Metabolic Engineering Of Bacillus Subtilis PY For Riboflavin Production

Based on the riboflavin (VB₂) biosynthetic reactions, B. subtilis PY strain was constructed to study the effect of heterologous rib operon on riboflavin biosynthesis. Then metabolic engineering of B. subtilis PY for riboflavin production were studied by over-expression of zwf gene, expression of heterologous vgb (Vitreoscilla hemoglobin) gene and deletion of ptsG gene in B. subtilis PY, respectively. The main results presented in this work are as follows:Fragments containing the whole rib operons of B. cereus ATCC 10987, B. cereus ATCC 14579 and Geobacillus stearother mophilu were detected and annotated respectively from GenBank. Three heterologous rib operons were operative in B. subtilis that was identified by riboflavin auxotroph complementary experiments.Three fragments of P43-rib, which were expressed in B. subtilis RH13, were respectively constructed by replacing native prompters of the three heteroglous rib operons with constitutively expressed strong promoter P43. It indicated that expression of P43-rib_{B.cereus ATCC 14579B} enhances riboflavin production most efficiently. Subsequently, P43-rib_{B.cereus ATCC 14579B} was integrated into the chromosome of B. subtilis RH33, yielding transformant B. subtilis PY. 4.3 g/l of riboflavin was achieved in batch culture of B. subtilis PY with 8 % glucose as carbon source, which was 27 % enhancement compared to the host strain.The effect of zwf gene (encoding glucose 6-phosphate dehydrogenase) amplification on riboflavin production of strain B. subtilis PY was studied in this thesis. Metbolites analysis indicated that the flux of PP pathway increased. With the increasing of the intracellular concentration of precursor ribulose 5-phosphate (Ru5P), the riboflavin production of B. subtilis achieved 5.4 g/l in batch fermentation medium containing 8 % glucose, which was 25 % enhancement compared to the host strain.ptsG gene of B. subtilis PY, which encoding EII in glucose phosphotransferase system,was inactivated. Compared to the host strain, ptsG-disrupted stain accumulated less acid, and directed more carbon source to biomass and riboflavin biosynthetic pathway, while the glucose uptake rate was declined. 22% more biomass and 19 % higher riboflavin were achieved, compared to the host strain in batch culture with 8 % glucose. However, the fermentation cycle was prolonged from 48 h to 72 h. A metabolic flux distribution analysis indicated that both the fluxes in EMP pathway and also in the fluxes of overflow metabolism decreased in ptsG-disrupted strain while the riboflavin yield was increased from 0.01 mol riboflavin/mol glucose to 0.016 mol riboflavin/mol glucose.Vitreoscilla hemoglobin (VHb) gene was integrated into the chromosome of B. subtilis PY, and its influence on physiological characters and riboflavin production has been investigated. The presence of VHb not only enhanced efficiency of oxidative phosphorylation, but increased the ratio of ATP/ADP from 1.3 to 3.2, which as a result improved the biomass and riboflavin production. Compared with the control strain under fed-batch fermentation in 5 l fermentor, 28 % higher cell growth density and about 20 % improvement of the yield of riboflavin were obtained. A metabolic flux distribution analysis indicated that VHb had significantly enhanced the fluxes through PPP and pathways for biomass biosynthesis. The overflow metabolism was also decreased.