Outer Membrane Protein AlkL Boosts Biocatalytic Oxyfunctionalization of Hydrophobic Substrates in Escherichia coli

Author:

Julsing Mattijs K.,Schrewe Manfred,Cornelissen Sjef,Hermann Inna,Schmid Andreas,Bühler Bruno

Abstract

ABSTRACTThe 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 gcdw−1) have been observed for the ω-oxyfunctionalization of dodecanoic acid methyl ester by recombinantEscherichia colicontaining the alkane monooxygenase AlkBGT ofPseudomonas putidaGPo1. 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 recombinantPseudomonas fluorescensCHA0 instead ofE. coliresulted in a similar activity increase. However, substrate mass transfer into cells was still found to be limiting. Remarkably, the coexpression of thealkLgene ofP. putidaGPo1 encoding an outer membrane protein with so-far-unknown function increased the dodecanoic acid methyl ester oxygenation activity of recombinantE. coli28-fold. In a two-liquid-phase bioreactor setup, a 62-fold increase to a maximal activity of 87 U gcdw−1was achieved, enabling the accumulation of high titers of terminally oxyfunctionalized products. Coexpression ofalkLalso 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.

Publisher

American Society for Microbiology

Subject

Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology

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