JP2007174957A - Yeast for producing hyaluronic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing hyaluronic acid, with which hyaluronic acid is produced in an in vivo system using yeast and which is industrially advantageous with respect to safety, cost, etc., to obtain transformed yeast and an expression recombinant vector for producing hyaluronic acid. <P>SOLUTION: The method for producing hyaluronic acid comprises: (1) a process for transforming yeast by using an expression recombinant vector containing a DNA encoding a protein having hyaluronate synthase activity under control of a promoter functioning in yeast; (2) a process for culturing a transformant obtained by transformation; and (3) a process for separating hyaluronic acid produced by the transformant. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing hyaluronic acid using yeast, a transformed yeast having hyaluronic acid-producing ability, and a method for producing them.

In 1934, MEYER and PALMER et al. Discovered a novel glycosaminoglycan (mucopolysaccharide) from the vitreous body of cattle eyes (see Non-Patent Document 1). They showed that this substance is a linear polysaccharide having a repeating structure of disaccharides in which glucuronic acid and N-acetylglucosamine are β1,3 and β1,4 linked (see Non-Patent Document 2).
Later, in the 1950s and 1960s, research on the biosynthesis of hyaluronic acid was conducted by cell-free experiments using group A streptococci, and uridine-5'-diphospho-glucuronic acid (hereinafter referred to as UDP) was used to elongate the hyaluronic acid chain. -Glucuronic acid or UDP-GlcA) and uridine-5'-diphospho-N-acetylglucosamine (hereinafter sometimes referred to as UDP-N-acetylglucosamine or UDP-GlcNAc) The activity of hyaluronic acid synthase localized in the streptococcal cell membrane was shown (see Non-Patent Document 3).
It has been difficult to solubilize and purify hyaluronic acid synthase as a stable active form for many years, but in 1993, the hyaluronic acid synthase gene (hasA) of Streptococcus was isolated. Since then, cloning of eukaryotic hyaluronan synthase gene has been reported (see Non-Patent Documents 5 to 10), and Chlorella virus PBCV-1 (see Non-Patent Document 11). And hyaluronic acid synthase gene derived from Pasteurella multocida (see Non-patent Document 12), and an active recombinant enzyme has been obtained.
With the progress of these studies, a wide range of physiological functions of hyaluronic acid have been elucidated, and unique physical properties and biological functions have been clarified. High molecular weight hyaluronic acid is used for the treatment of osteoarthritis, an ophthalmic surgical adjuvant, adhesion prevention, wound healing promotion effect and the like. In addition, low molecular weight hyaluronic acid has been reported to have a bioactive effect. And it is expected to be applied to biomaterials and new medical uses.
To date, hyaluronic acid has been produced by extraction from animal tissue or microbial fermentation. However, extraction from animal tissues is concerned about the risk of contamination with prions, viruses, etc. in mad cow disease, for example. Animal cells are difficult to maintain and require an expensive medium and have a slow growth rate. On the other hand, microbial fermentation has problems that some of these producing bacteria have pathogenicity to human resident bacteria and animals / humans. In addition, Escherichia coli, which is a prokaryotic organism, has problems such that protein processing does not occur, inclusion bodies (inclusion bodies) may be formed, and protease degradation occurs. On the other hand, in order to solve the above-mentioned problems, hyaluronic acid has been generated using genetic recombination using Bacillus as a host with high safety (Patent Document 13). However, when the prokaryotic Bacillus is used as a host, it is considered that it is not suitable for the expression of hyaluronic acid synthase derived from eukaryotes such as humans and mice. In the invention described in Patent Document 1, hyaluronic acid is produced in plants by introducing a hyaluronic acid synthase gene into plants and plant cells (Patent Document 1). Moreover, although there is an example in which hyaluronic acid was synthesized in vitro using DG42 (XeHAS) expressed in yeast, hyaluronic acid has not yet been produced in vivo (Non-patent Document 14).
2003 WO05 / 12529 Meyer, K. and Palmer, JW (1934) J. Biol. Chem., 107, 629-634 Weissman, B. and Meyer, K. (1954) J. Am. Chem. Soc., 76, 1753-1757 Markovitz, M., Cifonelli, JA and Dorfman, A. (1959) J. Biol. Chem., 234, 2343-2350 DeAngelis, PL, Papaconstantinou, J. and Weigel, PH (1993) J. Biol. Chem., 268, 14568-14571 Itano, N. and Kimata, K. (1996) J. Biol. Chem., 271, 9875-9878 Itano, N. and Kimata, K. (1996) Biochem. Biophys. Res. Commun. 222, 816-820 Spicer, AP, Augustine, ML and McDonald, J. A (1996) J. Biol. Chem., 271, 23400-23406 Spicer, AP, Olson, JS and McDonald, JA (1997) J. Biol. Chem., 272, 8957-8961 Shyjan AM, Heldin, P., Butcher EC, Yoshino T. and Briskin, MJ (1998) J. Biol. Chem., 271, 23395-23399 Watanabe, K. and Yamaguchi, Y. (1996) J. Biol. Chem., 271, 22945-22948 DeAngelis, PL, Jing, W. Graves, MV, Burbank, DE and vam Etten, JL (1998) Science, 278, 1800-1804 DeAngelis, PL, Jing, W. Drake, RR and Achyuthan, AM (1998) J. Biol. Chem., 273, 8454-8458 WO2003 / 54163 (Special Table 2005-525091) Paul L. DeAgelis and Ann Mary Achyuthan (1996) Vol. 271, No. 39,23657-23660

If hyaluronic acid can be produced in yeast with a long and safe history of food and brewing, it is considered to be particularly advantageous from an industrial point of view in terms of safety and cost.
INDUSTRIAL APPLICABILITY The present invention produces hyaluronic acid in an in vivo system using yeast, industrially advantageous hyaluronic acid production method from the viewpoint of safety and low cost, transformed yeast, and recombinant expression for production of hyaluronic acid The main challenge is to provide vectors.

Hyaluronic acid synthase generally has a plurality of transmembrane regions and membrane-bound regions. When a membrane-bound protein such as hyaluronic acid synthase is expressed as a recombinant enzyme by introduction of a foreign gene, the transmembrane region and the membrane-bound region often cannot maintain the correct structure. For example, it is suggested that active hyaluronan synthase forms a complex with one hyaluronan synthase and about 14-18 molecules of cardiolipin, a phospholipid present in the membrane, and affects hyaluronan synthase activity. (Paul L. DeAngelis and Ann Mary Achyuthan (1996) J. Biol. Chem., 271, 23657-23660).

As a result of intensive studies, the present inventors have found that the above technically difficult problems can be solved by the following means, and have reached the present invention. That is, the present invention relates to the following matters.
1. (1) a step of transforming yeast using a recombinant vector for expression containing a DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter capable of functioning in yeast, (2) step (1 ) Culturing the transformant obtained in (3), and (3) separating hyaluronic acid produced by the transformant.
2. (1) The DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter that can function in yeast is the same as at least one DNA encoding a protein having exogenous sugar nucleotide synthase activity Or a step of transforming yeast using at least one recombinant expression vector contained in a separate vector, (2) a step of culturing the transformant obtained in step (1), (3) the transformation The manufacturing method of hyaluronic acid including the process of isolate | separating the hyaluronic acid produced by the body.
3. Item 3. The method for producing hyaluronic acid according to Item 1 or 2, wherein the promoter is an inducible promoter.
4). Item 4. The method for producing hyaluronic acid according to Item 3, wherein the inducible promoter is a promoter having a stress-inducible or cold-inducible function.
5. Item 4. The method for producing hyaluronic acid according to any one of Items 1 to 3, wherein the promoter is a promoter having a culture environment stress-inducing function of the HSP12 gene.
6). Item 3. The method for producing hyaluronic acid according to item 1 or 2, wherein the DNA encoding a protein having hyaluronic acid synthase activity is the following DNA (a) or (b) or (c):
(A) DNA consisting of the base sequence represented by SEQ ID NO: 1 or 3.
(B) A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence of (a) and encodes a protein having hyaluronic acid synthase activity.
(C) A complementary strand of the DNA of (a) or (b) above.
7). Item 3. The method for producing hyaluronic acid according to item 1 or 2, wherein the protein having hyaluronic acid synthase activity is the following protein (a) or (b):
(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 2 or 4.
(B) A protein having an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having hyaluronic acid synthase activity.
8). Item 3. The method for producing hyaluronic acid according to Item 2, wherein the sugar nucleotide is UDP-glucuronic acid and / or UDP-N-acetylglucosamine.
9. Proteins with sugar nucleotide synthase activity are glutamine: fructose-6-phosphate amide transferase, UDP-N-acetylglucosamine diphosphorylase, phosphoacetylglucosamine mutase, glucosamine-6-phosphate N-acetyltransferase, glucosamine-1 phosphate N -Acetyltransferase, phosphoglucomutase, N-acetylglucosamine kinase, hexokinase, N-acetylglucosamine deacetylase, UDP-glucose dehydrogenase, UDP glucose-1-phosphate uridylyltransferase, inositol oxygenase, glucuronokinase and glucuronic acid-1 phosphorus Item 9. The hair according to any one of Items 1 to 8, which is one or more proteins selected from the group consisting of acid uridyltransferases. Manufacturing method of Ron acid.
10. Item 9. The method for producing hyaluronic acid according to any one of Items 1 to 8, wherein the protein having sugar nucleotide synthase activity is UDP-glucose dehydrogenase and / or glutamine: fructose-6-phosphate amide transferase.
11. Item 11. The method for producing hyaluronic acid according to any one of Items 2 to 10, wherein the DNA encoding a protein having sugar nucleotide synthase activity is a DNA derived from chlorella virus and / or Arabidopsis thaliana.
12 Item 12. The production of hyaluronic acid according to any one of Items 2 to 11, wherein the DNA encoding a protein having sugar nucleotide synthase activity is the following DNA (a), (b) or (c): Method.
(A) DNA consisting of the base sequence represented by SEQ ID NO: 5, 7, 9, 11, 13, or 15.
(B) DNA that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and encodes a protein having UDP-glucose dehydrogenase activity.
(C) A complementary strand of the DNA of (a) or (b) above.
13. Item 12. The method for producing hyaluronic acid according to any one of Items 2 to 11, wherein the protein having sugar nucleotide synthase activity is the following protein (a) or (b):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, 14, or 16;
(B) A protein comprising an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having UDP-glucose dehydrogenase activity.
14 Item 12. The production of hyaluronic acid according to any one of Items 2 to 11, wherein the DNA encoding a protein having sugar nucleotide synthase activity is the following DNA (a), (b) or (c): Method.
(A) DNA consisting of the base sequence represented by SEQ ID NO: 17, 19, or 21.
(B) a DNA that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and encodes a protein having glutamine: fructose-6-phosphate amide transferase activity .
(C) A complementary strand of the DNA of (a) or (b) above.
15. Item 12. The method for producing hyaluronic acid according to any one of Items 2 to 11, wherein the protein having sugar nucleotide synthase activity is the following protein (a) or (b):
(A) A protein consisting of the amino acid sequence represented by SEQ ID NO: 18, 20, or 22.
(B) A protein having an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having glutamine: fructose-6-phosphate amide transferase activity.
16. Item 16. The method for producing hyaluronic acid according to any one of Items 1 to 15, wherein the protein is induced to be produced at a temperature of 4 ° C to 20 ° C of the culture solution.
17. Item 17. The method for producing hyaluronic acid according to any one of Items 1 to 16, wherein the protein is induced to be produced by setting the NaCl concentration in the culture solution to 500 mM or more.
18. A transformed yeast having the ability to produce hyaluronic acid transformed with a recombinant vector for expression containing a DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter capable of functioning in yeast.
19. DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter capable of functioning in yeast and at least one DNA encoding a protein having exogenous sugar nucleotide synthase activity are the same or different A transformed yeast having the ability to produce hyaluronic acid transformed with at least one recombinant expression vector contained in the vector.
20. Item 20. The transformed yeast according to Item 18 or 19, wherein the promoter is an inducible promoter.
21. Item 20. The transformed yeast according to Item 18 or 19, wherein the inducible promoter is a promoter having a stress-inducible or cold-inducible function.
22. Item 20. The transformed yeast according to any one of Items 18 to 20, wherein the promoter is a promoter having a culture environment stress-inducing function of the HSP12 gene.
23. Item 20. The transformed yeast according to Item 18 or 19, wherein the DNA encoding a protein having hyaluronic acid synthase activity is the following DNA (a) or (b) or (c):
(A) DNA consisting of the base sequence represented by SEQ ID NO: 1 or 3.
(B) A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence of (a) and encodes a protein having hyaluronic acid synthase activity.
(C) A complementary strand of the DNA of (a) or (b) above.
24. Item 20. The transformed yeast according to Item 18 or 19, wherein the protein having hyaluronic acid synthase activity is the following protein (a) or (b):
(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 2 or 4.
(B) A protein having an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having hyaluronic acid synthase activity.
25. Item 20. The transformed yeast according to Item 19, wherein the sugar nucleotide is UDP-glucuronic acid and / or UDP-N-acetylglucosamine.
26. Proteins with sugar nucleotide synthase activity are glutamine: fructose-6-phosphate amide transferase, UDP-N-acetylglucosamine diphosphorylase, phosphoacetylglucosamine mutase, glucosamine-6-phosphate N-acetyltransferase, glucosamine-1 phosphate N -Acetyltransferase, phosphoglucomutase, N-acetylglucosamine kinase, hexokinase, N-acetylglucosamine deacetylase, UDP-glucose dehydrogenase, UDP-glucose-1-phosphate uridylyltransferase, inositol oxygenase, glucuronokinase and glucuronic acid-1 Item 20. The transformed yeast according to Item 19, which is one or more proteins selected from the group consisting of phosphate uridyltransferase.
27. Item 20. The transformed yeast according to Item 19, wherein the protein having sugar nucleotide synthase activity is UDP-glucose dehydrogenase.
28. Item 28. The transformed yeast according to any one of Items 19 to 27, wherein the DNA encoding a protein having sugar nucleotide synthase activity is a DNA derived from chlorella virus and / or Arabidopsis thaliana.
29. Item 30. The transformed yeast according to any one of Items 21 to 29, wherein the DNA encoding a protein having sugar nucleotide synthase activity is the following DNA (a), (b) or (c):
(A) DNA consisting of the base sequence represented by SEQ ID NO: 5, 7, 9, 11, 13, or 15
(B) DNA that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and encodes a protein having UDP-glucose dehydrogenase activity.
(C) A complementary strand of the DNA of (a) or (b) above.
30. Item 29. The transformed yeast according to any one of Items 19 to 28, wherein the protein having sugar nucleotide synthase activity is the following protein (a) or (b):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, 14, or 16;
(B) A protein comprising an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having UDP-glucose dehydrogenase activity.
31. Item 29. The transformed yeast according to any one of Items 19 to 28, wherein the DNA encoding a protein having sugar nucleotide synthase activity is the following DNA (a), (b) or (c):
(A) DNA consisting of the base sequence represented by SEQ ID NO: 17, 19, or 21.
(B) a DNA that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and encodes a protein having glutamine: fructose-6-phosphate amide transferase activity .
(C) A complementary strand of the DNA of (a) or (b) above.
32. Item 29. The transformed yeast according to any one of Items 19 to 28, wherein the protein having sugar nucleotide synthase activity is the following protein (a) or (b):
(A) A protein consisting of the amino acid sequence represented by SEQ ID NO: 18, 20, or 22.
(B) A protein having an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having glutamine: fructose-6-phosphate amide transferase activity.
33. Item 33. A hyaluronic acid-containing product obtained from the transformed yeast according to any one of Items 18 to 32.
34. A recombinant vector for expression for production of hyaluronic acid, comprising DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter that can function in yeast.
35. DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter capable of functioning in yeast and at least one DNA encoding a protein having sugar nucleotide synthase activity in the same or separate vectors A recombinant expression vector for production of hyaluronic acid or a combination thereof.
36. Item 36. The recombinant vector for expression for producing hyaluronic acid according to item 34 or 35, wherein the promoter is an inducible promoter.
37. Item 37. The recombinant expression vector for producing hyaluronic acid according to Item 36, wherein the inducible promoter is a promoter having a stress-inducible or cold-inducible function.
38. Item 38. The recombinant expression vector for production of hyaluronic acid according to Item 36 or 37, wherein the promoter is a promoter having a culture environment stress-inducing function of the HSP12 gene.
39. The expression for producing hyaluronic acid according to item 34 or 35, wherein the DNA encoding a protein having hyaluronic acid synthase activity is the following DNA of (a), (b) or (c): Recombinant vector.
(A) DNA consisting of the base sequence represented by SEQ ID NO: 1 or 3.
(B) A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence of (a) and encodes a protein having hyaluronic acid synthase activity.
(C) A complementary strand of the DNA of (a) or (b) above.
40. Item 37. The recombinant expression vector for production of hyaluronic acid according to item 35 or 36, wherein the protein having hyaluronic acid synthase activity is the following protein (a) or (b):
(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 2 or 4.
(B) A protein having an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having hyaluronic acid synthase activity.
41. Item 37. A recombinant expression vector for producing hyaluronic acid according to Item 36, wherein the sugar nucleotide is UDP-glucuronic acid and / or UDP-N-acetylglucosamine.
42. Proteins with sugar nucleotide synthase activity are glutamine: fructose-6-phosphate amide transferase, UDP-N-acetylglucosamine diphosphorylase, phosphoacetylglucosamine mutase, glucosamine-6-phosphate N-acetyltransferase, glucosamine-1 phosphate N -Acetyltransferase, phosphoglucomutase, N-acetylglucosamine kinase, hexokinase, N-acetylglucosamine deacetylase, UDP-glucose dehydrogenase, UDP-glucose-1-phosphate uridylyltransferase, inositol oxygenase, glucuronokinase and glucuronic acid-1 Item 36. The hyaluron according to item 35, which is one or more proteins selected from the group consisting of phosphate uridyltransferase. The recombinant expression vector for the production.
43. Item 36. The recombinant vector for expression for producing hyaluronic acid according to Item 35, wherein the protein having sugar nucleotide synthase activity is UDP-glucose dehydrogenase.
44. Item 44. The recombinant vector for expression for hyaluronic acid production according to any one of Items 35 to 43, wherein the DNA encoding a protein having sugar nucleotide synthase activity is DNA derived from chlorella virus and / or Arabidopsis thaliana.
45. Item 45. The hyaluronic acid production product according to any one of items 35 to 44, wherein the DNA encoding a protein having sugar nucleotide synthase activity is the following DNA (a), (b), or (c): Recombinant vector for expression.
(A) DNA comprising the base sequence represented by SEQ ID NO: 5, 7, 9, 11, 13, or 15.
(B) DNA that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and encodes a protein having UDP-glucose dehydrogenase activity.
(C) A complementary strand of the DNA of (a) or (b) above.
46. Item 46. The recombinant vector for expression for hyaluronic acid production according to any one of Items 35 to 45, wherein the protein having sugar nucleotide synthase activity is the following protein (a) or (b):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, 14, or 16;
(B) A protein comprising an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having UDP-glucose dehydrogenase activity.
47. Item 52. The hyaluronic acid production product according to any one of items 35 to 45, wherein the DNA encoding a protein having sugar nucleotide synthase activity is the following DNA (a), (b), or (c): Recombinant vector for expression.
(A) DNA consisting of the base sequence represented by SEQ ID NO: 17, 19, or 21.
(B) a DNA that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and encodes a protein having glutamine: fructose-6-phosphate amide transferase activity .
(C) A complementary strand of the DNA of (a) or (b) above.
48. Item 46. The recombinant vector for expression for hyaluronic acid production according to any one of Items 35 to 45, wherein the protein having sugar nucleotide synthase activity is the following protein (a) or (b):
(A) A protein consisting of the amino acid sequence represented by SEQ ID NO: 18, 20, or 22.
(B) A protein having an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having glutamine: fructose-6-phosphate amide transferase activity.
49. 49. A method for producing a transformed yeast having hyaluronic acid-producing ability, comprising a step of transforming a yeast using the vector according to any one of Items 34 to 48.
50. Item 18. A cosmetic composition comprising hyaluronic acid obtained by the method for producing hyaluronic acid according to any one of items 1 to 17 as an active ingredient.
51. Item 18. A functional food comprising hyaluronic acid obtained by the method for producing hyaluronic acid according to any one of items 1 to 17 as an active ingredient.

INDUSTRIAL APPLICABILITY According to the present invention, hyaluronic acid is produced in an in vivo system using yeast, and a hyaluronic acid production method, a transformed yeast, and an expression set for hyaluronic acid production that are industrially advantageous in terms of safety and cost. It is possible to provide a replacement vector.

As host systems for producing proteins by genetic recombination, plants, plant cultured cells, microorganisms such as Escherichia coli and yeast, animal cell cultures, genetically modified animals, and the like are currently available. Escherichia coli has problems such that protein processing does not occur, inclusion bodies (inclusion bodies) may be formed, and protease degradation occurs. Animal cells are difficult to maintain on a large scale, require expensive media, and are slow to grow, making large-scale production difficult. There is also a risk of contamination with viruses and cancer genes. In the case of transgenic animals, there are ethical issues in addition to maintenance issues.
On the other hand, yeast has established genetic engineering techniques and information as a model system for eukaryotic cells, and is easy to culture and can produce target substances in large quantities at low cost. In addition, the expression system using a microorganism as a host has the problems described above, whereas yeast is useful as a protein expression system. Since yeast has no hyaluronic acid-degrading enzyme (hyaluronidase), it was found that the problem of degradation of synthesized hyaluronic acid was solved and high molecular weight hyaluronic acid could be stably produced.
Yeast is also a promising substance production system that is familiar to us historically, such as wine, beer, liquor, bread, etc., and has no concerns about infectious viruses because it has been proven safe.

  Furthermore, the present inventors facilitated the maintenance of the correct higher-order structure of the membrane-bound protein by inducing and expressing hyaluronic acid synthase using an inducing factor such as environmental stress including low temperature, We also found that the cost burden was reduced.

In a preferred embodiment of the present invention, a hyaluronic acid synthase gene is expressed using a recombinant expression vector having a hyaluronic acid synthase gene under the control of an inducible promoter. When constitutive promoter is used and cultured at room temperature, hyaluronic acid synthase is relatively unstable, and when cultured yeast is cultured, hyaluronic acid synthase activity increases temporarily but then tends to decrease. When a stress-responsive inducible promoter is used, for example, if yeast is grown in a medium and environmental stress such as low temperature is induced during the logarithmic growth phase to induce the expression of hyaluronic acid synthase, The activity is maintained and hyaluronic acid can be efficiently produced. Using a constitutive promoter, for example, by culturing at a low temperature, the hyaluronic acid synthase can be cultured under stable conditions, and the instability of the hyaluronic acid synthase activity can be eliminated. Since the expression level of the promoter decreases, the hyaluronic acid production efficiency tends to decrease.
Yeast is useful as a protein expression system derived from eukaryotes.
In yeast, as in other organisms, there are constitutive expression systems that always produce proteins and inducible expression systems that produce proteins in larger quantities under certain conditions.
Constitutive expression systems are characterized by simple expression, and inducible expression systems are often used for the purpose of separating growth processes and protein production processes, for example, when producing proteins that adversely affect host cells. It is done. It is also well known that the inducible expression system is more powerful.
In yeast, a promoter that is induced in response to various stresses (changes in the external environment), such as the HSP12 promoter, is known.
Low temperature is a type of stress and induces various promoters in addition to HSP12.
Protein production at low temperature is selective because it is an environment where only some promoters respond to low temperature or maintain transcriptional activity even at low temperature, and the degradation of the produced protein is suppressed. There is a merit that high efficiency of protein higher-order structure formation (folding) is good. Therefore, a protein can be produced very efficiently by producing the protein at a low temperature.
There has been no actual example of producing a saccharide that yeast does not produce at all like the hyaluronic acid of the present invention, and two kinds of sugars such as hyaluronic acid are several tens or hundreds in a certain order. However, there is no example of producing a continuous polymer material.
In addition, hyaluronic acid synthase generally has a plurality of transmembrane regions and membrane-bound regions. When a membrane-bound protein such as hyaluronic acid synthase is expressed from a different gene from the host that expresses the gene, the transmembrane region and the membrane-bound region often cannot maintain the correct structure. In particular, an active hyaluronic acid synthase may form a complex with one hyaluronic acid synthase and about 14 to 18 molecules of cardiolipin, which is a phospholipid present in the membrane, and affect hyaluronic acid synthase activity. Has been suggested.

  In a preferred embodiment of the present invention, (1) a yeast is transformed with a recombinant expression vector containing a DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter that can function in yeast. A method for producing hyaluronic acid, comprising a step, (2) a step of culturing a transformant obtained by transformation, and (3) a step of separating hyaluronic acid produced by the transformant.

  In addition, (1) for expression comprising DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter that can function in yeast and DNA encoding a protein having exogenous sugar nucleotide synthase activity A step of transforming yeast using a recombinant vector, (2) a step of culturing a transformant obtained by transformation, and (3) a step of separating hyaluronic acid produced by the transformant. , A method for producing hyaluronic acid.

  Moreover, it is a cosmetic composition characterized by containing hyaluronic acid obtained by the method for producing hyaluronic acid as an active ingredient.

  Moreover, it is a functional food characterized by containing hyaluronic acid obtained by the method for producing hyaluronic acid as an active ingredient.

  Furthermore, a transformed yeast having the ability to produce hyaluronic acid transformed with a recombinant expression vector containing a DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter that can function in yeast. is there.

  A recombinant expression vector comprising a DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter that can function in yeast, and a DNA encoding a protein having an exogenous sugar nucleotide synthase activity Is a transformed yeast having the ability to produce hyaluronic acid transformed with

  Furthermore, it is a hyaluronic acid-containing product obtained from the transformed yeast.

  Further, the present invention is a recombinant expression vector for production of hyaluronic acid containing a DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter that can function in yeast.

  Also, expression for hyaluronic acid production comprising DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter that can function in yeast, and DNA encoding a protein having sugar nucleotide synthase activity Recombinant vector.

  Furthermore, a step of transforming yeast using a recombinant expression vector for production of hyaluronic acid containing DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter capable of functioning in yeast. A method for producing a transformed yeast having the ability to produce hyaluronic acid.

Furthermore, expression for the production of hyaluronic acid, comprising DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter that can function in yeast, and DNA encoding a protein having sugar nucleotide synthase activity This is a method for producing a transformed yeast having the ability to produce hyaluronic acid, comprising the step of transforming the yeast using the recombinant vector for use.
The present invention will be described in detail below.
Hyaluronic acid synthase In one preferred embodiment of the present invention, a DNA encoding a protein having a hyaluronic acid synthase activity under the control of a promoter capable of functioning in yeast and a protein having a sugar nucleotide synthase activity are encoded. Using the DNA to be transformed, yeast is transformed.

The protein having hyaluronic acid synthase activity of the present invention synthesizes hyaluronic acid having a polymer structure composed of repeating structures of glucuronic acid and N-acetylglucosamine using UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. .
The protein having hyaluronic acid synthase activity of the present invention is not particularly limited as long as it has the above-mentioned properties, and is hyaluronic acid synthase derived from animals, microorganisms or viruses (hereinafter abbreviated as HAS). Etc. can be used. Specifically, hyaluronic acid synthase derived from vertebrates such as humans, mice, rabbits, chickens, cows, and Xenopus, hyaluronic acid synthase derived from microorganisms such as Streptococcus and Pasteurella, and viruses such as Chlorella virus Hyaluronic acid synthase or the like can be used.

More specifically, HAS (A98R) from Chlorella virus PBCV-1 strain, HAS1, HAS2 and HAS3 from human hyaluronan synthase (hHAS), HAS1, HAS2 from mouse hyaluronan synthase (mHAS) And HAS3, chicken hyaluronan synthase (gHAS) HAS1, HAS2 and HAS3, rat hyaluronan synthase (rHAS) HAS2, bovine hyaluronan synthase (bHAS) HAS2, derived from Xenopus laevis Hyaluronic acid synthase (xHAS) HAS1, HAS2 and HAS3, Hyaluronic acid synthase derived from Pasteurella multocida (pmHAS), Streptococcus pyogenes-derived hyaluronic acid synthase (spHAS), hyaluronic acid synthase derived from Streptococcus equilisimiris (seHAS), etc. Can be given. Some hyaluronic acid synthase (HAS) genes have various types such as HAS1, HAS2, and HAS3, but the type is not particularly limited.
The HAS described above can be used, but a chlorella virus-derived HAS is preferred, and a chlorella virus-derived HAS represented by a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4 is particularly preferred.
In addition, the protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or 4 is a hyaluronic acid synthesis consisting of an amino acid sequence in which one or several or plural, for example, one or several amino acids are deleted, substituted, inserted or added. It may be a protein that has been mutated to such an extent that it does not lose its activity. For example, at least one, preferably about 1 to 10, and more preferably 1 to 5 amino acids of the amino acid sequence represented by SEQ ID NO: 2 or 4 may be deleted. At least 1, preferably about 1 to 10, more preferably 1 to 5 amino acids may be added to the amino acid sequence represented, or at least one of the amino acid sequences represented by SEQ ID NO: 2 or 4, Preferably about 1 to 10 amino acids, more preferably 1 to 5 amino acids may be substituted with other amino acids, but there is no particular limitation thereto. Such mutations occur in nature as well as artificial mutations. As an example, it has been reported that hyaluronic acid synthase derived from Pasteurella multocida has hyaluronic acid synthase activity even when approximately 270 amino acids, which appear to be membrane-bound and transmembrane regions, are deleted (Jing et al., 2000, Glycobiology, 10, 883-889). The number of mutated amino acids is not limited as long as hyaluronic acid synthesis activity is not lost. Moreover, it may be a part of the protein consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4 and a protein having hyaluronic acid synthesis activity.

The DNA encoding the protein having hyaluronic acid synthase activity of the present invention is hyaluronic acid having a polymer structure composed of a repeating structure of glucuronic acid and N-acetylglucosamine using UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. DNA encoding a protein having an enzymatic activity to be synthesized.
The DNA encoding the protein having hyaluronic acid synthase activity of the present invention is not particularly limited as long as it has the above-mentioned properties, and is hyaluronic acid synthase derived from animals, microorganisms and viruses (hereinafter abbreviated as HAS). It is possible to use a gene or the like. Specifically, hyaluronic acid synthase genes derived from vertebrates such as humans, mice, rabbits, chickens, cows, and Xenopus, hyaluronic acid synthase genes derived from microorganisms such as Streptococcus and Pasteurella, and viruses such as chlorella virus It is possible to use a derived hyaluronic acid synthase gene or the like.

More specifically, HAS (A98R) gene derived from Chlorella virus PBCV-1 strain, HAS1, HAS2 and HAS3 of human-derived hyaluronic acid synthase (hHAS) gene, mouse hyaluronic acid synthase (mHAS) gene HAS1, HAS2 and HAS3, HAS1, HAS2 and HAS3 of chicken hyaluronic acid synthase (gHAS) gene, HAS2 of rat hyaluronic acid synthase (rHAS) gene, hyaluronic acid synthase (bHAS) gene from bovine HAS2, Xenopus hyaluronan synthase (xHAS) genes HAS1, HAS2 and HAS3, Pasteurella multocida hyaluronan synthase (pmHAS) gene, Streptococcus pyogenes-derived hyaluronic acid synthase (spHAS) gene, Streptococcus equilisimilis Hyaluronic acid synthase (seHAS) gene. Some hyaluronic acid synthase (HAS) genes have various types such as HAS1, HAS2, and HAS3, but the type is not particularly limited.
The HAS gene described above can be used, but the HAS gene derived from chlorella virus is preferable, and the HAS gene derived from chlorella virus represented by the DNA consisting of the base sequence represented by SEQ ID NO: 1 or 3 is particularly preferable. Good.
Further, it is a DNA encoding a protein that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1 or 3, and has hyaluronic acid synthase activity. May be.
In the above, “stringent conditions” means that only a base sequence encoding a polypeptide having a hyaluronic acid synthase activity equivalent to the hyaluronic acid synthase having the base sequence represented by SEQ ID NO: 1 or 3 is the specific sequence. A base sequence that encodes a polypeptide that forms a hybrid (so-called specific hybrid) and does not have equivalent activity means a condition that does not form a hybrid (so-called non-specific hybrid) with the specific sequence. Those skilled in the art can easily select such conditions by changing the temperature during the hybridization reaction and washing, the salt concentration of the hybridization reaction solution and the washing solution, and the like. Specifically, it is 42 ° C. in 6 × SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 × SSPE (3 M NaCl, 0, ₂ M NaH ₂ PO ₄ , 20 mM EDTA · 2Na, pH 7.4). Examples of the stringent conditions of the present invention include, but are not limited to, the conditions of hybridization with, and further washing with 0.5 × SSC at 42 ° C.

The stringent conditions are preferably highly stringent conditions. The highly stringent conditions are not particularly limited as long as the gene encoding A98R does not hybridize. For example, washing is performed at 60 ° C. at a salt concentration corresponding to 0.1 × SSC and 0.1% SDS. It is a condition that is called.
In order to produce hyaluronic acid in a sugar nucleotide yeast, it is necessary to supply a sufficient amount of UDP-glucuronic acid and UDP-N-acetylglucosamine which are substrates for hyaluronic acid synthase. Among yeasts, Saccharomyces yeasts are useful because they have a long and safe history of wine, beer, liquor, bread, etc., and have established techniques and information for genetic engineering as a model system for eukaryotic cells. However, it has been reported that some Saccharomyces yeasts lack the UDP-glucuronic acid synthesis pathway (The Journal of Biological Chemistry 1996, Vol.271, No.39, 23657-23660). In order to improve the productivity of these sugar nucleotides, it is necessary to introduce an enzyme involved in the sugar nucleotide synthesis pathway, that is, a protein having sugar nucleotide synthase activity into yeast.
Furthermore, in the present invention, a repeated structure of glucuronic acid and N-acetylglucosamine using the increased UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates by simultaneously introducing a protein having hyaluronic acid synthase activity into yeast. It has been found that hyaluronic acid having a polymer structure consisting of can be synthesized.
Enzyme involved in sugar nucleotide synthesis pathway In the present invention, a DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter that can function in yeast and a protein having sugar nucleotide synthase activity Transformation of yeast is performed using DNA encoding.
The protein having sugar nucleotide synthase activity of the present invention may be an enzyme that catalyzes a reaction involved in a sugar nucleotide synthesis metabolic pathway. For example, as sugar nucleotides, UDP-N-acetylglucosamine, UDP-glucuronic acid, UDP-N-acetylgalactosamine, UDP-glucose, UDP-galactose, UDP-xylose, GDP-fucose, GDP-mannose, CMP-neuraminic acid, etc. Any protein having a synthase activity of Of these sugar nucleotides, a protein having a synthase activity of UDP-N-acetylglucosamine, UDP-glucuronic acid, UDP-N-acetylgalactosamine, UDP-glucose, UDP-galactose, UDP-xylose is preferable. More preferably, a protein having a synthase activity of UDP-N-acetylglucosamine or UDP-glucuronic acid is preferable.

Specifically, the protein having sugar nucleotide synthase activity of the present invention includes glutamine: fructose-6-phosphate amide transferase, UDP-N-acetylglucosamine diphosphorylase, phosphoacetylglucosamine mutase, glucosamine-6-phosphate N-acetyl. Transferase, glucosamine-1 phosphate N-acetyltransferase, phosphoglucomutase, N-acetylglucosamine kinase, hexokinase, N-acetylglucosamine deacetylase, UDP-glucose dehydrogenase, UDP glucose-1 phosphate uridylyltransferase, inositol oxygenase, glucose Examples include chronokinase and glucuronic acid-1-phosphate uridyltransferase. One or more proteins selected from the group consisting of these proteins having sugar nucleotide synthase activity may be expressed in yeast. UDP-glucose dehydrogenase is particularly preferred.
Further, at least UDP-glucose dehydrogenase may be selected as a protein having sugar nucleotide synthase activity, and may be expressed in yeast cells in combination with other proteins having sugar nucleotide synthase activity.
Proteins having sugar nucleotide synthase activity other than UDP-glucose dehydrogenase include UDP-N acetylglucosamine diphosphorylase, phosphoacetylglucosamine mutase, glucosamine-6 phosphate N acetyltransferase, glucosamine-1 phosphate N-acetyltransferase, Phosphoglucomutase, N-acetylglucosamine kinase, hexokinase, N-acetylglucosamine deacetylase, glutamine: fructose-6-phosphate amide transferase, UDP glucose-1 phosphate uridylyltransferase, inositol oxygenase, glucuronokinase and glucuronate-1 phosphorus Examples thereof include one or more proteins selected from the group consisting of acid uridyltransferases.
In order to exert the effect of the present invention, if at least UDP-glucose dehydrogenase is selected as a protein having sugar nucleotide synthase activity, an effect superior to the conventional one can be obtained. More preferably, both UDP-glucose dehydrogenase (hereinafter abbreviated as UGD) and glutamine: fructose-6-phosphate amide transferase (hereinafter abbreviated as GFAT) may be selected.
UDP-glucose dehydrogenase The protein having UDP-glucose dehydrogenase activity of the present invention may be any protein that synthesizes UDP-glucuronic acid using UDP-glucose as a substrate.
The protein having UDP-glucose dehydrogenase activity of the present invention is not particularly limited as long as it has the above properties, and examples thereof include eukaryote-derived, prokaryotic-derived, and virus-derived UGD. As eukaryotes, UGD such as human, bovine, mouse, poplar, sugarcane and Arabidopsis is used, UGD such as Escherichia coli, Pasteurella multocida and lactic acid bacteria is used as prokaryote, and UGD such as chlorella virus is used as virus. However, it is not limited to these.
The UGD described above can be preferably used, but more preferably UGD derived from chlorella virus or Arabidopsis thaliana. Particularly preferably, UGD derived from a chlorella virus represented by a protein consisting of the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, 14, or 16 or SEQ ID NO: 5, 7, 9, 11, 13, or 15 UGD derived from chlorella virus represented by a protein having the amino acid sequence shown is preferred.
The protein consisting of the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, 14, or 16 is UDP- consisting of an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added. It may be a protein mutated to such an extent that it does not lose glucose dehydrogenase activity. For example, at least one, preferably about 1 to 10, more preferably 1 to 5 amino acids of the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, 14, or 16 may be deleted. Alternatively, at least 1, preferably about 1 to 10, more preferably 1 to 5 amino acids may be added to the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, 14, or 16. Alternatively, at least one, preferably about 1 to 10, more preferably 1 to 5 amino acids in the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, 14, or 16 are substituted with other amino acids. However, it is not particularly limited to this. Such mutations occur in nature as well as artificial mutations. The number of mutated amino acids is not particularly limited as long as UGD activity is not lost. Further, the protein may be a part of a protein consisting of the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, 14, or 16 and having UDP-glucose dehydrogenase activity. .
The DNA encoding a protein having UDP-glucose dehydrogenase activity of the present invention is a DNA encoding a protein having an enzyme activity for synthesizing UDP-glucuronic acid using UDP-glucose as a substrate.
The DNA encoding the protein having UDP-glucose dehydrogenase activity of the present invention is not particularly limited as long as it has the above properties, and examples thereof include UGD genes such as eukaryotes, prokaryotes, and viruses. Examples of eukaryotes include human, bovine, mouse, poplar, sugarcane and Arabidopsis, prokaryotes include E. coli, Pasteurella multocida and lactic acid bacteria, and viruses include, but are not limited to, chlorella virus.

The UGD gene described above is preferably used, but more preferably a UGD gene derived from chlorella virus or Arabidopsis thaliana, particularly preferably a chlorella virus-derived UGD comprising the base sequence represented by SEQ ID NO: 5 or 7 It is a UGD gene derived from Arabidopsis thaliana consisting of the gene or the nucleotide sequence shown in SEQ ID NOs: 9, 11, 13, and 15.
Further, it hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence represented by SEQ ID NO: 5, 7, 9, 11, 13, or 15, and has a UDP-glucose dehydrogenase activity. It may be a DNA encoding a protein having
In the above, “stringent conditions” encodes a polypeptide having UDP-glucose dehydrogenase activity equivalent to the UDP-glucose dehydrogenase having the nucleotide sequence shown in SEQ ID NO: 5, 7, 9, 11, 13, or 15. Only the base sequence forms a hybrid (so-called specific hybrid) with the specific sequence, and the base sequence encoding a polypeptide having no equivalent activity does not form a hybrid (so-called non-specific hybrid) with the specific sequence. means. Those skilled in the art can easily select such conditions by changing the temperature during the hybridization reaction and washing, the salt concentration of the hybridization reaction solution and the washing solution, and the like. Specifically, it is 42 ° C. in 6 × SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 × SSPE (3 M NaCl, 0, ₂ M NaH ₂ PO ₄ , 20 mM EDTA · 2Na, pH 7.4). Examples of the stringent conditions of the present invention include, but are not limited to, the conditions in which the cells are hybridized with and washed with 0.5 × SSC at 42 ° C.

The stringent conditions are preferably highly stringent conditions. High stringent conditions are conditions in which washing is performed at a salt concentration corresponding to, for example, 0.1 × SSC and 0.1% SDS at 60 ° C.
Glutamine: fructose-6-phosphate amide transferase The protein having glutamine: fructose-6-phosphate amide transferase activity according to the present invention comprises glucosamine-6-phosphate using L-glutamine and fructose-6-phosphate as substrates. What is necessary is just to synthesize | combine an acid.
The protein having glutamine: fructose-6-phosphate amide transferase (GFAT) activity of the present invention is not particularly limited as long as it has the above-mentioned properties, and examples thereof include eukaryotic origin, prokaryotic origin, virus origin, etc. It is done. Examples of eukaryotes include humans, mice, corn, Arabidopsis thaliana, nematodes and yeasts, prokaryotes include Bacillus subtilis and Escherichia coli, and viruses include, but are not limited to, chlorella virus.
The GFAT described above can be preferably used, but GFAT derived from chlorella virus or Arabidopsis thaliana is more preferable. Particularly preferred is GFAT derived from chlorella virus represented by a protein comprising the amino acid sequence represented by SEQ ID NO: 18 or 20, or GFAT derived from Arabidopsis thaliana represented by a protein comprising the amino acid sequence represented by SEQ ID NO: 10.
The protein comprising the amino acid sequence represented by SEQ ID NO: 18, 20, or 22 is a glutamine: fructose-6-phosphate amide transferase comprising an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted, or added. It may be a protein that has been mutated to such an extent that it does not lose its activity. For example, at least one, preferably about 1 to 10, more preferably 1 to 5 amino acids of the amino acid sequence represented by SEQ ID NO: 18, 20, or 22 may be deleted, or SEQ ID NO: 18, At least 1, preferably about 1 to 10, more preferably 1 to 5 amino acids may be added to the amino acid sequence represented by 20 or 22, or the amino acid represented by SEQ ID NO: 18, 20 or 22 Although at least 1, preferably about 1 to 10, and more preferably 1 to 5 amino acids in the sequence may be substituted with other amino acids, it is not particularly limited thereto. Such mutations occur in nature as well as artificial mutations. The number of mutated amino acids is not particularly limited as long as GFAT activity is not lost. As an example of natural mutation, there is an example in which at least 2% of GFAT of the chlorella virus Hirosaki strain of SEQ ID NO: 17 and GFAT of the known chlorella virus PBCV-1 strain and K2 strain are mutated at the amino acid level. Further, it may be a part of the protein consisting of the amino acid sequence represented by SEQ ID NO: 18, 20, or 22 and having a glutamine: fructose-6-phosphate amide transferase activity.
The DNA encoding the protein having glutamine: fructose-6-phosphate amide transferase activity of the present invention is a protein having an enzyme activity for synthesizing glucosamine-6-phosphate using L-glutamine and fructose-6-phosphate as substrates. DNA encoding
The DNA encoding the protein having glutamine: fructose-6-phosphate amide transferase (GFAT) activity of the present invention is not particularly limited as long as it has the above properties, eukaryotic origin, prokaryotic origin, virus origin, etc. GFAT gene. Examples of eukaryotes include humans, mice, corn, Arabidopsis thaliana, nematodes and yeasts, examples of prokaryotes include Bacillus subtilis and Escherichia coli, and examples of viruses include chlorella virus. Some GFAT genes have various types such as GFAT1 and GFAT2, but the type is not particularly limited.

The GFAT gene described above can be preferably used, but more preferably a GFAT gene derived from chlorella virus or Arabidopsis thaliana, and particularly preferably a GFAT gene derived from a chlorella virus consisting of a base sequence represented by SEQ ID NOs: 17 and 19, Alternatively, an Arabidopsis derived GFAT gene consisting of the base sequence represented by SEQ ID NO: 21 is preferred.
A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence represented by SEQ ID NO: 17, 19 or 21, and encodes a protein having GFAT activity. Also good.
In the above, “stringent conditions” means a polyamine having glutamine: fructose-6-phosphate amide transferase activity equivalent to glutamine: fructose-6-phosphate amide transferase having the nucleotide sequence shown in SEQ ID NO: 5, 7 or 9. Only a base sequence encoding a peptide forms a hybrid (so-called specific hybrid) with the specific sequence, and a base sequence encoding a polypeptide having no equivalent activity hybridizes with the specific sequence (so-called non-specific hybrid). It means the condition that does not form. Those skilled in the art can easily select such conditions by changing the temperature during the hybridization reaction and washing, the salt concentration of the hybridization reaction solution and the washing solution, and the like. Specifically, hybridization was performed at 42 ° C. in 6 × SSC (0.9 M NaCl, 0.09 M trisodium citrate) or 6 × SSPE (3 M NaCl, 0, 2 M NaH 2 PO 4, 20 mM EDTA · 2Na, pH 7.4). Further, the condition of washing at 42 ° C. with 0.5 × SSC is an example of the stringent condition of the present invention, but is not limited thereto.

The stringent conditions are preferably highly stringent conditions. High stringent conditions are conditions in which washing is performed at a salt concentration corresponding to, for example, 0.1 × SSC and 0.1% SDS at 60 ° C.
Recombinant expression vector and DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter that can function in transformant yeast, and if necessary, exogenous sugar nucleotide synthase activity A yeast having the ability to produce hyaluronic acid can be obtained by transforming the host with a recombinant vector for expression containing DNA encoding the protein.
The term “protein having an exogenous sugar nucleotide synthase activity” means a protein having an exogenous sugar nucleotide synthase activity newly introduced into yeast from the outside, unlike an endogenous protein. As a specific method of “newly introducing from the outside”, transformation may be performed using a recombinant expression vector or the like. The “newly introduced gene from outside” includes the transformation of an originally endogenous gene using an external recombinant vector for expression. Since the presence of a protein having hyaluronic acid synthase activity in yeast has not been confirmed to date, it is obvious that it is exogenous even if not described as exogenous.

  The transformed yeast as described above can produce hyaluronic acid. This will be specifically described below.

  Examples of the yeast include Saccharomyces cerevisiae, experimental yeast, brewing yeast, edible yeast, and industrial yeast.

  Alternatively, hyaluronic acid may be produced by culturing a transformant obtained by the transformation and separating the hyaluronic acid produced by the transformant.

  As a recombinant vector for expression, a vector usually used for producing transformed yeast can be used.

  Such a vector is not particularly limited as long as it contains a promoter sequence that can be transcribed in yeast and a terminator sequence including a polyadenylation site necessary for stabilization of the transcription product. For example, plasmids “pESP Vector”, “pESC Vector”, “pAUR123”, “pYES”, “pLTex321s” and the like can be used.

When yeast is used as a host, transformation can be performed on yeast by the spheroplast method, the lithium acetate method, the electroporation method, or the like. An expression set for producing hyaluronic acid, comprising DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter that can function in yeast, and DNA encoding a protein having sugar nucleotide synthase activity This can be done by introducing a replacement vector. Each DNA may be introduced into yeast by one expression vector under the control of one promoter, or introduced into yeast using one or more expression vectors under the control of separate promoters. Also good. The yeast introduced with the expression vector is selected on the basis of auxotrophy such as uracil auxotrophy. Alternatively, a yeast transformation method of substitution / insertion into a chromosome using a vector such as a YIp system or a DNA sequence homologous to an arbitrary region in a chromosome may be used.
It is also conceivable to control the expression of DNA encoding a protein having hyaluronic acid synthase activity and DNA encoding a protein having sugar nucleotide synthase activity with an inducible promoter. Examples of inducible promoters are described below.

As the inducible promoter, any inducible promoter that functions in yeast cells can be used. For example, a GAL1 promoter induced by galactose, a CUP1 promoter induced by copper, a heat shock protein induced by heat shock A gene promoter, a PHO5 promoter induced by a low phosphate concentration, or the like can be used. Furthermore, in order to more stably express the DNA encoding the protein having hyaluronic acid synthase activity and the DNA encoding the protein having sugar nucleotide synthase activity in yeast, the use of an insulator or the target organelle In order to localize a protein having hyaluronic acid synthase activity and a protein having sugar nucleotide synthase activity, a signal peptide may be added, or a part of the enzyme may be substituted and deleted.
The low temperature inducible promoter means a promoter that exhibits high promoter activity at a temperature lower than the optimum culture temperature of yeast by comparing the promoter activity at the optimum culture temperature of yeast. Specifically, a low temperature inducible promoter exhibits a promoter activity three times or more at a temperature lower than the optimal culture temperature of yeast as compared with the promoter at the optimal culture temperature of yeast. Here, the optimal culture temperature of yeast is about 30 ° C. The temperature lower than the optimum culture temperature for yeast means a temperature lower than 30 ° C., for example, about 10 ° C., but is not limited to this temperature as long as it is a temperature of 20 ° C. or lower.
The heat shock protein 12 promoter is a stress-inducible promoter, and is induced by, for example, heat shock, osmotic stress, high concentration alcohol, high salt concentration, oxidative stress, glucose starvation and the like. Therefore, for example, when the HSP12 promoter is used, as long as the transcriptional activity of the promoter increases, the protein may be produced by induction of the promoter by any of these stresses. Furthermore, when a stress-inducible promoter other than HSP12 is used, as long as the transcriptional activity of the promoter increases, any of these stresses may cause the protein to be produced by induction of the promoter.

  As the promoter, a constitutive promoter may be used in addition to the inducible promoter. Examples of promoters that are constantly expressed in yeast include ADH promoter, TEF promoter, GPD promoter and the like.

DNA encoding a protein having hyaluronic acid synthase activity is preferably under the control of an inducible promoter, but should be under the control of a constitutive promoter (especially a promoter whose expression efficiency is not significantly reduced at low temperatures). You can also. DNA encoding a protein having sugar nucleotide synthase activity may be under the control of either a constitutive promoter or an inducible promoter.
Recovery of hyaluronic acid High-purity hyaluronic acid can be obtained by known purification methods such as culturing at any point and sterilization, followed by precipitation with an organic solvent such as alcohol and desalting by ultrafiltration. As a method for isolating or obtaining hyaluronic acid produced from transformed yeast that has expressed hyaluronic acid synthase activity protein and exogenous sugar nucleotide synthase activity protein at the same time and acquired hyaluronic acid production ability Examples of the method include the following methods.

Hyaluronic acid accumulated in the culture medium of transformed yeast cells may be purified using a known separation method. For example, hyaluronic acid accumulated in transformed yeast may be purified using a known separation method. You may do it. Alternatively, the transformed yeast may be dried and then pulverized and extracted with a suitable organic solvent. The yeast extract containing hyaluronic acid is filtered to obtain a hyaluronic acid filtered solution that does not contain yeast cells. This solution is purified by diafiltration to remove low molecular weight impurities. It is possible to separate hyaluronic acid by diafiltration of a filtered solution containing dissolved hyaluronic acid with pure water and continuously discarding the filtrate. When a medical product is required, a step of precipitating nucleic acid from the solution may be further performed. This step can be performed, for example, by adding a cationic surfactant such as a quaternary ammonium compound of cetylpyridinium chloride.

The culture conditions are not particularly limited, and a culture medium containing a large amount of GlcNAc and a saccharide serving as a raw material for producing glucuronic acid so as to increase the production amount of UDP-GlcNAc and UDP-glucuronic acid serving as a raw material for hyaluronic acid. It is preferred to use.
Use of hyaluronic acid The hyaluronic acid obtained by the present invention can be usefully used for cosmetics and pharmaceutical ingredients, biomaterials and the like. Specifically, it can be usefully used as a moisturizing ingredient in cosmetics, a therapeutic agent for arthritis, chronic rheumatism, burns or cuts, an eye drop ingredient, or the like. Moreover, you may utilize as a functional food useful in preventing and / or improving skin aging.

  You may manufacture the cosmetics composition which contains the hyaluronic acid obtained by the manufacturing method of the hyaluronic acid of this invention as an active ingredient. For example, it can be applied in the form of a liquid agent such as an aqueous solution, oil agent, emulsion, suspension, etc., a semi-solid agent such as gel or cream, or a solid agent such as powder, granule, capsule, microcapsule or solid. It is prepared in these forms by a conventionally known method, and lotion, emulsion, gel, cream, ointment, plaster, haptic, aerosol, suppository, injection, powder, granule, tablet, pill , Various dosage forms such as syrups and lozenges. These can be applied to the body by applying, sticking, spraying, drinking and the like. Among these dosage forms, lotions, emulsions, creams, ointments, plasters, haptics, aerosols and the like are particularly suitable for external preparations for skin. Cosmetics include skin lotions, cosmetics, emulsions, creams, packs, and other skin cosmetics, makeup base lotions, makeup creams, emulsions, creams or ointment foundations, lipsticks, eye colors, cheek colors, etc. It can be made into cosmetics for body, such as makeup cosmetics, hand cream, leg cream, and body lotion, bathing agents, oral cosmetics, and hair cosmetics. Usually, these dosage forms can be produced according to the formulation method used in cosmetics.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.
Example 1 Method for Isolating Chlorella Virus-Derived Hyaluronic Acid Synthase Gene PCR primers were prepared in order to isolate the chlorella virus hyaluronic acid synthase gene (cvHAS) by PCR. Primers are already disclosed in Chlorella virus genome sequence information (Kutish et al, 1996, Virology, 223, 303-317) (Li et al, 1995, Virology, 212, 134-150) (Li et al, 1997 , Virology, 237, 360-377) (Lu et al, 1995, Virology, 206, 339-352) (Lu et al, 1996, Virology, 216, 102-123) and identification information for chlorella virus-derived hyaluronic acid synthesis genes Designed based on (Graves et al, 1999, virology, 257, 15-23) (DeAngelis et al, 1997, Science, 278, 1800-1803) and added restriction enzyme sites necessary for introduction into expression vectors What was made was produced. The restriction enzyme site added a SacI site to the 3 ′ primer.
5 ′ primer (SEQ ID NO: 23)
5'- ATG GGT AAA AAT ATA ATC ATA ATG GTT TCG-3 '
3 'primer (SEQ ID NO: 24)
5 '-AAT TGA GCT CTC ACA CAG ACT GAG CAT TGG TAG −3'
The genomic DNA of the chlorella virus CVHI1 strain was used as a PCR template. PCR was performed using a reaction program of 25 cycles of 94 ° C for 2 minutes (94 ° C for 15 seconds, 55 ° C for 30 seconds, 68 ° C for 2 minutes) using KOD-plus- (Toyobo Co., Ltd.) as the DNA polymerase. The obtained PCR fragment was inserted into the SmaI site and SacI site of the multicloning site of the expression vector pLTex321s. From the inserted fragment, the DNA sequence of the CVHI1 strain-derived hyaluronic acid synthase gene cvHAS-HI (SEQ ID NO: 1) was confirmed by a DNA sequencer.
Example 2 Method for Isolating Chlorella Virus-Derived UDP-Glucose Dehydrogenase Gene PCR primers were prepared for isolating the chlorella virus UDP-glucose dehydrogenase gene (cvUGD) by PCR. Primers are designed based on the already known chlorella virus genome sequence information (Landstein. D et al., Virology. 1998 Oct 25; 250 (2): 388-96.) And introduced into the expression vector. Were prepared by adding the necessary restriction enzyme sites. The restriction enzyme site added a SacI site to the 3 ′ primer.
5 'primer (SEQ ID NO: 25)
5'- AATTGGTACCATGTCACGA ATCGCAGTCGT-3 '
3 'primer (SEQ ID NO: 26)
5 '-AATTTCTAGATTAGTTATCGCCATATACAT-3'
The genomic DNA of the chlorella virus CVHI1 strain was used as a PCR template. PCR was performed using a reaction program of 25 cycles of 94 ° C. for 2 minutes (94 ° C. for 15 seconds, 55 ° C. for 30 seconds, 68 ° C. for 2 minutes) using KOD-plus-Toyobo Co., Ltd. as the DNA polymerase. The obtained PCR fragment was inserted into the KpnI site and the XbaI site of the multicloning site of the expression vector pAUR123 (Takara Bio Inc.). From the inserted fragment, the DNA sequence of the CVHI1 strain-derived UDP-glucose dehydrogenase gene cv-UGD (SEQ ID NO: 7) was confirmed by a DNA sequencer.
Example 3 Transformation of cvHAS-Hirosaki gene into yeast (BY4743)
pLT321-cvHAS-Hirosaki plasmid DNA was prepared and introduced into yeast (BY4743) using the YEASTMAKER ^™ Yeast Transformation System2 (CLONTECH) kit. The culture is collected by centrifugation (5000 rpm, 1 min, 4 ° C), the supernatant is removed, the cells are resuspended in 1.0 ml of 0.9% NaCl solution, and the SD plate medium (0.67% Yeast nitrogen base w / o AA, 2% glucose, 0.02% adenine sulfate, 0.02% L-tryptophan, 0.02% L-histidine, 0.03% L-lysine monohydrochloride, 0.03% L-methionine, 0.03% L-leucine and 2% (Including agar) and cultured at 30 ° C. for 3 days to obtain a yeast (cvHAS yeast) containing pLT321-cvHAS-Hirosaki.
Example 4 Transformation of cvHAS-Hirosaki gene and cvUGD gene into yeast (BY4743)
pLT321-cvHAS-Hirosaki and pAUR123-cvUGD plasmid DNAs were prepared and introduced into yeast (BY4743) using the YEASTMAKER ^™ Yeast Transformation System 2 (CLONTECH) kit. The culture is collected by centrifugation (5000 rpm, 1 min, 4 ° C), the supernatant is removed, the cells are resuspended in 1.0 ml of 0.9% NaCl solution, and 0.5 μg / ml Aureobasidin A containing SD Plate medium (0.67% Yeast nitrogen base w / o AA, 2% glucose, 0.02% adenine sulfate, 0.02% L-tryptophan, 0.02% L-histidine, 0.03% L-lysine monohydrochloride, 0.03% L-methionine, -0.03% L-leucine) and cultured for 3 days at 30 ° C. to obtain yeast (cvHAS-cvUGD yeast) containing pLT321-cvHAS-Hirosaki and pAUR123-cvUGD.
Example 5 Hyaluronic acid production by cvHAS yeast
SD medium (0.67%) in 3 ml / 18 ml test tube in which 300 μl of cvHAS yeast was controlled at around pH 6.5
Yeast nitrogen base w / o AA, 2% glucose, 0.02% adenine sulfate, 0.02% L-tryptophan, 0.02% L-histidine, 0.03% L-lysine monohydrochloride, 0.03% L-methionine, 0.03% L-leucine Inoculated) and cultured at 30 ° C. overnight. After culturing, the 18 ml test tube was transferred to a shaking incubator set at 10 ° C in advance, and shaking culture (induction) was further performed at 10 ° C for 6 days. After 24 hours of induction at low temperature, HA with and without HA material (SD medium containing 100 mM UDP-N acetylglucosamine (UDP-GlcNAc) and 100 mM UDP-glucuronic acid (UDP-GlcA) and SD medium not containing) It was investigated whether fermentation production was possible. The culture solution (3 mL) was centrifuged (1,3000 rpm, 10 minutes) and separated into cells and supernatant. The supernatant was heated at 95 ° C. for 5 minutes, centrifuged (1,3000 rpm, 10 minutes), and the supernatant was recovered as a crude purified solution. The crude purified solution was diluted 100 times with water and used as a measurement sample. Hyaluronic acid was quantified using a hyaluronic acid plate “Chugai” (Chugai Diagnostics Science Co., Ltd.). The results are shown in FIG.
Example 6 Comparison of hyaluronic acid production by cvHAS yeast and cvHAS-cvUGD yeast
See Example 5 for hyaluronic acid production by cvHAS yeast. 3 ml / 18 ml test tube SD medium (0.67% Yeast nitrogen base w / o AA, 2% glucose, 0.02% adenine sulfate, 0.02% L-tryptophan, 0.02) in which 300 μl of cvHAS-cvUGD yeast was controlled at around pH 6.5 % L-histidine, 0.03% L-lysine monohydrochloride, 0.03% L-methionine, 0.03% L-leucine and 0.5 μg / ml Aureobasidin (no HA ingredients added) and inoculated overnight at 30 ° C did. After culturing, the 18-ml test tube was transferred to a shaking incubator that had been set to 10 ° C in advance, and shaking culture (induction) was further conducted at 10 ° C for 6 days to investigate whether or not HA fermentation production was possible. . The culture solution (3 mL) was centrifuged (1,3000 rpm, 10 minutes) and separated into cells and supernatant. The supernatant was heated at 95 ° C. for 5 minutes, centrifuged (1,3000 rpm, 10 minutes), and the supernatant was recovered as a crude purified solution. The crude purified solution was diluted 100 times with water and used as a measurement sample. Hyaluronic acid was quantified using a hyaluronic acid plate “Chugai” (Chugai Diagnostics Science Co., Ltd.). The result is shown in FIG.

INDUSTRIAL APPLICABILITY According to the present invention, hyaluronic acid is produced in an in vivo system using yeast, and a method for producing hyaluronic acid that is industrially advantageous in terms of safety and cost, transformed yeast, and expression for production of hyaluronic acid It is possible to provide a recombinant vector, which is expected to greatly contribute to the industry.

Hyaluronic acid production by cvHAS yeast and cvHAS-cvUGD yeast Hyaluronic acid production by cvHAS yeast and cvHAS-cvUGD yeast

Claims (51)

(1) a step of transforming yeast using a recombinant vector for expression containing a DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter capable of functioning in yeast, (2) step (1 ) Culturing the transformant obtained in (3), and (3) separating hyaluronic acid produced by the transformant. (1) The DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter that can function in yeast is the same as at least one DNA encoding a protein having exogenous sugar nucleotide synthase activity Or a step of transforming yeast using at least one recombinant expression vector contained in a separate vector, (2) a step of culturing the transformant obtained in step (1), (3) the transformation The manufacturing method of hyaluronic acid including the process of isolate | separating the hyaluronic acid produced by the body. The method for producing hyaluronic acid according to claim 1 or 2, wherein the promoter is an inducible promoter. The method for producing hyaluronic acid according to claim 3, wherein the inducible promoter is a promoter having a stress-inducible or cold-inducible function. The method for producing hyaluronic acid according to any one of claims 1 to 3, wherein the promoter is a promoter having a culture environment stress-inducing function of the HSP12 gene. The method for producing hyaluronic acid according to claim 1 or 2, wherein the DNA encoding a protein having hyaluronic acid synthase activity is the following DNA (a) or (b) or (c):
(A) DNA consisting of the base sequence represented by SEQ ID NO: 1 or 3.
(B) A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence of (a) and encodes a protein having hyaluronic acid synthase activity.
(C) A complementary strand of the DNA of (a) or (b) above. The method for producing hyaluronic acid according to claim 1 or 2, wherein the protein having hyaluronic acid synthase activity is the following protein (a) or (b):
(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 2 or 4.
(B) A protein having an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having hyaluronic acid synthase activity. The method for producing hyaluronic acid according to claim 2, wherein the sugar nucleotide is UDP-glucuronic acid and / or UDP-N-acetylglucosamine. Proteins with sugar nucleotide synthase activity are glutamine: fructose-6-phosphate amide transferase, UDP-N-acetylglucosamine diphosphorylase, phosphoacetylglucosamine mutase, glucosamine-6-phosphate N-acetyltransferase, glucosamine-1 phosphate N -Acetyltransferase, phosphoglucomutase, N-acetylglucosamine kinase, hexokinase, N-acetylglucosamine deacetylase, UDP-glucose dehydrogenase, UDP glucose-1-phosphate uridylyltransferase, inositol oxygenase, glucuronokinase and glucuronic acid-1 phosphorus The protein according to any one of claims 1 to 8, wherein the protein is one or more proteins selected from the group consisting of acid uridyltransferases. The method for producing hyaluronic acid. The method for producing hyaluronic acid according to any one of claims 1 to 8, wherein the protein having sugar nucleotide synthase activity is UDP-glucose dehydrogenase and / or glutamine: fructose-6-phosphate amide transferase. The method for producing hyaluronic acid according to any one of claims 2 to 10, wherein the DNA encoding a protein having sugar nucleotide synthase activity is DNA derived from chlorella virus and / or Arabidopsis thaliana. The DNA encoding a protein having sugar nucleotide synthase activity is the following DNA (a), (b) or (c): Production method.
(A) DNA consisting of the base sequence represented by SEQ ID NO: 5, 7, 9, 11, 13, or 15.
(B) DNA that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and encodes a protein having UDP-glucose dehydrogenase activity.
(C) A complementary strand of the DNA of (a) or (b) above. The method for producing hyaluronic acid according to any one of claims 2 to 11, wherein the protein having sugar nucleotide synthase activity is the following protein (a) or (b):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, 14, or 16;
(B) A protein comprising an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having UDP-glucose dehydrogenase activity. The DNA encoding a protein having sugar nucleotide synthase activity is the following DNA (a), (b) or (c): Production method.
(A) DNA consisting of the base sequence represented by SEQ ID NO: 17, 19, or 21.
(B) a DNA that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and encodes a protein having glutamine: fructose-6-phosphate amide transferase activity .
(C) A complementary strand of the DNA of (a) or (b) above. The method for producing hyaluronic acid according to any one of claims 2 to 11, wherein the protein having sugar nucleotide synthase activity is the following protein (a) or (b):
(A) A protein consisting of the amino acid sequence represented by SEQ ID NO: 18, 20, or 22.
(B) A protein having an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having glutamine: fructose-6-phosphate amide transferase activity. The method for producing hyaluronic acid according to any one of claims 1 to 15, wherein the protein is induced and produced at a temperature of the culture solution of 4 ° C to 20 ° C. The method for producing hyaluronic acid according to any one of claims 1 to 16, wherein the protein is induced to be produced by setting the NaCl concentration in the culture solution to 500 mM or more. A transformed yeast having the ability to produce hyaluronic acid transformed with a recombinant vector for expression containing a DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter capable of functioning in yeast. DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter capable of functioning in yeast and at least one DNA encoding a protein having exogenous sugar nucleotide synthase activity are the same or different A transformed yeast having the ability to produce hyaluronic acid transformed with at least one recombinant expression vector contained in the vector. The transformed yeast according to claim 18 or 19, wherein the promoter is an inducible promoter. 20. The transformed yeast according to claim 18 or 19, wherein the inducible promoter is a promoter having a stress-inducible or cold-inducible function. The transformed yeast according to any one of claims 18 to 20, wherein the promoter is a promoter having a culture environment stress-inducing function of the HSP12 gene. The transformed yeast according to claim 18 or 19, wherein the DNA encoding a protein having hyaluronic acid synthase activity is the following DNA (a) or (b) or (c):
(A) DNA consisting of the base sequence represented by SEQ ID NO: 1 or 3.
(B) A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence of (a) and encodes a protein having hyaluronic acid synthase activity.
(C) A complementary strand of the DNA of (a) or (b) above. The transformed yeast according to claim 18 or 19, wherein the protein having hyaluronic acid synthase activity is the following protein (a) or (b):
(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 2 or 4.
(B) A protein having an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having hyaluronic acid synthase activity. The transformed yeast according to claim 19, wherein the sugar nucleotide is UDP-glucuronic acid and / or UDP-N-acetylglucosamine. Proteins with sugar nucleotide synthase activity are glutamine: fructose-6-phosphate amide transferase, UDP-N-acetylglucosamine diphosphorylase, phosphoacetylglucosamine mutase, glucosamine-6-phosphate N-acetyltransferase, glucosamine-1 phosphate N -Acetyltransferase, phosphoglucomutase, N-acetylglucosamine kinase, hexokinase, N-acetylglucosamine deacetylase, UDP-glucose dehydrogenase, UDP-glucose-1-phosphate uridylyltransferase, inositol oxygenase, glucuronokinase and glucuronic acid-1 The transformation according to claim 19, which is one or more proteins selected from the group consisting of phosphate uridyltransferase. Mother. The transformed yeast according to claim 19, wherein the protein having sugar nucleotide synthase activity is UDP-glucose dehydrogenase. 28. The transformed yeast according to any one of claims 19 to 27, wherein the DNA encoding a protein having sugar nucleotide synthase activity is a DNA derived from chlorella virus and / or from Arabidopsis thaliana. 30. The transformed yeast according to any one of claims 21 to 29, wherein the DNA encoding a protein having sugar nucleotide synthase activity is the following DNA (a), (b) or (c): .
(A) DNA consisting of the base sequence represented by SEQ ID NO: 5, 7, 9, 11, 13, or 15
(B) DNA that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and encodes a protein having UDP-glucose dehydrogenase activity.
(C) A complementary strand of the DNA of (a) or (b) above. 29. The transformed yeast according to any one of claims 19 to 28, wherein the protein having sugar nucleotide synthase activity is the following protein (a) or (b):
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, 14, or 16;
(B) A protein comprising an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having UDP-glucose dehydrogenase activity. 29. The transformed yeast according to any one of claims 19 to 28, wherein the DNA encoding a protein having sugar nucleotide synthase activity is the following DNA (a) or (b) or (c): .
(A) DNA consisting of the base sequence represented by SEQ ID NO: 17, 19, or 21.
(B) a DNA that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and encodes a protein having glutamine: fructose-6-phosphate amide transferase activity .
(C) A complementary strand of the DNA of (a) or (b) above. 29. The transformed yeast according to any one of claims 19 to 28, wherein the protein having sugar nucleotide synthase activity is the following protein (a) or (b):
(A) A protein consisting of the amino acid sequence represented by SEQ ID NO: 18, 20, or 22.
(B) A protein having an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having glutamine: fructose-6-phosphate amide transferase activity. A hyaluronic acid-containing product obtained from the transformed yeast according to any one of claims 18 to 32. A recombinant vector for expression for production of hyaluronic acid, comprising DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter that can function in yeast. DNA encoding a protein having hyaluronic acid synthase activity under the control of a promoter capable of functioning in yeast and at least one DNA encoding a protein having sugar nucleotide synthase activity in the same or separate vectors A recombinant expression vector for production of hyaluronic acid or a combination thereof. 36. The recombinant expression vector for production of hyaluronic acid according to claim 34 or 35, wherein the promoter is an inducible promoter. 37. The recombinant expression vector for producing hyaluronic acid according to claim 36, wherein the inducible promoter is a promoter having a stress-inducible or cold-inducible function. The recombinant vector for expression for hyaluronic acid production according to claim 36 or 37, wherein the promoter is a promoter having a function of inducing culture environment stress of the HSP12 gene. The DNA encoding a protein having hyaluronic acid synthase activity is the following DNA (a) or (b) or (c): Recombinant vector for expression.
(A) DNA consisting of the base sequence represented by SEQ ID NO: 1 or 3.
(B) A DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence of (a) and encodes a protein having hyaluronic acid synthase activity.
(C) A complementary strand of the DNA of (a) or (b) above. 37. The recombinant expression vector for production of hyaluronic acid according to claim 35 or 36, wherein the protein having hyaluronic acid synthase activity is the following protein (a) or (b):
(A) A protein comprising the amino acid sequence represented by SEQ ID NO: 2 or 4.
(B) A protein having an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having hyaluronic acid synthase activity. The recombinant vector for expression for producing hyaluronic acid according to claim 36, wherein the sugar nucleotide is UDP-glucuronic acid and / or UDP-N-acetylglucosamine. Proteins with sugar nucleotide synthase activity are glutamine: fructose-6-phosphate amide transferase, UDP-N-acetylglucosamine diphosphorylase, phosphoacetylglucosamine mutase, glucosamine-6-phosphate N-acetyltransferase, glucosamine-1 phosphate N -Acetyltransferase, phosphoglucomutase, N-acetylglucosamine kinase, hexokinase, N-acetylglucosamine deacetylase, UDP-glucose dehydrogenase, UDP-glucose-1-phosphate uridylyltransferase, inositol oxygenase, glucuronokinase and glucuronic acid-1 36. One or more proteins selected from the group consisting of uridyl phosphate phosphotransferases. The recombinant expression vectors for Lelong acid production. 36. The recombinant expression vector for producing hyaluronic acid according to claim 35, wherein the protein having sugar nucleotide synthase activity is UDP-glucose dehydrogenase. 44. The recombinant vector for expression for producing hyaluronic acid according to any one of claims 35 to 43, wherein the DNA encoding a protein having sugar nucleotide synthase activity is DNA derived from chlorella virus and / or Arabidopsis thaliana. 45. The hyaluronic acid production according to any one of claims 35 to 44, wherein the DNA encoding a protein having sugar nucleotide synthase activity is the following DNA (a), (b) or (c): Recombinant vector for expression for.
(A) DNA consisting of the base sequence represented by SEQ ID NO: 5, 7, 9, 11, 13, or 15.
(B) DNA that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and encodes a protein having UDP-glucose dehydrogenase activity.
(C) A complementary strand of the DNA of (a) or (b) above. The recombinant vector for expression for hyaluronic acid production according to any one of claims 35 to 45, wherein the protein having sugar nucleotide synthase activity is the following protein (a) or (b): .
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 6, 8, 10, 12, 14, or 16;
(B) A protein comprising an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having UDP-glucose dehydrogenase activity. 46. The hyaluronic acid production according to any one of claims 35 to 45, wherein the DNA encoding a protein having sugar nucleotide synthase activity is the following DNA (a), (b) or (c): Recombinant vector for expression for.
(A) DNA consisting of the base sequence represented by SEQ ID NO: 17, 19, or 21.
(B) a DNA that hybridizes with a DNA comprising a base sequence complementary to the DNA comprising the base sequence of (a) under stringent conditions and encodes a protein having glutamine: fructose-6-phosphate amide transferase activity .
(C) A complementary strand of the DNA of (a) or (b) above. The recombinant vector for expression for hyaluronic acid production according to any one of claims 35 to 45, wherein the protein having sugar nucleotide synthase activity is the following protein (a) or (b): .
(A) A protein consisting of the amino acid sequence represented by SEQ ID NO: 18, 20, or 22.
(B) A protein having an amino acid sequence in which one or several or a plurality of amino acids are deleted, substituted or added in the amino acid sequence (a) and having glutamine: fructose-6-phosphate amide transferase activity. 49. A method for producing a transformed yeast having the ability to produce hyaluronic acid, comprising a step of transforming the yeast using the vector according to any one of claims 34 to 48. A cosmetic composition comprising, as an active ingredient, hyaluronic acid obtained by the method for producing hyaluronic acid according to any one of claims 1 to 17. A functional food comprising, as an active ingredient, hyaluronic acid obtained by the method for producing hyaluronic acid according to any one of claims 1 to 17.

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