Synthesis and Accumulation of Aromatic Aldehydes in an Engineered Strain of Escherichia coli
Synthesis and Accumulation of Aromatic Aldehydes in an Engineered Strain of Escherichia coli
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DOI：
10.1021/ja506664a
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发表时间：
2014-08-20
影响因子：
15
通讯作者：
Prather, Kristala L. J.
中科院分区：
文献类型：
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作者：
Kunjapur, Aditya M.;Tarasova, Yekaterina;Prather, Kristala L. J.
芳香醛在许多应用中都很有用，特别是作为香料、香料和药物前体。然而，微生物合成的醛阻碍了快速，内源性和多余的醛转化为相应的醇。我们报道了一株具有还原芳香醛（RARE）的大肠杆菌K-12 MG1655菌株的构建，该菌株作为芳香醛生物合成的平台。6个对模型底物苯甲醛有活性的基因被合理地定位为缺失：3个编码醛酮还原酶的基因和3个编码醇脱氢酶的基因。在RARE菌株中表达重组羧酸还原酶并在生长过程中添加苯甲酸酯，培养24 h后苯甲醛仍然存在，苯甲醛转化为苯甲醇的转化率低于12%。虽然个体过表达结果表明，所有6个基因都可以在体内还原苯甲醛，但对亚群缺失菌株进行的额外实验显示，在测试条件下，其中两个基因缺失是不必要的。接下来，研究了该工程菌株从香草酸中生产香兰素，并成功地防止了副产物香草醇的形成。引入了一种直接从葡萄糖生物合成香兰素的途径，当使用RARE菌株时，香兰素滴度比野生型菌株提高了55倍。最后，通过在RARE菌株中表达重组丙酮酸脱羧酶突变体，证实了从苯甲醛和葡萄糖合成手性药物中间体l -苯基乙酰甲醇（L-PAC）。除了允许芳香醛作为最终产物在大肠杆菌中积累之外，RARE菌株还扩展了可以通过醛中间体产生的微生物化学物质的类别。
Aromatic aldehydes are useful in numerous applications, especially as flavors, fragrances, and pharmaceutical precursors. However, microbial synthesis of aldehydes is hindered by rapid, endogenous, and redundant conversion of aldehydes to their corresponding alcohols. We report the construction of an Escherichia coli K-12 MG1655 strain with reduced aromatic aldehyde reduction (RARE) that serves as a platform for aromatic aldehyde biosynthesis. Six genes with reported activity on the model substrate benzaldehyde were rationally targeted for deletion: three genes that encode aldo-keto reductases and three genes that encode alcohol dehydrogenases. Upon expression of a recombinant carboxylic acid reductase in the RARE strain and addition of benzoate during growth, benzaldehyde remained in the culture after 24 h, with less than 12% conversion of benzaldehyde to benzyl alcohol. Although individual overexpression results demonstrated that all six genes could contribute to benzaldehyde reduction in vivo, additional experiments featuring subset deletion strains revealed that two of the gene deletions were dispensable under the conditions tested. The engineered strain was next investigated for the production of vanillin from vanillate and succeeded in preventing formation of the byproduct vanillyl alcohol. A pathway for the biosynthesis of vanillin directly from glucose was introduced and resulted in a 55-fold improvement in vanillin titer when using the RARE strain versus the wild-type strain. Finally, synthesis of the chiral pharmaceutical intermediate L-phenylacetylcarbinol (L-PAC) was demonstrated from benzaldehyde and glucose upon expression of a recombinant mutant pyruvate decarboxylase in the RARE strain. Beyond allowing accumulation of aromatic aldehydes as end products in E. coli, the RARE strain expands the classes of chemicals that can be produced microbially via aldehyde intermediates.