The Characteristics Of Prephenate Dehydratase For Chinese Huangjiu Yeast In 2-phenylethanol Synthetic Pathway

2-Phenylethanol?2-PE?,is a kind of fragrant constituent of rose flowers,which have attracted a great deal of attention in recent years.It is also reported that 2-PE in wine mainly comes from Saccharomyces cerevisiae.Improving the ability of yeast to produce 2-PE in the process of Huangjiu brewing is not only conducive to improving the flavour of Huangjiu.Its high aroma intensity can also give Huangjiu unique flavor,but the batches of Huangjiu vary in quantity and is not stable.In this study,S.cerevisiae HJ with high yield of 2-PE in the batch culture was taken as object.The key synthetic enzymes in the 2-PE metabolism pathway were identified through sequence alignment.The main results are as follows:Then Blast tool was used to analyze the PHA2p similarity between Chinese Huangjiu yeast HJ and Model S.cerevisiae W303.Compared with S.cerevisiae W303,3 mutation sites?I161K?L239P?Q250H?in pha2 was identified in S.cerevisiae HJ.The digested PCR products ligated with pET-28a and transformed into E.coli BL21 cells to overexpress PHA2p.Purified enzyme was used to determine activities at different pHs and temperatures.The tolerance of 2-PE,and the ability to inhibit the feedback of phenylalanine?L-Phe?were also measured.The result shows that:The optimal temperature for activity was 45?,The optimum pH was 8.2.There was good activity with pH ranged from 6.2 to 8.7 or temperature ranged from 40 to 60?.Kinetic tests was carried out using the Prephenate as the substrate.The result shows that:The recombinant protein PHA2p_W303303 showed K_m of 0.16 mmol?L^-1,k_cat/K_m of 26.50L?mmol^-1?min^-1.PHA2p_HJJ showed K_m of 0.16 mmol?L^-1,k_cat/K_m of 36.48 L?mmol^-1?min^-1.PHA2p_HJJ and PHA2p_W303303 showed K_m of 0.27 mmol?L^-1,k_cat/K_m of 2.82 and 2.91L?mmol^-1?min^-1with the existence of L-Phe.It is also confirmed that S.cerevisiae PHA2p will be inhibitated in the absence of exogenous L-Phe.The result also shows that the special enzymes activity of PHA2p in Chinese Huangjiu yeast higher 22.6%than in model strain S.cerevisiae W303.L-Phe acts as a feedback inhibitor of both activities of the PHA2p_HJJ and PHA2p_W303.It provides a new target for the transformation of aromatic amino acid metabolic pathway in Saccharomyces cerevisiae.The Chinese huangjiu yeast that carrying the mutant pha2 gene and the yeast W303 were cultured under the condition of normol and huangjiu simulated culture environment respectively,and the contents of L-Phe and 2-PE were monitored.Compared with mode yeast W303,Chinese huangjiu yeast HJ could have good activity of Ehrlich pathway.Chinese Huangjiu yeast can greater use shikimic acid pathway to produce 2-PE under the condition of exogenous existence of L-Phe.2-PE content products of Huangjiu yeast was mainly produced by shikimic acid pathway.