Kinetic Analysis of Terminal and Unactivated CH Bond Oxyfunctionalization in Fatty Acid Methyl Esters by Monooxygenase‐Based Whole‐Cell Biocatalysis

Schrewe, Manfred; Magnusson, Anders O.; Willrodt, Christian; Bühler, Bruno; Schmid, Andreas

doi:10.1002/adsc.201100440

Cited by 50 publications

(93 citation statements)

References 71 publications

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“…The PCR product was subcloned in pSMART-HCKan (Lucigen Corporation, Middleton, WI). After restriction with SalI, alkL was cloned downstream of alkG into pBT10 (55).…”

Section: Methodsmentioning

confidence: 99%

“…Finally, coexpression of alkL, encoding an uncharacterized outer membrane protein, was investigated. The selection of AlkL was based on earlier observations showing a positive effect of an unidentified component of the alkane degradation pathway on the biotransformation of fatty acids (54), FAMEs (55), and alkanes (22). Via the characterization and comparison of factors influencing microbial uptake of hydrophobic molecules, this work identifies AlkL as a factor boosting the intracellular availability of long-chain alkylic substrates.…”

mentioning

confidence: 99%

“…Uptake has recently been found to limit the selective -oxyfunctionalization of renewable fatty acid methyl esters (FAMEs) catalyzed by recombinant Escherichia coli W3110 (pBT10), when the fatty acid chain length exceeded nine carbon atoms (55). The alkane monooxygenase complex AlkBGT catalyzing this reaction ( Fig.…”

mentioning

confidence: 99%

“…1) originates from the OCT plasmid of Pseudomonas putida GPo1 (11) and is responsible for the first enzymatic step in alkane degradation. AlkBGT catalyzes the NADH-dependent -hydroxylation of a large variety of alkyl substrates and also was reported to catalyze alcohol oxidation (38,55,66). Whereas the oxygenation activity of AlkBGT-containing E. coli W3110 (pBT10) toward dodecanoic acid methyl ester (DAME) (1.9 U g cdw Ϫ1 ) and nonanoic acid methyl ester (NAME) (104 U g cdw Ϫ1 ) differed by a factor of 55, activities obtained with an enriched enzyme preparation differed only by a factor of 2.6, indicating poor uptake of the more hydrophobic substrate DAME into E. coli cells (55).…”

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confidence: 99%

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

Julsing

Schrewe

Cornelissen

et al. 2012

Appl Environ Microbiol

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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 -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, ...

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“…The PCR product was subcloned in pSMART-HCKan (Lucigen Corporation, Middleton, WI). After restriction with SalI, alkL was cloned downstream of alkG into pBT10 (55).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Julsing

Schrewe

Cornelissen

et al. 2012

Appl Environ Microbiol

Self Cite

110

140

View full text Add to dashboard Cite

show abstract

“…Non-heme iron-dependent ω-oxygenases are a large family of enzymes that catalyze the incorporation of O 2 into various substrates including fatty acids [26]. The family includes lipoxygenases, as well as the AlkBGT system from Pseudomonas putida GPo1, which is famous for terminal alkane-, fatty ester-, and fatty acid-hydroxylation [26,27]. Lipoxygenases (EC 1.13.11) are dioxygenases that catalyze the O 2 -dependent hydroperoxidation of unsaturated fatty acids with a cis,cis-pentadiene motif.…”

Section: Introductionmentioning

confidence: 99%