产PHB隐藏嗜酸菌DX1-1选育及代谢途径研究
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摘要
聚β-羟基丁酸(PHB)作为微生物合成的可降解材料,除了具有与化学合成高分子相似的性质外,还具有一般化学合成高分子没有的性质,如光学活性好、透氧性低、抗紫外线辐射、生物可降解性、生物组织相容性、压电性和抗凝血性等,具有广阔的应用前景,越来越受到人们的关注。
本论文针对产PHB兼养嗜酸菌Acidiphilium cryptum DX1-1开展了(1)菌种的分离、生理生化鉴定；(2)菌种的诱变选育和产PHB条件优化；(3)PHB的分离提取和性质研究；(4)代谢途径研究；(5)微异养条件下积累PHB相关基因表达和发酵条件的研究。取得如下主要结果。
完成了菌种和胞内聚合物的分离鉴定。从取自江西德兴铜矿的酸性矿坑废水中分离出一株菌株(DX1-1),对其形态特征、生理生化、底物利用等特性进行了分析,结果表明该菌具有兼性菌的典型特征。该菌的生长温度范围是20-35℃,pH范围是2.0-5.0。经系统发育分析确定该菌是一株Acidiphilium菌,进一步分析了其全细胞脂肪酸和DNA G+C%,中国典型培养物保藏中心将其定为隐藏嗜酸菌(Acidiphilium cryptum)。透射电镜显示该菌胞内可积累大量颗粒状聚合物。对该聚合物进行红外、紫外吸收光谱检测,结果显示该聚合物为PHB。利用四种方法对胞内聚合物PHB进行提取与纯化,确定出最好的提取方法是氯仿-次氯酸钠法,纯度达到97%,提取率达72%,分子量M=326 kD。对样品PHB进行了傅立叶红外光谱(FT-IR)分析、气相色谱质谱联用(GC-MS)、核磁共振(NMR)分析和微量热分析。
对菌株DX1-1积累PHB的培养条件进行了优化并对菌种进行了诱变改良。确定了最佳培养条件为葡萄糖40g/L、硝酸钾浓度15 g/L以及初始pH 3.0,此试验将菌株DX1-1积累PHB的产量提高到19.75g/L。采用紫外线照射和放射性元素钴60辐射诱变方法,对菌株DX1-1进行了诱变改良。结果显示钴60最佳诱变剂量为100Gy,紫外诱变的最佳条件为15W、30 cm、60 s,紫外诱变的效果比钴60诱变的效果好。诱变后筛选得到的一株菌UV60-3,PHB产量达到28.56g/L,并且可稳定遗传。对菌株UV60-3积累PHB的营养条件做了检测,结果显示最佳的氮源为硫酸铵,最佳浓度为30 g/L,此时PHB产量可达30.57 g/L。
对不同培养条件下菌株的蛋白质表达差异做了研究。研究了起始C/N比分别为24、7.5、2.4、1.2时菌株DX1-1积累PHB的情况,结果显示起始C/N比为2.4时PHB的产量为0.88g/1g细胞干重,所需要的时间也最短。对不同C/N比条件下菌株DX1-1蛋白质表达差异进行分析,结果显示酮醇酸还原异构酶、醇醛酮还原酶、磷酸甘油酸变位酶、葡萄糖磷酸化酶、苹果酸脱氢酶和烯醇化酶的表达在C/N为2.4时有明显提高。蛋白质差异表达的方法没能找到PHB合成相关的蛋白质。但是此结果显示PHB的积累除与糖代谢相关外还与胞内蛋白质代谢、脂质代谢、核酸代谢和转录翻译等因子具有重要关系。用Real-Time PCR方法对挑取的19个蛋白质点在RNA水平上进行检测,结果显示19个蛋白质的表达情况与对应基因的转录情况基本一致。
对隐藏嗜酸菌PHB代谢途径作了分析。首次以已报道全基因组序列的Acidiphilium cryptum JF-5的功能基因为参考,选择了PHB代谢相关的13个基因作为研究对象,用Real-Time PCR方法对不同C/N比情况下这些基因的转录情况进行了研究。结果显示β-羟基丁酸聚合酶和乙酰辅酶A合成酶在PHB代谢中起了非常重要的作用。菌株DX1-1积累PHB的起始底物是乙酸,并且找到了一条完整的PHB代谢途径。对13个基因的启动子序列进行了预测,模体模型为-35区：n-G-A-n-n-A-C-A和-10区：n-G-A-n-n-A-C-A,中间间隔平均为20 bp,该模体序列与E. coli并无明显的相似性,表明他们可能有较低的转录活性或者他们有不同的调节机制。
对微异养条件下DX1-1以硫为能源积累PHB的情况做了研究。DX1-1能以硫为能源缓慢生长,但加入少量葡萄糖后,生长周期明显缩短,细胞密度大大提高。结果显示,以硫为能源的培养基中,加入0.1%的葡萄糖,PHB的产量达到13.51g/L,与1%葡萄糖为能源时PHB产量相接近。用Real-Time PCR方法对硫、葡萄糖、硫+葡萄糖等六种培养条件下的5个PHB代谢相关关键基因,10个碳固定相关基因和7个硫代谢相关基因的表达情况进行分析,结果显示葡萄糖的加入可以诱导硫代谢相关基因和碳固定相关基因的表达；仅以单质硫能源不加葡萄糖时,PHB代谢相关关键基因和碳固定相关基因的表达均出现下调,但是硫代谢相关基因却有不同程度的上调趋势,这一结果与该菌在该状态下仅仅维持生命的生长状态相符合。
PHB is a kind of degradable microorganism material, besides the similar characteristics with chemosynthetic macromolecules, it also shows some other characteristics that are not obtained by ordinary chemosynthetic macromolecules, such as, good optical property, low oxygen permibility, anti-ultraviolet radiation, bio-degradability, bio-compatibility, piezoelectricity, and anti-cruor, etc.. It has thus a promising prospect in its application and has attracted more and more attention.
A new bacterial strain, designated as strain DX1-1, was isolated from an acid mine drainage of Dexing Copper Mine, Jiangxi Province, China. Basing on morphology, biochemical and physiological characterization, strain DX1-1 was identified as facultative autotrophic bacterium. It grew at temperature ranging from 20℃to 35℃and at initial pH values ranging from 2.0 to 5.0. It was affiliated to the genus Acidiphilium according to the phylogenetic tree derived from 16S rRNA sequence alignment. The capacity of producing polymer granules of strain DX1-1 was found by Transmission Electron Microscopy (TEM). The polymer granules was extracted and detected by ultraviolet-visible (UV) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy. The results show that it has the same character with poly-β-hydroxybutyrate (PHB). It suggests that strain DX1-1 might have the potential of producing biodegradable materials (PHB).
Four methods were employed to extract PHB from strain DX1-1. There is advantage and disadvantage for each method. Chloroform-sodium hypochlorite method is the best in extracting PHB form Acidiphilium cryptum DX1-1. The extraction rate reaches 73%, the purification rate is 97% and molecular weight is 326 kD. The PHB extracted by this method was then analyzed by UV spectroscopy, FT-IR spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. The results show that PHB from strain DX1-1 have the same biochemical structure and character with PHB standard. Mass spectrometer (MS) analysis reveals that the long chain of PHB is destroyed when treated by chloroform-sodium hypochlorite. The differential scanning calorimetry (DSC) of PHB shows PHB from Acidiphilium cryptum DX1-1 has low degree of crystallinity which makes the PHB have a wider range of applications.
The culture condition of strain DX1-1 for producing PHB was optimized. Glucose and KNO3 were found to be the best carbon and nitrogen substrates for the production of PHB. The L16 (43) orthogonal experiment improved the yield to 19.56g/L when 40g/L glucose as carbon source,15g/L KNO3 as nitrogen source and pH 3.0 were adopted. Then the cells growth curve and PHB production curve were measured under the optimum culture condition. It was found that the process of PHB accumulating was a growth-associated process. The strain DX1-1 was irradiated respectively by UV and Co60 to improve PHB production. The results indicated that the effect of UV better than using Co60. One strain of the UV mutagenized called UV60-3 has the highest PHB production yield, showing final PHB concentration of 28.56 g/L. Further research about the best culture condition of the mutant was done. The optimum nitrogen source was 30g/L (NH4)2SO4, the final PHB concentration reaches to 30.57g/L.
The time, yield and the expression of the PHB accumulation-related genes of Acidiphilium cryptum DX1-1 were investigated under four different initial C/N ratios 1.2,2.4,7.5, and 24. The results of time and yield of PHB accumulation show that the initial C/N ratio 2.4 was optimum for strain DX1-1 to accumulate PHB, both higher and lower initial C/N ratios did not favor that process. Total proteins and intracellular proteins were extracted from strain DX1-1 cultivated under different initial C/N ratios. The protein plots in 2-DE maps were abundant, dispersed and clear, suggesting that the 2-DE technique developed in this study could absolutely meet the requirement of further proteomics study. Eleven proteins pots in total proteins and 9 proteins pots in intracellular proteins which resolved differentially expression were chosen. The 2-DE and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF/TOF MS) were used to identify the proteins. No proteins which catalyze PHB metabolsim were found by this method. But the results show that PHB metabolism is related to metabolism of proteins, fatty acids, and nucleic acids and factor of transcription besides carbon metabolism. The mRNAs of the 19 genes encoding those proteins were detected by Real-Time PCR. The results show that the changes of expression for the 19 genes were consistent with the changes in their mRNAs.
Based on the genome of Acidiphilium cryptum JF-5, thirteen PHB accumulation related genes in strain JF-5 were chosen and successfully cloned from strain DX1-1. The differential expressions of the 13 functional genes, in different C/N ratios as cited above, were then studied by Real-time PCR. The results show that all the 13 genes were most upregulated when the initial C/N ratio was 2.4, and among which the gene Acry_3030 encoding poly-β-hydroxybutyrate polymerase and Acry_0626 encoding acetyl-CoA synthetase were much more upregulated than the other genes, which prove that they play the most important role for PHB accumulation and acetate is the main initial substance for PHB accumulation for strain DX1-1. Potential regulatory motifs analysis shows that the genes related to PHB accumulation are regulated by different promoters and that the motif has weak similarity to the model promoters, suggesting that PHB-metabolism in Acidiphilium cryptum may be mediated by a different mechanism.
Acidiphilium cryptum DX1-1 can grow slowly with sulfur as energy source. However, growth cycle shortened and cell density increased with glucose added. It was found that PHB reached 13.51g/l with 0.1% glucose added when sulfur as energy source. The expression of genes related to PHB metabolism, carbon fixation and sulfur metabolism in different culture media were detected by Real-time PCR. The results show that glucose could induce the expression of genes related to sulfur metabolism and carbon fixation. When sulfur was used as the only energy sources, the expression of the genes related to PHB metabolism and carbon fixation down-regulared while the genes related to sulfur metabolism up-regulated, which was according with the growth state of keeping the cells alive.
本论文针对产PHB兼养嗜酸菌Acidiphilium cryptum DX1-1开展了(1)菌种的分离、生理生化鉴定；(2)菌种的诱变选育和产PHB条件优化；(3)PHB的分离提取和性质研究；(4)代谢途径研究；(5)微异养条件下积累PHB相关基因表达和发酵条件的研究。取得如下主要结果。
完成了菌种和胞内聚合物的分离鉴定。从取自江西德兴铜矿的酸性矿坑废水中分离出一株菌株(DX1-1),对其形态特征、生理生化、底物利用等特性进行了分析,结果表明该菌具有兼性菌的典型特征。该菌的生长温度范围是20-35℃,pH范围是2.0-5.0。经系统发育分析确定该菌是一株Acidiphilium菌,进一步分析了其全细胞脂肪酸和DNA G+C%,中国典型培养物保藏中心将其定为隐藏嗜酸菌(Acidiphilium cryptum)。透射电镜显示该菌胞内可积累大量颗粒状聚合物。对该聚合物进行红外、紫外吸收光谱检测,结果显示该聚合物为PHB。利用四种方法对胞内聚合物PHB进行提取与纯化,确定出最好的提取方法是氯仿-次氯酸钠法,纯度达到97%,提取率达72%,分子量M=326 kD。对样品PHB进行了傅立叶红外光谱(FT-IR)分析、气相色谱质谱联用(GC-MS)、核磁共振(NMR)分析和微量热分析。
对菌株DX1-1积累PHB的培养条件进行了优化并对菌种进行了诱变改良。确定了最佳培养条件为葡萄糖40g/L、硝酸钾浓度15 g/L以及初始pH 3.0,此试验将菌株DX1-1积累PHB的产量提高到19.75g/L。采用紫外线照射和放射性元素钴60辐射诱变方法,对菌株DX1-1进行了诱变改良。结果显示钴60最佳诱变剂量为100Gy,紫外诱变的最佳条件为15W、30 cm、60 s,紫外诱变的效果比钴60诱变的效果好。诱变后筛选得到的一株菌UV60-3,PHB产量达到28.56g/L,并且可稳定遗传。对菌株UV60-3积累PHB的营养条件做了检测,结果显示最佳的氮源为硫酸铵,最佳浓度为30 g/L,此时PHB产量可达30.57 g/L。
对不同培养条件下菌株的蛋白质表达差异做了研究。研究了起始C/N比分别为24、7.5、2.4、1.2时菌株DX1-1积累PHB的情况,结果显示起始C/N比为2.4时PHB的产量为0.88g/1g细胞干重,所需要的时间也最短。对不同C/N比条件下菌株DX1-1蛋白质表达差异进行分析,结果显示酮醇酸还原异构酶、醇醛酮还原酶、磷酸甘油酸变位酶、葡萄糖磷酸化酶、苹果酸脱氢酶和烯醇化酶的表达在C/N为2.4时有明显提高。蛋白质差异表达的方法没能找到PHB合成相关的蛋白质。但是此结果显示PHB的积累除与糖代谢相关外还与胞内蛋白质代谢、脂质代谢、核酸代谢和转录翻译等因子具有重要关系。用Real-Time PCR方法对挑取的19个蛋白质点在RNA水平上进行检测,结果显示19个蛋白质的表达情况与对应基因的转录情况基本一致。
对隐藏嗜酸菌PHB代谢途径作了分析。首次以已报道全基因组序列的Acidiphilium cryptum JF-5的功能基因为参考,选择了PHB代谢相关的13个基因作为研究对象,用Real-Time PCR方法对不同C/N比情况下这些基因的转录情况进行了研究。结果显示β-羟基丁酸聚合酶和乙酰辅酶A合成酶在PHB代谢中起了非常重要的作用。菌株DX1-1积累PHB的起始底物是乙酸,并且找到了一条完整的PHB代谢途径。对13个基因的启动子序列进行了预测,模体模型为-35区：n-G-A-n-n-A-C-A和-10区：n-G-A-n-n-A-C-A,中间间隔平均为20 bp,该模体序列与E. coli并无明显的相似性,表明他们可能有较低的转录活性或者他们有不同的调节机制。
对微异养条件下DX1-1以硫为能源积累PHB的情况做了研究。DX1-1能以硫为能源缓慢生长,但加入少量葡萄糖后,生长周期明显缩短,细胞密度大大提高。结果显示,以硫为能源的培养基中,加入0.1%的葡萄糖,PHB的产量达到13.51g/L,与1%葡萄糖为能源时PHB产量相接近。用Real-Time PCR方法对硫、葡萄糖、硫+葡萄糖等六种培养条件下的5个PHB代谢相关关键基因,10个碳固定相关基因和7个硫代谢相关基因的表达情况进行分析,结果显示葡萄糖的加入可以诱导硫代谢相关基因和碳固定相关基因的表达；仅以单质硫能源不加葡萄糖时,PHB代谢相关关键基因和碳固定相关基因的表达均出现下调,但是硫代谢相关基因却有不同程度的上调趋势,这一结果与该菌在该状态下仅仅维持生命的生长状态相符合。
PHB is a kind of degradable microorganism material, besides the similar characteristics with chemosynthetic macromolecules, it also shows some other characteristics that are not obtained by ordinary chemosynthetic macromolecules, such as, good optical property, low oxygen permibility, anti-ultraviolet radiation, bio-degradability, bio-compatibility, piezoelectricity, and anti-cruor, etc.. It has thus a promising prospect in its application and has attracted more and more attention.
A new bacterial strain, designated as strain DX1-1, was isolated from an acid mine drainage of Dexing Copper Mine, Jiangxi Province, China. Basing on morphology, biochemical and physiological characterization, strain DX1-1 was identified as facultative autotrophic bacterium. It grew at temperature ranging from 20℃to 35℃and at initial pH values ranging from 2.0 to 5.0. It was affiliated to the genus Acidiphilium according to the phylogenetic tree derived from 16S rRNA sequence alignment. The capacity of producing polymer granules of strain DX1-1 was found by Transmission Electron Microscopy (TEM). The polymer granules was extracted and detected by ultraviolet-visible (UV) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy. The results show that it has the same character with poly-β-hydroxybutyrate (PHB). It suggests that strain DX1-1 might have the potential of producing biodegradable materials (PHB).
Four methods were employed to extract PHB from strain DX1-1. There is advantage and disadvantage for each method. Chloroform-sodium hypochlorite method is the best in extracting PHB form Acidiphilium cryptum DX1-1. The extraction rate reaches 73%, the purification rate is 97% and molecular weight is 326 kD. The PHB extracted by this method was then analyzed by UV spectroscopy, FT-IR spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. The results show that PHB from strain DX1-1 have the same biochemical structure and character with PHB standard. Mass spectrometer (MS) analysis reveals that the long chain of PHB is destroyed when treated by chloroform-sodium hypochlorite. The differential scanning calorimetry (DSC) of PHB shows PHB from Acidiphilium cryptum DX1-1 has low degree of crystallinity which makes the PHB have a wider range of applications.
The culture condition of strain DX1-1 for producing PHB was optimized. Glucose and KNO3 were found to be the best carbon and nitrogen substrates for the production of PHB. The L16 (43) orthogonal experiment improved the yield to 19.56g/L when 40g/L glucose as carbon source,15g/L KNO3 as nitrogen source and pH 3.0 were adopted. Then the cells growth curve and PHB production curve were measured under the optimum culture condition. It was found that the process of PHB accumulating was a growth-associated process. The strain DX1-1 was irradiated respectively by UV and Co60 to improve PHB production. The results indicated that the effect of UV better than using Co60. One strain of the UV mutagenized called UV60-3 has the highest PHB production yield, showing final PHB concentration of 28.56 g/L. Further research about the best culture condition of the mutant was done. The optimum nitrogen source was 30g/L (NH4)2SO4, the final PHB concentration reaches to 30.57g/L.
The time, yield and the expression of the PHB accumulation-related genes of Acidiphilium cryptum DX1-1 were investigated under four different initial C/N ratios 1.2,2.4,7.5, and 24. The results of time and yield of PHB accumulation show that the initial C/N ratio 2.4 was optimum for strain DX1-1 to accumulate PHB, both higher and lower initial C/N ratios did not favor that process. Total proteins and intracellular proteins were extracted from strain DX1-1 cultivated under different initial C/N ratios. The protein plots in 2-DE maps were abundant, dispersed and clear, suggesting that the 2-DE technique developed in this study could absolutely meet the requirement of further proteomics study. Eleven proteins pots in total proteins and 9 proteins pots in intracellular proteins which resolved differentially expression were chosen. The 2-DE and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF/TOF MS) were used to identify the proteins. No proteins which catalyze PHB metabolsim were found by this method. But the results show that PHB metabolism is related to metabolism of proteins, fatty acids, and nucleic acids and factor of transcription besides carbon metabolism. The mRNAs of the 19 genes encoding those proteins were detected by Real-Time PCR. The results show that the changes of expression for the 19 genes were consistent with the changes in their mRNAs.
Based on the genome of Acidiphilium cryptum JF-5, thirteen PHB accumulation related genes in strain JF-5 were chosen and successfully cloned from strain DX1-1. The differential expressions of the 13 functional genes, in different C/N ratios as cited above, were then studied by Real-time PCR. The results show that all the 13 genes were most upregulated when the initial C/N ratio was 2.4, and among which the gene Acry_3030 encoding poly-β-hydroxybutyrate polymerase and Acry_0626 encoding acetyl-CoA synthetase were much more upregulated than the other genes, which prove that they play the most important role for PHB accumulation and acetate is the main initial substance for PHB accumulation for strain DX1-1. Potential regulatory motifs analysis shows that the genes related to PHB accumulation are regulated by different promoters and that the motif has weak similarity to the model promoters, suggesting that PHB-metabolism in Acidiphilium cryptum may be mediated by a different mechanism.
Acidiphilium cryptum DX1-1 can grow slowly with sulfur as energy source. However, growth cycle shortened and cell density increased with glucose added. It was found that PHB reached 13.51g/l with 0.1% glucose added when sulfur as energy source. The expression of genes related to PHB metabolism, carbon fixation and sulfur metabolism in different culture media were detected by Real-time PCR. The results show that glucose could induce the expression of genes related to sulfur metabolism and carbon fixation. When sulfur was used as the only energy sources, the expression of the genes related to PHB metabolism and carbon fixation down-regulared while the genes related to sulfur metabolism up-regulated, which was according with the growth state of keeping the cells alive.
引文
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