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.

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 -oxyfunctionalization of dodecanoic acid methyl ester by recombinant Escherichia coli containing the alkane monooxygenase AlkBGT of Pseudomonas putida GPo1. 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 recombinant Pseudomonas fluorescens CHA0 instead of E. coli resulted in a similar activity increase. However, substrate mass transfer into cells was still found to be limiting. Remarkably, the coexpression of the alkL gene of P. putida GPo1 encoding an outer membrane protein with so-far-unknown function increased the dodecanoic acid methyl ester oxygenation activity of recombinant E. coli 28-fold. In a two-liquid-phase bioreactor setup, a 62-fold increase to a maximal activity of 87 U g cdw ؊1 was achieved, enabling the accumulation of high titers of terminally oxyfunctionalized products. Coexpression of alkL also 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.T he outer membrane of Gram-negative bacteria serves as an efficient barrier for hydrophobic molecules (12,36,44). Different approaches have been described to overcome uptake limitations in whole-cell biotransformations. The two-liquid-phase concept applied in stirred-tank reactors can increase mass transfer by ensuring maximal substrate availability and typically allows in situ product extraction (13,20,33,37,40,47,53,72). Next to that, the addition of rhamnolipids, synthetic surfactants, and cosolvents has been described as enhancing biotransformation rates. Rhamnolipids are synthesized by several Pseudomonas species in order to facilitate the uptake of hydrophobic compounds (46). Rhamnolipids as well as synthetic surfactants (e.g., Triton X-100) solubilize hydrophobic substrates in the aqueous phase and interact with bacterial membranes (1, 39). Similarly, cosolvents, such as dimethyl sulfoxide (DMSO), or chelating agents, such as EDTA, can be used to enhance substrate solubility and/or membrane permeability (44). Further strategies to improve rates for hydrophobic substrate bioconversions include the use of host strains capable of growth on and thus efficient uptake of hydrophobic substrates, such as hydrocarbons, or the transfer of respective properties, ...

Direct and regiospecific amino‐functionalization of non‐activated carbon could be achieved using one recombinant microbial catalyst. The presented proof of concept shows that heterologous pathway engineering allowed the construction of a whole‐cell biocatalyst catalyzing the terminal amino‐functionalization of fatty acid methyl esters (e.g., dodecanoic acid methyl ester) and alkanes (e.g., octane). By coupling oxygenase and transaminase catalysis in vivo, both substrates are converted with absolute regiospecificity to the terminal amine via two sequential oxidation reactions followed by an amination step. Such demanding chemical three‐step reactions achieved with a single catalyst demonstrate the tremendous potential of whole‐cell biocatalysts for the production of industrially relevant building blocks.

The oxyfunctionalization of unactivated C−H bonds can selectively and efficiently be catalyzed by oxygenase-containing whole-cell biocatalysts. Recombinant Escherichia coli W3110 containing the alkane monooxygenase AlkBGT and the outer membrane protein AlkL from Pseudomonas putida GPo1 have been shown to efficiently catalyze the terminal oxyfunctionalization of renewable fatty acid methyl esters yielding bifunctional products of interest for polymer synthesis. In this study, AlkBGTL-containing E. coli W3110 is shown to catalyze the multistep conversion of dodecanoic acid methyl ester (DAME) via terminal alcohol and aldehyde to the acid, exhibiting Michaelis-Menten-type kinetics for each reaction step. In two-liquid phase biotransformations, the product formation pattern was found to be controlled by DAME availability. Supplying DAME as bulk organic phase led to accumulation of the terminal alcohol as the predominant product. Limiting DAME availability via application of bis(2-ethylhexyl)phthalate (BEHP) as organic carrier solvent enabled almost exclusive acid accumulation. Furthermore, utilization of BEHP enhanced catalyst stability by reducing toxic effects of substrate and products. A further shift towards the overoxidized products was achieved by co-expression of the gene encoding the alcohol dehydrogenase AlkJ, which was shown to catalyze efficient and irreversible alcohol to aldehyde oxidation in vivo. With DAME as organic phase, the aldehyde accumulated as main product using resting cells containing AlkBGT, AlkL, as well as AlkJ. This study highlights the versatility of whole-cell biocatalysis for synthesis of industrially relevant bifunctional building blocks and demonstrates how integrated reaction and catalyst engineering can be implemented to control product formation patterns in biocatalytic multistep reactions.

Abstract:The alkane monooxygenase AlkBGT from Pseudomonas putida GPo1 constitutes a versatile enzyme system for the w-oxyfunctionalization of medium chain-length alkanes. In this study, recombinant Escherichia coli W3110 expressing alkBGT was investigated as whole-cell catalyst for the regioselective biooxidation of fatty acid methyl esters to terminal alcohols. The w-functionalized products are of general economic interest, serving as building blocks for polymer synthesis. The whole-cell catalysts proved to functionalize fatty acid methyl esters with a medium length alkyl chain specifically at the w-position. The highest specific hydroxylation activity of 104 U g CDW À1was obtained with nonanoic acid methyl ester as substrate using resting cells of E. coli W3110 (pBT10). In an optimized set-up, maximal 9-hydroxynonanoic acid methyl ester yields of 95% were achieved. For this specific substrate, apparent whole-cell kinetic parameters were determined with a V max of 204 AE 9 Ug CDW À1 , a substrate uptake constant (K S ) of 142 AE 17 mM, and a specificity constant V max /K S of 1.4 U g CDW À1 mm À1 for the formation of the terminal alcohol. The same E. coli strain carrying additional alk genes showed a different substrate selectivity. A comparison of biocatalysis with whole cells and enriched enzyme preparations showed that both substrate availability and enzyme specificity control the efficiency of the whole-cell bioconversion of the longer and more hydrophobic substrate dodecanoic acid methyl ester. The efficient coupling of redox cofactor oxidation and product formation, as determined in vitro, combined with the high in vivo activities make E. coli W3110 (pBT10) a promising biocatalyst for the preparative synthesis of terminally functionalized fatty acid methyl esters.