WO1999024558A2

WO1999024558A2 - Novel high-fermenting microorganisms

Info

Publication number: WO1999024558A2
Application number: PCT/EP1998/007011
Authority: WO
Inventors: Oscar Johannes Maria Goddijn; Jan Pen
Original assignee: Mogen International N.V.
Priority date: 1997-10-30
Filing date: 1998-10-30
Publication date: 1999-05-20
Also published as: WO1999024558A3; AU2412499A

Abstract

This invention describes novel microorganisms with an increased carbohydrate metabolism and/or fermentative capacity caused by the expression of a heterologous enzyme coding for trehalose phosphate synthase (TPS).

Description

NOVEL HIGH- ERMENTING MICROORGANISMS

FIELD OF THE INVENTION

The present invention relates to improvement of fermenting capacity of microorganisms, specifically yeast and more specifically Saccharomyces .

BACKGROUND ART Yeast strains for example belonging to the genus Saccharomyces are used worldwide in the production of ethanol, both as endproduct and for brewing purposes, and leavening of bread. Such yeasts are capable of fermenting sugars to approximately equimolar amounts of carbondioxide (C0₂) and ethanol under anaerobic conditions. Baker's yeast ( Saccharomyces cerevisiae) is commercially available as cream yeast (15%-21% dry matter) , compressed yeast (26%-33% dry matter) , active dry yeast (92%-94% dry matter) or instant dry yeast (94%-97% dry matter) . The past decades, one of the most important goals in yeast research has been the improvement of the fermentative capacity of baker's yeast, resulting in improved C0₂-production rates. For this purpose, both classical hybridisation and molecular genetic techniques have been used.

One of the targets of the research has been the carbohydrate metabolism in yeast. It has appeared that hexokinase functions as a signal molecule in the carbohydrate metabolism in the sense that a slower rate of hexose phosphorylation also reduces the first steps in the glycolysis (Hohmann, S. et al . , Curr. Genet. .23., 281-289, 1993; van Dam, K. et al . , Ant . V . Leeuwenh . 63., 315-321, 1993). This research has been done with yeast defective in trehalose metabolism (ggsl- mutant, described in Thevelein, J.M., Ant . V . Leeuwenh . 62_, 109-130, 1992) . It appeared that the defective gene was TPS1, coding for trehalose-6-phosphate synthase (Bell, . et al . , Eur. J. Biochem. 209, 951-959, 1992) . On basis of this research three possible models which may account for the interaction between a hexose transporter, hexose phosphorylating enzyme and the trehalose synthesizing complex have been proposed (Thevelein, J. ., Hohmann, S., TIBS 2_0, 3-10, 1995). Each of the models is able to explain some observations, none of the models, however, is definitely established nor supported by experimental evidence. It has been shown (EP 577 915) that ggsl- mutants (ggsl-1) show higher invertase and alpha-glucosidase activity and hence suppress the limiting step in yeast fermentation processes. However, the mechanism of action has not been unraveled. There still remains a need for means of regulating carbohydrate metabolism, especially in yeast, resulting in strains having a high capacity of fermentation. Furthermore, elucidation of the controlling mechanism on glycolysis and other metabolic processes is an object of ongoing research.

SUMMARY OF THE INVENTION

It has now been found that microorganisms having an increased fermentation capacity can be obtained by providing them with a recombinant DNA capable of expressing TPS, this recombinant DNA preferably being of heterologous origin, preferably selected from the group of bacterial, fungal, plant, animal and human DNA, more preferably from Escherichia coll .

More specifically the microorganism is a yeast, preferably a yeast of a strain of Saccharomyces, more preferably Saccharomyces cerevisiae . Another object of the invention is to provide a microorganism having an altered carbohydrate metabolism and/or fermentation capacity characterized in that this alteration is caused by a recombinant DNA expressing a protein which influences the endogenous level of trehalose-6-phosphate . Also object of the invention is a method for modifying the carbohydrate metabolism of a microorganism and/or the fermentation capacity of said microorganism by providing it with a recombinant DNA expressing TPS. The recombinant DNA in this method will preferably be a heterologous DNA sequence, preferably selected from the group of bacterial, fungal, plant, animal and human DNA, more preferably from Escherichia coli .

Further object of the invention are microorganisms having a modified carbohydrate metabolism and/or fermentation capacity characterized in that this alteration is caused by a recombinant DNA expressing a product which influences the endogenous level of trehalose-6- phosphate, the product preferably selected from the group consisting of TPS, TPP, trehalase, trehalose phosphorylase, trehalose hydrolase and anti-sense trehalase. Specifically the invention describes a method for providing microorganisms having an increased fermentation capacity by being provided with a recombinant DNA capable of expressing TPS, this recombinant DNA preferably being of heterologous origin, preferably selected from the group of bacterial, fungal, plant, animal and human DNA, more preferably from Escheri chia coli .

More specifically the microorganism used in this method is a yeast, preferably a yeast of a strain of Saccharomyces, more preferably Saccharomyces cerevisiae . Another object of the invention is improved dough for use in bakery, comprising a yeast according to this invention. Also comprised is a method for baking using said dough, and bread or other bakery products baked by this method. A further object of the invention is a method for ethanol production with the microorganisms of the invention. Also a method for beer brewing or brewing other alcoholic beverages forms part of the invention. Accordingly, also the beverages produced by such a method are comprised in this invention.

DESCRIPTION OF THE FIGURES

Figure 1 : Measurement of the fermentation capacity of a reference strain (Mog2) and an isogenic strain which contains a cassette expressing TPS (Mog4) . Fermentation capacity is measured by ethanol production versus time in an anaerobic reaction vessel under C0₂. Concentration of biomass was 2.00 g/1. The figure shows the results of a single comparison. Measurements of both strains have been taken in duplo .

DETAILED DESCRIPTION OF THE INVENTION Surprisingly, we have now found that yeasts expressing a heterologous gene for TPS are showing a higher fermentative capacity, resulting in increased C0₂ and ethanol production rates by having an increased carbohydrate metabolic capacity. In our experiments no difference was seen in batch cultures and levels of trehalose remained unaltered. However, in chemostate cultures, oscillations in metabolism were observed as noted by changes in oxygen consumption. When the yeasts were cultured in an anaerobic culture after preculturing in aerobic culture under limited sugar conditions, which is a good model for the industrial production of baker's yeast in fed-batch cultures, these strains showed a dramatic increase in fermentation capacity. It is known that trehalose synthesis in yeast is dependent on a complex of three enzymes, trehalose phosphate phosphatase (TPP) , trehalose phosphate synthase (TPS) and a third enzyme, which harbours homologous regions to both TPP and TPS, a so-called bipartite enzyme (TPS/P) . Said third enzyme has also been thought to have regulatory functions (Thevelein, J.M., Hohmann, S., TIBS 2_0, 3-10, 1995). These three enzymes interact with each other to produce trehalose-6- phosphate and, subsequently, trehalose from UDP-glucose and glucose-6- phosphate. This complex is suggested to play a role in sugar sensing and signalling. Disturbance of this complex, modification of its activity, or altered regulation of the activity by introduction of TPS appears to result in an increase of the glycolysis and thus in an increased rate of C0₂ and ethanol production.

The present invention provides a transformed microorganism, preferably a yeast, which is able to express a TPS gene. This TPS gene is preferably of heterologous origin. By heterologous DNA is meant DNA not originating from the same yeast genus. For example, heterologous DNA is used when Saccharomyces is transformed with DNA not originating from Saccharomyces . The heterologous DNA may be of any origin, for instance, bacterial, fungal, plant, animal or human DNA. Preferably the TPS gene is derived from Escherichia coli . This enzyme has been described in EP 0 784 095. Also other genes coding for TPS are available at this moment as can be learnt from WO 97/42326. Preferably the expression of TPS is under control of a constitutive promoter. Thus, in the case of overexpressing the endogenous TPS gene a recombinant DNA construct should be introduced which is able to place the endogenous gene under control of a constitutive promoter. By a constitutive promoter is meant a promoter which effects expression of a gene independently of environmental conditions, for example the alcohol dehydrogenase promoter (ADH1 - promoter) similar to that described by Bennetzen, J.C. and Hall, B.D. (J. Biol. Chem. 257, 3018), the glyceraldehyde-3 -phosphate dehydrogenase promoter (GAPDH-promoter) , similar to that as described by Holland and Holland (Holland and Holland, (1980), J. Biol . Chem. 255, 2596-2605) . Use of such a promoter effects expression under the conditions of fed-batch fermentation production processes as well as under dough conditions. The transformed yeast according to this invention can be used as a starting strain for strain improvement procedures, such as mutation, mass mating and protoplast fusion. The resulting strains are considered to form part of this invention.

After introduction of gene coding for TPS an increase in gas production may be observed in doughs .

The invention not only applies to dough, but to any fermentation process, for example fermentation systems for industrial ethanol production from hydrolysed starch. Transformed yeast strains of this invention therefor include not only strains of baker's yeast, but also, for example, beer, whiskey and wine yeast strains.

Next to TPS other enzymes known to be part of or influence the trehalose synthesis pathway can be used to mimic the effects of expression of TPS. It is thought that the effects of TPS on the carbohydrate metabolism of the yeast are produced by its effect on the amounts of intracellular trehalose-6-phosphate (T-6-P) . TPS is capable of forming T-6-P from glucose- 6 -phosphate and UDP-glucose. It is envisaged that any enzymes which are able to form T-6-P or to prevent its degradation would have the same effects . Thus production of an anti-sense trehalose phosphate phosphatase (as-TPP) which would result in the inhibition of degradation of T-6-P and thus in increased T-6-P levels, would have a similar effect.

Trehalase is an enzyme which is capable of degrading trehalose. It has been shown in plants (WO 97/42326) that expression of a DNA construct that prevents the functioning of endogenous trehalase (as-trehalase) yields phenotypical changes which are also seen with expression of heterologous TPS. Thus, it is envisaged that anti-sense expression of treahalase in yeast would give similar results as the expression of TPS.

Budapest treaty deposits were made with the Centraalbureau voor Schimmelcultures (Baarn, the Netherlands) for yeast strain S. cerevisiae harbouring pMOG1199 under number CBS 923.97 and S. cerevisiae harbouring pMOG1198 under number CBS 922.97 at Monday, 7 July 1997.

EXPERIMENTAL PART DNA manipulations

For all DNA procedures (DNA isolation from E. coli , restriction, ligation, transformation, etc.) references is made to the handbook of Sambrook et al . (Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular cloning; a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) .

Strains

E. coli K-12 strain DH5-alpha is used for cloning. Yeast strain Saccharomyces cerevisiae CEN.PK113-3C MATa trpl-289 AL2-8^C SUC2 is used in all examples described.

Construction of pMOG1199

A DNA fragment harbouring the TPS E. coli coding sequence including the plant 3' poly adenylation signal was obtained by digesting pMOG799 (PCT/EP 97/02497) with the restriction enzymes Smal and Pstl. This fragment was inserted in the yeast shuttle vector p424 GPD (Mumberg, D., Muller, R. and Funk, M. , Gene 156, 119-122, 1995), also digested with Smal and Pstl, yielding pMOG1199.

Construction of PMOG1198

Similar to the cloning of pMOG1199, a DNA fragment harbouring the TPP E. coli coding sequence was obtained tailored in such a way that Smal and Pstl sites are present at the terminal ends. This fragment was inserted in the yeast shuttle vector p424 GPD (Mumberg, D., Muller, R. and Funk, M. , Gene 156 (1995) 119-122), also digested with Smal and Pstl, yielding pMOG1198.

Electroporation to Yeast

Both constructs pMOG1198 and pMOG1199 were transferred to yeast strain Saccharomyces cerevisiae CEN.PK113-3C MATa trpl-289 MAI-2-8^c SUC2 by electroporation (In: Evans, I.H., Methods in Molecular Biology: Yeast protocols pp.139-145, 1996, Humana Press, Totowa, New Jersey) CULTURING METHODS

Strain maintenance

Precultures of strains were grown to stationary phase in shake-flask cultures on mineral medium containing 2% (w/v) glucose. After adding glycerol (30% v/v) , 2 ml aliquots were stored in sterile vials at - 80 °C. These frozen stock cultures were used to inoculate precultures for batch and chemostat cultivation.

Media A defined mineral medium containing vitamins was prepared as described by Verduyn et al . (1992) was used. For chemostat cultivation, the glucose concentration in reservoir media was 7.5 g.l^"1 (0.25 mol C.l^" X

Shake-flask cultivation

Precultures were prepared by inoculating 100 ml mineral medium (0.3% w/v glucose) with 1 ml frozen stock culture. Cultures were incubated on an orbital shaker (200 rpm) at 30°C for 1 day. For growth curves, 4 ml of preculture was inoculated in a 500 ml Erlenmeyer flask with 100 ml mineral medium (2% w/v glucose or 1% v/v ethanol, pH 6.0) and then shaken (200 rpm) at 30°C. Optical-density measurements were performed at appropriate time intervals as described by Weusthuis, R.A. et al . (Is the Kluyver effect in yeast caused by product inhibition? Microbiology 140: 1723-1729, 1994) .

Chemostat cultivation in fermenters

Aerobic chemostat cultivation was performed at 30°C in laboratory fermenters (Applikon, Schiedam, The Netherlands) , at a stirrer speed of 800 rpm and a dilution rate of 0.10 h¹. The working volume of the cultures was kept at 1.0 1 by a peristaltic effluent pump coupled to an electrical level sensor. This set-up ensured that under all growth conditions, biomass concentrations in samples taken directly from the cultures differed by less than 1% from biomass concentrations in samples taken from the effluent line. The pH was kept at 5.0 + 0.1 by an ADI 1030 biocontroller, via the automatic addition of 2 mol/1^"1

KOH. The fermenter was flushed with air at a flow rate of 0.5 1/min^"1 using a Brooks 5876 mass-flow controller. The dissolved oxygen concentration was continuously monitored with an oxygen electrode (Ingold, 34 100 3002) and remained above 60% of air saturation. Chemostat cultures were routinely checked for purity using phase- contrast microscopy.

ANALYSES

Gas analysis The exhaust gas was cooled in a condenser (2°C) and dried with a Perma Pure dryer (PD-625-12P) . 0₂ and C0₂ concentrations were determined with a Servomex 1100A analyzer and a Beckman model 864 infrared detector, respectively. The exhaust gas flow rate was measured as described by Weusthuis et al . , (1994) . Specific rates of C0₂ production and 0₂ consumption were calculated according to the method of van Urk,H. et al . (Metabolic responses of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 upon transition from glucose limitation to glucose excess. Yeast 4_: 283-291,1988).

Determination of culture dry weight

Culture samples (10 ml) were filtered over preweighed nitrocellulose filters (pore size 0.45 μm; Gelman Sciences). After removal of medium, the filters were washed with demineralized water, dried in a Sharp R- 4700 microwave oven for 20 min at 360 W output, and weighed. Parallel samples varied by less than 1%.

Determination of fermentative capacity Samples containing exactly 100 mg dry weight of biomass from a steady- state chemostat were harvested by centrifugation at 5000g for 5 min, washed once and resuspended in 5 ml 0.9% (w/v) NaCl solution. Subsequently, these cell suspensions were introduced into a thermostated (30°C) vessel containing 10 ml 5-fold concentrated mineral medium, set at pH 5.6. The volume was adjusted to 40 ml with demineralized water. After 10 min incubation a 10 ml glucose pulse (100 g.l^"1) was given and samples (1 ml) were taken at appropriate time intervals. The working volume was 50 ml with a 10 ml headspace which was continuously flushed with C0₂ gas at a flow rate of approximately 10 ml.h^"1. The ethanol concentration in the supernatant was determined with a colorimetric assay according to Verduyn, C. et al . (Colorimetric alcohol assays with alcohol oxidase. J. Microbiol. Meth. 2_ : 15-25, 1984) using partially purified alcohol oxidase from Hansenula polymorpha. The fermentative capacity is expressed as mmol ethanol produced- (g dry weight) ^_1 -h^"1.

Metabolite analysis

Glucose in reservoir media and supernatants was determined enzymatically using the GOD-PAP method (Merck systems kit 14144. The ethanol concentration in the medium was determined with a colorimetric assay according to Verduyn, C. et al . (1984) using partially purified alcohol oxidase from Hansenula polymorpha.

EXAMPLE 1

Growth-velocities and yield of the different transgenic Saccharomyces strains were determined in shake-flask experiments using mineral medium and glucose or ethanol as carbon source. Strains used are a wild-type Saccharomyces strain (prototrophic growth, WT) , an identical strain harbouring an empty expression vector (EV) , harbouring pM0G1199 (TPS) or pMOG1198 (TPP) . Velocities of growth are depicted in Table 1.

The final optical density of the batch-grown cultures has been measured as an indication for biomass yield. Results are depicted in Table 2.

Table 2

Final OD WT EV TPS TPP

Glucose (n=2) 6.3 ± 6.0 ± 5.0 + 5.3 ± 0.1 0.2 0.2 0.4

As can be concluded from these data, the transgenic strains harbouring

TPS and TPP grow only slightly less fast on glucose compared to the wild-type and empty vector strain resulting a slightly reduced final biomass in batch-grown cultures. These data suggest there is no strong effect on processes like aerobic fermentation. Using ethanol as a carbon source, no significant difference was found between the strains tested. EXAMPLE 2

The different yeast strains were also compared on growth characteristics in continuous fed aerobic chemostat cultures. When grown in steady state, no differences were noted between the different strains in biomass yield or production of metabolites. Remarkably, the TPS transgenic Saccharomyces strains revealed a persistent and characteristic metabolic oscillation as noted by continuous measuring the soluble oxygen concentration in the cultures. This oscillation was not comparable to that observed when spontaneous synchronisation of the cell-cycle occurs in some wild-type Saccharomyces strains, indicating that some form of metabolic regulation is disturbed in the transgenic strains.

EXAMPLE 3 The TPS transgenic yeast strain was also tested in a system that reflects the conditions occurring during the preparation of dough. For this purpose, the strain was precultured in aerobic, sugar limited chemostat cultures. This preculture reflects the industrial production process of bakers-yeast in fed-batch cultures. Subsequently the culture was transferred to anaerobic conditions in the presence of sugar to monitor the production of ethanol and C0₂. The results of those experiments are depicted in figure 1.

Strikingly, in repeated separate experiments, an increase of 30-40% of the fermentation capacity was noted in the transgenic TPS strain compared to the wild-type and control strains.

BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

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Mogen International N.V. RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT Einsteinweg 97 issued pursuant to Rule 1 .1 by the 2333 CB LEIDEN INTERNATIONAL DEPOSITARY AUTHORITY Nederland identified at the bottom of this page

name and address of depositor

Where Rule 6. (d) applies, such date is the date on which the status of international depositary authority was acquired.

Form BP/4 (sole page) 12 BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Mogen International N.V. RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT Einstein eg 97 issued pursuant to Rule 7 .1 by the 2333 CB LEIDEN INTERNATIONAL DEPOSITARY AUTHORITY Nederland identified at the bottom of this page

name and address of depositor

Where Rule 6.4(d) applies, such date is the date on which the status of international depositary authority was acquired.

Claims

1. Microorganism having modified carbohydrate metabolism and/or fermentation capacity characterized in that it comprises a recombinant DNA capable of expressing trehalose phosphate synthase (TPS) .

2. Microorganism according to claim 1, characterized in that the recombinant DNA is heterologous, preferably selected from the group of bacterial, fungal, plant, animal and human DNA, more preferably from Escherichia coli .

3. Microorganism according to claim 1 or 2 , characterized in that it is a yeast, preferably a yeast of a strain of Saccharomyces, more preferably Saccharomyces cerevisiae .

4. Microorganism having a modified carbohydrate metabolism and/or fermentation capacity characterized in that this alteration is caused by a recombinant DNA expressing a product which influences the endogenous level of trehalose-6 -phosphate, the product preferably selected from the group consisting of TPP, TPS, trehalase, and anti- sense trehalase.

5. Method for producing a microorganism having an increased carbohydrate metabolism and/or fermentation capacity characterized in that the microorganism is transformed with a recombinant DNA capable of expression of an expression product which influences the endogenous level of trehalose-6-phosphate .

6. Method according to claim 5, characterized in that the product is selected from the group consisting of TPP, TPS, trehalase, and anti-sense trehalase.

7. Use of a microorganism according to any of claims 1-4 in a fermentation process.

Dough comprising a microorganism according to any of claims 1-4.

9. Method for baking, characterized in that a dough according to claim 8 is used.

10. Bread or other bakery products, characterized in that they are produced from the dough according to claim 8 and/or through the method according to claim 9.

11. Method for the production of ethanol, characterized in that a microorganism according to any of claims 1-4 is used.

12. Method for producing alcoholic beverages, characterized in that a microorganism according to any of claims 1-4 is used in the fermentation .

13. Beer or other alcoholic beverages, characterized in that they are produced according to the method of claim 12.

14. Microorganism as deposited under number CBS 923.97 at the Centraal Bureau of Schimmelcultures on July 7, 1997.