Improved Herbicide Resistance of 4-Hydroxyphenylpyruvate Dioxygenase from Sphingobium sp. TPM-19 through Directed Evolution

Abstract

4-Hydroxyphenylpyruvate dioxygenase (HPPD) has attracted extensive interest as a promising target for the genetic engineering of herbicide-resistant crops. However, naturally occurring HPPDs are generally very sensitive to HPPD inhibitors. In this study, random mutagenesis was performed to increase the HPPD inhibitors’ resistance of Sphingobium sp. HPPD (SpHPPD). Two mutants, Q258M and Y333F, with improved resistance were obtained. Subsequently, a double-mutant (Q258M/Y333F) was generated through combined mutation. Q258M/Y333F exhibited the highest resistance to four HPPD inhibitors [topramezone, mesotrione, tembotrione, and diketonitrile (DKN)]. The enzyme fitness of Q258M/Y333F to topramezone, mesotrione, tembotrione, and DKN was increased by 4.0-, 4.1-, 4.2-, and 3.2-folds, respectively, in comparison with that of the wild-type. Molecular modeling and docking revealed that Q258M mutation leads to the decrease of enzyme-inhibitor-binding strength by breaking the hydrogen bond between the enzyme and the inhibitor, and Y333F mutation changes the conformational balance of the C-terminal helix H11, which hinders the binding of the inhibitor to the enzyme and thus would contribute to improved herbicide resistance. This study helps to further elucidate the structural basis for herbicide resistance and provides better genetic resources for the genetic engineering of herbicide-resistant crops.

Mutations of Asn321 and Glu322 Improve Resistance of 4-Hydroxyphenylpyruvate Dioxygenase SpHPPDm to Topramezone

“…The half maximal inhibitory concentration (IC 50 ) value of topramezone against SpHPPDm mutants was determined as previously described. 24 The concentrations of topramezone and 4-HPP ranged from 0 to 100 μM and from 0 to 500 μM, respectively.…”

“…The HPPD activity of purified proteins was measured as previously described. 24 The 1.0 mL enzyme reaction mixture consists of 2.0 mM 4-HPP, 1.0 mM FeSO 4 , 4.0 mM sodium ascorbate, 50 mM PBS buffer (pH 7.4), and an appropriate amount of purified protein. The enzymatic reaction was carried out at 35 °C for 10 min and terminated by the addition of 10% (v/v) trifluoroacetic acid.…”

“…Previously, we obtained a mutant of SpHPPD with increased topramezone resistance using 400 mM topramezone as a selection pressure. 24 In this study, we increased the topramezone concentration to 900 mM to screen mutants with higher resistance. A random mutant library of SpHPPDm was constructed by epPCR using E. coli BL21-(DE3) cells as the host.…”

“…A mutant of SpHPPD, named SpHPPDm, was obtained through directed evolution. 24 SpHPPDm has two mutation sites, Q258M (glutamine → methionine) and Y333F (tyrosine → phenylalanine), and its resistance to topramezone increased 4.7-fold when compared to SpHPPD.…”

Emerging Strategies for the Bioremediation of the Phenylurea Herbicide Diuron

“…Many different enzymatic processes are involved in biodegradation by microorganisms, including hydroxylation, demethylation, dechlorination, and oxidation (Liu et al, 2020;Bhatt et al, 2021e). Different biodegradation processes are associated with different enzymes, such as hydrolases, esterases, dehydrogenases, laccases, and lignin peroxidases (Maqbool et al, 2016;Lin et al, 2020;Mishra et al, 2020).…”