Maximization of cell viability rather than biocatalyst activity improves whole-cell ω-oxyfunctionalization performance.

It is a common misconception in whole-cell biocatalysis to refer to an enzyme as the biocatalyst, thereby neglecting the structural and metabolic framework provided by the cell. Here, the low whole-cell biocatalyst stability, that is, the stability of specific biocatalyst activity, in a process for the terminal oxyfunctionalization of renewable fatty acid methyl esters was investigated. This reaction, which is difficult to achieve by chemical means, is catalyzed by Escherichia coli featuring the monooxygenase system AlkBGT and the uptake facilitator AlkL from Pseudomonas putida GPo1. Corresponding products, that is, terminal alcohols, aldehydes, and acids, constitute versatile bifunctional building blocks, which are of special interest for polymer synthesis. It could clearly be shown that extensive dodecanoic acid methyl ester uptake mediated by high AlkL levels leads to whole-cell biocatalyst toxification. Thus, cell viability constitutes the primary factor limiting biocatalyst stability and, as a result, process durability. Hence, a compromise had to be found between low biocatalyst activity due to restricted substrate uptake and poor biocatalyst stability due to AlkL-mediated toxification. This was achieved by the fine-tuning of heterologous alkL expression, which, furthermore, enabled the identification of the alkBGT expression level as another critical factor determining biocatalyst stability. Controlled synthesis of AlkL and reduced alkBGT expression finally enabled an increase of product titers by a factor of 4.3 up to 229 g Lorg-1 in a two-liquid phase bioprocess setup. Clearly, ω-oxyfunctionalization process performance was determined by cell viability and thus biocatalyst stability rather than the maximally achievable specific biocatalyst activity. Biotechnol. Bioeng. 2017;114: 874-884. © 2016 Wiley Periodicals, Inc.

Efficient production of the Nylon 12 monomer ω-aminododecanoic acid methyl ester from renewable dodecanoic acid methyl ester with engineered Escherichia coli.

The expansion of microbial substrate and product scopes will be an important brick promoting future bioeconomy. In this study, an orthogonal pathway running in parallel to native metabolism and converting renewable dodecanoic acid methyl ester (DAME) via terminal alcohol and aldehyde to 12-aminododecanoic acid methyl ester (ADAME), a building block for the high-performance polymer Nylon 12, was engineered in Escherichia coli and optimized regarding substrate uptake, substrate requirements, host strain choice, flux, and product yield. Efficient DAME uptake was achieved by means of the hydrophobic outer membrane porin AlkL increasing maximum oxygenation and transamination activities 8.3 and 7.6-fold, respectively. An optimized coupling to the pyruvate node via a heterologous alanine dehydrogenase enabled efficient intracellular L-alanine supply, a prerequisite for self-sufficient whole-cell transaminase catalysis. Finally, the introduction of a respiratory chain-linked alcohol dehydrogenase enabled an increase in pathway flux, the minimization of undesired overoxidation to the respective carboxylic acid, and thus the efficient formation of ADAME as main product. The completely synthetic orthogonal pathway presented in this study sets the stage for Nylon 12 production from renewables. Its effective operation achieved via fine tuning the connectivity to native cell functionalities emphasizes the potential of this concept to expand microbial substrate and product scopes.

Reaction and catalyst engineering to exploit kinetically controlled whole-cell multistep biocatalysis for terminal FAME oxyfunctionalization.

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.

Whole-cell biocatalysis for selective and productive C-O functional group introduction and modification.

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.

Outer membrane protein AlkL boosts biocatalytic oxyfunctionalization of hydrophobic substrates in Escherichia coli.

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 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.