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首页> 外文期刊>Applied Microbiology >Outer Membrane Protein AlkL Boosts Biocatalytic Oxyfunctionalization of Hydrophobic Substrates in Escherichia coli
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Outer Membrane Protein AlkL Boosts Biocatalytic Oxyfunctionalization of Hydrophobic Substrates in Escherichia coli

机译：外膜蛋白AlkL增强大肠杆菌中疏水性底物的生物催化氧化功能。

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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 ω-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.
机译：微生物细胞的外膜形成疏水化合物的有效屏障，可能导致疏水性底物的吸收限制。对于含有恶臭假单胞菌GPo1的烷烃单加氧酶AlkBGT的重组大肠杆菌，十二烷酸甲酯的ω-氧官能化已观察到低的生物转化活性（1.9 U g_（cdw）〜（Δ1））。本研究使用脂肪酸甲酯的氧合作为模型反应，研究了改善疏水性底物细菌吸收的策略。混合表面活性剂和助溶剂以改善底物的增溶作用不会导致增加氧合速率。 EDTA的添加使初始十二烷酸甲酯的氧合活性增加了2.8倍。使用重组荧光假单胞菌CHA0代替大肠杆菌会导致类似的活性增加。然而，仍然发现底物向细胞内的质量转移是有限的。值得注意的是，恶臭假单胞菌GPo1的alkL基因的共表达编码了一种具有未知功能的外膜蛋白，使重组大肠杆菌的十二烷酸甲酯的氧合活性增加了28倍。在两相生物反应器中，最大活性达到87 U g_（cdw）〜（？1），增加了62倍，从而使高滴度的末端氧官能化产物得以积累。 alkL的共表达还增加了对天然AlkBGT底物辛烷和壬烷的氧化活性，这首次显示出清晰的证据表明AlkL在烷烃降解中具有重要作用。这项研究表明，AlkL是提高涉及疏水性脂族底物的全细胞生物转化效率的有效工具，因此具有广泛的应用潜力。

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