Engineering Corynebacterium glutamicum for hydroxylation of fatty acids

Abstract

Cytochrome P450 oxygenases (CYPs) are heme-containg enzymes that are able to catalyze oxygenation of a variety of organic substances. Among them, the CYP102A subfamily, a group of self-sufficient P450 monooxygenases that constitute both FMN/FAD-containing reductase domain and a heme domain, is one of the well-characterized. In this study, a whole-cell biocatalyst based on CYP102A3 of Bacillus subtilis was constructed and its catalytic properties have been investigated. The CYP102A3 gene was functionally expressed in an amino acid producer Corynebacterium glutamicum ATCC13032 leading to a specific enzyme activity of ca. 0.15U/mg protein. However, hydroxylation rate of the recombinant whole-cell biocatalyst toward dodecanoic acid remained below 0.4μmol/g cell dry weight (CDW)/h. To improve the whole-cell catalytic activity, the recombinant C. glutamicum cells were subjected to ethambutol treatment, which inhibits arabinosyltransferases involved in the biosynthesis of cell wall arabinogalactan and mycolate layers and thereby results in an increase of substrate permeability of cell envelops. The ethambutol-mediated cell wall modification enabled ca. 3-fold increase of hydroxylation rate toward dodecanoic acid. This study showed potential of the C. glutamicum-based biocatalyst for hydroxylation of fatty acids.;시토크롬 P450 (CYPs)은 heme그룹을 포함하는 효소군으로 다양한 유기물질의 산화반응을 촉매한다. CYP102A계열은 자급자족형으로 reductase영역과 heme영역이 하나로 이루어져 있다. 본 연구는 Bacillus subtilis 유래의 CYP102A3 유전자를 보효소 재생효율이 우수한 아미노산 생성 균주인 Corynebacterium glutamicum ATCC13032에 발현하여 생촉매 효율을 조사하였다. 재조합 C. glutamicum에서 발현된 CYP102A3의 specific activity는 0.15U/mg protein 이었다. dodecanoic acid를 이용한 전세포 생촉매의 hydroxylation rate는 0.4μmol/g cell dry weight (CDW)/h였다. 전세포 촉매 활성을 높이기 위해 재조합 C. glutamicum에 ethambutol처리를 하여 arabinogalactan층을 저해하여 기질 투과성을 높이기로 하였다. 그 결과 ethambutol이 처리된 세포는 hydroxylation rate 가 처리하지 않은 세포에 비해 3배정도 높게 나오는 것을 볼 수 있었다. 종합적으로 재조합 C. glutamicum은 지방산의 P450 수산화반응에 가능성 있는 생촉매라 사료된다.