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[Preprint]. 2023 Aug 30:2023.08.29.555398.
doi: 10.1101/2023.08.29.555398.

Shifting Redox Reaction Equilibria on Demand Using an Orthogonal Redox Cofactor

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Shifting Redox Reaction Equilibria on Demand Using an Orthogonal Redox Cofactor

Derek Aspacio et al. bioRxiv. .

Update in

Abstract

Natural metabolism relies on chemical compartmentalization of two redox cofactors, NAD+ and NADP+, to orchestrate life-essential redox reaction directions. However, in whole cells the reliance on these canonical cofactors limits flexible control of redox reaction direction as these reactions are permanently tied to catabolism or anabolism. In cell-free systems, NADP+ is too expensive in large scale. We have previously reported the use of nicotinamide mononucleotide, (NMN+) as a low-cost, noncanonical redox cofactor capable of specific electron delivery to diverse chemistries. Here, we present Nox Ortho, an NMNH-specific water-forming oxidase, that completes the toolkit to modulate NMNH/NMN+ ratio. This work uncovers an enzyme design principle that succeeds in parallel engineering of six butanediol dehydrogenases as NMN(H)-orthogonal biocatalysts consistently with a 103 - 106 -fold cofactor specificity switch from NAD(P)+ to NMN+. We combine these to produce chiral-pure 2,3-butanediol (Bdo) isomers without interference from NAD(H) or NADP(H) in vitro and in E. coli cells. We establish that NMN(H) can be held at a distinct redox ratio on demand, decoupled from both NAD(H) and NADP(H) redox ratios in vitro and in vivo.

Keywords: Escherichia coli; Nicotinamide mononucleotide; biomimetic cofactor; butanediol; cell-free biomanufacturing; noncanonical redox cofactor; water-forming NADH oxidase (NOX).

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.. Overview of the four proposed Bdo stereo-upgrading systems for testing two orthogonal cofactor’s crosstalk.
(A, C) Stereo-upgrading systems of m-Bdo to SS-Bdo where NAD(H) or NADP(H) drive the oxidation step and NMN(H) drives the reduction step. (B, D) Stereo-upgrading systems of m-Bdo to RR-Bdo where NMN(H) drives the oxidation step and NAD(H) or NADP(H) drives the reduction step. (E) Illustration of when crosstalk between the oxidation and reduction steps occurs, reactions are reversible and incomplete. The four systems above cannot be achieved with a single cofactor or multiple rapidly cross talking cofactors. Red and blue signify oxidation and reduction respectively. The two-color cofactor pool boxes represent rapid crosstalk and fully reversible reaction steps. (F) The typical stereospecificity and chiral substrate preference of Bdhs from different enzyme classes used. (G) Single cofactor reaction of the Bdhs with no cofactor recycling reactions driving each step. (H) Single cofactor reaction with L. brevis Nox WT recycling NADH to NAD+ to drive the oxidation step. (I) Single cofactor reaction with B. subtilis Gdh WT recycling NAD+ to NADH to drive the reduction step. The reaction does not proceed past the first oxidation step. The substrate for all experiments is 5 g/L m-Bdo and conversion is measured after 48 h at 30 °C. Bars represent the mean of three independent replicates with error bars of one standard deviation. White diamonds indicate the values of individual replicates.
Figure 2.
Figure 2.. Directed evolution of Ll Nox to exclude NADH.
(A) Schematic of the high-throughput growth-based selection platform workflow for NMNH-utilizing enzymes. The designed site saturated mutagenesis library was introduced to the engineered E. coli strain, MX502 where growth depends upon Nox NMNH-oxidase activity. Fast growing variants were characterized by specific activity assay. (B) The apparent catalytic efficiencies of Ll Nox WT (grey) and Nox Ortho (yellow) towards NADH, NADPH and NMNH. Assay at 37 °C in 50 mM Tris-Cl, pH 7.0 with varying reduced cofactor concentration. (C) Model of Nox Ortho binding pose with NMNH and FAD revealed novel hydrogen bond formation. (D) Ll Nox WT (grey) and Nox Ortho (yellow) with NADH bound revealed a conformational change in NADH binding pose. Mutations on Nox Ortho exclude NADH from its native binding mode to become exposed to solvent, consistent with the decreased catalytic efficiency for NADH observed. Data are presented as the mean of three replicates (n=3) ± one standard deviation.
Figure 3.
Figure 3.. Engineering Bdhs to utilize NMN(H).
(A) Kp m-Bdh Ortho predicted interactions with NMN+. (B) Apparent catalytic efficiencies of Kp m-Bdh WT (grey) and Kp m-Bdh Ortho (green) with each cofactor. The substrate was 50 mM m-Bdo with varying cofactor concentration. (C) Specific activities of six Bdh homologs based on analogous mutation transfer from Kp m-Bdh Ortho (Kp m-Bdh sites). Summary of measured chiral substrate and product preference in R/S-Ac feeding experiments (Fig S4). n.t denotes not tested. The substrate was 10 mM R/S-Ac with 0.2 mM reduced cofactor. (D) Ser S-Bdh Ortho predicted interactions with NMN+. (E) Apparent catalytic efficiencies of Ser S-Bdh WT (grey) and Ser S-Bdh Ortho (purple) with each cofactor. Substrate for Ser S-Bdh assays was 50 mM SS-Bdo with varying cofactor concentration. Bars are the average of at least three independent replicates with error bars of one standard deviation. Individual replicates are shown as white diamonds. All activity assays of Bdhs conducted at 30 °C in 50 mM Tris-Cl at pH 8.0.
Figure 4.
Figure 4.. Orthogonal redox driving forces enable Bdo stereo-upgrading in four cell-free systems.
(A-C) m-Bdo to SS-Bdo system that pairs an NAD(H) driven oxidation step and NMN(H) driven reduction step. (D-F) m-Bdo to RR-Bdo system that pairs an NMN(H) driven oxidation step and NAD(H) driven reduction step. (G-I) m-Bdo to SS-Bdo system that pairs an NADP(H) driven oxidation step and NMN(H) driven reduction step. (J-L) m-Bdo to RR-Bdo system that pairs an NMN(H) driven oxidation step and NADP(H) driven reduction step. (A, D, G, J) Reaction pathway maps. (B, E, H, K) Concentration of Bdo and Ac isomers. (C, F, I, L) Concentration ratio of each redox cofactors’ cognate reduced and oxidized species on a log10 scale. Bars represent the average of three independent replicates with error bars calculated by propagation of error from the standard deviation of oxidized and total cofactor concentration measurements. White diamonds with black outline represent the values of individual replicates. Grey outlined diamonds represent replicates whose reduced cofactor concentration is below limit of quantification (L.o.Q., see Methods). Ortho represents the cofactor engineered variants. Gluc, gluconic acid. All reactions incubated shaking for 24 h or 72 h at 30 °C for SS-Bdo or RR-Bdo production, respectively. Samples for redox ratio measurement were taken from the same reactions at the times listed in Methods.
Figure 5.
Figure 5.. Resting cell stereo-upgrading of m-Bdo to SS-Bdo in E. coli.
(A) Gene deletions and reaction pathways in the whole cell chassis, strain MX 102 R0. (B-D) m-Bdo to SS-Bdo system using NAD(H) driven oxidation step paired with an NMN(H) driven reduction step in vivo. (B) Reaction pathway details of the NAD(H) driven oxidation step. (C) Concentration of Bdo and Ac stereoisomers when 0 mM or 10 mM NMN+ is supplemented to the media, sampled after 24 h at 30 °C. (D) Product purity of SS-Bdo in different NMN+ supplementation conditions. (E-G) m-Bdo to SS-Bdo system using NADP(H) driven oxidation step paired with an NMN(H) driven reduction step in vivo. (E) Reaction pathway details of the NADP(H) driven oxidation step. (F) Concentration of Bdo and Ac stereoisomers when 0 mM or 10 mM NMN+ is supplemented to the media, sampled after 152 h at 18 °C. (G) Product purity of SS-Bdo in different NMN+ supplementation conditions. Bars represent an average of three biological replicates with error bars of one standard deviation. White diamonds represent the values of individual replicates. Product purity calculated as the percentage of SS-Bdo in the total amount of products formed (R-Ac, S-Ac, RR-Bdo, SS-Bdo, m-Bdo). Ortho represents the cofactor engineered variants. Gluc, gluconic acid; 6-P-Gluc, 6-phosphogluconate; Glucose-6P, glucose-6-phosphate; NaMN, nicotinic acid mononucleotide.

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