The selective hydroxylation of benzene to phenol, without the formation of side products resulting from overoxidation, is catalyzed by cytochrome P450BM3 with the assistance of amino acid derivatives as decoy molecules. The catalytic turnover rate and the total turnover number reached 259 min P450BM3 and 40 200 P450BM3 when N-heptyl-l-proline modified with l-phenylalanine (C7-l-Pro-l-Phe) was used as the decoy molecule. This work shows that amino acid derivatives with a totally different structure from fatty acids can be used as decoy molecules for aromatic hydroxylation by wild-type P450BM3. This method for non-native substrate hydroxylation by wild-type P450BM3 has the potential to expand the utility of P450BM3 for biotransformations.

An Escherichia coli whole-cell biocatalyst for the direct hydroxylation of benzene to phenol has been developed. By adding amino acid derivatives as decoy molecules to the culture medium, wild-type cytochrome P450BM3 (P450BM3) expressed in E.coli can be activated and non-native substrates hydroxylated, without supplementing with NADPH. The yield of phenol reached 59 % when N-heptyl-l-prolyl-l-phenylalanine (C7-Pro-Phe) was employed as the decoy molecule. It was shown that decoy molecules, especially those lacking fluorination, reached the cytosol of E. coli, thus imparting in vivo catalytic activity for the oxyfunctionalisation of non-native substrates to intracellular P450BM3.

To survive in the iron-devoid environment of their host, pathogenic bacteria have devised multifarious cunning tactics such as evolving intricate heme transport systems to pirate extracellular heme. Yet, the potential of heme transport systems as antimicrobial targets has not been explored. Herein we developed a strategy to deliver antimicrobials by exploiting the extracellular heme acquisition system protein A (HasA) of Pseudomonas aeruginosa. We demonstrated that, analogous to heme uptake, HasA can specifically traffic an antimicrobial, gallium phthalocyanine (GaPc), into the intracellular space of P. aeruginosa via the interaction of HasA with its outer membrane receptor HasR. HasA enables water-insoluble GaPc to be mistakenly acquired by P. aeruginosa, permitting its sterilization (>99.99%) by irradiation with near-infrared (NIR) light, irrespective of antibiotic resistance. Our findings substantiate that bacterial heme uptake via protein−protein recognition is an attractive target for antimicrobials, enabling specific and effective sterilization.

Highly effective dipeptidic decoy molecules, which stimulate the direct hydroxylation of benzene by wild-type cytochrome P450BM3, were successfully developed through a rationally designed screening method. Extensive synthesis and step-wise screening of over 600 dipeptide derivatives were performed for the efficient evolution of decoy molecules. In the presence of N-(3-cyclopentyl)propanoyl-L-pipecolyl-L-phenylalanine (3CPPA-Pip-Phe), one of the most effective decoy molecules discovered herein, the catalytic turnover frequency and total turnover number for benzene hydroxylation reached 405 min −1 P450BM3 −1 and 54,500 P450BM3 −1 , respectively. Furthermore, the decoy molecules developed in this work drastically accelerated the hydroxylation of other non-native substrates, such as anisole and toluene, as well as nonaromatic compounds, such as cyclohexane, propane, and ethane. Using Nenanthoyl-L-pipecolyl-L-phenylalanine (C7AM-Pip-Phe), the hydroxylation rate for ethane to ethanol reached 82.7 min −1 P450BM3 −1 .

The benzylic hydroxylation of non-native substrates was catalysed by cytochrome P450BM3, wherein “decoy molecules” controlled the stereoselectivity of the reactions.

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A new type of high‐pressure reactor based on liquid‐flow pressurisation using a HPLC pump has been developed. This high‐pressure reactor allows the easy and safe performance of reactions with gaseous alkanes under high‐pressures up to 10 MPa (100 atm), without the need for high‐pressure gas cylinders. The amount of substrate gas required for a single reaction is very small compared with reactions using a conventional autoclave, which, when using expensive substrate gasses, such as 13C‐labelled ethane, becomes critical. Employing this high‐pressure reactor in conjunction with cytochrome P450BM3 and the assistance of decoy molecules, the direct hydroxylation of gaseous alkanes was drastically improved. At 5 MPa the TOF of propane hydroxylation increased 10‐fold, reaching 2200 min−1. Hydroxylation of ethane was also substantially accelerated at 5 MPa, reaching a TOF of 28 min−1.

Haem substitution is an effective approach to tweak the function of haemoproteins. Herein, we report a facile haem substitution method for self-sufficient cytochrome P450BM3 (CYP102A1) from Bacillus megaterium utilising the transpeptidase Sortase A from Staphylococcus aureus. We successfully constructed Mn-substituted BM3 and investigated its catalytic activity.