Optimization Of Fermentation Conditions Of Saccharomyces Cerevisiae And The Effect Of Ergosterol Content By Overexpression Of Erg1

Ergosterol,is a precursor substance for the synthesis of vitamin D₂that is widely used in the pharmaceutical,food,and feed industries.As the research of ergosterol in domestic is late,and the demand is large,which is often in short supply.At present,the main method for ergosterol production in domestic is to use microbial fermentation,the major strains used are usually yeast,large fungi and molds.Its advantage is fast reproduction,easy to cultivate,large yield,safety and speed,but the common problem is that the ergosterol content varies greatly between strains,and the strain yield has not been considerable.Therefore,improving the ergosterol content of microbial sources is still an urgent problem.In this study,the changes of ergosterol content in Saccharomyces cerevisiae were performed by microbial physiological and biochemical as well as molecular biological techniques.The main study results are as follows:（1）Twenty strains with morphological characteristics similar to yeast were isolated and purified from grape pericarp,and ergosterol was extracted and detected in each of the 20strains,and eventually a strain of yeast numbered SC19 with high ergosterol yield was obtained.SC19 was identified as a yeast species of Saccharomyces cerevisiae by PCR of ITS gene and sequencing,whose ergosterol yield was 4.51%.The optimum absorbance value of ergosterol was verified to be 281 nm by high performance liquid chromatography（HPLC）,and the UV method,which is a fast and simple detection method,was finally chosen for the ergosterol yield detection considering the cost and time consumption.（2）The content of ergosterol in SC19 was extracted and detected using the saponification extraction method,and the highest ergosterol content of 4.51%was reached in the yeast SC19when the fermentation reached 72 h.Four-factor,three-level orthogonal experiments were conducted for KOH concentration（A）,extraction solvent ether volume（B）,saponification temperature（C）and saponification time（D）in the saponification extraction method,respectively.Analysis of variance（ANOVA）was performed using SPASS software,and the optimal combination was A₂B₃C₂D₁,that is,KOH concentration of 50%,ether volume of 15m L,saponification temperature of 80℃,saponification time of 1 h,and ergosterol content reached 5.46%under the optimal saponification conditions mentioned above.（3）By studying the influence of different components in the medium on the growth of yeast SC19 and its ergosterol content,the following results were obtained:the highest ergosterol content was reached when fructose was used as the carbon source,and the ergosterol content reached 6.86%;when NH₄NO₃was used as the nitrogen source,the growth of yeast SC19 was better and the ergosterol content was as high as 5.52%;when the concentration of KH₂PO₄was 0.1%and 0.2%,the growth of yeast SC19 was the best,and the content of ergosterol was 4.05%,and 3.94%respectively;when Mg SO₄was chosen as mental ion,it not only promoted the growth of yeast SC19,but also the content of ergosterol was much higher than other metal ions,reaching to 5.66%.Based on the above results,fructose（A）,NH₄NO₃（B）,Mg SO₄（C）and KH₂PO₄（D）were selected as medium components.A four-factor,three-level experiment was designed using SPASS,and the optimal medium combination was A₁B₃C₃D₃,that is,0.5%for fructose,3%for NH₄NO₃,0.4%for Mg SO₄,and0.2%for KH₂PO₄.After optimization,the ergosterol content in yeast SC19 reached 6.71%.（4）Design of specific primers using Snapgene.The erg1 gene was successfully cloned from yeast SC19 by PCR technique.The cloning plasmid p MD-18T-erg1 and the expression plasmid p YES2-erg1 were successfully constructed,and finally transferred into INVSC1yeast with clearer genetic background,and the successful overexpression of erg1 gene was verified by Western blot,and the engineering strain YIS7 with erg1 overexpression was successfully constructed.（5）The growth and ergosterol content of yeast SC19,INVSC1 and engineered strain YIS7 were studied using the optimized saponification extraction method and medium composition,and the results were as follows:the ergosterol content in engineered strain YIS7was 4.81%which obviously higher than that in yeast INVSC1,an increase of 0.89%.This indicates that the overexpression of erg1 gene has a certain promotion effect on ergosterol content in yeast.