JP5099512B2 - Highly efficient production method of glycolipid - Google Patents

Description

The present invention relates to improvements in microbial production process by genetic recombination technology, and more particularly, Pseudozyma (Pseudozyma) for increased production of glycolipids consisting of the genes encoding the mitochondrial ADP / ATP carrier protein from a eukaryotic microorganism belonging to the genus The present invention relates to a genetic material, an expression vector introduced with the genetic material, a microorganism transformed with the expression vector, and a method for efficiently producing metabolites such as glycolipids by culturing the transformant.

  Substance production processes using microorganisms have long been active in the brewing and food industry, and in particular, ethanol production technology by yeast fermentation has been used for the production of various fermented foods and fermented alcoholic beverages since ancient times. In addition, the glutamic acid fermentation technology developed in Japan can be cited as a representative example of a practical substance production process.

  In recent years, attention has been focused on technologies and businesses that utilize plant biomass resources, which are alternative and reproducible resources for solving global environmental problems. Substance production from plant biomass resources has zero emissions of carbon dioxide even in the provisions set forth in the Kyoto Protocol. For this reason, effective use of plant biomass resources has already been studied for use in new energy sources such as power generation and transportation fuel from the viewpoint of dispersion and diversification of energy supply and countermeasures against global warming. However, if new technologies that enable not only energy but also conversion to various chemical product groups can be established, large energy savings and creation of new businesses are expected in a wide range of manufacturing industries.

  In Europe and the United States, systematic chemical production (biorefinery) from biomass resources has been strongly promoted as an important national policy. The US has set a goal of converting 25% of all chemicals to biomass by 2020, and the EU is also working on a project to convert 30% by 2025. Under these circumstances, attempts are being made to innovate chemical manufacturing processes using biorefinery throughout the world. For example, a microorganism production process of 1,3-propanediol (see, for example, Non-Patent Document 1 and Patent Document 1) is a successful example of a biorefinery that has recently been established using genetic recombination technology.

  By the way, in order to achieve biorefinery, a skillful control technique of intracellular metabolic pathways that incorporate gene recombination techniques is required. For example, when only the synthesis reaction for producing the target compound is strengthened, the overall balance of the intracellular metabolic pathway is distorted, and efficient substance production cannot be achieved. In some cases, it is also expected to adversely affect cell growth. Therefore, in order to achieve biorefinery, a technique for achieving sophisticated metabolic control after understanding complicated metabolic pathways in microbial cells is required.

  Glycolipids are substances in which 1 to several tens of monosaccharides are bound to lipids, are involved in information transmission between cells in vivo, and play an important role in maintaining the functions of the nervous system and immune system. Etc. are being revealed. Glycolipids are amphiphilic substances that have both hydrophilic properties derived from the properties of sugars and lipophilic properties derived from the properties of lipids. It is called a substance. Until the petrochemical industry prospered, surfactants derived from biological components such as lecithin and saponin were used. Synthetic surfactants have been developed in recent years due to the development of the petrochemical industry, and their production has dramatically increased, making it an indispensable substance for daily life. As a result, environmental pollution has spread and social problems have arisen. For this reason, it is desired to develop a highly biodegradable surfactant that is highly safe and can reduce the burden on the environment.

  Conventionally, surfactants (biosurfactants) produced by microorganisms are classified into five types of surfactants of glycolipid type, acyl peptide type, phospholipid type, fatty acid type and polymer type. Among these, glycolipid-based surfactants are the most studied, and many types of surfactants from bacteria and yeast have been reported.

As the bacterium, rhamnolipid by Pseudomonas genus (see non-patent document 2), trehalose lipid by Rhodococcus genus (see non-patent document 3) and the like are known. However, in all cases, the production amount is 15 g / L or less.

As said yeast, the sophorose lipid by a Candida genus, the mannosyl erythritol lipid (for example, refer patent document 2), etc. are known.

With respect to the sophorose lipid, it has been reported that an efficient sophorose lipid production of 180 g / L can be produced in 200 hours by the fed -batch culture method of glucose and oleic acid using Candida bombicola (non-patent document). Reference 4).

Regarding the mannosyl erythritol lipid (MEL), Candida sp. It is possible to produce 35 g / L (production rate: 0.3 g / L / h, raw material yield: 70 mass%) of MEL from 5 mass% soybean oil using B-7 strain in 5 days. Have been reported (see Non-Patent Documents 5 and 6). Also, using Candida antarctica T-34 strain , it is possible to produce 38g / L (production rate: 0.2g / L / h, raw material yield: 48% by mass) of 8% by weight soybean oil in 8 days. (See Non-Patent Documents 7 and 8). Similarly, 110 g / L (production rate: 0.2 g / L / h, raw material yield) from 25% by mass of peanut oil after 24 days by Caddida antarctica T-34 strain using a total of 3 sequential feedings at 6-day intervals. It has been reported that production of MEL at a rate of 44 mass% is possible (see Non-Patent Document 9).

Production of 13.9 g / L (production rate: 0.57 g / L / h, raw material yield: 92% by mass) of MEL from 80% by mass of vegetable oil using Pseudozyma aphidis strain by fed-batch culture for 24 hours Has been reported to be possible (see Non-Patent Document 10).

Among the genus Pseudozyma , Pseudozyma antarctica and Pseudozyma aphidis have particularly high biosurfactant productivity among known biosurfactant-producing bacteria and production yeasts, and it is clear that they have high industrial utility value. Have been devised (see, for example, Patent Document 3). Up to now, in the production method of mannosyl erythritol lipid, attempts have been made to improve production efficiency (production rate, yield relative to raw material, and yield) by optimizing production conditions and searching for high-producing bacteria. A relatively high production volume is obtained.

  As described above, the production technique of glycolipid type biosurfactant using yeast has been repeatedly improved by improving the culture technique. On the other hand, the advancement of glycolipid biosurfactant production technology by gene recombination technology has not progressed. This is because the genetic engineering basic technology of glycolipid type biosurfactant-producing yeast has not been established.

  Many microbial processes have been improved through the introduction of gene recombination technology, and recent research for biorefinery, in particular, has made use of gene recombination technology. Similarly, the production technology of glycolipid type biosurfactant is expected to improve the production technology of glycolipid type biosurfactant by introducing gene recombination technology.

Recently, with the ultimate goal of improving glycolipid biosurfactant production technology through genetic recombination technology, comprehensive genes have been defined to clarify the characteristics of the yeast of the genus Pseudozyma , a glycolipid biosurfactant-producing yeast. Analysis was performed (see Non-Patent Document 11). The analysis results showed the characteristics of Pseudozyma yeast at the gene level, but most were limited to partial genetic information with unknown functions.

The basis of genetic recombination technology for yeasts of the genus Pseudozyma , a glycolipid-type biosurfactant-producing yeast, includes development of host cells, development of gene expression vectors, and improvement of transformation technology. Recently, because of its importance as a host for the production of heterologous proteins and for the development of technology for producing highly functional glycolipids, efficient transformation technology by electroporation of yeasts of the genus Pseudozyma , and the expression of foreign genes using gene expression vectors Expression has also been demonstrated, and the basic technology of gene recombination is being prepared although there is room for improvement (see Patent Document 4 and Non-Patent Document 12).

Considering the situation mentioned above, Pseudozyma yeasts are also able to control intracellular metabolism by genetic recombination technology due to recent progress in genetic engineering research, and improved glycolipid biosurfactant production technology How to select the target gene for the drug is the key to the advancement of glycolipid biosurfactant production technology.

Japanese Patent Laid-Open No. 2003-507022 JP 2002-45195 A JP 2007-185142 A Japanese Patent Application No. 2007-010470 T. Willke and K. D. Vorlop: Appl. Microbiol. Biotechnol., 66, 131 (2004). Q. Wang, X. Fang, B. Bai, X. Liang, P. J. Shuler, W. A. 3rd Goddard and Y. Tang., 98, 842 (2007). F. J. Aranda, J. A. Teruel, M. J. Espuny, A. Marques, A. Manresa, E. Palacios-Lidon and A. Ortiz: Biochim. Biophys. Acta., 1768, 2596 (2007). H. J. Daniel, R. T. Otto, M. Binder, M. Reuss and C. Syldatk: Appl. Microbiol. Biotechnol., 51, 40 (1999). T. Nakahara, H. Kawasaki, T. Sugisawa, Y. Takamori and T. Tabuchi: J. Ferment. Technol., 61, 19 (1983). H. Kawasaki, T. Nakahara, M. Oogaki and T. Tabuchi: J. Ferment. Technol., 61, 143 (1983). D. Kitamoto, S. Akiba, C. Hioki and T. Tabuchi: Agric. Biol. Chem., 54, 31 (1990). D. Kitamoto, K. Haneishi, T. Nakahara and T. Tabuchi: Agric. Biol. Chem., 54, 37 (1990). D. Kitamoto, K. Fujishiro, H. Yanagishita, T. Nakane and T. Nakahara: Biotechnol. Lett., 14, 305 (1992). U. Rau, L. A. Naguyen, H. Roeper, H. Koch and S. Lang: Appl. Microbiol. Biotechnol., (2005). T. Morita, M. Konishi, T. Fukuoka, T. Imura, and D. Kitamoto .: Yeast, 23, 661 (2006). T. Morita, H. Habe, T. Fukuoka, T. Imura, D. Kitamoto .: J Biosci Bioeng., 104, 517 (2007).

  As shown in the above background art, glycolipid biosurfactant is a highly functional material that can be mass-produced from biomass resources in a bioprocess, and is expected to be put to practical use in a wide range of industrial fields. On the other hand, in order to spread and expand it, further improvement in production efficiency and diversification of structure and functions, that is, expansion of product lineup is required.

If a genetic recombination technique is used, it is possible to modify microorganisms to be used at the metabolic level, so that a dramatic improvement in the bioprocess can be expected. Therefore, the object of the present invention is to challenge the genetic recombination of yeasts of the genus Pseudozyma using the currently available research materials in order to apply high-functional glycolipid biosurfactants to a wide range of industrial fields. The aim is to develop elemental technology for dramatically improving glycolipid biosurfactant production technology by improving efficiency and diversifying structural functions.

In view of the above technical problems, the present inventors have found genes that contribute to the production of glycolipids, particularly glycolipid-type biosurfactants, from partial gene information of yeasts of the genus Pseudozyma . Sequence analysis of the gene, cloning of the gene, and eukaryotic transformant using the gene expression vector were obtained, and Pseudozyma yeast was successfully improved by gene recombination technology. Furthermore, it was clarified that the gene is a gene encoding a mitochondrial ADP / ATP carrier protein, and it was confirmed that the production efficiency of glycolipid biosurfactant of the obtained Pseudozyma genus yeast transformant was improved, The present invention has been completed.

  In addition, it cannot be predicted at all that mitochondrial ADP / ATP carrier protein is forcibly expressed in cells, and the production efficiency of glycolipid biosurfactant cannot be predicted. In addition, the relationship between mitochondrial function and glycolipid biosynthesis was found for the first time by the present invention.

  Mitochondrial ADP / ATP carrier protein is responsible for the final stage of ATP metabolism in mitochondria, which is the place where organisms acquire energy, and the regulation of this gene expression regulates intracellular redox balance or ATP energy flow. Although it could be expected to be disturbed, it could never be expected to be involved in glycolipid production.

That is, the present invention is as follows.
(1) A genetic material for enhancing production of glycolipid, comprising a gene encoding a mitochondrial ADP / ATP carrier protein.

(2) A genetic material for enhancing production of glycolipid, comprising a gene encoding a protein of the following a) to c):
a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1,
b) a protein having 40% or more identity with the amino acid sequence shown in SEQ ID NO: 1 and having the ability to transport ATP to the cytoplasm,
c) A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 1, and having the ability to transport ATP to the cytoplasm.

(3) A genetic material for enhancing production of glycolipid, characterized by comprising any of the following genes a) or b).
a) a gene consisting of the base sequence shown in SEQ ID NO: 2;
b) A gene encoding a protein in which one or several genes are deleted, substituted and / or added in the gene shown in SEQ ID NO: 2 and have the ability to transport ATP to the cytoplasm.

(4) A recombinant vector comprising the gene according to any one of (1) to (3) above.

(5) A transformant, wherein a host cell having the ability to produce glycolipid is transformed with the recombinant vector described in (4) above.

(6) The transformant according to (5) above, wherein the host cell having the ability to produce glycolipid is a microorganism belonging to fungi.

(7) fungi microorganism belonging to the, Saccharomyces (Saccharomyces) genus or characterized in that it is a Pseudozyma (Pseudozyma) microorganism belonging to the genus transformant according to (6).

(8) A method for producing a glycolipid, comprising culturing the transformant according to any one of (5) to (7), and collecting glycolipid from the obtained culture.

According to the present invention, an ADP / ATP carrier protein and a gene thereof have been identified as a protein that is involved in the production of glycolipids of yeast of the genus Pseudozyma and enhances the production efficiency and a gene encoding the protein. Thereby, an expression vector containing the gene is provided, and a glycolipid, particularly a glycolipid type biosurfactant, can be produced with high efficiency by culturing a microorganism transformed with the expression vector.

On the other hand, mitochondria produce ATP by glycolysis and electron transfer circuit under aerobic conditions, and are responsible for respiratory function. The ADP / ATP carrier protein transports ATP, an energy substance produced in the mitochondria, to the cytoplasm.
An increase in respiratory activity means a change in mitochondrial function, and more specifically, an activation of mitochondrial membrane potential. More specifically, it means a change in the balance between the coenzyme and hydrogen ions, that is, a change in the intracellular redox balance. Since there is no knowledge about the correlation between the biosynthetic pathway of glycolipid biosurfactant and the redox balance or ATP flow, improving the production efficiency of glycolipid biosurfactant is an unexpected effect. However, this time, the expression of ADP / ATP carrier protein gene showed a significant effect on the production of glycolipid biosurfactant in yeasts belonging to the genus Pseudozyma, and the knowledge that the protein and its gene are involved in the production of glycolipid biosurfactant was obtained. However, according to this finding, since the ATP / ADP carrier protein and its gene are universally present in eukaryotes, the production efficiency of the biosurfactant can be improved by the expression of the ADP / ATP carrier protein gene. It strongly suggests that it is not limited to Pseudozyma yeast.

Hereinafter, the present invention will be described in more detail.
The present invention uses a gene encoding a mitochondrial ADP / ATP carrier protein as a genetic material for enhancing glycolipid production.

  ATP, an energy currency common to all living organisms, is essential for living organisms, and it is no exaggeration to say that most biological reactions are moving to acquire this energy. The metabolic reaction to acquire ATP varies depending on the organism and growth environment, but it is generally produced by the glycolysis system, citrate circuit, and electron transfer circuit under aerobic conditions. Is an organelle.

  One of the main features of mitochondrial function is oxidative phosphorylation (a process in which energy from fuel metabolism such as glucose or fatty acids is converted to ATP), which then converts various energies Used to drive the required biosynthetic reactions and other metabolic activities.

  The mitochondrial ADP / ATP carrier protein has a function of transporting ATP produced in the mitochondria to the cytoplasm at the final stage of mitochondrial ATP metabolism. This protein is recognized in most organisms.

The higher-order structure of this mitochondrial ADP / ATP carrier protein has been analyzed in detail by X-ray crystal structure analysis, and the precise reaction mechanism including the primary structural features has been clarified. Specifically, the amino acid sequence consisting of RRRMMM and PX (D / E) XX (K / R) is conserved, and the analysis result of hydrophobic / hydrophilic balance is the presence of six transmembrane domains. (E. Pebay-Peyroula, C. Dahout-Gonzalez, R. Kahn, V. Trezeguet, GJ Lauquin, G. Brandolin., 426, 39 (2003)).
In the mitochondrial ADP / ATP carrier protein localized in the mitochondrial membrane, information such as amino acid residues important for binding to ADP and formation of ATP, and conserved amino acid sequences essential for localization in the mitochondrial membrane are examined in detail. Therefore, it is possible to determine whether or not a protein with unknown function is a mitochondrial ADP / ATP carrier protein from the characteristics of its primary structure.

A specific example of a gene encoding a mitochondrial ADP / ATP protein used in the present invention is a gene extracted and isolated from Pseudozyma antarctic T-34 using a genetic engineering technique.
This strain is a microorganism isolated from the leaf surface as a glycolipid-type biosurfactant-producing yeast (see Non-Patent Document 7).

The gene encoding the mitochondrial ADP / ATP protein derived from Pseudozyma antarctic T-34 is a gene that is cultured under conditions for producing glycolipid biosurfactant and is expressed at a relatively high frequency in this culture. It has been found.
That is, the present invention extracts Pseudozyma antarctic T-34 messenger RMA cultured as described above, constructs a cDNA library, transforms E. coli, and randomly collects plasmids from the resulting colonies to insert inserts. The sequence was determined and a sequence library was constructed. Most of the genes in this sequence library are unknown in function, and their functions were analyzed sequentially against the database, but the mitochondrial ADP / ATP carrier with known insert sequences found relatively frequently in the above colonies. This is due to the fact that it was identified as a gene sequence encoding a protein and confirmed that the gene has a glycolipid production enhancing action.

  In addition, when the sequence of a function unknown gene having relatively high sequence homology with the gene encoding mitochondrial ADP / ATP carrier protein does not include the entire coding region, an undecoded sequence according to a known method in the art. The undecoded sequence was analyzed by, for example, inverse PCR or primer walking.

The gene encoding the mitochondrial ADP / ATP carrier protein of Pseudozyma antarctic T-34 thus obtained is shown in SEQ ID NO: 2, and the amino acid of the mitochondrial ADP / ATP carrier protein encoded by the gene is shown in SEQ ID NO: 1. Respectively.

The gene encoding the mitochondrial ADP / ATP carrier protein used in the present invention is not limited to the one derived from Pseudozyma antarctic T-34. FIG. 1 shows an alignment of amino acid sequences of Pseudozyma antarctic and other eukaryotic mitochondrial ADP / ATP carrier proteins. Table 1 in Example 1 shows mitochondrial ADP / ATP carrier proteins (derived from Pseudozyma antarctic T-34). The sequence homology (Identity) of each eukaryotic mitochondrial ADP / ATP carrier protein to SEQ ID NO: 1) is shown. As is clear from this, the mitochondrial ADP / ATP carrier protein has a very high similarity in each eukaryote. Therefore, in the present invention, genes encoding mitochondrial ADP / ATP carrier proteins derived from other eukaryotes can also be used as genetic material for enhancing production of glycolipids.
The amino acid sequences of these other eukaryotic mitochondrial ADP / ATP carrier proteins are shown in SEQ ID NOs: 13-18. Of these, the base sequence of the gene encoding the mitochondrial ADP / ATP carrier protein is shown in SEQ ID NOS: 19 to 21 only from Saccharomyces cerevisiae.

Further, even if a gene has an unknown function, it can be said that it has the same function as long as the encoded amino acid sequence has at least 40% identity with the amino acid sequence of SEQ ID NO: 1. Even an unknown gene is included in the gene encoding the mitochondrial ADP / ATP carrier protein of the present invention, and can be used as a genetic material for enhancing production of glycolipid. Genes encoding these mitochondrial ADP / ATP carrier proteins can be easily obtained, for example, by designing and synthesizing primers from the sequences and performing PCR using genomic DNA as a template. Chemical synthesis is also possible.
On the other hand, based on the contents disclosed in SEQ ID NO: 1, the function of mitochondrial ADP / ATP carrier protein can be achieved by adding, deleting, inserting, or substituting one to several amino acids using ordinary gene recombination techniques. Obtaining retained derivatives is easy for those skilled in the art. Therefore, derivatives retaining the function of mitochondrial ADP / ATP carrier protein obtained by such conventional means are also included in the scope of the present invention.

  When enhancing the production of glycolipid using the gene encoding the mitochondrial ADP / ATP carrier protein of the present invention, the gene encoding the mitochondrial ADP / ATP carrier protein is inserted downstream of the promoter in an appropriate expression vector. To construct a mitochondrial ADP / ATP carrier protein expression vector. An expression vector for inserting a gene encoding a mitochondrial ADP / ATP carrier protein may be derived from a plasmid, cosmid or virus.

Next, host cells having the ability to produce glycolipids are transformed using this recombinant expression vector. The resulting transformant has enhanced glycolipid production ability. Next, the transformant is cultured in a medium, and glycolipids are collected from the culture containing the transformant. In this case, a transformant may be separated from the culture, and glycolipids may be collected from the separated transformant.
The method for culturing the transformant is not particularly limited, and may be cultured by a known method. For example, in the case of a transformant belonging to the genus Pseudozyma , it is usually performed at pH 5-8, preferably pH 6, 20-35 ° C., preferably 22-28 ° C., for 3-7 days. The glycolipid produced by the culture can be recovered from the culture solution according to a conventional method.

  The expression vectors and host microorganisms shown in the following examples are merely examples of many vectors and host cells suitable for the expression of the mitochondrial ADP / ATP carrier protein of the present invention, and those skilled in the art will be familiar with conventional means in the art. Thus, a functional mitochondrial ADP / ATP carrier protein expression vector can be constructed in any host cell. The promoter may be appropriately selected from known ones, or may be newly prepared or synthesized by selecting one that can be used from various genes. Thus, the expression vectors of the present invention are not limited to the exemplary plasmids described herein, but can be modified using conventional techniques (eg, exchanging promoters) to provide different types of microorganisms and other cells. Expression vectors can be constructed that are functional within and / or capable of successfully expressing mitochondrial ADP / ATP carrier proteins.

The host cell used for transformation with the expression vector carrying the gene encoding the mitochondrial ADP / ATP carrier protein of the present invention may be any cell having glycolipid production ability, such as yeast, mold, etc. Eukaryotic microorganisms, and cells of higher organisms generally used may be used. Such cells of higher organisms include animal cells or cultured plant cells.
Preferred examples of the microorganism are strains belonging to the genus Saccharomyces (for example, S. cerevisiae ) and strains belonging to the genus Pseudozyma (for example, P. antarctica ).

When a strain belonging to the genus Pseudozyma is used as a host, the DNA described in SEQ ID NO: 2 is amplified by PCR, once ligated to an E. coli cloning vector, and then ligated to a gene expression vector applicable to a strain belonging to the genus Pseudozyma . Examples of the gene expression vector to be used include pUXV1 ATCC 77463, pUXV2 ATCC 77464, pUXV5 ATCC 77468, pUXV6 ATCC 77469, pUXV7 ATCC 77470, pUXV8 ATCC 77471, pUXV3 ATCC 77465, pU2X1 ATCC 77466, pU2X2 ATCC 77467 and the like.

  As used herein, the term “vector” refers to a DNA derived from a nucleic acid sequence, eg, a plasmid, cosmid, virus, or synthesized by chemical or enzymatic means, in which one or more nucleic acid fragments are contained. Inserted or cloned, where the nucleic acid encodes a particular gene. A vector can contain one or more unique restriction sites for this purpose and can replicate autonomously in a host or organism that is designed to reproduce the cloned sequence. Vectors can have a linear, circular or supercoiled configuration and can be complexed with other vectors or other materials for specific purposes. The components of the vector include, but are not limited to, DNA molecules including DNA; sequences encoding excision proteins or other desired products; and regulatory elements for transcription, translation, RNA stability and replication. Can be included.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. The plasmids, various restriction enzymes, T4 DNA ligase, and other enzymes used in the following experimental examples are all commercially available and were used according to the supplier's instructions. In addition, DNA cloning, construction of each plasmid, host transformation, transformant culture, and culture recovery were performed according to methods known to those skilled in the art or methods described in the literature.

Example 1
"Cloning and nucleotide sequence analysis of gene encoding mitochondrial ADP / ATP carrier protein of Pseudozyma ntarctic T-34 "
( Pseudozyma ntarctic T-34 culture)
a) Pseudozyma ntarctic T-34 strain stored in a storage medium (malt extract 3 g / L, yeast extract 3 g / L, peptone 5 g / L glucose 10 g / L, agar 30 g / L), glucose 20 g / L, A platinum loop is inoculated into a test tube containing 4 mL of a liquid medium composed of 1 g / L of yeast extract, 1 g / L of sodium nitrate, 0.5 g / L of potassium dihydrogen phosphate, and 0.5 g / L of magnesium sulfate. , Shaking culture at 30 ° C., then
b) The obtained bacterial cell culture solution has a composition of soybean oil 40 g / L, yeast extract 1 g / L, sodium nitrate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and magnesium sulfate 0.5 g / L. A Sakaguchi flask containing 20 mL of liquid medium was inoculated and cultured at 30 ° C. with shaking.
The following tests were performed using the bacterial cell culture solution obtained by each culture of a) and b).

(Analysis of gene sequence)
RNA was extracted from Pseudozyma antarctica T-34 cultured under glycolipid biosurfactant production conditions (cultured in b) above, and then ligated to a cloning vector for E. coli to construct a cDNA library. The DNA sequence (insert) contained in the plasmid DNA extracted from the obtained Escherichia coli transformant, that is, the gene sequence of Pseudozyma antarctica T-34 expressed under glycolipid biosurfactant production conditions was decoded. Furthermore, duplicated genes were collected as one gene, and a gene database was finally created. At this time, since the rate of gene duplication indicates the expression frequency (expression level in the cell), it is possible to estimate a gene having a high expression frequency under glycolipid biosurfactant production conditions using the gene database. It has become possible. Furthermore, when the function of each gene in the gene database was estimated by BLAST analysis, most of the genes were found to have unknown functions.

As a result of analyzing the deduced amino acid sequence of the gene of unknown function found in this study against a database, it showed relatively high homology with DNA encoding mitochondrial ADP / ATP carrier protein. The homology of each known organism with the mitochondrial ADP / ATP carrier protein is as follows (Table 1), and the results of comparing the sequences are shown in FIG.

  Furthermore, the results of the hydropathy analysis (analysis of the hydrophilic / hydrophobic balance based on the primary structure) (FIG. 2A) agreed well with the profile known to be common to all mitochondrial ADP / ATP carrier proteins. That is, it has 6 transmembrane domains with almost the same level of new aqueous / hydrophobic balance (FIG. 2B). In addition, the amino acid sequences constituting these six transmembrane domains are shown in FIG. The six transmembrane domains are known to be structural features necessary for the protein to pass through the outer mitochondrial membrane and localize to the inner membrane.

  Furthermore, in the deduced amino acid sequence of the DNA, it corresponds to an amino acid sequence highly conserved in mitochondrial ADP / ATP carrier protein, that is, RRRMMM and PX (D / E) XX (K / R) in three places Has an array. Furthermore, a repeat region of 3 times with 15% or more homology was also observed in the internal sequence. In addition, all important conserved amino acid residues revealed from the results of crystal structure analysis of mitochondrial ADP / ATP carrier protein were also observed. Details of the conserved amino acid sequence are set forth in FIG.

(Example 2)
"Expression of mitochondrial ADP / ATP carrier protein gene in S. cerevisiae strain and its effect on respiratory activity"
In order to confirm that a gene of unknown function that shows relatively high homology with DNA encoding mitochondrial ADP / ATP carrier protein actually functions as mitochondrial ADP / ATP carrier protein, mitochondrial ADP / ATP carrier of S. cerevisiae It was verified whether expression of the gene in a protein-deficient strain ( ΔAAC1 strain) and phenotype complementation (recovery) by the gene could be achieved . Here, complementation (recovery) of the phenotype was determined using as an index the poor growth on a glycerol medium known as a phenotype of S. cerevisiae mitochondrial ADP / ATP carrier protein deficiency.

(Construction of gene expression vector for S. cerevisiae )
A gene expression vector for S. cerevisiae was constructed by the following procedure. Pseudozyma antarctic T-34 genomic DNA was extracted according to a standard method and used as a template. PCR was performed using oligonucleotide primers (SEQ ID NOs: 3 and 4) designed based on the nucleotide sequence of SEQ ID NO: 2, and mitochondrial ADP / ATP carrier A gene of unknown function that showed relatively high homology with the DNA encoding the protein was amplified. The amplified gene was linked to the multiple cloning site of the expression vector pAUR123 for S. cerevisiae , and a gene expression vector pAUR123-PaAAC capable of expressing the gene under the control of the ADH promoter was constructed.

(Transformation of S. cerevisiae )
Using the constructed gene expression vector pAUR123-PaAAC or a control vector pAUR123 containing no insert, S. cerevisiae transformants were obtained by the lithium acetate method. Protocols such as selection of transformants were performed according to the instructions of the supplier of the expression vector pAUR123.

(Phenotypic complementation experiment of S. cerevisiaeΔAAC1 strain ( AAC1 gene disruption strain))
Each transformant introduced with the constructed gene expression vector pAUR123-PaAAC or the control vector pAUR123 containing no insert was cultured in a liquid medium containing glycerol as the sole carbon source, and the results of observation of the growth were observed. As shown in FIG. According to FIG. 3, it can be seen that the ΔAAC1 strain clearly grows slower in a liquid medium containing glycerol than the parent strain when a control vector is introduced. On the other hand, it can be seen that the growth of the ΔAAC1 strain introduced with the constructed gene expression vector in a liquid medium containing glycerol has recovered to the same level or higher as that of the parent strain. This result shows that the gene contained in the constructed gene expression vector, i.e., a gene of unknown function that shows relatively high homology with the DNA encoding the mitochondrial ADP / ATP carrier protein of Pseudozyma antarctic T-34, is actually S. It is expressed in cerevisiae cells and functions as a mitochondrial ADP / ATP carrier protein. Such a phenotypic complementation experiment is one of the conventional methods for specifying the function of a gene, and from this result, it was proved that the unknown gene is a mitochondrial ADP / ATP carrier protein. The function-unknown gene was named PaAAC .

(Respiratory activity of S. cerevisiae transformants)
Furthermore, in order to examine the respiratory activity of each transformant introduced with the constructed gene expression vector pAUR123-PaAAC or the control vector pAUR123 containing no insert, a rhodamine 123 staining experiment was performed on each transformant. Rhodamine 123 is a mitochondrial staining reagent, but its fluorescence intensity (degree of staining) depends on the mitochondrial membrane potential. It is known that mitochondrial membrane potential is respiratory activity, and cells having high respiratory activity have high staining intensity, and cells having low respiratory activity have low staining intensity. FIG. 4 shows the results of actually staining each transformant and observing it under a fluorescence microscope. According to FIG. 4, it can be seen that the staining intensity of the cells into which the constructed gene expression vector has been introduced is remarkably high. This result shows that the respiratory activity of the S. cerevisiae strain was improved by the expression of the gene. From the function of the mitochondrial ADP / ATP carrier protein, it is expected that ATP produced in the mitochondria flows in the cytoplasm in large quantities, and further, from this experiment result, the hydrogen ion concentration gradient through the mitochondrial membrane also fluctuates. Can be easily guessed. Further, the phenomenon observed here is not limited to microorganisms, but the same effect can be expected for a wide range of organisms including higher organisms.

(Example 3)
“Forced expression of mitochondrial ADP / ATP carrier protein gene in Pseudozyma antarctic T-34 and its effect on glycolipid biosurfactant production efficiency”
In order to investigate whether mitochondrial ADP / ATP carrier protein is involved in the production of glycolipid biosurfactant in Pseudozyma antarctic T-34, we constructed a gene expression vector for Pseudozyma antarctic T-34 and analyzed the characteristics of Pseudozyma antarctic T-34. Transformants were obtained and the productivity of glycolipid biosurfactant was evaluated.

( Construction of gene expression vector for Pseudozyma antarctic T-34)
A gene expression vector for Pseudozyma antarctic T-34 was constructed by the following procedure. Pseudozyma antarctic T-34 genomic DNA was extracted according to a standard method and used as a template. PCR was performed using oligonucleotide primers (SEQ ID NO: 5 and 6) designed based on the nucleotide sequence of SEQ ID NO: 2, and mitochondrial ADP / ATP carrier The DNA encoding the protein was amplified. Amplified gene is an expression vector for filamentous fungi Ustilago maydis, ligated into the cloning site of pUXV1 known to be also applicable to Pseudozyma spp yeast, capable of expressing the gene under the control of the gap promoter A gene expression vector pUXV1-PaAAC was constructed.

( Transformation of Pseudozyma antarctic T-34)
Using the constructed gene expression vector pUXV1-PaAAC or the control vector pUXV1 containing no insert, a transformant of Pseudozyma antarctic T-34 was obtained by electroporation. For selection of transformants, hygromycin B was used.

(Production of glycolipid biosurfactant by transformant of Pseudozyma antarctic T-34)
After culturing a) for 1 day, culturing b) for 7 days, adding an equal amount of ethyl acetate to the obtained bacterial cell culture, stirring sufficiently, and then separating the ethyl acetate layer . The amount of produced glycolipid biosurfactant contained in the ethyl acetate layer was quantified by high performance liquid chromatography. As a comparative example, a transformant of Pseudozyma antarctica strain T-34 obtained by transforming with a control vector was cultured under the same conditions, and the production amount of glycolipid biosurfactant was quantified by high performance liquid chromatography in the same manner. . The results are shown in FIG. The peak detected by high performance liquid chromatography coincided with that of a known mannosyl erythritol lipid. According to FIG. 5, the glycolipid biosurfactant production capacity of the Pseudozyma antarctica T -34 transformant carrying the gene expression vector pUXV1-PaAAC is about that of the transformant of the Pseudozyma antarctica T -34 strain carrying the control vector pUXV1. It reaches twice as much. Although this result was initially unexpected, it was confirmed that mitochondrial ADP / ATP carrier protein is involved in biosynthesis of glycolipid biosurfactant, which can improve the production efficiency of glycolipid biosurfactant. .

Example 4
“Mitochondrial ADP / ATP carrier protein mutant (amino acid substitution mutation) and its effect on glycolipid biosurfactant production”
In order to investigate in more detail that mitochondrial ADP / ATP carrier protein is involved in the production of glycolipid biosurfactant in Pseudozyma antarctic T-34 strain, an amino acid substitution mutant of mitochondrial ADP / ATP carrier protein was prepared and Pseudozyma antarctic The effect on the productivity of glycolipid biosurfactant was evaluated by introducing it into T-34.

(Construction of mitochondrial ADP / ATP carrier protein gene containing amino acid substitution mutants)
A gene expression vector containing an amino acid substitution mutant of mitochondrial ADP / ATP carrier protein was constructed by the following procedure. Pseudozyma antarctic T-34 genomic DNA was extracted according to a standard method and used as a template. PCR was performed using the oligonucleotide primer for creating a mutant designed based on the nucleotide sequence of SEQ ID NO: 2, and amino acid substitution mutants were included. DNA encoding mitochondrial ADP / ATP carrier protein was amplified. The mutation site of amino acid substitution in the base sequence of SEQ ID NO: 2 is shown in FIG. The mutation site targets a region essential for the expression of mitochondrial ADP / ATP carrier protein activity. With the combination of SEQ ID NOs: 7 and 8, a mutant (R90I) gene in which the 90th arginine residue was replaced with an isoleucine residue was amplified. In the combination of SEQ ID NOs: 9 and 10, a mutant (L136S) gene in which the 136th leucine residue was replaced with a serine residue was amplified. In the combination of SEQ ID NOs: 11 and 12, a mutant (R247I / R248I) gene in which the 247th arginine residue was replaced with an isoleucine residue and the 248th arginine residue was replaced with an isoleucine residue was amplified. Each amplified gene is linked to the cloning site of pUXV1, and gene expression vectors that can express the gene under the control of the gap promoter (pUXV1-PaAAC-R90I, pUXV1-PaAAC-L136S, pUXV1-PaAAC-R247I / R248I) was constructed to produce a transformant of Pseudozyma antarctic T-34.

(Effects of mitochondrial ADP / ATP carrier protein variants (amino acid substitution mutations) on glycolipid biosurfactant productivity)
After culturing a) for 1 day, culturing b) for 7 days, adding an equal amount of ethyl acetate to the obtained bacterial cell culture, stirring sufficiently, and then separating the ethyl acetate layer . The amount of produced glycolipid biosurfactant (mannosylerythritol lipid) contained in the ethyl acetate layer was quantified by high performance liquid chromatography. As a comparative example, a transformant of Pseudozyma antarctica strain T-34 obtained by transforming with a control vector was cultured under the same conditions, and the production amount of glycolipid biosurfactant was quantified by high performance liquid chromatography in the same manner. . The results are shown in FIG. According to FIG. 7, even when DNA encoding a mitochondrial ADP / ATP carrier protein containing the above amino acid substitution mutant is introduced, the ability to produce glycolipid biosurfactant is not improved. This result shows that the production efficiency of glycolipid biosurfactant was improved by the normal functional expression of mitochondrial ADP / ATP carrier protein.

The above results indicate that by controlling the expression of mitochondrial ADP / ATP carrier protein, it is possible to control the redox balance and ATP flow in the cell without seriously affecting the life activity. Genes and proteins are one of the target genes to improve the production technology of highly functional glycolipid biosurfactants expected to be put to practical use in a wide range of industries by gene recombination technology. It is shown that the production efficiency can be greatly improved by introducing Pseudozyma into yeast. Since this technology may be able to control the intracellular redox balance and ATP flow of organisms, it is expected to be applied to microbial processes that make use of genetic recombination technologies such as biorefinery that is being studied around the world. it can.

(Example 5)
"Mitochondrial ADP / ATP carrier protein expression and production of glycolipid biosurfactant in Pseudozyma yeast"
Transformants of Pseudozyma rugulosa and Pseudozyma fusiformata were obtained by electroporation using the above gene expression vector pUXV1-PaAAC or the control vector pUXV1 which does not contain an insert. For selection of transformants, hygromycin B was used.

(Production of glycolipid biosurfactant by Pseudozyma yeast transformants)
After culturing a) for 1 day, culturing b) for 7 days, adding an equal amount of ethyl acetate to the obtained bacterial cell culture, stirring sufficiently, and then separating the ethyl acetate layer . The amount of produced glycolipid biosurfactant contained in the ethyl acetate layer was quantified by high performance liquid chromatography. As a comparative example, transformants of Pseudozyma rugulosa and Pseudozyma fusiformata obtained by transforming with a control vector were cultured under the same conditions, and the production amount of glycolipid biosurfactant (mannosyl erythritol lipid) was similarly measured by high performance liquid chromatography. Quantified graphically. The results are shown in FIG. The peak detected by high performance liquid chromatography coincided with that of a known mannosyl erythritol lipid. According to FIG. 8, glycolipid biosurfactant production capacity transformants Pseudozyma rugulosa and Pseudozyma Fusiformata for holding the gene expression vector pUXV1-PaAAC is, Pseudozyma rugulosa and Pseudozyma Fusiformata transformant which holds the vector PUXV1 as a control Reaches about 1.2 times or 1.9 times. From the above results, not only Pseudozyma antarctica but also known mannosylerythritol lipid-producing bacteria can be expected to improve glycolipid production efficiency by forced expression of PaAAC gene (mitochondrial ADP / ATP carrier protein gene).

The deduced amino acid sequence encoded by P. antarctica 's unknown function gene (PsAAC) is encoded by S. cerevisiae mitochondrial ADP / ATP carrier proteins (AAC1, AAC2 and AAC3), Kluyveromyces lactis mitochondrial ADP / ATP carrier protein (KlAAC), Neurospora The figure which arranged for comparison with the mitochondria ADP / ATP carrier protein (NcAAC) of crassa and human mitochondrial ADP / ATP carrier protein (ANT1), and also showed the result of having analyzed the characteristic on the primary structure. The deduced amino acid sequence encoded by the P. antarctica unknown function gene (PsAAC) and the results of hydropathy analysis of the mitochondrial ADP / ATP carrier protein (AAC2) of S. cerevisiae , and the deduced amino acid encoded by the unknown function gene of P. antarctica The figure revealing that the sequence has the same secondary structural features as the mitochondrial ADP / ATP carrier protein. It is possible to introduce a gene expression vector capable of expressing a P. antarctica function unknown gene (PsAAC) into a mitochondrial ADP / ATP carrier protein deficient strain of S. cerevisiae to complement the phenotype of the deficient strain. Figure clarified that the unknown function gene is a mitochondrial ADP / ATP carrier protein. S. cerevisiae strain carrying a gene expression vector capable of expressing P. antarctica function-unknown gene (PsAAC) was stained with rhodamine 123 and observed under a fluorescence microscope, and the respiratory activity of the transformant was improved. The figure which shows having done. The figure which shows that the glycolipid type | mold biosurfactant production efficiency is improving in the P. antarctica transformant holding the gene expression vector which can express the function unknown gene (PsAAC) of P. antarctica . The figure which shows the base sequence of the gene (PsAAC) which codes a mitochondrial ADP / ATP carrier protein, and its variation | mutation location. The figure which shows the result of having investigated the glycolipid type | mold biosurfactant productivity of the transformant which introduce | transduced the mitochondrial ADP / ATP carrier protein gene (PsAAC) which is an amino acid substitution mutant. The figure which shows the result of having investigated the effect with respect to glycolipid type | mold biosurfactant productivity of the mitochondrial ADP / ATP carrier protein gene (PsAAC) in Pseudozyma rugulosa and Pseudozyma fusiformata.

Claims (4)

Culturing a transformant in which a host cell having glycolipid production ability is transformed with a recombinant vector containing a gene encoding a mitochondrial ADP / ATP carrier protein, and collecting the glycolipid from the obtained culture A method for producing a glycolipid, which is characterized.   The method for producing a glycolipid according to claim 1, wherein the gene encoding the mitochondrial ADP / ATP carrier protein is the gene described in 1) or 2) below.
1) Gene encoding the following protein a) or b)
  a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1,
  b) a mitochondrial ADP / ATP carrier protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 1;
2) Any of the following genes c) or d)
  c) a gene consisting of the base sequence shown in SEQ ID NO: 2,
  d) A gene in which one or several genes are deleted, substituted and / or added in the gene shown in SEQ ID NO: 2 and which encodes a mitochondrial ADP / ATP carrier protein.   The method for producing a glycolipid according to claim 1 or 2, wherein the host cell having the ability to produce glycolipid is a microorganism belonging to a fungus.   The method for producing a glycolipid according to any one of claims 1 to 3, wherein the microorganism belonging to the fungus is a microorganism belonging to the genus Saccharomyces or Pseudozyma.