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

Abstract

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.

Advances in flavonoid glycosyltransferase research: integrating recent findings with long-term citrus studies

“…Other putative secondary product GT clones from grapefruit were heterologously expressed and the resultant proteins tested for activity. One showed low activity with flavonols and another shows activity with catechol (Devaiah et al 2016). …”

“…It is intriguing to consider that this may be due to the importance of bitter compound production to protect very young tissues and fruits from herbivory while flavonol glucosylation is increased during expansion and growth of tissues, especially those that would need protection from damaging UV radiation due to more exposure to direct sunlight. Another grapefruit clone showed trace activity with flavonols and also showed highest level of gene expression in stage 5 younger leaves and stage 3 stems with only a trace of expression in stage 3 and 4 roots (Daniel et al 2011;Devaiah et al 2016). A catechol-GT was expressed in leaves of all stages with highest expression in stage 5 young leaves and stage 4 roots (Daniel et al 2011;Devaiah et al 2016).…”

“…Another grapefruit clone showed trace activity with flavonols and also showed highest level of gene expression in stage 5 younger leaves and stage 3 stems with only a trace of expression in stage 3 and 4 roots (Daniel et al 2011;Devaiah et al 2016). A catechol-GT was expressed in leaves of all stages with highest expression in stage 5 young leaves and stage 4 roots (Daniel et al 2011;Devaiah et al 2016).…”

“…This may seem an onerous task, but improvements in heterologous protein expression systems (e.g. Ohashi et al 2015;Devaiah et al 2016) and screening assays coupled with the many putative GTs annotated in citrus genome and EST databases provide significant opportunities.…”

The Effect of Recombinant Tags on Citrus paradisi Flavonol-Specific 3-O Glucosyltransferase Activity

“…It did not interact with flavonols containing methoxy groups, as seen by the lack of significant activity when assayed with 4′-methoxy-flavonol. Cp3GT was previously shown to be inactive when methoxy groups were placed on the flavonoid backbone [23,24,35]. There also was no significant activity with the flavanone naringenin which lacks a 3-OH group; this was also noted in previous studies [23,24,35].…”