KR102323926B1 - New yeast strain fermenting arabinose to produce ethanol - Google Patents

Abstract

The present invention relates to a novel yeast strain Saccharomyces cerevisiae that produces ethanol by fermenting arabinose and an optimal culture method for increasing the production of ethanol using the same, the strain of the present invention is an improvement in the consumption rate and fermentation ability of arabinose compared to the existing strain, and has a feature that the production capacity of ethanol as a biofuel is greatly improved.

Description

New yeast strain fermenting arabinose to produce ethanol by fermenting arabinose

The present invention relates to a novel yeast strain Saccharomyces cerevisiae that produces ethanol by fermenting arabinose and an optimal culture method for increasing the production of ethanol using the same.

Fermentation technology of pentose sugar, such as lignocellulose, has potential as a core technology needed to convert biomass using agricultural by-products into biofuels. In particular, the pectin-rich biomass from fruit by-products has a low content of lignin, and the production amount is increasing with the recent increase in processed fruit products, so the possibility of being used as a raw material for biofuels is increasing.

L-arabinose is one of the most abundant pentose sugars in pectin and is converted to D-xylulose-5-phosphate in the same pathway as xylose. In this pathway, L-arabinose is converted to D-xylulose through two redox processes, respectively. At this time, NAD(P)H-specific aldose reductase (AR or XR), NAD ⁺ specific L-arabitol-4- dehydrogenase (LAD), NAD(P)H-specific L-xylulose reductase (LXR or ALX), and NAD ⁺ specific xylitol dehydrogenase (XDH) are involved. The resulting D-xylulose is phosphorylated by xylulokinase (XK) and converted to D-xylulose-5 phosphate (Richard P, Putkonen M, Vaananen R, Londesborough J, Penttila M (2002) The missing link in the fungal L-arabinose catabolic pathway, identification of the L-xylulose reductase gene. Biochemistry 41:6432-6437). Although some naturally-derived strains have been found to have the ability to metabolize arabinose, most of these strains have low resistance to interfering substances such as ethanol and furfural, thus limiting their industrial use (Hahn-Hagerdal B, Karhumaa K, Fonseca C, Spencer-Martins I, Gorwa-Grauslund MF (2007) Towards industrial pentose-fermenting yeast strains. Appl Microbiol Biotechnol 74:937-953).

Accordingly, the present inventors discovered that, while studying a novel strain producing biofuel by fermenting arabinose, the production of ethanol usable as a biofuel is greatly increased when some metabolic pathways of Saccharomyces cerevisiae are adjusted. , the present invention has been completed.

It is an object of the present invention to provide a Saccharomyces cerevisiae strain having an ethanol production ability by fermenting arabinose.

Another object of the present invention is to provide a method for producing ethanol using the strain.

In order to achieve the above object, the present invention provides a Saccharomyces cerevisiae strain that produces ethanol by fermenting arabinose (L-asabinose).

In one embodiment of the present invention, the Saccharomyces cerevisiae is a strain (YE01) in which an arabinose metabolic pathway is constructed by introducing the LAD1 gene of richoderma reesei and the ALX1 gene of Ambrosiozyma monospora (YE01) Or it may include a strain (YE02) in which the PHO13 gene has been removed from the strain. In particular, the YE02 strain from which the PHO13 gene was removed was entrusted with KACC 933328P on August 28, 2019.

The introduction and removal of the gene may be performed according to a known method, and the method is not limited thereto. In an embodiment of the present invention, although the LiAc/SS-DNA/PEG method and the CRISPR/Cas9 technology were partially changed, other known gene editing technology or transformation technology may be used.

As used herein, the term "arabinose" is one of the pentose sugars, contains an aldehyde group, is a white crystalline powder, and the chemical formula is C ₅ H ₁₀ O ₅ . D-type or L-type exists in nature, and L-arabinose is actually more common than D-arabinose in nature and is known as a constituent of biopolymers such as hemicellulose and pectin. L-arabinose is known to be effective in preventing adult diseases such as obesity, diabetes, and hyperlipidemia as an alternative sugar that can replace sugar.

In addition, the present invention Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) culturing a strain in a medium to ferment arabinose; And it provides a method for producing ethanol comprising the step of recovering the ethanol from the medium or the strain according to the fermentation.

The medium used for the culture should meet the requirements of the particular strain in an appropriate way. Sugar sources that can be used include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid, etc. , fatty acids such as linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid, but are not limited thereto. These substances can be used individually or as a mixture.

On the other hand, the pH of the culture can be adjusted by using a basic compound such as sodium hydroxide, potassium hydroxide, ammonia, or an acid compound such as phosphoric acid or sulfuric acid in an appropriate manner. In addition, an antifoaming agent such as a fatty acid polyglycol ester may be used to inhibit the formation of bubbles. Oxygen or oxygen-containing gas (eg air) is injected into the culture to maintain aerobic conditions. The temperature of the culture may be usually between 20°C and 45°C. Fermentation is continued until the desired amount of arabinose production is maximized. For this purpose, the fermentation can usually be achieved in 10 to 100 hours. Erythritol may be excreted into the culture medium or contained in cells.

As a method for recovering ethanol that can be used in the present invention, methods known in the art such as centrifugation, filtration, anion exchange chromatography, crystallization and high performance liquid chromatography (HPLC) or GC/MS analysis may be used, but these examples is not limited to

The present invention relates to a transformed Saccharomyces cerevisiae strain, which is a yeast strain with improved fermentability of arabinose, and a culturing method thereof, and according to the strain and culturing method of the present invention, In comparison, the production of ethanol is significantly increased, and the ethanol can be used in various industrial fields such as biofuels.

1 shows the Fungal native pathway for arabinose metabolism. AR(XR), aldose reductase; LAD, L-arabitol-4-dehydrogenase; ALX, L-xylulose reductase; XDH, D-xylitol reductase; XK, xylokinase.
Figure 2 shows the effect of PHO13 gene deletion in arabinose fermentation. A and B show the fermentation profiles of YE01 and YE02 under aerobic conditions (250 rpm), and C shows the relationship between arabinose consumption rate and PHO13 removal under various oxygen conditions. (Error bars are standard deviations between two biological replicates) D is the relative expression level of TAL1 confirmed by ACT1, and E is the relative expression level of TAL1 confirmed by extracellular sedoheptulose concentration in arabinose metabolism. All experiments were performed at an initial OD of 10 in YP medium containing 40 g/L arabinose as the sole carbon source under aerobic conditions (250 rpm). Error bars represent standard deviations between three biological replicates. nd: not detected.
3 is a result of confirming the amount of intermediate metabolites selected from YE01 and YE02 grown in YP medium containing 40 g/L L-arabinose under aerobic conditions (250 rpm) for 24 hours and 48 hours of culture. Each bar is a dotted line. The mean and standard deviation of the plots are indicated. P-values were calculated via t-test between the two strains (YE01 and YE02): * and ** indicate significant differences at p <0.05 and p < 0.01, respectively. At 48 hours of incubation, the p-values of trehalose (B) and orotic acid (E) were 0.2 and 0.15, respectively.
Figure 4 shows the genetic composition of the arabinose fermentation strain. YE02 (A) and YE01 strains (B) were transformed with DY02 and DY01 as hosts, respectively.
Figure 5 is the effect of PHO13 gene deletion (pho13Δ) on arabinose fermentation profile under various aerobic conditions; Profile results in anaerobic (A and D), microaerobic (B and E) and aerobic (C and F). Arabinose metabolism of YE01 strain (A, B, and C) and YE01 pho13Δ mutant, YE02 strain (D, E and F), respectively, with an initial OD value of 10 in YP medium containing 40 g/L arabinose as a carbon source. tested. Error bars represent the standard deviation of two biological replicates.
6 is a hierarchical clustering and heat map projection result of intracellular metabolite profiles of YE01 and YE02 cultured in YP medium containing 40 g/L L-arabinose under aerobic conditions at 24 and 48 hours of culture. Intracellular metabolites were detected by GC/MS and determined as relative content ratios. For hierarchical cluster analysis, Euclidean distance was used as a distance measure (BioVinci, BioTuring, San Diego, CA, USA). The row Z-score of each feature is indicated by a red-blue color scale. Here, the red color of the tile indicates high abundance and the blue color indicates low abundance.

Hereinafter, the present invention will be described in detail.

The present invention relates to a Saccharomyces cerevisiae strain, which is a transformed yeast strain having an ethanol production ability through fermentation of arabinose, and the strain is reported to the National Academy of Agricultural Sciences on August 28, 2019. It was deposited under the accession number KACC 933328P.

In addition, in order to improve the ethanol production capacity of the Saccharomyces cerevisiae strain, some genes are introduced or removed to control the metabolic process of the strain. The strain of the present invention is a strain (YE01) in which an arabinose metabolic pathway is constructed by introducing the LAD1 gene derived from trichoderma reesei and the ALX1 gene derived from Ambrosiozyma monospora (YE01). Cassettes for, FBA1p-LAD1-FBA1t and PGK1p-ALX1-CYC1t cassettes were introduced into the arabinose fermenting yeast S. cerevisiae (S. cerevisiae). In addition, in order to further improve the arabinose-producing ability, a strain (YE02) in which the PHO13 gene was further removed from the YE01 strain was prepared.

The metabolic pathway of the Saccharomyces cerevisiae of the present invention may be shown as shown in FIG. 1 . D-xylulose can be produced by fermenting L-arabinose even in a natural state, but the yield is not high and it is difficult to use industrially due to various interfering substances. Therefore, in one embodiment of the present invention, the Saccharomyces cerevisiae strain introduces the LAD1 gene that promotes the production of L-xylulose from L-arabitol, and the ALX1 gene that promotes the production of xylitol from L-xylulose, The production of xylitol from L-arabinose was promoted (YE01).

In addition, by further removing the PHO13 gene, it was possible to activate the TAL1 gene and further promote arabinose fermentation. The TAL1 gene is a gene expressing transaldolase that converts sedoheptulose-7-phosphate to erythrose-4-phosphate. In the strain from which PHO13 is removed, the accumulation of sedoheptulose is significantly reduced. As confirmed in an embodiment of the present invention, when PHO13 is removed, the expression of the TAL1 gene is increased (FIG. 2D), and the accumulation amount of sedoheptulose is decreased (FIG. 2E).

In addition, based on the fact that when the PHO13 gene is removed, the amount of trehalose biosynthesis in Saccharomyces cerevisiae cultured in xylose is increased, the pentose phosphorylation pathway is strengthened, and the TCA cycle can be weakened, It was confirmed that the accumulation of sedoheptulose and the production of glucoheptonic acid were low in the YE02 strain from which PHO13 was removed, and through this, it was confirmed that the phosphorylation pathway of pentose sugar could be strengthened through the deletion of the PHO13 gene as in the arabinose fermentation pathway. (Figures 2 and 3)

That is, the present invention confirmed that by removing the PHO13 gene, the consumption rate of arabinose in Saccharomyces cerevisiae can be increased and the ethanol production can be maximized. In particular, in one embodiment of the present invention, when Saccharomyces cerevisiae was cultured under aerobic conditions, it was confirmed that the effect of increasing the ethanol production by removing the PHO13 gene was maximized. (Table 2)

Hereinafter, the present invention will be described in more detail by way of Examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited to these examples.

<Example 1> Materials and methods

<1-1> strain production

Yeast transformation was performed by the LiAc/SS-DNA/PEG method utilized in previous studies (Gietz RD et al, (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature protocols 2:31) and CRISPR/Cas9 technology was used with some modifications (Kim SR et al, (2015) Environmental Microbiology 81:1601-1609). Briefly, the FBA1p-LAD1-FBA1t DNA cassette and the PGK1p-ALX1-CYC1t DNA cassette were introduced into DY01 and DY02, yeast (S. cerevisiae) having xylose fermentation ability, respectively, and the LAD1 gene included in the cassette was Trichoderma reesei In RUT C-30 (ATCC 56765), the ALX1 gene was derived from Ambrosiozyma monospora (ATCC 18855). The DY01 will the introduction of TDH3p-XYL1-TDH3t, PGK1p- XYL2-PGK1t cassette in ALD6 locus introducing TEF1p-XYL3-TEF1t cassette in the INT # 1 spot, DY02 is TDH3p-XYL1-TDH3t the ALD6 locus, Introduce the PGK1p-XYL2-PGK1t cassette and , A TEF1p -XYL3-TEF1t cassette was introduced into the PHO13 gene position. In S. cerevisiae of the present invention, LAD1 and ALX1 introduced into DY01 were named YE01, and LAD1 and ALX1 introduced into DY02 were named YE02, respectively. The gene construction and introduction process is specifically described in FIG. 4, and information on plasmids and strains used in the study is shown in Table 1 below.

lasmid or strain Description Reference Plasmids pRR6-X123 pRS306 TDH3 _P -XYL1-TDH3 _T - PGK1 _P -XYL2-PGK1 _T -TDH3 _P -XYL3-TDH3 _T Kim et al. (2012) pRS41N-Cas9 A single-copy plasmid with a natNT marker and Cas9 Kim et al. (2015) pRS42H-ALD6.1 pRS42H plasmid with a gRNA block containing 20-bp ALD6 sequence (ALD6.1) this study pRS42K-PHO13.1 pRS42K plasmid with a gRNA block containing 20-bp PHO13 sequence (PHO13.1) this study pRS42H-INT#1 pRS42H plasmid with a gRNA block containing 20-bp near HIP1 sequence (INT#1) this study pRS403-PGK1p-CYC1t pRS403 plasmid with a PGK1promoter and CYC1 terminator this study pRS42H-SOR1.1 pRS42H plasmid with a gRNA block containing 20-bp SOR1 sequence (SOR1.1) this study strains DY01 (X123) S. cerevisiae D452-2 ald6::XYL12, XYL3 ¹⁾ this study DY02 (X123 pho13△ ) S. cerevisiae D452-2 ald6::XYL12, pho13::XYL3 this study YE01 (X123, LAD1, ALX1) S. cerevisiae DY01 sor1:: FBA1p-LAD1-FBA1t-PGK1p-ALX1-CYC1t this study X123, LAD1 pho13△ S. cerevisiae DY02 int#1:: FBA1p-LAD1-FBA1t ¹⁾ this study YE02 (X123, LAD1, ALX1 pho13△ ) S. cerevisiae DY02 int#1:: FBA1p-LAD1-FBA1t, sor1::PGK1p-ALX1-CYC1t this study

<1-2> Fermentation conditions and flask fermentation

Yeast was cultured in 10 mL of YP (10 g/L yeast extract and 20 g/L Bacto™ Peptone) medium containing 20 g/L glucose at 30°C for 24 hours. _{To increase the number of cells, the initial cell concentration was adjusted to OD 600} = 0.1 in 50 mL of YP medium containing 20 g/L glucose, and inoculated once more, and then fermented at 30 °C for 24 hours. Thereafter, the initial cell concentration _{was adjusted to OD 600} = 10 and inoculated into 20 mL of YP medium containing 40 g/L arabinose using a 100-mL Erlenmeyer flask. The initial cell concentration _{was adjusted to OD 600} = 10 for extracellular metabolite analysis, and 100 mL-serum bottles were used for anaerobic fermentation.

<1-3> Metabolite analysis through HPLC and GC/MS

The concentrations of L-arabinose, L-arabitol, and ethanol of the present invention were analyzed using HPLC 1260 series, Agilent Technologies, USA). Reference was made. Briefly, vacuum-dried samples were pretreated via methoxyamination and trimethylsilylation prior to GC/MS analysis. For methoxyamination, methoxyamine hydrochloride in 40 μL of pyrimidine was added to the sample and incubated at 30 °C for 90 minutes. For trimethylsilylation, 40 μL of N-methyl-N- (trimethyl silyl) trifluoroacetamide was applied to the sample and incubated at 37° C. for 30 minutes. GC/MS analysis was performed using a GC Agilent 6890 equipped with an Agilent 5973 mass screening detector, 1 μL of pretreated sample was used and separated by RTX-5Sil MS column in splitless mode. After maintaining the initial oven temperature at 50 °C for 1 minute, it was raised to 330 °C at a rate of 20 °C/min, and maintained for 2 minutes from the time the final temperature was reached. Helium gas was used as a carrier gas at a constant flow rate of 1.5 mL/min, and the temperatures of the ion source and delivery line were set to 250 °C and 280 °C, respectively. The mass detector was detected within the mass range of 50 - 800 m/z in scan mode.

<1-4> Transcriptional analysis using RT-qPCR

RT-qPCR of the TAL1 gene was performed as previously described (Kim SR et al, (2015) Environmental Microbiology 81:1601-1609). All cells were cultured in YP medium containing 40 g/L arabinose and cDNA solution was ^{used for qPCR with primers and iQ TM} SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). This was performed using the CFX Connect ^TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), and the cDNA samples of RT-qPCR were biologically and technically repeated three times.

<Example 2> Experimental results

<2-1> Fermentation characteristics of arabinose fermentation strains (YE01, YE02)

The fermentation characteristics of the two strains (YE01 and YE02) under various oxygen conditions are shown in Table 1 and FIG. 2 . The arabinose consumption rates of YE01 were 0.002, 0.020, and 0.021 g DCW/h under anaerobic, microaerobic and aerobic conditions, respectively. YE02 strain showed no significant difference from YE01 under anaerobic and microaerobic conditions, but showed a significant difference compared with YE01 in arabinose consumption rate of 0.051 g DCW/h under aerobic conditions (Tables 1 and 2).

That is, as shown in Table 1 below, the maximum ethanol production and specific ethanol productivity of YE02 under aerobic conditions increased 2 times and 3.5 times, respectively, compared to YE01. Arbitol production was the lowest under aerobic conditions, but there was no significant difference between the two strains.

These results showed that the arabinose consumption rate and specific ethanol productivity of the YE02 strain were the highest compared to the previously reported strain introducing the fungal arabinose metabolic pathway.

strain Genetic Backgrounds Heterologous gene Aeration
(rpm) Time
(h) Initial arabinose concentration
(g/L) Consumed
Arabinose
(g/L) q _arabinose Ethanol
(g/L) Arabitol
(g/L) Glycerol
(g/L) Y _ethanol V _ethanol P _ethanol Ref Fungal arabinose pathway YE01 XYL123 sor1::LAD1-ALX1 AN 672 40 6.9 0.002 1.5 8.4 0.6 0.51 0.01 0.94 this study 80 48 40 6.3 0.020 1.0 14.0 0.3 0.30 0.02 3.42 250 72 40 13.1 0.021 1.3 7.5 0.7 0.13 0.02 2.05 YE02 XYL123, pho13Δ sort1::LAD1 int#1::ALX1 AN 672 40 8.3 0.002 4.6 10.1 0.5 0.55 0.01 1.27 this study 80 48 40 7.2 0.022 1.1 13.1 0.3 0.25 0.02 3.43 250 24 40 18.6 0.051 2.6 8.4 0.0 0.16 0.11 7.13 H2561 XYL123 LAD1, LXR1 AN 70 50 - - 0.1 - - - - 0.35 Richard et al. (2003) 424A(LNH-ST)/pLXR _NAD- LAD XYL123 LAD1, ALX1 OL 96 45 40.5 - 9.0 13.5 0.6 0.19 0.10 1.66 Bera et al. (2010) Bacterial arabinose pathway JBY25-4M CEN.PK2-1C araABD, GAL2 OL - 20 - - 6.0 - - 0.30 - 60 Becker et al. (2003) BWY1-45 CEN.PK2-1C araABD, GAL2 AN 150 30 23.1 0.11 9.0 2.5 1.4 0.39 - 36 Wiedemann et al. (2008) IMS0002 xylA, XKS1 araABD AN 72 20 20.7 - 9.0 <0.02 1.4 0.45 - - Wisselink et al. (2007) BSW3AP CEN.PK102-3A araABD OL 120 20 18.6 0.7 6.9 0.13 - 0.43 - - Wang et al. (2013) BSW3AG CEN.PK102-3A araABD, GAL2 AN 80 20 - 0.61 - - 1.4 0.43 - 270 Wang et al. (2013)

Parameters: q _arabinose , rate of arabinose consumption (g DCW/h); Y _ethanol , ethanol yield (g ethanol/g arabinose); V _ethanol , ethanol volume production (g ethanol/h); and P _ethanol , specific ethanol productivity (mg ethanol/g DCW/h).

<2-2> Metabolic change of TAL1 gene by deletion of PHO13 gene

To confirm the effect of the PHO13 gene on arabinose fermentation through intracellular metabolite analysis in the YE02 strain from which the PHO13 gene was removed, the metabolite changes of the YE01 and YE02 strains under aerobic conditions were compared. 95 metabolites were identified in both strains collected from samples 24 and 48 hours after incubation, and there were many metabolites related to central carbon metabolism in the YE01 strain compared with the YE02 strain (FIG. 3). However, more metabolites related to the nitrogen metabolic pathway were identified in YE02, a strain from which PHO13 was removed.

That is, when S. cerevisiae was grown in a culture medium containing xylose, trehalose biosynthesis was increased due to the deletion of the PHO13 gene, thus upregulating the pentose phosphorylation pathway and downregulating the TCA cycle. Through the experiment, it was found that compared to YE01 strains low sedoheptulose accumulation and glucoheptonic acid accumulated amount of TAL1 YE02 strain the gene is activated, as in the xylose fermentation in arabinose fermentation process through which the pentose phosphorylation path by being PHO13 gene is removed reinforcement was confirmed. (Figures 2 and 3)

That is, through the above results, it was confirmed that the YE02 strain in which the PHO13 gene is removed can activate the metabolic pathway for ethanol production by activating transaldolase together with NADH production, especially under aerobic conditions.

National Academy of Agricultural Sciences KACC93328P 20190828

Claims (4)

LAD1 and ALX1 genes were introduced and the PHO13 gene was removed to improve TAL1 (transaldolase) gene expression and arabinose fermentation ability under aerobic conditions, Saccharomyces cerevisiae YE02 strain (KACC 93328P). delete 1) Saccharomyces cerevisiae of claim 1 ( Saccharomyces cerevisiae ) YE02 strain (KACC 93328P) fermenting in a medium under aerobic conditions; and
2) A method for producing ethanol comprising the step of recovering ethanol from the medium or the strain according to the fermentation. delete

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