For the generation of bulk chemicals to be used at a ton scale, the industry still primarily relies on petrol-based resources and classical chemical synthetic strategies, simply because economic factors outrun ecologic criteria. However, as our society strives for a more circular (bio)economy, there's a pressing need to reassess and potentially overhaul these practices in favour of more sustainable synthetic strategies.

Aliphatic compounds with terminal functionalization are ubiquitous starting materials for many materials such as polymers, cosmetics, or additives in dyes and glues. While different strategies have been devised to produce such compounds through biotechnological means, a significant gap in the mid-chain-length substrate range remains. We have tackled this issue by using oxygenases for the terminal hydroxylation of alcohols.

To this end, cytochrome P450 monooxygenases (CYPs) bear potential as extremely versatile and yet selective oxyfunctionalization catalysts. One interesting CYP subfamily is the CYP153 family. These enzymes are described as hydroxylases for medium-length alkanes, with a high regioselectivity for terminal hydroxylation of linear alkanes. This family can also use cyclic alkanes as substrates, and di-hydroxylate alkanes/alcohols and catalyze the epoxidation of alkenes.

In an efficient CYP catalysis, the electron supply plays a pivotal role. We have laid focus on a pair of Ferredoxin and Ferredoxin reductase that has been found together with a CYP153A and shows great potential for productive electron supply towards the target CYP. Furthermore, methods of creating chimeric fusion proteins give access to productive enzymes.

Different CYP153 enzymes were artificially fused to an electron transport system and produced in Escherichia coli. In whole-cell biotransformation, different compounds were used to test for reaction capacity (e.g. hydroxylation or epoxidation). Whilst colorimetric assays were used for general activity assessment, substrate and product quantification was achieved by liquid- and gas chromatographic methods. Reaction engineering addressed challenges that have to be met in a whole-cell format, as e.g. the tolerance of the host cells towards substrates and products, and methods to tackle downstream processing issues.

Substrate screening revealed a highly versatile reactivity throughout our CYP panel, of which some examples will be highlighted. From our collection of CYP153s, candidates able to perform different reactions were identified and their production optimized. Results from whole-cell biotransformations gave valuable insight into the factors attributing to reactivity and toxicity[1] of the target products.