Richard Peter, Viljanen Kaarina, Penttilä Merja
VTT Technical Research Centre of Finland Ltd, Tietotie 2, P.O. box 1000, 02044 VTT Espoo, Finland.
AMB Express. 2015 Feb 18;5:12. doi: 10.1186/s13568-015-0103-x. eCollection 2015.
The S. cerevisiae PAD1 gene had been suggested to code for a cinnamic acid decarboxylase, converting trans-cinnamic acid to styrene. This was suggested for the reason that the over-expression of PAD1 resulted in increased tolerance toward cinnamic acid, up to 0.6 mM. We show that by over-expression of the PAD1 together with the FDC1 the cinnamic acid decarboxylase activity can be increased significantly. The strain over-expressing PAD1 and FDC1 tolerated cinnamic acid concentrations up to 10 mM. The cooperation of Pad1p and Fdc1p is surprising since the PAD1 has a mitochondrial targeting sequence and the FDC1 codes for a cytosolic protein. The cinnamic acid decarboxylase activity was also seen in the cell free extract. The activity was 0.019 μmol per minute and mg of extracted protein. The overexpression of PAD1 and FDC1 resulted also in increased activity with the hydroxycinnamic acids ferulic acid, p-coumaric acid and caffeinic acid. This activity was not seen when FDC1 was overexpressed alone. An efficient cinnamic acid decarboxylase is valuable for the genetic engineering of yeast strains producing styrene. Styrene can be produced from endogenously produced L-phenylalanine which is converted by a phenylalanine ammonia lyase to cinnamic acid and then by a decarboxylase to styrene.
酿酒酵母的PAD1基因被认为编码一种肉桂酸脱羧酶，可将反式肉桂酸转化为苯乙烯。提出这一观点的原因是PAD1的过表达导致对肉桂酸的耐受性增强，可达0.6 mM。我们发现，通过同时过表达PAD1和FDC1，肉桂酸脱羧酶活性可显著提高。过表达PAD1和FDC1的菌株能耐受高达10 mM的肉桂酸浓度。Pad1p和Fdc1p的协同作用令人惊讶，因为PAD1具有线粒体靶向序列，而FDC1编码一种胞质蛋白。在无细胞提取物中也观察到了肉桂酸脱羧酶活性。该活性为每分钟0.019 μmol每毫克提取蛋白。PAD1和FDC1的过表达还导致对阿魏酸、对香豆酸和咖啡酸等羟基肉桂酸的活性增加。单独过表达FDC1时未观察到这种活性。一种高效的肉桂酸脱羧酶对于生产苯乙烯的酵母菌株的基因工程很有价值。苯乙烯可由内源性产生的L-苯丙氨酸产生，L-苯丙氨酸由苯丙氨酸解氨酶转化为肉桂酸，然后由脱羧酶转化为苯乙烯。
