脱羧酶基因ARO10的过量表达及其对Saccharomy cescerevisiae的β-苯乙醇合成代谢影响
Over-expression of the Decarboxylase Gene ARO10 and its influence on β-phenethyl alcohol biosynthesis in Saccharomyces cerevisiae
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摘要: 为了验证脱羧酶在S.cerevisiaeβ-苯乙醇合成途径中的调控作用,本文从S.cerevisiae S288C中克隆脱羧酶基因ARO10,并构建由3-磷酸甘油酸激酶基因PGK1组成型强启动子控制的ARO10基因表达载体pYES2-Ppgk-ARO10,将重组载体导入S.cerevisiae S288C,研究ARO10基因过量表达对重组菌株中β-苯乙醇合成的影响。经摇瓶实验测定,携带pYES2-Ppgk-ARO10的转化子SP10在发酵60h时β-苯乙醇产量达到最大量1.0g/L,较野生型的对照菌提高16.3%。研究结果表明,脱羧酶是S.cerevisiae S288C中β-苯乙醇生物合成途径的关键酶之一,增加ARO10基因表达量有利于提高β-苯乙醇产量,研究为构建β-苯乙醇高产工程菌株奠定了重要基础。Abstract: In order to verify the key role that the decarboxylase probably played in the synthesis pathway of β-phenethyl alcohol in S.cerevisiae, the decarboxylase gene ARO10 was cloned from S.cerevisiae S288C and then inserted into a constitutive expression vector controlled by the promoter of phosphoglyceate kinase gene PGK1, generating a recombinant vector pYES2- Ppgk- ARO10. After pYES2- Ppgk- ARO10 was introduced into S.cerevisiae S288C by the lithium acetate transformation method, the influence of gene ARO10 over-expression on the β- phenethyl alcohol production in the recombinant strain was evaluated. A maximum yield up to 1.0g /L of β-phenethyl alcohol was achieved when the recombinant strain SP10 was incubated for 60h in flask fermentation experiments, which was 16.3% higher than that of the wild strain.The results indicated that ARO10 over-expression significantly increased the β- phenethyl alcohol production in S. cerevisiae S288C, which demonstrated that the decarboxylase played a key role in the biosynthesis of β- phenethyl alcohol. This work established an important foundation for construction of the β-phenethyl alcohol high-producing genetically engineered strains.