Production of 10R-hydroxy fatty acids from linoleic acid, α-linolenic acid, hempseed oil hydrolyzate by recombinant cells expressing PpoC from Aspergillus nidulans

다국어 초록 (Multilingual Abstract)

The first and second preferred substrates of recombinant Escherichia coli cells expressing 10R-dioxygenase (PpoC) from Aspergillus nidulans and the purified enzyme were linoleic acid and α-linolenic acid, respectively. PpoC in cells showed higher thermal and reaction stabilities compared to purified PpoC. Thus, 10R-hydroxy unsaturated fatty acids were produced from linoleic acid, α-linolenic acid, and hempseed oil hydrolyzate containing linoleic acid and α-linolenic acid as substrates by whole recombinant cells expressing PpoC. The optimal reaction conditions for the production of 10R-hydroxy-8E,12Z-octadecadienoic acid (10R-HODE) were pH 8.0, 30 °C, 250 rpm, 5% (v/v) dimethyl sulfoxide, 5 g/L linoleic acid, 60 g/L cells in 100-ml baffled flask. Under these conditions, whole recombinant cells expressing PpoC produced 2.7 g/L 10R-HODE from 5 g/L linoleic acid for 40 min, with a conversion yield of 54% (w/w) and a productivity of 4.0 g/L/h; produced 2.2 g/L 10R-hydroxy-8E,12Z,15Z-octadecatrienoic acid (10R-HOTrE) from 3 g/L α-linolenic acid for 30 min, with a conversion yield of 72% (w/w) and a productivity of 4.3 g/L/h; and produced 1.8 g/L 10R-HODE and 0.5 g/L 10R-HOTrE from 5 g/L hempseed oil hydrolyzate containing 2.5 g/L linoleic acid and 1.0 g/L α-linolenic acid for 30 min, with a conversion yield of 74% and 51% (w/w), respectively, and a productivity of 3.6 and 1.0 g/L/h, respectively. To the best of our knowledge, this is the first report on the biotechnological production of 10R-hydroxy unsaturated fatty acids.

국문 초록 (Abstract)

Aspergillus nidulans 유래의 linoleate 10R-dioxygenase (PpoC)를 발현시킨 재조합 세포와 정제된 효소가 선호하는 기질은 리놀레산, 두 번째로 알파-리놀렌산이었다. 세포 내의 PpoC는 정제된 PpoC에 비해 높은 열 안정성과 반응 안정성을 보였다. 그러므로 PpoC를 발현시킨 재조합 세포를 이용하여 리놀레산, 알파-리놀렌산, 그리고 리놀레산과 알파-리놀렌산을 포함하는 대마씨유 가수분해물을 기질로 하여 10R-수산화 불포화 지방산을 생산하였다. 10R-Hydroxy-8E,12Z-octadecadienoic acid (10R-HODE) 의 생산에 대한 최적 조건은 100-ml baffled flask에서 pH 8.0, 30 °C, 250 rpm, 5% (v/v) dimethyl sulfoxide, 5 g/L substrate, 60 g/L cells이었다. 이 최적 조건에서 PpoC를 발현시킨 재조합 세포는 리놀레산으로부터 40분 동안 10R-HODE를 2.7 g/L 생산하였고 54%의 전환율과 4.0 g/L/h의 생산성을 나타냈으며, 알파-리놀렌산으로부터 30분 동안 10R-hydroxy-8E,12Z,15Z-octadecatrienoic acid (10R-HOTrE)를 2.1 g/L 생산하였으며 42%의 전환율과 4.1 g/L/h의 생산성을 나타냈다. 2.5 g/L 리놀레산과 1.0 g/L 알파-리놀렌산을 포함하는 5 g/L 대마씨유 가수분해물로부터는 30분 동안 1.8 g/L 10R-HODE와 0.5 g/L 10R-HOTrE를 생산하였으며, 각각 72%, 50%의 전환율과 3.6, 1.0 g/L/h의 생산성을 나타냈다. 우리가 아는 한, 이는 10R-수산화 불포화 지방산의 생물공학적 생산에 대한 처음 보고되는 것이다.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Materials and methods 3
• 2.1. Materials 3
• 2.2. Microorganisms, media, and culture conditions 3
• 2.3. Gene cloning 4
• 2.4. Enzyme purification 5
• 2.5. Determination of specific activities of PpoC enzyme and recombinant cells for unsaturated fatty acids 5
• 2.6. Determination of stabilities of PpoC in cells and purified PpoC 6
• 2.7. Effects of pH, temperature, reducing agent, and solvent on the activity of recombinant cells 6
• 2.8. Optimization of the concentrations of cells and substrate for the production of 10R-hydroxy unsaturated fatty acids 7
• 2.9. Production of 10R-hydroxy unsaturated fatty acids from linoleic acid, α-linolenic acid, and hempseed oil hydrolyzate 8
• 2.10. Analytical methods 8
• 3. Results 10
• 3.1. Substrate specificities of recombinant cells expressing PpoC from A. nidulans and the purified PpoC and identification of their products 10
• 3.2. Stabilities of PpoC from A. nidulans in recombinant cells and the purified PpoC 19
• 3.3. Effects of pH, temperature, reducing agent, and solvent on the production of 10R-HODE from linoleic acid by whole recombinant cells expressing PpoC from A. nidulans 22
• 3.4. Optimization of the concentrations of cells and substrate for the production of 10R-hydroxy unsaturated fatty acids by whole recombinant cells expressing PpoC from A. nidulans 27
• 3.5. Production of 10R-hydroxy unsaturated fatty acids from linoleic acid, α-linolenic acid, and hempseed oil hydrolyzate by whole recombinant cells expressing PpoC from A. nidulans under the optimized conditions 32
• 4. Discussion 35
• References 40
• Abstract (in Korean) 44