Asymmetric whole-cell bioreduction of ( R )-carvone by recombinant Escherichia coli with in situ substrate supply and product removal

Castiglione, Kathrin; Fu, Yilei; Polte, Ingmar; Leupold, Simeon; Meo, Andrea; Weuster‐Botz, Dirk

doi:10.1016/j.bej.2016.10.002

Cited by 34 publications

(42 citation statements)

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“…FDHs from Mycobacterium 12 , Saccharomyces 9 , and Candida 8,40 have been previously engineered to produce NADPH. Of these, MvaFDH 4M displayed one of the highest catalytic efficiency (Table S1), and has been used to regenerate NADPH in cellular and cell-free systems 41,42 . On the other hand, most mutagenesis data are available for PseFDH 5 , including residues involved in cofactor specificity (A198G, D221Q, R222, H223) as well as in maintaining activity and stability (C255) 21 .…”

Section: Molecular Basis Of Psefdh V9 Specificity Towards Nadp +mentioning

confidence: 99%

In vivo selection for formate dehydrogenases with high efficiency and specificity towards NADP⁺

Ramirez

Calvó-Tusell

Stoffel

et al. 2020

Preprint

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CGAR) 2 A b s t r a c t Efficient regeneration of cofactors is vital for the establishment of continuous biocatalytic processes.Formate is an ideal electron donor for cofactor regeneration due to its general availability, low reduction potential, and benign byproduct (CO 2 ). However, formate dehydrogenases (FDHs) are usual specific to NAD + , such that NADPH regeneration with formate is challenging. Previous studies reported naturally occurring FDHs or engineered FDHs that accept NADP + , but these enzymes show low kinetic efficiencies and specificities. Here, we harness the power of natural selection to engineer FDH variants to simultaneously optimize three properties: kinetic efficiency with NADP + , specificity towards NADP + , and affinity towards formate. By simultaneously mutating multiple residues of FDH from Pseudomonas sp. 101, which exhibits no initial activity towards NADP + , we generate a library of >10 6 variants. We introduce this library into an E. coli strain that cannot produce NADPH. By selecting for growth with formate as sole NADPH source, we isolate several enzyme variants that support efficient NADPH regeneration. We find that the kinetically superior enzyme variant, harboring five mutations, has 5-fold higher efficiency and 13-fold higher specificity than the best enzyme previously engineered, while retaining high affinity towards formate.By using molecular dynamics simulations, we reveal the contribution of each mutation to the superior kinetics of this variant. We further determine how non-additive epistatic effects improve multiple parameters simultaneously. Our work demonstrates the capacity of in vivo selection to identify superior enzyme variants carrying multiple mutations which would be almost impossible to find using conventional screening methods.3 I n t r o d u c t i o n Co-factor regeneration is vital for the continuous operation of biocatalytic processes taking place either within a living cell or in a cell free system 1 . A considerable amount of research has therefore been invested in developing and optimizing enzymatic systems for the in situ regeneration of key cofactors such as ATP, NADH, and NADPH 2,3 . Formate has been long considered as a suitable reducing agent for the regeneration of NADH both in vivo and in vitro 2,4 . This is due to several properties: (i) abundance of formate dehydrogenases (FDHs) that can efficiently transfer reducing power from formate to NAD + 5 ; (ii) formate oxidation is practically irreversible, increasing the efficiency of NADH regeneration; (iii) formate, a small molecule, can easily cross membranes, thus being accessible within cellular compartments; and (iv) the byproduct of formate oxidation, CO 2 , is non-toxic and can be easily expelled from the system.Many biocatalytic processes rely on NADPH rather than NADH 6, 7 . Therefore, in the last 20 years multiple studies aimed at identifying FDHs that can naturally accept NADP + or engineering NAD-dependent FDHs to accept the phosphorylated cofactor [8][9][10][11][12][13][14][15][16] . Whi...

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Section: Molecular Basis Of Psefdh V9 Specificity Towards Nadp +mentioning

confidence: 99%

In vivo selection for formate dehydrogenases with high efficiency and specificity towards NADP⁺

Ramirez

Calvó-Tusell

Stoffel

et al. 2020

Preprint

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“…Furthermore, in order to reduce toxicity on the biocatalyst and to overcome these low substrate solubilities, biocatalytic transformations on a laboratory or industrial scale are often conducted in aqueous biphasic systems. [37] Therefore, only catalytic activities using low substrate concentrations are of interest. The specific activities for the WT enzymes and all hybrids with an increased NADH activity are shown in Figure 3.…”

Section: Enhanced Dihydrocarvone Synthesismentioning

confidence: 99%

Loop Swapping as a Potent Approach to Increase Ene Reductase Activity with Nicotinamide Adenine Dinucleotide (NADH)

et al. 2019

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The asymmetric reduction of alkenes is a widely used transformation in industry. Ene reductases (ERs) are (βα) 8 -barrel folded enzymes capable of catalyzing this hydrogenation reaction. At the expense of nicotinamide coenzymes, ERs can reduce a wide range of electron-deficient alkenes in an anti-specific manner and with high regio-and stereoselectivities. However, a cost-effective industrial use of these enzymes is hampered, since most ERs prefer nicotinamide adenine dinucleotide phosphate (NADPH) to the more stable and less expensive non-phosphorylated nicotinamide adenine dinucleotide (NADH) as coenzyme. Here, we demonstrate an approach to both modify the biocatalysts coenzyme selectivity and strongly increase the activity and affinity with NADH. By swapping loop regions of the cyanobacterial NostocER1 for the corresponding regions of two NADH-favoring ERs, a strong alteration of the biocatalyst's coenzyme binding was achieved. This made possible a transfer of the respective donor-ER kinetic parameters to NostocER1. Additionally, outperformance of both donors in terms of activity was achieved through combinatorial swapping of loops of both species. These findings demonstrate the high potential of loop swapping as protein engineering approach to selectively optimize the coenzyme binding of ERs.

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“…The monoterpenoid (2 R ,5 R )‐dihydrocarvone serves as a chiral building block for the synthesis of natural products such as sesquiterpenes or striatenic acid, copolymers, and antimalarial drugs . It is naturally present in dill oil and caraway seeds, but cannot be obtained through extraction .…”

Section: Introductionmentioning

confidence: 99%

“…On a large scale, it is produced either by carvone hydrogenation or by limonene oxidation . Because of its industrial relevance, its production through several strategies has been investigated . Among those strategies, the use of biocatalysts has been described, and here the ene‐reductases (ERs) acting on ( R )‐carvone are preferred (Scheme ) .…”

Section: Introductionmentioning

confidence: 99%

“…Despite the catalytic power and selectivity of those enzymes, the employment of ERs as biocatalysts poses several challenges that need to be addressed . Firstly, these enzymes require reducing power that is naturally obtained from NAD(P)H. This cofactor can be recycled in situ in whole‐cell and cell‐free systems or can be replaced by nicotinamide cofactor mimics in cell‐free systems . Secondly, in this case the substrate and product (carvone and dihydrocarvone) are poorly soluble in water and so cosolvents are necessary and need to be implemented in the system used .…”

Section: Introductionmentioning

confidence: 99%

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Asymmetric Reduction of (R)‐Carvone through a Thermostable and Organic‐Solvent‐Tolerant Ene‐Reductase

et al. 2020

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Ene‐reductases allow regio‐ and stereoselective reduction of activated C=C double bonds at the expense of nicotinamide adenine dinucleotide cofactors [NAD(P)H]. Biological NAD(P)H can be replaced by synthetic mimics to facilitate enzyme screening and process optimization. The ene‐reductase FOYE‐1, originating from an acidophilic iron oxidizer, has been described as a promising candidate and is now being explored for applied biocatalysis. Biological and synthetic nicotinamide cofactors were evaluated to fuel FOYE‐1 to produce valuable compounds. A maximum activity of (319.7±3.2) U mg−1 with NADPH or of (206.7±3.4) U mg−1 with 1‐benzyl‐1,4‐dihydronicotinamide (BNAH) for the reduction of N‐methylmaleimide was observed at 30 °C. Notably, BNAH was found to be a promising reductant but exhibits poor solubility in water. Different organic solvents were therefore assayed: FOYE‐1 showed excellent performance in most systems with up to 20 vol% solvent and at temperatures up to 40 °C. Purification and application strategies were evaluated on a small scale to optimize the process. Finally, a 200 mL biotransformation of 750 mg (R)‐carvone afforded 495 mg of (2R,5R)‐dihydrocarvone (>95 % ee), demonstrating the simplicity of handling and application of FOYE‐1.

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Asymmetric whole-cell bioreduction of ( R )-carvone by recombinant Escherichia coli with in situ substrate supply and product removal

Cited by 34 publications

References 50 publications

In vivo selection for formate dehydrogenases with high efficiency and specificity towards NADP⁺

In vivo selection for formate dehydrogenases with high efficiency and specificity towards NADP⁺

Loop Swapping as a Potent Approach to Increase Ene Reductase Activity with Nicotinamide Adenine Dinucleotide (NADH)

Asymmetric Reduction of (R)‐Carvone through a Thermostable and Organic‐Solvent‐Tolerant Ene‐Reductase

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Asymmetric whole-cell bioreduction of ( R )-carvone by recombinant Escherichia coli with in situ substrate supply and product removal

Cited by 34 publications

References 50 publications

In vivo selection for formate dehydrogenases with high efficiency and specificity towards NADP+

In vivo selection for formate dehydrogenases with high efficiency and specificity towards NADP+

Loop Swapping as a Potent Approach to Increase Ene Reductase Activity with Nicotinamide Adenine Dinucleotide (NADH)

Asymmetric Reduction of (R)‐Carvone through a Thermostable and Organic‐Solvent‐Tolerant Ene‐Reductase

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In vivo selection for formate dehydrogenases with high efficiency and specificity towards NADP⁺

In vivo selection for formate dehydrogenases with high efficiency and specificity towards NADP⁺