Multistep biocatalysis for the sequential oxy- and amino-functionalization of renewable dodecanoic acid methyl ester
Strain engineering and process design
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Beschreibung
Toolbox-like whole-cell biocatalysts harboring heterologous pathways provide novel approaches for the development of sustainable products and processes for chemical and pharmaceutical industry. To fully exploit the potential of this approach, concerted strategies, combining biocatalyst, reaction and process engineering have to be pursued.
By following such an systems biotechnology / synthetic biology approach, an Escherichia coli-based bioprocess for the multistep conversion of renewable dodecanoic acid methyl ester (DAME) to 12-aminododecanoic acid methyl ester (ADAME), a valuable monomer for the synthesis of the high performance polymer Polyamide 12 was designed in the frame of this thesis. The respective synthetic pathway primarily is composed of the alkane monooxygenase complex AlkBGT from Pseudomonas putida GPo1 and the ω-transaminase from Chromobacterium violaceum 2025 (CV2025) for the sequential conversion of DAME to its terminal alcohol, aldehyde, and finally amine. Further biocatalyst engineering enabled facilitated DAME transfer over cellular membranes via the hydrophobic porin AlkL and intracellular supply of the transamination cosubstrate L-alanine via a heterologous alanine dehydrogenase regenerating L-alanine from pyruvate. Finally, the introduction of the alcohol dehydrogenase AlkJ and fine-tuning of multiple gene expression enhanced fluxes through the artificial pathway. This and the parallel design of a two-liquid phase biotransformation setup for preparative synthesis making use of oleic acid as reactive in situ extractant for toxic ADAME enabled almost exclusive ADAME formation without significant accumulation of intermediates.
Autor*in
Nadine Ladkau
Themen in »Multistep biocatalysis for the sequential oxy- and amino-functionalization of renewable dodecanoic acid methyl ester«
bioprocess design multistep biocatalysis two-liquid phase system whole-cell biotransformation