title: Adaptive Engineering of Phytochelatin-based Heavy Metal Tolerance.
abstract: Metabolic engineering approaches are increasingly employed for environmental applications. Because phytochelatins (PC) protect plants from heavy metal toxicity, strategies directed at manipulating the biosynthesis of these peptides hold promise for the remediation of soils and groundwaters contaminated with heavy metals. Directed evolution of Arabidopsis thaliana phytochelatin synthase (AtPCS1) yields mutants that confer levels of cadmium tolerance and accumulation greater than expression of the wild-type enzyme in Saccharomyces cerevisiae, Arabidopsis, or Brassica juncea. Surprisingly, the AtPCS1 mutants that enhance cadmium tolerance and accumulation are catalytically less efficient than wild-type enzyme. Metabolite analyses indicate that transformation with AtPCS1, but not with the mutant variants, decreases the levels of the PC precursors, glutathione and γ-glutamylcysteine, upon exposure to cadmium. Selection of AtPCS1 variants with diminished catalytic activity alleviates depletion of these metabolites, which maintains redox homeostasis while supporting PC synthesis during cadmium exposure. These results emphasize the importance of metabolic context for pathway engineering and broaden the range of tools available for environmental remediation.
authors.affiliation: From the Department of Biology, Washington University, St. Louis, Missouri 63130, the Donald Danforth Plant Science Center, St. Louis, Missouri 63132.
authors.forename: Rebecca E
authors.initials: RE
authors.lastname: Cahoon
authors.affiliation: the Donald Danforth Plant Science Center, St. Louis, Missouri 63132.
authors.forename: W Kevin
authors.initials: WK
authors.lastname: Lutke
authors.affiliation: From the Department of Biology, Washington University, St. Louis, Missouri 63130.
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Cameron
authors.affiliation: the Donald Danforth Plant Science Center, St. Louis, Missouri 63132, the Department of Biology, Genetics Institute, University of Florida, Gainesville, Florida 32610, and.
authors.forename: Sixue
authors.initials: S
authors.lastname: Chen
authors.affiliation: From the Department of Biology, Washington University, St. Louis, Missouri 63130.
authors.forename: Soon Goo
authors.initials: SG
authors.lastname: Lee
authors.affiliation: the Donald Danforth Plant Science Center, St. Louis, Missouri 63132.
authors.forename: Rebecca S
authors.initials: RS
authors.lastname: Rivard
authors.affiliation: the Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104.
authors.forename: Philip A
authors.initials: PA
authors.lastname: Rea
authors.affiliation: From the Department of Biology, Washington University, St. Louis, Missouri 63130, the Donald Danforth Plant Science Center, St. Louis, Missouri 63132, the Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 jjez@biology2.wustl.edu.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Jez
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002104:Cadmium / Q000378:metabolism / Q000633:toxicity; D020134:Catalytic Domain / Q000235:genetics; D002614:Chelating Agents / Q000378:metabolism; D019020:Directed Molecular Evolution; D052918:Environmental Restoration and Remediation; D000075322:Heavy Metal Poisoning; D060847:Metabolic Engineering; D019216:Metals, Heavy / Q000378:metabolism*; D008958:Models, Molecular; D009149:Mustard Plant / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D054811:Phytochelatins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011041:Poisoning / Q000378:metabolism; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D002614:Chelating Agents; D019216:Metals, Heavy; D011994:Recombinant Proteins; D002104:Cadmium; D054811:Phytochelatins; D019881:Aminoacyltransferases; C093784:glutathione gamma-glutamylcysteinyltransferase
keywords: enzyme catalysis; heavy metal tolerance; metabolic engineering; plant biochemistry; plant molecular biology; protein engineering