Heterotrophic and phototrophic multi-step biocatalysis for the production of nylon building blocks from renewables

Multi-step whole-cell biocatalysis is a versatile approach to facilitate the sustainable synthesis of value-added compounds, such as polyamides (PA) building blocks, from renewables. Currently, PAs are produced via multiple process steps, energy-, and waste- intensive reaction processes, heavily based on fossil resources. ¹ Whereas whole-cell biocatalysis benefits from highly specific enzymatic reactions under mild conditions, resulting in less by-product formation. It also enables multiple reactions in a single process step via the design of orthologous pathways (in-vivo cascades) thereby avoiding intermediary purification steps. ^{2; 3} Nylon 6 (PA6), an industrially widely used PA, can be synthesised from 6-aminohexanoic acid (6-AHA), which not only can serve as key monomer for PA6, but also constitutes a crucial pharmaceutical agent for the treatment of aneurysmal subarachnoid haemorrhages.

Our research aims to engineer biocatalysts capable of efficiently producing 6-AHA through an enzymatic cascade assembled from genes of diverse microbial origin. The goal is to maximize, scale up, and compare production efficiencies of heterotrophic strains, with a focus on Escherichia coli JM101, Pseudomonas taiwanensis VLB120, as well as the phototrophic cyanobacterium Synechocystis sp. PCC 6803 as host strains, the latter with the goal to enable light-driven 6-AHA synthesis. Substrates for this process are plant oil-derived hexanoic acid methyl esters or hexanoic acid, which can be produced from lignocellulosic biomass via an anaerobic digestion process.

To quantitatively analyse, detect bottle necks in, and to optimize the transgenic cascade, the enzymes are tested individually and in combinations. Promising in-vivo enzyme activities for the terminal functionalization of hexanoic acid methyl ester inter alia by the alkane monooxygenase AlkBGT will be reported.

We aim at the implementation of the resulting reaction cascade in a suitable bio-process concept mitigating substrate toxicity. The toxic effect of the apolar substrate HAME is likely caused by accumulation in the cell membrane. ³ Thus, various feeding strategies and a two-phase reactor approaches will be presented.

1. Bretschneider, L.; Wegner, M.; Bühler, K.; Bühler, B.; Karande, R. (2021) DOI: 10.1111/1751-7915.13744

2. Ladkau N.; Schmid, A.; Bühler, B. (2014) DOI: 10.1016/j.copbio.2014.06.003

3. Schrewe, M.; Julsing, M. K.; Bühler, B.; Schmid, A. (2013) DOI: 10.1039/c3cs60011d