- Research article
- Open Access
Search for genes responsible for the remarkably high acetic acid tolerance of a Zygosaccharomyces bailii-derived interspecies hybrid strain
- Margarida Palma†1,
- Filipa de Canaveira Roque†1,
- Joana Fernandes Guerreiro1,
- Nuno Pereira Mira1,
- Lise Queiroz1 and
- Isabel Sá-Correia1Email author
© Palma et al. 2015
- Received: 22 April 2015
- Accepted: 8 December 2015
- Published: 16 December 2015
Zygosaccharomyces bailii is considered the most problematic acidic food spoilage yeast species due to its exceptional capacity to tolerate high concentrations of weak acids used as fungistatic preservatives at low pH. However, the mechanisms underlying its intrinsic remarkable tolerance to weak acids remain poorly understood. The identification of genes and mechanisms involved in Z. bailii acetic acid tolerance was on the focus of this study. For this, a genomic library from the highly acetic acid tolerant hybrid strain ISA1307, derived from Z. bailii and a closely related species and isolated from a sparkling wine production plant, was screened for acetic acid tolerance genes. This screen was based on the transformation of an acetic acid susceptible Saccharomyces cerevisiae mutant deleted for the gene encoding the acetic acid resistance determinant transcription factor Haa1.
The expression of 31 different DNA inserts from ISA1307 strain genome was found to significantly increase the host cell tolerance to acetic acid. The in silico analysis of these inserts was facilitated by the recently available genome sequence of this strain. In total, 65 complete or truncated ORFs were identified as putative determinants of acetic acid tolerance and an S. cerevisiae gene homologous to most of them was found. These include genes involved in cellular transport and transport routes, protein fate, protein synthesis, amino acid metabolism and transcription. The role of strong candidates in Z. bailii and S. cerevisiae acetic acid tolerance was confirmed based on homologous and heterologous expression analyses.
ISA1307 genes homologous to S. cerevisiae genes GYP8, WSC4, PMT1, KTR7, RKR1, TIF3, ILV3 and MSN4 are proposed as strong candidate determinants of acetic acid tolerance. The ORF ZBAI_02295 that contains a functional domain associated to the uncharacterised integral membrane proteins of unknown function of the DUP family is also suggested as a relevant tolerance determinant. The genes ZbMSN4 and ZbTIF3, encoding a putative stress response transcription factor and a putative translation initiation factor, were confirmed as determinants of acetic acid tolerance in both Z. bailii and S. cerevisiae. This study provides valuable indications on the cellular components, pathways and processes to be targeted in order to control food spoilage by the highly acetic acid tolerant Z. bailii and Z. bailii-derived strains. Additionally, this information is essential to guide the improvement of yeast cells robustness against acetic acid if the objective is their use as cell factories.
- Zygosaccharomyces bailii
- Acetic acid tolerance genes
- Yeast hybrid strains
- Food spoilage yeasts
- Weak acid food preservatives
Zygosaccharomyces bailii is considered the most problematic spoilage yeast found in the food and beverage industry, particularly in acidic foods, soft drinks, fruit juices, dairy products and salad dressings [1, 2]. This yeast species ability to cause spoilage derives from its outstanding intrinsic capacity to resist to weak acids widely used as fungistatic preservatives, such as acetic, propionic, benzoic and sorbic acids [1–4]. Understanding the mechanisms of weak acid resistance is central to the development and implementation of more effective food and beverage preservation strategies in order to minimise economic losses. Although Z. bailii is the spoilage yeast that exhibits the highest level of tolerance to acetic acid, most of the scientific contributions on the mechanisms underlying adaptation and resistance to acetic acid in yeast have been focused on the more susceptible experimental model Saccharomyces cerevisiae [5–9]. At a pH equal or below its pKa (4.75), acetic acid is mainly in the undissociated form that can diffuse across the plasma membrane. Once in the near neutral cytosol, acetic acid dissociates leading to the accumulation of protons and acetate. The acidification of the cytosol leads to the inhibition of metabolic activity and to the dissipation of the proton gradient across plasma membrane required for secondary transport [10, 11]. To counteract this effect, the plasma membrane proton-pumping ATPase (PM H+-ATPase) is activated in acetic acid-stressed S. cerevisiae cells . The involvement of Z. bailii PM H+-ATPase in the active export of protons from cells challenged by weak acid preservatives, namely benzoic acid, was also demonstrated, suggesting that both yeasts share this response mechanism . Given that the charged acetate counterion is not able to easily cross the hydrophobic plasma membrane lipid bilayer, it accumulates in the cell interior leading to increased oxidative stress and turgor pressure, among other effects [3, 9]. To counteract these effects, the plasma membrane multidrug resistance (MDR) transporters of the Major Facilitator Superfamily (MFS) Tpo2 and Tpo3 were hypothesized to play a role in the extrusion of acetate from acetic acid-challenged S. cerevisiae cells . To date, no acetate export system was described in Z. bailii. S. cerevisiae response to acetic acid-induced stress involves several transcriptional regulators [4, 9]. The expression of the transcription factor encoding gene HAA1 was found to markedly decrease the duration of the adaptation period of a yeast cell population suddenly exposed to toxic concentrations of acetic acid, by decreasing the loss of cell viability that occurs during this phase of growth latency . Haa1 is considered one of the key players in the control of S. cerevisiae response to acetic acid due to its role in the direct, or indirect, regulation of approximately 80 % of acetic acid-responsive genes , several of them required for maximal tolerance to acetic acid . These genes code for protein kinases, MDR transporters, transcription factors and proteins involved in lipid metabolism and nucleic acid processing .
In order to identify determinants of Z. bailii tolerance to acetic acid at the genome level we used in this study a genomic library previously prepared from the highly acetic acid tolerant strain ISA1307, an interspecies hybrid between Z. bailii and a closely related species which was isolated from a continuous production plant of sparkling wine [14, 15]. This genomic library was used to rescue the high susceptibility phenotype of S. cerevisiae BY4741_haa1Δ. This mutant, deleted for HAA1 gene, was chosen due to its very high susceptibility to acetic acid to avoid the use of the much higher concentrations required to inhibit the parental strain growth. With this approach we also expected to identify the functional homologue of S. cerevisiae HAA1 gene in this hybrid strain. During the development of this study, our laboratory carried out the genome sequencing, assembly and annotation of ISA1307 . This hybrid strain has been on the focus of several physiological studies, some of them aiming at the understanding of the mechanisms underlying its remarkable intrinsic resistance to acetic acid. Differently from S. cerevisiae, the Z.bailii-derived hybrid strain ISA1307 co-consumes glucose and acetic acid when cultivated in glucose medium supplemented with a sublethal growth inhibitory concentration of acetic acid [16, 17]. Quantitative proteomic studies on the adaptive response of this strain indicate that in glucose and acetic acid cultures the acid is channelled through the TCA cycle . After glucose exhaustion, acetic acid being present as the sole carbon source, the content of several proteins involved in gluconeogenesis and pentose phosphate pathway was however found to increase .
The screening of ISA1307 genomic library for genes required for tolerance to acetic acid successfully pointed out several strong candidates, but since this is a hybrid strain difficult to be genetically manipulated, in order to confirm the role of the selected genes in acetic acid tolerance we have explored another strain, Z. bailii IST302. This strain was isolated from spontaneous fermentation of wine must and, contrarily to ISA1307 and Z. bailii CLIB213T , does not flocculate and proved to be more susceptible to genetic engineering than these reference strains. The genome of Z. bailii IST302 was recently sequenced and annotated in our laboratory (unpublished data), rendering possible the work here reported.
The knowledge gathered during the present study was based on the expression of ISA1307 genomic library in S. cerevisiae, the availability of the genome sequences of the hybrid strain ISA1307 and Z. bailii IST302 and the expression of selected ISA1307 and IST302 gene sequences in S. cerevisiae and Z. bailii strains and led to the identification of a number of Z. bailii genes involved in tolerance to acetic acid. Strong candidates for determinants of acetic acid tolerance in Z. bailii or S. cerevisiae are here proposed.
Selection of S. cerevisiae transformants with increased tolerance to acetic acid through the expression of an ISA1307 genomic library
In silico analysis of ISA1307 genomic DNA fragments whose expression was found to be required for increased acetic acid tolerance in S. cerevisiae
The in silico analysis of the 31 selected genomic DNA fragments from the Z. bailii-derived interspecies hybrid strain ISA1307 led to the identification of a number of ORFs which are putative candidate determinants of tolerance to the acid. Since we found that the expression of DNA inserts containing only one putative truncated ORF also led to the increase of haa1Δ tolerance to acetic acid, the incomplete ORFs were also considered for further analysis and the presence of conserved functional domains in the identified inserts was investigated. However, the truncated ORFs comprising less than 15 % of the complete sequence, which contained limited portions of conserved domains or no conserved domains, were disregarded at this phase, resulting in the selection of 32 complete and 33 truncated putative candidate ORFs (Additional file 2).
ORFs with homology to S. cerevisiae genes
For each ISA1307 selected ORF, the predicted S. cerevisiae homologous gene and the corresponding functional description were gathered (Additional file 2). References to previous studies in which the deletion of the gene was already described as leading to an increase or decrease of yeast susceptibility to acetic acid or other weak acids are also indicated. Since the strain ISA1307 is an interspecies hybrid between Z. bailii and a closely related species , Additional file 2 also includes information regarding the nucleotide sequence identity of each ORF present in the different DNA inserts with Z. bailii CLIB213T genome  to identify the ORFs that likely are from Z. bailii. Interestingly, about one half of the selected DNA inserts that were able to rescue the haa1∆ mutant susceptibility phenotype to acetic acid is, presumably, derived from the parental Z. bailii species (99–100 % identity with CLIB213T genome) while the other half is, apparently, derived from the other yeast species closely related to Z. bailii (93–98 % identity with CLIB213T genome).
ORFs with no homology with S. cerevisiae genes
Genomic DNA inserts from strain ISA1307 genomic library required for acetic acid tolerance containing the putative ORFs for which weak or no homology with S. cerevisiae genes was found. Information about other ORFs present in the same DNA insert is provided, as well as the best blast hits and the predicted functional and transmembrane domains of the corresponding protein
ORFs in the same inserta
Best blast hits
3.13 (4069 bp)
ZBAI_06221 (40 %)
ZBAI_06218 (47 %)
Glutathione S-transferase from Macrophomina phaseolina (charcoal rot fungus) and putative uncharacterized proteins from Torulaspora delbrueckii and several fungi.
Domains related with the Glutathione S-transferase family. In particular, the portion which is present in the DNA library fragment possesses a C-terminal alpha helical domain of the GST family.
18.9 (4511 bp)
ZBAI_03401 (28 %)
Two putative uncharacterised proteins, one from Z. rouxii and the other from T. delbrueckii.
ZBAI_03404 (30 %)
E13 (4470 bp)
Putative and described pyruvate decarboxylases from other yeasts and several other different organisms.
Pyrimidine binding domain (PYR) of pyruvate decarboxylase.
Weak similarities with several helicase-like proteins encoded by the Y’ element of subtelomeric regions from S. cerevisiae and S. kudriavzevii.
Domain characteristic from DNA and RNA helicases.
S06 (4413 bp)
Similarities with hypothetical proteins from Z. rouxii, most of them being uncharacterised and others sharing some homology with membrane proteins from DUP240 and DUP380 gene families, such as COS8, COS9 and YHL044W.
Domain related with DUP family, which consists of several yeast proteins of unknown functions.
Weak similarities with several hypothetical proteins from Z. rouxii which present no similarity with proteins from other species.
PRK15313 domain related to autotransport protein MisL (provisional).
V14 (2907 bp)
ZBAI_09509 (99 %)
Putative uncharacterised proteins mainly from other yeasts, such as Z. rouxii, T. delbrueckii and Lachancea thermotolerans species, and fungi.
Zinc-finger related domains.
The ORF ZBAI_02295 is required for tolerance to acetic acid
The results of the expression experiments support the idea that the ORF ZBAI_02295 should be considered as a strong candidate determinant of tolerance to acetic acid in strain ISA1307. On the contrary, the expression of ORF ZBAI_02296 had a negative effect in S. cerevisiae tolerance to acetic acid and for this reason it was not considered as a candidate determinant.
Selection of strong candidate determinants of acetic acid tolerance
Genomic DNA inserts from strain ISA1307 genomic library containing the strong candidate determinants of tolerance to acetic acid proposed in this work
Identity (%) with Z. bailii CLIB213Tb
S.cerevisiae putative homologue
Identity (%) with S. cerevisiae c
Size (aa) ISA1307 / S. cerevisiae / Alignmentd
Alteration in yeast deletion mutant susceptibility to weak organic acidsf
8.23 (2782 bp)
556 / 497 / 503
GTPase-activating protein involved in the regulation of ER to Golgi vesicle transportCellular transport and transport routes
18.22 (2803 bp)
ZBAI_04770 (41 %)
761 / 817 / 746
Acetic acid (S) 
23.1 (2277 bp)
ZBAI_09707 (40 %)
854 / 605 / 632
ER membrane protein involved in the translocation of soluble secretory proteins and insertion of membrane proteins into the ER membrane; may also have a role in the stress response
Cellular transport and transport routes
23.17 (1812 bp)
ZBAI_09663 (66 %)
583 / 585 / 584
Dihydroxyacid dehydratase, catalyzes third step in the common pathway leading to biosynthesis of branched-chain amino acids
Amino acid metabolism
B02 (3640 bp)
ZBAI_03527 (77 %)
574 / 630 / 590
Transcriptional activator that regulates the general stress response of S. cerevisiae
2,4-D (S) 
B18 (2917 bp)
433 / 436 / 453
Translation initiation factor eIF-4B
S07 (2860 bp)
ZBAI_05420 (57 %)
514 / 517 / 525
Putative mannosyltransferase involved in protein glycosylation
S06 (4413 bp)
178 / - / -
261 / - / -
Y08 (2104 bp)
ZBAI_01926 (34 %)
1555 / 1562 / 1571
RING domain E3 ubiquitin ligase; involved in the ubiquitin-mediated degradation of non-stop proteins
In summary, and taking into account the aforementioned criterion, the following S. cerevisiae gene homologues were selected and proposed herein as strong candidate determinants of acetic acid tolerance in the strain ISA1307: GYP8 and WSC4 (cellular transport and transport routes), PMT1, KTR7 and RKR1 (protein fate), TIF3 (protein synthesis), ILV3 (amino acid metabolism) and MSN4 (transcription).
Complementation of acetic acid susceptibility of S. cerevisiae mutants deleted for genes homologous to ISA1307 tolerance candidate genes
As previously mentioned, when expressed in the yeast host cell the inserts containing MSN4 and TIF3 homologues were among the best suppressors of the acetic acid susceptibility phenotype of haa1∆ mutant, also leading to a remarkable increase of acetic acid tolerance in the parental strain. Given that the role of these genes in the context of S. cerevisiae tolerance to weak acids was already reported in literature, they were selected for further experiments.
Expression of Z. bailii genes ZbMSN4 and ZbTIF3 in Z. bailii and S. cerevisiae strains
In order to get clues on the genes/proteins involved in the high intrinsic tolerance to acetic acid of the Z. bailii-derived interspecies hybrid strain ISA1307, we have explored a previously constructed genomic library from this strain, as well as the available information on its full genome sequence which was released and annotated during the progress of this study . Considering the experimental design of the screening carried out to search for genes required for maximal tolerance to acetic acid, it was not expected that the identification of ISA1307 HAA1 homologue would fail. Indeed, based on the information retrieved from the PEDANT database, two copies of a putative HAA1-homologue are present in the genome of this hybrid strain (ZBAI_01494 and ZBAI_05761) sharing, respectively, 50 and 47 % identity with S. cerevisiae HAA1 nucleotide sequence (pairwise alignment performed at http://www.ebi.ac.uk/Tools/psa/ (European Bioinformatics Institute)). The fact is that when the genomic library used in the screening was constructed , it was assumed that the size of ISA1307 genome was similar to the size of S. cerevisiae genome (12 Mb). However, following genome sequencing, the genome size of this hybrid strain was found to have approximately 22 Mb . This means that the genomic library tested is poorly representative of the full ISA1307 genome and thus a full functional HAA1 gene sequence may be missing in the library. Also, we cannot exclude at this time the possibility that ISA1307 Haa1 proteins do not have in this highly acetic acid tolerant strain the important role demonstrated for the S. cerevisiae homologue or that the HAA1 homologous genes in this strain are not functional.
The genes emerging from this study derive at similar proportions from each of the two parental strains of the hybrid strain ISA1307. Remarkably, the differences registered in the two alleles of ISA1307, at the nucleotide level, have apparently no impact in the amino acid sequence of the encoded proteins identified in our study as potential determinants of tolerance to acetic acid. In fact, only six gene pairs in ISA1307 whole genome exhibit nucleotide differences (rate of non-synonymous substitutions (dN) and synonymous substitutions (dS) above 1) with an impact in the amino acid sequence of the encoded proteins .
The mechanisms behind yeast adaptation and tolerance to weak acids stress are multifactorial . In other words, yeast tolerance does not rely in a single key cellular process, so it would be expectable that the candidate determinants of acetic acid tolerance emerging from this study are related to different functions in cell. Indeed, the genes identified in our screening as strong candidate determinants of acetic acid tolerance in the highly tolerant strain ISA1307 include genes presumably involved in functions such as cellular transport and transport routes, protein fate, protein synthesis, amino acid metabolism and transcription, as detailed below. The ORFs identified in this study that share poor or no homology with S. cerevisiae genes should also be considered as relevant, in particular ZBAI_09903 that holds a pyrimidine binding domain of pyruvate decarboxylase required for the conversion of pyruvate into acetaldehyde and ethanol. Interestingly, a pyruvate decarboxylase enzyme, involved in pyruvate fermentation to acetaldehyde and ethanol, was recently reported as being up-regulated in acetic acid-grown ISA1307 cells compared to cells grown on glucose . Moreover, the deletion of PDC1, coding for S. cerevisiae pyruvate decarboxylase, was found to lead to a susceptibility phenotype in cells exposed to acetic and propionic acids [7, 8].
Among the strong candidate determinants of acetic acid tolerance we identified GYP8 and WSC4 homologues, putatively involved in cellular transport, in particular vesicular transport. The processes of transport vesicle formation, targeting, docking and tethering require complex molecular machinery. Different Ras-like GTPases contribute to these activities by acting as key regulators in assembling specific protein complexes at different donor and target membranes [21–23]. In S. cerevisiae, GYP8 encodes a GTPase-activating protein for Ypt/Rab GTPases, which plays an essential function in endoplasmic reticulum to Golgi vesicular protein transport. S. cerevisiae gyp8Δ was found to be sensitive to acetic acid and the expression of the Z. bailii GYP8 homologue, sharing 99 % identity with Z. bailii CLIB213T, led to a suppression of the mutant phenotype providing evidences of a similar function in Z. bailii. To our best knowledge, this is the first time that this gene is related with increased tolerance to acetic acid in yeast. Also in the context of cellular transport, we found that the expression of WSC4 homologue (presumably derived from the non-Z. bailii parental strain) suppresses the susceptibility phenotype of S. cerevisiae wsc4Δ mutant. S. cerevisiae Wsc4 is specifically involved in the translocation of soluble secretory proteins and insertion of membrane proteins into the ER membrane. In addition to its role in protein trafficking activity, Wsc4 was also described to play a role in S. cerevisiae response to a number of stresses, namely to heat shock, exposure to ethanol or 4-nitroquinoline 1-oxide (4-NQO), a DNA-damaging agent . To our knowledge, there is no published information about the increased susceptibility to weak acids of wsc4Δ mutant, but the WSC4 gene was found to be up-regulated after exposure to acetic acid by two-fold in S. cerevisiae . Moreover, it is known that this gene is co-regulated by the weak acid-responsive transcription factors Haa1 and Rim101 in response to acetic acid stress .
Genes homologous to S. cerevisiae RKR1, PMT1 and KTR7, putatively involved in modification and degradation of proteins, were also considered strong candidate determinants of acetic acid tolerance. In S. cerevisiae, both PMT1 and KTR7 code for mannosyltransferases that have been described as key enzymes for protein glycosylation known to be essential for cell wall rigidity [25, 26]. The remodelling of cell wall is considered a common response to several environmental stresses . In the specific case of weak acid stress, such remodelling is known to be an essential adaptive response that presumably leads to the reduction of the diffusion rate of the undissociated weak acid form into the cell interior, limiting the futile cycle resulting from the continuous uptake of the lipophilic form by passive diffusion followed by the active expulsion of the counterion through specific transporters [27, 28]. Remarkably, the family-related S. cerevisiae gene KTR4, encoding another mannosyltransferase involved in protein glycosylation, was previously identified as a determinant of acetic acid tolerance .
The ILV3 homologue was also identified as a strong candidate determinant of acetic acid tolerance. In S. cerevisiae, ILV3 codes for a dihydroxyacid dehydratase that catalyzes the third step in the pathways leading to biosynthesis of branched-chain amino acids . Interestingly, the content of Ilv3 was found to be higher in acetic acid-challenged yeast cells, compared with unstressed cells .
Among the DNA inserts identified during the present work, the best inserts whose expression confers a high protective effect against acetic acid stress in both the parental and the deletion mutant strains are S06 and those containing MSN4 or TIF3 homologues.
The ORF ZBAI_02295, one of two ORFs included in S06 insert and presumably involved in membrane trafficking, was identified as being responsible for the considerable increase in the acetic acid tolerance of the haa1Δ mutant, but not of the parental strain, thereby suggesting that the expression of ZBAI_02295 can support the function of Haa1 targets in S. cerevisiae but cannot, at least individually, further enhance the acetic acid tolerance of the parental strain. The contribution of the encoding protein, containing a functional domain associated to the uncharacterised DUP family to acetic acid tolerance, is still unclear. DUP proteins were previously suggested as being connected to membrane trafficking and hypothesized as being involved in the trafficking of membrane transporters [20, 31]. Results gathered from large-scale chemical genomics screenings suggest that intracellular trafficking processes are required for weak acid tolerance . Moreover, the genes of the DUP240 family in S. cerevisiae YAR028W and YAR029W were found to be up-regulated, their transcription being Haa1 dependent, in cells exposed to acetic acid stress , reinforcing the idea that this family of proteins is involved in yeast response and tolerance to acetic acid and that the expression of ZBAI_02295 in haa1Δ can compensate the abolishment of YAR028W and YAR029W transcription and therefore elevate the acetic acid susceptibility of the mutant up to the level of the parental strain. This increase of tolerance was not observed in the parental cells maybe because the putative membrane protein originated from the ORF ZBAI_02295 does not bring any advantage in cells where the Haa1 regulon is transcribed. The fact that the expression of S06 insert, which contains ZBAI_02295 and ZBAI_02296, leads to the increase of the acetic acid tolerance of the parental strain may be due to the conjugated effect of both ORFs and also to the different conditions used, namely the medium composition and the cloning vector. The expression of the genes from S06 DNA insert under the control of their native promoters compared with the individual expression of each gene under the GAL1 inducible promoter can be different and it is likely that the overproduction of the two putative membrane proteins encoded by these ORFs might be deleterious to the host cell.
The relevance of TIF3 homologue as a putative translation initiation factor in yeast tolerance to acetic acid may rely on the fact that stressed cells generate stress-responsive programmes consisting on a specific up-regulation of translation initiation towards the induction of specific adaptation proteins, while a rapid inhibition of protein synthesis in general occurs, in order to rationalize the consumption of resources . Using the non-flocculating Z. bailii strain IST302, it was possible to confirm that the increased expression of ZbTIF3 in this same strain enhances its tolerance to acetic acid.
ISA1307 MSN4 gene is homologous to S. cerevisiae paralogous genes encoding the transcription factors Msn2 and Msn4. These transcription factors are involved in the regulation of genes of the general stress response and, despite their high homology, both proteins play non-redundant and condition-specific roles in gene expression regulation in S. cerevisiae . Interestingly, it was found that both the Haa1 and Msn2/4 regulons share a total of 23 acetic acid-activated genes . For this reason, it is likely that the strain ISA1307 Msn4 homologue has the ability to suppress haa1Δ susceptibility phenotype in S. cerevisiae. Remarkably, ISA1307 Msn4 homologue was indeed one of the best acetic acid susceptibility suppressors found in the screening. Moreover, the expression of ZbMSN4 from IST302 either in S. cerevisiae parental strains BY4741 and W303-1A or the derived deletion mutants msn4Δ and msn2Δmsn4Δ confers increased protection against acetic acid, indicating that ZbMsn4 has a role similar to its homologous S. cerevisiae protein. In addition, the increased expression of ZbMSN4 in Z. bailii IST302 also elevated this strain’s tolerance to acetic acid, confirming this putative transcription factor as an important determinant of acetic acid resistance in Z. bailii.
In the present study we have identified several strong candidate determinants of tolerance to acetic acid in the intrinsically highly acetic acid tolerant Z. bailii-derived hybrid strain ISA1307. Specifically, this is the case of the following S. cerevisiae homologues presumably involved in different cellular processes - GYP8 and WSC4 (cellular transport and transport routes), PMT1, KTR7 and RKR1 (protein fate), TIF3 (protein synthesis), ILV3 (amino acid metabolism), MSN4 (transcription) - and of ZBAI_02295 (homologous to membrane proteins of unknown function and presumably involved in cellular trafficking). The overexpression of ZbMSN4 and ZbTIF3 genes, which were confirmed as playing a relevant role in acetic acid tolerance in yeast through their homologous and heterologous expression in Z. bailii and S. cerevisiae strains, may be useful considering the improvement of yeast cell robustness against acetic acid if the objective is their use as cell factories.
Strains and growth media
The Saccharomyces cerevisiae deletion mutant strain BY4741_haa1Δ, derived from the parental strain BY4741 (MATa, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0) and obtained from the EUROSCARF collection was used as the host strain for the screening of acetic acid determinants of tolerance in a Zygosaccharomyces bailii-derived interspecies hybrid strain ISA1307 using a previously constructed genomic library . Strains gyp8Δ, ktr7Δ, rkr1Δ, msn4Δ, pmt1Δ, tif3Δ and wsc4Δ derived from the same S. cerevisiae parental strain were used for phenotype complementation assays. Since the deletion of ILV3 gene in the haploid background strain BY4741 is lethal, the ilv3Δ mutant used in phenotype complementation assays was derived from the diploid parental strain BY4743 (MATa/a, his3Δ1/his3Δ1, leu2Δ0/leu2Δ0, lys2Δ0/LYS2, MET15/met15Δ0, ura3Δ0/ura3Δ0) with one copy of ILV3 deleted. S. cerevisiae strains W303-1A (MATa, leu2Δ3, 112 trp1Δ1, can1Δ100, ura3Δ1, ade2Δ1, his3Δ11,15) and derived double deletion mutant msn2Δmsn4Δ  were used for complementation assays of MSN4 from Z. bailii IST302 (ZbMSN4). This strain was isolated from a spontaneous fermentation of grape must from Douro wine-producing areas and its genome was recently sequenced and annotated in our laboratory (unpublished results). The taxonomic identification of Z. bailii IST302 was confirmed by comparing the partial 26S ribosomal DNA sequence (Additional file 3) with other DNA sequences from Z. bailii strains using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI). Strain IST302 was used for the amplification of ZbMSN4 and ZbTIF3 genes that were cloned into pZ3 bT. This strain was also used as a host cell for the expression of an extra copy of its ZbMSN4 and ZbTIF3 genes. Escherichia coli XL1-Blue was used for plasmid maintenance and general cloning procedures. E. coli cells were grown in Luria-Bertani medium (LB), supplemented with 150 μg/ml ampicillin when required. Yeast strains were batch-cultured at 30 °C with orbital agitation (250 rpm) in liquid mineral medium (MM) that contains, per litre: 1.7 g yeast nitrogen base without amino acids or (NH4)2SO4 (Difco Laboratories, Detroit, Mich.), 20 g glucose (Merck) and 2.65 g (NH4)2SO4 (Merck). Supplementation was needed for auxotrophic strains either with 20 mg methionine, 20 mg histidine, 60 mg leucine and 20 mg uracil for growth of BY4741 and BY4743 background strains (MM4), or with 20 mg histidine, 60 mg leucine, 40 mg tryptophan, 80 mg adenine and 20 mg uracil for growth of W303-1A background strains (MM5) (all from Sigma, Spain). For transformants selection, yeast strains were grown in MM4 or MM5 media without uracil supplementation. YPD medium (2 % glucose (Merck), 1 % yeast extract (Difco) and 2 % peptone (Difco)) was used for yeast cells’ maintenance and to grow cells for transformation experiments. Solid media were obtained by adding 20 g of agar to each litre of the corresponding liquid media. All strains were maintained at –80 °C in appropriate media supplemented with 15 % glycerol (v/v).
Screening of the genomic library from Z. bailii-derived interspecies hybrid strain ISA1307
Transformation of S. cerevisiae BY4741_haa1Δ with strain ISA1307 genomic library
The ISA1307 strain genomic library constructed by Rodrigues et al.  in the pRS316 vector  was used in this work. This plasmid is a shuttle vector for S. cerevisiae and E. coli, containing origins of replication for both species and Ampr and URA3 genes as selection markers. Moreover, this cloning vector is a centromeric plasmid, possessing an S. cerevisiae centromere (CEN6). The choice for a low copy plasmid is useful for isolation of genes that may have a toxic effect when present in multiple copies. For library plasmid DNA isolation, E. coli cells containing the genomic library were grown at 26 °C on LB medium supplemented with ampicillin until late stationary-phase and plasmid DNA was extracted from these cells using the QIAprep® Spin Maxiprep Kit according to the manufacturer’s instructions. S. cerevisiae BY4741_haa1Δ strain was transformed with library plasmid DNA, using the lithium acetate method . The transformation mixture was plated onto solid MM4 medium without uracil and plates were incubated at 30 °C for 3 days.
Screening of the transformants for acetic acid tolerance
Selection of the transformants showing more tolerance to acetic acid was performed in 96-well microplates containing liquid MM4 medium without uracil at pH 4.0. At the end of the day, a first plate containing 200 μl of liquid medium was inoculated with cells taken from the different yeast transformants under study, and incubated at 30 °C overnight with orbital agitation at 250 rpm. On the next morning, each inoculum was diluted and used to inoculate a microplate containing the same liquid media supplemented with 60 mM of acetic acid, at an initial OD600nm of about 0.05. This plate was incubated at 30 °C for 24 h under stirring at 250 rpm and after that time period the culture OD600 nm was measured (VersaMax microplate reader, Molecular Devices). The tolerance of transformants to acetic acid was based on these values of growth observed after 24 h of incubation. Candidates were considered susceptible to acetic acid when OD600nm reached values below 0.250, while candidates with an intermediate tolerance phenotype to acetic acid exhibited an OD600nm between 0.250 and 0.350 and the tolerant candidates an OD600nm above 0.350. S. cerevisiae BY4741_haa1Δ was used as the negative control as it is particularly susceptible to 60 mM acetic acid, pH 4.0, being unable to grow within the period of incubation used.
Comparison of the tolerance of the selected candidates to acetic acid
A confirmation screening of acetic acid tolerance phenotype was performed with the isolation of total DNA from yeast transformants selected as the most tolerant to acetic acid. DNA was used to transform E. coli XL1-Blue strain competent cells by electroporation using Bio-Rad Gene Pulser II (400 Ω, 25 μF, 2.5 kV). The transformants obtained were selected for ampicillin resistance and cultivated for 12 h in 3 ml of LB liquid medium with ampicillin at 37 °C with an orbital agitation of 250 rpm. For plasmid isolation from E. coli a QIAprep® Spin Miniprep Kit was used, according to the manufacturer’s instructions. The extracted DNA was then used to transform the parental strain S. cerevisiae BY4741 and the derived deletion mutant BY4741_haa1Δ, using the lithium acetate method . Growth curves of these strains in liquid media, either in the absence or presence of acetic acid were determined. Cells were grown in MM4 medium without uracil at pH 4.0 until exponential phase (OD600nm of 0.5 ± 0.05) and then re-inoculated at an OD600nm of 0.05, in 50 ml of fresh medium, either or not supplemented with 60 mM of acetic acid. Growth was followed by measuring culture OD600nm during batch cultivation at 30 °C with an orbital agitation of 250 rpm. The parental strain S. cerevisiae BY4741 and the mutant BY4741_haa1Δ both transformed with the empty vector were used as controls. The suppressors of acetic acid susceptibility phenotype of BY4741_haa1Δ were selected and submitted to further sequence analysis. Finally, an additional confirmation step of the phenotype of BY4741_haa1Δ cells transformed with the sequenced suppressors was performed by repeating their growth curves in control conditions and in the presence of 60 mM of acetic acid in 96-well microplates (FilterMax F5, Multi-mode microplate reader, Molecular Devices), at 30 °C. Microplate reads were carried out every 10 min using a 595 nm absorbance filter. An orbital agitation of three seconds was performed prior to each read. The growth curves of S. cerevisiae BY4741 parental strain transformed with these same suppressors were repeated as well. The plates were inoculated with cells in exponential phase (OD600nm of 0.5 ± 0.05) at an initial OD600nm of 0.05. In order to be comparable, all the growth curves must come from the same plate, since the level of inhibition of the acid is hardly reproducible from plate to plate due to the extremely small volume contained in each well. So, all the parental strain transformants were grown in the same plate, and the same was done for the transformants of the deletion mutant BY4741_haa1Δ. For comparison purposes between the transformants from the two different backgrounds, the growth curve of the parental strain with the empty vector was included in the plates with the BY4741_haa1Δ transformants.
In silico analysis of the selected plasmid DNA inserts from strain ISA1307 genomic library
The complete DNA sequence of the selected plasmid inserts from strain ISA1307 genomic library was obtained in two steps. First, approximately 1000 bp of the DNA sequence ends of each plasmid insert were sequenced using vector specific primers (forward and reverse). Second, the complete sequence of the plasmid insert was acquired by alignment of the sequenced ends against the genome sequence of the strain ISA1307 available in the PEDANT database (http://pedant.helmholtz-muenchen.de/genomes.jsp?Category=fungal) . This sequence was part of a high-throughput DNA sequencing project, based on Illumina paired-end sequencing, carried out by our group . The in silico analysis of each DNA plasmid insert from the genomic library was performed also using the PEDANT database and the genomic browser GBrowse_syn (http://mips.helmholtz-muenchen.de/gbrowse2/cgi-bin/gbrowse_syn/zbailii)  that returns all the putative open reading frames (ORFs) present in the DNA fragment based on sequence homology with S. cerevisiae. Additionally, this tool provides the synteny alignments for the Z. bailii-derived interspecies hybrid strain ISA1307 genome versus the genomes of Z. bailii CLIB213T, Z. rouxii and S. cerevisiae. When the plasmid DNA insert contained truncated ORFs, the presence of specific functional domains was assessed using the Conserved Domain Database of the NCBI . The description of each gene was based on the information gathered in Saccharomyces Genome Database (www.yeastgenome.org) for the corresponding putative homologue.
Cloning and expression of ZBAI_02295 and ZBAI_02296 in S. cerevisiae
The pGREG506 plasmid from the DRAG & DROP collection  was used to individually clone by homologous recombination and express the genes ZBAI_02295 and ZBAI_02296 identified in the S06 DNA fragment from ISA1307 genomic library. That cloning vector was acquired from Euroscarf and contains a HIS3 gene under the control of a galactose inducible promoter (GAL1) and the yeast selectable marker URA3. During homologous recombination the HIS3 gene is replaced by the gene of interest. ZBAI_02295 and ZBAI_02296 DNA fragments were generated by PCR using genomic DNA extracted from the strain ISA1307 and the specific primers ZBAI_02295_FWD, ZBAI_02295_REV, ZBAI_02296_FWD and ZBAI_02296_REV, whose sequences are listed in Additional file 4. The cDNA from each gene was co-transformed into the parental strain BY4741 and derived deletion mutant haa1Δ with the pGREG506 vector, previously cut with SalI restriction enzyme. The recombinant plasmids pGREG506_ ZBAI_02295 and pGREG506_ ZBAI_02296 were obtained through homologous recombination in yeast. Correct cloning of each ORF was confirmed by DNA sequencing. Additionally, S. cerevisiae BY4741 parental and derived deletion mutant haa1Δ strains were transformed with the cloning vector pGREG506. To have the adequate control cells, both strains were transformed as well with the empty vector pGREG506 with the HIS3 gene deleted (pGREG506_noHIS3, obtained by digestion of pGREG506 with the restriction enzyme SalI), since in the recombinant vector the HIS3 gene is substituted by the insert of interest. The susceptibility to acetic acid of the parental strain BY4741 and the derived deletion mutant haa1Δ, harbouring each one of the four described plasmids (pGREG506_ ZBAI_02295, pGREG506_ ZBAI_02296, pGREG506 and pGREG506_noHIS3) was assessed. This was possible by comparing their growth curves in liquid MM4-U medium supplemented with 1 % of galactose (w/v) pH 4.0, at 30 °C with orbital agitation (250 rpm), either or not supplemented with 60 mM acetic acid, as previously described.
Complementation of S. cerevisiae deletion mutants by the corresponding Z. bailii ISA1307 single genes present in genomic library plasmids
The genomic library plasmids 8.23, 18.22, 23.1, B02, B18, S07 and Y08 carrying the single S. cerevisiae putative homologous genes GYP8, PMT1, WSC4, MSN4, TIF3, KTR7 or RKR1, respectively, were transformed in S. cerevisiae BY4741 EUROSCARF mutants lacking the respective gene. The complementation of library plasmid 23.17 carrying ILV3 was performed using the diploid BY4743 parental strain with one copy of the essential ILV3 gene deleted, because the haploid BY4741_ilv3Δ is unviable. Both S. cerevisiae parental strains transformed with the empty vector were used as control. The transformants were batch-cultivated in MM4-U medium at pH 4.0, either or not supplemented with 60 mM of acetic acid, as previously described. Since S. cerevisiae tif3Δ is highly susceptible to acetic acid, a growth medium containing a lower concentration of acetic acid (40 mM) was used. The complementation assays using the diploid strains derived from BY4743 were performed with a higher concentration (65 mM) of acetic acid.
Cloning and expression of ZbMSN4 in S. cerevisiae
The gene ZbMSN4 amplified from Z. bailii IST302 was cloned by homologous recombination into pGREG506 using the strategy above described for the cloning of ZBAI_02295 and ZBAI_02296. The primers ZbMSN4_pGREG_FWD and ZbMSN4_pGREG_REV (Additional file 4) were used for the amplification of ZbMSN4 coding region. This DNA fragment and the pGREG506 vector previously digested with SalI restriction enzyme were co-transformed into S. cerevisiae BY4741 parental strain. The obtained recombinant plasmid pGREG506_ZbMSN4 was digested with SpeI and AscI restriction enzymes to remove the GAL1 promoter from the vector. The digested vector and S. cerevisiae MSN4 promoter (approximately 1000 bp upstream the start codon) that was amplified with primers ZbMSN4prom_FWD and ZbMSN4prom_REV (Additional file 4) were co-transformed into S. cerevisiae BY4741 parental cells. The recombinant vector pGREG506_promMSN4Sc_ZbMSN4 obtained by homologous recombination was sequenced to confirm the correct cloning of the promoter region and the gene and transformed afterwards into S. cerevisiae BY4741_msn4Δ, W303-1A parental and derived double deletion mutant msn2∆msn4∆ strains. The susceptibility to acetic acid of the parental strains BY4741 and W303-1A and of the corresponding derived deletion mutants msn4Δ and msn2∆msn4Δ, harbouring pGREG506_promMSN4Sc_ZbMSN4 and also pGREG506_noHIS3 was assessed by comparing their growth curves in liquid MM4-U (BY4741 background strains) and MM5-U (W303-1A background strains), either or not supplemented with the appropriate acetic acid concentration.
Cloning and expression of an extra copy of ZbMSN4 and ZbTIF3 in Z. bailii IST302
The expression of an extra copy of ZbMSN4 and ZbTIF3 genes in the parental strain Z. bailii IST302 was performed by cloning these genes by homologous recombination into the centromeric expression vector pZ3 bT  linearized with XbaI. The genes ZbMSN4 and ZbTIF3 and their corresponding promoters (approximately 1000 bp upstream the start codon) were amplified from strain IST302 genomic DNA using the primers ZbMSN4_FWD, ZbMSN4_REV, ZbTIF3_FWD and ZbTIF3_REV (Additional file 4) and co-transformed with the linearized pZ3 bT into S. cerevisiae BY4741. Selection of the transformants holding the recombinant vectors pZ3 bT_ ZbMSN4 and pZ3 bT_ ZbTIF3 was performed in YPD plates containing G418 (200 mg/L). The correct recombination was confirmed by sequencing the obtained plasmids. The recombinant plasmids and the empty plasmid were used to transform Z. bailii IST302 using a commercial yeast transformation kit (MP Biomedicals, California) with minor modifications. The susceptibility to acetic acid of Z. bailii IST302 cells transformed with pZ3 bT or with the recombinant vectors pZ3 bT_ ZbMSN4 and pZ3 bT_ ZbTIF3 was assessed by comparing their growth curves in liquid MM either or not supplemented with 180 or 220 mM acetic acid.
All the supporting data of this article is included within the article and in its additional files. The partial sequence of the 26S ribosomal RNA gene of Z. bailii strain IST302 (Additional file 3) is deposited in GeneBank (accession number: KU194200).
We thank Dr. Paola Branduardi (University of Milano-Bicocca) for providing pZ3 bT plasmid and Dr. Marian Carlson (Columbia University) for the double deletion S. cerevisiae mutant msn2Δmsn4Δ.
Funding received by iBB-Institute for Bioengineering and Biosciences from FCT-Portuguese Foundation for Science and Technology (UID/BIO/04565/2013) and from Programa Operacional Regional de Lisboa 2020 (Project N. 007317) is acknowledged. FCT supported post-doctoral grant to MP (SFRH/BPD/73306/2010) and PhD grants to FCR (SFRH/BD/82226/2011) and JFG (SFRH/BD/80065/2011). LQ was an Erasmus exchange student from Institut Universitaire de Technologie de Dijon, France.
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