Identification, recombinant expression, and biochemical analysis of putative secondary product glucosyltransferases from Citrus paradisi

Authors: DEVAIAH, SHIVAKUMAR - East Tennessee State University; Owens, Daniel; SIBHATU, MEBRAHTU - East Tennessee State University; SARKAR, TAPASREE - East Tennessee State University; STRONG, CHRISTY - East Tennessee State University; MALLAMPALLI, VENKATA K.P.S. - East Tennessee State University; ASIAGO, JOSEPHAT - East Tennessee State University; COOKE, JENNIFER - East Tennessee State University; KISER, STARLA - East Tennessee State University; LIN, ZHANGFAN - East Tennessee State University; WAMUCHO, ANYE - East Tennessee State University; HAYFORD, DEBORAH - East Tennessee State University; WILLIAMS, BRUCE - East Tennessee State University; LOFTIS, PETRI - East Tennessee State University; Berhow, Mark; PIKE, LEE - East Tennessee State University; MCINTOSH, CECILIA - East Tennessee State University

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/18/2016
Publication Date: 3/9/2016
Citation: Devaiah, S.P., Owens, D.K., Sibhatu, M.B., Sarkar, T.R., Strong, C.L., Mallampalli, V., Asiago, J., Cooke, J., Kiser, S., Lin, Z., Wamucho, A., Hayford, D., Williams, B.E., Loftis, P., Berhow, M.A., Pike, L.M., McIntosh, C.A. 2016. Identification, recombinant expression, and biochemical analysis of putative secondary product glucosyltransferases from Citrus paradisi. Journal of Agricultural and Food Chemistry. 64(9):1957-1969.

Interpretive Summary: Glucosyltransferases are enzymes that attach the sugar glucose to larger molecules. Flavonoids are a class of natural products that are extremely common in citrus species, such as grapefruit. The way that glucose is added to certain flavonoids can produce compounds that are either very bitter or tasteless. Therefore, the activity of glucosyltransferases in citrus species can directly impact the taste characterisitics of fruit, juices, jellies, and other citrus products. In this work, potential glucosyltransferase enzymes were identified in grapefruit, bacteria (E.coli ) or yeast (P.pastoris) used as a system to generate enough enzyme for experiments, and individual enzymes tested for their ability to add glucose to a variety of different flavonoid and other compounds. The compound to which glucose was added could be assigned for some of the identified glucosyltransferases, and the characteristics of the enzymes were examined. This work is a preliminary step in a long term goal of affecting the taste characteristics of grapefruit products by manipulating glucosyltransferases.

Technical Abstract: Flavonoid and limonoid glycosides influence taste properties as well as marketability of citrus fruit and products, particularly in grapefruit. In this work, nine grapefruit putative natural product glucosyltransferases (PGTs) were resolved by either using degenerate primers against the semi-conserved 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 E. coli and/or P. 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 activity having been identified.