关键酶泛素化位点对柚皮素生物合成的影响

Effect of key enzymes ubiquitination sites on the biosynthesis of naringenin

黄酮类化合物具有多种生物活性,在食品、药品、化妆品等领域都有重要应用。柚皮素是多种重要黄酮类化合物生物合成的平台化合物。泛素化是蛋白质翻译后修饰的重要一环,参与调控细胞的生命活动。泛素化的蛋白质通过泛素-蛋白酶体系统降解,对维持细胞正常生理活动具有重要意义,对外源蛋白的表达和积累也可能具有显著影响。文中利用荧光双分子互补法在酿酒酵母Saccharomyces cerevisiae中建立了泛素化修饰的实时原位检测体系,以荧光强度表征蛋白的泛素化修饰程度。应用该方法获得了柚皮素合成途径中5个关键酶的潜在泛素化位点。将相关泛素化位点的赖氨酸突变为精氨酸,用于降低关键酶的泛素化修饰程度。其中,酪氨酸解氨酶FjTAL、查尔酮合成酶SjCHS、SmCHS突变体表现为荧光下降,表明其泛素化水平有所降低。发酵结果表明,表达酪氨酸解氨酶FjTAL突变体FjTAL-K487R的酿酒酵母在发酵72 h后获得了74.2 mg/L的对香豆酸产量,相较于原始FjTAL提高了32.3%,而表达查尔酮合成酶突变体的酿酒酵母产量没有明显变化。结果表明,对柚皮素生物合成途径相关蛋白的潜在泛素化位点进行突变,能够提高对香豆酸产量,对柚皮素生物合成有积极影响。

Flavonoids have a variety of biological activities and have important applications in food,medicine,cosmetics,and many other fields.Naringenin is a platform chemical for the biosynthesis of many important flavonoids.Ubiquitination plays a pivotal role in the post-translational modification of proteins and participates in the regulation of cellular activities.Ubiquitinated proteins can be degraded by the ubiquitin-protease system,which is important for maintaining the physiological activities of cells,and may also exert a significant impact on the expression of exogenous proteins.In this study,a real-time in-situ detection system for ubiquitination modification has been established in Saccharomyces cerevisiae by using a fluorescence bimolecular complementation approach.The ubiquitination level of protein was characterized by fluorescence intensity.By using the approach,the potential ubiquitination sites of proteins involved in the naringenin biosynthesis pathway have been obtained.The lysine residues of the relevant ubiquitination sites were mutated to arginine to reduce the ubiquitination level.The mutants of tyrosine ammonia-lyase(FjTAL)and chalcone synthase(SjCHS,SmCHS)showed decreased fluorescence,suggested that a decreased ubiquitination level.After fermentation verification,the S.cerevisiae expressing tyrosine ammonia-lyase FjTAL mutant FjTAL-K487R accumulated 74.2 mg/L p-coumaric acid at 72 h,which was 32.3%higher than that of the original FjTAL.The strains expressing chalcone synthase mutants showed no significant change in the titer of naringenin.The results showed that mutation of the potential ubiquitination sites of proteins involved in the naringenin biosynthesis pathway could increase the titer of p-coumaric acid and have positive effect on naringenin biosynthesis.