Screening Of Organic Tolerant Organisms And Homologous Expression Of Lipase Gene In Burkholderia Sp.SIB005

Organic solvent tolerant microorganism (OSTM) is a novel extremophile and it hasn't been systematically studied until 1980s. Relying on certain mechanisms, the OSTM is able to effectively defend and decrease the toxicity from organic solvents, which enable the OSTM to be potentially applied in the industrial fields such as whole-cell catalysis and environmental treatment, etc.In this study, Burkholderia sp.SIB005 was obtained through targeted screening with a capacity of producing lipase and organic solvent tolerance of benzene and some hydrocarbons. Due to the complicated lipase secretion mechanism of Burkholderia cepacia and the necessity for the involvement of homologous chaperone protein of lipase, therefore, we choose homologous ways to further enhance the lipase activity of Burkholderia sp.SIB005.To achieve the overexpression of lipase, lipase gene from Burkholderia sp.SIB005 was cloned, along with the upstream promoter region and chaperonin gene.Two constructs,pBBR22b-lipAB and pBBR22b-lipA, were established based on low copy plasmid pBBR22b. The piasmid pBBR22b-lipAB harbored lipase gene, its chaperonin gene and upstream promoter region of lipase; pBBR22b-lipA harbored lipase gene and upstream promoter region of lipase. These vectors were transformed into E.coli DH5a at the first place, and then they were transferred into strain SIB005 via electro-transformation. Under the same conditions, activities of lipase expressed from the two engineered systems were 88% and 25% more active relative to that from the wide type Burkholderia sp.SIB005. From this stidy, the chaperon of lipase is indispensable for overexpression of lipase gene. Moreover homologous expression of lipase in Burkholderia sp.SIB005 has been achieved through increasing the copy numbers of lipase gene and promoters, and the addition of chaperonin gene can further enhance the expression.