Bacterial whole cell bioconversions with oxygenases

In biocatalytic reactions with oxygenases whole cells are preferred over isolated enzymes primarily because cells are capable of regenerating NAD(P)H. Cells may also confer higher oxygenase stability by providing a protected compartment (soluble enzymes) or a better organization of its components (membrane-bound enzymes). Additionally, cells contain the necessary machinery to scavenge reactive oxygen species originating from uncoupling, which could inactivate the enzyme. Despite these advantages, important issues such as substrate/product toxicity, low substrate uptake, byproduct formation, limited oxygen transfer and reduced cofactor availability should be overcome.^147-149

1.3.1 Pseudomonas and E. coli as cell factories

The selection of the ideal host strain relies on high cell growth rates in simple media and access to knowledge and tools for its genetic manipulation. Other strain-related factors directly influencing the efficiency of a biocatalytic process include tolerance towards the substrate/product(s), stable recombinant protein expression, high NAD(P)H regeneration rate, low byproduct formation and the possibility of reuse for multiple reaction cycles.¹⁵⁰ In this

sense, Pseudomonas strains possess desirable features such as: 1) simple nutrient demand, 2) robustness to tolerate and modify several toxic aliphatic, aromatic and heterocyclic compounds, 3) efficient cofactor supply, and 4) inherent self-immobilization ability which facilitates the formation of stable and catalytically active biofilms.^151-154

Pseudomonas putida and related strains are nowadays used in diverse industrial applications, including the synthesis of bio-based materials, de novo synthesis and biotransformation of fine chemicals and pharmaceuticals.¹⁵³ Most of these processes exploit the unique xenobiotic-degrading enzymatic machinery of pseudomonads and are thus based on metabolically-engineered overproducing strains. The application of Pseudomonas strains in recombinant oxygenase-based biotransformations is still on the research level. In general, Pseudomonas strains seem more advantageous than E. coli because they have a network of glucose metabolism with multiple reduction and oxidation steps leading to a more efficient generation of redox power.¹⁵⁵ In terms of solvent tolerance, Pseudomonas putida strains are able to grow in the presence of high organic solvent concentrations (e.g. 6 % (v/v) 1-butanol).¹⁵⁶ However, solvent-tolerant strains also need more energy and cofactor to sustain defense mechanisms when exposed to toxic compounds.^157-159 Considering that the carbon source is one of the highest operation costs in a bioprocess, the use of a Pseudomonas biocatalyst is economically justified if it exhibits not only a significantly higher solvent tolerance compared to other industrial production organisms (e.g., E. coli), but a similar product yield on carbon source as well.¹⁶⁰ Oxygen transfer is also a critical factor during reactions with the strictly aerobic pseudomonads;

therefore, the process design should allow sufficient aeration to support both cell growth/maintenance and redox catalysis. Additional issues that should be addressed for industrial applications have been discussed elsewhere.¹⁶¹

In a recent study on the synthesis of (S)-styrene oxide from styrene in a biphasic system, recombinant E. coli (containing styrene monooxygenase/reductase) and Pseudomonas sp. strain VLB120∆C were compared.¹⁶⁰ This pseudomonad is a solvent-tolerant strain expressing styrene monooxygenase and reductase constitutively but lacking styrene oxide isomerase, which mediates the conversion of the target product styrene oxide to phenylacetaldehyde.

Biotransformations in E. coli resulted in higher specific activities, product yields on glucose and volumetric productivities than the investigated Pseudomonas strain. Nevertheless, the pseudomonad provided the advantages of tolerance towards higher product titers and higher process durability. In contrast to E. coli, the pseudomonad did not accumulate the byproduct 2-phenylethanol because it was able to degrade it.¹⁶⁰ Moreover, Pseudomonas sp. produced ten times less acetate than E. coli (0.3 vs. 3.6 g l^-1). Acetate formation is known to reduce the cofactor

regeneration yield per glucose consumed and to negatively influence the proton gradient across the cytoplasmic membrane.^{162, 163}

A step forward in the application of pseudomonads in redox biocatalysis consists of the establishment of biofilm-forming and engineered Pseudomonas strains for efficient styrene oxide production in novel continuous reactors. The bioprocess resulted in high volumetric productivities as well as improved tolerance and robustness comparable to those of planktonic cultures.^164-166

1.3.2 Growing and resting cells as whole cell biocatalysts

One key question in whole cell biocatalysis is whether to use growing or resting cells. Growing cells are capable of constant oxygenase synthesis and they should have a more active metabolism for cofactor regeneration. However, most of the available energy and cofactor are used for biomass formation rather than for redox biocatalysis. These cells also have low durability and low tolerance to substrate/product toxicity. Metabolically active resting cells have lower carbon and energy demands than growing cells, thus the cofactor formed during central carbon catabolism can be exploited more efficiently for biocatalysis rather than for cell growth.

This results in higher specific activities and product yields on energy source. In addition, resting cells display a higher biomass durability which might enable their reuse, provided high oxygenase activities are retained over time. ^167-170

Most limitations observed in whole biotransformations with resting cells depend on factors intrinsically associated to the biocatalyst (e.g., enzyme stability, enzyme kinetics, uncoupling) rather than on the metabolic capacity of the host strain.¹⁶⁷ For example, in the oxygenase-mediated epoxidation of styrene in biphasic medium, maximal specific activities with resting E.

coli cells doubled those of growing cells in a similar setup.¹⁷¹ Toxicity was not a problem and the enzyme was not deactivated, but product formation rates decreased steadily mainly due to product inhibition.¹⁶⁷ In another study with E. coli containing a recombinant Baeyer-Villiger monooxygenase, space-time yields of non-growing cells were 20 times higher than those of growing cells. Here the duration and rate of the oxidation reaction were limited by the intracellular stability of the oxygenase and the rate of substrate transport across the cell membrane.¹⁶⁹ Therefore, strategies to minimize product inhibitory effects on the biocatalyst include the application of two-liquid phase systems and in situ product removal techniques. The use of solvent tolerant strains can also contribute to avoid inhibition by efficient transport of the product out of the cell.¹⁷²