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Key Enzyme Activities and Anthocyanin Production Instability in Grape Cell Culture

Chien-Kuo Wang, Department of Biotechnology and Bioinformatics, Asia University, 500 Liu-Fong Rd, Wu-Fong Shiang, Taichung, Taiwan and Michael L. Shuler, School of Chemical and Biomolecular Engineering, Cornell University, 120 Olin Hall, Ithaca, NY 14853.

Plant cell culture is an attractive alternative source of many compounds that can not be readily chemically synthesized. In the past few years, efforts in enhancing the productivity of plant secondary metabolites have made commercial production of desired compounds more achievable. A major challenge remaining is that the production of secondary metabolites of interest can vary substantially within a subculture and over weeks and months; in other words, production instability. The types of production instability of interested compounds by plant cell suspension culture have been concluded into three categories. The first one is a gradual decrease in productivity with time in culture. The second type of the production instability is characterized by increases and decreases in the overall productivity of a culture over time, and the third type of instability, within-subculture variability, is characterized by a wide range of productivity in almost identical flasks inoculated from the same cell mixture with the same amount of inoculum and cultured under identical conditions. We will use anthocyanin production in grape cell culture as a model to delineate the causes of production instability in plant cell culture. UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) activity were first investigated, because it can stabilized the formation of anthocyanin and it was the only enzyme that was not expressed at measurable levels in white grape in previous studies. The results showed that the enzyme activities increased in parallel with the anthocyanin productivity, and restriction of gas exchange significantly reduced the enzyme activities by 75% for UFGT. Phenylalanine ammonialyase (PAL) and chalcone isomerase (CHI) activity were investigated subsequently. Again, the results show that the enzyme activities increases in parallel with the anthocyanin productivity, and restriction of gas exchange did have great impact on these two enzyme activities.More interestingly, once the gas restriction was lifted from the non-anthocyanin producing cultures, the enzyme activities and anthocyanin production usually returned to the level of their counterparts without gas restriction. Since gas exchange rates often change upon scale-up of cultures, this factor may be critical in observed instabilities in these cultures.