微生物高效生产γ-聚谷氨酸和β-聚苹果酸的研究
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摘要
γ-聚谷氨酸(Poly-γ-glutamatic acid, β-PGA)和β-聚苹果酸[poly(β-L-malic acid),PMLA]是两种受到人们关注的生物可降解水溶性聚合物材料,由于其优良的特性在医药和其他广泛领域有极大应用潜力。本文对于两种生物聚合物的微生物合成进行了系统研究。
首先,开展了对微生物合成γ-PGA的研究。以直接利用葡萄糖合成γ-PGA为目的,筛选获得一株谷氨酸非依赖型γ-PGA生产菌株,经过VITEK系统鉴定和16srDNA序列分析鉴定,命名为Bacillus subtilis ClO。对C10的发酵生产γ-PGA的培养条件进行了优化,发现柠檬酸能够作为C10菌株合成γ-PGA前体；在此基础上,以柠檬酸为底物在发酵罐上进行了间歇发酵和流加发酵培养,通过在发酵中后期流加柠檬酸和氯化铵溶液,γ-PGA产量达到38.2g/L。为了寻找有效提高γ-PGA产量的因素,研究了多种有机酸对于γ-PGA合成的影响,发现草酸能够在低浓度范围内显著促进菌体生长和γ-PGA合成。对关键节点酶活性的影响与代谢途径进行分析,结果表明草酸不进入γ-PGA的合成途径,而柠檬酸可以直接通过TCA循环进入γ-PGA的合成途径,间接作为谷氨酸合成的底物；草酸使丙酮酸脱氢酶活性增加是影响从葡萄糖从头合成谷氨酸的重要因素。对添加柠檬酸和草酸条件下的B. subtilis C10合成γ-PGA的代谢流进行分析,柠檬酸条件下,进入EMP途径的流量减弱,代谢流向HMP途径迁移；而在α-酮戊二酸节点碳流大部分流向γ-PGA合成途径；草酸条件下,α-酮戊二酸节点碳流大部分流向TCA循环,要提高γ-PGA的产量,必须弱化α-酮戊二酸脱氢酶的活性。
其次,开展了出芽短梗霉合成β-聚苹果酸的研究。分离了一株高产PMLA的生产菌株,对筛选到的菌株进行ITS序列分析鉴定,确定属于A ureobasidium Pullulans,命名为ZD-3d.对产物进行FT-IR和13C-NMR鉴定,确定为PMLA; PMLA酸水解产物为苹果酸、马来酸和富马酸；对PMLA的酸水解过程进行了研究,建立了PMLA水解释放苹果酸的动力学模型。接着对发酵培养条件进行了优化,发现富马酸能够促进菌体合成PMLA,5g/L富马酸能够使PMLA产量达到62.27g/L；碳酸钙能够调节菌体合成胞外多糖(exopolysaccharide, EPS)和PMLA的碳流方向,添加碳酸钙强烈刺激菌体合成PMLA,而抑制普鲁兰多糖的产生。对出芽短梗霉ZD-3d在1OL发酵罐上进行放大培养,PMLA最终浓度57.2g/L. PMLA的最大产率为0.35g/L/h,转化率为0.47g/g葡萄糖。
本论文对于γ-聚谷氨酸和β-聚苹果酸在相应菌株中的生物合成与代谢机理提出了一些新的研究视角,也为未来工业化生产这两种重要的生物聚合物提供一些新的策略。
As two novel biodegradable biomaterials, Poly-γ-glutamatic acid (γ-PGA) and poly (β-L-malic acid)(PMLA) have great potential applications in medicine and a wide range of other areas because of their excellent features. Microbial syntheses of the two biopolymers have been studied systematically in the present work.
Firstly, a glutamic acid-independent strain was screened out from sauce samples to biosynthesize γ-PGA without extrogeneous glutamic acid addition. According to the VITEK system identification and16srDNA sequence analysis, this new isolated strain was nominated as Bacillus subtilis C10. The metabolic characteristics and culture conditions of C10were investigated for enhanced γ-PGA production. The results showed that extrogeneous citric acid could be uasd as precuisor for γ-PGA synthesis, and one fed-batch strategy was examined to further improve the productivity. The mixture solution containing citric acid and ammonium chloride was fed in the later culture stage of fermentation, which resulted in high γ-PGA(38.2g/L) production. The factors influencing the endogenous glutamic acid supply and the biosynthesis of γ-PGA were also investigated in this strain, especially the effects of five different organic acids as possible precursors on the production of γ-PGA. To understand the possible mechanism for the improved γ-PGA biosynthesis by the tested organic acids, the activities of four key intracellular enzymes were measured. The result indicated that the increased bio activity of pyruvate dehydrogenase by oxalic acid was important for de novo sythesis of glutamic acid from glucose. According to metabolic pathway analyses, citric acid directly entered into the biosynthetic pathway of γ-PGA through the TCA cycle as indirect substrate of glutamate synthetase, while oxalic acid did not enter the biosynthetic pathway of γ-PGA. Metabolic flux analyses of γ-PGA synthesis in strain C10were carried out to explain the different effects caused by the addition of these organic acids. The feeding of citric acid reduced the flux entering the EMP pathway and increased the flux to γ-PGA synthesis pathway in α-ketoglutaric acid node. With the addition of oxalic acid, more carbon flux entered the TCA cycle in this strain. In order to improve the production of γ-PGA, the α-ketoglutaric acid dehydrogenase activity should be weakened in this glutamic acid-independent γ-PGA producer.
Secondly, a new strain with the distinguished high productivity of PMLA was isolated from fresh plant samples. According to morphological characteristics and phylogenetic analyses of the internal transcribed spacer sequences, one PMLA-producing strain (ZD-3d) was characterized as the candidate of Aureobasidium pullulans. As expected, PMLA can be hydrolyszed into malic acid, maleic acid and fumaric acid, and the corresponding kinetics of PMLA acid hydrolysis was modeled to simulate the whole degradation process. Further culture condition optimization brought about the highest PMLA concentration (62.27g/L) in the shake flask scale. In addition, the contribution of the carbon flux to exopolysaccharide (EPS) and PMLA could be regulated by the addition of CaCO3in the medium. This high-level fermentation process was further scaled up in the10L bench-top fermentor with a high PMLA concentration (57.2g/L), productivity (0.35g/L/h) and yield (0.47g/g glucose).
The present work made some deep insights into the biosynthesis and regulation of γ-PGA and PMLA in their corresponding strains and promises some novel strategies for industrial production of these two important biopolymers in the future.
首先,开展了对微生物合成γ-PGA的研究。以直接利用葡萄糖合成γ-PGA为目的,筛选获得一株谷氨酸非依赖型γ-PGA生产菌株,经过VITEK系统鉴定和16srDNA序列分析鉴定,命名为Bacillus subtilis ClO。对C10的发酵生产γ-PGA的培养条件进行了优化,发现柠檬酸能够作为C10菌株合成γ-PGA前体；在此基础上,以柠檬酸为底物在发酵罐上进行了间歇发酵和流加发酵培养,通过在发酵中后期流加柠檬酸和氯化铵溶液,γ-PGA产量达到38.2g/L。为了寻找有效提高γ-PGA产量的因素,研究了多种有机酸对于γ-PGA合成的影响,发现草酸能够在低浓度范围内显著促进菌体生长和γ-PGA合成。对关键节点酶活性的影响与代谢途径进行分析,结果表明草酸不进入γ-PGA的合成途径,而柠檬酸可以直接通过TCA循环进入γ-PGA的合成途径,间接作为谷氨酸合成的底物；草酸使丙酮酸脱氢酶活性增加是影响从葡萄糖从头合成谷氨酸的重要因素。对添加柠檬酸和草酸条件下的B. subtilis C10合成γ-PGA的代谢流进行分析,柠檬酸条件下,进入EMP途径的流量减弱,代谢流向HMP途径迁移；而在α-酮戊二酸节点碳流大部分流向γ-PGA合成途径；草酸条件下,α-酮戊二酸节点碳流大部分流向TCA循环,要提高γ-PGA的产量,必须弱化α-酮戊二酸脱氢酶的活性。
其次,开展了出芽短梗霉合成β-聚苹果酸的研究。分离了一株高产PMLA的生产菌株,对筛选到的菌株进行ITS序列分析鉴定,确定属于A ureobasidium Pullulans,命名为ZD-3d.对产物进行FT-IR和13C-NMR鉴定,确定为PMLA; PMLA酸水解产物为苹果酸、马来酸和富马酸；对PMLA的酸水解过程进行了研究,建立了PMLA水解释放苹果酸的动力学模型。接着对发酵培养条件进行了优化,发现富马酸能够促进菌体合成PMLA,5g/L富马酸能够使PMLA产量达到62.27g/L；碳酸钙能够调节菌体合成胞外多糖(exopolysaccharide, EPS)和PMLA的碳流方向,添加碳酸钙强烈刺激菌体合成PMLA,而抑制普鲁兰多糖的产生。对出芽短梗霉ZD-3d在1OL发酵罐上进行放大培养,PMLA最终浓度57.2g/L. PMLA的最大产率为0.35g/L/h,转化率为0.47g/g葡萄糖。
本论文对于γ-聚谷氨酸和β-聚苹果酸在相应菌株中的生物合成与代谢机理提出了一些新的研究视角,也为未来工业化生产这两种重要的生物聚合物提供一些新的策略。
As two novel biodegradable biomaterials, Poly-γ-glutamatic acid (γ-PGA) and poly (β-L-malic acid)(PMLA) have great potential applications in medicine and a wide range of other areas because of their excellent features. Microbial syntheses of the two biopolymers have been studied systematically in the present work.
Firstly, a glutamic acid-independent strain was screened out from sauce samples to biosynthesize γ-PGA without extrogeneous glutamic acid addition. According to the VITEK system identification and16srDNA sequence analysis, this new isolated strain was nominated as Bacillus subtilis C10. The metabolic characteristics and culture conditions of C10were investigated for enhanced γ-PGA production. The results showed that extrogeneous citric acid could be uasd as precuisor for γ-PGA synthesis, and one fed-batch strategy was examined to further improve the productivity. The mixture solution containing citric acid and ammonium chloride was fed in the later culture stage of fermentation, which resulted in high γ-PGA(38.2g/L) production. The factors influencing the endogenous glutamic acid supply and the biosynthesis of γ-PGA were also investigated in this strain, especially the effects of five different organic acids as possible precursors on the production of γ-PGA. To understand the possible mechanism for the improved γ-PGA biosynthesis by the tested organic acids, the activities of four key intracellular enzymes were measured. The result indicated that the increased bio activity of pyruvate dehydrogenase by oxalic acid was important for de novo sythesis of glutamic acid from glucose. According to metabolic pathway analyses, citric acid directly entered into the biosynthetic pathway of γ-PGA through the TCA cycle as indirect substrate of glutamate synthetase, while oxalic acid did not enter the biosynthetic pathway of γ-PGA. Metabolic flux analyses of γ-PGA synthesis in strain C10were carried out to explain the different effects caused by the addition of these organic acids. The feeding of citric acid reduced the flux entering the EMP pathway and increased the flux to γ-PGA synthesis pathway in α-ketoglutaric acid node. With the addition of oxalic acid, more carbon flux entered the TCA cycle in this strain. In order to improve the production of γ-PGA, the α-ketoglutaric acid dehydrogenase activity should be weakened in this glutamic acid-independent γ-PGA producer.
Secondly, a new strain with the distinguished high productivity of PMLA was isolated from fresh plant samples. According to morphological characteristics and phylogenetic analyses of the internal transcribed spacer sequences, one PMLA-producing strain (ZD-3d) was characterized as the candidate of Aureobasidium pullulans. As expected, PMLA can be hydrolyszed into malic acid, maleic acid and fumaric acid, and the corresponding kinetics of PMLA acid hydrolysis was modeled to simulate the whole degradation process. Further culture condition optimization brought about the highest PMLA concentration (62.27g/L) in the shake flask scale. In addition, the contribution of the carbon flux to exopolysaccharide (EPS) and PMLA could be regulated by the addition of CaCO3in the medium. This high-level fermentation process was further scaled up in the10L bench-top fermentor with a high PMLA concentration (57.2g/L), productivity (0.35g/L/h) and yield (0.47g/g glucose).
The present work made some deep insights into the biosynthesis and regulation of γ-PGA and PMLA in their corresponding strains and promises some novel strategies for industrial production of these two important biopolymers in the future.
引文
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