Heterologous Expression of Propionibacterium acnes Isomerase in Mouse (Mus musculus) Cells and Production of t10c12-conjugated Linoleic Acid
WANG Chao1,2, WANG Yang-Yang1,2, RAO Yu1,3, LI Mei-Juan3, LIU Zong-Ping1,2, LI Shi-Li4,*, GOU Ke-Mian1,2,*
1 Institute of Comparative Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China; 2 Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China; 3 State Key Laboratory of Agribiotechnologies, China Agricultural University, Beijing 100193, China; 4 Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75390, USA
Abstract：Trans 10, cis 12-conjugated linoleic acid (t10c12-CLA) can be obtained from linoleic acid by Propionibacterium acnes isomerase (PAI). Animals fed t10c12-CLA reduced fat deposition and milk fat content, and the dynamic balance of energy was affected. In this study, the codon optimized pai gene was transfected into mouse (Mus musculus) 3T3 cells. It was confirmed by gas chromatography that PAI could efficiently catalyze the substrate linoleic acid into t10c12-CLA. The content of t10c12-CLA was high, and reached 7.09% of the total amount of poly-unsaturated fatty acids in cells. When compared with the control cell line transfected with pIRES2-AcGFP1, the cell lines transfected with pai gene and synthesized t10c12-CLA showed linoleic acid accumulation (12.46% vs 14.38%~15.54%)(P<0.05) and arachidonic acid decrease (13.31% vs 9.03%~11.13%)(P<0.05), while stearic acid (14.07% vs 11.85%~12.54%)(P<0.05) and oleic acid (20.29% vs 14.89%~16.87%)(P<0.05) decreased simultaneously. T10c12-CLA produced in pai transfected cells could up-regulate the gene expression of stearoyl-CoA desaturase 1 (scd1) (P<0.05), which may be the reason for the decrease of stearic acid content in transgenic cells. However, exogenous t10c12-CLA did not down-regulate the expression of fatty acid desaturase 1 (fads1) and fads2 genes. The results of high pressure gas chromatography showed that the exogenous t10c12-CLA blocked the metabolism of linoleic acid to downstream arachidonic acid (P<0.05), that is, t10c12-CLA may competitively bind to FADS2/FADS1 enzyme system, so as to inhibit the normal metabolism of linoleic acid to arachidonic acid. This study confirmed that microbial pai gene could be normally expressed in mammalian cells and produce t10c12-CLA, which can provide a reference basis for the development of pai transgenic animals
王超, 王洋洋, 饶喻, 李美娟, 刘宗平, 李世丽, 苟克勉. 痤疮丙酸杆菌异构酶在小鼠细胞中异源表达及t10c12-共轭亚油酸的制备[J]. 农业生物技术学报, 2021, 29(12): 2304-2311.
WANG Chao, WANG Yang-Yang, RAO Yu, LI Mei-Juan, LIU Zong-Ping, LI Shi-Li, GOU Ke-Mian. Heterologous Expression of Propionibacterium acnes Isomerase in Mouse (Mus musculus) Cells and Production of t10c12-conjugated Linoleic Acid. 农业生物技术学报, 2021, 29(12): 2304-2311.
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