RECOMBINANT YEAST FOR THE PRODUCTION OF OLIGOPEPTIDE

Abstract

The invention relates to a recombinant yeast wherein the PEP4 gene is inactivated. Said yeast is useful for the production of oligopeptides.

Claims

1. Yeast genetically modified by inactivation of the PEP4 gene and of at least one gene involved in glutathione degradation through the γ-GT or DUG pathway.

2. Yeast according to claim 1, wherein said gene involved in glutathione degradation through the γ-GT or DUG pathway is selected from ECM38, DUG1, DUG2 and DUGS.

3. Yeast according to claim 1, wherein said inactivation of the PEP4 gene is obtained by total or partial gene deletion, or by mutagenesis or by insertion of exogenous DNA.

4. Yeast according to claim 1, which is haploid or diploid and wherein one or both the PEP4 gene copies are inactivated.

5. Yeast according to claim 1, which belongs to a genus selected from Saccharomyces and Pichia.

6. Yeast according to claim 5, which is selected from S. cerevisiae and P. pastoris.

7. Yeast according to claim 1, which is further genetically modified by introduction of one or more additional copies of the GSH1 or GSH2 gene.

8. Yeast according to claim 1, which belongs to the species S. cerevisiae, a strain whereof is deposited at CNCM—Institute Pasteur with registration number CNCM 1-5574 or CNCM 1-5575.

9. Fermentative process for the production of glutathione, comprising the following steps: (i) culturing a yeast as defined in claim 1, thereby forming a biomass; and (ii) separating and purifying glutathione from the biomass.

10. Biomass for glutathione production, which is obtainable by the process according to claim 9, step (i).

11. (canceled)

12. Yeast according to claim 3, wherein said inactivation of the PEP4 gene is obtained by homologous recombination with exogenous DNA.

Description

BRIEF DESCRIPTION OF FIGURES

[0045] FIG. 1: deletion of the DUG2 gene by substitution with the URA3 gene of K. lactis flanked by 2 repeated loxP sequences.

[0046] FIG. 2: deletion of the PEP4 gene by substitution with the KanMX4 gene flanked by two FRT sequences and two regions homologous with the PEP4 gene.

[0047] FIG. 3: stability of GSH—the graph shows the level of γ-Glu-Cys dipeptide present in the biomasses of S. cerevisiae at different times

[0048] FIG. 4: stability of GSH—the graph shows the level of γ-Glu-Cys dipeptide present in the biomasses of P. pastoris at different times.

EXAMPLE 1 (COMPARATIVE)

[0049] The yeast Saccharomyces cerevisiae strain NCYC2958 is cultured as described in EP1391517, Example 3; at the end of fermentation the yeast is centrifuged, and then washed in the centrifuge with demineralised water. The resulting biomass is dispersed in 10 volumes of an aqueous solution containing glucose and the other nutrients described, to increase the reduced glutathione content of the biomass; at the end of said procedure the whole broth is centrifuged and the biomass is washed with demineralised water to eliminate the supernatant.

[0050] The GSH-enriched yeast biomass then undergoes thermoacid lysis followed by microfiltration through ceramic membranes with a porosity of 0.2 microns, as described in Example 1 of EP1391517. The resulting almost clear solution is applied on a column of ion-exchange resin, then on adsorbent resin, and finally concentrated by nanofiltration, as described in paragraphs [0060] and [0061] of said patent.

[0051] Reduced glutathione in powder form is obtained from the purified aqueous solution by spray-drying; the resulting product complies with the purity specifications laid down in the European Pharmacopoeia.

EXAMPLE 2 CONSTRUCTION OF A RECOMBINANT STRAIN OF S. CEREVISIAE WITH DELETION OF ECM38 AND DUG2

[0052] Starting with strain BY4742, the previously known glutathione degradation pathways, encoded by the ECM38 and DUG2 genes, were inactivated by engineering activities conducted as described in the prior art (Ganguli et al. 2007, Genetics, Baudouin-Cornu et al., 2012, J Biol Chem).

[0053] The DUG2 gene was eliminated in strain BY4742 by substitution with the URA3 gene of Kluyveromyces lactis (homologue of the URA3 gene of Saccharomyces cerevisiae), flanked by 2 repeated loxP sequences (FIG. 1).

[0054] A DNA fragment comprising the LoxP-URA3-LoxP cassette and flanked by regions 5′ and 3′ of the DUG2 gene was used to transform strain BY4742; the transformants, selected for their ability to grow on uracil-free synthetic medium, were purified and analysed to confirm the substitution of the DUG2 gene with the URA3 marker. An expression cassette containing the GSH1 and GSH2 genes, which catalyse the 2 enzymes required for glutathione biosynthesis, was then inserted in the locus that initially contained the DUG2 gene.

[0055] The ECM38 gene was eliminated (in the strain already deleted for DUG2), by substitution with the LEU2 gene marker of Kluyveromyces lactis (homologue of the LEU2 gene of Saccharomyces cerevisiae), following the same steps as described for DUG2. Finally, subsequent recombinase induction eliminated the 2 URA3 and LEU2 marker genes.

[0056] A strain was thus obtained which, as well as having the DUG2 and ECM38 genes (responsible for glutathione degradation) deleted, also contains additional copies of the genes GSH1 and GSH2 that increase glutathione biosynthesis and production.

EXAMPLE 3 CONSTRUCTION OF A RECOMBINANT PEP4-DELETED STRAIN OF S. CEREVISIAE

[0057] The microorganism of the previous example is transformed with a DNA fragment containing a sequence (KanMX4) that confers resistance to compound G418. As a result of the transformation, said sequence is inserted in the place of the endogenous PEP4 gene, thereby inducing its knockout. For haploid strains, the result is the knockout of the only copy of the PEP4 gene existing in the genome of the microorganism; for diploid yeasts, the process is repeated to eliminate the second copy of the PEP4 gene too.

[0058] The DNA fragment used for the transformation contains the sequence of the KanMX4 gene (810 bp), flanked by two FRT (Flippase Recognition Target) recombination sequences and two regions homologous with the PEP4 gene (first part of FIG. 2), which serve to allow site-specific recombination of the fragment in the PEP4 locus.

[0059] The KanMX4 gene is obtained by amplification from plasmid pWKW (Storici et al. 1999, Yeast 15:271-283), using the binding sites of primers P1 and P2.

[0060] Two different DNA fragments, each of which is obtained via a specific pair of oligonucleotides, are used to knock out each of the two copies of the PEP4 gene present in the genome of the microorganism.

[0061] The following oligonucleotides are used for amplification of the first fragment and knockout of the first copy of the PEP4 gene:

TABLE-US-00001 FOR1 (SEQ ID NO: 1) TTGTTATCTACTTATAAAAGCTCTCTAGATGGCAGAAAAGGATAGGGCG GAGAAGTAAGAAAAGTTTAGCAAAAATAGGCGTATCACGAG REV1 (SEQ ID NO: 2) AAAGAAAAAAAAAAAGCCTAGTGACCTAGTATTTAATCCAAATAAAATT CAAACAAAAACCAAAACTAACTCGATGATAAGCTGTCAAAC

[0062] The following oligonucleotides are used for amplification of the second fragment and knockout of the second copy of the PEP4 gene:

TABLE-US-00002 FOR2 (SEQ ID NO: 3) TCAAATTGCTTTGGCCAAACCAACCGCATTGTTGCCCAAATCGTAAATA GAATAGTATTTACGCAAGAAGAAAAATAGGCGTATCACGAG REV2 (SEQ ID NO: 4) ATGTTCAGCTTGAAAGCATTATTGCCATTGGCCTTGTTGTTGGTCAGCG CCAACCAAGTTGCTGCAAAAGTCGATGATAAGCTGTCAAAC

[0063] Fragments 1 and 2 thus obtained are purified and used for transformation of the microorganism by the lithium acetate method (Kawai et al. 2010 Bioeng bugs 1(6) 395-403).

[0064] The yeast is transformed with fragment 1 and plated on YPD medium containing selection agent G418; 3 G418-resistant colonies are obtained and isolated. To verify the transformation and recombination of fragment 1 in the PEP4 locus, the 3 colonies are analysed by PCR amplification using the following primers and conditions:

TABLE-US-00003 (SEQ ID NO: 5) F1 TGATTTCAAATGTTTCTAGAGCGCA (SEQ ID NO: 6) R1 AATGCTGAAATTGGGGCCAA (SEQ ID NO: 7) F2 GCGTTCAAGTAATTTGTCAATGGAA (SEQ ID NO: 8) R2 TTTGAGAAGCCTACCACGTAAGG (SEQ ID NO: 9) K1 R1 TACAATCGATAGATTGTCGCAC works with F1 and R1 (SEQ ID NO: 10) K2 F2 AGTCGTCACTCATGGTGATT works with F2 and R2

[0065] The PCR products are analysed by 0.8% gel electrophoresis which identifies a 953 bp fragment and a 720 bp fragment, as expected.

[0066] The 3 transformants are inoculated into liquid YPD medium and left to grow under stirring at 200 rpm, 30° C., for 20 hours. During cell incubation, the endogenous recombination system of S. cerevisiae Flp/FRT is activated, leading to excision of the heterologous KanMX4 gene (Park Y N et al. Yeast 28(9) 673-681, 2011). Each of the 3 cultures, suitably diluted, is plated on YPD medium (in the absence of selective agent G418). The colonies grown on the plates are then transferred by replica-plating to plates of YPD+G418 medium. The colonies that fail to grow even on said plates are those which, due to the Flp/FRT recombination, have lost the heterologous KanMX4 gene. Said colonies are isolated from the original YPD plates and analysed by PCR using the following primers and conditions:

TABLE-US-00004 (SEQ ID NO: 11) F1 TGATTTCAAATGTTTCTAGAGCGCA (SEQ ID NO: 12) R2 TTTGAGAAGCCTACCACGTAAGG

[0067] The PCR products are analysed by 0.8% gel electrophoresis which identifies a 600 bp fragment, as expected, confirming the knockout of the first copy of the PEP4 gene.

EXAMPLE 4 CONSTRUCTION OF A RECOMBINANT DIPLOID STRAIN OF S. CEREVISIAE

[0068] Construction of strains GN2363 (from GN2361), GN2364 (from GN2362) and GN2376 (from GN2373). The procedure is conducted on the original strains GN2361, GN2362 and GN2373 as described in experiment 2, obtaining the corresponding PEP4-deleted strains: GN2363, GN2364 and GN2376.

[0069] The procedure proved replicable and applicable to various strains of yeast.

[0070] Yeast GN2363 is deposited and registered at the Collection Nationale de Cultures de Microorganismes—Institut Pasteur (Paris, International Depositary Authority under the Budapest Treaty), under registration number CNCM 1-5574.

[0071] Yeast GN2364 is deposited and registered at the Collection Nationale de Cultures de Microorganismes—Institut Pasteur (Paris, International Depositary Authority under the Budapest Treaty), under registration number CNCM 1-5575.

EXAMPLE 5 CULTIVATION OF YEAST ON A LABORATORY SCALE AND GSH STABILITY TEST

[0072] Strains GN2361 and GN2363 (original and recombinant) are cultured under the same conditions using a growth process in liquid culture, in an Erlenmeyer flask, comprising a vegetative stage followed by a productive stage.

[0073] The vegetative stage is obtained by inoculating 0.5 ml of a stock of cells (frozen and stored at −80° C.) into 20 ml of vegetative medium (1% yeast extract, 2% peptone, 2% glucose). The cultures are left to grow at 28° C. for 16 hours under stirring at 200 rpm. At the end of the incubation period, 10 ml of the vegetative culture is inoculated into 90 ml of productive medium (2% yeast extract, 8% glucose, 0.2% cysteine, 0.2% glycine, 0.2% L-glutamate). The cultures are left to grow at 28° C. for 48 hours under stirring at 250 rpm.

[0074] At the end of the incubation period the culture is divided into 2 equal aliquots to obtain 2 equal samples for use in the stability tests.

[0075] For each culture, one of the aliquots is immediately subjected to heat lysis, and its glutathione and γ-Glu-Cys dipeptide content analysed by the HPLC method. The second aliquot is incubated at 25° C. for 24 hours. After the incubation period the sample is subjected to heat lysis, and its glutathione and γ-Glu-Cys dipeptide content analysed.

[0076] The results are set out in Table 1, which shows the mean value obtained from 4 independent samples.

TABLE-US-00005 TABLE 1 Time GSH % GSH γ-GC % γ-GC strain (hours) mg/L residue mg/L increase GN2361 0 1083 100 42.7 0 24 1025 95 90.7 112 GN2363 0 949 100 12.0 0 24 927 98 14.3 19

[0077] The results demonstrate that PEP4-deleted strain GN2363 produces a smaller amount of γ-Glu-Cys dipeptide, and this remains constant even after 24 hours' incubation at 25° C. Original strain GN2361 (which contains the PEP4 gene) presents a 112% increase in the amounts of γ-Glu-Cys dipeptide, as well as exhibiting greater GSH degradation (95% GSH residue vs 98%).

EXAMPLE 6 CULTIVATION OF YEAST ON A LABORATORY SCALE, AND TEST OF GLUTATHIONE STABILITY IN THE BIOMASS

[0078] Strains GN2362 and GN2364 (original and recombinant) are cultured, and the stability test on the GSH and γ-Glu-Cys dipeptide content conducted, on a laboratory scale, using the same procedures as described in Example 4.

[0079] The results are set out in Table 2, which indicates the mean value obtained from 4 independent samples.

TABLE-US-00006 TABLE 2 Time GSH % GSH γ-GC % γ-GC Strain (hours) mg/L residue mg/L increase GN2362 0 1147 100 42.1 0 24 1095 95 79.3 88 GN2364 0 924 100 13.6 0 24 890 96 15.5 14

[0080] The results demonstrate that strain GN2364 (Apep4 corresponding to GN2362) produces a smaller amount of γ-Glu-Cys dipeptide than the parent strain. The increase in γ-Glu-Cys is considerably lower in strain GN2364 than parent strain GN2362 (14% vs 88% after 24 hours' incubation).

EXAMPLE 7 CULTIVATION OF YEAST ON A LABORATORY SCALE AND GSH STABILITY TEST

[0081] Strains GN2373 and GN2376 (original and recombinant) are cultured, and the stability test on the GSH and γ-Glu-Cys dipeptide content conducted, on a laboratory scale, using the same procedures as described in Example 4.

[0082] The results are set out in Table 3, which indicates the mean value obtained from 4 independent samples.

TABLE-US-00007 TABLE 3 Time GSH % GSH γ-GC % γ-GC Strain (hours) mg/L residue mg/L increase GN2373 0 1056 100 41.5 0 24 1054 100 71.0 71 GN2376 0 926 100 20.9 0 24 918 99 20.1 −3

[0083] The results demonstrate that recombinant strain GN2376 produces a smaller amount of γ-Glu-Cys dipeptide, which remains constant after 24 hours' incubation at 25° C. Original strain GN2373 (which still contains the PEP4 gene) presents a 71% increase in the amount of γ-Glu-Cys dipeptide.

EXAMPLE 8 CULTIVATION OF HAPLOID S. CEREVISIAE ON A LABORATORY SCALE AND GSH STABILITY TEST

[0084] Strains GN2357 and GN2357-Apep4 are cultured, and the stability test on the GSH and γ-Glu-Cys dipeptide content conducted, on a laboratory scale, using the same procedures as described in Example 4.

[0085] The results are set out in Table 4, which shows the mean value obtained from 4 independent samples.

TABLE-US-00008 TABLE 4 Time GSH % GSH γ-GC % γ-GC Strain (hours) mg/L residue mg/L increase GN2357 0 594.1 100 77.0 0 24 565.7 95 112.5 46 GN2357-Δpep4 0 683.1 100 20.8 0 24 661.4 97 21.3 2

[0086] The results demonstrate that strain GN2357-Apep4 produces a smaller amount of γ-Glu-Cys dipeptide, which remains constant even after 24 hours' incubation at 25° C. Instead, the strain which still contains the PEP4 gene presents a 46% increase in the amount of γ-Glu-Cys dipeptide.

EXAMPLE 9 CULTIVATION OF YEAST ON A PILOT SCALE AND GSH STABILITY TEST

[0087] Strains GN2361 and the corresponding GN2363 (recombinant Apep4) are cultured by a growth process in liquid medium, comprising a pre-vegetative stage and a vegetative stage in an Erlenmeyer flask, and a fermentative stage and productive stage in a bioreactor.

[0088] The pre-vegetative stage is conducted as described in Example 4.

[0089] The vegetative stage is conducted by transferring 0.1 ml of pre-vegetative culture into 400 ml of vegetative medium (1% yeast extract, 2% peptone, 2% glucose) in an Erlenmeyer flask. The culture is incubated at 28° C. for 24 hours under stirring at 240 rpm.

[0090] The fermentative stage is conducted by transfer into a 7 L bioreactor containing productive medium (yeast extract, glucose, ammonium, phosphate, sulphate and vitamin and mineral supplements) at 28° C., gassed (1-2 VVM air) and stirred (600-1200 rpm).

[0091] The biomass of the fermentative culture is harvested, concentrated to half its volume by centrifugation, and reintroduced into a 7 L bioreactor containing productive medium (glucose, ammonium, phosphate, sulphate, cysteine, glycine and glutamic acid) at 28° C., gassed (1 VVM air) and stirred (600 rpm).

[0092] At the end of the incubation period the culture is divided into 4 equal aliquots to obtain 4 equal samples for use in the stability tests.

[0093] For each culture, one aliquot is immediately subjected to heat lysis, and its glutathione and γ-Glu-Cys dipeptide content is analysed by the HPLC method. The remaining 3 aliquots are incubated at 25° C. for 24, 48 and 72 hours respectively. After each incubation period the sample is subjected to heat lysis, and its glutathione and γ-Glu-Cys dipeptide content is analysed.

[0094] The results are set out in Table 1, which shows the data obtained with the original strain GN2361 and the data from two independent tests with the corresponding genetically modified yeast GN2363.

TABLE-US-00009 TABLE 5 Strain Time (h) GSH % γ-GC (mg/l) γ-GC % GN2361 0 100 1326 100 24 68 2983 225 48 44 2932 221 GN2363 trial 1 0 100 100 100 24 92 130 130 48 71 135 135 GN2363 trial 2 0 100 180 100 24 93 278 154 48 84 270 150 GSH and γ-GC: HPLC titer of glutathione and γ-glutamyl-cysteine

[0095] The data demonstrate increased stability of glutathione in the genetically modified biomasses, with less overall degradation (% titer reduction) and enzymatic degradation almost eliminated (limited increase of γ-GC).

EXAMPLE 10 CULTIVATION OF YEAST ON A PILOT SCALE AND GSH STABILITY TEST

[0096] Strain GN2362 and its corresponding strain GN2364 (modified Apep4) are cultured as described in Example 9.

[0097] The results are set out in the table below and in FIG. 3.

TABLE-US-00010 TABLE 6 Strain Time (h) GSH % γ-GC (mg/l) γ-GC % GN2362 0 100 1503 100 24 70 4170 277 48 56 4451 296 GN2364 0 100 548 100 24 99 428 78 48 90 300 55 GSH and γ-GC: HPLC titer of glutathione and γ-glutamyl-cysteine

[0098] The data demonstrate the greater stability of glutathione in the genetically modified biomasses, with less overall degradation (% titer reduction) and enzymatic degradation almost eliminated (limited increase of γ-GC). γ-GC degrades slowly by a chemical process.

[0099] In GN2362, cell lysis rapidly releases proteinase A, while the growth of γ-Glu-Cys is faster, then slowly declines due to spontaneous degradation.

In GN2364 the growth of γ-Glu-Cys is slower with both whole and lysed cells.

EXAMPLE 11 FERMENTATION OF PICHIA PASTORIS AND GLUTATHIONE STABILITY TEST

[0100] The strains Pichia pastoris X-33 (which contains PEP4), SMD1168H (which does not contain PEP4) and GN2364 (recombinant S. cerevisiae, described above) are cultured in a suitable medium for 48 h, at 28° C. and 250 rpm. At the end of fermentation the cell biomass is harvested by centrifugation and resuspended in dH.sub.2O, obtaining one suspension for each strain.

[0101] A stock solution of glutathione in dH.sub.2O is prepared at the concentration of 150 g/l. One aliquot of the stock solution is added to the cell biomass suspension, obtaining a final GSH concentration of 10 g/l. The cell biomass with added GSH is divided into 1.5 ml aliquots, which are incubated at a controlled temperature of 25° C., with stirring at 900 rpm. The formation of γ-Glu-Cys is monitored for up to 96 hours, analysing samples incubated for different times by HPLC analysis. The resulting data are set out in FIG. 4.

[0102] The data demonstrate the degradation of glutathione to give γ-Glu-Cys by the Pichia X-33 strain, whereas the two yeasts devoid of the PEP4 gene, Pichia SMD1168H and Saccharomyces GN2364, exhibit the same behaviour and do not increase the production of γ-Glu-Cys.