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FEMS Yeast Res
FEMS Yeast Res
femsyr
FEMS Yeast Research
1567-1356
1567-1364
Oxford University Press

39270658
10.1093/femsyr/foae028
foae028
Research Article
AcademicSubjects/SCI01150
Isolation and characterization of Saccharomyces cerevisiae mutants with increased cell wall chitin using fluorescence-activated cell sorting
https://orcid.org/0000-0001-8414-0972
Chuene Lesiba Tyrone South African Grape and Wine Research Institute, University of Stellenbosch, Matieland, Postcode 7600, South Africa

Ndlovu Thulile South African Grape and Wine Research Institute, University of Stellenbosch, Matieland, Postcode 7600, South Africa

https://orcid.org/0000-0002-6266-0210
Rossouw Debra South African Grape and Wine Research Institute, University of Stellenbosch, Matieland, Postcode 7600, South Africa

https://orcid.org/0000-0002-5093-1327
Naidoo-Blassoples Rene Kathleen South African Grape and Wine Research Institute, University of Stellenbosch, Matieland, Postcode 7600, South Africa

https://orcid.org/0000-0001-5764-4542
Bauer Florian Franz South African Grape and Wine Research Institute, University of Stellenbosch, Matieland, Postcode 7600, South Africa

Corresponding author. South African Grape and Wine Research Institute, University of Stellenbosch, Matieland, Postcode 7600, South Africa. E-mail: fb2@sun.ac.za
2024
12 9 2024
12 9 2024
24 foae02818 7 2024
29 8 2024
11 9 2024
24 9 2024
© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Abstract

Yeast cell wall chitin has been shown to bind grape pathogenesis-related chitinases that are the primary cause of protein haze in wines, suggesting that yeast cell walls may be applied for haze protection. Here, we present a high-throughput screen to identify yeast strains with high cell wall chitin using a reiterative enrichment strategy and fluorescence-activated cell sorting of cells labelled with either GFP-tagged chitinase or Calcofluor white. To assess the validity of the strategy, we first used a pooled deletion strain library of Saccharomyces cerevisiae. The strategy enriched for deletion mutants with genes that had previously been described as having an impact on chitin levels. Genes that had not previously been linked to chitin biosynthesis or deposition were also identified. These genes are involved in cell wall maintenance and/or membrane trafficking functions. The strategy was then applied to a mutagenized population of a commercial wine yeast strain, S. cerevisiae EC1118. Enriched mutant strains showed significantly higher cell wall chitin than the wild type and significantly reduced the activity of chitinases in synthetic model wine, suggesting that these strains may be able to reduce haze formation in wine.

A new reiterative mutant enrichment strategy has been established and validated to isolate and identify high-chitin mutants, while potentially identifying new genes that have an impact on chitin content or deposition.

Saccharomyces cerevisiae
chitin
cell wall
mutagenesis
enrichment
chitinases
National Research Foundation of South Africa UID 83471 Royal Society 10.13039/501100000288
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pmcIntroduction

Yeast cell walls act as protective barriers against environmental perturbations and consist of interconnected layers of polymers and macromolecules, principally chitin, glucans, and mannoproteins, which provide structure and rigidity to the cell wall. The cell wall is responsible for the maintenance of cell shape, prevention of lysis, and the regulation of the uptake of substances from the environment (Hapala et al. 2013, Qiu et al. 2018). Yeast cell wall chitin, a nonbranched β-1,4-linked homopolymer of N-acetylglucosamine (GlcNAc) joined through glycosidic linkages, is a structural component of the cell wall. Together with β-1,3-linked glucan, chitin plays important roles in cellular development, structural morphogenesis, spore formation, and the maintenance of cell wall integrity (CWI; Vishukumar et al. 2017). Chitin metabolism and incorporation of chitin into the cell wall are spatially and temporally regulated during the cell and life cycles of yeast (Molon et al. 2018). Data also show that chitin is generally present in larger amounts in hyphal cell walls compared to planktonic yeast cells and may account for more than 2% of the cell wall’s dry weight in the latter (Latge 2010, Schiavone et al. 2014).

From a biotechnological perspective, yeast cell wall chitin has been linked to protein haze protection in wine, likely by binding and thereby reducing the concentration of chitinases, which are major contributors of haze (Marangon et al. 2011, Ndlovu et al. 2018, 2019). These chitinases are classified as grape-derived pathogenesis-related (PR) proteins, which are very stable and remain present in wine after processing. Of the PR proteins in wine, chitinases are, however, less stable when compared to other PR proteins such as thaumatin-like proteins (Albuquerque et al. 2021). Haze in wine is undesirable as it results in a milky or cloudy appearance of the wine and is unappealing to consumers (Van Sluyter et al. 2015). Based on findings by Ndlovu et al. (2019), chitinases bind to yeast cell wall chitin and are likely eliminated at the end of fermentation alongside the biomass, leading to haze reduction. It would therefore be desirable to select industrial wine yeast strains with high cell wall chitin for use in fermentations to remove chitinases and reduce wine haze formation. This would present a more cost-effective and environmentally sustainable alternative to traditional wine fining agents such as bentonite clay, which is expensive to use and dispose of (Van Sluyter et al. 2015, Cosme et al. 2020).

Here, we describe a strategy for the identification of yeast mutants with increased chitin levels. The strategy is based on labelling yeast populations with either Calcofluor white (CW), a chitin binding agent, or grape chitinases that have been tagged with green fluorescent protein (GFP). Labelled populations are subjected to flow cytometry and sorting of 5% of the population with the highest fluorescence (Black et al. 2011, Bernardo et al. 2014, González-Cabaleiro et al. 2017). The sorted 5% of the population is reinoculated into new growth medium, and again sorted at the end of the growth phase. This process is reiterated several times, after which randomly selected colonies are evaluated for cell wall chitin levels. The strategy was first evaluated using the EUROSCARF Saccharomyces cerevisiae pooled deletion mutant library. In a second approach, a commercial wine yeast strain (EC1118) was mutagenized using ethyl methanesulfonate (EMS) before undergoing the same procedure as described above.

After deletion library enrichment, strains with deletions in the genes IRC8, CTS1, TUS1, ACE2, and VTC3 were enriched. All of these strains indeed displayed significantly higher chitin than the wild type (WT). Several of these genes are involved in cell wall maintenance, cell wall remodelling, and membrane-related trafficking functions (Butler and Thiele 1991, Destruelle et al. 1994, Cohen et al. 1999, O’Conallain et al. 1999, Schmelzle et al. 2002, Alvaro et al. 2007). This served to validate the labelling and enrichment strategy employed here.

Similarly, the mutagenized wine yeast yielded mutants with variable, but significantly increased chitin levels when compared to the corresponding WT strain. The data showed that these strains were able to reduce chitinase activity levels when added to model wines containing chitinases, suggesting that the application of such strains in wine fermentations, either as live cultures or as additives (cell wall extracts/derivates of high-chitin mutants), is likely to reduce the risk of wine protein haze formation.The method of sequential enrichment described here can be applied to the study of other cell wall phenotypes of interest by screening genome-wide libraries or mutants using fluorescent dyes or protein, in combination with flow cytometry.

Materials and methods

Yeast strains

Experiments were performed using a pooled collection of the S. cerevisiae strain BY4742 EUROSCARF yeast deletion library [individually deleted in all nonessential Open Reading Frames (ORFs) identified in this organism; EUROSCARF, Frankfurt, Germany (Giaever and Nislow 2014; http://web.uni-frankfurt.de/fb15/mikro/euroscarf/); MATα; his3Δ1; leu2Δ0; lysΔ0; ura3Δ0] (≈6000) (Brachmann et al. 1998). The industrial strain S. cerevisiae EC1118® from Lallemand Inc., Montreal, QC, Canada, was used during this study as the parental commercial wine yeast.

Growth conditions

Pure cultures: Cells were stored at −80°C as glycerol stocks. Prior to use, cells were plated for 30–48 h on Wallerstein Laboratory nutrient agar, and then 5 ml precultures were grown overnight for 12 h until the mid-log growth phase had been reached, at a temperature of 30°C with agitation (50 rpm). Yeast peptone dextrose (YPD, pH 7.4) was used as a liquid medium. The optical density (OD600nm) was determined for each of the three biological repeats prior to inoculation into triplicate flasks containing 100 ml YPD broth. The starting inoculation density was at an OD600nm of 0.1 and was measured using a Jenway Genova 7206 spectrophotometer (Cole-Parmer Ltd).

Pool of mutants: Prior to enrichment, cells were grown in 5–10 ml YPD for at least 24–48 h. These 48-h cultures were used to inoculate 5 ml YPD to a starting OD600nm of 0.1 and the newly inoculated cells were cultured for 6–12 h. Following this, the same labelling procedure highlighted below was applied.

Labelling of cells with CW or GFP-tagged chitinase

Cells were stained with CW as described by de Groot et al. (2001). About 200 µl of the cell culture grown in YPD broth was centrifuged and the cells were washed with phosphate-buffered saline (PBS, pH 7.4; Na2HPO4). Cells were stained with 10 µl CW after the addition of 10 µl 10% KOH following the manufacturer’s instructions (Sigma–Aldrich, St. Louis, MO, USA). GFP-tagged chitinase was produced as described by Ndlovu et al. (2018). In short, grape berry chitinase class IVD fragment (accession number: AF532966.1) amplified from grape berry cDNA and GFP was polymerase chain reaction (PCR) amplified from pKEN mut 2 vector (Addgene, Cambridge, MA) were cloned into a shuttle vector pJET1.2/blunt (CloneJET™ PCR Cloning Kit, Thermo Fisher Scientific). The cassettes were then released from the pJET1.2/blunt™ and into the pET14b vector with the chitinase gene cloned upstream of the GFP gene. A protocol described by Lee and Coleman (2007) was used to overexpress the grape chitinase protein in Escherichia col i Rosetta 2(DE3) pLysS. The crude protein extract was concentrated using Amicon Ultra-15 Centrifugal Filter columns (Millipore™, Merck, Ireland, catalogue number UFC901096) and the concentrated enzyme was dissolved in enzyme storage buffer (50 mM potassium phosphate buffer, pH 7.0, containing 150 mM KCl, 1 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), and 10% glycerol).

Cell enrichment: cell sorting using flow cytometry

A BD fluorescence-activated cell sorting (FACS) Aria flow cytometer (BD FACSMelody, CAF Microscopy Unit, Stellenbosch University) was used for the cell sorting to separate the populations of cells into subpopulations based on fluorescent labelling (Ibrahim and van den Engh 2007). GFP-tagged chitinase and CW, which both bind to the yeast cell wall chitin, were used for the cell sorting of yeast cells with high chitin levels. Based on the fluorescence levels of individual cells, up to 1 million cells representing, first, the 5% of cells displaying the highest chitin levels were collected in PBS buffer, followed by new growth in YPD broth for 16 h, or until an OD600 of 1 was reached. The cycle was repeated six times. Second, in each round of sequential enrichment, 5% of cells displaying the lowest chitin levels were collected in PBS and freeze cultured using 50% glycerol. The excitation details were as follows: for GFP, 488 nm excitation laser and detection in the fluorescein isothiocyanate (FITC) channel with a 502 long-pass filter and 530/30 band-pass filter was used, while for CW, the excitation was carried out by using violet laser at a wavelength of 405 nm. The enrichment process is illustrated in Fig. 1. For the data analysis, FITC-area geometric mean of fluorescence intensity was used for the quantification of fluorescence produced by 50 000 cells and was expressed in arbitrary units (a.u.).

Figure 1. Enrichment strategy for the selection of mutants with high cell wall chitin. Cell populations were cultured in YPD overnight, fluorescently labelled with GFP-tagged chitinase or CW; and subjected to five/six successive enrichment cycles using flow cytometry to enrich for high-chitin mutants.

Chitin quantification

Cells were grown in YPD as described by de Groot et al. (2001), and equal numbers of cells based on OD600nm were washed and resuspended in PBS buffer before adding GFP-tagged chitinase or before staining with CW. Labelled cells were then subjected to flow cytometry as described above to quantify chitin fluorescent levels and numerical values were computed using FlowJo software (version 10.8.1)

Confocal microscopy

To show the differences in the chitin levels for the sorted cells, images were taken for cells at an OD of 0.1 for both populations of high chitin levels and low chitin levels. Z sectioning image acquisition was performed on a Carl Zeiss Confocal LSM 780 ELYRA S1 with Super-Resolution Structured Illumination Microscopy (SR-SIM super-resolution) platform and the Z-series images were captured at 0.5 µm intervals. The excitation laser used was a violet laser with 407 nm wavelength and the emission filter used was the Pacific Blue channel with a 450/40 band-pass filter, for CW-stained cells. Images were presented in a maximum intensity projection after being processed and background subtracted using the Zeiss Zen lite® 2011 software.

Strain identification of enriched yeast deletion library

General molecular biology protocols for DNA extraction and processing were performed. After the fifth round of enrichment using the EUROSCARF deletion pool, yeast cells were grown in YPD agar and individual colonies were further cultured in YPD broth. To identify randomly selected deletion mutants, DNA was extracted from the cultures and the identity of individual colonies was verified by sequencing the unique UPTAGs and DOWNTAGs coded in each deletion strain as detailed by Parisi et al. (2019) wih few changes.

Ethyl methanesulphonate mutagenesis

In this study, EMS was used to mutagenize S. cerevisiae EC1118 populations following the protocol described by Winston (2008) at a concentration of 5% (v/v) with minor modifications.

Chitinase enzyme assay

A chitinase assay kit (Sigma–Aldrich) with substrate 4-nitrophenyl N-acetyl-β-d-glucosaminide suitable for exochitinase activity detection was used. The ability of yeast cell wall chitin to remove chitinases was evaluated using commercial chitinases (Sigma–Aldrich) that were added in a model wine containing 12% ethanol and 4 g/l of tartaric acid at pH 3.3 (Ndlovu et al. 2018). The final concentration of chitinases prior to addition in a model wine was 0.2 mg/ml, and equal volumes were added to S. cerevisiae EC1118 mutants and WT S. cerevisiae EC1118 cells at the same biomass concentration, mixed, and incubated for 12 h. After 12 h of incubation, the supernatant was obtained by centrifugation (2 min, 5000 rpm) and the activity of unbound chitinases, which cleaves p-nitrophenol from the substrate to produce measurable yellow pigment upon the addition of a stop solution sodium carbonate, was measured by the VICTOR Nivo® Multimode Plate Reader. The chitinase enzyme assay was carried out according to the manufacturer’s instructions (Sigma–Aldrich).

Data analysis and statistics

Numerical analysis of the data was performed using XLSTATS software version 2021.5 to compute significant differences in growth for each strain compared to the WT. Tukey pairwise comparison was the preferred method to compare the differences in growth rate (µmax per hour) of strains relative to their WT. Significant differences are represented by P < .05 and nonsignificant differences are represented by P > .05.

Results

Screening of pooled yeast deletion mutant library

The FACS-based strategy was applied to the pooled deletion mutant library over five cycles of enrichment. Confocal microscopy images confirm the powerful separation of cells with high chitin comparing the 5% of cells with the highest chitin levels (cells that were used to start the next enrichment cycle) with the 5% of cells with lowest fluorescence levels (Fig. 2A and C, respectively). The data also suggest some differences between GFP-chitinase- and CW-labelled cells (Fig. 2B). GFP-chitinase-labelled cells are labelled more homogenously, and lateral chitin appears more prominent. This suggests that chitinases may bind less efficiently to bud scar chitin, while CW binds all chitin, and therefore emphasizes the scars more strongly.

Figure 2. Confocal microscopy [confocal SR-SIM super-resolution fluorescence microscope (scale bar = 10 µm)] images of yeast cells taken immediately after the fifth round of cell sorting using a BD fluorescence-activated cell sorting Aria flow cytometer representing (A) 5% of the population with high chitin levels after the fifth round of enrichment and, (B) the binding of GFP-labelled chitinases to the same population as panel (A), and (C) 5% of cells representing the population (isolated from the same population as panel A) with low chitin from the fifth round of enrichment.

A total of 137 and 112 randomly selected yeast mutant colonies from the GFP-chitinase and CW-based sorting, respectively, were isolated after the fifth enrichment cycle and screened for chitin levels (Table 1). Close to 42% and 54% of the mutant yeast strains screened using GFP chitinase and CW, respectively, showed at least a 2-fold increase in fluorescence relative to the parental strain, BY4742, while 21% and 20% of the GFP-chitinase- and CW-sorted mutant cells, respectively, showed at least a 3-fold increase in fluorescence. These data also suggests that both CW and GFP-tagged chitinase can enrich for mutants with higher cell wall chitin.

Table 1. Total numbers of colonies and chitin fold changes relative to parental strain WT BY4742 for selected isolates from the pooled deletion library after five cycles of enrichment.

	GFP chitinase	CW	
Total colonies screened	137	112	
2-fold increase	58	60	
≥3-fold increase	29	23	

Eighteen colonies from the pooled deletion library selected from both GFP-chitinase and CW enrichment strategies and displaying higher chitin levels when compared to parental WT strain, as well as two colonies displaying WT levels of chitin, were identified by their encoded tags. Table 2 provides the names of the genes identified from these mutants. Several gene deletion mutants were present more than once in this small sample, indicating successful enrichment of specific mutant strains. The screen identified deletions of the genes ACE2, TUS1/SOP10, CTS1, VTC3/PHM2, YGP1, and IRC8, which are associated with cell wall maintenance or remodelling or membrane trafficking functions. TUS1 deletion has previously been shown by Lesage et al. (2005) to lead to higher chitin deposition, but the higher chitin levels have not previously been described in BY4742Δace2, BY4742Δirc8, BY4742Δcts1, and BY4742Δvtc3.

Table 2. Yeast deletion mutant strains from the EUROSCARF library identified after sorting for high chitin levels.

ORF	Gene	Molecular function	Description	
YPL019C *4 (4G)	VTC3/PHM2	Unknown	Subunit of the vacuolar transporter chaperone (VTC) complex involved in membrane trafficking (Cohen et al. 1999).	
YLR131C *1 (1G)	ACE2	Sequence-specific DNA binding	Transcription factor required for septum destruction after cytokinesis; Ace2 transcriptional activator of CTS1 gene (endochitinase) (Butler and Thiele 1991).	
YLR425W *3 (1C, 2G)	TUS1/SOP10	CWI pathway	Guanine nucleotide exchange factor (GEF) that modulates Rho1p activity; involved in the cell integrity signalling pathway; interacts with Rgl1p (Schmelzle et al. 2002).	
YLR286C *5 (3C, 2G)	CTS1	Endochitinase activity	Endochitinase, required for cell separation after mitosis; transcriptional activation during the G1 phase of the cell cycle is mediated by transcription factor Ace2p (O’Conallain et al. 1999).	
YNL160W *2 (1C, 1G)	YGP1	Unknown	Cell wall-related secretory glycoprotein; induced by nutrient deprivation-associated growth arrest and upon entry into the stationary phase (Destruelle et al. 1994).	
YJL051W *3 (2C, 1G)	IRC8	Unknown	Bud tip localized protein of unknown function (Alvaro et al. 2007)	
The mutants were obtained from both the GFP-chitinase and CW screens.

* Indicates the number of the identified deletion mutant strain from the overall 18 sequenced colonies. C = Calcofluor white; G = GFP chitinase.

All identified mutants were labelled with both CW and GFP chitinase. The data (Fig. 3A and B) show a clear correlation between the two labelling methods, with BY4742∆ace2 showing the highest fluorescence levels, followed by BY4742Δirc8. However, there is some level of variation in relative chitin concentrations between strains in the two methods, for example in the case of Δirc8. This difference was consistently observed in this strain, suggesting differences in the efficiency of chitinase and CW binding. As described above (Fig. 2), we observed that GFP chitinases were less efficient in highlighting bud scars, perhaps because of being sterically hindered in the denser chitin network of the scar.

Figure 3. (A) Chitin fluorescence levels quantified using flow cytometry with CW staining. Chitin fluorescence levels are observed in all deletion mutant strains with the exception of BY4742∆ygp1, which showed levels similar to the WT. Fluorescence intensity is expressed in a.u. (B) GFP chitinase levels (arbitrary unit = a.u.) bound to yeast strains quantified using a BD fluorescence-activated cell sorting Aria flow cytometer.

Isolation of industrial wine yeast mutant strains with high chitin levels

After the successful application of the strategy to the deletion mutant library, we investigated the broader use of this enrichment strategy in the wine yeast strain S. cerevisiae EC1118. The strains were subjected to random mutagenesis using EMS (Godfroy et al. 2015, Ang et al. 2019). The mutated population were subjected to the same enrichment strategy as described above. Table 1 confirms that the use of either GFP-tagged chitinase or CW can effectively facilitate the enrichment of cell populations with high chitin content, and therefore, the subsequent enrichment for high-chitin mutants was carried out using CW only. Figure 4 shows the distribution of population-wide CW fluorescence across the six rounds of successive enrichment. The data indicate an increase in average fluorescence after each round (shift of peak to the right), suggesting the progressive increase in cells with higher average chitin content, until round 5. There appears to be no further increase for round 6, suggesting that an enrichment threshold may have been reached.

Figure 4. Flow cytometry data showing overlays of multiple, whole-population fluorescent peaks obtained from each enrichment round. ‘Round’ in the figure legends represents colour-aligned peaks of enrichment rounds of a total yeast cell population sorted for high chitin levels. The evolution of the cell wall chitin increases per enrichment round for the S. cerevisiae wine yeast mutant EC1118 culture using CW. Enrichment rounds 1–6 are shown here and the cell counts enriched per round is ~50 000 cells.

Quantifying cell wall chitin of selected isolates

Cell wall chitin levels of six selected strains after each round of enrichment were quantified individually (Fig. 5). The naming convention of strains after mutagenesis in each round of enrichment was as follows: ESC: (E) for S. cerevisiae EC1118, (S) for round of sorting, and (C) for colony number, e.g. ES1C1 refers to S. cerevisiae EC1118 colony number 1 from the first round of enrichment. The data show a number of strains with WT chitin levels throughout the enrichment rounds. Nevertheless, the two strains with the highest chitin contents were present after the final enrichment round, suggesting that the sequential enrichment for mutants with higher chitin levels can indeed be achieved in the mutant population.

Figure 5. The representation of CW fluorescence levels for yeast strains in each round of enrichment in comparison to the WT S. cerevisiae EC1118. The data represent the mean ± standard deviation (n = 3). Fluorescence intensity is expressed in arbitrary units (a.u.). Lighter grey mutants were selected for investigating the impact of chitin level on growth of strains.

Several mutant strains with different levels of chitin were selected for growth comparisons, ES5C1, ES6C1, ES1C4, ES2C2, ES4C5, ES6C4, and ES6C6. Two strains with chitin levels similar to the WT that had also been selected after the final two rounds of enrichment were included into the growth comparisons as controls. The data (Fig. 6) suggest that the high-chitin mutants grew slower than the WT. The two strains with chitin levels similar to the WT, however, showed similar growth to the WT (P > .05), suggesting that the sequential passage through the FACS had not led to an enrichment of slower growing cells per se (Table 3). While all strains with higher chitin levels displayed significantly slower growth (P < .05), there was no linear correlation between growth rate and the specific chitin levels of each strain. The data therefore suggest that increased chitin levels reduce cellular growth, but that other factors than total cell wall chitin concentration also play a role in the growth performance of the strains.

Figure 6. The growth of mutant strains from each round of enrichment in comparison to the WT S. cerevisiae EC1118. The data represent the mean ± standard deviation (n = 3). The growth was monitored in YPD; the * symbol represents a significant difference (P > .05) in growth rate to the WT and the # symbol represents cells that had no significant difference (P < .05) to the WT. The dotted lines represent the strains ES5C1 and ES6C1 that have chitin levels similar to the WT (also in dotted lines), while all solid lines represent mutants with higher chitin levels.

Table 3. Analysis of the statistical differences between the mutant and WT strains using Tukey mean comparisons (µmax per hour).

Strain	Mean (average) µmax per hour (h−1)	
ES5C1	0.279 ± 0.012a	
WT	0.279 ± 0.015a	
ES6C1	0.258 ± 0.012ab	
ES6C6	0.199 ± 0.012bc	
ES2C2	0.195 ± 0.012c	
ES4C5	0.192 ± 0.012c	
ES6C4	0.187 ± 0.012c	
ES1C4	0.175 ± 0.012c	
Various alphabetical symbols (a, b, and c) represents statistical differences (P < .05).

Mutants with various cell wall chitin levels were selected for further analysis. To investigate binding of chitinases to cells of these mutants, identical number of cells for each mutant were added to a solution containing equal amounts of chitinases. After the exposure, remaining chitinase activity was quantified in the supernatant. The data in Fig. 7(A) show that all S. cerevisiae EC1118 high-chitin mutants reduced chitinase activity more efficiently than the WT strain. However, the correlation again did not directly align with the relative chitin quantification shown in Fig. 7(B), suggesting the existence of other factors such as differences in cell wall structure and chitin deposition that may play a major role in the success of chitinase binding.

Figure 7. (A) Chitinase enzyme activity of unbound chitinases in the supernatant after incubation with high-chitin strains and the control (WT S. cerevisiae EC1118) [data represent mean ± standard deviation (n = 2)] and (B) their corresponding chitin levels [data represent mean ± standard deviation (n = 3)]. Fluorescence intensity is expressed in a.u.

Discussion

The study presents a method of enrichment of strains with high levels of cell wall chitin. Some general considerations regarding this strategy can be made. The enrichment of mutants at each single enrichment cycle likely enriched for two types of cells: (i) older cells with more bud scars and thus high chitin fluorescence and (ii) high-chitin mutants that display higher chitin fluorescence independent of age. As the data from the enriched deletion library clearly show, the enrichment strategy over several cycles leads to a selection of relevant mutations, and eliminated strains that would have been selected based on the age of individual cells. However, the growth phenotype observed in the high-chitin mutant strains may work as a counterselection force, since the culture growth during each cycle will favour faster growing strains. This may be a reason for the data in Fig 4, which suggest that the sixth round of enrichment did not lead to a further increase in average chitin levels. Nevertheless, the method efficiently selected mutants with significantly higher chitin levels.

Enrichment of the EUROSCARF pooled deletion library identified the strains BY4742Δtus1, BY4742Δace2, BY4742Δcts1, BY4742Δirc8, and BY4742Δvtc3. Lesage et al. (2005) had previously reported high levels of chitin deposition in Δtus1, confirming the validity of our approach. Our data show that deletion of the ACE2 (Δace2), CTS1 (Δcts1), IRC8 (Δirc8), and VTC3 (Δvtc3) genes also results in increased cell wall chitin. In a study by Arias et al. (2011) on genome-wide survey of yeast mutations leading to activation of the yeast cell integrity mitogen-activated-protein-kinase (MAPK) pathway, about 30% of the mutants identified had increased chitin levels.

The same enrichment method was applied to a population of a randomly mutagenized wine yeast strain and our data clearly show that we were able to isolate mutants with significantly increased levels of chitin fluorescence. In these mutants, a general correlation between high chitin and slower growth was observed. However, this correlation between high chitin fluorescence and growth did not apply linearly, suggesting that other factors such as the specific deposition of chitin play a role in modulating the impact of increased chitin deposition. The data suggest an increased metabolic burden that may be due to the demands of higher chitin synthesis, and/or that higher chitin levels result in higher cell wall rigidity, which may impede remodelling required for growth and morphogenic processes such as budding (Queiroz et al. 2019, 2021). The slow growth is not linked to the enrichment process since cells with the same levels of cell wall chitin as the WT but isolated at the end of the process grew similarly to the WT. In the future, the ‘high-chitin’ mutants will be investigated for any unique patterns of chitin deposition on bud scars or lateral chitin as well as the degree of linkage to other cell wall components, which might influence the final metabolic burden and physical constraint that might be exerted by excess chitin.

The ability of high-chitin mutants to reduce chitinases suggests a potential application to reduce protein haze in wine. However, while there was a clear link between high chitin and chitinase reduction in our model wine, this relation was not linear. Queiroz et al. (2021) reported that yeast cells might undergo cell wall reorganization/remodelling particularly on chitin–glucan linkages that help to resist cell wall lysis by external glucanases. Chitinases and glucanases are both plant PR proteins capable of breaking down the fungal cell wall (Maglovski et al. 2017, Schmaltz et al. 2020). Therefore, based on this and the findings of this study, we hypothesize that chitin–glucan remodelling will impact the binding of PR proteins to the cell wall. This shows that the use of these strains in the form of inactivated/dead yeast or yeast hulls will therefore be ideal for the reduction of chitinases.

Conclusion

In summary, a novel genetic high-throughput screen was successfully used to sort deletion mutants from the EUROSCARF library and mutagenized wine yeast strains with high cell wall chitin content. The identified EUROSCARF library mutant strains were affected in genes related to chitin synthesis, cell wall biogenesis, morphogenesis, and cell wall integrity-related signal transduction. Slower growth correlating with high cell wall chitin was observed, although this reduction in growth was not directly proportional to chitin levels. The method presented here is readily adaptable to the study of other cell wall phenotypes of interest through screening of genome-wide libraries or through the screening of high-chitin mutants using fluorescence dyes following chemical mutagenesis.

Acknowledgements

Confocal fluorescence microscopy and FACS experiments were carried out at the Central Analytical Facility (CAF, Stellenbosch University) with the help of Mrs L. Engelbrecht, Ms Dumisile Lumkwana, and Ms Rozanne Adams.

Conflict of interest

None declared.

Funding

This work was financially supported by the SARChI grant (UID 83471) to F.F.B. from the National Research Foundation of South Africa and FLAIR fellowship of Royal Society to D.R.
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References

Albuquerque  W, Ghezellou  P, Li  B  et al.  Identification of intact peptides by top-down peptidomics reveals cleavage spots in thermolabile wine proteins. Food Chem. 2021;363 :130437. 10.1016/j.foodchem.2021.130437.34214891
Alvaro  D, Lisby  M, Rothstein  R.  Genome-wide analysis of Rad52 foci reveals diverse mechanisms impacting recombination. PLoS Genet. 2007;3 :e228. 10.1371/journal.pgen.0030228.18085829
Ang  FS, Khaw  SY, Few  LL  et al.  Isolation of a stable astaxanthin-hyperproducing mutant of Xanthophyllomyces dendrorhous through random mutagenesis. Appl Biochem Microbiol. 2019;55 :255–63. 10.1134/S0003683819030025.
Arias  P, Díez-Muñiz  S, García  R  et al.  Genome-wide survey of yeast mutations leading to activation of the yeast cell integrity MAPK pathway: novel insights into diverse MAPK outcomes. BMC Genomics. 2011;12 :390. 10.1186/1471-2164-12-390.21810245
Bernardo  SM, Allen  CP, Waller  A  et al.  An automated high-throughput cell-based multiplexed flow cytometry assay to identify novel compounds to target Candida albicans virulence-related proteins. PLoS One. 2014;9 :e110354. 10.1371/journal.pone.0110354.25350399
Black  CB, Duensing  TD, Trinkle  LS  et al.  Cell-based screening using high-throughput flow cytometry. Assay Drug Dev Technol. 2011;9 :13–20. 10.1089/adt.2010.0308.21050072
Brachmann  CB, Davies  A, Cost  GJ  et al.  Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast. 1998;14 :115–32. 10.1002/(SICI)1097-0061(19980130)14:2<115::AID-YEA204>3.0.CO;2-2.9483801
Butler  G, Thiele  DJ.  ACE2, an activator of yeast metallothionein expression which is homologous to SWI5. Mol Cell Biol. 1991;11 :476–85.1986241
Cohen  A, Perzov  N, Nelson  H  et al.  A novel family of yeast chaperons involved in the distribution of V-ATPase and other membrane proteins. J Biol Chem. 1999;274 :26885–93. 10.1074/jbc.274.38.26885.10480897
Cosme  F, Fernandes  C, Ribeiro  T  et al.  White wine protein instability: mechanism, quality control and technological alternatives for wine stabilisation—an overview. Beverages. 2020;6 :1–28. 10.3390/beverages6010019.
de Groot  PWJ, Ruiz  C, de Aldana  CRV  et al.  A genomic approach for the identification and classification of genes involved in cell wall formation and its regulation in Saccharomyces cerevisiae. Comp Funct Genomics. 2001;2 :124–42. 10.1002/cfg.85.18628907
Destruelle  M, Holzer  H, Klionsky  DJ.  Identification and characterization of a novel yeast gene: the YGP1 gene product is a highly glycosylated secreted protein that is synthesized in response to nutrient limitation. Mol Cell Biol. 1994;14 :2740–54.8139573
Giaever  G, Nislow  C.  The yeast deletion collection: a decade of functional genomics. Genetics. 2014;197 :451–65. 10.1534/Genetics.114.161620.24939991
Godfroy  O, Peters  AF, Coelho  SM  et al.  Genome-wide comparison of ultraviolet and ethyl methanesulphonate mutagenesis methods for the brown alga Ectocarpus. Mar Geonomics. 2015;24 :109–13. 10.1016/j.margen.2015.03.007.
González-Cabaleiro  R, Mitchell  AM, Smith  W  et al.  Heterogeneity in pure microbial systems: experimental measurements and modelling. Front Microbiol. 2017;8 :1813. 10.3389/fmicb.2017.01813.28970826
Hapala  I1, Griač  P, Nosek  J  et al.  Yeast membranes and cell wall: from basics to applications. Curr Genet. 2013;59 :167–9. 10.1007/s00294-013-0408-8.24057126
Ibrahim  SF, van den Engh  G.  Flow cytometry and cell sorting. Adv Biochem Eng Biotechnol. 2007;106 :19–39.17728993
Latge  JP.  Tasting the fungal cell wall. Cell Microbiol. 2010;12 :863–72. 10.1111/j.1462-5822.2010.01474.x.20482553
Lee  P, Colman  RF.  Expression, purification, and characterization of stable, recombinant human adenylosuccinate lyase. Protein Exp Purif. 2007;51 :227–34. 10.1016/j.pep.2006.07.023.
Lesage  G, Shapiro  J, Specht  CA  et al.  An interactional network of genes involved in chitin synthesis in Saccharomyces cerevisiae. BMC Genet. 2005;6 :8. 10.1186/1471-2156-6-8.15715908
Maglovski  M, Gregorová  Z, Rybanský  L  et al.  Nutrition supply affects the activity of pathogenesis-related β-1,3-glucanases and chitinases in wheat. Plant Growth Regul. 2017;81 :443–53. 10.1007/s10725-016-0222-7.
Marangon  M, Van Sluyter  SC, Neilson  KA  et al.  Roles of grape thaumatin-like protein and chitinase in white wine haze formation. J Agric Food Chem. 2011;59 :733–40. 10.1021/jf1038234.21189017
Molon  M, Woznicka  O, Zebrowski  J.  Cell wall biosynthesis impairment affects the budding lifespan of the Saccharomyces cerevisiae yeast. Biogerontology. 2018;19 :67–79. 10.1007/s10522-017-9740-6.29189912
Ndlovu  T, Buica  A, Bauer  FF.  Chitinases and thaumatin-like proteins in Sauvignon Blanc and Chardonnay musts during alcoholic fermentation. Food Microbiol. 2019;78 :201–10. 10.1016/j.fm.2018.10.018.30497604
Ndlovu  T, Divol  B, Bauer  FF.  Yeast cell wall chitin reduces wine haze formation. Appl Environ Microbiol. 2018;84 :e00668 18. 10.1128/AEM.00668-18.29703738
O’Conallain  C, Doolin  MT, Taggart  C  et al.  Regulated nuclear localisation of the yeast transcription factor Ace2p controls expression of chitinase (CTS1) in Saccharomyces cerevisiae. Mol Gen Genet. 1999;262 :275–82. 10.1007/s004380051084.10517323
Parisi  K, Doyle  SR, Lee  E  et al.  Screening the Saccharomyces cerevisiae nonessential gene deletion library reveals diverse mechanisms of action for antifungal plant defensins. Antimicrob Agents Chemother. 2019;63 :e01097–19.
Qiu  Z, Wu  X, Gao  W  et al.  High temperature induced disruption of the cell wall integrity and structure in Pleurotus ostreatus mycelia. Appl Microbiol Biotechnol. 2018;102 :6627–36.29846777
Queiroz  MG, Elsztein  C, de Morais Jr  MA  The effects of the Ncw2 protein of Saccharomyces cerevisiae on the positioning of chitin in response to cell wall damage. Antonie Van Leeuwenhoek. 2019;113 :265–77. 10.1007/s10482-019-01335-y.31598818
Queiroz  MG, Elsztein  C, Strahl  S  et al.  The Saccharomyces cerevisiae Ncw2 protein works on the chitin/β-glucan organisation of the cell wall. Antonie Van Leeuwenhoek. 2021;114 :1141–53. 10.1007/s10482-021-01584-w(0123456789().,-w.33945065
Schiavone  M, Vax  A, Formosa  C  et al.  A combined chemical and enzymatic method to determine quantitatively the polysaccharide components in the cell wall of yeasts. FEMS Yeast Res. 2014;14 :933–47. 10.1111/1567-1364.12182.25041403
Schmaltz  S, Aita  BC, Alves  EA  et al.  Ultrasound-assisted fermentation for production of β-1,3-glucanase and chitinase by Beauveria bassian. J Chem Tech Biotech. 2021;96 :88–98. 10.1002/jctb.6514.
Schmelzle  T, Helliwell  SB, Hall  MN.  Yeast protein kinases and the RHO1 exchange factor TUS1 are novel components of the cell integrity pathway in yeast. Mol Cell Biol. 2002;22 :1329–39. 10.1128/MCB.22.5.1329-1339.2002.11839800
Van Sluyter  SCV, Mcrae  JM, Falconer  RJ  et al.  Wine protein haze: mechanisms of formation and advances in prevention. J Agric Food Chem. 2015;63 :4020–30. 10.1021/acs.jafc.5b00047.25847216
Vishukumar  A, Simenel  C, Garnaud  C  et al.  The dual activity responsible for the elongation and branching of β-(1,3)-glucan in the fungal cell wall. mBio. 2017;8 :e00619–17.28634239
Winston  F.  EMS and UV mutagenesis in yeast. Curr Protoc Mol Biol. 2008;82 :1–5. 10.1002/0471142727.mb1303bs82.
