Outer Membrane Protein AlkL Boosts Biocatalytic Oxyfunctionalization of Hydrophobic Substrates in Escherichia coli
Outer Membrane Protein AlkL Boosts Biocatalytic Oxyfunctionalization of Hydrophobic Substrates in Escherichia coli
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DOI：
10.1128/aem.00949-12
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发表时间：
2012-08-01
影响因子：
4.4
通讯作者：
Buehler, Bruno
中科院分区：
文献类型：
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作者：
Julsing, Mattijs K.;Schrewe, Manfred;Buehler, Bruno
微生物细胞的外膜对疏水化合物形成有效的屏障，潜在地导致对疏水底物的吸收限制。恶臭假单胞菌GPo1烷氧基单加氧酶AlkBGT的重组大肠杆菌对十二酸甲酯的氧化反应活性较低(1.9U g(CDW)(-1))。本研究以脂肪酸甲酯氧化为模型反应，研究了提高细菌对疏水底物摄取的策略。混合表面活性剂和助溶剂以改善底物增溶并不会导致氧合速率增加。EDTA的加入使十二酸甲酯的初始氧化活性提高了2.8倍。用重组荧光假单胞菌CHA0代替大肠杆菌，其活性也有类似的提高。然而，底物向细胞内的传质仍然是有限的。值得注意的是，恶臭假单胞菌GPo1编码一种功能未知的外膜蛋白的alkL基因的共表达使重组大肠杆菌的十二酸甲酯氧化活性提高了28倍。在两液相生物反应器装置中，最大活性达到87Ug(CDW)(-1)，增加了62倍，从而能够积累高滴度的末端含氧官能化产物。AlkL的共表达也增加了对天然AlkBGT底物辛烷和正庚烷的氧化活性，首次显示出AlkL在烷烃降解中的显著作用。这项研究表明，AlkL是一种有效的工具，可以提高涉及疏水脂肪底物的全细胞生物转化的生产率，因此具有广泛的应用潜力。
The outer membrane of microbial cells forms an effective barrier for hydrophobic compounds, potentially causing an uptake limitation for hydrophobic substrates. Low bioconversion activities (1.9 U g(cdw)(-1)) have been observed for the omega-oxyfunctionalization of dodecanoic acid methyl ester by recombinant Escherichia coli containing the alkane monooxygenase AlkBGT of Pseudomonas putida GPo1. Using fatty acid methyl ester oxygenation as the model reaction, this study investigated strategies to improve bacterial uptake of hydrophobic substrates. Admixture of surfactants and cosolvents to improve substrate solubilization did not result in increased oxygenation rates. Addition of EDTA increased the initial dodecanoic acid methyl ester oxygenation activity 2.8-fold. The use of recombinant Pseudomonas fluorescens CHA0 instead of E. coli resulted in a similar activity increase. However, substrate mass transfer into cells was still found to be limiting. Remarkably, the coexpression of the alkL gene of P. putida GPo1 encoding an outer membrane protein with so-far-unknown function increased the dodecanoic acid methyl ester oxygenation activity of recombinant E. coli 28-fold. In a two-liquid-phase bioreactor setup, a 62-fold increase to a maximal activity of 87 U g(cdw)(-1) was achieved, enabling the accumulation of high titers of terminally oxyfunctionalized products. Coexpression of alkL also increased oxygenation activities toward the natural AlkBGT substrates octane and nonane, showing for the first time clear evidence for a prominent role of AlkL in alkane degradation. This study demonstrates that AlkL is an efficient tool to boost productivities of whole-cell biotransformations involving hydrophobic aliphatic substrates and thus has potential for broad applicability.