ε-Caprolactone is a monomer of poly(ε-caprolactone) which has been widely used in tissue engineering due to its biodegradability and biocompatibility. To meet the massive demand for this monomer, an efficient whole-cell biocatalytic approach was constructed to boost the ε-caprolactone production using cyclohexanol as substrate. Combining an alcohol dehydrogenase (ADH) with a cyclohexanone monooxygenase (CHMO) in Escherichia coli, a self-sufficient NADPH-cofactor regeneration system was obtained. Furthermore, some improved variants with the better substrate tolerance and higher catalytic ability to ε-caprolactone production were designed by regulating the ribosome binding sites. The best mutant strain exhibited an ε-caprolactone yield of 0.80 mol/mol using 60 mM cyclohexanol as substrate, while the starting strain only got a conversion of 0.38 mol/mol when 20 mM cyclohexanol was supplemented. The engineered whole-cell biocatalyst was used in four sequential batches to achieve a production of 126 mM ε-caprolactone with a high molar yield of 0.78 mol/mol.
| [1] |
Ban N, Nissen P, Hansen J, . The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution. Science, 2000, 289: 905-920.
|
| [2] |
Espah Borujeni A, Channarasappa AS, Salis HM. Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites. Nucleic Acids Res, 2014, 42: 2646-2659.
|
| [3] |
France SP, Hepworth LJ, Turner NJ, . Constructing biocatalytic cascades: in vitro and in vivo approaches to de novo multi-enzyme pathways. ACS Catalysis, 2017, 7: 710-724.
|
| [4] |
Gandomkar S, Żądło-Dobrowolska A, Kroutil W. Extending designed linear biocatalytic cascades for organic synthesis. ChemCatChem, 2019, 11: 225-243.
|
| [5] |
Hildebrand F, Kohlmann C, Franz A, . Synthesis, characterization and application of new rhodium complexes for indirect electrochemical cofactor regeneration. Adv Synth Catal, 2008, 350: 909-918.
|
| [6] |
Hollmann F, Arends IWCE, Holtmann D. Enzymatic reductions for the chemist. Green Chem, 2011, 13: 2285-2314.
|
| [7] |
Karande R, Salamanca D, Schmid A, . Biocatalytic conversion of cycloalkanes to lactones using an in-vivo cascade in Pseudomonas taiwanensis VLB120. Biotechnol Bioeng, 2018, 115: 312-320.
|
| [8] |
Kohl A, Srinivasamurthy V, Bottcher D, . Co-expression of an alcohol dehydrogenase and a cyclohexanone monooxygenase for cascade reactions facilitates the regeneration of the NADPH cofactor. Enzyme Microb Technol, 2018, 108: 53-58.
|
| [9] |
Lee WH, Park JB, Park K, . Enhanced production of ɛ-caprolactone by overexpression of NADPH-regenerating glucose 6-phosphate dehydrogenase in recombinant Escherichia coli harboring cyclohexanone monooxygenase gene. Appl Microbiol Biotechnol, 2007, 76: 329-338.
|
| [10] |
Mallin H, Wulf H, Bornscheuer UT. A self-sufficient Baeyer-Villiger biocatalysis system for the synthesis of ɛ-caprolactone from cyclohexanol. Enzyme Microb Technol, 2013, 53: 283-287.
|
| [11] |
Ménil S, Petit J-L, Courvoisier-Dezord E, . Tuning of the enzyme ratio in a neutral redox convergent cascade: a key approach for an efficient one-pot/two-step biocatalytic whole-cell system. Biotechnol Bioeng, 2019, 116: 2852-2863.
|
| [12] |
Milker S, Goncalves LCP, Fink MJ et al (2017) Escherichia coli fails to efficiently maintain the activity of an important flavin monooxygenase in recombinant overexpression. Front Microbiol 8
|
| [13] |
De Novo DNA (2018) https://salislab.net/software/. Accessed 20 Oct 2018
|
| [14] |
Pathak VM, Navneet (2017) Review on the current status of polymer degradation: a microbial approach. Biores Bioprocessing 4
|
| [15] |
Quinto T, Köhler V, Ward TR. Recent trends in biomimetic NADH regeneration. Top Catal, 2014, 57: 321-331.
|
| [16] |
Salis HM, Mirsky EA, Voigt CA. Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol, 2009, 27: 946-950.
|
| [17] |
Scherkus C, Schmidt S, Bornscheuer UT, . A fed-batch synthetic strategy for a three-step enzymatic synthesis of poly-ε-caprolactone. ChemCatChem, 2016, 8: 3446-3452.
|
| [18] |
Scherkus C, Schmidt S, Bornscheuer UT, . Kinetic insights into ε-caprolactone synthesis: improvement of an enzymatic cascade reaction. Biotechnol Bioeng, 2017, 114: 1215-1221.
|
| [19] |
Schmidt S, Büchsenschütz HC, Scherkus C, . Biocatalytic access to chiral polyesters by an artificial enzyme cascade synthesis. ChemCatChem, 2015, 7: 3951-3955.
|
| [20] |
Schmidt S, Scherkus C, Muschiol J, . An enzyme cascade synthesis of ε-caprolactone and its oligomers. Angew Chem Int Edit, 2015, 54: 2784-2787.
|
| [21] |
Silva ALP, Batista PK, Filho AD, . Rapid conversion of cyclohexenone, cyclohexanone and cyclohexanol to ε-caprolactone by whole cells of Geotrichum candidum CCT 1205. Biocatal Biotransfor, 2017, 35: 185-190.
|
| [22] |
Spaans S, Weusthuis R, VanDer OJ, . NADPH-generating systems in bacteria and archaea. Front Microbiol, 2015
|
| [23] |
Staudt S, Bornscheuer UT, Menyes U, . Direct biocatalytic one-pot-transformation of cyclohexanol with molecular oxygen into ɛ-caprolactone. Enzyme Microb Technol, 2013, 53: 288-292.
|
| [24] |
Sun T, Li B, Nie Y et al (2017) Enhancement of asymmetric bioreduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine to corresponding (S)-enantiomer by fusion of carbonyl reductase and glucose dehydrogenase. Bioresources and Bioprocessing 4
|
| [25] |
ten Brink GJ, Arends IWCE, Sheldon RA. The Baeyer−Villiger reaction: new developments toward greener procedures. Chem Rev, 2004, 104: 4105-4124.
|
| [26] |
Tolia NH, Joshua-Tor L. Strategies for protein coexpression in Escherichia coli. Nat Methods, 2006, 3: 55-64.
|
| [27] |
Wang M, Chen B, Fang Y, . Cofactor engineering for more efficient production of chemicals and biofuels. Biotechnol Adv, 2017, 35: 1032-1039.
|
| [28] |
Wang X, Saba T, Yiu HHP, . Cofactor NAD(P)H regeneration inspired by heterogeneous pathways. Chem, 2017, 2: 621-654.
|
| [29] |
Weckbecker A, Gröger H, Hummel W. Wittmann C, Krull R. Regeneration of nicotinamide coenzymes: principles and applications for the synthesis of chiral compounds. Biosystems engineering I: creating superior biocatalysts, 2010, Berlin, Heidelberg: Springer.
|
| [30] |
Willetts AJ, Knowles CJ, Levitt MS, . Biotransformation of endo-bicyclo[2.2.1]heptan-2-ols and endo-bicyclo[3.2.0]hept-2-en-6-ol into the corresponding lactones. J Chem Soc Perk T, 1991, 1: 1608-1610.
|
| [31] |
Wu H, Tian C, Song X, . Methods for the regeneration of nicotinamide coenzymes. Green Chem, 2013, 15: 1773-1789.
|
| [32] |
Xu MQ, Li FL, Yu WQ, . Combined cross-linked enzyme aggregates of glycerol dehydrogenase and NADH oxidase for high efficiency in situ NAD+ regeneration. Int J Biol Macromol, 2019, 144: 1013-1021.
|
| [33] |
Zhang C, Song W, Liu J, . Production of enantiopure (R)-or (S)-2-hydroxy-4-(methylthio)butanoic acid by multi-enzyme cascades. Biores Bioprocess, 2019
|
Funding
the National Key R&D Program of China(2018YFA0901504)
the Natural Science Foundation of China(Grants 21878104 and U1701243)
the Project on the Integration of Industry, Education, and Research of Guangzhou, China(Grants 201903010086)
the Young Talents in Scientific and Technological Innovation of the Special Support Plan
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