The cytosolic NADP-isocitrate dehydrogenase (IDH) of oleaginous fungi provides the reducing power for fatty acid synthesis. Previous studies have shown that IDH4 from Mortierella alpina (MaIDH4) has a positive effect on lipid accumulation. To investigate the properties of MaIDH4, it was heterologously expressed and purified in Escherichia coli and Pichia pastoris expression systems respectively. The pure enzyme was successfully obtained in both systems. The effects of different expression systems on enzyme activity and yield were also investigated. Firstly, MaIDH4 was cloned into the expression vectors pET28a(+) and pPink-HC-3CZHEK to construct recombinant plasmids, which were then transformed into E. coli BL21 (DE3) and PichiaPinkTM Strain 2 respectively for induction of expression. Finally, the MaIDH4 recombinase carrying a 10×His tag was obtained by purification with a nickel column. The specific activity of the pure enzymes were 34.10 and 43.00 U/mg, with enzyme yields of 10.28 and 159.12 U/L, respectively. The analysis of the specific activity and enzyme yield of the pure enzyme indicated that the host of Pichia pastoris was more suitable for the heterologous expression of MaIDH4 than the E. coli expression system.
WANG Xuxu
,
SUN Xiaoqi
,
CHEN Haiqin
,
ZHAO Jianxin
,
CHEN Wei
,
TANG Xin
. Heterologous expression and activity analysis of NADP-isocitrate dehydrogenase from Mortierella alpina[J]. Food and Fermentation Industries, 2023
, 49(2)
: 13
-19
.
DOI: 10.13995/j.cnki.11-1802/ts.031576
[1] RATLEDGE C, WYNN J P.The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms[J].Advances in Applied Microbiology, 2002, 51:1-52.
[2] HIGASHIYAMA K, FUJIKAWA S, PARK E Y, et al.Production of arachidonic acid by Mortierella fungi[J].Biotechnology and Bioprocess Engineering, 2002, 7(5):252-262.
[3] CHANG L L, LU H Q, CHEN H Q, et al.Lipid metabolism research in oleaginous fungus Mortierella alpina:Current progress and future prospects[J].Biotechnology Advances, 2022, 54:107794.
[4] RATLEDGE C.The role of malic enzyme as the provider of NADPH in oleaginous microorganisms:A reappraisal and unsolved problems[J].Biotechnology Letters, 2014, 36(8):1 557-1 568.
[5] LOSMAN J A, KAELIN W G.What a difference a hydroxyl makes:Mutant IDH, (R)-2-hydroxyglutarate, and cancer[J].Genes & Development, 2013, 27(8):836-852.
[6] YANG E S, RICHTER C, CHUN J S, et al.Inactivation of NADP(+)-dependent isocitrate dehydrogenase by nitric oxide[J].Free Radical Biology and Medicine, 2002, 33(7):927-937.
[7] CONTRERAS-SHANNON V, LIN A P, MCCAMMON M T, et al.Kinetic properties and metabolic contributions of yeast mitochondrial and cytosolic NADP(+)-specific isocitrate dehydrogenases[J].Journal of Biological Chemistry, 2005, 280(6):4 469-4 475.
[8] WYNN J P, KENDRICK A, RATLEDGE C.Sesamol as an inhibitor of growth and lipid metabolism in Mucor circinelloides via its action on malic enzyme[J].Lipids, 1997, 32(6):605-610.
[9] HAO G F, CHEN H Q, WANG L, et al.Role of malic enzyme during fatty acid synthesis in the oleaginous fungus Mortierella alpina[J].Applied and Environmental Microbiology, 2014, 80(9):2 672-2 678.
[10] ZHAO L N, TANG X, LUAN X, et al.Role of pentose phosphate pathway in lipid accumulation of oleaginous fungus Mucor circinelloides[J].RSC Advances, 2015, 5(118):97 658-97 664.
[11] HAO G F, CHEN H Q, GU Z N, et al.Metabolic engineering of Mortierella alpina for enhanced arachidonic acid production through the NADPH-supplying strategy[J].Applied and Environmental Microbiology, 2016, 82(11):3 280-3 288.
[12] TANG X, SUN X Q, WANG X X, et al.Characterization of NAD+/NADP+-specific isocitrate dehydrogenases from oleaginous fungus Mortierella alpina involved in lipid accumulation[J].Frontiers in Nutrition, 2021, 8:746342.
[13] 刘丽琴, 陈婷婷, 李少伟, 等.大肠杆菌表达系统在基因工程疫苗研发中的应用与策略优化[J].中国新药杂志, 2020, 29(21):2 434-2 442.
LIU L Q, CHEN T T, LI S W, et al.Escherichia coli expression system applied in the development of recombinant human vaccines and its potential improvement[J].Chinese Journal of New Drugs, 2020, 29(21):2 434-2 442.
[14] YANG D, PARK S Y, PARK Y S, et al.Metabolic engineering of Escherichia coli for natural product biosynthesis[J].Trends in Biotechnology, 2020, 38 (7):745-765.
[15] CREGG J M.Introduction:Distinctions between Pichia pastoris and other expression systems[J].Methods in molecular biology (Clifton, N.J.), 2007, 389:1-10.
[16] WYNN J P, RATLEDGE C.Malic enzyme is a major source of NADPH for lipid accumulation by Aspergillus nidulans[J].Microbiology, 1997, 143(1):253-257.
[17] LI B Z, CAO Y Q, ZHOU L J, et al.A novel protein expression system-PichiaPink(TM)-and a protocol for fast and efficient recombinant protein expression[J].African Journal of Biotechnology, 2011, 10(83):19 464-19 472.
[18] HIGGINS D R.Overview of protein expression in Pichia pastoris[J].Current Protocols in Protein Science, 2001, Chapter 5: Unit5.7.
[19] INAN M, ARYASOMAYAJULA D, SINHA J, et al.Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase[J].Biotechnology and Bioengineering, 2006, 93(4):771-778.
[20] CREGG J M, CEREGHINO J L, SHI J, et al.Recombinant protein expression in Pichia pastoris[J].Molecular Biotechnology, 2000, 16(1):23-52.
[21] FERRER-MIRALLES N, DOMINGO-ESPÍN J, CORCHERO J L, et al.Microbial factories for recombinant pharmaceuticals[J].Microbial Cell Factories, 2009, 8:17.
[22] 胡立强, 郑文, 钟艺, 等.抗病毒蛋白RC28在大肠杆菌和毕赤酵母中的表达及活性比较[J].中国生物工程杂志, 2017, 37(1):14-20.
HU L Q, ZHENG W, ZHONG Y, et al.Comparison of expression and activity of antiviral protein RC28 in Escherichia coli and Pichia pastoris[J].China Biotechnology, 2017, 37(1):14-20.
[23] 胡莉, 王小红.抗黄曲霉毒素B1单链抗体在大肠杆菌和毕赤酵母中的表达和活性研究[J].生物学杂志, 2018, 35(1):26-31.
HU L, WANG X H.Comparison of expression and activity of anti-AFB1 scFv in Escherichia coli and Pichia pastoris[J].Journal of Biology, 2018, 35(1):26-31.
[24] 周晨妍, 刘振华, 王丹丹, 等.木聚糖酶Xyn43A基因在大肠杆菌及毕赤酵母中的表达比较[J].食品与发酵工业, 2016, 42(6):15-19.
ZHOU C Y, LIU Z H, WANG D D, et al.Expression of xylanase Xyn43A gene in Escherichia coli and Pichia pastoris[J].Food and Fermentation Industries, 2016, 42(6):15-19.
