JP2006246701A - Acetic acid bacterium having enhanced enzyme activity of central metabolic system and method for producing vinegar using the acetic acid bacterium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an acetic acid bacterium having improved growth ability in the presence of acetic acid by utilizing a protein having functions to improve acetic acid tolerance at a practical level, to provide a new method for breeding by which the growth ability of the acetic acid bacterium in the presence of the acetic acid can be improved and to further provide a means for producing vinegar or acetic acid at a high concentration by using the acetic acid bacterium. <P>SOLUTION: The acetic acid bacterium having the improved growth ability in the presence of the acetic acid is characterized in that the one or two or more enzyme activities selected from 13 species of central metabolic system enzyme groups are enhanced. A method for breeding the acetic acid bacterium and the method for producing the vinegar using the acetic acid bacterium are provided. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a microorganism overexpressing a gene encoding a central metabolic enzyme derived from a microorganism and a related enzyme protein, particularly an acetic acid bacterium belonging to the genus Gluconacetobacter and Acetobacter, and The present invention relates to a method for efficiently producing vinegar using these microorganisms.

Acetic acid bacteria are microorganisms widely used for vinegar production, and particularly acetic acid bacteria belonging to the genus Acetobacter and Glucon acetobacter are used for industrial acetic acid fermentation.
In acetic acid fermentation, ethanol in the medium is oxidized by acetic acid bacteria and converted to acetic acid. As a result, acetic acid accumulates in the medium, but acetic acid is also inhibitory to acetic acid bacteria. As the concentration of acetic acid increases and the concentration of acetic acid in the medium increases, the growth ability and fermentation ability of acetic acid bacteria gradually decrease.

  Therefore, in acetic acid fermentation, it is required to develop acetic acid bacteria that do not decrease the growth ability and fermentation ability even at higher acetic acid concentrations. As one means, a gene involved in acetic acid resistance (acetic acid resistance gene) is required. Attempts have been made to clone and breed and improve acetic acid bacteria using the acetic acid resistance gene.

  As for the knowledge about the acetic acid resistance gene of acetic acid bacteria so far, a cluster is formed as a complementary gene that can restore acetic acid sensitivity by mutating the acetic acid resistance of acetic acid bacteria of Acetobacter genus. Three genes (aarA, aarB, aarC) have been cloned (see, for example, Non-Patent Document 1).

  Of these, the aarA gene is a gene encoding citrate synthase, and the aarC gene was presumed to be an enzyme encoding an enzyme related to acetate utilization, but the function of the aarB gene is unknown. (For example, refer nonpatent literature 2).

  A transformed strain obtained by cloning a gene fragment containing these three acetate-resistant genes into a multicopy plasmid and transforming it into a Gluconacetobacter xylinus IFO3288 strain has an improved level of acetate resistance. Whether or not the actual performance of acetic acid fermentation is improved is not clear (see, for example, Patent Document 3).

  On the other hand, an example has been disclosed in which the final acetic acid concentration has been improved in acetic acid fermentation by introducing a gene encoding membrane-bound aldehyde dehydrogenase (ALDH) cloned from acetic acid bacteria into acetic acid bacteria. (For example, refer to Patent Document 2). However, since ALDH is an enzyme having a function of oxidizing acetaldehyde and not directly related to acetic acid resistance, it cannot be determined that the gene encoding ALDH is truly an acetic acid resistance gene.

In addition to the above, an aconitase gene (see Patent Document 3) is known as a gene that improves acetic acid resistance. Overexpression of this gene has been found to improve the acetic acid resistance of acetic acid bacteria, but has not succeeded in imparting sufficient acetic acid resistance.
From such a situation, it is possible to obtain a novel gene encoding a protein having a function capable of improving acetic acid resistance at a practical level, and to breed an acetic acid bacterium having a higher fermentative ability using the obtained gene. It was desired.

Japanese Patent Laid-Open No. 60-9488 JP-A-2-2364 JP 2003-289867 A “Journal of Bacteriology”, 172, p. 2096-2104, 1990 “Journal of Fermentation and Bioengineering”, vol. 76, p. 270-275, 1993

As described above, there have been no reports of successful development of practical acetic acid bacteria having high acetic acid resistance by elucidating acetic acid resistance at the gene level.
Therefore, the present invention utilizes a protein having a function capable of improving acetic acid resistance at a practical level, and has improved the ability to grow in the presence of acetic acid, and the ability of acetic acid bacteria to grow in the presence of acetic acid. By providing a new breeding method that can be improved, and by using an acetic acid bacterium that has been improved in the presence of acetic acid, vinegar can be produced efficiently and means that can produce acetic acid at a high concentration Is intended to provide.

In order to achieve the above object, the present inventor decided to examine means for improving acetic acid resistance of acetic acid bacteria from a viewpoint different from the conventional one. In the process, the present inventors paid attention to ATP production and a metabolic system related thereto, which have been hardly studied so far.
Since the production of ATP during acetic acid fermentation is based on ethanol oxidation, enzymes such as alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and glucose dehydrogenase (GDH) have been conventionally used as enzymes involved in acetic acid resistance. The pyrroloquinoline quinone (PQQ) -dependent dehydrogenase localized in the membrane and the redox system of PQQ are important, and little attention has been paid to the rest.
The inventor of the present invention pays attention to the fact that growth and fermentation are suppressed when the concentration of acetic acid is increased even when ethanol oxidation is sufficiently advanced as acetic acid fermentation proceeds, and an ATP generation system other than ethanol oxidation is important. I thought it was.

In acetic acid bacteria, ATP production is attributed to the reaction by the action of ATPase linked to the generation of a proton gradient generated by the redox reaction of the coenzyme by ubiquinol oxidase. In addition to PQQ, the supply of reducing power to ubiquinol oxidase is known to involve reactions involving NAD and FAD, but the importance of these has not been known.
Therefore, the present inventor paid attention to a central metabolic system such as a glycolysis system, a pentose phosphate pathway, an Entner-Dordorf pathway, and a TCA circuit as an enzyme involved in the reduction reaction of NAD in the metabolic pathway of acetic acid bacteria.

NADH produced by the central metabolic system such as glycolysis, pentose phosphate pathway, Entner-Dodorf pathway, and TCA cycle is regenerated to NAD, and NADH dehydrogenase that transmits electrons to ubiquinol oxidase, especially simultaneously with electron transfer We focused on the type I NADH dehydrogenase complex, which itself has the ability to generate a proton gradient.
Furthermore, as an enzyme involved in the reduction reaction of FAD in the central metabolic system, attention was also paid to dehydrogenases having FAD as a coenzyme, such as succinate dehydrogenase, lactate dehydrogenase, and malatequinone dehydrogenase.
Then, the enzyme activities related to the central metabolic system related to ATP production of acetic acid bacteria were measured, compared with those during acetic acid fermentation and those during non-fermentation, and changes in acetic acid fermentation were observed. It has been found for the first time that the activity decreases markedly during acetic acid fermentation, especially with the progress of acetic acid fermentation.

Based on these findings, it is concluded that in order to improve the acetic acid tolerance of acetic acid bacteria, that is, the growth in the presence of acetic acid and the ability to ferment acetic acid, it is important to enhance the activity of the enzyme that decreases during acetic acid fermentation. Reached.
And the novel gene which codes the protein which shows the activity of each enzyme in which the fall of activity was observed at the time of acetic acid fermentation was discovered by the cloning from acetic acid bacteria. Then, the gene was linked to a plasmid vector, transformed into acetic acid bacteria to amplify the copy number, and the growth in a medium containing acetic acid was compared with the original strain. As a result, it was found that in the transformed strain with the amplified copy number, the enzyme activity increased, and the growth in a medium containing a high concentration of acetic acid was improved accordingly. Furthermore, by culturing in a medium containing alcohol using the transformed strain, the acetic acid production rate and acetic acid concentration that can be accumulated increased, so that acetic acid resistance was improved, and vinegar had an efficient production capacity. In addition, it was found that a novel acetic acid bacterium capable of producing a high concentration of acetic acid could be bred.
The present invention is based on such knowledge.

That is, the present invention provides the following (1) to (17).
(1) NAD-dependent isocitrate dehydrogenase, succinyl CoA synthase, fumarate hydratase, malate quinone oxidoreductase, NAD-dependent malate dehydrogenase (decarboxylation), FAD-dependent lactate dehydrogenase, NADH Dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, enolase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, and phosphogluconate dehydratase An acetic acid bacterium having improved growth ability in the presence of acetic acid, wherein one or more enzyme activities selected from the group of central metabolic enzymes consisting of

(2) NAD-dependent isocitrate dehydrogenase, succinyl CoA synthase, fumarate hydratase, malate quinone oxidoreductase, NAD-dependent malate dehydrogenase (decarboxylation), FAD-dependent lactate dehydrogenase, NADH Dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, enolase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, and phosphogluconate dehydratase (1) The acetic acid according to (1), wherein the enzyme activity is enhanced by introducing into a cell and expressing a gene encoding a protein exhibiting one or more enzyme activities selected from the group of central metabolic enzymes consisting of A method for breeding acetic acid bacteria with improved viability in the presence.

(3) The method for breeding acetic acid bacteria according to (2), wherein the gene encoding the NAD-dependent isocitrate dehydrogenase is the DNA shown in the following (a) or (b):
(A) DNA consisting of base numbers 84 to 1112 among the base sequences set forth in SEQ ID NO: 1 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 1 in the sequence listing, DNA comprising a nucleotide sequence consisting of nucleotide numbers 84 to 1112 or DNA comprising a nucleotide sequence complementary to a part of the DNA under stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting NAD-dependent isocitrate dehydrogenase activity.

(4) The method for breeding acetic acid bacteria according to (2), wherein the gene encoding succinyl-CoA synthase is the DNA shown in the following (a) or (b):
(A) DNA consisting of base numbers 130 to 2218 among the base sequences set forth in SEQ ID NO: 3 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 3 in the sequence listing, DNA having a nucleotide sequence consisting of nucleotide numbers 130 to 2218 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions A DNA comprising a base sequence that hybridizes with a DNA encoding a protein exhibiting succinyl-CoA synthetase activity.

(5) Breeding of acetic acid bacteria according to (2), wherein the gene encoding fumarate hydratase is the DNA shown in the following (a), (b), (c) or (d) Method.
(A) DNA consisting of base numbers 23 to 1819 among the base sequences set forth in SEQ ID NO: 6 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 6 in the sequence listing, DNA having a nucleotide sequence consisting of nucleotide numbers 23 to 1819 or DNA having a nucleotide sequence complementary to a part of the DNA under stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting fumarate hydratase activity.
(C) DNA consisting of base numbers 85 to 1458 among the base sequences set forth in SEQ ID NO: 8 in the sequence listing.
(D) Among the nucleotide sequences set forth in SEQ ID NO: 8 in the sequence listing, DNA having a nucleotide sequence consisting of nucleotide numbers 85 to 1458 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting fumarate hydratase activity.

(6) The method for breeding acetic acid bacteria according to (2), wherein the gene encoding malate quinone oxidoreductase is the DNA shown in the following (a) or (b):
(A) DNA consisting of base numbers 182 to 1678 among the base sequences set forth in SEQ ID NO: 10 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 10 in the sequence listing, DNA comprising a nucleotide sequence consisting of nucleotide numbers 182-1678 or DNA comprising a nucleotide sequence complementary to a part of the DNA under stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting malate quinone oxidoreductase activity.

(7) Breeding of acetic acid bacteria according to (2), wherein the gene encoding NAD-dependent malate dehydrogenase (decarboxylation) is the DNA shown in (a) or (b) below Method.
(A) DNA consisting of base numbers 89 to 1891 among the base sequences set forth in SEQ ID NO: 12 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 12 of the Sequence Listing, DNA having a nucleotide sequence consisting of nucleotide numbers 89 to 1891 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions A DNA comprising a base sequence that hybridizes with a DNA encoding a protein exhibiting NAD-dependent malate dehydrogenase (decarboxylation) activity.

(8) The method for breeding acetic acid bacteria according to (2), wherein the gene encoding the FAD-dependent lactate dehydrogenase is the DNA shown in the following (a) or (b):
(A) DNA consisting of base numbers 46 to 1779 among the base sequences set forth in SEQ ID NO: 14 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 14 in the sequence listing, DNA having a nucleotide sequence consisting of nucleotide numbers 46 to 1779 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting FAD-dependent lactate dehydrogenase activity.

(9) The gene encoding NADH dehydrogenase is DNA encoding the subunit nuM of the NADH dehydrogenase complex shown in the following (a) or (b): Breeding method of acetic acid bacteria.
The method for breeding acetic acid bacteria according to (2), which is DNA.
(A) DNA consisting of base numbers 186 to 1727 among the base sequences set forth in SEQ ID NO: 16 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 16 in the sequence listing, it hybridizes under stringent conditions with DNA consisting of nucleotide numbers 186 to 1727 or with DNA complementary to a part of the DNA. DNA comprising a base sequence and encoding a protein exhibiting NADH dehydrogenase activity.

(10) The method for breeding acetic acid bacteria according to (2), wherein the gene encoding glyceraldehyde-3-phosphate dehydrogenase is the DNA shown in (a) or (b) below: .
(A) DNA consisting of base numbers 91 to 1113 among the base sequences set forth in SEQ ID NO: 18 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 18 in the sequence listing, DNA having a nucleotide sequence consisting of nucleotide numbers 91 to 1113 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions A DNA comprising a base sequence that hybridizes with a DNA encoding a protein exhibiting glyceraldehyde-3-phosphate dehydrogenase activity.

(11) The method for breeding acetic acid bacteria according to (2), wherein the gene encoding enolase is the DNA shown in the following (a) or (b):
(A) DNA consisting of base numbers 68 to 1393 among the base sequences set forth in SEQ ID NO: 20 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 20 in the sequence listing, DNA having a nucleotide sequence consisting of nucleotide numbers 68 to 1393 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting enolase activity.

(12) The method for breeding acetic acid bacteria according to (2), wherein the gene encoding glucose-6-phosphate dehydrogenase is the DNA shown in the following (a) or (b):
(A) DNA consisting of base numbers 71 to 1600 in the base sequence set forth in SEQ ID NO: 22 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 22 in the sequence listing, DNA having a nucleotide sequence consisting of nucleotide numbers 71 to 1600 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting glucose-6-phosphate dehydrogenase activity.

(13) The method for breeding acetic acid bacteria according to (2), wherein the gene encoding 6-phosphogluconolactonase is the DNA shown in the following (a) or (b):
(A) DNA consisting of base numbers 61 to 873 among the base sequences set forth in SEQ ID NO: 24 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 24 in the sequence listing, DNA having a nucleotide sequence consisting of nucleotide numbers 61 to 873 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions A DNA comprising a base sequence that hybridizes with a DNA encoding a protein exhibiting 6-phosphogluconolactonase activity.

(14) The method for breeding acetic acid bacteria according to (2), wherein the gene encoding 6-phosphogluconate dehydrogenase is the DNA shown in the following (a) or (b):
(A) DNA consisting of base numbers 72 to 1070 among the base sequences set forth in SEQ ID NO: 26 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 26 of the Sequence Listing, DNA having a nucleotide sequence consisting of nucleotide numbers 72 to 1070 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting phosphogluconate dehydrogenase activity.

(15) The method for breeding acetic acid bacteria according to (2), wherein the gene encoding phosphogluconate dehydratase is the DNA shown in the following (a) or (b):
(A) DNA consisting of base numbers 78 to 1916 among the base sequences set forth in SEQ ID NO: 28 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 28 of the Sequence Listing, DNA having a nucleotide sequence consisting of nucleotide numbers 78 to 1916 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting phosphogluconate dehydratase activity.

(16) The acetic acid bacterium having improved growth ability in the presence of acetic acid according to (1), wherein the acetic acid bacterium belongs to the genus Glucon acetobacter or genus Acetobacter.
(17) A method for producing vinegar, characterized in that the acetic acid bacteria having improved growth ability in the presence of acetic acid according to (1) are cultured in a medium containing alcohol and acetic acid is produced and accumulated in the medium. .

ADVANTAGE OF THE INVENTION According to this invention, the growth ability in the presence of acetic acid can be improved with respect to acetic acid bacteria. And the tolerance with respect to an acetic acid improves notably. As a result, it is possible to improve the rate of acetic acid fermentation in the medium and produce vinegar efficiently with a high concentration of acetic acid.
ADVANTAGE OF THE INVENTION According to this invention, the acetic acid bacterium in which the growth ability in the presence of acetic acid was remarkably improved is provided, and a method for efficiently breeding the acetic acid bacterium is provided. The acetic acid bacteria having improved growth in the presence of acetic acid can be used for the efficient production of vinegar and the production of acetic acid at a high concentration, and is useful.

Hereinafter, the present invention will be described in detail.
(1) Acetic acid bacterium having improved growth ability in the presence of acetic acid The acetic acid bacterium having improved growth ability in the presence of acetic acid according to the present invention has enhanced one or more enzyme activities selected from the group of central metabolic enzymes. It is characterized by.
In the present invention, a central metabolic enzyme group means a glycolytic system, a pentose phosphate pathway, an Entner-Dordorf pathway, a TCA cycle, and the like. Fumarate hydratase, malate quinone oxidoreductase, NAD-dependent malate dehydrogenase (decarboxylation), FAD-dependent lactate dehydrogenase, NADH dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, enolase , Glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, and phosphogluconate dehydratase.
Here, “enhanced enzyme activity” means that one or more enzyme activities selected from the group of central metabolic enzymes exceeds the activity of the same enzyme of the acetic acid bacteria of the parent strain (former strain). . Means more than 2 times. Specific means for enhancing the enzyme activity will be described in detail in (2) below.

The enzyme activity of these central metabolic enzymes in acetic acid bacteria can be measured, for example, by the following method.
First, acetic acid bacteria (former strain) is added to YPG medium (glucose 3%, yeast extract 0.5%, polypeptone 0.2%, pH 6.5) or YPGE medium (glucose 3%, yeast extract 0.5%, polypeptone). 0.2%, ethanol 2%, pH 6.5, ethanol concentration is controlled to 2% during culture). The crude enzyme solution prepared using the cells in the late logarithmic growth phase or the cells in the stationary phase is used as the measurement target.
Here, examples of acetic acid bacteria include acetic acid bacteria belonging to the genus Glucon acetobacter or genus Acetobacter, and specific examples are as described later in (2). Among them, it is preferable to use Glucon Acetobacter xylinus IFO3288 strain.

The crude enzyme solution was collected by centrifuging the culture solution, suspended in 50 mM potassium phosphate buffer (pH 7.5) and washed, and then 2% with a French press (20,000 psi). After the treatment, the supernatant prepared by removing undisrupted cells by centrifugation can be used. However, for malate quinone oxidoreductase and NADH dehydrogenase, the precipitate fraction obtained by ultracentrifugation (70,000 rpm, 1 hour) of the cell lysate is used as a crude enzyme solution.
The enzyme activity can be measured for each enzyme, for example, under the following conditions.

  NAD-dependent isocitrate dehydrogenase refers to an enzyme classified as EC: 1.1.1.41, and the enzyme activity is “Methods in Enza, except that NAD is used instead of NADP. It can be measured according to the method described in “Imology (Methods in Enzymology) Vol. 13, p. 30-p. 33, 1969”.

  Succinyl-CoA synthase refers to an enzyme classified as EC: 6.2.1.5, and its enzyme activity is “GTP is changed to ATP, except that the NADH solution concentration is changed to 1.6 mM”. It can be measured according to the method described in “Methods in Enzymology”, Volume 13, p.62-p.69, 1969 ”.

  Fumarate hydratase refers to an enzyme classified as EC: 4.2.1.2, whose enzymatic activity is “Methods in Enzymology, Vol. 13, p. 91-p. .99, 1969 ".

  Malate quinone oxidoreductase (malate quinone dehydrogenase) refers to an enzyme classified as EC: 1.1.9.16, whose enzyme activity is described in “European Journal of Biochemistry (Eur.J. Biochem.) 254, p.395-p.403, 1998 ”.

  NAD-dependent malate dehydrogenase (decarboxylation) refers to an enzyme classified as EC: 1.1.1.38, whose enzyme activity is described in “Biochemical Journal 97, p. .284-p.297, 1965 ".

  FAD-dependent lactate dehydrogenase refers to an enzyme classified as EC: 1.1.1.28, whose enzyme activity is “Biosci. Biotechnol. Biochem.” 68 Volume, p.516-p.522, 2004 ”can be measured.

  NADH dehydrogenase refers to an enzyme classified as EC: 1.6.5.3, and includes subunits nuroM and the like as NADH dehydrogenase complexes. The enzyme activity can be measured according to the method described in “Biochemistry Vol. 26, p. 7732-p. 7737, 1987”.

  Glyceraldehyde-3-phosphate dehydrogenase refers to an enzyme classified as EC: 1.2.1.12. The enzyme activity is “Methods in Enzymology 41”. Volume, p.264-p.273, 1975 "can be measured.

  Enolase refers to an enzyme classified as EC: 4.2.1.11, whose enzyme activity is “Methods in Enzymology, Vol. 9, p. 670-p. It can be measured according to the method described in “1966”.

  Glucose-6-phosphate dehydrogenase refers to an enzyme classified as EC: 1.1.1.149, whose enzyme activity is described in “Bioscience, Biotechnology and Biochemistry and Biochemistry”. Biochemistry) ", Vol. 67, No. 12, p. 2648-2651, 2003 ".

  6-phosphogluconolactonase refers to an enzyme classified as EC: 3.1.1.31, and its enzymatic activity is determined according to “Procedures Natural Academy Science USA (Proc. Natl. Acad. Sci. USA) ", Volume 82, No. 11, p. 3876-3878, 1985 ".

  6-phosphogluconate dehydrogenase refers to an enzyme classified as EC: 1.1.1.14, whose enzyme activity is described in “Bioscience, Biotechnology and Biochemistry”. Biochemistry) ", Vol. 67, No. 12, p. 2648-2651, 2003 ".

  Phosphogluconate dehydratase refers to an enzyme that has been classified as EC: 4.2.1.12, whose enzymatic activity is referred to as the activity of the entire Entner-Dordorf pathway as described in “Journal of Biotechnology, 105, p.11-31, 2003 ".

The acetic acid bacterium of the present invention has enhanced viability in the presence of acetic acid by enhancing one or more enzyme activities selected from the group of central metabolic enzymes as described above. That is, in the case of the original strain, growth is inhibited and growth is difficult to grow in the presence of acetic acid, but the acetic acid bacterium of the present invention can continue to grow without being inhibited even in the presence of acetic acid.
Here, with regard to the improvement of the growth ability in the presence of acetic acid, for example, the acetic acid bacterium of the original strain and the acetic acid bacterium with enhanced enzyme activity are combined with acetic acid, ethanol, and antibiotics corresponding to resistance genes ( Culturing is performed at 25 to 35 ° C. in a YPG medium containing ampicillin and the like, and growth (growth degree) in the medium can be confirmed by measuring and comparing the absorbance at 660 nm. Growth improvement in the presence of acetic acid means that the absorbance becomes 0.1 or more in a shorter time than the original strain.

Examples of such acetic acid bacteria of the present invention include the following 14 types of acetic acid bacteria. The enzyme activity of NAD-dependent isocitrate dehydrogenase is enhanced.
These acetic acid bacteria of the present invention were obtained from the National Institute of Advanced Industrial Science and Technology patent biological deposit center (February 1st, 1st, 1st, 1st, 6th, 1st, Tsukuba, Ibaraki, Japan) on February 2, 2005 or February 22, 2005. The deposit numbers are: Glucon Acetobacter xylinus IFO3288 / pICD for FERM ABP-10254, Glucon Acetobacter xylinus IFO3288 / pSUC for FERM ABP-10257, Glucon Acetobacter xylinus IFO3288 / ERFUP -10251, Glucon Acetobacter xylinus IFO3288 / pFUMC is FERM ABP-10252, Glucon Acetobacter xylinus IFO3288 / pMQO is FERM ABP-10219, Glucon acetobac -Xylinus IFO3288 / pMAL is FERM ABP-10218, Glucon Acetobacter xylinus IFO3288 / pLDH is FERM ABP-10217, Glucon Acetobacter xylinus IFO3288 / pNDH is FERM ABP-10255, Glucon Acetobacter LD / 88F ABP-10253, Glucon Acetobacter xylinus IFO3288 / pENO is FERM ABP-10221, Glucon Acetobacter xylinus IFO3288 / pG6P is FERM ABP-10215, Glucon Acetobacter xylinus IFO3288 / pPGL is FER102 AB Concon Xylinus IFO3288 / pPGD FERM ABP-10221, Gluconacetobacter-Kishirinusu IFO3288 / pPGH is FERM ABP-10256.

(2) Breeding method of the present invention The breeding method of the present invention is a method for breeding acetic acid bacteria having improved growth ability in the presence of acetic acid described in (1) above, and the specific center mentioned in (1) above. The enzyme activity is enhanced by introducing into a cell and expressing a gene encoding a protein showing one or more enzyme activities selected from the group of metabolic enzymes.
Specifically, the gene encoding a protein having one or more enzyme activities selected from the group of central metabolic enzymes means the 13 types of genes shown in the following (A) to (M).

(A) Gene encoding NAD-dependent isocitrate dehydrogenase As a gene encoding NAD-dependent isocitrate dehydrogenase, acetic acid can be used as long as it encodes a protein showing NAD-dependent isocitrate dehydrogenase activity. A gene derived from a fungus or Shewanella oneidensis can be used. As for the S01538 gene of Shewanella oneidensis, the nucleotide sequence of the gene and the amino acid sequence of the encoded protein are disclosed in the NCBI database as GI number 24347300.
Among these, in particular, a gene encoding an NAD-dependent isocitrate dehydrogenase derived from acetic acid bacteria (Acetobacter altoacetigenes MH-24), that is, a protein having an amino acid sequence described in SEQ ID NO: 2 in the sequence listing. The DNA to be encoded, specifically, the DNA consisting of base numbers 84 to 1112 among the base sequences set forth in SEQ ID NO: 1 in the sequence listing (a) is preferable.

Moreover, unless the original NAD-dependent isocitrate dehydrogenase activity of the protein having the amino acid sequence described in SEQ ID NO: 2 in the sequence listing is impaired, one or several amino acids are substituted or deleted at one or more positions. Or a DNA encoding the added protein, specifically, (b) a DNA consisting of a base sequence consisting of base numbers 84 to 1112 or a part of the DNA among the base sequences set forth in SEQ ID NO: 1 of the sequence listing A DNA comprising a base sequence that hybridizes under stringent conditions with a DNA consisting of a complementary base sequence and a DNA encoding a protein exhibiting NAD-dependent isocitrate dehydrogenase activity good.
The stringent condition referred to here is a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Although it is difficult to quantify these conditions clearly, for example, nucleic acids with high homology, for example, DNAs having homology of 70% or more hybridize and nucleic acids with lower homology than that. Examples include conditions that do not hybridize with each other, or normal hybridization washing conditions, such as conditions in which washing is performed at 60 ° C. with a salt concentration corresponding to 0.1% SDS with 1 × SSC.

  The amino acid sequence described in SEQ ID NO: 2 in the sequence listing shows 52% homology with the protein encoded by the S01538 gene in terms of amino acid sequence.

(B) Gene encoding succinyl-CoA synthetase As a gene encoding succinyl-CoA synthetase, genes derived from acetic acid bacteria or Escherichia coli can be used as long as they encode proteins showing succinyl-CoA synthetase activity. . The nucleotide sequence of the sucC gene and the sucD gene constituting the sucCsucD operon of Escherichia coli and the amino acid sequence of the protein encoded by the gene are published in the NCBI database. And the sucD gene is GI number 16128704.
Among these, in particular, a gene encoding a succinyl CoA synthase derived from an acetic acid bacterium (Acetobacter altoacetylenes MH-24), that is, a protein having an amino acid sequence described in SEQ ID NO: 4 in the sequence listing and a protein described in SEQ ID NO: 5 A DNA encoding a protein having an amino acid sequence, specifically, a DNA consisting of base numbers 130 to 2218 among the base sequences described in SEQ ID NO: 3 in the sequence listing (a) is preferable. Moreover, as long as the original succinyl CoA synthase activity of the protein having the amino acid sequence shown in SEQ ID NO: 4 and the amino acid sequence shown in SEQ ID NO: 6 is not impaired, one or several amino acids are present at one or more positions. DNA encoding a substituted, deleted or added protein, specifically, (b) DNA consisting of a base sequence consisting of base numbers 130 to 2218 among the base sequences set forth in SEQ ID NO: 3 of the sequence listing, A DNA comprising a base sequence that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to a part of the DNA, and a DNA encoding a protein exhibiting succinyl-CoA synthase activity good. The stringent conditions are the same as described in (A).

The DNA shown in the above (a) or (b) forms an operon of the SucC gene and the SucD gene, and the SucC portion corresponds to the base number 130 to 1341 portion of the base sequence described in SEQ ID NO: 3, The portion corresponds to the base numbers 1346 to 2218 in the base sequence.
The amino acid sequence described in SEQ ID NO: 4 in the sequence listing shows 53% homology with the protein encoded by the sucC gene of the sucCsucD operon.
The amino acid sequence described in SEQ ID NO: 5 in the sequence listing shows 64% homology in amino acid sequence with the protein encoded by the sucD gene of the operon.

(C) Gene encoding fumarate hydratase The gene encoding fumarate hydratase is a gene encoding a protein exhibiting fumarate hydratase activity. Can be used. The base sequence of the E. coli fumA gene and the amino acid sequence of the protein encoded by the gene have been published as GI number 16129570 in the NCBI database. The base sequence of the E. coli fumC gene and the amino acid of the protein encoded by the gene The sequence is published as GI number 16129959 in the NCBI database.
Among these, in particular, a DNA encoding a fumarate hydratase derived from acetic acid bacteria (Acetobacter altoacetigenes MH-24), that is, a DNA encoding a protein having an amino acid sequence described in SEQ ID NO: 7 in the sequence listing, Specifically, DNA consisting of base numbers 23 to 1819 is preferable among the base sequences set forth in SEQ ID NO: 6 in the sequence listing (a). In addition, one or several amino acids were substituted, deleted or added at one or a plurality of positions as long as the original fumarate hydratase activity of the protein having the amino acid sequence described in SEQ ID NO: 7 in the sequence listing was not impaired. DNA encoding a protein, specifically, (b) of the nucleotide sequence set forth in SEQ ID NO: 6 in the sequence listing, complementary to DNA consisting of the nucleotide sequence consisting of nucleotide numbers 23 to 1819 or a part of the DNA It may be a DNA comprising a base sequence that hybridizes with a DNA comprising a base sequence under stringent conditions and a DNA encoding a protein exhibiting fumarate hydratase activity. The stringent conditions are the same as described in (A).

  Similarly, a DNA encoding a protein having the amino acid sequence set forth in SEQ ID NO: 9, specifically, (c) a DNA consisting of base numbers 85 to 1458 among the base sequences set forth in SEQ ID NO: 8 in the sequence listing is preferable. . In addition, one or several amino acids were substituted, deleted or added at one or a plurality of positions as long as the original fumarate hydratase activity of the protein having the amino acid sequence described in SEQ ID NO: 9 in the sequence listing was not impaired. DNA encoding a protein, specifically, (d) of the base sequence set forth in SEQ ID NO: 8 in the sequence listing, is complementary to DNA consisting of a base sequence consisting of base numbers 85 to 1458 or a part of the DNA It may be a DNA containing a base sequence that hybridizes with a DNA comprising a base sequence under stringent conditions and a DNA encoding a protein exhibiting fumarate hydratase activity. The stringent conditions are the same as described in (A).

  The amino acid sequence described in SEQ ID NO: 7 in the sequence listing shows 63% homology with the protein encoded by the fumA gene. Further, the amino acid sequence described in SEQ ID NO: 9 in the sequence listing shows 55% homology with the protein encoded by the acetic acid bacteria-derived fumC gene.

(D) Gene encoding malate quinone oxidoreductase As a gene encoding malate quinone oxidoreductase, genes derived from acetic acid bacteria and Escherichia coli are used as long as they encode a protein exhibiting malate quinone oxidoreductase activity. be able to. The base sequence of the yojH gene of Escherichia coli and the amino acid sequence of the protein encoded by the gene are disclosed as GI number 16130147 in the NCBI database.
Among these, in particular, a gene encoding a malate quinone oxidoreductase derived from an acetic acid bacterium (Acetobacter altoacetylenes MH-24 strain), that is, a DNA encoding a protein having an amino acid sequence described in SEQ ID NO: 11 in the Sequence Listing, Specifically, DNA consisting of base numbers 182-1678 is preferred among the base sequences set forth in SEQ ID NO: 10 in the sequence listing (a).

  In addition, one or several amino acids are substituted, deleted or added at one or a plurality of positions as long as the original malate quinone oxidoreductase activity of the protein having the amino acid sequence described in SEQ ID NO: 11 in the sequence listing is not impaired. DNA encoding a protein, specifically, (b) of the base sequence set forth in SEQ ID NO: 10 of the sequence listing, complementary to DNA consisting of the base sequence consisting of base numbers 182-1678 or a part of the DNA It may be a DNA containing a base sequence that hybridizes with a DNA comprising a simple base sequence under stringent conditions, and a DNA encoding a protein exhibiting malate quinone oxidoreductase activity. The stringent conditions are the same as described in (A).

  The amino acid sequence set forth in SEQ ID NO: 11 in the Sequence Listing shows 59% homology with the protein encoding the yojH gene and the amino acid sequence.

(E) Gene encoding NAD-dependent malate dehydrogenase (decarboxylation) As a gene encoding NAD-dependent malate dehydrogenase (decarboxylation), NAD-dependent malate dehydrogenase (decarboxylation) A gene derived from acetic acid bacteria or Escherichia coli can be used as long as it is a gene encoding a protein exhibiting activity. The nucleotide sequence of the sfcA gene of Escherichia coli and the amino acid sequence of the protein encoded by the gene are disclosed as GI number 16129438 in the NCBI database.
Among these, in particular, a gene encoding an NAD-dependent malate dehydrogenase (decarboxylation) derived from acetic acid bacteria (Acetobacter altoacetylenes MH-24 strain), that is, the amino acid sequence described in SEQ ID NO: 13 in the sequence listing A DNA encoding a protein having a specific sequence, specifically, a DNA consisting of base numbers 89 to 1891 among (a) the base sequence set forth in SEQ ID NO: 12 in the sequence listing is preferable.

  In addition, as long as the original NAD-dependent malate dehydrogenase (decarboxylation) activity of the protein having the amino acid sequence shown in SEQ ID NO: 13 in the sequence listing is not impaired, one or several amino acids are present at one or more positions. DNA encoding a substituted, deleted or added protein, specifically, (b) DNA consisting of a base sequence consisting of base numbers 89 to 1891 among base sequences set forth in SEQ ID NO: 12 of the sequence listing, A DNA comprising a base sequence that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to a part of the DNA, and that exhibits NAD-dependent malate dehydrogenase (decarboxylation) activity It may be DNA encoding The stringent conditions are the same as described in (A).

  The amino acid sequence described in SEQ ID NO: 13 in the sequence listing shows 44% homology with the protein encoding the sfcA gene in amino acid sequence and is commonly conserved in NAD-dependent malate dehydrogenase (decarboxylation). The motif Pfam00390 also has a high homology of score 728.2.

(F) Gene encoding FAD-dependent lactate dehydrogenase As a gene encoding FAD-dependent lactate dehydrogenase, any gene that encodes a protein exhibiting FAD-dependent lactate dehydrogenase activity may be acetic acid bacteria or A gene derived from E. coli can be used. The base sequence of the dld gene of E. coli and the amino acid sequence of the protein encoded by the gene are disclosed as GI number 16130071 in the NCBI database.
Among these, in particular, it encodes a gene encoding an FAD-dependent lactate dehydrogenase derived from acetic acid bacteria (Acetobacter altoacetigenes MH-24), that is, a protein having the amino acid sequence described in SEQ ID NO: 15 in the sequence listing. DNA, specifically, (a) Of the base sequences set forth in SEQ ID NO: 14 in the sequence listing, DNA consisting of base numbers 46 to 1779 is preferred.

  In addition, as long as the original FAD-dependent lactate dehydrogenase activity of the protein having the amino acid sequence set forth in SEQ ID NO: 15 in the sequence listing is not impaired, 1 or several amino acids are substituted, deleted, or deleted at one or a plurality of positions. DNA encoding the added protein, specifically, among (b) the base sequence set forth in SEQ ID NO: 14 of the sequence listing, DNA consisting of the base sequence consisting of base numbers 46 to 1779, or a part of the DNA It may be a DNA containing a base sequence that hybridizes with a DNA comprising a complementary base sequence under stringent conditions, and a DNA encoding a protein exhibiting FAD-dependent lactate dehydrogenase activity. The stringent conditions are the same as described in (A).

  The amino acid sequence set forth in SEQ ID NO: 15 in the sequence listing shows 58% homology in amino acid sequence with the protein encoding the dld gene.

(G) Gene encoding NADH dehydrogenase The gene encoding NADH dehydrogenase is not particularly limited as long as it is a gene encoding a protein exhibiting NADH dehydrogenase activity. Can be used. In addition, all genes encoding NADH dehydrogenase complex may be used, but one or more genes of subunits can also be used. The nucleotide sequence of the nuM gene in the nuo operon of Escherichia coli and the amino acid sequence of the protein encoded by the gene are disclosed as GI number 16130212 in the NCBI database.
Among them, in particular, it has a gene (nuoM gene) encoding the subunit nuM of the NADH dehydrogenase complex derived from acetic acid bacteria (Gluconacetobacter xylinus IFO3288 strain), that is, the amino acid sequence described in SEQ ID NO: 17 in the Sequence Listing. A DNA encoding a protein, specifically, a DNA comprising base numbers 186 to 1727 among the base sequences set forth in SEQ ID NO: 16 in the (a) Sequence Listing is preferable.

  In addition, one or several amino acids were substituted, deleted or added at one or a plurality of positions as long as the original NADH dehydrogenase activity of the protein having the amino acid sequence described in SEQ ID NO: 17 in the sequence listing was not impaired. DNA encoding a protein, specifically, (b) out of the base sequence set forth in SEQ ID NO: 16 of the sequence listing, consisting of DNA consisting of base numbers 186 to 1727 or a base sequence complementary to part of the DNA It may be DNA containing a base sequence that hybridizes with DNA under stringent conditions, and may encode a protein that exhibits NADH dehydrogenase activity. The stringent conditions are the same as described in (A).

  The amino acid sequence described in SEQ ID NO: 17 in the sequence listing shows 36% homology with the protein encoding the nuM gene in amino acid sequence.

(H) Gene encoding glyceraldehyde-3-phosphate dehydrogenase The gene encoding glyceraldehyde-3-phosphate dehydrogenase exhibits glyceraldehyde-3-phosphate dehydrogenase activity. As long as it is a gene encoding a protein, a gene derived from acetic acid bacteria or Escherichia coli can be used. The base sequence of the E. coli gapA gene and the amino acid sequence of the protein encoded by the gene are disclosed as GI number 16129733 in the NCBI database.
Among these, in particular, it has a gene encoding glyceraldehyde-3-phosphate dehydrogenase derived from acetic acid bacteria (Acetobacter altoacetylenes MH-24 strain), that is, the amino acid sequence described in SEQ ID NO: 19 in the Sequence Listing. A DNA encoding a protein, specifically, a DNA consisting of base numbers 91 to 1113 among the base sequences described in SEQ ID NO: 18 in the sequence table (a) is preferable.

  Further, as long as the original glyceraldehyde-3-phosphate dehydrogenase activity of the protein having the amino acid sequence described in SEQ ID NO: 19 in the sequence listing is not impaired, one or several amino acids are substituted at one or a plurality of positions. DNA encoding a deleted or added protein, specifically, (b) DNA consisting of a base sequence consisting of base numbers 91 to 1113 among the base sequences set forth in SEQ ID NO: 18 of the sequence listing or the DNA A DNA comprising a base sequence that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to a part of the protein, and encoding a protein exhibiting glyceraldehyde-3-phosphate dehydrogenase activity It may also be DNA. The stringent conditions are the same as described in (A).

  The amino acid sequence set forth in SEQ ID NO: 19 in the sequence listing shows 48% homology with the protein encoding the gapA gene in amino acid sequence and is commonly conserved in glyceraldehyde-3-phosphate dehydrogenase. The motif Pfam00044 also has a high homology of score630.2.

(I) Gene Encoding Enolase As a gene encoding enolase, a gene derived from acetic acid bacteria or Escherichia coli can be used as long as it encodes a protein exhibiting enolase activity. The nucleotide sequence of the E. coli eno gene and the amino acid sequence of the encoded protein are disclosed as GI number 16130686 in the NCBI database.
Among these, in particular, a DNA encoding an enolase derived from an acetic acid bacterium (Acetobacter altoacetigenes MH-24 strain), that is, a DNA encoding a protein having the amino acid sequence described in SEQ ID NO: 21 in the Sequence Listing, specifically (A) Of the base sequences set forth in SEQ ID NO: 20 in the sequence listing, DNA consisting of base numbers 68 to 1393 is preferred.

  In addition, it encodes a protein in which one or several amino acids are substituted, deleted or added at one or a plurality of positions as long as the original enolase activity of the protein having the amino acid sequence described in SEQ ID NO: 2 in the sequence listing is not impaired Specifically, from (b) DNA consisting of a base sequence consisting of base numbers 68 to 1393 among base sequences set forth in SEQ ID NO: 20 of the sequence listing or a base sequence complementary to a part of the DNA DNA that includes a base sequence that hybridizes with a DNA under stringent conditions, and that encodes a protein exhibiting enolase activity. The stringent conditions are the same as described in (A).

  The amino acid sequence set forth in SEQ ID NO: 21 in the sequence listing shows 52% homology in amino acid sequence with the protein encoding the eno gene, and also has a high homology of score 678.2 with the motif Pfam00113 commonly conserved in enolase have.

(J) Gene encoding glucose-6-phosphate dehydrogenase The gene encoding glucose-6-phosphate dehydrogenase is a gene encoding a protein exhibiting glucose-6-phosphate dehydrogenase activity. If there are, genes derived from acetic acid bacteria or E. coli can be used. The base sequence of the zwf gene of Escherichia coli and the amino acid sequence of the protein encoded by the gene are published as GI number 16129805 in the NCBI database.
Among these, in particular, a gene encoding glucose-6-phosphate dehydrogenase derived from acetic acid bacteria (Acetobacter altoacetigenes MH-24), that is, a protein having an amino acid sequence described in SEQ ID NO: 23 in the sequence listing The DNA to be encoded, specifically, the DNA consisting of base numbers 71 to 1600 in the base sequence set forth in SEQ ID NO: 22 in the sequence table (a) is preferable.

  Further, unless the original glucose-6-phosphate dehydrogenase activity of the protein having the amino acid sequence set forth in SEQ ID NO: 23 in the sequence listing is impaired, one or several amino acids are substituted or missing at one or more positions. DNA encoding the lost or added protein, specifically, (b) DNA consisting of a base sequence consisting of base numbers 71 to 1600 of the base sequence set forth in SEQ ID NO: 22 of the sequence listing or one of the DNAs A DNA comprising a base sequence that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the DNA and encoding a protein exhibiting glucose-6-phosphate dehydrogenase activity. May be. The stringent conditions are the same as described in (A).

  The amino acid sequence set forth in SEQ ID NO: 23 in the sequence listing shows 39% homology with the protein encoding the zwf gene in amino acid sequence.

(K) Gene encoding 6-phosphogluconolactonase As a gene encoding 6-phosphogluconolactonase, if it is a gene encoding a protein showing 6-phosphogluconolactonase activity, acetic acid bacteria or A gene derived from Mesohizobium loti can be used. The base sequence of the Mesorhizobium loti ml16514 gene and the amino acid sequence of the protein encoded by the gene are disclosed in the NCBI database as GI number 13475444.
Among these, in particular, it encodes a gene encoding 6-phosphogluconolactonase derived from acetic acid bacteria (Acetobacter altoacetigenes MH-24), that is, a protein having the amino acid sequence described in SEQ ID NO: 25 in the Sequence Listing. DNA, specifically, (a) Of the base sequence set forth in SEQ ID NO: 24 in the sequence listing, DNA consisting of base numbers 61 to 873 is preferred.

  Moreover, as long as the original 6-phosphogluconolactonase activity of the protein having the amino acid sequence set forth in SEQ ID NO: 25 in the sequence listing is not impaired, one or several amino acids are substituted, deleted or deleted at one or a plurality of positions. DNA encoding the added protein, specifically, among (b) the base sequence set forth in SEQ ID NO: 24 of the sequence listing, DNA consisting of the base sequence consisting of base numbers 61 to 873, or a part of the DNA It may be a DNA containing a base sequence that hybridizes under stringent conditions with a DNA comprising a complementary base sequence, and may be a DNA encoding a protein exhibiting 6-phosphogluconolactonase activity. The stringent conditions are the same as described in (A).

  The amino acid sequence set forth in SEQ ID NO: 25 in the sequence listing shows 32% homology with the protein encoding the ml16514 gene in amino acid sequence.

(L) Gene encoding 6-phosphogluconate dehydrogenase The gene encoding 6-phosphogluconate dehydrogenase is acetic acid as long as it is a gene encoding a protein having 6-phosphogluconate dehydrogenase. A gene derived from a fungus or E. coli can be used. The base sequence of the gnd gene of Escherichia coli and the amino acid sequence of the protein encoded by the gene are disclosed in the NCBI database as GI number 16129970.
Among these, in particular, a gene encoding 6-phosphogluconate dehydrogenase derived from acetic acid bacteria (Acetobacter altoacetylenes MH-24 strain), that is, a protein having the amino acid sequence described in SEQ ID NO: 27 in the sequence listing is encoded. More specifically, (a) Of the base sequences set forth in SEQ ID NO: 26 in the sequence listing, DNA consisting of base numbers 72 to 1070 is preferable.

  Further, as long as the original 6-phosphogluconate dehydrogenase activity of the protein having the amino acid sequence described in SEQ ID NO: 27 in the sequence listing is not impaired, one or several amino acids are substituted or deleted at one or a plurality of positions. Or a DNA encoding the added protein, specifically, (b) a DNA consisting of a base sequence consisting of base numbers 72 to 1070 or a part of the DNA among the base sequences set forth in SEQ ID NO: 26 of the sequence listing It may be a DNA comprising a base sequence that hybridizes under stringent conditions with a DNA comprising a base sequence that is complementary to the DNA, and a DNA that encodes a protein exhibiting phosphogluconate dehydrogenase activity. The stringent conditions are the same as described in (A).

  The amino acid sequence set forth in SEQ ID NO: 27 in the sequence listing shows 35% homology with the protein encoding the gnd gene in amino acid sequence.

(M) Gene encoding phosphogluconate dehydratase As a gene encoding phosphogluconate dehydratase, a gene derived from acetic acid bacteria or Escherichia coli may be used as long as it is a gene encoding a protein exhibiting phosphogluconate hydratase activity. it can. The base sequence of the edd gene of Escherichia coli and the amino acid sequence of the protein encoded by the gene are disclosed as GI number 16129804 in the NCBI database.
Among these, in particular, a DNA encoding a phosphogluconate dehydratase derived from an acetic acid bacterium (Acetobacter altoacetylenes MH-24 strain), that is, a DNA encoding a protein having an amino acid sequence described in SEQ ID NO: 29 in the sequence listing, Specifically, DNA consisting of base numbers 78 to 1916 among the base sequences set forth in SEQ ID NO: 28 in the sequence listing is preferable.

  In addition, one or several amino acids were substituted, deleted or added at one or more positions as long as the original phosphogluconate dehydratase activity of the protein having the amino acid sequence described in SEQ ID NO: 29 in the sequence listing was not impaired. DNA encoding a protein, specifically, (b) of the base sequence set forth in SEQ ID NO: 28 in the sequence listing, complementary to DNA consisting of the base sequence consisting of base numbers 78 to 1916 or a part of the DNA It may be a DNA comprising a base sequence that hybridizes with a DNA comprising a base sequence under stringent conditions and a DNA encoding a protein exhibiting phosphogluconate dehydratase activity. The stringent conditions are the same as described in (A).

  The amino acid sequence set forth in SEQ ID NO: 29 in the sequence listing shows 58% homology with the protein encoding the edd gene in amino acid sequence.

The method for obtaining a gene encoding a protein exhibiting each enzyme activity selected from each of the central metabolic enzyme groups listed in the above (A) to (M) is not particularly limited, but as described above, Escherichia coli and Schwanella・ In microorganisms such as Shewanella oneidensis, Mesorhizobium loti, and acetic acid bacteria, the base sequence of each enzyme gene is clear, so these primers and probes designed from the sequence are used. It can be obtained from the genomic DNA of microorganisms.
For example, an oligonucleotide synthesized based on the base sequence of DNA constituting each gene is used as a primer, and acetic acid bacteria (for example, Acetobacter altoacetigenes MH-24 strain, Xyrinus IFO3288 (Gluconacetobacter xylinus IFO3288)), E. coli (eg, E. coli K-12 strain), Shewanella oneidensis (eg, MR-1 strain (ATCC 700550)), Mesorhizobium loti 30 (eg, MAsorhizobium loti 30) (Available from the National Institute of Agrobiological Sciences)), preferably acetic acid bacteria (especially Acetobacter altoacetigenes MH-24) and Gluconacetobacter xylinus IF 3288 (Gluconacetobacter xylinus IFO3288)) of DNA polymerase chain reaction (PCR reaction) (Trends Of Genetics (Trends Genet.) Using as template 5 vol, 185 pp, it can be obtained by 1989). The PCR reaction can be performed according to a conventional method using TaqDNA polymerase (Takara Shuzo) or KOD-Plus- (Toyobo) using a thermal cycler GeneAmp2400 manufactured by Applied Biosystems. .

It can also be obtained by hybridization using a genomic DNA library using an oligonucleotide synthesized based on the above base sequence as a probe. Oligonucleotides can be synthesized according to a conventional method using, for example, various commercially available DNA synthesizers.
On the other hand, it is not derived from the above-mentioned natural microorganisms, but can also be obtained by chemically synthesizing the base sequence of DNA constituting each gene with an automatic synthesizer or the like.

  In addition, a gene encoding a protein that is substantially the same as a protein exhibiting each enzyme activity is substituted with one or several amino acids at one or more positions as long as the original enzyme activity of the encoded protein is not impaired. A DNA encoding a deleted or added protein, that is, a DNA comprising a base sequence that hybridizes under stringent conditions with the original DNA or a DNA comprising a base sequence complementary to a part of the DNA. In addition, DNA encoding a protein exhibiting the original enzyme activity may be used. However, DNA encoding such a substantially identical protein may be detected at a specific site by, for example, site-directed mutagenesis. It can also be obtained by modifying the base sequence so that amino acids are deleted, substituted or added. The modified DNA as described above can also be obtained by a conventionally known mutation treatment.

  Furthermore, it is known that the amino acid sequence of a protein and the base sequence encoding it generally differ slightly between species, strains, mutants, and variants. Therefore, if the protein showing each enzyme activity can be obtained from all acetic acid bacteria, in particular, Gluconacetobacter or Acetobacter species, strains, mutants, and variants, DNA encoding substantially the same protein can be obtained from acetic acid bacteria belonging to the genus Gluconacetobacter or genus Acetobacter, or from mutated gluconacetobacter genus or acetic acid bacteria belonging to the genus Acetobacter, natural mutants or variants thereof. Can be isolated.

  Acquisition of a strain with enhanced enzyme activity can be achieved, for example, by introducing the above gene into a cell of acetic acid bacteria to obtain an analog-resistant mutant strain, or by a method of amplifying the copy number of the above gene in an acetic acid cell. In addition, the enzyme activity is enhanced by a method of transforming acetic acid bacteria using a recombinant DNA obtained by ligating a DNA fragment containing the structural gene of the above gene to a promoter sequence that functions efficiently in acetic acid bacteria. You can also.

Intracellular copy number amplification of the gene can be performed by introducing a multicopy vector carrying the gene into a cell of an Acetobacter bacterium. That is, it can be carried out by introducing a recombinant vector such as a plasmid or transposon holding the above gene into an acetic acid bacterium, that is, a cell of an acetic acid bacterium belonging to the genus Gluconacetobacter or Acetobacter.
Examples of plasmids include pUF106 (see, for example, “Cellulose, p. 153-158 (1989)”), pMV24 (see, for example, “Appl. Environ. Microbiol.”). 55, p.171-176 (1989)), pGI18 (see, for example, the present specification), pTA5001 (A), pTA5001 (B) (see, for example, JP-A-60-9488), and the like. PMVL1 (see, for example, "Agric. Biol. Chem.", 52, p. 3125-3129 (1988)). In addition, examples of transposons include Mu and IS1452.
The gene to be amplified in the cell is not particularly limited as long as it is a gene encoding a protein exhibiting one or more enzyme activities selected from the group of central metabolic enzymes as described above. These genes are particularly preferred because they are easier to express in acetic acid bacteria.

  The introduction of genes into gluconoacetobacter or acetic acid bacteria of the genus Acetobacter can be carried out by the calcium chloride method (Agricultural and Biological Chemistry 49, p.2091, 1985), The electroporation method (Biosci. Biotech. Biochem., 58, 974, 1994) can be used.

In order to insert a gene into a multicopy vector, first, the purified DNA is cleaved with an appropriate restriction enzyme, inserted into a restriction enzyme site of the appropriate vector DNA or a multicloning site, and then connected to the vector. Is done.
Here, when a gene is inserted into a multi-copy vector, it is necessary to select conditions under which the function of the gene is exhibited. Therefore, when a gene is inserted into a multicopy vector to obtain a recombinant vector, in addition to a promoter and a gene, a cis element such as an enhancer, a splicing signal, a poly A addition signal, a selection marker, a ribosome binding sequence (SD) Array) and the like.
Examples of the selection marker include a dihydrofolate reductase gene, a kanamycin resistance gene, a tetracycline resistance gene, an ampicillin resistance gene, and a neomycin resistance gene.

  In addition, in order to replace the promoter sequence of the enzyme gene on the chromosomal DNA with another promoter sequence that functions efficiently in acetic acid bacteria of the genus Acetobacter or Gluconacetobacter, construct a vector for homologous recombination, A vector may be used to cause homologous recombination in the chromosome of the microorganism. Examples of such promoter sequences include ampicillin resistance gene of plasmid pBR322 (manufactured by Takara Bio Inc.) of Escherichia coli, kanamycin resistance gene of plasmid pHSG298 (manufactured by Takara Bio Inc.), and chlorampheny of plasmid pHSG396 (manufactured by Takara Bio Inc.). Examples include promoter sequences derived from microorganisms other than acetic acid bacteria such as promoters of genes such as call resistance gene and β-galactosidase gene. The construction of a vector for homologous recombination is well known to those skilled in the art. Thus, by placing an endogenous enzyme gene in a microorganism under the control of a strong promoter, the copy number from the enzyme gene is amplified and expression is enhanced.

Here, examples of acetic acid bacteria to which genes are introduced include gluconoacetobacter or acetic acid bacteria belonging to the genus Acetobacter.
Examples of acetic acid bacteria belonging to the genus Gluconacetobacter include, for example, Gluconacetobacter xylinus, Gluconacetobacter europaeus, Gluconacetobacter diazotrocus, Gluconacetobacter diazotrocus Examples include Gluconacetobacter entanii, and specifically, Gluconacetobacter xylinus IFO3288, Gluconacetobacter europaeus DSM6160 (Gluconacetobacter europaeus DSM6160), Gluconacetobacter europaeus DSM6160 Gluconacetobacter diazotrophicus ATCC49037), Gluconacetobacter entanii February 1984 in Acetobacter altoacetigenes MH-24 (AIST Observatory, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st, 1st East, 1-chome Tsukuba, Ibaraki, Japan)) On the 23rd (original deposit), the deposit number FERM BP-491 can be used.

  Specific examples of the acetic acid bacteria belonging to the genus Acetobacter include Acetobacter aceti, and specifically, Acetobacter aceti. 1023 (Acetobacter aceti No. 1023) (incorporated administrative agency, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st, 1st, 1st East, Tsukuba City, Ibaraki, Japan) dated June 27, 1983 (original deposit) Accession number FERM BP-2287), Gluconacetobacter xylinus IFO3288 strain, Acetobacter aceti IFO3283 strain, etc. can be used.

  As described above, acetic acid bacteria whose acetic acid resistance is selectively enhanced by amplifying the copy number of the gene can be bred efficiently. The confirmation that the acetic acid resistance has been enhanced is as described in the above (1).

(3) Production method of vinegar according to the present invention The production method of vinegar according to the present invention is a medium containing alcohol containing the acetic acid bacteria having improved growth ability in the presence of acetic acid as described in (1) above. And acetic acid is produced and accumulated in the medium.

The culture in the medium containing alcohol of acetic acid bacteria in the production method of the present invention may be carried out under conditions that allow so-called acetic acid fermentation, and specifically, in the same manner as in the conventional vinegar production method by fermentation of acetic acid bacteria. Just do it.
As a medium containing alcohol, any medium used for acetic acid fermentation may be used. In addition to alcohol components such as ethanol, it contains a carbon source, a nitrogen source, an inorganic substance, etc., and if necessary, the strain used requires growth. What contains a suitable amount of nutrients can be used. The medium may be a synthetic medium or a natural medium. Examples of the carbon source include various carbohydrates including glucose and sucrose, and various organic acids. As the nitrogen source, natural nitrogen sources such as peptone and fermented bacterial cell decomposition products can be used.

The culture conditions are aerobic conditions such as stationary culture, shaking culture, and aeration and agitation culture, and the culture temperature is 25 to 35 ° C, usually 30 ° C. The pH of the medium is usually in the range of 2.5-7, preferably in the range of 2.7-6.5, and can be prepared with various acids, various bases, buffers and the like. Usually, by culturing for 1 to 21 days, a high concentration of acetic acid accumulates in the medium.
By such a method for producing vinegar according to the present invention, vinegar having a high acidity can be produced efficiently.

Hereinafter, the present invention will be specifically described by way of examples.
(Production Example 1) Preparation of E. coli-acetic acid shuttle vector pGI18 The acetic acid bacteria-E. Coli shuttle vector pGI18 is about 3 derived from Acetobacter altoacetigenes MH-24 strain (FERM BP-491). .1 kb plasmids pGI1 and pUC18.
That is, cells were collected from the culture solution of Acetobacter altoacetigenes MH-24 (FERM BP-491), lysed using sodium hydroxide or sodium dodecyl sulfate, After treatment with phenol, plasmid DNA was purified with ethanol.

The obtained plasmid was a circular double-stranded DNA plasmid having three recognition sites for HincII and one for SfiI, and the total length of the plasmid was about 31000 base pairs (bp). Moreover, it did not have recognition sites for EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, PstI, SphI and HindIII.
This plasmid was named pGI1 and was used to construct the vector pGI18.
The plasmid pGI1 obtained above was amplified by PCR using KOD-Plus- (manufactured by Toyobo Co., Ltd.) and cleaved with AatII. This fragment was inserted into the restriction enzyme AatII cleavage site of pUC18 to prepare plasmid pGI18 (FIG. 1).
Specifically, the PCR method was performed as follows. That is, PCR was performed under the following PCR conditions using plasmid pGI1 as a template and primers A (SEQ ID NO: 30) having a restriction enzyme AatII recognition site and primer B (SEQ ID NO: 31) as primers.
That is, the PCR method was carried out for 30 cycles, with 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 3 minutes as one cycle.

As shown in FIG. 1, the obtained plasmid pGI18 contained both pUC18 and pGI1, and the total length was about 5800 base pairs (5.8 kbp).
The base sequence of this plasmid pGI18 is shown in SEQ ID NO: 32 in the sequence listing.

(Example 1) Improvement of growth in the presence of acetic acid of Gluconacetobacter xylinus transformed with a NAD-dependent isocitrate dehydrogenase gene derived from Gluconacetobacter entanii (1) Cloning of NAD-Dependent Isoquine Dehydrogenase Gene Acetobacter altoacetigenes MH-24 (FERM BP-491), a strain of Gluconacetobacter entanii, 6 In YPG medium (containing 3% glucose, 0.5% yeast extract and 0.2% polypeptone) supplemented with% acetic acid and 4% ethanol, shaking culture was performed at 30 ° C. for 240 to 336 hours.
After completion of the culture, the culture solution was centrifuged (7,500 × g, 10 minutes) to obtain bacterial cells.

Chromosomal DNA was prepared from the obtained cells by the method disclosed in JP-A-60-9489.
That is, after washing the cells with TE buffer, the TES buffer solution was added to suspend the cells, lysozyme solution was added and allowed to stand, and EDTA solution was added and reacted at 37 ° C. for 20 minutes. After the reaction, sodium lauryl sulfate was added and the mixture was allowed to stand at 37 ° C. for 20 minutes. Next, centrifugation was performed to obtain a supernatant. Polyethylene glycol 6000 was added to the supernatant and allowed to stand overnight at 4 ° C., followed by centrifugation to obtain a precipitate containing DNA.

Using the chromosomal DNA obtained as described above as a template and as a primer, primer 1 (see the nucleotide sequence described in SEQ ID NO: 33 in the Sequence Listing) and primer 2 (see the nucleotide sequence described in SEQ ID NO: 34 in the Sequence Listing) Was used to obtain a DNA fragment containing a gene encoding NAD-dependent isocitrate dehydrogenase by PCR. The PCR method was performed 30 cycles, with 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 68 ° C. for 2 minutes as one cycle.
As a result, a DNA fragment consisting of the base sequence described in SEQ ID NO: 1 in the sequence listing was obtained. This base sequence described in SEQ ID NO: 1 contains the ORF of the NAD-dependent isocitrate dehydrogenase gene (corresponding to the amino acid sequence described in SEQ ID NO: 2) in the base numbers 84 to 1112 portion.
A DNA was prepared by inserting this DNA fragment into the restriction enzyme SmaI cleavage site of the acetic acid bacteria-E. Coli shuttle vector pGI18 by ligation.

  The DNA thus prepared was electroporated into Escherichia coli JM109 strain (see Biosci. Biotech. Biochem., 58, 974, 1994). Was transformed by. Transformants were selected on LB agar medium supplemented with 100 μg / ml ampicillin. The ampicillin resistant transformant grown on the above selective medium is extracted and analyzed by a conventional method, and a plasmid pICD in which a DNA fragment is inserted downstream of the pUC18-derived lac promoter in the same transcription direction as the lac promoter is retained. Clones were selected, and it was confirmed that the DNA having the base sequence described in Sequence Listing 1 was retained. Plasmid DNA was prepared from the resulting transformant by a conventional method to obtain pICD.

(2) Acquisition of transformant strain of acetic acid bacteria Using the pICD thus obtained, Gluconacetobacter xylinus IFO3288 was electroporated ("Proceeding of National Academy of Sciences"). Of USA (see Proc. Natl. Acad. Sci. USA, Vol. 87, p. 8130-8134 (1990)) ”. Transformants were selected on YPG medium supplemented with 100 μg / ml ampicillin and 2% acetic acid.
The ampicillin resistant transformant grown on the selective medium was analyzed by extracting the plasmid by a conventional method and confirmed to have pICD.
The resulting transformant, Gluconacetobacter xylinus IFO3288 / pICD strain, was established in 2005 by the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st, Higashi 1-chome, Tsukuba, Japan). It has been deposited on February 22, and its deposit number is FERM ABP-10254.

  The transformed strain of pICD and the original strain carrying only the vector pGI18 were cultured in YPG medium for 2 days, and the NAD-dependent isocitrate dehydrogenase activity of the resulting cell lysate was measured using NAD instead of NADP. The method was measured according to the method described in “Methods in Enzymology (Volume 13, p.30-p.33, 1969)”. The results are shown in Table 1.

As shown in Table 1, the NAD-dependent isocitrate dehydrogenase activity of the transformed strain was higher than that of the original strain. It was revealed that the enzyme activity was enhanced by introduction and expression.
In addition, the ampicillin-resistant transformant harboring the plasmid pICD obtained as described above was compared with the original strain into which only the shuttle vector pGI18 was introduced in the growth in YPG medium supplemented with acetic acid.
Specifically, shaking culture (150 rpm) is performed at 30 ° C. in 100 ml of YPG medium containing 3% acetic acid, 3% ethanol, and 100 μg / ml ampicillin, and the transformed strain and the original strain are grown in an acetic acid-added medium. Were compared by measuring the absorbance at 660 nm.
The results of each strain are shown in Table 2.

As is clear from Table 2, the transformed strain grew vigorously in the medium supplemented with 3% acetic acid, whereas the original strain hardly grew.
From this, it was confirmed that the acetic acid bacteria having enhanced NAD-dependent isocitrate dehydrogenase activity showed high growth ability in the presence of acetic acid and exhibited a growth promoting function.

(Example 2) Improvement of growth in the presence of acetic acid of Gluconacetobacter xylinus transformed with a succinyl CoA synthase gene derived from Gluconacetobacter entanii (1) Succinyl CoA synthase Gene Cloning Acetobacter altoacetigenes MH-24 (FERM) strain, which is a strain of Gluconacetobacter entanii prepared by the same method as described in Example 1 BP-491) as a template and as a primer, using primer 3 (see the nucleotide sequence described in SEQ ID NO: 35 of the Sequence Listing) and primer 4 (refer to the nucleotide sequence described in SEQ ID NO: 36 of the Sequence Listing). The PCR method was performed under the same conditions as in Example 1. Thus, a DNA fragment containing a gene encoding succinyl CoA synthase was obtained.
As a result, a DNA fragment consisting of the base sequence described in SEQ ID NO: 3 in the sequence listing was obtained. The base sequence described in SEQ ID NO: 3 is composed of two ORFs of the succinyl CoA synthase gene (corresponding to the sucC portion (corresponding to the amino acid sequence described in SEQ ID NO: 4)) and sucD in the base numbers 130 to 1341 and 1346 to 2218, respectively. Part (corresponding to the amino acid sequence described in SEQ ID NO: 5)).
A DNA was prepared by inserting this DNA fragment into the restriction enzyme SmaI cleavage site of the acetic acid bacteria-E. Coli shuttle vector pGI18 by ligation.

  Plasmid prepared by transforming the thus prepared DNA into Escherichia coli JM109 strain in the same manner as in Example 1, and inserting a DNA fragment in the same transcription direction as the lac promoter downstream of the lac promoter derived from pUC18 A transformant having pSUC was obtained, and plasmid DNA was prepared from the resulting transformant by a conventional method to obtain pSUC.

(2) Acquired Acetic Acid Bacteria Transformant Using the pSUC thus obtained, Gluconacetobacter xylinus IFO3288 was transformed in the same manner as in Example 1 to obtain a transformant. .
The resulting transformant, Gluconacetobacter xylinus IFO3288 / pSUC strain, was established in 2005 by the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st East, 1-chome, Tsukuba, Ibaraki, Japan). It has been deposited on February 22, and its deposit number is FERM ABP-10257.

  The transformed strain of pSUC and the original strain having only the vector pGI18 were cultured in YPG medium for 2 days. The succinyl-CoA synthase activity of the resulting cell disruption solution was changed from GTP to ATP and the NADH solution The measurement was carried out according to the method described in “Methods in Enzymology, Vol. 13, p. 62-p. 69, 1969” except that the concentration was changed to 1.6 mM. The results are shown in Table 3.

As shown in Table 3, since the activity of the succinyl CoA synthase of the transformed strain was higher than that of the original strain, the succinyl CoA synthase gene of acetic acid bacteria was introduced into the acetic acid bacteria and expressed. It was revealed that the enzyme activity was enhanced.
In addition, the ampicillin resistant transformant having the plasmid pSUC obtained as described above was measured for growth in a YPG medium supplemented with acetic acid at OD660 nm in the same manner as in Example 1, and the shuttle vector pGI18. Compared to the original strain that only introduced.
The results for each strain are shown in Table 4.

As a result, as shown in Table 4, the transformed strain grew vigorously in the medium supplemented with 3% acetic acid, while the original strain hardly grew.
From this, it was confirmed that the acetic acid bacteria having enhanced succinyl-CoA synthase activity showed high growth ability in the presence of acetic acid and exhibited a growth promoting function.

(Example 3) Improvement of growth in the presence of acetic acid of Gluconacetobacter xylinus transformed with a fumarate hydratase gene derived from Gluconacetobacter entanii (1) Fumarate hydratase Gene Cloning Acetobacter altoacetigenes MH-24 (FERM) strain, which is a strain of Gluconacetobacter entanii prepared by the same method as described in Example 1 BP-491) as a template, and as a primer, using primer 5 (see the nucleotide sequence described in SEQ ID NO: 37 in the Sequence Listing) and primer 6 (refer to the nucleotide sequence described in SEQ ID NO: 38 in the Sequence Listing). The PCR method was performed under the same conditions as in Example 1, A DNA fragment containing a gene encoding fumarate hydratase was obtained.
As a result, a DNA fragment consisting of the base sequence described in SEQ ID NO: 6 in the sequence listing was obtained. This base sequence described in SEQ ID NO: 6 contains the ORF of the fumarate hydratase A gene (corresponding to the amino acid sequence described in SEQ ID NO: 7) at base numbers 23 to 1819.
This DNA fragment was inserted into the restriction enzyme SmaI cleavage site of the acetic acid bacteria-E. Coli shuttle vector pGI18 by ligation to prepare DNA.

  Plasmid prepared by transforming the thus prepared DNA into Escherichia coli JM109 strain in the same manner as in Example 1, and inserting a DNA fragment in the same transcription direction as the lac promoter downstream of the lac promoter derived from pUC18 A transformant having pFUMA was obtained, and plasmid DNA was prepared from the resulting transformant by a conventional method to obtain pFUMA.

On the other hand, with the chromosomal DNA of Acetobacter altoacegenes MH-24 as a template and as a primer, primer 7 (see the nucleotide sequence described in SEQ ID NO: 39 in the Sequence Listing) and primer 8 (described in SEQ ID NO: 40 in the Sequence Listing) PCR was carried out under the same conditions as in Example 1 to obtain a DNA fragment containing a gene encoding fumarate hydratase.
As a result, a DNA fragment consisting of the base sequence described in SEQ ID NO: 8 in the sequence listing was obtained. This base sequence described in SEQ ID NO: 8 contains the ORF (corresponding to the amino acid sequence described in SEQ ID NO: 9) of the fumarate hydratase C gene at base numbers 85 to 1458.
A DNA was prepared by inserting the resulting DNA fragment into the restriction enzyme SmaI cleavage site of the acetic acid bacteria-E. Coli shuttle vector pGI18 by ligation.

  Plasmid prepared by transforming the thus prepared DNA into Escherichia coli JM109 strain in the same manner as in Example 1, and inserting a DNA fragment in the same transcription direction as the lac promoter downstream of the lac promoter derived from pUC18 A transformant harboring pFUMC was obtained, and plasmid DNA was prepared from the obtained transformant by a conventional method to obtain pFUMC.

(2) Acquisition of acetic acid bacteria transformed strain Gluconacetobacter xylinus IFO3288 was transformed in the same manner as in Example 1 using the thus obtained pFUMA and pFUMC to obtain a transformed strain. did.
The obtained transformant Gluconacetobacter xylinus IFO3288 / pFUMA strain and transformant JM109 / pFUMC strain are the National Institute of Advanced Industrial Science and Technology (AIST) No. 1 center 6) was deposited on February 22, 2005, and the respective deposit numbers are FERM ABP-10251 and FERM ABP-10252.

  The transformed strain of pFUMA, the transformed strain of pFUMC, and the original strain having only the vector pGI18 were cultured in YPG medium for 2 days, and the fumarate hydratase activity of the cell lysate of the obtained bacterial cells was determined as “Methods”. -In-enzymology (Methods in Enzymology) Vol. 13, p. 91-p. 99, 1969 "was measured. The results are shown in Table 5.

As shown in Table 5, since the fumarate hydratase activity of each transformed strain was higher than that of the original strain, the fumarate hydratase gene of acetic acid bacteria was introduced into acetic acid bacteria and expressed. It was revealed that the enzyme activity was enhanced.
In addition, for each of the ampicillin resistant transformant carrying the plasmid pFUMA and the ampicillin resistant transformant carrying the plasmid pFUMC obtained as described above, acetic acid was added in the same manner as in Example 1. Growth in the medium was measured at OD 660 nm and compared with the original strain into which only the shuttle vector pGI18 was introduced.
The results of each strain are shown in Table 6.

As a result, as shown in Table 6, the transformed strain grew vigorously in the medium supplemented with 3% acetic acid, whereas the original strain hardly grew.
From this, it was confirmed that the acetic acid bacteria having enhanced fumarate hydratase gene showed high growth ability in the presence of acetic acid and exhibited a growth promoting function.

(Example 4) Improvement of growth in the presence of acetic acid of Gluconacetobacter xylinus transformed with a malate quinone oxidoreductase gene derived from Gluconacetobacter entanii (1) Malate quinone Cloning of the Oxidoreductase (Dehydrogenase) Gene Acetobacter altoacetigenes MH-24 (Acetobacter altoacetigenes), a strain of Gluconacetobacter entanii prepared by a method similar to that described in Example 1 MH-24) strain (FERM BP-491) as a template and as a primer, primer 9 (see the nucleotide sequence described in SEQ ID NO: 41 of the sequence listing) and primer 10 (described in SEQ ID NO: 42 of the sequence listing) (See base sequence) A PCR method was performed under the same conditions as in Example 1 to obtain a DNA fragment containing a gene encoding malate quinone dehydrogenase.
As a result, a DNA fragment consisting of the base sequence described in SEQ ID NO: 10 in the sequence listing was obtained. This base sequence described in SEQ ID NO: 10 contains the ORF of the malate quinone oxidoreductase gene (corresponding to the amino acid sequence described in SEQ ID NO: 11) in the base numbers 182 to 1678.
A DNA was prepared by inserting this DNA fragment into the restriction enzyme SmaI cleavage site of the acetic acid bacteria-E. Coli shuttle vector pGI18 by ligation.

  Plasmid prepared by transforming the thus prepared DNA into Escherichia coli JM109 strain in the same manner as in Example 1, and inserting a DNA fragment in the same transcription direction as the lac promoter downstream of the lac promoter derived from pUC18 A transformant having pMQO was obtained, and plasmid DNA was prepared from the resulting transformant by a conventional method to obtain pMQO.

(2) Acquisition of transformant strain of acetic acid bacteria Using the pMQO thus obtained, Gluconacetobacter xylinus IFO3288 was transformed in the same manner as in Example 1 to obtain a transformant.
The resulting transformant, Gluconacetobacter xylinus IFO3288 / pMQO strain, was established in 2005 by the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st, 1st East, Tsukuba City, Ibaraki Prefecture, Japan). Deposited as of February 2nd, the deposit number is FERM ABP-10219.

  The transformed strain of pMQO and the original strain having only the vector pGI18 were cultured in YPG medium for 2 days, and the malate quinone oxidoreductase activity of the resulting cell lysate was determined according to “European Journal of Biotechnology”. Chemistry (Eur. J. Biochem.) 254, p.395-p.403, 1998 ”. The results are shown in Table 7.

As shown in Table 7, since the malate quinone oxidoreductase activity of each transformed strain was higher than that of the original strain, the malate quinone oxidoreductase gene of acetic acid bacteria was introduced into the acetic acid bacteria and expressed. It has been clarified that the enzyme activity is enhanced.
In addition, the ampicillin resistant transformant carrying the plasmid pMQO obtained as described above was measured for growth in an YPG medium supplemented with acetic acid by the same method as in Example 1 at OD660 nm, and the shuttle vector pGI18. Compared to the original strain that only introduced.
The results of each strain are shown in Table 8.

As a result, as shown in Table 8, the transformed strain grew vigorously in the medium supplemented with 3% acetic acid, whereas the original strain hardly grew.
From this, it was confirmed that the acetic acid bacteria with enhanced malate quinone oxidoreductase gene showed high growth ability in the presence of acetic acid and exhibited a growth promoting function.

(Example 5) Growth of Gluconacetobacter xylinus in the presence of acetic acid transformed with a NAD-dependent malate dehydrogenase (decarboxylation) gene derived from Gluconacetobacter entanii Improvement (1) Cloning of NAD-dependent malate dehydrogenase (decarboxylation) gene Acetobacter enthalii, a strain of Gluconacetobacter entanii, prepared in the same manner as described in Example 1 Using the chromosomal DNA of Altoacetogenes MH-24 (Acetobacter altoacetigenes MH-24) strain (FERM BP-491) as a template and as a primer, primer 11 (see the nucleotide sequence described in SEQ ID NO: 43 in the sequence listing) and primer 12 (See the nucleotide sequence described in SEQ ID NO: 44 in the sequence listing) PCR was carried out under the same conditions as in Example 1 to obtain a DNA fragment containing a gene encoding NAD-dependent malate dehydrogenase (decarboxylation).
As a result, a DNA fragment consisting of the base sequence described in SEQ ID NO: 12 in the sequence listing was obtained. The nucleotide sequence described in SEQ ID NO: 12 has 44% homology with sfcA encoding the NAD-dependent malate dehydrogenase (decarboxylation) gene of Escherichia coli at the base numbers 89 to 1891, and also has a NAD-dependent apple. The motif Pfam00390, which is commonly conserved in acid dehydrogenase (decarboxylation), also had a high homology of score 728.2. From this, it was confirmed that the base sequence described in SEQ ID NO: 12 contains the ORF of the NAD-dependent malate dehydrogenase (decarboxylation) gene (corresponding to the amino acid sequence described in SEQ ID NO: 13) in the base numbers 89 to 1891. did it.
A DNA was prepared by inserting this DNA fragment into the restriction enzyme SmaI cleavage site of the acetic acid bacteria-E. Coli shuttle vector pGI18 by ligation.

  Plasmid prepared by transforming the thus prepared DNA into Escherichia coli JM109 strain in the same manner as in Example 1, and inserting a DNA fragment in the same transcription direction as the lac promoter downstream of the lac promoter derived from pUC18 A transformant having pMAL was obtained, and plasmid DNA was prepared from the obtained transformant by a conventional method to obtain pMAL.

(2) Acquired Acetic Acid Bacteria Transformant Using the pMAL thus obtained, Gluconacetobacter xylinus IFO3288 was transformed in the same manner as in Example 1 to obtain a transformant.
The resulting transformant, Gluconacetobacter xylinus IFO3288 / pMAL strain, was established in 2005 by the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st, Higashi 1-chome, Tsukuba, Japan). Deposited as of February 2nd, the deposit number is FERM ABP-10218.

In addition, the ampicillin-resistant transformant harboring the plasmid pMAL obtained as described above was measured for growth in a YPG medium supplemented with acetic acid by the same method as in Example 1 at OD660 nm, and the shuttle vector pGI18. Compared to the original strain that only introduced.
The results for each strain are shown in Table 9.

As a result, as shown in Table 9, the transformed strain grew vigorously on the medium supplemented with 3% acetic acid, whereas the original strain hardly grew.
From this, it was confirmed that the acetic acid bacteria having enhanced NAD-dependent malate dehydrogenase (decarboxylation) gene showed high growth ability in the presence of acetic acid and exhibited a growth promoting function.

(Example 6) Improvement of growth in the presence of acetic acid of Gluconacetobacter xylinus transformed with a FAD-dependent lactate dehydrogenase gene derived from Gluconacetobacter entanii (1) FAD Cloning of Dependent Lactate Dehydrogenase Gene Acetobacter altoacetigenes MH-24 (Acetobacter altoacetigenes MH), a strain of Gluconacetobacter entanii prepared by the same method as described in Example 1 -24) Using the chromosomal DNA of the strain (FERM BP-491) as a template and as a primer, primer 13 (see the nucleotide sequence described in SEQ ID NO: 45 in the sequence listing) and primer 14 (bases described in SEQ ID NO: 46 in the sequence listing) PCR method) under the same conditions as in Example 1 To obtain a DNA fragment containing a gene encoding a FAD-dependent lactate dehydrogenase.
As a result, a DNA fragment consisting of the base sequence described in SEQ ID NO: 14 in the sequence listing was obtained. This base sequence described in SEQ ID NO: 14 contains the ORF of the FAD-dependent lactate dehydrogenase gene (corresponding to the amino acid sequence described in SEQ ID NO: 15) in the base numbers 46-1779 part.
A DNA was prepared by inserting the resulting DNA fragment into the restriction enzyme SmaI cleavage site of the acetic acid bacteria-E. Coli shuttle vector pGI18 by ligation.

  Plasmid prepared by transforming the thus prepared DNA into Escherichia coli JM109 strain in the same manner as in Example 1, and inserting a DNA fragment in the same transcription direction as the lac promoter downstream of the lac promoter derived from pUC18 A transformant having pLDH was obtained, and plasmid DNA was prepared from the resulting transformant by a conventional method to obtain pLDH.

(2) Acquisition of acetic acid bacteria transformed strain Using pLDH thus obtained, Gluconacetobacter xylinus IFO3288 was transformed in the same manner as in Example 1 to obtain a transformed strain. .
The resulting transformant, Gluconacetobacter xylinus IFO3288 / pLDH strain, was established in 2005 by the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st, East 1-chome, Tsukuba, Ibaraki, Japan). Deposited as of February 2nd, the deposit number is FERM ABP-10217.

  The transformed strain of pLDH and the original strain having only the vector pGI18 were cultured in YPG medium for 2 days, and the FAD-dependent lactate dehydrogenase activity of the cell disruption solution of the obtained bacterial cell was determined as “Bioscience / Biotechnology. Biochemistry (Biosci. Biotechnol. Biochem. 68), p.516-p.522, 2004 ”was measured. The results are shown in Table 10.

As shown in Table 10, the FAD-dependent lactate dehydrogenase activity of each transformed strain was higher than that of the original strain. It has been clarified that the enzyme activity is enhanced by expression.
For the ampicillin resistant transformant harboring the plasmid pLDH obtained as described above, growth in YPG medium supplemented with acetic acid was measured at OD660 nm in the same manner as in Example 1, and only the shuttle vector pGI18 was obtained. Compared with the original stock introduced.
The results for each strain are shown in Table 11.

As a result, as shown in Table 11, the transformed strain grew vigorously on the medium supplemented with 3% acetic acid, whereas the original strain hardly grew.
From this, it was confirmed that the acetic acid bacteria in which the FAD-dependent lactate dehydrogenase gene was enhanced showed high growth ability in the presence of acetic acid and exhibited a growth promoting function.

(Example 7) Growth of Gluconacetobacter xylinus in the presence of acetic acid transformed with a gene encoding subunit nuM of NADH dehydrogenase complex derived from Gluconacetobacter xylinus (1) Cloning of a gene encoding the subunit nuM of the NADH dehydrogenase complex Gluconacetobacter xylinus IFO3288 strain was transformed into YPG medium (3% glucose, 0.5% yeast extract, 0.2 In the same manner as described in Example 1 except that the bacterial strain was cultured at 30 ° C. for 24 hours in the same manner as described in Example 1, except that the prepared chromosomal DNA was used as a template and a primer. , Primer 15 (base sequence described in SEQ ID NO: 47 in the sequence listing) PCR) is carried out under the same conditions as in Example 1 using primer 16 and the primer 16 (see the nucleotide sequence described in SEQ ID NO: 48 in the sequence listing) to encode the subunit NUM of the NADH dehydrogenase complex. A DNA fragment containing the gene was obtained.
As a result, a DNA fragment consisting of the base sequence described in SEQ ID NO: 16 in the sequence listing was obtained. This base sequence described in SEQ ID NO: 16 contains the ORF of the subunit nuM gene of the NADH dehydrogenase complex (corresponding to the amino acid sequence described in SEQ ID NO: 17) in the base numbers 186 to 1727.
A DNA was prepared by inserting the resulting DNA fragment into the restriction enzyme SmaI cleavage site of the acetic acid bacteria-E. Coli shuttle vector pGI18 by ligation.

  Plasmid prepared by transforming the thus prepared DNA into Escherichia coli JM109 strain in the same manner as in Example 1, and inserting a DNA fragment in the same transcription direction as the lac promoter downstream of the lac promoter derived from pUC18 A transformant carrying pNDH was obtained, and plasmid DNA was prepared from the obtained transformant by a conventional method to obtain pNDH.

(2) Acquisition of acetic acid bacteria transformed strain Using pNDH thus obtained, Gluconacetobacter xylinus IFO3288 was transformed in the same manner as in Example 1 to obtain a transformed strain. .
The obtained transformant, Gluconacetobacter xylinus IFO3288 / pNDH strain, was established in 2005 by the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st, 1st East, Tsukuba City, Ibaraki Prefecture, Japan). It has been deposited on February 22, and its deposit number is FERM ABP-10255.

  The transformed strain of pNDH and the original strain having only the vector pGI18 were cultured in YPG medium for 2 days, and the NADH dehydrogenase activity of the cell disruption solution of the obtained bacterial cell was “Biochemistry 26, p. .7732-p.7373, 1987 ”. The results are shown in Table 12.

As shown in Table 12, since the NADH dehydrogenase activity of each transformed strain was higher than that of the original strain, the NADH dehydrogenase gene of acetic acid bacteria was introduced into the acetic acid bacteria and expressed. It was revealed that the enzyme activity was enhanced.
For the ampicillin resistant transformant harboring the plasmid pNDH obtained as described above, growth in YPG medium supplemented with acetic acid was measured at OD660 nm in the same manner as in Example 1, and only the shuttle vector pGI18 was obtained. Compared with the original stock introduced.
The results for each strain are shown in Table 13.

As a result, as shown in Table 13, the transformed strain grew vigorously in the medium supplemented with 3% acetic acid, whereas the original strain hardly grew.
From this, it was confirmed that the acetic acid bacteria with enhanced NADH dehydrogenase gene showed high growth ability in the presence of acetic acid and exhibited a growth promoting function.

(Example 8) Improvement of growth in the presence of acetic acid of Gluconacetobacter xylinus transformed with glyceraldehyde-3-phosphate dehydrogenase derived from Gluconacetobacter entanii (Gluconacetobacter xylinus) 1) Cloning of glyceraldehyde-3-phosphate dehydrogenase gene Acetobacter altoacetogenes, a strain of Gluconacetobacter entanii prepared by the same method as described in Example 1 Using chromosomal DNA of MH-24 (Acetobacter altoacetigenes MH-24) strain (FERM BP-491) as a template and as primers, primer 17 (see the nucleotide sequence described in SEQ ID NO: 49 in the sequence listing) and primer 18 (sequence listing) Example 1) using the base sequence described in SEQ ID NO: 50 PCR was carried out under the same conditions as above to obtain a DNA fragment containing a gene encoding glyceraldehyde-3-phosphate dehydrogenase.
As a result, a DNA fragment consisting of the base sequence described in SEQ ID NO: 18 in the sequence listing was obtained. The base sequence described in SEQ ID NO: 18 has 48% homology with gapA encoding glyceraldehyde-3-phosphate dehydrogenase gene of Escherichia coli at bases 91 to 1113, and glyceraldehyde-3 -The motif Pfam0444, which is commonly conserved in phosphate dehydrogenase, also had a high homology of score630.2. From this, it can be confirmed that the base sequence described in SEQ ID NO: 18 contains the ORF of the glyceraldehyde-3-phosphate dehydrogenase gene (corresponding to the amino acid sequence described in SEQ ID NO: 19) in the base numbers 91 to 1113. It was.
A DNA was prepared by inserting the resulting DNA fragment into the restriction enzyme SmaI cleavage site of the acetic acid bacteria-E. Coli shuttle vector pGI18 by ligation.

  Plasmid prepared by transforming the thus prepared DNA into Escherichia coli JM109 strain in the same manner as in Example 1, and inserting a DNA fragment in the same transcription direction as the lac promoter downstream of the lac promoter derived from pUC18 A transformant having pGLD was obtained, and plasmid DNA was prepared from the resulting transformant by a conventional method to obtain pGLD.

(2) Acquired Acetic Acid Bacteria Transformant Using the thus obtained pGLD, Gluconacetobacter xylinus IFO3288 was transformed in the same manner as in Example 1 to obtain a transformant.
The obtained transformant, Gluconacetobacter xylinus IFO3288 / pGLD strain, was established in 2005 by the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st, East 1-chome, Tsukuba, Ibaraki, Japan). Deposited as of February 22nd, the deposit number is FERM ABP-10253.

For the ampicillin resistant transformant harboring the plasmid pGLD obtained as described above, growth in YPG medium supplemented with acetic acid was measured at OD660 nm in the same manner as in Example 1, and only the shuttle vector pGI18 was obtained. Compared with the original stock introduced.
The results for each strain are shown in Table 14.

As a result, as shown in Table 14, the transformed strain grew vigorously in the medium supplemented with 3% acetic acid, whereas the original strain hardly grew.
From this, it was confirmed that the acetic acid bacteria having enhanced glyceraldehyde-3-phosphate dehydrogenase gene showed high growth ability in the presence of acetic acid and exhibited a growth promoting function.

(Example 9) Improvement of growth in the presence of acetic acid of Gluconacetobacter xylinus transformed with an enolase gene derived from Gluconacetobacter entanii (1) Cloning of enolase gene Example 1 Chromosome of Acetobacter altoacetigenes MH-24 (FERM BP-491), which is a strain of Gluconacetobacter entanii, prepared by the same method as described in 1) Similar to Example 1 using primer 19 (refer to the nucleotide sequence described in SEQ ID NO: 51 of the Sequence Listing) and primer 20 (refer to the nucleotide sequence described in SEQ ID NO: 52 of the Sequence Listing) using DNA as a template and primer. The PCR method under mild conditions to encode enolase A DNA fragment containing the gene was obtained.
As a result, a DNA fragment consisting of the base sequence described in SEQ ID NO: 20 in the sequence listing was obtained. The base sequence described in SEQ ID NO: 20 has 52% homology with E. coli eno at the base numbers 68 to 1393, and also has a high homology of score 678.2 with the motif Pfam00113 commonly conserved in enolase. Had. From this, it was confirmed that the base sequence described in SEQ ID NO: 20 contains the enolase gene ORF (corresponding to the amino acid sequence described in SEQ ID NO: 21) in the base numbers 68 to 1393.
A DNA was prepared by inserting this DNA fragment into the restriction enzyme SmaI cleavage site of the acetic acid bacteria-E. Coli shuttle vector pGI18 by ligation.

  Plasmid prepared by transforming the thus prepared DNA into Escherichia coli JM109 strain in the same manner as in Example 1, and inserting a DNA fragment in the same transcription direction as the lac promoter downstream of the lac promoter derived from pUC18 A transformant carrying pENO was obtained, and plasmid DNA was prepared from the obtained transformant by a conventional method to obtain pENO.

(2) Acquisition of acetic acid bacteria transformed strain Using pENO thus obtained, Gluconacetobacter xylinus IFO3288 was transformed in the same manner as in Example 1 to obtain a transformed strain. .
The resulting transformant, Gluconacetobacter xylinus IFO3288 / pENO strain, was established in 2005 by the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st, Higashi 1-chome, Tsukuba, Japan). Deposited as of February 2nd, the deposit number is FERM ABP-10211.

For the ampicillin resistant transformant harboring the plasmid pENO obtained as described above, growth in a YPG medium supplemented with acetic acid was measured at OD660 nm in the same manner as in Example 1, and only the shuttle vector pGI18 was obtained. Compared with the original stock introduced.
The results for each strain are shown in Table 15.

As a result, as shown in Table 15, the transformed strain grew vigorously in the medium supplemented with 3% acetic acid, whereas the original strain hardly grew.
From this, it was confirmed that the acetic acid bacteria with enhanced enolase gene showed high growth ability in the presence of acetic acid and exhibited a growth promoting function.

(Example 10) Improvement of growth of Gluconacetobacter xylinus transformed with glucose-6-phosphate dehydrogenase gene derived from Gluconacetobacter entanii in the presence of acetic acid (1 ) Cloning of glucose-6-phosphate dehydrogenase gene Acetobacter altoacetogenes MH-24, a strain of Gluconacetobacter entanii prepared by the same method as described in Example 1 Using the chromosomal DNA of (Acetobacter altoacetigenes MH-24) strain (FERM BP-491) as a template and as a primer, primer 21 (see the nucleotide sequence described in SEQ ID NO: 53 in the sequence listing) and primer 22 (SEQ ID NO: in the sequence listing) 54), and the same as in Example 1 The PCR method was carried out under conditions to obtain a DNA fragment containing a gene encoding glucose-6-phosphate dehydrogenase.
As a result, a DNA fragment consisting of the base sequence described in SEQ ID NO: 22 in the sequence listing was obtained. The base sequence described in SEQ ID NO: 22 contains the glucose-6-phosphate dehydrogenase gene ORF (corresponding to the amino acid sequence described in SEQ ID NO: 23) at bases 71 to 1600.
A DNA was prepared by inserting the resulting DNA fragment into the restriction enzyme SmaI cleavage site of the acetic acid bacteria-E. Coli shuttle vector pGI18 by ligation.

  Plasmid prepared by transforming the thus prepared DNA into Escherichia coli JM109 strain in the same manner as in Example 1, and inserting a DNA fragment in the same transcription direction as the lac promoter downstream of the lac promoter derived from pUC18 A transformant having pG6P was obtained, and plasmid DNA was prepared from the obtained transformant by a conventional method to obtain pG6P.

(2) Acquisition of Acetic Acid Bacteria Transformant Using the thus obtained pG6P, Gluconacetobacter xylinus IFO3288 was transformed in the same manner as in Example 1 to obtain a transformant.
The resulting transformant, Gluconacetobacter xylinus IFO3288 / pG6P, was established in 2005 at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1st, 1st, 1st East, Tsukuba City, Ibaraki Prefecture, Japan). Deposited as of February 2nd, the deposit number is FERM ABP-10215.

The transformed strain of pG6P and the original strain having only the vector pGI18 were cultured in YPG medium for 2 days. Technology and Biochemistry ”, Vol. 67, No. 12, p. 2648-2651, 2003 ".
The results are shown in Table 16.

As shown in Table 16, the glucose-6-phosphate dehydrogenase activity of each transformed strain was higher than that of the original strain. It has been clarified that the enzyme activity is enhanced by introducing it into a fungus and expressing it.
For the ampicillin resistant transformant harboring the plasmid pG6P obtained as described above, growth in YPG medium supplemented with acetic acid was measured at OD660 nm in the same manner as in Example 1, and only the shuttle vector pGI18 was obtained. Compared to the original stock introduced.
The results for each strain are shown in Table 17.

As a result, as shown in Table 17, the transformed strain grew vigorously on the medium supplemented with 3% acetic acid, whereas the original strain hardly grew.
From this, it was confirmed that the acetic acid bacteria with enhanced glucose-6-phosphate dehydrogenase gene showed high growth ability in the presence of acetic acid and exhibited a growth promoting function.

(Example 11) Improvement of growth in the presence of acetic acid of Gluconacetobacter xylinus transformed with 6-phosphogluconolactonase gene derived from Gluconacetobacter entanii (1) 6 -Cloning of the phosphogluconolactonase gene Acetobacter altoacetigenes MH-24 (Acetobacter altoacetigenes MH), a strain of Gluconacetobacter entanii prepared in the same manner as described in Example 1 -24) Using the chromosomal DNA of the strain (FERM BP-491) as a template and as a primer, primer 23 (see the nucleotide sequence described in SEQ ID NO: 55 in the sequence listing) and primer 24 (base listed in SEQ ID NO: 56 in the sequence listing) The same conditions as in Example 1) PCR was carried out to obtain a DNA fragment containing the gene encoding 6-phosphogluconolactonase.
As a result, a DNA fragment consisting of the base sequence described in SEQ ID NO: 24 in the sequence listing was obtained. The base sequence described in SEQ ID NO: 24 had 32% homology with mll 16514 of Mesozobium loti at base 61-873 parts. From this, it was confirmed that the base sequence described in SEQ ID NO: 24 contains the ORF of the 6-phosphogluconolactonase gene (corresponding to the amino acid sequence described in SEQ ID NO: 25) in the base numbers 61 to 873 portion.
A DNA was prepared by inserting the resulting DNA fragment into the restriction enzyme SmaI cleavage site of the acetic acid bacteria-E. Coli shuttle vector pGI18 by ligation.

  Plasmid prepared by transforming the thus prepared DNA into Escherichia coli JM109 strain in the same manner as in Example 1, and inserting a DNA fragment in the same transcription direction as the lac promoter downstream of the lac promoter derived from pUC18 A transformant carrying pPGL was obtained, and plasmid DNA was prepared from the resulting transformant by a conventional method to obtain pPGL.

(2) Acquired Acetic Acid Bacteria Transformant Using the thus obtained pPGL, Gluconacetobacter xylinus IFO3288 was transformed in the same manner as in Example 1 to obtain a transformant.
The resulting transformant, Gluconacetobacter xylinus IFO3288 / pPGL strain, was established in 2005 by the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st East, 1-chome, Tsukuba, Ibaraki, Japan). Deposited on February 2nd, the deposit number is FERM ABP-10223.

For the ampicillin resistant transformant harboring the plasmid pPGL obtained as described above, growth in a YPG medium supplemented with acetic acid was measured at OD660 nm in the same manner as in Example 1, and only the shuttle vector pGI18 was obtained. Compared to the original stock introduced.
The results for each strain are shown in Table 18.

As a result, as shown in Table 18, the transformed strain grew vigorously on the medium supplemented with 3% acetic acid, whereas the original strain hardly grew.
From this, it was confirmed that the acetic acid bacteria with enhanced 6-phosphogluconolactonase gene showed high growth ability in the presence of acetic acid and exhibited a growth promoting function.

(Example 12) Improvement of growth of Gluconacetobacter xylinus transformed with 6-phosphogluconate dehydrogenase gene derived from Gluconacetobacter entanii in the presence of acetic acid (1) 6 Cloning of Phosphogluconate Dehydrogenase Gene Acetobacter altoacetogenes MH-24 (Acetobacter), a strain of Gluconacetobacter entanii prepared by the same method as described in Example 1 altoacetigenes MH-24) strain (FERM BP-491) as a template and as a primer, primer 25 (see the nucleotide sequence described in SEQ ID NO: 57 of the sequence listing) and primer 26 (described in SEQ ID NO: 58 of the sequence listing) The same as in Example 1). A PCR method was performed under conditions to obtain a DNA fragment containing a gene encoding phosphogluconate dehydrogenase.
As a result, a DNA fragment consisting of the base sequence described in SEQ ID NO: 26 in the sequence listing was obtained. This base sequence described in SEQ ID NO: 26 contains the ORF of the phosphogluconate dehydrogenase gene (corresponding to the amino acid sequence described in SEQ ID NO: 27) at the base numbers 72 to 1070.
A DNA was prepared by inserting the resulting DNA fragment into the restriction enzyme SmaI cleavage site of the acetic acid bacteria-E. Coli shuttle vector pGI18 by ligation.

  Plasmid prepared by transforming the thus prepared DNA into Escherichia coli JM109 strain in the same manner as in Example 1, and inserting a DNA fragment in the same transcription direction as the lac promoter downstream of the lac promoter derived from pUC18 A transformant having pPGD was obtained, and plasmid DNA was prepared from the obtained transformant by a conventional method to obtain pPGD.

(2) Acquisition of transformant strain of acetic acid bacteria Using the thus obtained pPGD, Gluconacetobacter xylinus IFO3288 was transformed in the same manner as in Example 1 to obtain a transformant.
The resulting transformant, Gluconacetobacter xylinus IFO3288 / pPGD strain, was established in 2005 by the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st, East 1-chome, Tsukuba, Ibaraki, Japan). Deposited as of February 2nd, the deposit number is FERM ABP-10221.

  The transformed strain of pPGD and the original strain having only the vector pGI18 were cultured in YPG medium for 2 days, and the 6-phosphogluconate dehydrogenase activity of the resulting cell disruption solution of the cells was determined as “Bioscience / Biotechnology. E. and Biochemistry (Bioscience, Biotechnology and Biochemistry), Vol. 67, No. 12, p. 2648-2651, 2003 ". The results are shown in Table 19.

As shown in Table 19, the 6-phosphogluconate dehydrogenase activity of each transformed strain was higher than that of the original strain. It was revealed that the enzyme activity was enhanced by introduction and expression.
For the ampicillin resistant transformant harboring the plasmid pPGD obtained as described above, growth in a YPG medium supplemented with acetic acid was measured at OD660 nm in the same manner as in Example 1, and only the shuttle vector pGI18 was obtained. Compared with the original stock introduced.
The results for each strain are shown in Table 20.

As a result, as shown in Table 20, the transformed strain grew vigorously in the medium supplemented with 3% acetic acid, whereas the original strain hardly grew.
From this, it was confirmed that the acetic acid bacteria having enhanced 6-phosphogluconate dehydrogenase gene showed high growth ability in the presence of acetic acid and exhibited a growth promoting function.

(Example 13) Improvement of growth in the presence of acetic acid of Gluconacetobacter xylinus transformed with a phosphogluconate dehydratase gene derived from Gluconacetobacter entanii (1) Phosphogluconate dehydratase Gene Cloning Acetobacter altoacetigenes MH-24 (FERM) strain, which is a strain of Gluconacetobacter entanii prepared in the same manner as described in Example 1 BP-491) as a template, and as a primer, using primer 27 (see the nucleotide sequence described in SEQ ID NO: 59 of the Sequence Listing) and primer 28 (refer to the nucleotide sequence described in SEQ ID NO: 60 of the Sequence Listing). , Similar to Example 1 A PCR method was performed under conditions to obtain a DNA fragment containing a gene encoding phosphogluconate dehydratase.
As a result, a DNA fragment consisting of the base sequence described in SEQ ID NO: 28 in the sequence listing was obtained. The base sequence described in SEQ ID NO: 28 contains the ORF of the phosphogluconate dehydratase gene (corresponding to the amino acid sequence described in SEQ ID NO: 29) in the base numbers 78 to 1916.
A DNA was prepared by inserting this DNA fragment into the restriction enzyme SmaI cleavage site of the acetic acid bacteria-E. Coli shuttle vector pGI18 by ligation.

  Plasmid prepared by transforming the thus prepared DNA into Escherichia coli JM109 strain in the same manner as in Example 1, and inserting a DNA fragment in the same transcription direction as the lac promoter downstream of the lac promoter derived from pUC18 A transformant having pPGH was obtained, and plasmid DNA was prepared from the resulting transformant by a conventional method to obtain pPGH.

(2) Acquisition of Acetic Acid Bacteria Transformed Strain Gluconacetobacter xylinus IFO3288 was transformed with the PGH thus obtained in the same manner as in Example 1 to obtain a transformed strain.
The resulting transformant, Gluconacetobacter xylinus IFO3288 / pPGH strain, was established in 2005 by the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (1st, 1st, Higashi 1-chome, Tsukuba, Japan). It has been deposited on February 22, and its deposit number is FERM ABP-10256.

  The transformed strain of pPGH and the original strain having only the vector pGI18 were cultured in YPG medium for 2 days, and the phosphogluconate dehydratase activity of the resulting cell lysate was determined according to “Journal of Biotechnology (Journal of Biotechnology). Biotechnology), 105, p. 11-31, 2003 ". The results are shown in Table 21.

As shown in Table 21, since the phosphogluconate dehydratase activity of each transformed strain was higher than that of the original strain, the phosphogluconate dehydratase gene of acetic acid bacteria was introduced into the acetic acid bacteria and expressed. It was revealed that the enzyme activity was enhanced.
For the ampicillin resistant transformant harboring the plasmid pPGH obtained as described above, growth in YPG medium supplemented with acetic acid was measured at OD660 nm in the same manner as in Example 1, and only the shuttle vector pGI18 was obtained. Compared with the original stock introduced.
The results for each strain are shown in Table 22.

As a result, as shown in Table 22, the transformed strain grew vigorously on the medium supplemented with 3% acetic acid, whereas the original strain hardly grew.
From this, it was confirmed that the acetic acid bacteria with enhanced phosphogluconate hydratase gene showed high growth ability in the presence of acetic acid and exhibited a growth promoting function.

Example 14 Acetic Acid Fermentation Test of Gluconacetobacter xylinus Gluconacetobacter xylinus Gluconacetobacter xylinus Transformed with Gene Encoding Subunit nuM of NADH Dehydrogenase Complex Example 7 As for the transformed strain of Gluconacetobacter xylinus IFO3288 having ampicillin resistance and having the plasmid pNDH obtained in (1), Gluconacetobacter xylinus IFO3288 (Gluconacetobacter xylinus) having only the plasmid vector pGI18 of Production Example 1 The original strain was compared with the acetic acid fermentability.
Specifically, using a 5 liter mini jar (manufactured by Mitsuwa Chemical Co., Ltd .; KMJ-5A), acetic acid 4.9%, ethanol 2%, glucose 1.25%, yeast extract 0.125%, polypeptone 0. Aeration and agitation culture at 30 ° C., 500 rpm, and 0.3 vvm were performed in 1.7 L of a medium containing 05% ampicillin 100 μg / ml. During fermentation, the ethanol concentration in the medium was controlled to 2%.
The fermentation results are summarized in Table 23.

As is apparent from Table 23, when the specific growth rate was calculated from the maximum value of OD660 nm, which is the growth index, in the original strain and the transformed strain, the transformed strain grew 1.22 times faster than the original strain. It was. From this, it was found that the transformed strain had improved growth ability in the presence of acetic acid as compared with the original strain.
In addition, the average production rate was about 4.4% faster than that of the original strain, and it became clear that the fermentation time could be shortened.
Furthermore, since the finally reached acidity (final reached acidity) increased to 11.91% with respect to 11.41% of the original strain, the transformed strain was used to produce vinegar with high acidity. It became clear that it was available.

ADVANTAGE OF THE INVENTION According to this invention, the growth ability in the presence of acetic acid can be improved with respect to acetic acid bacteria. And the tolerance with respect to an acetic acid improves notably. As a result, it is possible to improve the rate of acetic acid fermentation in the medium and produce vinegar efficiently with a high concentration of acetic acid.
ADVANTAGE OF THE INVENTION According to this invention, the acetic acid bacterium in which the growth ability in the presence of acetic acid was remarkably improved is provided, and a method for efficiently breeding the acetic acid bacterium is provided. The acetic acid bacteria having improved growth in the presence of acetic acid can be used for the efficient production of vinegar and the production of acetic acid at a high concentration, and is useful.

It is the figure which showed the restriction enzyme map of plasmid pGI1 derived from Glucon Acetobacter enterii (Acetobacter altoacetigenes). It is the figure which showed the construction figure and restriction enzyme map of pGI18. FIG. 4 shows the positions of primers for PCR in Example 3.

Explanation of symbols

In FIG. 1, “Sfi I” and “Hinc II” indicate restriction enzyme recognition sites (the numbers in parentheses are the sequence numbers of the respective recognition sites). The numbers in parentheses indicate the base numbers shown in bp units. Furthermore, “pGI1” in the center indicates the name of the plasmid, and “3080 bp” indicates the total number of bases of the plasmid.
In FIG. 2, “Aat II” and “Sfi I” indicate restriction enzyme recognition sites. MCS is a multicloning site, Ampr is an ampicillin resistance gene site, and the numbers in parentheses are base numbers in bp units. Furthermore, “pUC18”, “pGI1” and “pGI18” in the center indicate plasmid names, and “2.7 kbp”, “3.1 kbp” and “5.8 kbp” indicate the total number of bases of the plasmid.
In FIG. 3, Primer 1 and Primer 2 indicate primer 1 and primer 2, respectively, and “Sfi I” and “Aat II” indicate restriction enzyme recognition sites.

Claims (17)

  NAD-dependent isocitrate dehydrogenase, succinyl CoA synthase, fumarate hydratase, malate quinone oxidoreductase, NAD-dependent malate dehydrogenase (decarboxylation), FAD-dependent lactate dehydrogenase, NADH dehydrogenase , Glyceraldehyde-3-phosphate dehydrogenase, enolase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, and phosphogluconate dehydratase An acetic acid bacterium having improved growth ability in the presence of acetic acid, wherein one or two or more enzyme activities selected from a group of metabolic enzymes are enhanced.   NAD-dependent isocitrate dehydrogenase, succinyl CoA synthase, fumarate hydratase, malate quinone oxidoreductase, NAD-dependent malate dehydrogenase (decarboxylation), FAD-dependent lactate dehydrogenase, NADH dehydrogenase , Glyceraldehyde-3-phosphate dehydrogenase, enolase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, and phosphogluconate dehydratase 2. In the presence of acetic acid according to claim 1, wherein the enzyme activity is enhanced by introducing into a cell and expressing a gene encoding a protein exhibiting one or more enzyme activities selected from a group of metabolic enzymes. Breeding method of acetic acid bacteria with improved growth ability. The method for breeding acetic acid bacteria according to claim 2, wherein the gene encoding the NAD-dependent isocitrate dehydrogenase is the DNA shown in (a) or (b) below.
(A) DNA consisting of base numbers 84 to 1112 among the base sequences set forth in SEQ ID NO: 1 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 1 in the sequence listing, DNA comprising a nucleotide sequence consisting of nucleotide numbers 84 to 1112 or DNA comprising a nucleotide sequence complementary to a part of the DNA under stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting NAD-dependent isocitrate dehydrogenase activity. The method for breeding acetic acid bacteria according to claim 2, wherein the gene encoding succinyl CoA synthase is the DNA shown in (a) or (b) below.
(A) DNA consisting of base numbers 130 to 2218 among the base sequences set forth in SEQ ID NO: 3 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 3 in the sequence listing, DNA having a nucleotide sequence consisting of nucleotide numbers 130 to 2218 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions A DNA comprising a base sequence that hybridizes with a DNA encoding a protein exhibiting succinyl-CoA synthetase activity. The method for breeding acetic acid bacteria according to claim 2, wherein the gene encoding fumarate hydratase is the DNA shown in (a), (b), (c) or (d) below.
(A) DNA consisting of base numbers 23 to 1819 among the base sequences set forth in SEQ ID NO: 6 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 6 in the sequence listing, DNA having a nucleotide sequence consisting of nucleotide numbers 23 to 1819 or DNA having a nucleotide sequence complementary to a part of the DNA under stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting fumarate hydratase activity.
(C) DNA consisting of base numbers 85 to 1458 among the base sequences set forth in SEQ ID NO: 8 in the sequence listing.
(D) Among the nucleotide sequences set forth in SEQ ID NO: 8 in the sequence listing, DNA having a nucleotide sequence consisting of nucleotide numbers 85 to 1458 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting fumarate hydratase activity. The method for breeding acetic acid bacteria according to claim 2, wherein the gene encoding malate quinone oxidoreductase is DNA shown in (a) or (b) below.
(A) DNA consisting of base numbers 182 to 1678 among the base sequences set forth in SEQ ID NO: 10 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 10 in the sequence listing, DNA comprising a nucleotide sequence consisting of nucleotide numbers 182-1678 or DNA comprising a nucleotide sequence complementary to a part of the DNA under stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting malate quinone oxidoreductase activity. The method for breeding acetic acid bacteria according to claim 2, wherein the gene encoding NAD-dependent malate dehydrogenase (decarboxylation) is the DNA shown in (a) or (b) below.
(A) DNA consisting of base numbers 89 to 1891 among the base sequences set forth in SEQ ID NO: 12 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 12 of the Sequence Listing, DNA having a nucleotide sequence consisting of nucleotide numbers 89 to 1891 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions A DNA comprising a base sequence that hybridizes with a DNA encoding a protein exhibiting NAD-dependent malate dehydrogenase (decarboxylation) activity. The method for breeding acetic acid bacteria according to claim 2, wherein the gene encoding the FAD-dependent lactate dehydrogenase is the DNA shown in (a) or (b) below.
(A) DNA consisting of base numbers 46 to 1779 among the base sequences set forth in SEQ ID NO: 14 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 14 in the sequence listing, DNA having a nucleotide sequence consisting of nucleotide numbers 46 to 1779 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting FAD-dependent lactate dehydrogenase activity. 3. The acetic acid bacterium according to claim 2, wherein the gene encoding NADH dehydrogenase is a DNA encoding the subunit nuM of the NADH dehydrogenase complex shown in the following (a) or (b): Breeding method.
(A) DNA consisting of base numbers 186 to 1727 among the base sequences set forth in SEQ ID NO: 16 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 16 in the sequence listing, it hybridizes under stringent conditions with DNA consisting of nucleotide numbers 186 to 1727 or with DNA complementary to a part of the DNA. DNA comprising a base sequence and encoding a protein exhibiting NADH dehydrogenase activity. The method for breeding acetic acid bacteria according to claim 2, wherein the gene encoding glyceraldehyde-3-phosphate dehydrogenase is the DNA shown in (a) or (b) below.
(A) DNA consisting of base numbers 91 to 1113 among the base sequences set forth in SEQ ID NO: 18 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 18 in the sequence listing, DNA having a nucleotide sequence consisting of nucleotide numbers 91 to 1113 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions A DNA comprising a base sequence that hybridizes with a DNA encoding a protein exhibiting glyceraldehyde-3-phosphate dehydrogenase activity. The method for breeding acetic acid bacteria according to claim 2, wherein the gene encoding enolase is the DNA shown in (a) or (b) below.
(A) DNA consisting of base numbers 68 to 1393 among the base sequences set forth in SEQ ID NO: 20 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 20 in the sequence listing, DNA having a nucleotide sequence consisting of nucleotide numbers 68 to 1393 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting enolase activity. The method for breeding acetic acid bacteria according to claim 2, wherein the gene encoding glucose-6-phosphate dehydrogenase is the DNA shown in (a) or (b) below.
(A) DNA consisting of base numbers 71 to 1600 in the base sequence set forth in SEQ ID NO: 22 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 22 in the sequence listing, DNA having a nucleotide sequence consisting of nucleotide numbers 71 to 1600 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting glucose-6-phosphate dehydrogenase activity. The method for breeding acetic acid bacteria according to claim 2, wherein the gene encoding 6-phosphogluconolactonase is the DNA shown in the following (a) or (b).
(A) DNA consisting of base numbers 61 to 873 among the base sequences set forth in SEQ ID NO: 24 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 24 in the sequence listing, DNA having a nucleotide sequence consisting of nucleotide numbers 61 to 873 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions A DNA comprising a base sequence that hybridizes with a DNA encoding a protein exhibiting 6-phosphogluconolactonase activity. The method for breeding acetic acid bacteria according to claim 2, wherein the gene encoding 6-phosphogluconate dehydrogenase is the DNA shown in (a) or (b) below.
(A) DNA consisting of base numbers 72 to 1070 among the base sequences set forth in SEQ ID NO: 26 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 26 of the Sequence Listing, DNA having a nucleotide sequence consisting of nucleotide numbers 72 to 1070 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting phosphogluconate dehydrogenase activity. The method for breeding acetic acid bacteria according to claim 2, wherein the gene encoding phosphogluconate dehydratase is the DNA shown in (a) or (b) below.
(A) DNA consisting of base numbers 78 to 1916 among the base sequences set forth in SEQ ID NO: 28 in the sequence listing.
(B) Among the nucleotide sequences set forth in SEQ ID NO: 28 of the Sequence Listing, DNA having a nucleotide sequence consisting of nucleotide numbers 78 to 1916 or DNA having a nucleotide sequence complementary to a part of the DNA and stringent conditions DNA comprising a base sequence that hybridizes with DNA and encoding a protein exhibiting phosphogluconate dehydratase activity.   The acetic acid bacterium having improved growth ability in the presence of acetic acid according to claim 1, wherein the acetic acid bacterium belongs to the genus Glucon acetobacter or genus Acetobacter.   A method for producing vinegar comprising culturing the acetic acid bacterium having improved growth ability in the presence of acetic acid according to claim 1 in a medium containing alcohol to produce and accumulate acetic acid in the medium.

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