Structure-guided engineering of a flavin-containing monooxygenase for the efficient production of indirubin

Abstract

Indirubin is a bisindole compound for the treatment of chronic myelocytic leukemia. Here, we presented a structure-guided method to improve the activity of a flavin-containing monooxygenase (bFMO) for the efficient production of indirubin in Escherichia coli. A flexible loop interlocked with the active pocket through a helix and the substrate tunnel rather than the active pocket in bFMO were identified to be two reconfigurable structures to improve its activity, resulting in K223R and N291T mutants with enhanced catalytic activity by 2.5- and 2.0-fold, respectively. A combined modification at the two regions (K223R/D317S) achieved a 6.6-fold improvement in catalytic efficiency (k_cat/K_m) due to enhancing π–π stacking interactions stabilization. Finally, an engineered E. coli strain was constructed by metabolic engineering, which could produce 860.7 mg/L (18 mg/L/h) indirubin, the highest yield ever reported. This work provides new insight into the redesign of FMOs to boost their activities and an efficient approach to produce indirubin.

Keywords

Flavin-containing monooxygenase / Indirubin / Loop region / Substrate tunnel / Structure-guided enzyme engineering / Microbial synthesis

References

[1] Alfieri A, Malito E, Orru R, Fraaije MW, Mattevi A. Revealing the moonlighting role of NADP in the structure of a flavin-containing monooxygenase. Proc Nat Acad Sci, 2008, 105(18): 6572.

[2] Anastas P, Eghbali N. Green chemistry: principles and practice. Chem Soc Rev, 2010, 39(1): 301-312.

[3] Apol E, Apostolov R, Berendsen H, Buuren A, Bjelkmar P, Drunen R, Feenstra KA, Fritsch S, Groenhof G, Junghans C, Kasson P, Larsson P, Meulenhoff P, Murtola T, Pall S, Pronk S, Schulz R, Shirts M, Sijbers A, Lindahl E (2013) Gromacs User Manual Version 4.6

[4] Batista PR, Wilter A, Durham EHAB, Pascutti PG. Molecular dynamics simulations applied to the study of subtypes of HIV-1 protease common to Brazil, Africa, and Asia. Cell Biochem Biophys, 2006, 44(3): 395-404.

[5] Berry A, Dodge TC, Pepsin M, Weyler W. Application of metabolic engineering to improve both the production and use of biotech indigo. J Ind Microbiol Biotechnol, 2002, 28(3): 127-133.

[6] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 72(1): 248-254.

[7] Cho HJ, Cho H, Kim K, Kim M, Kim S, Kang B. Structural and functional analysis of bacterial flavin-containing monooxygenase reveals its ping-pong-type reaction mechanism. J Struct Biol, 2011, 175: 39-48.

[8] Choi HS, Kim JK, Cho EH, Kim YC, Kim JI, Kim SW. A novel flavin-containing monooxygenase from Methylophaga sp. strain SK1 and its indigo synthesis in Escherichia coli. Biochem Biophys Res Commun, 2003, 306(4): 930-936.

[9] Dickey RM, Forti AM, Kunjapur AM. Advances in engineering microbial biosynthesis of aromatic compounds and related compounds. Bioresourc Bioprocess, 2021, 8(1): 91.

[10] Dong Q, Yuan S, Wu L, Su L, Zhao Q, Wu J, Huang W, Zhou J. Structure-guided engineering of a Thermobifida fusca cutinase for enhanced hydrolysis on natural polyester substrate. Bioresourc Bioprocess, 2020, 7(1): 37.

[11] Du J, Yang D, Luo ZW, Lee SY. Metabolic engineering of Escherichia coli for the production of indirubin from glucose. J Biotechnol, 2018, 267: 19-28.

[12] Efferth T, Li PCH, Konkimalla VSB, Kaina B. From traditional Chinese medicine to rational cancer therapy. Trends Mol Med, 2007, 13(8): 353-361.

[13] Gillam EMJ, Notley LM, Cai H, De Voss JJ, Guengerich FP. Oxidation of indole by cytochrome P450 enzymes. Biochemistry, 2000, 39(45): 13817-13824.

[14] Han GH, Shin H-J, Kim SW. Optimization of bio-indigo production by recombinant E. coli harboring fmo gene. Enzyme Microb Tech, 2008, 42(7): 617-623.

[15] Han GH, Gim GH, Kim W, Seo SI, Kim SW. Enhanced indirubin production in recombinant Escherichia coli harboring a flavin-containing monooxygenase gene by cysteine supplementation. J Biotechnol, 2013, 164(2): 179-187.

[16] Henzler-Wildman KA, Thai V, Lei M, Ott M, Wolf-Watz M, Fenn T, Pozharski E, Wilson MA, Petsko GA, Karplus M, Hübner CG, Kern D. Intrinsic motions along an enzymatic reaction trajectory. Nature, 2007, 450(7171): 838-844.

[17] Hoessel R, Leclerc S, Endicott JA, Nobel MEM, Lawrie A, Tunnah P, Leost M, Damiens E, Marie D, Marko D, Niederberger E, Tang W, Eisenbrand G, Meijer L. Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases. Nat Cell Biol, 1999, 1(1): 60-67.

[18] Hu S, Huang J, Mei L, Yu Q, Yao S, Jin Z. Altering the regioselectivity of cytochrome P450 BM-3 by saturation mutagenesis for the biosynthesis of indirubin. J Mol Cat B Enzym, 2010, 67(1): 29-35.

[19] Kim J, Lee J, Lee P-G, Kim E-J, Kroutil W, Kim B-g. Elucidating cysteine-assisted synthesis of indirubin by a flavin-containing monooxygenase. ACS Catal, 2019, 9(10): 9539-9544.

[20] Kokkonen P, Bednar D, Pinto G, Prokop Z, Damborsky J. Engineering enzyme access tunnels. Biotechnol Adv, 2019, 37(6

[21] Kreß N, Halder JM, Rapp LR, Hauer B. Unlocked potential of dynamic elements in protein structures: channels and loops. Curr Opin Chem Biol, 2018, 47: 109-116.

[22] Latimer LN, Russ ZN, Lucas J, Dueber JE. Exploration of acetylation as a base-labile protecting group in Escherichia coli for an indigo precursor. ACS Synth Biol, 2020, 9(10): 2775-2783.

[23] Leclerc S, Garnier M, Hoessel R, Marko D, Bibb JA, Snyder GL, Greengard P, Biernat J, Wu Y-Z, Mandelkow E-M, Eisenbrand G, Meijer L. Indirubins inhibit glycogen synthase kinase-3β and CDK5/p25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer’s disease. A property common to most cyclin-dependent kinase inhibitors?. J Biol Chem, 2001, 276(1): 251-260.

[24] Li Q-S, Schwaneberg U, Fischer P, Schmid RD. Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst. Chem Eur J, 2000, 6(9): 1531-1536.

[25] Liu Y, Xu G, Zhou J, Ni J, Zhang L, Hou X, Yin D, Rao Y, Zhao Y-L, Ni Y. Structure-guided engineering of d-Carbamoylase reveals a key loop at substrate entrance tunnel. ACS Catal, 2020, 10(21): 12393-12402.

[26] Loncar N, van Beek H, Fraaije M. Structure-based redesign of a self-sufficient Flavin-containing monooxygenase towards indigo production. Int J Mol Sci, 2019, 20: 6148.

[27] Lü H-t, Liu J, Deng R, Song J-y. Preparative isolation and purification of indigo and indirubin from Folium isatidis by high-speed counter-current chromatography. Phytochem Anal, 2012, 23(6): 637-641.

[28] Malumbres M, Barbacid M. Cell cycle, CDKs and cancer: a changing paradigm. Nat Rev Cancer, 2009, 9(3): 153-166.

[29] McClay K, Boss C, Keresztes I, Steffan RJ. Mutations of toluene-4-monooxygenase that alter regiospecificity of indole oxidation and lead to production of novel indigoid pigments. Appl Environ Microbiol, 2005, 71(9): 5476.

[30] Meng S, An R, Li Z, Schwaneberg U, Ji Y, Davari MD, Wang F, Wang M, Qin M, Nie K, Liu L. Tunnel engineering for modulating the substrate preference in cytochrome P450_BsβHI. Bioresourc Bioprocess, 2021, 8(1): 26.

[31] Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem, 2009, 30(16): 2785-2791.

[32] Nam S, Buettner R, Turkson J, Kim D, Cheng J, Muehlbeyer S, Hippe F, Vatter S, Merz K-H, Eisenbrand G, Jove R. Indirubin derivatives inhibit Stat3 signaling and induce apoptosis in human cancer cells. Proc Nat Acad Sci USA, 2005, 102: 5998-6003.

[33] Ni Z-F, Xu P, Zong M-H, Lou W-Y. Structure-guided protein engineering of ammonia lyase for efficient synthesis of sterically bulky unnatural amino acids. Bioresourc Bioprocess, 2021, 8(1): 103.

[34] Pavlova M, Klvana M, Prokop Z, Chaloupkova R, Banas P, Otyepka M, Wade RC, Tsuda M, Nagata Y, Damborsky J. Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate. Nat Chem Biol, 2009, 5(10): 727-733.

[35] Rapp LR, Marques SM, Zukic E, Rowlinson B, Sharma M, Grogan G, Damborsky J, Hauer B. Substrate anchoring and flexibility reduction in CYP153A_Ma.q leads to highly improved efficiency toward octanoic acid. ACS Catal, 2021, 11(5): 3182-3189.

[36] Rui L, Reardon KF, Wood TK. Protein engineering of toluene ortho-monooxygenase of Burkholderia cepacia G4 for regiospecific hydroxylation of indole to form various indigoid compounds. Appl Microbiol Biotechnol, 2005, 66(4): 422-429.

[37] Seyed MA, Jantan I, Bukhari SNA, Vijayaraghavan K. A comprehensive review on the chemotherapeutic potential of piceatannol for cancer treatment, with mechanistic insights. J Agric Food Chem, 2016, 64(4): 725-737.

[38] Souffrant MG, Yao X-Q, Momin M, Hamelberg D. N-glycosylation and Gaucher disease mutation allosterically alter active-site dynamics of acid-β-glucosidase. ACS Catal, 2020, 10(3): 1810-1820.

[39] Tan X, Zhang S, Song W, Liu J, Gao C, Chen X, Liu L, Wu J. A multi-enzyme cascade for efficient production of d-p-hydroxyphenylglycine from l-tyrosine. Bioresourc Bioprocess, 2021, 8(1): 41.

[40] van Berkel WJH, Kamerbeek NM, Fraaije MW. Flavoprotein monooxygenases, a diverse class of oxidative biocatalysts. J Biotechnol, 2006, 124(4): 670-689.

[41] Yin H, Chen H, Yan M, Li Z, Yang R, Li Y, Wang Y, Guan J, Mao H, Wang Y, Zhang Y. Efficient bioproduction of indigo and indirubin by optimizing a novel terpenoid cyclase xiaI in Escherichia coli. ACS Omega, 2021, 6(31): 20569-20576.

[42] Zhang X, Peng Y, Zhao J, Li Q, Yu X, Acevedo-Rocha CG, Li A. Bacterial cytochrome P450-catalyzed regio- and stereoselective steroid hydroxylation enabled by directed evolution and rational design. Bioresourc Bioprocess, 2020, 7(1): 2.

Funding

National Key Research and Development Program of China(No. SQ2020YFC210061)

the National Natural Science Foundation of China (No. 31500043)

Natural Science Foundation of Shanghai(No. 21ZR1417200)

the Open Funding Project of the State Key Laboratory of Bioreactor Engineering