TWI839924B - Freeze-thaw resistant yeast, preparation method and application thereof - Google Patents

Abstract

The present disclosure provides a yeast with freeze-thaw resistance, wherein the endogenous NTH1 gene and HSP12 gene are deleted. The present disclosure also provides a method for its preparation and its use for making dough.

Description

Freeze-thaw resistant yeast, preparation method and application thereof

This case relates to a yeast with freeze-thaw resistance. Specifically, it relates to a yeast with freeze-thaw resistance obtained by deleting the NTH1 gene and the HSP12 gene in the yeast ( Saccharomyces cerevisiae ), a preparation method thereof and its application.

In recent years, in order to save labor and time, improve product diversity, maintain product stability, ensure that products do not deteriorate during transportation, and increase product storage time, the bread industry has adopted a very effective solution to freeze dough. In addition, frozen dough can provide fresh bread to consumers through a simple process of thawing, fermentation and baking, so the production of frozen dough is one of the main development trends. The frozen dough market size was US$6.3877 billion in 2020 and is expected to reach US$7.5446 billion in 2026, with a compound annual growth rate of 4.3%. It can be seen that frozen dough still has a lot of room for development in the future.

However, when making frozen dough, the yeast contained in it is easily damaged by freeze during the freezing and thawing process, which in turn causes the yeast's gas production ability to decrease or even die, resulting in the dough being unable to expand after thawing, making it impossible to meet the quality requirements of subsequent bread production. Therefore, there is an urgent need to develop yeast with enhanced freeze resistance.

To achieve the above object, the present invention provides a yeast ( Saccharomyces cerevisiae ) in which the endogenous NTH1 gene and HSP12 gene of the yeast are deleted.

Preferably, the endogenous NTH1 gene and HSP12 gene are deleted by CRISPR-Cpf1.

Preferably, the yeast is a diploid yeast.

Preferably, the intracellular trehalose content is 112.9±5.4mg/g CDW (cell dry weight).

Preferably, the yeast was deposited at the Biological Resources Conservation and Research Center of the Food Industry Development Institute of the Republic of China on October 4, 2012, with the deposit number BCRC920129.

To achieve another purpose of the claimed invention, the present invention further provides a method for increasing the freeze-thaw resistance of yeast, comprising the following steps: (a) deleting the NTH1 gene and the HSP12 gene of a yeast to obtain a transgenic yeast; and (b) culturing the transgenic yeast.

Preferably, the NTH1 gene and the HSP12 gene of the yeast are deleted by CRISPR-Cpf1.

In order to achieve one purpose of the claimed invention, this case provides a dough comprising the aforementioned yeast.

In order to achieve another purpose of the claimed invention, the present invention further provides a method for making frozen dough, comprising preparing dough using the aforementioned yeast, and freezing the dough.

Figure 1 is a schematic diagram of the CRISPR-Cpf1 genome editing system cutting fragments.

Figure 2 shows the agarose gel electrophoresis colony PCR analysis of the genome-edited strain.

Figure 3 shows the results of cell viability tests of different strains after frozen storage.

Figure 4 shows the results of the dough expansion test after being frozen for 7 and 21 days.

The following is a detailed description of the embodiments with reference to the relevant drawings. However, these embodiments can be implemented in different forms, but this is not the only form of implementing or applying the specific embodiments of the invention claimed in this case, and should not be understood as a limitation on the above embodiments. The implementation method covers the features of multiple specific embodiments and the method steps and their sequence for constructing and operating these specific embodiments. However, other specific embodiments can also be used to achieve the same or equal functions and step sequences. On the contrary, these embodiments are provided so that this specification can be thoroughly and completely disclosed, so as to fully express the spirit of the present invention to those with ordinary knowledge in the technical field to which the present invention belongs. Similar component symbols in the drawings refer to similar components. In the following description, known functions or structures will not be described in detail to avoid unnecessary details in the embodiments.

Unless otherwise defined, all technical terms and terminology used herein have the same meaning as commonly understood by persons of ordinary skill in the art to which the present invention belongs. In the event of a conflict, this specification including the definitions shall prevail.

In the absence of a conflict with the context, a singular term used in this specification includes the plural form of the term; and a plural term used in this specification also includes the singular form of the term. In addition, in this specification and the scope of the patent application, the expressions "at least one" and "one or more" have the same meaning, both of which represent one, two, three or more.

Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate, the relevant numerical values in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently contains standard deviations caused by individual testing methods. Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range. Alternatively, the word "about" means that the actual value falls within the acceptable standard error of the mean, depending on the consideration of a person with ordinary knowledge in the art to which the present invention belongs. Except for the embodiments, or unless otherwise expressly stated, it is understood that all ranges, quantities, values and percentages used herein (for example, to describe material usage, time duration, temperature, operating conditions, quantity ratios and the like) are modified by "about". Therefore, unless otherwise stated, the numerical parameters disclosed in this specification and the scope of the patent application are approximate values and can be changed as needed. At least these numerical parameters should be understood as the indicated number of significant digits and the values obtained by applying the general rounding method. Here, the numerical range is expressed from one end point to another point or between two end points; unless otherwise stated, the numerical range described here includes the end points.

The following is an example of the specific technical features of the invention claimed in this case.

According to one embodiment, the NTH1 gene and the HSP12 gene in the yeast ( Saccharomyces cerevisiae ) are deleted by a gene editing system to prepare the yeast claimed in the present case. In this embodiment, the gene editing system can be any gene editing system of the existing technology, such as zinc finger nucleases (ZFNs), transcription activator-like nucleases (TALENs) and clustered regularly interspaced short palindromic repeat sequences (CRISPR) or large-range nucleases. Preferably, CRISPR is used to delete the NTH1 gene and the HSP12 gene. On the other hand, the yeast can be a haploid yeast or a diploid yeast. According to another embodiment of the present invention, after the NTH1 gene and the HSP12 gene are deleted, the intracellular trehalose content of the yeast is 80 to 140 mg/g CDW, preferably 112.9±5.4 mg/g CDW.

In a preferred embodiment, the yeast was deposited at the Biological Resources Conservation and Research Center of the Food Industry Development Institute of the Republic of China on October 4, 2012, with the deposit number BCRC920129.

According to another embodiment of the present invention, a method for increasing freeze-thaw resistance of yeast is provided, comprising: (a) deleting the NTH1 gene and the HSP12 gene of the yeast to obtain a transgenic yeast; and (b) culturing the transgenic yeast. Preferably, the NTH1 gene and the HSP12 gene of the yeast are deleted using the CRISPR-Cpf1 system.

According to another embodiment of the present invention, a dough is provided, the dough comprising starch, water and yeast obtained according to the above embodiment from which the NTH1 gene and HSP12 gene of the yeast are removed. In one embodiment, the starch is preferably wheat flour, barley flour, rice flour, cassava flour, potato flour, buckwheat flour, teff flour, soybean flour, sorghum flour, etc., more preferably wheat flour. The above dough may further comprise one or more of yeast activator (yeast food), fats, sugars, dairy products, egg products, salts, leavening agents, emulsifiers, enzymes, seasonings, preservatives, proteins, amino acids and spices.

According to another embodiment of the present invention, a method for producing frozen dough is provided, which comprises using the yeast from which the NTH1 gene and HSP12 gene of the yeast are removed, mixing with starch, water and other materials, stirring and mixing to obtain dough, and freezing the dough. Since the dough contains the edited yeast, it can have better anti-freeze-thaw ability, and can still have better swelling ability after the frozen dough is thawed.

The following is a specific example to illustrate the technical features of this case.

Clustered regularly interspaced short palindromic repeats (CRISPR) as a bacterial immune defense system have been developed as genome editing tools in various organisms. Class II CRISPR systems have been widely used for genome editing because of the inherent simplicity and flexibility of the sequence requirements of the single-stranded guide RNA (sgRNA). Using the Cas9 endonuclease from Streptococcus pyogenes, sgRNAs containing a 20-bp complementary sequence to the target sequence can guide Cas9 to target DNA with a protospacer adjacent motif (PAM) to generate double-strand breaks. Lethal bacteria prefer to repair DNA breaks through homologous recombination with homologous DNA templates.

Cpf1 is an RNA-guided endonuclease of the Class 2 CRISPR system, which cuts target DNA with different characteristics from Cas9, such as utilizing T-rich PAM, being guided by a single crRNA, and cutting at the sticky end, acting as an alternative to Cas9 for genome editing.

This case uses a visual recognition model to generate a CRISPR-Cpf1 genome editing system in brewing yeast, which presents red colonies when the ADE2 gene is deleted. In one embodiment of this case, a bread yeast strain lacking the trehalose gene NTH1 gene and/or the heat shock protein HSP12 gene was constructed by CRISPR-Cpf1 to study the intracellular trehalose content and cell viability after freezing and thawing. Finally, the edited yeast strain was used to prepare dough, and the fermentation ability of the frozen dough was analyzed.

Materials and methods

Strains and plasmids

The wild-type yeast BCRC21447 used in this study was obtained from the Biological Resources Collection and Research Center, Rotterdam, The Netherlands. The plasmid vectors pUDC175 and pUDE710 were generated by Dr. Jean-Marc Daran (Addgene plasmids #103019 and #103020). The genetic properties of the yeast and plasmids used in this case are summarized in Table 1.

Culture medium and growth conditions

DNA amplified E. coli Top10 strains were grown in LB medium (10 g/L trypticase, 5 g/L yeast extract, and 10 g/L NaCl) at 37°C. Yeasts were grown in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) at 30°C. When screening was required, antibiotics were added with 100 mg/L phenacyltransferase for E. coli and 200 mg/L G418 for yeast.

Plasma Construction

Yeast genomic DNA was isolated from yeast BCRC21447 using a DNA miniprep kit (D4301, Zymo Research Corp.). Primers used in this study were purchased from Genomics BioSci & Tech. Co., Ltd. Plasmid pBCoN1 was used to express the Fn Cpf1 cassette and contained the crRNA sequence crNTH1.2 targeting gene for NTH1 gene deletion. Plasmid pBCoH1 was used to express the Fn Cpf1 cassette and contained the crRNA sequence crHSP12.1 targeting gene for HSP12 gene deletion. Plasmid pBCoNH1 was used to express the Fn Cpf1 cassette and contained the crRNA sequences crNTH1.2 and crHSP12.1 targeting genes, respectively, for NTH1 and HSP12 gene deletion.

The crRNA sequence was designed by the CHOPCHOP website (http://chopchop.cbu.uib.no/) based on the following principles: (1) the PAM sequence of Fn Cpf1 of the crRNA is 5'-TTTV-3' (V = A/G/C), (2) the length of the direct repeat sequence (DR) is 19 nt, and (3) the crRNA has two DRs at the 5' and 3' ends.

Transformation and genome editing in yeast

All yeast transformations were performed using the lithium acetate protocol. Synthetic homologous DNA templates contained 60 bp of DNA upstream and downstream of the target gene (Table 2). Oligonucleotides were purchased from Integrated DNA Technologies (IDT) Inc. Because the ADE2 gene is essential for adenine biosynthesis and its deletion results in adenine trophobia and red colonies, genomic editing of ADE2 resulted in visible colored colonies. Genomic editing of the NTH1 gene and HSP12 gene was confirmed by colony PCR analysis and DNA sequencing using the nth1CK1/nth1CK2 and hsp12CK1/hsp12CK2 primer sets. The strains after editing were generally the same as before editing in terms of their physical characteristics.

Determination of intracellular trehalose content

The extraction method of intracellular trehalose from yeast cells is briefly described as follows. Yeast cells were harvested by centrifugation and washed twice with distilled water. Trehalose was extracted from 0.1 g of cell pellet with 4 mL of 5% (w/v) cold trichloroacetic acid for 45 minutes while shaking. The extraction was repeated once more, and the supernatants of the two extractions were combined and the trehalose content was determined using a trehalose assay kit (K-TREH, Megazyme Ltd.). Cell dry weight was determined by drying fresh yeast cells at 85°C overnight.

Cell viability assay

For freeze-thaw reactions, yeast cells were cultured in YPD medium at 30°C and harvested during the stationary phase. After microscopic counting of viable cells using a hemocytometer by trypan blue staining, the cells were aliquoted into cryovials and frozen at -20°C for different time periods. The frozen cell samples were thawed at 30°C for 30 minutes and the cell numbers were counted. The survival rate was calculated based on the viable cell numbers before and after freezing.

Determination of expansion capacity

The dough composition was 10 g wheat flour, 5 ml water, 1.25 g sugar, 0.25 g salt and 0.1 ml fresh yeast cells (0.3 g/ml). All materials were mixed well and each dough was placed in a 50 mL graduated cylinder. The height of the dough was recorded every 30 minutes for 3 hours. One set of dough was prepared with fresh yeast sample as a positive control, while another set of dough was prepared without any yeast sample as a negative control. The experimental dough with the requested yeast was stored at -20°C for 7 or 21 days. The frozen dough was thawed at 30°C for 30 minutes and then tested for fermentation ability. The height of the dough was measured from the scale surface of the graduated cylinder before and after fermentation, and the net increase in height was calculated.

Statistical analysis

All experiments were performed at least three times independently, and data reported are mean ± SD. Student's t-test confirmed the differences between the edited strain and the parental strain. Differences with p < 0.05 were considered statistically significant.

result

Generation of NTH1 gene-deficient strains and HSP12 gene-deficient strains by CRISPR-Cpf1

To better understand the content of the present invention, please refer to Figure 1 for the following explanation. Figure 1 is a schematic diagram of the CRISPR-Cpf1 genome editing system shearing fragments. The upper figure shows the relative positions of the (A) NTH1 gene and (B) HSP12 gene using CRISPR-Cpf1 in the brewing yeast system. The DNA sequencing spectrum shown in the lower figure is applicable to (A) △ NTH1 strain; and (B) △ HSP12 strain. Please also refer to Figure 2. Figure 2 is an agarose gel electrophoresis result of colony PCR analysis of the genome-edited strain. PCR amplified DNA containing (A) NTH1 gene amplified by nth1CK1/nth1CK2 primer set, and (B) HSP12 gene amplified by hsp12CK1/hsp12CK2 primer set. Lane 1: DNA marker; Lane 2: wild-type strain; Lanes 3-4: △ nth1 strain; Lanes 5-6: △ hsp12 strain; Lanes 7-10: strains with double deletion of NTH1 gene and HSP12 gene.

In order to improve the baking ability of yeast after long-term freezing in dough, the NTH1 gene and HSP12 gene of brewing yeast were partially deleted using the CRISPR-Cpf1 genome editing system. This study generated strains with NTH1 gene deletion (hereinafter labeled as △ nth1 ), HSP12 gene deletion (hereinafter labeled as △ hsp12 ), and NTH1 gene and HSP12 gene double deletion strains. Compared with the WT strain, PCR analysis of the △ nth1 strain showed that the size of the product deletion was 2,136bp. Similarly, PCR analysis of the △ hsp12 strain showed that the size of the product deletion was 210bp lower than that of the WT strain. PCR analysis of the △ nth1 /△ hsp12 strain confirmed that both genes were deleted (Figure 2).

Correlation test between NTH1 gene knockout and trehalose content

This experiment examined the effect of knocking out the NTH1 gene on the intracellular trehalose content. Trehalose is associated with freeze-thaw stress tolerance in yeast. To examine the intracellular trehalose content in different knockout strains, cells were harvested during the stationary phase (i.e., when trehalose synthesis is particularly intensive). The results showed that the trehalose content of the △ nth1 strain was 104±2.3mg/g cell dry weight (CDW), which was significantly higher than the wild-type strain of 45.4±2.3mg/g CDW, 2.3 times. However, the trehalose content did not increase in the HSP12 gene deletion strain. In addition, the intracellular trehalose content of the strain with double deletion of the NTH1 gene and the HSP12 gene was 112.9±5.4mg/g CDW, which was significantly higher than the wild-type strain by 2.5 times (Table 3). The trehalose content of all △ nth1 strains (△ nth1 and △ nth1 /△ HSP12 ) was significantly increased, indicating that the NTH1 gene is important for the trehalose content.

Viability test of NTH1 gene-deficient and/or HSP12 gene-deficient cells

In order to study the freezing tolerance of △ nth1 and/or △ hsp12 strains, the cell viability of the doughs added with different strains was analyzed after freezing for 7, 14 and 21 days. Please refer to Figure 3, which shows the cell viability test results of the frozen doughs added with different strains after freezing.

After freezing the frozen dough with different strains for 7 days, the cell viability of the wild-type strain (WT) was 42.6±1.58% and 74.7±4.26% without or with 30% glycerol as a cryoprotectant. However, it dropped to 9.15±1.82% after 21 days of frozen storage without adding a cryoprotectant. The cell viability of the Δnth1 strain was 87.17±8.32% and 65.46±6.41% after 7 and 14 days of frozen storage, respectively; however, it dropped sharply to 19.8±1.85% after 21 days. The results showed that the Δnth1 strain can maintain cell viability in short-term frozen storage, but cannot be frozen for a long time. After freezing for 7, 14 and 21 days, the cell viability of the △ hsp12 strain was 61.05±7.45%, 58.39±6.02% and 47.81±3.46, respectively. After freezing for 7 days, the viability of the △ hsp12 strain was lower than that of the △ nth1 strain, but the viability was better after freezing for 21 days. In addition, after freezing for 7, 14 and 21 days, the cell viability of the double gene deletion strain was 88.35±9.41%, 83.33±2.14% and 62.87±6.29%, respectively. The results showed that the double deletion of the NTH1 gene and the HSP12 gene increased the cell viability after long-term freezing. These results showed that trehalose content was positively correlated with the viability of yeast cells after short-term freezing, while the deletion of the HSP12 gene made them more resistant to long-term freezing.

In this case, an edited strain containing double gene deletions of genes encoding trehalose-degrading enzymes and membrane chaperones was constructed, and yeast was edited using the highly efficient CRISPR-Cpf1 genome editing system. The edited yeast increased the trehalose content and cell viability after 21 days of freezing. The dough of the edited yeast showed the ability to expand after freezing, indicating that precise editing of specific genes can improve the freezing tolerance of yeast.

The quality of frozen dough depends on the ability of yeast to produce carbon dioxide and to retain it after bread fermentation. Decreased yeast viability is considered one of the main factors leading to deterioration of dough quality. Therefore, many studies have focused on yeast strains that produce frozen dough with improved growth or higher fermentation rates.

In order to confirm the expansion ability of the dough after adding the edited yeast, please refer to Figure 4, which shows the expansion ability test results of the dough after being frozen for 7 days and 21 days.

Figure 4 (AD) shows the fermentation curves of bread dough used: (A) wild-type yeast; (B) △ nth1 yeast; (C) △ hsp12 yeast; and (D) △ nth1 /△ hsp12 yeast. The height of bread dough was measured after fermentation at 30°C for 0 to 180 minutes. (E) Relative net height of dough after 60 minutes of fermentation. Data are from three independent experiments and are expressed as mean ± SD. The symbol * indicates a statistically significant difference of the edited strain compared with the wild-type strain (** p < 0.01, * p < 0.05, using student t test).

As shown in Figure 4A to D, the wild-type yeast lost its fermentation ability after 7 and 21 days of freezing, while the △ nth1 yeast and △ hsp12 yeast were still able to ferment after 7 and 21 days of freezing, and the fermentation degree of the △ hsp12 yeast was slightly better than that of the △ nth1 yeast. The △ nth1 /△ hsp12 yeast had the best fermentation ability. The same result can be seen from Figure 4E, where the relative net height of the dough of the △ nth1 /△ hsp12 yeast was significantly higher than that of the other doughs. However, the fermentation ability of the dough with the △ nth1 and/or △ hsp12 gene missing tended to decline after 120 minutes.

The △ nth1 strain with a higher trehalose content (Table 3) can maintain cell viability during short-term frozen storage, but cannot survive long-term frozen storage (Figure 3). The △ hsp12 strain without increased trehalose content (Table 3) has a lower survival ability than the △ nth1 strain in short-term frozen storage, but has better survival ability in long-term frozen storage (Figure 3). Disruption of the NTH1 gene contributes to the accumulation of trehalose, while disruption of the HSP12 gene contributes to long-term survival under freezing stimulation. In this study, we demonstrated that strains with double deletion of the NTH1 gene and the HSP12 gene have a high content of trehalose and long-term viability under freezing conditions, which enhances the fermentation ability of frozen dough (Figure 4D). However, compared with the fresh dough of the wild-type strain ( FIG. 4A ), its fermentation ability and fermentation time still need to be improved ( FIG. 4D ).

In the present disclosure, a gene editing system is used to generate mutant strains with precise gene deletions. A preferred embodiment of the present invention is to manipulate the double knockout of the NTH1 gene and the HSP12 gene by simultaneously expressing Cpf1 and crRNAs based on editing of two loci. CRISPR can perform gene editing efficiently and accurately without the need to transfer foreign genes. Genome editing technology can be used to improve strain characteristics and will promote the application and development of yeast in the food industry.

【Biological material storage】

Deposited on October 4, 2012 at the Biological Resources Conservation and Research Center of the Food Industry Development Institute of the Republic of China, with the deposit number BCRC920129

TWI839924_111141425_SEQL.pdf

Claims (8)

A yeast ( Saccharomyces cerevisiae ), wherein endogenous NTH1 gene and HSP12 gene of the yeast are deleted by CRISPR-Cpf1. The yeast described in claim 1 is a diploid yeast. The yeast as described in claim 1, wherein the intracellular trehalose content is 80 to 140 mg/g cell dry weight. The yeast described in claim 1 was deposited at the Biological Resources Conservation and Research Center of the Food Industry Development Institute of the Republic of China on October 4, 2012, with the deposit number BCRC920129. A method for increasing the freeze-thaw resistance of yeast comprises the following steps: (a) deleting the NTH1 gene and the HSP12 gene of a yeast by CRISPR-Cpf1 to obtain a transgenic yeast; and (b) culturing the transgenic yeast. A dough comprising starch, water and the yeast as described in any one of claims 1 to 4. The dough as described in claim 6, wherein the starch is wheat flour, barley flour, rice flour, tapioca flour, potato flour, buckwheat flour, teff flour, soybean flour or sorghum flour. A method for producing frozen dough, comprising preparing a dough using the yeast described in any one of claims 1 to 4; and freezing the dough.