Biology subjects

Black, W. B.

Publications and source records attributed to Black, W. B..

6 recordsLinked to original sources

bioRxiv2025

Reprogramming the Rossmann Fold Signature Motif Creates Orthogonal Redox Biocatalysts

Biological reducing power is carried by nicotinamide adenine dinucleotide (phosphate) (NAD(P)/H), which supports cellular functions and cannot be specifically directed to engineered metabolic pathways. Nicotinamide mononucleotide (NMN(H)) has emerged as an orthogonal redox cofactor to address this challenge, yet strategies remain elusive for creating NMN(H)-specific enzymes that no longer interact with the cellular NAD(P)/H pools. Previous designs avoided perturbing the most ancient and conserved GxGxxG motif in Rossmann fold enzymes, the root cause of persistent NAD(P)/H recognition. Herein, we demonstrated that this motif, though long considered essential, is in fact mutable to yield active NMN+-specific enzymes. This is implemented on two unrelated model enzymes chosen to garner orthogonal reducing power from either the cheap electron source phosphite or glycolysis. On phosphite dehydrogenase (PTDH), variants NRC-01 and NRC-02 eliminated electron leaking to numerous NAD(P)H-dependent side reactions in whole cells and crude cell lysates while driving NMNH-dependent biotransformation with [~]240-fold higher productivity than existing catalysts. On glyceraldehyde-3-phosphate dehydrogenase (GapA), variant RSQ featured a [~]2.9x104-fold cofactor specificity switch to NMN+ from NAD+. Combined Rosetta modeling, systematic structural alignment, and experimental results revealed that Rossmann fold reprogramming, paired with engineered structural reinforcement, may offer a general route to orthogonal redox biocatalysts.

molecular biology↗

bioRxiv2025

A Sequence Motif Enables Widespread Use of Non-Canonical Redox Cofactors in Natural Enzymes

Non-canonical redox cofactors (NRCs) are promising alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) for biomanufacturing due to low cost and exquisite electron delivery control, yet their adoption is limited by the scarcity of compatible enzymes. Here, we screened the aldehyde dehydrogenase (ALDH) protein family and identified a conserved RH/QxxR sequence motif that enables widespread NRC activity among natural enzymes. Bos taurus ALDH3a1 and Pseudanabaena biceps ALDH exhibit unprecedented turnover with nicotinamide mononucleotide (NMN+), with kcat values matching or exceeding that of NAD+ and surpassing most engineered NRC-active enzymes by 10 to 105-fold, based on the relative NRC to native activity. Structural and dynamic analyses reveal this motif reinforces cofactor positioning and pre-organizes the active site without dependence on the adenosine monophosphate moiety of NAD+. When introduced into diverse ALDH scaffolds, the RH/QxxR motif enhances NMN+ activity up to 60-fold. In addition to NMN+, this motif also supports activity across multiple non-nucleotide, simple synthetic NRCs such as 1-(2-carbamoylmethyl)nicotinamide (AmNA+). These findings elucidate Natures solution to the engineering challenge of obtaining NRC-active enzymes and offers a blueprint to mine latent evolutionary plasticity in natural enzymes that serve as superior engineering starting points.

synthetic biology↗

bioRxiv2025

Activation-Free Upgrading of Carboxylic Acids to Aldehydes and Alcohols

Advances in organic and gas waste valorization have enabled high-yield production of carboxylic acids, positioning them as promising feedstocks for the bioeconomy. However, carboxylic acids must be activated before downstream use, typically requiring ATP, CoA, or reduced ferredoxin to overcome unfavorable thermodynamics. These activators are costly to generate and divert carboxylic acids into CO2-releasing pathways, reducing carbon efficiency. Here, we demonstrate that aldehyde dehydrogenases (ALDHs) can directly reduce carboxylic acids to aldehydes without prior activation, a process previously thought to be biologically inaccessible. Screening 133 ALDHs revealed that this activity is remarkably widespread within the protein family, enabling production of aliphatic aldehydes and alcohols, diols, and aromatic alcohols, at titers >1 g/L, in some cases, after optimization of thermodynamic driving forces. Additionally, we applied this system to upgrade waste-derived carboxylic acid effluent streams from wastewater sludge, food waste, and waste gas (CO2). This activation-free process, termed "reverse aldehyde oxidation" (rAOX), establishes a broadly applicable, energy-efficient platform for carboxylic acid valorization at 100% carbon yield. Analogous to the reverse tricarboxylic acid cycle (rTCA) and reverse {beta}-oxidation (rBOX), rAOX exemplifies that metabolic reactions classically defined as unidirectional may have unexpected plasticity to operate in reverse and open new avenues in biomanufacturing.

synthetic biology↗

bioRxiv2023

Shifting Redox Reaction Equilibria on Demand Using an Orthogonal Redox Cofactor

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.

synthetic biology↗

bioRxiv2020

Metabolic engineering of Escherichia coli for optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactor

Background Noncanonical redox cofactors are emerging as important tools in cell-free biosynthesis to increase the economic viability, to enable exquisite control, and to expand the range of chemistries accessible. However, these noncanonical redox cofactors need to be biologically synthesized to achieve full integration with renewable biomanufacturing processes.Results In this work, we engineered Escherichia coli cells to biosynthesize the noncanonical cofactor nicotinamide mononucleotide (NMN+), which has been efficiently used in cell-free biosynthesis. First, we developed a growth-based screening platform to identify effective NMN+ biosynthetic pathways in E. coli. Second, we explored various pathway combinations and host gene disruption to achieve an intracellular level of ~1.5 mM NMN+, a 130-fold increase over the cell’s basal level, in the best strain, which features a previously uncharacterized nicotinamide phosphoribosyltransferase (NadV) from Ralstonia solanacearum. Last, we revealed mechanisms through which NMN+ accumulation impacts E. coli cell fitness, which sheds light on future work aiming to improve the production of this noncanonical redox cofactor.Conclusion These results further the understanding of effective production and integration of NMN+ into E. coli. This may enable the implementation of NMN+-directed biocatalysis without the need for exogenous cofactor supply.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsNAD+nicotinamide adenine dinucleotideNADP+nicotinamide adenine dinucleotide phosphateP2NA+3-carbomoyl-1-phenethylpyridin-1-ium chlorideNMN+nicotinamide mononucleotideNadVnicotinamide phosphoribosyltransferasesNadE*nicotinamide mononucleotide synthasePncCnicotinamide mononucleotideNaMN+nicotinic acid mononucleotideNRnicotinamide ribosidePnuCnicotinamide riboside transporterPnuC*mutant nicotinamide riboside transporterNrk1nicotinamide riboside kinase from Saccharomyces cerevisiaeNadRnicotinamide riboside kinase (Salmonella enterica)NAnicotinamideLC-MSliquid chromatography-mass spectrometryNaADnicotinic acid adenine dinucleotidePCRpolymerase chain reactionIPTGisopropyl-β-D-thiogalactopyranosider.p.m.rotations per minuteView Full Text

synthetic biology↗

bioRxiv2020

Nicotinamide mononucleotide redox cofactor system enables aldehyde accumulation in Escherichia coli

It is challenging to biosynthesize industrially important aldehydes, which are readily consumed by the numerous alcohol dehydrogenases (ADHs) in cells. In this work, we demonstrate that a nicotinamide mononucleotide (NMN+)-dependent redox cofactor cycling system enables aldehyde accumulation in Escherichia coli crude lysates and whole cells. By specifically delivering reducing power to a recombinant enoate reductase, but not to endogenous ADHs, we convert citral to citronellal with minimal byproduct formation (98% and 83% product purity in crude lysate- and whole cell-based biotransformation, respectively). We envision the systems universal application to lower the noise in biomanufacturing by silencing the hosts metabolic background.

bioengineering↗