WO2024252885A1 - Production method for cellobiose - Google Patents

Abstract

Provided is a low-cost, favorably efficient production method for cellobiose. The method includes a step for reacting sucrose in a reaction liquid that contains glucose isomerase and yeast that has been made to express sucrose phosphorylase and cellobiose phosphorylase on the surface thereof.

Description

Method for producing cellobiose

The present invention relates to a method for producing cellobiose from sucrose using yeast that expresses an enzyme on its surface.

　Enzymatic degradation and enzymatic synthesis methods have been known as methods for producing cellobiose. For example, an enzymatic degradation method is known in which a cellulase preparation is reacted with cellulose to obtain cellobiose (Patent Document 1, Patent Document 2). For example, an enzymatic synthesis method is known in which starch is used as a starting material and α-glucan phosphorylase and cellobiose phosphorylase are allowed to act on the starch to obtain cellobiose (Non-Patent Document 1), and a method is known in which sucrose is used as a starting material and sucrose phosphorylase, glucose isomerase, and cellobiose phosphorylase are allowed to act on the sucrose to obtain cellobiose (Patent Document 3, Patent Document 4).

In the enzymatic decomposition method, cellulose is used as the raw material. Cellulose is the main component of plant cell walls, but it usually exists as a mixture with hemicellulose, lignin, etc. Therefore, when using cellulose in such plant cell walls as a raw material, it is first necessary to remove the hemicellulose, lignin, etc. by methods such as alkali treatment, which poses the problem of high production costs for the raw material.

The enzymatic synthesis method has low conversion efficiency from starch to glucose 1-phosphate and from sucrose to cellobiose, and requires reactions to be carried out under mild conditions (around 30°C) using enzymes, resulting in a slow reaction rate and making it unsuitable for low-cost cellobiose production.

Japanese Unexamined Patent Publication No. 2-295492 Japanese Patent Application Publication No. 8-89274 Japanese Patent Application Publication No. 3-130086 Special table 2019-507603 publication

Suzuki et al., J. Appl. Glycosci., (2010), 57:113-119

The object of the present invention is to provide a method for producing cellobiose from sucrose with a high conversion rate and at low cost.

The present inventors have discovered that by allowing sucrose to coexist in a reaction solution containing yeast expressing sucrose phosphorylase (hereinafter sometimes referred to as "SP") and cellobiose phosphorylase (hereinafter sometimes referred to as "CBP") and glucose isomerase (hereinafter sometimes referred to as "XI"), it is possible to produce cellobiose at a high conversion rate and at low cost, and have completed the present invention.

That is, the present invention includes the following inventions.
[Item 1] A method for producing cellobiose, comprising a reaction step of allowing sucrose to coexist in a reaction solution containing a recombinant yeast expressing sucrose phosphorylase and cellobiose phosphorylase and glucose isomerase.
[Item 2] The production method according to Item 1, wherein the recombinant yeast is a yeast of the genus Saccharomyces, Pichia, or Yarrowia.
[Item 3] The production method according to Item 1 or 2, wherein the sucrose phosphorylase is derived from a bacterium of the genus Bifidobacterium, Leuconostoc, or Streptococcus.
[Item 4] The method according to any one of Items 1 to 3, wherein the sucrose phosphorylase comprises a secreted sucrose phosphorylase secreted and expressed from a recombinant yeast, and an tethered sucrose phosphorylase expressed in a state tethered to the cell wall of the recombinant yeast.
[Item 5] The method according to any one of Items 1 to 4, wherein the cellobiose phosphorylase is derived from a bacterium of the genus Clostridium, Ruminococcus, Cellulomonas, or Thermotoga.
[Item 6] The method according to any one of Items 1 to 5, wherein the recombinant yeast is a recombinant yeast that expresses both sucrose phosphorylase and cellobiose phosphorylase in a single cell.
[Item 7] The method according to any one of Items 1 to 6, wherein the recombinant yeast is a combination of a recombinant yeast expressing sucrose phosphorylase and a recombinant yeast expressing cellobiose phosphorylase.
[Item 8] The method according to any one of Items 1 to 7, wherein the recombinant yeast is a yeast transformed with an expression vector carrying a sucrose phosphorylase gene.
[Item 9] The production method described in Item 8, wherein the expression vector comprises a combination of a first expression vector carrying both a sucrose phosphorylase gene and a yeast cell wall domain gene, and a second expression vector carrying only the sucrose phosphorylase gene but not the yeast cell wall domain gene.
[Item 10] The method according to any one of Items 1 to 9, wherein the glucose isomerase is derived from a bacterium of the genus Streptomyces.
[Item 11] The method according to any one of Items 1 to 10, further comprising treating the recombinant yeast at 45° C. to 75° C. for 15 to 150 minutes before allowing sucrose to coexist in the reaction solution.
[Item 12] The method according to any one of claims 1 to 11, further comprising culturing yeast cells using glycerol as a carbon source prior to the reaction step.
[Item 13] The method according to any one of Items 1 to 12, further comprising a step of separating the produced cellobiose from the reaction solution after the reaction step.
[Item 14] The method according to any one of Items 1 to 13, further comprising recovering the recombinant yeast from the reaction solution after the reaction step and subjecting it to another reaction step.

The present invention makes it possible to produce cellobiose from sucrose with a high conversion rate and low cost.

FIG. 1 is a graph showing the results of measuring the sucrose phosphorylase activity of a recombinant yeast strain expressing sucrose phosphorylase. FIG. 2 is a graph showing the changes over time in the concentrations of sucrose, glucose, fructose, and cellobiose in the reaction supernatant in an experiment to produce cellobiose from sucrose using a recombinant yeast strain expressing sucrose phosphorylase in combination with a recombinant yeast strain expressing cellobiose phosphorylase. The graph in Figure 2(a) shows the results when a strain expressing cellobiose phosphorylase (CsCBP) derived from Clostridium stercorarium was used, the graph in Figure 2(b) shows the results when a strain expressing cellobiose phosphorylase (CuCBP) derived from Cellulomonas uda was used, the graph in Figure 2(c) shows the results when a strain expressing cellobiose phosphorylase (TaCBP) derived from Thermosipho africanus was used, and the graph in Figure 2(d) shows the results when a strain expressing cellobiose phosphorylase (TnCBP) derived from Thermotoga neapolitana was used. Ibid. Figure 3 is a graph showing the time course of the concentrations of sucrose, glucose, fructose, and cellobiose in the reaction supernatant in an experiment to produce cellobiose from sucrose using a single recombinant yeast strain co-expressing sucrose phosphorylase and cellobiose phosphorylase. The graph in Figure 3(a) shows the results when a recombinant yeast with Saccharomyces cerevisiae as the host was used, and the graph in Figure 3(b) shows the results when a recombinant yeast with Pichia pastoris as the host was used. Figure 4 is a graph showing the time course of the concentrations of sucrose, glucose, fructose, and cellobiose in the reaction supernatant of an experiment to produce cellobiose from sucrose using a single recombinant yeast strain co-expressing sucrose phosphorylase and cellobiose phosphorylase with Pichia pastoris as the host. The graph in Figure 4(a) shows the results when the Pp-SP2CBP strain was cultured using glucose as the carbon source, and the graph in Figure 4(b) shows the results when the Pp-SP2CBP strain was cultured using glycerol as the carbon source. Figure 5 is a graph showing the time course of sucrose, glucose, fructose, and cellobiose concentrations in the reaction supernatant of a repeated experiment of cellobiose production from sucrose using a single recombinant yeast strain co-expressing sucrose phosphorylase and cellobiose phosphorylase. The graph in Figure 5(a) shows the results when recombinant yeast with Saccharomyces cerevisiae as the host was cultured using glucose as the carbon source, and the graph in Figure 5(b) shows the results when recombinant yeast with Pichia pastoris as the host was cultured using glycerol as the carbon source.

The present invention will be described in detail below with reference to specific embodiments. However, the present invention is not limited to the following embodiments, and can be implemented in any form without departing from the spirit of the present invention. All patent and non-patent documents described in this specification are incorporated by reference in their entirety into this application.

In this specification, "identity" of amino acid sequences means the proportion of identical amino acid residues, and "similarity" means the proportion of identical or similar amino acid residues. Homology and identity of amino acid sequences can be determined, for example, by the BLAST method (NCBI PBLAST default conditions). Furthermore, for example, when it is expressed as "80% or more homology," it clearly includes the case of "80% or more identity."

In addition, as used herein, the term "similar amino acid residues" refers to amino acid residues having side chains with similar chemical properties (e.g., charge or hydrophobicity). Examples of similar amino acid residues include the following combinations:
(1) Amino acid residues having an aliphatic side chain: glycine (Gly or G), alanine (Ala or A), valine (Val or V), leucine (Leu or L), and isoleucine (Ile or I) residues.
(2) Amino acid residues having aliphatic hydroxyl side chains: serine (Ser or S) and threonine (Thr or T) residues.
(3) Amino acid residues having amide-containing side chains: asparagine (Asn or N) and glutamine (Gln or Q) residues.
(4) Amino acid residues having aromatic side chains: phenylalanine (Phe or F), tyrosine (Tyr or Y), and tryptophan (Trp or W) residues.
(5) Amino acid residues having basic side chains: lysine (Lys or K), arginine (Arg or R), and histidine (His or H) residues.
(6) Amino acid residues having acidic side chains: aspartic acid (Asp or D) and glutamic acid (Glu or E) residues.
(7) Amino acid groups having sulfur-containing side chains: cysteine (Cys or C) and methionine (Met or M) residues.
Furthermore, the combination of (1) and a methionine (Met or M) residue, and the combination of (4) and a histidine (His or H) residue are also treated as similar amino acid residues.

One aspect of the present invention relates to a method for producing cellobiose. The method includes a step of allowing sucrose to coexist in a reaction solution containing a recombinant yeast expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP) and glucose isomerase (XI).

The yeast species is not particularly limited as long as it is capable of expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP). Examples include yeasts of the genus Saccharomyces, Pichia, or Yarrowia. In particular, to prevent sucrose from being decomposed by the action of invertase, it is preferable to use yeasts that do not have invertase activity on the cell surface, such as Pichia pastoris. For yeasts that have strong invertase activity on the cell surface, such as Saccharomyces cerevisiae, it is preferable to use a strain in which invertase activity has been lost by gene knockout or the like. In addition, since Pichia pastoris has been classified and renamed as Komagataella pastoris and Komagataella phaffii, Pichia pastoris is a synonym for both Komagataella pastoris and Komagataella phaffii.

The origin (species) of sucrose phosphorylase (SP) is not particularly limited. Examples include those derived from bacteria of the genus Bifidobacterium, Leuconostoc, or Streptococcus. Of these, those derived from bacteria of the genus Bifidobacterium are preferred, and those derived from Bifidobacterium longum are particularly preferred. The amino acid sequence of sucrose phosphorylase (SP) from Bifidobacterium longum is shown in SEQ ID NO: 34, and an example of a base sequence encoding this is shown in SEQ ID NO: 5. Examples of sucrose phosphorylase (SP) derived from Bifidobacterium longum include proteins consisting of an amino acid sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more homology (preferably identity) with SEQ ID NO:34.

When sucrose phosphorylase (SP) is expressed in recombinant yeast, it can be in the form of a secreted enzyme secreted and expressed from the recombinant yeast, or in the form of an tethered enzyme expressed in a state tethered to the cell wall of the recombinant yeast, but either form is acceptable. However, in the present invention, it is preferable to allow the secreted sucrose phosphorylase (SP) secreted and expressed from the recombinant yeast and the tethered sucrose phosphorylase (SP) expressed in a state tethered to the cell wall of the recombinant yeast to coexist in the reaction solution. This makes it possible to improve the conversion efficiency by sucrose phosphorylase (SP). The reason for this is unclear, but it is speculated to be due to the formation of a dimer between the secreted sucrose phosphorylase (SP) and the tethered sucrose phosphorylase (SP).

The origin (species) of cellobiose phosphorylase (CBP) is not particularly limited, but examples include those derived from bacteria of the genus Clostridium, Ruminococcus, Cellulomonas, or Thermotoga. Among these, those derived from bacteria of the genus Clostridium are preferred, and those derived from Clostridium stercorarium are particularly preferred. The amino acid sequence of cellobiose phosphorylase (CBP) from Clostridium stercorarium is shown in SEQ ID NO: 35, and an example of the base sequence encoding this is shown in SEQ ID NO: 6. Examples of cellobiose phosphorylase (CBP) derived from Clostridium stercorarium include proteins consisting of an amino acid sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more homology (preferably identity) with SEQ ID NO:35.

When expressing cellobiose phosphorylase (CBP) in recombinant yeast, it may be in the form of a secreted enzyme secreted and expressed from the recombinant yeast, or in the form of an tethered enzyme expressed in a state tethered to the cell wall of the recombinant yeast, either of which may be used. However, in the present invention, it is preferable to express it in the form of tethered cellobiose phosphorylase (CBP) expressed in a state tethered to the cell wall of the recombinant yeast.

As the recombinant yeast, a recombinant yeast that co-expresses sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP) in a single cell may be used, or a recombinant yeast that expresses sucrose phosphorylase (SP) may be used in combination with a recombinant yeast that expresses cellobiose phosphorylase (CBP). In addition, when a secreted enzyme and an tethered enzyme are used in combination as sucrose phosphorylase (SP) and/or cellobiose phosphorylase (CBP), a recombinant yeast that co-expresses the secreted enzyme and the tethered enzyme in a single cell may be used, or a recombinant yeast that expresses the secreted enzyme may be used in combination with a recombinant yeast that expresses the tethered enzyme.

The method for preparing recombinant yeast expressing sucrose phosphorylase (SP) and/or cellobiose phosphorylase (CBP) is not particularly limited. For example, one or more types of yeast are transformed with one or more expression vectors carrying the sucrose phosphorylase (SP) gene and/or the cellobiose phosphorylase (CBP) gene. Examples of expression vectors include plasmids, cosmids, lambda phages, etc., with plasmids being preferred. It is also preferred that the sucrose phosphorylase (SP) gene and/or cellobiose phosphorylase (CBP) gene are operably linked to regulatory sequences such as a promoter and a terminator, and carried in the expression vector in the form of an expression cassette that can be expressed autonomously in yeast cells. Such expression vectors can be constructed using standard genetic engineering techniques known to those skilled in the art, by referring appropriately to literature such as Michael R. Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, (2012), Cold Spring Harbor Laboratory.

The recombinant yeast is preferably pre-cultured in the presence of a carbon source prior to the reaction. There are no particular limitations on the type of carbon source used when culturing the recombinant yeast cells, and it may be selected appropriately depending on the type of yeast. Examples include glucose and glycerol. Of these, in the present invention, it is preferable to use at least glycerol as a carbon source. This can improve the conversion rate of sucrose to cellobiose.

It is preferable to subject the recombinant yeast to a pretreatment prior to the reaction to stop the uptake of glucose. An example of such a pretreatment is a heat treatment in which the yeast is exposed to a certain temperature for a certain period of time. In this case, the treatment conditions are sufficient as long as they can stop the uptake of glucose by the yeast used. The heating temperature can be, for example, 45°C or higher, or 50°C or higher, or 55°C or higher, and can be, for example, 75°C or lower, or 70°C or lower, or 65°C or lower. The heating time can be, for example, 15 minutes or more, or 45 minutes or more, or 70 minutes or more, and can be, for example, 150 minutes or less, 120 minutes or less, or 100 minutes or less. In a preferred example, the temperature can be about 60°C, and the time can be about 90 minutes. One example of such a pretreatment method is shaking a suspension of the transformed yeast under the above conditions.

In the present invention, glucose isomerase (XI) is allowed to coexist in a reaction solution containing recombinant yeast expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP). The amount of recombinant yeast cells in the reaction solution is not particularly limited, but can be, for example, 25 g wet cells/L or more, 50 g wet cells/L or more, or 75 g wet cells/L or more, and can be, for example, 200 g wet cells/L or less, 150 g wet cells/L or less, or 100 g wet cells/L or less.

Glucose isomerase (also known as xylose isomerase, XI) is, but is not limited to, for example, that derived from yeast of the genus Streptomyces. Among them, that derived from Streptomyces griseofuscus is particularly preferred. Note that various commercially available glucose isomerase products can be used.

Glucose isomerase (XI) may be expressed in yeast or other microorganisms in the same manner as other enzymes, but it is preferable to use the isolated glucose isomerase (XI) enzyme itself. The isolated glucose isomerase (XI) enzyme may be either a commercially available product or one obtained by culturing a microorganism that produces these enzymes. It may also be either a purified product or an unpurified product. In this case, it is preferable to add the isolated glucose isomerase (XI) enzyme to a reaction solution containing recombinant yeast that expresses sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP) and allow them to coexist.

The amount of glucose isomerase (XI) is not particularly limited, but is usually preferably 0.01 units or more, 0.1 units or more, or 1.0 units or more per mole of sucrose. Here, 1 unit (1 U) means the amount of enzyme that breaks down 1 μmol of fructose in 1 wt/vol % sucrose at 50°C and pH 7.0 per minute.

In the present invention, the reaction is carried out in the presence of sucrose in a reaction solution containing recombinant yeast expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP) and glucose isomerase (XI). Sucrose may be naturally occurring or chemically synthesized. There are no particular limitations on the amount of sucrose, but from the viewpoint of productivity, a concentration as high as possible is preferable. However, since the solubility of the cellobiose produced is about 15 to 20%, if it is desired to keep it dissolved, it is preferable to use a sucrose concentration of about 10 to 20%.

The conditions for the cellobiose synthesis reaction are not limited, but may be, for example, as follows. That is, the reaction temperature may be, for example, 35°C or higher, 45°C or higher, or 50°C or higher, and, for example, 60°C or lower, or 70°C or lower. The reaction time may be, for example, 60 minutes or more, 12 hours or more, or 24 hours or more, and may be, for example, 36 hours or less, or 48 hours or less. The pressure during the reaction is not particularly limited, and may be reduced pressure, normal pressure, or pressurized, but is usually carried out under normal pressure. The atmosphere during the reaction is also not particularly limited, and any atmosphere may be used as long as the enzyme activity is maintained even in an atmosphere in which the recombinant yeast used cannot grow.

After the reaction is completed, cellobiose can be obtained by separating and purifying it from the reaction solution as necessary. There are no particular limitations on the method of separation and purification, and any method can be used, including, for example, filtration separation, centrifugation, membrane separation, etc. These methods allow efficient separation of the used bacteria and cellobiose. Furthermore, in order to remove impurities from the obtained cellobiose or its solution, it may be purified to a higher degree by treatment with an ion exchange resin or recrystallization.

After the reaction is completed, the recombinant yeast may be separated and recovered from the reaction solution as necessary and reused in the cellobiose synthesis reaction. Even when the recombinant yeast of the present invention is separated and recovered from the reaction solution and reused in this way, the enzyme activity may be maintained, and high cellobiose synthesis efficiency may be repeatedly obtained. That is, according to a preferred embodiment, the cellobiose production method of the present invention further includes recovering the recombinant yeast from the reaction solution after the reaction step and subjecting it to another reaction step. The method for separating and recovering the recombinant yeast from the reaction solution is not limited, but examples include centrifugation. The number of times the recombinant yeast is separated and recovered and reused is not particularly limited, but according to one embodiment, it can be subjected to a reaction, for example, two or more times, three or more times, four or more times, or five or more times. The activity of the recombinant yeast when reused is not particularly limited, but according to one embodiment, it is preferable that 60% or more, 70% or more, 80% or more, or 90% or more of the activity in the previous reaction is maintained in the next reaction.

The present invention will be described in more detail below with reference to examples. However, these examples are merely examples shown for the convenience of explanation, and the present invention is in no way limited to these examples.

All PCR methods shown in this example were performed using KOD One ^™ PCR Master Mix (Toyobo Co., Ltd.) All gene introduction into yeasts shown in this example was performed by the lithium acetate method.

[Example Group I: Cellobiose production using transformed yeast (yeast displaying sucrose phosphorylase on the surface, yeast displaying cellobiose phosphorylase on the surface, and yeast displaying sucrose phosphorylase + cellobiose phosphorylase on the surface)]

A. Preparation of transformed yeast Various recombinant yeast strains expressing two enzymes, sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP), were constructed in yeast Saccharomyces cerevisiae BY4741ΔSUC2 strain by the lithium acetate method using the prepared plasmids. The details are described below.

Bacterial strains and medium As the bacterial strains, the yeast Saccharomyces cerevisiae BY4741 strain (MATα his3 leu2 met15 ura3 strain) and the yeast Saccharomyces cerevisiae BY4741ΔSUC2 strain (MATα his3 leu2 met15 ura3 suc2 strain) were used. The yeast Saccharomyces cerevisiae BY4741 strain was obtained from ATCC (American Type Culture Collection), and the yeast Saccharomyces cerevisiae BY4741ΔSUC2 strain was obtained from Yeast deletion MAT-A complete set (Thermo Fisher Scientific).

Construction of Plasmids Containing Expression Cassettes Plasmids containing the following expression cassettes X1 to X9 were prepared by the following procedure.
X1: SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + BlSP (SEQ ID NO: 5) + SED1 anchoring region (SEQ ID NO: 3) + DIT1 terminator (SEQ ID NO: 4)
X2: SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + BlSP (SEQ ID NO: 5) + DIT1 terminator (SEQ ID NO: 4)
X3: SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + CsCBP (SEQ ID NO: 6) + SED1 anchoring region (SEQ ID NO: 3) + DIT1 terminator (SEQ ID NO: 4)
X4: SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + CuCBP (SEQ ID NO: 7) + SED1 anchoring region (SEQ ID NO: 3) + DIT1 terminator (SEQ ID NO: 4)
X5: SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + TaCBP (SEQ ID NO: 8) + SED1 anchoring region (SEQ ID NO: 3) + DIT1 terminator (SEQ ID NO: 4)
X6: SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + TnCBP (SEQ ID NO: 9) + SED1 anchoring region (SEQ ID NO: 3) + DIT1 terminator (SEQ ID NO: 4)
X7: SPI1 promoter (SEQ ID NO: 10) + SPI1 secretion signal (SEQ ID NO: 11) + BlSP (SEQ ID NO: 5) + GCW30 anchoring region (SEQ ID NO: 12) + AOX1 terminator (SEQ ID NO: 13)
X8: SPI1 promoter (SEQ ID NO: 10) + SPI1 secretion signal (SEQ ID NO: 11) + BlSP (SEQ ID NO: 5) + AOX1 terminator (SEQ ID NO: 13)
X9: SPI1 promoter (SEQ ID NO: 10) + SPI1 secretion signal (SEQ ID NO: 11) + CsCBP (SEQ ID NO: 6) + GCW30 anchoring region (SEQ ID NO: 12) + AOX1 terminator (SEQ ID NO: 13)

(Preparation Example 1-1: Preparation of vector plasmid containing Bifidobacterium longum-derived sucrose phosphorylase (BISP) surface expression cassette X1)
A DNA fragment containing the coding region of the sucrose phosphorylase (BISP) gene derived from Bifidobacterium longum was prepared by gene synthesis. Next, vector plasmid pIEG-SSSD (a surface expression vector having an expression cassette in which the SED1 promoter derived from Saccharomyces cerevisiae, the secretory signal peptide sequence of SED1 derived from Saccharomyces cerevisiae, the coding region of the endoglucanase II (TrEGII) gene derived from Trichoderma reesei, the coding region (SED1 anchoring region) of the SED1 gene derived from Saccharomyces cerevisiae, and the terminator region of the DIT1 gene derived from Saccharomyces cerevisiae are arranged in this order: Applied Microbiology and Biotechnology, Vol.105, 2021, The resulting vector plasmid was amplified by PCR using primer pair SED1a-F (SEQ ID NO: 14) and SED1ss-R (SEQ ID NO: 15) with pIBISP-SSSD (SEQ ID NO: 5895-5904) as a template, and a DNA fragment containing the entire length of the vector plasmid excluding the coding region of the TrEGII gene was prepared. These fragments were ligated by the In-Fusion method. The resulting plasmid containing expression cassette X1 (SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + BlSP (SEQ ID NO: 5) + SED1 anchoring region (SEQ ID NO: 3) + DIT1 terminator (SEQ ID NO: 4)) was named pIBlSP-SSSD.

(Preparation Example 1-2: Preparation of vector plasmid containing Bifidobacterium longum-derived sucrose phosphorylase (BISP) secretion expression cassette X2)
Using the plasmid pIBlSP-SSSD as a template, the primer pair DIT1t-F (SEQ ID NO: 16) and BlSP-R (SEQ ID NO: 17) were used to amplify by PCR to prepare a DNA fragment containing the entire plasmid excluding the SED1 anchoring region. Both ends of this fragment were ligated and circularized by the In-Fusion method. Next, using this plasmid as a template, the primer pair BlSP-F (SEQ ID NO: 18) and DIT1t-R (SEQ ID NO: 19) were used to amplify by PCR to prepare a DNA fragment containing the coding region of the sucrose phosphorylase (BlSP) gene derived from Bifidobacterium longum and the terminator region of the DIT1 gene derived from Saccharomyces cerevisiae. In addition, vector plasmid pIL2-BG-SSS (a surface expression vector having an expression cassette in which the SED1 promoter derived from Saccharomyces cerevisiae, the secretory signal peptide sequence of SED1 derived from Saccharomyces cerevisiae, the coding region of the β-glucosidase 1 (AaBGL1) gene derived from Aspergillus aculeatus, the coding region (SED1 anchoring region) of the SED1 gene derived from Saccharomyces cerevisiae, and the terminator region of the α-agglutinin gene derived from Saccharomyces cerevisiae are arranged in this order: Metabolic Engineering, Vol. 57, 2020, Using pRS403-F (SEQ ID NO: 20) and pRS403-S (SEQ ID NO: 15) as a primer pair and pRS403-F (SEQ ID NO: 20), a DNA fragment was prepared that contained the entire length of the vector plasmid excluding the coding region of the β-glucosidase 1 (AaBGL1) gene derived from Aspergillus aculeatus, the coding region (SED1 anchoring region) of the SED1 gene derived from Saccharomyces cerevisiae, and the terminator region of the α-agglutinin gene derived from Saccharomyces cerevisiae. These fragments were ligated by the In-Fusion method. The resulting plasmid containing the expression cassette X2 (SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + BlSP (SEQ ID NO: 5) + DIT1 terminator (SEQ ID NO: 4)) was named pIL2-BlSP-SSD.

(Preparation Example 1-3: Preparation of vector plasmid containing Clostridium stercorarium-derived cellobiose phosphorylase (CsCBP) surface expression cassette X3)
A DNA fragment containing the coding region of the cellobiose phosphorylase (CsCBP) gene derived from Clostridium stercorarium was prepared by gene synthesis. Next, vector plasmid pIEG-SSSD (a surface expression vector having a cassette in which the SED1 promoter derived from Saccharomyces cerevisiae, the secretory signal peptide sequence of SED1 derived from Saccharomyces cerevisiae, the coding region of the endoglucanase II (TrEGII) gene derived from Trichoderma reesei, the coding region (SED1 anchoring region) of the SED1 gene derived from Saccharomyces cerevisiae, and the terminator region of the DIT1 gene derived from Saccharomyces cerevisiae are arranged in this order: Applied Microbiology and Biotechnology, Vol.105, 2021, Using the primer pair SED1a-F (SEQ ID NO: 14) and SED1ss-R (SEQ ID NO: 15) and PCR amplification was performed using the 5'-terminal end of the primer pair SED1a-F (SEQ ID NO: 14) and SED1ss-R (SEQ ID NO: 15) as a template to prepare a DNA fragment containing the full length of the vector plasmid excluding the coding region of the TrEGII gene. These fragments were ligated by the In-Fusion method. Next, using this plasmid as a template, a primer pair SED1p-F (SEQ ID NO: 21) and DIT1t-R (SEQ ID NO: 19) was used to amplify by PCR to prepare a DNA fragment containing the SED1 promoter derived from Saccharomyces cerevisiae, the secretory signal peptide sequence of SED1 derived from Saccharomyces cerevisiae, the coding region of cellobiose phosphorylase (CsCBP) derived from Clostridium stercorarium, the coding region (SED1 anchoring region) of the SED1 gene derived from Saccharomyces cerevisiae, and the terminator region of the DIT1 gene derived from Saccharomyces cerevisiae. Also, vector plasmid pIU5-CBH _D (a surface expression vector having a cassette in which the Saccharomyces cerevisiae-derived SED1 promoter, the coding region of the Talaromyces emersonii-derived cellobiohydrolase (TsCBH1) gene, the coding region of the Saccharomyces cerevisiae-derived SED1 gene (SED1 anchoring region), and the terminator region of the Saccharomyces cerevisiae-derived α-agglutinin gene are arranged in this order: Biotechnology and Bioengineering, Vol. 114, 2017, Using pRS403-F (SEQ ID NO: 20) and pRS403-R (SEQ ID NO: 22) as a template, the vector plasmid was amplified by PCR using primer pair pRS403-F (SEQ ID NO: 20) and pRS403-R (SEQ ID NO: 22) to prepare a DNA fragment containing the entire length of the vector plasmid excluding the expression cassette region. These fragments were ligated by the In-Fusion method. The resulting plasmid containing expression cassette X3 (SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + CsCBP (SEQ ID NO: 6) + SED1 anchoring region (SEQ ID NO: 3) + DIT1 terminator (SEQ ID NO: 4)) was named pIU5-CsCBP-SSSD.

(Preparation Example 1-4: Preparation of vector plasmid containing Cellulomonas uda-derived cellobiose phosphorylase (CuCBP) surface expression cassette X4)
A DNA fragment containing the coding region of the cellobiose phosphorylase (CuCBP) gene derived from Cellulomonas uda was prepared by gene synthesis. Next, the vector plasmid pIU5-CsCBP-SSSD was used as a template, and the primer pair SED1a-F (SEQ ID NO: 14) and SED1ss-R (SEQ ID NO: 15) were used to amplify by PCR to prepare a DNA fragment containing the full length of the vector plasmid excluding the coding region of the CsCBP gene. These fragments were ligated by the In-Fusion method. The resulting plasmid containing the expression cassette X4 (SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + CuCBP (SEQ ID NO: 7) + SED1 anchoring region (SEQ ID NO: 3) + DIT1 terminator (SEQ ID NO: 4)) was named pIU5-CuCBP-SSSD.

(Preparation Example 1-5: Preparation of vector plasmid containing Thermosipho africanus-derived cellobiose phosphorylase (TaCBP) surface expression cassette X5)
A DNA fragment containing the coding region of the Thermosipho africanus-derived cellobiose phosphorylase (TaCBP) gene was prepared by gene synthesis. Next, the vector plasmid pIU5-CsCBP-SSSD was used as a template, and the primer pair SED1a-F (SEQ ID NO: 14) and SED1ss-R (SEQ ID NO: 15) were used to amplify the vector plasmid by PCR, to prepare a DNA fragment containing the full length of the vector plasmid excluding the coding region of the CsCBP gene. These fragments were ligated by the In-Fusion method. The resulting plasmid containing the expression cassette X5 (SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + TaCBP (SEQ ID NO: 8) + SED1 anchoring region (SEQ ID NO: 3) + DIT1 terminator (SEQ ID NO: 4)) was named pIU5-TaCBP-SSSD.

(Preparation Example 1-6: Preparation of vector plasmid containing Thermotoga neapolitana-derived cellobiose phosphorylase (TnCBP) surface expression cassette X6)
A DNA fragment containing the coding region of the Thermotoga neapolitana-derived cellobiose phosphorylase (TnCBP) gene was prepared by gene synthesis. Next, the vector plasmid pIU5-CsCBP-SSSD was used as a template, and the primer pair SED1a-F (SEQ ID NO: 14) and SED1ss-R (SEQ ID NO: 15) were used to amplify the vector plasmid by PCR, to prepare a DNA fragment containing the full length of the vector plasmid excluding the coding region of the CsCBP gene. These fragments were ligated by the In-Fusion method. The resulting plasmid containing the expression cassette X6 (SED1 promoter (SEQ ID NO: 1) + SED1 secretion signal (SEQ ID NO: 2) + TnCBP (SEQ ID NO: 9) + SED1 anchoring region (SEQ ID NO: 3) + DIT1 terminator (SEQ ID NO: 4)) was named pIU5-TnCBP-SSSD.

(Preparation Example 1-7: Preparation of vector plasmid containing Bifidobacterium longum-derived sucrose phosphorylase (BISP) surface expression cassette X7)
Using the plasmid pIBlSP-SSSD as a template, amplified by PCR using the primer pair BlSP-NheI-F (SEQ ID NO: 23) and BlSP-XhoI-R (SEQ ID NO: 24), and further treated with NheI and XhoI, to prepare a DNA fragment containing the coding region of the BlSP gene. In addition, vector plasmid pIBG-PpSSG61 (a surface expression vector having an expression cassette in which the SPI1 promoter derived from Pichia pastoris, the secretory signal peptide sequence of SPI1 derived from Pichia pastoris, the coding region of the β-glucosidase 1 (AaBGL1) gene derived from Aspergillus aculeatus, the coding region of the GCW61 gene derived from Pichia pastoris (GCW61 anchoring region), and the terminator region of the AOX1 gene derived from Pichia pastoris are arranged in this order: Biotechnology and Bioengineering, Vol.120, 2023, 1097-1107) was treated with NheI and XhoI to prepare a DNA fragment containing the entire length of the vector plasmid excluding the coding region of the AaBGL1 gene. These fragments were ligated together. Next, this plasmid was used as a template and treated with XhoI and MluI to prepare a DNA fragment containing the entire length of the vector plasmid excluding the GCW61 anchoring region. Furthermore, plasmid pIBG-PpGMG30 (a surface expression vector having an expression cassette in which a GAPDH promoter derived from Pichia pastoris, a secretory signal peptide sequence of α-factor derived from Saccharomyces cerevisiae, a coding region of β-glucosidase 1 (AaBGL1) gene derived from Aspergillus aculeatus, a coding region of GCW30 gene derived from Pichia pastoris (GCW30 anchoring region), and a terminator region of AOX1 gene derived from Pichia pastoris are arranged in this order: Biotechnology and Bioengineering, Vol.120, 2023, 1097-1107) was treated with XhoI and MluI to prepare a DNA fragment containing the GCW30 anchoring region. These fragments were ligated together, and the resulting plasmid containing the expression cassette X7 (SPI1 promoter (SEQ ID NO: 10) + SPI1 secretion signal (SEQ ID NO: 11) + BlSP (SEQ ID NO: 5) + GCW30 anchoring region (SEQ ID NO: 12) + AOX1 terminator (SEQ ID NO: 13)) was named pIBlSP-PpSSG30.

(Preparation Example 1-8: Preparation of vector plasmid containing Bifidobacterium longum-derived sucrose phosphorylase (BISP) secretion expression cassette X8)
A DNA fragment containing the coding region of the BlSP gene was prepared by PCR amplification using the plasmid pIBlSP-PpSSG30 as a template and a primer pair BlSP-F2 (SEQ ID NO:25) and BlSP-R2 (SEQ ID NO:26). In addition, a surface expression vector having an expression cassette in which the SPI1 promoter derived from Pichia pastoris, the secretory signal peptide sequence of SPI1 derived from Pichia pastoris, the coding region of the endoglucanase II (TrEGII) gene derived from Trichoderma reesei, the coding region of the GCW34 gene derived from Pichia pastoris (GCW34 anchoring region), and the terminator region of the AOX1 gene derived from Pichia pastoris are arranged in this order: Biotechnology and Bioengineering, Vol. 120, 2023, 1097-1107) was used as a template, and a primer pair AOX1t-F (SEQ ID NO: 27) and SPI1s The vector plasmid was amplified by PCR using pIH-PpSS (SEQ ID NO: 28) and a DNA fragment containing the entire length of the vector plasmid excluding the coding region of the TrEGII gene and the GCW34 anchoring region was prepared. These fragments were ligated by the In-Fusion method. The resulting plasmid containing the expression cassette X8 (SPI1 promoter (SEQ ID NO: 10) + SPI1 secretion signal (SEQ ID NO: 11) + BlSP (SEQ ID NO: 5) + AOX1 terminator (SEQ ID NO: 13)) was named pIH-BlSP-PpSS.

(Preparation Example 1-9: Preparation of vector plasmid containing Clostridium stercorarium-derived cellobiose phosphorylase (CsCBP) surface expression cassette X9)
A DNA fragment containing the coding region of the CsCBP gene was prepared by amplifying the plasmid pIU5-CsCBP-SSSD as a template using the primer pair CsCBP-NheI-F (SEQ ID NO: 29) and CsCBP-XhoI-R (SEQ ID NO: 30) by PCR and further treating with NheI and XhoI. Also, a DNA fragment containing the full length of the vector plasmid excluding the coding region of the BlSP gene was prepared by treating the vector plasmid pIBlSP-PpSSG30 with NheI and XhoI. These fragments were ligated. Next, this plasmid was used as a template and amplified by PCR using the primer pair CsCBP-F (SEQ ID NO: 31) and AOX1t-R (SEQ ID NO: 32) to prepare a DNA fragment containing the coding region of the CsCBP gene, the GCW30 anchoring region, and the AOX1 terminator region. In addition, a vector plasmid pIZ-CBH1-PpSSG34 (a surface expression vector having an expression cassette in which the coding region of SPI1 derived from Pichia pastoris, the coding region of the cellobiohydrolase (TsCBH1) gene derived from Talaromyces emersonii, the coding region of the GCW34 gene derived from Pichia pastoris (GCW34 anchoring region), and the terminator region of the AOX1 gene derived from Pichia pastoris are arranged in this order: Biotechnology and Bioengineering, Vol. 120, 2023, 1097-1107) was used as a template, and a primer pair AOX1t-F2 (SEQ ID NO: 33) and SPI1s The vector was amplified by PCR using pIZ-CsCBP-PpSSG30 (SEQ ID NO: 28) and a DNA fragment was prepared containing the entire length of the vector plasmid excluding the coding region of the TsCBH1 gene, the GCW34 anchoring region, and the AOX1 terminator region. These fragments were ligated by the In-Fusion method. The resulting plasmid containing the expression cassette X9 (SPI1 promoter (SEQ ID NO: 10) + SPI1 secretion signal (SEQ ID NO: 11) + CsCBP (SEQ ID NO: 6) + GCW30 anchoring region (SEQ ID NO: 12) + AOX1 terminator (SEQ ID NO: 13)) was named pIZ-CsCBP-PpSSG30.

・Creation of transformed yeast
(Preparation Example 2-1: Production of sucrose phosphorylase surface-displaying yeast)
The plasmid pIBlSP-SSSD described in Preparation Example 1-1 was treated with NdeI, and then used in yeast Saccharomyces cerevisiae BY4741ΔSUC2 strain, which was transformed by the lithium acetate method. This transformed strain is referred to as the ΔSUC2-BlSP strain. Similarly, the plasmid pIBlSP-PpSSG30 described in Preparation Example 1-7 was treated with EcoRV, and then used in yeast Pichia pastoris CBS7435 strain, which was transformed by the lithium acetate method. This transformed strain is referred to as the Pp-BlSP strain.

Furthermore, the plasmid pIL2-BlSP-SSD described in Preparation Example 1-2 was treated with NdeI, and then used in the ΔSUC2-BlSP strain to transform it by the lithium acetate method. This transformed strain is called the ΔSUC2-BlSP2 strain. Similarly, the plasmid pIH-BlSP-PpSS described in Preparation Example 1-8 was treated with BsrGI, and then used in the Pp-BlSP strain to transform it by the lithium acetate method. This transformed strain is called the Pp-BlSP2 strain.

(Preparation Example 2-2: Production of cellobiose phosphorylase surface-displaying yeast)
The plasmids pIU5-CsCBP-SSSD, pIU5-CuCBP-SSSD, pIU5-TaCBP-SSSD, and pIU5-TnCBP-SSSD described in Preparation Examples 1-3 to 1-6 were treated with SpeI, and then used in yeast Saccharomyces cerevisiae BY4741ΔSUC2 strain to transform the strains by the lithium acetate method. These transformed strains are referred to as ΔSUC2-CsCBP strain, ΔSUC2-CuCBP strain, ΔSUC2-TaCBP strain, and ΔSUC2-TnCBP strain, respectively.

(Preparation Example 2-3: Production of yeast displaying sucrose phosphorylase + cellobiose phosphorylase on the surface)
The plasmid pIU5-CsCBP-SSSD described in Preparation Example 1-3 was treated with SpeI, and then used in the ΔSUC2-B1SP2 strain to transform the strain by the lithium acetate method. This transformed strain is referred to as the ΔSUC2-SP2CBP strain. Similarly, the plasmid pIZ-CsCBP-PpSSG30 described in Preparation Example 1-9 was treated with NsiI, and then used in the Pp-B1SP2 strain to transform the strain by the electroporation method. This transformed strain is referred to as the Pp-SP2CBP strain.

B. Pretreatment of transformed yeast Among the yeasts (strains) transformed in A, the ΔSUC2-BlSP strain, ΔSUC2-BlSP2 strain, ΔSUC2-CsCBP strain, ΔSUC2-CuCBP strain, ΔSUC2-TaCBP strain, ΔSUC2-TnCBP strain, and ΔSUC2-SP2CBP strains using Saccharomyces cerevisiae as a host were pretreated with 5 mL of YPD medium (1% dried yeast extract (Nacalai Tesque), Bacto ^™ Peptone (Life Technologies, Inc.)). The bacteria were transferred to 2% Nacalai Tesque (Nippon Biotechnology) and 2% D-glucose (Nacalai Tesque) and pre-cultured at 30°C and 200 rpm for 18 hours, and then inoculated into 50 mL of YPD medium to an OD600 of 0.05 and cultured with shaking at 30°C and 150 rpm for 48 hours. In addition, Pp-BlSP strain, Pp-BlSP2 strain, and Pp-SP2CBP strain, which use Pichia pastoris as a host, were transplanted into 5 mL of YPG medium (1% dry yeast extract (Nacalai Tesque), 2% Bacto ^TM Peptone (Life Technologies), 2% glycerol (Nacalai Tesque)), pre-cultured for 18 hours at 30 ° C. and 200 rpm, and then inoculated into 50 mL of YPG medium so that the OD600 was 0.05, and cultured with shaking at 30 ° C. and 150 rpm for 48 hours. These culture solutions were centrifuged to settle the cells, washed twice with pure water, and the supernatant was removed as much as possible to measure the weight of the solid wet cells (wet cells), and the same amount of pure water was added to obtain a cell suspension (about 500 g wet cells / L). The cell suspension was kept at 60° C. for 90 minutes to obtain pretreated cells.

C. Measurement of sucrose phosphorylase activity on the yeast cell surface 5 mL of reaction solution (composition: 10 g/L sucrose (Nacalai Tesque); 80 mM sodium phosphate buffer (pH 7.0); and pretreated cells of the Pp-BlSP strain or Pp-BlSP2 strain prepared in Preparation Example 2-1 (final cell concentration 100 g wet cells/L)) was prepared and reacted at 35 rpm and 60°C for 60 minutes. To stop the reaction, the reaction solution was kept at 100°C for 10 minutes, then cooled on ice for 10 minutes, centrifuged to precipitate the cells, etc., and the supernatant was collected and used for analysis of fructose concentration. The amount of enzyme that liberates 1 μmol of fructose in 1 minute is defined as 1 IU.

D. Cellobiose production from sucrose using yeast that displays sucrose phosphorylase on its surface in combination with yeast that displays cellobiose phosphorylase on its surface 5 mL of reaction solution (composition: 100 g/L sucrose (Nacalai Tesque); 80 mM sodium phosphate buffer (pH 7.0); 1% by weight magnesium sulfate; 1.5 IU/mL glucose isomerase (xylose isomerase, Godo Shusei Co., Ltd., Streptomyces griseosuchus (Streptomyces griseofuscus); pretreated cells of the ΔSUC2-BlSP2 strain prepared in Preparation Example 2-1 (final cell concentration 50 g wet cells/L); and pretreated cells of the ΔSUC2-CsCBP strain, ΔSUC2-CuCBP strain, ΔSUC2-TaCBP strain, or ΔSUC2-TnCBP strain prepared in Preparation Example 2-2 (final cell concentration 50 g wet cells/L)) were prepared and reacted at 35 rpm and 60°C for 24 hours. Sampling was performed twice, for 4 hours and 24 hours. To stop the reaction, the sampling liquid was placed in a sampling container and kept at 100°C for 10 minutes, then cooled on ice for 10 minutes, and centrifuged to precipitate the cells, etc., and the supernatant was collected and used for analysis.

E. 5 mL of cellobiose production reaction solution from sucrose using sucrose phosphorylase + cellobiose phosphorylase surface-displaying yeast (composition: 100 g / L sucrose (Nacalai Tesque); 80 mM sodium phosphate buffer (pH 7.0); 1 wt% magnesium sulfate; 1.5 IU / mL glucose isomerase (xylose isomerase, Godo Shusei Co., Ltd., derived from Streptomyces griseofuscus); and pretreated cells of the ΔSUC2-SP2CBP strain or Pp-BlSP2 strain prepared in Preparation Example 2-3 (final cell concentration 100 g wet cells / L)) were prepared and reacted at 35 rpm and 60 ° C. for 24 hours. Sampling was performed three times at 4 hours, 8 hours, and 24 hours. To terminate the reaction, the sampled liquid was placed in a sampling container, kept at 100° C. for 10 minutes, then cooled on ice for 10 minutes and centrifuged to precipitate the bacteria and the like, and the supernatant was collected and used for analysis.

F. Analysis HPLC was used to analyze the supernatants of C, D, and E. The analysis conditions were as follows:
Equipment: pump unit (Shimadzu LC-20AB), autosampler (Shimadzu SIL-20AC), column oven (Shimadzu CTO-20AC)
Column used: Sugar-D (COSMOSIL, Code: 05397-51, ID 4.6 x 250 mm)
Guard column: Sugar-D guard column (COSMOSIL, Code: 05397-81)
Mobile phase: acetonitrile/water = 70/30, flow rate 1.0 mL/min Column oven temperature: 30 ° C.
Detector: Refractive index detector (RID) (Shimadzu RID-10A)
Analysis time: 15 minutes. Calibration curve: Calibration was performed at three points of 1, 5, and 10 g/L using a sugar mixture (sucrose, glucose, fructose, and cellobiose).

G. The results of sucrose phosphorylase activity measurement of Result C are shown in Figure 1. Compared with the ΔSUC2-BlSP strain into which only the expression cassette X1 for expressing sucrose phosphorylase in a cell wall-anchored state was introduced, the ΔSUC2-BlSP2 strain into which the expression cassette X2 for secreting and expressing sucrose phosphorylase in addition to the expression cassette X1 was introduced, was found to exhibit significantly higher sucrose phosphorylase activity. This shows that it is preferable to use tethered and secreted sucrose phosphorylase in combination.

Figure 2 shows the time course of the sucrose, glucose, fructose, and cellobiose concentrations in the reaction supernatant of an experiment to produce cellobiose from sucrose using a combination of yeast D that displays sucrose phosphorylase and yeast D that displays cellobiose phosphorylase. When the ΔSUC-CsCBP strain was used as the yeast that displays cellobiose phosphorylase, the conversion rate of sucrose to cellobiose was approximately 30% 24 hours after the start of the reaction (Figure 2(a)). On the other hand, when the ΔSUC-CuCBP strain (Figure 2(b)), ΔSUC-TaCBP strain (Figure 2(c)), or ΔSUC-TnCBP strain (Figure 2(d)) was used as the yeast that displays cellobiose phosphorylase, the conversion rate was less than 10% even 24 hours after the start of the reaction. These results indicate that the CBP gene derived from Clostridium stercorarium is preferred.

Figure 3 shows the time course of the concentrations of sucrose, glucose, fructose, and cellobiose in the reaction supernatant of an experiment to produce cellobiose from sucrose using a sucrose phosphorylase + cellobiose phosphorylase surface-displaying yeast (ΔSUC2-SP2CBP strain) with Saccharomyces cerevisiae as the host. The graph in Figure 3(a) shows the results when the ΔSUC2-SP2CBP strain was used with Saccharomyces cerevisiae as the host, and the graph in Figure 3(b) shows the results when the Pp-SP2CBP strain was used with Pichia pastoris as the host. Compared to the experimental results in Figure 2(a), which used a recombinant yeast strain expressing only sucrose phosphorylase and a recombinant yeast strain expressing only cellobiose phosphorylase in combination with Saccharomyces cerevisiae as a host, Figure 3(a), which used a single recombinant yeast strain co-expressing sucrose phosphorylase and cellobiose phosphorylase with the same Saccharomyces cerevisiae as a host, shows that sucrose was converted to cellobiose at a high conversion rate (about 70%) despite the same total amount of yeast cells used in the reaction. This demonstrated that it is preferable to co-express sucrose phosphorylase and cellobiose phosphorylase in a single recombinant yeast strain. In addition, in Figure 3(b), when a single recombinant yeast strain co-expressing sucrose phosphorylase and cellobiose phosphorylase was used with Pichia pastoris as a host, the conversion rate of sucrose to cellobiose was improved to approximately 80%.

Figure 4 shows the time course of the concentrations of sucrose, glucose, fructose, and cellobiose in the reaction supernatant of an experiment to produce cellobiose from sucrose using a sucrose phosphorylase + cellobiose phosphorylase surface-displaying yeast (Pp-SP2CBP strain) with E. Pichia pastoris as the host. The graph in Figure 4(a) shows the results when the Pp-SP2CBP strain was cultured using glucose as the carbon source, and the graph in Figure 4(b) shows the results when the Pp-SP2CBP strain was cultured using glycerol as the carbon source. The conversion rate of sucrose to cellobiose in the Pp-SP2CBP strain was approximately 40% when it was cultured using glucose as the carbon source, but increased to approximately 80% when it was cultured using glycerol as the carbon source. This indicates that it is preferable to use glycerol as a carbon source when culturing a yeast that displays sucrose phosphorylase and cellobiose phosphorylase on the surface using Pichia pastoris as a host.

[Example Group II: Repeated cellobiose production by reusing transformed yeast (sucrose phosphorylase + cellobiose surface display yeast)]
A. Preparation of transformed yeast Among the yeasts (strains) transformed in Example Group I A, the ΔSUC2-SP2CBP strain using Saccharomyces cerevisiae as a host and the Pp-SP2CBP strain using Pichia pastoris as a host were used.

B. Pretreatment of transformed yeast The ΔSUC2-SP2CBP strain prepared in A was transferred to 5 mL of YPD medium (1% dried yeast extract (Nacalai Tesque), 2% Bacto ^™ Peptone (Life Technologies), 2% D-glucose (Nacalai Tesque)) and pre-cultured at 30°C and 200 rpm for 18 hours, and then inoculated into 50 mL of YPD medium to an OD600 of 0.05, and cultured with shaking at 30°C and 150 rpm for 48 hours. In addition, the Pp-SP2CBP strain was transferred to 5 mL of YPG medium (1% dried yeast extract (Nacalai Tesque), 2% Bacto ^TM Peptone (Life Technologies), 2% glycerol (Nacalai Tesque)), pre-cultured at 30 ° C. and 200 rpm for 24 hours, and then inoculated into 50 mL of YPG medium so that the OD600 was 0.05, and cultured with shaking at 30 ° C. and 150 rpm for 48 hours. These culture solutions were centrifuged to settle the cells, washed twice with pure water, and the weight of the solid wet cells was measured by removing as much of the supernatant as possible, and the same amount of pure water was added to obtain a cell suspension (about 500 g wet cells / L). The cell suspension was kept at 60 ° C. for 90 minutes to obtain pretreated cells.

C. 5 mL of cellobiose production reaction solution (composition: 100 g/L sucrose (Nacalai Tesque); 80 mM sodium phosphate buffer (pH 7.0); 1 wt% magnesium sulfate; 1.5 IU/mL glucose isomerase (xylose isomerase, Godo Shusei Co., Ltd., derived from Streptomyces griseofuscus); and pretreated cells of ΔSUC2-SP2CBP strain or Pp-SP2CBP strain prepared in G-1 (final cell concentration 100 g wet cells/L)) were prepared and reacted at 35 rpm and 60 ° C. for 24 hours (first time). The yeast cells were collected by centrifugation of the reacted liquid, resuspended in a new reaction liquid, and reacted at 35 rpm and 60 ° C. for 24 hours (second time). The third reaction was carried out in the same manner. Sampling was performed three times for each reaction, at 4 hours, 8 hours, and 24 hours. To stop the reaction, each sample was placed in a sampling container, kept at 100°C for 10 minutes, and then cooled on ice for 10 minutes and centrifuged to precipitate the bacteria and the like. The supernatant was collected and used for analysis.

D. Analysis The supernatant obtained in D above was analyzed by the method described in F of Example Group I.

E. Results The time course of the sucrose, glucose, fructose, and cellobiose concentrations in the supernatant obtained in D above is shown in FIG. 5(a) (ΔSUC2-SP2CBP strain) and FIG. 5(b) (Pp-SP2CBP strain). From these results, it was found that the method for producing cellobiose of the present invention, which includes a step of allowing sucrose to coexist in a reaction solution containing a recombinant yeast expressing sucrose phosphorylase and cellobiose phosphorylase and glucose isomerase, maintains a high conversion rate to cellobiose even when the step is repeated. In addition, when a sucrose phosphorylase + cellobiose phosphorylase surface-displaying yeast with Pichia pastoris cultured using glycerol as a carbon source was used as a transformed yeast, it was confirmed that the yeast of the Pp-SP2CBP strain maintained a high cellobiose conversion rate of about 80% even when the reaction was repeated three times at 60 ° C. for 24 hours.

The manufacturing method of the present invention can be used as a method for manufacturing cellobiose for use in food or pharmaceutical preparations.

Claims (14)

A method for producing cellobiose, comprising a reaction step of allowing sucrose to coexist in a reaction solution containing a recombinant yeast expressing sucrose phosphorylase and cellobiose phosphorylase and glucose isomerase. The method according to claim 1, wherein the recombinant yeast is a yeast of the genus Saccharomyces, Pichia, or Yarrowia. The method according to claim 1 or 2, wherein the sucrose phosphorylase is derived from a bacterium of the genus Bifidobacterium, Leuconostoc, or Streptococcus. The method for producing sucrose phosphorylase according to any one of claims 1 to 3, wherein the sucrose phosphorylase comprises a secreted sucrose phosphorylase secreted and expressed from the recombinant yeast, and an anchored sucrose phosphorylase expressed in a state anchored to the cell wall of the recombinant yeast. The method according to any one of claims 1 to 4, wherein the cellobiose phosphorylase is derived from a bacterium of the genus Clostridium, Ruminococcus, Cellulomonas, or Thermotoga. The method according to any one of claims 1 to 5, wherein the recombinant yeast expresses both sucrose phosphorylase and cellobiose phosphorylase in a single cell. The method according to any one of claims 1 to 6, wherein the recombinant yeast is a combination of a recombinant yeast expressing sucrose phosphorylase and a recombinant yeast expressing cellobiose phosphorylase. The method according to any one of claims 1 to 7, wherein the recombinant yeast is a yeast transformed with an expression vector carrying a sucrose phosphorylase gene. The method according to claim 8, wherein the expression vector comprises a combination of a first expression vector carrying both a sucrose phosphorylase gene and a yeast cell wall domain gene, and a second expression vector carrying only the sucrose phosphorylase gene but not the yeast cell wall domain gene. The method according to any one of claims 1 to 9, wherein the glucose isomerase is derived from a bacterium of the genus Streptomyces. The method according to any one of claims 1 to 10, further comprising a step of treating the recombinant yeast at 45°C to 75°C for 15 to 150 minutes before allowing sucrose to coexist in the reaction solution. The method according to any one of claims 1 to 11, further comprising a step of culturing yeast cells using glycerol as a carbon source prior to the reaction step. The method according to any one of claims 1 to 12, further comprising a step of separating the produced cellobiose from the reaction solution after the reaction step. The method according to any one of claims 1 to 13, further comprising recovering the recombinant yeast from the reaction solution after the reaction step and subjecting it to another reaction step.

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