Improving acid tolerance of Escherichia coli BL21 and its application for production of ω-hydroxyundec-9-enoic acid

Abstract

ω-Hydroxy carboxylic acids are found as signaling molecules and secondary metabolites in plants. The carboxylic acids are also widely used as starting materials for chemical synthesis. However, the compounds remained difficult to produce with high productivities. In this study, Escherichia coli-based biocatalyst was engineered to improve acid tolerance and used for biosynthesis of ω-hydroxyundec-9-enoic acid from ricinoleic acid and castor oil. It was found that glutamic acid dependent acid resistance (GDAR) system, which is known as the most effective system to acid tolerance, was not active in E. coli BL21(DE3). Therefore, DsrA and RcsB, important factors for activation of GDAR system, were introduced into E. coli BL21(DE3) to enhance the acid stress resistance. The recombinant E. coli containing DsrA and RcsB showed higher survival rate and specific growth rate than the E. coli wildtype under n-heptanoic acid stress as well as HCl stress. Furthermore, ω-hydroxyundec-9-enoic acid was produced with higher productivity in the biotransformation of ricinoleic acid and castor oil by the recombinant E. coli with DsrA and RcsB expressing an alcohol dehydrogenase and a Baeyer-Villiger monooxygenase than the recombinant E. coli control strain. This study may contribute to increasing productivity of ω-hydroxy carboxylic acid from plant oils and fatty acids as well as to improving acid tolerance of E. coli BL21(DE3). Keywords : n-heptanoic acid, Acid tolerance, Glutamic acid decarboxylase, High productivity, ω-hydroxyundec-9-enoic acid;ω-Hydroxy carboxylic acids는 식물 내에서 signaling molecules과 secondary metabolites로 사용된다. 또한 carboxylic acid는 화학적 합성을 위한 시작 물질로 사용되기도 한다. 그러나 이러한 물질들을 고수율로 얻어내기는 쉽지 않다. 그러므로 본 연구에서는 산에 대한 내성을 향상시키고, ricinoleic acid와 castor oil로부터 ω-hydroxyundec-9-enoic acid를 합성하기 위하여 Escherichia coli 기반의 생촉매를 개발하였다. Acid tolerance에 가장 효과적이라고 알려진 glutamic acid dependent acid resistance (GDAR) 시스템은 E. coli BL21(DE3)에서 작동하지 않는다는 것을 확인하였다. 그러므로 산에 대한 저항성을 향상시키기 위하여 GDAR 시스템의 활성에 중요한 인자인 DsrA와 RcsB를 E. coli BL21(DE3)에 도입하였다. DsrA와 RcsB가 도입된 유전자 재조합 대장균은 wild type 대장균에 비하여 HCl 뿐만 아니라 n-heptanoic acid stress 하에서 높은 생존율과 비성장속도를 보였다. 더불어, DsrA, RcsB가 도입된 균주는 alcohol dehydrogenase와 Baeyer-Villiger monooxygenase를 이용하여 ricinoleic acid와 castor oil로부터 ω-hydroxyundec-9-enoic acid를 높은 수율로 생산하였다. 본 연구는 E. coli BL21(DE3)의 내산성을 향상시킬 뿐만 아니라 식물성 오일 및 지방산으로부터의 ω-hydroxy carboxylic acid의 생산성을 향상시키는 것에 기여할 것이라 사료된다. 주요어 : n-heptanoic acid, 내산성, Glutamic acid decarboxylase, 고수율 생산, ω-hydroxyundec-9-enoic acid