WO2014109360A1

WO2014109360A1 - Method for highly efficiently producing heterologous protein

Info

Publication number: WO2014109360A1
Application number: PCT/JP2014/050233
Authority: WO
Inventors: 宏子北本; 渡部　貴志; 友岳森田; 基夫小板橋; 吉田　重信
Original assignee: 独立行政法人農業環境技術研究所; 独立行政法人産業技術総合研究所
Priority date: 2013-01-09
Filing date: 2014-01-09
Publication date: 2014-07-17
Also published as: JP6413117B2; JPWO2014109360A1

Abstract

Provided is a method which comprises developing a high-expression promoter of Pseudozyma antarctica, constructing a heterologous protein production system with the use of the obtained high-expression promoter, and then efficiently producing an enzyme using a strain belonging to the order Ustilaginales including a yeast of the genus Pseudozyma. The heterologous protein production method according to the present invention comprises: a step for culturing host cells, which have been transformed with an expression vector that contains a base sequence represented by SEQ ID NO:1 or SEQ ID NO:7 and a base sequence, said base sequence encoding a heterologous protein, functionally bound thereto, under such conditions as allowing the expression of the heterologous protein; and a step for harvesting the heterologous protein from a liquid culture medium.

Description

Highly efficient method for producing heterologous proteins

This study relates to a nucleic acid molecule containing the base sequence of the high expression promoter of the foliar yeast Pseudozyma antarctica, an expression vector containing the nucleic acid molecule, and a highly efficient method for producing a heterologous protein using the expression vector.

With the background of the depletion of fossil resources such as petroleum, the development of so-called biorefinery technology that uses biomass, which is one of the renewable resources, to produce energy and industrial raw materials is being promoted worldwide. In particular, for the production of useful proteins using microorganisms, an expression vector or gene fragment in which a strong gene expression promoter is linked upstream of the gene encoding this useful protein is introduced into the host cell. The transformant is used. Until now, host / expression vector systems have been constructed in many microorganisms, but the productivity of the intended useful protein may be low. In general, in order to improve the productivity of a target useful protein, a host vector system with higher expression efficiency is developed. For example, a means for discharging the produced target protein out of the cells and accumulating it in the medium can be said to be one of effective means for improving the protein production amount. It is also effective to use a promoter sequence or terminator sequence with high gene transcription activity to increase the target protein production rate by increasing the gene transcription rate. Furthermore, since the properties of microorganisms that serve as hosts vary widely, for example, the development of new host vector systems for various microorganisms with different assimilation and temperature sensitivity of carbon sources can improve the production of heterologous proteins. Therefore, it can be said that it is extremely important. These technological developments can not only produce heterogeneous proteins, but also create microorganisms that can produce functional chemicals by changing traits by producing heterologous proteins. This is an extremely important issue for the development of biorefinery technology.

When producing the desired heterologous protein, there are means for producing intracellularly (mainly using E. coli) and means for producing extracellularly (mainly using yeast and filamentous fungi). In general, since purification is easy and production costs can be kept low, it is considered that means for producing extracellularly is more practical. On the other hand, compared to filamentous fungi, since yeast is a unicellular organism, there is an advantage that it is easy to produce transformants and control culture. For this reason, there have been many studies using yeast as a host, but mainly ascomycetous yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, etc.) are used.

The genus Pseudozyma is a plant basidiomycetous yeast generally found from the surface of leaves and fruits. P. antartcica is found in rice stems and leaves, rice cakes, and brown rice, while P. aphidis, P. rugulosa, and P. tsukubaensis are found in the skins of vegetables and fruits such as pakchoi, mizuna and perilla. For example, in P. antarctica, tens of thousands to hundreds of thousands of live bacteria are isolated per gram of 籾, and when analyzing the raw weight of raw vegetables 10 mg, Pseudozyma yeast is isolated from almost all, Humans have a history of eating Pseudozyma yeast with plant foods. Pseudozyma yeast is known to produce lipase, and P. antartcica lipase (CALA, CALB) has abundant industrial use results such as use in detergents. In addition, it is known to produce glycolipid mannosyl erythritol lipids (MELs), a kind of biosurfactant with various physical properties and functions, and MELs derived from P. tsukubaensis から have a high skin protective effect. It is used as an additive for enhancing the functionality of commercial cosmetics. As described above, the yeast of the genus Pseudozyma can be expected as a promising host for producing high secretion of heterologous proteins since it has an excellent ability to produce substances. In addition, Pseudozyma yeasts are thought to have less protease secretion than the Ascomycetes fungi, so the produced heterologous proteins can be stably accumulated in the medium.

In order to highly express a heterologous protein using Pseudozyma yeast as a host, a strong expression promoter, secretion signal, and terminator that effectively function or express in the cell are required. In P. flocculosa and P. arcantarctica, promoters of heat shock protein 70 and glyceraldehyde-3-phosphate dehydrogenase derived from the phytopathogenic yeast Ustilago maydis (Non-patent document 1), β-glucosidase promoter derived from P. tsukubaensis (non-patented) There are reports using literature 2) and the P. flocculosa actin promoter (Non-patent literature 3). Various heterogeneous protein production systems using each promoter have been constructed, but have not yet been put into practical use. For this reason, the development of promoters with higher gene transcriptional activity, efficient secretion signals, and expression-inducible promoters that can control the timing of expression is necessary for the practical application of heterologous protein production technology using Pseudozyma yeast. It has become a challenge. Furthermore, by utilizing the characteristics of yeasts belonging to the genus Pseudozyma that can use various carbon sources, there is a need for the development of a promoter that can highly express the target gene when cheap raw resources such as plant biomass are used as raw materials.

On the other hand, polyethylene agricultural multi-films are consumed approximately 1.3 million tons annually in the world every year, and much labor is required for recovery processing. Therefore, agricultural multi-film made of biodegradable plastic has begun to be used. Currently, the market price of biodegradable mulch is about three times that of polyethylene mulch, but since it is poured and decomposed after use, there is no cost for stripping and disposal, and the unit area required for crop harvesting It is estimated that the cost related to multi-use is almost the same. If the usage increases in the future, the price can be expected to fall.

However, since biodegradable plastic materials are slowly decomposed in the environment, it may not be properly decomposed when they are used up. It is. Therefore, a method for quickly decomposing used materials by treating an enzyme that accelerates the degradation of biodegradable plastics has been proposed, and it has already been isolated from P. antarctica (Patent Document 1) separated from rice and barley. Powerful biodegradable plastic-degrading bacteria such as the selected filamentous fungus NITE 文献 P-573 (Patent Document 2) were selected, biodegradable plastic-degrading enzyme PaE derived from Pseudozyma antarctica, biodegradable plastic derived from filamentous fungus NITE P-573 Enzymes that efficiently decompose biodegradable plastic materials, such as the degrading enzyme PCLE, have been isolated and identified. In addition, by applying a PaE or PCLE solution to the surface of the biodegradable mulch, superior degradation characteristics with respect to the biodegradable mulch have been found. However, as described above, since the price of the current biodegradable plastic is almost the same as the price obtained by adding the stripping cost to polyethylene multi, it is desirable to reduce the cost of the enzyme as much as possible. If mass production of biodegradable plastic-degrading enzymes can be efficiently produced by gene recombination technology, lower-priced enzymes can be provided, and this will lead to the expansion of the use of agricultural multi-film made of biodegradable plastics. It is thought that you can.

Enzymes produced by gene recombination technology are widely used for processing fibers and additives for detergents. Upon introduction, it was confirmed that there was no problem even if it was irritating, allergenic, or discharged into the environment. In addition, based on the safety examination standards of the Ministry of Health, Labor and Welfare, the safety assessment standard for food additives manufactured using genetically modified microorganisms established by the Food Safety Committee of the Cabinet Office (decided on March 25, 2004) ) In principle, genetically modified additives that can be evaluated for safety are limited to the use of recombinants derived from non-pathogenic hosts with experience in the manufacture of additives or food experience. Has been. Enzymes that are applied directly to agricultural mulches after crop harvesting are not directly ingested, but are most safe and suitable for enzymes produced in an experienced host such as Pseudozyma yeast. Therefore, its mass production technology is required.

Here, in Cryptococcus sp. S-2 strain, which belongs to basidiomycetous yeast as well as Pseudozyma genus yeast, expression vectors using promoters upstream and downstream terminators of cutinase-like enzyme, amylase and xylanase have been developed. (Non-patent Document 4), which induces expression of heterologous protein genes downstream of the promoter in the presence of vegetable oil, maltose, and xylose, respectively.

However, this Cryptococcus sp. S-2 strain has been selected from soil and airborne yeast, and its origin is unknown. In addition, since the optimum culture temperature is 25 ° C. and it does not grow at a higher temperature (30 ° C. or higher), there is a demerit that when it is cultured in large quantities, a cooling cost for heat generation is required. P. antarctica has a history that has been derived from the plant surface and has been naturally ingested by humans as described above, and can grow even at 35 ° C. It has superior characteristics compared to Cryptococcus sp. S-2. Have. P. antarctica is the same basidiomycetous yeast as Cryptococcus sp. S-2, but is systematically far away, so when using a mechanism that induces gene expression in response to a specific stimulus. In addition, it is very unlikely that an expression vector developed in Cryptococcus sp. S-2 strain will work in Pseudozyma yeast. Therefore, it is necessary to develop a powerful gene expression system unique to Pseudozyma yeast. In addition, the method of mass production of P. antarctica-derived enzymes, such as biodegradable plastic degrading enzymes, is a method that consists only of genes derived from P. antarctica (self-cloning). high. Therefore, also from the above points, there is a demand for a technique for mass production of an enzyme that can use a host with a dietary experience such as yeast of the genus Pseudozyma.

Japanese Patent No. 4915593 Japanese Patent No. 5082125

The present invention developed a high-expression promoter of P. antarctica and constructed a heterologous protein production system using the obtained high-expression promoter, and the enzyme was efficiently used using the Ustilaginales (Krochomycetes) strain containing Pseudozyma yeast. It is an object of the present invention to provide a manufacturing method.

In order to achieve the above object, the present inventors have cultivated foliar yeast P. antarctica using various carbon sources, and as a result, when xylose, which is a constituent of herbaceous biomass, is used as a carbon source, We discovered for the first time that there are unknown proteins produced in large quantities. Further investigation was conducted, and the gene sequence (PANT_8c00118) of this unknown protein was identified from the whole genome sequence of the P. antarctica T34 strain. Furthermore, using the promoter base sequence and terminator sequence, the biodegradable plastic-degrading enzyme (PaE mass production system derived from P. antarctica) was successfully constructed, and the present invention was completed.
Since the novel protein is secreted and produced at a high concentration in the culture solution in the presence of xylose, the secretion amount of the heterologous protein into the culture solution can be controlled by using the secretory signal sequence.
Furthermore, by culturing in the presence of xylose, it was confirmed that other yeasts of the genus Psudozyma also secreted a protein with the same molecular weight of about 33 kDa in the culture medium as in the case of P. antarctica. .

The present invention provides a novel promoter nucleotide sequence upstream of the gene PANT_8c00118 derived from P. 18antarctica that induces the expression of downstream genes in the presence of xylose, in order to strongly and strictly control the expression of heterologous protein genes in Pseudozyma spp. An important feature is that it is used as a new promoter and enhances the secretion and productivity of heterologous proteins by placing or replacing the new signal sequence of PANT_8c00118 as a secretory signal sequence of the heterologous protein upstream of the target protein gene. And includes the following aspects.

(1) A nucleic acid molecule comprising the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 7.
(2) A nucleic acid molecule comprising a base sequence having at least 80% homology with the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 7, and having promoter activity.
(3) A nucleic acid molecule comprising the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 7.
(4) An expression vector comprising the nucleic acid molecule according to any one of (1) to (3) above or a fragment thereof having promoter activity.
(5) A base sequence shown in SEQ ID NO: 2 or SEQ ID NO: 8, or a base having terminator activity having at least 70% homology with the base sequence shown in SEQ ID NO: 2 or SEQ ID NO: 8. The expression vector according to (4), further comprising a sequence.
(6) The expression vector according to (4) or (5) above, further comprising a base sequence encoding a heterologous protein operably linked to the nucleic acid molecule.
(7) The expression vector according to (6) above, wherein the heterologous protein is a biodegradable plastic degrading enzyme PaE derived from yeast of the genus Pseudozyma or a biodegradable plastic degrading enzyme PCLE derived from the filamentous fungus NITE P-573.
(8) Adjacent to the upstream of the base sequence encoding the heterologous protein, at least the base sequence shown in SEQ ID NO: 3 or SEQ ID NO: 9, or the base sequence shown in SEQ ID NO: 3 or SEQ ID NO: 9 The expression vector according to (6) or (7) above, further comprising a base sequence having 90% homology and having a function as a secretion signal.
(9) A host cell transformed with the expression vector according to any one of (6) to (8) above.
(10) The host cell according to (9) above, which is a yeast of the genus Pseudozyma or a genus Ustilago.
(11) The host cell according to (9) above, which is Pseudozyma antarctica.
(12) a step of culturing the host cell according to any one of (9) to (11) above under conditions that allow the expression of the heterologous protein, and a step of recovering the heterologous protein from the culture solution. A method for producing a heterologous protein.
(13) The production method according to (12) above, wherein the host cell is cultured in a medium containing xylose (14) A protein encoded by the base sequence represented by SEQ ID NO: 4.
(15) A biodegradable plastic degradation preparation comprising the protein according to (14).

According to the present invention, by newly using the base sequence of the promoter upstream of the PANT_8c00118 gene of P. antarctica as a novel high expression promoter, heterologous proteins can be efficiently used with Pseudozyma yeast (for example, P. anatarctica) as a host. The remarkable effect is that it can be produced and accumulated in the medium. For example, in PaE, it was confirmed that the production amount was about 10 times higher than the production amount of the parent strain having an origin isolated from rice straw.
In addition, since the main component of the protein composition of the culture supernatant of the present invention is the target heterologous protein, it can be used directly as an enzyme solution by removing cells that are recombinants with a membrane filter or the like. .

Results of SDS-PAGE and CBB staining of P. antarctica broth with modified FMM using xylose as carbon source. M: marker, 1: reference strain P. antarctica JCM10317 strain, 2-10: isolated rice straw P. antarctica, 11: P. antarcticaT34 strain Results of SDS-PAGE and CBB staining of Pseudozyma yeast and Ustilago yeast culture with modified FMM using xylose as carbon source. M: Marker, 1: P. tsukubaensis, 2: P. thailandia, 3: P. aphidis, 4: P. prolifica, 5: P. flocculosa, 6: P. rugulosa, 7: Ustilago maydis MAFF235374, 8: U. maydis MAFF236375, 9: U. esuculenta, 10: P. antarctica T34 strain This shows the PANT_8c00118 gene sequence of P. antarctica T34 strain and the base sequence including the promoter region and terminator region (SEQ ID NO: 5). The base sequence in the frame is the base sequence of the structural gene of the PANT_8c00118 gene (SEQ ID NO: 4). Intron sequences are underlined and the start codon ATG and stop codon TGA are further framed. The production of pPAX1-neo is shown. The production of pPAX1-neo-PaCLE1 is shown. Evaluation of inducible production of recombinant bioplastic degradation enzyme PaE by xylose of pPAX1-neo-PaCLE1 transformant of P. antarctica T34 strain. Figure 2 shows halo formation by PBSA emulsion degradation. The PBSA emulsion decomposition | disassembly activity when using each saccharide | sugar source of the pPAX1-neo-PaCLE1 transformant of P. antarctica T34 strain is shown. Evaluation of inducible production of recombinant bioplastic degradation enzyme PaE by xylose of pPAX1-neo-PaCLE1 transformant of P.ulrugulosa JCM10323 strain. Figure 2 shows halo formation by PBSA emulsion degradation. The parental strain JCM10323 and transformants 1-7. Evaluation of inducible production of recombinant biodegradation enzyme PaE by xylose of pPAX1-neo-PaCLE1 transformants of P. tsukubaensis JCM10324 and P. aphidis JCM10318. PBSA emulsion degradation activity. Time course of dry cell weight and enzyme activity of pPAX1-neo-PaCLE1 (excluding UARS) transformants (GB4 (0) -PaE14) of P. antarctica GB-4 (0) strain in 5L Jar fermenter Indicates. PaE production in fed-batch culture (A), PaE production in high-density fed-batch culture (B). The production of pPAXn-PCLE is shown. Evaluation of inducible production of recombinant bioplastic degradation enzyme PaE by xylose of pPAXn-PCLE transformant of P. Pantarctica T34 strain. Figure 2 shows halo formation by PBSA emulsion degradation. The time-dependent changes in dry cell weight and enzyme activity of pPAXn-PCLE transformant (GB4 (0) -PCLE21 strain) of P. antarctica GB-4 (0) strain in 5L Jar fermenter are shown. The structural gene sequence of the xylose-inducible protein of P. antarctica GB-4 (0) strain and the base sequence including the promoter region and terminator region are shown (SEQ ID NO: 7). The base sequence in the frame is the base sequence of the structural gene of the xylose-derived protein. Intron sequences are underlined and the start codon ATG and stop codon TGA are further framed.

The present invention relates to a nucleic acid molecule comprising the base sequence shown in SEQ ID NO: 1. The base sequence shown in SEQ ID NO: 1 is a sequence existing upstream of a base sequence (SEQ ID NO: 4) encoding a protein having a molecular weight of about 33 kDa newly found in yeast of Pseudozyma antarctica.
The present invention further relates to a nucleic acid molecule comprising a base sequence having at least 80% homology with the base sequence shown in SEQ ID NO: 1 and having promoter activity. The base sequence is preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 and the base sequence shown in SEQ ID NO: 1. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology. Alternatively, as long as the base sequence has promoter activity in the protein production method defined in the present invention, one or several bases may be deleted, substituted or added. More preferably, the nucleic acid molecule of the present invention consists of the base sequence shown in SEQ ID NO: 1.

Furthermore, the present invention relates to a nucleic acid molecule comprising the base sequence shown in SEQ ID NO: 7. The base sequence shown in SEQ ID NO: 7 is a sequence existing upstream of the base sequence (SEQ ID NO: 10) encoding a protein of about 33 kDa molecular weight newly found in the yeast GB4 (0) strain of Pseudozyma antarctica. is there.
The present invention further relates to a nucleic acid molecule comprising a base sequence having at least 80% homology with the base sequence shown in SEQ ID NO: 7, and having promoter activity. The base sequence is preferably at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 and the base sequence shown in SEQ ID NO: 7. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology. Alternatively, as long as the base sequence has promoter activity in the protein production method defined in the present invention, one or several bases may be deleted, substituted or added. More preferably, the nucleic acid molecule of the present invention consists of the base sequence shown in SEQ ID NO: 7.

The present invention further relates to an expression vector comprising the above nucleic acid molecule or a fragment thereof having promoter activity. A fragment of the nucleic acid molecule of the present invention having promoter activity can be appropriately selected by a person skilled in the art by examining the promoter activity of the fragment according to a well-known method. Also, the length (base length) of the above fragment is not particularly limited as long as it has promoter activity, and is 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 bases or more. It is preferable. More preferably, the length of the fragment is 555, 560, 565, 570, 575, 580, 585, or 590 bases or more. Further, it may include a sequence in which sequences of 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 bases or more included in SEQ ID NO: 1 or SEQ ID NO: 7 are repeatedly linked. . Vectors suitable for gene expression are known in the art, and can be appropriately selected by those skilled in the art in consideration of combinations with host cells described below. As an expression vector, for example, a plasmid pUXV1 (available from ATCC) or a chromosomal transfer vector can be appropriately modified as necessary.

The expression vector of the present invention is a terminator sequence of the PANT_8c00118 gene defined in SEQ ID NO: 2, or a sequence defined in SEQ ID NO: 8 (terminator sequence of a PANT_8c00118 homologous gene derived from the GB-4 (0) strain of the same Pseudozyma antarctica) ) May be included. Alternatively, the expression vector of the present invention has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78 with the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 8. %, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, It may contain a nucleotide sequence having 95%, 96%, 97%, 98%, 99% homology and having terminator activity. As long as the base sequence has terminator activity in the protein production method defined in the present invention, one or several bases may be deleted, substituted or added.

Furthermore, the expression vector of the present invention may further comprise a base sequence encoding a heterologous protein operably linked to the nucleic acid molecule. In the present invention, “operably linked” means that transcription of a heterologous protein is regulated under the influence of the promoter activity of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 7 in an expression vector. It means that it is connected. The heterologous protein can be appropriately selected by those skilled in the art as to the protein that needs to be expressed, and is not limited to the biodegradable plastic degrading enzyme shown in the Examples. Preferable heterologous proteins include, for example, biodegradable plastic degrading enzyme PaE derived from yeast of the genus Pseudozyma or biodegradable plastic degrading enzyme PCLE derived from filamentous fungus NITE P-573. The above base sequence may be a cDNA base sequence.

The expression vector of the present invention is adjacent to the upstream of the base sequence encoding the heterologous protein, and is shown in SEQ ID NO: 3 or SEQ ID NO: 9, or shown in SEQ ID NO: 3 or SEQ ID NO: 9. A nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology with the nucleotide sequence and functioning as a secretion signal Further, it may be included. SEQ ID NO: 3 is considered to correspond to the secretory signal sequence of the protein encoded from the start codon of the PANT_8c00118 gene (SEQ ID NO: 4) to 102 bp, and SEQ ID NO: 9 is the same Pseudozyma antarctica GB-4 (0 It is considered that it corresponds to the secretory signal sequence of the protein encoded from the start codon of the PANT_8c00118 homologous sequence (SEQ ID NO: 10) derived from the strain to 103 bp. By including the sequence, as will be described later, the target heterologous protein can be highly secreted and produced outside the microbial cell using a host cell transformed with the expression vector.
In the expression vector of the present invention, for example, as described above, a promoter of a PANT_8c00118 gene, a heterologous protein cDNA base sequence, a terminator (eg, PANT_8c00118 gene terminator) gene base sequence is arranged, and a marker gene base sequence (eg, amino acid or It is possible to construct an expression cassette with a base synthesis gene and an antibiotic system gene).

The present invention further relates to a host cell transformed with the above expression vector. The host cell can be appropriately selected by those skilled in the art in consideration of the combination with the expression vector to be used, and P. antarctica and its related species Pseudozyma genus yeast, such as P. antarctica, P. tsukubaensis, Examples include yeasts of the genus Pseudozyma such as P. rugulosa and P. aphidis, and Ustilaginales such as Ustilago genus such as Ustilago maydis. Among the above host cells, yeasts belonging to the genus Pseudozyma are preferable, and Pseudozyma antarctica is particularly preferable.

The present invention further includes a step of culturing the above host cell containing a base sequence encoding a heterologous protein under conditions that allow expression of the heterologous protein, and a step of recovering the heterologous protein from the culture solution. The present invention relates to a method for producing a heterologous protein.
The culture conditions can determine the composition of the medium and the culture temperature depending on the type of the vector and the host cell, and culture can be performed at 25 to 40 ° C., preferably 30 ° C.
In the method of the present invention, the heterologous protein can be highly expressed by culturing host cells in a medium containing xylose. Xylose can be added to the medium in an amount of 2 to 12% by mass, preferably 4 to 8% by mass. When producing using a culture apparatus such as a jar, the culture is carried out while keeping the aeration conditions at 0.5-3 vvm (LPM / L), preferably 1-2 vvm, and maintaining the dissolved oxygen concentration at 20-50% of the saturation.
Also, by 24 hours at the beginning of the culture when the enzyme production efficiency is low, an inexpensive carbon source instead of xylose, for example, sucrose, glycerol is sufficient to obtain 2 to 4% by mass in the medium, or an equivalent amount of microbial cells, After growing the cells at high density using molasses, etc., xylose with a final concentration of 10-20% by mass is continuously fed to induce enzyme production. Production is possible.

The recovery of the heterologous protein from the culture solution can be performed by any method. For example, ammonium sulfate may be added to the supernatant of the culture solution to obtain a precipitate, or the culture solution may be concentrated using a ultrafiltration method using Asahi Kasei Microza or the like. Further, the precipitate may be dialyzed with a 20 mM Tris-HCl buffer or the like using a dialysis tube, and then the coexisting protein may be removed by passing through a DEAE-sepharose column, and if necessary, SP-sepharose. The enzyme may be purified through a column. Alternatively, concentration and contaminant removal may be performed in one step using the property (affinity) that the heterologous protein binds to the substrate.

The present invention further relates to a protein encoded by the base sequence represented by SEQ ID NO: 4 and a biodegradable plastic degradation preparation containing the protein. Biodegradable plastic degradation preparations can be prepared by those skilled in the art by well-known methods, and can also be mixed with well-known solvents, additives and the like.
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by this.

1. Acquisition of a gene whose expression is strongly induced by xylose When the present inventors were studying the production conditions of the biodegradable plastic-degrading enzyme produced by Pseudozyma antarctica, the reference strain Pseudozyma antarctica JCM10317 was used with various carbon sources. As a result, it was found that there is an unknown protein having a molecular weight of about 33 kDa that is strongly induced only in the presence of xylose and secreted in a large amount in the medium. Accordingly, it was confirmed whether a plurality of P. antarctica strains owned by the present inventors secrete unknown proteins having a molecular weight of about 33 kDa in the presence of xylose in the same manner as the reference strain.
What was cultured in YM medium at 30 ° C. for 24 hours was used as a preculture solution. In the main culture, a modified FMM was used to sterilize the carbon source (8% xylose) and other medium components separately and mix under sterilized conditions in order to avoid the formation of culture inhibitors such as browning and furfural. . Each composition is shown below.
YM medium (Yeast extract Malt extract Medium)
Yeast extract 0.3%
Malt extract 0.3%
Bacto peptone 0.5%
Glucose 1.0%

Modified FMM (Fungal minimum medium)
NaNO ₃ 0.2%
KH ₂ PO ₄ 0.06%
MgSO ₄・ 7H ₂ O 0.06%
Yeast extract 0.3%
Xylose 8.0%

20 ml of each main culture solution was placed in a 100 ml Erlenmeyer flask, inoculated with 200 μl of the aforementioned preculture solution, and cultured at 30 ° C. for 200 hours with shaking at 96 rpm. The resulting culture broth was electrophoresed by SDS-PAGE, and CBB staining was performed. As a result, among the 11 strains of P. antarctica tested, 10 strains had a molecular weight slightly lower than the 31 kDa marker position. It was confirmed that a large unknown protein of about 33 kDa was highly secreted (see FIG. 1). Among the tested samples, the P. antarctica T34 strain does not produce the biodegradable plastic-degrading enzyme PaE (thought to be expressed at this time) by xylose, so this was used thereafter.

Next, using 6 Pseudozyma yeasts and 3 Ustilago yeasts, which are closely related to P. antarctica, as in P. antarctica, we investigated whether an unknown protein with a molecular weight of about 33 kDa is secreted and produced by xylose. It was. As a result, P. tsukubaensis JCM10324 strain, P. aphidis JCM10318 strain, P. flocculosa JCM10321 strain, P. rugulosa JCM10323 strain, U. zeae (maydis) MAFF235374 strain, MAFF236375 strain were found in the culture solution as 31kDa protein molecular weight marker. It was found that a large amount of unknown protein was secreted, showing mobility corresponding to a position slightly larger than the band corresponding to (see Fig. 2). From this, it was found that the above yeast secreted and produced a protein with unknown function having a molecular weight of about 33 kDa in the presence of xylose as in the case of P. antarctica.

Next, a new gene sequence encoding this unknown protein was obtained. First, in order to obtain gene information of P. antarctica T-34 strain, DNA was extracted from the cells and physically fragmented, and then a Mate-pair library having an inserted fragment length of 3 to 4 kb was prepared. The obtained Mate-pair library was mixed with microbeads for DNA addition, and the Mate-pair fragment was amplified by emulsion PCR, and then the microbeads were filled in a picotiter plate and aligned. This picotiter plate was attached to a DNA sequencer (FLX Titanium), and the genomic DNA sequence was analyzed. The obtained primary data was used to assemble a sequence to obtain a contig sequence, and the putative translation region on the genomic DNA sequence was comprehensively extracted by homology search.

Subsequently, an unknown protein of about 33 kDa from the P. antarctica T34 strain was blotted onto a PVDF membrane from SDS-PAGE, and then the N-terminal amino acid sequence was analyzed. A 10-residue sequence (AATTLNAAIK: SEQ ID NO: 6) )was gotten. As a result of searching for genes having this sequence from the gene information of the prepared P. antarctica, the gene sequence PANT_8c00118 (see FIG. 3) matched 100%. The amino acid sequence was estimated from the gene sequence, and the molecular weight from the N-terminal amino acid sequence of the secreted protein was estimated to be 32,899.48 Da, which was consistent with the value of SDS-PAGE, confirming that the unknown protein was a protein encoded by PANT_8c00118 It was.
In addition, since this unknown protein is a secreted protein that is secreted into the culture solution at a very high concentration, the protein from the start codon is determined based on the N-terminal amino acid sequence of this protein purified from the culture solution. It was speculated that there was a strong secretion signal in 102 bp immediately before the gene sequence encoding the N-terminal amino acid sequence.

2. Construction of gene expression vector using promoter region of PANT_8c00118 gene A restriction enzyme site was added to the end of the promoter region and terminator region of the obtained PANT_8c00118 gene to construct plasmid pPAX1-neo shown in FIG.
This plasmid is based on the commercially available plasmid pUXV1 (available from ATCC). Instead of Pgap, a plurality of PANT_8c00118 gene promoter (SEQ ID NO: 1) and terminator (SEQ ID NO: 2) regions of P. antarctica T34 strain are used. In place of the hygromycin resistance gene, and a gene fragment in which the T34 strain-derived homocitrate synthase promoter and its downstream E. coli-derived neomycin resistance gene are linked.
FIG. 5 shows that this plasmid incorporates the cDNA gene PaCLE1 encoding the biodegradable plastic-degrading enzyme PaE derived from P. antarctica JCM10317. The constructed plasmid pPAX1-neo-PaCLE1 was introduced into the P. antarctica T34 strain according to the following method.

P. antarctica T34 strain was precultured in 5 ml YM medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% D-glucose) at 30 ° C. overnight. 100 μl of the preculture solution was inoculated into 50 ml of YM medium and cultured at 30 ° C. until OD660 = 1.3 to 1.5.
The culture solution was centrifuged at 3000 × g for 5 minutes at 4 ° C. to recover the cells. The collected cells were suspended in 20 ml of ice-cold 0.5 M sorbitol and centrifuged again at 4 ° C. and 3000 × g for 5 minutes to wash and collect the cells. Thereafter, the recovered cells were suspended in 10 ml of ice-cold 1M sorbitol, and centrifuged at 4 ° C. and 3000 × g for 5 minutes to recover the cells. The collected cells were suspended in 1 ml of ice-cold 1M sorbitol.
The above operation was performed on ice.
50 μl of the cell suspension and 50 μl of plasmid DNA (about 5 to 10 μg) were added to an electroporation cuvette (BIO-RAD) with a gap of 0.2 cm and incubated on ice for 5 minutes. A cuvette was set in E. coli pulser (BIO-RAD), and electroporation was performed under conditions of 1.5 kV, 25 kF, and 400 Ω. Thereafter, ice-cold YMS (YM + 1M sorbitol) was immediately added, mixed by pipetting, transferred to a 15 ml polyurethane tube, and incubated at 30 ° C. for 2 hours. Apply to a YMS plate containing antibiotic G418 (YMS + 0.05% G418, 2% agar) and incubate at 30 ° C until colonies form. The resulting transformant colonies contain the new antibiotic G418. Planted on YMS plate.

Transformants grown on the YMS medium containing antibiotics were added to FMM plates containing PBSA emulsion (0.1% yeast extract, 0.2% NaNO ₃ , 0.02% KH ₂ PO ₄ , 0.02% MgSO ₄ .7H ₂ O, 2 % Xylose, 1% PBSA emulsion (EM-301, Showa Denko KK, 2% agar), and statically cultured at 30 ° C. The T34 strain used as the parent strain does not produce biodegradable plastic-degrading enzyme PaE in the presence of xylose, but the transformant transforms the cloudy PBSA emulsion in the medium due to the activity of PaE induced by xylose. A transparent halo is formed (Figure 6).
Such a transformant was selected.

In order to confirm the substrate-inducibility of gene expression under the control of the PANT_8c00118 gene promoter, transformants incorporating the previously selected pPAX1-neo-PaCLE1 were transformed into 1% yeast extract solution with 6% of various sugar sources (glucose, When cultured in media supplemented with galactose, xylose, arabinose, sucrose, and lactose, respectively, it was revealed that PBSA emulsion degradation activity was strongly induced only when xylose was used (FIG. 7).
The enzyme activity was measured as follows. Place 1.9 ml of an aqueous solution in which PBSA emulsion is suspended in 20 mM Tris HCl buffer (pH 6.8) so that the absorbance (OD660) is approximately 0.65, and add 100 μl of the culture supernatant to 180 rpm at 30 ° C. The mixture was shaken for 15 minutes, and the amount of decrease in absorbance was measured. The unit of enzyme activity was defined as 1 U, which is the amount of enzyme that decreases the absorbance by 1.0 per minute.

3. Preparation of transformants in P. antarctica related species and confirmation of expression inducibility It was confirmed that expression induction of the prepared plasmid pPAX1-neo by xylose works well in P. antarctica. Transformants were produced from closely related species, and gene expression induction by xylose was confirmed.
When PPAX1-neo-PaCLE1 was incorporated into P. tsukubaensis JCM10324, P. aphidis JCM10318, and P. rugulosa JCM10323, which are yeasts belonging to the genus Pseudozyma, in the same manner as above, all strains of FMM containing PBSA emulsion On the plate, the emulsion was dissolved in the vicinity of the bacteria to obtain a transformant that formed halo (see FIG. 8, transformant of P. rugulosa JCM10323 strain), and 6% in 1% yeast extract It was confirmed that PBSA emulsion degradation activity was strongly induced in the culture filtrate by culturing in a liquid medium containing xylose (see FIG. 9, transformants of P. tsukubaensis JCM10324 and P. aphidis JCM10318). From these results, it was confirmed that the transformant induced and produced biodegradable plastic degrading enzyme PaE by xylose.
This result indicates that the promoter in the PANT_8c00118 sequence of P. antarctica can express the activity in general yeasts of the genus Pseudozyma. In addition, Ustilago and Pseudozyma are already included in the order of the Ustilaginales (Krochophyta), and the results of similarity comparison using the D1 / D2 sequence of rDNA have already shown that both genera are closely related. The results show the possibility of being used for these genetic manipulations.

4). Examination of mass production with Jar fermenter Subsequently, in order to demonstrate that the produced heterologous protein expression system is effective for mass production of heterologous proteins, a production test of PaE was conducted using a 5L scale Jar fermenter. .
Since pPAX1-neo-PaCLE1 has the U. maydis self-replicating gene UARS, it is difficult to obtain a transformant integrated into a chromosome even if it is linearized by treatment with a restriction enzyme. Therefore, UARS was deleted and linearized by treatment with the restriction enzyme SspI, and then a transformant GB4 (0) -PaE14 strain incorporated into the P. antarctica GB-4 (0) strain was prepared.

First, xylose fed-batch culture was performed using GB4 (0) -PaE14 strain. 3 L of medium with the following composition with low initial xylose concentration was added to 5 L Jar fermenter, inoculated with 30 ml of preculture, and cultured at 30 ° C. with stirring speed of 500 rpm and aeration rate of 8 LPM . Moreover, in order to prevent the culture solution from flowing out due to the formation of high bubbles, a 50-fold diluted disinfectant (Shin-Etsu Silicone KM-72F, Shin-Etsu Chemical Co., Ltd.) was added. In addition, the pH was lowered to 6.0 with aqueous ammonia to reduce the pH due to the use of ammonia ions, adding the nitrogen source. After 24 hours, the following feeding medium for inducing enzyme production was fed at 0.5 L / d.
Medium composition for Jar fermenter
Xylose 2%
NaNO ₃ 0.2%
MgSO ₄・ 7H ₂ O 0.04%
KH ₂ PO ₄ 0.04%
Yeast extract 0.2%
(NH ₄ ) ₂ SO ₄ 0.2%

Fed-batch medium composition for inducing enzyme production
Xylose 20%
YNB w / o AA & AS 0.085%
Yeast extract 0.2%

Enzyme activity increased with the addition of fed-batch medium, and after 72 hours, 56.9 U / mL of enzyme was produced in the culture supernatant, but the subsequent enzyme production rate slowed down to 104 hours. Later, the maximum activity was 64.1 U / mL (see FIG. 10A).
The cause was thought to be a lack of nutrient sources other than xylose in the medium. In addition, the protein production rate at the initial stage of the culture was low, and the protein production rate increased rapidly after 24 hours of culturing when the cells grew to some extent. It was decided to perform high-density fed-batch culture in a state where the protein production rate was kept high by performing only cell growth using the source and feeding xylose from 24 hours onward. The medium composition for high-density culture and the feed medium composition are shown below. Other culture conditions were the same as described above.

Medium composition for high density culture in Jar fermenter
Sucrose 3%
NaNO ₃ 0.2%
MgSO ₄・ 7H ₂ O 0.1%
KH ₂ PO ₄ 0.1%
Yeast extract 0.5%
(NH ₄ ) ₂ SO ₄ 1.0%

Fed-batch medium composition for high-density culture
Xylose 50%
YNB w / o AA & AS 0.085%
Yeast extract 0.2%

The dry cell weight grew to 22.6 g / L in 24 hours from the start of the culture, and then maintained a high protein production rate from the start of the culture to 104 hours by the addition of xylose. A very high concentration of PaE of mL (about 3 g / L) was obtained (see FIG. 10B). This value was about 10 times higher than that of the parent strain.

5. Heterologous protein production using the developed expression vector Next, in order to confirm that the developed expression vector is useful for heterologous protein production, the present inventors produced a biodegradable plastic PCLE derived from the filamentous fungus B47-9. Tried.
FIG. 11 shows pPAXn-PCLE in which the developed expression vector UARS was deleted by restriction enzyme SspI treatment and a cDNA gene sequence encoding PCLE (GenBank Accession No. AB823703) was incorporated. After pPAXn-PCLE was linearized by treatment with the restriction enzyme EcoRI, a transformant, T34-PCLE1 strain, incorporated into the P. antarctica T34 strain was prepared. When the parent T34 and T34-PCLE1 strains were inoculated on the FMM plate containing the PBSA emulsion described above, the T34-PCLE1 strain was decomposed by the production of PCLE, and the formation of halo was confirmed as shown in FIG. .

Furthermore, a transformant GB-4 (0) -PCLE3 strain in which pPAXn-PCLE was incorporated into the GB-4 (0) strain was prepared in the same manner. First, when flask culture was performed using the above-mentioned modified FMM, the PBSA emulsion degrading activity of the parent strain GB-4 (0) was 1.8 U / ml due to the activity of the parent strain derived PaE after 96 hours of shaking culture. On the other hand, GB-4 (0) -PCLE3 strain showed 2.6 U / ml, and the value was remarkably increased by the production of PCLE. In addition, the enzyme PCLE of the filamentous fungus B47-9 strain has a dramatic increase in PBSA emulsion degradation activity by the addition of CaCl ₂ (Suzuki K., et al., Appl. Microbiol. Biotechnol. DOI10.1007 / s00253). -013-5454-0), PBSA emulsion degradation activity with CaCl ₂ added to 1 mM was measured. The parent strain GB-4 (0) was 3.5 U / ml, whereas GB- The strain of 4 (0) -PCLE3 was 5.8 U / ml, and the effect of increasing PCLE activity by adding CaCl ₂ was confirmed.

Next, using the GB-4 (0) -PCLE3 strain described above, high-density culture using Jar-fermenter was performed under the culture conditions described in “4. Examination of mass production with Jar fermenter”. PBSA degradation activity is 22 U / ml in the CaCl ₂ no addition at 72 hours from the start, a high value of 30 U / ml in CaCl ₂ addition was confirmed (Figure 13). Since the value obtained when the parent strain GB-4 (0) was cultured was 17 U / ml without CaCl ₂ added, 5 U / ml of the subtraction is considered to be the degradation activity by PCLE. This value is 16.7 times the production amount (0.3 U / ml: see Japanese Patent No. 5082125) produced by culturing a wild-type filamentous fungus B47-9 strain in a flask for 3 days.

6). Acquisition of protein gene whose production is strongly induced by xylose of GB-4 (0) strain Next, the present inventors also strongly induced production by xylose in P. antarctica GB-4 (0) strain. An attempt was made to obtain the gene of the protein to be processed.
First, referring to the PANT_8c00118 gene promoter, structural gene, and terminator sequence (FIG. 3, SEQ ID NO: 5) of the P. antarctica T34 strain, the following 8 types of Primers were prepared.

XynF1: 5'-GAAGGCTGAAGCTTTGGCTCTGACAT-3 '
XynF2: 5'-CATGCTTGAAGCTCCAAGAAGATATAA-3 '
XynF3: 5'-CACTCGCAGCTGCCTTCGTGGGTGCAG-3 '
XynF4: 5'-GAAGGCAGTCTGCTCGGCCGCTCCCGA-3 '
XynR1: 5'-TGTGGTGTTTGTTTGGCGTTTTTGCTT-3 '
XynR2: 5'-ATCCCACGCGTACACCTTGCCCTTGTA-3 '
XynR3: 5'-CCCACGCAGTTGGACTGGGCCAGGCAG-3 '
XynR4: 5'-CGAGCGCGATTTTCTCCGAGTCTAAA-3 '

The gene sequence of 1954 residues (SEQ ID NO: 11) shown in FIG. 14 was obtained from the genomic DNA of GB-4 (0) by direct PCR. This gene sequence shows 88% homology with the PANT_8c00118 gene promoter, structural gene, and terminator sequence (FIG. 3, SEQ ID NO: 5) of the T34 strain as a whole, and the structural gene sequence encoding the protein (SEQ ID NO: 10) Showed 94% homology with the sequence of the T34 strain (SEQ ID NO: 4). In addition, the promoter region (SEQ ID NO: 7) shows 83% homology with that of the T34 strain (SEQ ID NO: 1), and the terminator region (SEQ ID NO: 8) has that of the T34 strain (SEQ ID NO: 2). ) And 72% homology. The signal sequence (SEQ ID NO: 9) showed 93% homology with that of the T34 strain (SEQ ID NO: 3).

Claims

A nucleic acid molecule comprising the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 7.
A nucleic acid molecule comprising a base sequence having at least 80% homology with the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 7, and having promoter activity.
A nucleic acid molecule consisting of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 7.
An expression vector comprising the nucleic acid molecule according to any one of claims 1 to 3 or a fragment thereof having promoter activity.
A nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 8, or a nucleotide sequence having at least 70% homology with the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 8 and having a terminator activity The expression vector according to claim 4 comprising:
The expression vector according to claim 4 or 5, further comprising a base sequence encoding a heterologous protein operably linked to the nucleic acid molecule.
The expression vector according to claim 6, wherein the heterologous protein is a biodegradable plastic degrading enzyme PaE derived from a yeast of the genus Pseudozyma or a biodegradable plastic degrading enzyme PCLE derived from a filamentous fungus NITE® P-573.
Adjacent to the upstream of the base sequence encoding the heterologous protein, at least 90% of the base sequence shown in SEQ ID NO: 3 or SEQ ID NO: 9, or the base sequence shown in SEQ ID NO: 3 or SEQ ID NO: 9 The expression vector according to claim 6 or 7, further comprising a base sequence having homology and having a function as a secretion signal.
A host cell transformed with the expression vector according to any one of claims 6 to 8.
The host cell according to claim 9, which is a yeast of the genus Pseudozyma or yeast of the genus Ustilago.
The host cell according to claim 9, which is Pseudozyma antarctica.
A method for producing a heterologous protein, comprising culturing the host cell according to any one of claims 9 to 11 under conditions that allow the heterologous protein to be expressed, and recovering the heterologous protein from the culture medium. Method.
The production method according to claim 12, wherein the host cells are cultured in a medium containing xylose.
A protein encoded by the base sequence shown in SEQ ID NO: 4.
A biodegradable plastic degradation preparation comprising the protein according to claim 14.