title: Surrogate biochemistry: use of Escherichia coli to identify plant cDNAs that impact metabolic engineering of carotenoid accumulation.
abstract: Carotenoids synthesized in plants but not animals are essential for human nutrition. Therefore, ongoing efforts to metabolically engineer plants for improved carotenoid content benefit from the identification of genes that affect carotenoid accumulation, possibly highlighting potential challenges when pyramiding traits represented by multiple biosynthetic pathways. We employed a heterologous bacterial system to screen for maize cDNAs encoding products that alter carotenoid accumulation either positively or negatively. Genes encoding carotenoid biosynthetic enzymes from the bacterium Erwinia uredovora were introduced into Escherichia coli cells that were subsequently transfected with a maize endosperm cDNA expression library; and these doubly transformed cells were then screened for altered carotenoid accumulation. DNA sequencing and characterization of one cDNA class conferring increased carotenoid content led to the identification of maize cDNAs encoding isopentenyl diphosphate isomerase. A cDNA that caused a reduced carotenoid content in E. coli was also identified. Based on DNA sequence analysis, DNA hybridization, and further functional testing, this latter cDNA was found to encode the small subunit of ADP-glucose pyrophosphorylase, a rate-controlling enzyme in starch biosynthesis that has been of interest for enhancing plant starch content.
authors.affiliation: Department of Biological Sciences, Lehman College, The City University of New York, 250 Bedford Park Blvd West, Bronx, New York 10468, USA.
authors.forename: C E
authors.initials: CE
authors.lastname: Gallagher
authors.forename: M
authors.initials: M
authors.lastname: Cervantes-Cervantes
authors.forename: E T
authors.initials: ET
authors.lastname: Wurtzel
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D015139:Blotting, Southern; D019748:Carbon-Carbon Double Bond Isomerases / Q000235:genetics* / Q000378:metabolism; D002338:Carotenoids / Q000096:biosynthesis*; D004269:DNA, Bacterial / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics*; D004885:Erwinia / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism*; D015723:Gene Library; D005818:Genetic Engineering; D051599:Glucose-1-Phosphate Adenylyltransferase; D006003:Glycogen / Q000096:biosynthesis; D045782:Hemiterpenes; D000077276:Lycopene; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D003313:Zea mays / Q000201:enzymology / Q000235:genetics*; D019207:beta Carotene / Q000096:biosynthesis
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D018076:DNA, Complementary; D018744:DNA, Plant; D045782:Hemiterpenes; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D019207:beta Carotene; D002338:Carotenoids; D006003:Glycogen; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase; D000077276:Lycopene