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6-Aminohexanoic acid (6AHA) is a vital polymer building block for Nylon 6 production and an FDA-approved orphan drug. However, its production from cyclohexane is associated with several challenges, including low conversion and yield, and severe environmental issues. We aimed at overcoming these challenges by developing a bioprocess for 6AHA synthesis. A mixed-species approach turned out to be most promising. Thereby, Pseudomonas taiwanensis VLB120 strains harbouring an upstream cascade converting cyclohexane to either є-caprolactone (є-CL) or 6-hydroxyhexanoic acid (6HA) were combined with Escherichia coli JM101 strains containing the corresponding downstream cascade for the further conversion to 6AHA. e-CL was found to be a better 'shuttle molecule' than 6HA enabling higher 6AHA formation rates and yields. Mixed-species reaction performance with 4 g l -1 biomass, 10 mM cyclohexane, and an airto-aqueous phase ratio of 23 combined with a repetitive oxygen feeding strategy led to complete substrate conversion with 86% 6AHA yield and an initial specific 6AHA formation rate of 7.7 AE 0.1 U g . The same cascade enabled 49% 7-aminoheptanoic acid yield from cycloheptane. This combination of rationally engineered strains allowed direct 6AHA production from cyclohexane in one pot with high conversion and yield under environmentally benign conditions.

6-hydroxyhexanoic acid (6HA) represents a polymer building block for the biodegradable polymer polycaprolactone. Alternatively to energy- and emission-intensive multistep chemical synthesis, it can be synthesized directly from cyclohexane in one step by recombinant Pseudomonas taiwanensis harboring a 4-step enzymatic cascade without the accumulation of any intermediate. In the present work, we performed a physiological characterization of this strain in different growth media and evaluated the resulting whole-cell activities. RB and M9* media led to reduced gluconate accumulation from glucose compared to M9 medium and allowed specific activities up to 37.5 ± 0.4 U gCDW−1 for 6HA synthesis. However, 50% of the specific activity was lost within 1 h in metabolically active resting cells, specifying growing cells, or induced resting cells as favored options for long-term biotransformation. Furthermore, the whole-cell biocatalyst was evaluated in a stirred-tank bioreactor setup with a continuous cyclohexane supply via the gas phase. At cyclohexane feed rates of 0.276 and 1.626 mmol min−1 L−1, whole-cell biotransformation occurred at first-order and zero-order rates, respectively. A final 6HA concentration of 25 mM (3.3 g L−1) and a specific product yield of 0.4 g gCDW−1 were achieved with the higher feed rate. Product inhibition and substrate toxification were identified as critical factors limiting biocatalytic performance. Future research efforts on these factors and the precise adjustment of the cyclohexane feed combined with an in situ product removal strategy are discussed as promising strategies to enhance biocatalyst durability and product titer and thus to enable the development of a sustainable multistep whole-cell process.

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