Isolation and Characterization of a Topramezone-Resistant 4-Hydroxyphenylpyruvate Dioxygenase from Sphingobium sp. TPM-19

Abstract

Topramezone is a 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor. Due to its broad-spectrum, high efficiency, and low toxicity, topramezone is a candidate herbicide for the construction of genetically modified (GM) herbicide-resistant crops. In the present study, we screened a topramezone-resistant isolate Sphingobium sp. TPM-19 and cloned a topramezone-resistant HPPD gene (SphppD) from this isolate. SpHPPD shared the highest similarity (53%) with an HPPD from Vibrio vulnificus CMCP6. SpHPPD was synthesized in Escherichia coli BL21­(DE3) and purified to homogeneity using Co2+-affinity chromatography. SpHPPD was found to be a monomer. The K m and k cat of SpHPPD for 4-hydroxyphenylpyruvate (4-HPP) were 82.8 μM and 15.0 s–1, respectively. SpHPPD showed high resistance to topramezone with half maximal inhibitory concentration (IC50) and K i values of 5.2 and 2.5 μM, respectively. Additionally, SpHPPD also showed high resistance to isoxaflutole (DKN) (IC50: 8.7 μM; K i: 6.0 μM) and mesotrione (IC50: 4.2 μM; K i: 1.3 μM) and moderate resistance to tembotrione (IC50: 2.5 μM; K i: 1.0 μM). The introduction of the SphppD gene into Arabidopsis thaliana enhanced obvious resistance against topramezone. In conclusion, this study provides a novel topramezone-resistant HPPD gene for the genetic engineering of GM herbicide-resistant crops.

Vibrio splendidus virulence to Apostichopus japonicus is mediated by hppD through glutamate metabolism and flagellum assembly

“…The 4-hppD catalytic reaction is the first committed reaction of the tyrosine catabolism pathway in essentially all aerobic organisms [ 38 ]. Homologues of 4-hppD have been identified from the bacterial pathogens Streptomyces avermitilis [ 39 , 40 ], Vibrio vulnificus [ 41 ], V. splendidus [ 17 ], Pseudomonas fluorescens [ 42 ] and Legionella pneumophila [ 43 , 44 ]. In our previous studies, the dual functions of 4-hppD for V. splendidus cytotoxicity and survival was determined by in vitro experiments [ 22 ].…”

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

“…The vector pET-29a(+) (Novagen, Madison, WI, USA) was used for protein expression. The recombinant plasmid pET29-SphppD (pET29a(+) derivative carrying SphppD) constructed in our previous work 12 was used as the template for SphppD amplification. E. coli BL21(DE3) was aerobically cultured at 37 °C in LB or TLB medium supplemented with 50 mg L −1 kanamycin, which was used as a host strain for the construction of the mutagenesis library and protein expression.…”

“…The half-maximal inhibitory concentration (IC 50 ) values and K i values were determined according to the standard reaction system and calculated as described previously. 12 Effects of Temperature and pH on Enzyme Activity and Stability. The effects of temperature on activities of the wild-type and mutants were measured at different temperatures ranging from 16 to 45 °C in 50 mM PBS buffer (pH 6.0).…”

“…TPM-19. 12 SpHPPD showed resistance to mesotrione, DKN, and tembotrione and had a relatively high catalytic efficiency (181.2 mM −1 s −1 ), thus having application potential in the construction of topramezone-resistant crops. However, our previous study showed that SphppD transgenic A. thaliana could tolerate the field application rate (25 g a.i./ha) of 1× topramezone but could not tolerate 4× topramezone, which is the recommended application rate.…”

“…However, our previous study showed that SphppD transgenic A. thaliana could tolerate the field application rate (25 g a.i./ha) of 1× topramezone but could not tolerate 4× topramezone, which is the recommended application rate. 12 In the present study, we used error-prone PCR (epPCR) to construct a SphppD random mutant library. Then, an efficient screening method based on the ability of the recombinant Escherichia coli to produce a red-brown pigment was established.…”