Improving The Resistance To Topramezone Of 4-Hydroxypyruvate Dioxygenase SpHPPD Through Directed Evolution

4-Hydroxyphenylpyruvate dioxygenase(HPPD,EC1.13.11.27)is a newly discovered herbicide target enzyme in recent years.Topramezone is one species of HPPD inhibitor herbicide of pyrazolones.With the advantages of broad-spectrum,high efficiency and safety,environmental friendliness,low resistance risk,and no cross-resistance with other herbicides,it is regarded as an ideal target herbicide to building herbicide-resistant transgenic plants.However,there are few Sp HPPD with high HPPD inhibitor resistance in nature,it is of great application value to obtain HPPD with high topramezone-resistance.Nowadays,directed evolution could effectively improve properties of enzyme in a relatively short period of time.Therefore,we can increase the topramezone-resistance of HPPD through directed evolution,which is conducive to alleviating the problems of weed resistance and provides more gene resources with high resistance for the construction of topramezone-resistant transgenic crops.In previous study,we cloned the gene of HPPD with high topramezone-resistance from Sphingobium sp.TPM-19,and obtained a mutant Q258M/Y333 F of Sp HPPD by directed evolution.In this study,the topramezone-resistance of Q258M/Y333 F was further improved by directed evolution.The major achievements are as follows:1、Construction of mutant library and screening of high topramezone-resistant mutantsIn this study,with the gene of Q258M/Y333 F as starting material,we established a random mutant library by ep PCR.Under the selection pressure of topramezone,four mutants contained in recombinant strains that could tolerate high concentration of topramezone were screened,which were named as K-19,K-28,K-63 and K-113,respectively.And their acid mutation sites were N321Y(K-19),E322R(K-28),Q166 R and E322V(K-63),G327 C and K249R(K-113).Using p ET-29 a to heterologous express four mutants,the specific enzyme activity value of purified mutants K-19,K-28,K-63 and K-113 showed that all mutants had high enzyme activity.Compared with Q258M/Y333 F,the topramezone-resistance of K-19,K-28,K-63 and K-113 increased by 2.0,0.6,0.8 and 2.0folds,respectively.The mesotrione-resistance of K-19,K-28 and K-113 increased by 1.4,0.8 and 0.6 folds,respectively,but the mesotrioneresistance of K-63 showed no change.The DKN-resistance of K-19 and K-113 increased by 1.0 and 1.7folds,respectively,but the DKN-resistance of K-28 decreased by about 20.7%,and the DKN-resistance of K-63 showed no change.2、Effects of mutation sites on resistance of mutants K-63 and K-113Mutants K-63 and K-113 contained two amino acid mutation sites respectively.In order to explain the effect of each mutation site to resistance and enzyme activity of the mutants,the single point mutants Q166 R and E322 V of K-63,K249 R and G327 C of K-113 were constructed by site-directed mutagenesis.The specific enzyme activities of purified Q166 R,E322V,K249 R and G327 C all had high enzyme activity..Compared with Q258M/Y333 F,the topramezone-resistance of Q166 R increased by 0.7 folds,While the topramezoneresistance of E322 V increased by 0.8 folds.The resistance of G327 C to topramezone,mesotrione and DKN increased by 1.8,0.6 and 1.7 folds,respectively.However,the resistance to the three inhibitors of K249 R showed no change.The above results suggested that all the single-point mutants had high enzyme activities,and the resistance change of K-63 was mainly affected by amino acid mutations at 322,while the resistance change of K-113 was only related to amino acid mutations at site 327.3、Three-dimensional structure simulation of mutants and the mechanism of resistance affected by each mutation sitesThe three-dimensional structure simulation of mutants showed that the structure of Sp HPPD was similar to that of Pseudomonas.fluorescens HPPD,which existed in the form of tetramer,each subunit contained two complementary domains composed of β-folding andα-helix,and the active center at the C-terminal was a bucket-like hydrophobic cavity.According to the results of analyzing the amino acid mutation sites of each mutant,we presumed that the amino acid mutations at sites 321 and 322 affected the flexibility of the active Loop region,thus restricting the conformation of H11.The amino acid at site 327 located on the H11 that acted as gating-control was Cys,its side chain was larger,which prevented the inhibitor from entering the active center.The amino acid mutation at 166 may affect the coordination between His157 on the A3-fold and Fe(Ⅱ),or affect the conformation of A3-fold locating at the top of the active center,resulting in the reduction of the top volume of the cavity and hindering the entry of the inhibitor.4、Combination mutagenesis of key pointsAccording to the results of the study on the resistance mechanism of each mutation site,it was speculated that 321 and 322 sites were the key sites affecting the resistance of Sp HPPD.Therefore,we constructed combination mutants N321Y/E322 R and N321Y/E322 V by combining key points 321 and 322.The specific enzyme activities of purified N321Y/E322 R and N321Y/E322 V all had high enzyme activity.Compared with Q258M/Y333 F,the resistance of N321Y/E322 R to topramezone,mesotrione and DKN increased by 4.3,3.3 and1.7 folds,respectively.The resistance of N321Y/E322 V to topramezone,mesotrione and DKN increased by 5.3,3.1 and 1.7 folds,respectively.The results showed that both N321Y/E322 R and N321Y/E322 V had high enzyme activities.Sites 321 and 322 are the key sites affecting the resistance of Sp HPPD,and the synergistic amino acid combination can improve the resistance of Sp HPPD.5、Saturation mutagenesis of key pointsIn order to explore the best synergistic combination of amino acid at 321 and 322 sites which could improve the topramezone-resistance of Sp HPPD for further,we established a saturation mutation library by saturating mutations at 321 and 322.Under the selection pressure of topramezone,three saturation mutants contained in recombinant strains that could tolerate high concentration of topramezone were screened,which were named as KB-31,KB-55 and KB-145,respectively.The specific enzyme activity of purified KB-31,KB-55 and K-145 indicated that all the saturation mutants had high enzyme activity.Compared with Q258M/Y333 F,the resistance of KB-31 to topramezone,mesotrione and DKN increased by5.8,3.5 and 1.0 folds,respectively.The resistance of KB-55 to three HPPH inhibitors increased by 6.0,3.8 and 2.1 folds,respectively.The resistance of KB-145 to three HPPD inhibitors increased by 9.3,4.9 and 2.3 folds,respectively.In summary,we have obtained several mutants with further improvement of topramezone-resistance.In addition,we found that 321 and 322 sites were the key sites affecting the resistance of Sp HPPD,and the amino acid combination on those two sites also affected the resistance of Sp HPPD to different inhibitors in various degree.