含透明颤菌血红蛋白基因重组菌的构建及其对菌株发酵产1，3-丙二醇的影响
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摘要
本文将编码透明颤菌血红蛋白的结构基因vgb引入1,3-丙二醇产生菌K.oxytoca M5al,同时还将编码甲酸脱氢酶基因fdh和vgb同时引入菌体表达。并考察了外源蛋白的表达对菌体发酵产1,3-丙二醇的影响。结果和结论如下:
发酵前对菌株进行驯化会明显提高菌株产1,3-丙二醇的能力,建立并比较了高甘油驯化策略和发酵液驯化策略。经驯化的菌株生长速度要高于未经驯化的菌株,且发酵液驯化的菌株生长速度高于高甘油驯化的菌株,相应发酵液驯化菌株合成的1,3-PD的能力和甘油的消耗速度均优于高甘油驯化菌株。摇瓶发酵45小时,高甘油液驯化菌株产1,3-PD浓度为6.58 g·l~(-1),相比原始菌产1,3-PD能力提高了41.2%;而发酵液驯化菌株产1,3-PD浓度为7.36 g·l~(-1),相比原始菌产1,3-PD能力提高了57.94%。经发酵液驯化后不仅提高了菌体对底物甘油的耐受能力,还提高了菌体对产物的耐受性能,从而提高菌体产1,3-PD的能力。
不同溶氧条件下摇瓶发酵考察表明,溶氧对最终的发酵结果有明显的影响,500ml的摇瓶装入100ml发酵液时发酵效果最好,摇瓶发酵产1,3-PD浓度为11.2g·l~(-1)。
透明颤菌血红蛋白和甲酸脱氢酶都成功在菌体内表达,CO差光谱分析和甲酸脱氢酶酶活分析表明所表达的外源蛋白都具有活性。构建了引入parDE基因的重组质粒pDV和pDFV,在原始菌中,其稳定性较未引入parDE基因的重组质粒pV和pFV大大提高。传代48小时后,pDV和pDFV质粒丢失率分别为23.3%、39.1%,与未引入parDE基因的质粒pV和pFV相比(质粒丢失率分别为69.6%、79.3%)稳定性明显提高。传代前后FDH酶活的测定结果表明,parDE基因的引入同样可以防止因质粒丢失而造成的酶活降低。
对重组菌在不同溶氧条件下发酵生产1,3-PD进行了初步考察。结果表明在K.oxytoca M5al内引入VHb并没有明显促进菌体生长,反而在一定程度上抑制了菌体生长;但低溶氧条件下VHb的表达提高了单位菌体合成1,3-PD的能力,摇瓶发酵45小时相比原始菌1,3-PD产量提高了43.7%。重组菌M5al-pDFV,摇瓶发酵45小时后产1,3-PD浓度达12.4 g·l~(-1),相比原始菌产1,3-PD提高了93.75%。菌体内NADH浓度测定表明重组菌M5al-pDV、M5al-pDFV体内NADH水平均高于原始菌。
The vgb gene coding for Vitreoscilla Hemoglobin was transferred into wild type strain K.oxytoca M5al, also the fdh gene coding for formate dehydrogenase and vgb were transferred into K.oxytoca M5al together. Then the influnce of heterogeneous proteins on the fementation of 1,3-propanediol was studied. The results were shown as followed:
We found that domestication of strain could obviously enhance the production of 1,3-propanediol. After domestication the cell growth was better than undomesticated strain, fermentation broth domestication had more influence on glycerol consumption and 1,3-propanediol production as well as on cell growth. After 45 hours’fermentation, the yield of 1,3-propanediol of glycerol domesticated strain and fermentation broth domesticated strain were 6.58 g·l~(-1) and 7.36 g·l~(-1),respectively. Compared with the wild type strain, the production of 1,3-propanediol was enhanced 41.2% and 57.94%,respectively. it stated clearly that fermentation broth domestication could not only enhance the endurence of substrate glycerol, but also enhanced endurence of products such as 1,3-propanediol, ethanol, lactate. So finally it could enhance the productivity of 1,3-propanediol.
The result of shaking flask fermentation under differents dissoved oxygen conditions showed that dissoved oxygen was a main factor that influnced the fermentation results, 500ml flask filled with 100ml medium would be the best dissoved oxygen condition, and the final concentration of 1,3-propanediol of 11.2g·l~(-1) was gained in this condition.
Vitreoscilla Hemoglobin and formate dehydrogenase were successfully expressed in K.oxytoca M5al, CO bonding experiment and formate dehydrogenase enzyme activity analysis showed that those heterogeneous proteins had fuctional activity. Recombinent plasmids pDV and pDFV were constructed which contained parDE gene, compared with plasmid pV or pFV, the stability of plasmid in recombinant strains contained parDE were obviously enhanced. Continuous passage 48 hours, only 23.3%、39.1% of plasmid pDV and pDFV were lost, while about 69.6%、79.3% of plasmid pV and pFV were lost. And it showed that the FDH activity were also less lost because of parDE gene.
The 1,3-PD production of recombinant strain M5al-pDV was higher than wild strain M5al while biomass was lower than M5al. Under high dissolved oxygen condition, after 45 hours, the yields of 1,3-PD of M5al and M5al-pDV were 9.27 g l~(-1) and 11.15 g l~(-1), respectively. The production of 1,3-PD of recombinant strain was 20.28% higher, and the ethanol also increased while the lactate concentration decreased. Under low dissolved oxygen condition, after 45 hours, the yields of 1,3-PD of M5al and M5al-pV were 6.4 g l~(-1) and 9.2 g l~(-1), respectively. The production of 1,3-PD of recombinant strain was 43.7% higher, and both lactate and ethanol concentrations decreased.
Recombinant strain M5al-pDFV was constructed which contain both Vitreoscilla Hemoglobin and formate dehydrogenase. After 45 hours’fermentation, the yields of 1,3-PD was 12.4 g·l~(-1),the production of 1,3-PD of M5al-pDFV was 93.75% higher compared with wild strain. The concentration of NADH in recobminent M5al-pDV、M5al-pDFV biomass were higher than wild strian, this may be the reason why the 1,3-PD production of recombinent strains increased.
发酵前对菌株进行驯化会明显提高菌株产1,3-丙二醇的能力,建立并比较了高甘油驯化策略和发酵液驯化策略。经驯化的菌株生长速度要高于未经驯化的菌株,且发酵液驯化的菌株生长速度高于高甘油驯化的菌株,相应发酵液驯化菌株合成的1,3-PD的能力和甘油的消耗速度均优于高甘油驯化菌株。摇瓶发酵45小时,高甘油液驯化菌株产1,3-PD浓度为6.58 g·l~(-1),相比原始菌产1,3-PD能力提高了41.2%;而发酵液驯化菌株产1,3-PD浓度为7.36 g·l~(-1),相比原始菌产1,3-PD能力提高了57.94%。经发酵液驯化后不仅提高了菌体对底物甘油的耐受能力,还提高了菌体对产物的耐受性能,从而提高菌体产1,3-PD的能力。
不同溶氧条件下摇瓶发酵考察表明,溶氧对最终的发酵结果有明显的影响,500ml的摇瓶装入100ml发酵液时发酵效果最好,摇瓶发酵产1,3-PD浓度为11.2g·l~(-1)。
透明颤菌血红蛋白和甲酸脱氢酶都成功在菌体内表达,CO差光谱分析和甲酸脱氢酶酶活分析表明所表达的外源蛋白都具有活性。构建了引入parDE基因的重组质粒pDV和pDFV,在原始菌中,其稳定性较未引入parDE基因的重组质粒pV和pFV大大提高。传代48小时后,pDV和pDFV质粒丢失率分别为23.3%、39.1%,与未引入parDE基因的质粒pV和pFV相比(质粒丢失率分别为69.6%、79.3%)稳定性明显提高。传代前后FDH酶活的测定结果表明,parDE基因的引入同样可以防止因质粒丢失而造成的酶活降低。
对重组菌在不同溶氧条件下发酵生产1,3-PD进行了初步考察。结果表明在K.oxytoca M5al内引入VHb并没有明显促进菌体生长,反而在一定程度上抑制了菌体生长;但低溶氧条件下VHb的表达提高了单位菌体合成1,3-PD的能力,摇瓶发酵45小时相比原始菌1,3-PD产量提高了43.7%。重组菌M5al-pDFV,摇瓶发酵45小时后产1,3-PD浓度达12.4 g·l~(-1),相比原始菌产1,3-PD提高了93.75%。菌体内NADH浓度测定表明重组菌M5al-pDV、M5al-pDFV体内NADH水平均高于原始菌。
The vgb gene coding for Vitreoscilla Hemoglobin was transferred into wild type strain K.oxytoca M5al, also the fdh gene coding for formate dehydrogenase and vgb were transferred into K.oxytoca M5al together. Then the influnce of heterogeneous proteins on the fementation of 1,3-propanediol was studied. The results were shown as followed:
We found that domestication of strain could obviously enhance the production of 1,3-propanediol. After domestication the cell growth was better than undomesticated strain, fermentation broth domestication had more influence on glycerol consumption and 1,3-propanediol production as well as on cell growth. After 45 hours’fermentation, the yield of 1,3-propanediol of glycerol domesticated strain and fermentation broth domesticated strain were 6.58 g·l~(-1) and 7.36 g·l~(-1),respectively. Compared with the wild type strain, the production of 1,3-propanediol was enhanced 41.2% and 57.94%,respectively. it stated clearly that fermentation broth domestication could not only enhance the endurence of substrate glycerol, but also enhanced endurence of products such as 1,3-propanediol, ethanol, lactate. So finally it could enhance the productivity of 1,3-propanediol.
The result of shaking flask fermentation under differents dissoved oxygen conditions showed that dissoved oxygen was a main factor that influnced the fermentation results, 500ml flask filled with 100ml medium would be the best dissoved oxygen condition, and the final concentration of 1,3-propanediol of 11.2g·l~(-1) was gained in this condition.
Vitreoscilla Hemoglobin and formate dehydrogenase were successfully expressed in K.oxytoca M5al, CO bonding experiment and formate dehydrogenase enzyme activity analysis showed that those heterogeneous proteins had fuctional activity. Recombinent plasmids pDV and pDFV were constructed which contained parDE gene, compared with plasmid pV or pFV, the stability of plasmid in recombinant strains contained parDE were obviously enhanced. Continuous passage 48 hours, only 23.3%、39.1% of plasmid pDV and pDFV were lost, while about 69.6%、79.3% of plasmid pV and pFV were lost. And it showed that the FDH activity were also less lost because of parDE gene.
The 1,3-PD production of recombinant strain M5al-pDV was higher than wild strain M5al while biomass was lower than M5al. Under high dissolved oxygen condition, after 45 hours, the yields of 1,3-PD of M5al and M5al-pDV were 9.27 g l~(-1) and 11.15 g l~(-1), respectively. The production of 1,3-PD of recombinant strain was 20.28% higher, and the ethanol also increased while the lactate concentration decreased. Under low dissolved oxygen condition, after 45 hours, the yields of 1,3-PD of M5al and M5al-pV were 6.4 g l~(-1) and 9.2 g l~(-1), respectively. The production of 1,3-PD of recombinant strain was 43.7% higher, and both lactate and ethanol concentrations decreased.
Recombinant strain M5al-pDFV was constructed which contain both Vitreoscilla Hemoglobin and formate dehydrogenase. After 45 hours’fermentation, the yields of 1,3-PD was 12.4 g·l~(-1),the production of 1,3-PD of M5al-pDFV was 93.75% higher compared with wild strain. The concentration of NADH in recobminent M5al-pDV、M5al-pDFV biomass were higher than wild strian, this may be the reason why the 1,3-PD production of recombinent strains increased.
引文
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