OPENPichia: licence-free Komagataella phaffii chassis strains and toolkit for protein expression.

The industrial yeast Komagataella phaffii (formerly named Pichia pastoris) is commonly used to synthesize recombinant proteins, many of which are used as human therapeutics or in food. However, the basic strain, named NRRL Y-11430, from which all commercial hosts are derived, is not available without restrictions on its use. Comparative genome sequencing leaves little doubt that NRRL Y-11430 is derived from a K. phaffii type strain deposited in the UC Davis Phaff Yeast Strain Collection in 1954. We analysed four equivalent type strains in several culture collections and identified the NCYC 2543 strain, from which we started to develop an open-access Pichia chassis strain that anyone can use to produce recombinant proteins to industry standards. NRRL Y-11430 is readily transformable, which we found to be due to a HOC1 open-reading-frame truncation that alters cell-wall mannan. We introduced the HOC1 open-reading-frame truncation into NCYC 2543, which increased the transformability and improved secretion of some but not all of our tested proteins. We provide our genome-sequenced type strain, the hoc1tr derivative that we named OPENPichia as well as a synthetic, modular expression vector toolkit under liberal end-user distribution licences as an unencumbered OPENPichia resource for the microbial biotechnology community.

GlycoVHH: optimal sites for introducing N-glycans on the camelid VHH antibody scaffold and use for macrophage delivery.

As small and stable high-affinity antigen binders, VHHs boast attractive characteristics both for therapeutic use in various disease indications, and as versatile reagents in research and diagnostics. To further increase the versatility of VHHs, we explored the VHH scaffold in a structure-guided approach to select regions where the introduction of an N-glycosylation N-X-T sequon and its associated glycan should not interfere with protein folding or epitope recognition. We expressed variants of such glycoengineered VHHs in the Pichia pastoris GlycoSwitchM5 strain, allowing us to pinpoint preferred sites at which Man5GlcNAc2-glycans can be introduced at high site occupancy without affecting antigen binding. A VHH carrying predominantly a Man5GlcNAc2 N-glycan at one of these preferred sites showed highly efficient, glycan-dependent uptake by Mf4/4 macrophages in vitro and by alveolar lung macrophages in vivo, illustrating one potential application of glyco-engineered VHHs: a glycan-based targeting approach for lung macrophage endolysosomal system delivery. The set of optimal artificial VHH N-glycosylation sites identified in this study can serve as a blueprint for targeted glyco-engineering of other VHHs, enabling site-specific functionalization through the rapidly expanding toolbox of synthetic glycobiology.

Yeast-produced fructosamine-3-kinase retains mobility after ex vivo intravitreal injection in human and bovine eyes as determined by Fluorescence Correlation Spectroscopy.

Globally, over 2 billion people suffer from vision impairment. Despite complex multifactorial etiology, advanced glycation end products are involved in the pathogenesis of many causative age- and diabetes-related eye diseases. Deglycating enzyme fructosamine-3-kinase (FN3K) was recently proposed as a potential therapeutic, but for further biopharmaceutical development, knowledge on its manufacturability and stability and mobility in the vitreous fluid of the eye is indispensable. We evaluated recombinant production of FN3K in two host systems, and its diffusion behavior in both bovine and human vitreous. Compared to Escherichia coli, intracellular production in Pichia pastoris yielded more and higher purity FN3K. The yeast-produced enzyme was used in a first attempt to use fluorescence correlation spectroscopy to study protein mobility in non-sonicated bovine vitreous, human vitreous, and intact bovine eyes. It was demonstrated that FN3K retained mobility upon intravitreal injection, although a certain delay in diffusion was observed. Alkylation of free cysteines was tolerated both in terms of enzymatic activity and vitreous diffusion. Ex vivo diffusion data gathered and the availability of yeast-produced high purity enzyme now clear the path for in vivo pharmacokinetics studies of FN3K.

An affinity-enhanced, broadly neutralizing heavy chain-only antibody protects against SARS-CoV-2 infection in animal models.

Broadly neutralizing antibodies are an important treatment for individuals with coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Antibody-based therapeutics are also essential for pandemic preparedness against future Sarbecovirus outbreaks. Camelid-derived single domain antibodies (VHHs) exhibit potent antimicrobial activity and are being developed as SARS-CoV-2­neutralizing antibody-like therapeutics. Here, we identified VHHs that neutralize both SARS-CoV-1 and SARS-CoV-2, including now circulating variants. We observed that the VHHs bound to a highly conserved epitope in the receptor binding domain of the viral spike protein that is difficult to access for human antibodies. Structure-guided molecular modeling, combined with rapid yeast-based prototyping, resulted in an affinity enhanced VHH-human immunoglobulin G1 Fc fusion molecule with subnanomolar neutralizing activity. This VHH-Fc fusion protein, produced in and purified from cultured Chinese hamster ovary cells, controlled SARS-CoV-2 replication in prophylactic and therapeutic settings in mice expressing human angiotensin converting enzyme 2 and in hamsters infected with SARS-CoV-2. These data led to affinity-enhanced selection of the VHH, XVR011, a stable anti­COVID-19 biologic that is now being evaluated in the clinic.

Exploration of Synergistic Action of Cell Wall-Degrading Enzymes against Mycobacterium tuberculosis.

The major global health threat tuberculosis is caused by Mycobacterium tuberculosis. M. tuberculosis has a complex cell envelope-a partially covalently linked composite of polysaccharides, peptidoglycan, and lipids, including a mycolic acid layer-which conveys pathogenicity but also protects against antibiotics. Given previous successes in treating Gram-positive and -negative infections with cell wall-degrading enzymes, we investigated such an approach for M. tuberculosis. In this study, we aimed to (i) develop an M. tuberculosis microtiter growth inhibition assay that allows undisturbed cell envelope formation to overcome the invalidation of results by typical clumped M. tuberculosis growth in surfactant-free assays, (ii) explore anti-M. tuberculosis potency of cell wall layer-degrading enzymes, and (iii) investigate the concerted action of several such enzymes. We inserted a bacterial luciferase operon in an auxotrophic M. tuberculosis strain to develop a microtiter assay that allows proper evaluation of cell wall-degrading anti-M. tuberculosis enzymes. We assessed growth inhibition by enzymes (recombinant mycobacteriophage mycolic acid esterase [LysB], fungal α-amylase, and human and chicken egg white lysozymes) and combinations thereof in the presence or absence of biopharmaceutically acceptable surfactant. Our biosafety level 2 assay identified both LysB and lysozymes as potent M. tuberculosis inhibitors but only in the presence of surfactant. Moreover, the most potent disruption of the mycolic acid hydrophobic barrier was obtained by the highly synergistic combination of LysB, α-amylase, and polysorbate 80. Synergistically acting cell wall-degrading enzymes are potently inhibiting M. tuberculosis, which sets the scene for the design of specifically tailored antimycobacterial (fusion) enzymes. Airway delivery of protein therapeutics has already been established and should be studied in animal models for active TB.

A Potential Role for Fructosamine-3-Kinase in Cataract Treatment.

Cataracts are the major cause of blindness worldwide, largely resulting from aging and diabetes mellitus. Advanced glycation end products (AGEs) have been identified as major contributors in cataract formation because they alter lens protein structure and stability and induce covalent cross-linking, aggregation, and insolubilization of lens crystallins. We investigated the potential of the deglycating enzyme fructosamine-3-kinase (FN3K) in the disruption of AGEs in cataractous lenses. Macroscopic changes of equine lenses were evaluated after ex vivo intravitreal FN3K injection. The mechanical properties of an equine lens pair were evaluated after treatment with saline and FN3K. AGE-type autofluorescence (AF) was measured to assess the time-dependent effects of FN3K on glycolaldehyde-induced AGE-modified porcine lens fragments and to evaluate its actions on intact lenses after in vivo intravitreal FN3K injection of murine eyes. A potential immune response after injection was evaluated by analysis of IL-2, TNFα, and IFNγ using an ELISA kit. Dose- and time-dependent AF kinetics were analyzed on pooled human lens fragments. Furthermore, AF measurements and a time-lapse of macroscopic changes were performed on intact cataractous human eye lenses after incubation with an FN3K solution. At last, AF measurements were performed on cataractous human eyes after crossover topical treatment with either saline- or FN3K-containing drops. While the lenses of the equine FN3K-treated eyes appeared to be clear, the saline-treated lenses had a yellowish-brown color. Following FN3K treatment, color restoration could be observed within 30 min. The extension rate of the equine FN3K-treated lens was more than twice the extension rate of the saline-treated lens. FN3K treatment induced significant time-dependent decreases in AGE-related AF values in the AGE-modified porcine lens fragments. Furthermore, in vivo intravitreal FN3K injection of murine eyes significantly reduced AF values of the lenses. Treatment did not provoke a systemic immune response in mice. AF kinetics of FN3K-treated cataractous human lens suspensions revealed dose- and time-dependent decreases. Incubation of cataractous human eye lenses with FN3K resulted in a macroscopic lighter color of the cortex and a decrease in AF values. At last, crossover topical treatment of intact human eyes revealed a decrease in AF values during FN3K treatment, while showing no notable changes with saline. Our study suggests, for the first time, a potential additional role of FN3K as an alternative treatment for AGE-related cataracts.

Development of a Counterselectable Transposon To Create Markerless Knockouts from an 18,432-Clone Ordered Mycobacterium bovis Bacillus Calmette-Guérin Mutant Resource.

Mutant resources are essential to improve our understanding of the biology of slow-growing mycobacteria, which include the causative agents of tuberculosis in various species, including humans. The generation of deletion mutants in slow-growing mycobacteria in a gene-by-gene approach in order to make genome-wide ordered mutant resources is still a laborious and costly approach, despite the recent development of improved methods. On the other hand, transposon mutagenesis in combination with Cartesian pooling-coordinate sequencing (CP-CSeq) allows the creation of large archived Mycobacterium transposon insertion libraries. However, such mutants contain selection marker genes with a risk of polar gene effects, which are undesired both for research and for use of these mutants as live attenuated vaccines. In this paper, a derivative of the Himar1 transposon is described which allows the generation of clean, markerless knockouts from archived transposon libraries. By incorporating FRT sites for FlpE/FRT-mediated recombination and I-SceI sites for ISceIM-based transposon removal, we enable two thoroughly experimentally validated possibilities to create unmarked mutants from such marked transposon mutants. The FRT approach is highly efficient but leaves an FRT scar in the genome, whereas the I-SceI-mediated approach can create mutants without any heterologous DNA in the genome. The combined use of CP-CSeq and this optimized transposon was applied in the BCG Danish 1331 vaccine strain (WHO reference 07/270), creating the largest ordered, characterized resource of mutants in a member of the Mycobacterium tuberculosis complex (18,432 clones, mutating 83% of the nonessential M. tuberculosis homologues), from which markerless knockouts can be easily generated.IMPORTANCE While speeding up research for many fields of biology (e.g., yeast, plant, and Caenorhabditis elegans), genome-wide ordered mutant collections are still elusive in mycobacterial research. We developed methods to generate such resources in a time- and cost-effective manner and developed a newly engineered transposon from which unmarked mutants can be efficiently generated. Our library in the WHO reference vaccine strain of Mycobacterium bovis BCG Danish targets 83% of all nonessential genes and was made publicly available via the BCCM/ITM Mycobacteria Collection. This resource will speed up Mycobacterium research (e.g., drug resistance research and vaccine development) and paves the way to similar genome-wide mutant collections in other strains of the Mycobacterium tuberculosis complex. The stretch to a full collection of mutants in all nonessential genes is now much shorter, with just 17% remaining genes to be targeted using gene-by-gene approaches, for which highly effective methods have recently also been described.

Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies.

Coronaviruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics. Here, we describe the isolation of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs neutralize MERS-CoV or SARS-CoV-1 S pseudotyped viruses, respectively. Crystal structures of these VHHs bound to their respective viral targets reveal two distinct epitopes, but both VHHs interfere with receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S and demonstrate that this cross-reactive VHH neutralizes SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a molecular basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these molecules may serve as useful therapeutics during coronavirus outbreaks.

Structural basis of the proinflammatory signaling complex mediated by TSLP.

Thymic stromal lymphopoietin (TSLP), a cytokine produced by epithelial cells at barrier surfaces, is pivotal for the development of widespread chronic inflammatory disorders such as asthma and atopic dermatitis. The structure of the mouse TSLP-mediated signaling complex reveals how TSLP establishes extensive interfaces with its cognate receptor (TSLPR) and the shared interleukin 7 receptor α-chain (IL-7Rα) to evoke membrane-proximal receptor-receptor contacts poised for intracellular signaling. Binding of TSLP to TSLPR is a mechanistic prerequisite for recruitment of IL-7Rα to the high-affinity ternary complex, which we propose is coupled to a structural switch in TSLP at the crossroads of the cytokine-receptor interfaces. Functional interrogation of TSLP-receptor interfaces points to putative interaction hotspots that could be exploited for antagonist design. Finally, we derive the structural rationale for the functional duality of IL-7Rα and establish a consensus for the geometry of ternary complexes mediated by interleukin 2 (IL-2)-family cytokines.