鹰嘴豆孢克鲁维酵母菊粉酶的分泌表达与应用研究

【作者】 俞静；

【导师】 吕红； 刘建平；

【作者基本信息】 复旦大学 ， 遗传学， 2010， 博士

【摘要】 通过转入菊粉酶基因表达质粒pUKD-S-aF-PinuT,成功地构建了分泌表达菊粉酶的重组乳酸克鲁维酵母(Kluyveromyces lactis),菊粉酶基因来源于鹰嘴豆孢克鲁维酵母(Kluyveromyces cicerisporus CBS4857, Y179)。研究发现Klhap1基因的突变能促进外源菊粉酶的分泌表达。发酵120h时,Klhap1△重组酵母发酵液酶活达到391 U/ml,是野生型重组酵母的2.2倍。荧光定量PCR数据显示Klhap1Δ重组酵母中的Kcinu mRNA水平明显比野生型重组酵母中的高。在非选择性培养基中生长50世代时,具有菊粉酶表达质粒的细胞在Klhap1Δ重组酵母和野生型重组酵母中比例分别达到99%、89%。以上结果说明,Klhapl基因的突变有利于菊粉酶在乳酸克鲁维酵母中更稳定、更高水平的表达。利用同源重组技术成功构建了hap1基因缺失突变的酿酒酵母(Saccharomyces cerevisiae)Y 16hap1Δ菌株,以lacZ为报告基因测定了不同启动子在Y16及Y16hap1△菌株中启动转录的效率,短型菊粉酶基因启动子PinuS与酿酒酵母翻译延伸因子基因启动子Ptef引导的β-半乳糖苷酶活性最高,长型菊粉酶基因启动子PinuL在Y16hap1△菌株中的转录效率比在Y16中的要低,这与乳酸克鲁维酵母中的结果相反,提示Klhap1基因与Schap1基因的功能有差异。上述研究使我们对鹰嘴豆孢克鲁维酵母菊粉酶基因在乳酸克鲁维酵母和酿酒酵母中外源表达的性质有更深入的了解,有助于酵母表达系统的开发与利用。提示了可以利用Klhap1Δ乳酸克鲁维酵母更高效地表达其他目的外源蛋白。构建了四种利用附加型载体表达菊粉酶的重组酿酒酵母,其中Y16/pHC-PinuLT分泌的菊粉酶酶活最高,发酵96h时达到最高(57U/m1),是Y16hap1Δ/pHC-PinuLT菌株产生的菊粉酶酶活的2倍。说明Schap1基因的突变降低了外源菊粉酶基因的表达。在菊芋培养液中发酵Y16/pHC-PinuLT时,添加一定量的尿素可以提高菊粉酶的产量。对工业酿酒酵母XN5菌株进行改造,在其基因组rDNA区整合菊粉酶基因表达单元,构建了重组酿酒酵母)CN5-αF-inu41,该菌株置于以菊粉为碳源的丰富培养基YEPI中发酵,72h时酶活达到最高值(29U/m1),)CN5-αF-inu 41工程菌产生的菊粉酶足够在48h内将95%的初始菊粉水解成还原糖,这为其发展成为高效利用菊芋原料发酵生产生物乙醇的酿酒酵母工程菌奠定了基础。利用第一部分研究中构建的分泌菊粉酶的重组酿酒酵母Y16/pHC-PInuLT菌株,敲除其基因组中的hxkl和hxk2,得到一株不能消耗果糖的酿酒酵母突变株Y16hxk1⊿/pHC-PInuLT在丰富培养基YEPD中发酵96h时,Schxk△重组酵母培养液中的菊粉酶酶活达到31U/ml,是野生型重组酵母的2.4倍。在以10%(w/v)菊粉为唯一碳源的培养基中发酵48h时,上清液果糖浓度达到9.0%(w/v),葡萄糖含量低于0.005%(w/v)。在以菊芋为唯一组分的菊芋培养基中发酵24h时,发酵液中果糖浓度达到9.2%(w/v),葡萄糖含量为0.10%(w/v)。结果说明以菊芋为培养基通过突变株Y16hxk1⊿/pHC-PInuLT的一步发酵,可产生高纯度的果糖。以菊芋为原料用天然高水平分泌菊粉酶的鹰嘴豆孢克鲁维Y179酵母发酵生产乙醇,从发酵工艺方面检测产乙醇过程并优化Y179的生产乙醇的发酵条件的各项参数。得到优化的发酵条件是：Y179在YEPD培养液中过夜培养36h后,以6%的接种量接入25%(w/v)菊芋液,30℃摇床培养8h后,静置培养。发酵40h左右,乙醇含量可以达到9-12%(v/v),糖醇转化率最高为75%左右。Y179在5L发酵罐中的发酵菊芋液生产乙醇过程如下,以约22%(w/v)糖含量的菊芋液为发酵液,30℃厌氧环境发酵,300rpm搅拌速度。发酵144h时,乙醇浓度达到12.34%(v/v),醪沼上清残糖0.35%(w/v),糖醇转化效率86.9%,糖利用率大于93.6%。用菊粉酶分泌表达酿酒酵母菌株Y16/pHC-PInuLT进行产乙醇研究。以约22%(w/v)糖含量的菊芋溶液为发酵液,30℃厌氧环境发酵,磁力搅拌器搅拌。发酵48h时,乙醇浓度达到10.5%(v/v),糖醇转化效率约85%。产乙醇的研究证明,利用鹰嘴豆孢克鲁维Y179酵母或菊粉酶分泌表达重组酿酒酵母Y16/pHC-PInuLT可一步糖化及发酵菊芋生产乙醇。表达菊粉酶的酿酒酵母与鹰嘴豆孢克鲁维酵母生产乙醇、果糖方法工艺简单,生产成本低,具有巨大的工业应用价值。更多还原

【Abstract】 Strains of Kluyveromyces lactis which secrete high level of inulinase were constructed by transforming of expression vector pUKD-S-αF-PinuT. The disruption of Klhapl increased heterologous expression of inulinase gene Kcinu derived from Kluyveromyces cicerisporus CBS4857. Inulinase activity in the supernatant of Klhap1Δstrain was 391 U/ml after cultured for 120 h, which was 2.2-fold that of the wildtype host. Comparing with the wildtype host, the relative KcINUl mRNA level of Klhap1Δstrain was increased significantly in the indicated culture time. After 50 generations of continuous cultured in non-selective medium, the percentage of expression plasmid-carrying cells were 99% and 89% for Klhap1Δand wildtype strains respectively. Based on these results, the disruption of Klhapl gene facilitated the high and stable expression of inulinase in K. lactis by certain mechanism.Saccharomyces cerevisiae strain Y16hap1Δwas obtained by homologous recombination of disruption cassette into strain Y16. Transcription level of different promoter in the Y16 and Y16 was determined by lacZ report gene.β-Galactosidase activities directed by PinuS and Ptef promoter were higher than other promoter tested. Transcription level of PinuL promoter in Y16hap1Δwas lower than that in the Y16 which is opposite to the result in K. lactis. This suggested that the function of Klhapl is different to that of Schapl in S. cerevisiae.The research gives us a better understanding about the property of heterologous inulinase expression in K. lactis and S. cerevisia and facilliates the development of yeast expression system. It indicated heterolgous target proteins could be more efficiently produced by using Klhap1ΔKluyveromyces lactic as host.Four recombinant S. cerevisiae of inulinase expression were constructed by introduction of episomal plasmid. Inulinase activities secreted by Y16/pHC-PinuLT were highest among these recombinant yeasts which reach 57U/ml after cultured for 96h. The inulinase activities were 2-fold that of Y16hap1Δ/pHC-PinuLT. The result show the disruption of ScHAPl gene impaired heterologous expression of inulinase. Inulinase activities secreted by Y16/pHC-PinuLT could be elevated by addition of urea to the raw Jerusalem artichoke medium. Recombinant diploid S. cerevisiae XN5-aF-inu 41 of inulinase secretion were constructed by integration of Kcinu into the rDNA locus in chromosome. Inulinase activity was 29 U/ml for XN5-aF-inu 41 after cultured in YEPI medium[1%(w/v) Yeast Extract,2%(w/v) Polypeptone, 10%(w/v) Inulin] for 72h. The inulinase produced by XN5-αF-inu 41 was sufficient to hydrolyze 95% of the initial inulin to reducing sugar in 48h. The results contribute to the development of ethanol production by inulinase secretion S. cerevisiae strains using Jerusalem artichoke as raw material. The genetic engineering stain of S. cerevisiae that is unable to consume fructose was then constructed by double disruption of the hexokinase gene hxkl and hxk2 in Y16/pHC-PInuLT strain constructed in the first part of the study. Inulinase activity in the culture supernatant of recombinant Schxk1Δhxk2Δstrain was 30.9 U ml-1 after cultured 96 h which was 2.4-fold that of Y16/pHC-PInuLT. After 48 h of fermentation in the medium initially containing 10%(w/v) inulin as sole carbon source by the recombinant Schxk1Δhxk2Δstrain, the fructose was accumulated to 9.0%(w/v) in the fermentation supernatant and the glucose content in the culture medium was less than 0.005%(w/v). When using wild Jerusalem artichoke as sole fermentation medium component without any supplementary, the fructose concentration of the fermentation supernatant reached 9.2%(w/v) and the glucose content was 0.10%(w/v) after 24 h incubation. The result suggested that fructose production of high purity can be achieved by one step fermentation of recombinant Schxk1Δhxk2Δyeast strain in the culture medium of Jerusalem artichoke as the sole component.Ethanol production from raw Jerusalem artichoke tubers by simultaneous saccharification and fermentation using yeast Y179 was studied. Kluyveromyces cicerisporus Strain Y179 could express high level of inulinase. The optimization of the fermentation process was achieved by research the impact of different fermentation parameter on ethanol production. Optimized fermentation conditions were as follow:Y179 was cultivated in YEPD medium for 36h.25%(w/v) wild artichoke medium was inoculated with 6%(v/v) inoculum of the 36h-precultured Y179 culture. After 8h of fermentation in rotary shaker, Y179 was then transferred to stationary cultivation at 30℃. Ethanol reached 9-11%(v/v) after 40h of fermentation. The highest conversion ration of carbohydrate to alcohol is about 75%. Jerusalem artichoke medium of 22%(w/v) total sugar content in a 5-L fermenter was inoculated with 10%(v/v) inoculum of 36 h-precultured Y179 cells. The batch fermentation was conducted at 30℃and with a stirring speed of 300 rpm under anaerobic condition. After 144 h of batch fermentation, ethanol concentration obtained from the fermentation broth was 12.34%(v/v) and the conversion efficiency of carbohydrate to ethanol was 86.9% of the theoretical ethanol yield. The cells utilized 93.6% of the total sugar during the fermentation.Ethanol production by inulinase-secretion S. cerevisiae Y16/pHC-PInuLT using crude Jerusalem artichoke medium of 22%(w/v) total sugar content was conducted with magnetic stirrer in anaerobic condition at 30℃. inoculated with 10%(v/v) inoculum of 24 h-precultured Y179 cells. After 48h of batch fermentation, ethanol concentration was 10.5%(v/v) and the conversion efficiency of carbohydrate to ethanol was 85% of the theoretical ethanol yield.The results of ethanol fermentation showed that K. cicerisporus Y179 and recombinant inulinase-secretion S. cerevisiae Y16/pHC-PInuLT are promising candidates for industrial application of fuel ethanol production in simultaneous saccharification and fermentation using crude Jerusalem artichoke.Ethanol and fructose production by S. cerevisiae and K. cicerisporus with inuliase activities is promising method which is simple and low cost for industrial application.更多还原