1,3-Propanediol oxidoreductase (PDOR) is a key regulating enzyme which catalyzes the rate-determining step in the biosynthesis of 1,3-propanediol (1,3-PD) from glycerol. Rational design of site-directed mutations was performed to achieve better catalytic activity, thermal stability, and pH tolerance, through calculating the free folding energy of Klebsiella pneumoniae PDOR(KpPDOR) with PoPMuSiC 2.1 and identifying functional hotspot around the catalytic center, as well as sequence conservation analysis with HotSpot Wizard 3.0. Two improved variants were obtained after directed evolution, induced expression, and purification. Their enzymatic properties are further investigated. V155N showed 1.7-fold and 2.5-fold reducing activity, compared with that of KpPDOR at pH 7.5 and 4.0 respectively. While the half-life of N262E at 37 ℃ lengthened 40%. Protein structure analysis further illustrated the improved properties. V155N introduced two hydrogen bonds into the β-sheet where it’s located, while N262E introduced another two hydrogen bonds, strengthening the rigidity of catalytic center. Thus, both variants improved the stability. The results of the conversion of glycerol into 1,3-PD by resting cells showed that the production capacity of 1,3-PD was 1.16 mmol/L of the engineered strain V155N, which was higher than that of the wild-type engineered strain (0.82 mmol/L). This study highlights a rational-design method which can be further applied in other industrial enzymes′ optimization.
FU Kaixuan
,
WANG Xueying
,
HUANG Yanzhe
,
XUE Haizhao
,
HU Yinghan
,
ZHAO Zongbao
. Rational design-based improvement of 1,3-propanediol oxidoreductase stability and activity[J]. Food and Fermentation Industries, 2023
, 49(16)
: 18
-25
.
DOI: 10.13995/j.cnki.11-1802/ts.035803
[1] PARK Y, KIM J S, LEE H E. Physical properties of the lithium bromide+1, 3-propanediol+water system[J]. International Journal of Refrigeration, 1997, 20(5):319-325.
[2] KONG P S, AROUA M K, DAUD W M A W. Conversion of crude and pure glycerol into derivatives: A feasibility evaluation[J]. Renewable and Sustainable Energy Reviews, 2016, 63:533-555.
[3] ZHOU S, LI L L, PERSEKE M, et al. Isolation and characterization of a Klebsiella pneumoniae strain from mangrove sediment for efficient biosynthesis of 1, 3-propanediol[J]. Science Bulletin, 2015, 60(5):511-521.
[4] SZYMANOWSKA-POWAŁOWSKA D, BIAŁAS W. Scale-up of anaerobic 1, 3-propanediol production by Clostridium butyricum DSP1 from crude glycerol[J]. BMC Microbiology, 2014, 14:45.
[5] DISHISHA T, PEREYRA L P, PYO S H, et al. Flux analysis of the Lactobacillus reuteri propanediol-utilization pathway for production of 3-hydroxypropionaldehyde, 3-hydroxypropionic acid and 1, 3-propanediol from glycerol[J]. Microbial Cell Factories, 2014, 13:76.
[6] MAERVOET V E T, DE MEY M, BEAUPREZ J, et al. Enhancing the microbial conversion of glycerol to 1, 3-propanediol using metabolic engineering[J]. Organic Process Research & Development, 2011, 15(1):189-202.
[7] LAMA S M, RO S M, SEOL E, et al. Characterization of 1, 3-propanediol oxidoreductase (DhaT) from Klebsiella pneumoniae J2B[J]. Biotechnology and Bioprocess Engineering, 2015, 20(6):971-979.
[8] RAVIKUMAR Y, NADARAJAN S P, YOO T H, et al. Unnatural amino acid mutagenesis-based enzyme engineering[J]. Trends in Biotechnology, 2015, 33(8):462-470.
[9] JIANG W, ZHUANG Y, WANG S Z, et al. Directed evolution and resolution mechanism of 1, 3-propanediol oxidoreductase from Klebsiella pneumoniae toward higher activity by error-prone PCR and bioinformatics[J]. PLoS One, 2015, 10(11): e0141837.
[10] PARVEZ A, RAVIKUMAR Y, BISHT R, et al. Functional and structural roles of the dimer interface in the activity and stability of Clostridium butyricum 1, 3-propanediol oxidoreductase[J]. ACS Synthetic Biology, 2022, 11(3):1261-1271.
[11] DEHOUCK Y, KWASIGROCH J M, GILIS D, et al. PoPMuSiC 2.1: A web server for the estimation of protein stability changes upon mutation and sequence optimality[J]. BMC Bioinformatics, 2011, 12:151.
[12] ZHANG S B, WU Z L. Identification of amino acid residues responsible for increased thermostability of feruloyl esterase A from Aspergillus niger using the PoPMuSiC algorithm[J]. Bioresource Technology, 2011, 102(2):2093-2096.
[13] QI X H, GUO Q, WEI Y, et al. Enhancement of pH stability and activity of glycerol dehydratase from Klebsiella pneumoniae by rational design[J]. Biotechnology Letters, 2012, 34(2):339-346.
[14] LIU B H, ZHANG J, FANG Z, et al. Enhanced thermostability of keratinase by computational design and empirical mutation[J]. Journal of Industrial Microbiology & Biotechnology, 2013, 40(7):697-704.
[15] GUO X, AN Y J, CHAI C C, et al. Construction of the R17L mutant of MtC1LPMO for improved lignocellulosic biomass conversion by rational point mutation and investigation of the mechanism by molecular dynamics simulations[J]. Bioresource Technology, 2020, 317:124024.
[16] UNGER T, JACOBOVITCH Y, DANTES A, et al. Applications of the Restriction Free (RF) cloning procedure for molecular manipulations and protein expression[J]. Journal of Structural Biology, 2010, 172(1):34-44.
[17] GUPTA A, DWIVEDI M, NAGANA GOWDA G A, et al. 1H NMR spectroscopy in the diagnosis of Klebsiella pneumoniae-induced urinary tract infection[J]. NMR in Biomedicine, 2006, 19(8):1055-1061.
[18] WU T K, LIU Y T, CHANG C H, et al. Site-saturated mutagenesis of histidine 234 of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase demonstrates dual functions in cyclization and rearrangement reactions[J]. Journal of the American Chemical Society, 2006, 128(19):6414-6419.
[19] XIE Y, AN J, YANG G Y, et al. Enhanced enzyme kinetic stability by increasing rigidity within the active site[J]. Journal of Biological Chemistry, 2014, 289(11):7994-8006.
[20] WANG Z F, YOU Y L, LI F F, et al. Research progress of NMR in natural product quantification[J]. Molecules, 2021, 26(20):6308.
[21] BRENIAUX M, DUTILH L, PETREL M, et al. Adaptation of two groups of Oenococcus oenistrains to red and white wines: The role of acidity and phenolic compounds[J]. Journal of Applied Microbiology, 2018, 125(4):1117-1127.
[22] PRASERTSAN P, SATTAYASAMITSATHIT S, METHACANON P. Enhance 1, 3-propanediol production from crude glycerol in batch and fed-batch fermentation with two-phase pH-controlled strategy[J]. Electronic Journal of Biotechnology, 2011, 14(6).DOI:10.2225/vol14-issue6-fulltext-6.
[23] RAVIKUMAR Y, NADARAJAN S P, HYEON YOO T, et al. Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications[J]. Biotechnology Journal, 2015, 10(12):1862-1876.
[24] BORNSCHEUER U T, HUISMAN G W, KAZLAUSKAS R J, et al. Engineering the third wave of biocatalysis[J]. Nature, 2012, 485(7397):185-194.
