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 ФC31 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. Saccharopolyspora spinosa, spinosad, Vitreoscilla hemoglobin, integrating vector, homologous recombination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
The coding sequence of Vitreoscilla hemoglobin (vhb) was cloned with PCR technique from Vitreoscilla stercoraria Pringsheim. The plant expression vector with vhb gene under the control of CaMV 35S promoter was constru...The coding sequence of Vitreoscilla hemoglobin (vhb) was cloned with PCR technique from Vitreoscilla stercoraria Pringsheim. The plant expression vector with vhb gene under the control of CaMV 35S promoter was constructed and used in the transformation of Petunia hybrida Vilm by the Agrobacterium mediated procedure. The results of PCR amplification and Southern hybridization indicated that the vhb gene had been integrated into the petunia genome and the vhb gene expression had been detected by RT-PCR amplification. In hydroponic culture the transgenic petunias grew much better than non-transgenic controls. For further analysis of hypoxia tolerance of transgenic petunia, the petunia plants with vhb gene were submerged into liquid MS medium. The transgenic plants survived in hypoxic condition and grew out of the liquid surface in a few weeks, while non-transgenic plants were still submerged and suffocated in culture solution without ability to grow out of liquid medium in submersed culture for four to five weeks. The vhb gene transformed petunia plants had been planted and tested in a simulated flooding condition, and showed obvious tolerance to water-logging. It seen is that hemoglobin gene from Vitreoscilla might have the potential use in molecular breeding for the improvement of plant resistance to hypoxia and flooding.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
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
The first successful Agrobacterium-mediated transformation of the green alga Haematococcus pluvialis using the vectors hosting the genes coding for VHB （ Vitreoscilla hemoglobin） and GLUT1 （glucose transporter 1 ）...The first successful Agrobacterium-mediated transformation of the green alga Haematococcus pluvialis using the vectors hosting the genes coding for VHB （ Vitreoscilla hemoglobin） and GLUT1 （glucose transporter 1 ） is reported here. The greenish yellow fluorescence of EGFP was observed when the zeocin-resistant cells were viewed with a fluorescent microscope. The functional expression of EGFP showed that the Agrobacterium-mediated transformation could efficiently transfer the exogenous gene into H. pluvialis. RT-PCR was used to successfully amplify the mRNA of VHB and GLUT1 genes from transformed cells, while Southern blots indicated the integration of VHB and GLUT1 genes into the genome of H. pluvialis. Transferring VHB and GLUT1 genes into this alga would pave the way for manip- ulation of many important pathways relevant to the food, pharmaceutical, and nutraceutical industries.展开更多
Exogenous Vitreoscilla globin gene (vgb), lytic genes of phage A with S amber mutation (S-RRz) and poly(B-hydroxybutyrate) (PHB) biosynthetic genes (phbCAB) were cloned into a same Escherichia coli cell, simultaneousl...Exogenous Vitreoscilla globin gene (vgb), lytic genes of phage A with S amber mutation (S-RRz) and poly(B-hydroxybutyrate) (PHB) biosynthetic genes (phbCAB) were cloned into a same Escherichia coli 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 ph&CAB 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
Salecan is a novel exopolysaccharide produced by the strain Agrobacterium sp. 7__X09, and it is composed of only glucose monomers. The unique chemical composition and excellent physicochemical properties make Salecan ...Salecan is a novel exopolysaccharide produced by the strain Agrobacterium sp. 7__X09, and it is composed of only glucose monomers. The unique chemical composition and excellent physicochemical properties make Salecan a promising material for applications in coagulation, lubrication, protection against acute liver injury, and alleviating constipation. In this study, we cloned the Vitreosci/la hemoglobin gene into a broad-host-range plasmid pCM158. Without antibiotic selection, there was negligible loss of the plasmid in the host Agrobacterium sp. ZX09 after one passage of cultivation. The expression of Vitreoscilla hemoglobin was demonstrated by carbon monoxide （CO） dif- ference spectrum. The engineered strain Agrobacterium sp. ZX09 increased Salecan yield by 30%. The other physi- ological changes included its elevated respiration rate and cellular invertase activity.展开更多
Objectives:This study used Deinococcus radiodurans,which is extremely resistant to oxidative damage,genotoxic chemicals,high levels of ionising and ultraviolet radiation and drying,and its Vitreoscilla haemoglobin(vgb...Objectives:This study used Deinococcus radiodurans,which is extremely resistant to oxidative damage,genotoxic chemicals,high levels of ionising and ultraviolet radiation and drying,and its Vitreoscilla haemoglobin(vgb)gene-cloned recombinant with the vgb−recombinant strain as a control.In addition to the conditions wherein bacteria have an optimum Cr(Ⅲ)biosorption capacity,the contribution of the vgb gene to the biosorption ability of the bacteria has been investigated by providing the organism with a more oxygenic environment.Methods:Bacteria were produced and metal stock solution was prepared.To determine the Cr(Ⅲ)removal capacities of wild and recombinant D.radiodurans strains,the residual metal concentration in aqueous media at the beginning and after biosorption was determined in Atomic Absorption Spectrophotometer.Some optimal conditions were created for the biosorption conditions to occur.Conclusions:The optimisation tests showed that Cr(Ⅲ)reached the highest biosorption capacity within 15 minutes at a metal concentration of 2,000 ppm,30°C,pH 5.0 and 150 rpm stirring speed in all the three bacteria.The vgb gene had no significant contribution to the biosorption capacity.展开更多
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 ФC31 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. Saccharopolyspora spinosa, spinosad, Vitreoscilla hemoglobin, integrating vector, homologous recombination
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.
文摘The coding sequence of Vitreoscilla hemoglobin (vhb) was cloned with PCR technique from Vitreoscilla stercoraria Pringsheim. The plant expression vector with vhb gene under the control of CaMV 35S promoter was constructed and used in the transformation of Petunia hybrida Vilm by the Agrobacterium mediated procedure. The results of PCR amplification and Southern hybridization indicated that the vhb gene had been integrated into the petunia genome and the vhb gene expression had been detected by RT-PCR amplification. In hydroponic culture the transgenic petunias grew much better than non-transgenic controls. For further analysis of hypoxia tolerance of transgenic petunia, the petunia plants with vhb gene were submerged into liquid MS medium. The transgenic plants survived in hypoxic condition and grew out of the liquid surface in a few weeks, while non-transgenic plants were still submerged and suffocated in culture solution without ability to grow out of liquid medium in submersed culture for four to five weeks. The vhb gene transformed petunia plants had been planted and tested in a simulated flooding condition, and showed obvious tolerance to water-logging. It seen is that hemoglobin gene from Vitreoscilla might have the potential use in molecular breeding for the improvement of plant resistance to hypoxia and flooding.
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 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 Project of the National High Technology Research and Development Program("863"Program)of China(Grant No.2012AA092103)Scientific Research Foundation of the Institute of Seawater Desalination&Multipurpose Utilization(Grant No.K-JBYWF-2015-T20)+1 种基金Project of the Third Institute of OceanographyState Oceanic Administration(Grant No.2014008)
文摘The first successful Agrobacterium-mediated transformation of the green alga Haematococcus pluvialis using the vectors hosting the genes coding for VHB （ Vitreoscilla hemoglobin） and GLUT1 （glucose transporter 1 ） is reported here. The greenish yellow fluorescence of EGFP was observed when the zeocin-resistant cells were viewed with a fluorescent microscope. The functional expression of EGFP showed that the Agrobacterium-mediated transformation could efficiently transfer the exogenous gene into H. pluvialis. RT-PCR was used to successfully amplify the mRNA of VHB and GLUT1 genes from transformed cells, while Southern blots indicated the integration of VHB and GLUT1 genes into the genome of H. pluvialis. Transferring VHB and GLUT1 genes into this alga would pave the way for manip- ulation of many important pathways relevant to the food, pharmaceutical, and nutraceutical industries.
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 A with S amber mutation (S-RRz) and poly(B-hydroxybutyrate) (PHB) biosynthetic genes (phbCAB) were cloned into a same Escherichia coli 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 ph&CAB only. The simultaneous expression of vgb and S- RRz in the recombinant VG1 (pTU14) showed a great potential for low-cost production of PHB.
基金supported by the Fundamental Research Funds for the Central Universities of China(No.30920130121013)the National Natural Science Foundation of China(No.31300111)
文摘Salecan is a novel exopolysaccharide produced by the strain Agrobacterium sp. 7__X09, and it is composed of only glucose monomers. The unique chemical composition and excellent physicochemical properties make Salecan a promising material for applications in coagulation, lubrication, protection against acute liver injury, and alleviating constipation. In this study, we cloned the Vitreosci/la hemoglobin gene into a broad-host-range plasmid pCM158. Without antibiotic selection, there was negligible loss of the plasmid in the host Agrobacterium sp. ZX09 after one passage of cultivation. The expression of Vitreoscilla hemoglobin was demonstrated by carbon monoxide （CO） dif- ference spectrum. The engineered strain Agrobacterium sp. ZX09 increased Salecan yield by 30%. The other physi- ological changes included its elevated respiration rate and cellular invertase activity.
基金This study was supported by Inonu University Scientific Research Projects Coordination Unit(project number:2012-192).
文摘Objectives:This study used Deinococcus radiodurans,which is extremely resistant to oxidative damage,genotoxic chemicals,high levels of ionising and ultraviolet radiation and drying,and its Vitreoscilla haemoglobin(vgb)gene-cloned recombinant with the vgb−recombinant strain as a control.In addition to the conditions wherein bacteria have an optimum Cr(Ⅲ)biosorption capacity,the contribution of the vgb gene to the biosorption ability of the bacteria has been investigated by providing the organism with a more oxygenic environment.Methods:Bacteria were produced and metal stock solution was prepared.To determine the Cr(Ⅲ)removal capacities of wild and recombinant D.radiodurans strains,the residual metal concentration in aqueous media at the beginning and after biosorption was determined in Atomic Absorption Spectrophotometer.Some optimal conditions were created for the biosorption conditions to occur.Conclusions:The optimisation tests showed that Cr(Ⅲ)reached the highest biosorption capacity within 15 minutes at a metal concentration of 2,000 ppm,30°C,pH 5.0 and 150 rpm stirring speed in all the three bacteria.The vgb gene had no significant contribution to the biosorption capacity.
