Matthias Bechtold

Systematic Optimization of Interface Interactions Increases the Thermostability of a Multimeric Enzyme

With their exceptional selectivity, biocatalysts play an increasing role in diverse chemical fields including food processing, production of fine, specialty, and bulk chemicals, and fuel production. A frequently remaining fundamental limitation is operational stability, in particular at higher reaction temperatures, which are often preferred because of an increase in reaction rate or reactant solubility, a decrease in medium viscosity or risk of microbial contamination, or a more favorable position of the reaction equilibrium. However, the structural integrity of an enzyme, especially of mesophilic origin, is often impaired under these high-temperature operational regimes. Inactivation typically starts with a loss of integrity of the quaternary (for multimeric enzymes) or tertiary structure (for monomeric enzymes) and is followed by an irreversible denaturation step. The situation can be improved by immobilization, additives, or enzyme engineering, preferably employing semi-rational approaches to prevent the often tedious development of high-throughput assay formats. Such approaches include increasing the similarity to a consensus amino acid sequence derived from sequence alignments or are based on available crystal structures, which can direct the variation of presumably very flexible amino acids identified by their high atomic displacement parameters (B-factors). In the case of multimeric enzymes, the placement of intersubunit ionic interactions or disulfide bridges was shown to prevent subunit dissociation and increase biocatalyst stability. However, successful placement of such strong links to prevent multimer dissociation as well as proper disulfide formation in model expression hosts such as E. coli is nontrivial. Consequently, generic strategies to systematically improve the thermostabilty of multimeric enzymes are still lacking. We reasoned that a systematic variation of the nonconserved residues of a protein–protein interface should rapidly reveal those positions in the amino acid sequence of an enzyme, the mutation of which can contribute to strengthening the inter-subunit interface and thus counter disintegration. To test this hypothesis, we selected the homodimeric dtagatose 3-epimerase of P. cichorii (PcDTE). PcDTE catalyzes the reversible C3-epimerization of all four ketohexose epimer pairs and is hence of strategic importance for preparative production of rare hexoses, which on industrial levels takes place at elevated temperatures to reduce viscosity and microbial contamination. However, wild-type (wt) PcDTE degrades rapidly at elevated temperatures. To exclude that disintegration of the tertiary structure of one of the monomers was actually the rate-determining step in thermal inactivation, we varied the 10 amino acids with the highest B-factors extracted from the analysis of 3 PcDTE crystal structures (PDB ID 2QUN, 2QUM, 2QUL) as described by Reetz et al. Of these, only substitution of K122 (K122V) showed a slight impact on the thermostability (conversion of d-fructose to d-psicose, T50 20 = 67.2 8C (+ 1.2 8C compared to wt), with T50 20 being the temperature at which 50% activity is lost after 20 minutes of incubation (see the Supporting Information for details). Interestingly, K122 is part of the dimer interface, whereas six of the other residues are not, suggesting that disintegration of the quaternary structure is the more likely rate-limiting step in thermal inactivation. As a first attempt towards stabilization of the dimeric interface of PcDTE, we introduced intersubunit disulfide bonds at positions F157 or position W160 and W262. These sites were selected by the program “MODIP” as the only potentially suitable locations at the interface, albeit already with a low quality ranking. Correspondingly, the subsequent mutagenesis and expression in E. coli did not lead to enzyme variants with increased stability (see Figure S1 in the Supporting Information). Instead, when we explored structure-guided systematic strengthening of interactions between the two subunits throughout the entire dimer interface, variants with improved thermostability could be readily isolated. For this, we conducted first a thorough analysis of one PcDTE crystal structure (PDB ID 2QUN) using the software PDBePISA to identify the residues involved in interface formation. As a homodimer, PcDTE has a virtually symmetric interaction pattern, suggesting a maximum of 44 sites for engineering (Figure S3 and Table S3). Three of the 44 residues make only negligible contributions to the buried surface area of the interface and were discarded. Ten highly conserved residues in the interface (Figure 1), likely to be crucial for function or structural integrity and thus unlikely to yield mutants with improved stability, were identified from a sequence alignment with 28 other DTE sequences (listed in the Uniprot database as DTEs, Figure S2) and discarded as well. Each of the 31 remaining sites was randomized in a first round of variation separately by site-saturation mutagenesis with NNK degeneracy primers allowing sampling of all [*] A. Bosshart, Prof. Dr. S. Panke, Dr. M. Bechtold Bioprocess Laboratory, Department of Biosystems Science and Engineering, ETH Zurich Mattenstrasse 26, 4058 Basel (Switzerland) E-mail: matthias.bechtold@bsse.ethz.ch Homepage: http://www.bsse.ethz.ch/bpl

A Separation‐Integrated Cascade Reaction to Overcome Thermodynamic Limitations in Rare‐Sugar Synthesis

Enzyme cascades combining epimerization and isomerization steps offer an attractive route for the generic production of rare sugars starting from accessible bulk sugars but suffer from the unfavorable position of the thermodynamic equilibrium, thus reducing the yield and requiring complex work-up procedures to separate pure product from the reaction mixture. Presented herein is the integration of a multienzyme cascade reaction with continuous chromatography, realized as simulated moving bed chromatography, to overcome the intrinsic yield limitation. Efficient production of D-psicose from sucrose in a three-step cascade reaction using invertase, D-xylose isomerase, and D-tagatose epimerase, via the intermediates D-glucose and D-fructose, is described. This set-up allowed the production of pure psicose (99.9%) with very high yields (89%) and high enzyme efficiency (300 g of D-psicose per g of enzyme).

Directed Divergent Evolution of a Thermostable D-Tagatose Epimerase towards Improved Activity for Two Hexose Substrates.

Functional promiscuity of enzymes can often be harnessed as the starting point for the directed evolution of novel biocatalysts. Here we describe the divergent morphing of an engineered thermostable variant (Var8) of a promiscuous D‐tagatose epimerase (DTE) into two efficient catalysts for the C3 epimerization of D‐fructose to D‐psicose and of L‐sorbose to L‐tagatose. Iterative single‐site randomization and screening of 48 residues in the first and second shells around the substrate‐binding site of Var8 yielded the eight‐site mutant IDF8 (ninefold improved kcat for the epimerization of D‐fructose) and the six‐site mutant ILS6 (14‐fold improved epimerization of L‐sorbose), compared to Var8. Structure analysis of IDF8 revealed a charged patch at the entrance of its active site; this presumably facilitates entry of the polar substrate. The improvement in catalytic activity of variant ILS6 is thought to relate to subtle changes in the hydration of the bound substrate. The structures can now be used to select additional sites for further directed evolution of the ketohexose epimerase.

Novel method for high-throughput colony PCR screening in nanoliter-reactors

We introduce a technology for the rapid identification and sequencing of conserved DNA elements employing a novel suspension array based on nanoliter (nl)-reactors made from alginate. The reactors have a volume of 35 nl and serve as reaction compartments during monoseptic growth of microbial library clones, colony lysis, thermocycling and screening for sequence motifs via semi-quantitative fluorescence analyses. nl-Reactors were kept in suspension during all high-throughput steps which allowed performing the protocol in a highly space-effective fashion and at negligible expenses of consumables and reagents. As a first application, 11 high-quality microsatellites for polymorphism studies in cassava were isolated and sequenced out of a library of 20 000 clones in 2 days. The technology is widely scalable and we envision that throughputs for nl-reactor based screenings can be increased up to 100 000 and more samples per day thereby efficiently complementing protocols based on established deep-sequencing technologies.

Simulated moving bed enantioseparation of amino acids employing memory effect-constrained chromatography columns.

Teicoplanin aglycone-based chromatography columns (Chirobiotic TAG) enable amino acid enantioseparation with aqueous mobile phases, which perfectly accommodates the distinct hydrophilicity of most amino acids. Therefore, this stationary phase constitutes a promising option in particular for preparative-scale separations that require high feed concentrations for economic operation. However, detailed studies revealed a solute-related memory effect when this column is subjected to high loadings of amino acids, conditions that prevail in SMB operation. High loadings yield an activation of the column as indicated by increased retention times when comparing finite injection chromatograms obtained before and after the column had been subjected to a concentrated amino acid feed. This effect can be slowly reversed by flushing the column with solvent devoid of amino acid. Obviously, the activation of the stationary phase needs to be accounted for in the determination of adsorption isotherms that are used for SMB design. In this work we introduce a perturbation method adapted specifically to capture the stationary phase behaviour at SMB-like conditions. The adsorption isotherms obtained from this method indeed allowed for accurate SMB design of a methionine enantioseparation as judged by the very good agreement of experimentally obtained and model-predicted purities. Furthermore, SMB operation over 3 days with constant purities (besides deviations originating from a dip in temperature) was accomplished indicating that the adsorption behaviour in the activated state is indeed time invariant and stable long-term SMB operation with these columns is principally feasible.

Multi-objective optimization for the economic production of d-psicose using simulated moving bed chromatography.

The biocatalytic production of rare carbohydrates from available sugar sources rapidly gains interest as a route to acquire industrial amounts of rare sugars for food and fine chemical applications. Here we present a multi-objective optimization procedure for a simulated moving bed (SMB) process for the production of the rare sugar d-psicose from enzymatically produced mixtures with its epimer d-fructose. First, model parameters were determined using the inverse method and experimentally validated on a 2-2-2-2 lab-scale SMB plant. The obtained experimental purities (PUs) were in excellent agreement with the simulated data derived from a transport-dispersive true-moving bed model demonstrating the feasibility of the proposed design. In the second part the performance of the separation was investigated in a multi-objective optimization study addressing the cost-contributing performance parameters productivity (PR) and desorbent requirement (DR) as a function of temperature. While rare sugar SMB operation under conditions of low desorbent consumption was found to be widely unaffected by temperature, SMB operation focusing on increased PR significantly benefited from high temperatures, with possible productivities increasing from 3.4kg(Lday)(-1) at 20°C to 5kg(Lday)(-1) at 70°C, indicating that decreased selectivity at higher temperatures could be fully compensated for by the higher mass transfer rates, as they translate into reduced switch times and hence higher PR. A DR/PR Pareto optimization suggested a similar but even more pronounced trend also under relaxed PU requirements, with the PR increasing from 4.3kg(Lday)(-1) to a maximum of 7.8kg(Lday)(-1) for SMB operation at 50°C when the PU of the non-product stream was reduced from 99.5% to 90%. Based on the in silico optimization results experimental SMB runs were performed yielding considerable PRs of 1.9 (30°C), 2.4 (50°C) and 2.6kg(Lday)(-1) (70°C) with rather low DR (27L per kg of rare sugar produced) on a lab-scale SMB installation.

Model-based identification of optimal operating conditions for amino acid simulated moving bed enantioseparation using a macrocyclic glycopeptide stationary phase.

Teicoplanin aglycone columns allow efficient separation of amino acid enantiomers in aqueous mobile phases and enable robust and predictable simulated moving bed (SMB) separation of racemic methionine despite a dependency of the adsorption behavior on the column history (memory effect). In this work we systematically investigated the influence of the mobile phase (methanol content) and temperature on SMB performance using a model-based optimization approach that accounts for methionine solubility, adsorption behavior and back pressure. Adsorption isotherms became more favorable with increasing methanol content but methionine solubility was decreased and back pressure increased. Numerical optimization suggested a moderate methanol content (25-35%) for most efficient operation. Higher temperature had a positive effect on specific productivity and desorbent requirement due to higher methionine solubility, lower back pressure and virtually invariant selectivity at high loadings of racemic methionine. However, process robustness (defined as a difference in flow rate ratios) decreased strongly with increasing temperature to the extent that any significant increase in temperature over 32°C will likely result in operating points that cannot be realized technically even with the lab-scale piston pump SMB system employed in this study.

Highly Efficient Production of Rare Sugars D-Psicose and L-Tagatose by Two Engineered D-Tagatose Epimerases

Rare sugars are monosaccharides that do not occur in nature in large amounts. However, many of them demonstrate high potential as low‐calorie sweetener, chiral building blocks or active pharmaceutical ingredients. Their production by enzymatic means from broadly abundant epimers is an attractive alternative to synthesis by traditional organic chemical means, but often suffers from low space‐time yields and high enzyme costs due to rapid enzyme degradation. Here we describe the detailed characterization of two variants of d‐tagatose epimerase under operational conditions that were engineered for high stability and high catalytic activity towards the epimerization of d‐fructose to d‐psicose and l‐sorbose to l‐tagatose, respectively. A variant optimized for the production of d‐psicose showed a very high total turnover number (TTN) of up to 108 catalytic events over a catalysts lifetime, determined under operational conditions at high temperatures in an enzyme‐membrane reactor (EMR). Maximum space‐time yields as high as 10.6 kg L−1 d−1 were obtained with a small laboratory‐scale EMR, indicating excellent performance. A variant optimized for the production of l‐tagatose performed less stable in the same setting, but still showed a very good TTN of 5.8 × 105 and space‐time yields of up to 478 g L−1 d−1. Together, these results confirm that large‐scale enzymatic access to rare sugars is feasible. Biotechnol. Bioeng. 2016;113: 349–358.