Amor Ilef Limem-Ben, Hehn Alain, Guedone Emmanuel, Ghedira Kamel, Engasser Jean-Marc, Chekir-Ghedrira Leila, Ghoul Mohamed
Laboratoire d'ingénierie des biomolécules, Nancy-Université, 2 avenue de la Forêt de Haye, B.P. 172, F-54505 Vandoeuvre-lès-Nancy, France.
Nat Prod Commun. 2010 Dec;5(12):1893-8.
To increase the biological activities of flavonoids and to enhance their stability and solubility by functionalization reactions (polymerization, esterification, alkylation, glycosylation and acylation), an increase in the number of hydroxyl groups in these molecules is needed. Hydroxylation reactions may be achieved using either chemical or enzymatic methods, the latter being more highly specific than the former. In our study, the flavonoid 3' hydroxylase (F3'H) from Gerbera hybrid, functionally expressed in Saccharomyces cerevisiae, was used to hydroxylate naringenin (the first flavonoid core synthesized in plants). Furthermore, we studied factors that may affect naringenin hydroxylation by recombinant cell-like yeast growth on selective or rich media and plasmid stability. The whole recombinant cells hydroxylated naringenin at position 3' to give eriodictyol. In a selective media, the yeast failed to grow to high cell densities (maximum 5 g/L), but the plasmid stability was nearly 90%, and naringenin hydroxylation reached 100%. In a rich complex media, the biomass reached 10 g/L, but the yield of naringenin hydroxylation reached only 71%, and the plasmid stability decreased. When yeast functionally expressing F3'H from Gerbera hybrid was used, in a selective media, 200 mg/L of eriodictyol from naringenin was produced.
为了通过官能团化反应（聚合、酯化、烷基化、糖基化和酰化）提高黄酮类化合物的生物活性，并增强其稳定性和溶解性，需要增加这些分子中羟基的数量。羟基化反应可以使用化学方法或酶促方法实现，后者比前者具有更高的特异性。在我们的研究中，在酿酒酵母中功能性表达的非洲菊黄酮3'羟化酶（F3'H）被用于将柚皮素（植物中合成的第一个黄酮类核心）羟基化。此外，我们研究了在选择性或丰富培养基上重组细胞样酵母生长以及质粒稳定性等可能影响柚皮素羟基化的因素。整个重组细胞将柚皮素在3'位羟基化生成圣草酚。在选择性培养基中，酵母无法生长到高细胞密度（最大5 g/L），但质粒稳定性接近90%，柚皮素羟基化率达到100%。在丰富的复合培养基中，生物量达到10 g/L，但柚皮素羟基化产率仅达到71%，质粒稳定性下降。当使用功能性表达非洲菊F3'H的酵母时，在选择性培养基中，从柚皮素产生了200 mg/L的圣草酚。
