The Expression Of Vitreoscilla Hemoglobin Gene In Bacillus Thuringiensis And Its Effects On Strain Fermentation Characters

In the process of Bacillus thuringiensis (Bt) fermentation, large amount of oxygen was needed to maintain cell metabolic level. VitreosciIla hemoglobin (VHb) is an oxygen-binding protein of prokaryotic origin. The purpose of this study is to express VHb gene (vgb) in Bt, and to improve cell growth and insecticidal crystal proteins (ICP) production by enhancing the oxygen usage ability of cell.1. Recombinant vector pEG491-vgb⁺ contain the vgb which was promoted by the promoter of luxS was constructed. By pEG491-vgb⁺the vgb was integrated in the chromosome of BMB171, the recombinant was named BMB171. cry1Ac10 containing plasmid pHT304-Kan^r-cry was transferred into BMB171-VHb⁺ by electroporation to construct BMB667-VHb⁺'a vgb and cry1Ac10 expressing Bt stain. The control stain BMB667-VHb was also constructed.2. In high, middle and low oxygen supply fermentation, compared with its control strain, the colony forming unit(CFU) of BMB667-VHb⁺ was 0.94, 1.23 and 1.59 fold; the ICP amount of BMB667-VHb⁺ was 1.22, 1.63 and 3.13 fold. The highest ICP amount of BMB667-VHb⁺ was appeared 4-8 h earlier than its control strain in middle and low oxygen supply fermentation. In the LC₅0 bioassay on Helicoverpa armigera of high and low volume flask culture, the LC₅0 of VHb expression strain reduced 36.6% and 10.0%, respectively.3. The critical oxygen concentration (COC) of BMB667-VHb⁺ and its control strain declined since the 4^th h. At the 4^th h, compared with the COC of BMB667-VHb(18%), that of BMB667-VHb⁺(12%) was reduced 33%. The highest oxygen uptake rate (OUR) of BMB667-VHb⁺ was 68.6 mmol/Lh, which was increased 73%, compared with that of the control strain (39.6 mmol/Lh). The maximum respiratory intensity (Q_o2) of BMB667-VHb⁺ was 18.6 mmol/gh, which was increased 20.4%, compared with that of the control strain (14.8 mmol/gh). Under the same fermentation condition, the VHb expression strain and its control strain absorbed 3.1% and 2.2% applied oxygen, respectively. VHb expression strain uptake oxygen 1.41 fold than its control stain. In continuous fermentation theμ_m of BMB667-VHb⁺ and BMB667-VHb^- was 0.783 h^-1 and 0.801 h^-1, and the glucose Ks of the 2 strains were 0.272 g/L and 0.297 g/L, respectively.4. These results demonstrated the vgb expressed after integrated into the chromosome of Bt. The cell density and ICP amount was improved by the vgb expression, and the increase on the latter were more obvious. The improve effects were enhanced when the oxygen supply decreased. The oxygen metabolic relating strain characters were changed: COC, OUR and Q_o2 were elevated and the oxygen absorbing ability was enhanced.5. In study how the agitation speed and aeration amount affect the turbidity value, an emendation formula was achieved. The turbidity values between the crystal-forming and non-crystal-forming fermentation were compared. The attribute ratio of viable cell, spore and crystal was 1, 1.3 and 0.5, respectively. By this ratio, the spore forming situation could be detected, and the terminal point of fermentation could be forecasted.