Protein Expression and Purification

Efficient production of human proteins for the development of tool compounds and biologics depends on a detailed understanding of the protein expression machinery in mammalian cells. Codon optimization is widely believed to enhance protein yield, yet its impact in homologous mammalian systems remains poorly defined. Here, we systematically compare five codon usage strategies reflecting common assumptions about rare codons, RNA stability, and synthesis efficiency. We developed pTipi, an efficient open-source mammalian expression vector, and evaluated its performance in antibody production. We generated plasmids for common epitope tag antibodies such as V5, anti-biotin and anti-His for distribution by Addgene. To compare codon usage schemes, we performed a bake-off of 18 human and murine Wnt pathway glycoproteins in mammalian cells. Small-scale expression screens revealed that codon optimization did not provide a general advantage over native coding sequences, while strategies prioritizing RNA stability consistently reduced expression. Interestingly, a skewed codon scheme using the most abundant codons produced yields comparable to native sequences and occasionally enhanced protein output. To enable flexible evaluation of codon strategies, we implemented a Golden Gate-compatible pTipi platform for efficient synthetic gene incorporation. We conclude that native codons are sufficient for robust homologous mammalian expression of glycoproteins, while selective codon skewing can be beneficial for some targets.

TBCK-related encephalopathy (TBCKE) is a neurodevelopmental disorder associated with biallelic mutations in TBCK. Despite the increasing number of reported cases worldwide, the biochemical and biophysical properties of TBCK remain unclear, hindering molecular understanding of its role in disease. Here, we present the successful expression, purification, and biochemical characterization of full-length human TBCK produced in Spodoptera frugiperda cells. Biochemical and biophysical analyses reveal that the catalytically inactive pseudokinase domain of TBCK lacks nucleotide binding, consistent with the absence of the canonical VAIK, HRD, and DFG motifs required for catalysis. These findings support that TBCK is a class I pseudokinase and provide a foundation for future structural and functional studies to elucidate its biological role.

T/A cloning is a popular method for generating recombinant DNA plasmids. This method relies on single A:T nucleotide base pairs between PCR product ends and vector. Theoretically, the directionality of insert ligation with relation to the vector is random. However, we have continuously observed directionality bias using the pGEM-T Vector System for T/A cloning in a Course-based Undergraduate Research Experience (CURE). Cloning of over 400 inserts has shown directional bias higher than 74% (p-value < 0.0005) "sense" to the T7 promoter of the vector. Awareness of biased insertion in our applications reduces time and cost in cloning and downstream analyses.

Using its 9 sequentially-placed cohesin (Coh) domains as bait, each CipA scaffoldin chain in a Clostridium thermocellum cellulosome binds to (and displays) some combination of 9 out of over [~]70 available dockerin (Doc) domain-bearing lignocellulose-degrading enzymes, at any time. Domains numbered Coh3 through Coh8 show over 94 % pairwise sequence identity with each other, but only [~] 61-76 % identity with Coh1, Coh2, and Coh9. Such identities are much lower ([~] 40-60 %) amongst Doc domains; however, amongst both Coh and Doc domains, polypeptide backbone folds are highly conserved, suggesting that loading preferences of enzyme-bearing Doc domains upon Coh domains must depend upon their relative abundances, and pairwise affinities. To explore this further, we used microscale thermophoresis (MST), size exclusion chromatography (SEC), native polyacrylamide gel electrophoresis (NPGE), mass spectrometry (MS) and bioinformatics-based approaches (BIBA), to examine 28 Coh-Doc pairwise interactions involving recombinant Coh [Coh1, Coh2, Coh3, Coh9] and enzyme-bearing Doc [Cel8A, Cel9F, Man26/5H, Cel9R, Xyn10C, Xyn11D, Xyn10Z] domains. Interactions were found to occur with varying affinities, suggesting that Coh1 prefers Xyn11D; Coh2 prefers Xyn10C; Coh3 prefers Cel9R; Coh9 prefers Cel9R; Coh1/Coh2 prefer Xyn partners; Coh3/Coh9 prefer Cel partners. Dual modes of binding are shown by Coh1 with Xyn10C and Xyn11D; Coh2 with Xyn10Z, Cel8A, and Cel9R; Coh3 with Cel8A, and Cel9R; and Coh9 with Xyn10C, suggesting that Doc domains use either of their two homologous helices (1 and 3) to bind to Coh domains, as earlier proposed. ImportanceBacteria such as Clostridium thermocellum use extracellular enzyme complexes called cellulosomes to degrade and use cellulose. Each complex uses a linear chain of nine cohesin (Coh) domains called a scaffoldin to bind to (and display) any nine of over seventy available xylan or cellulose-degrading enzymes that bear dockerin (Doc) domains. Understanding interactions between Coh and Doc domains facilitates an appreciation of how cellulosomes are assembled and supports the building of protein-engineered constructs that utilize such interactions for many conceivable enzymatic and other applications. The significance of the presented research lies in its demonstration of the differential modes and pairwise affinities of different Coh-Doc interactions, using recombinant protein constructs and a combination of quantitative, semi-quantitative and qualitative analytical methods.

The hydrolytic breakdown of cellobiose into glucose, catalysed by {beta}-glucosidases, is the last and rate-limiting step in cellulose saccharification for producing fermentable glucose in the bioethanol industry. This limitation arises because {beta}-glucosidase activity is inhibited by factors such as temperature, pH, and glucose accumulation in reactors. Enzyme inactivation leads to the buildup of cello-oligosaccharides, which, in turn, inhibit upstream cellulases. Therefore, glucose-tolerant {beta}-glucosidases are preferred for the formulation of industrial cellulase cocktails. In this study, we have recombinantly expressed, purified, and biochemically characterised a {beta}-glucosidase from the cellulolytic fungus Fusarium odoratissimum (FoBgl-WT). FoBgl-WT exhibits optimal cellobiose hydrolysis over a broad pH range (4.5-7.5), an important and industrially desirable property for its application in bioreactors. However, the glucose tolerance of FoBgl-WT was [~]0.56 M. Structure-based analyses were carried out to map the residues lining the active site of FoBgl, and their roles in stabilising the product glucose (or even the substrate, cellobiose) were elucidated through a series of site-specific mutations, followed by biochemical characterisation of the resulting FoBgl mutants. Among all the mutants generated, FoBgl-K256I-Y325F exhibits >2.5-fold greater glucose tolerance ([~]1.4 M) than FoBgl-WT. Further, we have observed that the FoBgl-K256W and FoBgl-K256I mutants exhibit improved kinetic properties, such as catalytic efficiencies. The structure-based rational engineering efforts improve glucose tolerance and the kinetic properties of FoBgl mutants, making it a useful and promising candidate enzyme for industrial cellulase cocktails.

We have developed a vector platform for delivery of foreign peptides by genetic modification of the temperate lambda ({lambda}) bacteriophage. This delivery platform is capable of displaying peptides or proteins on either terminus of the structural {lambda} head protein D, present in [~]420 copies per phage particle, and {lambda} side tail fiber (Stf), present at 12 copies per phage particle. Proteins and peptides can be easily fused for display through the low-cost and high-efficiency methods of recombineering and {lambda} prophage induction for recombinant phage preparation described here. To improve this vector technology for use in antigen selection and immunotherapy, we introduced several mutations in the bacterial host and resident prophage {lambda} that improve engineering, induction, phage stability, yield, fusion protein accommodation capacity, and longevity in animal systems. We tested the ability of this {lambda} display system to identify useful antigens and generate antibodies in a mouse model. We report its success as a new technology for both applications: the selection and delivery of therapeutic peptides and proteins.

Quality and price of fetal bovine serum (FBS) are traditionally determined by geographical origin and parameters listed in the Certificate of Analysis (CoA). Despite its central role in cell culture, selecting suitable FBS batches remains costly and labor-intensive due to substantial batch-to-batch variation. We propose a molecular assessment strategy based on transcriptomic and cytokine profiling of cells cultured in different FBS batches to evaluate performance more reliably. Analysis of differential gene expression in three cell lines - MRC-5, Jurkat, and THP-1 - enables batch grouping and reveals pathway-specific effects, with immune-related pathways showing the most pronounced variability. Although CoA parameters can stratify batches by origin, they do not consistently correlate with cytokine secretion or gene expression across cell lines. These findings demonstrate that geographical origin is an inadequate predictor of functional FBS performance and that molecular profiling provides a more robust and informative assessment.

This study explores computational design predictions related to experimental enzyme behavior by analyzing seven single-point mutants of {beta}-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 {Delta}TSE 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.

Cultivated meat has undeniable potential to address some of the current detrimental impacts of animal farming, while addressing food security worldwide. However, one of the main challenges in cultivated meat production is manufacturing cost. The main contributor to cost is the culture media which comprises expensive components such as growth factors and animal-derived proteins. This study investigated alternative, food grade, high protein extracts as serum replacements in serum-free media formulations. The extracts were chosen to represent various sustainable sources of proteins: marine (spirulina e.g. cyanobacterium), plant (faba bean) and insect (mealworm flour). Different processing methods and different solvents were investigated for production of cell culture-compatible extracts which were then tested with mouse myoblasts (C2C12) and primary porcine myosatellites (pMyoSCs). A serum-free medium formulation containing 2.6% v/v spirulina extract was found to support long term growth of C2C12 cells for [~]10 population doublings compared to only [~]2 in the control. The processing steps were optimized, showing that a glycerine solution was best for free amino acid and protein yield (4950 {micro}M total free amino acids, 11.45 mg/mL protein concentration). This solution had a positive effect on C2C12 cells, increasing their growth by up to 20% when added to the B8 medium. However, this benefit did not translate to pMyoSCs, which showed no significant growth increases in short-term screening. This work demonstrates a method for converting food grade protein powders into effective culture media supplements and highlights the potential of spirulina-based extracts for the use in cultivated meat. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/702276v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@206658org.highwire.dtl.DTLVardef@11f28ceorg.highwire.dtl.DTLVardef@b00fd6org.highwire.dtl.DTLVardef@dfaaf4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Created in BioRender. Gordon-Petrovskii, W. (2025) https://BioRender.com/by7khs1 C_FIG

BackgroundShigellosis morbidity and mortality, combined with the increase in multidrug-resistant infections make Shigella vaccine development a global imperative. Glycoconjugate vaccines that couple immunogenic O-antigen to protein derived from Shigella may provide broader protection across Shigella species and serogroups. Such an approach also circumvents immunotolerance arising from repeated use of the same carrier. Here we use bioconjugation, exploiting an oligosaccharyltransferase (OST) enzyme to couple O-antigen and carrier protein in vivo, to generate a "double-hit" Shigella glycoconjugate vaccine. MethodGlycoconjugates were synthesised in E. coli SDB1 cells expressing S. sonnei O-antigen, the OST PglS, and one of two Shigella carrier proteins. Recombinant glycoconjugate was purified using anion exchange chromatography and then used to immunise mice. Antibody responses were measured and compared by ELISA. ResultsWhen co-produced in E. coli, PglS was able to transfer the cloned S. sonnei O-antigen onto three carrier proteins, modified to accept glycans from the PglS transferase enzymes- the standard bioconjugate carrier ExoA and two immunogenic Shigella-specific outer membrane proteins, EmrK and MdtA. Production of MdtA or ExoA glycoconjugates for immunisation studies utilised successive rounds of anion exchange chromatography, to remove unglycosylated material and obtain highly purified glycoconjugate proteins for us in vaccination. Analysis of murine sera following immunisation revealed an IgG response was raised against both carrier protein and the S. sonnei O-antigen for each glycoconjugate. ConclusionA novel, conserved Shigella protein can be utilised as an effective carrier for the generation of a "double-hit", immunogenic Shigella glycoconjugate vaccine that elicits IgG responses to both carrier protein and S. sonnei O-antigen.

This study presents a multidisciplinary approach to evaluate the structure formation and digestion of lupin protein crosslinked with transglutaminase (TG). TG was applied at 0-10 U/g protein, and structural development was assessed by oscillatory rheology (G, G"), while SDS-PAGE and o-phthaldialdehyde (OPA) assays were used to evaluate protein participation and the reduction of free {varepsilon}-amino groups, respectively. Proteomics was further employed to characterise molecular features associated with crosslinking behaviour. Lupin protein showed a clear dose-dependent increase in gel strength during incubation, with G values reaching 214 {+/-} 43.9 Pa at 10 U/g TG, compared to 7.2 {+/-} 0.6 Pa in the untreated control. Across all conditions, G remained higher than G" throughout frequency sweeps, and low tan {delta} values confirmed the formation of elastic networks driven by covalent crosslinks. SDS-PAGE and OPA results consistently demonstrated efficient crosslink formation, which increased with both incubation time and TG dosage, with SDS-PAGE indicating involvement of specific protein fractions. Proteomic analysis revealed disordered structural domains in the protein are preferred regions to form crosslinks. Furthermore, TG treatment was found to slow the digestibility of the crosslinked lupin protein. Overall, this work demonstrates how integrating proteomic insights with functional measurements can guide the selection and optimisation of plant proteins for enzymatic structuring. The approach offers a rational pathway to enhance the functionality of alternative protein sources such as lupin, supporting the development of sustainable food systems, including applications in meat and dairy analogues.

Natural extracts could improve sperm storage and artificial insemination (AI). This study, for the first time, evaluates the suitability of a blueberry extract (Vaccinium corymbosum) obtained from pomace using a sustainable methodology as a supplement for bull semen extenders. Cryopreserved semen doses from eight bulls were combined in 9 pools (3 bulls/pool), supplemented with 0%, 1%, 5%, or 10% extract, and incubated up to 5 h at 38 {degrees}C. Motility was assessed hourly using OpenCASA, and the effects of treatment and time were evaluated using linear mixed-effects models. Motility was significantly better preserved with 1% extract (total and progressive motility, improved linear velocity and linearities, and decreased BCF and fractal dimension, related to hyperactivation). The effect of 5% was overall positive, but it was below 1%, whereas 10% mostly showed a negative effect. These results show that this natural extract could safely supplement bull semen extenders at least between 1% to 5%, and even help improve sperm motility. Therefore, this extract offers an opportunity to enhance cattle semen extenders using a sustainable approach, potentially improving reproductive outcomes.

GPI-anchored proteins are crucial cell surface proteins with diverse, organism-specific functions, in eukaryotes. They are produced when the GPI transamidase (GPIT), a five-subunit membrane-bound enzyme complex, attaches a pre-formed GPI anchor to the C-terminal end of nascent proteins on the lumenal face of the endoplasmic reticulum. This process requires the removal of a C-terminal signal sequence (SS) on the substrate protein by the action of an endopeptidase subunit of the GPIT, Gpi8/ PIG-K. Using an AMC-tagged peptide in a cell free (post-mitochondrial fraction) assay, this manuscript studies the steady state kinetics of enzymatic cleavage of the substrate by GPIT of the human pathogenic fungus, C. albicans. We show that Mn+2 enhances activity by improving substrate binding but plays no direct role in substrate cleavage per se. Molecular dynamics simulations suggest that the divalent cation binds at a site away from the active site but provides compactness and stability to Gpi8. It also enables a conformation in which a flexible loop (219-244 residues) in the vicinity of the catalytic pocket is able to interact with and position the scissile bond for cleavage by Cys202. Steady state kinetics also indicate that peptides of lengths 7-mer to 9-mer are better bound than 4-mer or 15-mer peptide substrates. A bulky residue at the site of cleavage reduces the catalytic activity of the GPIT. This is the first detailed steady state kinetics study on the endopeptidase activity of a GPIT from any organism.

Bacterial outer membrane vesicles (OMVs), are nano-sized, spherical structures released by Gram-negative bacteria that play diverse roles in bacterial physiology, including communication, nutrient acquisition, and host interactions. These vesicles bud from the bacterial outer membrane and contain lipopolysaccharides, periplasmic proteins, nucleotides, and other biomolecules. The Vesicle Nucleating peptide (VNp) is a short peptide tag that, when fused to the amino terminus of a protein of interest, promotes the formation of bespoke recombinant extracellular vesicles in Escherichia coli, enabling efficient production and simplified purification of recombinant proteins. Here, we characterise VNp-induced vesicles and compare their composition and organisation with naturally produced E. coli OMVs. While both vesicle types possess a single outer membrane-derived lipid bilayer, recombinant protein is highly enriched within the VNp vesicles compared to endogenous OMVs. VNp-fusions and periplasm-targeted recombinant proteins localize to distinct vesicle populations, with VNp-fusions showing markedly higher intra-vesicular concentrations and vesicular purity, compared to the OMV targeted protein. OmpX co-expression further enriched the VNp-fusion content of vesicles, further enhancing yield. The VNp-vesicle lumen is an oxidizing environment, thus supports formation of inter- and intra-molecular disulfide bonds within encapsulated proteins. Overall, VNp-induced vesicles represent a distinct class of recombinant extracellular vesicles that offer a simple and efficient route for producing and purifying concentrated, correctly folded recombinant proteins, expanding the utility of bacterial vesicle systems for biotechnological applications. Significance StatementBacterial extracellular vesicles are recognized as versatile tools for biotechnology yet engineering bacterial vesicle production in a controlled and efficient manner remains challenging. Here we describe how a short Vesicle Nucleating Peptide (VNp) tag, fused to a protein of interest, that can be used to program Escherichia coli to produce recombinant extracellular vesicles that are compositionally and structurally distinct from natural bacterial outer membrane vesicles (OMVs). VNp-induced vesicles are more homogeneous, and more highly enriched in target fusion proteins, providing a simple and efficient route for protein production and purification. The oxidizing lumen of these vesicles supports disulfide bond formation, and rapid compartmentalisation enables expression of otherwise challenging or toxic proteins. This work characterises a distinct class of recombinant bacterial vesicles and establishes a practical platform for producing correctly folded, concentrated, partially purified proteins in a self-packaged form, expanding the potential applications of bacterial extracellular vesicles in biotechnology and synthetic biology.

Extracellular vesicles are key intercellular messengers that modulate the function of target cells by carrying effectors, either at their surface or in their lumen. In the latter case, their action depends on the ability to deliver their content into the cytosol of target cells. How efficiently EVs deliver their content upon interaction with their target cell is thus a central question for understanding the functional impact of this mode of action. To address this question, signal-driven bimolecular interactions between two partners located respectively in the EV lumen and the target cell cytosol have become a widely used strategy to detect the cytosolic delivery EV content. However, the detection of cytosolic delivery with these assays was often tributary to the artificial enhancement of the fusion between EV and cell membranes, through for instance VSV-G fusogenic protein expression. Here we provide a robust and quantitative LUCiferase-based complementation assay (HiBiT/LgBiT), to quantify the Internalization and cytosolic Delivery of EV content: LUCID-EV. By optimizing the signal-to-noise ratio of the assay, the method for loading HiBiT fragment into EVs (fusion to a lipid-binding domain rather than to tetraspanins), and the intracellular position of LgBiT (associated to membranes), we could quantify cytosolic delivery from various non-VSV-G-expressing EVs into target immune dendritic cells. Importantly, this delivery did not involve the acidic late endosomes environment required for VSV-G-dependent EV cytosolic delivery. The limited efficacy of the process highlights the need for highly sensitive assays like the one described here. Further development of the LUCID-EV assay could help identifying EV/target cells pairs with enhanced cytosolic delivery properties and characterize the cellular route for delivery.

Heterotrophic marine microorganisms have the capability to degrade and metabolize laminarin, which is the most abundant source of energy and nutrients in the marine environment, via enzymes encoded by genes clustered in polysaccharide utilization loci (PULs). In this study, a PUL potentially responsible for laminarin utilization was identified in the genome of the marine bacterium Muricauda lutaonensis strain ISCAR-4703, with conserved synteny in the genus Muricauda. A GH17 laminarinase (MlGH17A) encoded in the newly identified PUL was cloned, produced, and characterized as an endo-acting laminarinase, exhibiting the ability to degrade laminarin and laminari-oligosaccharides with a degree of polymerization (DP) greater than four into laminaribiose, laminaritriose, and laminaritetraose, with laminaritriose as the main product making up >50% of the produced oligosaccharide products. The three-dimensional model of the enzyme revealed the presence of seven putative subsites, including four glycone subsites (-4 to -1) and three aglycone subsites (+1 to +3), with a wide cleft to accommodate branches at the -2 subsite, enabling it to act on {beta}-1,3 linked backbones in polysaccharides with {beta}-1,6 linked branches. This enzyme is, along with the recently characterized {beta}-1,3 glucanosyltransglycosylase (MlGH17B), conserved in several Muricauda species and is suggested to play a crucial role in the utilization of laminarin by these bacteria. ImportanceLaminarin, a {beta}-1,3-glucan with occasional {beta}-1,6 branching, is the most abundant source of energy and nutrients in the marine environment. In this study, polysaccharide utilization loci (PULs) for laminarin degradation were identified in various marine Muricauda species, encoding a range of glycoside hydrolases and transglycosylases. In Muricauda lutaonensis ISCAR-4703, the PUL included two GH17 enzymes, separated by a GH30 enzyme and a major facilitator superfamily (MFS) transporter, a feature observed in all corresponding Muricauda PULs. A novel endoacting laminarinase from the PUL, MlGH17A, was characterized and shown to hydrolyze laminarin into laminaribiose, laminaritriose, and laminaritetraose, with laminaritriose as main product. Bioinformatic analysis showed that the enzyme lacked the typical subdomain found in GH17 plant {beta}-glucanases, leading to a lower number of aglycone subsites (+1 to +3). Instead, MlGH17A possessed more glycone subsites (-1 to -4), attributed to the {beta}3-3 loop, which was longer than in GH17 plant {beta}-glucanases.

The protein sample preparation methods for shotgun proteomics are nowadays well-established unlike the ones for whole protein analysis. The goal of my work has been to create a simple methodology which provides a single uncomplicated sample preparation tool for these two fields. Nowadays the bulk of proteomics work is done using detergents for protein solubilization. The presented concept, which is based on unspecific adsorption of protein molecules on wide pore materials, allows for protein capture and clean-up from solutions of the most commonly used sodium dodecyl sulfate detergent. It could also be applied to proteins in detergent-free solutions. After the capture and clean-up, proteins could be either cleaved for the downstream peptide analysis or eluted for the whole protein analysis. If required, the eluted whole proteins could be recaptured and cleaved into peptides. Depending on the experimental goals, the sample preparation device could be fitted with embedded proteolytic enzymes to simplify routine sample processing and/or reversed phase media for the downstream peptide or protein separation.

Six-transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) has emerged as a promising therapeutic target for prostate cancer. We have optimized the expression and purification conditions of human STEAP1 to maximize the production of its homotrimeric form, which is crucial for metal ion reduction and maintaining cellular redox balance. Proteins obtained from these optimized conditions were complexed with both heme and flavin-adenine dinucleotide (FAD), two cofactors that are fundamental to STEAP functionality, suggesting native folding and interactions of the protein. In addition, we compared the impact of stable and transient expression systems on the protein quality of STEAP1. We found that stable expression promoted heme incorporation, improved expression homogeneity, and ensured correct protein orientation on cell surfaces. Our findings present effective strategies for optimizing the recombinant production of STEAP1, with potential applicability to other STEAP family proteins to facilitate therapeutic discovery.

Biomass separation represents a critical bottleneck in Komagataella phaffii-based biopharmaceutical processes, as typically high cell densities of 40 - 50 % create significant operational, technical and economic challenges for harvest operations. Yeast cell aggregation (flocculation) provides a solution to accelerate cell sedimentation by increasing particle size, thus allowing to improve biomass-supernatant separation efficiency during both natural gravity settling and (continuous) centrifugation operations. This study demonstrates successful engineering of K. phaffii strains with an inducible flocculation phenotype using CRISPR/Cas9-based genome editing to integrate the Saccharomyces cerevisiae FLO1 (ScFLO1) gene under control of various regulatory elements, including methanol-inducible and derepressible promoters. Flocculation strength could be enhanced by implementing transcriptional positive feedback circuits based on the methanol-inducible AOX1 promoter. To address methanol-free production requirements, we developed alternative systems to retrofit PAOX1-based ScFLO1 expression and exploited the derepressible PDF promoter, offering broader compatibility with biopharmaceutical manufacturing facilities. Flocculating cells cultivated in a bioreactor demonstrated significantly improved sedimentation behavior, with considerably lower supernatant turbidity after short low-speed centrifugation compared to non-flocculating controls. Crucially, cell flocculation had no negative impact on product amount and quality when expressing a multivalent NANOBODY(R) VHH molecule with pharmaceutical relevance. Thus, this work establishes the first genetically engineered flocculation system in K. phaffii compatible with recombinant protein production, providing the basis for an innovative approach to streamline harvest operations in biopharmaceutical processes.

The surface of gram-positive bacteria is a highly regulated environment with specific attachment of proteins required for viability. Sortase enzymes are cysteine transpeptidases that recognize and ligate substrates to the peptidoglycan layer in these microorganisms, which can be highly pathogenic (e.g., Staphylococcus aureus, Streptococcus pyogenes, etc.). As such, sortases represent a potentially novel target for antibiotic development. In addition, the catalytic activity of sortase enzymes is utilized in sortase-mediated ligation (SML) engineering approaches for a variety of uses. In SML experiments, engineered variants of Staphylococcus aureus sortase A (saSrtA) are the most widely used enzymes. One of the mutated amino acids in the previously engineered pentamutant (or saSrtA5M) enzyme is P94. Structural analyses of experimental saSrtA structures revealed that P94 interacts directly with Y187 when saSrtA is in its inactive conformation. While saSrtA5M, developed via directed evolution, contains a P94R mutation, we wanted to interrogate this position further and ask if other single P94 mutations may reveal a greater effect on activity and/or substrate specificity. We created 18 P94X mutations (excluding P94C), and tested relative activity using a fluorescence resonance energy transfer (FRET) assay for 4 substrate sequences: LPATG, LPETG, LPKTG, and LPSTG. We identified several P94 variants that outperformed the single mutant P94R for all peptides tested, including P94A, P94D, P94E, P94G, P94H, P94N, P94Q, P94S, and P94T. We further observed that the reactivity of substrates with variations in the central position of the pentapeptide recognition motif (LPXTG) can be sensitive to the identity of the P94X residue. We tested P94A and P94D saSrtA5M variants and found that, depending on LPXTG sequence, these variants could outperform saSrtA5M in activity > 3-fold. Finally, we compared saSrtA5M and P94D saSrtA5M in a model sortase-mediated ligation reaction using a LPKTG substrate and saw [~]2-fold greater product formation. Taken together, we characterized an important position that modulates substrate access and activity in saSrtA. Furthermore, we argue that future studies which combine rational design and high throughput approaches, e.g., directed evolution, may result in sortase variants with increased SML potential.