Genetic engineering of Pseudomonas putida KT2440 for rapid and high-yield production of vanillin from ferulic acid
Genetic engineering of Pseudomonas putida KT2440 for rapid and high-yield production of vanillin from ferulic acid
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DOI：
10.1007/s00253-013-5303-1
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发表时间：
2014-01-01
影响因子：
5
通讯作者：
Altenbuchner, Josef
中科院分区：
文献类型：
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作者：
Graf, Nadja;Altenbuchner, Josef
香兰素是当今使用的最重要的调味剂之一。这就是为什么人们在利用天然丰富的底物进行生物技术生产方面做出了许多努力。本研究对非致病恶臭假单胞菌菌株KT2440进行了阿魏酸转化为香草醛的遗传优化。香兰素脱氢酶基因(Vdh)的缺失不足以阻止香兰素的降解。转座子突变鉴定的钼酸盐转运蛋白的额外失活导致了一株不能以香兰素为唯一碳源生长的菌株。通过引入强大的tac启动子系统，通过提高阿魏酰辅酶A合成酶(FCS)和enoyl-CoA水合酶/缩醛酶(ECH)结构基因的染色体表达，优化了生物转化。进一步的基因工程导致了高的初始转化率，在短短3小时内摩尔香兰素的产率高达86%，同时伴随着非常低的副产物水平。据我们所知，这是迄今为止假单胞菌菌株获得的最高生产率和摩尔香兰素产量。由于其对阿魏酸的高度耐受性，开发的无质粒恶臭假单胞菌代表了一种很有希望的生物技术生产香兰素的候选菌株。
Vanillin is one of the most important flavoring agents used today. That is why many efforts have been made on biotechnological production from natural abundant substrates. In this work, the nonpathogenic Pseudomonas putida strain KT2440 was genetically optimized to convert ferulic acid to vanillin. Deletion of the vanillin dehydrogenase gene (vdh) was not sufficiant to prevent vanillin degradation. Additional inactivation of a molybdate transporter, identified by transposon mutagenesis, led to a strain incapable to grow on vanillin as sole carbon source. The bioconversion was optimized by enhanced chromosomal expression of the structural genes for feruloyl-CoA synthetase (fcs) and enoyl-CoA hydratase/aldolase (ech) by introduction of the strong tac promoter system. Further genetic engineering led to high initial conversion rates and molar vanillin yields up to 86 % within just 3 h accompanied with very low by-product levels. To our knowledge, this represents the highest productivity and molar vanillin yield gained with a Pseudomonas strain so far. Together with its high tolerance for ferulic acid, the developed, plasmid-free P. putida strain represents a promising candidate for the biotechnological production of vanillin.