Characterization of a recombinant 7,8-linoleate diol synthase from Glomerella cingulate

Abstract

A putative diol synthase from the fungus Glomerella cingulate was cloned and expressed in Escherichia coli. The putative diol synthase from G. cingulate was purified by His-Trap affinity chromatography with a specific activity of 0.87 U mg(-1), an eightfold purification, and a yield of 28%. One unit of activity was defined as the amount of enzyme required to produce 1 μmol of 7,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid (7,8-DiHODE) per min. The purified enzyme was estimated as a 127-kDa tetramer with a molecular mass of 510 kDa by gel filtration chromatography. The enzyme converted linoleic acid to a product, identified as 7S,8S-DiHODE by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. The specific activity and catalytic efficiency (k cat/K m) of 7,8-diol synthase from G. cingulate for the conversion of fatty acid to dihydroxy fatty acid followed the order linoleic acid > α-linolenic acid > oleic acid > palmitoleic acid, indicating that the enzyme is a 7,8-linoleate diol synthase (7,8-LDS). The activity of the enzyme for the conversion of 7,8-DiHODE from linoleic acid was maximal at pH 6.5, 40 °C, and 2.5% (v/v) dimethyl sulfoxide (DMSO). Under these conditions, 7,8-LDS from G. cingulate converted 1.0 mM linoleic acid to 0.62 mM 7,8-DiHODE for 30 min, with a conversion yield of 62% (mol/mol), via 8-hydroperoxy-9,12(Z,Z)-octadecadienoic acid (8-HPODE) as an intermediate. The accumulation of 8-HPODE was due to a higher 8-dioxygenase activity in the N-terminal domain than hydroperoxide isomerase activity in the C-terminal domain.

Production of 8,11‐dihydroxy and 8‐hydroxy unsaturated fatty acids from unsaturated fatty acids by recombinant Escherichia coli expressing 8,11‐linoleate diol synthase from Penicillium chrysogenum

“…The optimal pH and temperature for the conversion of unsaturated fatty acids to 5,8‐dihydroxy fatty acids by recombinant cells expressing 5,8‐LDS from A. nidulans were ranging from 7.0 and 35°C to 7.5 and 40°C, respectively . The maximum conversion rate of linoleic acid to 7,8‐DiHODE by 7,8‐LDS from G. cingulate occurred at pH 7.0 and 40°C . The optimal pH values of LDSs were similar.…”

“…The specific activity of recombinant cells expressing 5,8‐LDS from A. nidulans followed the order linoleic acid > α‐linolenic acid > oleic acid . The specific activity of 7,8‐LDS from Glomerella cingulate followed the order linoleic acid > α‐linolenic acid > oleic acid > palmitoleic acid . Therefore, the order of specific activity of the recombinant cells expressing 8,11‐LDS from P. chrysogenum was different from other LDSs.…”

“…10 The specific activity of 7,8-LDS from Glomerella cingulate followed the order linoleic acid > a-linolenic acid > oleic acid > palmitoleic acid. 17 Therefore, the order of specific activity of the recombinant cells expressing 8,11-LDS from P. chrysogenum was different from other LDSs.…”

“…[9][10][11] The maximum conversion rate of linoleic acid to 7,8-DiHODE by 7,8-LDS from G. cingulate occurred at pH 7.0 and 408C. 17 The optimal pH values of LDSs were similar. However, the optimal temperatures for recombinant cells expressing 8,11-LDS from P. chrysogenum for unsaturated fatty acids were lower than those of recombinant cells expressing 5,8-LDS from A. nidulans and 7,8-LDS from G. cingulate.…”

Production of 7,8-Dihydroxy Unsaturated Fatty Acids from Plant Oils by Whole Recombinant Cells Expressing 7,8-Linoleate Diol Synthase from Glomerella cingulata

“…Bacterial 7,10-oleate diol synthase from P. aeruginosa and fungal diol synthases, including 5,8-linoleate diol synthases (5,8-LDSs) from Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus, 7,8-LDSs from Gaeumannomyces graminis, Magnaporthe oryzae , and Glomerella cingulata , and 8,11-LDS from Penicillium chrysogenum, have been identified. However, the biotechnological production of 7,8-dihydroxy unsaturated fatty acids has not been attempted.…”