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.

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.

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.

Reference genome and comparative genome analysis for the WHO reference strain for Mycobacterium bovis BCG Danish, the present tuberculosis vaccine.

BACKGROUND: Mycobacterium bovis bacillus Calmette-Guérin (M. bovis BCG) is the only vaccine available against tuberculosis (TB). In an effort to standardize the vaccine production, three substrains, i.e. BCG Danish 1331, Tokyo 172-1 and Russia BCG-1 were established as the WHO reference strains. Both for BCG Tokyo 172-1 as Russia BCG-1, reference genomes exist, not for BCG Danish. In this study, we set out to determine the completely assembled genome sequence for BCG Danish and to establish a workflow for genome characterization of engineering-derived vaccine candidate strains. RESULTS: By combining second (Illumina) and third (PacBio) generation sequencing in an integrated genome analysis workflow for BCG, we could construct the completely assembled genome sequence of BCG Danish 1331 (07/270) (and an engineered derivative that is studied as an improved vaccine candidate, a SapM KO), including the resolution of the analytically challenging long duplication regions. We report the presence of a DU1-like duplication in BCG Danish 1331, while this tandem duplication was previously thought to be exclusively restricted to BCG Pasteur. Furthermore, comparative genome analyses of publicly available data for BCG substrains showed the absence of a DU1 in certain BCG Pasteur substrains and the presence of a DU1-like duplication in some BCG China substrains. By integrating publicly available data, we provide an update to the genome features of the commonly used BCG strains. CONCLUSIONS: We demonstrate how this analysis workflow enables the resolution of genome duplications and of the genome of engineered derivatives of the BCG Danish vaccine strain. The BCG Danish WHO reference genome will serve as a reference for future engineered strains and the established workflow can be used to enhance BCG vaccine standardization.