Novel microbial cultivation platforms are of increasing interest to researchers in academia and industry. The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups. Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish. It provides control over (co-)culturing conditions similar to a chemostat, while allowing organisms to be observed microscopically. The device was designed to be affordable, reusable, and above all, versatile. To test its functionality and general utility, we performed multiple experiments with Escherichia coli cells harboring synthetic gene circuits and were able to quantitatively study emerging expression dynamics in real-time via fluorescence microscopy. Furthermore, we demonstrated that the device provides a unique environment for the cultivation of nematodes, suggesting that the device could also prove useful in microscopy studies of multicellular microorganisms.
The enzyme system AlkBGT from Pseudomonas putida GPo1 can efficiently -functionalize fatty acid methyl esters. Outer membrane protein AlkL boosts this -functionalization. In this report, it is shown that whole cells of Escherichia coli expressing the AlkBGT system can also -oxidize ethyl nonanoate (NAEE). Coexpression of AlkBGT and AlkL resulted in 1.7-fold-higher -oxidation activity on NAEE. With this strain, initial activity on NAEE was 70 U/g (dry weight) of cells (g cdw ), 67% of the initial activity on methyl nonanoate. In time-lapse conversions with 5 mM NAEE the main product was 9-hydroxy NAEE (3.6 mM), but also 9-oxo NAEE (0.1 mM) and 9-carboxy NAEE (0.6 mM) were formed. AlkBGT also -oxidized ethyl, propyl, and butyl esters of fatty acids ranging from C 6 to C 10 . Increasing the length of the alkyl chain improved the -oxidation activity of AlkBGT on esters of C 6 and C 7 fatty acids. From these esters, application of butyl hexanoate resulted in the highest -oxidation activity, 82 U/g cdw . Coexpression of AlkL only had a positive effect on -functionalization of substrates with a total length of C 11 or longer. These findings indicate that AlkBGT(L) can be applied as a biocatalyst for -functionalization of ethyl, propyl, and butyl esters of medium-chain fatty acids. IMPORTANCEFatty acid esters are promising renewable starting materials for the production of -hydroxy fatty acid esters (-HFAEs).-HFAEs can be used to produce sustainable polymers. Chemical conversion of the fatty acid esters to -HFAEs is challenging, as it generates by-products and needs harsh reaction conditions. Biocatalytic production is a promising alternative. In this study, biocatalytic conversion of fatty acid esters toward -HFAEs was investigated using whole cells. This was achieved with recombinant Escherichia coli cells that produce the AlkBGT enzymes. These enzymes can produce -HFAEs from a wide variety of fatty acid esters. Medium-chain-length acids (C 6 to C 10 ) esterified with ethanol, propanol, or butanol were applied. This is a promising production platform for polymer building blocks that uses renewable substrates and mild reaction conditions. T he global demand for polymers is expected to grow in the coming years (1) and thus also the need for sustainable polymer production processes. -Hydroxy fatty acids (-HFAs) and dicarboxylic acids (DCAs) are building blocks of polymers such as polyesters and polyamides (1-3). These compounds can be produced from medium-chain-length fatty acids (MCFAs) by oxidation of the terminal methyl group, a reaction called -oxidation (4).A recent development is the production of fatty acids by processes using microbial chain elongation from organic waste material, yielding both odd-and even-chain-length fatty acids ranging from C 4 to C 9 (5, 6).Chemical -oxidation of nonactivated terminal methyl groups remains challenging due to the inert nature of these bonds. This results in poor selectivity (7,8). Biocatalytic -oxidation can be a solution for the terminal activation of fatty acid (est...
Direct and selective terminal oxidation of medium-chain n-alkanes is a major challenge in chemistry. Efforts to achieve this have so far resulted in low specificity and overoxidized products. Biocatalytic oxidation of medium-chain n-alkanes - with for example the alkane monooxygenase AlkB from P. putida GPo1- on the other hand is highly selective. However, it also results in overoxidation. Moreover, diterminal oxidation of medium-chain n-alkanes is inefficient. Hence, α,ω-bifunctional monomers are mostly produced from olefins using energy intensive, multi-step processes. By combining biocatalytic oxidation with esterification we drastically increased diterminal oxidation upto 92mol% and reduced overoxidation to 3% for n-hexane. This methodology allowed us to convert medium-chain n-alkanes into α,ω-diacetoxyalkanes and esterified α,ω-dicarboxylic acids. We achieved this in a one-pot reaction with resting-cell suspensions of genetically engineered Escherichia coli. The combination of terminal oxidation and esterification constitutes a versatile toolbox to produce α,ω-bifunctional monomers from n-alkanes.
SummaryThe AlkBGTL proteins coded on the alk operon from Pseudomonas putida GPo1 can selectively ω‐oxidize ethyl esters of C6 to C10 fatty acids in whole‐cell conversions with Escherichia coli. The major product in these conversions is the ω‐alcohol. However, AlkB also has the capacity to overoxidize the substrate to the ω‐aldehyde and ω‐acid. In this study, we show that alcohol dehydrogenase AlkJ and aldehyde dehydrogenase AlkH are able to oxidize ω‐alcohols and ω‐aldehydes of esterified fatty acids respectively. Resting E. coli expressing AlkBGTHJL enabled exclusive mono‐ethyl azelate production from ethyl nonanoate, with an initial specific activity of 61 U gcdw −1. Within 2 h, this strain produced 3.53 mM mono‐ethyl azelate, with a yield of 0.68 mol mol−1. This strain also produced mono‐ethyl dicarboxylic acids from ethyl esters of C6 to C10 fatty acids and mono‐methyl azelate from methyl nonanoate. Adding ethyl nonanoate dissolved in carrier solvent bis‐(2‐ethylhexyl) phthalate enabled an increase in product titres to 15.55 mM in two‐liquid phase conversions. These findings indicate that E. coli expressing AlkBGTHJL is an effective producer of mono‐esterified dicarboxylic acids from fatty acid esters.
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