Abstract:
The present invention relates to methods of producing, isolating, identifying and characterizing herbicide tolerant ACCase variants. In one embodiment, the invention encompasses a method of screening for an acetyle-CoA carboxylase (ACCase) enzyme which is tolerant to at least one herbicide, comprising: a) providing an ACCase-deficient yeast with a chimeric ACCase, said chimeric ACCase comprising: at least two regions that further comprise: i) an N-terminal region, said N-terminal region derived from yeast, fungi or monocot cytoplasmic ACCases, preferably a yeast ACCase; ii) a C-terminal region, said C-terminal region derived from monocot plastific ACCases and comprising an HSR; and iii) said N-terminal region comprises about 50% to about 60% of the chimeric ACCase; b) isolating herbicide tolerant yeast cells after culturing in the presence of at least one herbicide; and c) further comprising identifying the mutation(s) not present in chimeric ACCase prior to culturing, which confers tolerance to at least one herbicide.
Abstract:
The present invention relates to methods of producing, isolating, identifying and characterizing herbicide tolerant ACCase variants. In one embodiment, the invention encompasses a method of screening for an acetyle-CoA carboxylase (ACCase) enzyme which is tolerant to at least one herbicide, comprising: a) providing an ACCase-deficient yeast with a chimeric ACCase, said chimeric ACCase comprising: at least two regions that further comprise: i) an N-terminal region, said N-terminal region derived from yeast, fungi or monocot cytoplasmic ACCases, preferably a yeast ACCase; ii) a C-terminal region, said C-terminal region derived from monocot plastific ACCases and comprising an HSR; and iii) said N-terminal region comprises about 50% to about 60% of the chimeric ACCase; b) isolating herbicide tolerant yeast cells after culturing in the presence of at least one herbicide; and c) further comprising identifying the mutation(s) not present in chimeric ACCase prior to culturing, which confers tolerance to at least one herbicide.
Abstract:
Un procedimiento de producción de una enzima acetil-CoA carboxilasa (ACCasa) que es tolerante a al menos un herbicida, que comprende: a. proporcionar una levadura deficiente en ACCasa que comprende un ácido nucleico que codifica una ACCasa quimérica, comprendiendo dicha ACCasa quimérica una región N-terminal y una región Cterminal, en el que la región C-terminal comprende una región de sensibilidad a herbicidas (HSR) de una ACCasa plastídica de monocotiledónea de una Poaceae; b. poner en contacto dicha levadura con al menos un oligonucleótido mutagénico en condiciones que permitan la mutagénesis dirigida al sitio de al menos un codón del ácido nucleico que codifica la ACCasa quimérica, y hacer crecer la levadura mutagenizada formando de este modo una biblioteca de colonias de levadura mutagenizadas; c. cultivar las colonias de levadura mutagenizadas en presencia de al menos un herbicida inhibidor de ACCasa para formar colonias tratadas; y d. aislar de al menos una de dichas colonias tratadas, al menos una levadura mutagenizada que crece en presencia del herbicida, en el que la levadura mutagenizada que crece en presencia del herbicida comprende una ACCasa mutagenizada que tiene una tolerancia al herbicida que es mayor que la exhibida por la ACCasa quimérica de la etapa (a): en el que el procedimiento es un procedimiento de alto rendimiento en el que un trabajador capacitado puede generar y seleccionar al menos 500 diferentes ACCasas con HSR variante por mes, cada una de su propia colonia celular.
Abstract:
The present invention relates to methods of producing, isolating, identifying and characterizing herbicide tolerant ACCase variants. In one embodiment, the invention encompasses a method of screening for an acetyle-CoA carboxylase (ACCase) enzyme which is tolerant to at least one herbicide, comprising: a) providing an ACCase-deficient yeast with a chimeric ACCase, said chimeric ACCase comprising: at least two regions that further comprise: i) an N-terminal region, said N-terminal region derived from yeast, fungi or monocot cytoplasmic ACCases, preferably a yeast ACCase; ii) a C-terminal region, said C-terminal region derived from monocot plastific ACCases and comprising an HSR; and iii) said N-terminal region comprises about 50% to about 60% of the chimeric ACCase; b) isolating herbicide tolerant yeast cells after culturing in the presence of at least one herbicide; and c) further comprising identifying the mutation(s) not present in chimeric ACCase prior to culturing, which confers tolerance to at least one herbicide.
Abstract:
The present invention relates to methods of producing, isolating, identifying and characterizing herbicide tolerant ACCase variants. In one embodiment, the invention encompasses a method of screening for an acetyle-CoA carboxylase (ACCase) enzyme which is tolerant to at least one herbicide, comprising: a) providing an ACCase-deficient yeast with a chimeric ACCase, said chimeric ACCase comprising: at least two regions that further comprise: i) an N-terminal region, said N-terminal region derived from yeast, fungi or monocot cytoplasmic ACCases, preferably a yeast ACCase; ii) a C-terminal region, said C-terminal region derived from monocot plastific ACCases and comprising an HSR; and iii) said N-terminal region comprises about 50% to about 60% of the chimeric ACCase; b) isolating herbicide tolerant yeast cells after culturing in the presence of at least one herbicide; and c) further comprising identifying the mutation(s) not present in chimeric ACCase prior to culturing, which confers tolerance to at least one herbicide.
Abstract:
The present invention relates compositions and methods for identifying, isolating, and characterizing herbicide tolerant mutations in monocot plastidic acetyl-CoA carboxylases using a model system.