Efficient synthesis 1,4-cyclohexanedicarboxaldehyde by an engineered alcohol oxidase

Abstract

In this study, we selected and engineered a flavin adenine dinucleotide (FAD)-dependent alcohol oxidase (AOX) to produce 1,4-cyclohexanedicarboxaldehyde (CHDA), an initial raw material for spiral compounds, from 1,4-cyclohexanedimethanol (CHDM). First, the structure of alcohol oxidase from Arthrobacter cholorphenolicus (AcCO) was analyzed, and the mechanism of AcCO-catalyzed primary alcohol oxidation was elucidated, demonstrating that the energy barrier of the hydride (H⁻) transfer (13.4 kcal·mol⁻¹ and 20.4 kcal·mol⁻¹) decreases the catalytic efficiency of the primary alcohol oxidation reaction. Therefore, we designed a protein engineering strategy to adjust the catalytically active conformation to shorten the distance of hydride (H⁻) transfer and further decreased the core energy barrier. Following this strategy, variant W4 (S101A/H351V/N378S/Q329N) was obtained with 112.5-fold increased catalytic efficiency to produce CHDA compared to that of the wild-type strain. The 3 L scale preparation of CHDA reached a titer up to 29.6 g·L⁻¹ with a 42.2% yield by an Escherichia coli whole-cell catalyst, which demonstrates the potential of this system for industrial application.

Keywords

1,4-Cyclohexanedicarboxaldehyde / Alcohol oxidase / Primary alcohol oxidation reaction / Protein engineering

References

[1] Abad A, Almela C, Corma A, Garcia H. Unique gold chemoselectivity for the aerobic oxidation of allylic alcohols. Chem Commun, 2006, 14(30): 3178-3180.

[2] Akbulut G, Sonmez HB, Wudl F. Synthesis, characterization and properties of novel polyspiroacetals. J Polym Res, 2013, 20(3): 1-8.

[3] An JH, Nie Y, Xu Y. Structural insights into alcohol dehydrogenases catalyzing asymmetric reductions. Crit Rev Biotechnol, 2019, 39(3): 366-379.

[4] Asadi M, Bonke S, Poyzos A, Lupton DW. Fukuyama reduction and integrated thioesterification/fukuyama reduction of thioesters and acyl chlorides using continuous flow. ACS Catal, 2014, 4(6): 2070-2074.

[5] Bassanini I, Ferrandi EE, Riva S, Monti D. Biocatalysis with laccases: an updated overview. Catalysts, 2021, 11(1): 26.

[6] Best RB, Zhu X, Shim J, Lopes PEM, Mittal J, Feig M, MacKerell AD. Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone phi, psi and side-chain chi(1) and chi(2) dihedral angles. J Chem Theory Comput, 2012, 8(9): 3257-3273.

[7] Cleveland M, Lafond M, Xia FR, Chung R, Mulyk P, Hein JE, Brumer H. Two fusarium copper radical oxidases with high activity on aryl alcohols. Biotechnol Biofuels, 2021, 14(1): 138.

[8] Cruz P, Perez Y, del Hierro I, Fajardo M. Copper, copper oxide nanoparticles and copper complexes supported on mesoporous SBA-15 as catalysts in the selective oxidation of benzyl alcohol in aqueous phase. Micropor Mesopor Mat, 2016, 220: 136-147.

[9] Elmaaty TA, Castle LW. Chromate oxidation of α-nitro alcohols to α-nitro ketones: significant improvements to a classic method. Molecules, 2005, 10(12): 1458-1461.

[10] Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG. A smooth particle mesh ewald method. J Chem Phys, 1995, 103(19): 8577-8593.

[11] Feuerbacher N, Fritz V, Windscheidt J, Poetsch E. Nieger M (1999) Synthesis of rodlike dispiro hydrocarbon skeletons for new liquid crystal compounds. Synthesis, 1999, 1: 117-120.

[12] Gadda G. Choline oxidases. Enzymes, 2020, 47: 137-166.

[13] Heath RS, Birmingham WR, Thompson MP, Taglieber A, Daviet L, Turner NJ. An engineered alcohol oxidase for the oxidation of primary alcohols. ChemBioChem, 2019, 20(2): 276-281.

[14] Hess B, Bekker H, Berendsen HJC, Fraaije J. LINCS: a linear constraint solver for molecular simulations. J Comput Chem, 1997, 18(12): 1463-1472.

[15] Hess B, Kutzner C, van der Spoel D, Lindahl E. GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J Chem Theory Comput, 2008, 4(3): 435-447.

[16] Hossain MM, Shyu SG. Efficient and selective aerobic alcohol oxidation catalyzed by copper(II)/2,2,6,6,-tetramethylpiperidine-1-oxyl at room temperature. Adv Synth Catal, 2010, 352(17): 3061-3068.

[17] Krishnan A, McNeil BA, Stuart DT. Biosynthesis of fatty alcohols in engineered microbial cell factories: advances and limitations. Front Bioeng Biotech, 2020, 8(23): 2296-4185.

[18] Kunjapur AM, Prather KLJ. Microbial engineering for aldehyde synthesis. Appl Environ Microb, 2015, 81(6): 1892-1901.

[19] Kwit M, Plutecka A, Rychlewska U, Gawronski J, Khlebnikov AF, Kozhushkov SI, de Meijere A. Chiral macrocyclic aliphatic oligoimines derived from trans-1,2-diaminocyclohexane. Chem Eur J, 2007, 13(31): 8688-8695.

[20] Lee SB, Lee JC. Oxidation of benzylic alcohols with iodine and lithium carbonate in ionic liquid. B Kor Chem Soc, 2009, 30(12): 3107-3108.

[21] Marais L, Vosloo HCM, Swarts AJ. Homogeneous oxidative transformations mediated by copper catalyst systems. Coordin Chem Rev, 2021, 21(9): 3815-3820.

[22] Martinez-Montero L, Gotor V, Gotor-Fernandez V, Lavandera I. Stereoselective amination of racemic sec-alcohols through sequential application of laccases and transaminases. Green Chem, 2017, 19(2): 474-480.

[23] Mathieu Y, Offen WA, Forget SM, Ciano L, Viborg AH, Blagova E, Henrissat B, Walton PH, Davies GJ, Brumer H. Discovery of a fungal copper radical oxidase with high catalytic efficiency toward 5-hydroxymethylfurfural and benzyl alcohols for bioprocessing. Acs Catal, 2020, 10(5): 3042-3058.

[24] Mattey AP, Ford GJ, Citoler J, Baldwin C, Marshall JR, Palmer RB, Flitsch SL. Development of continuous flow systems to access secondary amines through previously incompatible biocatalytic cascades. Angew Chem Int Edit, 2021, 60(34): 18660-18665.

[25] Miao J, Ding Y, Li Y, Wang G, Cheng X, Jin M (2021) Method for preparing 1,4-cyclohexanedicarboxaldehyde from 3-cyclohexene-1-formaldehyde. China Patent CN112892601A, 4 Jun 2021

[26] Pellegrino S, Contini A, Clerici F, Gori A, Nava D, Gelmi ML. 1H-azepine-4-amino-4-carboxylic acid: a new α, α-disubstituted ornithine analogue capable of inducing helix conformations in short ala-aib pentapeptides. Chem Eur J, 2012, 18(28): 8705-8715.

[27] Pillai UR, Sahle-Demessie E. Selective oxidation of alcohols by molecular oxygen over a Pd/MgO catalyst in the absence of any additives. Green Chem, 2004, 6(3): 161-165.

[28] Raynal M, Ballester P, Vidal-Ferran A, van Leeuwen PWNM. Supramolecular catalysis. Part 2: artificial enzyme mimics. Chem Soc Rev, 2014, 43(5): 1734-1787.

[29] Sheldon RA. Fundamentals of green chemistry: efficiency in reaction design. Chem Soc Rev, 2012, 41(4): 1437-1451.

[30] Silbert SD, Serum EM, LaScala J, Sibi MP, Webster DC. Biobased, nonisocyanate, 2K polyurethane coatings produced from polycarbamate and dialdehyde cross-linking. ACS Sustain Chem Eng, 2019, 7(24): 19621-19634.

[31] Smitherman C, Rungsrisuriyachai K, Germann MW, Gadda G. Identification of the catalytic base for alcohol activation in choline oxidase. Biochemistry, 2015, 54(2): 413-421.

[32] Song W, Xu X, Gao C, Zhang Y, Wu J, Liu J, Liu LM. Open gate of Corynebacterium glutamicum threonine deaminase for efficient synthesis of bulky α-keto acids. ACS Catal, 2020, 10(17): 9994-10004.

[33] Tjallinks G, Martin C, Fraaije MW. Enantioselective oxidation of secondary alcohols by the flavoprotein alcohol oxidase from Phanerochaete chrysosporium. Arch Biochem Biophys, 2021, 704.

[34] Wen Z, Hu D, Hu BC, Zhang D, Huang JF, Wu MC. Structure-guided improvement in the enantioselectivity of an Aspergillus usamii epoxide hydrolase for the gram-scale kinetic resolution of ortho-trifluoromethyl styrene oxide. Enzyme Microb Tech, 2021, 146.

[35] Wu YY, Zhang S, Song W, Liu J, Chen XL, Hu GP, Wu J. Enhanced catalytic efficiency of l-amino acid deaminase achieved by a shorter hydride transfer distance. ChemCatChem, 2021, 13(21): 4557-4566.

[36] Xu J, Peng YZ, Wang ZG, Hu YJ, Fan JJ, Zheng H, Wu Q. Exploiting cofactor versatility to convert a FAD-dependent baeyer-villiger monooxygenase into a ketoreductase. Angew Chem Int Edit, 2019, 58(41): 14499-14503.

[37] Yamaguchi K, Mizuno N. Supported ruthenium catalyst for the heterogeneous oxidation of alcohols with molecular oxygen. Angew Chem Int Edit, 2002, 41(23): 4538-4546.

[38] Zhao YF, Lei MK, Wu YX, Zhang ZS, Wang CW. Efficient expression of codon-adapted human acetaldehyde dehydrogenase 2 cDNA with 6xHis tag in Pichia pastoris. Sci China Ser C, 2009, 52(10): 935-941.

[39] Zhao J, Gao P, Yu H, Li X, Zang J, Sun G, Liu Y (2017) Method of combined production of 1,4-cyclohexane dimethanol and cyclohexyl-1,4-diformaldehyde. China Patent CN106748704A, 31 May 2017

[40] Zhou WY, Tian P, Sun FA, He MY, Chen Q. Highly efficient transformation of alcohol to carbonyl compounds under a hybrid bifunctional catalyst originated from metalloporphyrins and hydrotalcite. J Catal, 2016, 335: 105-116.

[41] Zhou JY, Xu GC, Ni Y. Stereochemistry in asymmetric reduction of bulky-bulky ketones by alcohol dehydrogenases. ACS Catal, 2020, 10(19): 10954-10966.

Funding

National Key R & D Program of China(2021YFC2100100)

Innovative Research Group Project of the National Natural Science Foundation of China(22178146)

Natural Science Foundation of Jiangsu Province(BK20200622)