Direct Terminal Alkylamino‐Functionalization via Multistep Biocatalysis in One Recombinant Whole‐Cell Catalyst

Abstract

Direct and regiospecific amino‐functionalization of non‐activated carbon could be achieved using one recombinant microbial catalyst. The presented proof of concept shows that heterologous pathway engineering allowed the construction of a whole‐cell biocatalyst catalyzing the terminal amino‐functionalization of fatty acid methyl esters (e.g., dodecanoic acid methyl ester) and alkanes (e.g., octane). By coupling oxygenase and transaminase catalysis in vivo, both substrates are converted with absolute regiospecificity to the terminal amine via two sequential oxidation reactions followed by an amination step. Such demanding chemical three‐step reactions achieved with a single catalyst demonstrate the tremendous potential of whole‐cell biocatalysts for the production of industrially relevant building blocks.

Biochemical Analysis of Recombinant AlkJ from Pseudomonas putida Reveals a Membrane-Associated, Flavin Adenine Dinucleotide-Dependent Dehydrogenase Suitable for the Biosynthetic Production of Aliphatic Aldehydes

“…In particular, AlkJ, with its proven substrate specificity, seems to be predestined for the biocatalytic synthesis of OLAMe from HLAMe. OLAMe in turn can be enzymatically transaminated to 12-amino lauric acid methyl ester (ALAMe), a valuable building block for high-performance polymers (13).…”

“…Nevertheless, this catalytic activity is about 9 times lower than that for conversion of HLAMe into the aldehyde. This selective oxidation of the alcohol substrate by AlkJ should offer an opportunity to reduce the known overoxidation during the biocatalytic -functionalization of LAMe with the P. putida AlkBGT system, which was shown to yield only approximately equimolar amounts of aldehyde and (di-)carboxylic acid from LAMe (13). By sequestering the initial biocatalytic reaction product of AlkBGT, HLAMe, and accelerating its selective oxidation to OLAMe via AlkJ, the overall yield of aldehyde may be boosted, thus providing a more efficient substrate supply for the subsequent transamination to the industrially relevant ALAMe (Fig.…”

“…Efforts to employ the AlkBGT system in combination with a transaminase for the selective -amino functionalization of fatty acids have reached advanced stage, allowing, for example, the generation of -amino lauric acid methyl ester, which serves as precursor for high-performance polymers (13). However, during initial conversion of the substrate lauric acid methyl ester (LAMe) into the alcohol hydroxy-LAMe (HLAMe) by AlkBGT, considerable overoxidation to the aldehyde oxo-LAMe (OLAMe) and even to dodecanedioic acid monomethyl ester (DDAMe) was observed, which impairs the overall process yield (10) (Fig.…”

Efficient production of the Nylon 12 monomer ω-aminododecanoic acid methyl ester from renewable dodecanoic acid methyl ester with engineered Escherichia coli

“…However, the hitherto poor 79 performance of such pathways and their coupling to the microbial physiology represent major 80 challenges, which remain to be tackled. Recently, we combined oxygenase and transaminase 81 catalysis in such an orthogonal pathway for the conversion of plant oil derived dodecanoic acid 82 methyl ester (DAME) to 12-aminododecanoic acid methyl ester (ADAME), a monomer 83 suitable for Nylon 12 synthesis (Schrewe et al, 2013b). ADAME was produced in E. coli via a 84 three-step cascade, in which terminal DAME hydroxylation and alcohol oxidation both 85 catalyzed by the alkane monooxygenase AlkBGT from Pseudomonas putida GPo1 were 86 followed by terminal amination by means of the Chromobacterium violaceum -transaminase 87 (CV2025) (Fig.…”

Developments in Advanced Synthesis & Catalysis during 2013 and Plans for 2014

“…Details can be found in their communication. [4] . Also featured in that issue is a review by Wolfgang Kroutil and co-authors on C À C bond hydrolases.…”