Shifting redox reaction equilibria on demand using an orthogonal redox cofactor.

Nature's two redox cofactors, nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), are held at different reduction potentials, driving catabolism and anabolism in opposite directions. In biomanufacturing, there is a need to flexibly control redox reaction direction decoupled from catabolism and anabolism. We established nicotinamide mononucleotide (NMN+) as a noncanonical cofactor orthogonal to NAD(P)+. Here we present the development of Nox Ortho, a reduced NMN+ (NMNH)-specific oxidase, that completes the toolkit to modulate NMNH:NMN+ ratio together with an NMN+-specific glucose dehydrogenase (GDH Ortho). The design principle discovered from Nox Ortho engineering and modeling is facilely translated onto six different enzymes to create NMN(H)-orthogonal biocatalysts with a consistent ~103-106-fold cofactor specificity switch from NAD(P)+ to NMN+. We assemble these enzymes to produce stereo-pure 2,3-butanediol in cell-free systems and in Escherichia coli, enabled by NMN(H)'s distinct redox ratio firmly set by its designated driving forces, decoupled from both NAD(H) and NADP(H).

Protein stability prediction by fine-tuning a protein language model on a mega-scale dataset.

Protein stability plays a crucial role in a variety of applications, such as food processing, therapeutics, and the identification of pathogenic mutations. Engineering campaigns commonly seek to improve protein stability, and there is a strong interest in streamlining these processes to enable rapid optimization of highly stabilized proteins with fewer iterations. In this work, we explore utilizing a mega-scale dataset to develop a protein language model optimized for stability prediction. ESMtherm is trained on the folding stability of 528k natural and de novo sequences derived from 461 protein domains and can accommodate deletions, insertions, and multiple-point mutations. We show that a protein language model can be fine-tuned to predict folding stability. ESMtherm performs reasonably on small protein domains and generalizes to sequences distal from the training set. Lastly, we discuss our model's limitations compared to other state-of-the-art methods in generalizing to larger protein scaffolds. Our results highlight the need for large-scale stability measurements on a diverse dataset that mirrors the distribution of sequence lengths commonly observed in nature.

Docking carbocations into terpene synthase active sites using chemically meaningful constraints-The TerDockin approach.

Terpenes are a diverse class of natural products which have long been sought after for their chemical properties as medicine, perfumes, and for food flavoring. Computational docking studies of terpene mechanisms have been a challenge due to the lack of strong directing groups which many docking programs rely on. In this chapter, we dive into our computational method Terdockin (Terpene-Docking) as a successful methodology in modeling terpene synthase mechanisms. This method could also be used as inspiration for any multi-ligand docking project.

Discovery of Potent Glycosidases Enables Quantification of Smoke-Derived Phenolic Glycosides through Enzymatic Hydrolysis.

When grapes are exposed to wildfire smoke, certain smoke-related volatile phenols (VPs) can be absorbed into the fruit, where they can be then converted into volatile-phenol (VP) glycosides through glycosylation. These volatile-phenol glycosides can be particularly problematic from a winemaking standpoint as they can be hydrolyzed, releasing volatile phenols, which can contribute to smoke-related off-flavors. Current methods for quantitating these volatile-phenol glycosides present several challenges, including the requirement of expensive capital equipment, limited accuracy due to the molecular complexity of the glycosides, and the utilization of harsh reagents. To address these challenges, we proposed an enzymatic hydrolysis method enabled by a tailored enzyme cocktail of novel glycosidases discovered through genome mining, and the generated VPs from VP glycosides can be quantitated by gas chromatography-mass spectrometry (GC-MS). The enzyme cocktails displayed high activities and a broad substrate scope when using commercially available VP glycosides as the substrates for testing. When evaluated in an industrially relevant matrix of Cabernet Sauvignon wine and grapes, this enzymatic cocktail consistently achieved a comparable efficacy of acid hydrolysis. The proposed method offers a simple, safe, and affordable option for smoke taint analysis.

Acid-active proteases to optimize dietary protein digestibility: a step towards sustainable nutrition.

Introduction: Historically, prioritizing abundant food production often resulted in overlooking nutrient quality and bioavailability, however, environmental concerns have now propelled sustainable nutrition and health efficacy to the forefront of global attention. In fact, increasing demand for protein is the major challenge facing the food system in the 21st century with an estimation that 70% more food is needed by 2050. This shift has spurred interest in plant-based proteins for their sustainability and health benefits, but most alternative sources of protein are poorly digestible. There are two approaches to solve digestibility: improve the digestibility of food proteins or improve the digestive capacity of consumers. Enhancing nutrient digestibility and bioavailability across diverse protein sources is crucial, with proteases presenting a promising avenue. Research, inspired by the proteases of human breast milk, has demonstrated that exogenous microbial proteases can activate within the human digestive tract and substantially increase the digestion of targeted proteins that are otherwise difficult to fully digest. Methods: Here, we introduce the use of an acid-active family of bacterial proteases (S53) to improve the digestibility and nutritional quality of a variety of protein sources, evaluated using the INFOGEST 2.0 protocol. Results: Results from in vitro digestibility indicate that the most effective protease in the S53 family substantially improves the digestibility of an array of animal and plant-derived proteins-soy, pea, chickpea, rice, casein, and whey. On average, this protease elevated protein digestibility by 115% during the gastric phase and by 15% in the intestinal phase, based on the degree of hydrolysis. Discussion: The widespread adoption of these proteases has the potential to enhance nutritional value and contribute to food security and sustainability. This approach would complement ongoing efforts to improve proteins in the food supply, increase the quality of more sustainable protein sources and aid in the nourishment of patients with clinically compromised, fragile intestines and individuals like older adults and high-performance athletes who have elevated protein needs.

Missing a "Missing Self" Mechanism: Modeling and Detection of Ly49 Expression in Canine NK Cells.

NK cells are a key focus in immuno-oncology, based on their ability to eliminate malignant cells without prior sensitization. Dogs are valuable models for translational immunotherapy studies, especially for NK cells, where critical species differences exist between mice and humans. Given that the mechanism for recognition of "self" by canine NK cells is currently unknown, we sought to evaluate expression of Ly49 in canine NK cells using in silico and high-throughput techniques. We interrogated the identified polymorphism/mutation in canine Ly49 and assessed the potential impact on structure using computational modeling of three-dimensional protein structure and protein-protein docking of canine Ly49 with MHC class I (MHC-I). Bulk and single-cell RNA-sequencing analysis was performed to detect gene expression of Ly49/KLRA1 in resting and activated NK cells. Tertiary protein structure demonstrated significant structural similarity to the known murine system. Molecular docking of canine Ly49 with MHC-I was favorable, converging at a single low-energy conformation. RNA sequencing revealed expression of Ly49/KLRA1 in both resting and activated NK cells and demonstrated almost exclusive expression of the gene in the NK cluster at the single-cell level. Despite prior reports of a mutated, nonfunctional canine Ly49, our data support that the protein product is predicted to bind to MHC-I in a comparable conformation to the murine system and is expressed in canine NK cells with upregulation following activation. Taken together, these data suggest that Ly49 is capable of recognizing MHC-I and therefore regulating NK cell function in dogs.

Awakening the natural capability of psicose production in Escherichia coli.

Due to the rampant rise in obesity and diabetes, consumers are desperately seeking for ways to reduce their sugar intake, but to date there are no options that are both accessible and without sacrifice of palatability. One of the most promising new ingredients in the food system as a non-nutritive sugar substitute with near perfect palatability is D-psicose. D-psicose is currently produced using an in vitro enzymatic isomerization of D-fructose, resulting in low yield and purity, and therefore requiring substantial downstream processing to obtain a high purity product. This has made adoption of D-psicose into products limited and results in significantly higher per unit costs, reducing accessibility to those most in need. Here, we found that Escherichia coli natively possesses a thermodynamically favorable pathway to produce D-psicose from D-glucose through a series of phosphorylation-epimerization-dephosphorylation steps. To increase carbon flux towards D-psicose production, we introduced a series of genetic modifications to pathway enzymes, central carbon metabolism, and competing metabolic pathways. In an attempt to maximize both cellular viability and D-psicose production, we implemented methods for the dynamic regulation of key genes including clustered regularly interspaced short palindromic repeats inhibition (CRISPRi) and stationary-phase promoters. The engineered strains achieved complete consumption of D-glucose and production of D-psicose, at a titer of 15.3 g L-1, productivity of 2 g L-1 h-1, and yield of 62% under test tube conditions. These results demonstrate the viability of whole-cell catalysis as a sustainable alternative to in vitro enzymatic synthesis for the accessible production of D-psicose.

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.

Computational Drug Design of Novel Agonists of the µ -Opioid Receptor to Inhibit Pain Signaling.

Opioids such as Morphine, Codeine, Hydrocodone, and Oxycodone target the µ-opioid receptor, a G-protein-coupled receptor (GPCR), blocking the transmission of nociceptive signals. In this study, four opioids were analyzed for ADMET properties and molecular interactions with a GPCR crystal structure (PDB ID: 8EF6). This aided in the computational design of two novel drug candidates with improved docking scores and ADMET properties when compared to Hydrocodone. Homology analysis indicated that a Mus musculus (house mouse) animal model could be used in the preclinical studies of these drug candidates in the development of safer and more effective opioid drugs for pain management with reduced side effects.

Brewers' Spent Grain: An Unprecedented Opportunity to Develop Sustainable Plant-Based Nutrition Ingredients Addressing Global Malnutrition Challenges.

There is an urgent requirement to minimize food waste and create more sustainable food systems that address global increases in malnutrition and hunger. The nutritional value of brewers' spent grain (BSG) makes it attractive for upcycling into value-added ingredients rich in protein and fiber having a lower environmental impact than comparable plant-based ingredients. BSG is predictably available in large quantities globally and can therefore play a role in addressing hunger in the developing world via the fortification of humanitarian food aid products. Moreover, addition of BSG-derived ingredients can improve the nutritional profile of foods commonly consumed in more developed regions, which may aid in reducing the prevalence of dietary-related disease and mortality. Challenges facing the widespread utilization of upcycled BSG ingredients include regulatory status, variability of raw material composition, and consumer perception as low-value waste products; however, the rapidly growing upcycled food market suggests increasing consumer acceptability and opportunities for significant market growth via effective new product innovation and communication strategies.

Design to Data for mutants of B-glucosidase B from Paenibacillus polymyxa : V311D, F248N, Y166H, Y166K, M221K.

Engaging computational tools for protein design is gaining traction in the enzyme engineering community. However, current design and modeling algorithms have limited functionality predictive capacities for enzymes due to limitations of the dataset in terms of size and data quality. This study aims to expand training datasets for improved algorithm development with the addition of five rationally designed single-point enzyme variants. ß-glucosidase B variants were modeled in Foldit Standalone and then produced and assayed for thermal stability and kinetic parameters. Functional parameters: thermal stability (T M ) and Michaelis-Menten constants ( k cat , K M , and k cat /K M ) of five variants, V311D, Y166H, M221K, F248N, and Y166K, were added into the Design2Data database. As a case study, evaluation of this small mutant set finds mutational effect trends that both corroborate and contradict findings from larger studies examining the entire dataset.

Orthogonal glycolytic pathway enables directed evolution of noncanonical cofactor oxidase.

Noncanonical cofactor biomimetics (NCBs) such as nicotinamide mononucleotide (NMN+) provide enhanced scalability for biomanufacturing. However, engineering enzymes to accept NCBs is difficult. Here, we establish a growth selection platform to evolve enzymes to utilize NMN+-based reducing power. This is based on an orthogonal, NMN+-dependent glycolytic pathway in Escherichia coli which can be coupled to any reciprocal enzyme to recycle the ensuing reduced NMN+. With a throughput of >106 variants per iteration, the growth selection discovers a Lactobacillus pentosus NADH oxidase variant with ~10-fold increase in NMNH catalytic efficiency and enhanced activity for other NCBs. Molecular modeling and experimental validation suggest that instead of directly contacting NCBs, the mutations optimize the enzyme's global conformational dynamics to resemble the WT with the native cofactor bound. Restoring the enzyme's access to catalytically competent conformation states via deep navigation of protein sequence space with high-throughput evolution provides a universal route to engineer NCB-dependent enzymes.

Investigation of Acid-Base Catalysis in Halimadienyl Diphosphate Synthase Involved in Mycobacterium tuberculosis Virulence.

The devastating human pathogenMycobacterium tuberculosis (Mtb) is able to parasitize phagosomal compartments within alveolar macrophage cells due, in part, to the activity of its cell-surface lipids. Prominent among these is 1-tuberculosinyl-adenosine (1-TbAd), a derivative of the diterpenoid tuberculosinyl (halima-5,13-dienyl) diphosphate produced by the class II diterpene cyclase encoded by Rv3377c, termed here MtHPS. Given the demonstrated ability of 1-TbAd to act as a virulence factor for Mtb and the necessity for Rv3377c for its production, there is significant interest in MtHPS activity. Class II diterpene cyclases catalyze a general acid-base-mediated carbocation cascade reaction initiated by protonation of the terminal alkene in the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate and terminated by deprotonation of the final cyclized (and sometimes also rearranged) intermediate. Here, structure-guided mutagenesis was applied to characterize the various residues contributing to activation of the enzymatic acid, as well as identify the enzymatic base in MtHPS. Particularly given the ability of conservative substitution for the enzymatic base (Y479F) to generate an alternative product (labda-7,13-dienyl diphosphate) via deprotonation of an earlier unrearranged intermediate, further mutational analysis was carried out to introduce potential alternative catalytic bases. The results were combined with mechanistic molecular modeling to elucidate how these mutations affect the catalytic activity of this important enzyme. This not only provided detailed structure-function insight into MtHPS but also further emphasized the inert nature of the active site of MtHPS and class II diterpene cyclases more generally.

Structural plasticity enables evolution and innovation of RuBisCO assemblies.

Oligomerization is a core structural feature that defines the form and function of many proteins. Most proteins form molecular complexes; however, there remains a dearth of diversity-driven structural studies investigating the evolutionary trajectory of these assemblies. Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) is one such enzyme that adopts multiple assemblies, although the origins and distribution of its different oligomeric states remain cryptic. Here, we retrace the evolution of ancestral and extant form II RuBisCOs, revealing a complex and diverse history of oligomerization. We structurally characterize a newly discovered tetrameric RuBisCO, elucidating how solvent-exposed surfaces can readily adopt new interactions to interconvert or give rise to new oligomeric states. We further use these principles to engineer and demonstrate how changes in oligomerization can be mediated by relatively few mutations. Our findings yield insight into how structural plasticity may give rise to new oligomeric states.

Directed evolution of phosphite dehydrogenase to cycle noncanonical redox cofactors via universal growth selection platform.

Noncanonical redox cofactors are attractive low-cost alternatives to nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) in biotransformation. However, engineering enzymes to utilize them is challenging. Here, we present a high-throughput directed evolution platform which couples cell growth to the in vivo cycling of a noncanonical cofactor, nicotinamide mononucleotide (NMN+). We achieve this by engineering the life-essential glutathione reductase in Escherichia coli to exclusively rely on the reduced NMN+ (NMNH). Using this system, we develop a phosphite dehydrogenase (PTDH) to cycle NMN+ with ~147-fold improved catalytic efficiency, which translates to an industrially viable total turnover number of ~45,000 in cell-free biotransformation without requiring high cofactor concentrations. Moreover, the PTDH variants also exhibit improved activity with another structurally deviant noncanonical cofactor, 1-benzylnicotinamide (BNA+), showcasing their broad applications. Structural modeling prediction reveals a general design principle where the mutations and the smaller, noncanonical cofactors together mimic the steric interactions of the larger, natural cofactors NAD(P)+.

Foodomics: A Data-Driven Approach to Revolutionize Nutrition and Sustainable Diets.

Globally, we are failing to meet numerous nutritional, health, and environmental targets linked to food. Defining food composition in its full chemical and quantitative diversity is central to data-driven decision making for supporting nutrition and sustainable diets. "Foodomics"-the application of omics-technology to characterize and quantify biomolecules to improve wellbeing-has the potential to comprehensively elucidate what is in food, how this composition varies across the food system, and how diet composition as an ensemble of foods guides outcomes for nutrition, health, and sustainability. Here, we outline: (i) challenges of evaluating food composition; (ii) state-of-the-art omics technology and innovations for the analysis of food; and (iii) application of foodomics as a complementary data-driven approach to revolutionize nutrition and sustainable diets. Featuring efforts of the Periodic Table of Food Initiative, a participatory effort to create a globally shared foodomics platform, we conclude with recommendations to accelerate foodomics in ways that strengthen the capacity of scientists and benefit all people.

The pyruvate decarboxylase activity of IpdC is a limitation for isobutanol production by Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae contains an endogenous isobutanol synthesis pathway. The ipdC gene annotated as an indole-3-pyruvate decarboxylase (Kp-IpdC), was identified to catalyze the formation of isobutyraldehyde from 2-ketoisovalerate. RESULTS: Compared with 2-ketoisovalerate decarboxylase from Lactococcus lactis (KivD), a decarboxylase commonly used in artificial isobutanol synthesis pathways, Kp-IpdC has an 2.8-fold lower Km for 2-ketoisovalerate, leading to higher isobutanol production without induction. However, expression of ipdC by IPTG induction resulted in a low isobutanol titer. In vitro enzymatic reactions showed that Kp-IpdC exhibits promiscuous pyruvate decarboxylase activity, which adversely consume the available pyruvate precursor for isobutanol synthesis. To address this, we have engineered Kp-IpdC to reduce pyruvate decarboxylase activity. From computational modeling, we identified 10 amino acid residues surrounding the active site for mutagenesis. Ten designs consisting of eight single-point mutants and two double-point mutants were selected for exploration. Mutants L546W and T290L that showed only 5.1% and 22.1% of catalytic efficiency on pyruvate compared to Kp-IpdC, were then expressed in K. pneumoniae for in vivo testing. Isobutanol production by K. pneumoniae T290L was 25% higher than that of the control strain, and a final titer of 5.5 g/L isobutanol was obtained with a substrate conversion ratio of 0.16 mol/mol glucose. CONCLUSIONS: This research provides a new way to improve the efficiency of the biological route of isobutanol production.

Assessing Alkene Reactivity toward Cytochrome P450-Mediated Epoxidation through Localized Descriptors and Regression Modeling.

The prediction of sites of epoxidation by cytochrome P450s during metabolism is particularly important in drug design, as epoxides are capable of alkylating biological macromolecules. Reliable methods are needed to quantitatively predict P450-mediated epoxidation barriers for inclusion in high-throughput screening campaigns alongside protein-ligand docking. Utilizing the fractional occupation number weighted density (FOD) and orbital-weighted Fukui index (fw+) as descriptors of local reactivity and a data set of 36 alkene epoxidation barriers computed with density functional theory (DFT), we developed and validated a multiple linear regression model for the reliable estimation of epoxidation barriers using only substrate structures as input. Using our recommended level of theory (GFN2-xTB//GFN-FF), mean absolute errors in the training and test sets were found to be 0.66 and 0.70 kcal/mol, respectively, with coefficients of determination of ca. 0.80. We demonstrate the utility of this approach on three known substrates of CYP101A1 and further show that this approach is inappropriate for particularly electron-rich alkenes. By employing a modern semiempirical method on force-field-generated geometries, the required descriptors can be calculated on the millisecond timescale per structure, making the approach well suited for incorporation into high-throughput methodologies alongside docking.

Engineering Embden-Meyerhof-Parnas Glycolysis to Generate Noncanonical Reducing Power.

Noncanonical cofactors such as nicotinamide mononucleotide (NMN+) supplant the electron-transfer functionality of the natural cofactors, NAD(P)+, at a lower cost in cell-free biomanufacturing and enable orthogonal electron delivery in whole-cell metabolic engineering. Here, we redesign the high-flux Embden-Meyerhof-Parnas (EMP) glycolytic pathway to generate NMN+-based reducing power, by engineering Streptococcus mutans glyceraldehyde-3-phosphate dehydrogenase (Sm GapN) to utilize NMN+. Through iterative rounds of rational design, we discover the variant GapN Penta (P179K-F153S-S330R-I234E-G210Q) with high NMN+-dependent activity and GapN Ortho (P179K-F153S-S330R-I234E-G214E) with ~3.4 × 106-fold switch in cofactor specificity from its native cofactor NADP+ to NMN+. GapN Ortho is further demonstrated to function in Escherichia coli only in the presence of NMN+, enabling orthogonal control of glucose utilization. Molecular dynamics simulation and residue network connectivity analysis indicate that mutations altering cofactor specificity must be coordinated to maintain the appropriate degree of backbone flexibility to position the catalytic cysteine. These results provide a strategy to guide future designs of NMN+-dependent enzymes and establish the initial steps toward an orthogonal EMP pathway with biomanufacturing potential.

Head and Neck Injury Patterns among American Football Players.

IMPORTANCE: American football is a popular high-impact sport, leading to 2.7 million injuries in the United States annually. Recent evidence in football-related neurological damage has spurred national interest in player-safety. Football players injure their head and neck in up to 26% of total injuries. Variation in injury patterns between age groups and correlated hospitalizations for football-related head and neck injury has yet to be characterized. OBJECTIVE: Our aim is to evaluate injury patterns among American-football related head and neck trauma. METHODS: A retrospective cohort study of patients with football-related head and neck injury in the National Electronic Injury Surveillance System (NEISS). RESULTS: Nearly 100 000 ED visits for football-related head and neck injuries occur annually. Males comprised 95% of patients, with a median age of 13. The head comprised 70% of injuries followed by the face (13%). The most common diagnoses were concussions (39%), internal organ injury (26%), and lacerations (11%). Pediatric patients were more likely to sustain concussions while adults experienced more lacerations (P < .05%). Fractures and nerve damage were rare injuries but caused a disproportionate share of hospitalizations. CONCLUSION: Pediatric males are most likely to present for emergency care from football-related injury to the head and neck. Evaluating physicians can anticipate concussions, internal organ injury, and lacerations among presenting patients. Concussions, facial fractures, and nerve damage are injuries most likely to lead to hospitalization.