Lipid-bound [VHb（406）] and lipid-free [VHb（402）] wide type Vitreoscilla hemoglobin were separated from E. coli BL21（DE3）. The RZ is 3.30 for [VHb（406）] and 3.15 for [VHb（402）], respectively. VHb（406） shows...Lipid-bound [VHb（406）] and lipid-free [VHb（402）] wide type Vitreoscilla hemoglobin were separated from E. coli BL21（DE3）. The RZ is 3.30 for [VHb（406）] and 3.15 for [VHb（402）], respectively. VHb（406） shows a characteristic absorption band at 626 nm, while VHb（402） shows one at 644 nm, and it has more α-helix than VHb（402）. Data of Raman spectra experiment shows under the excitation wavelength 488 nm, υ4 vibration of both VHb（402） and VHb（406） had a pure and strong signal at 1375 cm-1, which proves that iron porphyrin of both the samples is at their trivalence oxidation state. And no matter lipid binds VHb or not, the vinyl of porphyrin is at cis state. The catalase activity of Vitreoscilla hemoglobin was explored with L-dopa and H2O2 as substrates. The results indicate that VHb（406） has more catalase activity than VHb（402）, VHb is a cell’s oxygen modulator and its catalase ability is modulated by the membrane.展开更多
Vitreoscilla hemoglobin is an oxygen-binding protein that promotes oxygen delivery and reduces oxygen consumption under low oxygen conditions to increase the effi ciency of cell respiration and metabolism.In this stud...Vitreoscilla hemoglobin is an oxygen-binding protein that promotes oxygen delivery and reduces oxygen consumption under low oxygen conditions to increase the effi ciency of cell respiration and metabolism.In this study,we introduced a Vitreoscilla hemoglobin gene(vgb)into Chlorella vulgaris by Agrobacterium tumefaciens-mediated transformation(ATMT).PCR analysis confi rmed that the vgb gene was successfully integrated into the Chlorella vulgaris genome.Analysis of biomass obtained in shake fl asks revealed transformant biomass concentrations as high as 3.28 g/L,which was 38.81% higher than that of the wild-type strain.Lutein content of transformants also increased slightly.Further experiments recovered a maximum lutein yield of 2.91 mg/L from the transformants,which was 36.77% higher than that of the wild-type strain.The above results suggest that integrated expression of the vgb gene may improve cell growth and lutein yield in Chlorella vulgaris,with applications to lutein production from Chlorella during fermentation.展开更多
Poly-γ-glutamic acid(γ-PGA)is a natural polymer with various applications,and its high-viscosity hinders ox-ygen transmission and improvement of synthesis level.Vitreoscilla hemoglobin(VHB)has been introduced into v...Poly-γ-glutamic acid(γ-PGA)is a natural polymer with various applications,and its high-viscosity hinders ox-ygen transmission and improvement of synthesis level.Vitreoscilla hemoglobin(VHB)has been introduced into various hosts as oxygen carrier,however,its expression strength and contact efficiency with oxygen hindered efficient oxygen transfer and metabolite synthesis.Here,we want to optimize the expression cassette of VHB for γ-PGA production.Firstly,our results implied that γ-PGA yields were enhanced when introducing twin-arginine translocation(Tat)signal peptides(SP_(YwbN),SP_(PhoD) and SP_(TorA))into VHB expression cassette,and the best per-formance was attained by SP YwbN from Bacillus subtilis,theγ-PGA yield of which was 18.53% higher than that of control strain,and intracellular ATP content and oxygen transfer coefficient(K_(L)a)were increased by 29.71% and 73.12%,respectively,indicating that VHB mediated by SP YwbN benefited oxygen transfer and ATP generation forγ-PGA synthesis.Furthermore,four promoters were screened,and P vgb was proven as the more suitable promoter for VHB expression andγ-PGA synthesis,andγ-PGA yield of attaining strain WX/pPvgb-YwbN-Vgb was further increased to 40.59 g/L by 10.18%.Finally,WX/pPvgb-YwbN-Vgb was cultivated in 3 L fermentor for fed-batch fermentation,and 46.39 g/Lγ-PGA was attained by glucose feeding,increased by 49.26%compared with the initial yield(31.01 g/L).Taken together,this study has attained an efficient VHB expression cassette for oxygen transfer andγ-PGA synthesis,which could also be applied in the production of other metabolites.展开更多
To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the co...To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the control of the promoter for the erythromycin resistance gene by splicing using overlapping extension PCR. This was cloned into the integrating vector pSET152, yielding the Vitreoscilla hemoglobin gene expression plasmid pSET152EVHB. This was then introduced into S. spinosa SP06081 by conjugal transfer, and integrated into the chromosome by site-specific recombination at the integration site ΦC31 on pSET152EVHB. The resultant conjugant, S. spinosa S078-1101, was genetically stable. The integration was further confirmed by PCR and Southern blotting analysis. A carbon monoxide differential spectrum assay showed that active Vitreoscilla hemoglobin was successfully expressed in S. spinosa S078-1101. Fermentation results revealed that expression of the Vitreoscilla hemoglobin gene significantly promoted spinosad biosynthesis under normal oxygen and moderately oxygen-limiting conditions (P<0.01). These findings demonstrate that integrating expression of the Vitreoscilla hemoglobin gene improves oxygen uptake and is an effective means for the genetic improvement of S. spinosa fermentation.展开更多
Exogenous Vitreoscilla globin gene (vgb), lytic genes of phage γ with S amber mutation (S^-RRz) and poly(fl-hydroxybutyrate) (PHB) biosynthetic genes (phbCAB) were cloned into a same Escherichia coil cell,simultaneou...Exogenous Vitreoscilla globin gene (vgb), lytic genes of phage γ with S amber mutation (S^-RRz) and poly(fl-hydroxybutyrate) (PHB) biosynthetic genes (phbCAB) were cloned into a same Escherichia coil cell,simultaneously or respectively. Six novel strains containing phbCAB and vgb with or without lytic genes were constructed. Strain VG1 (pTU14), in which vgb, phbCAB and S-RRz could all be successfully expressed, has superior characteristics in cell growth and PHB accumulation, while the results of strains containing vgb and phbCAB without S- RRz were not better than that of strains harbored phbCAB only. The simultaneous expression of vgb and S-RRz in the recombinant VG1 (pTU14) showed a great potential for low-cost production of PHB.展开更多
基金Supported by the National Natural Science Foundation of China(No.30670458)
文摘Lipid-bound [VHb（406）] and lipid-free [VHb（402）] wide type Vitreoscilla hemoglobin were separated from E. coli BL21（DE3）. The RZ is 3.30 for [VHb（406）] and 3.15 for [VHb（402）], respectively. VHb（406） shows a characteristic absorption band at 626 nm, while VHb（402） shows one at 644 nm, and it has more α-helix than VHb（402）. Data of Raman spectra experiment shows under the excitation wavelength 488 nm, υ4 vibration of both VHb（402） and VHb（406） had a pure and strong signal at 1375 cm-1, which proves that iron porphyrin of both the samples is at their trivalence oxidation state. And no matter lipid binds VHb or not, the vinyl of porphyrin is at cis state. The catalase activity of Vitreoscilla hemoglobin was explored with L-dopa and H2O2 as substrates. The results indicate that VHb（406） has more catalase activity than VHb（402）, VHb is a cell’s oxygen modulator and its catalase ability is modulated by the membrane.
基金Supported by the Ocean Public Welfare Scientific Research Special Appropriation Project(No.201005020)
文摘Vitreoscilla hemoglobin is an oxygen-binding protein that promotes oxygen delivery and reduces oxygen consumption under low oxygen conditions to increase the effi ciency of cell respiration and metabolism.In this study,we introduced a Vitreoscilla hemoglobin gene(vgb)into Chlorella vulgaris by Agrobacterium tumefaciens-mediated transformation(ATMT).PCR analysis confi rmed that the vgb gene was successfully integrated into the Chlorella vulgaris genome.Analysis of biomass obtained in shake fl asks revealed transformant biomass concentrations as high as 3.28 g/L,which was 38.81% higher than that of the wild-type strain.Lutein content of transformants also increased slightly.Further experiments recovered a maximum lutein yield of 2.91 mg/L from the transformants,which was 36.77% higher than that of the wild-type strain.The above results suggest that integrated expression of the vgb gene may improve cell growth and lutein yield in Chlorella vulgaris,with applications to lutein production from Chlorella during fermentation.
基金the National Natural Science Foundation of China(31972849)National Key Research and Development Program of China(2021YFC2101700)the Science and Technology Project of Hubei Tobacco Company(027Y2020-013).
文摘Poly-γ-glutamic acid(γ-PGA)is a natural polymer with various applications,and its high-viscosity hinders ox-ygen transmission and improvement of synthesis level.Vitreoscilla hemoglobin(VHB)has been introduced into various hosts as oxygen carrier,however,its expression strength and contact efficiency with oxygen hindered efficient oxygen transfer and metabolite synthesis.Here,we want to optimize the expression cassette of VHB for γ-PGA production.Firstly,our results implied that γ-PGA yields were enhanced when introducing twin-arginine translocation(Tat)signal peptides(SP_(YwbN),SP_(PhoD) and SP_(TorA))into VHB expression cassette,and the best per-formance was attained by SP YwbN from Bacillus subtilis,theγ-PGA yield of which was 18.53% higher than that of control strain,and intracellular ATP content and oxygen transfer coefficient(K_(L)a)were increased by 29.71% and 73.12%,respectively,indicating that VHB mediated by SP YwbN benefited oxygen transfer and ATP generation forγ-PGA synthesis.Furthermore,four promoters were screened,and P vgb was proven as the more suitable promoter for VHB expression andγ-PGA synthesis,andγ-PGA yield of attaining strain WX/pPvgb-YwbN-Vgb was further increased to 40.59 g/L by 10.18%.Finally,WX/pPvgb-YwbN-Vgb was cultivated in 3 L fermentor for fed-batch fermentation,and 46.39 g/Lγ-PGA was attained by glucose feeding,increased by 49.26%compared with the initial yield(31.01 g/L).Taken together,this study has attained an efficient VHB expression cassette for oxygen transfer andγ-PGA synthesis,which could also be applied in the production of other metabolites.
基金supported by the National Basic Research Program of China (Grant Nos. 2012CB722301 and 2011CB111605)the National High Technology Research and Development Project of China (Grant No. 2011AA10A203)the National Natural Science Foundation of China (Grant No. 31070006)
文摘To promote spinosad biosynthesis by improving the limited oxygen supply during high-density fermentation of Saccharopolyspora spinosa, the open reading frame of the Vitreoscilla hemoglobin gene was placed under the control of the promoter for the erythromycin resistance gene by splicing using overlapping extension PCR. This was cloned into the integrating vector pSET152, yielding the Vitreoscilla hemoglobin gene expression plasmid pSET152EVHB. This was then introduced into S. spinosa SP06081 by conjugal transfer, and integrated into the chromosome by site-specific recombination at the integration site ΦC31 on pSET152EVHB. The resultant conjugant, S. spinosa S078-1101, was genetically stable. The integration was further confirmed by PCR and Southern blotting analysis. A carbon monoxide differential spectrum assay showed that active Vitreoscilla hemoglobin was successfully expressed in S. spinosa S078-1101. Fermentation results revealed that expression of the Vitreoscilla hemoglobin gene significantly promoted spinosad biosynthesis under normal oxygen and moderately oxygen-limiting conditions (P<0.01). These findings demonstrate that integrating expression of the Vitreoscilla hemoglobin gene improves oxygen uptake and is an effective means for the genetic improvement of S. spinosa fermentation.
基金Supported by the National Natural Science Foundation of China (No. 29834103, 29876021).
文摘Exogenous Vitreoscilla globin gene (vgb), lytic genes of phage γ with S amber mutation (S^-RRz) and poly(fl-hydroxybutyrate) (PHB) biosynthetic genes (phbCAB) were cloned into a same Escherichia coil cell,simultaneously or respectively. Six novel strains containing phbCAB and vgb with or without lytic genes were constructed. Strain VG1 (pTU14), in which vgb, phbCAB and S-RRz could all be successfully expressed, has superior characteristics in cell growth and PHB accumulation, while the results of strains containing vgb and phbCAB without S- RRz were not better than that of strains harbored phbCAB only. The simultaneous expression of vgb and S-RRz in the recombinant VG1 (pTU14) showed a great potential for low-cost production of PHB.
