The Construction Of Inonotus Obliquus (LNUF008) Squalene Synthase Gene Engineering Strain And The Preliminary Study On The Fermentation Production Of Triterpene

Inonotus obliquus (I.obliquus) is a precious medicinal fungus, and it has been used in folk medicine to prevent the occurrence of a number of diseases, including tuberculosis, gastrointestinal cancer and cardiovascular disease in East Europe as far back as the 16th Century. The main active components in I.obliquus are the triterpenes, and the syntheses of these compounds require the key enzyme called squalene synthase (SQS). Due to the importance of triterpenes, there is a high demand for these compounds, and extracting the compounds from I.obliquus does not meet the demand since I.obliquus is also a rare fungus. Therefore, one way to overcome the shortage in supply of triterpene would be to improve the biosynthetic capacity of tritepene through genetic modification of I.obliquus.In this study, the SQS cDNA of I.obliquus was amplified from the plasmid pGAPZaA-SQS (which had been previously constructed in our laboratory) by a pair of SQS-specific primers, each contained a specific restriction site at the 5’end. At the same time, another pair of primers were used to amplify pAN7-1-△hph. These primers, each contained at the 5’end, the same specific restriction site as that in the SOS-specific primer. The two DNA fragments were ligated to each other after cleavage by the corresponding restriction enzymes, yielding the construct pAN7-1-SQS. Protoplasts of I.obliquus, which were prepared by digesting the hyphal pellicle with lywallzyme, were cotransformed with the linearized pAN7-1-SQS and pAN7-1, and positive transformants were selected on the basis of resistance to hygromycin B. Cross-intron primers were designed and used to verify the insertion of the SQS cDNA in the chromosomal DNA of the positive transformants. Subsequent DNA sequencing of the amplified product confirmed the identity of the DNA as SQS gene. Compared to wild-type I.obliquus, the level of total tripetenes in the genetically modified I.obliquus strain increased by about 34%, but the mycelial dry weight remained almost unchanged. This study has provided a possible approach to improve the yield of triterpene in I.obliquus by genetic engineering and established the foundation for further improving the value of I.obliquus.