High Cell Density Cultivation Combined with high Specific Enzyme Activity: Cultivation Protocol for the Production of an Amine Transaminase from Bacillus megaterium in E. coli.

High cell density cultivation is an established method for the production of various industrially important products such as recombinant proteins. However, these protocols are not always suitable for biocatalytic processes as the focus often lies on biomass production rather than high specific activities of the enzyme inside the cells. In contrast, a range of shake flask protocols are well known with high specific activities but rather low cell densities. To overcome this gap, we established a tailor-made fed-batch protocol combining both aspects: high cell density and high specific activities of heterologously produced enzyme. Using the example of an industrially relevant amine transaminase from Bacillus megaterium, we describe a strategy to optimize the cultivation yield based on the feed rate, IPTG concentration, and post-induction temperature. By adjusting these key parameters, we were able to increase the specific activity by 2.6-fold and the wet cell weight by even 17-fold compared to shake flasks. Finally, we were able to verify our established protocol by transferring it to another experimenter. With that, our optimization strategy can serve as a template for the production of high titers of heterologously produced, active enzymes and might enable the availability of these catalysts for upscaling biocatalytic processes.

Interdisciplinary development of an overall process concept from glucose to 4,5-dimethyl-1,3-dioxolane via 2,3-butanediol.

To reduce carbon dioxide emissions, carbon-neutral fuels have recently gained renewed attention. Here we show the development and evaluation of process routes for the production of such a fuel, the cyclic acetal 4,5-dimethyl-1,3-dioxolane, from glucose via 2,3-butanediol. The selected process routes are based on the sequential use of microbes, enzymes and chemo-catalysts in order to exploit the full potential of the different catalyst systems through a tailor-made combination. The catalysts (microbes, enzymes, chemo-catalysts) and the reaction medium selected for each conversion step are key factors in the development of the respective production methods. The production of the intermediate 2,3-butanediol by combined microbial and enzyme catalysis is compared to the conventional microbial route from glucose in terms of specific energy demand and overall yield, with the conventional route remaining more efficient. In order to be competitive with current 2,3-butanediol production, the key performance indicator, enzyme stability to high aldehyde concentrations, needs to be increased. The target value for the enzyme stability is an acetaldehyde concentration of 600 mM, which is higher than the current maximum concentration (200 mM) by a factor of three.

Carboligation of 5-(hydroxymethyl)furfural via whole-cell catalysis to form C12 furan derivatives and their use for hydrazone formation.

BACKGROUND: Biobased 5-(hydroxymethyl)furfural (5-HMF) is an important platform that offers numerous possibilities for upgrading to a range of chemical, material and fuel products. One reaction of special interest is the carboligation of 5-HMF into C12 compounds, including 5,5'-bis(hydroxymethyl)furoin (DHMF) and its subsequent oxidation to 5,5'-bis(hydroxymethyl)furil (BHMF), due to their potential applications as building blocks for polymers and hydrocarbon fuels. OBJECTIVES: This study was aimed at evaluating the use of whole cells of Escherichia coli carrying recombinant Pseudomonas fluorescens benzaldehyde lyase as biocatalysts for 5-HMF carboligation, recovery of the C12 derivatives DHMF and BHMF, and testing the reactivity of the carbonyl groups for hydrazone formation for potential use as cross-linking agents in surface coatings. The effects of different parameters on the reaction were investigated to find the conditions for achieving high product yield and productivity. RESULTS: The reaction with 5 g/L 5-HMF using 2 gCDW/L recombinant cells in 10% dimethyl carbonate, pH 8.0 at 30 °C resulted in DHMF yield of 81.7% (0.41 mol/mol) at 1 h, and BHMF yield of 96.7% (0.49 mol/mol) at 72 h reaction time. Fed-batch biotransformation generated a maximum DHMF concentration of 53.0 g/L (or 26.5 g DHMF/g cell catalyst) with productivity of 10.6 g/L.h, after five feeds of 20 g/L 5-HMF. Both DHMF and BHMF reacted with adipic acid dihydrazide to form hydrazone that was confirmed by Fourier-transform infrared spectroscopy and 1H NMR. CONCLUSION: The study demonstrates the potential application of recombinant E. coli cells for cost-effective production of commercially relevant products.

EnzymeML: seamless data flow and modeling of enzymatic data.

The design of biocatalytic reaction systems is highly complex owing to the dependency of the estimated kinetic parameters on the enzyme, the reaction conditions, and the modeling method. Consequently, reproducibility of enzymatic experiments and reusability of enzymatic data are challenging. We developed the XML-based markup language EnzymeML to enable storage and exchange of enzymatic data such as reaction conditions, the time course of the substrate and the product, kinetic parameters and the kinetic model, thus making enzymatic data findable, accessible, interoperable and reusable (FAIR). The feasibility and usefulness of the EnzymeML toolbox is demonstrated in six scenarios, for which data and metadata of different enzymatic reactions are collected and analyzed. EnzymeML serves as a seamless communication channel between experimental platforms, electronic lab notebooks, tools for modeling of enzyme kinetics, publication platforms and enzymatic reaction databases. EnzymeML is open and transparent, and invites the community to contribute. All documents and codes are freely available at https://enzymeml.org .

Effective Production of Selected Dioxolanes by Sequential Bio- and Chemocatalysis Enabled by Adapted Solvent Switching.

Most combinations of chemo- and biocatalysis take place in aqueous media or require a solvent change with complex intermediate processing. Using enzymes in the same organic solvent as the chemocatalyst eliminates this need. Here, it was shown that a complete chemoenzymatic cascade to form dioxolanes could be carried out in a purely organic environment. The result, including downstream processing, was compared with a classical mode, shifting solvent. First, a two-step enzyme cascade starting from aliphatic aldehydes to chiral diols (3,4-hexanediol and 4,5-octanediol) was run either in an aqueous buffer or in the potentially biobased solvent cyclopentyl methyl ether. Subsequently, a ruthenium molecular catalyst enabled the conversion to dioxolanes [e. g., (4S,5S)-dipropyl-1,3-dioxolane]. Importantly, the total synthesis of this product was not only highly stereoselective but also based on the combination of biomass, CO2 , and hydrogen, thus providing an important example of a bio-hybrid chemical.

Modulation of Transaminase Activity by Encapsulation in Temperature-Sensitive Poly(N-acryloyl glycinamide) Hydrogels.

Smart hydrogels hold much potential for biocatalysis, not only for the immobilization of enzymes, but also for the control of enzyme activity. We investigated upper critical solution temperature-type poly N-acryloyl glycinamide (pNAGA) hydrogels as a smart matrix for the amine transaminase from Bacillus megaterium (BmTA). Physical entrapment of BmTA in pNAGA hydrogels results in high immobilization efficiency (>89 %) and high activity (97 %). The temperature-sensitiveness of pNAGA is preserved upon immobilization of BmTA and shows a gradual deswelling upon temperature reduction. While enzyme activity is mainly controlled by temperature, deactivation tended to be higher for immobilized BmTA (≈62-68 %) than for free BmTA (≈44 %), suggesting a deactivating effect due to deswelling of the pNAGA gel. Although the deactivation in response to hydrogel deswelling is not yet suitable for controlling enzyme activity sufficiently, it is nevertheless a good starting point for further optimization.

The Effect of Visible Light on the Catalytic Activity of PLP-Dependent Enzymes.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes are a versatile class of biocatalysts and feature a variety of industrial applications. However, PLP is light sensitive and can cause inactivation of enzymes in certain light conditions. As most of the PLP-dependent enzymes are usually not handled in dark conditions, we evaluated the effect of visible light on the activity of PLP-dependent enzymes during production as well as transformation. We tested four amine transaminases, from Chromobacterium violaceum, Bacillus megaterium, Vibrio fluvialis and a variant from Arthrobacter species as well as two lysine decarboxylases, from Selenomonas ruminantium and the LDCc from Escherichia coli. It appeared that five of these six enzymes suffered from a significant decrease in activity by up to 90 % when handled in laboratory light conditions. Surprisingly, only the amine transaminase variant from Arthrobacter species appeared to be unaffected by light exposure and even showed an activation to 150 % relative activity over the course of 6 h regardless of the light conditions.

Applied biocatalysis beyond just buffers - from aqueous to unconventional media. Options and guidelines.

In nature, enzymes conventionally operate under aqueous conditions. Because of this, aqueous buffers are often the choice for reaction media when enzymes are applied in chemical synthesis. However, to meet the demands of an industrial application, due to the poor water solubility of many industrially relevant compounds, an aqueous reaction system will often not be able to provide sufficient substrate loadings. A switch to a non-aqueous solvent system can provide a solution, which is already common for lipases, but more challenging for biocatalysts from other enzyme classes. The choices in solvent types and systems, however, can be overwhelming. Furthermore, some engineering of the protein structure of biocatalyst formulation is required. In this review, a guide for those working with biocatalysts, who look for a way to increase their reaction productivity, is presented. Examples reported clearly show that bulk water is not necessarily required for biocatalytic reactions and that clever solvent systems design can support increased product concentrations thereby decreasing waste formation. Additionally, under these conditions, enzymes can also be combined in cascades with other, water-sensitive, chemical catalysts. Finally, we show that the application of non-aqueous solvents in biocatalysis can actually lead to more sustainable processes. At the hand of flowcharts, following simple questions, one can quickly find what solvent systems are viable.

Production of the Carboxylate Reductase from Nocardia otitidiscaviarum in a Soluble, Active Form for in†vitro Applications.

Accessing aldehydes from carboxylate moieties is often a challenging task. In this regard, carboxylate reductases (CARs) are promising catalysts provided by nature that are able to accomplish this task in just one step, avoiding over-reduction to the alcohol product. However, the heterologous expression of CARs can be quite difficult due to the excessive formation of insoluble protein, thus hindering further characterization and application of the enzyme. Here, the heterologous production of the carboxylate reductase from Nocardia otitidiscaviarum (NoCAR) was optimized by a combination of i) optimized cultivation conditions, ii) post-translational modification with a phosphopantetheinyl transferase and iii) selection of an appropriate expression strain. Especially, the selection of Escherichia coli tuner cells as host had a strong effect on the final 110-fold increase in the specific activity of NoCAR. This highly active NoCAR was used to reduce sodium benzoate to benzaldehyde, and it was successfully assembled with an in vitro regeneration of ATP and NADPH, being capable of reducing about 30 mM sodium benzoate with high selectivity in only 2 h of reaction.

An Enzymatic 2-Step Cofactor and Co-Product Recycling Cascade towards a Chiral 1,2-Diol. Part I: Cascade Design.

Alcohol dehydrogenases are of high interest for stereoselective syntheses of chiral building blocks such as 1,2-diols. As this class of enzymes requires nicotinamide cofactors, their application in biotechnological synthesis reactions is economically only feasible with appropriate cofactor regeneration. Therefore, a co-substrate is oxidized to the respective co-product that accumulates in equal concentration to the desired target product. Co-product removal during the course of the reaction shifts the reaction towards formation of the target product and minimizes undesired side effects. Here we describe an atom efficient enzymatic cofactor regeneration system where the co-product of the ADH is recycled as a substrate in another reaction set. A 2-step enzymatic cascade consisting of a thiamine diphosphate (ThDP)-dependent carboligase and an alcohol dehydrogenase is presented here as a model reaction. In the first step benzaldehyde and acetaldehyde react to a chiral 2-hydroxy ketone, which is subsequently reduced by to a 1,2-diol. By choice of an appropriate co-substrate (here: benzyl alcohol) for the cofactor regeneration in the alcohol dehydrogenases (ADH)-catalyzed step, the co-product (here: benzaldehyde) can be used as a substrate for the carboligation step. Even without any addition of benzaldehyde in the first reaction step, this cascade design yielded 1,2-diol concentrations of >100 mM with optical purities (ee, de) of up to 99%. Moreover, this approach overcomes the low benzaldehyde solubility in aqueous systems and optimizes the atom economy of the reaction by reduced waste production. The example presented here for the 2-step recycling cascade of (1R,2R)-1-phenylpropane-1,2-diol can be applied for any set of enzymes, where the co-products of one process step serve as substrates for a coupled reaction.

Stimulus-Responsive Regulation of Enzyme Activity for One-Step and Multi-Step Syntheses.

Multi-step biocatalytic reactions have gained increasing importance in recent years because the combination of different enzymes enables the synthesis of a broad variety of industrially relevant products. However, the more enzymes combined, the more crucial it is to avoid cross-reactivity in these cascade reactions and thus achieve high product yields and high purities. The selective control of enzyme activity, i.e., remote on-/off-switching of enzymes, might be a suitable tool to avoid the formation of unwanted by-products in multi-enzyme reactions. This review compiles a range of methods that are known to modulate enzyme activity in a stimulus-responsive manner. It focuses predominantly on in vitro systems and is subdivided into reversible and irreversible enzyme activity control. Furthermore, a discussion section provides indications as to which factors should be considered when designing and choosing activity control systems for biocatalysis. Finally, an outlook is given regarding the future prospects of the field.

Selective aerobic oxidation reactions using a combination of photocatalytic water oxidation and enzymatic oxyfunctionalisations.

Peroxygenases offer attractive means to address challenges in selective oxyfunctionalisation chemistry. Despite their attractiveness, the application of peroxygenases in synthetic chemistry remains challenging due to their facile inactivation by the stoichiometric oxidant (H2O2). Often atom inefficient peroxide generation systems are required, which show little potential for large scale implementation. Here we show that visible light-driven, catalytic water oxidation can be used for in situ generation of H2O2 from water, rendering the peroxygenase catalytically active. In this way the stereoselective oxyfunctionalisation of hydrocarbons can be achieved by simply using the catalytic system, water and visible light.

Citrate as Cost-Efficient NADPH Regenerating Agent.

The economically efficient utilization of NAD(P)H-dependent enzymes requires the regeneration of consumed reduction equivalents. Classically, this is done by substrate supplementation, and if necessary by addition of one or more enzymes. The simplest method thereof is whole cell NADPH regeneration. In this context we now present an easy-to-apply whole cell cofactor regeneration approach, which can especially be used in screening applications. Simply by applying citrate to a buffer or directly using citrate/-phosphate buffer NADPH can be regenerated by native enzymes of the TCA cycle, practically present in all aerobic living organisms. Apart from viable-culturable cells, this regeneration approach can also be applied with lyophilized cells and even crude cell extracts. This is exemplarily shown for the synthesis of 1-phenylethanol from acetophenone with several oxidoreductases. The mechanism of NADPH regeneration by TCA cycle enzymes was further investigated by a transient isotopic labeling experiment feeding [1,5-13C]citrate. This revealed that the regeneration mechanism can further be optimized by genetic modification of two competing internal citrate metabolism pathways, the glyoxylate shunt, and the glutamate dehydrogenase.

Enzymatic and Chemoenzymatic Three-Step Cascades for the Synthesis of Stereochemically Complementary Trisubstituted Tetrahydroisoquinolines.

Chemoenzymatic and enzymatic cascade reactions enable the synthesis of complex stereocomplementary 1,3,4-trisubstituted tetrahydroisoquinolines (THIQs) with three chiral centers in a step-efficient and selective manner without intermediate purification. The cascade employs inexpensive substrates (3-hydroxybenzaldehyde and pyruvate), and involves a carboligation step, a subsequent transamination, and finally a Pictet-Spengler reaction with a carbonyl cosubstrate. Appropriate selection of the carboligase and transaminase enzymes enabled the biocatalytic formation of (1R,2S)-metaraminol. Subsequent cyclization catalyzed either enzymatically by a norcoclaurine synthase or chemically by phosphate resulted in opposite stereoselectivities in the products at the C1 position, thus providing access to both orientations of the THIQ C1 substituent. This highlights the importance of selecting from both chemo- and biocatalysts for optimal results.

Reductive amination of ketones catalyzed by whole cell biocatalysts containing imine reductases (IREDs).

The asymmetric reductive amination of ketones represents an elegant and convenient way to obtain chiral amines. Recently, several examples have been reported in which isolated imine reductases (IREDs) have been used for this type of reaction leading to promising results. In this work we focus on the applicability of whole cell biocatalysts (recombinant E. coli cells heterologously overexpressing an IRED) to simplify its preparation and to cut on catalyst production costs. Thirteen IREDs were screened towards six different ketones, using methylamine as amine donor. The targeted amines were formed with low to very high conversions and good to excellent stereoselectivity, depending on both, the ketone amine pair used for the reaction, as well as the applied IRED. It was further proven that a micro-aqueous reaction environment was applicable showing similar activity trends for the various reductive aminations but predominantly reduced conversions. A preparative scale experiment in a buffered environment was conducted leading to 93% conversion and 99% stereoselectivity of the product (1S,3R)-N,3-dimethylcyclohexylamine. As the whole cells intrinsic glucose dehydrogenase could be used for cofactor regeneration, no enzyme addition had to be applied, making this biocatalyst formulation particularly cost efficient.

BioCatNet: A Database System for the Integration of Enzyme Sequences and Biocatalytic Experiments.

The development of novel enzymes for biocatalytic processes requires knowledge on substrate profile and selectivity; this can be derived from databases and from publications. Often, these sources lack time-course data for the substrate or product, and an unambiguous link between experiment and enzyme sequence. The lack of integrated, original data hampers the comprehensive analysis of enzyme kinetics and the evaluation of sequence-function relationships. In order to accelerate enzyme engineering, BioCatNet integrates protein sequence, protein structure, and experimental data for a given enzyme family. BioCatNet explicitly assigns the enzyme sequence to the experimental data, which consists of information on reaction conditions and time-course data. BioCatNet facilitates the consistent documentation of reaction conditions, the archiving of time-course data, and the efficient exchange of experimental data among collaborators. Data integration is demonstrated for three case studies by using the TEED (Thiamine diphosphate-dependent Enzymes Engineering Database).

Regio- and Stereoselective Aliphatic-Aromatic Cross-Benzoin Reaction: Enzymatic Divergent Catalysis.

The catalytic asymmetric synthesis of chiral 2-hydroxy ketones by using different thiamine diphosphate dependent enzymes, namely benzaldehyde lyase from Pseudomonas fluorescens (PfBAL), a variant of benzoylformate decarboxylase from Pseudomonas putida (PpBFD-L461A), branched-chain 2-keto acid decarboxylase from Lactococcus lactis (LlKdcA) and a variant of pyruvate decarboxylase from Acetobacter pasteurianus (ApPDC-E469G), was studied. Starting with the same set of substrates, substituted benzaldehydes in combination with different aliphatic aldehydes, PfBAL and PpBFD-L461A selectively deliver the (R)- and (S)-2-hydroxy-propiophenone derivatives, respectively. The (R)- and (S)-phenylacetylcarbinol (1-hydroxy-1-phenylacetone) derivatives are accessible in a similar way using LlKdcA and ApPDC-E469G, respectively. In many cases excellent stereochemical purities (>98 % enantiomeric excess) could be achieved. Hence, the regio- and stereochemistry of the product in the asymmetric aliphatic-aromatic cross-benzoin reaction can be controlled solely by choice of the appropriate enzyme or enzyme variant.

Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale.

Recent advances in biocatalysis have strongly boosted its recognition as a valuable addition to traditional chemical synthesis routes. As for any catalytic process, catalyst's costs and stabilities are of highest relevance for the economic application in chemical manufacturing. Employing biocatalysts as whole cells circumvents the need of cell lysis and enzyme purification and hence strongly cuts on cost. At the same time, residual cell wall components can shield the entrapped enzyme from potentially harmful surroundings and aid to enable applications far from natural enzymatic environments. Further advantages are the close proximity of reactants and catalysts as well as the inherent presence of expensive cofactors. Here, we review and comment on benefits and recent advances in whole cell biocatalysis.

Enantioselective, continuous (R)- and (S)-2-butanol synthesis: achieving high space-time yields with recombinant E. coli cells in a micro-aqueous, solvent-free reaction system.

The stereoselective production of (R)- or (S)-2-butanol is highly challenging. A potent synthesis strategy is the biocatalytic asymmetric reduction of 2-butanone applying alcohol dehydrogenases. However, due to a time-dependent racemisation process, high stereoselectivity is only obtained at incomplete conversion after short reaction times. Here, we present a solution to this problem: by using a continuous process, high biocatalytic selectivity can be achieved while racemisation is suppressed successfully. Furthermore, high conversion was achieved by applying recombinant, lyophilised E. coli cells hosting Lactobacillus brevis alcohol dehydrogenase in a micro-aqueous solvent-free continuous reaction system. The optimisation of residence time (τ) and 2-butanone concentration boosted both conversion (>99%) and enantiomeric excess (ee) of (R)-2-butanol (>96%). When a residence time of only τ=3.1 min was applied, productivity was extraordinary with a space-time yield of 2278±29g/(L×d), thus exceeding the highest values reported to date by a factor of more than eight. The use of E. coli cells overexpressing an ADH of complementary stereoselectivity yielded a synthesis strategy for (S)-2-butanol with an excellent ee (>98%). Although conversion was only moderate (up to 46%), excellent space-time yields of up to 461g/(L×d) were achieved. The investigated concept represents a synthesis strategy that can also be applied to other biocatalytic processes where racemisation poses a challenge.