Safety, immunogenicity and efficacy of an mRNA-based COVID-19 vaccine, GLB-COV2-043, in preclinical animal models.

Several COVID-19 vaccines, some more efficacious than others, are now available and deployed, including multiple mRNA- and viral vector-based vaccines. With the focus on creating cost-effective solutions that can reach the low- and medium- income world, GreenLight Biosciences has developed an mRNA vaccine candidate, GLB-COV2-043, encoding for the full-length SARS-CoV-2 Wuhan wild-type spike protein. In pre-clinical studies in mice, GLB-COV2-043 induced robust antigen-specific binding and virus-neutralizing antibody responses targeting homologous and heterologous SARS-CoV-2 variants and a TH1-biased immune response. Boosting mice with monovalent or bivalent mRNA-LNPs provided rapid recall and long-lasting neutralizing antibody titers, an increase in antibody avidity and breadth that was held over time and generation of antigen-specific memory B- and T- cells. In hamsters, vaccination with GLB-COV2-043 led to lower viral loads, reduced incidence of SARS-CoV-2-related microscopic findings in lungs, and protection against weight loss after heterologous challenge with Omicron BA.1 live virus. Altogether, these data indicate that GLB-COV2-043 mRNA-LNP vaccine candidate elicits robust protective humoral and cellular immune responses and establishes our mRNA-LNP platform for subsequent clinical evaluations.

Engineering E. coli for the biosynthesis of 3-hydroxy-γ-butyrolactone (3HBL) and 3,4-dihydroxybutyric acid (3,4-DHBA) as value-added chemicals from glucose as a sole carbon source.

3-hydroxy-γ-butyrolactone (3HBL) is a versatile chiral synthon, deemed a top value-added chemical from biomass by the DOE. We recently reported the first biosynthetic pathway towards 3HBL and its hydrolyzed form, 3,4-dihydroxybutyric acid (3,4-DHBA) in recombinant Escherichia coli using glucose and glycolic acid as feedstocks and briefly described their synthesis solely from glucose. Synthesis from glucose requires integration of the endogenous glyoxylate shunt with the 3,4-DHBA/3HBL pathway and co-overexpression of seven genes, posing challenges with respect to expression, repression of the glyoxylate shunt and optimal carbon distribution between the two pathways. Here we discuss engineering this integration. While appropriate media and over-expression of glyoxylate shunt enzymes helped overcome repression, two orthogonal expression systems were employed to address the expression and carbon distribution challenge. Synthesis of up to 0.3g/L of 3HBL and 0.7g/L of 3,4-DHBA solely from glucose was demonstrated, amounting to 24% of the theoretical maximum.

A platform pathway for production of 3-hydroxyacids provides a biosynthetic route to 3-hydroxy-γ-butyrolactone.

The replacement of petroleum feedstocks with biomass to produce platform chemicals requires the development of appropriate conversion technologies. 3-Hydroxy-γ-butyrolactone has been identified as one such chemical; however, there are no naturally occurring biosynthetic pathways for this molecule or its hydrolyzed form, 3,4-dihydroxybutyric acid. Here we design a novel pathway to produce various chiral 3-hydroxyacids, including 3,4-dihydroxybutyric acid, consisting of enzymes that condense two acyl-CoAs, stereospecifically reduce the resulting ß-ketone and hydrolyze the CoA thioester to release the free acid. Acetyl-CoA serves as one substrate for the condensation reaction, whereas the second is produced intracellularly by a pathway enzyme that converts exogenously supplied organic acids. Feeding of butyrate, isobutyrate and glycolate results in the production of 3-hydroxyhexanoate, 3-hydroxy-4-methylvalerate and 3,4-dihydroxybutyric acid+3-hydroxy-γ-butyrolactone, respectively, molecules with potential uses in applications from materials to medicines. We also unexpectedly observe the condensation reaction resulting in the production of the 2,3-dihydroxybutyric acid isomer, a potential value-added monomer.

Microbial chemical factories: recent advances in pathway engineering for synthesis of value added chemicals.

The dwindling nature of petroleum and other fossil reserves has provided impetus towards microbial synthesis of fuels and value added chemicals from biomass-derived sugars as a renewable resource. Microbes have naturally evolved enzymes and pathways that can convert biomass into hundreds of unique chemical structures, a property that can be effectively exploited for their engineering into Microbial Chemical Factories (MCFs). De novo pathway engineering facilitates expansion of the repertoire of microbially synthesized compounds beyond natural products. In this review, we visit some recent successes in such novel pathway engineering and optimization, with particular emphasis on the selection and engineering of pathway enzymes and balancing of their accessory cofactors.

Electrospray as a tool for drug micro- and nanoparticle patterning.

Carbamazepine (CBZ) microparticles of different sizes and shapes, including spheres, q-tips, elongated spheres, and tear-shaped particles, were formed by electrospraying solutions of different CBZ concentrations. The particle characteristics were determined by the interplay between jet formation, droplet breakup, solvent evaporation, and eventual particle solidification. The average particle size increased with increasing CBZ concentration, with particles of different shapes being observed for different CBZ concentrations. The cascade of sizes and shapes observed was interpreted in terms of Rayleigh instability theory as applied to charged jets and droplets, with the final sizes depending upon the time needed to evaporate the solvent sufficiently for CBZ to solidify; the lower the initial concentration of CBZ, the smaller the final droplets/particles that are formed.