Cloning Of Squalene Synthase Gene YisP From The Bacillus Strain SWB16and Construction Of The Mutant Deleted In The Cyclase Gene Sqhc

Squalene is widely distributed in animals, plants and microorganisms, which has many functions of biology e.g. antioxidant, anti-radiation and participating in cholesterol metabolism and can also improve the utilization of oxygen and detoxicate multiple fat-soluble toxins in vito. Because of the characteristic of anticancer, anti-aging and anti-radiation, squalene has been exploited for various applications. Currently, squalene is mainly extracted from shark liver, although it is also existed in other animals and plants, the low content make them hardly further applied in our daily life. So microorganism fermentation for production of squalene is expected to be an effective measure to replace the traditional method. In the majority of prokaryote, hopanoids are pentacyclic triterpenes that are important for the intergration of cell memberane, and squalene is the intermediate product of hopanoids metabolic pathways. However, because squalene usually directly participates in cyclization reaction of the next step and can’t be accumulated in organisms, to aquire a great quantity of squalene for further application, the gene which controls the cyclization must be modified by using molecular techniques.The genus Bacillus is widely distribute in nature and is one of the most studied microorganisms. The yisP gene that widespread in Bacillus strains is supposed to synthesize squalene, so Bacillus strains are expected to be the source materials to produce squalene. The gene sqhC synthetize the cyclase enzyme that catalyse the squalene chain to form cyclic terpene, therefore, construction of the sqhC deletion mutant can block the process of cyclization and then accumulate the intermediate product—squalene. In this study, a plant endogeny Bacillus SWB16was identified by morphology, physiological and biochemical characteristics and molecular techniques, and then specific primers were designed to clone the yisP gene. To construct the engineered strains producing squalene, we cloned the downstream gene sqhC and constructed the corresponding deletion mutation, which can probably provide well foundation for the following fermentation experiments and successfully applied for the production of squalene in industry.The experimental results are as follows:1. According to analysis of morphology, physiological and biochemical characteristics, SWB16was identified as Bacillus for the first step. As the phylogenetic analysis of16Sr DNA can’t distinguish the Bacillus subtilis group, according to phylogenetic analysis of gyrA, SWB16clustered with Bacillus amyloliquefaciens, so it can be identified as Bacillus amyloliquefaciens.2. The yisP gene of SWB16was cloned, illustrating that squalene is involved in the metabolic pathway of the Bacillus amyloliquefaciens SWB16. Additionally, the phylogenetic analysis of yisP coordinated to the results of gyrA, indicating that this gene can not only differentiate Bacillus strains, but also discriminate subspecies of genus Bacillus. Co-analyzed the partial sequences of yisP and gyrA gene, strain SWB16was ultimately identified as B. amyloliquefaciens subsp. plantarum.3. Compared with the similarities of gyrA gene between B. subtilis and B. amyloliquefaciens, yisP gene is more variable. What’s more, the similarities of yisP gene are much lower than gyrA within B. subtilis and B. amyloliquefaciens. These results indicated that yisP gene is conserved intraspecies and possess higher dissimilarity interspecies.4. The construction result of sqhC deletion mutation showed that the targeting vector homologous recombinated with the genome of SWB16. The fragment length changing from1461bp to2800bp by using the specific primers of sqhC gene for B. amyloliquefaciens, and the sequncing results revealed that this framgment indeed insert the neo gene between the front and back homologous arms of sqhC gene, suggesting that the deletion mutation is successful constructed.