Design to Data for mutants of β-glucosidase B from Paenibacillus polymyxa: Y333F, A88E, L219Q, A408H, Y173L, E340S, and Y422F

bioRxiv (Cold Spring Harbor Laboratory) · 2026-02-05

ABSTRACT This study explores computational design predictions related to experimental enzyme behavior by analyzing seven single-point mutants of β-glucosidase B (BglB) from Paenibacillus polymyxa : Y333F, A88E, L219Q, A408H, Y173L, E340S, and Y422F. Each mutation was modeled using Foldit Standalone, and mutant selections were based on predicted thermodynamic stability changes of interest. Six of the seven mutants in this set yielded soluble, expressed protein. Most variants had similar catalytic efficiency compared to the wild type with one exception. The melting temperatures for most variants were also similar to the wild type. Correlation analysis revealed weak but potentially informative relationships between predicted ΔTSE and (a) thermal stability and (b) catalytic efficiency. These results further support known limitations of TSE score as a tool for single point mutation design and add to a growing dataset being generated to build the next generation of functionally predictive protein models.

Gut Microbiota Mediate the Metabolism of Colonic Prostaglandins

Research Square · 2026-02-20

Basic importance: mechanistic molecular modeling of the <i>ent</i> -copalyl diphosphate synthase from <i>Arabidopsis thaliana</i> (AtCPS)

Organic & Biomolecular Chemistry · 2026-01-01 · 2 citations

(AtCPS), representative of plant class II diterpene cyclases more generally, as displacement of its (conserved) catalytic base leads to such alternative product outcomes. Here we characterize the mechanistic basis for the profound effects of these disruptions in AtCPS using the TerDockin computational approach, combining quantum chemical electronic structure calculations and docking of the relevant carbocation intermediates, to provide insight into the underlying enzymatic structure-function relationships. Our predictions help identify important bases in the wild type and mutant systems of AtCPS, which include trapped water and the conjugate base of the catalytic acid that initiates terpene cyclization.

Dock &amp; design: engineering specificity for an alternative pimaradiene outcome with the <i>ent</i> -kaurene synthase from <i>Bradyrhizobium japonicum</i>

Chemical Science · 2026-01-01

The complexity of the carbocation cascade reactions catalyzed by terpene synthases has hindered enzymatic engineering. Prospective application of the computationally inexpensive TerDockin approach enables rational design of product outcome.

Expression and characterization of SARS-CoV-2 spike protein in Thermothelomyces heterothallica C1

Vaccine · 2025-09-30

The COVID-19 pandemic demonstrated a pressing need for rapid, adaptive, and scalable manufacturing of vaccines and reagents. With the transition into an endemic disease and rising threats of other emerging pandemics, production of these biologicals requires a stable and sustainable supply chain and accessible distribution methods. In this study, we demonstrate the strength of an engineered filamentous fungal platform, Thermothelomyces heterothallica C1, for high volumetric productivity of the full-length spike glycoprotein. Spike protein produced in this system is highly thermostable and immunization of mice with spike made in C1 or mammalian platforms resulted in a similar humoral response. Additionally, it was shown that the native N-glycan profile can be redecorated with complex sialylated structures, if necessary, resulting in a more human-like glycan profile, without impacting binding characteristics as shown experimentally and in simulations. Through extensive physicochemical analysis, the C1-produced spike performs similarly to spike proteins produced in other commercially available systems. The data presented is evidence that C1 can be a strong platform for production of complex glycosylated recombinant proteins such as subunit antigen vaccines. • Engineered filamentous fungal platform, Thermothelomyces heterothallica C1 is suitable for high volumetric productivity of the full-length spike glycoprotein. • Spike protein produced in this system is highly thermostable and immunization of mice with spike made in C1 or mammalian platforms resulted in a similar humoral response. • The native N-glycan profile can be redecorated with complex sialylated structures, if necessary, resulting in a more human-like glycan profile. • C1 produced spike performs similarly to spike proteins produced in other commercially available systems. • This can be a useful platform for production of complex glycosylated recombinant proteins such as subunit antigen vaccines.

Activation-Free Upgrading of Carboxylic Acids to Aldehydes and Alcohols

bioRxiv (Cold Spring Harbor Laboratory) · 2025-07-28 · 2 citations

Abstract 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 CO 2 -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 &gt;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 (CO 2 ). 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 β-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.

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

bioRxiv (Cold Spring Harbor Laboratory) · 2025-08-02 · 1 citations

ABSTRACT 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 k cat values matching or exceeding that of NAD + and surpassing most engineered NRC-active enzymes by 10 to 10 5 -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 Nature’s 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.

Unleashing the innate ability of Escherichia coli to produce D-Allose

Metabolic Engineering · 2025-01-19 · 9 citations

D-allose is a rare monosaccharide, found naturally in low abundances. Due to its low-calorie profile and similar taste to sucrose, D-allose has the potential to become an ideal sugar substitute. D-allose also displays unique properties and health benefits that can be applied to various fields, including food and medicine. D-allose can be produced using two enzymatic steps in vitro : the epimerization of D-fructose, then the isomerization of the resulting D-psicose. This method suffers from poor yield due to the reversible nature of both reactions. We found that Escherichia coli possesses all of the required enzymes to convert D-glucose to D-allose with a thermodynamically favorable pathway, through a series of phosphorylation-epimerization-isomerization-dephosphorylation steps. To increase carbon flux toward D-allose production, the pathway genes were additionally expressed, and the competing pathways were removed. The engineered strains achieved production of D-allose, at a titer of 56.4 g L −1 , a productivity of 0.65 g L −1 hr − 1 , and a yield of 41.4% under test tube conditions. • The rare sugar D-allose was produced solely from glucose in E. coli. • E. coli can produce D-allose using only its native genes. • D-allose production was optimized by modifying pathway gene expression. • D-allose was co-produced with the sugar substitute D-psicose. • The maximum D-allose titer was 56.4 g L −1 in 120h.

Retrofitting Escherichia coli for de novo production of rare L-sorbose from abundant D-glucose

Metabolic Engineering · 2025-08-18

Monosaccharides exist in either “D” or “L” conformations, with L-sugars being much less abundant in nature and therefore classified as “rare sugars.” Rare sugars hold significant potential due to their unique interactions with biological systems, offering health, food, and crop benefits. One such sugar, L-sorbose, serves as a critical precursor to Vitamin C and offers a low-calorie, moderately sweet alternative to table sugar, being 60–70% as sweet but with only 25% of the caloric value. However, the broader study and application of rare sugars, including L-sorbose, are constrained by their high cost and limited availability. To address this challenge, we developed a biosynthetic strategy to convert the abundant and inexpensive D-sugar D-glucose into the rare L-sugar L-sorbose using microbial production. By utilizing phosphorylation and dephosphorylation steps to thermodynamically drive carbon flux, efficient production of 14.5 g L -1 L-sorbose was achieved under test tube conditions. Additionally, this pathway results in the co-production of D-sedoheptulose, a non-sweet, rare sugar shown to inhibit C6 sugar consumption in humans by modulating energy metabolism. The dual production of L-sorbose and D-sedoheptulose presents unique opportunities for applications in food and health sciences. This study demonstrates microbial production as a promising platform for rare L-sugar biosynthesis and provides a generalizable strategy for converting abundant D-sugars into underexplored L-sugars. Expanding access to L-sugars enables deeper investigations into their biological functions, metabolic pathways, and industrial applications. By advancing both fundamental sugar metabolism research and microbial production strategies, this study broadens the scope of rare sugar utilization. • Microbial conversion of D-glucose to rare L-sugar L-sorbose was developed • L-sorbose production achieved via thermodynamically driven biosynthesis • Rare sugar D-sedoheptulose co-produced • Platform expands access and study of rare L-sugars

International Research Collaborations Between Universities and Industry

International series in management science/operations research/International series in operations research & management science · 2025-01-01