FI84624C - Process for producing ethanol and a protein or peptide - Google Patents

Description

1 84624

This invention relates to fermentation processes and their products, and more particularly to the production of an alcohol such as ethanol using sugars in yeast.

In the production of alcohol by fermentation, the sugars in aqueous solution are converted to ethanol using yeast. Yeast grows during fermentation, and although a small portion of the yeast can be used in subsequent fermentation processes, the remaining yeast still forms an excess that must be eliminated. This extra yeast can be used for some purposes, such as animal feed and in the manufacture of yeast extracts, but the amount of excess yeast produced is still high and its market value is relatively low.

Fermentation processes of this type on an industrial scale form three broad categories: 1) Fermentation processes in which the resulting, finished, aqueous medium is the desired end product. This category includes conventional brewing processes for the production of beer (the term "beer" as used herein includes ale, stout, lager and other types of fermented beverages based on malt), cider and other fermented beverages.

2) Fermentation processes in which the desired end product is a distilled, potable alcoholic concentrate. This category includes fermentation processes for the production of whiskeys, brandies and other spirits, as well as alcohol used to fortify other beverages.

3) Fermentation processes for the production of alcohol for industrial use. This category includes fermentation processes carried out on an industrial scale in some countries to produce fuel alcohol.

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A characteristic feature of all these industrial processes is the production of excess yeast.

In recent years, considerable interest has been placed in the genetic modification of microorganisms to produce heterologous proteins and peptides, i.e., proteins and peptides that are not produced by the inherent genetic building blocks of these microorganisms. Numerous different microorganisms have been used for such genetic manipulation, and of these microorganisms, yeast has aroused some interest. However, the yeasts used in laboratory experiments are not usually those used in industrial-scale fermentation processes to produce alcohol, and the yeast growth conditions in the laboratory are significantly different from those observed in yeasts in the industrial alcohol fermentation process.

The present invention is based on the finding that a genetically modified yeast capable of expressing a heterologous protein or peptide can be used in an industrial fermentation process involving the production of alcohol. Surprisingly, it has been found that the use of such yeast is compatible with the conditions of the industrial fermentation process. This means that the excess yeast obtained in the fermentation process acts as a source of heterologous protein or peptide and thus its industrial value is greatly improved. In addition, because the production of alcohol remains the main goal of the fermentation process and because most of the traditional equipment can be used with little modification, the additional cost of producing a better yeast product is low, so this new process may be an economically viable way to obtain heterologous proteins or peptides without high levels. the dignity of the products.

Accordingly, the present invention provides a process for producing an ethanol as well as a yeast heterologous protein or peptide.

3,84624, which process comprises using an aqueous sugar-containing medium with a yeast strain genetically modified to be capable of expressing a heterologous protein or peptide, recovering the ethanol thus generated, and obtaining said heterologous protein or peptide from fermentation products. The essential features of the invention are set out in the appended claims.

Yeasts are a group of lower eukaryotic microorganisms whose biological and biochemical properties of the microorganisms vary considerably. Usually, the term "yeast" is used to describe those strains of Saccharomyces cerevisiae that are of commercial importance in the bakery, brewing, and bulb industries. Related yeasts are used in winemaking as well as in the production of sake and in the production of fuel alcohol using sucrose or hydrolysed starch.

All yeasts used in the brewing, bakery and bulb industries can be taxonomically classified as Saccharomyces cerevisiae yeasts. This classification includes surface-fermenting yeasts (S. cerevisiae) as well as bottom-bearing lager yeasts (S. uvarum or S. carlsbergensis).

Strictly speaking, brewer's yeast differs from all other yeasts in that it is a yeast strain used to make beer, i.e., a strain of yeast that is currently used in the brewing process. Due to their fermentability, such yeasts must be able to produce a beer that is acceptable in terms of taste when using hop-containing malt extract (bulb wort). The main products of this fermentation process are ethanol and carbon dioxide, which are essential ingredients in beer. However, not all yeasts of the species S. cerevisiae are able to meet these requirements. Indeed, a critical factor in this regard is believed to be the ability of the yeast strain to form quantitatively minor metabolic products, such as esters, acids, higher alcohols, and ketones, in tightly balanced proportions.

Yeast may be unsuitable for brewing use because one or more of these minor metabolic products 4 84624 are produced in excessive amounts, either in absolute terms or relative to other products. (Rainbow, C.A., 1970, in "The Yeasts," ed. Rose, A.H. & Harrison J.S. Vol. 3, p. 147.)

In general, the properties of bulb yeast distinguish this yeast from other yeasts. Most industrial yeast strains, unlike laboratory yeasts, are incapable of sexual reproduction; they are said to be gay. Industrial yeasts are usually aneuploid or polyploids, so the chances of phenotypic detection of gene mutations are slim. Most polyploid strains do not form spores, or the viability of the spores they produce is very low, so performing a sensible genetic analysis is frustrating.

Together, these factors tend to provide a phenotype stability in industrial yeasts that may contribute to their selection for industrial applications. Similarly, the number of genes associated with high ploidy may contribute to the general suitability of such strains for use in the fermentation process compared to haploids and diplodes with poor fermentability.

In addition, bulb yeasts have a certain technological behavior that makes them well suited to their usual environment, hop-containing brewery wort.

The mode of operation of the new process depends on the type of industrial fermentation process. If the fermentation process is designed to produce an aqueous, potable liquid, such as beer, at the end of fermentation, the spent liquid is separated from the yeast (and usually also from other solid material present in the spent medium). Under these conditions, it is clearly desirable, and usually indeed essential, that the heterologous protein or peptide be insoluble in the fermented liquid, as it is usually not acceptable for the heterologous protein or peptide to be present in the drinkable liquid. Under these conditions, the heterologous protein or peptide is available from about 5,84624 yeast cells. However, if the alcohol is recovered by distillation, as is the case with the second and third types of industrial fermentation processes mentioned above, it may be acceptable, and even desirable, for the protein or peptide to be present in solution in the fermented solution.

The yeast strain used in the new process must, of course, be suitable for the type of industrial fermentation process under consideration. This object can be safeguarded by performing genetic modification in a yeast strain known to have desirable properties, as it has been found that those desirable properties that make a yeast strain suitable for a particular type of industrial fermentation process are not usually lost during genetic modification. For example, if the fermentation process is a process for making beer, then the yeast strain selected for genetic modification is preferably a known bulb yeast strain currently used in such fermentation processes. As already mentioned above, industrial strains of such brewer's yeasts have properties that differ from those of "laboratory yeast", such as the ability to use hop-containing brewer's wort.

Brewery wort is mainly a hot water extract of malted barley, or other cereal products prepared by soaking and germination, to which hops have been added as a flavoring agent. The most important parameter for yeast growth and metabolism is the carbohydrate and nitrogen composition (as well as the amino acid composition). This varies from country to country as well as from brewery to brewery, see for example "Malting and Brewing Science", Vol. 2, Hopped Wort and Beer; authors Hough, J.S., Briggs, D.E., Steven R. and Young, T.W., 1982, Chapman and Hall, London, New York, pp. 456-498. In general, a brewer's wort contains a total of 5-10 g of fermentable sugars per 100 ml of wort, at least half of which is maltose.

Other factors affecting yeast growth and performance are: (1) Growth factors. These include substances such as biotin, 6,84624 thiamine, riboflavin, pyridoxine, pantothenic acid and nicotinic acid. In general, brewer's wort is rich in these factors, which are consumed during yeast growth.

(2) Minerals. The mineral need of brewer's yeast is reminiscent of the mineral need of most living organisms. The brewery satisfies these needs as it contains very small amounts of metal ions such as iron, potassium, magnesium, zinc, manganese and copper, which are essential for vital metabolic enzymes.

The most significant difference between the laboratory culture medium and the brewery wort is the sugar composition of the medium. Most laboratory vessels use glucose as the main source of carbon, while maltose is the main fermentable ingredient in wort.

Brewery fermentations are usually carried out as anaerobic (oxygen-free) fermentation processes. However, oxygen is the most important condition for yeast growth in the early stages of fermentation. Most laboratory fermentations are designed to maximize the yield of yeast mass, while beer fermentations focus on ethanol production as well as the aroma of the product. Thus, in beer fermentation, the amount of inoculation ("inoculation") is greater than what would normally be used in a laboratory. Thus, the number of cell doublings (cell generations) is reduced between two and four per fermentation event.

The fermentation of the beer wort usually takes place at a temperature in the range of 8-25 ° C, with temperatures at the upper end of this range, for example 15-25 ° C, being used when the product is ale type, and temperatures in the range 8-15 ° C being used when the product is lager. type. Under laboratory conditions, yeasts are cultured at significantly higher temperatures, e.g., 25-35 ° C.

Similarly, in cases where the industrial fermentation process is a process for the production of alcohol to be separated by distillation, it is necessary to use genetically modified yeast,

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7 84624 obtained from a strain suitable for such fermentation. In such fermentation processes, for example, cereals, potatoes, cassava, sugar cane or sugar beet can be used as a source of sugars, and this source may optionally be pretreated, for example, by chemical or enzymatic hydrolysis to convert the cellulose and / or starch in these raw materials into fermentability.

Genetic modification of yeast can be carried out in a known manner. Suitable methods have been described in the literature, and certain methods are given in the examples below.

Numerous different heterologous proteins or peptides can be selected for expression in yeast. Examples are enzymes such as beta-lactamase, beta-glucanase and beta-galactosidase. Other useful heterologous proteins and peptides include materials of human origin that may be useful for medical purposes, such as human serum albumin and immunoglobulins. The literature discloses methods for genetically modifying microorganisms to express such proteins and peptides.

A heterologous protein or peptide obtained for use with genetically modified yeast can be used in a number of different ways. In the simplest case, the yeast retains the protein or peptide in its cell and the cells are used as is. However, it is usually advisable to isolate this heterologous protein or peptide. If the yeast secretes this protein or peptide into the surrounding medium, the fermentation medium is treated to isolate the protein or peptide. As noted above, this method is generally inappropriate if the fermented medium is intended to be ingested, for example, as a refreshment. In this case, the desired protein or peptide is obtained from the yeast formed during the fermentation process. For example, yeast cells can be disrupted to release the contents of the cells, and then the protein or peptide can be isolated from the contents of the cells.

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The following examples illustrate the invention in more detail. The accompanying drawing shows gene maps showing the formation of the plasmid used in one example.

These examples describe the conversion of brewer's yeast to produce beta-lactamase and / or beta-glucanase as heterologous proteins, as well as the use of the modified yeast in the brewing process.

β-Lactamase is the name given to a group of enzyme-forming proteins that act to catalyze the hydrolysis of 6-aminopenicillanic acid or 7-amino-cephalosporanic acid and the amide bond in the β-lactam ring of their N-acyl derivatives. Said derivatives are penicillins and cephalosporins, commonly known as β-lactam antibiotics (Citri, N., 1971, "The Enzymes", 3rd edition, edited by P.A. Boyer, IV, page 23).

β-lactamase is widely found in different bacterial genera and has been found in both Gram-negative and Gram-positive bacteria. The gene that determines β-lactamase production has often been found to be associated with both chromosomal and extrachromosomal elements. In intestinal bacteria, the β-lactamase gene can often be obtained by infecting an extrachromosomal body in the form of a plasmid and providing a resistance factor (or R factor). One such R factor that has the β-lactamase gene and thus confers resistance to β-lactam antibiotics in its host bacterium is R1 (Meynell, E.

& Datta, N., 1966, Genetical Research, 1, p. 134). This plasmid was identified from a clinical isolate sample of Salmonella paratyphi B (Meynell, E. & Datta, N., 1966, Genetical Research, 1, p. 134). The species specificity of β-lactamase has been questioned, as R factors are able to mediate their own migration and thus the migration of the β-lactamase gene in the genus Enterobacteriaceae (intestinal bacteria) (Datta, N. & Richmond, MH, 1966, Biochemical Journal, 98 :, p. 204). .

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With the development of genetic modification technology (recombinant DNA technology), there has been a need for easily manipulable plasmid vectors for use in DNA cloning. The β-lactamase gene present in plasmid R1 factor has been introduced into new plasmids to form new cloning vectors. One such vector is RSF 2124 (So, M. et al., 1975, Molecular and General Genetics, 142, page 239), which was constructed from plasmid Col E1 and a derivative of R1 drd 19 (Meynell, E. &

Datta, N., 1967, Nature, 214, page 885).

Subsequently, RSF 2124 has been manipulated into the plasmid vector pBR322 (Bolivar, F. et al., 1977, Gene, 2_, page 95), which has been further manipulated into plasmid pAT153 (Twigg, AA & Sherratt, D., 1980, Nature, 283, page 216). . The β-lactamase gene of R1 is conserved in all of these plasmid vectors and is capable of regulating the production of the β-lactamase enzyme in Escherichia coli.

Further manipulation of plasmid cloning vectors derived from plasmid pBR322, which thus also comprise the R1 β-lactamase gene, has been necessary to generate plasmids suitable for yeast transformation (i.e., plasmids that can be implanted into yeast). Thus, for example, plasmid pAT153 (Twigg, AJ & Sherratt, D., 1980, Nature, 283, page 216) is attached to segments of yeast chromosomal DNA (the LEU-2 gene of Saccharomyces cerevisiae, which determines an enzyme involved in leucine biosynthesis, β isopropyl malate dehydrogenase) and 2 plasmid DNA (2 is a yeast endogenous plasmid) to form plasmid pJDB207 (Beggs, JD, 1981, "Molecular Genetics in Yeast", ed. von Wettstein, D., Stenderup, A., Kielland-Brandt, M. & Friis, J.,

Alfred Benzon Symposium No 16, Munksgaard, Copenhagen, page 383).

β-lactamase was the first heterologous protein to be expressed in the yeast S. cerevisiae (Hollenberg, CP, 1979, ICN-UCLA Symposium Molecular and Cellular Biology, 15 ^, page 325; Hollenberg, CP, 1979, "Plasmids of Medical, Environmental and Commercial Importance ", 10 84624 ed. Timmis, KN & Puhler, A., Elsevier, page 481). The bacterial ampicillin resistance gene that determines (3-lactamase production) was derived from plasmid pBR325, a derivative of plasmid pBR322, and was ultimately derived from Salmonella paratyphi-B (see previous references). Synthesized by S. cerevisiae 8 β-lactamase protein has been purified 100-fold compared to crude extracts and has been shown to have exactly the same enzymatic activity, molecular weight and binding to specific antibodies as the corresponding properties of the purified protein from E. coli (Roggenkamp, R. et al., 1981, PNAS USA, 7j), page 4466). the level of β-lactamase expression in yeast is low due to the weak function of the bacterial gene promoter (regulatory region in the gene); however, gene expression can be greatly enhanced by the use of the yeast ADH1 promoter (alcohol dehydrogenate) (Hollenberg, CP et al., 1983, "Gene Expression in Yeast", eds. Korhola, M. & Väisänen, E., Proceedings of the Alko Yeast Symposium , Helsinki, page 73).

Yeast transformation (i.e., introduction of DNA into yeast) may be a relatively inefficient procedure, with success depending on the appropriate selection system. Most plasmids currently used to transform yeast are selectable because they have a wild-type gene that complements the auxotrophic mutation in a selected recipient strain that was a haploid laboratory strain of S. cerevisiae. However, brewer's yeasts are prototrophic and have no auxotrophic needs. To select for transformants from brewer's yeast, it is essential that the cell have a dominant gene that confers the ability to grow under otherwise unusual conditions. CUP-1 is the dominant yeast gene that determines the production of a protein capable of chelating copper ions. This gene is cloned into yeast / E. coli mediator vector pJDB207 by inserting restriction endonuclease-Sau3A-derived DNA fragments from strain X2180-1A to generate plasmid pET13 (Henderson, R.C.A., 1983, "The Genetics and Applications of Copper Resistance in Yeast").

Ph.D. dissertation, University of Oxford). The accompanying drawings li 11 84624 show a gene map of plasmid pET13: 1. Plasmid pET13 has the yeast chromosomal genes LEU-2 and CUP-1 and the origin of DNA replication of the yeast 2 plasmid, as well as DNA from plasmid pAT153; thus, pET13: 1 contains a bacterial β-lactamase gene known to express β-lactamase in yeast. Henderson (1983) describes in great detail methods for transforming brewer's yeast (ale yeast and lager yeast) with plasmid pET13: 1. Similarly, he describes the screening of β-lactamase activity in brewer's yeast transformants using the starch iodide plate assay below.

It is clear that bacterial β-lactamase protein is generated in brewer's yeast transformed with plasmid pET13: 1, and this can be demonstrated by growing the transformants in a suitable indicator medium.

The following describes the genetic modification of a particular strain of brewer's yeast by introducing the plasmid pET13. The yeast used was NCYC 240, which is ale yeast and is publicly available from the National Collection of Yeast Cultures (Agricultural Research Council, Food Research Institute, Colney Lane, Norwich, England).

Before strain NCYC 240 could be transformed with plasmid pET13 (CUP-1 / β-lactamase), its sensitivity to copper was determined. For this purpose, NCYC 240 yeast was inoculated on YED-glucose or YED-glucose agar (1% w / v yeast extract, 2% w / v peptone, 2% w / v glucose, solidified on 2% w / v agar) and grown for 2 days 28 ° C. They were then replated on NEP agar plates (MgSO 4 .7H 2 O 2 g / l, (NH 4> 2SO 4 2 g / l, KH 2 PO 4 3 g / l,

CaCl2.2H2O 0.25 g / l, yeast extract 2 g / l, peptone 3 g / l, glucose 40 g / l, solidified on 2% agar. Naiki, N. & Yamagata, S., 1976, Plant and Cell Physiology, 1-7 / page 1281) with increasing concentrations of copper sulfate (CuSO 4). The tested strain did not grow in NEP medium containing 0.1 mM CuSO 4. Thus, it was concluded that a concentration of 12,84624 of CuSO 4 in NEP medium in excess of 0.1 mM is sufficient for the selection of brewer's yeast copper-resistant transformants.

Plasmid DNA for plasmid pET13 was isolated from Escherichia coli strain K-12 strain JA221 (recA1, leuB6, trg_E5, hsdR-, hsdM +, IacY. Beggs, JD, 1978, Nature, 275, page 104) clarified cell lysate cesium bromide chloride / etium cesium chloride / etium by centrifugation, using DB Clewell and D.R. Helinski's method (1967, Proceedings of the National Academy of Science, USA, 6_2, page 1159), modified by Zahn et al. (1977, Molecular and General Genetics, 153, page 45).

Samples of NCYC 240 yeast were pretreated for transformation with plasmid pET13: 1 by two methods in each case: (A) J.D. By the method of Beggs (1978, Nature, 275, page 104), and (B) R.C.A. By the method of Henderson (1983, "The Genetics and Applications of Copper Resistance in Yeast",

Ph.D. dissertation, University of Oxford), however, with the exception that the protoplastic enzyme used was Zymolyase (40 μg / ml) (Kirin Brewery Co. Ltd.). 100 of the yeast spheroplasts produced by Methods A and B were mixed with 15 of pET13: 1 DNA (approximately 250 ug of DNA / ml) and treated with polyethylene glycol (1 ml of 40% PEG 4000). In 10 mM CaCl 2, 10 mM Tris / HCl pH 7-6). After treatment with polyethylene glycol, the cells were pelleted by agitation and gently resuspended in 500 μl of NEP-glucose medium containing 1.2 M sorbitol. Incubation lasted for one hour at 28 ° C, after which the cells were added to 10 ml of molten NEP-glucose medium with 3% agar containing 0.3 mM CuSO 4; and 1.2 M sorbitol. This was then poured into NEP glucose medium with 2% agar containing 1.2 M sorbitol and 0.3 mM CuSO 4. Transformation plates were incubated for four to five days at 28 ° C, after which yeast colonies rising on selective copper medium were detached and transferred to NEP-glucose agar containing 0.3 mM CuSO 4. These transplanted colonies were termed putative

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i3 84624 pET13: 1 transformants and were assayed below to ensure that they were genuine brewer's yeast transformants. The frequency of transformation in each of these two methods A and B using NCYC 240 yeast was <4 transformants / μg DNA and 20 transformants / μg DNA, respectively.

In order to ensure that the yeast or bacterial strain is an authentic transformant, it is common to check for the presence of one or more genetic traits as determined by the introduced plasmid DNA. Therefore, the high level of copper resistance as well as β-lactamase activity of the putative transformants described above were determined, as the genes in plasmid pET13 determine each phenotype (copper resistance / β-lactamase activity). The following methods were used: (a) High level of copper resistance. The putative pET13: 1 transformants, transfected and grown on NEP-glucose agar containing 0.3 mM CuSO 4, were further cultured by replating the colonies in the same medium and NEP-glucose agar + 1 mM CuSO 4. Those grafted colonies grown on media containing both 0.3 mM and 1 mM CuSO 4 clearly had a high level of copper resistance. This property is thought to be a feature of plasmid transformants containing the CUP-1 gene, as the number of copies regulates copper resistance in yeast (Fogel, S. et al., 1983, Current Gelnetics, 1_, p. 347). It is not unreasonable to expect that plasmid transformants have a high level of copper resistance due to the numerous copies of the plasmid genome. Subsequently, those transplanted colonies with high levels of copper resistance were subjected to the β-lactamase assay.

(b) β-lactamase assay yeast / E. for the detection of β-lactamase produced by yeast strains containing coli mixed plasmids is routinely applied to yeast transformants. (Chevallier, M.R.

& Aigle, M., 1979), FEBS Letters, 108, p. 179). The experiment strictly adheres to the method described by Chevallier and Aigle (1979), which involves the following measures: 14 84624

The experiment is based on the fact that penicillinase (β-lactamase) hydrolyzes penicillin, resulting in a reducing compound, penicillinic acid. The reducing effect of penicillinic acid is visualized by the disappearance of the color of the dark blue iodine-starch complex incorporated in the solid agar medium. Thus, if β-lactamase-producing strains are placed in the test medium, a colorless ring will appear around the β-lactamase-producing strain.

Test medium: Yeast nitrogen base (Difco) 0.65% w / v, glucose 0.1% w / v, soluble starch 0.2% w / v, agar 2% w / v, buffered 0.02 M with phosphate to pH 6-7.

Test medium containing soft agar: as above, but containing 1% w / v agar.

Reagent: 3 mg / ml 15 mg / ml CI; 0.02 M phosphate buffer pH 7; 3 mg / ml ampicillin.

Plates containing the test medium are inoculated with a putative transformant of brewer's yeast. The plates are incubated at 30 ° C for 18 hours. Prepare a mixture of 4 ml of molten, soft agar-containing test medium and 1.5 ml of reagent. The mixture is mixed and carefully poured onto the test medium. The dark blue plates are left at 30 ° C for one hour, after which they are placed at 4 ° C. After about 24 hours, β-lactamase-producing strains have produced a well-defined, white (colorless) ring, whereas plasmid-free control strains show very little and limited color loss. Thus, β-lactamase-producing transformants have been clearly distinguished from strains that do not contain the β-lactamase gene.

(c) Hereditary instability. Yeast strains transformed with 2 μm-based plasmids such as pET13: 1 and pJDB207 (pJDB207 is the parent plasmid of pET13: 1) are characterized by the inheritability of the plasmid.

li is 84624 This! the consequence of the instability is that a small proportion of the yeast cells in the population produce plasmid-free daughter cells as the cell divides. In the case of plasmid pET13, plasmid-free cells can be detected on the basis that these cells are sensitive to copper (NEP-glucose agar + 0.3 mM CUSO4). Thus, copper-resistant transformants (see (a) above) are transferred to YED glucose medium and allowed to grow for 3-4 days at 27 ° C. Colonies grown on YED medium are then replated on the same medium and NEP-glucose agar + 0.3 mM CuSO 4. Colonies lacking the copper resistance plasmid pET13 do not grow on copper-supplemented medium. A variation of this method for assessing defective phenotypes of brewer's yeast transformants can be used, in which the putative transformants are first inoculated onto NEP-glucose medium (liquid medium without agar) where they are allowed to grow overnight at 27 ° C. The next day, cells can be plated on NEP glucose agar at an appropriate dilution to obtain individual colonies after three days of incubation at 27 ° C. The yeast colonies can then be replated on NEP-glucose agar and this same medium supplemented with 0.3 mM CuSO 4. Brewer's yeast transformants containing plasmid pET13 can be spontaneously separated from copper-resistant derivatives on the basis that copper resistance can be removed.

Methods (a), (b) and (c) together are sufficient to ascertain whether the putative copper-resistant transformant is genuine. It is also recommended to examine the morphology of all putative transformant cells by light microscopy. Careful comparison of the transformant with the parent strain (i.e., untransformed brewer's yeast) indicates whether the transformant is indeed a genetically modified yeast or a contaminant.

If desired, other methods can also be used to confirm plasmid transformants.

16,84624 The yeast transformant thus obtained, known as NCYC 240 (pET13: 1), was deposited in the National Collection of Yeast Cultures, Colney Lane, Norwich, NR4 7AU, England, on December 12, 1984, under number NCYC1545,

A single colony of yeast NCYC 240 (pET13: 1), which was identified as a true plasmid transformant by the above methods, was allowed to grow on NEP-glucose agar with 1 mM CuSO 4 and inoculated into 200 ml of NEP-glucose medium supplemented with 0.2 mM with CuSO 4 (liquid medium). The culture was incubated in a shake flask at 28 ° C for two days, after which all 200 ml of this was inoculated into 5 liters of this same liquid medium. These cultures were allowed to grow in shake flasks at 20 ° C for four days. The 5-liter cultures were then diluted, each to an estimated 45 liters of stock. The wort was allowed to run for seven days, the yeast was recovered and inoculated into the ale wort prepared as follows.

95% ale malt and 5% crystal malt were mashed in South Staffords hire water at 65.5 ° C for 90 minutes. Hops were added up to 36 EBU and caramel color up to 30 EBU.

The mixture was boiled for 90 minutes at 1 bar, then allowed to stand in a whirlpool for 30 minutes. At the time of use, the specific gravity of the wort was 1055 ° at 15 ° C.

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The yeast was compressed and inoculated at 5.7 kg / m 2 and had a maximum fermentation temperature of 16 ° C. The beer was placed in storage tanks after the specific gravity dropped to 1012 °. The beer was allowed to develop at -1 ° C for 3 days. The beer was filtered and diluted to a specific gravity of 1038 °, 1008 PG, 24 EBU bitter content and 20 EBU color units. The ethanol content was 4%.

It was found that the beer was drinkable.

The beer sample was dialyzed, after which it was lyophilized. The β-lactamase activity of the lyophilized beer was determined and 17,84624 was found to have no detectable β-lactamase activity.

Similar procedures were performed using NCYC 240 yeast; lacking plasmid pET13 (i.e., unmodified NCYC 240 yeast), with the exception that the original yeast culture on NEP-glucose medium did not contain copper sulfate. Beers produced with both NCYC 240 yeast and NCYC 240 (pET13: 1) yeast were found to be somewhat similar by conventional triangular flavor assay and aroma profile analysis (for a review of these methods, see RJ Anderson, 1983 Brewers Guardian, November, p. 25). ).

During brewing using both yeast forms, samples of those yeasts were analyzed to evaluate cell number and living cells. The results showed that there was little, if any, difference between the yeasts in this respect. In the case of modified yeast, the proportion of cells containing the plasmid (pET13: 1) was determined, and it was found that relatively few cells lacked the plasmid. Other factors were also monitored during fermentation, for both modified and unmodified yeast. These included: a decrease in the specific gravity of the wort over time, an increase in the number of cells over time, and the size of the final yeast catch. It was found that no significant difference was found between the use of modified yeast and unmodified yeast with respect to any of these factors.

Some of the modified yeast produced in the fermentation process was still used in a similar brewing process, while the excess yeast served as a source of 8-lactamase.

The 8-lactamase content was determined by a biological assay as well as an enzymatic assay. In such experiments, cells are recovered by centrifugation for 10 minutes, after which they are resuspended in 0.1 M phosphate / citrate buffer, pH 6.5, and disrupted using a Braun homogenizer. Glass aliquots and ie 84624 cell debris are removed by centrifugation (8000 x g, 10 minutes) and the supernatant is centrifuged again (1000 x g, 30 minutes). When using a biological assay to determine the resulting cell-free extracts, penicillin-sensitive E. coli cells are plated on soft agar containing 25 ug / ml ampicillin. 25 μl of the extract of yeast NCYC 240 and NCYC 240 (pET13: 1) is applied to these plates, which are then incubated at 37 ° C. Strong bacterial growth is observed in the vicinity of NCYC 240 (pET13: 1) yeast colonies, whereas no such growth is observed in the vicinity of NCYC 240 yeast colonies. This indicates that NCYC 240 (pET13: 1) cells obtained from beer fermentation produce a substance capable of degrading penicillin and thus allowing the growth of sensitive E. coli cells, whereas NCYC 240 cells (unmodified) do not have this activity. This activity can be considered to be related to β-lactamase protein. Further evidence that such activity is associated with β-lactamase protein in NCYC 240 (pET13: 1) cells is available from the results of enzymatic experiments. The first of these experiments used a qualitative paper disc detection system in which samples of yeast cell extracts are spotted on Cefinase plates impregnated with chromogenic cephalosporin, Nitrocefin, whereby Nitrocefin changes from yellow to red in the presence of Dickinson Systems, BB ) (CH 0'Callaghan et al., Antimicrobial Agents and Chemotherapy, 1972, 1, p. 283). Cell-free extracts of the fermentation yeast NCYC 240 (pET13: 1) change the color of the discs from yellow to red, whereas NCYC 240 (unmodified) shows no discoloration on the disc, indicating the presence of 8-lactamase protein in NCYC 240 (pET13: 1), but not in NCYC 240 yeast. The activity of β-lactamase in yeast cell extracts is quantified using the same chromogenic cephalosporin, Nitrocefin, and C.H. 0'Callaghan et al. (1972, Antimicrobial Agents and Chemotherapy, 1, p. 283). Enzyme reactions are performed at 37 ° C in a 1 cm cuvette with a total volume of 1 ml of Nitrocefin solution (51.6 μg Nitrocefin 87/312 per ml 0.05 M phosphate buffer, pH 7) to which is added 20 ml. ^ ul i9 84624 cell-free yeast extract. The change in optical density of the reaction mixture is determined at 386 nm and 482 nm using a Beckman DU 7 spectrophotometer. The cell-free crude extracts of NCYC 240 (pET13: 1) yeast obtained from beer fermentation are capable of destroying 4.87 nanomoles of Nitrocefin 87/312 per minute per milligram of protein at 37 ° C and pH 7.0 (protein estimates are obtained at 230 wavelengths). adsorption of UV and 260 nm ultraviolet radiation to VF Kaihi and RW Bernlohrr (1977, Analytical Biochemistry, 82, p. 362) in crude cell extracts of yeast NCYC 240 (unmodified) and in boiled yeasts of yeast NCYC 240 (pET13: 1). at 100 ° C) the extracts have no 3-lactamase activity.

Measures similar to those described above in connection with the yeast NCYC 240 have also been applied to the privately owned brewer's yeast stock, and the results obtained were very similar.

The following is a modification of NCYC 240 yeast to cause the yeast to produce another proteinaceous material, namely 8-glucanase. Endo-1,3-1,4-8-D-glucanase (EC 3.2.1.73) is an enzyme that catalyses the hydrolysis of alternating sequences of 8-1,3- and β-1,4-bound 8-D-glucan , and such glucans include barley β-glucan and lichenan. The unique function of this enzyme prevents it from hydrolyzing the repetitive sequences of β-1,3-bound glucan such as laminarin and the repetitive sequences of β-1,4-bound glucan such as carboxymethylcellulose (Barras, DR, 1969, Cellulases and Their Applications ", 156th meeting of the American Chemical Society, September 11-12, 1968, Atlantic City, .s. 105).

The Gram-positive bacterium Bacillus subtilis produces extracellular endo-1,3-1,4-D-glucanase, which behaves similarly to the above enzyme (Moscatelli, EA et al. 1961, Journal of Biological Chemistry, 236, p. 2858, Rickes, EL et al.

1962, Archives of Biochemistry and Biophysics, £ 9, p. 371).

20o 84624 The chromosomal β-glucanase gene of B. subtilis has been isolated by gene cloning from a strain of B. subtilis designated NCIB 8565 (Hinchliffe, E., 1984, Journal of General Microbiology, 130, p. 1285). The active gene was found to be located in a 3.5 kilobase pair restriction endonuclease Eco RI fragment of DNA expressing a functional enzyme in E. coli. The cloned β-glucanase gene was found to encode an enzyme specific for the hydrolysis of barley β-glucan and was found to be primarily extracytoplasmic in E. coli (Hinchliffe, 1984).

Subsequently, the β-glucanase gene cloned by deletion analysis was found to be located in a 1.4 kilobase pair restriction endonuclease-PvuI-ClaI DNA fragment. The same location has been found for the B. subtilis bacterial β-glucanase gene isolated from strain NCIB 2117 (Cantwell, B.A. & McConnell, D.J., 1983, Gene, ^ 3, p. 211). A more precise molecular structure obtained by DNA sequence analysis of the NCIB 2117 strain has recently been published (Murphy, N. et al. 1984, Nucleic Acids Research, 12, p.

5355).

Yeasts, including S. cerevisiae, produce numerous different types of β-glucanases; however, none of these are capable of hydrolyzing β-1,3-1,4-bound glucan (Abd-El-Al, A.T.H. & Phaff, H.J., 1968, Biochemical Journal, 109, p. 347). From this, it can be concluded that yeast does not produce endo-1,3-1,4-8-glucanase. Therefore, the cloned β-glucanase gene from B. subtilis has been implanted in S. cerevisiae yeast, and it has been shown that this gene is capable of encoding a biologically active protein in S. cerevisiae yeast, and that the activity of this enzyme is typical of B. subtilis and For an enzyme found in E. coli (Hinchliffe, E. & Box, WG, 1984,

Current Genetics, 8, p. 471). Expression of the cloned β-glucanase gene in S. cerevisiae yeast is inefficient, depending on the amounts of biologically active enzyme produced in B. subtilis and E. coli containing the cloned β-glucanase gene. However, the enzyme activity in yeast is

II

2i 84624 can be detected only in crude cell extracts of yeast containing the cloned gene (Hinchliffe, E. & Box, W.G., 1984). This may mean that the cell is unable to secrete the yeast-produced enzyme out of the cell, with the enzyme being intracellular in nature; unlike an enzyme produced by bacteria that is extracellular.

To introduce the B. subtilis β-glucanase gene into the brewer's yeast NCYC 240, the plasmid pET13: 1, which is capable of replication in both E. coli and S. cerevisiae yeast, was used as a mediator vector, as mentioned above. The 3.5 kilobase pair Eco R1 DNA fragment present in plasmid pEHB3 was subcloned in vitro by rearranging it to the only Bam HI site on plasmid pET131, as can be seen in more detail in the accompanying drawing. In the gene map of the drawing, the radially lined arcs represent DNA from B. subtilis containing the β-glucanase gene (8G), the broad, unfilled arcs represent chromosomal DNA indicating the location of the LEU-2 and CUP-1 genes (Henderson, RCA, 1983, "The Genetics and Applications of Copper Resistance in Yeast", Ph.D. dissertation, University of Oxford), and narrow black arc lines denote 2 μm plasmid DNA and thick black arc lines denote E. coli vector DNA sequences. Treatment with T4 DNA ligase following cleavage of the pEHB3 plasmid by Eco R1 (Hinchliffe, E., 1984, Journal of General Microbiology, 130, p. 1285) was performed at dilute DNA concentrations, thus contributing to the degradation of Eco R1. joining two products into a ring. One of these products is a DNA ring derived from the broad black arc of plasmid pEHB3. When these products were digested with restriction endonuclease BglII, this ring was cleaved at the BglII site and a linear 3.5 kb fragment was generated. Meanwhile, pET13 was cleaved and the resulting linear fragment was ligated with the linear fragment from pEHB3 using T4 DNA ligase. This digestion and ligation was performed at higher DNA concentrations, which facilitated the fusion of 22,84624 B. subtilis rearranged DNA to the Bam HI site of plasmid pET13: 1 (Henderson, RCA, 1983, "The Genetics and Applications of Copper Resistance in Yeast ", Ph.D. dissertation, University of Oxford). Ligation is performed because the endonucleases Bam HI and BglII provide competing adhesive ends that interlock to form Bam HI / BglII hybrid sites that are not recognized by either of the endonucleases Bam HI or BglII. Transformants were found in E. coli strain HB101 to be resistant to ampicillin, sensitive to tetracycline, and β-glucanase-positive in E. coli, so they could be isolated from plasmid pEHB3 in E. coli. The direction of insertion of the rearranged Eco RI fragment in pEHB10 was determined by restriction endonuclease digestion followed by agarose gel electrophoresis. The new plasmid is named pEHB10.

Plasmid DNA was isolated from the strain HB101 containing the hybrid plasmid pEHB10; this DNA was transformed into brewer's yeast NCYC 240 as described above. Copper resistance was also selected as described above. Plasmid transformants of yeast NCYC 240 were confirmed by a combination of high-level resistance assays and β-lactamase assays from which NCYC 240 (pEHB10) was obtained.

Individual colonies of yeast NCYC 240 (pEHB10), NCYC 240 (pET13: 1) and NCYC 240 were inoculated into 200 ml of NEP-glucose medium (supplemented with 0.2 mM CuSO4, if appropriate). The cultures were incubated with shaking at 27 ° C for 2 days, after which each was inoculated into 2 liters of the same medium. After allowing to grow for 3 days at 27 ° C, the cells were harvested by centrifugation and washed twice with 0.1 M phosphate / citrate buffer, pH 6.4, before disrupting the cells with a Braun homogenizer. Supernatants were further treated as described above, except that each supernatant was dialyzed overnight against 2 x 21 0.1 M phosphate / citrate buffer, pH 6.4. Crude cell extracts of these three 23,84624 NCYC 240 yeasts were then subjected to β-glucanase experiments according to Hinchliffern and Box (1984).

These experiments showed β-glucanase activity associated with cell-free extracts of yeast NCYC 240 (pEHBIO) (1.17 nanomoles of reducing sugar was released from barley β-glucan per minute per milligram of protein at 40 ° C and pH 6.2), and no activity from cell-free extracts of both NCYC 240 (pET13: 1) and NCYC 240 yeast.

The yeast transformant thus obtained, identified as NCYC 240 (pEHBIO), is deposited in the Yeast Collection of the National Collection of Yeast Cultures, Colney Lane, Norwich NR47AU, England, on December 12, 1984, under number 1546.

A sample of NCYC 240 (pEHBIO) yeast was cultured as described above and used in a brewing process identical to that described above for NCYC 240 (pET13: 1) yeast. The process yielded a drinkable beverage that was substantially free of endo-1,3-1,4-6-D-glucanase. Yeast from the brewing process was found to contain the plasmid pEHBIO, which determines the production of β-glucanase (1 nanomolar of reducing sugars was released from barley β-glucan per minute per milligram of protein at 40 ° C and pH 6.4), so part of the yeast could be recycled (i.e. used in subsequent brewing processes) and part of the yeast could be used as a source of enzyme. In addition, crude cell extracts of yeast NCYC 240 (pEHBIO) from the brewing process contain both β-lactamase enzyme activity (2.33 nanomoles of Nitrocefin 87/312 were destroyed per minute at 37 ° C and pH 7.0 per milligram of protein). ) and the activity of the enzyme 8-glucanase. This indicates the potential for the simultaneous production of more than one heterologous protein in a genetically modified brewer's yeast, such as NCYC 240 yeast.

Endo-1,3-1,4-β-D-glucanase from B. subtilis is currently marketed as an enzyme preparation that can be used to overcome the difficulties associated with the presence of unwanted β-glucan. Thus, this method described above can be used to produce an enzyme suitable for this same purpose.

Claims (9)

A process for the production of ethanol and a protein or peptide which is heterologous to yeast, comprising fermenting an aqueous sugar-containing medium with a yeast, then extracting the ethanol and other so-formed products, characterized by fermenting the aqueous , the sugar-containing medium with an industrial genetically engineered modification of a Saccharomyces cerevisiae or S. carlsbergensis yeast strain capable of extracting a heterologous protein or peptide, the ethanol is extracted in the form of an aqueous liquid which is essentially free from yeast and from said heterologous protein or peptide and containing substantially all water and ethanol from the fermented medium, and said heterologous protein or peptide is obtained as the protein or peptide residue in the yeast produced during the fermentation. Process according to Claim 1, characterized in that said aqueous liquid is drinkable. 26 84624 Process according to claim 1, characterized in that the ethanol is extracted from the fermented medium in the form of an ethanol distillate. 4. Process according to claim 2, characterized in that the aqueous sugar-containing medium contains maltose as the sugar mainly present. Process according to claim 4, characterized in that the aqueous sugar-containing medium is a barley malt-based beer wort. Process according to claim 2, 4 or 5, characterized in that the fermentation is carried out at 8-25 ° C. Process according to claim 3, characterized in that the aqueous sugar-containing medium is a fermentation medium for the production of potable distilled ethanol or motor ethanol. Process according to claim 7, characterized in that the medium is based on cereals, potatoes, cassava, sugarcane or sugar beet, optionally pretreated to convert cellulose and / or starch therein into fermentable sugars. Process according to any of claims 1-8, characterized in that the fermentation is a substantially anaerobic fermentation.