Formate dehydrogenase (FDH) is often used in enzymatic cofactor regeneration system in the fermentation industry. To engineer the cofactor specificity of FDH from Komagataella phaffii (KphFDH) and apply it to an in vitro NADPH cofactor regeneration system, this study identified D195, Y196 as mutation sites based on multiple sequence alignment and literature review, constructed 16 variants and characterized their specific activities and kinetics of enzymatic reaction. The optimal NADP+-specific variant was KphFDHD195Q/Y196R, which had a catalytic efficiency (kcat/Km) of 1.967 (mmol·s)-1 for NADP+, a ratio of catalytic efficiency ((kcat/KmNADP+)/(kcat/KmNAD+)) of 36.426, and a catalytic efficiency (kcat/Km) of 0.02 (mmol·s)-1 for formate when using NADP+ as cofactor. Finally, this variant was successfully applied to the regeneration of NADPH in the phenylpyruvic acid (PPA) reductive amination reaction, which almost completely reacted within 200 min. This study fills a research gap in cofactor-specific engineering of FDH from K. phaffii, provides a tool enzyme for the application of in vitro NADPH cofactor regeneration system in the fermentation industry, and also offers a candidate variant for further engineering its cofactor specificity.
HU Liyuan
,
LIU Luyao
,
BAI Zhonghu
,
YANG Yankun
,
JIN Jian
. Cofactor specificity engineering and application of formate dehydrogenase from Komagataella phaffii[J]. Food and Fermentation Industries, 2023
, 49(13)
: 9
-16
.
DOI: 10.13995/j.cnki.11-1802/ts.035349
[1] 黄志华, 刘铭, 王宝光, 等.甲酸脱氢酶用于辅酶NADH再生的研究进展[J].过程工程学报, 2006, 6(6):1011-1016.
HUANG Z H, LIU M, WANG B G, et al.Formate dehydrogenase and its application in cofactor NADH regeneration[J].The Chinese Journal of Process Engineering, 2006, 6(6):1011-1016.
[2] VAN DER DONK W A, ZHAO H M.Recent developments in pyridine nucleotide regeneration[J].Current Opinion in Biotechnology, 2003, 14(4):421-426.
[3] WICHMANN R, VASIC-RACKI D.Cofactor regeneration at the lab scale[J].Advances in Biochemical Engineering/Biotechnology, 2005, 92:225-260.
[4] 江金鹏, 吴旭日, 陈依军.解决氧化还原酶反应体系中辅酶问题的策略及其应用[J].生物工程学报, 2012, 28(4):410-419.
JIANG J P, WU X R, CHEN Y J.Strategy to solve cofactor issues in oxidoreductase catalyzed biocatalytic applications[J].Chinese Journal of Biotechnology, 2012, 28(4):410-419.
[5] TISHKOV V I, POPOV V O.Protein engineering of formate dehydrogenase[J].Biomolecular Engineering, 2006, 23(2-3):89-110.
[6] 蔡礼年, 黄春辉, 林陈水.毕赤酵母甲酸脱氢酶在大肠杆菌中的融合表达及其酶学性质[J].氨基酸和生物资源, 2015, 37(1):30-34.
CAI L N, HUANG C H, LIN C S.Fusion expression of formate dehydrogenase from Pichia pastoris in E.coli and its enzymatic property[J].Biotic Resources, 2015, 37(1):30-34.
[7] GAO X Z, HUANG F, FENG J H, et al.Engineering the meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum by site saturation mutagenesis for D-phenylalanine synthesis[J].Applied and Environmental Microbiology, 2013, 79(16):5078-5081.
[8] ZHANG D P, JING X R, ZHANG W L, et al.Highly selective synthesis of D-amino acids from readily available L-amino acids by a one-pot biocatalytic stereoinversion cascade[J].RSC Advances, 2019, 9(51):29927-29935.
[9] MOLLA G, MELIS R, POLLEGIONI L.Breaking the mirror:L-Amino acid deaminase, a novel stereoselective biocatalyst[J].Biotechnology Advances, 2017, 35(6):657-668.
[10] ANDREADELI A, PLATIS D, TISHKOV V, et al.Structure-guided alteration of coenzyme specificity of formate dehydrogenase by saturation mutagenesis to enable efficient utilization of NADP+[J].The FEBS Journal, 2008, 275(15):3859-3869.
[11] WU W H, ZHU D M, HUA L.Site-saturation mutagenesis of formate dehydrogenase from Candida bodinii creating effective NADP+-dependent FDH enzymes[J].Journal of Molecular Catalysis B:Enzymatic, 2009, 61(3-4):157-161.
[12] 倪敏君. 酿酒酵母甲酸脱氢酶及其辅酶专一性突变体在大肠杆菌中的高表达,纯化及性质研究[D].上海:华东理工大学, 2010.
NI M J.Expression, purification, and characterization of formate dehydrogenase from Saccharomyces cerevisiae and its two coenzyme-specificity mutants[D].Shanghai:East China University of Science and Technology, 2010.
[13] SEROV A E, POPOVA A S, FEDORCHUK V V, et al.Engineering of coenzyme specificity of formate dehydrogenase from Saccharomyces cerevisiae[J].The Biochemical Journal, 2002, 367(3):841-847.
[14] GUL-KARAGULER N, SESSIONS R B, CLARKE A R, et al.A single mutation in the NAD-specific formate dehydrogenase from Candida methylica allows the enzyme to use NADP[J].Biotechnology Letters, 2001, 23(4):283-287.
[15] ÖZGÜN G P, ORDU E B, TÜTÜNCÜ H E, et al.Site saturation mutagenesis applications on Candida methylica formate dehydrogenase[J].Scientifica, 2016, 2016:4902450.
[16] GALKIN A G, KUTSENKO A S, BAJULINA N P, et al.Site-directed mutagenesis of the essential arginine of the formate dehydrogenase active centre[J].Biochimica et Biophysica Acta, 2002, 1594(1):136-149.
[17] LAMZIN V S, DAUTER Z, POPOV V O, et al.High resolution structures of holo and apo formate dehydrogenase[J].Journal of Molecular Biology, 1994, 236(3):759-785.
[18] KATO N, SAHM H, WAGNER F.Steady-state kinetics of formaldehyde dehydrogenase and formate dehydrogenase from a methanol-utilizing yeast, Candida boidinii[J].Biochimica et Biophysica Acta (BBA)-Enzymology, 1979, 566(1):12-20.
[19] 程峰, 魏澜, 王成娇, 等.甲酸脱氢酶及其在手性生物制造中的应用[J].生物工程学报, 2022, 38(2):632-649.
CHENG F, WEI L, WANG C J, et al.Formate dehydrogenase and its application in biomanufacturing of chiral chemicals[J].Chinese Journal of Biotechnology, 2022, 38(2):632-649.
[20] LINDNER S N, RAMIREZ L C, KRÜSEMANN J L, et al.NADPH-auxotrophic E.coli:A sensor strain for testing in vivo regeneration of NADPH[J].ACS Synthetic Biology, 2018, 7(12):2742-2749.
