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Immunization with mosaic-8b (nanoparticles presenting 8 SARS-like betacoronavirus [sarbecovirus] receptor-binding domains [RBDs]) elicits more broadly cross-reactive antibodies than homotypic SARS-CoV-2 RBD-only nanoparticles and protects against sarbecoviruses. To investigate original antigenic sin (OAS) effects on mosaic-8b efficacy, we evaluated the effects of prior COVID-19 vaccinations in non-human primates and mice on anti-sarbecovirus responses elicited by mosaic-8b, admix-8b (8 homotypics), or homotypic SARS-CoV-2 immunizations, finding the greatest cross-reactivity for mosaic-8b. As demonstrated by molecular fate mapping, in which antibodies from specific cohorts of B cells are differentially detected, B cells primed by WA1 spike mRNA-LNP dominated antibody responses after RBD-nanoparticle boosting. While mosaic-8b- and homotypic-nanoparticles boosted cross-reactive antibodies, de novo antibodies were predominantly induced by mosaic-8b, and these were specific for variant RBDs with increased identity to RBDs on mosaic-8b. These results inform OAS mechanisms and support using mosaic-8b to protect COVID-19-vaccinated/infected humans against as-yet-unknown SARS-CoV-2 variants and animal sarbecoviruses with human spillover potential., Competing Interests: Declaration of interests P.J.B. and A.A.C. are inventors on U.S. Patent Application 17/523,813 filed by the California Institute of Technology that covers the mosaic nanoparticles described in this work. A.I.R. and J.D.P. are inventors on U.S. Patent Application 11,780,888 that covers the chimeric sequence of RBD fused to the HA2 stem of influenza hemagglutinin. A.J.G. is an inventor on a Fred Hutchinson Cancer Center-optioned technology related to DMS of the RBD of SARS-CoV-2 spike protein. L.M., S.B., R.C., C.S.-A., and I.G.F. are inventors on U.S. Patent Applications 16/952,983 and 17/651,476 filed by Ingenza Ltd. that cover Bacillus and Pichia strains established to manufacture endotoxin-free vaccine products. J.H.E. is an employee of HDT Bio that provided the repRNA-LION vaccine. D.H.F. is a co-founder of Orlance, Inc. that is developing gene gun delivery of DNA and repRNA vaccines. S.B., R.C., M.Q.-A., E.R., C.S.-A., L.M., and I.G.F. are employees of Ingenza Ltd. P.J.B. and G.D.V. are scientific advisors for Vaccine Company, Inc., and P.J.B. is a scientific advisor for Vir Biotechnology. N.P. is named on patents describing the use of nucleoside-modified mRNA in LNPs as a vaccine platform. N.P. served on the mRNA strategic advisory board of Sanofi Pasteur in 2022 and the mRNA technology advisory board of Pfizer in 2023 and is a member of the scientific advisory board of AldexChem and Bionet-Asia. P.J.C.L. is an employee of Acuitas Therapeutics, a company developing LNP delivery systems for RNA therapeutics., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Immunization with mosaic-8b [60-mer nanoparticles presenting 8 SARS-like betacoronavirus (sarbecovirus) receptor-binding domains (RBDs)] elicits more broadly cross-reactive antibodies than homotypic SARS-CoV-2 RBD-only nanoparticles and protects against sarbecoviruses. To investigate original antigenic sin (OAS) effects on mosaic-8b efficacy, we evaluated effects of prior COVID-19 vaccinations in non-human primates and mice on anti-sarbecovirus responses elicited by mosaic-8b, admix-8b (8 homotypics), or homotypic SARS-CoV-2 immunizations, finding greatest cross-reactivity for mosaic-8b. As demonstrated by molecular fate-mapping in which antibodies from specific cohorts of B cells are differentially detected, B cells primed by WA1 spike mRNA-LNP dominated antibody responses after RBD-nanoparticle boosting. While mosaic-8b- and homotypic-nanoparticles boosted cross-reactive antibodies, de novo antibodies were predominantly induced by mosaic-8b, and these were specific for variant RBDs with increased identity to RBDs on mosaic-8b. These results inform OAS mechanisms and support using mosaic-8b to protect COVID-19 vaccinated/infected humans against as-yet-unknown SARS-CoV-2 variants and animal sarbecoviruses with human spillover potential., Competing Interests: DECLARATION OF INTERESTS P.J.B. and A.A.C. are inventors on a US patent application (17/523,813) filed by the California Institute of Technology that covers the mosaic nanoparticles described in this work. A.I.R. and J.D.P. are inventors on a US patent (11,780,888) that covers the chimeric sequence of RBD fused to the HA2 stem of influenza hemagglutinin. A.J.G. is an inventor on a Fred Hutchinson Cancer Center-optioned technology related to DMS of the RBD of SARS-CoV-2 spike protein. L.M., S.B., R.C., C.S-A., and I.G.F. are inventors on U.S. Patent Applications (16/952,983) and (17/651,476) filed by Ingenza Ltd. that cover Bacillus and Pichia strains established to manufacture endotoxin-free vaccine products. J.H.E. is an employee of HDT Bio that provided the repRNA-LION vaccine. D.H.F. is a co-founder of Orlance, Inc. that is developing gene gun delivery of DNA and repRNA vaccines. S.B., R.C., M.Q.-A., E.R., C.S.-A., L.M., and I.G.F. are employees of Ingenza, LTD. P.J.B. and G.D.V. are scientific advisors for Vaccine Company, Inc, and P.J.B. is a scientific advisor for Vir Biotechnology. N.P. is named on patents describing the use of nucleoside-modified mRNA in lipid nanoparticles as a vaccine platform. N.P. served on the mRNA strategic advisory board of Sanofi Pasteur in 2022 and the mRNA technology advisory board of Pfizer in 2023 and is a member of the Scientific Advisory Board of AldexChem and Bionet-Asia. P.J.C.L. is an employee of Acuitas Therapeutics, a company developing LNP delivery systems for RNA therapeutics.

The methylotrophic yeast Pichia pastoris is commonly used for the production of recombinant proteins at scale. The identification of an optimally overexpressing strain following transformation can be time and reagent consuming. Fluorescent reporters like GFP have been used to assist identification of superior producers, but their relatively big size, maturation requirements and narrow temperature range restrict their applications. Here, we introduce the use of iLOV, a flavin-based fluorescent protein, as a fluorescent marker to identify P. pastoris high-yielding strains easily and rapidly. The use of this fluorescent protein as a fusion partner is exemplified by the production of the antimicrobial peptide NI01, a difficult target to overexpress in its native form. iLOV fluorescence correlated well with protein expression level and copy number of the chromosomally integrated gene. An easy and simple medium-throughput plate-based screen directly following transformation is demonstrated for low complexity screening, while a high-throughput method using fluorescence-activated cell sorting (FACS) allowed for comprehensive library screening. Both codon optimization of the iLOV_NI01 fusion cassettes and different integration strategies into the P. pastoris genome were tested to produce and isolate a high-yielding strain. Checking the genetic stability, process reproducibility and following the purification of the active native peptide are eased by visualization of and efficient cleavage from the iLOV reporter. We show that this system can be used for expression and screening of several different antimicrobial peptides recombinantly produced in P. pastoris., (© 2022 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Using directed evolution, a variant N-acetyl amino acid racemase (NAAAR G291D/F323Y) has been developed with up to 6-fold higher activity than the wild-type on a range of N-acetylated amino acids. The variant has been coupled with an enantiospecific acylase to give a preparative scale dynamic kinetic resolution which allows 98% conversion of N-acetyl-DL-allylglycine into D-allylglycine in 18 h at high substrate concentrations (50 g L(-1)). This is the first example of NAAAR operating under conditions which would allow it to be successfully used on an industrial scale for the production of enantiomerically pure α-amino acids. X-ray crystal analysis of the improved NAAAR variant allowed a comparison with the wild-type enzyme. We postulate that a network of novel interactions that result from the introduction of the two side chains is the source of improved catalytic performance.

Both D- and L-beta- and gamma-substituted alpha-amino acids can be interconverted to their respective L- and D- diastereoisomers by treatment with an enantioselective amino acid oxidase and a chemical reducing agent.

A wide variety of methods have been used to prepare unnatural amino acids. This review covers methods reported between 2000 and the early part of 2001. Some of the approaches discussed are applications of established methods, but emphasis has been given to new approaches that could be generally applicable to large-scale syntheses, including catalytic reactions such as the Strecker reaction and biological approaches.

The introduction of a stereogenic centre to produce an "unnatural" amino acid can be accomplished in a variety of ways ranging from asymmetric hydrogenation to biotransformations based on transaminase enzymes. Our transaminase approach can be used to access a wide variety of L- and D-amino acids from an alpha-keto acid substrate. It is run as a whole cell biotransformation and uses coupled enzyme systems. In addition, formation of amino acids with small side chains, such as 2-aminobutyrate, can cause significant isolation problems due to the presence of small amounts of other amino acids, such as alanine. The improvements we have made to the approach are illustrated with 2-aminobutyrate as the example. Aspartic acid is used as the amino donor and gives rise to the formation of pyruvate, a substrate for the transaminase enzymes. We have now developed an alternative approach where lysine is used as the amino donor to allow formation of a cyclic by-product that is removed from the equilibrium.

L-2-Aminobutyric acid was synthesised in a transamination reaction from L-threonine and L-aspartic acid as substrates in a whole cell biotransformation using recombinant Escherichia coli K12. The cells contained the cloned genes tyrB, ilvA and alsS which respectively encode tyrosine aminotransferase of E. coli, threonine deaminase of E. coli and alpha-acetolactate synthase of B. subtilis 168. The 2-aminobutyric acid was produced by the action of the aminotransferase on 2-ketobutyrate and L-aspartate. The 2-ketobutyrate is generated in situ from L-threonine by the action of the deaminase, and the pyruvate by-product is eliminated by the acetolactate synthase. The concerted action of the three enzymes offers significant yield and purity advantages over the process using the transaminase alone with an eight to tenfold increase in the ratio of product to the major impurity.

Transaminase enzymes are being increasingly applied to the large-scale synthesis of unnatural and nonproteinogenic amino acids. Typically displaying relaxed substrate specificity, rapid reaction rates and lacking the need for cofactor regeneration, they possess many characteristics that make them desirable as effective biocatalysts. By judiciously combining the transaminase reaction with additional enzymatic steps, this approach can be used very efficiently to prepare a broad range of D- and L-amino acids.

In Bacillus sphaericus and other Bacillus spp., D-amino acid transaminase has been considered solely responsible for biosynthesis of D-glutamate, an essential component of cell wall peptidoglycan, in contrast to the glutamate racemase employed by many other bacteria. We report here the cloning of the dat gene encoding D-amino acid transaminase and the glr gene encoding a glutamate racemase from B. sphaericus ATCC 10208. The glr gene encodes a 28. 8-kDa protein with 40 to 50% sequence identity to the glutamate racemases of Lactobacillus, Pediococcus, and Staphylococcus species. The dat gene encodes a 31.4-kDa peptide with 67% primary sequence homology to the D-amino acid transaminase of the thermophilic Bacillus sp. strain YM1.

The gene encoding the D-aminotransferase from Bacillus licheniformis was cloned and the complete DNA sequence was determined. The deduced D-aminotransferase protein sequence, consists of 283 amino acids and shows a high degree of homology with other Bacillus D-aminotransferases, branched chain aminotransferase of Escherichia coli and the 4-amino-benzoate-4-deoxychorismate lyase of Bacillus subtilis and Escherichia coli.

In this paper we describe the cloning and sequence analysis of the tyrB and aspC genes from Escherichia coli K12, which encode the aromatic aminotransferase and aspartate aminotransferase respectively. The tyrB gene was isolated from a cosmid carrying the nearby dnaB gene, identified by its ability to complement a dnaB lesion. Deletion and linker insertion analysis located the tyrB gene to a 1.7-kilobase NruI-HindIII-digest fragment. Sequence analysis revealed a gene encoding a 43 000 Da polypeptide. The gene starts with a GTG codon and is closely followed by a structure resembling a rho independent terminator. The aspC gene was cloned by screening gene banks, prepared from a prototrophic E. coli K12 strain, for plasmids able to complement the aspC tyrB lesions in the aminotransferase-deficient strain HW225. Sub-cloning and deletion analysis located the aspC gene on a 1.8-kilobase HincII-StuI-digest fragment. Sequence analysis revealed the presence of a gene encoding a 43 000 Da protein, the sequence of which is identical with that previously obtained for the aspartate aminotransferase from E. coli B. Considerable overproduction of the two enzymes was demonstrated. We compared the deduced protein sequences with those of the pig mitochondrial and cytoplasmic aspartate aminotransferases. From the extensive homology observed we are able to propose that the two E. coli enzymes possess subunit structures, subunit interactions and coenzyme-binding and substrate-binding sites that are very similar both to each other and to those of the mammalian enzymes and therefore must also have very similar catalytic mechanisms. Comparison of the aspC and tyrB gene sequences reveals that they appear to have diverged as much as is possible within the constraints of functionality and codon usage.