Ketogluconate production by Gluconobacter strains: enzymes and biotechnological applications.

Gluconobacter strains perform incomplete oxidation of various sugars and alcohols, employing regio- and stereoselective membrane-bound dehydrogenases oriented toward the periplasmic space. This oxidative fermentation process is utilized industrially. The ketogluconate production pathway, characteristic of these strains, begins with the conversion of d-glucose to d-gluconate, which then diverges and splits into 2 pathways producing 5-keto-d-gluconate and 2-keto-d-gluconate and subsequently 2,5-diketo-d-gluconate. These transformations are facilitated by membrane-bound d-glucose dehydrogenase, glycerol dehydrogenase, d-gluconate dehydrogenase, and 2-keto-d-gluconate dehydrogenase. The variance in end products across Gluconobacter strains stems from the diversity of enzymes and their activities. This review synthesizes biochemical and genetic knowledge with biotechnological applications, highlighting recent advances in metabolic engineering and the development of an efficient production process focusing on enzymes relevant to the ketogluconate production pathway in Gluconobacter strains.

Engineering Glucose Dehydrogenase to Favor Totally Synthetic Biomimetic Cofactors Containing Carboxyl Group.

The utilization of unnatural nicotinamide cofactors for reactions catalyzed by oxidoreductases has gained increasing interest. Totally synthetic nicotinamide cofactor biomimetics (NCBs) are cost-effective and convenient to synthesize. Thus, it has become increasingly important to develop enzymes that accept NCBs. Here, we have engineered SsGDH to favor a newly synthesized unnatural cofactor 3-carbamoyl-1-(4-carboxybenzyl) pyridin-1-ium (BANA+ ). Using in situ ligand minimization tool, sites 44 and 114 were identified as hotspots for mutagenesis. All the double mutants demonstrated 2.7-7.7-fold improvements in catalytic activity, and the best double mutant E44D/E114 L exhibited 10.6-fold increased catalytic efficiency toward BANA+ . These results provide valuable information for the rational engineering of oxidoreductases with versatile NCBs-dependency, as well as the design of novel biomimetic cofactors.

Constitutive glucose dehydrogenase elevates intracellular NADPH levels and luciferase luminescence in Bacillus subtilis.

BACKGROUND: Genetic modifications in Bacillus subtilis have allowed the conversion of myo-inositol into scyllo-inositol, which is proposed as a therapeutic agent for Alzheimer's disease. This conversion comprises two reactions catalyzed by two distinct inositol dehydrogenases, IolG and IolW. The IolW-mediated reaction requires the intracellular regeneration of NADPH, and there appears to be a limit to the endogenous supply of NADPH, which may be one of the rate-determining factors for the conversion of inositol. The primary mechanism of NADPH regeneration in this bacterium remains unclear. RESULTS: The gdh gene of B. subtilis encodes a sporulation-specific glucose dehydrogenase that can use NADP+ as a cofactor. When gdh was modified to be constitutively expressed, the intracellular NADPH level was elevated, increasing the conversion of inositol. In addition, the bacterial luciferase derived from Photorhabdus luminescens became more luminescent in cells in liquid culture and colonies on culture plates. CONCLUSION: The results indicated that the luminescence of luciferase was representative of intracellular NADPH levels. Luciferase can therefore be employed to screen for mutations in genes involved in NADPH regeneration in B. subtilis, and artificial manipulation to enhance NADPH regeneration can promote the production of substances such as scyllo-inositol.

The TonB-dependent uptake of pyrroloquinoline-quinone (PQQ) and secretion of gluconate by Escherichia coli K-12.

Glucose is taken up by Escherichia coli through the phosphotransferase system (PTS) as the preferred carbon source. PTS mutants grow with glucose as a carbon source only in the presence of pyrroloquinoline quinone (PQQ), which is needed as a redox cofactor for the glucose dehydrogenase Gcd. The membrane-anchored Gcd enzyme oxidises glucose to gluconolactone in the periplasm. For this reaction to occur, external supply of PQQ is required as E. coli is unable to produce PQQ de novo. Growth experiments show that PqqU (previously YncD) is the TonB-ExbBD-dependent transporter for PQQ through the outer membrane. PQQ protected the cells from the PqqU-dependent phage IsaakIselin (Bas10) by competition for the receptor protein. As a high affinity uptake system, PqqU allows E. coli to activate Gcd even at surrounding PQQ concentrations of about 1 nmoL/L. At about 30-fold higher PQQ concentrations, the activation of Gcd gets PqqU independent. Due to its small size, Pqq may also pass the outer membrane through porins. The PQQ-dependent production of gluconate has been demonstrated in many plant growth-promoting bacteria that solubilise phosphate minerals in the soil by secreting this acid. Under phosphate limiting conditions also E. coli induces the glucose dehydrogenase and secretes gluconate, even in absence of PTS, that is, even when the bacterium is unable to grow on glucose without PQQ.

Protocol for construction and characterization of direct electron transfer-based enzyme-electrode using gold binding peptide as molecular binder.

Here, we present a protocol for constructing direct electron transfer (DET)-based enzyme-electrodes using gold-binding peptide (GBP). We describe fusion of four GBPs to flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase gamma-alpha complex (GDHγα), as model oxidoreductase, to generate four GDHγα variants. We then detail the measurements of catalytic and bioelectrochemical properties of these GDHγα variants on electrode together with surface morphology of GDHγα variants immobilized on gold surface. This protocol is useful for construction and validation of enzyme-based electrocatalytic system. For complete details on the use and execution of this protocol, please refer to Lee et al. (2021).

A cytochrome b-glucose dehydrogenase chimeric enzyme capable of direct electron transfer.

The development of third generation biosensors depends on the availability of direct electron transfer (DET) capable enzymes. A successful strategy is to fuse a cytochrome domain to an enzyme to fulfil the function of a built-in redox mediator between the catalytic center and the electrode. In this study, we fused the cytochrome domain of Neurospora crassa CDH IIA (NcCYT) N-terminally to glucose dehydrogenase from Glomerella cingulata (GcGDH) to generate the chimeric enzyme NcCYT-GcGDH in a large amount for further studies. Heterologous expression in P. pastoris and chromatographic purification resulted in 1.8 g of homogeneous chimeric enzyme. Biochemical and electrochemical characterization confirmed that the chimeric enzyme is catalytically active, able to perform interdomain electron transfer (IET) and direct electron transfer (DET) via the fused cytochrome domain. The midpoint redox potential of the fused b-type cytochrome is 91 mV vs. SHE at pH 6.5 and the specific current obtained on a porous graphite electrode is 2.3 µA cm-2. The high current obtained on this simple, unmodified electrode at a rather low redox potential is a promising starting point for further optimization. The high yield of NcCYT-GcGDH and its high specific activity supports the application of the chimeric enzyme in bioelectrocatalytic applications.

A promoter engineering-based strategy enhances polyhydroxyalkanoate production in Pseudomonas putida KT2440.

Polyhydroxyalkanoate (PHA), a class of biopolyester synthesized by various bacteria, is considered as an alternative to petroleum-based plastics because of its excellent physochemical and material properties. Pseudomonas putida KT2440 can produce medium-chain-length PHA (mcl-PHA) from glucose, fatty acid and glycerol, and its whole-genome sequences and cellular metabolic networks have been intensively researched. In this study, we aim to improve the PHA yield of P. putida KT2440 using a novel promoter engineering-based strategy. Unlike previous studies, endogenous strong promoters screening from P. putida KT2440 instead of synthetic or exogenous promoters was applied to the optimization of PHA biosynthesis pathway. Based on RNA-seq and promoter prediction, 30 putative strong promoters from P. putida KT2440 were identified. Subsequently, the strengths of these promoters were characterized by reporter gene assays. Furthermore, each of 10 strong promoters screened by transcriptional level and GFP fluorescence was independently inserted into upstream of PHA synthase gene (phaC1) on chromosome. As a result, the transcriptional levels of the phaC1 and phaC2 genes in almost all of the promoter-substituted strains were improved, and the relative PHA yields of the three promoter-substituted strains KTU-P1C1, KTU-P46C1 and KTU-P51C1 were improved obviously, reaching 30.62 wt%, 33.24 wt% and 33.29 wt% [the ratio of PHA weight to cell dry weight (CDW)], respectively. By further deletion of the glucose dehydrogenase gene in KTU-P1C1, KTU-P46C1 and KTU-P51C1, the relative PHA yield of the resulting mutant strain KTU-P46C1-∆gcd increased by 5.29% from 33.24% to 38.53%. Finally, by inserting P46 into upstream of pyruvate dehydrogenase gene in the genome of KTU-P46C1-∆gcd, the relative PHA yield and CDW of the resulting strain KTU-P46C1A-∆gcd reached nearly 42 wt% and 4.06 g/l, respectively, which increased by 90% and 40%, respectively, compared with the starting strain KTU. In particular, the absolute PHA yield of KTU-P46C1A-∆gcd reached 1.7 g/l, with a 165% improvement compared with the strain KTU. Herein, we report the highest PHA yield obtained by P. putida KT2440 in shake-flask fermentation to date. We demonstrate for the first time the effectiveness of endogenous strong promoters for improving the PHA yield and biomass of P. putida KT2440. More importantly, our findings highlight great potential of this strategy for enhanced production of secondary metabolites and heterologous proteins in P. putida KT2440.

Orientated Immobilization of FAD-Dependent Glucose Dehydrogenase on Electrode by Carbohydrate-Binding Module Fusion for Efficient Glucose Assay.

The discovery or engineering of fungus-derived FAD-dependent glucose 1-dehydrogenase (FAD-GDH) is especially important in the fabrication and performance of glucose biosensors. In this study, a novel FAD-GDH gene, phylogenetically distantly with other FAD-GDHs from Aspergillus species, was identified. Additionally, the wild-type GDH enzyme, and its fusion enzyme (GDH-NL-CBM2) with a carbohydrate binding module family 2 (CBM2) tag attached by a natural linker (NL), were successfully heterogeneously expressed. In addition, while the GDH was randomly immobilized on the electrode by conventional methods, the GDH-NL-CBM2 was orientationally immobilized on the nanocellulose-modified electrode by the CBM2 affinity adsorption tag through a simple one-step approach. A comparison of the performance of the two electrodes demonstrated that both electrodes responded linearly to glucose in the range of 0.12 to 40.7 mM with a coefficient of determination R2 > 0.999, but the sensitivity of immobilized GDH-NL-CBM2 (2.1362 × 10-2 A/(M*cm2)) was about 1-fold higher than that of GDH (1.2067 × 10-2 A/(M*cm2)). Moreover, a lower detection limit (51 µM), better reproducibility (<5%) and stability, and shorter response time (≈18 s) and activation time were observed for the GDH-NL-CBM2-modified electrode. This facile and easy immobilization approach used in the preparation of a GDH biosensor may open up new avenues in the development of high-performance amperometric biosensors.

Site-specifically wired and oriented glucose dehydrogenase fused to a minimal cytochrome with high glucose sensing sensitivity.

Direct electron transfer based enzymatic biosensors are highly efficient systems where electrons are transferred directly from the enzyme's electroactive site to the electrode. One way of achieving it is by 'wiring' the enzyme to the electrode surface. The wiring of enzymes to electrode surfaces can be reached in many different ways but controlling its orientation towards the electrode surface is still a challenge. In this study we have designed a Flavin-adenine dinucleotide dependent glucose dehydrogenase that is fused to a minimal cytochrome with a site-specifically incorporated unnatural amino acid to control its orientation towards the electrode. Several site-specifically wired mutant enzymes were compared to each other and to a non-specifically wired enzyme using atomic force microscopy and electrochemical techniques. The surface and activity analyses suggest that the site-specific wiring through different sites maintains the correct folding of the enzyme and have a positive effect on the apparent electrochemical electron transfer rate constant kETapp. Electrochemical analysis revealed an efficient electron transfer rate with more than 15 times higher imax and 10-fold higher sensitivity of the site-specifically wired enzyme variants compared to the non-specifically wired ones. This approach can be utilized to control the orientation of other redox enzymes on electrodes to allow a significant improvement of their electron transfer communication with electrodes.

The wheat growth-promoting traits of Ochrobactrum and Pantoea species, responsible for solubilization of different P sources, are ensured by genes encoding enzymes of multiple P-releasing pathways.

Production and release of organic acids and phosphatase enzymes by microbes are important for inorganic and organic phosphorus cycling in soil. The presence of microorganisms with corresponding traits in the plant rhizosphere lead to improved plant P uptake and ultimately growth promotion. We studied the potential of two rhizosphere-competent strains, Pantoea sp. MR1 and Ochrobactrum sp. SSR, for solubilization of different organic and inorganic P sources in vitro. In a pot experiment we further revealed the impact of the two strains on wheat seedling performance in soil amended with either phytate, rock phosphate or K2HPO4 as solely P source. To directly link P-solubilizing activity to the strain-specific genetic potential, we designed novel primers for glucose dehydrogenase (gcd), phosphatase (pho) and phytase (phy) genes, which are related to the organic and inorganic P solubilization potential. Quantitative tracing of these functional genes in the inoculated soils of the conducted pot experiment further allowed to compare strain abundances in the soil in dependency on the present P source. We observed strain- and P source-dependent patterns of the P solubilization in vitro as well as in the pot experiment, whereby P release, particularly from phytate, was linked to the strain abundance. We further revealed that the activity of microbial phosphatases is determined by the interplay between functional gene abundance, available soil P, and substrate availability. Moreover, positive impacts of microbial seed inoculation on wheat root architecture and aboveground growth parameters were observed. Our results suggest that screening for rhizosphere-competent strains with gcd, pho and phy genes may help to identify new microbial taxa that are able to solubilize and mineralize inorganic as well as organic bound P. Subsequently, the targeted use of corresponding strains may improve P availability in agricultural soils and consequently reduce fertilizer application.

Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine.

BACKGROUND: Biosynthesis of L-tert-leucine (L-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of L-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. RESULTS: In this work, a novel fusion enzyme (GDH-R3-LeuDH) for the efficient biosynthesis of L-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH-R3-LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of L-tle by GDH-R3-LeuDH was all enhanced by twofold. Finally, the space-time yield of L-tle catalyzing by GDH-R3-LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). CONCLUSIONS: It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize L-tle and reach the highest space-time yield up to now. These results demonstrated the great potential of the GDH-R3-LeuDH fusion enzyme for the efficient biosynthesis of L-tle.

Efficient production of an ezetimibe intermediate using carbonyl reductase coupled with glucose dehydrogenase.

Ezetimibe is a top-selling hypolipidemic drug for the treatment of cardiovascular diseases. Biosynthesis of (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl]-4-phenyl-1,3-oxazolidin-2-one ((S)-ET-5) using carbonyl reductase has shown advantages including high catalytic efficiency, excellent stereoselectivity, mild reaction conditions, and environmental friendness, and was considered as the key step for ezetimibe production. The regeneration efficiency of the cofactor, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) is one of the main restricted factor. Recombinant Escherichia coli strain (smCR125) coexpressing carbonyl reductase (CR125) and glucose dehydrogenase were successfully constructed and applied for the production of (S)-ET-5 for the first time. Without extra addition of the coenzyme NADPH, the yield of 99.8% and the enantiomeric excess (e.e.) of 99.9% were achieved under ET-4 concentration of 200 g/L. Using a substrate fed-batch strategy, under the optimal conditions, the substrate ET-4 concentration was increased to 250 g/L with the yield of 98.9% and the e.e. of 99.9% after 12 hr reaction. The space-time yield of 494.5 g L-1 d-1 and the space-time yield per gram biocatalyst of 24.7 g L-1 d-1 g-1 DCW were achieved, which were higher than ever reported for the biosynthesis of the ezetimibe intermediate.

Identification of a novel type of glucose dehydrogenase involved in the mineral weathering ability of Collimonas pratensis strain PMB3(1).

The exact molecular mechanisms as well as the genes involved in the mineral weathering (MW) process by bacteria remain poorly characterized. To date, a single type of glucose dehydrogenase (GDH) depending on a particular co-factor named pyrroloquinoline quinone (PQQ) is known. These enzymes allow the production of gluconic acid through the oxidation of glucose. However, it remains to be determined how bacteria missing PQQ-dependent GDH and/or the related pqq biogenesis genes weather minerals. In this study, we considered the very effective mineral weathering bacterial strain PMB3(1) of Collimonas pratensis. Genome analysis revealed that it does not possess the PQQ-based system. The use of random mutagenesis, gene complementation and functional assays allowed us to identify mutants impacted in their ability to weather mineral. Among them, three mutants were strongly altered on their acidification and biotite weathering abilities (58% to 75% of reduction compared to WT) and did not produce gluconic acid. The characterization of the genomic regions allowed noticeably to the identification of a Glucose/Methanol/Choline oxidoreductase. This region appeared very conserved among collimonads and related genera. This study represents the first demonstration of the implication of a PQQ-independent GDH in the mineral weathering process and explains how Collimonas weather minerals.

Semi-Continuous Flow Biocatalysis with Affinity Co-Immobilized Ketoreductase and Glucose Dehydrogenase.

The co-immobilization of ketoreductase (KRED) and glucose dehydrogenase (GDH) on highly cross-linked agarose (sepharose) was studied. Immobilization of these two enzymes was performed via affinity interaction between His-tagged enzymes (six histidine residues on the N-terminus of the protein) and agarose matrix charged with nickel (Ni2+ ions). Immobilized enzymes were applied in a semicontinuous flow reactor to convert the model substrate; α-hydroxy ketone. A series of biotransformation reactions with a substrate conversion of >95% were performed. Immobilization reduced the requirement for cofactor (NADP+) and allowed the use of higher substrate concentration in comparison with free enzymes. The immobilized system was also tested on bulky ketones and a significant enhancement in comparison with free enzymes was achieved.

Creation of a novel DET type FAD glucose dehydrogenase harboring Escherichia coli derived cytochrome b562 as an electron transfer domain.

Fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) are the most popular and advanced enzymes for SMBG sensors because of their high substrate specificity toward glucose and oxygen insensitivity. However, this type of FADGDH hardly shows direct electron transfer (DET) ability. In this study, we developed a new DET-type FADGDH by harboring Cytochrome b562 (cyt b562) derived from Escherichia coli as the electron transfer domain. The structural genes encoding fusion enzymes composed of cyt b562 at either the N- or C-terminus of fungal FADGDH, (cyt b562-GDH or GDH-cyt b562), were constructed, recombinantly expressed, and characteristics of the fusion proteins were investigated. Both constructed fusion enzymes were successfully expressed in E. coli, as the soluble and GDH active proteins, showing cyt b562 specific redox properties. Thusconstructed fusion proteins showed internal electron transfer between FAD in FADGDH and fused cyt b562. Consequently, both cyt b562-GDH and GDH-cyt b562 showed DET abilities toward electrode. Interestingly, cyt b562-GDH showed much rapid internal electron transfer and higher DET ability than GDH-cyt b562. Thus, we demonstrated the construction and production of a new DET-type FADGDH using E.coli as the host cells, which is advantageous for future industrial application and further engineering.

Biosensing and electrochemical properties of flavin adenine dinucleotide (FAD)-Dependent glucose dehydrogenase (GDH) fused to a gold binding peptide.

In the present work, direct electron transfer (DET) based biosensing system for the determination of glucose has been fabricated by utilizing gold binding peptide (GBP) fused flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) from Burkholderia cepacia. The GBP fused FAD-GDH was immobilized on the working electrode surface of screen-printed electrode (SPE) which consists of gold working electrode, a silver pseudo-reference electrode and a platinum counter electrode, to develop the biosensing system with compact design and favorable sensing ability. The bioelectrochemical and mechanical properties of GBP fused FAD-GDH (GDH-GBP) immobilized SPE (GDH-GBP/Au) were investigated. Here, the binding affinity of GDH-GBP on Au surface, was highly increased after fusion of gold binding peptide and its uniform monolayer was formed on Au surface. In the cyclic voltammetry (CV), GDH-GBP/Au displayed significantly high oxidative peak currents corresponding to glucose oxidation which is almost c.a. 10-fold enhanced value compared with that from native GDH immobilized SPE (GDH/Au). As well, GDH-GBP/Au has shown 92.37% of current retention after successive potential scans. In the chronoamperometry, its steady-state catalytic current was monitored in various conditions. The dynamic range of GDH-GBP/Au was shown to be 3-30 mM at 30 °C and exhibits high selectivity toward glucose in whole human blood. Additionally, temperature dependency of GDH-GBP/Au on DET capability was also investigated at 30-70 °C. Considering this efficient and stable glucose sensing with simple and easy sensor fabrication, GDH-GBP based sensing platform can provide new insight for future biosensor in research fields that rely on DET.

A convenient and rapid method for detecting d-glucose in honey used smartphone.

Information concerning food composition, including information on its glucose content, is essential for modern food industry due to greater consumer awareness and expectations. In this work, the gene encoding d-glucose dehydrogenase (GDH) from Bacillus Natto was expressed in Escherichia coli BL21(DE3) firstly. Ni-IDA column was used for the purification of GDH. Then, the purified GDH was used to construct a color system with stable and effective measurement of concentration of d-glucose. The smart phone photographing and the software Microsoft Photoshop have been used in the system for determination of the color. The enzymatic analysis system can detect the concentration of d-glucose from 5 mM to 40 mM, and other various sugars has no interference to the system. The system was used to quantitatively detect the concentration of d-glucose in honey. The system can be used for convenient and rapid detection of d-glucose in food, especially for large numbers of samples.

Multicopy Chromosomal Integration Using CRISPR-Associated Transposases.

Controlling the copy number of gene expression cassettes is an important strategy to engineer bacterial cells into high-efficiency biocatalysts. Current strategies mostly use plasmid vectors, but multicopy plasmids are often genetically unstable, and their copy numbers cannot be precisely controlled. The integration of expression cassettes into a bacterial chromosome has advantages, but iterative integration is laborious, and it is challenging to obtain a library with varied gene doses for phenotype characterization. Here, we demonstrated that multicopy chromosomal integration using CRISPR-associated transposases (MUCICAT) can be achieved by designing a crRNA to target multicopy loci or a crRNA array to target multiple loci in the Escherichia coli genome. Within 5 days without selection pressure, E. coli strains carrying cargos with successively increasing copy numbers (up to 10) were obtained. Recombinant MUCICAT E. coli containing genomic multicopy glucose dehydrogenase expression cassettes showed 2.6-fold increased expression of this important industrial enzyme compared to E. coli harboring the conventional protein-expressing plasmid pET24a. Successful extension of MUCICAT to Tatumella citrea further demonstrated that MUCICAT may be generally applied to many bacterial species.

Caged Activators of Artificial Allosteric Protein Biosensors.

The ability of proteins to interconvert unrelated biochemical inputs and outputs underlays most energy and information processing in biology. A common conversion mechanism involves a conformational change of a protein receptor in response to a ligand binding or a covalent modification, leading to allosteric activity modulation of the effector domain. Designing such systems rationally is a central goal of synthetic biology and protein engineering. A two-component sensory system based on the scaffolding of modules in the presence of an analyte is one of the most generalizable biosensor architectures. An inherent problem of such systems is dependence of the response on the absolute and relative concentrations of the components. Here we use the example of two-component sensory systems based on calmodulin-operated synthetic switches to analyze and address this issue. We constructed "caged" versions of the activating domain thereby creating a thermodynamic barrier for spontaneous activation of the system. We demonstrate that the caged biosensor architectures could operate at concentrations spanning 3 orders of magnitude and are applicable to electrochemical, luminescent, and fluorescent two-component biosensors. We analyzed the activation kinetics of the caged biosensors and determined that the core allosteric switch is likely to be the rate limiting component of the system. These findings provide guidance for predictable engineering of robust sensory systems with inputs and outputs of choice.

Evolution of Glucose Dehydrogenase for Cofactor Regeneration in Bioredox Processes with Denaturing Agents.

Glucose dehydrogenase (GDH) is a general tool for driving nicotinamide (NAD(P)H) regeneration in synthetic biochemistry. An increasing number of synthetic bioreactions are carried out in media containing high amounts of organic cosolvents or hydrophobic substrates/products, which often denature native enzymes, including those for cofactor regeneration. In this work, we attempted to improve the chemical stability of Bacillus megaterium GDH (BmGDHM0 ) in the presence of large amounts of 1-phenylethanol by directed evolution. Among the resulting mutants, BmGDHM6 (Q252L/E170K/S100P/K166R/V72I/K137R) exhibited a 9.2-fold increase in tolerance against 10 % (v/v) 1-phenylethanol. Moreover, BmGDHM6 was also more stable than BmGDHM0 when exposed to hydrophobic and enzyme-inactivating compounds such as acetophenone, ethyl 2-oxo-4-phenylbutyrate, and ethyl (R)-2-hydroxy-4-phenylbutyrate. Coupled with a Candida glabrata carbonyl reductase, BmGDHM6 was successfully used for the asymmetric reduction of deactivating ethyl 2-oxo-4-phenylbutyrate with total turnover number of 1800 for the nicotinamide cofactor, thus making it attractive for commercial application. Overall, the evolution of chemically robust GDH facilitates its wider use as a general tool for NAD(P)H regeneration in biocatalysis.