Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
  • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
  • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
  • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts

Definitions

• the present invention refers to processes and additions of flocculant genes in microorganisms for the production of industrial raw materials and process using said microorganisms.
• this application refers to genetically modified microorganisms, a process to produce said organisms and fermentation processes using said organisms. These microorganisms flocculate after the end of the fermentation process, settling more quickly in the bottom of the fermentation vessel, thus separating from the final product obtained during fermentation. This consists in a purifying process.
• microorganisms to modify and produce medical or food raw materials is known since the early history of civilization, with a basic example in the process to ferment "juices" of materials rich in sugar, such as grape, barley and other elements which, after that process, are transformed by means of anaerobic reactions into other products such as wine, beer and other compounds.
• fermentation referred in biotechnology necessarily to anaerobic fermentation, in which microorganisms processed the raw material in the lack of air.
• the main products from anaerobic fermentation are alcohol, ethanol, alcoholic beverages and the bread industry.
• fermenters started to be used in aerobic fermentation processes.
• microorganisms are used to produce other substances such as proteins, pharmacologicals, industrial enzymes resulting from the expression of given genes naturally included in said microorganisms or introduced into these microorganisms by means of recombinant DNA skills.
• An important industrial problem for fermentation processes is to separate the microorganism from the final product of interest. This step is known as purification and is made by several ways. Purification skills are divided in two macro groups. The first skill involves the separation of the final product from larger components such as microorganisms. The second skill involves product purification after being recovered.
• centrifuges or rotation filtration under vacuum which, by means of the centrifuge force, force the accelerated separation of substances with different densities, thus separating the final product from microorganisms.
• decanting As decanting is natural, it becomes very slow, such as in the case of brandy production. Furthermore, it can cause early cell disruption, launching undesirable substances into certain processes, such as the wine manufacturing process.
• Cell disruption is another skill used when the product of interest is found within the cell, i. e. it has not been secreted. Disruption is made by means of chemical or mechanical agents breaking cell membranes, liberating the product into the medium.
• Adsorption chromatography is another very used process but, besides being expensive, it is highly specific for each kind of product of interest. All these processes become expensive for the delay to obtain the final product or for the high costs involved with the industrial plant required to process them.
• the phenomenon of flocculation can be used for various industrial purposes, being a part of the process to purify the product of interest.
• the combination of a conditional promoter with a flocculation gene optimizes the fermentation process, so to make use of the whole substract transformed into product as linked to the first purification stage.
• Genes intend to mean nucleotide sequences codifying peptides or proteins.
• Genes have a non-coding region called PROMOTER, subdividing genes into two classes: CONSTITUTIVE GENES, which are expressed apart from any stimulation and REGULATED GENES, depending on a stimulus to be expressed.
• each promoter has the own characteristics of the nucleotide sequence and position concerning the coding part, and can restrain or induce gene expression according to the components of the fermentation medium.
• ADH promoter adjacent to flocculation genes which are expressed in the lack of glucose and inhibited in the lack of said sugar.
• Watari refers to the flocculation gene FLO1 as found in yeasts.
• the main focus of Watari's patent is the gene, but Watari mentions in the examples the use of the coding part of the FLO1 gene with the promoter ADH for the production of beer with yeasts Saccharomyces cerevisiae.
• this reference concerns to beer production process, but said process with flocculants presents a few disadvantages, such as low fermentation speed; formation of lumps fluctuating on the surface and making fermentation and aeration of vats used in the process become difficult; loss of cells in the fermented lather and cloggings.
• Pereira (2000) has also presented a patent application (PI 0001122-3 A), in which he also uses the FLO1 gene with the ADH promoter, but applicable to the production of ethanol and wine.
• Said patent has three main limitations: i) it is limited to alcoholic fermentation, ii) it is limited to fermentation process (and does not cover microorganism and plasmides) and iii) it is limited to use in wild line microorganisms.
• That process is an attempt to enhance beer fermentation, by simply disrupting Lg-FLO1 gene or increasing its expression, by making use of the complete gene.
• the expression is enhanced or elliminated, it has only one way of operation during the whole fermentation: either it just flocculates or it never flocculates.
• Oliver's patent GB 2353798 also uses flocculation genes regulated by specific promoters. But the used flocculation gene is PKC1 with promoters SRB1 and PSA1. This construction is solely used for beer production. This combination of genes and promoters and their use for beer production are not the scope of our invention.
• the object of the present invention is to obtain a genetically modified microorganism which can have flocculation genes regulated by promoters which are started or restrained, depending on characteristics of chemical composition of the medium, pH or by physical excitations.
• the present application is different from the patents as mentioned above as state of the art for various reasons. First of all for the process, since all above patents described as state of the art refer to anaerobic fermentation processes, while the present application refers to aerobic fermentation processes. This difference brings unexpected effects with scientific and economical importance.
• the anaerobic fermentation process only allows the production of alcoholic products, such as wine, ethanol and alcoholic beverages in general.
• the aerobic fermentation process allows the production of other substances: e.g. proteins, pharmaceuticals, insuline, vaccines, industrial enzymes and enzymes in general. The production of these other substances results from the expression of given genes, naturally contained in said microorganisms or introduced into said microorganisms, by means of recombinant DNA skills.
• the present invention also refers to anaerobic fermentation processes but, in this case, we make use of combinations of flocculation genes, promoters and microorganisms which are different from those referred to in the patents of the state of the art. These differences also bring unexpected effects with scientific and economical importance. These different combinations of flocculation genes, promoters and microorganisms have also been applied to the patent applications for microorganisms, vectors, plasmids and cassettes, which can be used for both anaerobic and aerobic fermentation processes.
• the present application requires a patent not only for the process, but also for the genetically modified microorganism.
• a patent is also required for the vectors, plasmids and cassettes which are required to construct said genetically modified microorganism.
• Watari's and Oliver's patents claim just yeasts as genetically modified microorganisms.
• bacteriae, algae, protozoae, fungi and archae All these microorganisms have very different characteristics from yeasts, e. g. they are not eucariotes, their handling is simpler and they are able to survive under high temperatures and make photosynthesis.
• Pereira's patent does not claim the microorganism, vectors, plasmids and cassettes, but only claims a patent for the fermentation process. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
• Watari's patent mentions the use of FLO1 gene and ADH promoter for a yeast Saccharomyces cerevisiae strain W204 for beer fermentation.
• yeast Saccharomyces cerevisiae strain W204 for beer fermentation.
• FLO1 gene and ADH for yeasts with different characteristics from Saccharomyces cerevisiae, such as Pichia pastoris, Hansenula polymorpha, Saccharomyces fragilis, Saccharomyces ellipsoideus, Saccharomyces calsbergensis, Candida utilis, Candida lipolytica or Kluyveromyces lactis.
• Saccharomyces cerevisiae such as Pichia pastoris, Hansenula polymorpha, Saccharomyces fragilis, Saccharomyces ellipsoideus, Saccharomyces calsbergensis, Candida utilis, Candida lipolytica or Kluyveromyces lactis.
• yeasts are extremely more productive than Saccharomyces cerevisi
• Pichia pastoris and Hansenula polymorpha have very large productiveness for the production of proteins coded by genes located in given regions. They are used as highly efficient expression vectors, which are much more efficient than Saccharomyces cerevisiae.
• E. g. Saccharomyces ellipsoideus is used to ferment wines.
• E. g. Saccharomyces calsbergensis is used for "lager” beer production, while Saccharomyces cerevisiae is used for "ale” beer production.
• Candida utilis, Candida lipolytica and Kluyveromyces lactis are used to ferment ethanol.
• KIL-k2 From draught beer) Antonie van Leeuwenhoek, 1978, 44, 59 M. Richards 738. KIL-k2 (brewery contaminant) Antonie van Leeuwenhoek, 1978, 44, 59 A. P. Maule 761. KIL-k3 From palm wine), CBS 7903 J. Perm. Technol., 1985, 63, 421-429 N. Okafor 1001. KIL-k2 (brewing yeast) Antonie van Leeuwenhoek, 1978, 44, 59 1561. A8209B his4-864 KIL-k1 G. Fink via T. Young
• K398-4D a spol 1 ura3 ade6 arg4 aro7 asp5 met14 Iys2 pet17 trpl L. Johnston 1611.
• K382-23A a spoi l ura3 canl cyh2 ade2 his7 hom3 L. Johnston
• cdc9-7 (L94-4D) a cdc9-7 trpl ura3 L. Johnston 1670. cdc9-7 a adel ade2 ural his7 Iys2 tyrl gall cdc9-7 L. Johnston 1633. cdc9-7 rho- cdc9-7 rho- L. Johnston 1671. cdc9-8 a adel ade2 ural his7 Iys2 tyrl gah cdc9-8 L. Johnston
• cdc21 (MH21) cdc21/cdc21 gal1/gal1 ade1/ade1 +/ade2 his1/his7 trp2/+ +/ura1 +/Iys2 +/tyr1 2 ⁇ + L. Johnston 1718.
• JC2 (L31-9a [a]/L31-2c [alpha]) cdc9/cdc9 cdc28/cdc2 ⁇ ade/+ tyr1/tyr/1 canR/+
• alpha131-20 alpha ade2-R8 cyh2 canl leul ura3 L. Johnston 607.
• JCK5-5A alpha his4-A15 ade2-1 can(R) kar1-1 J. Conde
• g739-2a a rad50-1 canl hisl ade2 (or adeX) L. Johnston 1721. g739-2d alpha rad50-1 hom3-10 hisl trp2 L. Johnston
• K382-19D alpha spol 1 ura3 canl cyh2 ade2 his7 hom3 tyrl L.
• K382-23A a spol 1 ura3 canl cyh2 ade2 his7 hom3 L.
• Strain K Manchester brewery strain, 1 :5:4:2:1. 22 ⁇ .
• Strain R Sheffield brewery strain, 5:1 :1 :3:5.
• Strain V Burton-on-Trent brewery strain, 1 :5:5:3:1.
• Hybrid 1 (NCYC 227 x NCYC 22 ⁇ )
• Hybrid 15 (NCYC 227 x NCYC 232)
• Hybrid 18 (NCYC 220 x NCYC 232)
• Hybrid 24 (NCYC 222 x NCYC 221)
• Hybrid 30 (NCYC 223 x NCYC 221 )
• Hybrid 38 (NCYC 224 x NCYC 226)
• Hybrid 39 (NCYC 225 x NCYC 226)
• JB143 strain alpha Ieu2 ade2 65 ⁇ .
• JA36 strain a Ieu3 ade2 Iys10
• NCTC 466 ⁇ 3. A. Harden (1920). Carlsberg Laboratory strain 21 , NCTC 381. 84. H. J. Bunker (1945). NCTC 7043. 85. A. J. Kluyver (1939). NCTC 5916. 66. ATCC (1942). ATCC 7753, CBS 1321 , NCTC 6421.
• Saccharomyces chevalieri CBS 400, ATCC 9804, NCTC 2054. Type strain for Saccharomyces chevalieri. From wine.
• CBS (1997). CBS 2247, CL 504, CCRC 21961 , DBVPG 6172, IFO 1991 , NRRL YB-4237, NRRL YB-4254, VKPM Y 47.
• the most well-known flocculation genes are FLO1 or FLO1 S or FLO1 L, FLO2, FLO3, FLO4, FLO5, FLO6, FLO7, FLO8, FLO9, FLO10, FLO11 , PKC1 and Lg-FLO1.
• FLO1 or FLO1 S or FLO1 L FLO2, FLO3, FLO4, FLO5, FLO6, FLO7, FLO8, FLO9, FLO10, FLO11 , PKC1 and Lg-FLO1.
• genes FLO1 , FLO1s, FLO1 L and PKC1 have different characteristics from genes FLO1 , FLO1s, FLO1 L and PKC1. These different characteristics are, among others, the ability to deflocculate only with sugar, with no need of acids. This characteristic is very important to allow the process to be reversible by adding substract from the next fermentation cycle. Therefore, the yeast can be re-used with no need of one more stage in the production cycle which should occur at the time of adding the acid. Furthermore, the lack of need of adding acid makes the process become cheaper and simpler.
• HSP30 promoter together with flocculation genes generates a different effect, since this promoter is controlled by other factors, such as thermal shock, lack of nitrogen and other nutrients, fall of pH and high ethanol concentration. For these characteristics, it allows to control the end of the fermentation process and the start of flocculation through other mechanisms. It is therefore allowed to end fermentation not only as required when the glucose ends, but before or afterwards.
• a patent has also been claimed for the genetically modified microorganism, by using genes sfHou, fsulou, fsu2ou, tupl ou, cyc ⁇ ou, cka2 or FMC1 , which had not been the scope of any patent application in the state of the art, combining such genes with regulated promoters for effects characterizing the end of the fermentation process.
• these genes were regulated by a range of promoters, HSP30, pMET3, MOX and ADH.
• Some of the promoters with said characteristics are the following: ADH, Mox or HSP30p.
• Such promoters can be started or restrained at the end of a fermentation process, by adding flocculation genes to the coding part, causing the flocculation and settling of the microorganism in the bottom of the vessel in which said fermentation process is taking place. Said effect is interesting, among other things, to separate the microorganism from an eventual substance which production is desired.
• Such substances might be produced by the process of putting nutrients and microorganisms in a fermenter, in which microorganisms will produce interesting substances while being fed with nutrients. After nutrient consumption, the promoter activates flocculation genes and the microorganism would settle down in the bottom of the fermenter, separating from the substances of interest.
• the biological process under which the microorganism consumes nutrients can be ruled by aerobic or anaerobic breathing, and the substance of interest may be the result of an alcoholic fermentation or consequent substances from the transcription of specific genes.
• alcohol is produced from the conversion of sugar in a breathing process.
• the second process there is the transcription of a specific gene from the microorganism, and the final product is not the result of a metabolic cycle of cell breathing.
• Aerobic breathing consists of a fully different metabolic process. That difference allows the production of other substances than alcohol, such as pharmaceutical products (e. g. insuline, antibiotics, growth hormones and others), industrial and agricultural enzymes (such as xanthan gum, aminoacids, organic acids, flavors, vitamins, bioinsecticides and many others).
• pharmaceutical products e. g. insuline, antibiotics, growth hormones and others
• industrial and agricultural enzymes such as xanthan gum, aminoacids, organic acids, flavors, vitamins, bioinsecticides and many others.
• the anaerobic breathing process mentioned by Pereira (2000) is not appropriate to the production of said substances, but only alcohol.
• flocculation genes such as
• the gene FLO10 is deflocculated by the introduction of sugar again into the medium. This effect is important, since for other applications it allows to re-use microorganisms.
• the FLO1 gene can only be used for beer industry, since yeasts are not re-used there.
• the FLO1 gene can deflocculate with the introduction of sulfuric acid, but the FLO10 gene elliminates this costly stage, since it can already deflocculate in the next production stage, when sugar is re-introduced into the medium for the next fermentation.
• Another aspect is the use of promoters aside from ADH, such as MOX and HSP30p. These promoters have different characteristics. Mox, although also regulated by the presence of glucose such as ADH, has a more intense expression than ADH, causing higher flocculation rate.
• the HSP30p promoter has other interesting characteristics, since it can be activated not only by the lack of glucose, but also by the lack of nitrogen, or it can be activated by physical stimulation, such as thermal shock or pH fall.
• Another characteristic is the use of not only wild lines, such as claimed by Pereira, but also non-wild lines, with essential consequences to the economical feasibility of the project.
• the non-wild lines are most easily manipulated in laboratories, and can therefore be used in recombinant DNA skills, as well as the production in controlled aerobic fermentations for the production of pharmaceuticals, industrial enzymes and other products.
• non-wild lines have well-known markers, thus facilitating to select modified cells and avoiding the use of markers with higher public health risks.
• Another aspect of the wild lines is that they are diploid and not haploid, such as laboratory lines. Being diploid, wild yeasts have higher productiveness rate, but demand much more work to make a genetic modification, since two copies of the gene must be modified. In haploid yeasts, there is only one copy to be modified. Wild yeasts also have a different characteristic in terms of natural selection in fermentation tanks, besides being less stable to a plasmid change.
• One of the ways to practice the invention is to put in a plasmid promoters such as ADH, MOX or HSP30p together to the coding part of flocculation genes such as FLO1.
• FLO1 has insufficient flocculation index in various yeasts, since this gene presents a repeating portion and frequently suffers recombinations.
• FLO1L or FLO1 S gene has much higher efficacy.
• genes FLO1 , FLO1 L and FLO1S only deflocculate in the presence of an acid, such as sulfuric acid.
• the FL010 gene deflocculates with the presence of sugar, generating deflocculation by the raw material of the next producing cycle itself, making the fermentation process become simpler and cheaper.
• Cells are grown in a medium with the specific antibiotic of the marker to only select genetically modified yeasts.
• Said selected yeasts are then able to conditionally flocculate, following the expression characteristics of the promoter. They can flocculate when glucose ends, when nitrogen ends, when there is a thermal shock or an abrupt fall of pH.
• flocculation activators occur at the end of the fermentation process, to make best possible use of nutrients.
• the flocculation cassette has been introduced into the yeast by means of electroporation, using the following standards: 1.5 kV, 200 ohm and 1.2 cm tub.
• the transformed yeast has been selected for the lack of uridine and uracil in the culture medium.
• the fermentation process has taken place in a minimum medium supplemented with glucose. After 17 hours of culture, flocculation occurred.
• the use of the FLO10 gene makes the introduction of glucose after the occurrence of flocculation, making the flocculation process to be visibly reversible.
• the transformed strain has remained stable during culture.

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• 2002
  • 2002-07-08 BR BR0203754-8A patent/BR0203754A/pt not_active IP Right Cessation
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