还原型谷胱甘肽高产菌的胁迫生理特性与高密度发酵过程优化技术
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摘要
还原型谷胱甘肽(y-glutamyl-L-cysteinylglycine, GSH)是一种非蛋白巯基化合物,广泛分布在各种生物活细胞内。GSH在细胞内参与了许多生理活动并起到非常重要的作用,最主要三种功能：(1)作为抗氧化剂,维持细胞内氧化还原状态；(2)在高等真核细胞中作为免疫增加剂；(3)作为解毒剂。这些特点使GSH广泛应用在疾病治疗上,比如肝硬化、糖尿病、神经退行性疾病和抗衰老等,因此GSH认为是生物体内最重要的一种能自我生产的抗疾病防卫小分子化合物。目前GSH作为一种药物广泛使用在医疗行业,如果产品价格能进一步降低,它在食品和化妆品行业也有很好的应用前景,因此GSH的市场需求在进一步扩大。但是,我国发酵法生产GSH仍停留在工业化试验阶段,尚未有企业成功实现发酵法生产GSH。为此,本论文针对国内目前GSH发酵生产过程中发酵水平和分离纯化技术所遇到的难题,基于多尺度参数相关分析,对GSH高产菌开展菌种生理特性和发酵过程优化研究,具体研究结果如下：
1.GSH高产菌菌种鉴定和基本胁迫生理特性研究
以GSH高产菌株的总DNA为模板,采用真菌通用引物,对其26S rDNA D1/D1区进行特异扩增,产物回收后测序,测序结果与NCBI的DNA数据库中已知菌种的26S rDNA序列进行比对,发现GSH高产菌株是一株酿酒酵母菌,命名为酿酒酵母T65。对此高产菌株和酿酒酵母W303A进行高浓度乙醇、葡萄糖、高温、高渗透压、营养饥饿、低pH、氧化胁迫和抗生素G418和Zecin毒性的耐受性分析,发现此高产菌株对测定的胁迫条件均表现出相对较好的耐受性。另外它能在酵母SD-URA培养基中生长,说明此高产菌株不是尿嘧啶缺陷型。GSH高产菌的这些生理数据为下一步研究高密度发酵生产GSH提供生理生化基础指导。
2.基于在线活细胞生物量的GSH高密度发酵比生长速率控制策略研究
将活细胞生物量传感器(型号Biomass monitor 220)成功地应用在复合培养基高密度补料发酵生产GSH过程中,建立了在线测量的电容值与细胞干重、OD600、活细胞计数和菌体离心体积计数法(Packed mycelia volume)这四种离线生物量检测方法的线性关系,确定了它们之间的线性方程和相关系数,其中电容值与活细胞数的相关系数达到0.995。相比较这四种离线方法,利用电容法来检测证明是一种在发酵过程中更方便准确的检测生物量的方法。因此,在复合培养基高密度补料分批发酵生产GSH过程中,Biomass monitor 220是一个非常有用的在线检测活细胞生物量的工具。GSH发酵过程比生长速率(μ)可通过电容值进行在线直接估算。对电容、OUR和CER这三种比生长速率估算方法进行比较,发现利用电容值来估算比通过OUR或CER这两种在线间接估算方法更为可靠实用。进一步将控制比生长速率反馈补料策略应用到GSH生产中,建立了GSH发酵过程在线控制比生长速率的有效优化方法。将比生长速率控制在0.2 h-’时,细胞密度和GSH发酵水平分别达到120 g/l和1.8 g/l,分别比控制比生长速率在0.15 h-1时提高了86.7%和200%。
3.利用呼吸商(RQ)反馈控制技术建立了高密度补料发酵生产GSH过程中同时提高GSH产量和胞内GSH含量的发酵策略。
采用RQ反馈控制葡萄糖补料速率,PID控制器将RQ控制在设定值。当RQ控制在0.65时使补料阶段的GSH和生物量生产效率最高,分别达到46.9 mg/l/h和3.5g/1/h,同时提高了GSH产量和胞内GSH含量。RQ控制使GSH最终产量达到2.1g/1、生物量126 g/l、胞内GSH含量1.67%和GSH生产率55.3 mg/l/h,分别比传统的乙醇反馈控制提高了75%、11.5%、57.5%和82.5%。并且RQ控制使副产物乙醇含量低于0.3 g/l。与传统的乙醇反馈控制相比,RQ控制能够降低副产物的形成,缩短发酵时间,并且有更高的GSH产量和胞内GSH含量等优势。通过代谢流分析表明,GSH合成途径通量显著增加是RQ控制高产的直接原因。此外还对补料成分进行优化,将补料培养基从只添加葡萄糖改为同时添加葡萄糖和酵母粉,使发酵水平得到进一步提高,GSH发酵产量达到2.4 g/l。因此,RQ反馈控制这种简单有效的控制方法有可能替代GSH生产中的传统反馈控制方法。
4.GSH高产菌高产机理初步探索—γ-谷氨酰半胱氨酸合成酶(GSH1)酶活和基因(gshl)序列比较分析
对GSH高产菌株和野生菌株γ-谷氨酰半胱氨酸合成酶(GSH1)酶活和基因(gshl)序列比较分析,初步探索GSH高产菌高产的可能机理。结果表明：(1)高产菌GSH1酶活是野生菌的4倍,这表明有可能是高产菌的GSH1发生突变引起酶活变化。(2)对高产菌株中的gshl基因进行克隆测序,发现DNA序列上有13碱基发生突变,并引起相应的氨基酸序列的三处点突变,并都发生在此酶的保守结构域上和活性催化区域。(3)对GSH1的三维空间结构同源建模分析表明,这三处突变引起三维空间结构的改变,预测的结构势能也更低,这些结构上变化可能造成突变后的GSH1的空间结构更稳定,从而使酶活显著提高,最终细胞内的GSH含量明显提高。
5..建立了在不破碎酵母细胞条件下乙醇原位高效提取GSH方法。
对影响在不破碎酵母细胞条件下乙醇原位提取GSH的三个因素乙醇浓度、提取温度和提取时间进行考察,发现只有乙醇浓度是影响GSH提取效率的显著因素。接着采用单因素实验对乙醇浓度这个条件进行进一步优化,确定25%乙醇浓度提取效率最高,达到0.31%。把这个方法与目前GSH工业上常用的提取方法进行了比较,发现两者提取效率相当,但用乙醇提取的杂蛋白浓度更少。总体而言,这个方法环境友好和条件温和,有可能成为目前GSH工业上常用提取方法的一种替代方法。
Reduced glutathione (y-glutamyl-L-cysteinylglycine, GSH), the most abundant non-protein thiol compound, is distributed widely in living cells. Although GSH is involved in many physiological processes and plays important roles, it serves three key functions: an antioxidant which plays key roles in maintaining the redox homeostasis of the cell, an enhancer of immunity, and a detoxifier in higher eukaryotic organisms. These characteristics make GSH important in the treatment of many diseases, such as liver cirrhosis, pulmonary diseases, gastrointestinal and pancreatic inflammations, diabetes, neurodegenerative diseases, and aging. Thus, GSH is considered to be one of the most important self-generated defense molecules. GSH is now widely used as a medicine. It also has great potential as food additives and in the cosmetic industries, only if the production cost can be further decreased. The commercial demand for GSH is expanding. However, there is no fermentative production of GSH on an industrial scale in China. Based on multi-scale analysis, cell physiology, and process optimization of a GSH high-yield strain were investigated in this work.
1. Identification and stress resistance of GSH high-yield strain
The 26S rDNA fragment of GSH high-yield strain was amplified by PCR with the template of its genomic DNA and fungal 26S rDNA universal primers. After blast the sequence of this 26S rDNA fragment in GSH high-yield strain using GenBank database of NCBI, it was identified as Saccharomyces cerevisiae. Moreover, stress resistance of GSH high-yield strain and Saccharomyces cerevisiae W303A to ethanol, glucose, nutrient starvation, temperature, osmotic pressure, oxidative pressure, and G418 and Zecin toxicity were studied by cell growth assay. The results showed that GSH high-yield strain is more resistant to the tested stress conditions than Saccharomyces cerevisiae W303A. Furthermore, GSH high-yield strain can grow on yeast SD-URA medium, suggesting that it is not a uracil autotrophic mutant. These results serve as useful reference data for further studies of GSH high-cell-density cultivation.
2. Specific growth rates control strategy based on on-line viable-cell mass monitoring for GSH high-cell-density cultivation
An on-line monitoring of viable-cell mass in high-cell-density fed-batch cultivations of GSH high-yield strain grown on an industrial complex medium was performed with an in situ capacitance probe fitted to a 50-1 fermentor. Conventional off-line biomass determinations of several parameters, including dry cell weight (DCW), optical density at 600 nm wavelength (OD600 nm), packed mycelial volume (PMV) and number of colony forming units (CFU), were performed throughout the bioprocess and then compared with on-line viable-cell concentrations measured using a capacitance probe. Capacitance versus viable biomass and all off-line biomass assay values were compared during GSH fermentation in industrial complex culture media. As a result, the relationship between the number of colony forming units and capacitance with a correlation coefficient (R) of 0.995 was achieved. Simultaneously, compared with those determined by at-line indirect estimation methods including oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER), the specific growth rates (μ) estimated by on-line capacitance measurement could be more reliable during GSH fermentation. An on-lineμfeedback control strategy based on capacitance measurement was developed for GSH high-cell-density fermentation.μcontrolled at 0.2 h"1 achieved yeast dry weight (120 g/1) and GSH yield (1.8 g/1), which improved by 86.7% and 200%, respectively, compared withμcontrolled at 0.15 h-1. Therefore, it is concluded that a capacitance probe is a practical tool for real-time viable biomass monitoring in GSH high-cell-density fed-batch cultivation in a complex medium.
3. RQ feedback control for simultaneous improvement of GSH yield and GSH content
GSH production by high-cell-density fed-batch cultivation of GSH high-yield strain, a respiratory quotient (RQ) feedback control strategy was applied to determine glucose feeding rate(F) based on on-line off-gas monitoring. Glucose feed was manipulated by a classical proportional-integral-derivative (PID) controller to control RQ at its set-point. RQ controlled at 0.65 resulted in the highest GSH productivity (46.9 mg/1/h) and cell productivity (3.5 g/1/h) as well as improved GSH yield and intracellular GSH content. RQ feedback control achieved yeast dry weight (126 g/1), GSH yield (2.1 g/1), GSH content (1.67%) and GSH productivity (55.3 mg/1/h), which improved by 11.5%,75%,57.5% and 82.5%, respectively, compared with conventional ethanol feedback control in the GSH industry. Moreover, RQ feedback control reduced ethanol (byproduct) level to below 0.3 g/1. Advantages of RQ feedback control over the conventional ethanol feedback control in the GSH industry include lower byproduct concentration, shorter cultivation period, higher GSH content, and higher GSH yield. Based on metabolic flux analysis (MFA), it indicated that the reason of GSH over-production by RQ control was due to increase of metabolic flux of GSH biosynthesis pathway. Furthermore, GSH yield achieved 2.4 g/1 when feeding medium was changed from glucose (600 g/1) to glucose (600 g/1) and yeast extract (15 g/1). Hence, feedback control based on RQ is a promising method that poses as a simple and efficient alternative to conventional feed control techniques presently practiced in the GSH industry.
4. A possible molecular mechanism for GSH high-yield synthesis
y-glutamylcysteine synthetase (GSH1) catalytic activity of GSH high-yield strain achieved 4.1 mmol Pi/mg protein/min, which was-4-fold compared to that of wild-type strain (control). It may indicate that GSH1 mutation of GSH high-yield strain resulted in a significantly increased activity. DNA sequencing of GSH1 in GSH high-yield strain revealed that three amino acids were changed among GSH1 conserved domain and its catalytic subunit. The 3D structure of GSH1 in GSH high-yield strain was generated by homology modeling using Swiss-Model based on the crystal structure of GSH1 of S. cerevisiae (PDB code:3ig5A). The result showed that the structure of GSH1 was changed compared with control. Moreover, the Potential energy of GSH1 was lower than that of control, indicating that GSH1 is more stable than control, and may increase reaction rate with substrate. Examination of this model suggested that the structure change may affect substrate binding with GSH1, and regulate the enzymatic activity.
5. Efficient extraction of GSH by ethanol
GSH from fermentation broth of GSH high-yield strain was extracted with ethanol without disruption of the cells. The effects of ethanol concentration, extraction temperature and extraction time were assessed by using 23 full factorial designs (FFD). Preliminary studies showed that ethanol concentration had the most influence on GSH yield by ethanol extraction, based on the first order regression coefficients derived using MINITAB software, and an optimal ethanol concentration (25%, v/v) was obtained. However, compared to the conventional extraction technique (hot water extraction), there was no significant advantage in yield of GSH from yeast cells using ethanol extraction under these optimized conditions. But ethanol extraction has several advantages, such as lower energy consumption and lower protein concentration of extraction broth, which may reduce the complexity and cost of the purification process. Hence, ethanol extraction which does not disrupt yeast cells could be an inexpensive, simple and efficient alternative to conventional extraction techniques in the GSH industry.
1.GSH高产菌菌种鉴定和基本胁迫生理特性研究
以GSH高产菌株的总DNA为模板,采用真菌通用引物,对其26S rDNA D1/D1区进行特异扩增,产物回收后测序,测序结果与NCBI的DNA数据库中已知菌种的26S rDNA序列进行比对,发现GSH高产菌株是一株酿酒酵母菌,命名为酿酒酵母T65。对此高产菌株和酿酒酵母W303A进行高浓度乙醇、葡萄糖、高温、高渗透压、营养饥饿、低pH、氧化胁迫和抗生素G418和Zecin毒性的耐受性分析,发现此高产菌株对测定的胁迫条件均表现出相对较好的耐受性。另外它能在酵母SD-URA培养基中生长,说明此高产菌株不是尿嘧啶缺陷型。GSH高产菌的这些生理数据为下一步研究高密度发酵生产GSH提供生理生化基础指导。
2.基于在线活细胞生物量的GSH高密度发酵比生长速率控制策略研究
将活细胞生物量传感器(型号Biomass monitor 220)成功地应用在复合培养基高密度补料发酵生产GSH过程中,建立了在线测量的电容值与细胞干重、OD600、活细胞计数和菌体离心体积计数法(Packed mycelia volume)这四种离线生物量检测方法的线性关系,确定了它们之间的线性方程和相关系数,其中电容值与活细胞数的相关系数达到0.995。相比较这四种离线方法,利用电容法来检测证明是一种在发酵过程中更方便准确的检测生物量的方法。因此,在复合培养基高密度补料分批发酵生产GSH过程中,Biomass monitor 220是一个非常有用的在线检测活细胞生物量的工具。GSH发酵过程比生长速率(μ)可通过电容值进行在线直接估算。对电容、OUR和CER这三种比生长速率估算方法进行比较,发现利用电容值来估算比通过OUR或CER这两种在线间接估算方法更为可靠实用。进一步将控制比生长速率反馈补料策略应用到GSH生产中,建立了GSH发酵过程在线控制比生长速率的有效优化方法。将比生长速率控制在0.2 h-’时,细胞密度和GSH发酵水平分别达到120 g/l和1.8 g/l,分别比控制比生长速率在0.15 h-1时提高了86.7%和200%。
3.利用呼吸商(RQ)反馈控制技术建立了高密度补料发酵生产GSH过程中同时提高GSH产量和胞内GSH含量的发酵策略。
采用RQ反馈控制葡萄糖补料速率,PID控制器将RQ控制在设定值。当RQ控制在0.65时使补料阶段的GSH和生物量生产效率最高,分别达到46.9 mg/l/h和3.5g/1/h,同时提高了GSH产量和胞内GSH含量。RQ控制使GSH最终产量达到2.1g/1、生物量126 g/l、胞内GSH含量1.67%和GSH生产率55.3 mg/l/h,分别比传统的乙醇反馈控制提高了75%、11.5%、57.5%和82.5%。并且RQ控制使副产物乙醇含量低于0.3 g/l。与传统的乙醇反馈控制相比,RQ控制能够降低副产物的形成,缩短发酵时间,并且有更高的GSH产量和胞内GSH含量等优势。通过代谢流分析表明,GSH合成途径通量显著增加是RQ控制高产的直接原因。此外还对补料成分进行优化,将补料培养基从只添加葡萄糖改为同时添加葡萄糖和酵母粉,使发酵水平得到进一步提高,GSH发酵产量达到2.4 g/l。因此,RQ反馈控制这种简单有效的控制方法有可能替代GSH生产中的传统反馈控制方法。
4.GSH高产菌高产机理初步探索—γ-谷氨酰半胱氨酸合成酶(GSH1)酶活和基因(gshl)序列比较分析
对GSH高产菌株和野生菌株γ-谷氨酰半胱氨酸合成酶(GSH1)酶活和基因(gshl)序列比较分析,初步探索GSH高产菌高产的可能机理。结果表明：(1)高产菌GSH1酶活是野生菌的4倍,这表明有可能是高产菌的GSH1发生突变引起酶活变化。(2)对高产菌株中的gshl基因进行克隆测序,发现DNA序列上有13碱基发生突变,并引起相应的氨基酸序列的三处点突变,并都发生在此酶的保守结构域上和活性催化区域。(3)对GSH1的三维空间结构同源建模分析表明,这三处突变引起三维空间结构的改变,预测的结构势能也更低,这些结构上变化可能造成突变后的GSH1的空间结构更稳定,从而使酶活显著提高,最终细胞内的GSH含量明显提高。
5..建立了在不破碎酵母细胞条件下乙醇原位高效提取GSH方法。
对影响在不破碎酵母细胞条件下乙醇原位提取GSH的三个因素乙醇浓度、提取温度和提取时间进行考察,发现只有乙醇浓度是影响GSH提取效率的显著因素。接着采用单因素实验对乙醇浓度这个条件进行进一步优化,确定25%乙醇浓度提取效率最高,达到0.31%。把这个方法与目前GSH工业上常用的提取方法进行了比较,发现两者提取效率相当,但用乙醇提取的杂蛋白浓度更少。总体而言,这个方法环境友好和条件温和,有可能成为目前GSH工业上常用提取方法的一种替代方法。
Reduced glutathione (y-glutamyl-L-cysteinylglycine, GSH), the most abundant non-protein thiol compound, is distributed widely in living cells. Although GSH is involved in many physiological processes and plays important roles, it serves three key functions: an antioxidant which plays key roles in maintaining the redox homeostasis of the cell, an enhancer of immunity, and a detoxifier in higher eukaryotic organisms. These characteristics make GSH important in the treatment of many diseases, such as liver cirrhosis, pulmonary diseases, gastrointestinal and pancreatic inflammations, diabetes, neurodegenerative diseases, and aging. Thus, GSH is considered to be one of the most important self-generated defense molecules. GSH is now widely used as a medicine. It also has great potential as food additives and in the cosmetic industries, only if the production cost can be further decreased. The commercial demand for GSH is expanding. However, there is no fermentative production of GSH on an industrial scale in China. Based on multi-scale analysis, cell physiology, and process optimization of a GSH high-yield strain were investigated in this work.
1. Identification and stress resistance of GSH high-yield strain
The 26S rDNA fragment of GSH high-yield strain was amplified by PCR with the template of its genomic DNA and fungal 26S rDNA universal primers. After blast the sequence of this 26S rDNA fragment in GSH high-yield strain using GenBank database of NCBI, it was identified as Saccharomyces cerevisiae. Moreover, stress resistance of GSH high-yield strain and Saccharomyces cerevisiae W303A to ethanol, glucose, nutrient starvation, temperature, osmotic pressure, oxidative pressure, and G418 and Zecin toxicity were studied by cell growth assay. The results showed that GSH high-yield strain is more resistant to the tested stress conditions than Saccharomyces cerevisiae W303A. Furthermore, GSH high-yield strain can grow on yeast SD-URA medium, suggesting that it is not a uracil autotrophic mutant. These results serve as useful reference data for further studies of GSH high-cell-density cultivation.
2. Specific growth rates control strategy based on on-line viable-cell mass monitoring for GSH high-cell-density cultivation
An on-line monitoring of viable-cell mass in high-cell-density fed-batch cultivations of GSH high-yield strain grown on an industrial complex medium was performed with an in situ capacitance probe fitted to a 50-1 fermentor. Conventional off-line biomass determinations of several parameters, including dry cell weight (DCW), optical density at 600 nm wavelength (OD600 nm), packed mycelial volume (PMV) and number of colony forming units (CFU), were performed throughout the bioprocess and then compared with on-line viable-cell concentrations measured using a capacitance probe. Capacitance versus viable biomass and all off-line biomass assay values were compared during GSH fermentation in industrial complex culture media. As a result, the relationship between the number of colony forming units and capacitance with a correlation coefficient (R) of 0.995 was achieved. Simultaneously, compared with those determined by at-line indirect estimation methods including oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER), the specific growth rates (μ) estimated by on-line capacitance measurement could be more reliable during GSH fermentation. An on-lineμfeedback control strategy based on capacitance measurement was developed for GSH high-cell-density fermentation.μcontrolled at 0.2 h"1 achieved yeast dry weight (120 g/1) and GSH yield (1.8 g/1), which improved by 86.7% and 200%, respectively, compared withμcontrolled at 0.15 h-1. Therefore, it is concluded that a capacitance probe is a practical tool for real-time viable biomass monitoring in GSH high-cell-density fed-batch cultivation in a complex medium.
3. RQ feedback control for simultaneous improvement of GSH yield and GSH content
GSH production by high-cell-density fed-batch cultivation of GSH high-yield strain, a respiratory quotient (RQ) feedback control strategy was applied to determine glucose feeding rate(F) based on on-line off-gas monitoring. Glucose feed was manipulated by a classical proportional-integral-derivative (PID) controller to control RQ at its set-point. RQ controlled at 0.65 resulted in the highest GSH productivity (46.9 mg/1/h) and cell productivity (3.5 g/1/h) as well as improved GSH yield and intracellular GSH content. RQ feedback control achieved yeast dry weight (126 g/1), GSH yield (2.1 g/1), GSH content (1.67%) and GSH productivity (55.3 mg/1/h), which improved by 11.5%,75%,57.5% and 82.5%, respectively, compared with conventional ethanol feedback control in the GSH industry. Moreover, RQ feedback control reduced ethanol (byproduct) level to below 0.3 g/1. Advantages of RQ feedback control over the conventional ethanol feedback control in the GSH industry include lower byproduct concentration, shorter cultivation period, higher GSH content, and higher GSH yield. Based on metabolic flux analysis (MFA), it indicated that the reason of GSH over-production by RQ control was due to increase of metabolic flux of GSH biosynthesis pathway. Furthermore, GSH yield achieved 2.4 g/1 when feeding medium was changed from glucose (600 g/1) to glucose (600 g/1) and yeast extract (15 g/1). Hence, feedback control based on RQ is a promising method that poses as a simple and efficient alternative to conventional feed control techniques presently practiced in the GSH industry.
4. A possible molecular mechanism for GSH high-yield synthesis
y-glutamylcysteine synthetase (GSH1) catalytic activity of GSH high-yield strain achieved 4.1 mmol Pi/mg protein/min, which was-4-fold compared to that of wild-type strain (control). It may indicate that GSH1 mutation of GSH high-yield strain resulted in a significantly increased activity. DNA sequencing of GSH1 in GSH high-yield strain revealed that three amino acids were changed among GSH1 conserved domain and its catalytic subunit. The 3D structure of GSH1 in GSH high-yield strain was generated by homology modeling using Swiss-Model based on the crystal structure of GSH1 of S. cerevisiae (PDB code:3ig5A). The result showed that the structure of GSH1 was changed compared with control. Moreover, the Potential energy of GSH1 was lower than that of control, indicating that GSH1 is more stable than control, and may increase reaction rate with substrate. Examination of this model suggested that the structure change may affect substrate binding with GSH1, and regulate the enzymatic activity.
5. Efficient extraction of GSH by ethanol
GSH from fermentation broth of GSH high-yield strain was extracted with ethanol without disruption of the cells. The effects of ethanol concentration, extraction temperature and extraction time were assessed by using 23 full factorial designs (FFD). Preliminary studies showed that ethanol concentration had the most influence on GSH yield by ethanol extraction, based on the first order regression coefficients derived using MINITAB software, and an optimal ethanol concentration (25%, v/v) was obtained. However, compared to the conventional extraction technique (hot water extraction), there was no significant advantage in yield of GSH from yeast cells using ethanol extraction under these optimized conditions. But ethanol extraction has several advantages, such as lower energy consumption and lower protein concentration of extraction broth, which may reduce the complexity and cost of the purification process. Hence, ethanol extraction which does not disrupt yeast cells could be an inexpensive, simple and efficient alternative to conventional extraction techniques in the GSH industry.
引文
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