OLEAGINOUS YEAST VARIANT, METHOD FOR OBTAINING THEREOF AND USE THEREOF FOR LIPID PRODUCTION

Abstract

The present invention concerns an oleaginous yeast variant of the species Rhodosporidium azoricum characterized by higher biomass yields and intra- cellular lipid accumulation useful for the production of bio-fuels higher, in determined conditions, with respect to the wild type strain of the same species. Furthermore, the invention concerns a method through which said oleaginous yeast variant of the species Rhodosporidium azoricum was obtained. The invention further concerns the lipid production by means of said variant strain of oleaginous yeast of the species Rhodosporidium azoricum.

Claims

1. An oleaginous yeast variant of the species Rhodosporidium azoricum, characterized by an intracellular lipid accumulation, deposited at Leibniz-Institut DSMZ—Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Inhoffenstraβe 7 B 38124 Braunschweig (Germany), on Oct. 10, 2014, with the deposit number DSM 29495.

2. The oleaginous yeast variant according to claim 1, characterized by an intracellular lipid accumulation in an amount higher or equal to 60% of its cell dry weight, when cultured in culture media rich in nitrogen sources.

3. The oleaginous yeast variant according to claim 1, which accumulates lipids in an amount higher or equal to 40% of its cell dry weight when cultured in the presence of substrates derived from hydrolysis of lignocellulosic materials as nutrient source.

4. A method for obtaining an oleaginous yeast variant, comprising: i. exposing one or more cells of an oleaginous yeast strain to a mutagenic agent in order to generate random mutations in genomic DNA of the cells; ii. inoculating and cultivating in a culture medium the cells treated with the mutagenic agent, wherein the culture medium comprises a carbon source at a concentration from 20 g/L to 100 g/L, wherein at least one nitrogen source is present; and iii. separating yeast cells having a lower density from the cell culture.

5. The method according to claim 4, wherein the inoculating and cultivating is performed for a time of 16 to 48 hours, at a temperature of 10° C. to 40° C.

6. The method according to claim 4, wherein the separating comprises: a) collecting a sample from the culture and adding at least one thickening agent in a suitable amount, to obtain a cell suspension; b) centrifuging the cell suspension at a low speed, to obtain a centrifugation supernatant; c) collecting a higher fraction of the centrifugation supernatant in order to isolate one or more cells characterized by lower density from the rest of the cell culture; d) inoculating and cultivating the fraction of the supernatant isolated in c) in a culture medium, comprising a carbon source at a concentration from 20 g/L to 100 g/L, wherein at least one nitrogen source is present; e) repeating a) to d) for at least 2 times; and f) isolating one or more single mutant colonies.

7. The method according to claim 6, wherein a) is carried out by adding sorbitol to the culture sample, as a thickening agent, up to a concentration of 1M to 3M.

8. The method according to claim 6, wherein the inoculating and cultivating d) is performed for a time of 16 to 48 hours, at a temperature of 10° C. to 40° C.

9. The method according to claim 4, wherein the oleaginous yeast is Rhodosporidium azoricum.

10. The method according to claim 4, wherein the oleaginous yeast variant obtained a variant of the strain Rhodosporidium azoricum DMS 29495.

11. The method according to claim 4, wherein the at least one nitrogen source is replaced by: i. at least one sulfur source, organic or inorganic, at a concentration of 3 g/L to 40 g/L; or ii. at least one phosphorus source, organic or inorganic, at a concentration of 3 g/L to 40 g/L.

12. The method according to claim 4, wherein mutagenesis occurs: i. physically, through exposure of the yeast strain to ultraviolet radiation with a wavelength of 230 to 260 nm; or ii. chemically, through exposure to ethyl mesylate at a concentration of 0.1% to 10% (vol/vol) in the culture medium where the strain is inoculated and maintaining the culture in the presence of the mutagenic agent for a time of 10 minutes to 12 hours.

13. The method according to claim 6, wherein the culture volume collected during a) corresponds to an optical density of 50 OD.sub.660 to 200 OD.sub.660.

14. The method according to claim 6, wherein the centrifugation during b) is carried out for a time of 1 to 5 minutes at an acceleration of 500 g to 1000 g.

15. A method for producing a lipid by the cultivation of the oleaginous yeast variant Rhodosporidium azoricum DSM 29495, comprising: i. preparing a culture medium comprising: a carbon source at a concentration of 20 g/L to 100 g/L; and a nitrogen source, at a concentration of 3 g/L to 40 g/L; ii. cultivating the oleaginous yeast variant in the culture medium; iii. separating one or more cells of the yeast variant from the culture medium; and iv. extracting intracellular lipids accumulated within the cells of the yeast variant.

16. The method according to claim 15, wherein the culture is carried out: i. at a temperature of 10° C. to 40° C.; and/or ii. for a time of 50 hours to 200 hours; and/or iii. in aerobic conditions, blowing sterile air and under stirring ranging from 600 to 900 revolution per minute, modulated with the air flow so as to maintain the concentration of dissolved oxygen (DO.sub.2,) equal to 30% of the saturation value; and/or iv. at pH maintained between 4.5 and 7.0.

17. The method according to claim 15, wherein the culture is carried out starting from an inoculum in an amount of 1% to 5% (vol/vol) of the total volume of the medium, obtained from a previous culture of the yeast strain carried out in the same medium for a time of 6 to 24 hours.

18. The method according to claim 15, wherein the culture medium comprises glucose as the carbon source and (NH.sub.4).sub.2SO.sub.4 as the nitrogen source.

19. The method according to claim 15, wherein the culture medium comprises a lignocellulosic hydrolyzate as the carbon source.

20. The method according to claim 19, wherein the lignocellulosic hydrolyzate derives from the treatment of: a. one or more products deriving from cultures expressly cultivated for energetic use, comprising scraps, residues and waste deriving from the products or from the processing thereof; b. one or more products deriving from agriculture, comprising scraps, residues and waste deriving from the products or from the processing thereof; c. one or more products deriving from forestation or forestry, comprising scraps, residues and waste deriving from the products or from the processing thereof; d. one or more scraps of food and agricultural products for human alimentation or zootechnics; e. one or more paper industry residues, non-chemically treated; f. waste deriving from the recycling collection of urban solid waste; or g. algae.

21. The method according to claim 19, wherein the lignocellulosic hydrolyzate derives from the treatment of the species Arundo donax (common cane), comprising scraps, residues and waste deriving from the processing thereof.

22. The method according to claim 21, wherein the lignocellulosic hydrolyzate of Arundo donax (common cane) is obtained by hydrothermal treatment and subsequent enzymatic hydrolysis.

23. The method according to claim 15, wherein a culture grown-up for a time of 12 to 24 hours, after the inoculum is further carried out in fed-batch for a time of 90 hours to 200 hours, adding an aqueous glucose solution so as to obtain a stable concentration of glucose in the medium of 25 g/L to 50 g/L.

24. The method according to claim 19, wherein a culture grown-up for a time of 12 to 24 hours, after the inoculum is further carried out in fed-batch for a time of 90 hours to 200 hours, adding lignocellulosic hydrolyzate so as to obtain a stable concentration of total sugars of 25 g/L to 50 g/L.

Description

EXAMPLE 1

Obtainment of the Variant Rhodosporidium azoricum DSM 29495

[0114] Following it is reported a practical example of the method for obtaining variant strains of the wild-type strain of the species Rhodosporidium azoricum, through which the variant deposited at Leibniz-Institut DSMZ, with the deposit number DSM 29495, was obtained.

[0115] In such example, the random mutagenesis was obtained by UV irradiation.

[0116] A cell sample of a wild-type strain of the species Rhodosporidium azoricum was inoculated in the “YEPD” medium (yeast extract 10 g/L, Peptone 10 g/L, glucose g/L) up to achieving the growth exponential phase (10 OD.sub.660). After, cells were collected by centrifugation at 1500 g for 5 minutes, washed in sterile water, then resuspended in 1 mL of sterile water and placed on the bottom of an empty Petri plate.

[0117] Cells placed on the plate were exposed to a source of ultraviolet radiation, represented by a UV lamp of 15 Watt (wavelength 254 nm), put at a distance of 30 cm, for a time equal to 20 seconds, in order to obtain a residue vitality rate of 10%. After that cells were collected from the plate and used to inoculate a liquid culture of yeast cells in medium “B” (yeast extract 1 g/L, KH.sub.2PO.sub.4 1 g/L, MgSO.sub.4.7H.sub.2O 0.05 g/L, NaCl 0.01 g/L CaCl.sub.2.2H.sub.2O 0.01 g/L) containing (NH.sub.4).sub.2SO.sub.4 5 g/L and such culture was incubated at 30° C. for 24 hours.

[0118] After, a volume of culture equivalent to 100 OD.sub.660 was collected and cells, recovered by centrifugation, were resuspended in 5 mL of medium “YEPD”. To this volume, 5 mL of a sterile solution of sorbitol 2M was added and the so obtained cell suspension was centrifuged for 1 minute at 500 g. After centrifugation mL of suspension was sterilely collected from the higher fraction of supernatant, so isolating in this way those cells with the highest lipid content, less dense, than those cells without lipids (or with lipids accumulated in lower amount) denser, which so are located in the lower portion of the supernatant and in the centrifugation sediment. The collected sample was used to inoculate a new liquid culture. This culture series and isolation of the less dense fraction through centrifugation in a density gradient was repeated for 8 cycles, therefore the culture was spread on “YEPD” medium containing agar, in order to isolate single yeast colonies.

EXAMPLE 2

Genotypic Characterization of the Variant Rhodosporidium azoricum DSM 29495

[0119] In the present example the characterization process of the variant Rhodosporidium azoricum DSM 29495 is described, by sequencing of the genomic DNA and bioinformatic analysis of mutations present in comparison with the sequence of the genomic DNA of the wild-type strain of the same yeast species.

[0120] The genomic DNA was extracted from cultures of both strains (variant DSM 29495 and the wild-type strain) carried out in the medium “YEPD” (yeast extract 10 g/L, Peptone 10 g/L, glucose 20 g/L) for one night, and purified with the commercial kit DNeasy Blood & Tissue kit from Quiagen (cat. Num. 69504) following the instructions provided by the producer.

[0121] After checking the purity and integrity degree by electrophoresis, the genomic DNA of each strain was treated separately with the kit “TruSeq DNA Library Preparation Kit” by Illumina, by following the protocol combined with the kit. Briefly, DNA was processed in order to obtain fragments of dimensions comprised between 200 and 400 bp, then bound to 3′ and 5′ terminus to oligonucleotide adapters provided with the kit and them amplified by “Polymerase Chain Reaction” (PCR) by using as primer the same oligonucleotides. The products of gene amplification were quantified and checked with the device Bioanalyzer 2100 by Agilent Technology. Of each fragment obtained the DNA sequence was determined by using the HiSeq2500 sequencer by Illumina and the obtained sequences were optimized based on “phred” value [Ewing B., Hillier L., Wendl M. C., Green P. (1998), “Base-calling of automated sequencer traces using phred. I. Accuracy assessment”. Genome Res. 8 (3): 175-185]. The sequence analysis allowed the identification of 893 contiguous regions (“contigs”) of dimensions ranging from 120 to 440,000 bp, for a total of 22,722,315 bp. This value is in line with the dimension of the genomic DNA of yeasts filogenetically similar to the genus Rhodosporidium, therefore it is believed that the obtained libraries represent the complete genome of Rhodosporidium azoricum DSM 29495 and of the correspondent wild-type strain. Sequences of the two genomic DNAs were compared each other by using the software “Map Reads to Reference” and “Quality-based Variant Detection” by CLCbio.

[0122] The identified mutations were confirmed by verifying their presence in both sequencing directions and determining the accuracy through the “phred” value.

[0123] Overall in the genomic DNA of the variant of Rhodosporidium azoricum DSM 29495, 124 mutations were pointed out (comprising substitutions, deletions and nucleotide insertions) reported in Table I.

EXAMPLE 3

Culture in Flask

[0124] In this example, performances in terms of growth and lipid accumulation of the yeast variant strain Rhodosporidium azoricum DSM 29495 in a medium enriched in nitrogen sources, compared with a wild-type strain of the same species, were assessed. In two sterile flasks of 500 mL, 100 mL of culture medium “B” were put (yeast extract 1 g/L, KH.sub.2PO.sub.4 1 g/L, MgSO.sub.4.7H.sub.2O 0.05 g/L, NaCl 0.01 g/L CaCl.sub.2.2H.sub.2O 0.01 g/L) containing (NH.sub.4).sub.2SO.sub.4 5 g/L and its pH was adjusted at pH 6.0 by adding of the buffer MES 0.1 M.

[0125] In both flasks the culture medium was inoculated with a culture of the variant yeast strain Rhodosporidium azoricum DSM 29495 or of the wild-type strain of the same species, both preventively carried out in the medium “B” for about 24 hours, and inoculated in turn with samples of the two yeast strains maintained at −80° C. in a suspension containing glycerol 15% (vol/vol). The inoculated culture volume is settled in order to initially obtain 0.1 OD.sub.660.

[0126] After about 70 hours, after 90 hours and then 114 hours of growth, a culture sample was collected from both flasks and on such sample the biomass was determined (expressed as dry-weight of cells in g per liter of culture) and the amount of the accumulated lipids (expressed as lipid concentration in g/L of the culture and as percentage ratio between lipid weight and the total cells dry-weight).

[0127] The analysis results are reported in Table II.

TABLE-US-00002 TABLE II Culture- time at the moment Dry-weight Lipids of (g/L (g/L Strain sampling culture) culture) Lipids % Rhodosporidium 66 hours 11.17 2.58 23 azoricum wild- 90 hours 14.80 3.75 25 type 114 hours  15.15 3.97 26 Rhodosporidium 71 hours 12.40 3.62 29 azoricum DSM 90 hours 15.08 4.88 32 29495 114 hours  17.18 7.33 43

EXAMPLE 4

“Fed-Batch” Culture

[0128] In this example growth and lipid accumulation of the yeast variant strain Rhodosporidium azoricum DSM 29495 was assessed, compared with the wild-type strain of the same species, in a “fed-batch” fermentation.

[0129] In order to prepare the fermentation inoculum, 100 mL of “YEPD” medium (yeast extract 1 g/L, Peptone 10 g/L and glucose 20 g/L) were put in two 500 mL flasks.

[0130] Then, the culture medium was inoculated with cultures of the variant yeast strain Rhodosporidium azoricum DSM 29495 or of the wild-type strain of the same species, preventively carried out in the “YEPD” medium for about 12 hours, and inoculated in turn with samples of the two yeast strains maintained at −80° C. in a suspension containing glycerol 15% (vol/vol). The culture volume inoculated is settled in order to initially obtain 0.1 OD.sub.660.

[0131] The cultures were incubated at 30° C. under stirring for 24 hours.

[0132] Cell suspensions obtained were used to separately inoculate fermenters of 20 L, in which 6 L of medium containing glucose 50 g/L, Corn Steep Solid 5 g/L, yeast extract 2 g/L, KH.sub.2PO.sub.4 6 g/L, MgSO.sub.4.7H.sub.2O 0.3 g/L, NaCl 0.06 g/L CaCl.sub.2.2H.sub.2O 0.06 g/L and (NH.sub.4).sub.2SO.sub.4 5 g/L were placed.

[0133] The inoculum volume for each strain is that one necessary to obtain about 6 L of cell suspension having 0, 4 OD.sub.660. For the following growth phase in the “fed-batch” mode, each fermenter is fed with an aqueous solution of glucose 600 g/L according to the consumption kinetics of the carbon source from the two yeast strains, so as to maintain a stable concentration of glucose in the cultures equal to 30 g/l.

[0134] The growth occurred in aerobic conditions by air flow and variable stirring between 600 and 900 rpm modulated with the air flux so as to maintain the concentration of dissolved oxygen (DO.sub.2) equal to 30% of the saturation value, and at a pH of about 5.0, maintained through the addition, when necessary, of some drops of a solution of KOH 5 M or H.sub.2SO.sub.4 10% (vol/vol).

[0135] After 77 hours and after 140 hours of growth, from both the fermenters a culture sample was collected and on such samples the analyses described in the example 3 were carried out.

[0136] The results of the analysis are reported in Table III.

TABLE-US-00003 TABLE III Culture- time at the Dry- moment weight Lipids of (g/L (g/L Lipids Productivity Strain sampling culture) culture) % (g/L * h) Rhodosporidium  77 hours 70.80 22.80 32.20 0.29 azoricum wild- 140 hours 76.10 34.80 45.70 0.24 type Rhodosporidium  77 hours 64.90 33.10 51.00 0.43 azoricum DSM 140 hours 95.30 61.80 64.90 0.44 29495

[0137] Data reported in this table clearly demonstrate that the variant of Rhodosporidium azoricum DSM 29495, in the fermentation conditions described, produces and accumulates lipids in amounts significantly higher than the wild-type strain.

EXAMPLE 5

“Fed-Batch” Culture by Using a Lignocellulosic Hydrolyzate of Arundo donax as a Nutrient

[0138] In this example the production of biomass and lipid accumulation of the variant strain of the yeast Rhodosporidium azoricum DSM 29495 in a “fed-batch” fermentation was assessed, in which as growth substrate the lignocellulosic hydrolyzate deriving from the hydrothermal and enzymatic treatment of Arundo donax was used, compared to the wild-type strain of the same species.

[0139] The composition of the hydrolyzate of Arundo donax used comprises glucose 224.75 g/L, xylose 107.19 g/L, arabinose 3.94 g/L, cellobiose 16.65 g/L, glycerol 4.24 g/L acetic acid 9.80 g/L, 5-hydroxymethyl-furfural 0.74 g/L and a concentration of total nitrogen equal to 3.69 g/L. The analytic procedures are analogues to those described in the international patent application WO2014/102254.

[0140] The total nitrogen was determined by TOC/TN analyzer equipped with gas infrared detector (NDIR, non-dispersive infrared) and using the method of the catalytic oxidation developed by Shimadtzu.

[0141] In order to prepare the fermentation inoculum, in two flasks of 2 L, 600 mL of medium consisting of a mixture of 560 mL of “YEPD” medium (yeast extract 1 g/L, peptone 10 g/L and glucose 20 g/L) and 40 mL of lignocellulosic hydrolyzate from Arundo donax were predisposed.

[0142] The culture medium was inoculated with 10 mL of cultures of the variant yeast strain Rhodosporidium azoricum DSM 29495 or of the wild type strain of the same species, preventively carried out in the “YEPD” medium for about 12 hours, and inoculated in turn with samples of the two yeast strains maintained at −80° C. in suspension containing glycerol 15% (vol/vol).

[0143] The culture were incubated at 30° C. under stirring for 24 hours.

[0144] A volume equal to 550 mL of each cell suspension was separately inoculated in two fermenters in which a fermentation medium with the following composition was predisposed: lignocellulosic hydrolyzate of Arundo donax 127 mL/L, yeast extract 2 g/L, Corn Steep Liquor 3 g/L, KH.sub.2PO.sub.4 6 g/L, MgSO.sub.4.7H.sub.2O 0.3 g/L, NaCl 0.06 g/L CaCl.sub.2.2H.sub.2O 0.06 g/L e (NH.sub.4).sub.2SO.sub.4 4 g/L.

[0145] For the next step of growth, in each fermenter the lignocellulosic hydrolyzate from Arundo donax according to the consumption kinetics of the carbon source from the two yeast strains was fed, in order to maintain a constant concentration of glucose in the cultures equal to 30 g/L. As the hydrolyzate from Arundo donax had a total nitrogen content equal to 3.69 g/L, the feeding also brought such nutritional source to the culture in the fermenter.

[0146] The growth was carried out in aerobic conditions, by air insufflation and stirring ranging from 600 to 900 rpm, modulated with an air flux so as to maintain the concentration of dissolved oxygen (D0.sub.2) equal to 30% of the saturation value, and at pH equal to 6.0, maintained through the addition, when it is necessary, of some drops of a solution of KOH 5 M or H.sub.2SO.sub.4 10% (vol/vol).

[0147] After 74 hours and after 144 hours of growth, a culture sample was collected from both the fermenters and on said samples the analyses described in the example 3 were carried out.

[0148] The results of the analyses were reported in Table IV.

TABLE-US-00004 TABLE IV Culture- time at the Dry- moment weight Lipids of (g/L (g/L Lipids Productivity Strain sampling culture) culture) % (g/L * h) Rhodosporidium  74 hours 44.90 10.90 24.30 0.15 azoricum wild- 144 hours 46.40 15.20 32.80 0.11 type Rhodosporidium  74 hours 56.00 21.25 38.30 0.29 azoricum DSM 144 hours 73.30 33.10 45.20 0.23 29495