Method for producing cellobiose

Abstract

To provide a low-cost and efficient method for producing cellobiose. [Solution] A method for producing cellobiose, comprising the step of co-containing sucrose with a reaction solution containing yeast expressing sucrose phosphorylase and cellobiose phosphorylase on its surface and glucose isomerase.

Description

Technical Field

[0001] The present invention relates to a method for producing cellobiose from sucrose using yeast with enzyme surface display. Background Art

[0002] As methods for producing cellobiose, enzymatic decomposition methods and enzymatic synthesis methods have been conventionally known. As an enzymatic decomposition method, for example, a method of obtaining cellobiose by reacting a cellulase preparation with cellulose (Patent Document 1, Patent Document 2) is known. As enzymatic synthesis methods, for example, there are known a method of obtaining cellobiose by allowing α-glucan phosphorylase and cellobiose phosphorylase to act with starch as a starting material (Non-Patent Document 1), and a method of obtaining cellobiose by allowing sucrose phosphorylase, glucose isomerase and cellobiose phosphorylase to act with sucrose as a starting material (Patent Document 3, Patent Document 4).

[0003] The enzymatic decomposition method uses cellulose as a raw material. Cellulose is the main component of plant cell walls, but it usually exists as a mixture with hemicellulose, lignin and the like. Therefore, when cellulose in such plant cell walls is used as a raw material, hemicellulose, lignin and the like must first be removed by a method such as alkali treatment, which poses a problem that the production cost of the raw material increases.

[0004] In the enzymatic synthesis method, the conversion efficiency from starch to glucose 1-phosphate and the conversion efficiency from sucrose to cellobiose are low, and the reaction needs to be carried out under mild conditions (about 30°C) using enzymes, so the reaction rate is slow, which poses a problem that the method is not suitable for low-cost cellobiose production. Prior Art Documents Patent Documents

[0005] Patent Document 1 Japanese Unexamined Patent Publication No. Hei 2-295492 [Patent Document 2] Japanese Patent Application Publication No. 8-89274 [Patent Document 3] Japanese Patent Application Publication No. 3-130086 [Patent Document 4] Special Publication No. 2019-507603 [Non-patent literature]

[0006] [Non-Patent Document 1] Suzuki et al., J. Appl. Glycosci., (2010), 57:113-119 [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide a method for producing cellobiose from sucrose with a high conversion rate and low cost. [Means for solving the problem]

[0008] The inventors of the present invention have discovered that by co-producing sucrose 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"), cellobiose can be produced inexpensively with a high conversion rate, and have completed the present invention.

[0009] In other words, the present invention encompasses the following inventions. [Item 1] A method for producing cellobiose, comprising the step of co-containing sucrose in a reaction solution containing recombinant yeast expressing sucrose phosphorylase and cellobiose phosphorylase and glucose isomerase. [Item 2] The method of production described in Item 1, wherein the recombinant yeast is a yeast of the genus Saccharomyces, Pichia, or Yarrowia. [Item 3] The method for producing a product 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 for producing a sucrose phosphorylase according to any one of items 1 to 3, wherein the sucrose phosphorylase comprises secretory sucrose phosphorylase secreted and expressed from recombinant yeast, and anchored sucrose phosphorylase expressed while anchored to the cell wall of recombinant yeast. [Item 5] A method of production according to any one of items 1 to 4, wherein the cellobiose phosphorylase is derived from a bacterium of the genera Clostridium, Ruminococcus, Cellulomonas, or Thermotoga. [Item 6] The method for producing a recombinant yeast 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 for producing a recombinant yeast according to any one of items 1 to 6, wherein the recombinant yeast is a combination of recombinant yeast expressing sucrose phosphorylase and recombinant yeast expressing cellobiose phosphorylase. [Clause 8] The method for producing a recombinant yeast according to any one of Clauses 1 to 7, wherein the recombinant yeast is yeast transformed with an expression vector carrying a sucrose phosphorylase gene. [Clause 9] The method for producing a product 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 and not the yeast cell wall domain gene. [Item 10] The method for producing glucose isomerase according to any one of items 1 to 9, wherein the glucose isomerase is derived from bacteria of the genus Streptomyces. [Item 11] The method of production according to any one of items 1 to 10, further comprising the step of treating recombinant yeast at 45°C to 75°C for 15 to 150 minutes before introducing sucrose into the reaction solution. [Clause 12] The manufacturing method according to any one of claims 1 to 11, further comprising the step of culturing yeast cells using glycerol as a carbon source before the reaction step. [Item 13] The method of production according to any one of items 1 to 11, further comprising the step of separating the produced cellobiose from the reaction solution. [Effects of the Invention]

[0010] According to the present invention, it is possible to produce cellobiose from sucrose with a high conversion rate and low cost. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 is a graph showing the results of sucrose phosphorylase activity measurements in recombinant yeast strains expressing sucrose phosphorylase. [Figure 2-1] Figure 2 is a graph showing the time course of sucrose, glucose, fructose, and cellobiose concentrations in the reaction supernatant of a cellobiose production experiment from sucrose using recombinant yeast strains expressing sucrose phosphorylase and recombinant yeast strains expressing cellobiose phosphorylase. The graph in Figure 2(a) shows the results when using a strain expressing cellobiose phosphorylase (CsCBP) derived from Clostridium stercorarium, the graph in Figure 2(b) shows the results when using a strain expressing cellobiose phosphorylase (CuCBP) derived from Cellulomonas uda, the graph in Figure 2(c) shows the results when using a strain expressing cellobiose phosphorylase (TaCBP) derived from Thermosipho africanus, and the graph in Figure 2(d) shows the results when using a strain expressing cellobiose phosphorylase (TnCBP) derived from Thermotoga neapolitana. [Figure 2-2] Same as above. [Figure 3]Figure 3 is a graph showing time-course changes in the concentrations of sucrose, glucose, fructose, and cellobiose in the supernatant of a reaction solution from an experiment for producing cellobiose from sucrose using a single recombinant yeast strain that co-expresses sucrose phosphorylase and cellobiose phosphorylase. The graph in Figure 3(a) shows the case where recombinant yeast using *Saccharomyces cerevisiae* as the host is used, and the graph in Figure 3(b) shows the case where recombinant yeast using *Pichia pastoris* as the host is used. [Figure 4] Figure 4 is a graph showing time-course changes in the concentrations of sucrose, glucose, fructose, and cellobiose in the supernatant of a reaction solution from an experiment for producing cellobiose from sucrose using a single recombinant yeast strain that uses *Pichia pastoris* as the host and co-expresses sucrose phosphorylase and cellobiose phosphorylase. The graph in Figure 4(a) shows the results obtained when using the Pp-SP2CBP strain cultured with glucose as the carbon source, and the graph in Figure 4(b) shows the results obtained when using the Pp-SP2CBP strain cultured with glycerol as the carbon source. MODE FOR CARRYING OUT THE INVENTION

[0012] Hereinafter, the present invention will be described in detail 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 documents and non-patent documents described in the present specification are hereby incorporated by reference in their entireties into the present application.

[0013] In this specification, "identity" of an amino acid sequence refers to the percentage of matching amino acid residues, while "similarity" refers to the percentage of matching or similar amino acid residues. The homology and identity of amino acid sequences can be determined, for example, by the BLAST method (the default conditions for NCBI's PBLAST). Furthermore, when we express, for example, "80% or more homology," it is clear that this includes the case of "80% or more identity."

[0014] Furthermore, in this specification, "similar amino acid residues" means 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 with aliphatic side chains: 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 with basic side chains: lysine (Lys or K), arginine (Arg or R), and histidine (His or H) residues. (6) Amino acid residues with 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 methionine (Met or M), and the combination of (4) and histidine (His or H) residues are also treated as similar amino acid residues.

[0015] One aspect of the present invention relates to a method for producing cellobiose. Such a method includes the step of co-administering sucrose in a reaction solution containing recombinant yeast expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP), and glucose isomerase (XI).

[0016] The yeast species is not particularly limited as long as it can express sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP). Examples include yeasts of the genera Saccharomyces, Pichia, or Yarrowia. In particular, it is preferable to use yeast that does not have invertase activity on the cell surface in order to prevent sucrose from being degraded by the action of invertase, and an example is Pichia pastoris. For yeasts that have strong invertase activity on the cell surface, such as Saccharomyces cerevisiae, it is preferable to use strains that have lost invertase activity by methods such as gene knockout. Furthermore, since Pichia pastoris has been reclassified and renamed as Komagataella pastoris and Komagataella phaffii, Pichia pastoris is synonymous with both Komagataella pastoris and Komagataella phaffii.

[0017] The origin (species) of sucrose phosphorylase (SP) is not particularly limited. Examples include those derived from bacteria of the genera Bifidobacterium, Leuconostoc, or Streptococcus. Among 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 the nucleotide sequence encoding it is shown in SEQ ID NO: 5. Examples of sucrose phosphorylase (SP) derived from Bifidobacterium longum include proteins consisting of amino acid sequences having 80% or more, or 85% or more, or 88% or more, or 90% or more, or 93% or more, or 95% or more, or 97% or more, or 98% or more, or 99% or more homology (preferably identical) to SEQ ID NO: 34.

[0018] When expressing sucrose phosphorylase (SP) in recombinant yeast, two forms are possible: a secreted enzyme expressed from the recombinant yeast and a tethered enzyme expressed while anchored to the cell wall of the recombinant yeast. Either form is acceptable. However, in this invention, it is preferable to have both secreted sucrose phosphorylase (SP) expressed from the recombinant yeast and tethered sucrose phosphorylase (SP) expressed while anchored to the cell wall of the recombinant yeast coexist in the reaction solution. This makes it possible to improve the conversion efficiency by sucrose phosphorylase (SP). The reason for this is not clear, but it is presumed to be due to the formation of a dimer between secreted sucrose phosphorylase (SP) and tethered sucrose phosphorylase (SP).

[0019] The origin (species) of cellobiose phosphorylase (CBP) is not particularly limited, but examples include those derived from bacteria of the genera 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 nucleotide sequence encoding it is shown in SEQ ID NO: 6. Examples of cellobiose phosphorylase (CBP) derived from Clostridium stercorarium include proteins consisting of amino acid sequences having 80% or more, or 85% or more, or 88% or more, or 90% or more, or 93% or more, or 95% or more, or 97% or more, or 98% or more, or 99% or more homology (preferably identical) to the aforementioned SEQ ID NO: 35.

[0020] When expressing cellobiose phosphorylase (CBP) in recombinant yeast, there are two possible forms: a secreted enzyme expressed from the recombinant yeast, and a tethered enzyme expressed while anchored to the cell wall of the recombinant yeast. Either form is acceptable. However, in this invention, the tethered cellobiose phosphorylase (CBP) form, expressed while anchored to the cell wall of the recombinant yeast, is preferred.

[0021] As recombinant yeast, recombinant yeast that co-expresses sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP) in a single cell may be used, or recombinant yeast expressing sucrose phosphorylase (SP) and recombinant yeast expressing cellobiose phosphorylase (CBP) may be used in combination. Furthermore, when using both secreted and tethered enzymes as sucrose phosphorylase (SP) and / or cellobiose phosphorylase (CBP), recombinant yeast that co-expresses both secreted and tethered enzymes in a single cell may be used, or recombinant yeast expressing secreted enzymes and recombinant yeast expressing tethered enzymes may be used in combination.

[0022] The method for preparing recombinant yeast expressing sucrose phosphorylase (SP) and / or cellobiose phosphorylase (CBP) is not particularly limited. For example, one method can be used to transform one or more types of yeast 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., but plasmids are preferred. Furthermore, it is preferable to link the sucrose phosphorylase (SP) gene and / or cellobiose phosphorylase (CBP) gene with regulatory sequences such as promoters and terminators in an actionable manner and to support them on an expression vector in the form of an expression cassette that can be autonomously expressed in yeast cells. The construction of such expression vectors can be carried out using standard genetic engineering techniques known to those skilled in the art, by appropriately referring to literature such as Michael R. Green et al, Molecular Cloning: A Laboratory Manual, Fourth Edition, (2012), Cold Spring Harbor Laboratory.

[0023] Recombinant yeast is preferably pre-cultured in the presence of a carbon source prior to the reaction. The type of carbon source used when culturing recombinant yeast cells is not particularly limited and can be appropriately selected depending on the type of yeast. Examples include glucose and glycerol. In particular, in this invention, it is preferable to use at least glycerol as the carbon source. This improves the conversion rate from sucrose to cellobiose.

[0024] It is preferable to pre-treat the recombinant yeast prior to the reaction to stop glucose uptake. Such pre-treatment includes heat treatment, which involves exposing the yeast to a constant temperature for a certain period of time. In this case, the treatment conditions only need to be able to stop glucose uptake by the yeast used, and the heating temperature can be, for example, 45°C or higher, 50°C or higher, or 55°C or higher, or for example, 75°C or lower, 70°C or lower, or 65°C or lower. The heating time can be, for example, 15 minutes or more, 45 minutes or more, or 70 minutes or more, or for example, within 150 minutes, within 120 minutes, or within 100 minutes. In a preferred example, the temperature can be about 60°C and the time about 90 minutes. One example of such pre-treatment method is to shake the suspension of transformed yeast under the above conditions.

[0025] In this invention, glucose isomerase (XI) is coexisted 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 or more of wet cells / L, or 50 g or more of wet cells / L, or 75 g or more of wet cells / L, or for example, 200 g or less of wet cells / L, or 150 g or less of wet cells / L, or 100 g or less of wet cells / L.

[0026] The glucose isomerase (also known as xylose isomerase, XI) is not limited to any particular type, but examples include those derived from yeasts of the genus Streptomyces. Among these, those derived from Streptomyces griseofuscus are particularly preferred. Various commercially available glucose isomerases can be used.

[0027] Glucose isomerase (XI), like other enzymes, may be expressed in yeast or other microorganisms, but it is preferable to use the isolated glucose isomerase (XI) enzyme itself. The isolated glucose isomerase (XI) enzyme may be a commercially available product or obtained by culturing microorganisms that produce these enzymes. It may also be a purified or unpurified product. In this case, it is preferable to add the isolated glucose isomerase (XI) enzyme to a reaction solution containing recombinant yeast expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP) and allow them to coexist.

[0028] The amount of glucose isomerase (XI) is not particularly limited, but it is generally preferable to use 0.01 units or more, or 0.1 units or more, or 1.0 unit or more per mole of sucrose. Here, 1 unit (1 U) means the amount of enzyme that decomposes 1 μmol of fructose per minute at pH 7.0 into 1 wt / vol% sucrose at 50°C.

[0029] In this invention, the reaction is carried out in a reaction solution containing recombinant yeast expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP), and glucose isomerase (XI), with sucrose present in the same solution. The sucrose may be naturally occurring or chemically synthesized. The amount of sucrose is not particularly limited, but from the viewpoint of productivity, the highest possible concentration is preferred. However, since the solubility of the resulting cellobiose is about 15 to 20%, it is preferable to use a sucrose concentration of about 10 to 20% if it is desired to maintain a dissolved state.

[0030] The conditions for the cellobiose synthesis reaction are not limited, but can be as follows: The reaction temperature can be, for example, 35°C or higher, 45°C or higher, or 50°C or higher, or for example, 60°C or lower, or 70°C or lower. The reaction time can be, for example, 60 minutes or more, 12 hours or more, or 24 hours or more, or for example, 36 hours or less, or 48 hours or less. The pressure during the reaction is not particularly limited and can be under reduced pressure, atmospheric pressure, or pressurized pressure, but it is usually carried out under atmospheric pressure. The atmosphere during the reaction is also not particularly limited and can be any atmosphere as long as the enzyme activity is maintained even in an atmosphere where the recombinant yeast used cannot grow.

[0031] After the reaction is complete, cellobiose can be obtained by separating and purifying it from the reaction solution as needed. The separation and purification method is not particularly limited, and any method can be used, but examples include filtration, centrifugation, and membrane separation. These methods allow for efficient separation of the bacterial cells used from the cellobiose. Furthermore, 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 by recrystallization. [Examples]

[0032] The present invention will be described in more detail below with reference to examples, but these examples are merely illustrative examples for explanatory purposes, and the present invention is not limited in any sense to these examples.

[0033] Note that the PCR methods shown in this example are all KOD One TM The procedure was performed using PCR Master Mix (Toyobo). Furthermore, gene transfer into all yeast cells shown in this example was carried out using the lithium acetate method.

[0034] A. Preparation of transformed yeast Using a prepared plasmid, various recombinant yeast strains expressing two enzymes, sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP), were constructed in the yeast strain Saccharomyces cerevisiae BY4741ΔSUC2 via the lithium acetate method. These are described in detail below.

[0035] • Bacterial strains and culture media The yeast strains used were Saccharomyces cerevisiae strain BY4741 (MATα his3 leu2 met15 ura3 strain) and Saccharomyces cerevisiae strain BY4741ΔSUC2 (MATα his3 leu2 met15 ura3 suc2 strain). Saccharomyces cerevisiae strain BY4741 was obtained from the ATCC (American Type Culture Collection), and Saccharomyces cerevisiae strain BY4741ΔSUC2 was obtained from the Yeast deletion MAT-A complete set (Thermo Fisher Scientific).

[0036] • Construction of plasmids including expression cassettes Plasmids containing the following expression cassettes X1 to X9 were prepared according to the procedure below. 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)

[0037] (Preparation Example 1-1: Preparation of a vector plasmid containing a surface expression cassette X1 of sucrose phosphorylase (BlSP) derived from Bifidobacterium longum) DNA fragments containing the coding region of the sucrose phosphorylase (BlSP) gene derived from Bifidobacterium longum were prepared by gene synthesis. Next, the vector plasmid pIEG-SSSD (a surface expression vector having an expression cassette in which the SED1 promoter from Saccharomyces cerevisiae, the secretory signal peptide sequence of SED1 from Saccharomyces cerevisiae, the coding region of the endoglucanase II (TrEGII) gene from Trichoderma lisey, the coding region (SED1 anchoring region) of the SED1 gene from Saccharomyces cerevisiae, and the terminator region of the DIT1 gene from Saccharomyces cerevisiae are arranged in this order: Applied Microbiology and Biotechnology, Vol.105, 2021) Using 5895-5904) as a template, the DNA was amplified by PCR using primer pairs SED1a-F (SEQ ID NO: 14) and SED1ss-R (SEQ ID NO: 15) to prepare DNA fragments containing the full-length vector plasmid with the coding region of the TrEGII gene removed. These fragments were ligated by in-fusion. 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.

[0038] (Preparation Example 1-2: Preparation of a vector plasmid containing a Bifidobacterium longum-derived sucrose phosphorylase (BlSP) secretion expression cassette X2) Using the plasmid pIBlSP-SSSD as a template, the DNA was amplified by PCR using the primer pair DIT1t-F (SEQ ID NO: 16) and BlSP-R (SEQ ID NO: 17) to prepare a DNA fragment containing the full plasmid length excluding the SED1 anchoring region. The ends of this fragment were ligated and ring-closed using the in-fusion method. Next, using this plasmid as a template, the DNA was amplified by PCR using the primer pair BlSP-F (SEQ ID NO: 18) and DIT1t-R (SEQ ID NO: 19) to prepare a DNA fragment containing the coding region of the sucrose phosphorylase (BlSP) gene from Bifidobacterium longum and the terminator region of the DIT1 gene from Saccharomyces cerevisiae.Furthermore, the 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 acreatus, the coding region (SED1 anchoring region) of the SED1 gene derived from Saccharomyces cerevisiae, and the terminator region of the α-aglutinin gene derived from Saccharomyces cerevisiae are arranged in this order: Metabolic Engineering, Vol.57, 2020) Using 110-117) as a template, the DNA was amplified by PCR using the primer pair pRS403-F (SEQ ID NO: 20) and SED1ss-R (SEQ ID NO: 15) to prepare DNA fragments containing the full-length vector plasmid, excluding the coding region of the Aspergillus acleatus β-glucosidase 1 (AaBGL1) gene, the coding region (SED1 anchoring region) of the Saccharomyces cerevisiae SED1 gene, and the terminator region of the Saccharomyces cerevisiae α-aglutinin gene. These fragments were ligated by in-fusion. The resulting plasmid containing 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.

[0039] (Preparation Examples 1-3: Preparation of vector plasmids 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, the vector plasmid pIEG-SSSD (a surface expression vector having a cassette in which the SED1 promoter from Saccharomyces cerevisiae, the secretory signal peptide sequence of SED1 from Saccharomyces cerevisiae, the coding region of the endoglucanase II (TrEGII) gene from Trichoderma risei, the coding region (SED1 anchoring region) of the SED1 gene from Saccharomyces cerevisiae, and the terminator region of the DIT1 gene from Saccharomyces cerevisiae are arranged in this order: Applied Microbiology and Biotechnology, Vol.105, 2021) Using 5895-5904) as a template, the DNA was amplified by PCR using primer pairs SED1a-F (SEQ ID NO: 14) and SED1ss-R (SEQ ID NO: 15) to prepare DNA fragments containing the full-length vector plasmid with the coding region of the TrEGII gene removed. These fragments were ligated using the in-fusion method.Next, using this plasmid as a template, it was amplified by PCR using primer pairs SED1p-F (SEQ ID NO: 21) and DIT1t-R (SEQ ID NO: 19) to prepare DNA fragments containing the Saccharomyces cerevisiae-derived SED1 promoter, the Saccharomyces cerevisiae-derived SED1 secretion signal peptide sequence, the Clostridium stercorarium-derived cellobiose phosphorylase (CsCBP) coding region, the Saccharomyces cerevisiae-derived SED1 gene coding region (SED1 anchoring region), and the Saccharomyces cerevisiae-derived DIT1 gene terminator region. Additionally, the vector plasmid pIU5-CBH was prepared. D(A surface expression vector having a cassette in which the SED1 promoter from Saccharomyces cerevisiae, the coding region of the cellobiohydrolase (TsCBH1) gene from Talaromyces emersonii, the coding region (SED1 anchoring region) of the SED1 gene from Saccharomyces cerevisiae, and the terminator region of the α-aglutinin gene from Saccharomyces cerevisiae are arranged in this order: Biotechnology and Bioengineering, Vol.114, 2017) Using 1201-1207) as a template, the data was amplified by PCR using primer pairs pRS403-F (SEQ ID NO: 20) and pRS403-R (SEQ ID NO: 22) to prepare DNA fragments containing the full-length vector plasmid excluding the expression cassette region. These fragments were ligated by in-fusion. 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.

[0040] (Preparation Examples 1-4: Preparation of vector plasmids containing cellobiose phosphorylase (CuCBP) surface expression cassette X4 derived from Cellulomonas uda) A DNA fragment containing the coding region of the cellobiose phosphorylase (CuCBP) gene derived from Cellulomonas uda was prepared by gene synthesis. Next, using the vector plasmid pIU5-CsCBP-SSSD as a template, it was amplified by PCR using primer vs primer vs SED1a-F (SEQ ID NO: 14) and SED1ss-R (SEQ ID NO: 15) to prepare a DNA fragment containing the full-length vector plasmid with the coding region of the CsCBP gene removed. These fragments were ligated by in-fusion. The resulting plasmid containing 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.

[0041] (Preparation Examples 1-5: Preparation of vector plasmids containing Thermosipho africanus-derived cellobiose phosphorylase (TaCBP) surface expression cassette X5) A DNA fragment containing the coding region of the cellobiose phosphorylase (TaCBP) gene derived from Thermosipho africanus was prepared by gene synthesis. Next, using the vector plasmid pIU5-CsCBP-SSSD as a template, it was amplified by PCR using the primer pair SED1a-F (SEQ ID NO: 14) and SED1ss-R (SEQ ID NO: 15) to prepare a DNA fragment containing the full-length vector plasmid with the coding region of the CsCBP gene removed. These fragments were ligated by in-fusion. The resulting plasmid containing 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.

[0042] (Preparation Examples 1-6: Preparation of vector plasmids containing Thermotoga neapolitana-derived cellobiose phosphorylase (TnCBP) surface expression cassette X6) A DNA fragment containing the coding region of the cellobiose phosphorylase (TnCBP) gene derived from Thermotoga neapolitana was prepared by gene synthesis. Next, using the vector plasmid pIU5-CsCBP-SSSD as a template, it was amplified by PCR using the primer pair SED1a-F (SEQ ID NO: 14) and SED1ss-R (SEQ ID NO: 15) to prepare a DNA fragment containing the full-length vector plasmid with the coding region of the CsCBP gene removed. These fragments were ligated by in-fusion. The resulting plasmid containing 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.

[0043] (Preparation Examples 1-7: Preparation of vector plasmids containing Bifidobacterium longum-derived sucrose phosphorylase (BlSP) surface expression cassette X7) Using the plasmid pIBlSP-SSSD as a template, DNA fragments containing the coding region of the BlSP gene were prepared by amplification using the primer pairs BlSP-NheI-F (SEQ ID NO: 23) and BlSP-XhoI-R (SEQ ID NO: 24) via PCR, and further treatment with NheI and XhoI. Furthermore, the vector plasmid pIBG-PpSSG61 (a surface expression vector having an expression cassette in which the SPI1 promoter derived from Pichia pastoris, the secretion signal peptide sequence of SPI1 derived from Pichia pastoris, the coding region of the β-glucosidase 1 (AaBGL1) gene derived from Aspergillus acleatus, the coding region (GCW61 anchoring region) of the GCW61 gene derived from Pichia pastoris, 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 DNA fragments containing the full-length vector plasmid excluding the coding region of the AaBGL1 gene. These fragments were ligated. Next, using this plasmid as a template, DNA fragments containing the full-length vector plasmid, with the GCW61 anchoring region removed, were prepared by treatment with XhoI and MluI.Furthermore, plasmid pIBG-PpGMG30 (a surface expression vector having an expression cassette in which the GAPDH promoter from Pichia pastoris, the secretory signal peptide sequence of α-factor from Saccharomyces cerevisiae, the coding region of the β-glucosidase 1 (AaBGL1) gene from Aspergillus acreatus, the coding region (GCW30 anchoring region) of the GCW30 gene from Pichia pastoris, and the terminator region of the AOX1 gene from Pichia pastoris are arranged in this order: Biotechnology and Bioengineering, Vol.120, 2023, 1097-1107) was treated with XhoI and MluI to prepare DNA fragments containing the GCW30 anchoring region. These fragments were ligated. The plasmid containing the obtained 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.

[0044] (Preparation Examples 1-8: Preparation of vector plasmids containing Bifidobacterium longum-derived sucrose phosphorylase (BlSP) secretion expression cassette X8) Using plasmid pIBlSP-PpSSG30 as a template, the DNA fragment containing the coding region of the BlSP gene was amplified by PCR using primer pairs BlSP-F2 (SEQ ID NO: 25) and BlSP-R2 (SEQ ID NO: 26). Furthermore, using the vector plasmid pIH-EG-SSG34 (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 lisey, the coding region (GCW34 anchoring region) of the GCW34 gene derived from Pichia pastoris, 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) as a template, primer pair AOX1t-F (SEQ ID NO: 27) and SPI1s DNA fragments containing the full-length vector plasmid, excluding the coding region and GCW34 anchoring region of the TrEGII gene, were prepared by amplification using PCR with sR (SEQ ID NO: 28). These fragments were ligated using the in-fusion method. The resulting plasmid containing 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.

[0045] (Preparation Examples 1-9: Preparation of vector plasmids containing Clostridium stercorarium-derived cellobiose phosphorylase (CsCBP) surface expression cassette X9) Using the plasmid pIU5-CsCBP-SSSD as a template, DNA fragments containing the coding region of the CsCBP gene were prepared by PCR using the primer pair CsCBP-NheI-F (SEQ ID NO: 29) and CsCBP-XhoI-R (SEQ ID NO: 30), and further treated with NheI and XhoI. Additionally, DNA fragments containing the full-length vector plasmid with the coding region of the BlSP gene removed were prepared by treating the vector plasmid pIBlSP-PpSSG30 with NheI and XhoI. These fragments were ligated. Next, using this plasmid as a template, DNA fragments containing the coding region, GCW30 anchoring region, and AOX1 terminator region of the CsCBP gene were prepared by PCR using the primer pair CsCBP-F (SEQ ID NO: 31) and AOX1t-R (SEQ ID NO: 32). Furthermore, using the vector plasmid pIZ-CBH1-PpSSG34 (a surface expression vector having an expression cassette in which the coding region of the SPI1 gene derived from Pichia pastoris, the coding region of the cellobiohydrolase (TsCBH1) gene derived from Talaromyces emersonii, the coding region (GCW34 anchoring region) of the GCW34 gene derived from Pichia pastoris, 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) as a template, primer pair AOX1t-F2 (SEQ ID NO: 33) and SPI1s DNA fragments containing the full-length vector plasmid, excluding the coding region, GCW34 anchoring region, and AOX1 terminator region of the TsCBH1 gene, were prepared by amplification using PCR with sR (SEQ ID NO: 28). These fragments were ligated using the in-fusion method.The plasmid containing the obtained 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.

[0046] • Preparation of transformed yeast (Preparation Example 2-1: Production of yeast displaying sucrose phosphorylase on the surface) Plasmid pIBlSP-SSSD described in Preparation Example 1-1 was treated with NdeI and transformed into the yeast Saccharomyces cerevisiae strain BY4741ΔSUC2 by lithium acetate. This transformed strain is referred to as the ΔSUC2-BlSP strain. Similarly, plasmid pIBlSP-PpSSG30 described in Preparation Example 1-7 was treated with EcoRV and transformed into the yeast Pichia pastoris strain CBS7435 by lithium acetate. This transformed strain is referred to as the Pp-BlSP strain.

[0047] Furthermore, the plasmid pIL2-BlSP-SSD described in Preparation Example 1-2 was treated with NdeI and applied to the ΔSUC2-BlSP strain, which was transformed by the lithium acetate method. This transformed strain is referred to as the ΔSUC2-BlSP2 strain. Similarly, the plasmid pIH-BlSP-PpSS described in Preparation Example 1-8 was treated with BsrGI and applied to the Pp-BlSP strain, which was transformed by the lithium acetate method. This transformed strain is referred to as the Pp-BlSP2 strain.

[0048] (Preparation Example 2-2: Production of cellobiose phosphorylase-displaying yeast) Plasmids pIU5-CsCBP-SSSD, pIU5-CuCBP-SSSD, pIU5-TaCBP-SSSD, or pIU5-TnCBP-SSSD described in Preparation Examples 1-3 to 1-6 were treated with SpeI and transformed into the yeast Saccharomyces cerevisiae BY4741ΔSUC2 strain using the lithium acetate method. These transformed strains are referred to as ΔSUC2-CsCBP, ΔSUC2-CuCBP, ΔSUC2-TaCBP, and ΔSUC2-TnCBP strains, respectively.

[0049] (Preparation Example 2-3: Production of yeast surface displaying sucrose phosphorylase + cellobiose phosphorylase) The plasmid pIU5-CsCBP-SSSD described in Preparation Example 1-3 was treated with SpeI and applied to the ΔSUC2-BlSP2 strain, which was transformed 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 applied to the Pp-BlSP2 strain, which was transformed by the electroporation method. This transformed strain is referred to as the Pp-SP2CBP strain.

[0050] B. Pretreatment of transformed yeast Of the yeast strains transformed with A, the following strains, which use Saccharomyces cerevisiae as their host, were tested in 5 mL of YPD medium (1% dried yeast extract (Nacalai Tesque), Bacto TMThe cells were transplanted into Peptone (Life Technologies) 2%, D-glucose (Nacalai Tesque) 2%, pre-cultured at 30°C and 200 rpm for 18 hours, then inoculated into 50 mL of YPD medium to achieve an OD600 of 0.05, and cultured with shaking at 30°C and 150 rpm for 48 hours. In addition, Pp-BlSP strains, Pp-BlSP2 strains, and Pp-SP2CBP strains, which use Pichia pastoris as a host, were inoculated into 5 mL of YPG medium (dried yeast extract (Nacalai Tesque) 1%, Bacto TM The cells were transplanted into Peptone (Life Technologies) 2% and Glycerol (Nacalai Tesque) 2%, and pre-cultured at 30°C and 200 rpm for 18 hours. Then, they were inoculated into 50 mL of YPG medium to achieve an OD600 of 0.05, and cultured with shaking at 30°C and 150 rpm for 48 hours. These cultures were centrifuged to settle the cells, washed twice with pure water, and the supernatant was removed as much as possible. The weight of the solid, wet cells was measured, and an equal amount of pure water was added to obtain a cell suspension (approximately 500 g wet cells / L). The cell suspension was kept at 60°C for 90 minutes to be used as pre-treated cells.

[0051] C. Measurement of sucrose phosphorylase activity on the surface of yeast cells A 5 mL reaction solution (composition: 10 g / L sucrose (Nacalai Tesque); 80 mM sodium phosphate buffer (pH 7.0); and pre-treated cells of 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 held at 100°C for 10 minutes, then cooled on ice for 10 minutes, and centrifuged to precipitate the cells and other materials. The supernatant was collected and used for analysis of the fructose concentration. 1 IU is defined as the amount of enzyme that releases 1 μmol of fructose per minute.

[0052] Cellobiose production from sucrose using D. sucrose phosphorylase surface-displaying yeast and cellobiose phosphorylase surface-displaying yeast. 5 mL of 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, manufactured by Godo Shusei Co., Ltd., derived from Streptomyces griseofuscus); pre-treated cells of the ΔSUC2-BlSP2 strain prepared in Preparation Example 2-1 (final cell concentration 50 g wet cells / L); and pre-treated 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, at 4 hours and 24 hours. To stop the reaction, the sample solution 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 bacterial cells and other materials. The supernatant was collected and used for analysis.

[0053] Cellobiose production from sucrose using surface-displaying yeast with E. sucrose phosphorylase + cellobiose phosphorylase. A 5 mL 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, manufactured by Godo Shusei Co., Ltd., derived from Streptomyces griseofuscus); and pre-treated cells of the ΔSUC2-SP2CBP strain or Pp-BlSP2 strain prepared in Preparation Example 2-3 (final cell concentration 100 g wet cells / L)) was 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 stop the reaction, the sample solution was placed in a sampling container, held at 100°C for 10 minutes, then cooled on ice for 10 minutes, and centrifuged to precipitate the cells, etc. The supernatant was collected and used for analysis.

[0054] F. Analysis HPLC was used to analyze the supernatants of C, D, and E. The analytical 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 × 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: Differential refractive index detector (RID) (Shimadzu RID-10A) ·Analysis time: 15 minutes Calibration curve: A sugar mixture (sucrose, glucose, fructose, and cellobiose) was used to calibrate at three points: 1, 5, and 10 g / L.

[0055] G.Results Figure 1 shows the results of measuring the sucrose phosphorylase activity of cell C. Compared to the ΔSUC2-BlSP strain, which was introduced with only expression cassette X1 for tethered expression of sucrose phosphorylase in the cell wall, the ΔSUC2-BlSP2 strain, which was introduced with expression cassette X2 for secretory expression of sucrose phosphorylase in addition to expression cassette X1, showed significantly higher sucrose phosphorylase activity. This indicates that it is preferable to use both tethered and secretory forms of sucrose phosphorylase.

[0056] Figure 2 shows the time course of sucrose, glucose, fructose, and cellobiose concentrations in the reaction supernatant of a cellobiose production experiment from sucrose using both sucrose phosphorylase-presenting yeast and cellobiose-presenting yeast. When the ΔSUC-CsCBP strain was used as the cellobiose-presenting yeast, the conversion rate of sucrose to cellobiose was approximately 30% after 24 hours from 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 cellobiose-presenting yeast, the conversion rate was less than 10% even after 24 hours from the start of the reaction. This indicates that the CBP gene derived from Clostridium stercorarium is preferable.

[0057] Figure 3 shows the time course of sucrose, glucose, fructose, and cellobiose concentrations in the reaction supernatant of a cellobiose production experiment from sucrose using a surface-displaying yeast strain (ΔSUC2-SP2CBP) with sucrose phosphorylase + cellobiose phosphorylase in Saccharomyces cerevisiae as the host. The graph in Figure 3(a) shows the results when using the ΔSUC2-SP2CBP strain with Saccharomyces cerevisiae as the host, and the graph in Figure 3(b) shows the results when using the Pp-SP2CBP strain with Pichia pastoris as the host. In comparison with the experimental results in Figure 2(a) using recombinant yeast strains expressing only sucrose phosphorylase and recombinant yeast strains expressing only cellobiose phosphorylase in combination with Saccharomyces cerevisiae as the host, Figure 3(a), which uses a single recombinant yeast strain co-expressing both sucrose phosphorylase and cellobiose phosphorylase in the same Saccharomyces cerevisiae as the host, shows that sucrose is converted to cellobiose at a higher rate (approximately 70%), even though the total amount of yeast cells used in the reaction is the same. This indicates that it is preferable to co-express sucrose phosphorylase and cellobiose phosphorylase in a single recombinant yeast strain. Furthermore, in Figure 3(b), using a single recombinant yeast strain co-expressing sucrose phosphorylase and cellobiose phosphorylase in Pichia pastoris as the host, the conversion rate of sucrose to cellobiose improved to approximately 80%.

[0058] Figure 4 shows the time course of sucrose, glucose, fructose, and cellobiose concentrations in the reaction supernatant of a cellobiose production experiment from sucrose using sucrose phosphorylase + cellobiose phosphorylase surface-displaying yeast (Pp-SP2CBP strain) in Pichia pastoris (F) as the host. The graph in Figure 4(a) shows the results when using the Pp-SP2CBP strain cultured with glucose as the carbon source, and the graph in Figure 4(b) shows the results when using the Pp-SP2CBP strain cultured with glycerol as the carbon source. The conversion rate of sucrose to cellobiose in the Pp-SP2CBP strain was approximately 40% when cultured with glucose as the carbon source, but it improved to approximately 80% when cultured with glycerol as the carbon source. This indicates that when culturing sucrose phosphorylase + cellobiose phosphorylase surface-displaying yeast using Pichia pastoris as the host, it is preferable to use glycerol as the carbon source. [Industrial applicability]

[0059] The manufacturing method of the present invention can be used as a method for producing cellobiose for use in food or pharmaceutical preparations.

Claims

1. A method for producing cellobiose, comprising the step of co-containing sucrose in a reaction solution containing recombinant yeast expressing sucrose phosphorylase and cellobiose phosphorylase, and glucose isomerase.

2. The method for producing a product according to claim 1, wherein the recombinant yeast is a yeast belonging to the genera Saccharomyces, Pichia, or Yarrowia.

3. The method for producing a product according to claim 1 or 2, wherein the sucrose phosphorylase is derived from a bacterium of the genus Bifidobacterium, Leuconostoc, or Streptococcus.

4. The method for producing a product according to any one of claims 1 to 3, wherein the sucrose phosphorylase comprises secretory sucrose phosphorylase secreted and expressed from recombinant yeast, and anchored sucrose phosphorylase expressed while anchored to the cell wall of recombinant yeast.

5. The method for producing a product 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.

6. The method for producing a recombinant yeast according to any one of claims 1 to 5, wherein the recombinant yeast is a recombinant yeast that expresses both sucrose phosphorylase and cellobiose phosphorylase in a single cell.

7. The method for producing the recombinant yeast according to any one of claims 1 to 6, wherein the recombinant yeast is a combination of recombinant yeast expressing sucrose phosphorylase and recombinant yeast expressing cellobiose phosphorylase.

8. The method for producing a recombinant yeast according to any one of claims 1 to 7, wherein the recombinant yeast is yeast transformed with an expression vector carrying a sucrose phosphorylase gene.

9. The method for producing a product 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 and not the yeast cell wall domain gene.

10. The method for producing a product according to any one of claims 1 to 9, wherein the glucose isomerase is derived from a bacterium of the genus Streptomyces.

11. The method for producing the product according to any one of claims 1 to 10, further comprising the step of treating recombinant yeast at 45°C to 75°C for 15 to 150 minutes before introducing sucrose into the reaction solution.

12. The manufacturing method according to any one of claims 1 to 11, further comprising the step of culturing yeast cells using glycerol as a carbon source before the reaction step.

13. The method for producing a product according to any one of claims 1 to 12, further comprising the step of separating the produced cellobiose from the reaction solution.

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