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 -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.T he outer membrane of Gram-negative bacteria serves as an efficient barrier for hydrophobic molecules (12,36,44). Different approaches have been described to overcome uptake limitations in whole-cell biotransformations. The two-liquid-phase concept applied in stirred-tank reactors can increase mass transfer by ensuring maximal substrate availability and typically allows in situ product extraction (13,20,33,37,40,47,53,72). Next to that, the addition of rhamnolipids, synthetic surfactants, and cosolvents has been described as enhancing biotransformation rates. Rhamnolipids are synthesized by several Pseudomonas species in order to facilitate the uptake of hydrophobic compounds (46). Rhamnolipids as well as synthetic surfactants (e.g., Triton X-100) solubilize hydrophobic substrates in the aqueous phase and interact with bacterial membranes (1, 39). Similarly, cosolvents, such as dimethyl sulfoxide (DMSO), or chelating agents, such as EDTA, can be used to enhance substrate solubility and/or membrane permeability (44). Further strategies to improve rates for hydrophobic substrate bioconversions include the use of host strains capable of growth on and thus efficient uptake of hydrophobic substrates, such as hydrocarbons, or the transfer of respective properties, ...

Lactam formation from w-aminocarboxylic acids is thermodynamically unfavored in aqueous solution and therefore hard to achieve. In the present work w-laurolactam hydrolases from Acidovorax sp. T31 and Cupriavidus sp. U124 were investigated regarding their potential to catalyze lactam formation. Both enzymes are known to hydrolyze laurolactam to 12-aminododecanoic acid. The w-laurolactam hydrolase genes were expressed in Escherichia coli BL21 (DE3) and the catalytic activity of the respective proteins was investigated. As expected from thermodynamics, only laurolactam hydrolysis but not 12-aminododecanoic acid cyclization was observed in whole-cell biotransformations and cell extract assays. The utilization of 12-aminododecanoic acid methyl ester, as an activated form of 12-aminododecanoic acid, resulted in intramolecular amide bond formation with the product laurolactam. Maximum laurolactam formation rates of 13.5 and 14.3 U g CDW À1 and molar yields of 11.5% and 13.0% were achieved in biotransformations at pH 10 with recombinant E. coli harboring the w-laurolactam hydrolase from Cupriavidus sp. U124 and Acidovorax sp. T31, respectively. Furthermore, it was shown that under the harsh reaction conditions applied, the utilization of whole-cell biocatalysts enables 17.2-fold higher laurolactam formation activity in comparison to free enzymes in solution. This study shows that hydrolasecatalyzed laurolactam synthesis can be achieved in aqueous solution by selection of an appropriate substrate and reaction pH.