【作者】 葛少林；

【导师】 李寅；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 谷胱甘肽,γ-L-谷氨酰-L-半胱氨酰-甘氨酸(γ-L -glutamyl-L-cysteinyl-gly- cine,GSH),广泛存在于真核生物、革兰氏阴性菌和部分革兰氏阳性菌中,具有重要的生理功能和应用前景。微生物发酵法是目前谷胱甘肽生产的主要方法之一,它具有生产成本低,步骤简单、无需外源添加ATP等优点。发酵法所用菌株主要是经诱变育种得到各种酵母菌,包括酿酒酵母、产朊假丝酵母等。近几年,重组毕赤酵母开始应用于谷胱甘肽合成,由于该酵母遗传操作简便、极易在廉价培养基上实现高密度培养,因此已初步显示出其在谷胱甘肽生产上的应用价值与前景。用于谷胱甘肽生产的重组毕赤酵母菌株,是通过在毕赤酵母宿主中过表达催化谷胱甘肽合成酶的基因构建而成,而选择理想的(如催化效率高、无产物抑制等)谷胱甘肽合成酶进行过表达,也是提高毕赤酵母生产谷胱甘肽产率的先决条件。为得到相对理想的谷胱甘肽合成酶,最为简单实用的方法之一是在毕赤酵母中尝试表达各种微生物来源的谷胱甘肽合成酶基因,考察这些合成酶对于毕赤酵母生产谷胱甘肽的影响。之前的研究只报道过在毕赤酵母中表达来源于酿酒酵母模式菌株S288c的L-谷氨酰-L-半胱氨酸合成酶(γ-GCS)和谷胱甘肽合成酶(GS)基因。本论文考察了六种菌株来源的谷胱甘肽合成酶基因,研究了它们对毕赤酵母生产谷胱甘肽的影响。特别的,首次评估了革兰氏阳性菌来源的双功能谷胱甘肽合成酶基因(gshF)在毕赤酵母转化法生产谷胱甘肽中的应用潜力。具体研究内容包括:首先,考察了酿酒酵母模式菌S288c,安琪酵母工业菌株和产朊假丝酵母来源的γ-GCS基因gsh1的过表达对于谷胱甘肽产量的影响。通过将上述酵母的gsh1基因和酿酒酵母来源的GS的基因gsh2分别克隆并插入到毕赤酵母GS115基因组内进行表达。不加三种前体氨基酸摇瓶发酵结果发现它们的重组子合成的GSH分别是出发菌的2.2倍、4.3倍和4.4倍,加入三种前体氨基酸后,对照组及重组子合成GSH的稳定产量分别是116 mg/L、256 mg/L、426 mg/L和438 mg/L。结果表明来源于模式菌S288c和产朊假丝酵母的γ-GCS基因对谷胱甘肽合成的催化效率更高。其次,考察了来源于革兰氏阳性菌Listeria monocytogene、Lactobacillus plantarum和Streptococcus agalactia的双功能谷胱甘肽合成酶基因(gshF)对于毕赤酵母产谷胱甘肽的影响。将L. monocytogenes、L. plantarum和S. agalactiae来源的gshF分别克隆并插入到毕赤酵母GS115基因组内,得到三株重组菌株GS115/Lm-gshF GS115/Sa-gshF和GS115/ Lp-gshF。摇瓶发酵结果发现只有含L. monocytogene gshF基因的重组子GS115/Lmg-shF合成的GSH是出发菌的2.8倍,GS115/SagshF、GS115/ LpgshF两重组子合成的GSH与出发菌相比没有区别,在添加三种前体氨基酸的情况下,GS115/Lm-gshF合成GSH的最高产量达到336 mg/L,表明L. monocytogenes来源的gshF在毕赤酵母转化法生产谷胱甘肽上具有较好的应用潜力。更多还原

【Abstract】 Glutathione (γ-glutamyl-cysteinyl-glycine) (GSH) is the predominant low molecular weight peptide thiol present in living organisms. It plays a pivotal role in many metabolic processes and has a number of diverse applications. To date, GSH is mainly produced via fermentation. Yeasts, especially Saccharomyces cerevisiae and Candida utilis, are the most commonly used microorganisms in GSH production in an industrial scale. Most recently, recombinant Pichia pastoris has shown great potential in GSH production due to its easiness to achieve high density culture at low-cost media.The GSH producing recombinant P. pastoris strains are generated by over-expressing glutathione synthesis enzymes, and the GSH yield is mainly depende- nton the catalytic characteristics of these enzymes. In previous studies,γ-GCS and GS derived from S.cerevisiae S288c were chosen to be expressed in P. pastoris. However, S.cerevisiae S288c did not accumulate significant amount of intracellular GSH possibly due to feedback inhibition of GSH onγ-GCS. Therefore, we hypothesized that GSH yield of recombinant P. pastoris could be further improved by optimizing the catalysis characteristics of glutathione synthesis enzymes. To this end, in this work, glutatithione synthesis enzymes derived from a variety of strains was tested for their catalytic activity in P. pastoris, which includes following aspects.1) GSHI(encodingγ-GCS) derived from S.cerevisiae S288c, S.cerevisiae Angel and C. utilis, were co-expressed with S.cerevisiae GSHII in P. pastoris, respectively. The effects of different GSHI on GSH yield by recombinant P. pastoris were determined. Results showed that the GSH yields of the three strains are 2.2, 4.3 and 4.4 times the level of GS115. After feeding with three substrate amino acids, the GSH yields of control and recombinant strains reached 116 mg/L,256 mg/L, 426 mg/L and 438 mg/L, respectively.2) The gram positive bacteria bifunctional enzyme (encoded by gshF) was tested for their potential in GSH production in the context of recombinant P. pastoris. gshF genes derived from L. monocytogenes、L. plantarum and S. agalactiae were over-expressed in P. pastoris, respectively, thus resulting in the recombinant P. pastoris strains GS115/Lm-gshF, GS115/Sa-gshF and GS115/ LpgshF. Results showed that GS115/Lm-gshF produced more than twice as much GSH as GS115. No significant difference of GSH production was found between GS115/Sa-gshF, GS115/ Lp-gshF and GS115. SDS-PAGE results revealed that gshF from L. monocytogenes rather than L. plantarum and S. agalactiae was successfully expressed. After feeding with three substrate amino acids, the highest GSH production of GS115/ Lm-gshF reached 336 mg/L.更多还原