Identification, Recombinant Expression, and Biochemical Analysis of Putative Secondary Product Glucosyltransferases from Citrus paradisi

• Published in: Journal of agricultural and food chemistry Vol. 64; no. 9; pp. 1957 - 1969
• Main Authors: Devaiah, Shivakumar P, Owens, Daniel K, Sibhatu, Mebrahtu B, Sarkar, Tapasree Roy, Strong, Christy L, Mallampalli, Venkata K. P. S, Asiago, Josephat, Cooke, Jennifer, Kiser, Starla, Lin, Zhangfan, Wamucho, Anye, Hayford, Deborah, Williams, Bruce E, Loftis, Peri, Berhow, Mark, Pike, Lee M, McIntosh, Cecilia A
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 09.03.2016
• Subjects: Amino Acid Sequence; Citrus paradisi - enzymology; Coumarins - metabolism; Escherichia coli - metabolism; Flavonoids - metabolism; Gene Expression; Genes, Plant - genetics; Glucosyltransferases - analysis; Glucosyltransferases - genetics; Limonins - metabolism; Molecular Sequence Data; Phenols - metabolism; Phylogeny; Pichia - metabolism; Recombinant Proteins - genetics; Seeds - enzymology; Sequence Alignment; Substrate Specificity; flavor; glucosyltransferase; heterologous expression; flavonoid; Citrus paradisi
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/acs.jafc.5b05430

Flavonoid and limonoid glycosides influence taste properties as well as marketability of Citrus fruit and products, particularly grapefruit. In this work, nine grapefruit putative natural product glucosyltransferases (PGTs) were resolved by either using degenerate primers against the semiconserved PSPG box motif, SMART-RACE RT-PCR, and primer walking to full-length coding regions; screening a directionally cloned young grapefruit leaf EST library; designing primers against sequences from other Citrus species; or identifying PGTs from Citrus contigs in the harvEST database. The PGT proteins associated with the identified full-length coding regions were recombinantly expressed in Escherichia coli and/or Pichia pastoris and then tested for activity with a suite of substrates including flavonoid, simple phenolic, coumarin, and/or limonoid compounds. A number of these compounds were eliminated from the predicted and/or potential substrate pool for the identified PGTs. Enzyme activity was detected in some instances with quercetin and catechol glucosyltransferase activities having been identified.