酿酒酵母耐热元器件的筛选

Screening of Heat- resistant Device in Saccharomyces cerevisiae

以提高酿酒酵母耐热性、降低乙醇发酵过程控温能耗成本为目的,通过分析嗜热栖热菌(Thermus thermophiles)HB8热激蛋白基因,设计并构建了5个热激蛋白元器件,并导入酿酒酵母。通过梯度升温培养筛选出性能较好的耐热元器件FBA1p-groes-SLM5t,并利用恒定高温培养进一步验证了含有该元器件的酿酒酵母工程菌S.c-Gro ES具有良好的耐热性,研究表明在42℃培养48h的存活率是对照的3倍。此外,FBA1p-groes-SLM5t还能提高酵母的抗氧化性,42℃下菌株S.c-Gro ES的ROS水平比对照低37.6%,H2O2处理1 h后存活率是对照的1.62倍,说明耐热元器件在缓解热胁迫的同时对细胞的抗氧化性也有帮助。耐热工程酿酒酵母S.c-Gro ES,其40℃发酵乙醇产量相对于30℃对照和40℃对照分别提高了25%和13.8%。嗜热菌热激蛋白的引入可以明显提高酿酒酵母的耐热性及其乙醇合成效率。

In order to improve the thermotolerance of Saccharomyces cerevisiae and decrease the energy consumption cost for controlling temperature in ethanol fermentation process, 5heat shock protein （HSP） devices aredesigned and constructed, then transformed into S. cerevisiae through mining heat shock protein genes in Thermus thermophiles HB8.All the HSP devices could transcript normally at 42℃. The cell growth of the engineered yeast with heat-resistant device FBA1p-groes-SLM5tis improved 29.2% than the control under the graduallyenhanced high temperatureincubation.And thecellgrowth of S. c-GroES cultured at graduallyenhanced high temperature is nearly identical to the controlincubated at 30℃. Therefore, the heat-resistant deviceFBA1p-groes-SLM5t which endows yeast with better thermotolerant property is screened. Then, the thermotolerance of S. c-GroES is further verified through constant high temperature incubation. The engineered strain S. c-GroES shows better cell growth than the control by measurement of OD660 and cell viability under 37℃ （heat shock temperature）and 42 ℃ （heat lethal temperature）.For instance, the cell viability of S. c-GroESdisplays3 times higher than the control at 42 ℃,48h. Moreover, the cell morphology of S. c-GroESis normal after heat shocked which indicates that the metabolism of S. c-GroESis not damaged. The above results of high temperature incubationshow that the engineered S. cerevisiaewith heat-resistant device FBA1p-groes-SLM5tcould adapt to various high temperature fermentation type. Meanwhile, the S. cerevisiae with heat-resistant device FBA1p-groes-SLM5tisendowed with anti-oxidation. The ROS level of S. c-GroES is 36.7% lower than the control at 42 ℃. Additionally, after treated with H2O2 of final concentration of 2mM,the cell viability of S. c-GroES shows1.62 times higher than the control.These results indicate that heat-resistant device could not only improve the thermotolerance of S. cerevisiae but also help cell defense oxidative stress. Under the 40 ℃ethanol fermentation,the cell growth of S. c-GroES and the control isworse than the above graduallyenhanced high temperature incubation and constant high temperature incubation owing to the anaerobic and heat shock cultural condition. However, the cell growth of S. c-GroES is better than the control cultured at 40℃ and nearly the same to the control cultured at 30℃. Under the batch fermentation, the ethanol yield of the control cultured at 30℃ is lower than the control cultured at 40℃, 60 h in the YPD medium containing 40g/L glucose, which is the same to the previous research, while its OD660 shows excellent than other strains. That is because nutrition is used by the control cultured at 30 ℃ to cell growth and ethanol can be the carbon resource when lack of glucose. However, the ethanol yield of S. c-GroESisimproved by 25% and 13.8% than the control cultured at 30 ℃ and 40℃, respectively. Meanwhile,the ethanol yield of single cell of S. c-GroESwas improved than the control trough calculating owing to the protection of heat shocked cells by heat-resistant device FBA1p-groes-SLM5t. These results show that the thermotolerance and ethanol synthesis efficiency of S. cerevisiae could greatly be improved byintroducing heat shock protein from thermophilus. This method provides a platform to increase production efficiency and reduce energy consumption substantially. Moreover, properties of other strains can be also improved by this valuable method.