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Improving glyphosate oxidation activity of glycine oxidase from Bacillus cereus by directed evolution.

Zhan T, Zhang K, Chen Y, Lin Y, Wu G, Zhang L, Yao P, Shao Z, Liu Z - PLoS ONE (2013)

Bottom Line: Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling.Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities.These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, P. R. China.

ABSTRACT
Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg(51) mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops.

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Comparison of catalytic efficiency (the ratios of kcat/Km) of wild-type BceGO and evolved variants.
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pone-0079175-g001: Comparison of catalytic efficiency (the ratios of kcat/Km) of wild-type BceGO and evolved variants.

Mentions: Starting from mutant B1R, a second round of error-prone PCR was performed, and six mutants (B2R3, B2R6, B2R11, B2R14, B2R23 and B2R81) showed improved activity on glyphosate as compared with wild-type BceGO. For all the six variants, the changes of kcat,app parameters were prominent for the substitution of G60S introduced in the second round of random mutagenesis, and the kcat,app value of B2R23 was 11.08-fold that of the starting template (mutant B1R). These results demonstrated that BceGO had a considerable evolutionary landscape for improving kinetic efficiency on glyphosate. Further promotion of glyphosate oxidase activity by DNA shuffling was apparent in the third generation, obtaining four recombinants (B3S1, B3S4, B3S6 and B3S7) with a higher activity than those in the second generation. As shown in Table 3 and Figure 1, variation in the kinetic parameters of recombinants can reflect how the catalytic efficiency on glyphosate was improved: a significant decrease in Km for all recombinants, and a high kcat value for all mutants except B3S7. Especially, B3S1 demonstrated a 160-fold increase in substrate affinity for glyphosate, a 326-fold increase in catalytic efficiency towards glyphosate and a significant enhancement in the specificity constant over the wild-type BceGO, achieving the goal of efficient oxidation of glyphosate by evolution of glycine oxidase.


Improving glyphosate oxidation activity of glycine oxidase from Bacillus cereus by directed evolution.

Zhan T, Zhang K, Chen Y, Lin Y, Wu G, Zhang L, Yao P, Shao Z, Liu Z - PLoS ONE (2013)

Comparison of catalytic efficiency (the ratios of kcat/Km) of wild-type BceGO and evolved variants.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818420&req=5

pone-0079175-g001: Comparison of catalytic efficiency (the ratios of kcat/Km) of wild-type BceGO and evolved variants.
Mentions: Starting from mutant B1R, a second round of error-prone PCR was performed, and six mutants (B2R3, B2R6, B2R11, B2R14, B2R23 and B2R81) showed improved activity on glyphosate as compared with wild-type BceGO. For all the six variants, the changes of kcat,app parameters were prominent for the substitution of G60S introduced in the second round of random mutagenesis, and the kcat,app value of B2R23 was 11.08-fold that of the starting template (mutant B1R). These results demonstrated that BceGO had a considerable evolutionary landscape for improving kinetic efficiency on glyphosate. Further promotion of glyphosate oxidase activity by DNA shuffling was apparent in the third generation, obtaining four recombinants (B3S1, B3S4, B3S6 and B3S7) with a higher activity than those in the second generation. As shown in Table 3 and Figure 1, variation in the kinetic parameters of recombinants can reflect how the catalytic efficiency on glyphosate was improved: a significant decrease in Km for all recombinants, and a high kcat value for all mutants except B3S7. Especially, B3S1 demonstrated a 160-fold increase in substrate affinity for glyphosate, a 326-fold increase in catalytic efficiency towards glyphosate and a significant enhancement in the specificity constant over the wild-type BceGO, achieving the goal of efficient oxidation of glyphosate by evolution of glycine oxidase.

Bottom Line: Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling.Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities.These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, P. R. China.

ABSTRACT
Glyphosate, a broad spectrum herbicide widely used in agriculture all over the world, inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, and glycine oxidase (GO) has been reported to be able to catalyze the oxidative deamination of various amines and cleave the C-N bond in glyphosate. Here, in an effort to improve the catalytic activity of the glycine oxidase that was cloned from a glyphosate-degrading marine strain of Bacillus cereus (BceGO), we used a bacteriophage T7 lysis-based method for high-throughput screening of oxidase activity and engineered the gene encoding BceGO by directed evolution. Six mutants exhibiting enhanced activity toward glyphosate were screened from two rounds of error-prone PCR combined with site directed mutagenesis, and the beneficial mutations of the six evolved variants were recombined by DNA shuffling. Four recombinants were generated and, when compared with the wild-type BceGO, the most active mutant B3S1 showed the highest activity, exhibiting a 160-fold increase in substrate affinity, a 326-fold enhancement in catalytic efficiency against glyphosate, with little difference between their pH and temperature stabilities. The role of these mutations was explored through structure modeling and molecular docking, revealing that the Arg(51) mutation is near the active site and could be an important residue contributing to the stabilization of glyphosate binding, while the role of the remaining mutations is unclear. These results provide insight into the application of directed evolution in optimizing glycine oxidase function and have laid a foundation for the development of glyphosate-tolerant crops.

Show MeSH
Related in: MedlinePlus