Identification and use of an alkane transporter plug-in for applications in biocatalysis and whole-cell biosensing of alkanes.
Identification and use of an alkane transporter plug-in for applications in biocatalysis and whole-cell biosensing of alkanes.
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DOI：
10.1038/srep05844
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发表时间：
2014-07-28
影响因子：
4.6
通讯作者：
Baganz F
中科院分区：
文献类型：
--
作者：
Grant C;Deszcz D;Wei YC;Martínez-Torres RJ;Morris P;Folliard T;Sreenivasan R;Ward J;Dalby P;Woodley JM;Baganz F
全细胞装置在合成生物学和生物催化中的有效应用将始终需要考虑目标分子进入细胞的摄取。在这里，我们证明了来自恶臭假单胞菌GPo1的AlkL蛋白是一种烷烃进口蛋白，能够以工业相关的速率吸收C7-C16正构烷烃。在没有alkL表达的情况下，天然大肠杆菌正构烷烃摄取是C7-C16正构烷烃全细胞生物转化和C10和C11正构烷烃激活全细胞烷烃生物传感器的限速步骤。通过共表达alkL作为转运体插件，>C12烷烃生物氧化成脂肪醇和酸的特定产量提高了100倍。结果表明，alkL蛋白在过度表达时对宿主有毒，但当从能够控制诱导的载体上表达时，烷烃氧化的产量进一步提高了10倍（8 g/L和1.7 g/g总氧化产物）。利用控制表达载体对正辛烷活性进行进一步测试，结果显示，总氧化产物的最高氧化率为120 μmol/min/g和1 g/L/h。这是AlkL第一次被证明直接促进C10-C16烷烃的吸收，并且代表了报告中使用它所产生的产品收率的最高收益。
Effective application of whole-cell devices in synthetic biology and biocatalysis will always require consideration of the uptake of molecules of interest into the cell. Here we demonstrate that the AlkL protein from Pseudomonas putida GPo1 is an alkane import protein capable of industrially relevant rates of uptake of C7-C16 n-alkanes. Without alkL expression, native E.coli n-alkane uptake was the rate-limiting step in both the whole-cell bioconversion of C7-C16 n-alkanes and in the activation of a whole-cell alkane biosensor by C10 and C11 alkanes. By coexpression of alkL as a transporter plug-in, specific yields improved by up to 100-fold for bioxidation of >C12 alkanes to fatty alcohols and acids. The alkL protein was shown to be toxic to the host when overexpressed but when expressed from a vector capable of controlled induction, yields of alkane oxidation were improved a further 10-fold (8 g/L and 1.7 g/g of total oxidized products). Further testing of activity on n-octane with the controlled expression vector revealed the highest reported rates of 120 μmol/min/g and 1 g/L/h total oxidized products. This is the first time AlkL has been shown to directly facilitate enhanced uptake of C10-C16 alkanes and represents the highest reported gain in product yields resulting from its use.