BRPI0306254B1 - process for reducing the single cell protein nucleic acid content by expressing a heterologous nuclease - Google Patents

Abstract

"Process for Reducing Single Cell Protein Nucleic Acid Content by Expressing a Heterologous Nuclease". The present invention is a process for eliminating or significantly reducing the nucleic acid content of yeast single cell proteins as well as the transformation vector and the transgenic strain to improve the production of scp for animal or human consumption. an innovative alternative to solve the problem of world hunger.

Description

FIELD OF THE INVENTION FOR "PROCESS FOR REDUCING SINGLE CELL PROTEIN NUCLEIC ACID CONTENT BY EXPRESSION OF A HETEROLOGICAL NUCLEASE" FIELD OF THE INVENTION

The present invention is a process for eliminating or significantly reducing the nucleic acid content of single cell proteins as well as the transformation vector and the transgenic strain for the production of single cell proteins for animal or human consumption. .

BACKGROUND OF THE INVENTION

Single-cell proteins (SCP) are proteins derived from single cells, such as bacteria, yeast, fungi, algae or protozoa (SUZUKI, T., Microbiological conversion -Production of Single- Cell Protein Biomass handbook Kitani, O., Hall, CW (Eds.) Gordon and Breach Science Publishers, New York, 1989), cultivated on a large scale, for use as a protein source in food or feed (LITCHFIELD, JH, Single Cell Proteins, Science 219: 740, 1983).

The use of microorganisms as a food source, while it may seem unacceptable to some, may offer an innovative alternative to solve the problem of world hunger. For thousands of years, men have been intentionally or unintentionally consuming products such as alcoholic beverages, cheeses, yogurts, soy sauce and, together with these products, the microorganisms responsible for their production (TUSE, D. Single cell protein: current status). and future prospects, Crit Rev Food Sci. 19: 273, 1984).

The first use of SCP as a protein supplement was in Germany during World War I, cultivating "baker's yeast", S. cerevisiae, in a mixture of molasses and ammonium salt as sources of carbon and nitrogen, respectively. Later, also in Germany, during World War II, Candida utilis, known as "Torula yeast", was cultivated in liquid sulfite residues resulting from acid hydrolysis of wood (paper manufacture) and used as a protein source for humans and humans. animals (WILEY, AJ, In: Industrial Fermentations, LA Underkoffler, RJ Hickey, (Eds.) Chemical Publishing Co., New York, p. 307, 1954).

[005] One of the most important characteristics for the development of microorganisms as a food supplement is its ability to rapidly convert cheap raw materials into proteins and can reach high levels of protein by dry biomass, even when compared to foods such as milk (25). % - 30%), eggs (35%), etc. (BRESSANI, R., ELIAS, LG Processed vegetable protein mixtures for human consumption in developing countries. Adv, Food Res. 16: 1, 1368 and KIHLBERG, R. The microbes as a source of food. Annu. Rev. Microbiol. 26 : 427, 1972}.

On the other hand, the cost of producing protein in the form of SCP, or other unconventional forms such as seed extract, may be much lower than conventional protein production (WORGAN, JT Single cell protein, In: Protein in Human Nutrition, Porter and Rolls (Eds.), Academic Press, 1973) (Table 1).

Table 1 * $: value in dollars * lb: weight in pounds [007] The most significant cost in SCP production is raw material (43% - 77%), followed by substrate (29% -64%) and, Finally, the overall cost of maintenance (12% - 24%). When substrate is some form of waste (WSL and bagasse), there is a change in this order: substrate (17% - 26%) and maintenance (25% - 37%) (MOO-YOUNG, M. Economics of SCP production. Biochemistry 5: 6, 1977).

For non-solid substrates, of the total investment cost, fermentation is the largest at 43% - 52%, followed by drying, harvesting (8% - 14%) and media preparation (1.1%). - 16%). With the solid bagasse substrate, the data are more expensive for the preparation of culture medium (22%), but nevertheless cheaper for drying (4%) (MOO-YOUNG, M, supra).

Other factors affecting the economic viability of the SCP process are cost capital, plant location, reservoir for raw material and product, and maintenance of product viability, as well as cost of raw material (sources of carbon, energy, nitrogen and nutrient minerals), energy, water supply, waste treatment, labor and maintenance of fully aseptic conditions.

[010] The nutritional value of SCP varies according to the microorganism employed. Components such as vitamins, nitrogen, carbohydrates, fatty acids, cell wall components, nucleic acids, protein concentration and amino acids should be analyzed before the product is used for protein supplementation or feeding.

[011] Algae are microorganisms rich in vitamins A, B, C, D and E, proteins (40% - 60%), minerals (7%), fatty acids, chlorophyll and fibers and have a low concentration. of nucleic acids (4% - 6%) (BROCK, TD A textbook of industrial microbiology. Sunderland, MA: Sinauer Associates Inc., p. 306, 1989).

[012] While algae are rich in all types of vitamins, fungi provide only B-complex vitamins and a low concentration of nucleic acids (9.7%) (FRAZIER, WC WESTHOFF, DC Food microbiology. New Delhi: Tata McGraw Hill Publishing Company Limited, 398, 1990). In yeast the protein content can reach 48% of its dry weight. Other aspects such as lipid, carbohydrate and nucleic acid contents are also analyzed. Lipids such as linolenic, oleic and linoleic acids can reach values of 15%, 20% and 40% (SCHELL, P., AKIN, C., Functional properties of yeast grown on ethanol. J. Am. Oil Chem. Soc., 56: 82, 1979) and carbohydrates may vary between 20% and 35% of dry weight, depending on the nature of the medium used for growth, whereas the concentration of nucleic acids in yeast may vary from 8% to 13% (PHAFF, HJ (Enzymatic yeast cell wall degradation (Adv. Chem. Ser. 160: 244, 1977)).

[013] SCPs from bacteria have high protein content and some essential amino acids, especially methionine (2.2% - 3%). The protein composition of bacteria reaches 80% of the dry weight, however the nucleic acid content, especially RNA, is very high (15% - 16%) (SIGH, BD Biotechnology. New Delhi: Kalyani Publishers, p. 498). , 1998).

[014] Although algae is a great source of nutrients, there are some limitations for human consumption such as the presence of the cell wall, which cannot be digested due to the absence of cellulase enzyme (TREHAN, K. Biotechnology. New Delhi: Wiley Eastern Limited , pp. 79, 1993).

[015] The use of bacteria for the production of SCP is limited due to the high cost of processing as bacterial cells are very small in size and do not flocculate. In addition to this, there is the high content of nucleic acids and the psychological barrier to the use of bacteria as a food source (TREHAN, K, supra).

[016] Nucleic acids, when ingested by humans in large amounts, result in increased plasma level of uric acid, which causes a metabolic disorder that can lead to uricacidemia, kidney stones, and gout. Since the problems arising from the accumulation of uric acid in humans have been identified, several processes have been developed to eliminate or significantly reduce the nucleic acid content of SCPs.

[017] The World Health Organization has stipulated that the acceptable amount of nucleic acids for human consumption is at most two grams per day. Thus, the consumption of yeast or bacterial proteins should not exceed 10 g of yeast or bacterial protein in the form of cell biomass (KAPSIOTIS, GD, Single cell protein: Review and assessment. Food Nutr. 4: 2, 1978). SCRIMSHAW, NS, Single Cell Protein for Human Consumption - An overview In: Single Cell Protein II, SR Tannenbaum, DIC Wang, Eds., MIT Press, Cambridge, 24. 24. LITCHFIELD, JH, Food Technol. 31: 175, 1977; REED, G., Microbial biomass, single-cell protein, and other microbial products In: REED, G. (ed.) Prescott & Dunn's Industrial Microbiology 4th ed., Avi Publishing Company Inc., Westport, 1981. Chap. 13, p.541, 1981).

[018] Traditional treatments for reducing nucleic acid content are based on pH variation, followed by increasing or decreasing temperature, which leads to disruption of nucleic acids. Although reduction of nucleic acids is achieved, in the process of purification of cell proteins, considerable losses of protein mass occur.

[019] Temperature rise and pH variation (heat shock process) has also been shown to be effective in activating endogenous ribonucleases, although with unsatisfactory results (SINSKEY, AJ, TANNENBAUM, SR, Removal of nucleic acid in SCP. Single Cell Protein II (SR Tannenbaum, DIC Wang (Editors), MIT Press, Cambridge, Mass, p. 158, 1975).

[020] Other studies have demonstrated the use of an autolytic method that allowed an 85% reduction in nucleic acid present in Candida utilis (MAUL, SB, SINSKEY, AJ, TANNENBAUM, SR, New Process for reducing the nucleic acid content of yeast. Nature, 228: 181, 1970). This process, like the previous one, stimulates the action of ribonucleases, causing breakdowns of nucleic acids, but, on the other hand, activates proteases that eventually lead to a decrease in protein yield.

[021] In yeast, nucleic acid contents can also be reduced by controlling their growth rate (SINSKEY and TANNENBAUM, 1975. supra), which, however, also decreases productivity. Increasing pH may reduce RNA content (LINDBLOM, M., The influence of alkali and heat treatment on yeast protein. Biotechnol. Bioeng. 16: 1495, 1974; VANANUVAT, P., KINSELLA, JE Extraction of protein low in nucleic acid, from Saccharomyces fragilis grown continuously on crude lactose J. Agric Food Chem 23: 216, 1975 and HEDENSKOG, G., MORGREN, H. Some methods of processing for single cell protein Biotechnol Bioeng 15: 129 , 1973). However, this process promotes a reduction in the amounts of cysteine, cystine and serine and consequent protein denaturation.

[022] In recent years, however, an interesting strategy has been used to produce SCPs with reduced nucleic acid content: the addition of nucleases. These enzymes, which target the genetic material, are added to the SCP production pathways at the end of the process after cell lysis, or are induced to activity even internally, depending on the microorganism.

[023] MARTINEZ et al. (New insolubilized derivatives of ribonuclease and endonuclease for elimination of nucleic acids in single cell protein concentrates. Biotechnol. Appl. Biochem. 12: 643, 1990) used insoluble pancreatic ribonuclease and Staphylococcus aureus endonuclease to reduce the percentage of acid. nucleic acid of yeast SCP concentrates. Hydrolysis of nucleic acid in SCP is initiated with ribonuclease followed by the addition of the endonuclease derivative. This treatment demonstrated a 5% to 15% reduction in nucleic acids, with a 6% protein loss.

[024] Serratia marcescens bacterial nuclease, which acts nonspecifically against DNA and RNA, is also used to reduce the nucleic acid content of SCP. Known commercially as Benzonase, the use of this enzyme provided a 3% to 6% reduction in DNA against a 50% reduction in RNA, with negligible protein loss. Hydrolysis of nucleic acid in SCP was performed by adding Benzonase to the dorns, along with yeast strains (MORENO, JM, SANCHEZ-MONTERO, JM, BALLESTEROS, A., SINITERRA, JV. concentrates using immobilized benzonase (Appl. Biochem. Biotechnol. 31: 43, 1991).

[025] In BALAN, A. F., Suicidal yeast system based on degradation of genetic material for biosafety. Doctoral Thesis, Institute of Biomedical Sciences, University of São Paulo. P. 180, 1999, a strain of Saccharomyces cerevisiae was constructed, which produces the serratia marcescens nuclease internally and in a controlled manner from the expression of the nucA gene inserted into a yeast bacterial episomal plasmid. This nuclease has been shown to be able to nonspecifically "in vitro" and "in vivo" double-stranded DNA molecules so that yeast carrying plasmid with the nuclease gene die and can be released into the environment. without risks of horizontal genetic transfer to other organisms. These results opened the prospect of developing a biosafety system, but showed a low stability of the transgene produced by the described process.

[026] Heterologous protein expression systems from gene fusion allow high levels of heterologous proteins to be obtained which can be secreted into the medium or maintained within the cell.

[027] The major system for the secretion of heterologous proteins produced by S. cerevisiae utilizes the pheromone signal sequence or alpha factor of the yeast itself (BRAKE, AJ Secretion of heterologous proteins directed by the yeast α-Factor leader. PJ, BRAKE, A., VALENZUELA, P. (eds) Yeast Genetic Engineering Butterworth, Boston, 269, 1989; KURJAN, J., HERKOWITZ, I. Structure of a yeast pheromone gene (MF a): a putative precursor contains four tandem copies of mature a-factor (Cell 30: 933, 1982). In this system, the desired gene is cloned in the form of gene fusion with the sequence encoding the alpha factor signal peptide immediately after the dividing site (BRAKE, AJ, MERRYWEATHER, JP, COIT, DG, HEBERLEIN, UA, MASIARZ). , FR, MULLENBACH, GT, URDEA, MS, VALENZUELA, P., BARR, PJ (Factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 81: 4642, 1984) .

[028] Another system that takes advantage of fusion protein expression was built by SABIN, EA, LEE-NG, CT, SHUSTER, JR, BARR, PJ. High-level expression and in vivo processing of chimeric ubiquitin fusion proteins in Saccharomyces. cerevisiae. Bio / Technology. 7: 705, 1989, and is based on the in vivo processing of chimeric proteins between yeast ubiquitin (amino terminal) and heterologous protein (carboxy terminal) via the highly ubiquitin hydrolase enzyme specific for the Arg sequence Gly-Gly from the carboxy terminal end of ubiquitin itself (VARSHAVSKY, A., BACHAMAIR, A., FINLEY, D., GONDA, D., WUNNING, I. Targeting of proteins for degradation. In: BARR, PJ, BRAKE , A. and VALENZUELA, P. eds., Yeast Genetic Engineering (Butterworth, Boston, p. 109, 1989).

[029] This system was initially used by VARSHAVSKY et al. (1989, supra) for producing recombinant proteins with each of the different amino acid residues at their amino ends.

Subsequently, it has been found that this system may lead to very high levels of expression of processed heterologous proteins (SABIN et al., 1989, supra). Initially, high-level expression of human interferon gamma and alpha 1-proteinase inhibitor was obtained, which had amino ends equal to the authentic proteins. Several regions of HIV envelope and integrase proteins (BARR, PJ, POWER, MD, LEE-NG, CT, GIBSON, HL, LUCIW, PA were also expressed through this system. Expression of active human immunodefficiency virus reverse transcriptase in Saccharomyces cerevisiae Bio / Technology 5: 486, 1987).

The first recombinant proteins were obtained in yeast using gene promoters encoding glycolytic pathway enzymes, such as the production of human alpha interferon using the alcohol dehydrogenase I (ADH1) gene promoter (HITZEMAN, RA , HAGIE, FE, LEVINE, HL, GOEDDEL, DV, AMMERER, G., HALL, BD Expression of a human gene for interferon in yeast (Nature 293: 717, 1981).

[032] The phosphoglycerate kinase gene PGK promoter was used for the production of heterologous proteins (TUITE, MF, DOBSON, MJ, ROBERTS, NA, KING, RM, BURK, DC, KINGSMAN, SM, KINGSMAN, AJ Regulated. high efficiency expression of human interferon in Saccharomyces cerevisiae EMBO J. 1: 603, 1982; CHANG, CN, MATTEUCCI, M., PERRY, LJ, WULF, JJ, CHEN, CI, HITZEMANN, RA Saccharomyces cerevisiae secretes and correctly human processes interferon hybrid proteins containing yeast invertase signal peptides (Mol. Cel. Biol. 6: 1812, 1986) with expression levels similar to those obtained with the ADH1 promoter. Systems based on control by the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter gene (BITTER, GA, EGAN, KE Expression of heterologous genes in Saccharomyces cerevisiae using vectors the glyceraldehyde-3-phosphate dehydrogenase promoter gene. Gene 32: 263 , 1984), allowed the expression of Herpes virus thymidine kinase (ZHU, XL, WARD, C., WEISSBACH, A. Control of Herpes simplex virus thymidine kinase gene expression in Saccharomyces cerevisiae by yeast promoter sequence. Mol. Gen. Genet 194: 31, 1984) and HIV reverse transcriptase (BARR et al., Supra).

High levels of expression are obtained when small portions derived from these promoters are used (ROSENBERG, S., COIT, F., TEKAMP-OLSON, P. Glyceraldehyde-3-phosphate dehydrogenase derived expression cassettes for constitutive synthesis of heterologous proteins. Meth, Enzymol, 185: 341, 1990). Based on these characteristics hybrid promoters were constructed containing GAPDH promoter regions and inducible promoter regulatory regions such as GAL1 (BITTER, GA, EGAN, KE Expression of interferon-from hybrid yeast GPD promoters containing upstream regulatory sequences from GAL1-GAL10 intergenic region. Gene 69 : 193, 1988), GAL7 (WAGENBACH, M., O'RORURKE, K., VITEZ, L., WIECZOREK, A., HOFFMAN, S., DURFEE, S., TEDESCO, J., STELER, G. Synthesis of wild type and mutant hemoglobins in Saccharomyces cerevisiae. Bio / Technology. 9: 57, 1991) and ADH2 (COUSENS, LS, SHUSTER, J., GALLEGOS, C., KU, L., STEMPIEN, MM, URDEA, MS, SANCHEZ-FISHERMAN, R., TAYLOR, A., TEKAMP-OLSON, P. High-level expression of proinsulin in the yeast Saccharomyces cerevisiae Gene 61: 265, 1987).

[034] S. cerevisiae yeast has four alcohol dehydrogenase isoenzymes: ADHI, ADHII (CIRIACY, M., Isolation and characterization of further cis and trans acting regulatory elements involved in the synthesis of the repressible alcohol dehydrogenase (ADHII) in Saccharomyces Gen. Gen. 176: 427, 1979), ADHIII (YOUNG, ET, PILGRIM, DB, Isolation and DNA sequence of ADH3, a nuclear gene encoding the mitochondrial enzyme of alcohol-dehydrogenase in Saccharomyces cerevisiae. Mol. Cell Biol. 5: 3024, 1985) and ADHIV (PAQUIN, CE, WILLIAMSON, VM Ty insertion at two sites for most of the spontaneous antimycin Resistance mutations during growth at 15 ° C of a Saccharomyces cerevisiae strain lacking alcohol-dehydrogenase I Mol. Cell. Biol. 6: 70, 1986). The great advantage of using the ADH2 promoter is that it is repressed in the presence of glucose, its activity being induced about 100 times with the total consumption of all fermentable carbon sources (LUTSTORF, U., MEGNET, R. Multiple forms of alcohol dehydrogenase in Saccharomyces cerevisiae (Arch. Biochem. Biophys. 126: 933, 1968). In the regulatory region of this gene, a 22bp palindromic sequence was found (RUSSEL, DW, SMITH, M., WILLIAMSON, VM, YOUNG, ET Nucleotide sequence of the yeast alcohol dehydrogenase II gene. J. Biol. Chem. 258: 2674, 1983), which was identified as UAS (Upstream Activation Site), which is responsible for the induction of the ADH2 gene in cells grown in the absence of glucose.

The genes required for the induction of ADH2 have been identified and belong to two different types, some specific for the expression of ADH2 and others necessary for the expression of other glucose-suppressed genes (SHUSTER, JR Regulated transcriptional systems for the production of proteins in yeast: Regulation by carbon source In: BARR, PJ, BRAKE, A., VALENZÜELA, P. Yeast Genetic Engineering (Butterworth, Boston, p. 83, 1989).

The ADR1, ADR6 and ADR4 gene products are responsible for the specific action on the ADH2 gene promoter. For the ADR1 gene, two types of mutants are found, the recessive adr1 form, unable to induce ADH2 and the dominant form ADR1C, with high baseline levels of ADHII production in the presence of glucose. ADR1C mutants also produce more ADHII in non-fermentable carbon source media compared to wild lines (SHUSTER, 1989, supra).

[037] The ADR1 gene is constitutive (DENIS, CL, GALLO, C. Constitutive RNA synthesis for the yeast activator ADR1 and Identification of the ADR1-5C mutation: Implications in post-translational control of ADR1. Mol. Cell. Biol. 6 : 4026, 1986) and ADR1 protein activity is regulated by cyclic AMP-dependent protein kinase (CHERRY, JR, JOHNSON, TR, DOLLARD, CA, SHUSTER, JR, DENIS, CL Cyclic AMP-dependent protein kinase phosphorylates and inactivates the transcriptional activator ADR1 Cell 56: 409, 1989). ADR1C mutants show alterations in the ADR1 protein phosphorylation mode. The interaction of this protein with the 22 bp sequence corresponding to the UASadm has already been demonstrated (EISEN, A., TAYLOR, WE, BLUMBERG, H., YOUNG, ET. The yeast regulatory protein ADR1 binds in a zinc dependent manner to the regulatory upstream activating sequence of ADH2, Mol. Cell. Biol. 8: 4552, 1988).

[038] Another ADH2 transcriptional activator is the ADR6 gene, although its mechanism of action has not been fully clarified (TAGUSHI, AKW, YOUNG, ET The cloning and mapping of ADR6, the gene required for sporulation and expression of the alcohol -dehydrogenase II isozyme for Saccharomyces cerevisiae (Genetics 116: 531, 1987). The ADR4 gene participates in ADR1-dependent ADH2 activation (CIRIACY, 1979, supra).

The identification of the UAS regulatory regions of the ADH2 promoter allowed the use of segments of this promoter for the regulation of the expression of heterologous proteins containing these UASADH2. The first use of this promoter in the production of a heterologous protein involved the synthesis and secretion of human epithelial growth factor (hEGF) using the alpha factor leader sequence (SHUSTER, JR Regulated gene expression heterologous proteins in yeast.) In: STEWART, GG, RUSSEL, I., KLEIN, RD, HIEBSCH, RR Biological Research of Industrial Yeast II (CRC Press, Boca Raton, Florida, USA, p. 19, 1987).

In order to increase the yield of heterologous protein production, hybrid promoters containing UASAdh2 were fused with different glycolytic pathway gene promoters, which have higher transcription efficiency.

In particular, the hybrid promoter ADH2 / GAPDH, described by COUSENS et al. (1987, supra) for the synthesis of human insulin, which has been widely used in the synthesis of various recombinant proteins, such as HBsAg and other viral antigens (VALENZUELA, P., MEDINA, A., RUTTER WJ, AMMERER, G., HALL, BD Synthesis and assembly of hepatitis B virus surface antigen particles in yeast (Nature 2: 374, 1982). The ADH2 / GAPDH hybrid promoter is subject to glucose repression due to the UASADH2 sequence. This regulatory control has advantages over other expression systems in S. cerevisiae, such as GAL1,10 (GUARENTE, L., YOCUM, RR, GIFFORD , P. A GAL10 :: CYC1 Hybrid yeast promoter identifies the GAL4 regulatory region as an upstream site Proc. Natl. Acad. Sci. USA 79: 7410, 1982), PH05 (BOSTIAN, KA, LEMICE, JM, CANNON, LE , HALVORSON, HO Physiological control of repressible phosphatase gene transcripts in Saccharomyces cerevisiae (Mol. Cell. Biol. 3: 839, 1983), because in this system, the addition of an external inducer is not necessary to induce the expression of the heterologous protein, which It occurs spontaneously once all the glucose in the medium has been consumed, making the process much simpler and cheaper. Finally, ADH2 derepression does not require specific host lineages or mutations (COUSENS et al., 1987, supra), although some mutations may potentiate the system.

[042] In general, because expression systems use high copy number plasmids per cell, it is important to maintain a balance between UAS and their activating proteins, avoiding a likely limiting effect due to activator insufficiency (IRANI, M , TAYLOR, WE, YOUNG, ET Transcription of the ADHII gene in Saccharomyces cerevisiae is limited by positive factors that bind competitively to its intact promoter region on multicopy plasmids. Mol. Cell. Biol. 7: 1233, 1987). In the case of expression using ΌΑΞαοης, therefore, overexpression of ADR1 is recommended, which can be obtained by adding one copy of ADR1 to the expression vector or by integrating multiple copies of this gene into the host genome, or by integration of a copy of this gene into the genome but subjected to regulation of a strong promoter (SHUSTER, 1989, supra).

BRIEF DESCRIPTION OF THE FIGURES

[043] Figure 1: Schematic representation of the construction of the pUCC Nuc vector and obtaining the CNC fragment for integration into the Saccharomyces cerevisiae genome.

[044] Figure 2: Digestion of plasmid pUbNuc with Smal and NruI enzymes to obtain expression cassette. 1. non-cleaved pübNuc, 2. Smal / NruI cleaved pübNuc, 3. ÀHind (50 ng / pL) [045] Figure 3: Confirmation of plasmid construction pUCCNuc - 1. pUCCNuc uncleaved, 2. pUCC EcoRV linearized Nuc , 3. HindIII-digested pUCC Nuc (arrow shows 4540 bp CNC integrative fragment), 4. Sall-digested pUCC 5. XHind (50 ng / pL) [046] Figure 4: PCR reaction for confirmation of integration of the recombination CNC fragment into the V chromosome of transformed lines - 1. ÀHind (50 ng / pL), 2. S288C / CNC, 3. PE2-CNC, 4. JSC302 / CNC, 5. XHind (50 ng / pL) [047] Figure 5: Glucose consumption along the cell growth of different yeast strains Saccharomyces cerevisiae [048] Figure 6-A: Survival curve of S288C / CNC strain in YPD2% medium.

[049] Figure 6-B: Survival curve of the PE-2 / CNC strain in YPD2% medium.

[050] Figure 6-C: Survival curve of JSC302 / CNC strain in YPD2% medium.

[051] Figure 7: PCR Reaction Result to certify presence of integrated CNC fragment in different S. cerevisiae yeast transformants - 1. XHind (50 ng / pL), 2. S288C / CNC, 3. PE-2 / CNC, 4. JSC302 / CNC, 5. Negative Control, 6. XHind (50 ng / pL) [052] Figure 8: Dry mass of the different transformants and their controls as a function of time.

[053] Figure 9: Double-stranded DNA (dsDNA) content of transformed strains and their controls as a function of time.

[054] Figure 10: Protein concentration of transformed strains and their controls.

DESCRIPTION OF THE INVENTION

[055] The present invention has as its main object to provide a process for reducing the single cell protein nucleic acid content by expressing a heterologous nuclease, the expression vector and the transgenic strain. Thus, this process aims to stabilize the yeast expression system, consisting of the ADH2 / GAPDH hybrid promoter and the Serratia marcescens bacterium nucA gene by chromosomal integration into the yeast, comprising the following steps: (a) Construction of the pUCCNuc intermediate plasmid; (b) Obtaining the CNC integrative fragment containing the Serratia marcescens nucA gene expression cassette; (c) Yeast transformation with the CNC integrative fragment; (d) Selection of yeast transformants showing stability of Serratia marcescens nuclease production.

[056] The present invention has sought to develop a system for rendering Saccharomyces cerevisiae yeast capable of non-specifically self-degrading nucleic acids with the aim of reducing the DNA and RNA content in the SCP end product.

To this end, the expression cassette containing the serratia marcescens bacterium nuclease gene under the control of the S. cerevisiae ADH2 / GAPDH promoter was cloned into the pUCC plasmid, described by CAMARGO, ME, Yeast Transformation System and detection of recombinogenic activity. Doctoral thesis. ICB-USP, e.g. 97, 2000 (Figure 1).

[058] ADH2 promoter activity is suppressed by glucose (DENIS, CL, FERGUSON, J. and YOUNG, ET mRNAs for the fermentative alcohol dehydrogenase of Saccharomyces cerevisiae decrease on growth on a fermentable carbon source. J. Biol. Chem. 258: 1165, 1983; COUSENS et al., 1987, supra), so that yeast can achieve maximum cell growth and therefore produce a large amount of protein mass before the bacterial nuclease gene is expressed and causes the yeast to grow. your death. The main aspect of this system is the destruction of nuclease yeast genetic material, which will prevent the formation of nucleoprotein complexes normally produced at the time of cell lysis for SCP processing.

For the construction of the plasmid of interest, pUCCNuc, we started from the plasmid püb Nuc, constructed by BALAN (1999, supra), which carries a truncated form of the S. marcescens bacterium nucA gene, devoid of the coding sequence. of the signal peptide. This truncated version of the nucA gene is under transcription control of the S. cerevisiae hybrid promoter ADH2 / GAPDH, so that the nucA gene will only be expressed when there is no glucose in the yeast culture medium. As intermediate plasmid, plasmid pUCC was used (CAMARGO, 2000, supra).

[060] There is a great advantage in using a plasmid that has the ability to integrate into the host yeast genome as the problem of plasmid loss across cell divisions is minimized and heterologous information remains stable in the genome. of the organism for countless generations.

[061] A second advantage of the system employed in the present invention stems from the fact that only the gene of interest is added to the host cell genome, without any other undesirable gene sequence, such as bacterial antibiotic resistance markers, etc. . Since the purpose of the present invention is to suit the yeast for food purposes, this feature is of even greater relevance.

[062] Previous studies show that most of the methods used to reduce the nucleic acid content in the SCP end product, in addition to promoting its higher cost, also promote a decrease in its final yield.

[063] In the present invention, it was possible to stabilize the nuclease expression system in S. cerevisiae, achieving 100% stability.

In the following, the present invention will be explained in more detail with the aid of the description of its preferred embodiment.

Materials and Methods BACTERIAL TRANSFORMATION BY ELECTROPORATION PREPARING COMPETENT CELLS

From a pre-grown overnight culture, 1 mL was inoculated into 100 mL of LB medium (Luria broth, as described in SAMBROOK, J.; FRITSCH, EF; MANIATIS, T. Molecular Cloning: A laboratory manual. New York: Cold Spring Harbor, Cold Spring Harbor Laboratory Press, 1989), followed by incubation at 37 ° C with shaker shaking at 110 rpm until reaching an absorbance at 600 nm of about 0.5-0, 6 The suspension was then collected in centrifuge tubes, allowed to stand on ice for about 15 min - 30 min and centrifuged at 4500 g for 15 min at 4 ° C. The supernatant was removed and the pellet resuspended in 100 mL of sterile distilled water at 4 ° C and centrifuged. After centrifugation, the cells were resuspended in 50 mL of sterile ice-cold distilled water and followed by further centrifugation. The cells were resuspended in 2 mL of 10% glycerol at 4 ° C and centrifuged for the last time. Cells were resuspended in a final volume of 300 μύ with 10% ice-cold glycerol (concentration of 1-3 x 10 10 cells / mL). The cell suspension was aliquoted into 1.5 mL Eppendorf tubes in 40 μΒ aliquots and stored at -70 ° C.

TRANSFORMATION

[066] Cells were removed from the -70 ° C freezer, placed immediately on ice and used only after thawing. About 40 μL of the cell suspension was placed in frozen microcentrifuge tubes with 2 μΒ DNA. The BioRad "Gene-Pulser" electroporator was calibrated according to the cuvette used. The cell / DNA mixture was transferred to the cuvette at 4 ° C and inserted into the electroporator. Then the pulse was applied under the established conditions. Immediately after the pulse, the cuvettes were removed from the pulse application chamber and 1 mL of SOC medium (described in SAMBROOK et al., Supra) was added to the cells. The suspension was then transferred to a test tube and incubated at 37 ° C with 110 rpm shaking for 1 h. After this incubation time, in order to express the resistance gene present in the plasmid, the bacterial suspension was seeded in plates containing selective solid medium and the plates incubated in an oven at 37 ° C for 24 h.

SMALL SCALE PLASMIDIAL DNA PREPARATIONS

[067] E.coli DH5a clones (F '/ endAl, hsdR17 (rK-, mK +), supE44, thi-1, recAl, gyrA, relAl, (Nalr), relAl, Δ (lacZYA-argF) U169, (Φ 8011acZAM15) (SAMBROOK et al., Supra)) were inoculated into 5 mL LB medium containing ampicillin at the final concentration of 100 μg / mL medium. This culture was incubated at 37 ° C overnight (approximately 16 h) with shaking at 110 rpm. An aliquot of 3 mL was centrifuged in 2 Eppendorf-type tubes at 4500 g for 2 min, the supernatant discarded and the excess removed with a fine tip pipette. The pellet was resuspended at 300 μ! of solution I (25 mM Tris-HCl, pH 8.0; 10 mM EDTA; 50 mM glucose) with pipette. Then 300 μ were added! of solution II (0.2 N NaOH; 1% (w / v) SDS), and the tube was allowed to stand for 5 min at room temperature. 300 μΐ solution III (3 M sodium acetate, pH 4.8) was added and the tube was stored for 10 min on ice. After this time, the tubes were centrifuged for 15 min at 4 ° C at 10,000 g. The supernatant was recovered and to this was added 600 μΈ chloroform / isoamyl alcohol solution (24: 1 solution), mixed by inversion and centrifuged for 2 min at 4500 g at room temperature. The aqueous phase was then recovered and to this was added 600 μΐ of isopropanol. The solution was mixed by inversion and left for 30 min at room temperature. The solution was centrifuged at 10,000 g for 15 min at 4 ° C. The pellet was washed with 70% ethanol at 4 ° C and vacuum dried for 10 min. The dried residue was resuspended in 50 μΐϋ sterile bistilled water containing 1 μg RNAase. The material was submitted to electrophoretic run in 0.8% agarose gel. AVERAGE PLASMIDIAL DNA PREPARATIONS (MIDI PREPARATION) [068] E.coli clones were inoculated into 50 mL LB medium containing ampicillin at the final concentration of 100 μg / mL medium. This culture was incubated at 37 ° C overnight (approximately 16 h) with shaking at 110 rpm. The culture was centrifuged at 4 ° C for 5 min at 4500 g and the supernatant discarded. The pellet was resuspended in 1000 μl of pipette solution I. Then 2000 μ 2000ι of solution II was added, and the tube was allowed to stand for 5 min at 4 ° C. 1500 µl of solution III was added, and the tube was stored for 30 min at 4 ° C. After this time, the tube was centrifuged for 15 min at 4 ° C at 8500 g. The aqueous phase was then recovered and to this was added 8 mL of isopropanol. The solution was mixed by inversion and left for 30 min at room temperature. The solution was centrifuged at 10,000 g for 15 min at 4 ° C. The pellet was washed with 70% ethanol at 4 ° C and vacuum dried for 10 min. The dried residue was resuspended in 2 mL of buffered phenol and the tube was shaken vigorously and then centrifuged for 2 min at 4500 g. This operation was repeated using chloroform / isoamyl alcohol solution (24: 1 solution). The aqueous phase, containing the DNA, was transferred to another tube, to which 2 volumes of isopropanol were added at room temperature. After 15 min incubation, another centrifugation was performed for 15 min at 7800 g, and the obtained supernatant was discarded. The precipitate was dissolved in 0.4 ml TE elution buffer (20 mM Tris-HCl, pH 8.0; 20 mM EDTA) and transferred to the Eppendorf microtube. 60 μΕ of 3 M sodium acetate and 1 mL of ethanol -20 ° C were added. After incubation for 10 min at -20 ° C, 2 min was centrifuged at 15000 g. The final precipitate was dissolved in 0.2 mL TE, to which 20 μΕ RNAse A (1 mg / mL) was added and incubated for 30 min at 37 ° C. DNA precipitation was performed by adding 20 μΕ 3 M NaAc, 0.3 μΐ ethanol -20 ° C with incubation for 10 min at room temperature. Centrifuged for 2 min and the supernatant was discarded. Resuspension of the DNA was performed in 100 µL of -TR suspension buffer (10 mM Tris-HCl, pH 7.5; 1.0 mM EDTA).

[069] DNA electrophoretic run was performed on agarose gel (as described in SAMBROOK et al., Supra) and DNA treatment with enzymes, as recommended in New England Biolabs or Gibco-BRL catalogs. Resin absorption DNA elution was performed according to the BioAgency Geneclean II Kit protocol. DNA POLYMERASE (PCR) CHAIN AMPLIFICATION REACTION [070] Confirmation of the integration of the expression cassette (CNC fragment) into the V chromosome of S. cerevisiae yeast strains, in addition to stability analyzes, was obtained by Reaction DNA polymerase chain amplification (PCR). The template used was the total DNA obtained from the extraction of the different yeast transforming clones.

[071] For PCR reactions, the following primers were employed: 1. Sequence referring to the 3'-end of the CNC fragment, composed of a fragment of the internal region of the Saccharomyces cerevisiae CAN1 gene.

Reverse Amplification 5 'TCA AAG CTT GCA TAA ATC TG 3' 2. Sequence referring to the 5 'end of the CNC fragment, composed of a fragment of the internal region of the S. cerevisiae CAN1 gene.

Forward Amplification 5 'GTT GAA GCT TCA CAA ACA CAC 3' [072] All primers used in the reactions were synthesized by GIBCO-BRL. The reagents employed in the reactions, as well as the enzyme Platinum Pfx DNA polymerase, were sourced from INVITROGEN.

[073] Reactions followed the enzyme manufacturer's indications, including the program applied. The equipment used was DNA enzinae - MJ Research - model PTTC-200 Peltier Thermal Cycler.

DNA HYBRIDIZATION REACTION

[074] The DNA hybridization reaction followed the description of SAMBROOK et al., Supra. As a probe for hybridization, the Sall - SstII fragment corresponding to the nucA gene, taken from plasmid pUbNuc (Figure 1), was used.

Yeast Transformation

[075] Yeast transformation was performed as described in GIETZ, D .; St. Jean, A .; WOODS, R.E .; SHIESTL, R.H. Improved Method for efficient transformation of intact yeast cells. Nucl. Acid Res. 20: 1425, 1992.

SELECTION OF TRANSFORMERS

[07 6] For the selection of cells transformed with CNC integrative fragments, 60 μg / mL (final concentration) of a fungistatic drug in the medium, L-canavanine, was added, allowing direct selection of transformants. The insertion of the CNC fragment into the CAN1 gene interrupts the production of L-arginine permease, preventing the entry of this amino acid into the cell and consequently also preventing the entry of the toxic analog L-canavanine. Thus, recombinant strains lose sensitivity to this agent and form colonies on the medium containing L-canavanine. For selection of JSC 302 strain transformants, growth factors leucine, tryptophan, and uracil were also added to the final concentration. 40 pg / mL, since the strain has auxotrophic mutations for these.

ANALYSIS OF FRAGMENT STABILITY INTEGRATED IN THE Yeast GENOME

For the analysis of CNC integrative fragment stability in recombinant clones, the percentage loss of cloned information per generation was evaluated. Successive chimes were made on non-selective medium every 24 h for 5 days. The initial inoculum, as well as all subcultures, contained approximately 1 x 10 6 cells / mL complete medium (YPD), described in SHERMAN, F; FINK, GR; LAWRENCE, CW, Methods in Yeast Genetics: Laboratory Manual. New York: Cold Spring Harbor Laboratory, 1979. Every 24 h, cultures were washed, diluted and seeded in minimal solid medium (SD - SHERMAN supra) plus amino acids or fungistatic L-canavanine and YPD medium. After incubation in an oven at 28 ° C for 48 h, colonies were counted by comparing the number of colonies grown on SD plates with those grown on YPD plates. Transformant colonies grown on selective medium were subjected to PCR analysis to determine the presence of the CNC fragment. The estimated number of generations and plasmid loss per generation were calculated according to the formulas described in RUBIÓ, I.G.S., Construction of a multi-integrative expression vector for the genetic transformation of S. cerevisiae. São Paulo, 1996. 132 p., Master's Dissertation - Institute of Biomedical Sciences, University of São Paulo.

DOSAGE OF GLUCOSE CONCENTRATION IN FULL MEANS

[078] The consumption of glucose in the complete growth medium by S. cerevisiae strains was measured using the "Commercial Glucose-Enzyme Kit" (CELM - Cia. For this, the manufacturer's instructions were followed, dosing aliquots, diluted 10 and 20 times, for the first 12 consecutive hours and then, 24 hours, 48 hours and 72 hours in spectrophotometer, Abs505. All measurements were made in duplicates.

CULTURE OF TRANSFORMERS FOR QUICK ANALYSIS OF EXPRESSION OF RECOMBINANT PROTEIN IN LIQUID MEDIA

Following pre-inoculum growth, 1.5 x 10 6 cell / ml were inoculated into 25 ml YPD2% medium and incubated under the same conditions for 120 h. Every 12 h, aliquots were taken for total and viable cell count. These were detected in YPD2% Agar medium after two days of incubation in a 28 ° C oven. Survival rate (N) was calculated by the ratio of viable cell number (NV) to total cell number (NT).

TOTAL DRY MASS ANALYSIS OF TRANSFORMED LINES AND CONTROL

[080] For dry mass analysis, 10 mL aliquots of transformed yeast culture and control were collected over the growth time in YPD2% medium. Each 10ml aliquot was vacuum filtered with previously weighed membranes. The membranes were dried in a microwave oven for 18 min, power 135W, then transferred to desiccator for 10 min and then weighed on an analytical balance. To determine the dry mass, the following equation was used: MS = MMC - MM (1 - u% / 100) x 1000 / vol Where: MS: Dry mass MMC: Membrane cell mass after drying (g) MM: membrane mass (g) Vol: volume of sample suspension u: membrane moisture (%} dsDNA ANALYSIS OF TRANSFORMED LINES AND CONTROL

[081] Aliquots taken at 24 h, 72 h and 96 h were collected from transformed and control strains and their total DNAs extracted for analysis of double stranded DNA (dsDMA) content. This analysis was performed on the Eppendorf Biophotometer and the reading was taken at an absorbance of 260 nm (A260), where each absorbance unit equals 50 pg / mL DNA.

EXTRACTION AND QUANTIFICATION OF TOTAL Yeast PROTEINS

[082] Total protein extractions were performed in 24h, 72h and 96h aliquots of the transformed and control strains and subjected to total protein analysis by the Eppendorf Biophotometer apparatus.

[083] For total protein extraction aliquots of ImL were collected from cultures in Eppendorf-type tubes, followed by centrifugation. To the pellet was added 100 µl distilled water, 100 µl 0.2 M NaOH, followed by incubation at room temperature for 5 min. After incubation, these cells were centrifuged and the supernatant discarded again. 50 µl of SDS sample buffer was then added and the mixture was subjected to 100 ° C for 3 min (KUSHNIROV, VV Rapid and reliable protein extraction from yeast. Yeast 16: 857, 2000) and then the concentrations of Total proteins were analyzed in the Eppendorf Biophotometer apparatus at 280 nm wavelength. Sample buffer solution IX and 1 mg / mL BSA solution were used as blank.

RESULTS CONSTRUCTION OF THE pUCCNuc PLASMID FOR THE CHROMOSOME INTEGRATION OF THE SERRATIA MARCENSENS NUCLEASE GENE IN Saccharomyces cerevisiae [084] The expression cassette consisting of the hybrid promoter ADH2 / GAPDH of S. cerevisiae and the ubiquisia gene of S. cerevisiae from the S. marcescens nuclease and the S. cerevisiae alpha factor transcription terminator, was removed from plasmid pUbNuc after dividing with the enzymes Smal and NruI. This 3190 base pair (bp) fragment was ligated into the pUCC vector, previously cleaved with BstEII, to yield the pUCC Nuc plasmid (Figure 1).

Thus, expression of the cassette of interest is independent of insert orientation and allows rapid analysis of plasmid DNAs extracted from bacterial transformants using only one restriction enzyme. After confirmation of the insertion of the cassette of interest into the pUCC plasmid, it was resubmitted with the HindIII enzyme to remove the cassette, now flanked by sequences from the S. cerevisiae CAN1 gene.

[086] This last 4540 bp fragment, called CNC, was used to transform different strains of S. cerevisiae yeast. Obtaining the expression cassette containing the serratia marcescens nuca gene [087] To obtain the expression cassette, the plasmid pUb Nuc was cleaved by digestion with the restriction endonucleases Smal and NruI for which there are sites. flanking the cassette, releasing a 3190 bp fragment (Figure 2). Binding of FRAGMENT CORRESPONDING TO NUCLEASE EXPRESSION CASSETTE IN pUCC VECTOR AND E. coli CELL TRANSFORMATION [088] The 3190 bp fragment of plasmid pübNuc and the pUCC vector, previously linearized by BstEII digestion, were subjected to a mixture of binding to approximately 250 ng DNA and T4 DNA ligase. After an incubation period of 24 h at 16 ° C, the ligation mixture was used for transformation of the E. coli DH5a strain by the electroporation method.

874 AmpR transformants were obtained and of these 100 transformants were analyzed by restriction analysis.

Only one transformant presented the plasmid with insert of a 3190 bp fragment that corresponds to the nuclease expression cassette, all other results being recircularization of the pUCC plasmid without insertion of the desired fragment. The chimeric plasmid was called pUC CNuc (SEQ ID No 2) and was subjected to restriction analysis to confirm insertion of the expression cassette into the correct pUCC plasmid site (Figure 3). Yeast Transformation Saccharomyces cerevisiae [091] For the transformation of S. cerevisiae yeast, three different strains were chosen for comparison of nuclease expression and activity: S288C (MATcl, wt, CAN1, standard genome sequence sequenced in GOFFEAU A, BARRELL BG , BUSSEY H, DAVIS RW, DUJON B, FELDMANN H, GALIBERT F, HOHEISEL JD, JACQ C, JOHNSTON M, LOUIS EJ, MEWES HW, PHILIPPSEN P, TETTELIN H, OLIVER SG.

Life with 6000 genes. Science 274: 563, 1996), PE-2 (wt-ethanol production - Cans), JSC302 (MATa; leu2-3,112; trpl; ura 3-52; prbl-1122; pep4-3; prcl4Q7 :: DM15 (pGAPDH / ADR1, G418 *); cir1, CAN1). PE-2 is a strain used in alcohol production plants, S288C is the standard strain of S, cerevisiae and JSC302 is a protease synthesis deficient S. cerevisia mutant strain and with an additional copy of the ADR1 gene, which has potentiating action of UASADh;> · [092] The constructed pUCCWuc plasmid was used for the transformation of yeast strains after their treatment with HindIII enzyme and obtaining only the integrative CNC fragment.

[093] Results for the transformation of each lineage are presented in table 2.

Table 2 ANALYSIS OF Yeast Transformers Saccharomyces cerevisiae FOR VERIFICATION OF CNC FRAGMENT INTEGRATION IN THE GENOME

[094] To confirm the integration of the nuclease gene expression cassette into the host yeast genome, a hybridization reaction of some of the transformants obtained from each strain (S288C, PE-2 and JSC302) was performed using the gene. nucA, and we performed PCR reaction (Figure 4), which amplifies the sequence referring to the CNC fragment that has been integrated in the yeast genome, besides ensuring that the integration of the CNC fragment by recombination occurred in the desired chromosome.

Primers were constructed that recognize sequences upstream and downstream of the fragment binding site to make sure that the amplified sequence actually contained the expression cassette. The results presented here undoubtedly showed that in all three strains the CNC fragment was integrated into the correct chromosome (Figure 4).

DETERMINATION OF GLUCOSE CONSUMPTION

[096] Glucose consumption in the culture medium was determined so that the duration of glucose repression of the ADH2 / GAPDH promoter and the timing of its activation were known. For this, 1 x 10 6 cells / ml of S288C, PE-2 and JSC320 strains, without the integrated plasmid, were inoculated into the YPD2% culture medium and incubated for 72 h. Samples were taken at different times and used for glucose determination and cell number counting (Figure 5).

[097] S. cerevisiae PE-2 industrial yeast showed the best growth, with all glucose consumed within 14 h, as is the case with laboratory strains S288C and JSC302, which showed a total glucose consumption in the period at least 24 h and 48 h respectively.

[098] Consequently, the ADH2 / GAPDH promoter is deaerated from 14 h for industrial yeast PE-2, while for S288C and JSC302 yeast, promoter derepression with consequent nuclease expression will only begin from 24 h and 48 h, respectively. PRELIMINARY ANALYSIS OF NUCLE GENE EXPRESSION UNDER CONTROL OF THE HYBRID ADH2 / GAPDH PROMOTER IN S. CERevisiae LIQUID CROPS [099] The 60 transformants of the different S. cerevisiae strains, which had the CNC fragment integrated into their genome, were used for one. rapid analysis of expression in SD1% liquid culture medium (low glucose concentration allows ADH2 promoter to be decompressed faster, leading to nuclease expression) by reading optical density at 600nm to verify which transformants were who suffered the nuclease action more quickly. From this experiment, one clone from each strain was chosen for the following assays, which were performed in triplicates.

[100] After precultivation in YPD8I medium, 1 x 10 5 cells from each clone were transferred to 5 mL of YPD2% medium. The flasks were incubated on a shaker and the percentage of culture survivors was monitored by the ratio between cell number. viable and the number of total cells.

[101] As can be seen from Table 3, all transformants with the integrated CMC fragment had lower survival rates than control cells without the integrated fragment. This result becomes evident from 72 h of incubation on YPD2I medium.

Table 3 [102] The JSC302 strain carrier bearing the CNC fragment was more sensitive to the suicidal effect than the other strain S288C / CNC and PE-2 / CNC, as it achieved 0% survival much earlier in 96 h. This is probably due to the fact that this strain has in its genome an extra copy of the ADR1 gene, which is responsible for the up-regulation of the ADH2 promoter. SURVIVAL OF TRANSFORMERS AND H YPD2% CULTURE MEDICINE Saccharozoyces cerevisiae S288C LINE TEST

[103] The clone S288C / CNC was chosen for the assay because as can be seen earlier, the transformed strain that has integrated into its genome the CNC fragment containing the nuclease expression cassette grew less compared to the control cells.

[104] S. cerevisiae S288C / CNC transformants showed normal growth up to 60 h. From this moment on, the suicidal effect began to manifest itself, and by 120 h viable cells were no longer detected (Figure 6-A). Saccharomyces cerevisiae PE-2 LINEAGE TEST [105] The same conditions were used for the S. cerevisiae PE-2 and PE-2 / CNC cell assay.

[106] Overall, this strain behaved similarly to strain S288C during growth in complete YPD2% medium.

[107] Transformed control and PE-2 cells showed the same growth profile until 60 h of incubation, when only the transformed cells began to show viability, reaching total death shortly after the 140 h point. Compared to the S288C strain, the cell inactivation kinetics were lower for the PE-2 strain (Figure 6-B). Saccharomyces cerevisiae JSC302 LINE TEST [108] The JSC302 / CNC8 line began to show suicidal effect later than the other lines, that is, only after 72 h. However, from 72 h onwards, the drop in viability was much faster for this strain, which eventually reached full death earlier (96 h) than the other strains (Figure 6-C).

[109] As can be seen from the analysis of Figures 6 (A, B and C), the PE-2 strain, although presenting the highest glucose consumption rate, when carrying the integrated nucA expression cassette, was the one with the highest resistance to the lethal effect of nuclease, showing complete death in the longest time. Perhaps this strain has a more efficient double-stranded DNA repair mechanism than laboratory strains. It is also possible for the PE-2 strain to produce more proteases than the others, since it is a ploidy strain greater than 1.

[110] The S288C strain had a survival curve comparable to the PE-2 strain, but achieved full inactivation earlier (120 h).

[111] The JSC302 strain was the one that resisted the lethal nuclease effect for the longest time, as it began to inactivate only after 72 h, while the others began at 60 h. However, from this point on, the JSC302 strain suffered a much faster survival reduction than the other strains, so that it achieved complete inactivation in the shortest time (96 h), while the control cells remained viable until at least 144 hours under the conditions employed (Figure 6-C). The explanation may be due to the presence in the JSC302 lineage of a super copy of the ADR1 gene, whose product acts as a positive regulator of the ADH2 / GAPDH promoter. In addition, the JSC302 strain carries a mutation in the PEP4 gene that encodes an important yeast protease that could be destroying the nuclease in strains capable of producing this protease. However, this cannot be said to be the reason for the lack of isogenic controls consisting of the JSC302 strain without these genotypic changes. In any case, the inactivation of the JSC302 line, after 72 h, follows an exponential kinetics, without any curve inflection, unlike the other lines analyzed, which demonstrates its greater sensitivity to the suicidal effect.

STABILITY ANALYSIS OF TRANSFORMERS

[112] The stability of the S288C, PE-2 and JSC302 genome-integrated expression cassette during growth was analyzed after 100 successive spikes in complete YPD2% medium and sowing in SD8% solid medium plus canavanine by calculation of loss per generation (Table 4).

[113] The percentage loss of the canavanin resistance phenotype and therefore of the genome-integrated CNC fragment was zero in all transformant strains analyzed. The stability of the system is therefore 100%. At the end of consecutive subcultures, PCR analysis of the presence of the nucA gene in the transformed cells was performed (Figure 7), to detect the presence of the CNC fragment still integrated in the cell genome, as a guarantee that there was no deletion of the CAN1 gene and consequent false positive results when sowing in SD8% + Can medium.

Table 4 [114] Analysis of the stability of the CMC integrative fragment confirmed the high efficiency and stability of the system, as no loss of cloned information was observed, emphasized by the results obtained in complete nio without. no selective pressure in favor of transformants, [115] There is great industrial advantage in the use of an integral system as described herein, since the problem of plasmid loss during cell growth is minimized and heterologous information remains uneven. stable in the genome of the organism for countless generations.

DRY MASS ANALYSIS OF TRANSFORMED LINES

[116] In order to evaluate the protein yield of different strains under the influence of nuclease action, the dry mass of the transformant clones of the PE-2, S288C and JSC302 strains after 24h, 72h and 120h growth were analyzed. .

[117] Transformers S288C / CNC and PE-2 / CNC provided dry mass measurements with values similar to their controls. The same did not occur with the JSC302 / CNC strain, where the amount of dry mass, especially at the 120 h point was much lower than the control (Figure 8). In the dry mass analysis of the transformed strains, all of them provided a final dry mass concentration lower than the control (Figure 8).

[118] These results may be justified by the fact that nuclease is an enzyme that nonspecifically degrades DNA and RNA; the degradation of RNA molecules that would be translated into protein will no longer be, resulting in a decrease in cellular protein yield. Among the strains analyzed, the JSC302 / CNC transformed strain presented the lowest final dry mass, precisely because it is the strain that presented the highest sensitivity to the effect of nuclease, and the dry mass also includes nucleic acids.

DOSAGE OF DsDNA CONTENT IN TRANSFORMED LINES EXPRESSING NUCLEASIS

[119] Transformed strains, after initial inoculation of 1 x 10 6 cells / mL in YPD2% culture medium, had their total DNAs extracted after different growth times (24 h, 72 h and 120 h) and the measurement of DNA concentration in a spectrophotometer to assess whether degradation by heterologous nuclease action had occurred (Figure 9).

[120] Analysis of the amount of dsDNA from different transformed strains compared to control strains without the nuclease expression cassette (Figure 9) showed as expected a decrease in dsDNA in the transformed strains, mainly in the JSC302 strain, probably due to This strain has characteristics that enhance the action of nuclease. In contrast to the results obtained by BALAN (1999) where in 24 h all cells were dead, in the present invention it was observed that the decrease in the amount of dsDNA is gradual.

DOSAGE OF PROTEIN CONCENTRATION OF TRANSFORMERS

[121] Nuc + transformant clones and respective controls of the different S. cerevisiae yeast strains were analyzed for protein yield at 24 h, 72 h and 120 h. Protein extraction was performed according to KUSHNIROV, V.V. Rapid and reliable protein extraction from yeast. Yeast 16: 857, 2000. The results are shown in Figure 10. It can be seen that the PE-2 Nuc + strain compared to the control strain showed a marked decrease in protein concentration. The same occurred with the S288C Nuc + strain compared to its control, with higher protein concentration in the control cells, except for the 72 h time. However, for the JSC302 Nuc + strain, only at 24 h time a higher protein yield was observed for the control cells, whereas at 72 h and 120 h the protein concentrations of the two strains matched.

[122] A certain degree of decrease in protein concentration was expected to be unavoidable under conditions of nonspecific degradation of cellular RNAs. Ideally, therefore, a balance should be found where the reduction in protein content is minimal while the reduction in nucleic acid content is maximum. Of the strains analyzed, the one that showed to be closer to this equilibrium condition was the JSC302 strain, which, after 120 h, presented the lowest dsDNA content and no reduction of total proteins in relation to the control.

[123] A likely explanation lies in the fact that the JSC302 strain has genotypic characteristics which, in addition to enhancing nuclease expression, inhibit protease degradation of enzymes, thereby enhancing DNA and RNA degradation, decreasing the formation of nucleoprotein complexes, which are one of the main factors responsible for the decrease in SCP protein extraction yield.

[124] Previous studies have shown that most of the methods used to reduce the nucleic acid content in the SCP end product, in addition to promoting its higher cost, also promote a decrease in its final yield.

[125] The system described here provides a reduction in the costs of the SCP production process as it is the yeast itself that will destroy its nucleic acids at the end of the fermentation process.

[126] The results obtained in the present invention demonstrate the possibility of using this system in industrial fermentations, since the heterologous information cloned at chromosomal level is not lost along successive cell divisions, and 100% production stability has been achieved. of heterologous nuclease.

[127] All publications mentioned in the descriptive report are incorporated herein by reference. Various modifications and variations of the inventive system will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in conjunction with the specific preferred embodiment, it is to be understood that the invention as claimed should not be unduly limited to that specific embodiment.

Claims

Claims (9)

1. PROCESS FOR REDUCING SINGLE CELL PROTEIN NUCLEIC ACID CONTENT BY EXPRESSION OF A HYPEROLOGICAL NUCLEASE, comprising at least the following steps: (a) Construction of the pUCCNizc intermediate plasmid (SEQ ID2); (b) Obtaining from the intermediate plasmid pUCCNuc (SEQ ID2) the integrative CNC fragment (CAN-Nuc-CAN) containing the Serratia marcescens nucA gene expression cassette; (c) Yeast transformation with the CNC integrative fragment (CAN-Nuc-CAN); (d) Selection of yeast transformants showing stability of Serratia marcescens nuclease production. Process according to Claim 1, characterized in that said expression cassette comprises the hybrid promoter ADH2 / GAPDH (Saccharomyces cerevisiae and nuclease ubiquitin and Saccharomyces cerevisiae ubiquitin and dehydrogenase 2 / glyceraldehyde-3-phosphate dehydrogenase) promoter of Serratia marcescens and Saccharomyces cerevisiae alpha factor transcription terminator. Process according to Claim 1, characterized in that said yeast is Saccharomyces cerevisiae. Transformation vector, obtained from the intermediate plasmid pUCCNuc (SEQ ID2) according to the process defined in claims 1 to 3, characterized in that it comprises the serratia marcescens nucA gene devoid of the coding sequence of its signal peptide, under the commands of Saccharomyces cerevisiae transcription, interspersed between Saccharomyces cerevisiae chromosome sequences. Transgenic Saccharomyces cerevisiae CEPA, characterized in that it comprises the transformation vector, as defined in claim 4, integrated into one of its chromosomes. Transgenic CEPA according to Claim 5, characterized in that it contains, as a foreign gene sequence, exclusively the Serratia marcescens nucA gene. Transgenic CEPA according to Claim 5, characterized in that it has the same growth rate as that of the original strain, while nuclease expression remains suppressed. Transgenic CEPA according to claim 5, characterized in that it intracellularly expresses Serratia marcescens nuclease stably over 100 generations in non-selective medium. Transgenic CEPA according to Claim 8, characterized in that its nucleic acid content is reduced at the end of the fermentation process.