Improving the herbicide resistance of 4-hydroxyphenylpyruvate dioxygenase SpHPPD by directed evolution

Topramezone, a highly efficient 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibitor herbicide, is an ideal target for herbicide-resistant genetic engineering. However, there is still a lack of HPPD gene that is highly resistant to topramezone. In previous studies, we obtained a topramezone-resistant HPPD (SpHPPDm) gene from Sphingobium sp. TPM-19, however, its resistance strength still could not meet the requirements for construction of herbicide-resistant crop. In this study, random mutagenesis (error-prone PCR) was employed to improve the topramezone resistance of SpHPPDm. Two mutants with improved resistance, K-28 (E322R) and K-113 (K249R, G327C), were screened from the random mutation library of SpHPPDm. The catalytic efficiency (k_cat/K_m) of mutants K-28 and K-113 only slightly decreased by approximately 2%. The half-maximal inhibitory concentration (IC₅₀) of topramezone increased by 58.5% and 195.5% for mutants K-28 and K-113, respectively. Furthermore, mutant K-113 also showed significantly improved resistance to mesotrione and DKN (the active ingredient of isoxaflutole) with the IC₅₀ increasing by 60.3% and 167.5%, respectively; while mutant K-28 only showed increased resistance to mesotrione with IC₅₀ increasing by 77.6%, but reduced resistance to DKN with IC₅₀ declining by 20.9%. Site-directed mutation assays revealed that G327C, but not K249R, contributed to topramezone resistance in mutant K-113. This study provides genetic resources for the genetic engineering of HPPD-inhibitor-resistant crops and a basis for further research on HPPD resistance mechanisms.