耐酸酵母的选育及初步应用
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摘要
耐酸酵母因其可以在低pH值的酸性条件下生长和发酵,备受人们的关注。本文研究核心是筛选具有耐酸性能的酵母菌株,初步探讨其在通风及厌氧条件下的发酵性能及应用,并以此耐酸酵母菌株为出发菌株,通过代谢工程改造使其能够生产L-乳酸,并对重组菌L-乳酸发酵的主要影响因素进行了初步研究,从而为将来的工业化生产做一些基础性工作。主要研究内容包括以下方面:
从自然界中96个样品中筛选得到2株可在含68 g/L乳酸的YEPD培养基中生长良好的耐酸酵母菌株。经形态、生理生化和分子生物学鉴定后,一株其为分类地位应属于假丝酵母属(Candida sp.),另一株为东方伊萨酵母(Issatchenkia orientalis)。以筛选出来的耐酸酵母Candida sp.和I. orientalis为研究对象,进行发酵性能初探。发现通风发酵时,I. orientalis在可应用于利用木薯发酵产酒精的废液来生产单细胞蛋白,其菌体干重可达到7.40 g/L,且其酒糟废液pH由初始的3.9升至6.5以上。在对耐酸酵母在兼性厌氧条件下发酵性能及应用进行研究中,发现I. orientalis菌株能够较好的降解山葡萄酒中的苹果酸,发酵结束时,其苹果酸最终含量低于1 g/L,苹果酸降解了约80%,且pH值有略微升高。以筛选出来的耐酸酵母菌株为研究对象,通过代谢工程改造,通过表达外源L-乳酸脱氢酶基因(ldhA),增加其乳酸代谢途径,试图将代谢流引向L-乳酸形成方向。以本实验是保存的pYX212质粒为基础载体,构建了含有米根霉As3.819的乳酸脱氢酶编码基因(ldhA)及G418抗性基因的重组质粒pYX212-kanMX-ldhA,然后电转化入野生型耐酸酵母菌株中,筛选获得了一株具有产L-乳酸能力的重组菌株C4-2。对重组菌进行乳酸发酵实验发现,改变其初始糖浓度、pH值、通风发酵时间以及接种量,均能提高其乳酸产量,可达到42.4 g/L。再对乳酸脱氢酶基因的串联表达进行了初步探索,构建了串联重组表达质粒pYX212-TPI-ldhA-TTA-TPI-ldhA-TTB-kanMX,并成功转入耐酸酵母Candida sp.中,得到串联重组耐酸酵母C4-E,发现C4-E的乳酸产量略低于C4-2,说明糖酸转化率不高的原因不在于ldhA的拷贝数。
综上所述,本研究通过实验验证耐酸酵母在通风及厌氧条件下的应用前景,并对研究利用耐酸酵母进行有机酸生产具有重要启示。
Acid and yeast had got much attantion because of its caracter that it can be grown and fermented under acidic conditions. The objective of this work was to screen acid-resistant yeast and reseach its application in the fermentation, and we used the mtabolic engineering for producing L-lactic acid by wild acid-resistant yeast, and discussed primary factor of L-lactic acid fermentation of recombinant. It was basic work as the L-lactic acid industrial production in the future. The mainly researching contents are blow.
Two wild acid-resistant yeast strains isolated from 96 natural samples, was found to be able to grow well at YEPD medium(containing 68 g/L the lactic acid). Based on further molecular biological tests, one strain was primarily identified as Candida.sp; the other one was primarily identified as Issatchenkia orientalis.The acid-resistant yeast strains were taken as the further research strains. In the result of aerobic fermentation research, it was found that, I. orientalis could be used for producing the single-cell protein in the cassava-alcohol wastewater. The cell dry weight of it can be up to 7.40 g/L, and the pH of the wastewater pH can rose to 6.5 from 3.9. On the study of facultative anaerobic fermentation, the malic acid-degradation could better be operated in the wild grape wine by I. orientalis. At the end of fermentation, the malic acid content can be decreased to 1 g/L, and the malic acid had been degradated about 80%, and pH values had slightly increased. In this study, acid-resistant yeast which was screened out as the research object, by metabolic engineering to expression of exogenous L-lactate dehydrogenase gene(ldhA), increased the lactic acid metabolic pathway, and the metabolic flux lead to the direction of L-lactic acid formation,and in order to achieve the purpose of producing lactic acid at low pH. After that, the gene ldhA, encoding a lactate dehydrogenase from Rhizopus oryzae As3.819, was cloned into a yeast shuttle vector containing G418 resistance gene. The resultant plasmid pYX212-kanMX-ldhA was introduced into acid-resistant yeast strains by electroporation transformation. Subsequently, a recombinant yeast C4-2 was obtained which could produce L-lactic acid.It was shown by the fermentation results that the initial sugar concentration,pH value of medium and the time of ventilation fermentation could be the factors of effect the concentration of lactic acid. The highest yield was up to 42.4 g/L. The tandem expression of lactate dehydrogenase gene was studied in this work. The resultant tandem restructuring expression plasmid pYX212-TPI-ldhA-TTA-TPI-ldhA- TTB-kanMX was successfully constructed and introduced into acid-resistant yeast strains by electroporation transformation, and subsequently a recombinant yeast C4-E was obtained. It was shown by the fermentation results lactic acid yield by C4-E was slightly lower than that of C4-2. It showed that the copies of ldhA gene were not the main reason for the yield of L-lactic acid being low.
In summary, this study verified the prospect of acid-resistant yeast by experiments in ventilation and anaerobic conditions, and it will be important inspiration to study the organic acid production of yeasts.
从自然界中96个样品中筛选得到2株可在含68 g/L乳酸的YEPD培养基中生长良好的耐酸酵母菌株。经形态、生理生化和分子生物学鉴定后,一株其为分类地位应属于假丝酵母属(Candida sp.),另一株为东方伊萨酵母(Issatchenkia orientalis)。以筛选出来的耐酸酵母Candida sp.和I. orientalis为研究对象,进行发酵性能初探。发现通风发酵时,I. orientalis在可应用于利用木薯发酵产酒精的废液来生产单细胞蛋白,其菌体干重可达到7.40 g/L,且其酒糟废液pH由初始的3.9升至6.5以上。在对耐酸酵母在兼性厌氧条件下发酵性能及应用进行研究中,发现I. orientalis菌株能够较好的降解山葡萄酒中的苹果酸,发酵结束时,其苹果酸最终含量低于1 g/L,苹果酸降解了约80%,且pH值有略微升高。以筛选出来的耐酸酵母菌株为研究对象,通过代谢工程改造,通过表达外源L-乳酸脱氢酶基因(ldhA),增加其乳酸代谢途径,试图将代谢流引向L-乳酸形成方向。以本实验是保存的pYX212质粒为基础载体,构建了含有米根霉As3.819的乳酸脱氢酶编码基因(ldhA)及G418抗性基因的重组质粒pYX212-kanMX-ldhA,然后电转化入野生型耐酸酵母菌株中,筛选获得了一株具有产L-乳酸能力的重组菌株C4-2。对重组菌进行乳酸发酵实验发现,改变其初始糖浓度、pH值、通风发酵时间以及接种量,均能提高其乳酸产量,可达到42.4 g/L。再对乳酸脱氢酶基因的串联表达进行了初步探索,构建了串联重组表达质粒pYX212-TPI-ldhA-TTA-TPI-ldhA-TTB-kanMX,并成功转入耐酸酵母Candida sp.中,得到串联重组耐酸酵母C4-E,发现C4-E的乳酸产量略低于C4-2,说明糖酸转化率不高的原因不在于ldhA的拷贝数。
综上所述,本研究通过实验验证耐酸酵母在通风及厌氧条件下的应用前景,并对研究利用耐酸酵母进行有机酸生产具有重要启示。
Acid and yeast had got much attantion because of its caracter that it can be grown and fermented under acidic conditions. The objective of this work was to screen acid-resistant yeast and reseach its application in the fermentation, and we used the mtabolic engineering for producing L-lactic acid by wild acid-resistant yeast, and discussed primary factor of L-lactic acid fermentation of recombinant. It was basic work as the L-lactic acid industrial production in the future. The mainly researching contents are blow.
Two wild acid-resistant yeast strains isolated from 96 natural samples, was found to be able to grow well at YEPD medium(containing 68 g/L the lactic acid). Based on further molecular biological tests, one strain was primarily identified as Candida.sp; the other one was primarily identified as Issatchenkia orientalis.The acid-resistant yeast strains were taken as the further research strains. In the result of aerobic fermentation research, it was found that, I. orientalis could be used for producing the single-cell protein in the cassava-alcohol wastewater. The cell dry weight of it can be up to 7.40 g/L, and the pH of the wastewater pH can rose to 6.5 from 3.9. On the study of facultative anaerobic fermentation, the malic acid-degradation could better be operated in the wild grape wine by I. orientalis. At the end of fermentation, the malic acid content can be decreased to 1 g/L, and the malic acid had been degradated about 80%, and pH values had slightly increased. In this study, acid-resistant yeast which was screened out as the research object, by metabolic engineering to expression of exogenous L-lactate dehydrogenase gene(ldhA), increased the lactic acid metabolic pathway, and the metabolic flux lead to the direction of L-lactic acid formation,and in order to achieve the purpose of producing lactic acid at low pH. After that, the gene ldhA, encoding a lactate dehydrogenase from Rhizopus oryzae As3.819, was cloned into a yeast shuttle vector containing G418 resistance gene. The resultant plasmid pYX212-kanMX-ldhA was introduced into acid-resistant yeast strains by electroporation transformation. Subsequently, a recombinant yeast C4-2 was obtained which could produce L-lactic acid.It was shown by the fermentation results that the initial sugar concentration,pH value of medium and the time of ventilation fermentation could be the factors of effect the concentration of lactic acid. The highest yield was up to 42.4 g/L. The tandem expression of lactate dehydrogenase gene was studied in this work. The resultant tandem restructuring expression plasmid pYX212-TPI-ldhA-TTA-TPI-ldhA- TTB-kanMX was successfully constructed and introduced into acid-resistant yeast strains by electroporation transformation, and subsequently a recombinant yeast C4-E was obtained. It was shown by the fermentation results lactic acid yield by C4-E was slightly lower than that of C4-2. It showed that the copies of ldhA gene were not the main reason for the yield of L-lactic acid being low.
In summary, this study verified the prospect of acid-resistant yeast by experiments in ventilation and anaerobic conditions, and it will be important inspiration to study the organic acid production of yeasts.
引文
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2.高志勇.单细胞蛋白研究概况[J].畜牧与兽医, 2009, 41(8): 111—112.
3.王丹,马玉龙,刘宁普,等.红霉素废液生产单细胞蛋白的发酵条件研究[J].环境科学与技术, 2009, 32(12): 48—51.
4.孙靓,孙菲菲,黄艳燕,等.木薯淀粉发酵生产L-乳酸的培养条件优化[J].中国酿造, 2009, (7): 33—37.
5.李勇.碳水化合物加工技术[M].北京,化学工业出版社, 2004.
6.李延云,朱杰,聂宇燕.利用木薯酒精糟液生产生物蛋白饲料[J].中国饲料, 1998, (4): 39—40.
7.方志.山葡萄与山葡萄酒[J].酿酒科技, 2003, 120(6); 93—94.
8.王建钧,李志平.山葡萄酒的酿造[J].酿酒科技, 2001, 3, 58—59.
9.蒋志东,南海龙,李华.干红山葡萄酒的降酸研究[J].酿酒科技, 2008, (12); 47—49.
10.甄会英,王颉,李长文,等.苹果酸-乳酸发酵在葡萄酒酿造中的应用[J].酿酒科技, 2005, (3): 75—79.
11.李华,梁新红,郭安鹊,等.葡萄酒苹果酸-乳酸菌精氨酸代谢研究概况[J].微生物学报, 2006, 46(4): 663—667.
12.李艾,苏旭东,程淑梅,等.粟酒裂殖酵母降苹果酸基因克隆及其序列分析[J].酿酒科技, 2007, (7): 128—131.
13.王博彦,金其荣.发酵有机酸生产与应用手册[M].北京:中国轻工业出版社, 2000, 340—343.
14.张刚.乳酸细菌-基础、技术和应用[M].北京:化学工业出版社, 2006. 269—308.
15.欧提库尔,穆斯塔怕,张志东,等. L-乳酸的研究进展及其应用前景[J].新疆化工, 2005, (3): 5—11.
16. Maas R H, Bakker R R, Jansen M L, et al. Lactic acid production from lime-treated wheat straw by Bacillus coagulans: neutralization of acid by fed-batch addition of alkaline substrate. Appl Microbiol Biotechnol, 2008, 78(5):751—758.
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