AlkBGT

In this study, whole-cell biotransformation was conducted to produce nonanedioic acid from nonanoic acid by expressing the alkane hydroxylating system (AlkBGT) from Pseudomonas putida GPo1 in Escherichia coli. Following adaptive laboratory evolution, an efficient E. coli mutant strain, designated as MRE, was successfully obtained, demonstrating the fastest growth (27-fold higher) on nonanoic acid as the sole carbon source compared to the wild-type strain. Additionally, the MRE strain was engineered to block nonanoic acid degradation by deleting fadE...

Hydroxy fatty acids (HFAs) are valuable compounds that are widely used in medical, cosmetic and food fields. Production of ω-HFAs via bioconversion by engineered Escherichia coli has received a lot of attention because this process is environmentally friendly. In this study, a whole-cell bio-catalysis strategy was established to synthesize medium-chain ω-HFAs based on the AlkBGT hydroxylation system from Pseudomonas putida GPo1. The effects of blocking the β-oxidation of fatty acids (FAs) and enhancing the transportation of FAs on ω-HFAs bio-production were also investigated...

In this study, a signal peptide of AlkL was replaced with other signal peptides to improve the soluble expression and thereby facilitate the transport of dodecanoic acid methyl ester (DAME) substrate into the E. coli. Consequently, AlkL with signal peptide FadL (AlkLf ) showed higher transport activity toward DAME. Furthermore, the promoter optimization for the efficient heterologous expression of the transporter AlkLf and alkane monooxygenase (AlkBGT) system was conducted and resulted in increased ω-oxygenation activity of AlkBGT system...

Two-liquid-phase reaction media have long been used in bioconversions to supply or remove hydrophobic organic reaction substrates and products to reduce inhibitory and toxic effects on biocatalysts. In case of the terminal oxyfunctionalization of linear alkanes by the AlkBGT monooxygenase the excess alkane substrate is often used as a second phase to extract the alcohol, aldehyde, and acid products. However, the selection of other carrier phases or surfactants is complex due to a large number of parameters that are involved, such as biocompatibility, substrate bioavailability, and product extraction selectivity...

BACKGROUND: Numerous challenges remain to achieve industrially competitive space-time yields for bio-oxidations. The ability to rapidly screen bioconversion reactions for characterization and optimization is of major importance in bioprocess development and biocatalyst selection; studies at conventional lab scale are time consuming and labor intensive with low experimental throughput. The direct ω-oxyfunctionalization of aliphatic alkanes in a regio- and chemoselective manner is efficiently catalyzed by monooxygenases such as the AlkBGT enzyme complex from Pseudomonas putida under mild conditions...

Gas-liquid mass transfer of gaseous reactants is a major limitation for high space-time yields, especially for O2 -dependent (bio)catalytic reactions in aqueous solutions. Herein, oxygenic photosynthesis was used for homogeneous O2 supply via in situ generation in the liquid phase to overcome this limitation. The phototrophic cyanobacterium Synechocystis sp. PCC6803 was engineered to synthesize the alkane monooxygenase AlkBGT from Pseudomonas putida GPo1. With light, but without external addition of O2 , the chemo- and regioselective hydroxylation of nonanoic acid methyl ester to ω-hydroxynonanoic acid methyl ester was driven by O2 generated through photosynthetic water oxidation...

It is a common misconception in whole-cell biocatalysis to refer to an enzyme as the biocatalyst, thereby neglecting the structural and metabolic framework provided by the cell. Here, the low whole-cell biocatalyst stability, that is, the stability of specific biocatalyst activity, in a process for the terminal oxyfunctionalization of renewable fatty acid methyl esters was investigated. This reaction, which is difficult to achieve by chemical means, is catalyzed by Escherichia coli featuring the monooxygenase system AlkBGT and the uptake facilitator AlkL from Pseudomonas putida GPo1...

UNLABELLED: The enzyme system AlkBGT from Pseudomonas putida GPo1 can efficiently ω-functionalize fatty acid methyl esters. Outer membrane protein AlkL boosts this ω-functionalization. In this report, it is shown that whole cells of Escherichia coli expressing the AlkBGT system can also ω-oxidize ethyl nonanoate (NAEE). Coexpression of AlkBGT and AlkL resulted in 1.7-fold-higher ω-oxidation activity on NAEE. With this strain, initial activity on NAEE was 70 U/g (dry weight) of cells (gcdw), 67% of the initial activity on methyl nonanoate...

BACKGROUND: In recent years, there have been intensive efforts to develop synthetic microbial platforms for the production, biosensing and bio-remediation of fossil fuel constituents such as alkanes. Building predictable engineered systems for these applications will require the ability to tightly control and modulate the rate of import of alkanes into the host cell. The native components responsible for the import of alkanes within these systems have yet to be elucidated. To shed further insights on this, we used the AlkBGT alkane monooxygenase complex from Pseudomonas putida GPo1 as a reporter system for assessing alkane import in Escherichia coli...

An engineered reversal of the β-oxidation cycle was exploited to demonstrate its utility for the synthesis of medium chain (6-10-carbons) ω-hydroxyacids and dicarboxylic acids from glycerol as the only carbon source. A redesigned β-oxidation reversal facilitated the production of medium chain carboxylic acids, which were converted to ω-hydroxyacids and dicarboxylic acids by the action of an engineered ω-oxidation pathway. The selection of a key thiolase (bktB) and thioesterase (ydiI) in combination with previously established core β-oxidation reversal enzymes, as well as the development of chromosomal expression systems for the independent control of pathway enzymes, enabled the generation of C6-C10 carboxylic acids and provided a platform for vector based independent expression of ω-functionalization enzymes...

The oxyfunctionalization of unactivated C−H bonds can selectively and efficiently be catalyzed by oxygenase-containing whole-cell biocatalysts. Recombinant Escherichia coli W3110 containing the alkane monooxygenase AlkBGT and the outer membrane protein AlkL from Pseudomonas putida GPo1 have been shown to efficiently catalyze the terminal oxyfunctionalization of renewable fatty acid methyl esters yielding bifunctional products of interest for polymer synthesis. In this study, AlkBGTL-containing E. coli W3110 is shown to catalyze the multistep conversion of dodecanoic acid methyl ester (DAME) via terminal alcohol and aldehyde to the acid, exhibiting Michaelis-Menten-type kinetics for each reaction step...

This study evaluates the technical feasibility of biofilm-based biotransformations at an industrial scale by theoretically designing a process employing membrane fiber modules as being used in the chemical industry and compares the respective process parameters to classical stirred-tank studies. To our knowledge, catalytic biofilm processes for fine chemicals production have so far not been reported on a technical scale. As model reactions, we applied the previously studied asymmetric styrene epoxidation employing Pseudomonas sp...

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...