Engineering of Outer Membrane Vesicles of Escherichia coli as Nanobioreactors for Fatty Acid Biotransformation and Lactam Degradation

Abstract

Bacterial outer membrane vesicles (OMVs) are small unilamellar proteoliposomes (30-200 nm), which are filled with periplasmic content. These molecules are involved in various functions from cell−cell signaling to protein excretion [1]. Thereby, this study has focused on engineering of OMVs of Escherichia coli as nanoscale bioreactors for biotransformation of fatty acids and degradation of lactam-based antibiotics. First, OMVs of E. coli were engineered to include fatty acid double bond hydratases (i.e., OhyA) of Stenotrophomonas maltophilia [2]. The OhyA-containing OMVs, which are spherical in size of 20 to 100 nm, were able to convert oleic acid (Z-octadec-9-enoic acid) into 10-hydroxyoctadecanoic acid. The biotransformation rate of an OMV was 7.3 x 10-6 pmol/min/OMV, which is 10-fold lower than a single E. coli cell, expressing the OhyAs inside cells. However, the biotransformation rate of OMVs per µm3 reached 0.13 pmol/min/µm3, which is 1000-fold greater than that of the E. coli cell. The high biotransformation rates of OMVs per µm3 suggested efficient sorting of the enzymes into the OMVs as well as efficient interaction with oleic acid, which exist as uni- and bilamellar liposomes of predominantly spherical shape in size of 20 to 100 nm. The OMVs of E. coli were also engineered to include a class C β-lactamase from virulent clinical strain of Enterobacter aerogenes for degradation of lactam-based antibiotics such as cephamycins (cefoxitin and cefotetan), penicillins, and cephalosporins [3]. The lactam hydrolysis activity of recombinant OMVs containing the β-lactamase with a chromogenic β-lactam substrate (i.e., nitrocefin (3-(2, 4 dinitrostyrl)-(6R,7R-7-(2-thienylacetamido)-ceph-3-em-4-carboxylic acid, E-isomer)) as a substrate was estimated to 4.13 x 10-5 pmol/min/OMV, which is ca.5-fold lower than that of a single E. coli cell. However, the lactam hydrolysis activity of OMVs per µm3 was estimated to reach 0.25 pmol/min/µm3, which is ca. 1500-fold higher than that of the E. coli cell. This result indicated that the recombinant OMVs containing the β-lactamase were quite active nanoscale bioreactors, which can be used for degradation of lactam-based antibiotics. In summary, this study demonstrated that OMVs of E. coli could be engineered to include a variety of enzymes (e.g., fatty acid double bond hydratases and class C β-lactamases), which are able to carry out a number of biotransformations. Thereby, it is assumed that the OMVs of E. coli can be used as nanoscale bioreactors. ;Bacterial outer membrane vesicles (OMVs)이란 periplasmic contents로 채워져 세포 외막에서 budding되는 unilamellar proteoliposomes (30-200 nm의)이다. 이 분자들은 세포와 세포 사이의 신호 전달, 단백질 secretion과 같은 다양한 기능에 관여한다 [1]. 본 연구에서는 Escherichia coli에서 생성된 OMV를 지방산의 생물 전환 및 락탐 기반 항생제의 분해를 위한 nanoscale bioreactor로 기능하도록 개량하였다. 이를 위해 먼저, OMV내에 Stenotrophomonas maltophilia유래의 지방산 이중결합 수화효소(i.e., OhyA)를 발현시켜주었다 [2]. OhyA가 포함된 OMVs를 활용하여 oleic acid (Z-octadec-9-enoic acid)를 10-hydroxyoctadecanoic acid으로 전환하는 biotransformation을 진행하였다. 그 결과 OMV의 개수당 활성은 7.3 x 10-6 pmol/min/OMV 로 E. coli 보다 10배 가량 낮았다. 하지만, 부피를 고려하였을 때 OMV의 활성은 0.13 pmol/min/µm3로 E. coli보다 1000배가량 높았다. OMV의 높은 biotransformation 속도는 OMV 내부로 효소가 효율적으로 들어갔을 뿐만 아니라 기질인 oleic acid가 20-100 nm로 구형 모양의 단층 및 쌍층 liposome으로 존재하여 OMV와 효율적으로 상호 작용하기 때문이라 설명할 수 있다. 또한, OMV 내에 Enterobacter aerogenes유래의 class C β-lactamase를 발현시켜 cephamycin (cefoxitin, cefotetan), penicillins, cephalosporins와 같은 lactam계 항생제의 분해에 대한 활성을 확인하였다 [3]. Lactam substrate를(i.e., nitrocefin (3-(2, 4 dinitrostyrl)-(6R,7R-7-(2-thienylacetamido)-ceph-3-em-4-carboxylic acid, E-isomer)) 이용하여 β-lactamase를 발현시킨 OMV의 활성을 측정한 결과 OMV의 갯수당 활성은 4.13 x 10-5 pmol/min/OMV로 E. coli보다 5배 낮았다. 하지만 OhyA의 경우와 마찬가지로 부피를 고려한 결과 OMV의 µm3당 Lactam 분해 활성은 0.25 pmol/min/µm3로 E. coli cell보다 1500배 정도 높은 값을 보였다. 이를 통해 β-lactamase가 포함되어 있는 재조합 OMV가 Nanoscale bioreactor로서 활성을 가지고 lactam 기반 항생제의 분해에 사용될 수 있음을 증명하였다. 이 연구는 E. coli에서 생성된 OMV내에 다양한 효소 (e.g., fatty acid double bond hydratases and class C β-lactamases)가 존재하도록 개량하여 많은 biotransformation을 진행할 수 있음을 입증했다. 따라서, E. coli의 OMV는 nanoscale bioreactor로 사용될 수 있다고 기대된다.