fkp from Bacteriodes fragilis and improved the fucose importation by overexpressing the transporters. Then, the complete synthesis pathway of 2′-FL was constructed by introducing the heterologous fucosyltransferases from different sources, and it was found that the gene from Helicobacter pylori was the best one for 2′-FL synthesis. We also improved the substrate lactose importation by introducing heterologous lactose permeases and eliminated endogenous β-galactosidase (yesZ) to block the lactose degradation. Next, the production of 2′-FL and GDP-l-fucose was improved by fine-tuning the expression of cofactor guanosine 5′-triphosphate regeneration module genes gmd, ndk, guaA, guaC, ykfN, deoD, and xpt. Finally, a 3 L fed-batch fermentation was performed, and the highest 2′-FL titer reached 5.01 g/L with a yield up to 0.85 mol/mol fucose. We optimized the synthesis modules of 2′-FL in B. subtilis, and this provides a good starting point for metabolic engineering to further improve 2′-FL production in the future.

Streptomyces transglutaminase (TGase) is widely used to improve food texture properties. In this study, random mutagenesis and site-directed genetic modification were used to improve the production of TGase in Streptomyces mobaraensis. First, S. mobaraensis DSM40587 (smWT) was subjected to atmospheric and room-temperature plasma mutagenesis, and then a mutant (smY2019) with a 5.5-fold increase in TGase yield was screened from approximately 3000 × 25 (round) mutants. Compared to smWT, smY2019 exhibits a 3.2-fold higher TGase mRNA level and two site mutations within the −10 region of the TGase promoter. The recombinant expression analysis in the TGase-deficient S. mobaraensis suggests that the mutated TGase promoter is more robust than the wild-type one. Finally, we integrated two additional TGase expression cassettes into the smY2019 genome, yielding the recombinant strain smY2019-3C with a 103% increase in TGase production compared to smY2019. The smY2019-3C strain with 40 U/mL of TGase yield could be a suitable candidate for the industrial production of TGase.

Streptomyces mobaraensis transglutaminase (TGase) is extracellularly expressed as a zymogen and then activated by TGase-activating protease (TAP). In this study, we reported the strategy for improving TGase production via the regulation of TAP activity in S. mobaraensis. First, we analyzed the effects of three inorganic nitrogen sources on TGase production. With 30 mM nitrogen content, the time to the peak of TGase activity induced by (NH4)2SO4 or NH4Cl was 72 h, 12 h earlier than that of the fermentation without adding NH4+. SDS-PAGE analysis indicated that NH4+ accelerated the TGase activation in S. mobaraensis. Then, we examined the effect of NH4+ on TAP biosynthesis using a TGase-deficient S. mobaraensis strain. It showed that NH4+ enhanced the TAP activity at the early stage of the fermentation, which was dependent on the concentration and time of NH4+ addition. Last, the yield and productivity of S. mobaraensis TGase were increased by 1.18-fold and 2.1-fold, respectively, when optimal NH4+ addition (60 mM and 12 h) was used. The fermentation period was shortened from 84 to 48 h. The NH4+ addition also increased the storage stability of crude enzyme at room temperature. These findings will benefit the TGase production and its activation mechanism in S. mobaraensis.