Immobilization of engineered E. coli cells for asymmetric reduction of methyl acetoacetate to methyl-(R)-3-hydroxybutyrate

Abstract

The efficient asymmetric bio-synthesis of chiral β-hydroxy esters is of great importance for industrial production. In this work, a simple and productive engineered E.coli cell-immobilized strategy was applied for the asymmetric reduction of MAA to (R)-HBME with high enantioselectivity. Compared with the corresponding inactivated free cells, the alginate-immobilized cells remained 45% of initial activity at 50 ℃ and 65% after reuse of 10 times. After 60 days of storage at 4 ℃, the immobilized cells maintained more than 80% relative activity. Immobilization contributed significantly to the improvement of thermal stability, pH tolerance, storage stability and operation stability without affecting the yield of product. The immobilized recombinant E. coli cell had absolute enantioselectivity for the asymmetric reduction of MAA to (R)-HBME with e.e. > 99.9%. Therefore, microbial cell immobilization is a perspective approach in asymmetric synthesis of chiral β-hydroxy esters for industrial applications.

Keywords

Recombinant E. coli cell / Immobilization / Asymmetric synthesis / Methyl-(R)-3-Hydroxybutyrate

References

[1] Al-Hasawi ZM, Abdel-Hamid MI, Almutairi AW, Touliabah HE. Response of Pseudokirchneriella subcapitata in free and alginate immobilized cells to heavy metals toxicity. Molecules, 2020, 25(12): 2847.

[2] Bickerstaff GF. Immobilization of enzymes and cells. Met Mol Biol, 2006, 25: 232-232.

[3] Carvalho C. Enzymatic and whole cell catalysis: finding new strategies for old processes. Biotechnol Adv, 2011, 29(1): 75-83.

[4] Chen XH. Asymmetric synthesis of chiral alcohols catalyzed by Acetobacter sp. CCTCC M209061 and characterization of the anti-Prelog carbonyl reductase, 2013, Guangzhou: South China University of Technology.

[5] Chen XH, Wang XT, Lou WY, Li Y, Wu H, Zong MH, Smith TJ, Chen XD. Immobilization of Acetobacter sp. CCTCC M209061 for efficient asymmetric reduction of ketones and biocatalyst recycling. Microb Cell Fact, 2012, 11: 119-132.

[6] Domach MM. Perspectives and prospects for whole cell catalysis. Catal Lett, 2015, 145(1): 346-359.

[7] Donova MV, Egorova OV. Microbial steroid transformations: current state and prospects. Appl Microb Biotechnol, 2012, 94: 1423-1431.

[8] Erbeldinger M, Mesiano AJ, Russell AJ. Enzymatic catalysis of formation of z-aspartame in ionic liquid-an alternative to enzymatic catalysis in organic solvents. Biotechnol Progr, 2000, 16: 1129-1131.

[9] Greenberg N, Tartakovsky B, Yirme G, Ulitzur S, Sheintuch M. Observations and modeling of growth of immobilized microcolonies of luminous E.Coli. Chem Eng Sci., 1995, 51(5): 743-756.

[10] Güngörmüşler M, Tamayol A, Levin DB. Hydrogen production by immobilized cells of clostridium intestinale strain URNW using alginate beads. Appl Biochem Biotechnol, 2021, 193(5): 1558-1573.

[11] Guo M. Bioconversion from whole cell biocatalysis to metabolic engineering. China Biotechnol, 2010, 30: 110-115.

[12] Hollmann F, Arends IWCE, Holtmann D. Enzymatic reductions for the chemist. Green Chem, 2011, 13: 2285-2314.

[13] Huang J, Mei LH, Wu H, Lin DQ. Biosynthesis of γ-aminobutyric acid (GABA) using immobilized whole cells of Lactobacillus brevis. World J Microb Biotechnol, 2007, 23: 865-871.

[14] Lević S, Lijaković IP, Đorđević V, Rac V, Rakić V, Knudsen TŠ, Pavlović V, Bugarski B, Nedović V. Characterization of sodium alginate/D-limonene emulsions and respective calcium alginate/D-limonene beads produced by electrostatic extrusion. Food Hydrocol, 2015, 45: 111-123.

[15] Luo ZW, Lee SY. Biotransformation of p-xylene into terephthalic acid by engineered Escherichia coli. Nat Commun, 2017, 8: 15689-15877.

[16] Ou SY, Zhao J, Wang Y, Tian Y, Wang J. Preparation of octacosanol from filter mud produced after sugarcane juice clarification. Food Sci Technol, 2012, 45: 295-298.

[17] Pajic-Lijakovic I, Levic S, Hadnađev M, Stevanovic-Dajic Z, Radosevic R, Nedovic V, Bugarskia B. Structural changes of Ca-alginate beads caused by immobilized yeast cell growth. Biochem Eng J, 2015, 103: 32-38.

[18] Thomas SM, Dicosimo R, Nagarajan V. Biocatalysis: applications and potentials for the chemical industry. Trends Biotechnol, 2002, 20: 238-242.

[19] Wang XT, Chen XH, Xu Y, Lou WY, Wu H, Zong MH. Biocatalytic anti-Prelog stereoselective reduction of ethyl acetoacetate catalyzed by whole cells of Acetobacter sp. CCTCC M209061. J Biotechnol, 2013, 163: 292-300.

[20] Wei P, Xu P, Wang XT, Lou W, mZong MH, . Asymmetric reduction of ethyl acetoacetate catalyzed by immobilized Acetobacter sp. CCTCC M209061 cells in hydrophilic ionic liquid hybrid system. Biotechnol Biopro Eng, 2015, 20(2): 324-332.

[21] Wei P, Cui YH, Zong MH, . Enzymatic characterization of a recombinant carbonyl reductase from Acetobacter sp. CCTCC M20906. Bioresour Bioprocess, 2017

[22] Xie X, Tang Y. Efficient synthesis of simvastatin by use of whole-cell biocatalysis. Appl Environ Microb, 2007, 73: 2054-2067.

[23] Xu GC, Yu HL, Zhang XY, Xu JH. Access to optically active aryl halohydrins using a substrate-tolerant carbonyl reductase discovered from Kluyveromyces thermotolerans. ACS Catal, 2014, 2: 2566-2571.

Funding

National Key Research and Development Program of China(2021YFC2102800)

National Natural Science Foundation of China(21908070)