Chromohalobacter sp. HS-2 유래 수산화효소 유전자 내포 재조합 대장균을 이용한 hydroxybenzoate의 전환

Abstract

Chromohalobactersp. strain HS-2 was isolated from salted fermented clams and analyzed for the ability to grow on (hydroxy)benzoate as the sole carbon and energy source. HS-2 was characterized to be moderately halophilic, with an optimal NaCl concentration of 10%. The genes encoding the benzoate metabolism of HS-2 were cloned into a cosmid vector, sequenced, and then analyzed to reveal the benzoate dioxygenase (benABC), p-hydroxybenzoate hydroxylase gene (pobA) and m-hydroxybenzoate hydroxylase gene (mobA). The HS-2 genes involved in (hydroxy)benzoate degradation were clustered within approximately 39 kb region, and showed quite a different genetic organization from those of other benzoate catabolic genes. The benABC, pobA, mobA genes were cloned into the expression vector pEXP5-CT/TOPO. The expressed BenABC, PobA, MobA were analyzed by SDS-PAGE and the specific bands were confirmed. The HPLC and LC-MS/MS analysis revealed that resting cells of E. coli BL21 (DE3) harboring benABC, pobA, mobA oxidized 3-chlorobenzoate to 4-chlorocatechol, p-hydroxybenzoate to protocatechuate and m-hydroxybenzoate to protocatechuate, respectively. To enhance the conversion rate, pKJE7 chaperone (dnaK-dnaJ-grpE) were co-expressed with E. coli harboring pobA and mobA genes, respectively. The pobA and mobA genes expressed higher level of protein than without pKJE7. And bioconversion of high concentration of m, p-hydroxybenzoate to protocatechuate by co-expression of pobA with pKJE7.