Antonie van Leeuwenhoek (2014) 105:641–652 DOI 10.1007/s10482-014-0118-3
ORIGINAL PAPER
Presence of proline has a protective effect on weak acid stressed Saccharomyces cerevisiae D. Greetham • H. Takagi • T. P. Phister
Received: 12 November 2013 / Accepted: 17 January 2014 / Published online: 6 February 2014 Ó European Union 2014
Abstract Fermentation of sugars released from lignocellulosic biomass (LCMs) is a sustainable option for the production of bioethanol. LCMs release fermentable hexose sugars and the currently non-fermentable pentose sugars; ethanol yield from lignocellulosic residues is dependent on the efficient conversion of available sugars to ethanol, a sideproduct of the process is acetic acid production. Presence of acetic acid reduced metabolic output and growth when compared with controls; however, it was observed that incubation with proline had a protective effect, which was proline specific and concentration dependent; the protective effect did not extend to furan or phenolic stressed yeast cells. Proline accumulating strains displayed tolerance to acetic acid when compared with background strains, whereas, strains with a compromised proline
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0118-3) contains supplementary material, which is available to authorized users. D. Greetham (&) T. P. Phister School of Biosciences, University of Nottingham, Loughborough, Leicestershire LE12 5RD, UK e-mail: [email protected] H. Takagi Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Japan
metabolism displayed sensitivity. Sensitivity to weak acids appears to be reduced with the addition of proline; proline is an imino acid freely available as a nitrogen source in the aerobic phase of fermentations. Yeast strains with higher intracellular proline concentrations would be desirable for industrial bioethanol fermentations. Keywords Proline Weak acids Yeast Fermentation Microarrays
Introduction Short-chain weak organic acids are potent inhibitors of microbial growth with a wide range of applications in the food and beverage industries, they are released during industrial fermentation processes or by the deacetylation of xylan during pretreatment (Palmqvist et al. 1999). The action of acetic acid has been well documented, with stress beginning at 0.05 % (*8.3 mM) in a typical industrial yeast fermentation (Narendranath et al. 1997; Thomas et al. 2002). Acetic acid toxicity is pH dependent, as acetic acid in its undissociated form diffuses through the cell membrane with dissociation dependent upon cytosolic pH. Intracellular pH is maintained through ATPase transporter systems transporting protons across the cell membranes (Verduyn et al. 1990). Exposure to acetic acid in yeast stimulates a form of programmed cell
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death (PCD); PCD is initiated through a mitochondria specific caspase cascade and leads to cellular death (Madeo et al. 2004). This response to acetic acid is separate from acidifying the cytosol via passive diffusion of acetic acid through cell membranes. During fermentations, presence of low concentrations of acetic acid (\25 mM) stimulates ATP production, and the rate of ethanol production increases when compared with the unstressed control (Taherzadeh et al. 1996). At increasing acetic acid concentrations ([25 mM), this stimulation in ATP production is overtaken by a lowering in cytosolic pH and an increase in ATPase activity (Taherzadeh et al. 1996). Proline is an imino acid that can be a source of nitrogen in yeast, however, due to a requirement for oxygen metabolism is restricted to the early aerobic phase of fermentation (Ough and Amerine 1988). During early stages of fermentations, availability of nitrogen sources such as ammonia and glutamine results in transport of proline being suppressed (Soetens et al. 2001). During later stages of fermentation when such preferred nitrogen sources have declined and transport is no longer repressed, the absence of oxygen means that proline is no longer available as a nitrogen source to the cell (Poole 2002). Strains with the ability to utilise proline as a nitrogen source have been shown to produce twice the biomass of a reference strain (Martin et al. 2003). Addition of proline as an individual amino acid or in combination with other amino acids (glycine betaine and glycine) helps maintain viability of yeast through the course of a very high gravity fermentation when compared with control conditions (Thomas et al. 1994) Beyond the nutritional advantages of providing accessible nitrogen, enhanced proline uptake has been shown to convey a broad protective effect, such as growth advantages under oxidative stress Terao et al. (2003) and as a cryoprotectant Morita et al. (2002). Intracellular levels of proline have been correlated with stress resistance in yeast (Takagi et al. 2000), yeast strains which accumulate proline display improved viability to freezing or desiccation (Morita et al. 2002; Takagi et al. 2000; Poole et al. 2009). The specific role of proline as an osmoprotector and cryoprotector is currently unclear. We aimed to look at the importance of proline to the performance of yeast during bioethanol fermentations.
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Materials and methods Yeast strains and growth conditions Saccharomyces cerevisiae NCYC 2592 was obtained from the National Collection of Yeast Cultures (NCYC), Norwich, UK, S. cerevisiae BY4741 and selected deletion mutants were a kind gift from Professor Ed Louis (University of Nottingham, UK) and S. cerevisiae proline accumulating yeast strains were a kind gift from Professor Takagi (Nara Institute of Science and Technology, Japan) respectively. All strains were maintained on YPD containing 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, and 20 g/L agar. Cultures were cryopreserved in glycerol stock solution at -80 °C. Viability Methylene blue was dissolved in 2 % (w/v) sodium citrate solution to give a final concentration of 0.01 % (w/v). Cells were grown to mid-exponential phase at 30 °C, with shaking in YPD (determined by a reading of between 0.4 and 0.6 nm at OD 600, at this point the relevant concentration of inhibitory compound/proline was added to the relevant flask and cells were exposed for 15 min before harvesting. Cells were resuspended in methylene blue and a cell count was performed using a haemocytometer. The cell suspension was diluted to a final concentration of 1 9 107 cells per mL and mixed in a 1:1 ratio with citrate methylene blue. The cells were microscopically examined after 5 min at 409 magnification. Dead cells stain blue, while the viable ones were colourless, as live cells exclude the stain (Pierce 1970; Sami et al. 1994). Viability counts were performed in triplicate and compared in the presence and absence of inhibitory compounds as appropriate. Viability testing by methylene blue is the industry standard in brewing, however, reports have shown that methylene blue may overestimate yeast viability (Land 2001). Phenotypic microarray analysis Biolog growth medium was prepared using 60 g/L (w/ v) glucose supplemented with 6.7 g/L (w/v) minimal medium (YNB—yeast nitrogen base), and 0.2 lL of dye D (Biolog, US). Volumes of amino acids, inhibitory compounds such as acetic, formic acid, furfural etc. from 1 M stock solutions were added as appropriate and
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the final volume adjusted to 120 lL with the addition of sterile deionised water and aliquoted to individual wells in a 96 well microassay plate (Biolog, US). Saccharomyces cerevisiae strains were prepared for inoculation using the following protocol, cryopreserved yeast colonies were streaked on to YPD plates and incubated at 30 °C for 48 h. Representative colonies were grown in YPD broth at 30 °C for 48 h. Harvested cells were washed twice and the pellet re-suspended in sterile water in 20 9 100 mm test tubes and transmittance was adjusted to 62 % using a turbidimeter (Biolog, US). The cell suspension for inoculation was prepared by mixing 0.125 mL of cells with 2.65 mL of IFY buffer (Biolog, USA) and adjusted to a final volume of 3 mL by the addition of sterile deionised water. The resulting cell suspension (90 lL) was inoculated into each well in the Biolog bespoke 96-well plate. Oxygen absorbing packs and CO2 producing packs (Mitsubishi AnaeroPakTMSystem) were used to create anaerobic conditions which were monitored using an anaerobic indicator (Oxoid, UK). Plates were covered using sterile PM bags (Biolog, US), heat sealed and incubated for 96 h at 30 °C in an Omnilog reader (Biolog, US). The OmniLog reader read the plates every 15 min, converting pixel density to a signal value reflecting the conversion of tetrazolium dye from the oxidised to the reduced form. After completion of the run, the signal data was compiled and converted using Biolog software into MicrosoftÒ Excel compatible data. In all cases, a minimum of three replicate PM assay runs were conducted, and the mean of the signal values reported. Metabolic output as measured by the phenotypic microarray is defined here as redox signal intensity (Greetham, et al., unpublished). Measurement of yeast growth Cells were grown to exponential phase in YPD medium as monitored by OD600 readings, and diluted to a starting OD600 of 0.2 using YPD broth. Either 25 mM acetic acid or 10 mM formic acid was added to wells on a 96-well microtitre plate and the volume of each well was adjusted to 100 lL using sterile deionised water. Yeast growth (OD600) was measured every 15 min using a Tecan Infinite M200 Pro plate reader (Mannedorf, Switzerland), at 30 °C for 72 h. The assay was performed in triplicate and the average reading was plotted.
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Fermentation Fermentations were conducted in 180 mL fermentation vessels (FV). Cryopreserved yeast colonies were streaked onto YPD plates and incubated at 30 °C for 48 h. Colonies of NCYC2592 were used to inoculate 20 mL of YPD broth and incubated in an orbital shaker at 30 °C for 24 h. These were then transferred to 200 mL of YPD and grown for 48 h in a 500 mL conical flask shaking at 30 °C. Cells were harvested and washed three times with sterile deionised water and then re-suspended in 5 mL of sterile water. Under control conditions, 1.5 9 107 cells/mL were inoculated in 99.6 mL of medium containing 4 % glucose, 2 % peptone, 1 % yeast extract with 0.4 mL sterile deionised water. Under inhibitor stress, 1.5 9 107 cells.mL-1 were incubated in 99.6 mL of medium containing 4 % glucose, 2 % peptone, 1 % yeast extract with 25 mM acetic acid and 10 mM proline. Volumes of media were adjusted to account for the addition of acetic acid (*145 lL) and proline (200 lL) to ensure that all fermentations began with the same carbon load. pH was monitored with readings taken every 2 h. Anaerobic conditions were established using a sealed butyl plug (Fisher, Loughborough, UK) and aluminium caps (Fisher Scientific). A hypodermic needle attached with a Bunsen valve was pierced through the rubber septum to facilitate the release of CO2. Weight loss as a means of monitoring rates of fermentation was measured in triplicate at regular time points during the fermentation, fermentations were conducted at 30 °C, with orbital shaking at 200 rpm. Yeast strains BY4741 and pro1I150T Dput1 were prepared for fermentation experiment as described above for S. cerevisiae NCYC2592. Under inhibitor stress, 1.5 9 107 cells/mL were incubated in 99.6 mL of medium containing 4 % glucose, 2 % peptone, 1 % yeast extract with 10 mM acetic acid. Volumes of media were adjusted to account for the addition of acetic acid (*58 lL) to ensure that all fermentations began with the same carbon load.
Detection of glucose and ethanol from FV experiments via HPLC Glucose and ethanol were quantified by HPLC. The HPLC system included a Jasco AS-2055 Intelligent
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auto sampler (Jasco, Tokyo, Japan) and a Jasco PU1580 Intelligent pump (Jasco). The chromatographic separation was performed on a Rezex ROA H? organic acid column, 5 lm, 7.8 9 300 mm, (Phenomenex, Macclesfield, UK) at ambient temperature. The mobile phase was 0.005 N H2SO4 with a flow rate of 0.5 mL/min. For detection a Jasco RI-2031 Intelligent refractive index detector (Jasco) was employed. Data acquisition was via the Azur software (version 4.6.0.0, Datalys, St Martin D’heres, France) and concentrations were determined by peak area comparison with injections of authentic standards. The injected volume was 10 lL and analysis was completed in 28 min. All chemicals used were analytical grade ([95 % purity, Sigma-Aldrich, UK).
Results Effect of acetic acid and amino acids on metabolic output of yeast cells The effect of 25 mM acetic acid on metabolic output of yeast using be-spoke phenotypic microarray plate was measured and it was observed that the presence of 25 mM acetic acid reduced metabolic output when compared with unstressed controls (Fig. 1a). Addition of 10 mM proline improved metabolic output in the presence of acetic acid (Fig. 1b); despite there being no effect on metabolic output in unstressed cells. 25 mM acetic acid was chosen for the assays as it is released under relatively mild pretreatment methods (unpublished data) and 10 mM proline was chosen as this concentration has been shown to inhibit c-glutamyl kinase (GK) activity, proline synthesis is strictly regulated through feedback inhibition of GK by proline (Sekine et al. 2007). Assays looking for improved metabolic output in the presence of acetic acid with other amino acids failed to observe any improvement (Supplementary Fig. S1). Proline has been identified as an osmoprotectant (Kaino and Takagi 2009), however, other osmoprotectants such as glycerol (Meikle et al. 1988) or phosphatidylcholine (Kiewietdejonge et al. 2006) in our phenotypic microarray assays failed to protect the yeast cell against weak acid stress (data not shown).
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Presence of proline improved metabolic output at higher concentrations of acetic acid stressed yeast cells Presence of 10 mM proline improved metabolic output at higher acetic acid concentrations (50 and 75 mM acetic acid) (Figs. 1c, d) with a further improvement observed in assays with 20 mM proline (data not shown). Proline’s protective role appears to be weak acid specific The protective role of proline to stress caused by inhibitory compounds present in hydrolysates derived from lignocellulosic material was assessed (TomasPejo et al. 2008). It was observed that presence of proline improved metabolic output in the presence of formic acid (Fig. 2a), but not for coumaric or furoic acid (Fig. 2e, f) or cells under furfural, HMF or vanillin stress (Fig. 2b–d). Assays with strong acids failed to observe any improvement in metabolic output for citric acid stressed cells in the presence of proline (Fig. 2g). There was no improvement in metabolic output in assays with 20 mM proline for furfural, HMF, vanillin or citric acid stressed cells (data not shown). Presence of proline had little effect on pH in the absence or presence of acetic acid Acetic acid enters the cell much more readily as an uncharged undissociated ion than as a charged anion. We assessed if the presence of proline had an effect on the pH of fermentations with the addition of acetic acid or under control conditions. It was observed that pH dropped from an initial pH of 5.3–4.15 during fermentation under control conditions (Fig. 3). Addition of 25 mM acetic acid at the beginning of the fermentation elicited an immediate reduction in pH to pH 4.2; however, subsequently pH did not alter significantly during the time period of the fermentation (Fig. 3). Addition of 10 mM proline had no effect on the pH under control conditions or in the presence of acetic acid (data not shown). Presence of proline improved yeast growth and viability under weak acid stress We assessed the impact of proline on yeast growth under control and weak acid stressed conditions, it was
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Fig. 1 Phenotypic microarray analysis (redox signal intensity) for S. cerevisiae (NCYC 2592) on media containing 0–25 mM acetic acid and 10 mM proline. Plates were incubated at 30 °C and read for 40 h, under anaerobic conditions. a Redox signal intensity for S. cerevisiae (NCYC 2592) in the presence of 0–25 mM acetic acid. b Redox signal intensity for S. cerevisiae (NCYC 2592) in the presence of 0–25 mM acetic acid plus 10 mM proline. c Redox signal intensity for S. cerevisiae (NCYC 2592) in the presence of 50 mM acetic acid plus 10 mM proline. d Redox signal intensity for S. cerevisiae (NCYC 2592) in the presence of 75 mM acetic acid plus 10 mM proline. Data an average of triplicate values with standard deviation shown. Redox signal intensity is a measurement of metabolism using a redox sensitive redox dye
observed that the addition of 10 mM proline had little effect on growth under anaerobic conditions suggesting that in the absence of oxygen proline was not metabolized in this assay (Fig. 4a). Incubation with proline improved growth in acetic acid or formic acid stressed yeast when compared with acid stressed cells (Fig. 4b, c). There was no improvement in cellular growth with proline under furfural, HMF or vanillin stress (data not shown). Viability assays showed no effect with addition of proline under unstressed conditions (Fig. 4d), however, there was an improvement in weak acid stressed cells (Fig. 4d), with viability increasing in the
presence of increasing proline (Fig. 4d). In comparison, we failed to observe an improvement in viability for cells under furfural stress (Fig. 4d) or other stresses such as HMF, vanillin or citric acid (data not shown). Accumulation of proline promotes a weak acid tolerant phenotype Previous assays relied on the introduction of proline into the media and assessed for the impact on metabolic output of cells. We wanted to assess for the importance of proline in the cell and through selected yeast strains examined how yeast metabolize,
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Fig. 2 Phenotypic microarray analysis (redox signal intensity) for S. cerevisiae (NCYC 2592) on media 10 mM proline plus inhibitory compounds. a Redox signal intensity for S. cerevisiae (NCYC 2592) on media containing 10 mM formic acid plus 10 mM proline. b Redox signal intensity for S. cerevisiae (NCYC 2592) on media containing 10 mM furfural plus 10 mM proline. c Redox signal intensity for S. cerevisiae (NCYC 2592) on media containing 10 mM HMF plus 10 mM proline. d Redox signal intensity for S. cerevisiae (NCYC 2592) on media
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containing 10 mM vanillin plus 10 mM proline. e Redox signal intensity for S. cerevisiae (NCYC 2592) on media containing 10 mM coumaric acid plus 10 mM proline. f Redox signal intensity for S. cerevisiae (NCYC 2592) on media containing 10 mM feroic acid plus 10 mM proline and g redox signal intensity for S. cerevisiae (NCYC 2592) on media containing 10 mM citric acid plus 10 mM proline. Data an average of triplicate values with standard deviation shown
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Fig. 3 Effect of acetic acid and proline on pH during a fermentation. pH was monitored during a fermentation using 4 % YPD for S. cerevisiae NCYC 2592 in the presence of 25 mM acetic acid and 10 mM proline. Readings were taken every 2 h. Data an average of triplicate values with standard deviation shown
transport and recycle proline. This work was performed in the S. cerevisiae BY4741 reference strain background to facilitate use of knockout strains, phenotypic microarrays confirmed that presence of proline protects against acetic acid stress in this strain as observed with S. cerevisiae NCYC 2592 (Supplementary data S2). Analysis of strains with components of the proline metabolism pathway disrupted for acetic acid tolerance showed that a strain with a pro1 knockout was more sensitive to acetic acid when compared with the background strain (BY4741) (Fig. 5a). PRO1 encodes an enzyme which is the first step in the conversion of glutamate into proline (Brandriss 1979) and the null strain has been linked to sensitivity to heat, osmotic and other stresses (Morita et al. 2003). There was a slight increase in sensitivity to acetic acid in a pro2 disrupted strain; PRO2 encodes the enzyme required for the next step in proline biosynthesis (Fig. 5a). The final step which leads to the formation of proline is catalysed by Pro3p; however, pro3 is a lethal knockout in the S. cerevisiae S288C background (Giaever et al. 2002). There was a significant reduction in viability in pro1 and pro2 knockouts when compared with BY4741 under acetic acid stress (Fig. 4c). PUT1 encodes an enzyme which begins the conversion of proline into glutamate (PUT2 encodes the second step in this pathway) and a put1 disrupted strain has been shown previously to have elevated levels of proline (Takagi et al. 2000). Assays with this strain showed an increase in metabolic output and viability when compared with BY4741 in the presence of weak acids (Fig. 5a, c). The importance of transcriptional activation of PUT1 and PUT2 was also investigated; PUT3
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regulates PUT1 and PUT2 expression by binding to promoter sites, metabolic output of a put3 strain was slightly elevated under acetic acid stress when compared with BY4741 (Fig. 5a). Addition of 10 mM proline recovered the metabolic output in pro1 or pro2 disrupted strains in the presence of acetic acid when compared with no proline added (Fig. 5b), however, additional proline elicited little improvement in a put1 disrupted strain to acetic acid (Fig. 5b). Adenine can be converted to proline, and in the absence of oxygen proline accumulates due to the inability of the cell to convert proline into glutamate which is catalysed by the oxygen dependent enzyme proline oxidase (Put1p) (Brandriss and Falvey 1992). The importance of this pathway for weak acid tolerance was assessed by looking at the genes which regulate the process of converting adenine into proline. Adenine is converted to proline in a two-step process catalysed by Car1p and Car2p (Middelhoven 1964). It was observed that metabolic output in car1 and car2 disrupted strains was reduced in the presence of weak acids when compared with BY4741 (Supplementary Fig. S2A). We used an fps1 disrupted strain as a positive control in these experiments, this strain has been shown to be weak acid tolerant Mollapour et al. (2008) and in our assays metabolic output was increased in response to weak acids when compared with BY4741 (Supplementary Fig. S2A). Proline accumulating mutants are weak acid tolerant Proline accumulation leading to tolerance to oxidative stress has been observed in a strain with an altered Pro1p enzyme (Kaino and Takagi 2009). Tolerance to osmotic stress was also noted if this enzyme was expressed in a put1-disrupted strain (Sasano et al. 2012a, b). It was observed that the strains with higher intracellular proline levels (Sasano et al. 2012a, b) had significantly higher metabolic output under control conditions and were significantly more tolerant to 10 mM acetic acid than BY4741 (Fig. 6a, b). Proline accumulating yeast strains display acetic acid tolerance during fermentation The performance of pro1I150TDput1 was compared with the background strain BY4741 during fermentations in the presence of 10 mM acetic acid. Under
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Fig. 4 Effect of acetic acid and proline on the growth of S. cerevisiae NCYC2592. Growth was monitored on a TECAN plate reader at OD600 for 96 h with readings taken every 15 min for a control and 10 mM proline (b) control, 25 mM acetic acid
and 25 mM acetic acid and 10 mM proline (c) control, 10 mM formic acid and 10 mM formic acid. d The effect of inhibitory compounds and proline on yeast viability. Data an average of triplicate values with standard deviation shown
control conditions, glucose utilisation and production of ethanol were identical for BY4741 and pro1I150TDput1 (Fig. 6c, d), in the presence of 10 mM acetic acid, however, there was a delay in both glucose utilisation and production of ethanol in BY4741 when compared with pro1I150TDput1 (Fig. 6c, d). Indeed, the presence of acetic acid appeared to have no influence on the rate of fermentation for pro1I150TDput1 when compared with unstressed control conditions (Fig. 6c, d).
Discussion
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Proline is an imino acid freely available to yeast at relatively high concentrations during beverage fermentations; however, requirements for oxygen restrict proline utilisation as a nitrogen source to the early stages of fermentation. Previous work has shown that the presence of proline or arginine correlates to stress resistance in yeast, though its precise mode of action is unclear (Takagi et al. 2000); Morita et al. 2002).
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Fig. 5 Phenotypic microarray analysis (redox signal intensity) for S. cerevisiae (BY4741, put1, put2, pro1, pro2, and put3 to inhibitory compounds and proline. a Effect of 50 mM acetic acid, 75 mM acetic acid, 10 mM formic acid, 10 mM furfural or 10 mM vanillin defined as % redox signal intensity (%RSI) of stressed versus control. b Effect of 50 mM acetic acid and 50 mM acetic acid plus 10 mM proline defined as % RSI of stressed versus control. c The effect of 50 and 75 mM acetic acid on viability. Data an average of triplicate values with standard deviation shown
Disruption of key genes in the conversion of proline into glutamate leads to proline accumulation and improved viability to freezing and desiccation Takagi et al. (2000). Accumulation of proline has also been associated with tolerance to oxidative stress whereas accumulation of proline with trehalose improves fermentation rates when compared with accumulation of either proline or trehalose individually( Sasano et al. 2012a, b). Weak acids such as acetic and formic acid inhibit yeast fermentations reducing both growth and ethanol production. The inhibitory effects of weak acids have been linked to intracellular anion accumulation leading to a reduction in cytosolic pH affecting enzyme kinetics (Pereira et al. 2011). Low concentrations of weak acids can increase ethanol production at the
expense of cellular division, though the precise mode of action for this increase is unclear (Pereira et al. 2011). Pre-treatment processes also release phenolic compounds such as vanillin and these compounds have demonstrated inhibitory effects on yeast cells (Endo et al. 2008, 2009). Presence of proline made cells more tolerant to weak acids when compared with other amino acids, this appeared to be weak acid specific as proline had no impact on furanic or phenolic stressed yeast cells. Tolerance appeared to be concentration dependent as increasing concentrations of proline increased metabolic output and viability in the presence of weak acids. There was no change in pH in assays containing proline compared with absence of proline under control or weak acid stress conditions suggesting that
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Fig. 6 Phenotypic microarray analysis (redox signal intensity) for S. cerevisiae (BY4741, pro1I150T and pro1I150T Dput1) to acetic acid and proline. a Redox signal intensity under control conditions b Redox signal intensity in the presence of 25 mM acetic acid. c Glucose utilisation during fermentation by BY4741 and pro1I150T Dput1 under control and acetic acid stressed conditions d Production of ethanol during a fermentation of BY4741 and pro1I150T Dput1 under control and acetic acid stressed conditions. Data an average of triplicate values with standard deviation shown
the protective role of proline is response to weak acids rather than a response to a drop in pH. In yeast, proline is synthesized from glutamate in a three step process and recycled back to glutamate in a two-step process (Sasano et al. 2012a, b). It was observed that put1 disrupted strains were more tolerant to weak acids when compared with BY4741 (Fig. 5a), there was no improvement in tolerance to furfural or vanillin in this strain (data not shown). Put1p catalyses the first step in the conversion of proline into glutamate and put1 disrupted strains have higher levels of proline in their cells. There was also increased sensitivity in pro1 and pro2 disrupted
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strains, with pro1 in particular displaying sensitivity to weak acids (Fig. 5a). This sensitivity could be rescued by the addition of proline, suggesting that intracellular proline was the reason for weak acid sensitivity in these strains. PRO1 and PRO2 encode enzymes which convert glutamate into proline, pro1 and pro2 disrupted strains have been shown to be less tolerant to oxidative, osmotic and other stresses (Kaino and Takagi 2009). Strains incapable of converting adenine into proline also displayed sensitivity to weak acids; this pathway is of particular relevance during fermentations as further conversion of proline is not possible without the presence of
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oxygen leading to proline accumulation under anaerobic conditions. Disruption of proline regulator (PUT3) had little influence on weak acid tolerance. A put3 disrupted strain displayed a slight tolerance to weak acid when compared with BY4741 (Fig. 5a), though there is evidence that in the absence of Put3p, the Gal4p transcriptional activator is able to upregulate PUT2 (D’Alessio and Brandriss 2000). It would be interesting to investigate a gal4,put3 double deletion and assess for weak acid sensitivity. Previous work has shown that yeast strains with higher intracellular proline concentrations are osmotically and oxidatively tolerant (Sasano et al. 2012a, b). It was observed that these strains were weak acid tolerant when compared with BY4741; these strains displayed no increased tolerance to other inhibitory compounds. Fermentations using a proline accumulating yeast strain were significantly faster in the presence of acetic acid when compared with the background strain despite no observable differences under control conditions. On the basis of this data we have displayed the importance of proline in cells under weak acid stress. Presence of weak acids in hydrolysates from plant material is unavoidable as the processes involved in liberating fermentable sugars also generates weak acids. This paper is the first to show that proline has a role in how yeast cells cope with weak acid stress; this appears to be weak acid and proline specific. We observed that cells unable to synthesise proline are weak acid sensitive, whereas, those which have high intracellular proline concentrations are weak acid tolerant. Running a fermentation under sterile conditions on a large scale is not economically viable, and maintaining a low pH (\4.5) to prevent bacterial contamination is important. Fermentations of hydrolysates from lignocellulosic has shown that ethanol is produced during a fermentation with a starting pH of 4 (Kadar et al. 2007). Proline consumption has been observed in fermentations of lychee juice under control conditions, however, the poor nitrogen concentrations of this media led to this consumption (Chen et al. 2014). Hydrolysates from lignocellulosic material are generally low in nutrients and nitrogen with pretreated wheat straw only containing around 0.4 % total nitrogen on a dry weight basis (Jones and Ingledew 1994; Linde et al. 2006). Addition of proline
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into nitrogen deficient pretreatment hydrolysate at a relatively low pH (\4.0) could be economically more viable than raising the pH to above pH 5, addition of valine has been shown to reduce undesirable flavour compounds during wort fermentations (Krogerus and Gibson 2013) and though this consideration would not be relevant for bioethanol fermentations addition of amino acids such as proline could be industrially relevant. Acknowledgments This project is part financed by the European Regional Development Fund project EMX05568. The research reported here was supported (in full or in part) by the Biotechnology and Biological Sciences Research Council (BBSRC) Sustainable Bioenergy Centre (BSBEC), under the programme for ‘Lignocellulosic Conversion To Ethanol’ (LACE) [Grant Ref: BB/G01616X/1]. This is a large interdisciplinary programme and the views expressed in this paper are those of the authors alone, and do not necessarily reflect the views of the collaborators or the policies of the funding bodies.
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652 Kaino T, Takagi H (2009) Proline as a stress protectant in the yeast Saccharomyces cerevisiae: effects of trehalose and PRO1 gene expression on stress tolerance. Biosci Biotechnol Biochem 73(9):2131–2135 Kiewietdejonge A, Pitts M, Cabuhat L, Sherman C, Kladwang W, Miramontes G, Floresvillar J, Chan J, Ramirez RM (2006) Hypersaline stress induces the turnover of phosphatidylcholine and results in the synthesis of the renal osmoprotectant glcerophosphocholine in Saccharomyces cerevisiae. FEMS Yeast Res 6(2):205–217 Krogerus K, Gibson BR (2013) Influence of valine and other amino acids on total diacetyl and 2,3-pentanedione levels during fermentation of brewers wort. Appl Microbiol Biotechnol 97(15):6919–6930 Land M (2001) Citrate-buffered methylene violet stain as an alternative to conventional stains used to determine yeast viability 2001 subcommittee report P232–233. American Soc Brew Chem. doi:10.1094/ASBCJ-59-0232 Linde M, Galbe M, Zacchi G (2006) Steam pretreatment of acidsprayed and acid soaked barley straw for production of ethanol. Appl Biochem Biotechnol 129–132:546–562 Madeo F, Herker E, Wissing S, Jungwirth H, Eisenberg T, Frohlich KU (2004) Apoptosis in yeast. Curr Opin Microbiol 7(6):655–660 Martin O, Brandriss MC, Schnieder G, Bakalinksy AT (2003) Improved anaerobic use of arginine by Saccharomyces cerevisiae. Appl Environ Microbiol 69(3):1623–1628 Meikle AJ, Reed RH, Gadd GM (1988) Osmotic adjustment and the accumulation of organic colutes in whole cells and protoplasts of Saccharomyces cerevisiae. J Gen Microbiol 134(11):3049–3060 Middelhoven WJ (1964) The pathway of arginine breakdown in Saccharomyces cerevisiae. Biochim Biophys Acta 93: 650–652 Mollapour M, Shepherd A, Piper PW (2008) Novel stress responses facilitate Saccharomyces cerevisiae growth in the presence of the monocarboxylate preservatives. Yeast 25(3):169–177 Morita Y, Nakamori S, Takagi H (2002) Effect of proline and arginine metabolism on freezing stress of Saccharomyces cerevisiae. J Biosci Bioeng 94(5):390–394 Morita Y, Nakamori S, Takagi H (2003) L-proline accumulation and freeze tolerance of Saccharomyces cerevisiae are caused by a mutation in the PRO1 gene encoding gammaglutamyl kinase. Appl Environ Microbiol 69(1):212–219 Narendranath NV, Hynes SH, Thomas KC, Ingledew WM (1997) Effects of lactobacilli on yeast-catalyzed ethanol fermentations. Appl Environ Microbiol 63(11):4158–4163 Ough CS, Amerine MA (1988) Methods for analysis of musts and wines. Wiley, New York Palmqvist E, Grage H, Meinander NQ, Hahn-Hagerdahl B (1999) Main and interaction effects of acetic acid, furfural, and p-hydroxybenzoic acid on growth and ethanol productivity of yeasts. Biotechnol Bioeng 63(1):46–55 Pereira FB, Guimaraes PM, Gomes DG, Mira NP, Teixeira MC, Sa-Correia I, Domingues L (2011) Identification of candidate genes for yeast engineering to improve bioethanol
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Antonie van Leeuwenhoek (2014) 105:641–652 production in very high gravity and lignocellulosic biomass industrial fermentations. Biotechnol Biofuels 4(1):57 Pierce J (1970) Institute of brewing analysis committee: measurement of yeast viability. J Inst Brew 34:306–312 Poole K (2002). Enhancing yeast performance under oenological conditions by enabling proline utilization. Ph.D thesis, University of Adelaide Sami M, Ikeda M, Yabuuchi S (1994) Evaluation of the alkaline methylene blue staining method for yeast activity determination. J Ferment Bioeng 78(3):212–216 Sasano Y, Haitani Y, Hashida K, Ohtsu I, Shima J, Takagi H (2012a) Enhancement of the proline and nitric oxide synthetic pathway improves fermentation ability under multiple baking-associated stress conditions in industrial baker’s yeast. Microb Cell Fact 11:40 Sasano Y, Haitani Y, Hashida K, Ohtsu I, Shima J, Takagi H (2012b) Simultaneous accumulation of proline and trehalose in industrial baker’s yeast enhances fermentation ability in frozen dough. J Biosci Bioeng 113(5):592–595 Sekine T, Kawaguchi A, Hamano Y, Takagi J (2007) Desensitization of feedback inhibition of the Saccharomyces cerevisiae gamma-glutamyl kinase enhances proline accumulation and freezing tolerance. Appl Environ Microbiol 73(12): 4011–4019 Soetens O, De Craene JO, Andrea B (2001) Ubiquitin is required for sorting to the vacuole of the yeast general amino acid permease, Gap1. J Biol Chem 276(47):43949–43957 Taherzadeh MJ, Liden G, Gustafsson L, Niklasson C (1996) The effects of pantothenate deficiency and acetate addition on anaerobic batch fermentation of glucose by Saccharomyces cerevisiae. Appl Microbiol Biotechnol 46(2):176–182 Takagi H, Sakai K, Morida K, Nakamori S (2000) Proline accumulation by mutation or disruption of the proline oxidase gene improves resistance to freezing and desiccation stresses in Saccharomyces cerevisiae. FEMS Microbiol Lett 184(1):103–108 Terao Y, Nakamori S, Takagi H (2003) Gene dosage effect of Lproline biosynthetic enzymes on L-proline accumulation and freeze tolerance in Saccharomyces cerevisiae. Appl Environ Microbiol 69(11):6527–6532 Thomas KC, Hynes SH, Ingledew WM (1994) Effects of particulate materials and osmoprotectants on very-high-gravity ethanolic fermentation by Saccharomyces cerevisiae. Appl Environ Microbiol 60(5):1519–1524 Thomas KC, Hynes SH, Ingledew WM (2002) Influence of medium buffering capacity on inhibition of Saccharomyces cerevisiae growth by acetic and lactic acids. Appl Environ Microbiol 68(4):1616–1623 Tomas-Pejo E, Oliva JM, Ballesteros M, Olsson L (2008) Comparison of SHF and SSF processes from steamexploded wheat straw for ethanol production by xylosefermenting and robust glucose-fermenting Saccharomyces cerevisiae strains. Biotechnol Bioeng 100(6):1122–1131 Verduyn C, Postma E, Scheffers WA, van Dijken JP (1990) Energetics of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. J Gen Microbiol 136(3): 405–412
ORIGINAL PAPER
Presence of proline has a protective effect on weak acid stressed Saccharomyces cerevisiae D. Greetham • H. Takagi • T. P. Phister
Received: 12 November 2013 / Accepted: 17 January 2014 / Published online: 6 February 2014 Ó European Union 2014
Abstract Fermentation of sugars released from lignocellulosic biomass (LCMs) is a sustainable option for the production of bioethanol. LCMs release fermentable hexose sugars and the currently non-fermentable pentose sugars; ethanol yield from lignocellulosic residues is dependent on the efficient conversion of available sugars to ethanol, a sideproduct of the process is acetic acid production. Presence of acetic acid reduced metabolic output and growth when compared with controls; however, it was observed that incubation with proline had a protective effect, which was proline specific and concentration dependent; the protective effect did not extend to furan or phenolic stressed yeast cells. Proline accumulating strains displayed tolerance to acetic acid when compared with background strains, whereas, strains with a compromised proline
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0118-3) contains supplementary material, which is available to authorized users. D. Greetham (&) T. P. Phister School of Biosciences, University of Nottingham, Loughborough, Leicestershire LE12 5RD, UK e-mail: [email protected] H. Takagi Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma, Japan
metabolism displayed sensitivity. Sensitivity to weak acids appears to be reduced with the addition of proline; proline is an imino acid freely available as a nitrogen source in the aerobic phase of fermentations. Yeast strains with higher intracellular proline concentrations would be desirable for industrial bioethanol fermentations. Keywords Proline Weak acids Yeast Fermentation Microarrays
Introduction Short-chain weak organic acids are potent inhibitors of microbial growth with a wide range of applications in the food and beverage industries, they are released during industrial fermentation processes or by the deacetylation of xylan during pretreatment (Palmqvist et al. 1999). The action of acetic acid has been well documented, with stress beginning at 0.05 % (*8.3 mM) in a typical industrial yeast fermentation (Narendranath et al. 1997; Thomas et al. 2002). Acetic acid toxicity is pH dependent, as acetic acid in its undissociated form diffuses through the cell membrane with dissociation dependent upon cytosolic pH. Intracellular pH is maintained through ATPase transporter systems transporting protons across the cell membranes (Verduyn et al. 1990). Exposure to acetic acid in yeast stimulates a form of programmed cell
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death (PCD); PCD is initiated through a mitochondria specific caspase cascade and leads to cellular death (Madeo et al. 2004). This response to acetic acid is separate from acidifying the cytosol via passive diffusion of acetic acid through cell membranes. During fermentations, presence of low concentrations of acetic acid (\25 mM) stimulates ATP production, and the rate of ethanol production increases when compared with the unstressed control (Taherzadeh et al. 1996). At increasing acetic acid concentrations ([25 mM), this stimulation in ATP production is overtaken by a lowering in cytosolic pH and an increase in ATPase activity (Taherzadeh et al. 1996). Proline is an imino acid that can be a source of nitrogen in yeast, however, due to a requirement for oxygen metabolism is restricted to the early aerobic phase of fermentation (Ough and Amerine 1988). During early stages of fermentations, availability of nitrogen sources such as ammonia and glutamine results in transport of proline being suppressed (Soetens et al. 2001). During later stages of fermentation when such preferred nitrogen sources have declined and transport is no longer repressed, the absence of oxygen means that proline is no longer available as a nitrogen source to the cell (Poole 2002). Strains with the ability to utilise proline as a nitrogen source have been shown to produce twice the biomass of a reference strain (Martin et al. 2003). Addition of proline as an individual amino acid or in combination with other amino acids (glycine betaine and glycine) helps maintain viability of yeast through the course of a very high gravity fermentation when compared with control conditions (Thomas et al. 1994) Beyond the nutritional advantages of providing accessible nitrogen, enhanced proline uptake has been shown to convey a broad protective effect, such as growth advantages under oxidative stress Terao et al. (2003) and as a cryoprotectant Morita et al. (2002). Intracellular levels of proline have been correlated with stress resistance in yeast (Takagi et al. 2000), yeast strains which accumulate proline display improved viability to freezing or desiccation (Morita et al. 2002; Takagi et al. 2000; Poole et al. 2009). The specific role of proline as an osmoprotector and cryoprotector is currently unclear. We aimed to look at the importance of proline to the performance of yeast during bioethanol fermentations.
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Materials and methods Yeast strains and growth conditions Saccharomyces cerevisiae NCYC 2592 was obtained from the National Collection of Yeast Cultures (NCYC), Norwich, UK, S. cerevisiae BY4741 and selected deletion mutants were a kind gift from Professor Ed Louis (University of Nottingham, UK) and S. cerevisiae proline accumulating yeast strains were a kind gift from Professor Takagi (Nara Institute of Science and Technology, Japan) respectively. All strains were maintained on YPD containing 10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose, and 20 g/L agar. Cultures were cryopreserved in glycerol stock solution at -80 °C. Viability Methylene blue was dissolved in 2 % (w/v) sodium citrate solution to give a final concentration of 0.01 % (w/v). Cells were grown to mid-exponential phase at 30 °C, with shaking in YPD (determined by a reading of between 0.4 and 0.6 nm at OD 600, at this point the relevant concentration of inhibitory compound/proline was added to the relevant flask and cells were exposed for 15 min before harvesting. Cells were resuspended in methylene blue and a cell count was performed using a haemocytometer. The cell suspension was diluted to a final concentration of 1 9 107 cells per mL and mixed in a 1:1 ratio with citrate methylene blue. The cells were microscopically examined after 5 min at 409 magnification. Dead cells stain blue, while the viable ones were colourless, as live cells exclude the stain (Pierce 1970; Sami et al. 1994). Viability counts were performed in triplicate and compared in the presence and absence of inhibitory compounds as appropriate. Viability testing by methylene blue is the industry standard in brewing, however, reports have shown that methylene blue may overestimate yeast viability (Land 2001). Phenotypic microarray analysis Biolog growth medium was prepared using 60 g/L (w/ v) glucose supplemented with 6.7 g/L (w/v) minimal medium (YNB—yeast nitrogen base), and 0.2 lL of dye D (Biolog, US). Volumes of amino acids, inhibitory compounds such as acetic, formic acid, furfural etc. from 1 M stock solutions were added as appropriate and
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the final volume adjusted to 120 lL with the addition of sterile deionised water and aliquoted to individual wells in a 96 well microassay plate (Biolog, US). Saccharomyces cerevisiae strains were prepared for inoculation using the following protocol, cryopreserved yeast colonies were streaked on to YPD plates and incubated at 30 °C for 48 h. Representative colonies were grown in YPD broth at 30 °C for 48 h. Harvested cells were washed twice and the pellet re-suspended in sterile water in 20 9 100 mm test tubes and transmittance was adjusted to 62 % using a turbidimeter (Biolog, US). The cell suspension for inoculation was prepared by mixing 0.125 mL of cells with 2.65 mL of IFY buffer (Biolog, USA) and adjusted to a final volume of 3 mL by the addition of sterile deionised water. The resulting cell suspension (90 lL) was inoculated into each well in the Biolog bespoke 96-well plate. Oxygen absorbing packs and CO2 producing packs (Mitsubishi AnaeroPakTMSystem) were used to create anaerobic conditions which were monitored using an anaerobic indicator (Oxoid, UK). Plates were covered using sterile PM bags (Biolog, US), heat sealed and incubated for 96 h at 30 °C in an Omnilog reader (Biolog, US). The OmniLog reader read the plates every 15 min, converting pixel density to a signal value reflecting the conversion of tetrazolium dye from the oxidised to the reduced form. After completion of the run, the signal data was compiled and converted using Biolog software into MicrosoftÒ Excel compatible data. In all cases, a minimum of three replicate PM assay runs were conducted, and the mean of the signal values reported. Metabolic output as measured by the phenotypic microarray is defined here as redox signal intensity (Greetham, et al., unpublished). Measurement of yeast growth Cells were grown to exponential phase in YPD medium as monitored by OD600 readings, and diluted to a starting OD600 of 0.2 using YPD broth. Either 25 mM acetic acid or 10 mM formic acid was added to wells on a 96-well microtitre plate and the volume of each well was adjusted to 100 lL using sterile deionised water. Yeast growth (OD600) was measured every 15 min using a Tecan Infinite M200 Pro plate reader (Mannedorf, Switzerland), at 30 °C for 72 h. The assay was performed in triplicate and the average reading was plotted.
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Fermentation Fermentations were conducted in 180 mL fermentation vessels (FV). Cryopreserved yeast colonies were streaked onto YPD plates and incubated at 30 °C for 48 h. Colonies of NCYC2592 were used to inoculate 20 mL of YPD broth and incubated in an orbital shaker at 30 °C for 24 h. These were then transferred to 200 mL of YPD and grown for 48 h in a 500 mL conical flask shaking at 30 °C. Cells were harvested and washed three times with sterile deionised water and then re-suspended in 5 mL of sterile water. Under control conditions, 1.5 9 107 cells/mL were inoculated in 99.6 mL of medium containing 4 % glucose, 2 % peptone, 1 % yeast extract with 0.4 mL sterile deionised water. Under inhibitor stress, 1.5 9 107 cells.mL-1 were incubated in 99.6 mL of medium containing 4 % glucose, 2 % peptone, 1 % yeast extract with 25 mM acetic acid and 10 mM proline. Volumes of media were adjusted to account for the addition of acetic acid (*145 lL) and proline (200 lL) to ensure that all fermentations began with the same carbon load. pH was monitored with readings taken every 2 h. Anaerobic conditions were established using a sealed butyl plug (Fisher, Loughborough, UK) and aluminium caps (Fisher Scientific). A hypodermic needle attached with a Bunsen valve was pierced through the rubber septum to facilitate the release of CO2. Weight loss as a means of monitoring rates of fermentation was measured in triplicate at regular time points during the fermentation, fermentations were conducted at 30 °C, with orbital shaking at 200 rpm. Yeast strains BY4741 and pro1I150T Dput1 were prepared for fermentation experiment as described above for S. cerevisiae NCYC2592. Under inhibitor stress, 1.5 9 107 cells/mL were incubated in 99.6 mL of medium containing 4 % glucose, 2 % peptone, 1 % yeast extract with 10 mM acetic acid. Volumes of media were adjusted to account for the addition of acetic acid (*58 lL) to ensure that all fermentations began with the same carbon load.
Detection of glucose and ethanol from FV experiments via HPLC Glucose and ethanol were quantified by HPLC. The HPLC system included a Jasco AS-2055 Intelligent
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auto sampler (Jasco, Tokyo, Japan) and a Jasco PU1580 Intelligent pump (Jasco). The chromatographic separation was performed on a Rezex ROA H? organic acid column, 5 lm, 7.8 9 300 mm, (Phenomenex, Macclesfield, UK) at ambient temperature. The mobile phase was 0.005 N H2SO4 with a flow rate of 0.5 mL/min. For detection a Jasco RI-2031 Intelligent refractive index detector (Jasco) was employed. Data acquisition was via the Azur software (version 4.6.0.0, Datalys, St Martin D’heres, France) and concentrations were determined by peak area comparison with injections of authentic standards. The injected volume was 10 lL and analysis was completed in 28 min. All chemicals used were analytical grade ([95 % purity, Sigma-Aldrich, UK).
Results Effect of acetic acid and amino acids on metabolic output of yeast cells The effect of 25 mM acetic acid on metabolic output of yeast using be-spoke phenotypic microarray plate was measured and it was observed that the presence of 25 mM acetic acid reduced metabolic output when compared with unstressed controls (Fig. 1a). Addition of 10 mM proline improved metabolic output in the presence of acetic acid (Fig. 1b); despite there being no effect on metabolic output in unstressed cells. 25 mM acetic acid was chosen for the assays as it is released under relatively mild pretreatment methods (unpublished data) and 10 mM proline was chosen as this concentration has been shown to inhibit c-glutamyl kinase (GK) activity, proline synthesis is strictly regulated through feedback inhibition of GK by proline (Sekine et al. 2007). Assays looking for improved metabolic output in the presence of acetic acid with other amino acids failed to observe any improvement (Supplementary Fig. S1). Proline has been identified as an osmoprotectant (Kaino and Takagi 2009), however, other osmoprotectants such as glycerol (Meikle et al. 1988) or phosphatidylcholine (Kiewietdejonge et al. 2006) in our phenotypic microarray assays failed to protect the yeast cell against weak acid stress (data not shown).
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Presence of proline improved metabolic output at higher concentrations of acetic acid stressed yeast cells Presence of 10 mM proline improved metabolic output at higher acetic acid concentrations (50 and 75 mM acetic acid) (Figs. 1c, d) with a further improvement observed in assays with 20 mM proline (data not shown). Proline’s protective role appears to be weak acid specific The protective role of proline to stress caused by inhibitory compounds present in hydrolysates derived from lignocellulosic material was assessed (TomasPejo et al. 2008). It was observed that presence of proline improved metabolic output in the presence of formic acid (Fig. 2a), but not for coumaric or furoic acid (Fig. 2e, f) or cells under furfural, HMF or vanillin stress (Fig. 2b–d). Assays with strong acids failed to observe any improvement in metabolic output for citric acid stressed cells in the presence of proline (Fig. 2g). There was no improvement in metabolic output in assays with 20 mM proline for furfural, HMF, vanillin or citric acid stressed cells (data not shown). Presence of proline had little effect on pH in the absence or presence of acetic acid Acetic acid enters the cell much more readily as an uncharged undissociated ion than as a charged anion. We assessed if the presence of proline had an effect on the pH of fermentations with the addition of acetic acid or under control conditions. It was observed that pH dropped from an initial pH of 5.3–4.15 during fermentation under control conditions (Fig. 3). Addition of 25 mM acetic acid at the beginning of the fermentation elicited an immediate reduction in pH to pH 4.2; however, subsequently pH did not alter significantly during the time period of the fermentation (Fig. 3). Addition of 10 mM proline had no effect on the pH under control conditions or in the presence of acetic acid (data not shown). Presence of proline improved yeast growth and viability under weak acid stress We assessed the impact of proline on yeast growth under control and weak acid stressed conditions, it was
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Fig. 1 Phenotypic microarray analysis (redox signal intensity) for S. cerevisiae (NCYC 2592) on media containing 0–25 mM acetic acid and 10 mM proline. Plates were incubated at 30 °C and read for 40 h, under anaerobic conditions. a Redox signal intensity for S. cerevisiae (NCYC 2592) in the presence of 0–25 mM acetic acid. b Redox signal intensity for S. cerevisiae (NCYC 2592) in the presence of 0–25 mM acetic acid plus 10 mM proline. c Redox signal intensity for S. cerevisiae (NCYC 2592) in the presence of 50 mM acetic acid plus 10 mM proline. d Redox signal intensity for S. cerevisiae (NCYC 2592) in the presence of 75 mM acetic acid plus 10 mM proline. Data an average of triplicate values with standard deviation shown. Redox signal intensity is a measurement of metabolism using a redox sensitive redox dye
observed that the addition of 10 mM proline had little effect on growth under anaerobic conditions suggesting that in the absence of oxygen proline was not metabolized in this assay (Fig. 4a). Incubation with proline improved growth in acetic acid or formic acid stressed yeast when compared with acid stressed cells (Fig. 4b, c). There was no improvement in cellular growth with proline under furfural, HMF or vanillin stress (data not shown). Viability assays showed no effect with addition of proline under unstressed conditions (Fig. 4d), however, there was an improvement in weak acid stressed cells (Fig. 4d), with viability increasing in the
presence of increasing proline (Fig. 4d). In comparison, we failed to observe an improvement in viability for cells under furfural stress (Fig. 4d) or other stresses such as HMF, vanillin or citric acid (data not shown). Accumulation of proline promotes a weak acid tolerant phenotype Previous assays relied on the introduction of proline into the media and assessed for the impact on metabolic output of cells. We wanted to assess for the importance of proline in the cell and through selected yeast strains examined how yeast metabolize,
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Fig. 2 Phenotypic microarray analysis (redox signal intensity) for S. cerevisiae (NCYC 2592) on media 10 mM proline plus inhibitory compounds. a Redox signal intensity for S. cerevisiae (NCYC 2592) on media containing 10 mM formic acid plus 10 mM proline. b Redox signal intensity for S. cerevisiae (NCYC 2592) on media containing 10 mM furfural plus 10 mM proline. c Redox signal intensity for S. cerevisiae (NCYC 2592) on media containing 10 mM HMF plus 10 mM proline. d Redox signal intensity for S. cerevisiae (NCYC 2592) on media
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containing 10 mM vanillin plus 10 mM proline. e Redox signal intensity for S. cerevisiae (NCYC 2592) on media containing 10 mM coumaric acid plus 10 mM proline. f Redox signal intensity for S. cerevisiae (NCYC 2592) on media containing 10 mM feroic acid plus 10 mM proline and g redox signal intensity for S. cerevisiae (NCYC 2592) on media containing 10 mM citric acid plus 10 mM proline. Data an average of triplicate values with standard deviation shown
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Fig. 3 Effect of acetic acid and proline on pH during a fermentation. pH was monitored during a fermentation using 4 % YPD for S. cerevisiae NCYC 2592 in the presence of 25 mM acetic acid and 10 mM proline. Readings were taken every 2 h. Data an average of triplicate values with standard deviation shown
transport and recycle proline. This work was performed in the S. cerevisiae BY4741 reference strain background to facilitate use of knockout strains, phenotypic microarrays confirmed that presence of proline protects against acetic acid stress in this strain as observed with S. cerevisiae NCYC 2592 (Supplementary data S2). Analysis of strains with components of the proline metabolism pathway disrupted for acetic acid tolerance showed that a strain with a pro1 knockout was more sensitive to acetic acid when compared with the background strain (BY4741) (Fig. 5a). PRO1 encodes an enzyme which is the first step in the conversion of glutamate into proline (Brandriss 1979) and the null strain has been linked to sensitivity to heat, osmotic and other stresses (Morita et al. 2003). There was a slight increase in sensitivity to acetic acid in a pro2 disrupted strain; PRO2 encodes the enzyme required for the next step in proline biosynthesis (Fig. 5a). The final step which leads to the formation of proline is catalysed by Pro3p; however, pro3 is a lethal knockout in the S. cerevisiae S288C background (Giaever et al. 2002). There was a significant reduction in viability in pro1 and pro2 knockouts when compared with BY4741 under acetic acid stress (Fig. 4c). PUT1 encodes an enzyme which begins the conversion of proline into glutamate (PUT2 encodes the second step in this pathway) and a put1 disrupted strain has been shown previously to have elevated levels of proline (Takagi et al. 2000). Assays with this strain showed an increase in metabolic output and viability when compared with BY4741 in the presence of weak acids (Fig. 5a, c). The importance of transcriptional activation of PUT1 and PUT2 was also investigated; PUT3
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regulates PUT1 and PUT2 expression by binding to promoter sites, metabolic output of a put3 strain was slightly elevated under acetic acid stress when compared with BY4741 (Fig. 5a). Addition of 10 mM proline recovered the metabolic output in pro1 or pro2 disrupted strains in the presence of acetic acid when compared with no proline added (Fig. 5b), however, additional proline elicited little improvement in a put1 disrupted strain to acetic acid (Fig. 5b). Adenine can be converted to proline, and in the absence of oxygen proline accumulates due to the inability of the cell to convert proline into glutamate which is catalysed by the oxygen dependent enzyme proline oxidase (Put1p) (Brandriss and Falvey 1992). The importance of this pathway for weak acid tolerance was assessed by looking at the genes which regulate the process of converting adenine into proline. Adenine is converted to proline in a two-step process catalysed by Car1p and Car2p (Middelhoven 1964). It was observed that metabolic output in car1 and car2 disrupted strains was reduced in the presence of weak acids when compared with BY4741 (Supplementary Fig. S2A). We used an fps1 disrupted strain as a positive control in these experiments, this strain has been shown to be weak acid tolerant Mollapour et al. (2008) and in our assays metabolic output was increased in response to weak acids when compared with BY4741 (Supplementary Fig. S2A). Proline accumulating mutants are weak acid tolerant Proline accumulation leading to tolerance to oxidative stress has been observed in a strain with an altered Pro1p enzyme (Kaino and Takagi 2009). Tolerance to osmotic stress was also noted if this enzyme was expressed in a put1-disrupted strain (Sasano et al. 2012a, b). It was observed that the strains with higher intracellular proline levels (Sasano et al. 2012a, b) had significantly higher metabolic output under control conditions and were significantly more tolerant to 10 mM acetic acid than BY4741 (Fig. 6a, b). Proline accumulating yeast strains display acetic acid tolerance during fermentation The performance of pro1I150TDput1 was compared with the background strain BY4741 during fermentations in the presence of 10 mM acetic acid. Under
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Fig. 4 Effect of acetic acid and proline on the growth of S. cerevisiae NCYC2592. Growth was monitored on a TECAN plate reader at OD600 for 96 h with readings taken every 15 min for a control and 10 mM proline (b) control, 25 mM acetic acid
and 25 mM acetic acid and 10 mM proline (c) control, 10 mM formic acid and 10 mM formic acid. d The effect of inhibitory compounds and proline on yeast viability. Data an average of triplicate values with standard deviation shown
control conditions, glucose utilisation and production of ethanol were identical for BY4741 and pro1I150TDput1 (Fig. 6c, d), in the presence of 10 mM acetic acid, however, there was a delay in both glucose utilisation and production of ethanol in BY4741 when compared with pro1I150TDput1 (Fig. 6c, d). Indeed, the presence of acetic acid appeared to have no influence on the rate of fermentation for pro1I150TDput1 when compared with unstressed control conditions (Fig. 6c, d).
Discussion
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Proline is an imino acid freely available to yeast at relatively high concentrations during beverage fermentations; however, requirements for oxygen restrict proline utilisation as a nitrogen source to the early stages of fermentation. Previous work has shown that the presence of proline or arginine correlates to stress resistance in yeast, though its precise mode of action is unclear (Takagi et al. 2000); Morita et al. 2002).
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Fig. 5 Phenotypic microarray analysis (redox signal intensity) for S. cerevisiae (BY4741, put1, put2, pro1, pro2, and put3 to inhibitory compounds and proline. a Effect of 50 mM acetic acid, 75 mM acetic acid, 10 mM formic acid, 10 mM furfural or 10 mM vanillin defined as % redox signal intensity (%RSI) of stressed versus control. b Effect of 50 mM acetic acid and 50 mM acetic acid plus 10 mM proline defined as % RSI of stressed versus control. c The effect of 50 and 75 mM acetic acid on viability. Data an average of triplicate values with standard deviation shown
Disruption of key genes in the conversion of proline into glutamate leads to proline accumulation and improved viability to freezing and desiccation Takagi et al. (2000). Accumulation of proline has also been associated with tolerance to oxidative stress whereas accumulation of proline with trehalose improves fermentation rates when compared with accumulation of either proline or trehalose individually( Sasano et al. 2012a, b). Weak acids such as acetic and formic acid inhibit yeast fermentations reducing both growth and ethanol production. The inhibitory effects of weak acids have been linked to intracellular anion accumulation leading to a reduction in cytosolic pH affecting enzyme kinetics (Pereira et al. 2011). Low concentrations of weak acids can increase ethanol production at the
expense of cellular division, though the precise mode of action for this increase is unclear (Pereira et al. 2011). Pre-treatment processes also release phenolic compounds such as vanillin and these compounds have demonstrated inhibitory effects on yeast cells (Endo et al. 2008, 2009). Presence of proline made cells more tolerant to weak acids when compared with other amino acids, this appeared to be weak acid specific as proline had no impact on furanic or phenolic stressed yeast cells. Tolerance appeared to be concentration dependent as increasing concentrations of proline increased metabolic output and viability in the presence of weak acids. There was no change in pH in assays containing proline compared with absence of proline under control or weak acid stress conditions suggesting that
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Fig. 6 Phenotypic microarray analysis (redox signal intensity) for S. cerevisiae (BY4741, pro1I150T and pro1I150T Dput1) to acetic acid and proline. a Redox signal intensity under control conditions b Redox signal intensity in the presence of 25 mM acetic acid. c Glucose utilisation during fermentation by BY4741 and pro1I150T Dput1 under control and acetic acid stressed conditions d Production of ethanol during a fermentation of BY4741 and pro1I150T Dput1 under control and acetic acid stressed conditions. Data an average of triplicate values with standard deviation shown
the protective role of proline is response to weak acids rather than a response to a drop in pH. In yeast, proline is synthesized from glutamate in a three step process and recycled back to glutamate in a two-step process (Sasano et al. 2012a, b). It was observed that put1 disrupted strains were more tolerant to weak acids when compared with BY4741 (Fig. 5a), there was no improvement in tolerance to furfural or vanillin in this strain (data not shown). Put1p catalyses the first step in the conversion of proline into glutamate and put1 disrupted strains have higher levels of proline in their cells. There was also increased sensitivity in pro1 and pro2 disrupted
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strains, with pro1 in particular displaying sensitivity to weak acids (Fig. 5a). This sensitivity could be rescued by the addition of proline, suggesting that intracellular proline was the reason for weak acid sensitivity in these strains. PRO1 and PRO2 encode enzymes which convert glutamate into proline, pro1 and pro2 disrupted strains have been shown to be less tolerant to oxidative, osmotic and other stresses (Kaino and Takagi 2009). Strains incapable of converting adenine into proline also displayed sensitivity to weak acids; this pathway is of particular relevance during fermentations as further conversion of proline is not possible without the presence of
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oxygen leading to proline accumulation under anaerobic conditions. Disruption of proline regulator (PUT3) had little influence on weak acid tolerance. A put3 disrupted strain displayed a slight tolerance to weak acid when compared with BY4741 (Fig. 5a), though there is evidence that in the absence of Put3p, the Gal4p transcriptional activator is able to upregulate PUT2 (D’Alessio and Brandriss 2000). It would be interesting to investigate a gal4,put3 double deletion and assess for weak acid sensitivity. Previous work has shown that yeast strains with higher intracellular proline concentrations are osmotically and oxidatively tolerant (Sasano et al. 2012a, b). It was observed that these strains were weak acid tolerant when compared with BY4741; these strains displayed no increased tolerance to other inhibitory compounds. Fermentations using a proline accumulating yeast strain were significantly faster in the presence of acetic acid when compared with the background strain despite no observable differences under control conditions. On the basis of this data we have displayed the importance of proline in cells under weak acid stress. Presence of weak acids in hydrolysates from plant material is unavoidable as the processes involved in liberating fermentable sugars also generates weak acids. This paper is the first to show that proline has a role in how yeast cells cope with weak acid stress; this appears to be weak acid and proline specific. We observed that cells unable to synthesise proline are weak acid sensitive, whereas, those which have high intracellular proline concentrations are weak acid tolerant. Running a fermentation under sterile conditions on a large scale is not economically viable, and maintaining a low pH (\4.5) to prevent bacterial contamination is important. Fermentations of hydrolysates from lignocellulosic has shown that ethanol is produced during a fermentation with a starting pH of 4 (Kadar et al. 2007). Proline consumption has been observed in fermentations of lychee juice under control conditions, however, the poor nitrogen concentrations of this media led to this consumption (Chen et al. 2014). Hydrolysates from lignocellulosic material are generally low in nutrients and nitrogen with pretreated wheat straw only containing around 0.4 % total nitrogen on a dry weight basis (Jones and Ingledew 1994; Linde et al. 2006). Addition of proline
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into nitrogen deficient pretreatment hydrolysate at a relatively low pH (\4.0) could be economically more viable than raising the pH to above pH 5, addition of valine has been shown to reduce undesirable flavour compounds during wort fermentations (Krogerus and Gibson 2013) and though this consideration would not be relevant for bioethanol fermentations addition of amino acids such as proline could be industrially relevant. Acknowledgments This project is part financed by the European Regional Development Fund project EMX05568. The research reported here was supported (in full or in part) by the Biotechnology and Biological Sciences Research Council (BBSRC) Sustainable Bioenergy Centre (BSBEC), under the programme for ‘Lignocellulosic Conversion To Ethanol’ (LACE) [Grant Ref: BB/G01616X/1]. This is a large interdisciplinary programme and the views expressed in this paper are those of the authors alone, and do not necessarily reflect the views of the collaborators or the policies of the funding bodies.
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