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Willem P. C. Stemmer - One of the best experts on this subject based on the ideXlab platform.
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Improved Green Fluorescent Protein by Molecular Evolution Using DNA Shuffling
Nature Biotechnology, 2004Co-Authors: Allanah Crameri, Erik A. Whitehorn, Emily Tate, Willem P. C. StemmerAbstract:Green fluorescent protein (GFP) has rapidly become a widely used reporter of gene regulation. However, for many organisms, particularly eukaryotes, a stronger whole cell fluorescence signal is desirable. We constructed a synthetic GFP gene with improved codon usage and performed recursive cycles of DNA Shuffling followed by screening for the brightest E. coli colonies. A visual screen using UV light, rather than FACS selection, was used to avoid red-shifting the excitation maximum. After 3 cycles of DNA Shuffling, a mutant was obtained with a whole cell fluorescence signal that was 45-fold greater than a standard, the commercially available Clontech plasmid pGFP. The expression level in E. coli was unaltered at about 75% of total protein. The emission and excitation maxima were also unchanged. Whereas in E. coli most of the wildtype GFP ends up in inclusion bodies, unable to activate its chromophore, most of the mutant protein is soluble and active. Three amino acid mutations appear to guide the mutant protein into the native folding pathway rather than toward aggregation. Expressed in Chinese Hamster Ovary (CHO) cells, this shuffled GFP mutant showed a 42-fold improvement over wildtype GFP sequence, and is easily detected with UV light in a wide range of assays. The results demonstrate how molecular evolution can solve a complex practical problem without needing to first identify which process is limiting. DNA Shuffling can be combined with screening of a moderate number of mutants. We envision that the combination of DNA Shuffling and high throughput screening will be a powerful tool for the optimization of many commercially important enzymes for which selections do not exist.
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Molecular breeding of genes, pathways and genomes by DNA Shuffling
Journal of Molecular Catalysis B-enzymatic, 2002Co-Authors: Willem P. C. StemmerAbstract:Abstract Classical breeding is a gentle mutagenesis process that simply creates new combinations of a large number of DNA sequence polymorphisms that pre-exist in the population, thus allowing the evolutionary optimization of very complex genomes. We have developed and applied a wide variety of derivative processes called ‘molecular breeding’ to breed single genes, contiguous pathways, distributed pathways, and even whole microbial genomes. Libraries of clones that are created by breeding are phenotypically diverse because clones tend to differ by many amino acids due to the exchange of sequence blocks, yet an exceptionally high fraction of the library is functional because the natural sequence polymorphisms were preselected for compatibility with function. A wide variety of formats and applications of molecular breeding is described.
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Molecular breeding of genes, pathways and genomes by DNA Shuffling
Biotechnology and Bioprocess Engineering, 2002Co-Authors: Willem P. C. StemmerAbstract:Existing methods for optimization of sequences by random mutagenesis generate libraries with a small number of mostly deleterious mutations, resulting in libraries containing a large fraction of non-functional clones that explore only a small part of squence space. Large numbers of clones need to be screened to find the rare mutants with improvements. Library display formats are useful to screen very large libraries but impose screening limitations that limit the value of this approach for most commercial applications. By contrast, in both classical breeding and in DNA Shuffling, natural diversity is permutated by homologous recombination, generating libraries of very high quality, from which improved clones can be identified with a small number of complex screens. Given that this small number of screens can be performed under the conditions of actual use of the product, commercially relevant improvements can be reliably obtained.
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evolution of a human immunodeficiency virus type 1 variant with enhanced replication in pig tailed macaque cells by DNA Shuffling
Journal of Virology, 2002Co-Authors: Katja Pekrun, Willem P. C. Stemmer, Phillip A Patten, Riri Shibata, Tatsuhiko Igarashi, Margaret Reed, Liana Sheppard, Malcolm A Martin, Nay Wei SoongAbstract:DNA Shuffling facilitated the evolution of a human immunodeficiency virus type 1 (HIV-1) variant with enhanced replication in pig-tailed macaque peripheral blood mononuclear cells (pt mPBMC). This variant consists exclusively of HIV-1-derived sequences with the exception of simian immunodeficiency virus (SIV) nef. Sequences spanning the gag-protease-reverse transcriptase (gag-pro-RT) region from several HIV-1 isolates were shuffled and cloned into a parental HIV-1 backbone containing SIV nef. Neither this full-length parent nor any of the unshuffled HIV-1 isolates replicated appreciably or sustainably in pt mPBMC. Upon selection of the shuffled viral libraries by serial passaging in pt mPBMC, a species emerged which replicated at substantially higher levels (50 to 100 ng/ml p24) than any of the HIV-1 parents and most importantly, could be continuously passaged in pt mPBMC. The parental HIV-1 isolates, when selected similarly, became extinct. Analyses of full-length improved proviral clones indicate that multiple recombination events in the shuffled region and adaptive changes in the rest of the genome contributed synergistically to the improved phenotype. This improved variant may prove useful in establishing a pig-tailed macaque model of HIV-1 infection.
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Directed evolution of a fucosidase from a galactosidase by DNA Shuffling and screening
Proceedings of the National Academy of Sciences, 2002Co-Authors: J. H. Zhang, Glenn Dawes, Willem P. C. StemmerAbstract:An efficient -fucosidase was evolved by DNA shuff ling from the Escherichia coli lacZ -galactosidase. Seven rounds of DNA shuff ling and colony screening on chromogenic fucose substrates were performed, using 10,000 colonies per round. Compared with native -galactosidase, the evolved enzyme purified from cells from the final round showed a 1,000-fold increased substrate specificity for o-nitrophenyl fucopyranoside versus o-nitrophenyl galactopyr-anoside and a 300-fold increased substrate specificity for p-nitrophenyl fucopyranoside versus p-nitrophenyl galacto-pyranoside. The evolved cell line showed a 66-fold increase in p-nitrophenyl fucosidase specific activity. The evolved fuco-sidase has a 10-to 20-fold increased k cat ͞K m for the fucose substrates compared with the native enzyme. The DNA se-quence of the evolved fucosidase gene showed 13 base changes, resulting in six amino acid changes from the native enzyme. This effort shows that the library size that is required to obtain significant enhancements in specificity and activity by reiterative DNA shuff ling and screening, even for an enzyme of 109 kDa, is within range of existing high-throughput technology. Reiterative generation of libraries and stepwise accumulation of improvements based on addition of beneficial mutations appears to be a promising alternative to rational design.
Quanhong Yao - One of the best experts on this subject based on the ideXlab platform.
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identification of a phosphinothricin resistant mutant of rice glutamine synthetase using DNA Shuffling
Scientific Reports, 2015Co-Authors: Yongsheng Tian, Wei Zhao, Xiaojuan Xing, Rihe Peng, Quanhong YaoAbstract:To date, only bar/pat gene derived from Streptomyces has been used to generate the commercial PPT-resistant crops currently available in the market. The limited source of bar/pat gene is probably what has caused the decrease in PPT-tolerance, which has become the main concern of those involved in field management programs. Although glutamine synthetase (GS) is the target enzyme of PPT, little study has been reported about engineering PPT-resistant plants with GS gene. Then, the plant-optimized GS gene from Oryza sativa (OsGS1S) was chemically synthesized in the present study by PTDS to identify a GS gene for developing PPT-tolerant plants. However, OsGS1S cannot be directly used for developing PPT-tolerant plants because of its poor PPT-resistance. Thus, we performed DNA Shuffling on OsGS1S, and one highly PPT-resistant mutant with mutations in four amino acids (A63E, V193A, T293A and R295K) was isolated after three rounds of DNA Shuffling and screening. Among the four amino acids substitutions, only R295K was identified as essential in altering PPT resistance. The R295K mutation has also never been previously reported as an important residue for PPT resistance. Furthermore, the mutant gene has been transformed into Saccharomyces cerevisiae and Arabidopsis to confirm its potential in developing PPT-resistant crops.
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improved glyphosate resistance of 5 enolpyruvylshikimate 3 phosphate synthase from vitis vinifera in transgenic arabidopsis and rice by DNA Shuffling
Molecular Breeding, 2015Co-Authors: Yongsheng Tian, Wei Zhao, Xiaojuan Xing, Rihe Peng, Quanhong YaoAbstract:To date, only AroA variant derived from Agrobacterium tumefaciens CP4 has been used to generate the commercial glyphosate-resistant crops currently available in the market. This single source of the EPSPS gene may have caused the decrease in herbicide tolerance, which has become a major concern of those involved in field management programs. Therefore, it is of interest to increase aroA/EPSPS gene diversity and seek new glyphosate-tolerant genes for developing glyphosate-tolerant crops. In the current study, EPSPS gene from Vitis vinifera (VvEPSPS) was cloned using reverse transcription polymerase chain reaction. However, wild type VvEPSPS cannot be directly used for developing transgenic crops because of its extreme glyphosate sensitivity. Recent studies have demonstrated that DNA Shuffling is an effective strategy in producing multi-mutated EPSPS resourced from plants (EPSPS plant ) with improved glyphosate resistance in bacteria and plants. After performing DNA Shuffling on VvEPSPS gene, one highly glyphosate-resistant mutant with seven amino acid variations was isolated after five rounds of Shuffling and screening. The mutant showed seven amino acid changes in the EPSPS gene, namely, Q93R, T113A, P117L, G126A, C160Y, N239H, and V343A. The assay of glyphosate resistance further confirmed the potential of the VvEPSPS mutant in developing glyphosate-resistant crops.
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mutation by DNA Shuffling of 5 enolpyruvylshikimate 3 phosphate synthase from malus domestica for improved glyphosate resistance
Plant Biotechnology Journal, 2013Co-Authors: Yongsheng Tian, Wei Zhao, Rihe Peng, Aisheng Xiong, Hongjuan Han, Quanhong YaoAbstract:A new 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate-tolerant plants. However, wild-type MdEPSPS is not suitable for the development of transgenic glyphosate-tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA Shuffling on MdEPSPS, and one highly glyphosate-resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA Shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site-directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single-site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate-resistant crops.
Yongsheng Tian - One of the best experts on this subject based on the ideXlab platform.
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identification of a phosphinothricin resistant mutant of rice glutamine synthetase using DNA Shuffling
Scientific Reports, 2015Co-Authors: Yongsheng Tian, Wei Zhao, Xiaojuan Xing, Rihe Peng, Quanhong YaoAbstract:To date, only bar/pat gene derived from Streptomyces has been used to generate the commercial PPT-resistant crops currently available in the market. The limited source of bar/pat gene is probably what has caused the decrease in PPT-tolerance, which has become the main concern of those involved in field management programs. Although glutamine synthetase (GS) is the target enzyme of PPT, little study has been reported about engineering PPT-resistant plants with GS gene. Then, the plant-optimized GS gene from Oryza sativa (OsGS1S) was chemically synthesized in the present study by PTDS to identify a GS gene for developing PPT-tolerant plants. However, OsGS1S cannot be directly used for developing PPT-tolerant plants because of its poor PPT-resistance. Thus, we performed DNA Shuffling on OsGS1S, and one highly PPT-resistant mutant with mutations in four amino acids (A63E, V193A, T293A and R295K) was isolated after three rounds of DNA Shuffling and screening. Among the four amino acids substitutions, only R295K was identified as essential in altering PPT resistance. The R295K mutation has also never been previously reported as an important residue for PPT resistance. Furthermore, the mutant gene has been transformed into Saccharomyces cerevisiae and Arabidopsis to confirm its potential in developing PPT-resistant crops.
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improved glyphosate resistance of 5 enolpyruvylshikimate 3 phosphate synthase from vitis vinifera in transgenic arabidopsis and rice by DNA Shuffling
Molecular Breeding, 2015Co-Authors: Yongsheng Tian, Wei Zhao, Xiaojuan Xing, Rihe Peng, Quanhong YaoAbstract:To date, only AroA variant derived from Agrobacterium tumefaciens CP4 has been used to generate the commercial glyphosate-resistant crops currently available in the market. This single source of the EPSPS gene may have caused the decrease in herbicide tolerance, which has become a major concern of those involved in field management programs. Therefore, it is of interest to increase aroA/EPSPS gene diversity and seek new glyphosate-tolerant genes for developing glyphosate-tolerant crops. In the current study, EPSPS gene from Vitis vinifera (VvEPSPS) was cloned using reverse transcription polymerase chain reaction. However, wild type VvEPSPS cannot be directly used for developing transgenic crops because of its extreme glyphosate sensitivity. Recent studies have demonstrated that DNA Shuffling is an effective strategy in producing multi-mutated EPSPS resourced from plants (EPSPS plant ) with improved glyphosate resistance in bacteria and plants. After performing DNA Shuffling on VvEPSPS gene, one highly glyphosate-resistant mutant with seven amino acid variations was isolated after five rounds of Shuffling and screening. The mutant showed seven amino acid changes in the EPSPS gene, namely, Q93R, T113A, P117L, G126A, C160Y, N239H, and V343A. The assay of glyphosate resistance further confirmed the potential of the VvEPSPS mutant in developing glyphosate-resistant crops.
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mutation by DNA Shuffling of 5 enolpyruvylshikimate 3 phosphate synthase from malus domestica for improved glyphosate resistance
Plant Biotechnology Journal, 2013Co-Authors: Yongsheng Tian, Wei Zhao, Rihe Peng, Aisheng Xiong, Hongjuan Han, Quanhong YaoAbstract:A new 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate-tolerant plants. However, wild-type MdEPSPS is not suitable for the development of transgenic glyphosate-tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA Shuffling on MdEPSPS, and one highly glyphosate-resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA Shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site-directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single-site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate-resistant crops.
Rihe Peng - One of the best experts on this subject based on the ideXlab platform.
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identification of a phosphinothricin resistant mutant of rice glutamine synthetase using DNA Shuffling
Scientific Reports, 2015Co-Authors: Yongsheng Tian, Wei Zhao, Xiaojuan Xing, Rihe Peng, Quanhong YaoAbstract:To date, only bar/pat gene derived from Streptomyces has been used to generate the commercial PPT-resistant crops currently available in the market. The limited source of bar/pat gene is probably what has caused the decrease in PPT-tolerance, which has become the main concern of those involved in field management programs. Although glutamine synthetase (GS) is the target enzyme of PPT, little study has been reported about engineering PPT-resistant plants with GS gene. Then, the plant-optimized GS gene from Oryza sativa (OsGS1S) was chemically synthesized in the present study by PTDS to identify a GS gene for developing PPT-tolerant plants. However, OsGS1S cannot be directly used for developing PPT-tolerant plants because of its poor PPT-resistance. Thus, we performed DNA Shuffling on OsGS1S, and one highly PPT-resistant mutant with mutations in four amino acids (A63E, V193A, T293A and R295K) was isolated after three rounds of DNA Shuffling and screening. Among the four amino acids substitutions, only R295K was identified as essential in altering PPT resistance. The R295K mutation has also never been previously reported as an important residue for PPT resistance. Furthermore, the mutant gene has been transformed into Saccharomyces cerevisiae and Arabidopsis to confirm its potential in developing PPT-resistant crops.
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improved glyphosate resistance of 5 enolpyruvylshikimate 3 phosphate synthase from vitis vinifera in transgenic arabidopsis and rice by DNA Shuffling
Molecular Breeding, 2015Co-Authors: Yongsheng Tian, Wei Zhao, Xiaojuan Xing, Rihe Peng, Quanhong YaoAbstract:To date, only AroA variant derived from Agrobacterium tumefaciens CP4 has been used to generate the commercial glyphosate-resistant crops currently available in the market. This single source of the EPSPS gene may have caused the decrease in herbicide tolerance, which has become a major concern of those involved in field management programs. Therefore, it is of interest to increase aroA/EPSPS gene diversity and seek new glyphosate-tolerant genes for developing glyphosate-tolerant crops. In the current study, EPSPS gene from Vitis vinifera (VvEPSPS) was cloned using reverse transcription polymerase chain reaction. However, wild type VvEPSPS cannot be directly used for developing transgenic crops because of its extreme glyphosate sensitivity. Recent studies have demonstrated that DNA Shuffling is an effective strategy in producing multi-mutated EPSPS resourced from plants (EPSPS plant ) with improved glyphosate resistance in bacteria and plants. After performing DNA Shuffling on VvEPSPS gene, one highly glyphosate-resistant mutant with seven amino acid variations was isolated after five rounds of Shuffling and screening. The mutant showed seven amino acid changes in the EPSPS gene, namely, Q93R, T113A, P117L, G126A, C160Y, N239H, and V343A. The assay of glyphosate resistance further confirmed the potential of the VvEPSPS mutant in developing glyphosate-resistant crops.
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mutation by DNA Shuffling of 5 enolpyruvylshikimate 3 phosphate synthase from malus domestica for improved glyphosate resistance
Plant Biotechnology Journal, 2013Co-Authors: Yongsheng Tian, Wei Zhao, Rihe Peng, Aisheng Xiong, Hongjuan Han, Quanhong YaoAbstract:A new 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate-tolerant plants. However, wild-type MdEPSPS is not suitable for the development of transgenic glyphosate-tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA Shuffling on MdEPSPS, and one highly glyphosate-resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA Shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site-directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single-site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate-resistant crops.
Wei Zhao - One of the best experts on this subject based on the ideXlab platform.
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identification of a phosphinothricin resistant mutant of rice glutamine synthetase using DNA Shuffling
Scientific Reports, 2015Co-Authors: Yongsheng Tian, Wei Zhao, Xiaojuan Xing, Rihe Peng, Quanhong YaoAbstract:To date, only bar/pat gene derived from Streptomyces has been used to generate the commercial PPT-resistant crops currently available in the market. The limited source of bar/pat gene is probably what has caused the decrease in PPT-tolerance, which has become the main concern of those involved in field management programs. Although glutamine synthetase (GS) is the target enzyme of PPT, little study has been reported about engineering PPT-resistant plants with GS gene. Then, the plant-optimized GS gene from Oryza sativa (OsGS1S) was chemically synthesized in the present study by PTDS to identify a GS gene for developing PPT-tolerant plants. However, OsGS1S cannot be directly used for developing PPT-tolerant plants because of its poor PPT-resistance. Thus, we performed DNA Shuffling on OsGS1S, and one highly PPT-resistant mutant with mutations in four amino acids (A63E, V193A, T293A and R295K) was isolated after three rounds of DNA Shuffling and screening. Among the four amino acids substitutions, only R295K was identified as essential in altering PPT resistance. The R295K mutation has also never been previously reported as an important residue for PPT resistance. Furthermore, the mutant gene has been transformed into Saccharomyces cerevisiae and Arabidopsis to confirm its potential in developing PPT-resistant crops.
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improved glyphosate resistance of 5 enolpyruvylshikimate 3 phosphate synthase from vitis vinifera in transgenic arabidopsis and rice by DNA Shuffling
Molecular Breeding, 2015Co-Authors: Yongsheng Tian, Wei Zhao, Xiaojuan Xing, Rihe Peng, Quanhong YaoAbstract:To date, only AroA variant derived from Agrobacterium tumefaciens CP4 has been used to generate the commercial glyphosate-resistant crops currently available in the market. This single source of the EPSPS gene may have caused the decrease in herbicide tolerance, which has become a major concern of those involved in field management programs. Therefore, it is of interest to increase aroA/EPSPS gene diversity and seek new glyphosate-tolerant genes for developing glyphosate-tolerant crops. In the current study, EPSPS gene from Vitis vinifera (VvEPSPS) was cloned using reverse transcription polymerase chain reaction. However, wild type VvEPSPS cannot be directly used for developing transgenic crops because of its extreme glyphosate sensitivity. Recent studies have demonstrated that DNA Shuffling is an effective strategy in producing multi-mutated EPSPS resourced from plants (EPSPS plant ) with improved glyphosate resistance in bacteria and plants. After performing DNA Shuffling on VvEPSPS gene, one highly glyphosate-resistant mutant with seven amino acid variations was isolated after five rounds of Shuffling and screening. The mutant showed seven amino acid changes in the EPSPS gene, namely, Q93R, T113A, P117L, G126A, C160Y, N239H, and V343A. The assay of glyphosate resistance further confirmed the potential of the VvEPSPS mutant in developing glyphosate-resistant crops.
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mutation by DNA Shuffling of 5 enolpyruvylshikimate 3 phosphate synthase from malus domestica for improved glyphosate resistance
Plant Biotechnology Journal, 2013Co-Authors: Yongsheng Tian, Wei Zhao, Rihe Peng, Aisheng Xiong, Hongjuan Han, Quanhong YaoAbstract:A new 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Malus domestica (MdEPSPS) was cloned and characterized by rapid amplification of cDNA ends to identify an EPSPS gene appropriate for the development of transgenic glyphosate-tolerant plants. However, wild-type MdEPSPS is not suitable for the development of transgenic glyphosate-tolerant plants because of its poor glyphosate resistance. Thus, we performed DNA Shuffling on MdEPSPS, and one highly glyphosate-resistant mutant with mutations in eight amino acids (N63D, N86S, T101A, A187T, D230G, H317R, Y399R and C413A.) was identified after five rounds of DNA Shuffling and screening. Among the eight amino acid substitutions on this mutant, only two residue changes (T101A and A187T) were identified by site-directed mutagenesis as essential and additive in altering glyphosate resistance, which was further confirmed by kinetic analyses. The single-site A187T mutation has also never been previously reported as an important residue for glyphosate resistance. Furthermore, transgenic rice was used to confirm the potential of MdEPSPS mutant in developing glyphosate-resistant crops.