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Metabolic engineering 68, 199-209 (2021). doi:10.1016/j.ymben.2021.10.006, Published by Academic Press, Orlando, Fla.

A 6‐step cascade is distributed among P. taiwanensis and E. coli to synthesize the Nylon‐6 monomer 6‐aminohexanoic acid from cyclohexane under environmentally friendly conditions. The appropriate shuttle compound, avoidance of substrate toxicity, and mass transfer considerations finally enabled 86% 6‐aminohexanoic acid yield in a mixed‐species biotransformation., Summary 6‐Aminohexanoic acid (6AHA) is a vital polymer building block for Nylon 6 production and an FDA‐approved orphan drug. However, its production from cyclohexane is associated with several challenges, including low conversion and yield, and severe environmental issues. We aimed at overcoming these challenges by developing a bioprocess for 6AHA synthesis. A mixed‐species approach turned out to be most promising. Thereby, Pseudomonas taiwanensis VLB120 strains harbouring an upstream cascade converting cyclohexane to either є‐caprolactone (є‐CL) or 6‐hydroxyhexanoic acid (6HA) were combined with Escherichia coli JM101 strains containing the corresponding downstream cascade for the further conversion to 6AHA. ε‐CL was found to be a better ‘shuttle molecule’ than 6HA enabling higher 6AHA formation rates and yields. Mixed‐species reaction performance with 4 g l‐1 biomass, 10 mM cyclohexane, and an air‐to‐aqueous phase ratio of 23 combined with a repetitive oxygen feeding strategy led to complete substrate conversion with 86% 6AHA yield and an initial specific 6AHA formation rate of 7.7 ± 0.1 U gCDW ‐1. The same cascade enabled 49% 7‐aminoheptanoic acid yield from cycloheptane. This combination of rationally engineered strains allowed direct 6AHA production from cyclohexane in one pot with high conversion and yield under environmentally benign conditions.

Previous studies reported various and differing effects of the heterologous production of carbon-based molecules on photosynthetic and growth efficiency of cyanobacteria. The typically applied cultivation in batch mode, with continuously changing growth conditions, however, precludes a clear differentiation between the impact of cultivation conditions on cell physiology and effects related to the specific nature of the product and its synthesis pathway.

As photosynthetic microbes, cyanobacteria are attractive hosts for the production of high-value molecules from CO2 and light. Strategies for genetic engineering and tightly controlled gene expression are essential for the biotechnological application of these organisms. Numerous heterologous or native promoter systems were used for constitutive and inducible expression, yet many of them suffer either from leakiness or from a low expression output. Anyway, in recent years, existing systems have been improved and new promoters have been discovered or engineered for cyanobacteria. Moreover, alternative tools and strategies for expression control such as riboswitches, riboregulators or genetic circuits have been developed. In this mini-review, we provide a broad overview on the different tools and approaches for the regulation of gene expression in cyanobacteria and explain their advantages and disadvantages.

Microbial bioprocessing based on orthologous pathways constitutes a promising approach to replace traditional greenhouse gas- and energy-intensive production processes, e.g., for adipic acid (AA). We report the construction of a Pseudomonas taiwanensis strain able to efficiently convert cyclohexane to AA. For this purpose, a recently developed 6-hydroxyhexanoic acid (6HA) synthesis pathway was amended with alcohol and aldehyde dehydrogenases, for which different expression systems were tested. Thereby, genes originating from Acidovorax sp. CHX100 and the XylS/Pm regulatory system proved most efficient for the conversion of 6HA to AA as well as the overall cascade enabling an AA formation activity of up to 48.6 ± 0.2 U g

Solvent-tolerant bacteria represent an interesting option to deal with the substrate and product toxicity in bioprocesses. Recently, constitutive solvent tolerance was achieved for Pseudomonas taiwanensis VLB120 via knockout of the regulator TtgV, making tedious adaptation unnecessary. Remarkably, ttgV knockout increased styrene epoxidation activities of P. taiwanensis VLB120Δ C. With the aim to characterize and exploit the biocatalytic potential of P. taiwanensis VLB120Δ C and VLB120Δ CΔ ttgV, we investigated and correlated growth physiology, native styrene monooxygenase (StyAB) gene expression, whole-cell bioconversion kinetics, and epoxidation performance. Substrate inhibition kinetics was identified but was attenuated in two-liquid phase bioreactor setups. StyA fusion to the enhanced green fluorescent protein enabled precise enzyme level monitoring without affecting epoxidation activity. Glucose limitation compromised styAB expression and specific activities (30-40 U/g CDW for both strains), whereas unlimited batch cultivation enabled specific activities up to 180 U/g CDW for VLB120Δ CΔ ttgV strains, which is unrivaled for bioreactor-based whole-cell oxygenase biocatalysis. These extraordinarily high specific activities of constitutively solvent-tolerant P. taiwanensis VLB120∆ C∆ ttgV could be attributed to its high metabolic capacity, which also enabled high expression levels. This, together with the high product yields on glucose and biomass obtained qualifies the VLB120∆ ttgV strain as a highly attractive tool for the development of ecoefficient oxyfunctionalization processes and redox biocatalysis in general.

Background Cyanobacteria are ideal model organisms to exploit photosynthetically derived electrons or fixed carbon for the biotechnological synthesis of high value compounds and energy carriers. Much effort is spent on the rational design of heterologous pathways to produce value-added chemicals. Much less focus is drawn on the basic physiological responses and potentials of phototrophs to deal with natural or artificial electron and carbon sinks. However, an understanding of how electron sinks influence or regulate cellular physiology is essential for the efficient application of phototrophic organisms in an industrial setting, i.e., to achieve high productivities and product yields. Results The physiological responses of the cyanobacterium Synechocystis sp. PCC 6803 to electron sink variation were investigated in a systematic and quantitative manner. A variation in electron demand was achieved by providing two N sources with different degrees of reduction. By additionally varying light and CO2 availabilities, steady state conditions with strongly differing source–sink ratios were established. Balancing absorbed photons and electrons used for different metabolic processes revealed physiological responses to sink/source ratio variation. Surprisingly, an additional electron sink under light and thus energy limitation was found not to hamper growth, but was compensated by improved photosynthetic efficiency and activity. In the absence of carbon and light limitation, an increase in electron demand even stimulated carbon assimilation and growth. Conclusion The metabolism of Synechocystis sp. PCC 6803 is highly flexible regarding the compensation of additional electron demands. Under light limitation, photosynthesis obviously does not necessarily run at its maximal capacity, possibly for the sake of robustness. Increased electron demands can even boost photosynthetic activity and growth. Electronic supplementary material The online version of this article (10.1186/s13068-019-1378-y) contains supplementary material, which is available to authorized users.

Cyclohexanone monooxygenase (CHMO), a member of the Baeyer-Villiger monooxygenase family, is a versatile biocatalyst that efficiently catalyzes the conversion of cyclic ketones to lactones. In this study, an Acidovorax-derived CHMO gene was expressed in Pseudomonas taiwanensis VLB120. Upon purification, the enzyme was characterized in vitro and shown to feature a broad substrate spectrum and up to 100% conversion in 6 h. Further, we determined and compared the cyclohexanone conversion kinetics for different CHMO-biocatalyst formats, i.e., isolated enzyme, suspended whole cells, and biofilms, the latter two based on recombinant CHMO-containing P. taiwanensis VLB120. Biofilms showed less favorable values for K (9.3-fold higher) and k (4.8-fold lower) compared to corresponding K and k values of isolated CHMO, but a favorable K for cyclohexanone (5.3-fold higher). The unfavorable K and k values are related to mass transfer- and possibly heterogeneity issues and deserve further investigation and engineering, in order to exploit the high potential of biofilms regarding process stability. Suspended cells showed an only 1.8-fold higher K, but 1.3- and 4.2-fold higher k and K values than isolated CHMO. This together with the efficient NADPH regeneration via glucose metabolism makes this format highly promising from a kinetics perspective.

Amino acid hydroxylases depend directly on the cellular TCA cycle via their cosubstrate α-ketoglutarate (α-KG) and are highly useful for the selective biocatalytic oxyfunctionalization of amino acids. This study evaluates TCA cycle engineering strategies to force and increase α-KG flux through proline-4-hydroxylase (P4H). The genes sucA (α-KG dehydrogenase E1 subunit) and sucC (succinyl-CoA synthetase β subunit) were alternately deleted together with aceA (isocitrate lyase) in proline degradation-deficient Escherichia coli strains (ΔputA) expressing the p4h gene. Whereas, the ΔsucCΔaceAΔputA strain grew in minimal medium in the absence of P4H, relying on the activity of fumarate reductase, growth of the ΔsucAΔaceAΔputA strictly depended on P4H activity, thus coupling growth to proline hydroxylation. P4H restored growth, even when proline was not externally added. However, the reduced succinyl-CoA pool caused a 27% decrease of the average cell size compared to the wildtype strain. Medium supplementation partially restored the morphology and, in some cases, enhanced proline hydroxylation activity. The specific proline hydroxylation rate doubled when putP, encoding the Na+ /l-proline transporter, was overexpressed in the ΔsucAΔaceAΔputA strain. This is in contrast to wildtype and ΔputA single-knock out strains, in which α-KG availability obviously limited proline hydroxylation. Such α-KG limitation was relieved in the ΔsucAΔaceAΔputA strain. Furthermore, the ΔsucAΔaceAΔputA strain was used to demonstrate an agar plate-based method for the identification and selection of active α-KG dependent hydroxylases. This together with the possibility to waive selection pressure and overcome α-KG limitation in respective hydroxylation processes based on living cells emphasizes the potential of TCA cycle engineering for the productive application of α-KG dependent hydroxylases. Biotechnol. Bioeng. 2017;114: 1511-1520. © 2017 Wiley Periodicals, Inc.

Fatty acid methyl esters (FAMEs) originating from plant oils are most interesting renewable feedstocks for biofuels and bio-based materials. FAMEs can also be produced and/or functionalized by engineered microbes to give access to, e.g., polymer building blocks. Yet, they are often subject to hydrolysis yielding free fatty acids, which typically are degraded by microbes. We identified BioH as the key enzyme responsible for the hydrolysis of medium-chain length FAME derivatives in different E. coli K-12 strains. E. coli ΔbioH strains showed up to 22-fold reduced FAME hydrolysis rates in comparison with respective wild-type strains. Knockout strains showed, beside the expected biotin auxotrophy, unchanged growth behavior and biocatalytic activity. Thus, high specific rates (~80 U gCDW −1) for terminal FAME oxyfunctionalization catalyzed by a recombinant alkane monooxygenase could be combined with reduced hydrolysis. Biotransformations in process-relevant two-liquid phase systems profited from reduced fatty acid accumulation and/or reduced substrate loss via free fatty acid metabolization. The BioH knockout strategy was beneficial in all tested strains, although its effect was found to differ according to specific strain properties, such as FAME hydrolysis and FFA degradation activities. BioH or functional analogs can be found in virtually all microorganisms, making bioH deletion a broadly applicable strategy for efficient microbial bioprocessing involving FAMEs.

Whole-cell biocatalysis for C-H oxyfunctionalization depends on and is often limited by O2 mass transfer. In contrast to oxygenases, molybdenum hydroxylases use water instead of O2 as an oxygen donor and thus have the potential to relieve O2 mass transfer limitations. Molybdenum hydroxylases may even allow anaerobic oxyfunctionalization when coupled to anaerobic respiration. To evaluate this option, the coupling of quinoline hydroxylation to denitrification is tested under anaerobic conditions employing Pseudomonas putida (P. putida) 86, capable of aerobic growth on quinoline. P. putida 86 reduces both nitrate and nitrite, but at low rates, which does not enable significant growth and quinoline hydroxylation. Introduction of the nitrate reductase from Pseudomonas aeruginosa enables considerable specific quinoline hydroxylation activity (6.9 U gCDW -1 ) under anaerobic conditions with nitrate as an electron acceptor and 2-hydroxyquinoline as the sole product (further metabolization depends on O2 ). Hydroxylation-derived electrons are efficiently directed to nitrate, accounting for 38% of the respiratory activity. This study shows that molybdenum hydroxylase-based whole-cell biocatalysts enable completely anaerobic carbon oxyfunctionalization when coupled to alternative respiration schemes such as nitrate respiration.

Photoautotrophic organisms are promising hosts for biocatalytic oxyfunctionalizations because they supply reduction equivalents as well as O2 via photosynthetic water oxidation. Thus far, research on photosynthesis-driven bioprocesses mainly focuses on strain development and the proof of principle in small-scale biocatalytic reaction setups. This study investigates the long-term applicability of the previously developed cyanobacterial strain Synechocystis sp. PCC 6803_BGT harboring the alkane monooxygenase system AlkBGT catalyzing terminal alkyl group oxyfunctionalization. For the regiospecific ω-hydroxylation of nonanoic acid methyl ester (NAME), this biocatalyst showed light intensity-independent hydroxylation activity and substantial hydrolysis of NAME to nonanoic acid. Substrate mass transfer limitation, substrate hydrolysis, as well as reactant toxicity were overcome via in situ substrate supply by means of a two-liquid phase system. The application of diisononyl phthalate as organic carrier solvent enabled 1.7-fold increased initial specific activities (5.6 ± 0.1 U/gCDW ) and 7.6-fold increased specific yields on biomass (3.8 ± 0.1 mmolH-NAME /gCDW ) as compared with single aqueous phase biotransformations. Finally, the whole-cell biotransformation system was successfully scaled from glass tubes to a stirred-tank photobioreactor. This is the first study reporting the application of the two-liquid phase concept for efficient phototrophic whole-cell biocatalysis.

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.

The microbial production of isoprenoids has recently developed into a prime example for successful bottom-up synthetic biology or top-down systems biology strategies. Respective fermentation processes typically rely on growing recombinant microorganisms. However, the fermentative production of isoprenoids has to compete with cellular maintenance and growth for carbon and energy. Non-growing but metabolically active E. coli cells were evaluated in this study as alternative biocatalyst configurations to reduce energy and carbon loss towards biomass formation. The use of non-growing cells in an optimized fermentation medium resulted in more than fivefold increased specific limonene yields on cell dry weight and glucose, as compared to the traditional growing-cell-approach. Initially, the stability of the resting-cell activity was limited. This instability was overcome via the optimization of the minimal fermentation medium enabling high and stable limonene production rates for up to 8 h and a high specific yield of ≥50 mg limonene per gram cell dry weight. Omitting MgSO4 from the fermentation medium was very promising to prohibit growth and allow high productivities. Applying a MgSO4 -limitation also improved limonene formation by growing cells during non-exponential growth involving a reduced biomass yield on glucose and a fourfold increase in specific limonene yields on biomass as compared to non-limited cultures. The control of microbial growth via the medium composition was identified as a key but yet underrated strategy for efficient isoprenoid production. Biotechnol. Bioeng. 2016;113: 1305-1314. © 2015 Wiley Periodicals, Inc.

The current industrial production of polymer building blocks such as e-caprolactone (e-CL) and 6-hydroxyhexanoic acid (6HA) is a multi-step process associated with critical environmental issues such as the generation of toxic waste and high energy consumption. Consequently, there is a demand for more eco-efficient and sustainable production routes. This study deals with the generation of a platform organism that converts cyclohexane to such polymer building blocks without the formation of byproducts and under environmentally benign conditions. Based on kinetic and thermodynamic analyses of the individual enzymatic steps, a 4-step enzymatic cascade in Pseudomonas taiwanensis VLB120 is rationally engineered via stepwise biocatalyst improvement on the genetic level. It is found that the intermediate product cyclohexanol severely inhibits the cascade which could be optimized by enhancing the expression level of downstream enzymes. The integration of a lactonase enables exclusive 6HA formation without side products. The resulting biocatalyst shows a high activity of 44.8 ± 0.2 U gCDW-1 and fully converts 5 mm cyclohexane to 6HA within 3 h. This platform organism can now serve as a basis for the development of greener production processes for polycaprolactone and related polymers.

Metabolic engineering strategies mark a milestone for the fermentative production of bulk and fine chemicals. Yet, toxic products and volatile reaction intermediates with low solubilities remain challenging. Prominent examples are artificial multistep pathways like the production of perillyl acetate (POHAc) from glucose via limonene. For POHAc, these limitations can be overcome by mixed-culture fermentations. A limonene biosynthesis pathway and cytochrome P450 153A6 (CYP153A6) as regioselective hydroxylase are used in two distinct recombinant E. coli. POHAc formation from glucose in one recombinant cell was hindered by ineffective coupling of limonene synthesis and low rates of oxyfunctionalization. The optimization of P450 gene expression led to the formation of 6.20 ± 0.06 mg gcdw−1 POHAc in a biphasic batch cultivation with glucose as sole carbon and energy source. Increasing the spatial proximity between limonene synthase and CYP153A6 by a genetic fusion of both enzymes changed the molar limonene/POHAc ratio from 3.2 to 1.6. Spatial separation of limonene biosynthesis from its oxyfunctionalization improved POHAc concentration 3.3-fold to 21.7 mg L−1 as compared to a biphasic fermentation. Mixed-cultures of E. coli BL21 (DE3) containing the limonene biosynthesis pathway and E. coli MG1655 harboring either CYP153A6, or alternatively a cymene monooxygenase, showed POHAc formation rates of 0.06 or 0.11 U gcdw−1, respectively. This concept provides a novel framework for fermentative syntheses involving toxic, volatile, or barely soluble compounds or pathway intermediates. Biotechnol. Bioeng. 2015;112: 1738–1750. © 2015 Wiley Periodicals, Inc.

The formation of stable emulsions in biphasic biotransformations catalyzed by microbial cells turned out to be a major hurdle for industrial implementation. Recently, a cost-effective and efficient downstream processing approach, using supercritical carbon dioxide (scCO2 ) for both irreversible emulsion destabilization (enabling complete phase separation within minutes of emulsion treatment) and product purification via extraction has been proposed by Brandenbusch et al. (2010). One of the key factors for a further development and scale-up of the approach is the understanding of the mechanism underlying scCO2 -assisted phase separation. A systematic approach was applied within this work to investigate the various factors influencing phase separation during scCO2 treatment (that is pressure, exposure of the cells to CO2 , and changes of cell surface properties). It was shown that cell toxification and cell disrupture are not responsible for emulsion destabilization. Proteins from the aqueous phase partially adsorb to cells present at the aqueous-organic interface, causing hydrophobic cell surface characteristics, and thus contribute to emulsion stabilization. By investigating the change in cell-surface hydrophobicity of these cells during CO2 treatment, it was found that a combination of catastrophic phase inversion and desorption of proteins from the cell surface is responsible for irreversible scCO2 mediated phase separation. These findings are essential for the definition of process windows for scCO2 -assisted phase separation in biphasic whole-cell biocatalysis.

The efficient coupling of biotransformation steps to an existing fermentation pathway is an interesting strategy to expand the product portfolio of Corynebacterium glutamicum as whole-cell biocatalyst. This is especially challenging if the biotransformation step comprises a direct link to central metabolism, as in the case of α-ketoglutarate-dependent oxygenase catalysis. Aiming at trans-4-hydroxy-L-proline (Hyp) production from glucose in a minimal medium, the proline-4-hydroxylase gene from Dactylosporangium sp. strain RH1 was introduced into a proline-producing, isoleucine-bradytroph C. glutamicum strain. The production of proline was found to be induced by isoleucine limitation. Proline and Hyp production were found to depend differently on isoleucine limitation. Severe isoleucine limitation was shown to result in proline accumulation and low hydroxylation rates both in batch and continuous cultivation set-ups. The investigation of different steady states with various glucose/isoleucine molar ratios revealed that optimal conditions for Hyp production are met around a molar ratio of 46:1, where isoleucine limitation is sufficient to trigger proline production but the hydroxylation rate is high enough to convert the majority of formed proline to Hyp. A high cell-density fed-batch set-up was designed, capable of producing 7.1 g L−1 of Hyp from glucose in 23 h with 98.5% conversion of proline to Hyp. Reaction engineering, specifically the fine-tuning of the glucose/isoleucine concentration ratio, enabled control of the fermentation profile and thus the accumulation of the desired product Hyp from glucose in minimal and defined media. Biotechnol. Bioeng. 2015;112: 322–330. © 2014 Wiley Periodicals, Inc.

The efficiency and productivity of cellular biocatalysts play a key role in the industrial synthesis of fine and bulk chemicals. This study focuses on optimizing the synthesis of (S)-limonene from glycerol and glucose as carbon sources using recombinant Escherichia coli. The cyclic monoterpene limonene is extensively used in the fragrance, food, and cosmetic industries. Recently, limonene also gained interest as alternative jet fuel of biological origin. Key parameters that limit the (S)-limonene yield, related to genetics, physiology, and reaction engineering, were identified. The growth-dependent production of (S)-limonene was shown for the first time in minimal media. E. coli BL21 (DE3) was chosen as the preferred host strain, as it showed low acetate formation, fast growth, and high productivity. A two-liquid phase fed-batch fermentation with glucose as the sole carbon and energy source resulted in the formation of 700 mg L(org) (-1) (S)-limonene. Specific activities of 75 mU g(cdw) (-1) were reached, but decreased relatively quickly. The use of glycerol as a carbon source resulted in a prolonged growth and production phase (specific activities of ≥50 mU g(cdw) (-1) ) leading to a final (S)-limonene concentration of 2,700 mg L(org) (-1) . Although geranyl diphosphate (GPP) synthase had a low solubility, its availability appeared not to limit (S)-limonene formation in vivo under the conditions investigated. GPP rerouting towards endogenous farnesyl diphosphate (FPP) formation also did not limit (S)-limonene production. The two-liquid phase fed-batch setup led to the highest monoterpene concentration obtained with a recombinant microbial biocatalyst to date.

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.

A systematic and powerful knowledge-based framework exists for improving the activity and stability of chemical catalysts and for empowering the commercialization of respective processes. In contrast, corresponding biotechnological processes are still scarce and characterized by case-by-case development strategies. A systematic understanding of parameters affecting biocatalyst efficiency, that is, biocatalyst activity and stability, is essential for a rational generation of improved biocatalysts. Today, systematic approaches only exist for increasing the activity of whole-cell biocatalysts. They are still largely missing for whole-cell biocatalyst stability. In this review, we structure factors affecting biocatalyst stability and summarize existing, yet not completely exploited strategies to overcome respective limitations. The factors and mechanisms related to biocatalyst destabilization are discussed and demonstrated inter alia based on two case studies. The factors are similar for processes with different objectives regarding target molecule or metabolic pathway complexity and process scale, but are in turn highly interdependent. This review provides a systematic for the stabilization of whole-cell biocatalysts. In combination with our knowledge on strategies to improve biocatalyst activity, this paves the way for the rational design of superior recombinant whole-cell biocatalysts, which can then be employed in economically and ecologically competitive and sustainable bioprocesses.

Oxygenase-containing cyanobacteria constitute promising whole-cell biocatalysts for oxyfunctionalization reactions. Photosynthetic water oxidation thereby delivers the required cosubstrates, that is activated reduction equivalents and O2 , sustainably. A recombinant Synechocystis sp. PCC 6803 strain showing unprecedentedly high photosynthesis-driven oxyfunctionalization activities is developed, and its technical applicability is evaluated. The cells functionally synthesize a heterologous cytochrome P450 monooxygenase enabling cyclohexane hydroxylation. The biocatalyst-specific reaction rate is found to be light-dependent, reaching 26.3 ± 0.6 U gCDW-1 (U = μmol min-1 and cell dry weight [CDW]) at a light intensity of 150 µmolphotons m-2 s-1 . In situ substrate supply via a two-liquid phase system increases the initial specific activity to 39.2 ± 0.7 U gCDW-1 and stabilizes the biotransformation by preventing cell toxification. This results in a tenfold increased specific product yield of 4.5 gcyclohexanol gCDW-1 as compared to the single aqueous phase system. Subsequently, the biotransformation is scaled from a shake flask to a 3 L stirred-tank photobioreactor setup. In situ O2 generation via photosynthetic water oxidation allows a nonaerated process operation, thus circumventing substrate evaporation as the most critical factor limiting the process performance and stability. This study for the first time exemplifies the technical applicability of cyanobacteria for aeration-independent light-driven oxyfunctionalization reactions involving highly toxic and volatile substrates.

Microbial physiology plays a crucial role in whole-cell biotransformation, especially for redox reactions that depend on carbon and energy metabolism. In this study, regio- and enantio-selective proline hydroxylation with recombinant Escherichia coli expressing proline-4-hydroxylase (P4H) was investigated with respect to its interconnectivity to microbial physiology and metabolism. P4H production was found to depend on extracellular proline availability and on codon usage. Medium supplementation with proline did not alter p4h mRNA levels, indicating that P4H production depends on the availability of charged prolyl-tRNAs. Increasing the intracellular levels of soluble P4H did not result in an increase in resting cell activities above a certain threshold (depending on growth and assay temperature). Activities up to 5-fold higher were reached with permeabilized cells, confirming that host physiology and not the intracellular level of active P4H determines the achievable whole-cell proline hydroxylation activity. Metabolic flux analysis revealed that tricarboxylic acid cycle fluxes in growing biocatalytically active cells were significantly higher than proline hydroxylation rates. Remarkably, a catalysis-induced reduction of substrate uptake was observed, which correlated with reduced transcription of putA and putP , encoding proline dehydrogenase and the major proline transporter, respectively. These results provide evidence for a strong interference of catalytic activity with the regulation of proline uptake and metabolism. In terms of whole-cell biocatalyst efficiency, proline uptake and competition of P4H with proline catabolism are considered the most critical factors.

Pseudomonas putida KT2440 is frequently used in biotechnical research and applications due to its metabolic versatility and organic solvent resistance. A major drawback for a broad application is the inability of the bacterium to survive and grow under anoxic conditions, which prohibits the production of oxygen-sensitive proteins and metabolites. To develop a P. putida strain, which is able to survive under anoxic conditions, the enzymatic systems of anaerobic nitrate and nitrite respiration were introduced into KT2440. For this purpose, two cosmids encoding all structural, maturation and regulatory genes for P. aeruginosa nitrate reductase (pNAR) and nitrite- and nitric oxide reductase (pNIR-NOR) were stably maintained in P. putida KT2440. Transcriptome analyses revealed expression of the encoded nar, nir and nor operons and accessory genes under anoxic conditions. The produced enzyme systems efficiently reduced nitrate or nitrite, respectively, sustaining anaerobic life of recombinant KT2440. Interestingly, anaerobic life of P. putida induced genes involved in arginine-fermentation and genes encoding a putative copper stress resistance operon.

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.

Selection of the ideal microbe is crucial for whole-cell biotransformations, especially if the target reaction intensively interacts with host cell functions. Asymmetric styrene epoxidation is an example of a reaction which is strongly dependent on the host cell owing to its requirement for efficient cofactor regeneration and stable expression of the styrene monooxygenase genes styAB. On the other hand, styrene epoxidation affects the whole-cell biocatalyst, because it involves toxic substrate and products besides the burden of additional (recombinant) enzyme synthesis. With the aim to compare two fundamentally different strain engineering strategies, asymmetric styrene epoxidation by StyAB was investigated using the engineered wild-type strain Pseudomonas sp. strain VLB120ΔC, a styrene oxide isomerase (StyC) knockout strain able to accumulate (S)-styrene oxide, and recombinant E. coli JM101 carrying styAB on the plasmid pSPZ10. Their performance was analyzed during fed-batch cultivation in two-liquid phase biotransformations with respect to specific activity, volumetric productivity, product titer, tolerance of toxic substrate and products, by-product formation, and product yield on glucose. Thereby, Pseudomonas sp. strain VLB120ΔC proved its great potential by tolerating high styrene oxide concentrations and by the absence of by-product formation. The E. coli-based catalyst, however, showed higher specific activities and better yields on glucose. The results not only show the importance but also the complexity of host cell selection and engineering. Finding the optimal strain engineering strategy requires profound understanding of bioprocess and biocatalyst operation. In this respect, a possible negative influence of solvent tolerance on yield and activity is discussed.

Mammalian cytochrome P450 enzymes are of special interest as biocatalysts for fine chemical and drug metabolite synthesis. In this study, the potential of different recombinant microorganisms expressing rat and human cyp1a1 genes is evaluated for such applications. The maximum specific activity for 7-ethoxyresorufin O-deethylation and gene expression levels were used as parameters to judge biocatalyst performance. Under comparable conditions, E. coli is shown to be superior over the use of S. cerevisiae and P. putida as hosts for biocatalysis. Of all tested E. coli strains, E. coli DH5α and E. coli JM101 harboring rat CYP1A1 showed the highest activities (0.43 and 0.42 U g CDW −1 , respectively). Detection of active CYP1A1 in cell-free E. coli extracts was found to be difficult and only for E. coli DH5α, expression levels could be determined (41 nmol g CDW −1 ). The presented results show that efficient expression of mammalian cyp1a1 genes in recombinant microorganisms is troublesome and host-dependent and that enhancing expression levels is crucial in order to obtain more efficient biocatalysts. Specific activities currently obtained are not sufficient yet for fine chemical production, but are sufficient for preparative-scale drug metabolite synthesis.

Biocatalytic hydrocarbon oxyfunctionalizations are typically accomplished using oxygenases in living bacteria as biocatalysts. These processes are often limited by either oxygen mass transfer, cofactor regeneration, and/or enzyme instabilities due to the formation of reactive oxygen species. Here, we discuss an alternative approach based on molybdenum (Mo)-containing dehydrogenases, which produce, rather than consume, reducing equivalents in the course of substrate hydroxylation and use water as the oxygen donor. Mo-containing dehydrogenases have a high potential for overcoming limitations encountered with oxygenases. In order to evaluate the suitability and efficiency of a Mo-containing dehydrogenase-based biocatalyst, we investigated quinaldine 4-oxidase (Qox)-containing Pseudomonas strains and the conversion of quinaldine to 4-hydroxyquinaldine. Host strain and carbon source selection proved to be crucial factors influencing biocatalyst efficiency. Resting P. putida KT2440 (pKP1) cells, grown on and induced with benzoate, showed the highest Qox activity and were used for process development. To circumvent substrate and product toxicity/inhibition, a two-liquid phase approach was chosen. Without active aeration and with 1-dodecanol as organic carrier solvent a productivity of 0.4 g l (tot) (-1) h(-1) was achieved, leading to the accumulation of 2.1 g l (tot) (-1) 4-hydroxyquinaldine in 6 h. The process efficiency compares well with values reported for academic and industrially applied biocatalytic oxyfunctionalization processes emphasizing the potential and feasibility of the Qox-containing recombinant cells for heteroaromatic carbon oxyfunctionalizations without the necessity for active aeration.

Biphasic hydrocarbon functionalizations catalyzed by recombinant microorganisms have been shown to be one of the most promising approaches for replacing common chemical synthesis routes on an industrial scale. However, the formation of stable emulsions complicates downstream processing, especially phase separation. This fact has turned out to be a major hurdle for industrial implementation. To overcome this limitation, we used supercritical carbon dioxide (scCO(2)) for both phase separation and product purification. The stable emulsion, originating from a stereospecific epoxidation of styrene to (S)-styrene oxide, a reaction catalyzed by recombinant Escherichia coli, could be destabilized efficiently and irreversibly, enabling complete phase separation within minutes. By further use of scCO(2) as extraction agent, the product (S)-styrene oxide could be obtained with a purity of 81% (w/w) in one single extraction step. By combining phase separation and product purification using scCO(2), the number of necessary workup steps can be reduced to one. This efficient and easy to use technique is generally applicable for the workup of biphasic biocatalytic hydrocarbon functionalizations and enables a cost effective downstream processing even on a large scale.

Styrene can efficiently be oxidized to ( S )-styrene oxide by recombinant Escherichia coli expressing the styrene monooxygenase genes styAB from Pseudomonas sp. strain VLB120. Targeting microbial physiology during whole-cell redox biocatalysis, we investigated the interdependency of styrene epoxidation, growth, and carbon metabolism on the basis of mass balances obtained from continuous two-liquid-phase cultures. Full induction of styAB expression led to growth inhibition, which could be attenuated by reducing expression levels. Operation at subtoxic substrate and product concentrations and variation of the epoxidation rate via the styrene feed concentration allowed a detailed analysis of carbon metabolism and bioconversion kinetics. Fine-tuned styAB expression and increasing specific epoxidation rates resulted in decreasing biomass yields, increasing specific rates for glucose uptake and the tricarboxylic acid (TCA) cycle, and finally saturation of the TCA cycle and acetate formation. Interestingly, the biocatalysis-related NAD(P)H consumption was 3.2 to 3.7 times higher than expected from the epoxidation stoichiometry. Possible reasons include uncoupling of styrene epoxidation and NADH oxidation and increased maintenance requirements during redox biocatalysis. At epoxidation rates of above 21 μmol per min per g cells (dry weight), the absence of limitations by O 2 and styrene and stagnating NAD(P)H regeneration rates indicated that NADH availability limited styrene epoxidation. During glucose-limited growth, oxygenase catalysis might induce regulatory stress responses, which attenuate excessive glucose catabolism and thus limit NADH regeneration. Optimizing metabolic and/or regulatory networks for efficient redox biocatalysis instead of growth (yield) is likely to be the key for maintaining high oxygenase activities in recombinant E. coli .

Utilization of solvent tolerant bacteria as biocatalysts has been suggested to enable or improve bioprocesses for the production of toxic compounds. Here, we studied the relevance of solvent (product) tolerance and inhibition, carbon metabolism, and the stability of biocatalytic activity in such a bioprocess. Styrene degrading Pseudomonas sp. strain VLB120 is shown to be solvent tolerant and was engineered to produce enantiopure (S)-styrene oxide from styrene. Whereas glucose as sole source for carbon and energy allowed efficient styrene epoxidation at rates up to 97 micromol/min/(g cell dry weight), citrate was found to repress epoxidation by the engineered Pseudomonas sp. strain VLB120DeltaC emphasizing that carbon source selection and control is critical. In comparison to recombinant Escherichia coli, the VLB120DeltaC-strain tolerated higher toxic product levels but showed less stable activities during fed-batch cultivation in a two-liquid phase system. Epoxidation activities of the VLB120DeltaC-strain decreased at product concentrations above 130 mM in the organic phase. During continuous two-liquid phase cultivations at organic-phase product concentrations of up to 85 mM, the VLB120DeltaC-strain showed stable activities and, as compared to recombinant E. coli, a more efficient glucose metabolism resulting in a 22% higher volumetric productivity. Kinetic analyses indicated that activities were limited by the styrene concentration and not by other factors such as NADH availability or catabolite repression. In conclusion, the stability of activity of the solvent tolerant VLB120DeltaC-strain can be considered critical at elevated toxic product levels, whereas the efficient carbon and energy metabolism of this Pseudomonas strain augurs well for productive continuous processing.

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.

Background Understanding the metabolism of the microbial host is essential for the development and optimization of whole-cell based biocatalytic processes, as it dictates production efficiency. This is especially true for redox biocatalysis where metabolically active cells are employed because of the cofactor/cosubstrate regenerative capacity endogenous in the host. Recombinant Escherichia coli was used for overproducing proline-4-hydroxylase (P4H), a dioxygenase catalyzing the hydroxylation of free l-proline into trans-4-hydroxy-l-proline with a-ketoglutarate (a-KG) as cosubstrate. In this whole-cell biocatalyst, central carbon metabolism provides the required cosubstrate a-KG, coupling P4H biocatalytic performance directly to carbon metabolism and metabolic activity. By applying both experimental and computational biology tools, such as metabolic engineering and 13C-metabolic flux analysis (13C-MFA), we investigated and quantitatively described the physiological, metabolic, and bioenergetic response of the whole-cell biocatalyst to the targeted bioconversion and identified possible metabolic bottlenecks for further rational pathway engineering. Results A proline degradation-deficient E. coli strain was constructed by deleting the putA gene encoding proline dehydrogenase. Whole-cell biotransformations with this mutant strain led not only to quantitative proline hydroxylation but also to a doubling of the specific trans-4-l-hydroxyproline (hyp) formation rate, compared to the wild type. Analysis of carbon flux through central metabolism of the mutant strain revealed that the increased a-KG demand for P4H activity did not enhance the a-KG generating flux, indicating a tightly regulated TCA cycle operation under the conditions studied. In the wild type strain, P4H synthesis and catalysis caused a reduction in biomass yield. Interestingly, the ΔputA strain additionally compensated the associated ATP and NADH loss by reducing maintenance energy demands at comparably low glucose uptake rates, instead of increasing the TCA activity. Conclusions The putA knockout in recombinant E. coli BL21(DE3)(pLysS) was found to be promising for productive P4H catalysis not only in terms of biotransformation yield, but also regarding the rates for biotransformation and proline uptake and the yield of hyp on the energy source. The results indicate that, upon a putA knockout, the coupling of the TCA-cycle to proline hydroxylation via the cosubstrate a-KG becomes a key factor constraining and a target to further improve the efficiency of a-KG-dependent biotransformations. Electronic supplementary material The online version of this article (doi:10.1186/s12934-015-0298-1) contains supplementary material, which is available to authorized users.

Variability in whole-cell biocatalyst performance represents a critical aspect for stable and productive bioprocessing. In order to investigate whether and how oxygenase-catalyzed reactions are affected by such variability issues in solvent-tolerant Pseudomonas, different inducers, expression systems, and host strains were tested for the reproducibility of xylene and styrene monooxygenase catalyzed hydroxylation and epoxidation reactions, respectively. Significantly higher activity variations were found for biocatalysts based on solvent-tolerant Pseudomonas putida DOT-TIE and S12 compared with solvent-sensitive P. putida KT2440, Escherichia coli JM101, and solvent-tolerant Pseudomonas taiwanensis VLB120. Specific styrene epoxidation rates corresponded to cellular styrene monooxygenase contents. Detected variations in activity strictly depended on the type of regulatory system employed, being high with the alk- and low with the lac-system. These results show that the occurrence of clonal variability in recombinant gene expression in Pseudomonas depends on the combination of regulatory system and host strain, does not correlate with a general phenotype such as solvent tolerance, and must be evaluated case by case.

Styrene is efficiently converted into (S)-styrene oxide by growing Escherichia coli expressing the styrene monooxygenase genes styAB of Pseudomonas sp. strain VLB120 in an organic/aqueous emulsion. Now, we investigated factors influencing the epoxidation activity of recombinant E. coli with the aim to improve the process in terms of product concentration and volumetric productivity. The catalytic activity of recombinant E. coli was not stable and decreased with reaction time. Kinetic analyses and the independence of the whole-cell activity on substrate and biocatalyst concentrations indicated that the maximal specific biocatalyst activity was not exploited under process conditions and that substrate mass transfer and enzyme inhibition did not limit bioconversion performance. Elevated styrene oxide concentrations, however, were shown to promote acetic acid formation, membrane permeabilization, and cell lysis, and to reduce growth rate and colony-forming activity. During biotransformations, when cell viability was additionally reduced by styAB overexpression, such effects coincided with decreasing specific epoxidation rates and metabolic activity. This clearly indicated that biocatalyst performance was reduced as a result of product toxicity. The results point to a product toxicity-induced biological energy shortage reducing the biocatalyst activity under process conditions. By reducing exposure time of the biocatalyst to the product and increasing biocatalyst concentrations, volumetric productivities were increased up to 1,800 µmol/min/liter aqueous phase (with an average of 8.4 g/Laq ·h). This represents the highest productivity reported for oxygenase-based whole-cell biocatalysis involving toxic products. © 2006 Wiley Periodicals, Inc.

Oxygenases catalyze, among other interesting reactions, highly selective hydrocarbon oxyfunctionalizations, which are important in industrial organic synthesis but difficult to achieve by chemical means. Many enzymatic oxygenations have been described, but few of these have been scaled up to industrial scales, due to the complexity of oxygenase based biocatalysts and demanding process implementation. We have combined recombinant whole-cell catalysis in a two-liquid phase system with fed-batch cultivation in an optimized medium and developed an industrially feasible process for the kinetically controlled and complex multistep oxidation of pseudocumene to 3,4-dimethylbenzaldehyde using the xylene monooxygenase of Pseudomonas putida mt-2 in Escherichia coli. Successful scale up to 30 L working volume using downscaled industrial equipment allowed a productivity of 31 g L−1 d−1 and a product concentration of 37 g L−1. These performance characteristics meet present industry requirements. Product purification resulted in the recovery of 469 g of 3,4-dimethyl- benzaldehyde at a purity of 97% and an overall yield of 65%. This process illustrates the general feasibility of industrial biocatalytic oxyfunctionalization. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 833–842, 2003.

The two-liquid phase concept was used to develop a whole cell biocatalytic system for the efficient multistep oxidation of pseudocumene to 3,4-dimethylbenzaldehyde. Recombinant Escherichia coli cells were employed to express the Pseudomonas putida genes encoding xylene monooxygenase, which catalyzes the multistep oxygenation of one methyl group of toluene and xylenes to corresponding alcohols, aldehydes, and acids. A fed-batch based two-liquid phase bioconversion was established with bis(2-ethylhexyl)- phthalate as organic carrier solvent and a phase ratio of 0.5; the product formation pattern, the impact of the nutrient feeding strategy, and the partitioning behavior of the reactants were studied. On the basis of the favorable conditions provided by the two-liquid phase system, engineering of the initial pseudocumene concentration allowed exploiting the complex kinetics of the multistep reaction for the exclusive production of 3,4-dimethyl- benzaldehyde. Further oxidation of the product to 3,4-dimethylbenzoic acid could be inhibited by suitable concentrations of pseudocumene or 3,4-dimethylbenzyl alcohol. The optimized biotransformation setup includes a completely defined medium with high iron content and a nutrient feeding strategy that avoids severe glucose limitation as well as high inhibitory glucose levels. Using such a system on a 2-liter scale, we were able to produce, within 14.5 h, 30 g of 3,4-dimethylbenzaldehyde as predominant reactant in the organic phase and reached a maximal productivity of 1.6 g per liter liquid volume per hour. The present study implicates that the two-liquid phase concept is an efficient tool to exploit the kinetics of multistep biotransformations in general.

For whole-cell biotransformations involving toxic organic compounds, two-liquid phase setups are typically applied employing an apolar extractive phase. Bis(2-ethylhexyl)phthalate (BEHP) has proven to be an ideal solvent for stereospecific styrene epoxidation with recombinant E. coli, providing excellent extractive properties and a high biocompatibility. In eco-efficiency evaluations, BEHP, however, has been identified as a critical factor regarding costs and environmental impact. In this study, the constitutive solvent tolerance of Pseudomonas taiwanensis VLB120ΔCΔttgV is shown to enable high specific activities (up to 180 U gCDW-1 ) and extensive reduction of the BEHP amount in two-liquid phase setups and thus to constitute an excellent tool to improve the environmental and economic efficiency of such processes. At a 90% reduction of carrier solvent use and accordingly increased aqueous styrene concentrations, this strain still showed reasonably high specific styrene epoxidation activities (100 U gCDW-1 ), while the solvent-adaptable wildtype strain immediately was toxified. A moderate 55% reduction of BEHP enabled a specific activity of 150 U gCDW-1 and thus represents a good trade-off between volumetric productivity maximization and environmental impact minimization. These results for the first time show a clear benefit of a solvent-tolerant compared to solvent-sensitive host strains and how such a benefit can be achieved.

The application of whole cells as biocatalysts is often limited by the toxicity of organic solvents, which constitute interesting substrates/products or can be used as a second phase for in situ product removal and as tools to control multistep biocatalysis. Solvent-tolerant bacteria, especially Pseudomonas strains, are proposed as promising hosts to overcome such limitations due to their inherent solvent tolerance mechanisms. However, potential industrial applications suffer from tedious, unproductive adaptation processes, phenotypic variability, and instable solvent-tolerant phenotypes. In this study, genes described to be involved in solvent tolerance were identified in Pseudomonas taiwanensis VLB120, and adaptive solvent tolerance was proven by cultivation in the presence of 1% (vol/vol) toluene. Deletion of ttgV , coding for the specific transcriptional repressor of solvent efflux pump TtgGHI gene expression, led to constitutively solvent-tolerant mutants of P. taiwanensis VLB120 and VLB120Δ C . Interestingly, the increased amount of solvent efflux pumps enhanced not only growth in the presence of toluene and styrene but also the biocatalytic performance in terms of stereospecific styrene epoxidation, although proton-driven solvent efflux is expected to compete with the styrene monooxygenase for metabolic energy. Compared to that of the P. taiwanensis VLB120Δ C parent strain, the maximum specific epoxidation activity of P. taiwanensis VLB120Δ C Δ ttgV doubled to 67 U/g of cells (dry weight). This study shows that solvent tolerance mechanisms, e.g., the solvent efflux pump TtgGHI, not only allow for growth in the presence of organic compounds but can also be used as tools to improve redox biocatalysis involving organic solvents.

The utilization of the cellular metabolism for cofactor regeneration is a common motivation for the application of whole cells in redox biocatalysis. Introduction of an active oxidoreductase into a microorganism has profound consequences on metabolism, potentially affecting metabolic and biotransformation efficiency. An ambitious goal of systems biotechnology is to design process-relevant and knowledge-based engineering strategies to improve biocatalyst performance. Metabolic flux analysis (MFA) has shown that the competition for NAD(P)H between redox biocatalysis and the energy metabolism becomes critical during asymmetric styrene epoxidation catalyzed by growing Escherichia coli containing recombinant styrene monooxygenase. Engineering TCA-cycle regulation allowed increased TCA-cycle activities, a delay of acetate formation, and enhanced NAD(P)H yields during batch cultivation. However, at low biomass and product concentrations, the cellular metabolism of both the mutants as well as the native host strains could cope with increased NADH demands during continuous two-liquid phase biotransformations, whereas elevated but still subtoxic product concentrations were found to cause a significantly increased NAD(P)H demand and a compromised efficiency of metabolic operation. In conclusion, operational conditions determine cellular energy and NAD(P)H demands and thus the biocatalytic efficiency of whole-cell redox biocatalysts.

Living microbial cells are considered to be the catalyst of choice for selective terpene functionalization. However, such processes often suffer from side product formation and poor substrate mass transfer into cells. For the hydroxylation of (S)-limonene to (S)-perillyl alcohol by Pseudomonas putida KT2440 (pGEc47ΔB)(pCom8-PFR1500), containing the cytochrome P450 monooxygenase CYP153A6, the side products perillyl aldehyde and perillic acid constituted up to 26% of the total amount of oxidized terpenes. In this study, it is shown that the reaction rate is substrate-limited in the two-liquid phase system used and that host intrinsic dehydrogenases and not CYP153A6 are responsible for the formation of the undesired side products. In contrast to P. putida KT2440, E. coli W3110 was found to catalyze perillyl aldehyde reduction to the alcohol and no oxidation to the acid. Furthermore, E. coli W3110 harboring CYP153A6 showed high limonene hydroxylation activities (7.1 U g CDW-1). The outer membrane protein AlkL was found to enhance hydroxylation activities of E. coli twofold in aqueous single-phase and fivefold in two-liquid phase biotransformations. In the latter system, E. coli harboring CYP153A6 and AlkL produced up to 39.2 mmol (S)-perillyl alcohol L tot-1 within 26 h, whereas no perillic acid and minor amounts of perillyl aldehyde (8% of the total products) were formed. In conclusion, undesired perillyl alcohol oxidation was reduced by choosing E. coli's enzymatic background as a reaction environment and co-expression of the alkL gene in E. coli represents a promising strategy to enhance terpene bioconversion rates.

Background Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase. Results For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly soluble steroids in biphasic systems. Employing a biphasic system with the organic solvent and Y. lipolytica carbon source ethyl oleate for the whole-cell bioconversion of progesterone, the initial specific hydroxylation rate in a 1.5 L stirred tank bioreactor was further increased 2-fold. Furthermore, the product formation was significantly prolonged as compared to the aqueous system. Co-expression of the human CPR gene led to a 4-10-fold higher specific activity, compared to the co-overexpression of the native Y. lipolytica CPR gene. Multicopy transformants showed a 50-70-fold increase of activity as compared to single copy strains. Conclusions Alkane-assimilating yeast Y. lipolytica, coupled with the described expression strategies, demonstrated its high potential for biotransformations of hydrophobic substrates in two-liquid biphasic systems. Especially organic solvents which can be efficiently taken up and/or metabolized by the cell might enable more efficient bioconversion as compared to aqueous systems and even enable simple, continuous or at least high yield long time processes.

In an earlier study, biocatalytic carbon oxyfunctionalization with water serving as oxygen donor, e.g., the bioconversion of quinaldine to 4-hydroxyquinaldine, was successfully achieved using resting cells of recombinant Pseudomonas putida, containing the molybdenum-enzyme quinaldine 4-oxidase, in a two-liquid phase (2LP) system (Ütkür et al. J Ind Microbiol Biotechnol 38:1067–1077, 2011). In the study reported here, key parameters determining process performance were investigated and an efficient and easy method for product recovery was established. The performance of the whole-cell biocatalyst was shown not to be limited by the availability of the inducer benzoate (also serving as growth substrate) during the growth of recombinant P. putida cells. Furthermore, catalyst performance during 2LP biotransformations was not limited by the availability of glucose, the energy source to maintain metabolic activity in resting cells, and molecular oxygen, a possible final electron acceptor during quinaldine oxidation. The product and the organic solvent (1-dodecanol) were identified as the most critical factors affecting biocatalyst performance, to a large extent on the enzyme level (inhibition), whereas substrate effects were negligible. However, none of the 13 alternative solvents tested surpassed 1-dodecanol in terms of toxicity, substrate/product solubility, and partitioning. The use of supercritical carbon dioxide for phase separation and an easy and efficient liquid–liquid extraction step enabled 4-hydroxyquinaldine to be isolated at a purity of >99.9% with recoveries of 57 and 84%, respectively. This study constitutes the first proof of concept on an integrated process for the oxyfunctionalization of toxic substrates with a water-incorporating hydroxylase.

Metabolically active resting (i.e., nongrowing) bacterial cells have a high potential in cofactor-dependent redox biotransformations. Where growing cells require carbon and energy for biomass production, resting cells can potentially exploit their metabolism more efficiently for redox biocatalysis allowing higher specific activities and product yields on energy source. Here, the potential of resting recombinant E. coli containing the styrene monooxygenase StyAB was investigated for enantioselective styrene epoxidation in a two-liquid phase setup. Resting cells indeed showed twofold higher specific activities as compared to growing cells in a similar setup. However, product formation rates decreased steadily resulting in lower final product concentrations. The low intrinsic stability of the reductase component StyB was found to limit overall biocatalyst stability. Such limitation by enzyme stability was overcome by increasing intracellular StyB levels. Beyond that, product inhibition was identified as a limiting factor, whereas complete toxification of the bacterial cells, as it was observed with growing cells, and deactivation of the multicomponent enzyme system did not occur. The resting cell setup allowed high product yields on glucose of more than 5 mol mol(glucose)(-1), which makes the use of resting cells a promising approach for ecologically as well as economically sustainable oxygenase-based whole-cell biocatalysis.

Cell physiology is a critical factor determining the efficiency of reactions performed by microbial biocatalysts. In order to develop an efficient biotransformation procedure for the hydroxylation of (S)-limonene to (S)-perillyl alcohol by recombinant Pseudomonas putida cells harboring the cytochrome P450 monooxygenase CYP153A6, physiological parameters were optimized. The previously reported synthesis of (S)-perillyl alcohol by P. putida GPo12 was based on complex and sensitive octane feeding strategies (van Beilen et al. in Appl Environ Microbiol 71:1737-1744, 2005), indicating the pivotal role of cell physiology. In contrast to previous findings, the screening of different carbon sources showed that glycerol and citrate are suitable alternatives to octane allowing high specific limonene hydroxylation activities. The use of P. putida KT2440 as an alternative host strain and citrate as the carbon source improved practical handling and allowed a 7.5-fold increase of the specific activity (to 22.6 U g (CDW) (-1) ). In two-liquid-phase biotransformations, 4.3 g of (S)-perillyl alcohol L (tot) (-1) were produced in 24 h, representing a sixfold improvement in productivity compared to previously reported results. It is concluded that, for selective cytochrome P450-based hydrocarbon oxyfunctionalizations by means of living microbial cells, the relationship between cell physiology and the target biotransformation is crucial, and that understanding the relationship should guide biocatalyst and bioprocess design.

Whole-cell redox biocatalysis relies on redox cofactor regeneration by the microbial host. Here, we applied flux balance analysis based on the Escherichia coli metabolic network to estimate maximal NADH regeneration rates. With this optimization criterion, simulations showed exclusive use of the pentose phosphate pathway at high rates of glucose catabolism, a flux distribution usually not found in wild-type cells. In silico, genetic perturbations indicated a strong dependency of NADH yield and formation rate on the underlying metabolic network structure. The linear dependency of measured epoxidation activities of recombinant central carbon metabolism mutants on glucose uptake rates and the linear correlation between measured activities and simulated NADH regeneration rates imply intracellular NADH shortage. Quantitative comparison of computationally predicted NADH regeneration and experimental epoxidation rates indicated that the achievable biocatalytic activity is determined by metabolic and enzymatic limitations including non-optimal flux distributions, high maintenance energy demands, energy spilling, byproduct formation, and uncoupling. The results are discussed in the context of cellular optimization of biotransformation processes and may guide a priori design of microbial cells as redox biocatalysts.

Oxidoreductases catalyze a large variety of regio-, stereo-, and chemoselective hydrocarbon oxyfunctionalizations, reactions, which are important in industrial organic synthesis but difficult to achieve by chemical means. This review summarizes process implementation aspects for the in vivo application of the especially versatile enzyme class of oxygenases, capable of specifically introducing oxygen from molecular oxygen into a large range of organic molecules. Critical issues such as reaching high enzyme activity and specificity, product degradation, cofactor recycling, reactant toxicity, and substrate and oxygen mass transfer can be overcome by biochemical process engineering and biocatalyst engineering. Both strategies provide a growing toolset to facilitate process implementation, optimization, and scale-up. Major advances were achieved via heterologous overexpression of oxygenase genes, directed evolution, metabolic engineering, and in situ product removal. Process examples from industry and academia show that the combined use of different concepts enables efficient oxygenase-based whole-cell catalysis of various commercially interesting reactions such as the biosynthesis of chiral compounds, the specific oxyfunctionalization of complex molecules, and also the synthesis of medium-priced chemicals. Better understanding of the cell metabolism and future developments in both biocatalyst and bioprocess engineering are expected to promote the implementation of many and various industrial biooxidation processes.

During the last few decades, the microbial degradation pathways of aromatic and aliphatic hydrocarbons have received a lot of scientific interest because of the high potential of the enzyme systems involved for environmental (43) and preparative applications (38, 59). These pathways are usually initiated by an oxygenase-catalyzed chemo-, regio-, and stereoselective hydroxylation of the hydrocarbons, a reaction for which often no organic chemical counterpart is known (9, 13). The xylene degradation pathway of Pseudomonas putida mt-2 and its initiating oxygenase, the xylene monooxygenase (XMO), are among the best-studied examples of aromatic hydrocarbon degradation (5, 36, 57, 61). The enzymes for xylene degradation are encoded on a catabolic plasmid, the TOL plasmid pWW0. XMO is the first enzyme in the upper degradation pathway for toluene and xylenes, in which a carboxylic acid is formed (1, 16, 58). The upper pathway also involves benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, which catalyze the oxidation of benzyl alcohols via benzaldehydes to benzoic acids (46-48). The carboxylic acid is then transformed to substrates of the Krebs cycle through the meta cleavage pathway (10, 14, 36, 55). XMO consists of two polypeptide subunits, encoded by xylM and xylA (16, 52). XylA, the NADH:acceptor reductase component, is an electron transport protein transferring reducing equivalents from NADH to XylM (45). XylM, the hydroxylase component, is located in the membrane, and its activity depends on phospholipids and ferrous ion, with a pH optimum of 7 (44, 62). The substrate spectrum of XMO was investigated, with focus on preparative applications. XMO expressed in Escherichia coli oxidizes toluene and xylenes but also m- and p-ethyl-, methoxy-, nitro-, and chloro-substituted toluenes, as well as m-bromo-substituted toluene, to the corresponding benzyl alcohol derivatives (21, 65). Furthermore, styrene is transformed into S-styrene oxide with an enantiomeric excess (ee) of 95% (64, 65). The one-step oxygenation of styrene catalyzed by recombinant XMO in growing cells of E. coli was applied to produce S-styrene oxide on a 2-liter scale with hexadecane as the second organic phase (30). The wild-type strain P. putida mt-2 was used to oxidize methyl groups on aromatic heterocyclics to the corresponding carboxylic acids (20). In large-scale fermentations, a 5-methyl-2-pyrazinecarboxylic acid titer up to 20 g liter−1 was reached. This system exploits the inability of the wild-type strain to further degrade heteroaromatic carboxylic acids. In P. putida mt-2, all three enzyme activities of the upper xylene degradation pathway are responsible for the three-step oxidation. Early reports suggested that XMO also catalyzes alcohol and aldehyde oxidations (15, 16). Later, such activities were attributed to dehydrogenases present in the E. coli host (16, 44). Recently, we verified by in vivo experiments that XMO indeed catalyzes the oxidation of benzyl alcohols and benzaldehydes, both via a monooxygenation type of reaction (4). E. coli cells expressing XMO genes under the control of the alk regulatory system (12, 51, 62, 67) were used for these experiments. Potential preparative in vivo applications of XMO are hampered by low water solubilities and high toxicities of possible substrates and products, limiting the performance of aqueous systems. Nonconventional reaction media such as an aqueous-organic two-liquid-phase system are promising alternatives (8, 40). A second immiscible phase can act as a reservoir for substrate and products, regulating the concentration of such compounds in the biocatalyst microenvironment, minimizing toxicity and simplifying product recovery (24, 60, 63). In the present study, we characterized the multistep oxidation of substrates such as pseudocumene and toluene by whole cells of E. coli containing XMO with the aims of clarifying the natural role of such a multistep catalysis and identifying possible applications. The biotechnological conversion of pseudocumene is of special interest because a controlled regio- and chemospecific multistep oxidation of only one methyl group is difficult to achieve by purely chemical methods. We determined the kinetics of the one-enzyme multistep reaction and analyzed the whole-cell biocatalyst in a two-liquid-phase biotransformation on a 2-liter scale. Our results indicate that, depending on the reaction conditions, product formation may be directed to one specific product, either benzylic alcohols, aldehydes, or acids.