Phenotypic evaluation and characterization of 21 industrial Saccharomyces cerevisiae yeast strains

Phenotypic evaluation and characterization of 21 industrial Saccharomyces cerevisiae yeast strains Abstract Microorganisms have been studied and used extensively to produce value-added fuels and chemicals. Yeasts, specifically Saccharomyces cerevisiae, receive industrial attention because of their well-known ability to ferment glucose and produce ethanol. Thousands of natural or genetically modified S. cerevisiae have been found in industrial environments for various purposes. These industrial strains are isolated from industrial fermentation sites, and they are considered as potential host strains for superior fermentation processes. In many cases, industrial yeast strains have higher thermotolerance, increased resistances towards fermentation inhibitors and increased glucose fermentation rates under anaerobic conditions when compared with laboratory yeast strains. Despite the advantages of industrial strains, they are often not well characterized. Through screening and phenotypic characterization of commercially available industrial yeast strains, industrial fermentation processes requiring specific environmental conditions may be able to select an ideal starting yeast strain to be further engineered. Here, we have characterized and compared 21 industrial S. cerevisiae strains under multiple conditions, including their tolerance to varying pH conditions, resistance to fermentation inhibitors, sporulation efficiency and ability to ferment lignocellulosic sugars. These data may be useful for the selection of a parental strain for specific biotechnological applications of engineered yeast. Yeast, Saccharomyces cerevisiae, industrial yeast, laboratory yeast, hydrolysate, biotechnology INTRODUCTION Yeasts have served many important industrial purposes for humans over thousands of years. Specifically, the Saccharomyces cerevisiae yeast species has acted as the primary species to produce wine, beer and bread (Legras et al.2007: 2091–102). Although the production of baked goods and fermented foods or beverages has historically been the major industrial application for S. cerevisiae, new avenues of yeast fermentation capabilities were discovered in recent decades. These new opportunities are due in part to rapid and significant advances in our understanding of yeast genetics and physiology (Walker 1998: 362). Through our improved understanding of yeast hardiness and functions, we have exploited the robustness of S. cerevisiae to produce various value-added chemicals and fuels (Turner et al.2018). Industrial strains of S. cerevisiae are known to have higher tolerances against harsh industrial environments, such as lower pH, fermentation inhibitors, osmolality and higher temperature. With these desired phenotypes of industrial strains, it is feasible to have an improved engineered industrial strain for sustainable biofuel or biochemical production from lignocellulosic hydrolysates. Although several microbial species are used for industrial production of value-added products, yeasts are perhaps the most well studied. Yeasts can natively ferment glucose to produce ethanol, and this capability has been used for wine production for thousands of years (McGovern et al.1997: 3–21; Cavalieri et al.2003: S226–32). In recent decades, the yeast S. cerevisiae has been utilized extensively for biofuel production (Nissen et al.2000: 69–77). The majority of bioethanol produced by S. cerevisiae is from the fermentation of sugarcane or corn-derived glucose (Wheals et al.1999: 482–7). Although industrial yeast fermentation has resulted in the annual production of more than 50 billion liters ethanol production in the United States alone, the availability of corn and sugarcane is a limiting step in using biofuels as a total replacement for fossil fuels (Chum et al.2014: 205–23). Therefore, abundant lignocellulosic crops are considered as feasible alternative feedstocks for the production of bio-based fuels and chemicals (Carroll and Somerville 2009: 165–82). Although S. cerevisiae is well-studied with many genetic manipulation tools available (Ostergaard, Olsson and Nielsen 2000: 34–50; DiCarlo et al.2013: 4336–43), it does not efficiently produce fuels and chemicals from processed lignocellulosic hydrolysates due, in part, to the harsh environment present in the hydrolysate. Specifically, the low pH of lignocellulosic hydrolysates coupled with the presence of many known and unknown fermentation inhibitors acts as major hurdles to efficiently produce fuels and chemicals by engineered S. cerevisiae (Jayakody et al.2013: 6589–600; Jonsson, Alriksson and Nilvebrant 2013: 16). In general, the inhibitory effects of lignocellulosic hydrolysates are not well understood. Several attempts to overcome lignocellulosic hydrolysate toxicity were not successful, and this may be due to inhibition of desired yeast phenotypes caused by the fermentation conditions and that the strains are lacking resistance toward the inhibitors’ toxicity (Almeida et al.2007: 340–9). One possible solution is to modify the pretreatment process of lignocellulosic hydrolysate to reduce the inhibitors or to balance the pH. However, this solution can result in reduced fermentable sugar availability (Persson et al.2002: 5318–25) or increased production costs (Rivard et al.1996: 183–91). Owed to these hurdles, developing an engineered microbe from a parental strain that is natively resistant to the many fermentation inhibitors present in hydrolysates to efficiently ferment lignocellulosic hydrolysates may be a more cost-effective alternative (Brandberg, Franzen and Gustafsson 2004: 122–5). In particular, laboratory strains are generally regarded as possessing lower tolerance to fermentation inhibitors as compared to industrial yeast strains and, therefore, industrial strains may be ideal for producing fuels and chemicals from lignocellulosic hydrolysates (Martıín and Jönsson 2003: 386–95). However, yeast strains have widely varying inhibitor tolerances and fermentation profiles. It is challenging and time consuming to select and characterize the best strains for their resistances toward common fermentation inhibitors present in lignocellulosic hydrolysates such as hydroxymethylfurfural (HMF), furfural and acetate. Thus, developing a dataset of industrial strains, ranked for their characteristic and tolerances under certain industrial conditions, was the main goal in the present study. This dataset will be provide useful guidance for selecting an ideal candidate strain for specific industrial fermentation processes. Here, we have selected 21 S. cerevisiae yeast strains that are publicly available from the American Type Culture Collection (ATCC), and screened each strain to compare their fermentation phenotypes in a variety of conditions. MATERIALS AND METHODS Industrial strain backgrounds All S. cerevisiae strains were obtained from the ATCC. In total, 21 strains were selected. A list containing the ATCC nomenclature for each strain, the original isolation source of the strain and a representative literature citation for each strain is shown in Table 1. The well studied control strain, J3 or ATCC4124, was bold in the table. In some instances, the original isolation source or any peer-reviewed reference was not available. Table 1. Listing of all 21 S. cerevisiae strains that were screened in this study, along with their associated ATCC number, their origin of isolation, and a relevant reference if available. Code name  ATCC number  Isolation origin  Reference(s)  Jin 01 or J1  ATCC 2360  Kefir  Billen and Lichstein (1949: 215–21)  Jin 02 or J2  ATCC 4098  German white wine  Grewal and Miller (1972: 1897–905)  Jin 03 or J3  ATCC 4124  Molasses distillery  Ness et al. (1993: 89–94)  Jin 04 or J4  ATCC 4126  Amylo process  Bazua and Wilke (1977: 105–18)  Jin 05 or J5  ATCC 4127  American Concord grapes  N/A  Jin 06 or J6  ATCC 4921  French wine  N/A  Jin 08 or J8  ATCC 7754  Fleischmann bakers’ yeast  Snell, Eakin and Williams (1940: 175–8)  Jin 09 or J9  ATCC 9763  Distillery  Tseng and Phillips (1982: 1319–25)  Jin 11 or J11  ATCC 20598  N/A  Heady (1982)  Jin 12 or J12  ATCC 24855  Egyptian distillery  Ismail and Ali (1971a: 346–9) and Ismail and Ali (1971b: 350–4)  Jin 13 or J13  ATCC 24858  N/A  Ismail and Ali (1971a: 346–9) and Ismail and Ali (1971b: 350–4)  Jin 14 or J14  ATCC 24860  N/A  Jeppsson, Yu and Hahn-Hagerdal (1996: 1705–9)  Jin 15 or J15  ATCC 26422  Sake  Suizu et al. (1994: 119–24)  Jin 17 or J17  ATCC 46523  Baker's yeast  Middelhoven and Arkesteyn (1981: 121–31)  Jin 18 or J18  ATCC 56069  Fermented banana  N/A  Jin 19 or J19  ATCC 60222  Egyptian baker's yeast  N/A  Jin 20 or J20  ATCC 60223  Alsa Briochin bakers’ yeast  N/A  Jin 21 or J21  ATCC 60493  Canned strawberries  Put, Jong and Sand (1977)  Jin 24 or J24  ATCC 66348  Japanese soil  Tokuoka et al. (1985: 411–27)  Jin 25 or J25  ATCC 66349  Japanese soil  Tokuoka et al. (1985: 411–27)  Jin 26 or J26  ATCC 96581  Spent sulfite liquor fermentation  Linden, Peetre and Hahn-Hagerdal (1992: 1661–9)  Code name  ATCC number  Isolation origin  Reference(s)  Jin 01 or J1  ATCC 2360  Kefir  Billen and Lichstein (1949: 215–21)  Jin 02 or J2  ATCC 4098  German white wine  Grewal and Miller (1972: 1897–905)  Jin 03 or J3  ATCC 4124  Molasses distillery  Ness et al. (1993: 89–94)  Jin 04 or J4  ATCC 4126  Amylo process  Bazua and Wilke (1977: 105–18)  Jin 05 or J5  ATCC 4127  American Concord grapes  N/A  Jin 06 or J6  ATCC 4921  French wine  N/A  Jin 08 or J8  ATCC 7754  Fleischmann bakers’ yeast  Snell, Eakin and Williams (1940: 175–8)  Jin 09 or J9  ATCC 9763  Distillery  Tseng and Phillips (1982: 1319–25)  Jin 11 or J11  ATCC 20598  N/A  Heady (1982)  Jin 12 or J12  ATCC 24855  Egyptian distillery  Ismail and Ali (1971a: 346–9) and Ismail and Ali (1971b: 350–4)  Jin 13 or J13  ATCC 24858  N/A  Ismail and Ali (1971a: 346–9) and Ismail and Ali (1971b: 350–4)  Jin 14 or J14  ATCC 24860  N/A  Jeppsson, Yu and Hahn-Hagerdal (1996: 1705–9)  Jin 15 or J15  ATCC 26422  Sake  Suizu et al. (1994: 119–24)  Jin 17 or J17  ATCC 46523  Baker's yeast  Middelhoven and Arkesteyn (1981: 121–31)  Jin 18 or J18  ATCC 56069  Fermented banana  N/A  Jin 19 or J19  ATCC 60222  Egyptian baker's yeast  N/A  Jin 20 or J20  ATCC 60223  Alsa Briochin bakers’ yeast  N/A  Jin 21 or J21  ATCC 60493  Canned strawberries  Put, Jong and Sand (1977)  Jin 24 or J24  ATCC 66348  Japanese soil  Tokuoka et al. (1985: 411–27)  Jin 25 or J25  ATCC 66349  Japanese soil  Tokuoka et al. (1985: 411–27)  Jin 26 or J26  ATCC 96581  Spent sulfite liquor fermentation  Linden, Peetre and Hahn-Hagerdal (1992: 1661–9)  View Large Table 1. Listing of all 21 S. cerevisiae strains that were screened in this study, along with their associated ATCC number, their origin of isolation, and a relevant reference if available. Code name  ATCC number  Isolation origin  Reference(s)  Jin 01 or J1  ATCC 2360  Kefir  Billen and Lichstein (1949: 215–21)  Jin 02 or J2  ATCC 4098  German white wine  Grewal and Miller (1972: 1897–905)  Jin 03 or J3  ATCC 4124  Molasses distillery  Ness et al. (1993: 89–94)  Jin 04 or J4  ATCC 4126  Amylo process  Bazua and Wilke (1977: 105–18)  Jin 05 or J5  ATCC 4127  American Concord grapes  N/A  Jin 06 or J6  ATCC 4921  French wine  N/A  Jin 08 or J8  ATCC 7754  Fleischmann bakers’ yeast  Snell, Eakin and Williams (1940: 175–8)  Jin 09 or J9  ATCC 9763  Distillery  Tseng and Phillips (1982: 1319–25)  Jin 11 or J11  ATCC 20598  N/A  Heady (1982)  Jin 12 or J12  ATCC 24855  Egyptian distillery  Ismail and Ali (1971a: 346–9) and Ismail and Ali (1971b: 350–4)  Jin 13 or J13  ATCC 24858  N/A  Ismail and Ali (1971a: 346–9) and Ismail and Ali (1971b: 350–4)  Jin 14 or J14  ATCC 24860  N/A  Jeppsson, Yu and Hahn-Hagerdal (1996: 1705–9)  Jin 15 or J15  ATCC 26422  Sake  Suizu et al. (1994: 119–24)  Jin 17 or J17  ATCC 46523  Baker's yeast  Middelhoven and Arkesteyn (1981: 121–31)  Jin 18 or J18  ATCC 56069  Fermented banana  N/A  Jin 19 or J19  ATCC 60222  Egyptian baker's yeast  N/A  Jin 20 or J20  ATCC 60223  Alsa Briochin bakers’ yeast  N/A  Jin 21 or J21  ATCC 60493  Canned strawberries  Put, Jong and Sand (1977)  Jin 24 or J24  ATCC 66348  Japanese soil  Tokuoka et al. (1985: 411–27)  Jin 25 or J25  ATCC 66349  Japanese soil  Tokuoka et al. (1985: 411–27)  Jin 26 or J26  ATCC 96581  Spent sulfite liquor fermentation  Linden, Peetre and Hahn-Hagerdal (1992: 1661–9)  Code name  ATCC number  Isolation origin  Reference(s)  Jin 01 or J1  ATCC 2360  Kefir  Billen and Lichstein (1949: 215–21)  Jin 02 or J2  ATCC 4098  German white wine  Grewal and Miller (1972: 1897–905)  Jin 03 or J3  ATCC 4124  Molasses distillery  Ness et al. (1993: 89–94)  Jin 04 or J4  ATCC 4126  Amylo process  Bazua and Wilke (1977: 105–18)  Jin 05 or J5  ATCC 4127  American Concord grapes  N/A  Jin 06 or J6  ATCC 4921  French wine  N/A  Jin 08 or J8  ATCC 7754  Fleischmann bakers’ yeast  Snell, Eakin and Williams (1940: 175–8)  Jin 09 or J9  ATCC 9763  Distillery  Tseng and Phillips (1982: 1319–25)  Jin 11 or J11  ATCC 20598  N/A  Heady (1982)  Jin 12 or J12  ATCC 24855  Egyptian distillery  Ismail and Ali (1971a: 346–9) and Ismail and Ali (1971b: 350–4)  Jin 13 or J13  ATCC 24858  N/A  Ismail and Ali (1971a: 346–9) and Ismail and Ali (1971b: 350–4)  Jin 14 or J14  ATCC 24860  N/A  Jeppsson, Yu and Hahn-Hagerdal (1996: 1705–9)  Jin 15 or J15  ATCC 26422  Sake  Suizu et al. (1994: 119–24)  Jin 17 or J17  ATCC 46523  Baker's yeast  Middelhoven and Arkesteyn (1981: 121–31)  Jin 18 or J18  ATCC 56069  Fermented banana  N/A  Jin 19 or J19  ATCC 60222  Egyptian baker's yeast  N/A  Jin 20 or J20  ATCC 60223  Alsa Briochin bakers’ yeast  N/A  Jin 21 or J21  ATCC 60493  Canned strawberries  Put, Jong and Sand (1977)  Jin 24 or J24  ATCC 66348  Japanese soil  Tokuoka et al. (1985: 411–27)  Jin 25 or J25  ATCC 66349  Japanese soil  Tokuoka et al. (1985: 411–27)  Jin 26 or J26  ATCC 96581  Spent sulfite liquor fermentation  Linden, Peetre and Hahn-Hagerdal (1992: 1661–9)  View Large General flask fermentations Yeast cells were cultured in YP medium (10 g/L yeast extract and 20 g/L peptone) containing glucose (YPD), xylose (YPX), cellobiose (YPC), maltose, mannose or sucrose. Concentrations of the sugars were displayed as numbers following their initials (e.g. YPD160 refers to YP medium containing 160 g/L of glucose). Stock cultures were maintained on YPD20 agar (YP medium containing 20 g/L of glucose) plates at 4°C. Yeast precultures were grown in 5 mL YPD20 and harvested at the mid-exponential phase. Fermentations were conducted with an initial cell optical density (OD600) of ∼ 1.0 and at a volume of 50 mL in 250 mL Erlenmeyer Pyrex® flasks (Corning, MA). Flasks were shaken at 100 RPM on an Innova 2300 shaker (New Brunswick Scientific, CT) in a 30°C incubation room. Optical density was measured via NanoDrop 2000C (Thermo Fisher Scientific, MA) or BioMate 3 UV-visible spectrophotometer (Thermo Fisher Scientific, MA). Metabolite concentrations such as glucose, xylose, cellobiose, xylitol, glycerol, acetate and ethanol were monitored by a 1200 Infinity series HPLC system (Agilent Technologies, CA) equipped with a refractive index detector using a Rezex ROA-Organic Acid H+ (8%) column (Phenomenex Inc., CA). The column was eluted with 0.005 N H2SO4 at a flow rate of 0.6 mL/min at 50°C. The construction and evaluation of xylose-fermenting strains The pSR6-X123 plasmid (Kim et al.2012: 336–43) containing Scheffersomyces stipitis XYL1, XYL2 and XYL3 genes was linearized by XcmI digestion and introduced into the URA3 locus of all industrial yeast strains to allow for xylose utilization. A standard high-efficiency lithium acetate transformation method was used for the chromosomal introduction of the linearized plasmid (Daniel Gietz and Woods 2002: 87–96). The colonies with xylose assimilation pathway were selected on YP medium with 40 g/L xylose (YPX40) agar plates. Eight successful transformants of each strain were evaluated in 5 mL culture of YPX40 at 30°C. The transformant from each strain with the highest xylose uptake rate and ethanol productivity was selected for further study. Comparisons between selected transformants of each strain were conducted in 50 mL YPX40 following the protocol as listed in the ‘general flask fermentations’ sub-section, with the exception that xylose was used as the carbon source in place of glucose. The construction and evaluation of cellobiose-fermenting strains The pRS425-BTT plasmid (Ha et al.2013: 525–31) containing Neurospora crassa β-glucosidase (gh1-1) and cellodextrin transporter (cdt-1) genes was linearized and introduced into all industrial yeast strains to allow for cellobiose utilization. Several transformants of each strain were screened in 5 mL of YP medium containing 40 g/L of cellobiose (YPC40) at 30°C. The transformant from each strain with the highest cellobiose uptake rate and ethanol productivity was selected for further study. Comparisons between selected transformants of each strain were conducted in 50 mL YPC40, following the protocol as listed in the ‘general flask fermentations’ sub-section, with the exception that cellobiose was used as the carbon source in place of glucose. Gas pressure analysis Strains were evaluated for their gas pressure production in sealed glass bottles, with a higher gas pressure indicative of higher CO2 production and in-turn higher ethanol production. Jars with a maximum volume of 100 mL were filled with 20 mL of either YP medium containing 20 g/L of glucose, YP medium containing 20 g/L of glucose and 25% hydrolysate or Verduyn's (Verduyn et al.1992: 501–17) medium with 20 g/L of glucose and 0.628 g/L of complete supplement mixture (MP Biomedicals). The initial pH value of each medium was adjusted to 6 with concentrated NaOH or HCl, as needed. The fermentation bottles were shaken at 100 RPM on an Innova 2300 shaker (New Brunswick Scientific, CT) in a 30°C incubation room. Cell optical density (OD600) was measured as discussed in the ‘general flask fermentations’ sub-section, and the initial OD600 was ∼1.0. gas pressure was monitored with RF gas production modules (ANKOM Technology, NY) as pounds per square inch every 5 min. Growth assays under various pH levels OD600 was monitored for determining the cell growth in YP medium containing 20 g/L of glucose with an initial pH value adjusted to 4.5, 5.0, 5.5, 6.0 or 6.5. A 200 μL initial volume of medium was placed into individual wells of Costar 96-well flat-bottom polystyrene plates with lids placed on top (Corning, MA). The initial OD600 was 1.0, and microplates were incubated at 30°C and 200 RPM in a Symphony incubating microplate shaker (VWR, PA). An aliquot of 50 μL of mineral oil was placed on top of each well to prevent media evaporation. Wells containing media with mineral oil, but without the addition of yeast, were used as negative controls and as a blank by which the negative control OD was subtracted from the OD of experimental wells to account for any increased absorbance due to the oil. A Synergy HT plate reader (Biotek, Winooski, VT) was used to measure the OD600. Tolerance assays Precultures of S. cerevisiae strains were grown at 30°C in YPD20. Cells were harvested at the mid-exponential phase and adjusted to an initial OD600 of 1.0 for further use. Ten-fold serial dilutions were made in water, 10 μL of the 10−1, 10−2, 10−3, 10−4, 10−5 and 10−6 dilutions were spotted on YPD20 agar plates. The agar plates included the following: YPD20 plates incubated at 30°C, 37°C, 42°C and 45°C for heat tolerance; YPD20 plates with inhibitors, 2 g/L of HMF, 1 g/L of furfural or 20% hydrolysates at 30°C for inhibitor tolerance; and YPD20 medium containing 1 g/L, 2 g/L or 3 g/L of acetate at 30°C for organic acid tolerance. Visible-light pictures of the spotting assay were taken at 48 h. Top-performing strains were determined based on the number of colonies formed compared to the ATCC 4124 (Jin 03) control strain at equivalent dilutions. Relative genome content and ploidy analysis by flow cytometry Precultures of S. cerevisiae strains were grown at 30°C in YP medium containing 20 g/L of glucose (YPD20) and harvested at mid-exponential phase. Cells were collected once they reached OD600 ∼1.0. Samples were then prepared as previously described (Haase 2004: Unit 7.23) for flow cytometry. Briefly, cells were first fixed and permeabilized with cold 70% ethanol, and then washed with sodium citrate containing RNaseA. SYTOX® Green dye (Life Technologies) was used to stain the nucleic acid. The DNA contents of the stained cells were analyzed and detected by the excitation and emission spectra of the SYTOX® Green/DNA complex using an LSR II Flow Cytometry Analyzer (BD Biosciences, CA). The absolute ploidy of the industrial strains was estimated by comparing its DNA content to the control strains, the known haploid, diploid, triploid and tetraploid laboratory strains, L6437, L6438, L6439 and L6440, respectively, from Gerald Fink's laboratory (Galitski et al.1999: 251–4). The relative DNA content was calculated based on the average intensity of SYTOX® Green dye from the mid-log phase cells. From the two peaks of flow cytometry data (Fig. S1, Supporting Information), the first peak was used for the calculation, because the average intensity values of the first peak were correlated with the ploidy. An example of a comparison of ATCC 4124 with other control strains that are known for haploid, diploid, triploid and tetraploid was shown. In addition, the ploidy can be confirmed and correlates well with the genome sequencing data from previous studies of ATCC 4124 (Zhang et al.2014: 7694–701; Kim et al.2017: 176–85). Mating-type test A halo assay based on yeast pheromone response was used to determine strain mating-type (Meissner et al.2010: 2425–33). Mating type tester strains (DBY7730 for MATα and DBY7442 for MATa) (Julius et al.1983: 839–52) were first spread on YPD 20 g/L agar plates. Each industrial strain was then spotted on the lawn of each tester strain, and the plates were incubated overnight. The presence of a halo around a strain spot was used to score its mating type. Sporulation efficiency determination A single colony of each strain was obtained from YPD20 plates. The colonies were then cultured in the sporulation medium (1% potassium acetate, 0.1% yeast extract, 0.05% glucose and 2% agar). Cell division and sporulation occurred within 3–7 days and their sporulation efficiency were evaluated by the population of the tetrads. Heat map A heat map was used to summarize the performances of a specific test. No growth was scaled at −3. Any performance that was 10% lower than the ATCC 4124 control strain was scaled at −2. Performance from 5% to 10% lower was scaled at −1, performance within 5% lower or higher was scaled at 0, performance 5% higher was scaled +1, and performance greater than 10% higher was scaled at +2. RESULTS Phenotypic characterization of the industrial strains under common, industrially relevant stressors Osmotic stress is one of the common environmental stressors that yeasts encounter in the industrial-scale fermentation environment. Therefore, 20 industrial strains were selected and compared with a laboratory strain (D452-2) and an industrial strain (ATCC 4124) due to its well-known characteristic for lignocellulosic fermentation (Ho, Chen and Brainard 1998: 1852–9; Casey et al.2010: 385–93; Bera et al.2011: 617–26). They were evaluated for their tolerance against a high ethanol concentration from the high glucose fermentation (160 g/L), which is comparable to the total sugar content that would be present in sugar cane juice and plant hydrolysates (Soccol et al.2010: 4820–5). As expected at 12 h, all industrial strains outperformed the laboratory strain, D452-2. Other industrial strains consumed glucose rapidly and performed similarly to ATCC 4124. When comparing the ethanol productivity and ethanol yield, ATCC 4098 (Jin 02), ATCC 56069 (Jin 18), ATCC 60493 (Jin 21) and ATCC 66348 (Jin 24) showed a better ethanol productivity and ethanol yield as compared with ATCC 4124 (Jin 03) (Fig. 1; Table 2). Table 2. Fermentation data in YP medium with 160 g/L of glucose. The produced ethanol (g/L), the ethanol yield (g ethanol/g glucose consumed), and the ethanol productivity (g/L/h) at 12 h were listed in this table. Code name  Strains  Ethanol produced (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J1  ATCC 2360  53.606  0.431  4.467  J2  ATCC 4098  58.452  0.423  4.871  J3  ATCC 4124  55.746  0.400  4.645  J4  ATCC 4126  50.107  0.418  4.176  J5  ATCC 4127  57.015  0.410  4.751  J6  ATCC 4921  51.870  0.421  4.323  J8  ATCC 7754  57.645  0.427  4.804  J9  ATCC 9763  50.468  0.418  4.206  J11  ATCC 20598  48.097  0.418  4.008  J12  ATCC 24855  47.401  0.406  3.950  J13  ATCC 24858  48.647  0.400  4.054  J14  ATCC 24860  50.576  0.407  4.215  J15  ATCC 26422  43.300  0.404  3.608  J17  ATCC 46523  46.969  0.419  3.914  J18  ATCC 56069  62.944  0.423  5.245  J19  ATCC 60222  57.956  0.413  4.830  J20  ATCC 60223  50.250  0.408  4.188  J21  ATCC 60493  59.631  0.407  4.969  J24  ATCC 66348  62.655  0.414  5.221  J25  ATCC 66349  57.541  0.408  4.795  J26  ATCC 96581  43.154  0.416  3.596    D452-2  29.811  0.415  2.484  Code name  Strains  Ethanol produced (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J1  ATCC 2360  53.606  0.431  4.467  J2  ATCC 4098  58.452  0.423  4.871  J3  ATCC 4124  55.746  0.400  4.645  J4  ATCC 4126  50.107  0.418  4.176  J5  ATCC 4127  57.015  0.410  4.751  J6  ATCC 4921  51.870  0.421  4.323  J8  ATCC 7754  57.645  0.427  4.804  J9  ATCC 9763  50.468  0.418  4.206  J11  ATCC 20598  48.097  0.418  4.008  J12  ATCC 24855  47.401  0.406  3.950  J13  ATCC 24858  48.647  0.400  4.054  J14  ATCC 24860  50.576  0.407  4.215  J15  ATCC 26422  43.300  0.404  3.608  J17  ATCC 46523  46.969  0.419  3.914  J18  ATCC 56069  62.944  0.423  5.245  J19  ATCC 60222  57.956  0.413  4.830  J20  ATCC 60223  50.250  0.408  4.188  J21  ATCC 60493  59.631  0.407  4.969  J24  ATCC 66348  62.655  0.414  5.221  J25  ATCC 66349  57.541  0.408  4.795  J26  ATCC 96581  43.154  0.416  3.596    D452-2  29.811  0.415  2.484  View Large Table 2. Fermentation data in YP medium with 160 g/L of glucose. The produced ethanol (g/L), the ethanol yield (g ethanol/g glucose consumed), and the ethanol productivity (g/L/h) at 12 h were listed in this table. Code name  Strains  Ethanol produced (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J1  ATCC 2360  53.606  0.431  4.467  J2  ATCC 4098  58.452  0.423  4.871  J3  ATCC 4124  55.746  0.400  4.645  J4  ATCC 4126  50.107  0.418  4.176  J5  ATCC 4127  57.015  0.410  4.751  J6  ATCC 4921  51.870  0.421  4.323  J8  ATCC 7754  57.645  0.427  4.804  J9  ATCC 9763  50.468  0.418  4.206  J11  ATCC 20598  48.097  0.418  4.008  J12  ATCC 24855  47.401  0.406  3.950  J13  ATCC 24858  48.647  0.400  4.054  J14  ATCC 24860  50.576  0.407  4.215  J15  ATCC 26422  43.300  0.404  3.608  J17  ATCC 46523  46.969  0.419  3.914  J18  ATCC 56069  62.944  0.423  5.245  J19  ATCC 60222  57.956  0.413  4.830  J20  ATCC 60223  50.250  0.408  4.188  J21  ATCC 60493  59.631  0.407  4.969  J24  ATCC 66348  62.655  0.414  5.221  J25  ATCC 66349  57.541  0.408  4.795  J26  ATCC 96581  43.154  0.416  3.596    D452-2  29.811  0.415  2.484  Code name  Strains  Ethanol produced (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J1  ATCC 2360  53.606  0.431  4.467  J2  ATCC 4098  58.452  0.423  4.871  J3  ATCC 4124  55.746  0.400  4.645  J4  ATCC 4126  50.107  0.418  4.176  J5  ATCC 4127  57.015  0.410  4.751  J6  ATCC 4921  51.870  0.421  4.323  J8  ATCC 7754  57.645  0.427  4.804  J9  ATCC 9763  50.468  0.418  4.206  J11  ATCC 20598  48.097  0.418  4.008  J12  ATCC 24855  47.401  0.406  3.950  J13  ATCC 24858  48.647  0.400  4.054  J14  ATCC 24860  50.576  0.407  4.215  J15  ATCC 26422  43.300  0.404  3.608  J17  ATCC 46523  46.969  0.419  3.914  J18  ATCC 56069  62.944  0.423  5.245  J19  ATCC 60222  57.956  0.413  4.830  J20  ATCC 60223  50.250  0.408  4.188  J21  ATCC 60493  59.631  0.407  4.969  J24  ATCC 66348  62.655  0.414  5.221  J25  ATCC 66349  57.541  0.408  4.795  J26  ATCC 96581  43.154  0.416  3.596    D452-2  29.811  0.415  2.484  View Large Fermentation inhibitors and acetic acid are commonly found in lignocellulosic hydrolysates (Jonsson, Alriksson and Nilvebrant 2013: 16), and they limit the cell growth and viability. Therefore, growth assays on agar plates with the supplementation of fermentation inhibitors, acetic acid or lignocellulosic hydrolysates were used to test the tolerance against fermentation inhibitors. Also, agar plates were incubated at several temperatures (30°C, 37°C, 42°C and 45°C) to test their heat tolerance (Fig. 3), 42°C and 45°C not shown. High temperature is one of the potential stressors in an industrial-scale environment. The result shows ATCC 7754 (Jin 08), ATCC 9763 (Jin 09), ATCC 24858 (Jin 13), ATCC 46523 (Jin 17), ATCC 66348 (Jin 24) and ATCC 66349 (Jin 25) possess slightly better heat tolerance as compared with ATCC 4124. However, none of the strains could grow at 42°C or 45°C (data not shown). For the acetic acid tolerance, ATCC 9763 (Jin 09), ATCC 24858 (Jin 13), ATCC JIN 46523 (Jin 17) and ATCC 66348 (Jin 24) showed better tolerance against the organic acid when compared with ATCC 4124 (Fig. 5). Lastly, ATCC 4098 (Jin 02), ATCC 4127 (Jin 05), ATCC 9763 (Jin 09), ATCC 66348 (Jin 24) and ATCC 96581 (Jin 26) showed a greater tolerance against hydrolysates and/or specific fermentation inhibitors among all the strains (Figs 1 and 4). Figure 1. View largeDownload slide A heat map indicating the relative performance of our industrial strain screening under various conditions. ATCC 4124 is the control strain, and the z-score was scaled at 0. Performance for a specific test that was 10% lower than the ATCC 4124 control strain was scaled at −2. Performance 5% to 10% lower was scaled at −1, performance within 5% lower or 5% higher was scaled at 0, performance 5% higher was scaled +1, and performance greater than 10% higher was scaled at +2. No growth was scaled at −3 (gray color). Abbreviations: Glu, Glucose; YPD20, YP medium with 20g/L glucose; HMF, hydroxymethylfurfural; Xyl, xylose; Cel, cellobiose; GA, gas analysis; CSM, complete supplement mixture. Figure 1. View largeDownload slide A heat map indicating the relative performance of our industrial strain screening under various conditions. ATCC 4124 is the control strain, and the z-score was scaled at 0. Performance for a specific test that was 10% lower than the ATCC 4124 control strain was scaled at −2. Performance 5% to 10% lower was scaled at −1, performance within 5% lower or 5% higher was scaled at 0, performance 5% higher was scaled +1, and performance greater than 10% higher was scaled at +2. No growth was scaled at −3 (gray color). Abbreviations: Glu, Glucose; YPD20, YP medium with 20g/L glucose; HMF, hydroxymethylfurfural; Xyl, xylose; Cel, cellobiose; GA, gas analysis; CSM, complete supplement mixture. A low-pH resistant yeast strain is beneficial for an industrial setting because low pH combined with organic acid will severely inhibit the growth and metabolism of most yeast. Therefore, the performance of the industrial strains was also tested under various pH values. The results showed that ATCC 4127 (Jin 05), ATCC 9763 (Jin 09), ATCC 46523 (Jin 17), ATCC 56069 (Jin 18) and ATCC 66348 (Jin 24) have a higher tolerance than other industrial strains and ATCC 4124 (Jin 03) (Fig. 1). Combining all findings, ATCC 9763 (Jin 09), ATCC 46523 (Jin 17) and ATCC 66348 (Jin 24) could be preferential strains for industrial lignocellulosic hydrolysate fermentations. Determination of the ploidy and the sporulation efficiency of industrial yeast strains It is challenging to introduce genetic perturbations into industrial yeasts due in part to their complex genome structure, such as aneuploidy, polyploidy or another chromosomal rearrangement (Argueso et al.2009: 2258–70; Akao et al.2011: 423–34; Borneman et al.2011: e1001287; Babrzadeh et al.2012: 485–94; Borneman et al.2012: 88–96; Dunn et al.2012: 908–24). It could be easier to manipulate a haploid strain than a diploid or even a triploid strain. Therefore, the ploidy of the industrial strains was estimated by the relative DNA content measured by flow cytometry, and the selected haploid was confirmed with a mating type test. Among all the industrial strains (Fig. 2), ATCC 60222 (Jin 19) and ATCC 60223 (Jin 20) have the highest relative DNA content, which correlates with the higher number of ploidy. ATCC 20598 (Jin 11) was believed to be a haploid with MATa, after the mating type test confirmation, and a lower relative DNA content was measured. Therefore, it would be easier to choose ATCC 20598 (Jin 11) for further genetic manipulation. Previous results (Fig. 1) suggested ATCC 9763 (Jin 09) and ATCC 46523 (Jin 17) as good candidates for lignocellulosic fermentation, but their ploidy is believed to be diploid or triploid based on the relative DNA content, causing the strains to be more difficult to undergo directed genetic manipulation compared to haploid strains. However, ATCC 66348 (Jin 24) is also a good candidate for lignocellulosic fermentations (Fig. 1), and its relative DNA content indicates that it may be a haploid strain that would make it more ammenable to genetic manipulation than diploid or triploid strains. Figure 2. View largeDownload slide The relative DNA content of industrial strains was measured by flow cytometry. Strains with known ploidy were used as a control and colored in black. Relative DNA content correlating to approximately 1N to 2N, 2N to 3N, and 3N to 4N is shown in red, blue, or pink respectively. Figure 2. View largeDownload slide The relative DNA content of industrial strains was measured by flow cytometry. Strains with known ploidy were used as a control and colored in black. Relative DNA content correlating to approximately 1N to 2N, 2N to 3N, and 3N to 4N is shown in red, blue, or pink respectively. Figure 3. View largeDownload slide Temperature tolerance from the spotting assay on YPD medium incubated at 30°C or 37°C. Red bars indicate strains that showed the consistently best growth in the highlighted condition. Red numbers across the top indicate strain number, e.g. 1 is J1, 2 is J2 and so forth. Figure 3. View largeDownload slide Temperature tolerance from the spotting assay on YPD medium incubated at 30°C or 37°C. Red bars indicate strains that showed the consistently best growth in the highlighted condition. Red numbers across the top indicate strain number, e.g. 1 is J1, 2 is J2 and so forth. Figure 4. View largeDownload slide Inhibitor tolerance from the spotting assay on YPD medium containing 2 g/L HMF, 1 g/L furfural, or 20% hydrolysate. Red bars indicate strains which showed the consistently best growth in the highlighted condition. Red numbers across the top indicate strain number, e.g. 1 is J1, 2 is J2 and so forth. Figure 4. View largeDownload slide Inhibitor tolerance from the spotting assay on YPD medium containing 2 g/L HMF, 1 g/L furfural, or 20% hydrolysate. Red bars indicate strains which showed the consistently best growth in the highlighted condition. Red numbers across the top indicate strain number, e.g. 1 is J1, 2 is J2 and so forth. Figure 5. View largeDownload slide Acetic acid tolerance from spotting assay on YPD medium containing 1, 2 or 3 g/L acetate. Red bars indicate strains which showed the consistently best growth in the highlighted condition. Red numbers across the top indicate strain number, e.g. 1 is J1, 2 is J2 and so forth. Figure 5. View largeDownload slide Acetic acid tolerance from spotting assay on YPD medium containing 1, 2 or 3 g/L acetate. Red bars indicate strains which showed the consistently best growth in the highlighted condition. Red numbers across the top indicate strain number, e.g. 1 is J1, 2 is J2 and so forth. Mating, sporulation and isolation of haploids are one of the alternative ways to modify yeasts to achieve certain desired phenotypes. The anticipated phenotypes of polyploidy industrial strains could be hybridized with another industrial strain once a pool of spores (haploid) was generated after sporulation (Fukuda et al.2016: 45). Crossbreeding could be done by mating the spores (haploid) with the opposite mating type. Sporulation efficiency is used to measure the ratio of tetrads produced by the strain. It is the first step to evaluate the feasibility of a strain for mating experiments and to measure the ratio of tetrads produced by strain. When the cells are cultivated in sporulation medium with limiting nutrients for 5–7 days, yeast cells will start to sporulate as a response to nutrient deprivation and stress. Among all the industrial strains, ATCC 9763 (Jin 09), ATCC 46523 (Jin 17) and ATCC 6022 (Jin 20) had the highest sporulation efficiency (Table 3), and these three strains could be used for mating and crossbreeding experiments. However, the ATCC 66348 (Jin 24) has lower sporulation efficiency, leading to the complication of manipulating this strain. Lastly, ATCC 9763 (Jin 09) and ATCC 46523 (Jin 17) could be considered as the next preferable host strains for further strain engineering due to their high sporulation efficiency and reduced ploidy in the genome. Table 3. Sporulation efficiency of each industrial strain; ATCC 20598 was unable to be induced to sporulate;—indicates no sporulation was detected,+ indicates ∼25% sporulation efficiency, ++ indicates ∼50% sporulation efficiency, and +++ indicates ∼75% or greater sporulation efficiency. Code name  Strains  Sporulation efficiency  J1  ATCC 2360  +  J2  ATCC 4098  +  J3  ATCC 4124  +  J4  ATCC 4126  ++  J5  ATCC 4127  +  J6  ATCC 4921  +  J8  ATCC 7754  ++  J9  ATCC 9763  +++  J11  ATCC 20598  −  J12  ATCC 24855  ++  J13  ATCC 24858  ++  J14  ATCC 24860  +  J15  ATCC 26422  +  J17  ATCC 46523  +++  J18  ATCC 56069  +  J19  ATCC 60222  +++  J20  ATCC 60223  +++  J21  ATCC 60493  +  J24  ATCC 66348  +  J25  ATCC 66349  ++  J26  ATCC 96581  +  Code name  Strains  Sporulation efficiency  J1  ATCC 2360  +  J2  ATCC 4098  +  J3  ATCC 4124  +  J4  ATCC 4126  ++  J5  ATCC 4127  +  J6  ATCC 4921  +  J8  ATCC 7754  ++  J9  ATCC 9763  +++  J11  ATCC 20598  −  J12  ATCC 24855  ++  J13  ATCC 24858  ++  J14  ATCC 24860  +  J15  ATCC 26422  +  J17  ATCC 46523  +++  J18  ATCC 56069  +  J19  ATCC 60222  +++  J20  ATCC 60223  +++  J21  ATCC 60493  +  J24  ATCC 66348  +  J25  ATCC 66349  ++  J26  ATCC 96581  +  View Large Table 3. Sporulation efficiency of each industrial strain; ATCC 20598 was unable to be induced to sporulate;—indicates no sporulation was detected,+ indicates ∼25% sporulation efficiency, ++ indicates ∼50% sporulation efficiency, and +++ indicates ∼75% or greater sporulation efficiency. Code name  Strains  Sporulation efficiency  J1  ATCC 2360  +  J2  ATCC 4098  +  J3  ATCC 4124  +  J4  ATCC 4126  ++  J5  ATCC 4127  +  J6  ATCC 4921  +  J8  ATCC 7754  ++  J9  ATCC 9763  +++  J11  ATCC 20598  −  J12  ATCC 24855  ++  J13  ATCC 24858  ++  J14  ATCC 24860  +  J15  ATCC 26422  +  J17  ATCC 46523  +++  J18  ATCC 56069  +  J19  ATCC 60222  +++  J20  ATCC 60223  +++  J21  ATCC 60493  +  J24  ATCC 66348  +  J25  ATCC 66349  ++  J26  ATCC 96581  +  Code name  Strains  Sporulation efficiency  J1  ATCC 2360  +  J2  ATCC 4098  +  J3  ATCC 4124  +  J4  ATCC 4126  ++  J5  ATCC 4127  +  J6  ATCC 4921  +  J8  ATCC 7754  ++  J9  ATCC 9763  +++  J11  ATCC 20598  −  J12  ATCC 24855  ++  J13  ATCC 24858  ++  J14  ATCC 24860  +  J15  ATCC 26422  +  J17  ATCC 46523  +++  J18  ATCC 56069  +  J19  ATCC 60222  +++  J20  ATCC 60223  +++  J21  ATCC 60493  +  J24  ATCC 66348  +  J25  ATCC 66349  ++  J26  ATCC 96581  +  View Large Evaluation of the newly constructed, xylose-fermenting industrial yeast strains Because xylose is the second-most abundant sugar in lignocellulosic hydrolysates, it is important to have an efficient xylose-fermenting strain for an efficient industrial fermentation. To examine the effect of different strain backgrounds on the efficiency of xylose fermentation, all the strains were engineered to express a xylose fermentation pathway (xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XK) encoded by XYL1, XYL2 and XYL3, respectively), because S. cerevisiae cannot natively metabolize xylose. ATCC 4124 (Jin 03) was known to have an efficient xylose fermentation after strain engineering from previous studies (Ho et al.1998: 1852–9; Casey et al.2010: 385–93; Bera et al.2011: 617–26). Therefore, the industrial strains constructed with the xylose pathway were compared with ATCC 4124 (Jin 03) with the xylose pathway. At least eight transformants for each ATCC strain were selected and screened in 5 mL YPX40. The final list of the best xylose-fermenting strains was summarized in Fig. 1 and Table 4. All the transformants were labeled, such as (J3-02), referring to the #2 colony of the ATCC 4124 (Jin 03) strain, (J18-01) referring to the #1 colony of the ATCC 56069 (Jin 18) strain and so forth. Overall, only the transformants from ATCC 4127 (J5-08), ATCC 46523 (J17-01) and ATCC 66348 (J24-11) were found to consume xylose and produce ethanol more efficiently than the best transformant from ATCC 4124 (J3-02) at 72 h. Also, ATCC 20598 (Jin 11) and ATCC 96581 (Jin 26) failed to assimilate xylose. Even though ATCC 20598 (Jin 11) is a haploid and presumably more amenable to manipulation, it failed to ferment xylose, possibly due to the lack of strain fitness or other complex genetic problems. Combined with the previous results, ATCC 66348 (Jin 24) consistently exhibited the most desirable phenotypes for lignocellulosic hydrolysates fermentations, which included the tolerances against fermentation inhibitors and higher xylose assimilation rate. However, it may be challenging to work with ATCC 66348 (Jin 24) due to its potential diploid nature (Fig. 2) and low sporulation efficiency (Table 3). Table 4. Fermentation data of the selected xylose-fermenting transformants from the industrial strains grown in YPX40 medium and measured at 72 h into the fermentations. J3-02 refers to the #2 colony of Jin 03 (ATCC 4124) strain; J18-01 refers to the #1 colony of Jin 18 (ATCC 56069) strain, and so forth. Code name  Strains  Xylose consumption rate (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J2-08  ATCC 4098  0.489  0.200  0.098  J3-02  ATCC 4124  0.533  0.305  0.163  J3-07  ATCC 4124  0.509  0.244  0.124  J4-01  ATCC 4126  0.493  0.217  0.107  J5-01  ATCC 4127  0.494  0.238  0.118  J5-03  ATCC 4127  0.509  0.221  0.112  J5-06  ATCC 4127  0.511  0.214  0.109  J5-07  ATCC 4127  0.503  0.192  0.096  J5-08  ATCC 4127  0.544  0.181  0.098  J17-01  ATCC 46523  0.553  0.247  0.137  J18-01  ATCC 56069  0.526  0.224  0.118  J24-05  ATCC 66348  0.475  0.197  0.094  J24-11  ATCC 66348  0.535  0.246  0.132  J24-12  ATCC 66348  0.515  0.211  0.108  J24-15  ATCC 66348  0.514  0.211  0.108  Code name  Strains  Xylose consumption rate (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J2-08  ATCC 4098  0.489  0.200  0.098  J3-02  ATCC 4124  0.533  0.305  0.163  J3-07  ATCC 4124  0.509  0.244  0.124  J4-01  ATCC 4126  0.493  0.217  0.107  J5-01  ATCC 4127  0.494  0.238  0.118  J5-03  ATCC 4127  0.509  0.221  0.112  J5-06  ATCC 4127  0.511  0.214  0.109  J5-07  ATCC 4127  0.503  0.192  0.096  J5-08  ATCC 4127  0.544  0.181  0.098  J17-01  ATCC 46523  0.553  0.247  0.137  J18-01  ATCC 56069  0.526  0.224  0.118  J24-05  ATCC 66348  0.475  0.197  0.094  J24-11  ATCC 66348  0.535  0.246  0.132  J24-12  ATCC 66348  0.515  0.211  0.108  J24-15  ATCC 66348  0.514  0.211  0.108  View Large Table 4. Fermentation data of the selected xylose-fermenting transformants from the industrial strains grown in YPX40 medium and measured at 72 h into the fermentations. J3-02 refers to the #2 colony of Jin 03 (ATCC 4124) strain; J18-01 refers to the #1 colony of Jin 18 (ATCC 56069) strain, and so forth. Code name  Strains  Xylose consumption rate (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J2-08  ATCC 4098  0.489  0.200  0.098  J3-02  ATCC 4124  0.533  0.305  0.163  J3-07  ATCC 4124  0.509  0.244  0.124  J4-01  ATCC 4126  0.493  0.217  0.107  J5-01  ATCC 4127  0.494  0.238  0.118  J5-03  ATCC 4127  0.509  0.221  0.112  J5-06  ATCC 4127  0.511  0.214  0.109  J5-07  ATCC 4127  0.503  0.192  0.096  J5-08  ATCC 4127  0.544  0.181  0.098  J17-01  ATCC 46523  0.553  0.247  0.137  J18-01  ATCC 56069  0.526  0.224  0.118  J24-05  ATCC 66348  0.475  0.197  0.094  J24-11  ATCC 66348  0.535  0.246  0.132  J24-12  ATCC 66348  0.515  0.211  0.108  J24-15  ATCC 66348  0.514  0.211  0.108  Code name  Strains  Xylose consumption rate (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J2-08  ATCC 4098  0.489  0.200  0.098  J3-02  ATCC 4124  0.533  0.305  0.163  J3-07  ATCC 4124  0.509  0.244  0.124  J4-01  ATCC 4126  0.493  0.217  0.107  J5-01  ATCC 4127  0.494  0.238  0.118  J5-03  ATCC 4127  0.509  0.221  0.112  J5-06  ATCC 4127  0.511  0.214  0.109  J5-07  ATCC 4127  0.503  0.192  0.096  J5-08  ATCC 4127  0.544  0.181  0.098  J17-01  ATCC 46523  0.553  0.247  0.137  J18-01  ATCC 56069  0.526  0.224  0.118  J24-05  ATCC 66348  0.475  0.197  0.094  J24-11  ATCC 66348  0.535  0.246  0.132  J24-12  ATCC 66348  0.515  0.211  0.108  J24-15  ATCC 66348  0.514  0.211  0.108  View Large Evaluation of the newly constructed, cellobiose-fermenting industrial yeast strains Cellobiose is commonly found in cellulose, galactan, and red seaweed after hydrolysis. However, yeast cannot naturally metabolize cellobiose. Previous studies reported a high-affinity cellodextrin transporter (cdt-1) and an intracellular β-glucosidase (gh1-1) from Neurospora crassa were introduced into S. cerevisiae strains, and the resulting strains could ferment cellobiose efficiently (Galazka et al.2010: 84–6). In addition, cellobiose and xylose could be co-fermented simultaneously without glucose suppression from the sequential fermentation of glucose first and xylose second, because glucose was hydrolyzed intracellularly (Ha et al.2013: 525–31). With all the benefits of cellobiose fermentation, the cellobiose pathway was introduced into the industrial strains. However, only a few transformants were obtained, and only the transformants from ATCC 4124 (Jin 03), ATCC 9763 (Jin 09) and ATCC 24858 (Jin 13) could ferment cellobiose efficiently (Fig. 1; Table 5). When compared with the transformant of ATCC 4124 (Jin 03) at 37 h, only the transformant from ATCC 9763 (Jin 09) was comparable. The others were either not able to metabolize cellobiose or grew poorly. Combing the results, ATCC 9763 (Jin 09) has the most desirable phenotypes for cellobiose fermentation and tolerances against high temperatures, fermentation inhibitors and organic acids. Table 5. Fermentation data of the selected cellobiose fermenting transformants from the industrial strains grown in YPC80 medium and measured at 37 h into the fermentation. Code name  Strains  Cellobiose consumption rate (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J3  ATCC 4124  2.316  0.369  0.855  J9  ATCC 9763  2.377  0.338  0.803  J13  ATCC 24858  1.709  0.180  0.307  Code name  Strains  Cellobiose consumption rate (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J3  ATCC 4124  2.316  0.369  0.855  J9  ATCC 9763  2.377  0.338  0.803  J13  ATCC 24858  1.709  0.180  0.307  View Large Table 5. Fermentation data of the selected cellobiose fermenting transformants from the industrial strains grown in YPC80 medium and measured at 37 h into the fermentation. Code name  Strains  Cellobiose consumption rate (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J3  ATCC 4124  2.316  0.369  0.855  J9  ATCC 9763  2.377  0.338  0.803  J13  ATCC 24858  1.709  0.180  0.307  Code name  Strains  Cellobiose consumption rate (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J3  ATCC 4124  2.316  0.369  0.855  J9  ATCC 9763  2.377  0.338  0.803  J13  ATCC 24858  1.709  0.180  0.307  View Large Phenotyping the industrial strains under minimal media using gas pressure analysis Strains were evaluated for their ability to increase gas pressure in a sealed glass bottle, with a higher gas pressure indicative of higher CO2 production and in-turn higher ethanol production. The yeast strains were grown in YP medium containing 20 g/L of glucose, YP medium containing 20 g/L of glucose and 25% (final concentration) hydrolysate or Verduyn's (Verduyn et al.1992: 501–17) medium with 20 g/L of glucose and 0.628 g/L of complete supplement mixture (MP Biomedicals, CA) to compare the industrial strains’ behaviors among different media composition. Once the fermentation started in a sealed bottle, the attached RF gas production modules (ANKOM Technology, NY) would monitor the gas production as pounds per square inch, recording every 5 min. For the YPD20 with 25% hydrolysate (Fig. 1), ATCC 60493 (Jin 21) produced greater gas pressure, likely through CO2 production, which correlates with higher ethanol production when compared with the ATCC 4124 (Jin 03) strain. There are no significant differences among strains in the YPD20 condition. The industrials strains ATCC 2360 (Jin 01), ATCC 4127 (Jin 05), ATCC 7754 (Jin 08), ATCC 24858 (Jin 13), ATCC 38544 (Jin 16), ATCC 46523 (Jin 17), ATCC 56069 (Jin 18), ATCC 60222 (Jin 19) and ATCC 60493 (Jin 21) all performed better than ATCC 4124 (Jin 03) in Verduyn's medium, suggesting that the ATCC4124 (Jin 03) control strain is not ideal for growth in certain minimal media, such as Verdyn's medium. Taking into account the relative performances of the strains regarding low pH tolerance, fermentation inhibitor resistance, and xylose fermentation rates, we narrowed down five industrial strains and ATCC 4124 (Jin 03) to conduct further experiments. The selected industrial strains were ATCC 9763 (Jin 09), ATCC 24858 (Jin 13), ATCC 46523 (Jin 17), ATCC 56069 (Jin 18) and ATCC 66348 (Jin 24). Synthetic complete minimal medium with and without CSM (Complete Supplement Mixture, MP Biomedicals, CA) was used to evaluate the strains (Fig. 6). Of the selected industrial strains, ATCC 56069 (Jin 18) significantly underperformed compared to the ATCC 4124 (Jin 03) control in either condition. However, the other four industrial strains outperformed the ATCC 4124 (Jin 03) control with CSM. Under the nutrient-limiting medium (SC minimal medium, SCD), ATCC 24848 (Jin 13) and ATCC 66348 (Jin 24) had the highest CO2 production as compared with other industrial strains, suggesting that these two strains required fewer nutrients for optimal growth and ethanol production. Overall, ATCC 56069 (Jin 18) was the worst strain in the minimal medium in terms of gas production. Figure 6. View largeDownload slide A heat map indicating the relative performance of industrial strain screening on mannose, maltose, and sucrose condition. The ranking of their performances was analyzed in this figure. Performance for a specific test that was 10% lower than the ATCC 4124 control strain was scaled at −2. Performance 5%–10% lower was scaled at −1, performance within 5% lower or higher was scaled at 0, performance 5% higher was scaled +1, and performance greater than 10% higher was scaled at +2. No growth was scaled at -3 (gray color). Abbreviations: SM, synthetic complete medium with complete supplement; SCD, synthetic complete medium without complete supplement; GA, gas analysis; Man, Mannose; Malt, Maltose; Sucro, Sucrose. Figure 6. View largeDownload slide A heat map indicating the relative performance of industrial strain screening on mannose, maltose, and sucrose condition. The ranking of their performances was analyzed in this figure. Performance for a specific test that was 10% lower than the ATCC 4124 control strain was scaled at −2. Performance 5%–10% lower was scaled at −1, performance within 5% lower or higher was scaled at 0, performance 5% higher was scaled +1, and performance greater than 10% higher was scaled at +2. No growth was scaled at -3 (gray color). Abbreviations: SM, synthetic complete medium with complete supplement; SCD, synthetic complete medium without complete supplement; GA, gas analysis; Man, Mannose; Malt, Maltose; Sucro, Sucrose. Assimilation of other sugars Mannose, maltose and sucrose utilization are also interesting to certain industries. For example, mannose is a sugar hydrolyzed from plant hemicellulose and seaweed, maltose is a disaccharides breakdown product from starch, and sucrose is found in sugarcane juice. To expand the feasibility of our selected strains and utilize a variety of sources for the substrates (sugars), we examined the fermentation capability of the industrial yeast strains by measuring their ethanol production rate and specific growth rate in YP medium with mannose 100 g/L, maltose 100 g/L or sucrose 100 g/L. When compared with ATCC 4124 (Jin 03) (Fig. 6), ATCC 46523 (Jin 17) has the highest specific growth rate under both mannose and maltose conditions. Both ATCC 56069 (Jin 18) and ATCC 66348 (Jin 24) have a higher specific growth rate in the sucrose condition. In terms of ethanol productivities, ATCC 46523 (Jin 17) and ATCC 66348 (Jin 24) are worse performing than ATCC 4124 (Jin 03) under the sucrose condition. ATCC 24858 (Jin 13) and ATCC 56069 (Jin 18) are worse than ATCC 4124 (Jin 03) under the mannose condition, with only ATCC 66348 (Jin 24) being slightly better. Under the maltose condition, ATCC 56069 (Jin 18) and ATCC 66348 (Jin 24) could not consume maltose, and no ethanol was produced. The ethanol productivity of ATCC 24858 (Jin 13) was slightly better than ATCC 4124 (Jin 03) under the maltose condition. DISCUSSION In broad terms, S. cerevisiae can be divided up into two major categories, industrial or laboratory strains. As the name implies, industrial yeast strains are considered as such due to their ability to resist harsh industrial fermentation conditions, which includes fermentation inhibitor-laden lignocellulosic hydrolysates. However, industrial yeasts are polyploid strains in many cases, whereas laboratory yeasts are most commonly haploid strains (Hansen and Kielland-Brandt 1996: 1–12; Walker 1998: 362). The increased ploidy can aid the resistance of the yeast strain to fermentation conditions, but can also increase the difficulty of introducing targeted genetic perturbations. Fortunately, with the recent development of the CRISPR/Cas9 gene editing system, engineering polyploid yeast strains has become increasingly less laborious and easier as compared with traditional engineering methods (Ryan et al.2014). With the CRISPR/Cas9 system in mind, identifying the phenotypic characteristics of industrial yeast strains would be beneficial, and superior strains could be achievable shortly with the recent developments. In this study, a review and analysis of 21 industrial S. cerevisiae yeast strains was conducted. Compared to the control strain ATCC 4124 (JIN 03), several strains appeared to excel in three conditions amenable to lignocellulosic hydrolysate fermentations: a rapid xylose fermentation rate, resistance under low pH conditions and tolerance against fermentation inhibitors. Of the 21 industrial yeast strains in this study, five strains (ATCC numbers 4127, 4921, 56069, 60222 and 60223) have no peer-reviewed literature citing the ATCC nomenclature and, to our knowledge, have not been used in any major laboratory- or industrial-scale studies. Despite this, these five strains also did not have any significantly desired phenotypes compared to the highly studied ATCC 4124 control strain. Several interesting trends appeared at the end of the screening process. We observed that only three strains (ATCC 56069, 60493 and 66348) could exceed the ethanol productivities of the ATCC 4124 (Jin 03) control strain in high concentrations of glucose (160 g/L). This result suggests that some industrial strains are considerably high osmotolerance and are resistant to a higher concentration of ethanol, whereas many other industrial yeasts do not have this tolerance phenotype. As a result, our dataset screened out the three strains that are better than the ATCC 4124 strain in that regard. In addition, we observed that some strains were more affected by higher temperature (37°C) fermentation than that of the control, suggesting some industrial strains are relatively more sensitive to elevated temperatures than others. Regarding the fermentation inhibitors commonly found in lignocellulosic hydrolysates, such as acetic acid, furfural and HMF, most strains were as tolerant as the ATCC 4124 control. Interestingly, ATCC 4098 (Jin 02), ATCC 4127 (Jin 05), ATCC 9763 (Jin 09), ATCC 24858 (Jin 13), ATCC 46523 (Jin 17) and ATCC 66348 (Jin 24) grew to a higher colony count than the control strain in the presence of these inhibitors, suggesting that they may be preferential strains for industrial lignocellulosic hydrolysate fermentations. It was reported that most industrial strains are diploid, aneuploid and occasionally polyploid (Argueso et al.2009: 2258–70; Akao et al.2011: 423–34; Borneman et al.2011: e1001287; Babrzadeh et al.2012: 485–94; Borneman et al.2012: 88–96; Dunn et al.2012: 908–24). As expected, only one industrial strain in this study, ATCC 20598 (Jin 11), was confirmed to be haploid, while the other strains are either diploid or polyploid. Many studies suggested that increased ploidy and increased resistances toward environmental stressors are correlated. However, we do not see the correlation in our results. The best-selected strains in this study do not contain the highest predicted ploidy. The strains with the highest ploidy, ATCC 60222 (Jin 19) and ATCC 60223 (Jin 20), performed worse than other industrial strains in most conditions in this study. In terms of xylose fermentation capability, most transformants from other industrials trains expressing the xylose fermentation pathway performed comparably to the control ATCC 4124 strain after a heterologous xylose fermentation pathway consisting of XYL1, XYL2 and XYL3 was introduced and expressed into the industrial strains. Interestingly, transformants from ATCC 4127 (Jin 05), ATCC 56069 (Jin 18) and ATCC 66348 (Jin 24) were found to ferment xylose more rapidly and produce ethanol than the ATCC 4124 control. On the other hand, most of the transformants expressing the cellobiose fermentation pathway performed similarly or worse than ATCC 4124 (Jin 03). Taking into account the relative performances of the strains in terms of low pH tolerance, fermentation inhibitor resistance and xylose fermentation rates, we identified ATCC 66348 (Jin24) as the overall top-performing best strain to conduct further experiments or genetic improvements for the efficient bioconversion of lignocellulosic hydrolysates. CONCLUSIONS Collectively, this study has provided a useful dataset to refer to when choosing an industrial S. cerevisiae for unique fermentation purposes, especially lignocellulosic hydrolysates. This study builds on previous studies that have also aimed to evaluate a variety of industrial yeast strains with the intent to improve the available dataset of industrial yeast phenotypes (Martıín and Jönsson 2003: 386–95; Li et al.2015: 266–74). Selecting the best host for lignocellulosic hydrolysates is one of the motivations in this study, and ATCC 66348 (Jin 24), originally isolated from Japanese soil, was chosen as the best overall, broadly applicable candidate for future lignocellulosic hydrolysate studies. SUPPLEMENTARY DATA Supplementary data is available at FEMSYR online. Acknowledgements We thank the American Type Culture Collection (ATCC) for continued maintenance of their yeast strain collections. FUNDING This work was supported by the Agriculture and Food Research Initiative Competitive Grant [No. 2015-67011-22806] from the United States Department of Agriculture, National Institute of Food and Agriculture to [TLT]. Conflict of interest. None declared. REFERENCES Akao T, Yashiro I, Hosoyama A et al.   Whole-genome sequencing of sake yeast Saccharomyces cerevisiae Kyokai no. 7. DNA Res  2011; 18: 423– 34. 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Phenotypic evaluation and characterization of 21 industrial Saccharomyces cerevisiae yeast strains

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Abstract

Abstract Microorganisms have been studied and used extensively to produce value-added fuels and chemicals. Yeasts, specifically Saccharomyces cerevisiae, receive industrial attention because of their well-known ability to ferment glucose and produce ethanol. Thousands of natural or genetically modified S. cerevisiae have been found in industrial environments for various purposes. These industrial strains are isolated from industrial fermentation sites, and they are considered as potential host strains for superior fermentation processes. In many cases, industrial yeast strains have higher thermotolerance, increased resistances towards fermentation inhibitors and increased glucose fermentation rates under anaerobic conditions when compared with laboratory yeast strains. Despite the advantages of industrial strains, they are often not well characterized. Through screening and phenotypic characterization of commercially available industrial yeast strains, industrial fermentation processes requiring specific environmental conditions may be able to select an ideal starting yeast strain to be further engineered. Here, we have characterized and compared 21 industrial S. cerevisiae strains under multiple conditions, including their tolerance to varying pH conditions, resistance to fermentation inhibitors, sporulation efficiency and ability to ferment lignocellulosic sugars. These data may be useful for the selection of a parental strain for specific biotechnological applications of engineered yeast. Yeast, Saccharomyces cerevisiae, industrial yeast, laboratory yeast, hydrolysate, biotechnology INTRODUCTION Yeasts have served many important industrial purposes for humans over thousands of years. Specifically, the Saccharomyces cerevisiae yeast species has acted as the primary species to produce wine, beer and bread (Legras et al.2007: 2091–102). Although the production of baked goods and fermented foods or beverages has historically been the major industrial application for S. cerevisiae, new avenues of yeast fermentation capabilities were discovered in recent decades. These new opportunities are due in part to rapid and significant advances in our understanding of yeast genetics and physiology (Walker 1998: 362). Through our improved understanding of yeast hardiness and functions, we have exploited the robustness of S. cerevisiae to produce various value-added chemicals and fuels (Turner et al.2018). Industrial strains of S. cerevisiae are known to have higher tolerances against harsh industrial environments, such as lower pH, fermentation inhibitors, osmolality and higher temperature. With these desired phenotypes of industrial strains, it is feasible to have an improved engineered industrial strain for sustainable biofuel or biochemical production from lignocellulosic hydrolysates. Although several microbial species are used for industrial production of value-added products, yeasts are perhaps the most well studied. Yeasts can natively ferment glucose to produce ethanol, and this capability has been used for wine production for thousands of years (McGovern et al.1997: 3–21; Cavalieri et al.2003: S226–32). In recent decades, the yeast S. cerevisiae has been utilized extensively for biofuel production (Nissen et al.2000: 69–77). The majority of bioethanol produced by S. cerevisiae is from the fermentation of sugarcane or corn-derived glucose (Wheals et al.1999: 482–7). Although industrial yeast fermentation has resulted in the annual production of more than 50 billion liters ethanol production in the United States alone, the availability of corn and sugarcane is a limiting step in using biofuels as a total replacement for fossil fuels (Chum et al.2014: 205–23). Therefore, abundant lignocellulosic crops are considered as feasible alternative feedstocks for the production of bio-based fuels and chemicals (Carroll and Somerville 2009: 165–82). Although S. cerevisiae is well-studied with many genetic manipulation tools available (Ostergaard, Olsson and Nielsen 2000: 34–50; DiCarlo et al.2013: 4336–43), it does not efficiently produce fuels and chemicals from processed lignocellulosic hydrolysates due, in part, to the harsh environment present in the hydrolysate. Specifically, the low pH of lignocellulosic hydrolysates coupled with the presence of many known and unknown fermentation inhibitors acts as major hurdles to efficiently produce fuels and chemicals by engineered S. cerevisiae (Jayakody et al.2013: 6589–600; Jonsson, Alriksson and Nilvebrant 2013: 16). In general, the inhibitory effects of lignocellulosic hydrolysates are not well understood. Several attempts to overcome lignocellulosic hydrolysate toxicity were not successful, and this may be due to inhibition of desired yeast phenotypes caused by the fermentation conditions and that the strains are lacking resistance toward the inhibitors’ toxicity (Almeida et al.2007: 340–9). One possible solution is to modify the pretreatment process of lignocellulosic hydrolysate to reduce the inhibitors or to balance the pH. However, this solution can result in reduced fermentable sugar availability (Persson et al.2002: 5318–25) or increased production costs (Rivard et al.1996: 183–91). Owed to these hurdles, developing an engineered microbe from a parental strain that is natively resistant to the many fermentation inhibitors present in hydrolysates to efficiently ferment lignocellulosic hydrolysates may be a more cost-effective alternative (Brandberg, Franzen and Gustafsson 2004: 122–5). In particular, laboratory strains are generally regarded as possessing lower tolerance to fermentation inhibitors as compared to industrial yeast strains and, therefore, industrial strains may be ideal for producing fuels and chemicals from lignocellulosic hydrolysates (Martıín and Jönsson 2003: 386–95). However, yeast strains have widely varying inhibitor tolerances and fermentation profiles. It is challenging and time consuming to select and characterize the best strains for their resistances toward common fermentation inhibitors present in lignocellulosic hydrolysates such as hydroxymethylfurfural (HMF), furfural and acetate. Thus, developing a dataset of industrial strains, ranked for their characteristic and tolerances under certain industrial conditions, was the main goal in the present study. This dataset will be provide useful guidance for selecting an ideal candidate strain for specific industrial fermentation processes. Here, we have selected 21 S. cerevisiae yeast strains that are publicly available from the American Type Culture Collection (ATCC), and screened each strain to compare their fermentation phenotypes in a variety of conditions. MATERIALS AND METHODS Industrial strain backgrounds All S. cerevisiae strains were obtained from the ATCC. In total, 21 strains were selected. A list containing the ATCC nomenclature for each strain, the original isolation source of the strain and a representative literature citation for each strain is shown in Table 1. The well studied control strain, J3 or ATCC4124, was bold in the table. In some instances, the original isolation source or any peer-reviewed reference was not available. Table 1. Listing of all 21 S. cerevisiae strains that were screened in this study, along with their associated ATCC number, their origin of isolation, and a relevant reference if available. Code name  ATCC number  Isolation origin  Reference(s)  Jin 01 or J1  ATCC 2360  Kefir  Billen and Lichstein (1949: 215–21)  Jin 02 or J2  ATCC 4098  German white wine  Grewal and Miller (1972: 1897–905)  Jin 03 or J3  ATCC 4124  Molasses distillery  Ness et al. (1993: 89–94)  Jin 04 or J4  ATCC 4126  Amylo process  Bazua and Wilke (1977: 105–18)  Jin 05 or J5  ATCC 4127  American Concord grapes  N/A  Jin 06 or J6  ATCC 4921  French wine  N/A  Jin 08 or J8  ATCC 7754  Fleischmann bakers’ yeast  Snell, Eakin and Williams (1940: 175–8)  Jin 09 or J9  ATCC 9763  Distillery  Tseng and Phillips (1982: 1319–25)  Jin 11 or J11  ATCC 20598  N/A  Heady (1982)  Jin 12 or J12  ATCC 24855  Egyptian distillery  Ismail and Ali (1971a: 346–9) and Ismail and Ali (1971b: 350–4)  Jin 13 or J13  ATCC 24858  N/A  Ismail and Ali (1971a: 346–9) and Ismail and Ali (1971b: 350–4)  Jin 14 or J14  ATCC 24860  N/A  Jeppsson, Yu and Hahn-Hagerdal (1996: 1705–9)  Jin 15 or J15  ATCC 26422  Sake  Suizu et al. (1994: 119–24)  Jin 17 or J17  ATCC 46523  Baker's yeast  Middelhoven and Arkesteyn (1981: 121–31)  Jin 18 or J18  ATCC 56069  Fermented banana  N/A  Jin 19 or J19  ATCC 60222  Egyptian baker's yeast  N/A  Jin 20 or J20  ATCC 60223  Alsa Briochin bakers’ yeast  N/A  Jin 21 or J21  ATCC 60493  Canned strawberries  Put, Jong and Sand (1977)  Jin 24 or J24  ATCC 66348  Japanese soil  Tokuoka et al. (1985: 411–27)  Jin 25 or J25  ATCC 66349  Japanese soil  Tokuoka et al. (1985: 411–27)  Jin 26 or J26  ATCC 96581  Spent sulfite liquor fermentation  Linden, Peetre and Hahn-Hagerdal (1992: 1661–9)  Code name  ATCC number  Isolation origin  Reference(s)  Jin 01 or J1  ATCC 2360  Kefir  Billen and Lichstein (1949: 215–21)  Jin 02 or J2  ATCC 4098  German white wine  Grewal and Miller (1972: 1897–905)  Jin 03 or J3  ATCC 4124  Molasses distillery  Ness et al. (1993: 89–94)  Jin 04 or J4  ATCC 4126  Amylo process  Bazua and Wilke (1977: 105–18)  Jin 05 or J5  ATCC 4127  American Concord grapes  N/A  Jin 06 or J6  ATCC 4921  French wine  N/A  Jin 08 or J8  ATCC 7754  Fleischmann bakers’ yeast  Snell, Eakin and Williams (1940: 175–8)  Jin 09 or J9  ATCC 9763  Distillery  Tseng and Phillips (1982: 1319–25)  Jin 11 or J11  ATCC 20598  N/A  Heady (1982)  Jin 12 or J12  ATCC 24855  Egyptian distillery  Ismail and Ali (1971a: 346–9) and Ismail and Ali (1971b: 350–4)  Jin 13 or J13  ATCC 24858  N/A  Ismail and Ali (1971a: 346–9) and Ismail and Ali (1971b: 350–4)  Jin 14 or J14  ATCC 24860  N/A  Jeppsson, Yu and Hahn-Hagerdal (1996: 1705–9)  Jin 15 or J15  ATCC 26422  Sake  Suizu et al. (1994: 119–24)  Jin 17 or J17  ATCC 46523  Baker's yeast  Middelhoven and Arkesteyn (1981: 121–31)  Jin 18 or J18  ATCC 56069  Fermented banana  N/A  Jin 19 or J19  ATCC 60222  Egyptian baker's yeast  N/A  Jin 20 or J20  ATCC 60223  Alsa Briochin bakers’ yeast  N/A  Jin 21 or J21  ATCC 60493  Canned strawberries  Put, Jong and Sand (1977)  Jin 24 or J24  ATCC 66348  Japanese soil  Tokuoka et al. (1985: 411–27)  Jin 25 or J25  ATCC 66349  Japanese soil  Tokuoka et al. (1985: 411–27)  Jin 26 or J26  ATCC 96581  Spent sulfite liquor fermentation  Linden, Peetre and Hahn-Hagerdal (1992: 1661–9)  View Large Table 1. Listing of all 21 S. cerevisiae strains that were screened in this study, along with their associated ATCC number, their origin of isolation, and a relevant reference if available. Code name  ATCC number  Isolation origin  Reference(s)  Jin 01 or J1  ATCC 2360  Kefir  Billen and Lichstein (1949: 215–21)  Jin 02 or J2  ATCC 4098  German white wine  Grewal and Miller (1972: 1897–905)  Jin 03 or J3  ATCC 4124  Molasses distillery  Ness et al. (1993: 89–94)  Jin 04 or J4  ATCC 4126  Amylo process  Bazua and Wilke (1977: 105–18)  Jin 05 or J5  ATCC 4127  American Concord grapes  N/A  Jin 06 or J6  ATCC 4921  French wine  N/A  Jin 08 or J8  ATCC 7754  Fleischmann bakers’ yeast  Snell, Eakin and Williams (1940: 175–8)  Jin 09 or J9  ATCC 9763  Distillery  Tseng and Phillips (1982: 1319–25)  Jin 11 or J11  ATCC 20598  N/A  Heady (1982)  Jin 12 or J12  ATCC 24855  Egyptian distillery  Ismail and Ali (1971a: 346–9) and Ismail and Ali (1971b: 350–4)  Jin 13 or J13  ATCC 24858  N/A  Ismail and Ali (1971a: 346–9) and Ismail and Ali (1971b: 350–4)  Jin 14 or J14  ATCC 24860  N/A  Jeppsson, Yu and Hahn-Hagerdal (1996: 1705–9)  Jin 15 or J15  ATCC 26422  Sake  Suizu et al. (1994: 119–24)  Jin 17 or J17  ATCC 46523  Baker's yeast  Middelhoven and Arkesteyn (1981: 121–31)  Jin 18 or J18  ATCC 56069  Fermented banana  N/A  Jin 19 or J19  ATCC 60222  Egyptian baker's yeast  N/A  Jin 20 or J20  ATCC 60223  Alsa Briochin bakers’ yeast  N/A  Jin 21 or J21  ATCC 60493  Canned strawberries  Put, Jong and Sand (1977)  Jin 24 or J24  ATCC 66348  Japanese soil  Tokuoka et al. (1985: 411–27)  Jin 25 or J25  ATCC 66349  Japanese soil  Tokuoka et al. (1985: 411–27)  Jin 26 or J26  ATCC 96581  Spent sulfite liquor fermentation  Linden, Peetre and Hahn-Hagerdal (1992: 1661–9)  Code name  ATCC number  Isolation origin  Reference(s)  Jin 01 or J1  ATCC 2360  Kefir  Billen and Lichstein (1949: 215–21)  Jin 02 or J2  ATCC 4098  German white wine  Grewal and Miller (1972: 1897–905)  Jin 03 or J3  ATCC 4124  Molasses distillery  Ness et al. (1993: 89–94)  Jin 04 or J4  ATCC 4126  Amylo process  Bazua and Wilke (1977: 105–18)  Jin 05 or J5  ATCC 4127  American Concord grapes  N/A  Jin 06 or J6  ATCC 4921  French wine  N/A  Jin 08 or J8  ATCC 7754  Fleischmann bakers’ yeast  Snell, Eakin and Williams (1940: 175–8)  Jin 09 or J9  ATCC 9763  Distillery  Tseng and Phillips (1982: 1319–25)  Jin 11 or J11  ATCC 20598  N/A  Heady (1982)  Jin 12 or J12  ATCC 24855  Egyptian distillery  Ismail and Ali (1971a: 346–9) and Ismail and Ali (1971b: 350–4)  Jin 13 or J13  ATCC 24858  N/A  Ismail and Ali (1971a: 346–9) and Ismail and Ali (1971b: 350–4)  Jin 14 or J14  ATCC 24860  N/A  Jeppsson, Yu and Hahn-Hagerdal (1996: 1705–9)  Jin 15 or J15  ATCC 26422  Sake  Suizu et al. (1994: 119–24)  Jin 17 or J17  ATCC 46523  Baker's yeast  Middelhoven and Arkesteyn (1981: 121–31)  Jin 18 or J18  ATCC 56069  Fermented banana  N/A  Jin 19 or J19  ATCC 60222  Egyptian baker's yeast  N/A  Jin 20 or J20  ATCC 60223  Alsa Briochin bakers’ yeast  N/A  Jin 21 or J21  ATCC 60493  Canned strawberries  Put, Jong and Sand (1977)  Jin 24 or J24  ATCC 66348  Japanese soil  Tokuoka et al. (1985: 411–27)  Jin 25 or J25  ATCC 66349  Japanese soil  Tokuoka et al. (1985: 411–27)  Jin 26 or J26  ATCC 96581  Spent sulfite liquor fermentation  Linden, Peetre and Hahn-Hagerdal (1992: 1661–9)  View Large General flask fermentations Yeast cells were cultured in YP medium (10 g/L yeast extract and 20 g/L peptone) containing glucose (YPD), xylose (YPX), cellobiose (YPC), maltose, mannose or sucrose. Concentrations of the sugars were displayed as numbers following their initials (e.g. YPD160 refers to YP medium containing 160 g/L of glucose). Stock cultures were maintained on YPD20 agar (YP medium containing 20 g/L of glucose) plates at 4°C. Yeast precultures were grown in 5 mL YPD20 and harvested at the mid-exponential phase. Fermentations were conducted with an initial cell optical density (OD600) of ∼ 1.0 and at a volume of 50 mL in 250 mL Erlenmeyer Pyrex® flasks (Corning, MA). Flasks were shaken at 100 RPM on an Innova 2300 shaker (New Brunswick Scientific, CT) in a 30°C incubation room. Optical density was measured via NanoDrop 2000C (Thermo Fisher Scientific, MA) or BioMate 3 UV-visible spectrophotometer (Thermo Fisher Scientific, MA). Metabolite concentrations such as glucose, xylose, cellobiose, xylitol, glycerol, acetate and ethanol were monitored by a 1200 Infinity series HPLC system (Agilent Technologies, CA) equipped with a refractive index detector using a Rezex ROA-Organic Acid H+ (8%) column (Phenomenex Inc., CA). The column was eluted with 0.005 N H2SO4 at a flow rate of 0.6 mL/min at 50°C. The construction and evaluation of xylose-fermenting strains The pSR6-X123 plasmid (Kim et al.2012: 336–43) containing Scheffersomyces stipitis XYL1, XYL2 and XYL3 genes was linearized by XcmI digestion and introduced into the URA3 locus of all industrial yeast strains to allow for xylose utilization. A standard high-efficiency lithium acetate transformation method was used for the chromosomal introduction of the linearized plasmid (Daniel Gietz and Woods 2002: 87–96). The colonies with xylose assimilation pathway were selected on YP medium with 40 g/L xylose (YPX40) agar plates. Eight successful transformants of each strain were evaluated in 5 mL culture of YPX40 at 30°C. The transformant from each strain with the highest xylose uptake rate and ethanol productivity was selected for further study. Comparisons between selected transformants of each strain were conducted in 50 mL YPX40 following the protocol as listed in the ‘general flask fermentations’ sub-section, with the exception that xylose was used as the carbon source in place of glucose. The construction and evaluation of cellobiose-fermenting strains The pRS425-BTT plasmid (Ha et al.2013: 525–31) containing Neurospora crassa β-glucosidase (gh1-1) and cellodextrin transporter (cdt-1) genes was linearized and introduced into all industrial yeast strains to allow for cellobiose utilization. Several transformants of each strain were screened in 5 mL of YP medium containing 40 g/L of cellobiose (YPC40) at 30°C. The transformant from each strain with the highest cellobiose uptake rate and ethanol productivity was selected for further study. Comparisons between selected transformants of each strain were conducted in 50 mL YPC40, following the protocol as listed in the ‘general flask fermentations’ sub-section, with the exception that cellobiose was used as the carbon source in place of glucose. Gas pressure analysis Strains were evaluated for their gas pressure production in sealed glass bottles, with a higher gas pressure indicative of higher CO2 production and in-turn higher ethanol production. Jars with a maximum volume of 100 mL were filled with 20 mL of either YP medium containing 20 g/L of glucose, YP medium containing 20 g/L of glucose and 25% hydrolysate or Verduyn's (Verduyn et al.1992: 501–17) medium with 20 g/L of glucose and 0.628 g/L of complete supplement mixture (MP Biomedicals). The initial pH value of each medium was adjusted to 6 with concentrated NaOH or HCl, as needed. The fermentation bottles were shaken at 100 RPM on an Innova 2300 shaker (New Brunswick Scientific, CT) in a 30°C incubation room. Cell optical density (OD600) was measured as discussed in the ‘general flask fermentations’ sub-section, and the initial OD600 was ∼1.0. gas pressure was monitored with RF gas production modules (ANKOM Technology, NY) as pounds per square inch every 5 min. Growth assays under various pH levels OD600 was monitored for determining the cell growth in YP medium containing 20 g/L of glucose with an initial pH value adjusted to 4.5, 5.0, 5.5, 6.0 or 6.5. A 200 μL initial volume of medium was placed into individual wells of Costar 96-well flat-bottom polystyrene plates with lids placed on top (Corning, MA). The initial OD600 was 1.0, and microplates were incubated at 30°C and 200 RPM in a Symphony incubating microplate shaker (VWR, PA). An aliquot of 50 μL of mineral oil was placed on top of each well to prevent media evaporation. Wells containing media with mineral oil, but without the addition of yeast, were used as negative controls and as a blank by which the negative control OD was subtracted from the OD of experimental wells to account for any increased absorbance due to the oil. A Synergy HT plate reader (Biotek, Winooski, VT) was used to measure the OD600. Tolerance assays Precultures of S. cerevisiae strains were grown at 30°C in YPD20. Cells were harvested at the mid-exponential phase and adjusted to an initial OD600 of 1.0 for further use. Ten-fold serial dilutions were made in water, 10 μL of the 10−1, 10−2, 10−3, 10−4, 10−5 and 10−6 dilutions were spotted on YPD20 agar plates. The agar plates included the following: YPD20 plates incubated at 30°C, 37°C, 42°C and 45°C for heat tolerance; YPD20 plates with inhibitors, 2 g/L of HMF, 1 g/L of furfural or 20% hydrolysates at 30°C for inhibitor tolerance; and YPD20 medium containing 1 g/L, 2 g/L or 3 g/L of acetate at 30°C for organic acid tolerance. Visible-light pictures of the spotting assay were taken at 48 h. Top-performing strains were determined based on the number of colonies formed compared to the ATCC 4124 (Jin 03) control strain at equivalent dilutions. Relative genome content and ploidy analysis by flow cytometry Precultures of S. cerevisiae strains were grown at 30°C in YP medium containing 20 g/L of glucose (YPD20) and harvested at mid-exponential phase. Cells were collected once they reached OD600 ∼1.0. Samples were then prepared as previously described (Haase 2004: Unit 7.23) for flow cytometry. Briefly, cells were first fixed and permeabilized with cold 70% ethanol, and then washed with sodium citrate containing RNaseA. SYTOX® Green dye (Life Technologies) was used to stain the nucleic acid. The DNA contents of the stained cells were analyzed and detected by the excitation and emission spectra of the SYTOX® Green/DNA complex using an LSR II Flow Cytometry Analyzer (BD Biosciences, CA). The absolute ploidy of the industrial strains was estimated by comparing its DNA content to the control strains, the known haploid, diploid, triploid and tetraploid laboratory strains, L6437, L6438, L6439 and L6440, respectively, from Gerald Fink's laboratory (Galitski et al.1999: 251–4). The relative DNA content was calculated based on the average intensity of SYTOX® Green dye from the mid-log phase cells. From the two peaks of flow cytometry data (Fig. S1, Supporting Information), the first peak was used for the calculation, because the average intensity values of the first peak were correlated with the ploidy. An example of a comparison of ATCC 4124 with other control strains that are known for haploid, diploid, triploid and tetraploid was shown. In addition, the ploidy can be confirmed and correlates well with the genome sequencing data from previous studies of ATCC 4124 (Zhang et al.2014: 7694–701; Kim et al.2017: 176–85). Mating-type test A halo assay based on yeast pheromone response was used to determine strain mating-type (Meissner et al.2010: 2425–33). Mating type tester strains (DBY7730 for MATα and DBY7442 for MATa) (Julius et al.1983: 839–52) were first spread on YPD 20 g/L agar plates. Each industrial strain was then spotted on the lawn of each tester strain, and the plates were incubated overnight. The presence of a halo around a strain spot was used to score its mating type. Sporulation efficiency determination A single colony of each strain was obtained from YPD20 plates. The colonies were then cultured in the sporulation medium (1% potassium acetate, 0.1% yeast extract, 0.05% glucose and 2% agar). Cell division and sporulation occurred within 3–7 days and their sporulation efficiency were evaluated by the population of the tetrads. Heat map A heat map was used to summarize the performances of a specific test. No growth was scaled at −3. Any performance that was 10% lower than the ATCC 4124 control strain was scaled at −2. Performance from 5% to 10% lower was scaled at −1, performance within 5% lower or higher was scaled at 0, performance 5% higher was scaled +1, and performance greater than 10% higher was scaled at +2. RESULTS Phenotypic characterization of the industrial strains under common, industrially relevant stressors Osmotic stress is one of the common environmental stressors that yeasts encounter in the industrial-scale fermentation environment. Therefore, 20 industrial strains were selected and compared with a laboratory strain (D452-2) and an industrial strain (ATCC 4124) due to its well-known characteristic for lignocellulosic fermentation (Ho, Chen and Brainard 1998: 1852–9; Casey et al.2010: 385–93; Bera et al.2011: 617–26). They were evaluated for their tolerance against a high ethanol concentration from the high glucose fermentation (160 g/L), which is comparable to the total sugar content that would be present in sugar cane juice and plant hydrolysates (Soccol et al.2010: 4820–5). As expected at 12 h, all industrial strains outperformed the laboratory strain, D452-2. Other industrial strains consumed glucose rapidly and performed similarly to ATCC 4124. When comparing the ethanol productivity and ethanol yield, ATCC 4098 (Jin 02), ATCC 56069 (Jin 18), ATCC 60493 (Jin 21) and ATCC 66348 (Jin 24) showed a better ethanol productivity and ethanol yield as compared with ATCC 4124 (Jin 03) (Fig. 1; Table 2). Table 2. Fermentation data in YP medium with 160 g/L of glucose. The produced ethanol (g/L), the ethanol yield (g ethanol/g glucose consumed), and the ethanol productivity (g/L/h) at 12 h were listed in this table. Code name  Strains  Ethanol produced (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J1  ATCC 2360  53.606  0.431  4.467  J2  ATCC 4098  58.452  0.423  4.871  J3  ATCC 4124  55.746  0.400  4.645  J4  ATCC 4126  50.107  0.418  4.176  J5  ATCC 4127  57.015  0.410  4.751  J6  ATCC 4921  51.870  0.421  4.323  J8  ATCC 7754  57.645  0.427  4.804  J9  ATCC 9763  50.468  0.418  4.206  J11  ATCC 20598  48.097  0.418  4.008  J12  ATCC 24855  47.401  0.406  3.950  J13  ATCC 24858  48.647  0.400  4.054  J14  ATCC 24860  50.576  0.407  4.215  J15  ATCC 26422  43.300  0.404  3.608  J17  ATCC 46523  46.969  0.419  3.914  J18  ATCC 56069  62.944  0.423  5.245  J19  ATCC 60222  57.956  0.413  4.830  J20  ATCC 60223  50.250  0.408  4.188  J21  ATCC 60493  59.631  0.407  4.969  J24  ATCC 66348  62.655  0.414  5.221  J25  ATCC 66349  57.541  0.408  4.795  J26  ATCC 96581  43.154  0.416  3.596    D452-2  29.811  0.415  2.484  Code name  Strains  Ethanol produced (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J1  ATCC 2360  53.606  0.431  4.467  J2  ATCC 4098  58.452  0.423  4.871  J3  ATCC 4124  55.746  0.400  4.645  J4  ATCC 4126  50.107  0.418  4.176  J5  ATCC 4127  57.015  0.410  4.751  J6  ATCC 4921  51.870  0.421  4.323  J8  ATCC 7754  57.645  0.427  4.804  J9  ATCC 9763  50.468  0.418  4.206  J11  ATCC 20598  48.097  0.418  4.008  J12  ATCC 24855  47.401  0.406  3.950  J13  ATCC 24858  48.647  0.400  4.054  J14  ATCC 24860  50.576  0.407  4.215  J15  ATCC 26422  43.300  0.404  3.608  J17  ATCC 46523  46.969  0.419  3.914  J18  ATCC 56069  62.944  0.423  5.245  J19  ATCC 60222  57.956  0.413  4.830  J20  ATCC 60223  50.250  0.408  4.188  J21  ATCC 60493  59.631  0.407  4.969  J24  ATCC 66348  62.655  0.414  5.221  J25  ATCC 66349  57.541  0.408  4.795  J26  ATCC 96581  43.154  0.416  3.596    D452-2  29.811  0.415  2.484  View Large Table 2. Fermentation data in YP medium with 160 g/L of glucose. The produced ethanol (g/L), the ethanol yield (g ethanol/g glucose consumed), and the ethanol productivity (g/L/h) at 12 h were listed in this table. Code name  Strains  Ethanol produced (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J1  ATCC 2360  53.606  0.431  4.467  J2  ATCC 4098  58.452  0.423  4.871  J3  ATCC 4124  55.746  0.400  4.645  J4  ATCC 4126  50.107  0.418  4.176  J5  ATCC 4127  57.015  0.410  4.751  J6  ATCC 4921  51.870  0.421  4.323  J8  ATCC 7754  57.645  0.427  4.804  J9  ATCC 9763  50.468  0.418  4.206  J11  ATCC 20598  48.097  0.418  4.008  J12  ATCC 24855  47.401  0.406  3.950  J13  ATCC 24858  48.647  0.400  4.054  J14  ATCC 24860  50.576  0.407  4.215  J15  ATCC 26422  43.300  0.404  3.608  J17  ATCC 46523  46.969  0.419  3.914  J18  ATCC 56069  62.944  0.423  5.245  J19  ATCC 60222  57.956  0.413  4.830  J20  ATCC 60223  50.250  0.408  4.188  J21  ATCC 60493  59.631  0.407  4.969  J24  ATCC 66348  62.655  0.414  5.221  J25  ATCC 66349  57.541  0.408  4.795  J26  ATCC 96581  43.154  0.416  3.596    D452-2  29.811  0.415  2.484  Code name  Strains  Ethanol produced (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J1  ATCC 2360  53.606  0.431  4.467  J2  ATCC 4098  58.452  0.423  4.871  J3  ATCC 4124  55.746  0.400  4.645  J4  ATCC 4126  50.107  0.418  4.176  J5  ATCC 4127  57.015  0.410  4.751  J6  ATCC 4921  51.870  0.421  4.323  J8  ATCC 7754  57.645  0.427  4.804  J9  ATCC 9763  50.468  0.418  4.206  J11  ATCC 20598  48.097  0.418  4.008  J12  ATCC 24855  47.401  0.406  3.950  J13  ATCC 24858  48.647  0.400  4.054  J14  ATCC 24860  50.576  0.407  4.215  J15  ATCC 26422  43.300  0.404  3.608  J17  ATCC 46523  46.969  0.419  3.914  J18  ATCC 56069  62.944  0.423  5.245  J19  ATCC 60222  57.956  0.413  4.830  J20  ATCC 60223  50.250  0.408  4.188  J21  ATCC 60493  59.631  0.407  4.969  J24  ATCC 66348  62.655  0.414  5.221  J25  ATCC 66349  57.541  0.408  4.795  J26  ATCC 96581  43.154  0.416  3.596    D452-2  29.811  0.415  2.484  View Large Fermentation inhibitors and acetic acid are commonly found in lignocellulosic hydrolysates (Jonsson, Alriksson and Nilvebrant 2013: 16), and they limit the cell growth and viability. Therefore, growth assays on agar plates with the supplementation of fermentation inhibitors, acetic acid or lignocellulosic hydrolysates were used to test the tolerance against fermentation inhibitors. Also, agar plates were incubated at several temperatures (30°C, 37°C, 42°C and 45°C) to test their heat tolerance (Fig. 3), 42°C and 45°C not shown. High temperature is one of the potential stressors in an industrial-scale environment. The result shows ATCC 7754 (Jin 08), ATCC 9763 (Jin 09), ATCC 24858 (Jin 13), ATCC 46523 (Jin 17), ATCC 66348 (Jin 24) and ATCC 66349 (Jin 25) possess slightly better heat tolerance as compared with ATCC 4124. However, none of the strains could grow at 42°C or 45°C (data not shown). For the acetic acid tolerance, ATCC 9763 (Jin 09), ATCC 24858 (Jin 13), ATCC JIN 46523 (Jin 17) and ATCC 66348 (Jin 24) showed better tolerance against the organic acid when compared with ATCC 4124 (Fig. 5). Lastly, ATCC 4098 (Jin 02), ATCC 4127 (Jin 05), ATCC 9763 (Jin 09), ATCC 66348 (Jin 24) and ATCC 96581 (Jin 26) showed a greater tolerance against hydrolysates and/or specific fermentation inhibitors among all the strains (Figs 1 and 4). Figure 1. View largeDownload slide A heat map indicating the relative performance of our industrial strain screening under various conditions. ATCC 4124 is the control strain, and the z-score was scaled at 0. Performance for a specific test that was 10% lower than the ATCC 4124 control strain was scaled at −2. Performance 5% to 10% lower was scaled at −1, performance within 5% lower or 5% higher was scaled at 0, performance 5% higher was scaled +1, and performance greater than 10% higher was scaled at +2. No growth was scaled at −3 (gray color). Abbreviations: Glu, Glucose; YPD20, YP medium with 20g/L glucose; HMF, hydroxymethylfurfural; Xyl, xylose; Cel, cellobiose; GA, gas analysis; CSM, complete supplement mixture. Figure 1. View largeDownload slide A heat map indicating the relative performance of our industrial strain screening under various conditions. ATCC 4124 is the control strain, and the z-score was scaled at 0. Performance for a specific test that was 10% lower than the ATCC 4124 control strain was scaled at −2. Performance 5% to 10% lower was scaled at −1, performance within 5% lower or 5% higher was scaled at 0, performance 5% higher was scaled +1, and performance greater than 10% higher was scaled at +2. No growth was scaled at −3 (gray color). Abbreviations: Glu, Glucose; YPD20, YP medium with 20g/L glucose; HMF, hydroxymethylfurfural; Xyl, xylose; Cel, cellobiose; GA, gas analysis; CSM, complete supplement mixture. A low-pH resistant yeast strain is beneficial for an industrial setting because low pH combined with organic acid will severely inhibit the growth and metabolism of most yeast. Therefore, the performance of the industrial strains was also tested under various pH values. The results showed that ATCC 4127 (Jin 05), ATCC 9763 (Jin 09), ATCC 46523 (Jin 17), ATCC 56069 (Jin 18) and ATCC 66348 (Jin 24) have a higher tolerance than other industrial strains and ATCC 4124 (Jin 03) (Fig. 1). Combining all findings, ATCC 9763 (Jin 09), ATCC 46523 (Jin 17) and ATCC 66348 (Jin 24) could be preferential strains for industrial lignocellulosic hydrolysate fermentations. Determination of the ploidy and the sporulation efficiency of industrial yeast strains It is challenging to introduce genetic perturbations into industrial yeasts due in part to their complex genome structure, such as aneuploidy, polyploidy or another chromosomal rearrangement (Argueso et al.2009: 2258–70; Akao et al.2011: 423–34; Borneman et al.2011: e1001287; Babrzadeh et al.2012: 485–94; Borneman et al.2012: 88–96; Dunn et al.2012: 908–24). It could be easier to manipulate a haploid strain than a diploid or even a triploid strain. Therefore, the ploidy of the industrial strains was estimated by the relative DNA content measured by flow cytometry, and the selected haploid was confirmed with a mating type test. Among all the industrial strains (Fig. 2), ATCC 60222 (Jin 19) and ATCC 60223 (Jin 20) have the highest relative DNA content, which correlates with the higher number of ploidy. ATCC 20598 (Jin 11) was believed to be a haploid with MATa, after the mating type test confirmation, and a lower relative DNA content was measured. Therefore, it would be easier to choose ATCC 20598 (Jin 11) for further genetic manipulation. Previous results (Fig. 1) suggested ATCC 9763 (Jin 09) and ATCC 46523 (Jin 17) as good candidates for lignocellulosic fermentation, but their ploidy is believed to be diploid or triploid based on the relative DNA content, causing the strains to be more difficult to undergo directed genetic manipulation compared to haploid strains. However, ATCC 66348 (Jin 24) is also a good candidate for lignocellulosic fermentations (Fig. 1), and its relative DNA content indicates that it may be a haploid strain that would make it more ammenable to genetic manipulation than diploid or triploid strains. Figure 2. View largeDownload slide The relative DNA content of industrial strains was measured by flow cytometry. Strains with known ploidy were used as a control and colored in black. Relative DNA content correlating to approximately 1N to 2N, 2N to 3N, and 3N to 4N is shown in red, blue, or pink respectively. Figure 2. View largeDownload slide The relative DNA content of industrial strains was measured by flow cytometry. Strains with known ploidy were used as a control and colored in black. Relative DNA content correlating to approximately 1N to 2N, 2N to 3N, and 3N to 4N is shown in red, blue, or pink respectively. Figure 3. View largeDownload slide Temperature tolerance from the spotting assay on YPD medium incubated at 30°C or 37°C. Red bars indicate strains that showed the consistently best growth in the highlighted condition. Red numbers across the top indicate strain number, e.g. 1 is J1, 2 is J2 and so forth. Figure 3. View largeDownload slide Temperature tolerance from the spotting assay on YPD medium incubated at 30°C or 37°C. Red bars indicate strains that showed the consistently best growth in the highlighted condition. Red numbers across the top indicate strain number, e.g. 1 is J1, 2 is J2 and so forth. Figure 4. View largeDownload slide Inhibitor tolerance from the spotting assay on YPD medium containing 2 g/L HMF, 1 g/L furfural, or 20% hydrolysate. Red bars indicate strains which showed the consistently best growth in the highlighted condition. Red numbers across the top indicate strain number, e.g. 1 is J1, 2 is J2 and so forth. Figure 4. View largeDownload slide Inhibitor tolerance from the spotting assay on YPD medium containing 2 g/L HMF, 1 g/L furfural, or 20% hydrolysate. Red bars indicate strains which showed the consistently best growth in the highlighted condition. Red numbers across the top indicate strain number, e.g. 1 is J1, 2 is J2 and so forth. Figure 5. View largeDownload slide Acetic acid tolerance from spotting assay on YPD medium containing 1, 2 or 3 g/L acetate. Red bars indicate strains which showed the consistently best growth in the highlighted condition. Red numbers across the top indicate strain number, e.g. 1 is J1, 2 is J2 and so forth. Figure 5. View largeDownload slide Acetic acid tolerance from spotting assay on YPD medium containing 1, 2 or 3 g/L acetate. Red bars indicate strains which showed the consistently best growth in the highlighted condition. Red numbers across the top indicate strain number, e.g. 1 is J1, 2 is J2 and so forth. Mating, sporulation and isolation of haploids are one of the alternative ways to modify yeasts to achieve certain desired phenotypes. The anticipated phenotypes of polyploidy industrial strains could be hybridized with another industrial strain once a pool of spores (haploid) was generated after sporulation (Fukuda et al.2016: 45). Crossbreeding could be done by mating the spores (haploid) with the opposite mating type. Sporulation efficiency is used to measure the ratio of tetrads produced by the strain. It is the first step to evaluate the feasibility of a strain for mating experiments and to measure the ratio of tetrads produced by strain. When the cells are cultivated in sporulation medium with limiting nutrients for 5–7 days, yeast cells will start to sporulate as a response to nutrient deprivation and stress. Among all the industrial strains, ATCC 9763 (Jin 09), ATCC 46523 (Jin 17) and ATCC 6022 (Jin 20) had the highest sporulation efficiency (Table 3), and these three strains could be used for mating and crossbreeding experiments. However, the ATCC 66348 (Jin 24) has lower sporulation efficiency, leading to the complication of manipulating this strain. Lastly, ATCC 9763 (Jin 09) and ATCC 46523 (Jin 17) could be considered as the next preferable host strains for further strain engineering due to their high sporulation efficiency and reduced ploidy in the genome. Table 3. Sporulation efficiency of each industrial strain; ATCC 20598 was unable to be induced to sporulate;—indicates no sporulation was detected,+ indicates ∼25% sporulation efficiency, ++ indicates ∼50% sporulation efficiency, and +++ indicates ∼75% or greater sporulation efficiency. Code name  Strains  Sporulation efficiency  J1  ATCC 2360  +  J2  ATCC 4098  +  J3  ATCC 4124  +  J4  ATCC 4126  ++  J5  ATCC 4127  +  J6  ATCC 4921  +  J8  ATCC 7754  ++  J9  ATCC 9763  +++  J11  ATCC 20598  −  J12  ATCC 24855  ++  J13  ATCC 24858  ++  J14  ATCC 24860  +  J15  ATCC 26422  +  J17  ATCC 46523  +++  J18  ATCC 56069  +  J19  ATCC 60222  +++  J20  ATCC 60223  +++  J21  ATCC 60493  +  J24  ATCC 66348  +  J25  ATCC 66349  ++  J26  ATCC 96581  +  Code name  Strains  Sporulation efficiency  J1  ATCC 2360  +  J2  ATCC 4098  +  J3  ATCC 4124  +  J4  ATCC 4126  ++  J5  ATCC 4127  +  J6  ATCC 4921  +  J8  ATCC 7754  ++  J9  ATCC 9763  +++  J11  ATCC 20598  −  J12  ATCC 24855  ++  J13  ATCC 24858  ++  J14  ATCC 24860  +  J15  ATCC 26422  +  J17  ATCC 46523  +++  J18  ATCC 56069  +  J19  ATCC 60222  +++  J20  ATCC 60223  +++  J21  ATCC 60493  +  J24  ATCC 66348  +  J25  ATCC 66349  ++  J26  ATCC 96581  +  View Large Table 3. Sporulation efficiency of each industrial strain; ATCC 20598 was unable to be induced to sporulate;—indicates no sporulation was detected,+ indicates ∼25% sporulation efficiency, ++ indicates ∼50% sporulation efficiency, and +++ indicates ∼75% or greater sporulation efficiency. Code name  Strains  Sporulation efficiency  J1  ATCC 2360  +  J2  ATCC 4098  +  J3  ATCC 4124  +  J4  ATCC 4126  ++  J5  ATCC 4127  +  J6  ATCC 4921  +  J8  ATCC 7754  ++  J9  ATCC 9763  +++  J11  ATCC 20598  −  J12  ATCC 24855  ++  J13  ATCC 24858  ++  J14  ATCC 24860  +  J15  ATCC 26422  +  J17  ATCC 46523  +++  J18  ATCC 56069  +  J19  ATCC 60222  +++  J20  ATCC 60223  +++  J21  ATCC 60493  +  J24  ATCC 66348  +  J25  ATCC 66349  ++  J26  ATCC 96581  +  Code name  Strains  Sporulation efficiency  J1  ATCC 2360  +  J2  ATCC 4098  +  J3  ATCC 4124  +  J4  ATCC 4126  ++  J5  ATCC 4127  +  J6  ATCC 4921  +  J8  ATCC 7754  ++  J9  ATCC 9763  +++  J11  ATCC 20598  −  J12  ATCC 24855  ++  J13  ATCC 24858  ++  J14  ATCC 24860  +  J15  ATCC 26422  +  J17  ATCC 46523  +++  J18  ATCC 56069  +  J19  ATCC 60222  +++  J20  ATCC 60223  +++  J21  ATCC 60493  +  J24  ATCC 66348  +  J25  ATCC 66349  ++  J26  ATCC 96581  +  View Large Evaluation of the newly constructed, xylose-fermenting industrial yeast strains Because xylose is the second-most abundant sugar in lignocellulosic hydrolysates, it is important to have an efficient xylose-fermenting strain for an efficient industrial fermentation. To examine the effect of different strain backgrounds on the efficiency of xylose fermentation, all the strains were engineered to express a xylose fermentation pathway (xylose reductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XK) encoded by XYL1, XYL2 and XYL3, respectively), because S. cerevisiae cannot natively metabolize xylose. ATCC 4124 (Jin 03) was known to have an efficient xylose fermentation after strain engineering from previous studies (Ho et al.1998: 1852–9; Casey et al.2010: 385–93; Bera et al.2011: 617–26). Therefore, the industrial strains constructed with the xylose pathway were compared with ATCC 4124 (Jin 03) with the xylose pathway. At least eight transformants for each ATCC strain were selected and screened in 5 mL YPX40. The final list of the best xylose-fermenting strains was summarized in Fig. 1 and Table 4. All the transformants were labeled, such as (J3-02), referring to the #2 colony of the ATCC 4124 (Jin 03) strain, (J18-01) referring to the #1 colony of the ATCC 56069 (Jin 18) strain and so forth. Overall, only the transformants from ATCC 4127 (J5-08), ATCC 46523 (J17-01) and ATCC 66348 (J24-11) were found to consume xylose and produce ethanol more efficiently than the best transformant from ATCC 4124 (J3-02) at 72 h. Also, ATCC 20598 (Jin 11) and ATCC 96581 (Jin 26) failed to assimilate xylose. Even though ATCC 20598 (Jin 11) is a haploid and presumably more amenable to manipulation, it failed to ferment xylose, possibly due to the lack of strain fitness or other complex genetic problems. Combined with the previous results, ATCC 66348 (Jin 24) consistently exhibited the most desirable phenotypes for lignocellulosic hydrolysates fermentations, which included the tolerances against fermentation inhibitors and higher xylose assimilation rate. However, it may be challenging to work with ATCC 66348 (Jin 24) due to its potential diploid nature (Fig. 2) and low sporulation efficiency (Table 3). Table 4. Fermentation data of the selected xylose-fermenting transformants from the industrial strains grown in YPX40 medium and measured at 72 h into the fermentations. J3-02 refers to the #2 colony of Jin 03 (ATCC 4124) strain; J18-01 refers to the #1 colony of Jin 18 (ATCC 56069) strain, and so forth. Code name  Strains  Xylose consumption rate (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J2-08  ATCC 4098  0.489  0.200  0.098  J3-02  ATCC 4124  0.533  0.305  0.163  J3-07  ATCC 4124  0.509  0.244  0.124  J4-01  ATCC 4126  0.493  0.217  0.107  J5-01  ATCC 4127  0.494  0.238  0.118  J5-03  ATCC 4127  0.509  0.221  0.112  J5-06  ATCC 4127  0.511  0.214  0.109  J5-07  ATCC 4127  0.503  0.192  0.096  J5-08  ATCC 4127  0.544  0.181  0.098  J17-01  ATCC 46523  0.553  0.247  0.137  J18-01  ATCC 56069  0.526  0.224  0.118  J24-05  ATCC 66348  0.475  0.197  0.094  J24-11  ATCC 66348  0.535  0.246  0.132  J24-12  ATCC 66348  0.515  0.211  0.108  J24-15  ATCC 66348  0.514  0.211  0.108  Code name  Strains  Xylose consumption rate (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J2-08  ATCC 4098  0.489  0.200  0.098  J3-02  ATCC 4124  0.533  0.305  0.163  J3-07  ATCC 4124  0.509  0.244  0.124  J4-01  ATCC 4126  0.493  0.217  0.107  J5-01  ATCC 4127  0.494  0.238  0.118  J5-03  ATCC 4127  0.509  0.221  0.112  J5-06  ATCC 4127  0.511  0.214  0.109  J5-07  ATCC 4127  0.503  0.192  0.096  J5-08  ATCC 4127  0.544  0.181  0.098  J17-01  ATCC 46523  0.553  0.247  0.137  J18-01  ATCC 56069  0.526  0.224  0.118  J24-05  ATCC 66348  0.475  0.197  0.094  J24-11  ATCC 66348  0.535  0.246  0.132  J24-12  ATCC 66348  0.515  0.211  0.108  J24-15  ATCC 66348  0.514  0.211  0.108  View Large Table 4. Fermentation data of the selected xylose-fermenting transformants from the industrial strains grown in YPX40 medium and measured at 72 h into the fermentations. J3-02 refers to the #2 colony of Jin 03 (ATCC 4124) strain; J18-01 refers to the #1 colony of Jin 18 (ATCC 56069) strain, and so forth. Code name  Strains  Xylose consumption rate (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J2-08  ATCC 4098  0.489  0.200  0.098  J3-02  ATCC 4124  0.533  0.305  0.163  J3-07  ATCC 4124  0.509  0.244  0.124  J4-01  ATCC 4126  0.493  0.217  0.107  J5-01  ATCC 4127  0.494  0.238  0.118  J5-03  ATCC 4127  0.509  0.221  0.112  J5-06  ATCC 4127  0.511  0.214  0.109  J5-07  ATCC 4127  0.503  0.192  0.096  J5-08  ATCC 4127  0.544  0.181  0.098  J17-01  ATCC 46523  0.553  0.247  0.137  J18-01  ATCC 56069  0.526  0.224  0.118  J24-05  ATCC 66348  0.475  0.197  0.094  J24-11  ATCC 66348  0.535  0.246  0.132  J24-12  ATCC 66348  0.515  0.211  0.108  J24-15  ATCC 66348  0.514  0.211  0.108  Code name  Strains  Xylose consumption rate (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J2-08  ATCC 4098  0.489  0.200  0.098  J3-02  ATCC 4124  0.533  0.305  0.163  J3-07  ATCC 4124  0.509  0.244  0.124  J4-01  ATCC 4126  0.493  0.217  0.107  J5-01  ATCC 4127  0.494  0.238  0.118  J5-03  ATCC 4127  0.509  0.221  0.112  J5-06  ATCC 4127  0.511  0.214  0.109  J5-07  ATCC 4127  0.503  0.192  0.096  J5-08  ATCC 4127  0.544  0.181  0.098  J17-01  ATCC 46523  0.553  0.247  0.137  J18-01  ATCC 56069  0.526  0.224  0.118  J24-05  ATCC 66348  0.475  0.197  0.094  J24-11  ATCC 66348  0.535  0.246  0.132  J24-12  ATCC 66348  0.515  0.211  0.108  J24-15  ATCC 66348  0.514  0.211  0.108  View Large Evaluation of the newly constructed, cellobiose-fermenting industrial yeast strains Cellobiose is commonly found in cellulose, galactan, and red seaweed after hydrolysis. However, yeast cannot naturally metabolize cellobiose. Previous studies reported a high-affinity cellodextrin transporter (cdt-1) and an intracellular β-glucosidase (gh1-1) from Neurospora crassa were introduced into S. cerevisiae strains, and the resulting strains could ferment cellobiose efficiently (Galazka et al.2010: 84–6). In addition, cellobiose and xylose could be co-fermented simultaneously without glucose suppression from the sequential fermentation of glucose first and xylose second, because glucose was hydrolyzed intracellularly (Ha et al.2013: 525–31). With all the benefits of cellobiose fermentation, the cellobiose pathway was introduced into the industrial strains. However, only a few transformants were obtained, and only the transformants from ATCC 4124 (Jin 03), ATCC 9763 (Jin 09) and ATCC 24858 (Jin 13) could ferment cellobiose efficiently (Fig. 1; Table 5). When compared with the transformant of ATCC 4124 (Jin 03) at 37 h, only the transformant from ATCC 9763 (Jin 09) was comparable. The others were either not able to metabolize cellobiose or grew poorly. Combing the results, ATCC 9763 (Jin 09) has the most desirable phenotypes for cellobiose fermentation and tolerances against high temperatures, fermentation inhibitors and organic acids. Table 5. Fermentation data of the selected cellobiose fermenting transformants from the industrial strains grown in YPC80 medium and measured at 37 h into the fermentation. Code name  Strains  Cellobiose consumption rate (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J3  ATCC 4124  2.316  0.369  0.855  J9  ATCC 9763  2.377  0.338  0.803  J13  ATCC 24858  1.709  0.180  0.307  Code name  Strains  Cellobiose consumption rate (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J3  ATCC 4124  2.316  0.369  0.855  J9  ATCC 9763  2.377  0.338  0.803  J13  ATCC 24858  1.709  0.180  0.307  View Large Table 5. Fermentation data of the selected cellobiose fermenting transformants from the industrial strains grown in YPC80 medium and measured at 37 h into the fermentation. Code name  Strains  Cellobiose consumption rate (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J3  ATCC 4124  2.316  0.369  0.855  J9  ATCC 9763  2.377  0.338  0.803  J13  ATCC 24858  1.709  0.180  0.307  Code name  Strains  Cellobiose consumption rate (g/L)  Ethanol yield (g/g)  Ethanol productivity (g/L/h)  J3  ATCC 4124  2.316  0.369  0.855  J9  ATCC 9763  2.377  0.338  0.803  J13  ATCC 24858  1.709  0.180  0.307  View Large Phenotyping the industrial strains under minimal media using gas pressure analysis Strains were evaluated for their ability to increase gas pressure in a sealed glass bottle, with a higher gas pressure indicative of higher CO2 production and in-turn higher ethanol production. The yeast strains were grown in YP medium containing 20 g/L of glucose, YP medium containing 20 g/L of glucose and 25% (final concentration) hydrolysate or Verduyn's (Verduyn et al.1992: 501–17) medium with 20 g/L of glucose and 0.628 g/L of complete supplement mixture (MP Biomedicals, CA) to compare the industrial strains’ behaviors among different media composition. Once the fermentation started in a sealed bottle, the attached RF gas production modules (ANKOM Technology, NY) would monitor the gas production as pounds per square inch, recording every 5 min. For the YPD20 with 25% hydrolysate (Fig. 1), ATCC 60493 (Jin 21) produced greater gas pressure, likely through CO2 production, which correlates with higher ethanol production when compared with the ATCC 4124 (Jin 03) strain. There are no significant differences among strains in the YPD20 condition. The industrials strains ATCC 2360 (Jin 01), ATCC 4127 (Jin 05), ATCC 7754 (Jin 08), ATCC 24858 (Jin 13), ATCC 38544 (Jin 16), ATCC 46523 (Jin 17), ATCC 56069 (Jin 18), ATCC 60222 (Jin 19) and ATCC 60493 (Jin 21) all performed better than ATCC 4124 (Jin 03) in Verduyn's medium, suggesting that the ATCC4124 (Jin 03) control strain is not ideal for growth in certain minimal media, such as Verdyn's medium. Taking into account the relative performances of the strains regarding low pH tolerance, fermentation inhibitor resistance, and xylose fermentation rates, we narrowed down five industrial strains and ATCC 4124 (Jin 03) to conduct further experiments. The selected industrial strains were ATCC 9763 (Jin 09), ATCC 24858 (Jin 13), ATCC 46523 (Jin 17), ATCC 56069 (Jin 18) and ATCC 66348 (Jin 24). Synthetic complete minimal medium with and without CSM (Complete Supplement Mixture, MP Biomedicals, CA) was used to evaluate the strains (Fig. 6). Of the selected industrial strains, ATCC 56069 (Jin 18) significantly underperformed compared to the ATCC 4124 (Jin 03) control in either condition. However, the other four industrial strains outperformed the ATCC 4124 (Jin 03) control with CSM. Under the nutrient-limiting medium (SC minimal medium, SCD), ATCC 24848 (Jin 13) and ATCC 66348 (Jin 24) had the highest CO2 production as compared with other industrial strains, suggesting that these two strains required fewer nutrients for optimal growth and ethanol production. Overall, ATCC 56069 (Jin 18) was the worst strain in the minimal medium in terms of gas production. Figure 6. View largeDownload slide A heat map indicating the relative performance of industrial strain screening on mannose, maltose, and sucrose condition. The ranking of their performances was analyzed in this figure. Performance for a specific test that was 10% lower than the ATCC 4124 control strain was scaled at −2. Performance 5%–10% lower was scaled at −1, performance within 5% lower or higher was scaled at 0, performance 5% higher was scaled +1, and performance greater than 10% higher was scaled at +2. No growth was scaled at -3 (gray color). Abbreviations: SM, synthetic complete medium with complete supplement; SCD, synthetic complete medium without complete supplement; GA, gas analysis; Man, Mannose; Malt, Maltose; Sucro, Sucrose. Figure 6. View largeDownload slide A heat map indicating the relative performance of industrial strain screening on mannose, maltose, and sucrose condition. The ranking of their performances was analyzed in this figure. Performance for a specific test that was 10% lower than the ATCC 4124 control strain was scaled at −2. Performance 5%–10% lower was scaled at −1, performance within 5% lower or higher was scaled at 0, performance 5% higher was scaled +1, and performance greater than 10% higher was scaled at +2. No growth was scaled at -3 (gray color). Abbreviations: SM, synthetic complete medium with complete supplement; SCD, synthetic complete medium without complete supplement; GA, gas analysis; Man, Mannose; Malt, Maltose; Sucro, Sucrose. Assimilation of other sugars Mannose, maltose and sucrose utilization are also interesting to certain industries. For example, mannose is a sugar hydrolyzed from plant hemicellulose and seaweed, maltose is a disaccharides breakdown product from starch, and sucrose is found in sugarcane juice. To expand the feasibility of our selected strains and utilize a variety of sources for the substrates (sugars), we examined the fermentation capability of the industrial yeast strains by measuring their ethanol production rate and specific growth rate in YP medium with mannose 100 g/L, maltose 100 g/L or sucrose 100 g/L. When compared with ATCC 4124 (Jin 03) (Fig. 6), ATCC 46523 (Jin 17) has the highest specific growth rate under both mannose and maltose conditions. Both ATCC 56069 (Jin 18) and ATCC 66348 (Jin 24) have a higher specific growth rate in the sucrose condition. In terms of ethanol productivities, ATCC 46523 (Jin 17) and ATCC 66348 (Jin 24) are worse performing than ATCC 4124 (Jin 03) under the sucrose condition. ATCC 24858 (Jin 13) and ATCC 56069 (Jin 18) are worse than ATCC 4124 (Jin 03) under the mannose condition, with only ATCC 66348 (Jin 24) being slightly better. Under the maltose condition, ATCC 56069 (Jin 18) and ATCC 66348 (Jin 24) could not consume maltose, and no ethanol was produced. The ethanol productivity of ATCC 24858 (Jin 13) was slightly better than ATCC 4124 (Jin 03) under the maltose condition. DISCUSSION In broad terms, S. cerevisiae can be divided up into two major categories, industrial or laboratory strains. As the name implies, industrial yeast strains are considered as such due to their ability to resist harsh industrial fermentation conditions, which includes fermentation inhibitor-laden lignocellulosic hydrolysates. However, industrial yeasts are polyploid strains in many cases, whereas laboratory yeasts are most commonly haploid strains (Hansen and Kielland-Brandt 1996: 1–12; Walker 1998: 362). The increased ploidy can aid the resistance of the yeast strain to fermentation conditions, but can also increase the difficulty of introducing targeted genetic perturbations. Fortunately, with the recent development of the CRISPR/Cas9 gene editing system, engineering polyploid yeast strains has become increasingly less laborious and easier as compared with traditional engineering methods (Ryan et al.2014). With the CRISPR/Cas9 system in mind, identifying the phenotypic characteristics of industrial yeast strains would be beneficial, and superior strains could be achievable shortly with the recent developments. In this study, a review and analysis of 21 industrial S. cerevisiae yeast strains was conducted. Compared to the control strain ATCC 4124 (JIN 03), several strains appeared to excel in three conditions amenable to lignocellulosic hydrolysate fermentations: a rapid xylose fermentation rate, resistance under low pH conditions and tolerance against fermentation inhibitors. Of the 21 industrial yeast strains in this study, five strains (ATCC numbers 4127, 4921, 56069, 60222 and 60223) have no peer-reviewed literature citing the ATCC nomenclature and, to our knowledge, have not been used in any major laboratory- or industrial-scale studies. Despite this, these five strains also did not have any significantly desired phenotypes compared to the highly studied ATCC 4124 control strain. Several interesting trends appeared at the end of the screening process. We observed that only three strains (ATCC 56069, 60493 and 66348) could exceed the ethanol productivities of the ATCC 4124 (Jin 03) control strain in high concentrations of glucose (160 g/L). This result suggests that some industrial strains are considerably high osmotolerance and are resistant to a higher concentration of ethanol, whereas many other industrial yeasts do not have this tolerance phenotype. As a result, our dataset screened out the three strains that are better than the ATCC 4124 strain in that regard. In addition, we observed that some strains were more affected by higher temperature (37°C) fermentation than that of the control, suggesting some industrial strains are relatively more sensitive to elevated temperatures than others. Regarding the fermentation inhibitors commonly found in lignocellulosic hydrolysates, such as acetic acid, furfural and HMF, most strains were as tolerant as the ATCC 4124 control. Interestingly, ATCC 4098 (Jin 02), ATCC 4127 (Jin 05), ATCC 9763 (Jin 09), ATCC 24858 (Jin 13), ATCC 46523 (Jin 17) and ATCC 66348 (Jin 24) grew to a higher colony count than the control strain in the presence of these inhibitors, suggesting that they may be preferential strains for industrial lignocellulosic hydrolysate fermentations. It was reported that most industrial strains are diploid, aneuploid and occasionally polyploid (Argueso et al.2009: 2258–70; Akao et al.2011: 423–34; Borneman et al.2011: e1001287; Babrzadeh et al.2012: 485–94; Borneman et al.2012: 88–96; Dunn et al.2012: 908–24). As expected, only one industrial strain in this study, ATCC 20598 (Jin 11), was confirmed to be haploid, while the other strains are either diploid or polyploid. Many studies suggested that increased ploidy and increased resistances toward environmental stressors are correlated. However, we do not see the correlation in our results. The best-selected strains in this study do not contain the highest predicted ploidy. The strains with the highest ploidy, ATCC 60222 (Jin 19) and ATCC 60223 (Jin 20), performed worse than other industrial strains in most conditions in this study. In terms of xylose fermentation capability, most transformants from other industrials trains expressing the xylose fermentation pathway performed comparably to the control ATCC 4124 strain after a heterologous xylose fermentation pathway consisting of XYL1, XYL2 and XYL3 was introduced and expressed into the industrial strains. Interestingly, transformants from ATCC 4127 (Jin 05), ATCC 56069 (Jin 18) and ATCC 66348 (Jin 24) were found to ferment xylose more rapidly and produce ethanol than the ATCC 4124 control. On the other hand, most of the transformants expressing the cellobiose fermentation pathway performed similarly or worse than ATCC 4124 (Jin 03). Taking into account the relative performances of the strains in terms of low pH tolerance, fermentation inhibitor resistance and xylose fermentation rates, we identified ATCC 66348 (Jin24) as the overall top-performing best strain to conduct further experiments or genetic improvements for the efficient bioconversion of lignocellulosic hydrolysates. CONCLUSIONS Collectively, this study has provided a useful dataset to refer to when choosing an industrial S. cerevisiae for unique fermentation purposes, especially lignocellulosic hydrolysates. This study builds on previous studies that have also aimed to evaluate a variety of industrial yeast strains with the intent to improve the available dataset of industrial yeast phenotypes (Martıín and Jönsson 2003: 386–95; Li et al.2015: 266–74). Selecting the best host for lignocellulosic hydrolysates is one of the motivations in this study, and ATCC 66348 (Jin 24), originally isolated from Japanese soil, was chosen as the best overall, broadly applicable candidate for future lignocellulosic hydrolysate studies. SUPPLEMENTARY DATA Supplementary data is available at FEMSYR online. Acknowledgements We thank the American Type Culture Collection (ATCC) for continued maintenance of their yeast strain collections. FUNDING This work was supported by the Agriculture and Food Research Initiative Competitive Grant [No. 2015-67011-22806] from the United States Department of Agriculture, National Institute of Food and Agriculture to [TLT]. Conflict of interest. None declared. REFERENCES Akao T, Yashiro I, Hosoyama A et al.   Whole-genome sequencing of sake yeast Saccharomyces cerevisiae Kyokai no. 7. DNA Res  2011; 18: 423– 34. 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