Expression Of Vitreoscilla Hemoglobin In Poly γ-glutamic Acid-producing Bacillus Licheniformis WX-02

Polyγ-glutamic acid(γ-PGA) is a biopolymer material that has potential applications in a wide range of industry and agriculture.Bacillus licheniformis WX-02 is a kind of areobacterium that can produceγ-PGA,the accumulation ofγ-PGA in the culture medium causes a significant viscosity increase that reduces dissolved oxygen(DO) in the fermentation broth.As a result,DO becomes one of the restricted factors that affects the increase ofγ-PGA yield and biomass.Vitreoscilla hemoglobin(VHb) is an oxygen-binding proteins of prokaryotic origin.This study was carried out to improve the transmission of oxygen by expression of Vitreoscilla hemoglobin gene(vgb) in Bacillus licheniformis WX-02 in order to promote synthesis ofγ-PGA and cell growth.An homologous recombination expression vector pDG1730-vgb which contains theα-amylase gene(amyE) for chromosomal intergration,was introduced into Bacillus licheniformis WX-02 by Spizizen natural genetic transformention,PCR analysis indicated that vgb has been successfully intergrated into the chromosome;CO binding difference spectrum analysis revealed the recombinate strain Bacillus licheniformis M2 has expressed Vitreoscilla hemoglobin with bio-activation;Shask flask fermentation experiments preliminarily showed that:the strains was cultivated at 37℃,180 r/min for 36 h,the biomass andγ-PGA yield of M2 were 4.5 g/L and 27.71 g/L,which were 6.43%and 6.74%higher than those of WX-02 when 50mL culture broth was in the 250 mL flask, comparatively the biomass andγ-PGA yield of M2 were 4.17 g/L and 26.59 g/L,then 15.02%and 21.7%higher than WX-02 with 100mL culture broth in the 250 mL flask, these results demonstrate the biomass andγ-PGA yield was improved by the vgb expression,and the increase on the latter were more obvious.The improve effects were enhanced following the oxygen supply decreased.It was shown by the results of batch cultures in a 3 L bioreactor that biomass andγ-PGA obtained in the recombinant M2 were 25.5%and 20%higher than those of the control respectively when agitation speed was 600 r/min and ventilation volume was 2 L/min,which further comferms the positive promotion effects of VHb on cell growth andγ-PGA producing.Another integrative expression vector psacB-vgb which contains the levansucrase gene(sacB) for chromosomal intergration was constructed,it will be integrated into the chromosome of Bacillus subtilis 003 in the next experimentation.