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
AbstractTrans 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
WANG Chao,WANG Yang-Yang,RAO Yu, et al. Heterologous Expression of Propionibacterium acnes Isomerase in Mouse (Mus musculus) Cells and Production of t10c12-conjugated Linoleic Acid[J]. 农业生物技术学报, 2021, 29(12): 2304-2311.
[1] Agatha G, Voigt A, Kauf E, et al.2004. Conjugated linoleic acid modulation of cell membrane in leukemia cells[J]. Cancer Letter, 209(1): 87-103. [2] Amarù D L, Field C J.2009. Conjugated linoleic acid decreases MCF-7 human breast cancer cell growth and insulin-like growth factor-1 receptor levels[J]. Lipids, 44(5): 449-458. [3] Castro-Webb N, Ruiz-Narváez E A, Campos H.2012. Cross-sectional study of conjugated linoleic acid in adipose tissue and risk of diabetes[J]. American Journal of Clinic Nutrition, 96(1): 175-181. [4] Cho H J, Kim E J, Lim S S, et al.2006. Trans-10, cis-12, not cis-9, trans-11, conjugated linoleic acid inhibits G1-S progression in HT-29 human colon cancer cells[J]. Journal of Nutrition, 136(4): 893-898. [5] Choi Y, Kim Y C, Han Y B, et al.2000. The trans-10, cis-12 isomer of conjugated linoleic acid downregulates stearoyl-CoA desaturase 1 gene expression in 3T3-L1 adipocytes[J]. Journal of Nutrition, 130(8): 1920-1924. [6] Choi Y, Park Y, Pariza M W, et al.2001. Regulation of stearoyl-CoA desaturase activity by the trans-10, cis-12 isomer of conjugated linoleic acid in HepG2 cells[J]. Biochemical and Biophysical Research Communications, 284(3): 689-693. [7] Choi Y, Park Y, Storkson J M, et al.2002. Inhibition of stearoyl-CoA desaturase activity by the cis-9, trans-11 isomer and the trans-10, cis-12 isomer of conjugated linoleic acid in MDA-MB-231 and MCF-7 human breast cancer cells[J]. Biochemical and Biophysical Research Communications, 294(4): 785-790. [8] Deng M D, Grund A D, Schneider K J, et al.2007. Linoleic acid isomerase from Propionibacterium acnes: Purification, characterization, molecular cloning, and heterologous expression[J]. Applied Biochemistry and Biotechnology, 143(3): 199-211. [9] Eder K, Slomma N, Becker K.2002. Trans-10, cis-12 conjugated linoleic acid suppresses the desaturation of linoleic and alpha-linolenic acids in HepG2 cells[J]. Journal of Nutrition, 132(6): 1115-1121. [10] Evans M, Geigerman C, Cook J, et al.2000. Conjugated linoleic acid suppresses triglyceride accumulation and induces apoptosis in 3T3-L1 preadipocytes[J]. Lipids, 35(8): 899-910. [11] Hornung E, Krueger C, Pernstich C, et al.2005. Production of (10E,12Z)-conjugated linoleic acid in yeast and tobacco seeds[J]. Biochimica et Biophysica Acta, 1738(1-3): 105-114. [12] Jupp J, Hillier K, Elliott D H, et al.2007. Colonic expression of leukotriene-pathway enzymes in inflammatory bowel diseases[J]. Inflammatory Bowel Diseases, 13(5): 537-546. [13] Kim J H, Kim Y, Kim Y J, et al.2016. Conjugated linoleic acid: Potential health benefits as a functional food ingredient[J]. Annual Review of Food Science and Technology, 7: 221-244. [14] Kohno-Murase J, Iwabuchi M, Endo-Kasahara S, et al.2006. Production of trans-10, cis-12 conjugated linoleic acid in rice[J]. Transgenic Research, 15(1): 95-100. [15] Li S L, Ma S Y, Xu B R, et al.2015. Effects of trans-10, cis-12-conjugated linoleic acid on mice are influenced by the dietary fat content and the degree of murine obesity[J]. European Journal of Lipid Science and Technology, 117(12): 1908-1918. [16] Ochoa J J, Farquharson A J, Grant I, et al.2004. Conjugated linoleic acids (CLAs) decrease prostate cancer cell proliferation: Different molecular mechanisms for cis-9, trans-11 and trans-10, cis-12 isomers[J]. Carcinogenesis, 25(7): 1185-1191. [17] Rao Y, Li S L, Li M J, et al.2020. A single amino acid substitution in the FAD-binding domain causes the inactivation of Propionibacterium Acnes isomerase[J]. Bioscience, Biotechnology and Biochemistry, 84(4): 789-796. [18] Rosberg-Cody E, Stanton C, O'Mahony L, et al.2011. Recombinant lactobacilli expressing linoleic acid isomerase can modulate the fatty acid composition of host adipose tissue in mice[J]. Microbiology, 157(Pt 8): 609-615. [19] Schley P D, Brindley D N, Field C J.2007. (n-3) PUFA alter raft lipid composition and decrease epidermal growth factor receptor levels in lipid rafts of human breast cancer cells[J]. Journal of Nutrition, 137(3): 548-553. [20] Smith W L.2008. Nutritionally essential fatty acids and biologically indispensable cyclooxygenases[J]. Trends in Biochemical Sciences, 33(1): 27-37. [21] Subbaiah P V, Gould I G, Lal S, et al.2011. Incorporation profiles of conjugated linoleic acid isomers in cell membranes and their positional distribution in phospholipids[J]. Biochimica et Biophysica Acta, 1811(1): 17-24. [22] Yamasaki M, Miyamoto Y, Chujo H, et al.2005. Trans10, cis12-conjugated linoleic acid induces mitochondria-related apoptosis and lysosomal destabilization in rat hepatoma cells[J]. Biochimica et Biophysica Acta, 1735(3): 176-184.
