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

The expansion of microbial substrate and product scopes will be an important brick promoting future bioeconomy. In this study, an orthogonal pathway running in parallel to native metabolism and converting renewable dodecanoic acid methyl ester (DAME) via terminal alcohol and aldehyde to 12-aminododecanoic acid methyl ester (ADAME), a building block for the high-performance polymer Nylon 12, was engineered in Escherichia coli and optimized regarding substrate uptake, substrate requirements, host strain choice, flux, and product yield. Efficient DAME uptake was achieved by means of the hydrophobic outer membrane porin AlkL increasing maximum oxygenation and transamination activities 8.3 and 7.6-fold, respectively. An optimized coupling to the pyruvate node via a heterologous alanine dehydrogenase enabled efficient intracellular L-alanine supply, a prerequisite for self-sufficient whole-cell transaminase catalysis. Finally, the introduction of a respiratory chain-linked alcohol dehydrogenase enabled an increase in pathway flux, the minimization of undesired overoxidation to the respective carboxylic acid, and thus the efficient formation of ADAME as main product. The completely synthetic orthogonal pathway presented in this study sets the stage for Nylon 12 production from renewables. Its effective operation achieved via fine tuning the connectivity to native cell functionalities emphasizes the potential of this concept to expand microbial substrate and product scopes.

Single active-site mutants are sufficient to enhance serine:pyruvate α-transaminase activity in an ω-transaminase.

We have analyzed the natural evolution of transaminase structure and sequence between an α-transaminase serine-pyruvate aminotransferase and an ω-transaminase from Chromobacterium violaceum with < 20% sequence identity, and identified the active-site regions that are least conserved structurally. We also show that these structural changes correlate strongly with transaminase substrate specificity during evolution and therefore might normally be presumed to be essential determinants of substrate specificity. However, key residues are often conserved spatially during evolution and yet originate from within a different region of the sequence via structural reorganizations. In the present study, we also show that α-transaminase-type serine-pyruvate aminotransferase activity can be engineered into the CV2025 ω-transaminase scaffold with any one of many possible single-point mutations at three key positions, without the requirement for significant backbone remodeling, or repositioning of the residue from a different region of sequence. This finding has significant implications for enzyme redesign in which solutions to substrate specificity changes may be found more efficiently than is achieved by engineering in all sequence and structure determinants identified by correlation to substrate specificity.

The microbial cell-functional unit for energy dependent multistep biocatalysis.

Whole-cell biocatalysis has emerged as an important tool for the synthesis of value-added fine and bulk chemicals as well as pharmaceuticals. Especially, the rapid development of recombinant DNA technologies resulted in a shift from the exploitation of natural enzymes and pathways to the design of recombinant cell factories comprising heterologous enzymes and/or synthetic, orthologous pathways for the synthesis of industrially relevant compounds. This review discusses recent developments and concepts applied in the frame of multistep whole-cell biocatalysis along with representative examples.