Nicotinamide mononucleotide (NMN) is a precursor of NAD+, its efficient bio-production through microbial fermentation has rarely been reported. Bacillus subtilis was postulated to lack the inherent ability of NMN biosynthesis, however, certain level of NMN (53.67 mg/L) was detected in the fermentation broth containing nicotinamide. An endogenous enzyme (PncB) previously annotated as a nicotinic acid phosphotransferase was identified to be involved in the key synthetic reaction, since the deletion of pncB revoked the production of NMN, while the enhanced expression of PncB significantly increased the titer of NMN (110.0 mg/L). The structural simulation of PncB revealed the possible catalytic mechanisms of NMN forming. The NMN synthesis was further boosted by removing a possible deamidase (CinA), the yield of extracellular NMN reached 202.9 mg/L. After introducing the NMN transporter PnuC into the host cell, the fermentation level of NMN increased by another 65.34 %. And, with 20 g/L glucose and 10 g/L NAM supplemented in the medium, a maximal of 1.21 g/L of NMN was accumulated, signifying the potential of biological production of NMN using food-grade Bacillus subtilis.

Abstract Background l‑Fucose 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)‑l‑fucose. Therefore, a more direct approach for biomanufacturing l‑fucose could be the enzymatic degradation of GDP‑l‑fucose. 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 GDP‑l‑fucose. Results Herein, we constructed a de novo l‑fucose synthetic route in Bacillus subtilis by introducing heterologous GDP‑l‑fucose synthesis pathway and engineering GDP‑mannose mannosyl hydrolase (WcaH). WcaH displays a high binding affinity but low catalytic activity for GDP‑l‑fucose, therefore, a substrate simulation‑based structural analysis of the catalytic center was employed for the rational design and mutagenesis of selected positions on WcaH to enhance its GDP‑l‑fucose‑splitting efficiency. Enzyme mutants were evaluated in vivo by inserting them into an artificial metabolic pathway that enabled B. subtilis to yield l‑fucose. WcaHR36Y/N38R was found to produce 1.6 g/L l‑fucose during shake‑flask growth, which was 67.3% higher than that achieved by wild‑type WcaH. The accumulated l‑fucose 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 l‑fucose. Additionally, we found an efficient GDP‑mannose mannosyl hydrolase mutant for L‑fucose biosynthesis that directly hydrolyzes GDP‑l‑fucose. The engineered strain system established in this study is expected to provide new solutions for l‑fucose or its high value‑added derivatives production.

Microbial synthesis of 2'-fucosyllactose (2'-FL) has received much attention in recent years. In this study, Bacillus subtilis ATCC 6051a, a nonpathogenic GRAS (generally recognized as safe) organism, was engineered to produce 2'-FL. After a synthetic pathway comprising six genes was incorporated into the host cell genome, only a low level of 2'-FL (120 mg/L) was initially detected in recombinant cell culture. Comparison of heterologous lactose transporters confirmed the superior role of LacY from Escherichia coli for efficient production of 2'-FL in B. subtilis. There are two beta-galactosidases, GanA and YesZ, in B. subtilis. Deletion of ganA and yesZ caused by insertion of the lacY cassette led to 2'-FL accumulation in shaking-flask culture at concentrations of 3.13 g/L and 5.56 g/L, respectively, which increased to 6.13 g/L in the double-deletion strain 164FL-GY. In addition, enhanced xylose metabolism in 164FL-GY further increased the concentration of 2'-FL to 7.14 g/L. In fed-batch fermentation, the highest productivity of 0.56 g/L.h was achieved in glycerol and xylose containing medium using engineered B. subtilis , and the accumulated 2'-FL reached 31.2 g/L. This is the first report of recombinant B. subtilis for high-level production of 2'-FL via a de novo synthesis pathway.

Citramalic acid (citramalate) is important for the chemical synthesis of methylmethacrylate, a bulk monomer used for manufacturing biodegradable polymers. Escherichia coli strains have been engineered for the biological production of citramalate since a rare mutant of citramalate synthase was invented as a mesophilic catalyzer to form the chemical. However, acidic byproducts are routinely excreted by E. coli host cells during fermentation, which could significantly reduce the carbon conversion efficiency of biosynthetic chemicals, including citramalate. Herein, an unusual mutant called S17-3 was reported, and this mutant demonstrated superb recovery efficacy from glucose to citramalate and secreted a very low level of fermentation byproducts during the fermentative production of citramalate. However, S17-3 tends to produce a large amount of colonic acid (CA). Genetically manipulating the regulators involved in CA synthesis revealed that the deletion of rcsD could significantly improve the citramalate titer. Fed-batch fermentation under optimized conditions led to a maximum titer of 46.2 g/L citramalate accumulated in the broth, with a conversion efficiency of 0.80 g citramalate per gram glucose and a close-to-the-theoretical mole transversion efficiency (mol/mol) of 0.97. This study facilitates the development of a practical bioproduction of citramalate using E. coli strains.