RESUMO
Synthesis of the tyrosine derived cyanogenic glucoside dhurrin in Sorghum bicolor is catalyzed by two multifunctional, membrane bound cytochromes P450, CYP79A1 and CYP71E1, and a soluble UDPG-glucosyltransferase, UGT85B1 (Tattersall, D.B., Bak, S., Jones, P.R., Olsen, C.E., Nielsen, J.K., Hansen, M.L., Høj, P.B., Møller, B.L., 2001. Resistance to an herbivore through engineered cyanogenic glucoside synthesis. Science 293, 1826-1828). All three enzymes retained enzymatic activity when expressed as fluorescent fusion proteins in planta. Transgenic Arabidopsis thaliana plants that produced dhurrin were obtained by co-expression of CYP79A1/CYP71E1-CFP/UGT85B1-YFP and of CYP79A1/CYP71E1/UGT85B1-YFP but not by co-expression of CYP79A1-YFP/CYP71E-CFP/UGT85B1. The lack of dhurrin formation upon co-expression of the two cytochromes P450 as fusion proteins indicated that tight interaction was necessary for efficient substrate channelling. Transient expression in S. bicolor epidermal cells as monitored by confocal laser scanning microscopy showed that UGT85B1-YFP accumulated in the cytoplasm in the absence of CYP79A1 or CYP71E1. In the presence of CYP79A1 and CYP71E1, the localization of UGT85B1 shifted towards the surface of the ER membrane in the periphery of biosynthetic active cells, demonstrating in planta dhurrin metabolon formation.
Assuntos
Arabidopsis/metabolismo , Nitrilas/metabolismo , Sorghum/metabolismo , Arabidopsis/enzimologia , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Citosol/enzimologia , Regulação Enzimológica da Expressão Gênica , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sorghum/citologia , Sorghum/enzimologiaRESUMO
The broad range of environmental conditions under which Debaryomyces hansenii can grow, and its production of lipolytic and proteolytic enzymes, have promoted its widespread use. The present work represents a preliminary characterization of D. hansenii for heterologous expression and secretion of green fluorescent protein (GFP). Six heterologous expression vectors were used to address protein production efficiency under regulated expression conditions. Protein expression in D. hansenii seems to be similar to that in Saccharomyces cerevisiae, with transcription being controlled by almost all of the S. cerevisiae and D. hansenii inducible promoters tested, with the exception of the alcohol dehydrogenase 2 gene promoter from S. cerevisiae. Extracellular protein levels in D. hansenii were lower than in S. cerevisiae when Saccharomyces signal peptides were used.
Assuntos
Regulação Fúngica da Expressão Gênica , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Álcool Desidrogenase/genética , Citocromos c/genética , Vetores Genéticos , Glicerolfosfato Desidrogenase/genética , Proteínas de Fluorescência Verde , Proteínas de Choque Térmico/genética , Regiões Promotoras Genéticas , Proteínas Quinases/genética , Sinais Direcionadores de Proteínas , Transcrição GênicaRESUMO
The isolation of elements driving high-level expression of foreign genes in mycobacteria would significantly aid characterization of mycobacterial antigens and recombinant vaccine development. Mycobacterium smegmatis is a widely employed host for recombinant mycobacterial gene expression. This report describes the identification of strong promoter elements of M. smegmatis. Fluorescence-activated cell sorting was employed to isolate DNA fragments permitting high-level expression of the Aequorea victoria green fluorescent protein within recombinant M. smegmatis. Ten postulated M. smegmatis promoters were identified which showed activity two to six times that of the strong beta-lactamase promoter of Mycobacterium fortuitum. The utility of one of these promoters for the over-expression of foreign genes in mycobacteria was demonstrated by the efficient purification of the Mycobacterium leprae 35-kDa antigen from recombinant M. smegmatis.