Biocatalysis is an enabling technology adding to organic oxidation chemistry. Especially the high selectivity of enzymatic oxidation coevally operating under mild conditions and not necessitating problematic solvents makes it a very valuable tool for (green) chemistry. The present state of the art in the use of enzymes and microorganisms for catalytic oxidation and oxyfunctionalization chemistry is reviewed.

During the last decades, biocatalysis became of increasing importance for chemical and pharmaceutical industries. Regarding regio- and stereospecificity, enzymes have shown to be superior compared to traditional chemical synthesis approaches, especially in C–O functional group chemistry. Catalysts established on a process level are diverse and can be classified along a functional continuum starting with single-step biotransformations using isolated enzymes or microbial strains towards fermentative processes with recombinant microorganisms containing artificial synthetic pathways. The complex organization of respective enzymes combined with aspects such as cofactor dependency and low stability in isolated form often favors the use of whole cells over that of isolated enzymes. Based on an inventory of the large spectrum of biocatalytic C–O functional group chemistry, this review focuses on highlighting the potentials, limitations, and solutions offered by the application of self-regenerating microbial cells as biocatalysts. Different cellular functionalities are discussed in the light of their (possible) contribution to catalyst efficiency. The combined achievements in the areas of protein, genetic, metabolic, and reaction engineering enable the development of whole-cell biocatalysts as powerful tools in organic synthesis.

A key limitation of whole‐cell redox biocatalysis for the production of valuable, specifically functionalized products is substrate/product toxicity, which can potentially be overcome by using solvent‐tolerant micro‐organisms. To investigate the inter‐relationship of solvent tolerance and energy‐dependent biocatalysis, we established a model system for biocatalysis in the presence of toxic low logP_ow solvents: recombinant solvent‐tolerant Pseudomonas putida DOT‐T1E catalyzing the stereospecific epoxidation of styrene in an aqueous/octanol two‐liquid phase reaction medium. Using ¹³C tracer based metabolic flux analysis, we investigated the central carbon and energy metabolism and quantified the NAD(P)H regeneration rate in the presence of toxic solvents and during redox biocatalysis, which both drastically increased the energy demands of solvent‐tolerant P. putida. According to the driven by demand concept, the NAD(P)H regeneration rate was increased up to eightfold by two mechanisms: (a) an increase in glucose uptake rate without secretion of metabolic side products, and (b) reduced biomass formation. However, in the presence of octanol, only ∼ 1% of the maximally observed NAD(P)H regeneration rate could be exploited for styrene epoxidation, of which the rate was more than threefold lower compared with operation with a non‐toxic solvent. This points to a high energy and redox cofactor demand for cell maintenance, which limits redox biocatalysis in the presence of octanol. An estimated upper bound for the NAD(P)H regeneration rate available for biocatalysis suggests that cofactor availability does not limit redox biocatalysis under optimized conditions, for example, in the absence of toxic solvent, and illustrates the high metabolic capacity of solvent‐tolerant P. putida. This study shows that solvent‐tolerant P. putida have the remarkable ability to compensate for high energy demands by boosting their energy metabolism to levels up to an order of magnitude higher than those observed during unlimited growth.

The outer membrane of microbial cells forms an effective barrier for hydrophobic compounds, potentially causing an uptake limitation for hydrophobic substrates. Low bioconversion activities (1.9 U g_cdw⁻¹) have been observed for the ω-oxyfunctionalization of dodecanoic acid methyl ester by recombinantEscherichia colicontaining the alkane monooxygenase AlkBGT ofPseudomonas putidaGPo1. Using fatty acid methyl ester oxygenation as the model reaction, this study investigated strategies to improve bacterial uptake of hydrophobic substrates. Admixture of surfactants and cosolvents to improve substrate solubilization did not result in increased oxygenation rates. Addition of EDTA increased the initial dodecanoic acid methyl ester oxygenation activity 2.8-fold. The use of recombinantPseudomonas fluorescensCHA0 instead ofE. coliresulted in a similar activity increase. However, substrate mass transfer into cells was still found to be limiting. Remarkably, the coexpression of thealkLgene ofP. putidaGPo1 encoding an outer membrane protein with so-far-unknown function increased the dodecanoic acid methyl ester oxygenation activity of recombinantE. coli28-fold. In a two-liquid-phase bioreactor setup, a 62-fold increase to a maximal activity of 87 U g_cdw⁻¹was achieved, enabling the accumulation of high titers of terminally oxyfunctionalized products. Coexpression ofalkLalso increased oxygenation activities toward the natural AlkBGT substrates octane and nonane, showing for the first time clear evidence for a prominent role of AlkL in alkane degradation. This study demonstrates that AlkL is an efficient tool to boost productivities of whole-cell biotransformations involving hydrophobic aliphatic substrates and thus has potential for broad applicability.