Screening of Heat-resistant Device in Saccharomyces cerevisiae

Abstract

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-SLM5 tis improved 29. 2% than the control under the graduallyenhanced high temperatureincubation. And thecellgrowth of S. c-Gro ES cultured at graduallyenhanced high temperature is nearly identical to the controlincubated at 30℃. Therefore,the heat-resistant device FBA1pgroes-SLM5 t which endows yeast with better thermotolerant property is screened. Then,the thermotolerance of S.c-Gro ES is further verified through constant high temperature incubation. The engineered strain S. c-Gro ES 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-Gro ESdisplays3 times higher than the control at 42 ℃,48 h. Moreover,the cell morphology of S. c-Gro ESis normal after heat shocked which indicates that the metabolism of S. c-Gro ESis not damaged. The above results of high temperature incubationshow that the engineered S. cerevisiaewith heat-resistant device FBA1p-groes-SLM5 tcould adapt to various high temperature fermentation type. Meanwhile,the S. cerevisiae with heat-resistant device FBA1p-groesSLM5 tisendowed with anti-oxidation. The ROS level of S. c-Gro ES is 36. 7% lower than the control at 42 ℃.Additionally,after treated with H2O2 of final concentration of 2m M,the cell viability of S. c-Gro ES 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-Gro ES 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-Gro ES 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 40 g / 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-Gro ESisimproved by 25% and 13. 8% than the control cultured at 30 ℃ and 40℃,respectively. Meanwhile,the ethanol yield of single cell of S. c-Gro ESwas improved than the control trough calculating owing to the protection of heat shocked cells by heat-resistant device FBA1p-groes-SLM5 t. 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.