Toxic and Useful: Harnessing yeast killer toxins for product recovery

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Loan, Jian-Wei Liu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8904913/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background: Saccharomyces cerevisiae is widely used in biotechnology to produce proteins and other biomolecules. Recovery of intracellular products, however, remains a key bottleneck for industrial applications. The robust cell wall demands energy-intensive chemical or mechanical disruption that increase processing costs and can damage sensitive targets. Results: In this study, we investigated yeast killer toxins as a genetically encoded tool to enhance membrane permeability and facilitate intracellular product recovery. Five killer toxins with distinct modes of action were expressed intracellularly and their effects on cell survival, membrane permeability, susceptibility to lysis and enhancement of intracellular product recovery were examined. In combination with common mechanical disruption methods, toxin expression enhanced total intracellular protein recovery up to 6-fold relative to the control strain without a killer toxin expressed. Conclusion: These results establish killer toxin expression as a tuneable strategy for enhanced product recovery from yeast, that complements and augment conventional bioprocessing techniques. Saccharomyces cerevisiae Killer toxins membrane integrity product recovery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Many yeast species, collectively known as ‘killer yeasts’ secrete protein toxins that either inhibit or kill susceptible fungi while protecting themselves from the effect of their own toxin through immunity domains coded on the protein. To date, a wide variety of killer toxins have been identified across different genera of yeast including Saccharomyces cerevisiae , Kluyveromyces lactis and Zygosaccharomyces bailii , which exhibit remarkable diversity in their molecular size, mode of action, and stability under varying pH and thermal conditions. These toxins can range from small peptides to multimeric protein complexes and affect susceptible fungi through pore formation in the plasma membrane, enzymatic degradation of the cell wall, cell cycle inhibition, or protein synthesis disruption. The structural, genetic and functional diversity of these toxins has been comprehensively reviewed recently by S. Billerbeck et al( 1 ). To date the use of protein toxins in industrial applications has been limited to biocontrol, including inhibiting growth of spoilage microorganisms in food and beverage industries to improve food safety, development of antifungal therapeutics for clinical and veterinary applications and protection of plants against fungal pathogens( 2 – 10 ). Beyond these conventional roles, the use of toxins in other biotechnological applications remains limited. One unexplored application is the use of their membrane permeabilisation properties to improve intracellular product release. Currently, recovery of intracellular products from yeast heavily relies on either mechanical disruption techniques such as bead milling, high-pressure homogenization, ultrasonication or chemical disruption methods such as lysis with solvents, detergents, or alkali treatment ( 11 ). While these methods can efficiently lyse cells, they are time- and energy-intensive, incur high capital and operating costs and can potentially damage sensitive target molecules ( 11 , 12 ). Development of new techniques such as hydrodynamic cavitation or the combined use of established techniques aim to mitigate these drawbacks but remain constrained by their own challenges in scalability, cost and efficiency ( 13 , 14 ). Yeast killer toxins that compromise cell wall or plasma membrane integrity could offer a novel biological strategy to enhance product recovery by ‘priming’ the cells for lysis. Toxins such as HM-1 from Cyberlindnera mrakii and HSK from C. saturnus inhibit β-glucan synthases and impede cell wall construction( 15 ). Zymocin from Kluyveromyces lactis has chitinase activity, while K2 from S. cerevisiae satellite virus M2 and Zygocin from Zygosaccharomyces bailii disrupt the plasma membrane( 16 – 20 ). By targeting distinct layers of the cell envelope, killer toxins create a controlled weakened state that may facilitate efficient release of intracellular products. By targeting the cell envelope of susceptible microorganisms, these toxins compromise cellular integrity, increasing susceptibility to intracellular leakage and ultimately, could enhance product recovery. Baker's yeast ( Saccharomyces cerevisiae ) is one of the most widely used microbial cells to produce an array of products ranging from biopharmaceuticals to food grade ingredients. S. cerevisiae is genetically tractable, robust, fast growing and possesses well-characterised cellular machinery capable of synthesising complex proteins, lipids, and other natural products( 21 – 23 ). However, like most yeast, it has a tough multilayered cell wall that creates a significant bottleneck in product recovery and downstream processing. In the present study, S . cerevisiae was engineered to express different yeast killer toxins and the effects of toxin expression on cell physiology and cell wall integrity were investigated. We evaluate the potential of yeast killer toxins as novel strategy for enhancing product recovery based on our findings presented here. Materials and Methods Strains and plasmids All strains employed in this study have been developed from the parent S. cerevisiae strain BY4741 (genotype: MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 ) (ATCC no. 201388). The five different yeast killer toxins expressed separately in BY4741 were killer toxin K2 (UniProt: A0A291FEA7) from Saccharomyces killer virus M2-4, HM-1 (UniProt: P10410) from Cyberlindnera mrakii , HSK (UniProt: Q00948) from Cyberlindnera saturnus , Zygocin (UniProt: Q8NJU1) from Zygosaccharomyces bailii , Zymocin (UniProt: P09805) from Kluyveromyces lactis . The native signal peptide of the K2 killer toxin was replaced with truncated α mating factor to remove its immunity domain ( 24 ) and Zymocin was reduced to only its endogenous signal peptide and chitinase activity α domain ( 25 ). For assessing recombinant protein recovery from yeast supernatant, the different toxin strains were co-transformed with plasmid containing fluorescent reporter protein mCherry (UniProt: X5DSL3) under constitutive promoter TDH3. All genes were codon optimised for S. cerevisiae using IDT codon optimisation tool. The Kozak sequence AAACA was introduced at the 5’ end of the start codon to enhance expression. Plasmids were designed and assembled in silico using the PYEAST digital toolkit( 26 ). The plasmids employed in this study have been summarised in Tables S1. Media and culture conditions Chemicals were purchased from Sigma Aldrich unless indicated otherwise. BY4741 was cultivated in YPD media containing yeast extract (10 g/L), peptone (20 g/L) and dextrose (20 g/L) prior to transformation. Toxin-containing plasmids were constructed in yeast with DNA fragments flanked by overlapping sequences to enable assembly by homologous recombination in vivo. Transformation with the empty vector (pESC-His) and the DNA fragment mix was carried out using a yeast transformation kit, following the manufacturer's instructions. The resulting transformants were maintained on yeast synthetic complete (SC) agar plates containing yeast nitrogen base without amino acids (6.7 g/L) dextrose (20 g/L), agar (20 g/L) and yeast synthetic dropout medium supplements (1.92 g/L) (Merck, USA) without uracil and/or histidine as required. Chi.Bio reactors ( 27 ) (University of Oxford) were used to measure growth kinetics. The control strain (BY4741 containing empty vector pESC-His) and strains containing the toxin genes were initially grown in 7 ml SC medium containing dextrose (SCD medium) in 50 ml tubes (CELLSTAR® CELLREACTOR Tube) at 30°C, at 250 rpm for 16–18 h to attain exponential phase. Cultures were then diluted to an O.D. 600nm value of 0.2–0.4 in 25 ml SC induction (SCI) medium containing galactose (20 g/L) and raffinose (10 g/L) for toxin expression and incubated at 30°C, with maximum stirring speed for 24 h in Chi.Bio reactors prior to harvesting. Spot assay BY4741 strains containing pESC-His or toxin genes were grown in SCD medium at 30°C, 250 rpm for 16–18 h. Cultures were normalised to an equal O.D. 600nm of 12 and 10-fold serial dilutions were prepared in SCI medium. To assess the temperature range of the toxins, 5 µL aliquots of dilutions from 10 − 2 to 10 − 6 were spotted on SCI medium agar plates and incubated for 3 days at either 30°C or 37°C. To assess the pH range, 5 µL from these dilutions were spotted on SCI medium agar plates with adjusted to 4.6 or 5.5 and incubated at 30°C for 3 days. Oxygen transfer rate measurements Oxygen transfer rate was measured continuously in a 96 well microtiter plate using a prototype micro-scale Transfer rate Online Measurement device (microTOM), manufactured by Kuhner shaker (Switzerland). Strains were inoculated into SCI medium at an initial O.D. 600nm of 0.2 and grown at 30°C for 36 h at 300 rpm and 50 mm shaking diameter. For all other cell physiology studies and co-transformation study, the strains were first grown in SCD medium at 30°C, 250 rpm for 16–18 h to accumulate sufficient biomass. Overnight cultures were harvested by centrifugation and then resuspended in 20 ml SCI medium for toxin expression and incubated at 30°C, 250 rpm for 18 h before analysis. Fluorescence staining Following incubation in SCI medium for 18 h, 1 ml of yeast cultures were pelleted and washed with Hank’s buffered salt solution (HBSS, Thermo Fisher Scientific). The samples were adjusted to O.D. 600nm of 0.5 in HBBS and SYTOX™ Green (Thermo Fisher Scientific) was added at final concentration of 1 µM. The yeast samples were incubated in the dark at room temperature for 20 min without shaking. After incubation, the samples were washed twice with HBBS to remove excess dye and resuspended in HBSS prior to flow cytometric analysis. Flow cytometric analysis Flow cytometric analysis with SYTOX™ Green-stained BY4741 strains was performed on a LSR II flow cytometer (BD Biosciences). Heat-treated cells (100°C, 5 min) transformed with an empty vector were included as a positive control, representing maximal membrane permeability (100%). Yeast cells were differentiated based on forward scattering (FSC-A) and side scattering (SSC-A) signals. Fluorescent signals were acquired using a 488 nm excitation laser and 530/30 bandpass (BP) collection filter. Samples were recorded at a flow rate of 12.5 µL min − 1 . Subsequently, the acquired data was analysed using FlowJo software v10.8 (FlowJo LLC). Cell samples were normalised to 10 000 events and cell debris and doublets were gated out of the flow cytometry data. Scanning Electron Microscopy (SEM) Yeast cells cultured in SCI medium for 18 h were pelleted and washed with Milli-Q water to remove residual media components. The pellet was resuspended in freshly prepared formaldehyde-alcohol-acetic acid (FAA) fixative solution with the composition: 10% formalin (37–40% w/v formaldehyde), 5% glacial acetic acid, 50% ethanol, and 35% distilled water (v/v/v/v)( 28 ). Samples were gently agitated for 1 h on a shaker at room temperature. Following fixation, cells were subjected to an ethanol dehydration gradient consisting of 10%, 30%, 50%, 70%, 90%, and 100% ethanol (v/v). At each gradient, samples were incubated for 15 minutes at room temperature before progressing to the next concentration. The final dehydration step involved two washes in 100% anhydrous ethanol for 15 min each. Following dehydration, samples were mounted onto SEM stubs with silica chips and coated with approximately 10 nm layer of gold using a sputter coater (Quorum MiniQ S). Prepared samples were imaged using a scanning electron microscope (ZEISS UltraPlus analytical FESEM) at an accelerating voltage of 5 kV. Sample preparation for proteomic analysis After 16 h incubation in SCI medium, 400 µl of culture supernatant from BY4741 strains containing pESC-His or toxin genes was collected. Proteins in the culture supernatant were precipitated with 1600 µl cold acetone at -20°C overnight. The precipitated pellet was air-dried and resuspended in 40 µl 8M urea. Protein solution (10 µl) was reduced using 1 µl of 15% (w/v) DTT at room temperature for 30 min and subsequently alkylated with 1 µl of 40% acrylamide and incubated at room temperature for 30 min. Digestion was performed by adding 47 µl of trypsin solution (0.2 µg of trypsin in 25 mM ammonium bicarbonate) and incubating at 37°C for 3 h. Digestion was stopped by adding 1 µl of 10% formic acid. Samples were passed through a 0.22 µm filter before liquid chromatography mass spectrometry (LC-MS) analysis. LC-MS proteomic analysis Tryptic peptides were separated on a nano column (Aurora Elite™ XT 15×75 C18 UHPLC column, Ionopticks) on UltimateTM 3000 RSLC nano LC system (Thermo Fisher Scientific). Mobile phase A comprised of water with 0.1% (v/v) formic acid and mobile phase B consisted of 80% (v/v) acetonitrile with 0.8% (v/v) formic acid. Tryptic peptides were separated on a gradient of 8–30% solvent B for 30 min, 30% – 50% solvent B for 8 min and 50% – 97.5% for 6 min. The eluted peptides were ionised with using a Nanospray Flex Ion Source (Thermo Fisher Scientific) at 1.8 kV with capillary temperature 300°C and analysed using Orbitrap Fusion MS (Thermo Fisher Scientific) operated in top speed mode with a 3 s cycle for both the MS and MS/MS scans. Full MS survey scans of peptide precursors were acquired from scan range of 400 to 1500 m/z at a resolution of 120,000 (at 200 m/z) with an automatic gain target (AGC) set as 4 x 10 5 and a maximum injection time of 50 ms. Most abundant precursors (charge state 2 + − 7 + with intensity greater than 1 x 10 5 ) were isolated in the quadrupole (1.6 m/z window) and fragmented by high- energy collision dissociation with collision energy of 28%. Fragments were detected in ion trap detector with AGC target of 4 x 10 3 , with a maximum injection time of 300 ms and a dynamic exclusion on 15 s. Proteomic data analysis Proteins/ peptides were identified and their relative abundance was quantified using Proteome Discoverer 2.2 (Thermo Fisher Scientific) and Sequest HT search engine. The spectrum data was searched against recombinant killer toxins, mCherry and S. cerevisiae proteome database (Proteome ID: UP000002311_559292). Precursor mass tolerance was set to 10 ppm and product ions were searched at 0.6 Da. Three missed tryptic cleavages were allowed. Variable modification included oxidation (+ 15.995 Da), deamidation (+ 0.984 Da), amidation (− 0.984 Da), and propionamidation (+ 71.037 Da). Peptide spectral matches were validated using the Percolar algorithm, based on q-values and 1% False Discovery Rate (FDR). Relative abundance is calculated from precursor abundance intensity and is normalized from total peptide amount. Downstream data analysis was performed in R studio where the data was filtered to retain proteins with High combined FDR confidence, classified as Master proteins, with more than 1 unique peptide and ‘High’ found status. Mechanical Disruption and Spectrophotometric analysis BY4741 strains containing pESC-His or toxin genes co-transformed with mCherry were grown in SCI medium for 18 h, harvested, washed and resuspended in HBSS buffer to an O.D. 600nm of 0.5. Mechanical disruption was achieved using either bead-milling or freeze-thawing, as specified below: For bead-milling, acid-washed glass beads (422–600 µm) were added to samples. The samples were subjected to mechanical disruption using Ratek multi tube vortex mixer (Model # MTV1) for 20 min with intermittent cycle of 35 seconds on / 5 seconds off, at a speed setting of 7.5 (instrument dial setting). For freeze-thawing, the samples were frozen at -80°C and subsequently thawed rapidly at 37°C. Following disruption by either method, the samples were centrifuged at 1000 x g for 10 min at room temperature and the supernatant was carefully collected. Cellular debris from the supernatant was removed by filtration using a 0.22 µm pore size filter. mCherry fluorescence in filtered supernatant samples were then analysed using Fluostar Omega spectrophotometer (BMG LABTECH). For each assay, 100 µL of supernatant samples were aliquoted into a black, flat-bottom 96 well plate (Costar #3916). HBSS buffer was used as a blank for background fluorescence correction. mCherry fluorescence was measured using excitation and emission wavelengths of 585 nm and 620 nm, respectively. The instrument gain was optimised prior to data acquisition to ensure the signals were within the detector’s linear range. All samples and blanks were measured in triplicates, and the analyses were performed at room temperature. Data Processing and Analysis All statistical analysis, data processing, and visualization were conducted using a combination of Python (version 3.12.9) and R (version 4.4.2). Statistical tests such as ANOVA, Tukey’s, Games-Howell and Welch’s t-test were conducted based on data distribution using standard libraries and packages (Pandas, NumPy, statsmodels and SciPy for Python and lmFit, limma, eBayes for R). Data visualisation was performed using Matplotlib seaborn and ggplot2. Custom scripts were developed and executed in Jupyter Notebook and RStudio environments. Each experiment had 3 biological replicates, and the statistical significance threshold was defined as p < 0.05. Results and Discussion Assessing cytotoxicity of yeast expressing killer toxins The growth effect of toxins expressed under the control of the GAL1 promoter was evaluated through a 10-fold serial-dilution spot assay on SCI agar medium (Fig. 1 A). All toxin-expressing strains displayed growth inhibition at lower dilution compared to the empty-vector control at 30°C, which showed the colony formation up to 10 − 5 dilution. HM-1- and HSK-producing strains exhibited complete growth inhibition at 10 − 2 dilution, whereas truncated K2 showed visible impact on growth at 10 − 3 dilution, and zygocin- and zymocin-producing strains showed visible growth suppression at 10 − 4 dilution. To assess the functional range of these toxins in terms of growth temperature and pH, spot assays were repeated at 37°C and at pH 4.4 or 5.5. All toxins except zymocin exhibited the same growth inhibitory profile at 37°C as at 30°C, indicating temperature stability of the effect of the toxins between 30°C and 37°C. Zymocin showed enhanced potency, with complete growth inhibition at 10 − 2 dilution at 37°C, suggesting optimal activity of this toxin at elevated temperature. pH 4.6 and 5.5 produced indistinguishable inhibition patterns, demonstrating broad pH tolerance of these toxins (Figure S1 ). As the optimal growth temperature for S. cerevisiae is 30°C, all subsequent experiments were carried out at this temperature. Growth kinetics of the toxin expressing strains further revealed toxin-dependent physiological effects on cellular proliferation in the presence of galactose (Fig. 1 B). The control strain had a relatively short lag phase and reached an O.D. 600nm of 5.22 ± 0.51 by 24 h with minimal replicate variability. In contrast, the zygocin-producing strain had a pronounced lag phase of ~ 7 h and plateaued at a lower O.D. 600nm of 3.23 ± 0.24, indicating moderate stress. Both zymocin and truncated K2 strains exhibited further extended lag phases of ~ 8 h and 14 h, before achieving O.D. 600nm 1.5 ± 0.11 and 1.3 ± 0.10 respectively, suggesting considerable stress on the cells, consistent with the absence of cognate immunity factors that normally protect killer strains from their own toxins ( 24 , 25 ). HM-1 and HSK strains remained essentially quiescent throughout the 24 h, with O.D. 600nm values below 0.7 ± 0.07, indicating near-complete growth inhibition, which aligned with the spot assay results. The spot assay and growth kinetics demonstrated that intracellular expression of the killer toxins in toxin-sensitive BY4741 yeast exerted varying degrees of antiproliferative effects, establishing that each toxin preserved its characteristic biological activity when expressed in a heterologous host, consistent with its known killer activity. Metabolic activity of different toxin expressing strains was compared by continuously measuring oxygen transfer rate (OTR) over a 36 h period. OTR is a suitable parameter to monitor the physiological state of a microbial culture, since metabolic activity in aerobic cultivations is reflected in oxygen consumption. The OTR and cumulative oxygen transfer (COT) revealed notable differences between the control strain and toxin expressing strains. The control strain grew exponentially for 18 h, reaching a peak OTR of 4.2 mmol L − 1 h − 1 and declining to 2 mmol L − 1 h − 1 by 23 h (Fig. 2 A). Strains expressing HM-1 and HSK showed negligible OTR over 36 h (Fig. 2 B-C, 2 E-F), consistent with their impaired growth caused by toxin expression. Truncated K2, zygocin and zymocin expressing cells exhibited delayed growth and reduced OTRs, relative to the control, indicating a pronounced effect of toxin expression. Rare survivors emerging by toxin resistance or gene silencing by mutation or plasmid loss likely contribute to the observed late-stage OTR increase, which was not investigated further in the current study ( 29 ). These OTR profiles showed that intracellular toxin expression imposes toxin-dependent constraints on the overall respiratory metabolism that align with growth defects observed in growth kinetics and antiproliferative characteristics demonstrated in the growth assays. Effect of toxin expression on cell membrane integrity The impact of different toxins on the cell membrane integrity was evaluated with SYTOX™ Green, which only enters cells with compromised membranes, coupled with flow cytometric analysis. Heat-treated empty vector control cells served as a positive control, that demonstrated complete (100%) membrane permeability. The percentage of BY4741 cells with compromised cell membranes was significantly different between the empty vector control strain and the strains expressing different toxins (Fig. 3 ). Strains expressing HSK and HM-1 exhibited elevated SYTOX™ Green uptake with ~ 81.3% and 58% of the population staining positive. This corresponded to 20-fold and 14-fold increase in membrane permeability relative to control strain, reflecting their known inhibition of β-glucan synthase ( 11 , 25 ). Zygocin also caused significant membrane damage, with 70.9% of the cell population staining positive (17-fold increase compared to control), attributed to its ability to form pores in the cell membrane ( 16 , 25 ). In comparison, truncated K2 and zymocin caused moderate but statistically significant cell membrane damage, as indicated by 30% and 16.2% SYTOX™ Green positive cells (7-fold and 4-fold increase relative to the control strain). One-way ANOVA confirmed significant impact of toxins on membrane permeability and post hoc Welch’s t-test demonstrated that the percentage of SYTOX™ Green positive cells of each toxin strain significantly differed from that of the empty vector control strain without heat-treatment ( p value < 0.05; n = 3). The morphological effect of toxins on yeast cells was observed using Scanning Electron Microscopy (SEM) (Fig. 4 ). The empty vector control strain had the classic budding yeast morphology with cells retaining their smooth ovoid shape, characteristic bud and birth scars with little evidence of cell collapse or cell shrinkage (Fig. 4 A) ( 30 ). In contrast, cells expressing different toxins exhibited varying degrees of morphological change. HSK and zygocin expressing cells exhibited extensive cell damage, characterised by regional invaginations and structural collapse along with scattered cellular debris, which are hallmarks of cytosolic content release (Fig. 4 B & E), suggesting that these strains exhibited higher functional toxin activity. HM-1-expressing strain showed moderate cell damage, with smaller serrations and cellular debris (Fig. 4 C). Cells expressing truncated K2 and zymocin displayed cells with prominent surface wrinkling accompanied with minor tears in the cell wall and no detectable extracellular debris, consistent with their lower levels of membrane permeabilisation (Fig. 4 D & F). Proteomic profiling of the culture supernatant was performed to examine the consequences of toxin expression at the proteome level. Detected proteins were annotated by subcellular localization based on Gene Ontology (GO) Cellular Component (CC) terms, allowing supernatant protein abundance to be partitioned into intracellular and extracellular components (Fig. 5 A). In strains expressing HM-1, HSK, and zygocin, proteins normally localized intracellularly contributed more than 90% of total supernatant protein abundance, indicating that the leaked proteome was dominated by cytoplasmic components (Fig. 5 A). This strong enrichment of intracellular proteins in the supernatant is consistent with markedly increased membrane permeability, extensive membrane rupture, and large-scale structural failure of the cellular envelope ( 15 , 19 ). Surprisingly, strains expressing truncated K2 and zymocin also showed higher contribution of intracellular proteins to total supernatant protein abundance, despite relatively less membrane permeability detected by flow cytometry, a potential consequence of low level cumulative release of proteins from structurally weakened cells. Toxin-induced leakage of cellular content was further characterised by quantifying the number of intracellular proteins detected in the supernatant. An increase in the number of intracellular proteins was detected in the supernatant of all toxin-expressing strains relative to that of the empty vector control strain, indicating enhanced membrane permeability and leakage of a wider variety of proteins (Fig. 5 B). Assessing recombinant protein recovery from yeast cell culture supernatant Following confirmation of toxin-driven cell membrane permeabilisation, each strain was co-transformed with the mCherry reporter gene and its release into the culture supernatant was quantified to assess the degree of toxin-driven protein leakage. Spectrophotometric analysis of filtered culture supernatant demonstrated varying levels of mCherry leakage in toxin expressing strains (Fig. 6 A). The highest mCherry levels were observed in strains expressing toxins HSK and zygocin, which were consistent with previously measured cell membrane permeabilisation (Fig. 2 A). The HM-1 strain showed intermediate mCherry concentration in the culture supernatant, while truncated K2 and zymocin showed significantly lower levels, parallelling their low membrane permeability. These patterns were further supported by proteomic detection of mCherry in the culture supernatant (Figure S2). Two mechanical cell disruption methods, bead-milling and freeze-thawing, were paired with toxin expression to evaluate their ability to improve mCherry release in the culture supernatant compared to empty vector control. Bead-milling produced a measurable increase in mCherry fluorescence in the supernatant of all strains. The expression of toxins increased overall mCherry fluorescence in the supernatant by 1.5-to-6-fold relative to control (Fig. 5 B). A similar trend was observed when the toxin-expressing strains were subject to freeze-thawing where mCherry release in the culture supernatant increased by 2-to-4.5-fold in comparison to control strain (Fig. 5 C). Even with strain-to-strain variation in mCherry expression (Figure S3), toxin-expressing strains with high cell membrane permeability exhibited a significant fold increase in mCherry release relative to empty vector control strain, whereas toxin-expressing strains with lower membrane permeability displayed a moderate yet significant fold change. These data aligned with the cell damage observed with respective toxins through SYTOX™ screening and SEM analysis. These results indicate that toxin expression increases the cells’ susceptibility to mechanical disruption, leading to dramatic increases in protein release through effects on cell membrane permeability. Notably, even strains with lower membrane permeability, such as strains expressing truncated K2 and zymocin, showed considerable increases in mCherry release when paired with mechanical disruption, compared to the empty vector control strain. This combinatorial approach provides a practical strategy to increase cell susceptibility to lysis and thereby improve intracellular product recovery in yeast by up to 6-fold. Overall, the experimental results demonstrate that toxin expression disrupts cell structure and exerts significant physiological effects. Cell membrane integrity assays and scanning electron microscopy reveal pronounced cell wall damage in strains exhibiting high membrane permeability, characterised by cell rupture, collapse, and leakage of intracellular content. Both high- and low-permeability strains displayed slower growth and increased extracellular protein leakage; however, strains with lower membrane permeability show comparatively less overt physical damage, suggesting intracellular stress and perturbation of cellular homeostasis. Mechanistically, these effects may arise from additional cellular processes, including potential overloading of the host secretory pathway due to toxin precursor processing through the endoplasmic reticulum and Golgi apparatus. In parallel, toxin-induced remodelling of the cell wall or trafficking machinery, through interactions with chitin or β-glucans could further influence protein export ( 17 , 20 , 24 ). Taken together, these findings highlight the multifaceted impact of toxin expression on yeast cells, with damage ranging from overt structural compromise to more subtle intracellular dysfunction, thereby advancing our understanding of toxin-mediated cellular perturbations. Conclusion This study showcases a novel strategy for harnessing yeast killer toxins in different biotechnological applications. The findings demonstrate the efficacy of these toxins as tools to increase cell membrane permeability that can improve intracellular product release. Expression of these toxins in S. cerevisiae produced disruptive physiological effects similar to those observed when applied to susceptible yeast strains. Comprehensive analyses including membrane integrity assay, proteomics and scanning electron microscopy clearly established the impact of these toxins on yeast morphology, physiology and function. Co-expression with a fluorescent reporter protein further validated the effectiveness of this strategy, where toxin expression augmented the effect of mechanical disruption, increasing product release by up to 6-fold. Importantly, the results also highlighted the versatility of this approach. Different toxins can be leveraged based on application requirements. Potent toxins can be employed where severe cell membrane disruption is needed. In contrast, milder toxins can be used for more controlled cell membrane permeabilisation, which, when combined with milder downstream disruption processes offer a strategy that is more focused on membrane weakening prior to collecting cells for product recovery, which may be advantageous for the recovery of products sensitive to harsher lysis conditions. Although this research demonstrated the use of toxins for product release, further optimisation is required. Fine-tuning toxin expression or employing combinatorial toxin strategy, could aid in precisely tailoring membrane permeabilisation to suit diverse bioprocessing requirements, potentially reducing the reliance on other disruption strategies. Overall, this research unlocks the potential for yeast killer toxins as effective biological agents for programmable cell permeabilisation and provides a new avenue for enhancing intracellular product release across a range of industrial biotechnology applications. Declarations Data Availability The datasets supporting the conclusions of this article are available in the CSIRO Data Access Portal, https://doi.org/10.25919/f4g8-qj41. Acknowledgments The authors would like to thank Dr. Phil Hands at Black Mountain MicroImaging Centre, CSIRO for providing technical advice and guidance in using Scanning Electron Microscope and Microscopy Australia (ROR: 042mm0k03) at the Centre for Advanced Microscopy, The Australian National University, a facility enabled by NCRIS and university support. In addition, we acknowledge the expertise of Michael Devoy and Fei-Ju Li at Cytometry, Histology and Spatial Multiomics (CHASM) facility of the John Curtin School of Medical Research, The Australian National University. The graphical abstract was created with BioRender.com. This work was performed on the traditional lands of the Ngunnawal and Ngambri people. Author contributions A.S., T.L. and S.O. conceived the study. A.S. carried out the investigation and collected the data. A.S. and M.N. analysed the proteomics data, and A.S. and D.W. analysed the OTR data; A.S. analysed all remaining data. J-W.L. acquired the proteomic data and did the preliminary proteomic analysis. A.S. wrote the manuscript. S.O. and T.L. contributed to the reviewing and editing the manuscript and supervised the research. All the authors read and approved the final manuscript. Funding This research was supported by Nourish Ingredients (Canberra, Australia). Ethics Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Billerbeck S, Walker RSK, Pretorius IS. Killer yeasts: expanding frontiers in the age of synthetic biology. Vol. 42, Trends in Biotechnology. Elsevier Ltd; 2024. p. 1081–96. Séguy N, Cailliez JC, Polonelli L, Dei-Cas E, Camus D. 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Available from: https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2020.589468 Walker RSK, Pretorius IS. Applications of yeast synthetic biology geared towards the production of biopharmaceuticals. Vol. 9, Genes. MDPI AG; 2018. Prins RC, Billerbeck S. The signal peptide of yeast killer toxin K2 confers producer self-protection and allows conversion into a modular toxin-immunity system. Cell Rep. 2024 Jul 23;43(7). Wemhoff S, Klassen R, Meinhardt F. Site-directed mutagenesis of the heterotrimeric killer toxin zymocin identifies residues required for early steps in toxin action. Appl Environ Microbiol. 2014;80(20):6549–59. Madika A, Suri A, Purohit A, Van Raad D, Norman M, Hartley CJ, et al. PYEAST – Python Enabled Automated Strain Transformation. bioRxiv [Internet]. 2025 Jan 1;2025.05.19.655004. Available from: http://biorxiv.org/content/early/2025/05/21/2025.05.19.655004.abstract Steel H, Habgood R, Kelly C, Papachristodoulou A. In situ characterisation and manipulation of biological systems with Chi.Bio. PLoS Biol. 2020 Jul 30;18:e3000794. Robinow CF. Chapter 1 The Preparation of Yeasts for Light Microscopy. In: Prescott DM, editor. Methods in Cell Biology [Internet]. Academic Press; 1975. p. 1–22. Available from: https://www.sciencedirect.com/science/article/pii/S0091679X08603143 Hu KKY, Suri A, Dumsday G, Haritos VS. Cross-feeding promotes heterogeneity within yeast cell populations. Nat Commun [Internet]. 2024;15(1):418. Available from: https://doi.org/10.1038/s41467-023-44623-y Powell CD, Quain DE, Smart KA. Chitin scar breaks in aged Saccharomyces cerevisiae. Microbiology (N Y) [Internet]. 2003;149(11):3129–37. Available from: https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.25940-0 Additional Declarations No competing interests reported. Supplementary Files SupplementaryToxicandUsefulHarnessingyeastkillertoxinsforproductrecovery.docx Schematic.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 24 Mar, 2026 Reviews received at journal 19 Mar, 2026 Reviews received at journal 17 Mar, 2026 Reviewers agreed at journal 17 Mar, 2026 Reviewers agreed at journal 17 Mar, 2026 Reviewers invited by journal 15 Mar, 2026 Editor assigned by journal 20 Feb, 2026 Submission checks completed at journal 20 Feb, 2026 First submitted to journal 17 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8904913","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":606493709,"identity":"91e6186a-f32d-4969-8560-7a6b32971a40","order_by":0,"name":"Ankita Suri","email":"","orcid":"","institution":"Commonwealth Scientific and Industrial Research Organisation","correspondingAuthor":false,"prefix":"","firstName":"Ankita","middleName":"","lastName":"Suri","suffix":""},{"id":606493710,"identity":"8edb47b4-19d4-40b5-a91a-36a6e2885026","order_by":1,"name":"Michael Norman","email":"","orcid":"","institution":"Commonwealth Scientific and Industrial Research Organisation","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Norman","suffix":""},{"id":606493711,"identity":"1bc6dce0-c3f9-41a2-b47e-d14a40b25e40","order_by":2,"name":"David Wollborn","email":"","orcid":"","institution":"Commonwealth Scientific and Industrial Research Organisation","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Wollborn","suffix":""},{"id":606493712,"identity":"59d2ea78-a1fd-4e38-9804-e1749570af26","order_by":3,"name":"Thomas D. Loan","email":"","orcid":"","institution":"Australian National University","correspondingAuthor":false,"prefix":"","firstName":"Thomas","middleName":"D.","lastName":"Loan","suffix":""},{"id":606493715,"identity":"8621fd0a-2cbc-4df7-a0a8-8d8e2dea17d8","order_by":4,"name":"Jian-Wei Liu","email":"","orcid":"","institution":"Commonwealth Scientific and Industrial Research Organisation","correspondingAuthor":false,"prefix":"","firstName":"Jian-Wei","middleName":"","lastName":"Liu","suffix":""},{"id":606493720,"identity":"4648c646-6fb1-4dd6-ae23-205fd700ab54","order_by":5,"name":"Shoko Okada","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2UlEQVRIiWNgGAWjYDACCQZmEGXAwJDA+ADEJUkLswHJWtiIUc/AID+7+bDBBwYbY/725GcVP/5Y5PMzMD/Dq9fgzrHkxBkMaWYSZ56Z3extk7Cc2cBmhl+LRI7xYR6GwzYMNxLMbjM2SBgYHGDAr0V+BlSL/I30b8UMf0Ba2L/h99SNHONkoBYzgxs5ZswMbCAtPAQcdiMt2XCGQZqx4Zk3xZJAvxhINvMUW+B3WPJhiQ8VNobzjqdv/PDjT50BP3v7xht4HQaxC5nDTFj9KBgFo2AUjAICAADXR0F+zEPJ4gAAAABJRU5ErkJggg==","orcid":"","institution":"Commonwealth Scientific and Industrial Research Organisation","correspondingAuthor":true,"prefix":"","firstName":"Shoko","middleName":"","lastName":"Okada","suffix":""}],"badges":[],"createdAt":"2026-02-18 01:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8904913/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8904913/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104774201,"identity":"8ff6e166-ca81-4475-a3b2-9e2260a99752","added_by":"auto","created_at":"2026-03-17 06:16:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":189837,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth inhibition of toxin-expressing strains assessed by spot assay and growth kinetics. A Serial dilution spot assay of S. cerevisiae BY4741 expressing\u0026nbsp;different yeast killer toxins. Cultures were serially diluted 10-fold (10⁻² to 10⁻⁶) and spotted onto selective SCI agar plates. Plates were incubated at 30\u0026nbsp;°C or 37\u0026nbsp;°C for72 h. B Growth kinetics of S. cerevisiae expressing different killer toxins. Cultures were induced in SC induction medium, and optical density (O.D.₆₀₀\u003csub\u003enm\u003c/sub\u003e) was monitored over 24 h. Curves represent the mean ± SD of biological replicates.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8904913/v1/7089d2b2e3cc99e5e0406a66.png"},{"id":104783206,"identity":"89626ce0-72b0-413d-b009-7813bde9bf4b","added_by":"auto","created_at":"2026-03-17 07:58:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":156514,"visible":true,"origin":"","legend":"\u003cp\u003eOxygen transfer rates and cumulative oxygen transfer for six different S. cerevisiae strains cultivated in 96-well square well plates in replicates, n=4. The error is displayed as shadow in the respective colour. Cells were inoculated into SCI media at a starting O.D.₆₀₀\u003csub\u003enm\u003c/sub\u003e of 0.2. Temperature 30 °C, shaking speed 300 rpm, shaking diameter 50mm. A/M empty vector control strain, B/E producing Hm-1, C/F producing HSK, G/J producing Zygocin, H/K producing truncated (trunc) K2, I/L producing Zymocin.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8904913/v1/9c6bc500bbfa94f236a071cd.png"},{"id":104783198,"identity":"096cc881-f37b-47f9-9551-9406794218f6","added_by":"auto","created_at":"2026-03-17 07:58:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":28586,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of cell membrane permeability of S. cerevisiae strains expressing different killer toxins. Bar represents the mean percentage of cells stained with SYTOX™ Green (mean± SD, n=3) relative to the heat-treated empty vector strain set at 100%. * represents significance of each toxin compared to the empty-vector control strain without heat-treatment (p value \u0026lt;0.05; n=3).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8904913/v1/d75c0258cc3861293fdc4048.png"},{"id":104782603,"identity":"31bcb591-d7ab-446d-8c17-0a67e86e940b","added_by":"auto","created_at":"2026-03-17 07:57:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":423203,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8904913/v1/24325dd77880515c008bdcfc.png"},{"id":104782573,"identity":"7c4a6d46-9973-4f87-97c9-1debb9507fe7","added_by":"auto","created_at":"2026-03-17 07:57:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":75463,"visible":true,"origin":"","legend":"\u003cp\u003eProteomic profiling reveals distinct secretome alterations in toxin-expressing yeast strains. A Percentage of protein abundance that is contributed by intracellular proteins B Total number of distinct intracellular proteins secreted based on Gene Ontology (GO) Cellular Component terms in toxin-expressing strains (mean± SD, n=3).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8904913/v1/5d371050cd547eb948e14250.png"},{"id":104808656,"identity":"d51ea290-802e-4593-8e6d-31e4fc68f58b","added_by":"auto","created_at":"2026-03-17 12:39:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":53762,"visible":true,"origin":"","legend":"\u003cp\u003emCherry fluorescence in culture supernatant. A mCherry fluorescence measured in the culture supernatant of empty vector control and toxin-expressing yeast strains in the absence of mechanical cell disruption. B mCherry fluorescence measured in the culture supernatant after bead-milling and C mCherry fluorescence measured in the culture supernatant after freeze-thaw treatment of yeast cell cultures. Data represent mean ± SD from three biological replicates (n=3). Statistical analysis used in panel A - C used Welch’s one way ANOVA followed by Games-Howell comparison against control strain; where * represents p \u0026lt;0.05 and † represents 0.05 ≤ p \u0026lt; 0.10.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8904913/v1/fccc9fd69657ade5ed346bde.png"},{"id":104809797,"identity":"6187d7f7-76a5-4d37-810b-0cefa3f74287","added_by":"auto","created_at":"2026-03-17 12:53:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1321054,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8904913/v1/8629ed74-31dd-4ca4-8e58-5226cf2ee658.pdf"},{"id":104808583,"identity":"d5f389a1-44b7-42ad-bdb1-033e18cec81c","added_by":"auto","created_at":"2026-03-17 12:38:51","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3609632,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryToxicandUsefulHarnessingyeastkillertoxinsforproductrecovery.docx","url":"https://assets-eu.researchsquare.com/files/rs-8904913/v1/5d2964827dc7dd7403e283af.docx"},{"id":104783344,"identity":"676b0395-5862-48fc-8d09-1a982ef994a1","added_by":"auto","created_at":"2026-03-17 07:58:42","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":120824,"visible":true,"origin":"","legend":"","description":"","filename":"Schematic.docx","url":"https://assets-eu.researchsquare.com/files/rs-8904913/v1/05cfaa74fc4adf878d0815d3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Toxic and Useful: Harnessing yeast killer toxins for product recovery","fulltext":[{"header":"Background","content":"\u003cp\u003eMany yeast species, collectively known as \u0026lsquo;killer yeasts\u0026rsquo; secrete protein toxins that either inhibit or kill susceptible fungi while protecting themselves from the effect of their own toxin through immunity domains coded on the protein. To date, a wide variety of killer toxins have been identified across different genera of yeast including \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, \u003cem\u003eKluyveromyces lactis\u003c/em\u003e and \u003cem\u003eZygosaccharomyces bailii\u003c/em\u003e, which exhibit remarkable diversity in their molecular size, mode of action, and stability under varying pH and thermal conditions. These toxins can range from small peptides to multimeric protein complexes and affect susceptible fungi through pore formation in the plasma membrane, enzymatic degradation of the cell wall, cell cycle inhibition, or protein synthesis disruption. The structural, genetic and functional diversity of these toxins has been comprehensively reviewed recently by S. Billerbeck et al(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo date the use of protein toxins in industrial applications has been limited to biocontrol, including inhibiting growth of spoilage microorganisms in food and beverage industries to improve food safety, development of antifungal therapeutics for clinical and veterinary applications and protection of plants against fungal pathogens(\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6 CR7 CR8 CR9\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Beyond these conventional roles, the use of toxins in other biotechnological applications remains limited. One unexplored application is the use of their membrane permeabilisation properties to improve intracellular product release.\u003c/p\u003e \u003cp\u003eCurrently, recovery of intracellular products from yeast heavily relies on either mechanical disruption techniques such as bead milling, high-pressure homogenization, ultrasonication or chemical disruption methods such as lysis with solvents, detergents, or alkali treatment (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). While these methods can efficiently lyse cells, they are time- and energy-intensive, incur high capital and operating costs and can potentially damage sensitive target molecules (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Development of new techniques such as hydrodynamic cavitation or the combined use of established techniques aim to mitigate these drawbacks but remain constrained by their own challenges in scalability, cost and efficiency (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eYeast killer toxins that compromise cell wall or plasma membrane integrity could offer a novel biological strategy to enhance product recovery by \u0026lsquo;priming\u0026rsquo; the cells for lysis. Toxins such as HM-1 from \u003cem\u003eCyberlindnera mrakii\u003c/em\u003e and HSK from \u003cem\u003eC. saturnus\u003c/em\u003e inhibit β-glucan synthases and impede cell wall construction(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Zymocin from \u003cem\u003eKluyveromyces lactis\u003c/em\u003e has chitinase activity, while K2 from \u003cem\u003eS. cerevisiae\u003c/em\u003e satellite virus M2 and Zygocin from \u003cem\u003eZygosaccharomyces bailii\u003c/em\u003e disrupt the plasma membrane(\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). By targeting distinct layers of the cell envelope, killer toxins create a controlled weakened state that may facilitate efficient release of intracellular products. By targeting the cell envelope of susceptible microorganisms, these toxins compromise cellular integrity, increasing susceptibility to intracellular leakage and ultimately, could enhance product recovery.\u003c/p\u003e \u003cp\u003eBaker's yeast (\u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e) is one of the most widely used microbial cells to produce an array of products ranging from biopharmaceuticals to food grade ingredients. \u003cem\u003eS. cerevisiae\u003c/em\u003e is genetically tractable, robust, fast growing and possesses well-characterised cellular machinery capable of synthesising complex proteins, lipids, and other natural products(\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). However, like most yeast, it has a tough multilayered cell wall that creates a significant bottleneck in product recovery and downstream processing.\u003c/p\u003e \u003cp\u003eIn the present study, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae\u003c/em\u003e was engineered to express different yeast killer toxins and the effects of toxin expression on cell physiology and cell wall integrity were investigated. We evaluate the potential of yeast killer toxins as novel strategy for enhancing product recovery based on our findings presented here.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eStrains and plasmids\u003c/p\u003e \u003cp\u003eAll strains employed in this study have been developed from the parent \u003cem\u003eS. cerevisiae\u003c/em\u003e strain BY4741 (genotype: MATa \u003cem\u003ehis3Δ1 leu2Δ0 met15Δ0 ura3Δ0\u003c/em\u003e) (ATCC no. 201388). The five different yeast killer toxins expressed separately in BY4741 were killer toxin K2 (UniProt: A0A291FEA7) from \u003cem\u003eSaccharomyces\u003c/em\u003e killer virus M2-4, HM-1 (UniProt: P10410) from \u003cem\u003eCyberlindnera mrakii\u003c/em\u003e, HSK (UniProt: Q00948) from \u003cem\u003eCyberlindnera saturnus\u003c/em\u003e, Zygocin (UniProt: Q8NJU1) from \u003cem\u003eZygosaccharomyces bailii\u003c/em\u003e, Zymocin (UniProt: P09805) from \u003cem\u003eKluyveromyces lactis\u003c/em\u003e. The native signal peptide of the K2 killer toxin was replaced with truncated α mating factor to remove its immunity domain (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) and Zymocin was reduced to only its endogenous signal peptide and chitinase activity α domain (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). For assessing recombinant protein recovery from yeast supernatant, the different toxin strains were co-transformed with plasmid containing fluorescent reporter protein mCherry (UniProt: X5DSL3) under constitutive promoter TDH3. All genes were codon optimised for \u003cem\u003eS. cerevisiae\u003c/em\u003e using IDT codon optimisation tool. The Kozak sequence AAACA was introduced at the 5\u0026rsquo; end of the start codon to enhance expression. Plasmids were designed and assembled in silico using the PYEAST digital toolkit(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The plasmids employed in this study have been summarised in Tables S1.\u003c/p\u003e \u003cp\u003eMedia and culture conditions\u003c/p\u003e \u003cp\u003eChemicals were purchased from Sigma Aldrich unless indicated otherwise. BY4741 was cultivated in YPD media containing yeast extract (10 g/L), peptone (20 g/L) and dextrose (20 g/L) prior to transformation. Toxin-containing plasmids were constructed in yeast with DNA fragments flanked by overlapping sequences to enable assembly by homologous recombination \u003cem\u003ein vivo.\u003c/em\u003e Transformation with the empty vector (pESC-His) and the DNA fragment mix was carried out using a yeast transformation kit, following the manufacturer's instructions. The resulting transformants were maintained on yeast synthetic complete (SC) agar plates containing yeast nitrogen base without amino acids (6.7 g/L) dextrose (20 g/L), agar (20 g/L) and yeast synthetic dropout medium supplements (1.92 g/L) (Merck, USA) without uracil and/or histidine as required.\u003c/p\u003e \u003cp\u003eChi.Bio reactors (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) (University of Oxford) were used to measure growth kinetics. The control strain (BY4741 containing empty vector pESC-His) and strains containing the toxin genes were initially grown in 7 ml SC medium containing dextrose (SCD medium) in 50 ml tubes (CELLSTAR\u0026reg; CELLREACTOR Tube) at 30\u0026deg;C, at 250 rpm for 16\u0026ndash;18 h to attain exponential phase. Cultures were then diluted to an O.D.\u003csub\u003e600nm\u003c/sub\u003e value of 0.2\u0026ndash;0.4 in 25 ml SC induction (SCI) medium containing galactose (20 g/L) and raffinose (10 g/L) for toxin expression and incubated at 30\u0026deg;C, with maximum stirring speed for 24 h in Chi.Bio reactors prior to harvesting.\u003c/p\u003e \u003cp\u003eSpot assay\u003c/p\u003e \u003cp\u003eBY4741 strains containing pESC-His or toxin genes were grown in SCD medium at 30\u0026deg;C, 250 rpm for 16\u0026ndash;18 h. Cultures were normalised to an equal O.D.\u003csub\u003e600nm\u003c/sub\u003e of 12 and 10-fold serial dilutions were prepared in SCI medium. To assess the temperature range of the toxins, 5 \u0026micro;L aliquots of dilutions from 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e to 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e were spotted on SCI medium agar plates and incubated for 3 days at either 30\u0026deg;C or 37\u0026deg;C. To assess the pH range, 5 \u0026micro;L from these dilutions were spotted on SCI medium agar plates with adjusted to 4.6 or 5.5 and incubated at 30\u0026deg;C for 3 days.\u003c/p\u003e \u003cp\u003eOxygen transfer rate measurements\u003c/p\u003e \u003cp\u003eOxygen transfer rate was measured continuously in a 96 well microtiter plate using a prototype micro-scale Transfer rate Online Measurement device (microTOM), manufactured by Kuhner shaker (Switzerland). Strains were inoculated into SCI medium at an initial O.D.\u003csub\u003e600nm\u003c/sub\u003e of 0.2 and grown at 30\u0026deg;C for 36 h at 300 rpm and 50 mm shaking diameter.\u003c/p\u003e \u003cp\u003eFor all other cell physiology studies and co-transformation study, the strains were first grown in SCD medium at 30\u0026deg;C, 250 rpm for 16\u0026ndash;18 h to accumulate sufficient biomass. Overnight cultures were harvested by centrifugation and then resuspended in 20 ml SCI medium for toxin expression and incubated at 30\u0026deg;C, 250 rpm for 18 h before analysis.\u003c/p\u003e \u003cp\u003eFluorescence staining\u003c/p\u003e \u003cp\u003eFollowing incubation in SCI medium for 18 h, 1 ml of yeast cultures were pelleted and washed with Hank\u0026rsquo;s buffered salt solution (HBSS, Thermo Fisher Scientific). The samples were adjusted to O.D.\u003csub\u003e600nm\u003c/sub\u003e of 0.5 in HBBS and SYTOX\u0026trade; Green (Thermo Fisher Scientific) was added at final concentration of 1 \u0026micro;M. The yeast samples were incubated in the dark at room temperature for 20 min without shaking. After incubation, the samples were washed twice with HBBS to remove excess dye and resuspended in HBSS prior to flow cytometric analysis.\u003c/p\u003e \u003cp\u003eFlow cytometric analysis\u003c/p\u003e \u003cp\u003eFlow cytometric analysis with SYTOX\u0026trade; Green-stained BY4741 strains was performed on a LSR II flow cytometer (BD Biosciences). Heat-treated cells (100\u0026deg;C, 5 min) transformed with an empty vector were included as a positive control, representing maximal membrane permeability (100%). Yeast cells were differentiated based on forward scattering (FSC-A) and side scattering (SSC-A) signals. Fluorescent signals were acquired using a 488 nm excitation laser and 530/30 bandpass (BP) collection filter. Samples were recorded at a flow rate of 12.5 \u0026micro;L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Subsequently, the acquired data was analysed using FlowJo software v10.8 (FlowJo LLC). Cell samples were normalised to 10 000 events and cell debris and doublets were gated out of the flow cytometry data.\u003c/p\u003e \u003cp\u003eScanning Electron Microscopy (SEM)\u003c/p\u003e \u003cp\u003eYeast cells cultured in SCI medium for 18 h were pelleted and washed with Milli-Q water to remove residual media components. The pellet was resuspended in freshly prepared formaldehyde-alcohol-acetic acid (FAA) fixative solution with the composition: 10% formalin (37\u0026ndash;40% w/v formaldehyde), 5% glacial acetic acid, 50% ethanol, and 35% distilled water (v/v/v/v)(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Samples were gently agitated for 1 h on a shaker at room temperature. Following fixation, cells were subjected to an ethanol dehydration gradient consisting of 10%, 30%, 50%, 70%, 90%, and 100% ethanol (v/v). At each gradient, samples were incubated for 15 minutes at room temperature before progressing to the next concentration. The final dehydration step involved two washes in 100% anhydrous ethanol for 15 min each. Following dehydration, samples were mounted onto SEM stubs with silica chips and coated with approximately 10 nm layer of gold using a sputter coater (Quorum MiniQ S).\u003c/p\u003e \u003cp\u003ePrepared samples were imaged using a scanning electron microscope (ZEISS UltraPlus analytical FESEM) at an accelerating voltage of 5 kV.\u003c/p\u003e \u003cp\u003eSample preparation for proteomic analysis\u003c/p\u003e \u003cp\u003eAfter 16 h incubation in SCI medium, 400 \u0026micro;l of culture supernatant from BY4741 strains containing pESC-His or toxin genes was collected. Proteins in the culture supernatant were precipitated with 1600 \u0026micro;l cold acetone at -20\u0026deg;C overnight. The precipitated pellet was air-dried and resuspended in 40 \u0026micro;l 8M urea. Protein solution (10 \u0026micro;l) was reduced using 1 \u0026micro;l of 15% (w/v) DTT at room temperature for 30 min and subsequently alkylated with 1 \u0026micro;l of 40% acrylamide and incubated at room temperature for 30 min. Digestion was performed by adding 47 \u0026micro;l of trypsin solution (0.2 \u0026micro;g of trypsin in 25 mM ammonium bicarbonate) and incubating at 37\u0026deg;C for 3 h. Digestion was stopped by adding 1 \u0026micro;l of 10% formic acid. Samples were passed through a 0.22 \u0026micro;m filter before liquid chromatography mass spectrometry (LC-MS) analysis.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS proteomic analysis\u003c/h2\u003e \u003cp\u003eTryptic peptides were separated on a nano column (Aurora Elite\u0026trade; XT 15\u0026times;75 C18 UHPLC column, Ionopticks) on UltimateTM 3000 RSLC nano LC system (Thermo Fisher Scientific). Mobile phase A comprised of water with 0.1% (v/v) formic acid and mobile phase B consisted of 80% (v/v) acetonitrile with 0.8% (v/v) formic acid. Tryptic peptides were separated on a gradient of 8\u0026ndash;30% solvent B for 30 min, 30% \u0026ndash; 50% solvent B for 8 min and 50% \u0026ndash; 97.5% for 6 min. The eluted peptides were ionised with using a Nanospray Flex Ion Source (Thermo Fisher Scientific) at 1.8 kV with capillary temperature 300\u0026deg;C and analysed using Orbitrap Fusion MS (Thermo Fisher Scientific) operated in top speed mode with a 3 s cycle for both the MS and MS/MS scans. Full MS survey scans of peptide precursors were acquired from scan range of 400 to 1500 m/z at a resolution of 120,000 (at 200 m/z) with an automatic gain target (AGC) set as 4 x 10\u003csup\u003e5\u003c/sup\u003e and a maximum injection time of 50 ms. Most abundant precursors (charge state 2\u0026thinsp;+\u0026thinsp;\u0026minus;\u0026thinsp;7\u0026thinsp;+\u0026thinsp;with intensity greater than 1 x 10\u003csup\u003e5\u003c/sup\u003e) were isolated in the quadrupole (1.6 m/z window) and fragmented by high- energy collision dissociation with collision energy of 28%. Fragments were detected in ion trap detector with AGC target of 4 x 10\u003csup\u003e3\u003c/sup\u003e, with a maximum injection time of 300 ms and a dynamic exclusion on 15 s.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProteomic data analysis\u003c/h3\u003e\n\u003cp\u003eProteins/ peptides were identified and their relative abundance was quantified using Proteome Discoverer 2.2 (Thermo Fisher Scientific) and Sequest HT search engine. The spectrum data was searched against recombinant killer toxins, mCherry and \u003cem\u003eS. cerevisiae\u003c/em\u003e proteome database (Proteome ID: UP000002311_559292). Precursor mass tolerance was set to 10 ppm and product ions were searched at 0.6 Da. Three missed tryptic cleavages were allowed. Variable modification included oxidation (+\u0026thinsp;15.995 Da), deamidation (+\u0026thinsp;0.984 Da), amidation (\u0026minus;\u0026thinsp;0.984 Da), and propionamidation (+\u0026thinsp;71.037 Da). Peptide spectral matches were validated using the Percolar algorithm, based on q-values and 1% False Discovery Rate (FDR). Relative abundance is calculated from precursor abundance intensity and is normalized from total peptide amount. Downstream data analysis was performed in R studio where the data was filtered to retain proteins with High combined FDR confidence, classified as Master proteins, with more than 1 unique peptide and \u0026lsquo;High\u0026rsquo; found status.\u003c/p\u003e \u003cp\u003eMechanical Disruption and Spectrophotometric analysis\u003c/p\u003e \u003cp\u003eBY4741 strains containing pESC-His or toxin genes co-transformed with mCherry were grown in SCI medium for 18 h, harvested, washed and resuspended in HBSS buffer to an O.D.\u003csub\u003e600nm\u003c/sub\u003e of 0.5. Mechanical disruption was achieved using either bead-milling or freeze-thawing, as specified below:\u003c/p\u003e \u003cp\u003eFor bead-milling, acid-washed glass beads (422\u0026ndash;600 \u0026micro;m) were added to samples. The samples were subjected to mechanical disruption using Ratek multi tube vortex mixer (Model # MTV1) for 20 min with intermittent cycle of 35 seconds on / 5 seconds off, at a speed setting of 7.5 (instrument dial setting). For freeze-thawing, the samples were frozen at -80\u0026deg;C and subsequently thawed rapidly at 37\u0026deg;C. Following disruption by either method, the samples were centrifuged at 1000 x g for 10 min at room temperature and the supernatant was carefully collected. Cellular debris from the supernatant was removed by filtration using a 0.22 \u0026micro;m pore size filter. mCherry fluorescence in filtered supernatant samples were then analysed using Fluostar Omega spectrophotometer (BMG LABTECH). For each assay, 100 \u0026micro;L of supernatant samples were aliquoted into a black, flat-bottom 96 well plate (Costar #3916). HBSS buffer was used as a blank for background fluorescence correction. mCherry fluorescence was measured using excitation and emission wavelengths of 585 nm and 620 nm, respectively. The instrument gain was optimised prior to data acquisition to ensure the signals were within the detector\u0026rsquo;s linear range. All samples and blanks were measured in triplicates, and the analyses were performed at room temperature.\u003c/p\u003e \u003cp\u003eData Processing and Analysis\u003c/p\u003e \u003cp\u003eAll statistical analysis, data processing, and visualization were conducted using a combination of Python (version 3.12.9) and R (version 4.4.2). Statistical tests such as ANOVA, Tukey\u0026rsquo;s, Games-Howell and Welch\u0026rsquo;s t-test were conducted based on data distribution using standard libraries and packages (Pandas, NumPy, statsmodels and SciPy for Python and lmFit, limma, eBayes for R). Data visualisation was performed using Matplotlib seaborn and ggplot2. Custom scripts were developed and executed in Jupyter Notebook and RStudio environments. Each experiment had 3 biological replicates, and the statistical significance threshold was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eAssessing cytotoxicity of yeast expressing killer toxins\u003c/p\u003e \u003cp\u003eThe growth effect of toxins expressed under the control of the \u003cem\u003eGAL1\u003c/em\u003e promoter was evaluated through a 10-fold serial-dilution spot assay on SCI agar medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). All toxin-expressing strains displayed growth inhibition at lower dilution compared to the empty-vector control at 30\u0026deg;C, which showed the colony formation up to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e dilution. HM-1- and HSK-producing strains exhibited complete growth inhibition at 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e dilution, whereas truncated K2 showed visible impact on growth at 10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e dilution, and zygocin- and zymocin-producing strains showed visible growth suppression at 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e dilution.\u003c/p\u003e \u003cp\u003eTo assess the functional range of these toxins in terms of growth temperature and pH, spot assays were repeated at 37\u0026deg;C and at pH 4.4 or 5.5. All toxins except zymocin exhibited the same growth inhibitory profile at 37\u0026deg;C as at 30\u0026deg;C, indicating temperature stability of the effect of the toxins between 30\u0026deg;C and 37\u0026deg;C. Zymocin showed enhanced potency, with complete growth inhibition at 10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e dilution at 37\u0026deg;C, suggesting optimal activity of this toxin at elevated temperature. pH 4.6 and 5.5 produced indistinguishable inhibition patterns, demonstrating broad pH tolerance of these toxins (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). As the optimal growth temperature for \u003cem\u003eS. cerevisiae\u003c/em\u003e is 30\u0026deg;C, all subsequent experiments were carried out at this temperature.\u003c/p\u003e \u003cp\u003eGrowth kinetics of the toxin expressing strains further revealed toxin-dependent physiological effects on cellular proliferation in the presence of galactose (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The control strain had a relatively short lag phase and reached an O.D.\u003csub\u003e600nm\u003c/sub\u003e of 5.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 by 24 h with minimal replicate variability. In contrast, the zygocin-producing strain had a pronounced lag phase of ~\u0026thinsp;7 h and plateaued at a lower O.D.\u003csub\u003e600nm\u003c/sub\u003e of 3.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24, indicating moderate stress. Both zymocin and truncated K2 strains exhibited further extended lag phases of ~\u0026thinsp;8 h and 14 h, before achieving O.D. \u003csub\u003e600nm\u003c/sub\u003e 1.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 and 1.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 respectively, suggesting considerable stress on the cells, consistent with the absence of cognate immunity factors that normally protect killer strains from their own toxins (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). HM-1 and HSK strains remained essentially quiescent throughout the 24 h, with O.D. \u003csub\u003e600nm\u003c/sub\u003e values below 0.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, indicating near-complete growth inhibition, which aligned with the spot assay results. The spot assay and growth kinetics demonstrated that intracellular expression of the killer toxins in toxin-sensitive BY4741 yeast exerted varying degrees of antiproliferative effects, establishing that each toxin preserved its characteristic biological activity when expressed in a heterologous host, consistent with its known killer activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMetabolic activity of different toxin expressing strains was compared by continuously measuring oxygen transfer rate (OTR) over a 36 h period. OTR is a suitable parameter to monitor the physiological state of a microbial culture, since metabolic activity in aerobic cultivations is reflected in oxygen consumption. The OTR and cumulative oxygen transfer (COT) revealed notable differences between the control strain and toxin expressing strains. The control strain grew exponentially for 18 h, reaching a peak OTR of 4.2 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and declining to 2 mmol L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e by 23 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Strains expressing HM-1 and HSK showed negligible OTR over 36 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C,\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F), consistent with their impaired growth caused by toxin expression. Truncated K2, zygocin and zymocin expressing cells exhibited delayed growth and reduced OTRs, relative to the control, indicating a pronounced effect of toxin expression. Rare survivors emerging by toxin resistance or gene silencing by mutation or plasmid loss likely contribute to the observed late-stage OTR increase, which was not investigated further in the current study (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). These OTR profiles showed that intracellular toxin expression imposes toxin-dependent constraints on the overall respiratory metabolism that align with growth defects observed in growth kinetics and antiproliferative characteristics demonstrated in the growth assays.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eEffect of toxin expression on cell membrane integrity\u003c/p\u003e \u003cp\u003eThe impact of different toxins on the cell membrane integrity was evaluated with SYTOX\u0026trade; Green, which only enters cells with compromised membranes, coupled with flow cytometric analysis. Heat-treated empty vector control cells served as a positive control, that demonstrated complete (100%) membrane permeability. The percentage of BY4741 cells with compromised cell membranes was significantly different between the empty vector control strain and the strains expressing different toxins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Strains expressing HSK and HM-1 exhibited elevated SYTOX\u0026trade; Green uptake with ~\u0026thinsp;81.3% and 58% of the population staining positive. This corresponded to 20-fold and 14-fold increase in membrane permeability relative to control strain, reflecting their known inhibition of β-glucan synthase (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Zygocin also caused significant membrane damage, with 70.9% of the cell population staining positive (17-fold increase compared to control), attributed to its ability to form pores in the cell membrane (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). In comparison, truncated K2 and zymocin caused moderate but statistically significant cell membrane damage, as indicated by 30% and 16.2% SYTOX\u0026trade; Green positive cells (7-fold and 4-fold increase relative to the control strain). One-way ANOVA confirmed significant impact of toxins on membrane permeability and post hoc Welch\u0026rsquo;s t-test demonstrated that the percentage of SYTOX\u0026trade; Green positive cells of each toxin strain significantly differed from that of the empty vector control strain without heat-treatment (\u003cem\u003ep\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe morphological effect of toxins on yeast cells was observed using Scanning Electron Microscopy (SEM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The empty vector control strain had the classic budding yeast morphology with cells retaining their smooth ovoid shape, characteristic bud and birth scars with little evidence of cell collapse or cell shrinkage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). In contrast, cells expressing different toxins exhibited varying degrees of morphological change. HSK and zygocin expressing cells exhibited extensive cell damage, characterised by regional invaginations and structural collapse along with scattered cellular debris, which are hallmarks of cytosolic content release (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB \u0026amp; E), suggesting that these strains exhibited higher functional toxin activity. HM-1-expressing strain showed moderate cell damage, with smaller serrations and cellular debris (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Cells expressing truncated K2 and zymocin displayed cells with prominent surface wrinkling accompanied with minor tears in the cell wall and no detectable extracellular debris, consistent with their lower levels of membrane permeabilisation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD \u0026amp; F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProteomic profiling of the culture supernatant was performed to examine the consequences of toxin expression at the proteome level. Detected proteins were annotated by subcellular localization based on Gene Ontology (GO) Cellular Component (CC) terms, allowing supernatant protein abundance to be partitioned into intracellular and extracellular components (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In strains expressing HM-1, HSK, and zygocin, proteins normally localized intracellularly contributed more than 90% of total supernatant protein abundance, indicating that the leaked proteome was dominated by cytoplasmic components (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This strong enrichment of intracellular proteins in the supernatant is consistent with markedly increased membrane permeability, extensive membrane rupture, and large-scale structural failure of the cellular envelope (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Surprisingly, strains expressing truncated K2 and zymocin also showed higher contribution of intracellular proteins to total supernatant protein abundance, despite relatively less membrane permeability detected by flow cytometry, a potential consequence of low level cumulative release of proteins from structurally weakened cells. Toxin-induced leakage of cellular content was further characterised by quantifying the number of intracellular proteins detected in the supernatant. An increase in the number of intracellular proteins was detected in the supernatant of all toxin-expressing strains relative to that of the empty vector control strain, indicating enhanced membrane permeability and leakage of a wider variety of proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAssessing recombinant protein recovery from yeast cell culture supernatant\u003c/p\u003e \u003cp\u003eFollowing confirmation of toxin-driven cell membrane permeabilisation, each strain was co-transformed with the mCherry reporter gene and its release into the culture supernatant was quantified to assess the degree of toxin-driven protein leakage. Spectrophotometric analysis of filtered culture supernatant demonstrated varying levels of mCherry leakage in toxin expressing strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). The highest mCherry levels were observed in strains expressing toxins HSK and zygocin, which were consistent with previously measured cell membrane permeabilisation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The HM-1 strain showed intermediate mCherry concentration in the culture supernatant, while truncated K2 and zymocin showed significantly lower levels, parallelling their low membrane permeability. These patterns were further supported by proteomic detection of mCherry in the culture supernatant (Figure S2).\u003c/p\u003e \u003cp\u003eTwo mechanical cell disruption methods, bead-milling and freeze-thawing, were paired with toxin expression to evaluate their ability to improve mCherry release in the culture supernatant compared to empty vector control. Bead-milling produced a measurable increase in mCherry fluorescence in the supernatant of all strains. The expression of toxins increased overall mCherry fluorescence in the supernatant by 1.5-to-6-fold relative to control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). A similar trend was observed when the toxin-expressing strains were subject to freeze-thawing where mCherry release in the culture supernatant increased by 2-to-4.5-fold in comparison to control strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Even with strain-to-strain variation in mCherry expression (Figure S3), toxin-expressing strains with high cell membrane permeability exhibited a significant fold increase in mCherry release relative to empty vector control strain, whereas toxin-expressing strains with lower membrane permeability displayed a moderate yet significant fold change. These data aligned with the cell damage observed with respective toxins through SYTOX\u0026trade; screening and SEM analysis.\u003c/p\u003e \u003cp\u003eThese results indicate that toxin expression increases the cells\u0026rsquo; susceptibility to mechanical disruption, leading to dramatic increases in protein release through effects on cell membrane permeability. Notably, even strains with lower membrane permeability, such as strains expressing truncated K2 and zymocin, showed considerable increases in mCherry release when paired with mechanical disruption, compared to the empty vector control strain. This combinatorial approach provides a practical strategy to increase cell susceptibility to lysis and thereby improve intracellular product recovery in yeast by up to 6-fold.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, the experimental results demonstrate that toxin expression disrupts cell structure and exerts significant physiological effects. Cell membrane integrity assays and scanning electron microscopy reveal pronounced cell wall damage in strains exhibiting high membrane permeability, characterised by cell rupture, collapse, and leakage of intracellular content. Both high- and low-permeability strains displayed slower growth and increased extracellular protein leakage; however, strains with lower membrane permeability show comparatively less overt physical damage, suggesting intracellular stress and perturbation of cellular homeostasis. Mechanistically, these effects may arise from additional cellular processes, including potential overloading of the host secretory pathway due to toxin precursor processing through the endoplasmic reticulum and Golgi apparatus. In parallel, toxin-induced remodelling of the cell wall or trafficking machinery, through interactions with chitin or β-glucans could further influence protein export (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Taken together, these findings highlight the multifaceted impact of toxin expression on yeast cells, with damage ranging from overt structural compromise to more subtle intracellular dysfunction, thereby advancing our understanding of toxin-mediated cellular perturbations.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study showcases a novel strategy for harnessing yeast killer toxins in different biotechnological applications. The findings demonstrate the efficacy of these toxins as tools to increase cell membrane permeability that can improve intracellular product release. Expression of these toxins in \u003cem\u003eS. cerevisiae\u003c/em\u003e produced disruptive physiological effects similar to those observed when applied to susceptible yeast strains. Comprehensive analyses including membrane integrity assay, proteomics and scanning electron microscopy clearly established the impact of these toxins on yeast morphology, physiology and function. Co-expression with a fluorescent reporter protein further validated the effectiveness of this strategy, where toxin expression augmented the effect of mechanical disruption, increasing product release by up to 6-fold.\u003c/p\u003e \u003cp\u003eImportantly, the results also highlighted the versatility of this approach. Different toxins can be leveraged based on application requirements. Potent toxins can be employed where severe cell membrane disruption is needed. In contrast, milder toxins can be used for more controlled cell membrane permeabilisation, which, when combined with milder downstream disruption processes offer a strategy that is more focused on membrane weakening prior to collecting cells for product recovery, which may be advantageous for the recovery of products sensitive to harsher lysis conditions. Although this research demonstrated the use of toxins for product release, further optimisation is required. Fine-tuning toxin expression or employing combinatorial toxin strategy, could aid in precisely tailoring membrane permeabilisation to suit diverse bioprocessing requirements, potentially reducing the reliance on other disruption strategies.\u003c/p\u003e \u003cp\u003eOverall, this research unlocks the potential for yeast killer toxins as effective biological agents for programmable cell permeabilisation and provides a new avenue for enhancing intracellular product release across a range of industrial biotechnology applications.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this article are available in the CSIRO Data Access Portal, https://doi.org/10.25919/f4g8-qj41.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank Dr. Phil Hands at Black Mountain MicroImaging Centre, CSIRO for providing technical advice and guidance in using Scanning Electron Microscope and Microscopy Australia (ROR: 042mm0k03) at the Centre for Advanced Microscopy, The Australian National University, a facility enabled by NCRIS and university support. In addition, we acknowledge the expertise of Michael Devoy and Fei-Ju Li at Cytometry, Histology and Spatial Multiomics (CHASM) facility of the John Curtin School of Medical Research, The Australian National University. The graphical abstract was created with BioRender.com. This work was performed on the traditional lands of the Ngunnawal and Ngambri people.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.S., T.L. and S.O. conceived the study. A.S. carried out the investigation and collected the data. A.S. and M.N. analysed the proteomics data, and A.S. and D.W. analysed the OTR data; A.S. analysed all remaining data. J-W.L. acquired the proteomic data and did the preliminary proteomic analysis. A.S. wrote the manuscript. S.O. and T.L. contributed to the reviewing and editing the manuscript and supervised the research. All the authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Nourish Ingredients (Canberra, Australia).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBillerbeck S, Walker RSK, Pretorius IS. Killer yeasts: expanding frontiers in the age of synthetic biology. Vol. 42, Trends in Biotechnology. 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Available from: https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.25940-0\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Saccharomyces cerevisiae, Killer toxins, membrane integrity, product recovery","lastPublishedDoi":"10.21203/rs.3.rs-8904913/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8904913/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Saccharomyces cerevisiae is widely used in biotechnology to produce proteins and other biomolecules. Recovery of intracellular products, however, remains a key bottleneck for industrial applications. The robust cell wall demands energy-intensive chemical or mechanical disruption that increase processing costs and can damage sensitive targets.\u003c/p\u003e\n\u003cp\u003eResults: In this study, we investigated yeast killer toxins as a genetically encoded tool to enhance membrane permeability and facilitate intracellular product recovery. Five killer toxins with distinct modes of action were expressed intracellularly and their effects on cell survival, membrane permeability, susceptibility to lysis and enhancement of intracellular product recovery were examined. In combination with common mechanical disruption methods, toxin expression enhanced total intracellular protein recovery up to 6-fold relative to the control strain without a killer toxin expressed.\u003c/p\u003e\n\u003cp\u003eConclusion: These results establish killer toxin expression as a tuneable strategy for enhanced product recovery from yeast, that complements and augment conventional bioprocessing techniques.\u003c/p\u003e","manuscriptTitle":"Toxic and Useful: Harnessing yeast killer toxins for product recovery","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-17 06:16:27","doi":"10.21203/rs.3.rs-8904913/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-24T16:00:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-19T12:34:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-18T03:28:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291437330108295635744041093108848239676","date":"2026-03-18T01:59:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"100767051686192304104068768414215123817","date":"2026-03-18T01:34:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-16T00:12:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-20T06:55:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-20T06:50:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2026-02-18T00:49:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c33204c0-b570-4303-8367-9359ea01cfb1","owner":[],"postedDate":"March 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T13:10:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-17 06:16:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8904913","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8904913","identity":"rs-8904913","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}