Enhanced Fermentative Capacity of Yeasts Engineered in Storage Carbohydrate Metabolism Roberto Perez-Torrado Dept. de Biotecnologıa, Inst. de Agroquımica y Tecnologıa de Alimentos, IATA-CSIC, Valencia, Spain
Emilia Matallana Dept. de Biotecnologıa, Inst. de Agroquımica y Tecnologıa de Alimentos, IATA-CSIC, Valencia, Spain Dept. de Bioquımica y Biologıa Molecular, Universitat de Vale`ncia, Valencia, Spain DOI 10.1002/btpr.1993 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com)
During yeast biomass production, cells are grown through several batch and fed-batch cultures on molasses. This industrial process produces several types of stresses along the process, including thermic, osmotic, starvation, and oxidative stress. It has been shown that Saccharomyces cerevisiae strains with enhanced stress resistance present enhanced fermentative capacity of yeast biomass produced. On the other hand, storage carbohydrates have been related to several types of stress resistance in S. cerevisiae. Here we have engineered industrial strains in storage carbohydrate metabolism by overexpressing the GSY2 gene, that encodes the glycogen synthase enzyme, and deleting NTH1 gene, that encodes the neutral trehalase enzyme. Industrial biomass production process simulations were performed with control and modified strains to measure cellular carbohydrates and fermentation capacity of the produced biomass. These modifications increased glycogen and trehalose levels respectively during bench-top trials of industrial biomass propagation. We finally show that these strains display an improved fermentative capacity than its parental strain after biomass production. Modification of storage carbohydrate content increases fermentation or metabolic C 2014 capacity of yeast which can be an interesting application for the food industry. V American Institute of Chemical Engineers Biotechnol. Prog., 000:000–000, 2014 Keywords: S. cerevisiae, storage carbohydrates, fermentative capacity
Introduction The yeast biomass production industry represents the largest bulk production of any single-celled microorganism in the world. To allow long-term storage of the starters, active dry yeasts have been developed in addition to the classical fresh pressed yeasts.1 Due to its seasonal use just after grape harvesting, the stability of the starter is particularly relevant to wine industry. The same principles of baker’s yeasts manufacture are followed in the main stage of yeast biomass production.2–5 Several batch stages in increasing culture volumes on aerated molasses are followed by several fedbatch stages where highest yield of biomass is achieved because the low input of sugars promotes a respiratory metabolism and ethanol formation is avoided. These industrial processes are technologically optimized for the highest biomass yields but poorly characterized from the point of view of yeast molecular adaptation to the adverse growth conditions. However, this aspect is critical for a good performance of the final product.6 The use of bench-top trials reproducing both industrial biomass yield and growth rate can overcome the difficulties in the study of yeasts under real industrial conditions and it allows the application of
Correspondence concerning this article should be addressed to Roberto Perez-Torrado at [email protected] C 2014 American Institute of Chemical Engineers V
molecular tools for the understanding and improvement of yeasts behavior.7–12 A great body of knowledge is already available regarding the molecular responses of laboratory strains of Saccharomyces cerevisiae to different stresses.13 Also, several approaches to the characterization of stress response under industrial conditions have been carried out and some correlations have been found between stress resistance of several yeast strains and their suitability for industrial processes.2,14,15 Among other molecules, yeast storage carbohydrates (glycogen and trehalose) have been highlighted due to their relationship with stress resistance. Glycogen is the main carbon source and energy reserve in many organisms, including yeast.16 Glycogen plays an important role during starvation, during adaptation to respiratory metabolism, in emergence from stationary phase, and during cell sporulation and spore germination.16 The amount of glycogen accumulated by a yeast cell depends on environmental conditions and on the cell growth phase.17 Glycogen also is linked to yeast viability,17 suggesting that it also has a function during the yeast stress response.18 Regulation of glycogen metabolism in yeast is mediated by glycogen synthase encoded by the GSY2 gene. GSY2 is transcriptionally regulated under stress conditions by Msn2p/Msn4p.19 The main function of trehalose is to act as a reserve carbohydrate but also as a protective molecule in stress 1
2
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response, reviewed in Ref. 20. This second effect can be achieved by two different ways, i.e., by protecting membrane integrity through the union with phospholipids21 and by preserving the native conformation of proteins and preventing aggregation of partially denatured proteins.22 When yeast cells suffer several stress types as thermal or osmotic stress, they accumulate big amounts of trehalose.23 This accumulation can be easily explained by the described mechanisms of gene expression induction and activation of the trehalose synthase complex, but surprisingly, the trehalose degradation metabolism is also induced under those conditions.23–25 The best characterized trehalase activity is the neutral trehalase activity encoded by the NTH1 gene. The function of Nth1p is to hydrolyze intracellular trehalose both in normal growth and under different stress conditions.26 In this work, we have focused on the study of the effect of increased carbohydrate levels during bench-top trials of yeast biomass propagation and their effect in yeast fermentative capacity, an important parameter in several industrial applications. In order to study the effect of increased glycogen level, we constructed a strain overexpressing GSY2 gene and to study the effect of increased trehalose level we constructed a strain without NTH1 gene. We tested whether the modified strains can increase the levels of the corresponding storage carbohydrate. The increased utility of yeast produced using these two strains is demonstrated by the enhanced fermentative capacity.
Materials and Methods Yeast strains and plasmids S. cerevisiae industrial strain T73 (CECT1894) is a natural diploid strain isolated from Alicante (Spain) musts27 and has been commercialized by Lallemand Inc. (Montreal, Canada). This strain has been previously used in several studies11,28 and has proven to be a good wine yeast model. We constructed two different derivatives of strain T73. Strain TGsy228 was obtained by overexpressing the GSY2 yeast gene in a T73ura3-derivative strain.29 Overexpression was achieved by inserting a 3.8-kb SalI fragment containing the gene into the multicopy vector YEp352 carrying the selectable marker URA3. Strain TDnth1 was obtained by sequential deletion of the two copies of the NTH1 gene in strain T73ura3. Disruption was carried out by homologous recombination at both ends of the NTH1 open reading frame of an integration cassette carrying a kanR marker gene flanked by loxP sites. The cassette was amplified by PCR using the pUG6 plasmid as a template with oligos that carry sequences homologous to NTH1 gene sides. T73ura3 strain was transformed with the cassette following the procedure of lithium acetate, selecting for geneticin resistance colonies. Excision of the marker was induced by expression of Cre recombinase introduced in the same strain,30 allowing repeated disruptions. Integration of the cassette at the NTH1 locus and further excision of the kanR marker were confirmed by PCR and Southern blot analysis. Uracil prototrophy was restored by introducing a 1.1-kb HindIII linear fragment containing the URA3 gene. Industrial production conditions Experiments were carried out as previously described.11 YPD precultures were used to inoculate (OD600 0.1) industrial media. Molasses medium (diluted to 60 g L21 sucrose
for batch or 100 g L21 sucrose for fed-batch) was supplemented with 7.5 g L21 (NH4)2SO2; 3.5 g L21 KH2PO4; 0.75 g L21 MgSO47H2O; 10 mL L21 vitamin solution (50 mg L21 D-biotin; 1 g L21 calcium pantothenate; 1 g L21 thiamine hydrochloride); 1 mL L21 antifoam 204 (Sigma). Molasses and mineral solutions were autoclaved separately, and the vitamin solution was filter sterilized (0.2 lm) prior to use in the molasses medium. Fermentations were performed in a bioreactor BIOFLO III (NBS, NJ). Initial pH was 4.5 and it was allowed to freely vary between 4 and 5 during the batch step. In the fed batch process, pH was automatically maintained at 4.5 with 42.5% H3PO4 and 1M NaOH. Cell growth was followed by measuring the OD600 and the cell dry weight. Air flux of 0.5–1.5 L min21 was maintained. Dissolved O2 was followed with an O2 electrode (Mettler Toledo, USA) and maintained above 20% by a PID control system that allowed the automatic modification of the agitation speed between the range limits of 300–500 rpm. Determination of storage carbohydrate content The method of Parrou and Francois31 for the determination of storage carbohydrates content was used as follows. Cells (10 mg [dry weight]) were collected by centrifugation (3,000g; 2 min) from laboratory cultures or microvinification experiments at several growth stages and were washed with cold distilled water. Cells were stored at 220 C. Then, they were thawed, resuspended in 0.25 mL of 0.25M Na2CO3, transferred to screw-cap tubes, and incubated at 95 C for 4 h. Cell extracts were neutralized by adding 0.15 mL of 1M acetic acid and 0.6 mL of 0.2M Na acetate (pH 5.2). The sample was used to determine glycogen content by enzymatic breakage with a commercial Aspergillus niger amyloglucosidase (1.2 U/mL) (Boehringer Mannheim GmbH) at 57 C overnight under rotation in a hybridization oven. Controls were prepared without enzyme and treated as described above. Samples were centrifuged at 12,000g for 30 s, and the released glucose in supernatants was determined with glucose oxidase-peroxidase (Boehringer Mannheim GmbH). Trehalose was measured after enzymatic degradation with commercial trehalase (Sigma). Released glucose was determined by the glucose oxidase/peroxidase assay. The amount of trehalose or glycogen is expressed as mg of glucose (mg of cells dry weight)21. Dehydration and measurement of fermentative capacity Fermentative or metabolic capacity is defined as the amount of CO2 produced for a given number of cells in a period of time. To determine the fermentative capacity of dry cells, yeast biomass was dehydrated overnight under air flux in an oven at 39 C. For determination of fermentative capacity, 107 cells/mL were inoculated in bottles with YPGF medium (1% yeast extract, 2% peptone, 10% glucose, 10% fructose) and incubated with gentle shaking (65 rpm) at 30 C. The exact number of cells was determined by recounting in a Neubauer chamber and viability was determined by plating cells in YPD plates. CO2 production was measured every 20 min for 3 h in a Chittick instrument (American association of cereal Chemist, 12-10). The fermentative capacity for all strains in the freshly produced biomass (control condition) was higher than 0.15 mL CO2 (107 cells)21 min21, a similar value than produced by baker yeasts.32
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Figure 1. Glycogen accumulation during biomass propagation bench-top trials. Data from experiments with the control strain T73 (circles) and GSY2 overexpressing strain TGsy2 (rhombus) are shown. Along the process, samples were taken to measure glycogen levels for both strains. Average of two independent experiments and standard deviations are shown.
3
Figure 2. Trehalose accumulation during biomass propagation bench-top trials. Control strain T73 (circles) and NTH1 deletant strain TDnth1 performed a yeast biomass production. Along the process, samples were taken to measure trehalose levels for both strains. Average of two independent experiments and standard deviations are shown.
Experiments were carried out in triplicate and results were expressed as a percentage of the control condition.
Results Increased carbohydrate content during yeast biomass production In order to investigate the effect of increased carbohydrate content on industrial yeast during biomass propagation process, we constructed two modified strains with the objective of increasing trehalose or glycogen content. To increase the content of glycogen, we constructed the TGsy2 strain which contains an overexpression of the GSY2 gene. On the other hand, to increase the content of trehalose we constructed the TDnth1 strain which contains a deletion of the gene NTH1. The effects of these metabolic modifications were investigated by carrying out bench-top trials of biomass propagation cultures with molasses media. The performance of engineered strains during bench-top trials of biomass production was similar. Specific growth rates in the batch were similar (0.40 6 0.03, 0.39 6 0.02, and 0.37 6 0.04 h21 for T73, TGsy2, and TDnth1 strain, respectively), reaching no significantly different biomass levels (results not shown). Figure 1 shows the glycogen content during the biomass production process for the modified strain TGsy2 and the control strain T73. We can observe that the modified strain was able to accumulate higher glycogen amount than the control strain, reaching up to 70% more in the last phase of the process. In the case of trehalose, we compared the accumulation of this metabolite using TDnth1 strain and the control strain T73. As can be seen in Figure 2, the modified strain was able to increase the trehalose content during the biomass production process. TDnth1 accumulated two times more trehalose at some time points, although the final levels were 30% increased level compared to T73 control strain. Storage carbohydrate engineered strains increase fermentative capacity of yeast cells In order to check the physiological effect of storage carbohydrate modifications and their consequent increase in trehalose or glycogen yeast cell content, experiments of biomass propagation followed by dehydration were performed in order to assay the fermentative capacity of dry yeasts. The
Figure 3. Dried biomass from wild type T73 (white bars), GSY2 overexpressing strain TGsy2 (gray bars), and NTH1 deletant strain TDnth1 (black bars) were analyzed for fermentative capacity with respect to the freshly obtained product of bench-top trial fermentations. Average of three independent experiments and standard deviations are shown.
comparison of these strains can provide us information on the suitability and benefits of these modifications for an industrial application. No growth, lag phase, or cell viability differences were found between the modified and the reference strains (data not shown). Assays were performed for dry yeast using the same cell number for each strain, and the fermentative capacity of freshly obtained biomass was taken as 100%. Figure 3 shows the percentage of fermentative capacity for the modified and the parental strains after 3 h. As can be seen, the remaining fermentative capacity was low for dry cells in control strains (20%) but the GSY2 overexpressing strain (45%) and, especially, the NTH1 knockout strain (90 %) maintained a high activity percentage compared to reference strain. These data correlate storage carbohydrate levels with increased fermentative capacity of yeast
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produced in conditions.
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bench-top
trials
reproducing
industrial
Discussion The yeast biomass propagation process that produces starters for different foods is a well-established industry optimized to obtain high biomass yields and good reserve and protection carbohydrate accumulation, allowing complete consumption of nutrients by cells before entering the stationary phase.33 We previously described the suitability of bench-top simulation of yeast biomass propagation in combination with molecular techniques to study the effects in the complex and changing industrial environment.11 We have observed that several stresses are affecting yeast performance during these industrial conditions. Thus, increased content of storage carbohydrates, two molecules closely related with stress resistance can be highly interesting to preserve yeast cells from stressing industrial conditions. In fact, reserve carbohydrate utilization by yeast cells has been described in industrial conditions.34 Here, we described two metabolic modifications that can be of interest for the food industries where increased carbohydrate levels are valuable characteristic of the potentially relevant strains. The diminished loss of fermentative capacity of the TGsy2 and, especially, the TDnth1 strain suggests a long-term effect of the improved response to endogenous challenges. The increase of fermentative capacity is a valuable characteristic in industrial conditions and other studies have been performed to enhance it, for example decreasing tryptophan metabolism in cold fermentations.35 Regarding TGsy2 strain, the main conclusion is that this strain displays an industrially interesting property of increased resistance under glucose deprivation conditions that occurs when biomass is obtained and dried. This phenotype of strain TGsy2 indicates the importance of glycogen as an energy reserve and the requirement of glycogen mobilization for displaying higher performance after glucose exhaustion. Although it has been previously shown that increased glycogen was beneficial to increase viability in conditions of carbohydrate deprivation,28 this is the first time that an increased fermentative capacity is described. In fact, other studies showed the relation of glycogen with the resistance to stress, specially glucose deprivation where the metabolic utilization of glycogen as a carbon source has important role to keep yeast cell performance.25,36 The data presented for TDnth1 strain is remarkable due to the high fermentative capacity conserved after the production of dry yeast cells. The important implication in stress resistance of trehalose can explain this enhanced phenotype. In fact, yeast biomass production plants use trehalose levels as an indicator of biomass vitality.37 It has been described that trehalose is very important to resist cellular negative effects of desiccation as oxidative stress.38 Since desiccation is one of the most important stresses occurring during the production of dry biomass, this property of trehalose can explain the enhanced fermentative capacity observed in the TDnth1 strain.
Conclusions In conclusion, glycogen and trehalose increased levels are interesting to enhance some properties of industrially produced biomass as fermentative capacity. Also, it is possible
that this modifications help yeast cells to cope with other stressful situations that cells encounter during industrial applications, as prolonged yeast starvation, maturation after biomass production, storage, or final stages of wine production. Experiments in industrial plant condition should be carried out to evaluate the potential benefit of the strains described in this work. Thus, the engineered yeast presented in this work can be of interest to food industry and benefit at several levels.
Acknowledgments This work was supported by grants AGL2002-01109, AGL 2005-00508 from the “Ministerio de Educacion y Ciencia” (MEC) and GVACOMP2007-157 from the “Generalitat Valenciana”. R.P-T. was supported by a predoctoral fellowship from “Generalitat Valenciana”. The authors declare no conflict of interest.
Literature Cited 1. Rosen K. Preparation of yeast for industrial use in the production of beverages. In: Cantarelli C, Lanzarini G, editors. Biotechnology Applications in Beverage Production. London: Elsevier Applied Science; 1989:169–187. 2. Beudeker RF, van Damm HW, van der Plaat JB, Vellenga K. Developments in baker’s yeast production. In: Verachtert H, deMot R, editors. Yeast: Biotechnology and Biocatalysis. New York and Basel: Marcel Dekker Inc.; 1990:103–146. 3. Gomez-Pastor R, Perez-Torrado R, Matallana, E. Improving yield of industrial biomass propagation by increasing the Trx2p dosage. Bioeng Bugs. 2010;1:352–353. 4. Pereira MD, Eleutherio ECA, Panek AD. Acquisition of tolerance against oxidative damage in Saccharomyces cerevisiae. BMC Microbiol. 2001;1:11. 5. Rose AH, Vijaylakshimi G. Baker’s yeasts. In: Rose AH, Harrison JS, editors. The Yeasts, Vol.5. 2nd ed., London: Academic Press; 1993:357–397. 6. Walker GM. Yeast Physiology and Biotechnology. Chichester: Wiley; 1998. 7. Gomez-Pastor R, Perez-Torrado R, Cabiscol E, Matallana E. Transcriptomic and proteomic insights of the wine yeast biomass propagation process. FEMS Yeast Res. 2010;10:870–884. 8. Gomez-Pastor R, Perez-Torrado R, Cabiscol E, Ros J, Matallana E. Reduction of oxidative cellular damage by overexpression of the thioredoxin TRX2 gene improves yield and quality of wine yeast dry active biomass. Microb Cell Fact. 2010;12:9 9. Gomez-Pastor R, Perez-Torrado R, Cabiscol E, Ros J, Matallana E. Engineered Trx2p industrial yeast strain protects glycolysis and fermentation proteins from oxidative carbonylation during biomass propagation. Microb Cell Fact. 2012:9:11–14. 10. Gomez-Pastor R, Perez-Torrado R, Matallana E. Modification of the TRX2 gene dose in Saccharomyces cerevisiae affects hexokinase 2 gene regulation during wine yeast biomass production. Appl Microbiol Biotechnol. 2012;94:773–787. 11. Perez-Torrado R, Bruno-Barcena JM, Matallana E. Monitoring stress-related genes during the process of biomass propagation of Saccharomyces cerevisiae strains used for wine making. Appl Environ Microbiol. 2005;71:6831–6837. 12. Perez-Torrado R, Gomez-Pastor R, Larsson C, and Matallana E. Fermentative capacity of dry active wine yeast requires a specific oxidative stress response during industrial biomass growth. Appl Microbiol Biotechnol. 2009;81:951–60 13. Hohmann S, Mager WH. Yeast Stress Responses. Topics in Current Genetics Series, Vol.1. Heidelberg: Springer Verlag; 2003. 14. Jamieson DJ. Oxidative stress responses of the yeast Saccharomyces cerevisiae. Yeast. 1998;14:1511–27. 15. Perez-Torrado R, Carrasco P, Aranda A, Gimeno-Alca~niz JV, Perez-Ortın JE, Matallana E, del Olmo M. Study of the first
Biotechnol. Prog., 2014, Vol. 00, No. 00
16. 17.
18. 19. 20. 21. 22. 23. 24.
25.
26.
27. 28.
hours of microvinification by the use of osmotic stress-response genes as probes. System Appl Microbiol. 2002;25:153–161. Lillie SH, Pringle JR. Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation. J Bacteriol. 1980;143:1384–1394. Sillje HHW, Paalman JWG, Schure EG, Olsthoorn SQB, Verkleij AJ, Boonstra J, Verrips CT. Function of trehalose and glycogen in cell cycle progression and cell viability in Saccharomyces cerevisiae. J Bacteriol. 1999;181:396–400. Hottinger T, Boller T, Wiemken A. Rapid changes of heat and desiccation tolerance correlated with changes of trehalose content in Saccharomyces cerevesiae. FEBS Lett. 1987;220:113–115. Smith A, Ward MP, Garrett S. Yeast PKA represses Msn2p/Msn4pdependent gene expression to regulate growth, stress response and glycogen accumulation. EMBO J. 1998;17:3556–3564. Elbein AD, Pan YT, Pastuszak ICarroll D. New insights on trehalose: a multifunctional molecule. Glycobiol. 2003;13:17R–7R. Crowe JH, Hoekstra FACrowe LM. Anhydrobiosis. Ann Rev Physiol. 1992;54:579–599. Singer MA, Lindquist S. Multiple effects of trehalose on protein folding in vitro and in vivo. Mol Cell. 1998;1:639–648. Blomberg A. Metabolic surprises in Saccharomyces cerevisiae during adaptation to saline conditions: questions, some answers and a model. FEMS Microbiol Lett. 2000;182:1–8. Garre E, Perez-Torrado R, Gimeno-Alca~niz JV, Matallana E. Acid trehalase is involved in intracellular trehalose mobilization during postdiauxic growth and severe saline stress in Saccharomyces cerevisiae. FEMS Yeast Res. 2009;9:52–62. Parrou JL, Teste MA, Franc¸ois J. Effects of various types of stress on the metabolism of reserve carbohydrates in Saccharomyces cerevisiae: genetic evidence for a stress-induced recycling of glycogen and trehalose. Microbiol. 1997;143:1891–1900. San Miguel PF, Arg€uelles JC. Differential changes in the activity of cytosolic and vacuolar trehalases along the growth cycle of Saccharomyces cerevisiae. Biochim Biophys Acta. 1994;1200: 155–160. Querol A, Barrio E.Ramon D. A Comparative-study of different methods of yeast-strain characterization. System Appl Microbiol. 1992;15:439–446. Perez-Torrado R, Gimeno-Alca~niz JV, Matallana E. Wine yeast strains engineered for glycogen overproduction display enhanced
5
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
viability under glucose deprivation conditions. Appl Environ Microbiol. 2002;68:3339–3344. Puig S, Ramon D, Perez-Ortın JE. Optimized method to obtain stable food-safe recombinant wine yeast strains. J Agric Food Chem. 1998;46:1689–1693. Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 1996;24:2519–2524. Parrou JL, Francois J. A simplified procedure for a rapid and reliable assay of both glycogen and trehalose in whole yeast cells. Anal Biochem. 1997;248:186–188. Panadero J, Hernandez-Lopez MJ, Prieto JA, Randez-Gil F. Overexpression of the calcineurin target CRZ1 provides freeze tolerance and enhances the fermentative capacity of baker’s yeast. Appl Environ Microbiol. 2007;73:4824–4831 Jorgensen H, Olsson L, Ronnow B, Palmqvist EA. Fed-batch cultivation of baker’s yeast followed by nitrogen or carbon starvation: effects on fermentative capacity and content of trehalose and glycogen. Appl Environ Microbiol. 2002;59:310–317. Novo MT, Beltran G, Torija MJ, Poblet M, Roze`s N, Guillamon JM, Mas A. Changes in wine yeast storage carbohydrate levels during preadaptation, rehydration and low temperature fermentations. Int J Food Microbiol. 2003;86:153–161. Lopez-Malo M, Garcıa-Rios E, Chiva R, Guillamon JM, MartıRaga M. Effect of deletion and overexpression of tryptophan metabolism genes on growth and fermentation capacity at low temperature in wine yeast. Biotechnol Prog. 2014;30:776–783. Franc¸ois JM, Parrou JL. Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev. 2001;25:125–45. Degre R. Selection and commercial cultivation of wine yeast and bacteria. In: Fleet GH, editor. Wine Microbiology and Biotechnology. Switzerland: Harwood Academic Publishers; 1993: 421–447. Franc¸a MB, Panek AD, Eleutherio EC. Oxidative stress and its effects during dehydration. Comp Biochem Physiol A Mol Integr Physiol. 2007;146:621–631.
Manuscript received Jul. 28, 2014, and revision received Sept. 9, 2014.
Emilia Matallana Dept. de Biotecnologıa, Inst. de Agroquımica y Tecnologıa de Alimentos, IATA-CSIC, Valencia, Spain Dept. de Bioquımica y Biologıa Molecular, Universitat de Vale`ncia, Valencia, Spain DOI 10.1002/btpr.1993 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com)
During yeast biomass production, cells are grown through several batch and fed-batch cultures on molasses. This industrial process produces several types of stresses along the process, including thermic, osmotic, starvation, and oxidative stress. It has been shown that Saccharomyces cerevisiae strains with enhanced stress resistance present enhanced fermentative capacity of yeast biomass produced. On the other hand, storage carbohydrates have been related to several types of stress resistance in S. cerevisiae. Here we have engineered industrial strains in storage carbohydrate metabolism by overexpressing the GSY2 gene, that encodes the glycogen synthase enzyme, and deleting NTH1 gene, that encodes the neutral trehalase enzyme. Industrial biomass production process simulations were performed with control and modified strains to measure cellular carbohydrates and fermentation capacity of the produced biomass. These modifications increased glycogen and trehalose levels respectively during bench-top trials of industrial biomass propagation. We finally show that these strains display an improved fermentative capacity than its parental strain after biomass production. Modification of storage carbohydrate content increases fermentation or metabolic C 2014 capacity of yeast which can be an interesting application for the food industry. V American Institute of Chemical Engineers Biotechnol. Prog., 000:000–000, 2014 Keywords: S. cerevisiae, storage carbohydrates, fermentative capacity
Introduction The yeast biomass production industry represents the largest bulk production of any single-celled microorganism in the world. To allow long-term storage of the starters, active dry yeasts have been developed in addition to the classical fresh pressed yeasts.1 Due to its seasonal use just after grape harvesting, the stability of the starter is particularly relevant to wine industry. The same principles of baker’s yeasts manufacture are followed in the main stage of yeast biomass production.2–5 Several batch stages in increasing culture volumes on aerated molasses are followed by several fedbatch stages where highest yield of biomass is achieved because the low input of sugars promotes a respiratory metabolism and ethanol formation is avoided. These industrial processes are technologically optimized for the highest biomass yields but poorly characterized from the point of view of yeast molecular adaptation to the adverse growth conditions. However, this aspect is critical for a good performance of the final product.6 The use of bench-top trials reproducing both industrial biomass yield and growth rate can overcome the difficulties in the study of yeasts under real industrial conditions and it allows the application of
Correspondence concerning this article should be addressed to Roberto Perez-Torrado at [email protected] C 2014 American Institute of Chemical Engineers V
molecular tools for the understanding and improvement of yeasts behavior.7–12 A great body of knowledge is already available regarding the molecular responses of laboratory strains of Saccharomyces cerevisiae to different stresses.13 Also, several approaches to the characterization of stress response under industrial conditions have been carried out and some correlations have been found between stress resistance of several yeast strains and their suitability for industrial processes.2,14,15 Among other molecules, yeast storage carbohydrates (glycogen and trehalose) have been highlighted due to their relationship with stress resistance. Glycogen is the main carbon source and energy reserve in many organisms, including yeast.16 Glycogen plays an important role during starvation, during adaptation to respiratory metabolism, in emergence from stationary phase, and during cell sporulation and spore germination.16 The amount of glycogen accumulated by a yeast cell depends on environmental conditions and on the cell growth phase.17 Glycogen also is linked to yeast viability,17 suggesting that it also has a function during the yeast stress response.18 Regulation of glycogen metabolism in yeast is mediated by glycogen synthase encoded by the GSY2 gene. GSY2 is transcriptionally regulated under stress conditions by Msn2p/Msn4p.19 The main function of trehalose is to act as a reserve carbohydrate but also as a protective molecule in stress 1
2
Biotechnol. Prog., 2014, Vol. 00, No. 00
response, reviewed in Ref. 20. This second effect can be achieved by two different ways, i.e., by protecting membrane integrity through the union with phospholipids21 and by preserving the native conformation of proteins and preventing aggregation of partially denatured proteins.22 When yeast cells suffer several stress types as thermal or osmotic stress, they accumulate big amounts of trehalose.23 This accumulation can be easily explained by the described mechanisms of gene expression induction and activation of the trehalose synthase complex, but surprisingly, the trehalose degradation metabolism is also induced under those conditions.23–25 The best characterized trehalase activity is the neutral trehalase activity encoded by the NTH1 gene. The function of Nth1p is to hydrolyze intracellular trehalose both in normal growth and under different stress conditions.26 In this work, we have focused on the study of the effect of increased carbohydrate levels during bench-top trials of yeast biomass propagation and their effect in yeast fermentative capacity, an important parameter in several industrial applications. In order to study the effect of increased glycogen level, we constructed a strain overexpressing GSY2 gene and to study the effect of increased trehalose level we constructed a strain without NTH1 gene. We tested whether the modified strains can increase the levels of the corresponding storage carbohydrate. The increased utility of yeast produced using these two strains is demonstrated by the enhanced fermentative capacity.
Materials and Methods Yeast strains and plasmids S. cerevisiae industrial strain T73 (CECT1894) is a natural diploid strain isolated from Alicante (Spain) musts27 and has been commercialized by Lallemand Inc. (Montreal, Canada). This strain has been previously used in several studies11,28 and has proven to be a good wine yeast model. We constructed two different derivatives of strain T73. Strain TGsy228 was obtained by overexpressing the GSY2 yeast gene in a T73ura3-derivative strain.29 Overexpression was achieved by inserting a 3.8-kb SalI fragment containing the gene into the multicopy vector YEp352 carrying the selectable marker URA3. Strain TDnth1 was obtained by sequential deletion of the two copies of the NTH1 gene in strain T73ura3. Disruption was carried out by homologous recombination at both ends of the NTH1 open reading frame of an integration cassette carrying a kanR marker gene flanked by loxP sites. The cassette was amplified by PCR using the pUG6 plasmid as a template with oligos that carry sequences homologous to NTH1 gene sides. T73ura3 strain was transformed with the cassette following the procedure of lithium acetate, selecting for geneticin resistance colonies. Excision of the marker was induced by expression of Cre recombinase introduced in the same strain,30 allowing repeated disruptions. Integration of the cassette at the NTH1 locus and further excision of the kanR marker were confirmed by PCR and Southern blot analysis. Uracil prototrophy was restored by introducing a 1.1-kb HindIII linear fragment containing the URA3 gene. Industrial production conditions Experiments were carried out as previously described.11 YPD precultures were used to inoculate (OD600 0.1) industrial media. Molasses medium (diluted to 60 g L21 sucrose
for batch or 100 g L21 sucrose for fed-batch) was supplemented with 7.5 g L21 (NH4)2SO2; 3.5 g L21 KH2PO4; 0.75 g L21 MgSO47H2O; 10 mL L21 vitamin solution (50 mg L21 D-biotin; 1 g L21 calcium pantothenate; 1 g L21 thiamine hydrochloride); 1 mL L21 antifoam 204 (Sigma). Molasses and mineral solutions were autoclaved separately, and the vitamin solution was filter sterilized (0.2 lm) prior to use in the molasses medium. Fermentations were performed in a bioreactor BIOFLO III (NBS, NJ). Initial pH was 4.5 and it was allowed to freely vary between 4 and 5 during the batch step. In the fed batch process, pH was automatically maintained at 4.5 with 42.5% H3PO4 and 1M NaOH. Cell growth was followed by measuring the OD600 and the cell dry weight. Air flux of 0.5–1.5 L min21 was maintained. Dissolved O2 was followed with an O2 electrode (Mettler Toledo, USA) and maintained above 20% by a PID control system that allowed the automatic modification of the agitation speed between the range limits of 300–500 rpm. Determination of storage carbohydrate content The method of Parrou and Francois31 for the determination of storage carbohydrates content was used as follows. Cells (10 mg [dry weight]) were collected by centrifugation (3,000g; 2 min) from laboratory cultures or microvinification experiments at several growth stages and were washed with cold distilled water. Cells were stored at 220 C. Then, they were thawed, resuspended in 0.25 mL of 0.25M Na2CO3, transferred to screw-cap tubes, and incubated at 95 C for 4 h. Cell extracts were neutralized by adding 0.15 mL of 1M acetic acid and 0.6 mL of 0.2M Na acetate (pH 5.2). The sample was used to determine glycogen content by enzymatic breakage with a commercial Aspergillus niger amyloglucosidase (1.2 U/mL) (Boehringer Mannheim GmbH) at 57 C overnight under rotation in a hybridization oven. Controls were prepared without enzyme and treated as described above. Samples were centrifuged at 12,000g for 30 s, and the released glucose in supernatants was determined with glucose oxidase-peroxidase (Boehringer Mannheim GmbH). Trehalose was measured after enzymatic degradation with commercial trehalase (Sigma). Released glucose was determined by the glucose oxidase/peroxidase assay. The amount of trehalose or glycogen is expressed as mg of glucose (mg of cells dry weight)21. Dehydration and measurement of fermentative capacity Fermentative or metabolic capacity is defined as the amount of CO2 produced for a given number of cells in a period of time. To determine the fermentative capacity of dry cells, yeast biomass was dehydrated overnight under air flux in an oven at 39 C. For determination of fermentative capacity, 107 cells/mL were inoculated in bottles with YPGF medium (1% yeast extract, 2% peptone, 10% glucose, 10% fructose) and incubated with gentle shaking (65 rpm) at 30 C. The exact number of cells was determined by recounting in a Neubauer chamber and viability was determined by plating cells in YPD plates. CO2 production was measured every 20 min for 3 h in a Chittick instrument (American association of cereal Chemist, 12-10). The fermentative capacity for all strains in the freshly produced biomass (control condition) was higher than 0.15 mL CO2 (107 cells)21 min21, a similar value than produced by baker yeasts.32
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Figure 1. Glycogen accumulation during biomass propagation bench-top trials. Data from experiments with the control strain T73 (circles) and GSY2 overexpressing strain TGsy2 (rhombus) are shown. Along the process, samples were taken to measure glycogen levels for both strains. Average of two independent experiments and standard deviations are shown.
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Figure 2. Trehalose accumulation during biomass propagation bench-top trials. Control strain T73 (circles) and NTH1 deletant strain TDnth1 performed a yeast biomass production. Along the process, samples were taken to measure trehalose levels for both strains. Average of two independent experiments and standard deviations are shown.
Experiments were carried out in triplicate and results were expressed as a percentage of the control condition.
Results Increased carbohydrate content during yeast biomass production In order to investigate the effect of increased carbohydrate content on industrial yeast during biomass propagation process, we constructed two modified strains with the objective of increasing trehalose or glycogen content. To increase the content of glycogen, we constructed the TGsy2 strain which contains an overexpression of the GSY2 gene. On the other hand, to increase the content of trehalose we constructed the TDnth1 strain which contains a deletion of the gene NTH1. The effects of these metabolic modifications were investigated by carrying out bench-top trials of biomass propagation cultures with molasses media. The performance of engineered strains during bench-top trials of biomass production was similar. Specific growth rates in the batch were similar (0.40 6 0.03, 0.39 6 0.02, and 0.37 6 0.04 h21 for T73, TGsy2, and TDnth1 strain, respectively), reaching no significantly different biomass levels (results not shown). Figure 1 shows the glycogen content during the biomass production process for the modified strain TGsy2 and the control strain T73. We can observe that the modified strain was able to accumulate higher glycogen amount than the control strain, reaching up to 70% more in the last phase of the process. In the case of trehalose, we compared the accumulation of this metabolite using TDnth1 strain and the control strain T73. As can be seen in Figure 2, the modified strain was able to increase the trehalose content during the biomass production process. TDnth1 accumulated two times more trehalose at some time points, although the final levels were 30% increased level compared to T73 control strain. Storage carbohydrate engineered strains increase fermentative capacity of yeast cells In order to check the physiological effect of storage carbohydrate modifications and their consequent increase in trehalose or glycogen yeast cell content, experiments of biomass propagation followed by dehydration were performed in order to assay the fermentative capacity of dry yeasts. The
Figure 3. Dried biomass from wild type T73 (white bars), GSY2 overexpressing strain TGsy2 (gray bars), and NTH1 deletant strain TDnth1 (black bars) were analyzed for fermentative capacity with respect to the freshly obtained product of bench-top trial fermentations. Average of three independent experiments and standard deviations are shown.
comparison of these strains can provide us information on the suitability and benefits of these modifications for an industrial application. No growth, lag phase, or cell viability differences were found between the modified and the reference strains (data not shown). Assays were performed for dry yeast using the same cell number for each strain, and the fermentative capacity of freshly obtained biomass was taken as 100%. Figure 3 shows the percentage of fermentative capacity for the modified and the parental strains after 3 h. As can be seen, the remaining fermentative capacity was low for dry cells in control strains (20%) but the GSY2 overexpressing strain (45%) and, especially, the NTH1 knockout strain (90 %) maintained a high activity percentage compared to reference strain. These data correlate storage carbohydrate levels with increased fermentative capacity of yeast
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produced in conditions.
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bench-top
trials
reproducing
industrial
Discussion The yeast biomass propagation process that produces starters for different foods is a well-established industry optimized to obtain high biomass yields and good reserve and protection carbohydrate accumulation, allowing complete consumption of nutrients by cells before entering the stationary phase.33 We previously described the suitability of bench-top simulation of yeast biomass propagation in combination with molecular techniques to study the effects in the complex and changing industrial environment.11 We have observed that several stresses are affecting yeast performance during these industrial conditions. Thus, increased content of storage carbohydrates, two molecules closely related with stress resistance can be highly interesting to preserve yeast cells from stressing industrial conditions. In fact, reserve carbohydrate utilization by yeast cells has been described in industrial conditions.34 Here, we described two metabolic modifications that can be of interest for the food industries where increased carbohydrate levels are valuable characteristic of the potentially relevant strains. The diminished loss of fermentative capacity of the TGsy2 and, especially, the TDnth1 strain suggests a long-term effect of the improved response to endogenous challenges. The increase of fermentative capacity is a valuable characteristic in industrial conditions and other studies have been performed to enhance it, for example decreasing tryptophan metabolism in cold fermentations.35 Regarding TGsy2 strain, the main conclusion is that this strain displays an industrially interesting property of increased resistance under glucose deprivation conditions that occurs when biomass is obtained and dried. This phenotype of strain TGsy2 indicates the importance of glycogen as an energy reserve and the requirement of glycogen mobilization for displaying higher performance after glucose exhaustion. Although it has been previously shown that increased glycogen was beneficial to increase viability in conditions of carbohydrate deprivation,28 this is the first time that an increased fermentative capacity is described. In fact, other studies showed the relation of glycogen with the resistance to stress, specially glucose deprivation where the metabolic utilization of glycogen as a carbon source has important role to keep yeast cell performance.25,36 The data presented for TDnth1 strain is remarkable due to the high fermentative capacity conserved after the production of dry yeast cells. The important implication in stress resistance of trehalose can explain this enhanced phenotype. In fact, yeast biomass production plants use trehalose levels as an indicator of biomass vitality.37 It has been described that trehalose is very important to resist cellular negative effects of desiccation as oxidative stress.38 Since desiccation is one of the most important stresses occurring during the production of dry biomass, this property of trehalose can explain the enhanced fermentative capacity observed in the TDnth1 strain.
Conclusions In conclusion, glycogen and trehalose increased levels are interesting to enhance some properties of industrially produced biomass as fermentative capacity. Also, it is possible
that this modifications help yeast cells to cope with other stressful situations that cells encounter during industrial applications, as prolonged yeast starvation, maturation after biomass production, storage, or final stages of wine production. Experiments in industrial plant condition should be carried out to evaluate the potential benefit of the strains described in this work. Thus, the engineered yeast presented in this work can be of interest to food industry and benefit at several levels.
Acknowledgments This work was supported by grants AGL2002-01109, AGL 2005-00508 from the “Ministerio de Educacion y Ciencia” (MEC) and GVACOMP2007-157 from the “Generalitat Valenciana”. R.P-T. was supported by a predoctoral fellowship from “Generalitat Valenciana”. The authors declare no conflict of interest.
Literature Cited 1. Rosen K. Preparation of yeast for industrial use in the production of beverages. In: Cantarelli C, Lanzarini G, editors. Biotechnology Applications in Beverage Production. London: Elsevier Applied Science; 1989:169–187. 2. Beudeker RF, van Damm HW, van der Plaat JB, Vellenga K. Developments in baker’s yeast production. In: Verachtert H, deMot R, editors. Yeast: Biotechnology and Biocatalysis. New York and Basel: Marcel Dekker Inc.; 1990:103–146. 3. Gomez-Pastor R, Perez-Torrado R, Matallana, E. Improving yield of industrial biomass propagation by increasing the Trx2p dosage. Bioeng Bugs. 2010;1:352–353. 4. Pereira MD, Eleutherio ECA, Panek AD. Acquisition of tolerance against oxidative damage in Saccharomyces cerevisiae. BMC Microbiol. 2001;1:11. 5. Rose AH, Vijaylakshimi G. Baker’s yeasts. In: Rose AH, Harrison JS, editors. The Yeasts, Vol.5. 2nd ed., London: Academic Press; 1993:357–397. 6. Walker GM. Yeast Physiology and Biotechnology. Chichester: Wiley; 1998. 7. Gomez-Pastor R, Perez-Torrado R, Cabiscol E, Matallana E. Transcriptomic and proteomic insights of the wine yeast biomass propagation process. FEMS Yeast Res. 2010;10:870–884. 8. Gomez-Pastor R, Perez-Torrado R, Cabiscol E, Ros J, Matallana E. Reduction of oxidative cellular damage by overexpression of the thioredoxin TRX2 gene improves yield and quality of wine yeast dry active biomass. Microb Cell Fact. 2010;12:9 9. Gomez-Pastor R, Perez-Torrado R, Cabiscol E, Ros J, Matallana E. Engineered Trx2p industrial yeast strain protects glycolysis and fermentation proteins from oxidative carbonylation during biomass propagation. Microb Cell Fact. 2012:9:11–14. 10. Gomez-Pastor R, Perez-Torrado R, Matallana E. Modification of the TRX2 gene dose in Saccharomyces cerevisiae affects hexokinase 2 gene regulation during wine yeast biomass production. Appl Microbiol Biotechnol. 2012;94:773–787. 11. Perez-Torrado R, Bruno-Barcena JM, Matallana E. Monitoring stress-related genes during the process of biomass propagation of Saccharomyces cerevisiae strains used for wine making. Appl Environ Microbiol. 2005;71:6831–6837. 12. Perez-Torrado R, Gomez-Pastor R, Larsson C, and Matallana E. Fermentative capacity of dry active wine yeast requires a specific oxidative stress response during industrial biomass growth. Appl Microbiol Biotechnol. 2009;81:951–60 13. Hohmann S, Mager WH. Yeast Stress Responses. Topics in Current Genetics Series, Vol.1. Heidelberg: Springer Verlag; 2003. 14. Jamieson DJ. Oxidative stress responses of the yeast Saccharomyces cerevisiae. Yeast. 1998;14:1511–27. 15. Perez-Torrado R, Carrasco P, Aranda A, Gimeno-Alca~niz JV, Perez-Ortın JE, Matallana E, del Olmo M. Study of the first
Biotechnol. Prog., 2014, Vol. 00, No. 00
16. 17.
18. 19. 20. 21. 22. 23. 24.
25.
26.
27. 28.
hours of microvinification by the use of osmotic stress-response genes as probes. System Appl Microbiol. 2002;25:153–161. Lillie SH, Pringle JR. Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation. J Bacteriol. 1980;143:1384–1394. Sillje HHW, Paalman JWG, Schure EG, Olsthoorn SQB, Verkleij AJ, Boonstra J, Verrips CT. Function of trehalose and glycogen in cell cycle progression and cell viability in Saccharomyces cerevisiae. J Bacteriol. 1999;181:396–400. Hottinger T, Boller T, Wiemken A. Rapid changes of heat and desiccation tolerance correlated with changes of trehalose content in Saccharomyces cerevesiae. FEBS Lett. 1987;220:113–115. Smith A, Ward MP, Garrett S. Yeast PKA represses Msn2p/Msn4pdependent gene expression to regulate growth, stress response and glycogen accumulation. EMBO J. 1998;17:3556–3564. Elbein AD, Pan YT, Pastuszak ICarroll D. New insights on trehalose: a multifunctional molecule. Glycobiol. 2003;13:17R–7R. Crowe JH, Hoekstra FACrowe LM. Anhydrobiosis. Ann Rev Physiol. 1992;54:579–599. Singer MA, Lindquist S. Multiple effects of trehalose on protein folding in vitro and in vivo. Mol Cell. 1998;1:639–648. Blomberg A. Metabolic surprises in Saccharomyces cerevisiae during adaptation to saline conditions: questions, some answers and a model. FEMS Microbiol Lett. 2000;182:1–8. Garre E, Perez-Torrado R, Gimeno-Alca~niz JV, Matallana E. Acid trehalase is involved in intracellular trehalose mobilization during postdiauxic growth and severe saline stress in Saccharomyces cerevisiae. FEMS Yeast Res. 2009;9:52–62. Parrou JL, Teste MA, Franc¸ois J. Effects of various types of stress on the metabolism of reserve carbohydrates in Saccharomyces cerevisiae: genetic evidence for a stress-induced recycling of glycogen and trehalose. Microbiol. 1997;143:1891–1900. San Miguel PF, Arg€uelles JC. Differential changes in the activity of cytosolic and vacuolar trehalases along the growth cycle of Saccharomyces cerevisiae. Biochim Biophys Acta. 1994;1200: 155–160. Querol A, Barrio E.Ramon D. A Comparative-study of different methods of yeast-strain characterization. System Appl Microbiol. 1992;15:439–446. Perez-Torrado R, Gimeno-Alca~niz JV, Matallana E. Wine yeast strains engineered for glycogen overproduction display enhanced
5
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
viability under glucose deprivation conditions. Appl Environ Microbiol. 2002;68:3339–3344. Puig S, Ramon D, Perez-Ortın JE. Optimized method to obtain stable food-safe recombinant wine yeast strains. J Agric Food Chem. 1998;46:1689–1693. Guldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 1996;24:2519–2524. Parrou JL, Francois J. A simplified procedure for a rapid and reliable assay of both glycogen and trehalose in whole yeast cells. Anal Biochem. 1997;248:186–188. Panadero J, Hernandez-Lopez MJ, Prieto JA, Randez-Gil F. Overexpression of the calcineurin target CRZ1 provides freeze tolerance and enhances the fermentative capacity of baker’s yeast. Appl Environ Microbiol. 2007;73:4824–4831 Jorgensen H, Olsson L, Ronnow B, Palmqvist EA. Fed-batch cultivation of baker’s yeast followed by nitrogen or carbon starvation: effects on fermentative capacity and content of trehalose and glycogen. Appl Environ Microbiol. 2002;59:310–317. Novo MT, Beltran G, Torija MJ, Poblet M, Roze`s N, Guillamon JM, Mas A. Changes in wine yeast storage carbohydrate levels during preadaptation, rehydration and low temperature fermentations. Int J Food Microbiol. 2003;86:153–161. Lopez-Malo M, Garcıa-Rios E, Chiva R, Guillamon JM, MartıRaga M. Effect of deletion and overexpression of tryptophan metabolism genes on growth and fermentation capacity at low temperature in wine yeast. Biotechnol Prog. 2014;30:776–783. Franc¸ois JM, Parrou JL. Reserve carbohydrates metabolism in the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev. 2001;25:125–45. Degre R. Selection and commercial cultivation of wine yeast and bacteria. In: Fleet GH, editor. Wine Microbiology and Biotechnology. Switzerland: Harwood Academic Publishers; 1993: 421–447. Franc¸a MB, Panek AD, Eleutherio EC. Oxidative stress and its effects during dehydration. Comp Biochem Physiol A Mol Integr Physiol. 2007;146:621–631.
Manuscript received Jul. 28, 2014, and revision received Sept. 9, 2014.
