JP2018157814A - A new strain of Pseudosima Antactica - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel strain of Pseudozaima antactica. SOLUTION: The Pseudozyma antactica GB-4 (0) strain enables the production of auxotrophic mutants and transformants and efficient protein production. And P. It will be possible to produce auxotrophic mutants and transformants that have not been realized in the past for Pseudozaima spp. Other than Antactica. In addition, it is possible to stabilize biodegradable plastic-degrading enzymes based on the findings obtained in the course of a series of experiments. [Selection diagram] None

Description

  The present invention relates to novel strains and auxotrophic mutants of Pseudozyma antarctica, and P. cerevisiae expressing a protein of interest derived from these strains. The present invention relates to an antitactic transformant, a method for producing a target protein using the transformant, and a method for obtaining a stabilized biodegradable plastic degrading enzyme.

  It has been found that many yeasts that can be obtained from the foliage have biodegradable plastic degradation ability (Patent Document 1). Of these, the foliar yeast of the genus Pseudozyma is superior. Antactica is particularly excellent in biodegradable plastic decomposing ability, and biodegradable plastic degrading enzyme PaE has been isolated and identified from this yeast (Non-patent Document 1). And the technique of mass-producing PaE using the strain with high PaE production ability is also developed (patent document 2). Furthermore, P.I. Attempts have also been made to introduce a gene into Antactica to produce a heterologous protein (Patent Document 3).

  In addition, in the case of producing proteins by culturing microorganisms that have been modified with foreign genes, facilities and production, purification, and waste liquid treatment processes are required to completely prevent leakage of the recombinant microorganisms. Therefore, careful handling is necessary, and it is difficult to prove that microorganisms have been completely removed in the filtration process for removing microorganisms, which is generally used especially when the scale is large, and the bacteria are sterilized in a further purification process. There is a need. On the other hand, when a protein is produced using a self-cloning strain cloned using the gene of the host microorganism itself, the microorganism is managed as a recombinant by the installation of the above equipment and the production process. Is not required, and can be handled in the same way as wild strains.

Patent No. 4915593 Patent No. 5849297 International Publication No. 2014/109360

Shinozaki et al., Appl. Microbiol. Biotechnol. (2013), Vol. 97, pp. 2951-2959

  In the conventional method for culturing yeast having biodegradable plastic degrading ability, a high aeration amount is required for the culture solution of the yeast, and the enzyme productivity decreases when the aeration amount decreases. The cost of aeration accounts for a very large proportion of the electrical cost for culture, and there is a technical limit to increasing the aeration rate when the capacity of the culture apparatus is increased. Therefore, P.P. can efficiently produce proteins such as biodegradable plastic degrading enzymes even with a low air flow rate. There is a need for new strains of Antactica.

  P.P. In order to use Antactica as a host for producing a protein derived from a foreign gene, a plasmid containing an antibiotic resistance gene and a gene cassette that expresses the protein of interest is introduced and transformed to grow on a medium using the antibiotic. When the body is selected, when the transformant obtained by this conventional method is cultured in a medium not containing the antibiotic, the once introduced plasmid falls out of the cell body during the culture. In order to solve this problem, P.I. When a gene cassette that expresses an antibiotic resistance gene and a protein of interest is inserted on the Antactica chromosome, it can be expected that the gene is retained even in a medium that does not contain antibiotics. However, in many cases, the genes inserted into the cells after culture were not found. This is due to the fact that P. cerevisiae forming the conventional pseudomycelium In an antactica, it is difficult to isolate one cell into which a gene has been introduced or This is thought to be because the growth of cells that do not contain the transgene predominates during the process of growth of antactica. Thus, conventionally, a protein derived from a foreign gene could not be stably produced from a transformant. P. The same is true when creating an auxotrophic mutant to create a self-cloning strain of Antactica. If a conventional strain was used as a parent strain, it would probably be in the process of growing by forming pseudohyphae. Only the strains having the wild-type genome could be obtained because the growth of the bacterial cells having the genome not containing the gene defect was dominant. Therefore, the introduced gene or the gene having mutation can be stably maintained. There is a need for new strains of Antactica.

As a result of intensive studies to solve the above problems, the present inventors have expressed proteins efficiently and can stably maintain introduced genes or genes with mutations (ie, auxotrophic mutants and transformants). It is possible to produce strains such as A new strain of Antactica was found and the present invention was completed. As a result of examining the characteristics of the new strain and the culture conditions for obtaining the mutant strain, It was also possible to produce auxotrophic mutants and transformants that had not been realized in the past for pseudozaima species other than Antactica. In addition, in the course of a series of experiments, a method for stabilizing a biodegradable plastic degrading enzyme was found and the present invention was completed. That is, the present invention relates to P.I. Novel antactica strain, auxotrophic mutant thereof, auxotrophic mutant of Pseudozyma spp., And transformant of Pseudozyma sp. That expresses the target protein derived from these strains and the same The present invention provides a method for producing a target protein and a method for stabilizing a biodegradable plastic degrading enzyme.
[1] Pseudozyma antarctica GB-4 (0) strain (Accession number NITE P-02392).
[2] An auxotrophic mutant of Pseudozyma antactica GB-4 (0) strain (Accession Number NITE P-02392).
[3] The auxotrophic mutant according to [2], which is lysine-requiring or uracil-requiring.
[4] An auxotrophic mutant of the genus Pseudozyma that has a mutation in at least part of the base sequence of LYS2, LYS12, and / or LYS20, or at least one of the base sequences of URA3 and / or URA5 An auxotrophic mutant characterized by having a mutation in the part.
[5] a deletion of at least a part of the gene encoding Met1 to Glu195 of LYS12 protein, or a substitution at position 113, substitution at position 867, and / or position 614 in the URA3 nucleotide sequence The auxotrophic mutant according to [4] above, which has a deletion of T.
[6] The auxotrophic mutant according to [4] or [5] above, wherein the pseudozaima bacterium is a strain of the genus pseudozaima that does not form pseudohyphae.
[7] A transformant of Pseudozyma that expresses the target protein,
The transformant is a transformant of an auxotrophic mutant,
The transformant has at least one gene cassette;
The gene cassette contains a gene that encodes the protein of interest, a promoter of XYN1 or FRE1 of Pseudozyma sp., And a gene that complements the auxotrophy of the auxotrophic mutant, and encodes the protein of interest. A transformant, wherein the gene is located downstream of the promoter.
[8] The transformant according to [7], wherein the target protein is a protein of the genus Pseudozyma or a protein of an organism other than the genus Pseudozyma.
[9] The transformant according to [7] or [8], wherein a codon used in the gene encoding the target protein is optimized to a codon used by Pseudozyma sp.
[10] Pseudozyma antactica GB-4 (0) L1-S12 strain (Accession number NITE P-02393), GB-4 (0) PGB480 strain (Accession number NITE P-02638), GB-4 (0) PGB469 strain (Transfer of Pseudozyma antactica selected from the group consisting of (Accession No. NITE P-02639) and GB-4 (0) PGB474 strain (Accession No. NITE P-02640).
[11] A method for producing a target protein,
A process of preparing an auxotrophic mutant of pseudozaima,
Introducing at least one gene cassette into the auxotrophic mutant; and
Culturing the auxotrophic mutant introduced with the gene cassette to produce the protein of interest,
Including
The gene cassette contains a gene that encodes the protein of interest, a promoter of XYN1 or FRE1 of Pseudozyma sp., And a gene that complements the auxotrophy of the auxotrophic mutant, and encodes the protein of interest. A method wherein the gene is located downstream of said promoter.
[12] A method for obtaining a stabilized biodegradable plastic degrading enzyme,
Preparing a solution containing the biodegradable plastic-degrading enzyme;
Adding ethanol to the biodegradable plastic-degrading enzyme-containing solution to stabilize or precipitate the biodegradable plastic-degrading enzyme; and
Recovering the stabilized or precipitated biodegradable plastic-degrading enzyme;
A method comprising the steps of:
[13] The method according to [12], further including a step of concentrating the biodegradable plastic degrading enzyme before the ethanol addition step.
[14] The method according to [12] or [13], further including the step of producing the biodegradable plastic degrading enzyme according to the method according to [11].

  In accordance with the present invention, P.I. The novel strain of Antactica makes it possible to produce stable transformants or stable auxotrophic mutants. P.P. It is also possible to produce auxotrophic mutants of Pseudozyma species other than Antactica. Then, various auxotrophic mutants of Pseudozyma sp. Can produce transformants or self-cloning strains that do not rely on antibiotic resistance genes. Further, a gene cassette comprising a gene encoding a target protein, a promoter of XYN1 or FRE1 of Pseudozyma spp., And a gene that complements the auxotrophy of an auxotrophic mutant, and encodes the target protein A target protein can be efficiently produced by introducing a gene cassette in which a gene is located downstream of the promoter into an auxotrophic mutant of the genus Pseudozyma. In addition, by using ethanol to purify the biodegradable plastic degrading enzyme, it can be expected to denature other proteins coexisting with the bactericidal effect of the solution containing the biodegradable plastic degrading enzyme. While preventing the conversion body from mixing, the stability of the solution can be improved.

P. at low air flow. The PaE production ability by an antactica is shown. An optical microscopic observation photograph of pseudozaima is shown. The structure of the PaE high expression cassette using PaLYS12 as a marker is shown. P. using non-homologous end joining (NHEJ). The production scheme of the PaURA3-PaCLE1 gene sequence fragment in Antactica is shown. The gene fragment production scheme for self-cloning strain preparation is shown. The structure of the plasmid for preparing the gene cassette (PaCLE1 and PaURA3 connected) used for preparing the donor strain is shown. The construction scheme of the gene sequence for the reporter assay is shown. The evaluation of various promoters using PaE as a reporter is shown.

Hereinafter, the present invention will be described in more detail.
The new strain of Pseudozyma antactica (P. antactica) of the present invention is GB-4 (0) strain, which was first deposited at the Patent Microorganism Depositary of the National Institute of Technology and Evaluation for Product Evaluation for the first time just prior to the filing of this patent application. (Accession number NITE P-02392). “Pseudozyma antactica” described in the present specification is a kind of Pseudozaima (yeast) that can inhabit the leaves of gramineous plants such as rice and wheat, and produces biodegradable plastic degrading enzymes and the like. To get. The P.A. The Antactica GB-4 (0) strain was kept under the control of the present inventors until deposited, and even if there was a request for transfer, the present inventors were not willing to respond to the request. However, it was never actually transferred to another person.

  In certain embodiments, the present invention provides a P.I. The present invention relates to an auxotrophic mutant of Antactica GB-4 (0) strain. The “auxotrophic mutant” described in the present specification refers to a strain in which auxotrophy is changed by mutation. In addition, a “mutation” or “mutation” described in the present specification is a deletion or substitution of one or more bases in a specific gene so that a change occurs in the amino acid sequence of the protein encoded by the gene. And / or added.

In the auxotrophic mutant of the present invention, an enzyme that synthesizes specific nutrients (such as amino acids and bases) does not function, and therefore cannot grow in an environment lacking such nutrients.
The auxotrophic mutant strain may have any nutritional requirement changed and need not be particularly limited, but may be, for example, lysine auxotrophic or uracil auxotrophic. When the auxotrophic mutant is lysine auxotrophic, the mutant is a gene encoding an enzyme involved in biosynthesis of lysine in Pseudozyma, such as LYS1, LYS2, LYS4, LYS9, LYS12, and / Or at least a part of the base sequence of LYS20 may have a mutation. When the auxotrophic mutant is uracil auxotrophic, the mutant is a gene encoding an enzyme involved in biosynthesis of uracil in pseudozaima, for example, at least one of the base sequences of URA3 and / or URA5. It may have a mutation in the part. P. The base sequences of the genes encoding the enzymes involved in the biosynthesis of antactica lysine and the enzymes involved in the biosynthesis of uracil, and their deduced amino acid sequences are as follows.

-LYS1 sequence
(SEQ ID NO: 1)
・ Deduced amino acid sequence of LYS1 protein
MSAAANRQPLYLRCEMKPAEHRAALTPTTAKKLIEAGFDITVESDPQRIFDDKEYADVGCTIAKHNTFHSLPKHIPIIGLKELEEPGPDLPHTHIQFAHCYKKQAGWVDVLGRFKRGGGKLYDLEFLEDSNGRRVAAFGWHAGFAGAALGLLALAEQVQGAERRLGPQKAYPNEQALIAHAKQQIEHIRNSRADGKVKALVVGALGRCGRGAIDFFEKAGVAPEDIVRWDMKETSAKHGPYQELLDVDIFVNCIYLTSKIPPFLDEATIQAAGAERRLGVVVDVSCDTTNPNNPLPIYSINTTFDKPTVDVDTGAGNPALSVISIDHLPTLLPRESSEGFSNDLLPSLLQLPLVLGKDTTQLDTLEEGKGAVWQRAEKLFRHHLADAEKNGA (SEQ ID NO: 2)

-LYS2 sequence
(SEQ ID NO: 3)
・ Deduced amino acid sequence of LYS2 protein
(SEQ ID NO: 4)

-LYS4 sequence
(SEQ ID NO: 5)
-Deduced amino acid sequence of LYS4 protein
(SEQ ID NO: 6)

-LYS9 sequence
(SEQ ID NO: 7)
・ Deduced amino acid sequence of LYS9 protein
(SEQ ID NO: 8)

LYS12 sequence (underlined intron)
ATGACGTCGACCGCCGCCAAGATCCTCAAGAATGCCACTTCGGGCGTGCTGCGCCTGGGCATGATGCCCGCAGACGGCATTGGACGCGAGGTGCTCCCTTGCGCGCAGCGTGTGCTCCAGAACACGCCCGGCGCACCCAAGTTTGAGTTTGTGCACCTCGACGCTGGCTTCGAGCACTTCCAGAAGACCGGCTCGGCGCTTCCCGAGGAGACCGTGCGTGCACTCAAGGACGACTGCCACGGAGCCATGTTCGGCTCCGTCTCGAGCCCGTCGCACAAGGTCGAGGGCTACAGCTCGCCCATCGTCAAGCTCAGGAAGGAGCTCGACCTGTACGCCAACATTCGCCCCGTCATCGGCGTGCGCGGCACCAAGGACGACGACAAGTTCATCGACAGCGTCATCATTCGCGAGAACACCGAGTGCCTG GTAAGTAGCTGATCCGTCGTCGAGGAGAGCGAAAGATGAGATACTGACGCAGAACGGTCCCTCCCCAACTCGCTTCCACGCGAGCAG TACATCAAGTCCGAGACGAGCGAGAGCGGTCCGGACGGTCAGATTGCGCGCGCCATCCGCCAGATCACCGAGAAGGCTTCGACGCGCATCGGCAAGAAGGCGTTTGAAGTCGCGATTGCGCGCAAGGAGCTGCGCAAGGTGAACCAGCCGTCGTACGAGCCTACCGTGACGATCTGCCACAAGTCCAACGTGCTGAGCGTCACCGACGGTCTCTTCCGCACCTCGGTCCGCGGCGTGTACGAGAAGGACCAGAAGGCCAACGGAGGCGCCGGCCGATACGAGGGCGTCAAGCTCAACGAGCAGCTCGTCGACTCCATGGTCTACCGCCTCTTCCGCGAGCCCGAGGTGTTTGACGTGGTCGTGGCGCCCAACCTGTACGGTGACATTGTCTCGGATGCTGCCGCCGCCCTCGTGGGCTCCCTTGGCCTCGTCCCCTCCGTCAACGCCGGCGACAACTTCTTCATG GTAAGCCCCATCCTCCGTC TTCTCCTTCCTTCCAGCCCCGAGCGCCTACTTACATTTTGCCCCGCCTTGTTCTTCCCTTGTGCTGCGCATCGTAG GGCGAGCCGGTCCACGGTTCGGCGCCCGATATCGCGGGTCAGGGCAAGGCGAACCCGATCGCCTCGATCCGCTCCGCCGCCCTCATGCTCGAATACATGGGCTACACCGAGCCTGCGCTCAAGATCTACACGGCCGTCGACCAGGTCATCCGCGAGGGCAAGTACCTCACCCCAGACCTCGGCGGATCCGCCACCACCACCCAGTGCGAAGAGGCCATCCTCAAAAAGATCAACGCCTAG (SEQ ID NO: 9)
-Deduced amino acid sequence of LYS12 protein
MTSTAAKILKNATSGVLRLGMMPADGIGREVLPCAQRVLQNTPGAPKFEFVHLDAGFEHFQKTGSALPEETVRALKDDCHGAMFGSVSSPSHKVEGYSSPIVKLRKELDLYANIRPVIGVRGTKDDDKFIDSVIIRENTECLYIKSETSESGPDGQIARAIRQITEKASTRIGKKAFEVAIARKELRKVNQPSYEPTVTICHKSNVLSVTDGLFRTSVRGVYEKDQKANGGAGRYEGVKLNEQLVDSMVYRLFREPEVFDVVVAPNLYGDIVSDAAAALVGSLGLVPSVNAGDNFFMGEPVHGSAPDIAGQGKANPIASIRSAALMLEYMGYTEPALKIYTAVDQVIREGKYLTPDLGGSATTTQCEEAILKKINA (SEQ ID NO: 10)

-LYS20 sequence
(SEQ ID NO: 11)
-Deduced amino acid sequence of LYS20 protein
MCDHSEPSAIQQVNGTAPDTHVAIDTSAPNTNGAANGANGALATADNGNVGSVQPFNTRYSDFLSNVSNFKIIESTLREGEQFANAFFDTETKIKIAKALDKFGVDCIELTSPAASEQSRKDCEAICKLGLKAKVITHIRCHMDDARIAVETGVDGVDVVMGTSKFLREFSHGKDMTYITKTAIEVIQFVKSKGIEIRFSTEDSFRSDLVDLLSIYQAVDKVGVNRVGIADTVGVANPRQVYDLVRTLRGVVGCDIECHFHNDTGCAIANAYTALEAGATHVDTSVLGIGERNGIPSLGGFIARMYTADREYVMSKYNLKALREVENLVAEAVQIQVPFNNYVTGYCAFTHKAGIHAKAILANPSTYEIIRPEDFGMSRYVSIGHRLTGWNAVKSRCEQLELNLTEDQVKEVTAKIKQLADVREQTMEDVDSILRNYARAIENGTA (SEQ ID NO: 12)

・ URA3 sequence (underlined intron)
ATGTCGAGCATCACCCTCCAGACGTACGCATCACGCGCGGCCAAGCAGCCCAACGCCGCCGCCAAGGCGCTGCTCGAGTGCATCGAGCGCAAGCAGTCCAACCTGTGCGTCTCGGTCGACGTGACCAACAAGCAGGACCTGCTCGACGTGTGCGATGCCGTCGGCCCAGACGTGTGCCTGGTCAAGACGCACATCGACATTGTCGAGGATTTTGACATCGACCTCGTCCAGCAGCTCACCGCGCTCAGCCAGAAGCACGACTTTCTCATCTTCGAGGACCGCAAGTTTGCCGACATTG GTAAGTCCATGCGATCGTCCACTCGAAGTGCCTGTGCACCCCGCTGACACGAGCTTACCCTGGCGCATTTGCATCTACAG (SEQ ID NO: 13)
・ Deduced amino acid sequence of URA3 protein
MSSITLQTYASRAAKQPNAAAKALLECIERKQSNLCVSVDVTNKQDLLDVCDAVGPDVCLVKTHIDIVEDFDIDLVQQLTALSQKHDFLIFEDRKFADIGNTASLQYSSGVHKIASWSHITNAHLVPGPGVISGLAKVGQPLGRGLLLLAEMSSAGALTKGSYTQACVDEAKKDTTGFVCGFIAMSRVDEREGPNTERDLLILTPGVGLDVKGDAMGQQYRTPDQVIRESGCDVIIVGRGIYGALMTEEGKADKKAAFDKVRQQGSRYKKAGWEAYLARLGQK (SEQ ID NO: 14)

  The mutation site in the gene is not particularly limited as long as it reduces the function of the protein encoded by the gene. For example, the mutation in the gene is a mutation of the gene encoding Met1-Glu195 of the LYS12 protein. URA3 113th C substitution (eg substitution to G), 867th C substitution (eg substitution to T), and / or 614th It may be a deletion of T. In addition, in this specification, the name of the gene homolog known by Saccharomyces cerevisiae (Saccharomyces cerevisiae) may be used as it is as the name of the gene of the genus Pseudozyma. And in particular, P.I. When referring to an atactica gene, the gene name may be prefixed with “Pa”. For example, S.M. P. cerevisiae showing homology to LYS12 or URA3 of cerevisiae. The antitactic gene may be simply referred to as “LYS12” and “URA3”, respectively, or may be referred to as “PaLYS12” and “PaURA3”.

  In one aspect, the present invention relates to an auxotrophic mutant of Pseudozyma, wherein the auxotrophic mutant has a mutation in at least a part of the LYS2, LYS12, and / or LYS20 base sequence, or It has a mutation in at least a part of the URA3 and / or URA5 base sequence. The type of mutation in the gene is not particularly limited as long as it reduces the function of the protein encoded by the gene. For example, at least a part of the gene encoding Met1 to Glu195 of the LYS12 protein is missing. URA3, 113th C substitution (eg, substitution with G), 867th C substitution (eg, substitution with T), and / or 614th T deletion, Also good. As a parent strain for producing the auxotrophic mutant, for example, P.I. Antactica, P.A. Flocculosa, P.M. Rugulosa, P.A. Aphidis, and P.I. Although various strains of Pseudozyma spp. Such as Tsukubaensis can be employed, Pseudozyma spp. That do not form pseudohyphae are preferred. The “pseudomycelium” described herein is a mycelium produced by germination of a fungus, in which a daughter cell extends into an elongated cell in close contact with the mother cell at one end, and from these ends a new elongated shape is obtained by further budding. A long filamentous structure formed by repeating the process of generating cells. The parent strain is particularly preferably P. Antactica GB-4 (0) strain, P.I. Antactica OMM62-2 strain (Accession No. NITE P-02239), P.I. Flocculosa JCM10321 strain, Tsukubaensis JCM10324 strain.

  Pseudozyma sp. As a method for obtaining an auxotrophic mutant from the Antactica GB-4 (0) strain, various methods known in the art can be employed without particular limitation. For example, the auxotrophic mutant may be obtained by inducing mutation by ultraviolet irradiation or by screening mutants produced in the natural environment.

  In one aspect, the present invention relates to a transformant of Pseudozyma that expresses a protein of interest. The transformant is prepared by introducing a gene cassette that expresses the protein of interest into an auxotrophic mutant of the genus Pseudozyma, and has at least one gene cassette. The gene cassette contains a gene that encodes the target protein, a promoter of XYN1 or FRE1 of Pseudozyma sp., And a gene that complements the auxotrophy of the auxotrophic mutant. The encoding gene is located downstream of the promoter. XYN1 of the genus Pseudozyma is a gene encoding xylanase, and FRE1 of the genus Pseudozyma is highly homologous to FRE1 of S. cerevisiae encoding a ferric reductase-like protein. It is a gene. Moreover, the gene that complements the auxotrophy of the auxotrophic mutant may be located downstream of the promoter of the auxotrophic complementary gene, or may be located downstream of other promoters. The gene cassette may optionally contain a terminator (eg, a terminator such as XYN1 or FRE1 of Pseudozyma spp.) Downstream of the gene encoding the protein of interest. When all the genes constituting the gene cassette are derived from the same species as the auxotrophic mutant to be transformed, the produced transformant is a self-cloning strain. Self-cloning strains are not required to be strictly managed as recombinants and can be handled in the same way as wild-type strains.

  The transformant having a plurality of the gene cassettes may be produced by introducing a plurality of gene cassettes by chance when introducing the gene cassette into the auxotrophic mutant, It may be prepared by once introducing a transformant having one gene cassette and then repeatedly introducing the same gene cassette. A transformant having a plurality of the gene cassettes is expected to express the target protein in a larger amount.

  In one aspect, the present invention relates to a method for producing a protein of interest, the step of preparing an auxotrophic mutant of Pseudozyma spp., The step of introducing at least one gene cassette into the auxotrophic mutant, And culturing an auxotrophic mutant strain into which the gene cassette has been introduced to produce the protein of interest. The gene cassette contains a gene that encodes the protein of interest, a promoter of XYN1 or FRE1 of Pseudozyma sp., And a gene that complements the auxotrophy of the auxotrophic mutant, and encodes the protein of interest. A gene is located downstream of the promoter. Moreover, the gene that complements the auxotrophy of the auxotrophic mutant may be located downstream of the promoter of the auxotrophic complementary gene, or may be located downstream of other promoters. The gene cassette may optionally contain a terminator (eg, a terminator such as XYN1 or FRE1 of Pseudozyma spp.) Downstream of the gene encoding the protein of interest.

  As the protein of interest to be expressed in the transformant of the genus Pseudozyma of the present invention or the protein of interest produced by the method of the present invention, various proteins can be employed without any particular limitation, and proteins of the genus Pseudozyma are employed. Alternatively, proteins from organisms other than Pseudozyma may be employed. The protein of the genus Pseudozyma is, for example, cutinase-like enzyme 1 (CLE1) which is a biodegradable plastic degrading enzyme (P. antactica CLE1 is also referred to as “PaE” or “PaCLE1 protein”), lipase A, or lipase. B may be sufficient. Examples of proteins of organisms other than the Pseudozyma species include not only luciferase, biodegradable plastic-degrading enzymes of different types of yeasts and fungi, or enzymes such as amylase, protease, lipase, and cellulase, but also antigen recognition sites of antibodies, etc. It may be a useful protein. In addition, as a codon used in the gene encoding the target protein, various codons can be adopted without particular limitation as long as the transformant can be used. May be optimized for codons frequently used by Pseudozyma and / or optimized to give the desired GC content. The codon frequently used by Pseudozyma sp. Is, for example, P. cultivated in a medium containing 8% xylose or 8% glucose. From the gene expression profile obtained by analyzing the total RNA of the Antactica GB-4 (0) strain with a microarray, the top 20 genes whose transcription amount is high only when cultured in a medium containing xylose are selected. It may be identified by examining the frequency of codon usage in the gene. The GC content may be, for example, 45 to 75%, preferably 50 to 70%, more preferably 55 to 67%. By optimizing the codon and / or the GC content, the expression efficiency of the target protein can be increased.

  P. of the present invention. As specific examples of transformants of Antactica, P. Examples include the L1-S12 strain (accession number NITE P-02393) derived from the L1 strain, which is a lysine-requiring mutant of the Antactica GB-4 (0) strain. In the L1 strain as a host, 7827 bp including 672 bp in the first half (5 ′ end side) of the PaLYS12 nucleotide sequence is deleted, and the intracellular lysine biosynthetic pathway does not function. The L1-S12 strain is a A gene encoding PaE (PaCLE1 protein), a biodegradable plastic degrading enzyme of Antactica, A gene cassette provided between the promoter and terminator of the antitactic xylanase gene PaXYN1 and having a sequence in which PaLYS12 is arranged between the promoter and terminator of PaLYS12 is introduced into the L1 strain, After selection, it is a PaE high expression strain obtained by screening using biodegradable plastic degrading enzyme activity (PaE activity) as an index. The gene encoding PaE constituting the gene cassette (PaCLE1), PaXYN1 promoter and terminator, PaLYS12, and PaLYS12 promoter and terminator are all P.P. Since it is a gene derived from Antactica, the L1-S12 strain is a self-cloning strain.

  P. of the present invention. As another specific example of the transformant of Antactica, P. GB-4 (0) PGB480 (Accession No. NITE P-02638) and GB-4 (0) PGB474 (Accession No. NITE) derived from the L1 strain, which is a lysine-requiring mutant of the Antactica GB-4 (0) strain P-02640), and P.I. Examples include GB-4 (0) PGB469 strain (accession number NITE P-02639) derived from PGB371 strain, which is a uracil-requiring mutant strain of Antactica GB-4 (0) strain. The gene cassette introduced into these transformants is P. a. The transformant is a self-cloning strain because it was composed only of DNA sequences derived from Antactica.

  In one aspect, the present invention relates to a method for obtaining a stabilized biodegradable plastic degrading enzyme, the step of preparing a solution containing the biodegradable plastic degrading enzyme, and a solution containing the biodegradable plastic degrading enzyme. And adding ethanol to stabilize or precipitate the biodegradable plastic-degrading enzyme, and recovering the stabilized or precipitated biodegradable plastic-degrading enzyme. Many proteins are denatured by ethanol and inactivate by forming a precipitate in a solution. However, the biodegradable plastic degrading enzyme is temporarily inactivated by forming a precipitate by ethanol, If the plastic-degrading enzyme is redissolved in water or buffer, the enzyme will regain activity. In addition, the biodegradable plastic degrading enzyme can be stably stored in an ethanol solution. Thus, if ethanol is used, P.I. It is possible to efficiently purify and concentrate the biodegradable plastic-degrading enzyme recovered in the culture solution of yeast such as Antactica and store it stably. The final concentration after the addition of ethanol is not particularly limited as long as the enzyme can be recovered by precipitation when purifying and concentrating the biodegradable plastic-degrading enzyme. For example, the final concentration is about 50% to about 99%. It may be present, preferably about 75% to about 90%. The precipitate of the biodegradable plastic degrading enzyme may be recovered by centrifugation or filtration (filtering) and stored in a precipitated state. In addition, when the purpose is to stably preserve the activity of the biodegradable plastic-degrading enzyme, the enzyme may or may not be precipitated, and the final concentration after addition of the ethanol is, for example, about 1 % To 90%, preferably about 5% to 70%, particularly preferably about 10% to 50%. The biodegradable plastic degrading enzyme that does not form a precipitate but is stabilized may be recovered as it is as a solution after addition of ethanol, and stored in a solution state.

  The step of adding ethanol to the solution containing the biodegradable plastic degrading enzyme has a final concentration of about 10% to about 90% (preferably about 30 to about 80%, particularly preferably about 65% to about 75%). Adding ethanol so that the final concentration is about 50% to about 99% (preferably about 75% to about 90%) may be included. In general, ethanol has a bactericidal action, and particularly when the ethanol concentration is about 65% to about 75%, the bactericidal action with ethanol is most effectively achieved. In order to collect the biodegradable plastic-degrading enzyme by precipitation after sterilizing by temporarily adjusting the ethanol concentration of the solution to a concentration in a range where the bactericidal action is exhibited, such as about 65% to about 75%. By adjusting to a predetermined concentration of P.P. While purifying biodegradable plastic-degrading enzymes produced in the culture medium of yeast such as Antactica, killing the cells producing the biodegradable plastic-degrading enzymes that may be mixed in the culture solution I can expect to be able to. The biodegradable plastic-degrading enzyme may be used to decompose biodegradable plastic agricultural multi-films, etc. that have been used in outdoor fields. It can be expected that the bacterial cells are prevented from diffusing into the natural world.

  The acquisition method of the present invention may include a step of concentrating the biodegradable plastic degrading enzyme before the ethanol addition step. As the means for concentrating, means usually used in the art can be used without particular limitation. For example, in the concentration step, the solution containing the biodegradable plastic degrading enzyme is subjected to ultrafiltration. A step of providing, a step of adding ammonium sulfate to a solution containing the biodegradable plastic-degrading enzyme to form a precipitate and collecting the precipitate, or a step of subjecting the solution containing the biodegradable plastic-degrading enzyme to freeze-drying, etc. Or any combination of these steps. Although it is technically possible to add ethanol to a microorganism culture solution containing a biodegradable plastic-degrading enzyme, for example, to a final concentration of about 90%, a large amount of ethanol is required. On the other hand, if the biodegradable plastic-degrading enzyme is concentrated by ultrafiltration, ammonium sulfate, or freeze-drying before adding ethanol, it is sufficient to use a relatively small amount of ethanol, so the ethanol addition operation is easy. become. The final concentration after the addition of ammonium sulfate is not particularly limited as long as the biodegradable plastic-degrading enzyme can be recovered by precipitation, but may be, for example, about 40% saturation to about 100% saturation, preferably about 50%. Saturated to about 80% saturated.

  The acquisition method of the present invention may further include a step of producing the biodegradable plastic degrading enzyme according to the method of producing the target protein of the present invention.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, the scope of the present invention is not limited to these Examples.

[Example 1] P.I. A new strain of Antactica (1) Protein production ability of Antactica GB-4 (0) strain under low aeration conditions Rice husks from Ibaraki, Japan (Agricultural Bioresource Genebank, JP No. 203119) were used as a biodegradable plastic polybutyl. As a bacterium having an activity of degrading succinate (PBS) multi-film and polybutylene succinate-co-adipate (PBSA) multi-film, that is, a bacterium that produces PaE which is a biodegradable plastic degrading enzyme. Antactica GB-4 (0) strain (Accession number NITE P-02392) and Antactica GB-4 (1) W strain (Accession No. FERM P-22155) was isolated.

  FIG. 1 shows the PaE activity in the culture solution when these strains are jar-cultured with different aeration rates. When cultured with an aeration rate of 10 LPM, P.P. The culture solution of P. It showed almost the same PaE activity as the culture solution of Antactica GB-4 (1) W strain. On the other hand, when cultured with an aeration rate of 2 LPM, P.P. The PaE activity in the culture solution of Antactica GB-4 (1) W strain was decreased compared to the case where the culture was performed with an aeration rate of 10 LPM. The PaE activity in the culture solution of the Antactica GB-4 (0) strain was hardly changed. Therefore, P.I. It was found that the Antactica GB-4 (0) strain has a high ability to produce proteins even under culture conditions with a low aeration rate.

(2) P.I. A. Transgenicity holding capacity of Antactica GB-4 (0) strain Antactica GB-4 (0) strain and P. aeruginosa A plasmid (pUXV1-neo) containing a neomycin resistance gene was introduced into the Antactica T-34 strain for transformation. When the transformation efficiency (number of transformants per 1 μg of DNA) was calculated, P. p. In the antitactic T-34 strain, it was 1.4 transformant / μg DNA, whereas P. a. In the Antactica GB-4 (0) strain, it was 57.4 transformant / μg DNA. Therefore, P.I. The Antactica GB-4 (0) strain was found to have high transformation efficiency.

  When the morphology of each microbial cell was observed with an optical microscope equipped with a differential interference device (for details, see Example 3 (6) below), P.I. The Antactica T-34 strain forms pseudohyphae during growth (FIG. 2F). The Antactica GB-4 (0) strain was found to have detached daughter cells from the mother cells after budding and growing with little formation of pseudomycelia (FIG. 2A). In the case of strains that form pseudohyphae, multiple cells linked from the pseudohyphae can form during growth. Therefore, after genetic manipulation, colonies that have grown in the selective medium contain a mixture of transformants that retain the introduced gene derived from linked cells and non-transformants that do not retain the gene. it seems to do. On the other hand, P.I. Since the antitactic GB-4 (0) strain hardly forms pseudohyphae, it is considered that only the cells carrying the introduced gene are likely to grow on the selective medium. Thus, P.I. Since the antitactic GB-4 (0) strain has a high protein production ability and an excellent transgene retention ability, this strain can be used for producing a desired protein. P.P. The Antactica GB-4 (0) strain can be used for the production of an auxotrophic mutant, and the mutant can be used for producing a desired protein.

[Example 2] P.I. Preparation of lysine-requiring mutant of Antactica (1) Purpose An auxotrophic mutant that disrupts the synthetic gene of Antactica nutrients is prepared. By using a gene that complements this lost auxotrophy as a marker for a gene transfer vector, a self-cloning strain can be selected. First, a lysine-requiring mutant is prepared.

(2) Material The composition of the medium used in this example was as follows.
YM medium Yeast extract (Difco Laboratories) 0.3% by mass
Malt extract (Difco Laboratories) 0.3% by mass
Peptone (Difco Laboratories) 0.5% by mass
Glucose 1.0% by mass
SD medium Bacto yeast nitrogen base containing no amino acid and (NH ₄ ) ₂ SO ₄ (Difco Laboratories) 0.17% by mass
Glucose 2.0% by mass
(NH ₄ ) ₂ SO ₄ 0.5% by mass
SD w / o AA & AS medium Bacto yeast nitrogen base containing no amino acid and (NH ₄ ) ₂ SO ₄ (Difco Laboratories) 0.17% by mass
Glucose 2.0% by mass
SD NaNO ₃ medium Bacto yeast nitrogen base containing no amino acid and (NH ₄ ) ₂ SO ₄ (Difco Laboratories) 0.17% by mass
NaNO ₃ 0.2% by mass
Glucose 2.0% by mass

The base sequences of the primers used in this example were as follows.
・ Lys12-pro-618 F
5'-GACATATGAGTGGACAGGCGTGTTCCACAGACAG-3 '(SEQ ID NO: 15)
・ Lys12-ter + 1782 R
5'-GAGAATTCCGAGAAGTTCACCTCCATGGACGAGC-3 '(SEQ ID NO: 16)
・ Lys20-21-618 F1
5'-TGTGGCAAAAAGCGGCCCAGCGCCA-3 '(SEQ ID NO: 17)
・ Lys20-21 + 11802 R4
5'-TCTGAAGCAGGTCAAGACTTTTGTT-3 '(SEQ ID NO: 18)
・ LYS2 pro F
5'-GATGTGGAGTCCATTGCTGTTGTCGAC-3 '(SEQ ID NO: 19)
・ LYS2 ter R
5'-CAAATCCCTTCTTTCGTCCCTCGGCT-3 '(SEQ ID NO: 20)

(3) Acquisition of auxotrophic mutants by ultraviolet irradiation The antitactic GB-4 (0) strain was inoculated into a test tube containing 5 mL of YM medium, and cultured with shaking at 30 ° C. and 160 rpm for 24 hours. 1 mL of this culture solution was added to a petri dish containing 30 mL of cold sterilized water, and irradiated with ultraviolet rays for 60 seconds while stirring with a stirrer. The whole amount of cells irradiated with ultraviolet rays by centrifugation was collected, resuspended in 5 mL of YM medium, and then subjected to recovery culture at 30 ° C. and 160 rpm for 3 hours. After 1 mL of the culture solution after recovery culture was collected, the cells were washed twice with sterilized water, and then a medium (SD w / o AA & AS) in which ammonium sulfate serving as a nitrogen source was removed from a minimal medium (SD) based on Wickerham's synthetic medium. Medium) In addition to a test tube containing 5 mL, it was cultured with shaking at 30 ° C. and 160 rpm for 4.5 hours to promote nitrogen starvation and cessation of cell growth. Cells subjected to nitrogen starvation treatment were collected, and the cells were washed twice with sterilized water, and then resuspended in 4 mL of SD NaNO ₃ medium using sodium nitrate as a nitrogen source of the SD medium. This was cultured with shaking at 30 ° C. and 160 rpm for 3 hours to promote the growth of cells that were not auxotrophic. 500 μL of a nystatin solution (100 μg / mL) was added to this culture solution, and further cultured with shaking at 30 ° C. and 160 rpm for 1 hour, thereby killing proliferating cells that were not auxotrophic (nystatin concentration). After the nystatin concentration treatment solution was diluted 10 ³ times with cold sterilized water, 50 μL each was applied to a YM agar medium (YM medium containing 2% by mass of agar). When this was statically cultured at 30 ° C. for 2 days, about 200 colonies per petri dish appeared. About 3600 colonies that had grown, replicas were produced on YM agar medium and SD agar medium (SD medium containing 2% by mass of agar). As a result, three strains that could grow on the YM agar medium but not on the SD medium were obtained, and these were transplanted to a minimal medium to which various amino acids were added. Then, since all the strains recovered their growth only on the medium supplemented with lysine, it was considered to be a lysine-requiring mutant. Each strain was designated as P. They were named Antactica GB-4 (0) -L1 strain, GB-4 (0) -L8 strain, and GB-4 (0) -L14 strain.

(4) Identification of mutant gene of lysine-requiring mutant strain obtained Based on gene information related to lysine synthesis of S. cerevisiae, Homologous genes PaLYS1, PaLYS2, PaLYS4, PaLYS9, PaLYS12, and PaLYS20 were found from the genomic information of the Antactica GB-4 (0) strain. P. Can each gene amplified by PCR from the genomic DNA of the Antactica GB-4 (0) strain be introduced into the three obtained strains (L1, L8, and L14 strains) to restore growth in the minimal medium? I checked. As a result, the L1 strain was identified as a PaLYS12 mutant because its growth was restored when the PaLYS12 gene fragment (Lys12-pro-618F and Lys12-ter + 1782R was used as primers) was introduced. The L8 strain was identified as a PaLYS20 mutant because its growth was restored when the PaLYS20 gene fragment (Lys20-21-618 F1 and Lys20-21 + 11802 R4 were used as primers) was introduced. The L14 strain was identified as a PaLYS2 mutant because its growth was restored when the PaLYS2 gene fragment (using LYS2 pro F and LYS2 ter R as a primer) was introduced.

  In addition, the genome sequence of the L1 strain was decoded and compared with the genome sequence of the parent strain GB-4 (0) strain. As a result, in the L1 strain, PaLYS1, PaLYS2, PaLYS4, PaLYS9, and PaLYS20 were healthy, but 672 bp (underlined part is an intron) shown below in the first half of the PaLYS12 base sequence (5 ′ end side) and the gene It was found that 7827 bp including the upstream sequence was deleted.

ATGACGTCGACCGCCGCCAAGATCCTCAAGAATGCCACTTCGGGCGTGCTGCGCCTGGGCATGATGCCCGCAGACGGCATTGGACGCGAGGTGCTCCCTTGCGCGCAGCGTGTGCTCCAGAACACGCCCGGCGCACCCAAGTTTGAGTTTGTGCACCTCGACGCTGGCTTCGAGCACTTCCAGAAGACCGGCTCGGCGCTTCCCGAGGAGACCGTGCGTGCACTCAAGGACGACTGCCACGGAGCCATGTTCGGCTCCGTCTCGAGCCCGTCGCACAAGGTCGAGGGCTACAGCTCGCCCATCGTCAAGCTCAGGAAGGAGCTCGACCTGTACGCCAACATTCGCCCCGTCATCGGCGTGCGCGGCACCAAGGACGACGACAAGTTCATCGACAGCGTCATCATTCGCGAGAACACCGAGTGCCTG GTAAGTAGCTGATCCGTCGTCGAGGAGAGCGAAAGATGAGATACTGACGCAGAACGGTCCCTCCCCAACTCGCTTCCACGCGAGCAG TACATCAAGTCCGAGACGAGCGAGAGCGGTCCGGACGGTCAGATTGCGCGCGCCATCCGCCAGATCACCGAGAAGGCTTCGACGCGCATCGGCAAGAAGGCGTTTGAAGTCGCGATTGCGCGCAAGGAGCTGCGCAAGGTGAACCAGCCGTCGTACGAG (SEQ ID NO: 21)

The deduced amino acid sequence of the defective portion is as follows.
MTSTAAKILKNATSGVLRLGMMPADGIGREVLPCAQRVLQNTPGAPKFEFVHLDAGFEHFQKTGSALPEETVRALKDDCHGAMFGSVSSPSHKVEGYSSPIVKLRKELDLYANIRPVIGVRGTKDDDKFIDSVIIRENTECLYIKSETSESGPDGQIARAIRQITEKASTRIGKKAFEVARKE

[Example 3] Production of biodegradable plastic-degrading enzyme PaE high-production self-cloning strain using lysine-requiring strain (1) Purpose Using the above-mentioned L1 strain as a host and using PaLYS12 as a gene transfer marker, the same species A high protein production cassette by self-cloning is constructed by introducing a cassette for high protein production consisting only of the genes of the cells. P. In Antactica, it is known that expression of a gene under the control of PaXYN1 promoter is strongly induced in the presence of xylose (International Publication No. 2014/109360). Therefore, a self-cloning strain that produces PaE under the control of the PaXYN1 promoter is prepared.

(2) Material The composition of the medium used in this example was as follows.
SDP agar medium Bacto yeast nitrogen base containing no amino acid and (NH ₄ ) ₂ SO ₄ (Difco Laboratories) 0.17% by mass
Glucose 2.0% by mass
0.5M Na ₂ HPO ₄ solution 5mL / L
Agar 2.0% by mass
-XY medium Yeast extract (Difco Laboratories) 1 mass%
6% by mass of xylose
・ 3xFMM (Fungal minimum medium) 8% xylose medium 0.2% by mass of NaNO ₃
KH ₂ PO ₄ 0.06% by mass
MgSO ₄ · 7H ₂ O 0.06% by mass
Yeast extract (Difco Laboratories) 0.3% by mass
Xylose 8.0% by mass
YPX medium Yeast extract (Difco Laboratories) 1% by mass
Bactopeptone (Difco Laboratories) 2% by mass
8% by mass of xylose
· 3xFMM (Fungal minimum medium) -0.5 % PBSA emulsion, 8% xylose agar NaNO ₃ 0.2 wt%
KH ₂ PO ₄ 0.06% by mass
MgSO ₄ · 7H ₂ O 0.06% by mass
Yeast extract (Difco Laboratories) 0.3% by mass
PBSA emulsion * 0.5% by mass
Xylose 8.0% by mass
Agar 2% by mass
* Bionole emulsion EM-301 (PBSA) (Showa Denko)

The base sequences of the primers used in this example were as follows.
・ PLYS12 F-1
5'-ACGGCCAGTGAATTCGTACATATAGTTGGCCGCTGCATAT-3 '(SEQ ID NO: 23)
・ TLYS12-TXYN R
5'-AAAGAGAACGAGAAGTTCACCTCCATGGACGAGC-3 '(SEQ ID NO: 24)
・ PXYN F
5'-ACCGAGCTCGAATTCCACGCCACGTTCGATACCGAC-3 '(SEQ ID NO: 25)
・ PXYN-PaEG R
5'-AACTGCATCGTAAGGTTTGTTTGGCGTTTTGGG-3 '(SEQ ID NO: 26)
・ TXYN-PaEG F
5'-AGGGATAATCCGTCGTCACCGGCTTGCCTGTAT-3 '(SEQ ID NO: 27)
・ TXYN-TLYS12 R
5'-ACTTCTCGTTCTCTTTTTGCCAGGTGTTGGACT-3 '(SEQ ID NO: 28)
・ PaEG F
5'-ACCTTACGATGCAGTTCAAGTCGACCTTTGCCG-3 '(SEQ ID NO: 29)
・ PaEG R
5'-ACGACGGATTATCCCTGAAGAGCCTTGATACCG-3 '(SEQ ID NO: 30)
・ M13 F
5'-GTAAAACGACGGCCAG-3 '(SEQ ID NO: 31)
・ M13 R
5'-GGAAACAGCTATGACCATG-3 '(SEQ ID NO: 32)
・ LYS12 F-1000
5'-GTACATATAGTTGGCCGCTGCATAT-3 '(SEQ ID NO: 33)
・ PXYN-1513 F
5'-CACGCCACGTTCGATACCGAC-3 '(SEQ ID NO: 34)

(3) Production of gene cassette for high expression of PaE Using the genomic DNA of the Antactica GB-4 (0) strain as a template and using the primers shown in Table 1, the lysine synthetic gene PaLYS12 and its promoter and terminator, PaXYN1 promoter (PXYN), PaXYN1 terminator (TXYN), and PaE The gene fragment for self-cloning of PaCLE1, which is a gene encoding the gene, was amplified.

The gene sequences of PaLYS12 (including promoter and terminator), PXYN, TXYN, and PaCLE1 are as follows.
PaLYS12 (including promoter and terminator) (2781 bp (structural gene region 1313 bp, promoter region 999 bp, and terminator region 469 bp); underlined portion is intron)
GTAAGTAGCTGATCCGTCGTCGAGGAGAGCGAAAGATGAGATACTGACGCAGAACGGTCCCTCCCCAACTCGCTTCCACGCGAGCAG TACATCAAGTCCGAGACGAGCGAGAGCGGTCCGGACGGTCAGATTGCGCGCGCCATCCGCCAGATCACCGAGAAGGCTTCGACGCGCATCGGCAAGAAGGCGTTTGAAGTCGCGATTGCGCGCAAGGAGCTGCGCAAGGTGAACCAGCCGTCGTACGAGCCTACCGTGACGATCTGCCACAAGTCCAACGTGCTGAGCGTCACCGACGGTCTCTTCCGCACCTCGGTCCGCGGCGTGTACGAGAAGGACCAGAAGGCCAACGGAGGCGCCGGCCGATACGAGGGCGTCAAGCTCAACGAGCAGCTCGTCGACTCCATGGTCTACCGCCTCTTCCGCGAGCCCGAGGTGTTTGACGTGGTCGTGGCGCCCAACCTGTACGGTGACATTGTCTCGGATGCTGCCGCCGCCCTCGTGGGCTCCCTTGGCCTCGTCCCCTCCGTCAACGCCGGCGACAACTTCTTCATG GTAAGCCCCATCCTCCGTCTTCTCCTTCCTTCCAGCCCCGAGCGCCTACTTACATTTTGCCCCGCCTTGTTCTTCCCTTGTGCTGCGCATCGTAG (SEQ ID NO: 35)

・ PXYN (1513 bp)
(SEQ ID NO: 36)

・ TXYN (300bp)
TCCGTCGTCACCGGCTTGCCTGTATGATGCATCGGGACCCGAGTCCACCATGCAATCCAGATACGAGTGTTGCTCTCTTCTTGGCTCACTTTACGCGTCTGTTTGAGTGGGGCGCGGGGAGGAAAAAAGGCTGAGTTCGCTGGGCCACGCGCGTGGAAGCAAGACTGCTGCTGAGCCCTTTTTCTCTTGCGCTGAGAGAGTCGCGGCGTGCGGCCCCTTTTTTTATTAATACTAATATTATAGACTCTGAAGGAAATCGCGCTCGTGGTCAGTCGAGTCCAACACCTGGCAAAAAGAGAA (SEQ ID NO: 37)

・ PaCLE1 (675bp)
(SEQ ID NO: 38)

  The above four gene fragments were ligated to plasmid pUC19 cut with EcoRI using In-fusion HD cloning kit (Clontech). The prepared plasmid was introduced into Escherichia coli DH5α by the heat shock method, seeded on ampicillin-containing LB agar medium, and cultured at 37 ° C. For colonies that grew overnight, colony PCR was performed using the primer set used for the preparation of the above gene fragments, and colonies into which the four gene fragments were correctly introduced were selected, and a gene fragment of about 5.3 kbp was selected. A plasmid pPaLYS12X-PaEG containing was prepared. The structure of the cassette for high expression of PaE in the plasmid is shown in FIG. The gene PaCLE1 encoding PaE is located between the xylanase promoter and terminator, and the base sequence of PaLYS12 (including two introns) is located between the promoter and terminator of PaLYS12.

(4) Production of PaE high-producing strain using lysine-requiring mutant strain The gene fragment was amplified by PCR using the plasmid pPaLYS12X-PaEG as a template and M13 F and M13 R as primers. 5 μg of the obtained gene fragment was introduced into the L1 strain, which is a lysine-requiring mutant of the GB-4 (0) strain, by electroporation, and applied on an SD (SDP) agar medium containing disodium phosphate. Cultured at 30 ° C. Two days later, about 20 large colonies appeared per plate (about 100 small colonies that seemed to be false positives). Of these, 80 colonies were randomly picked on a new SDP agar medium. Each of the transformed transformants was inoculated with 1 platinum loop into a test tube containing 5 mL of YX medium, and cultured with shaking at 30 ° C. and 160 rpm for 3 days.

  For each transformed strain, the culture was repeated three times independently, and the PaE enzyme activity (PaE activity) of the culture supernatant was examined. The PaE activity was measured according to a conventional method. That is, the PBSA emulsion was suspended in 20 mM Tris-HCl buffer (pH 6.8) so that the absorbance (OD660) was about 0.65. 1.9 mL of the aqueous suspension was put into a test tube having a diameter of 13 mm, and the culture supernatant was appropriately diluted, added with 100 μL, shaken at 30 ° C. and 180 rpm for 15 minutes, and the absorbance of the aqueous suspension was measured. As a unit of enzyme activity, the amount of enzyme that decreases the absorbance by 1.0 per minute was defined as 1 U.

A plurality of transformants having high PaE activity values were selected, and these strains and GB-4 (0) strain were cultured in flasks, respectively, and PaE activity and dry cell mass were compared. Specifically, 1 platinum ear of the selected strain or GB-4 (0) strain was inoculated into a test tube containing 5 mL of YM medium, and precultured at 30 ° C. and 160 rpm for 24 hours. 200 μL of the preculture solution was inoculated into a 100 mL Erlenmeyer flask containing 20 mL of 3 × FMM-8% xylose medium, and cultured with shaking at 30 ° C. and 140 rpm for 96 hours. Three cultures were independently repeated for each strain, and the PaE activity of each culture supernatant was measured. Moreover, the microbial cell of each strain | stump | stock after culture | cultivation was collect | recovered by centrifugation, it was made to dry, and the mass was measured. These results are shown in Table 2.

  The culture supernatants of the L1-S9 strain, L1-S12 strain, L1-S24 strain, L1-S33 strain, L1-S49 strain, and L1-S76 strain are more PaE than the culture supernatant of the GB-4 (0) strain. Since the activity was high, these strains can be used as a gene fragment-introduced strain for high expression of PaE. In particular, the culture supernatants of the L1-S33 strain, the L1-S49 strain, and the L1-S76 strain showed high PaE activity, and the culture supernatant of the L1-S12 strain showed the highest PaE activity. Conventionally, in studies such as mold, it is known that the expression level of a gene may vary depending on the insertion position of a foreign gene into the chromosome. The reason for the difference is considered to be that the insertion position of the gene cassette used in this example into the chromosome was different for each strain.

(5) Preparation of another PaE high-producing strain using a lysine-requiring mutant strain. A gene fragment for gene introduction containing only the sequence derived from the Antactica GB-4 (0) strain was prepared by the following two methods.
(I) Plasmid pPaLYS12X-PaEG prepared in item (3) above was amplified with E. coli DH5α, the amplified plasmid was extracted and treated with restriction enzyme EcoRI. This gene fragment was separated by agarose gel electrophoresis, and only the 5268 bp gene fragment containing the cassette for high expression of PaE was recovered and purified.
(Ii) The gene fragment was amplified by PCR using the plasmid pPaLYS12X-PaEG prepared in item (3) above as a template and LYS12 F-1000 and PXYN-1513 F as primers. The amplified gene fragments were separated by agarose gel electrophoresis, and only the 5262 bp gene fragment containing the cassette for high expression of PaE was recovered and purified.

  The gene fragment obtained by the above method (i) or (ii) is introduced into the L1 strain, which is a lysine-requiring mutant of the GB-4 (0) strain, and applied on an SDP agar medium at 30 ° C. Cultured. Strains that showed good growth on the medium ((i) 183 strain and (ii) 130 strain) were inoculated into 3 × FMM-0.5% PBSA emulsion / 8% xylose agar medium, and 2 to 3 at 30 ° C. The size of halo after daily culture was measured. Each was performed three times, and a strain having an average value of the difference between the diameter of the halo and the diameter of the colony ([halo diameter]-[colony diameter]) of 3.5 or more was selected ((i) 115 strains and ( ii) 76 strains).

  The selected strain was transferred to a YPX medium and cultured in a 2 mL deep well plate for 66 hours, and the PaE activity of the supernatant was evaluated. For the measurement of PaE activity, the method described in (4) above is partially modified (using 25 mM HEPES buffer (pH 7.3) instead of Tris-HCl buffer and using a microplate instead of a test tube). Carried out. A plurality of transformants ((i) 4 strains and (ii) 8 strains) having high PaE activity values were selected.

  A total of 12 selected transformants and a strain (L1-Lys12-1) in which only the LYS12 gene was introduced into the L1 strain as a control were inoculated in a volume of 200 μL each in a 100 mL flask containing 20 mL of YPX medium, and 30 ° C. And cultured with shaking at 200 rpm for 72 hours. Three cultures were independently repeated for each strain, and the PaE activity of each culture supernatant was measured. PaE activity was measured by partially modifying the method described in (4) above (using 25 mM HEPES buffer (pH 7.3) instead of Tris-HCl buffer). Moreover, the microbial cell of each strain | stump | stock after culture | cultivation was collect | recovered by centrifugation, it was made to dry, and the mass was measured. These results are shown in Table 3.

  Regardless of the method (i) or (ii) described above, the culture supernatant of the strain into which the gene fragment has been introduced has a PaE activity higher than that of the L1-Lys12-1 strain, which is a control. Since it was high, these strains can be used as gene fragment-introduced strains for high expression of PaE. In particular, the culture supernatants of the L1-1-3 strain (GB-4 (0) PGB474 strain) and the L1-2107 strain (GB-4 (0) PGB480 strain) were compared with other transformants. It showed high PaE activity.

Example 4 Production of uracil auxotrophic mutant of Antactica (1) Purpose S. cerevisiae cannot grow on SD agar medium supplemented with 5-fluoroorotic acid (5-FOA), which is an intermediate metabolite of the pyrimidine biosynthetic pathway, and uracil, and has a mutation in the uracil biosynthesis-related gene (URA3). Only uracil-requiring mutants can grow. Utilizing this property, the S. s. A method for obtaining a uracil-requiring mutant of S. cerevisiae has been reported (Boeke JD et al., Mol. Gen. Genet. (1984), 197, 345). S. When a uracil-requiring strain of cerevisiae is used as a host and a uracil biosynthetic gene is used as a marker gene for gene transfer, the marker gene can be removed from the transformant (Christine Guthrie, Methods, Methods). in enzyme morphology, vol. 194, Guide to yeast genetics and molecular biology, p. 293-298). Therefore, transformation and removal of the marker gene can be repeated to introduce a plurality of genes onto the chromosome of the same individual. On the other hand, P.I. For antactica, no uracil-requiring mutant has been reported yet. Therefore, in this embodiment, P.I. An attempt was made to obtain a uracil-requiring mutant of Antactica. Such mutant strains may be used for useful breeding such as self-cloning.

(2) Material The composition of the medium used in this example was as follows.
YPD medium yeast extract (Difco Laboratories) 1% by mass
Bactopeptone (Difco Laboratories) 2% by mass
Glucose 2% by mass
-YM agar medium YM medium containing 2.0% by mass of agar-SD agar medium SD medium containing 2.0% by mass of agar-SD + U + FOA medium Bacto yeast nitrogen without amino acids and (NH ₄ ) ₂ SO ₄ 0.17% by mass (manufactured by BD bioscience)
(NH ₄ ) ₂ SO ₄ 0.5% by mass
Glucose 2% by mass
Uracil (Sigma-Aldrich) 1000mg / L
5′-fluoroorotic acid (manufactured by Wako) 1000 mg / L
Agar 2% by mass

The base sequences of the primers used in this example were as follows.
・ PaURA 3-651
5'-CGAGCTGCGACACCTCGACGTAGCG-3 '(SEQ ID NO: 39)
・ PaURA3 + 1449c
5'-GTGGCCATTGGCCGTGACAACAGTA-3 '(SEQ ID NO: 40)
・ PaURA3-300
5'-GCTGCAGCTGCAGCTCTCCAAGCTG-3 '(SEQ ID NO: 41)
・ PaURA3 + 150
5'-GTGCGATGCCGTCGGCCCAGAC-3 '(SEQ ID NO: 42)

(3) P.I. Extraction of tactical URA3 homologous sequence (PaURA3) From the genomic sequence of the Antactica GB-4 (0) strain, The gene sequence encoding a protein having 82% homology with the Ura3 protein derived from cerevisiae was extracted. This was named PaURA3. The base sequence of PaURA3 is as described above (SEQ ID NO: 13).

(4) P.P. caused by ultraviolet (UV) irradiation. Acquisition of uracil-requiring mutant strain of Antactica GB-4 (0) strain 1) Acquisition of PaURA3 mutation candidate strain Antactica GB-4 (0) strain was cultured overnight at 30 ° C. and 150 rpm in a test tube containing 2 mL of YPD liquid medium. The cells were centrifuged, suspended in a liquid minimal medium so that the turbidity (600 nm) was 1.0, and 200 μL of this suspension was applied to SD + U + FOA medium. This was irradiated with light from a UV lamp for 3 minutes and then cultured at 30 ° C. for 5 days. As a result, 12 colonies were obtained.

2) Selection of PaURA3 mutant The growth of 12 colonies obtained in 1) above was observed on a YM agar medium or an SD agar medium. Since one of these strains (No. 8) grew in any medium and another one (No. 9) did not grow in any medium, these two strains were not the desired auxotrophic mutants. It was judged. The other strains were expected to be uracil-requiring because they did not grow on the SD agar medium but grew on the YM agar medium and also on the SD + U + FOA medium.

  PaURA3 gene fragment was introduced into 10 strains that showed uracil requirement and applied to an SD agar medium to observe the appearance of transformants. Specifically, P. coli cultured in YPD liquid medium. Antitactic cells were suspended in a transformation buffer containing 40% by weight polyethylene glycol 3350 (Sigma-Aldrich) 0.2 M lithium acetate and 0.1 M dithiothreitol. The PaURA3 gene fragment was prepared by PCR from genomic DNA of GB-4 (0) strain using PaURA3-651 and PaURA3 + 1449c as primers. P. After adding single-stranded salmon sperm DNA (ssDNA) (manufactured by Sigma-Aldrich) and PaURA3 gene fragment to the cell suspension of Antactica and allowing to stand at 37 ° C. for 60 minutes, it was applied to an SD agar medium, Static culture was performed at 30 ° C.

  When a transformant appears, it is considered that the uracil requirement is complemented by the introduced PaURA3, and thus the obtained mutant strain can be regarded as a mutant in which PaURA3 does not function. As a result of the introduction of the PaURA3 gene fragment, the 3 strains (No. 1, No. 2, and No. 7) appeared stably with transformants, and the others did not show much transformants. The three strains were found to be uracil-requiring mutant strains having mutations in PaURA3. No. The strain 2 was named PGB045 because it had a good appearance efficiency of colonies that were transformants, and was used for the subsequent studies. As a result of analyzing the sequence around PaURA3 with the primers PaURA3-300 or PaURA3 + 150, it was found that in the PGB045 strain, the 867th C of the PaURA3 gene was changed to T, and a stop codon was generated. For the remaining 7 strains, mutations in PaURA5, not PaURA3, are presumed.

(5) Acquisition of PaURA3 natural mutants In the production of mutants by UV irradiation, there may be mutations in chromosomal sites other than the target gene, resulting in adverse effects on enzyme production, growth, and stress resistance. There is. On the other hand, in the case of a PaURA3 mutant obtained from a natural environment with a low mutation frequency, there is a high possibility that sites other than PaURA3 on the chromosome are healthy. However, when the wild type GB-4 (0) strain is simply applied on the SD + U + FOA medium, there are no growing colonies, and a natural PaURA3 mutant cannot be obtained. Therefore, the composition of the medium was prepared so that the selection efficiency of the natural mutant strain was increased.

  To the double concentration of YM medium, uracil and FOA were added so that the final concentrations were (a) 250 mg / mL and 1000 mg / mL, or (b) 600 mg / L and 2000 mg / L, respectively. YM + U + FOA) was prepared. P. The Antactica GB-4 (0) strain was cultured with shaking in 5 mL of YM medium at 30 ° C. and 160 rpm for 24 hours, and 50 μL of the culture solution was applied onto each YM + U + FOA agar medium having a diameter of 9 cm. Forty colonies appeared from a) and ten colonies appeared from medium (b). Two of the colonies in the medium (a) did not grow on the SD medium, and all of the colonies in the medium (b) did not grow on the SD medium.

  A PaURA3 gene fragment was prepared by PCR using PaURA3 + 1449c and PaURA3-300 as primers, and transformed into colony-derived cells that appeared on the YM + U + FOA medium. As a result, one strain derived from the colony of the medium (a) and two strains derived from the colony of the medium (b) recovered the growth on the SD medium after transformation. These three strains derived from GB-4 (0) strain in which PaURA3 complemented uracil requirement were considered PaURA3 mutant strains, and were named PGB371 strain, PGB373 strain, and PGB378 strain, respectively.

  As a result of analyzing the sequence around PaURA3 with the primer PaURA3-300 or PaURA3 + 150, the PGB371 strain selected from the YM + U + FOA medium (a) had a frame shift due to the deletion of the 614th base T of the PaURA3 gene. In PGB373 and PGB378 selected from the YM + U + FOA medium (b), the 113th C of PaURA3 was changed to G, and as a result, the serine of the 38th amino acid sequence was changed to tryptophan. PGB373 and PGB378 are obtained by culturing GB-4 (0) strain in one container containing YM liquid medium and then applying it to the same selective medium. It is possible that the child is a descendant. For the remaining 7 strains (PGB372, 374, 375, 376, 377, 379, 380, 381, and 382), mutations in PaURA5 are presumed instead of PaURA3.

(6) Acquisition of further uracil-requiring mutants An attempt was made to obtain a uracil-requiring mutant strain for Pseudozyma spp. Other than the Antactica GB-4 (0) strain. First, various bacteria were cultured with shaking in 5 mL of YM medium at 30 ° C. and 160 rpm for 24 hours, and the morphology when grown on the YM medium was observed with an optical microscope equipped with a differential interference device. P. Antactica GB-4 (0) strain (FIG. 2A), P.I. Antactica OMM62-2 strain (Accession No. NITE P-02239) (FIG. 2B), P.I. Froculosa JCM10321 strain (FIG. 2C) Tsukubaensis strain JCM10324 (FIG. 2I) did not form pseudohyphae, whereas Antactica JCM3941 strain (FIG. 2D), P.I. Antactica GB-4 (1) W strain (FIG. 2E), Antactica T-34 strain (FIG. 2F), P.I. Legrosa JCM10323 strain (FIG. 2G) Aphidis JCM10318 strain (FIG. 2H) formed pseudohyphae.

Next, 50 μL of the culture solution in the YM medium was applied onto a 9 cm caliber YM + U + FOA agar medium (b; containing 600 mg / L uracil and 2000 mg / L FOA). Then, a strain that did not grow on the SD medium, that is, a uracil-requiring mutant was selected from the colonies that grew on the medium (b). The number of obtained mutant strains is shown in Table 4 below together with the observation results of the parent strain morphology.

  As shown in Table 4, P.I. A uracil-requiring mutant strain could be obtained even when a pseudozaima genus other than the Antactica GB-4 (0) strain was used. In particular, strains of the genus Pseudozyma that do not form pseudohyphae have higher acquisition efficiency of uracil-requiring mutants than strains that form pseudohyphae. In the same manner as in the method described in (4) of this example, P.I. PaURA3 was introduced into a uracil auxotrophic mutant obtained from Antactica OMM62-2. That is, when the PaURA3 gene fragment amplified from GB-4 (0) strain genomic DNA by PCR using PaURA3 + 1449c and PaURA3-300 as primers was transformed into the above mutant strain, 2 strains out of 6 strains were found on the SD medium. Since it came to grow, it turned out that the said 2 strain | stump | stock is a natural mutant which has a variation | mutation in URA3.

  In addition, P.P. In the antitactic T-34 strain, a uracil-requiring mutant strain could not be produced so far. However, one uracil-requiring mutant strain could be obtained by selection using YM + U + FOA agar medium (b), although the frequency was low. When this strain was transformed with the PaURA3 gene fragment amplified from the GB-4 (0) strain genomic DNA, it grew on the SD medium, so that the strain is a natural mutant having a mutation in URA3. I understood it. Furthermore, P.I. Since 6 strains that showed uracil requirement of Tsukubaensis JCM10324 strain were transformed with PaURA3 gene fragment amplified from GB-4 (0) strain genomic DNA, 2 strains began to grow on SD medium. The two strains were found to be natural mutants having mutations in URA3.

  Thus, the use of YM + U + FOA medium (b) makes it possible to obtain various uracil-requiring mutant strains of pseudozaima. It is also possible to select those obtained by mutating the URA3 homologous gene of each bacterium from the obtained uracil-requiring mutant strains. The uracil-requiring requirement of the selected URA3 mutant strains is GB-4 (0 It can also be complemented with a PaURA3 gene fragment amplified from strain genomic DNA. Therefore, it was shown that URA3 mutant strains were obtained for various Pseudozyma species, and that transformation by introduction of a foreign gene using PaURA3 as a gene transfer marker was possible.

[Example 5] Production of biodegradable plastic-degrading enzyme PaE high-production self-cloning strain using uracil requirement (1) Purpose The uracil-requiring mutant strain of the Antactica GB-4 (0) strain can be used for genetic manipulation (self-cloning) without using a gene derived from a heterologous organism. In this example, a self-cloning strain that produces a large amount of biodegradable plastic-degrading enzyme PaE is prepared.

(2) Material The composition of the medium used in this example was as follows.
3xFMM-PBSA emulsion 8% xylose plate medium (as described in Example 3 (2))

The base sequences of the primers used in this example were as follows.
・ PaCLE1-627
5'-GATTCGATACATGGCGTCTCTG-3 '(SEQ ID NO: 43)
・ PaURA3 (+ 900-932) -PaCLE1 + 1426c
5'-GAGGCCTACCTCGCCCGTCTTGGTCAGAAGTAGCACTACCTGTTGCGTTCGTA-3 '(SEQ ID NO: 44)
PaURA3-300 (as described in Example 4 (2))
・ PaXYN1-609
5'-GGCTGAAGCTTTGGCTCTGACA-3 '(SEQ ID NO: 45)
・ PaCLE1 + 25c-PaXYN1-1c
5'-CGGCAAAGGTCGACTTGAACTGCATCGTAAGGTTTGTTTGGCGTTTTGGG-3 '(SEQ ID NO: 46)
・ PaCLE1 + 1
5'-ATGCAGTTCAAGTCGACCTTTGCCG-3 '(SEQ ID NO: 47)
・ PaURA3-200
5'-CCCATTTCTTCCGGTTCACGTGAGC-3 '(SEQ ID NO: 48)
・ PaURA3-100
5'-TTTGCCTTTGCATGACATTCGCACG-3 '(SEQ ID NO: 49)
・ SIu-PaCLE1-1575
5'-TCATCGATGAATTCGCGTACGATATGCAGCCAATC-3 '(SEQ ID NO: 50)
・ SIdc-PaCLE1 + 1426c
5'-CCAGTGTCGAAAACGCACTACCTGTTGCGTTCGTA-3 '(SEQ ID NO: 51)
・ HindIII-PaURA3-651
5'-CCGCCAGCTGAAGCTCGAGCTGCGACACCTCGA-3 '(SEQ ID NO: 52)
・ XbaI-PaURA3 + 1449c
5'-CGCCTTTTCGTCTAGGTGGCCATTGGCCGTGAC-3 '(SEQ ID NO: 53)
・ NdeIu-PaCLE1 + 176
5'-GACAATATGTCCATACCGCCGTTTCGGGTGGCTCC-3 '(SEQ ID NO: 54)
・ NdeIdc-PaCLE1 + 1426c
5'-TGAGAGTGCACCATACACTACCTGTTGCGTTCGTA-3 '(SEQ ID NO: 55)
・ PaCLE1-1574
5'-CGTACGATATGCAGCCAATCGC-3 '(SEQ ID NO: 56)
PaURA3-651 (as described in Example 4 (2))

(3) Preparation of gene sequence for self-cloning Using PaURA3 as a selection marker, In order to introduce a gene fragment for high expression of PaE composed only of an anti-tactica-derived sequence, non-homologous end joining (NHEJ) was used for the PaURA3 gene and PaCLE1 gene including the respective promoter region and terminator region. Application is made by applying a gene transfer method (Hoshida et al., Yeast (2014), 31, 29-46). According to this method, two kinds of gene fragments introduced from the outside can be connected in vivo, and only a strain having the connected gene fragments can be grown on the medium and collected.

  First, P.I. From the chromosomal DNA of the Antactica GB-4 (0) strain, PaCLE1 gene fragment was amplified using PaCLE1-627 and PaURA3 (+ 900-932) -PaCLE1 + 1426c as primers. In this PaCLE1 gene fragment, a part of the gene sequence of PaURA3 is added in the reverse direction on the 3 ′ end side of the PaCLE1 gene (upper left of FIG. 4). Next, P.I. From the chromosomal DNA of the Antactica GB-4 (0) strain, PaURA3 gene fragment was amplified using PaURA3-300 and PaURA3 + 899c as primers. This PaURA3 gene fragment lacks the 3 'terminal sequence of the PaURA3 gene (upper right of FIG. 4), and does not function as PaURA3 as it is. A strain containing both gene fragments introduced by introducing these two gene fragments into the PGB050 strain having mutations in PaURA3 and joined by non-homologous end joining (NHEJ) in the introduced cells, that is, a strain having functional PaURA3 (FIG. 4) was obtained as a transformant grown on an SD medium. The connection between PaCLE1 and PaURA3 was evaluated by colony PCR, and the transformant in which the connection was recognized was named PGB357.

(4) Preparation of self-cloning strain It is known that in Pseudozyma spp. (Yeast), a gene under the control of PaXYN1 promoter is strongly induced in the presence of xylose. Therefore, it was decided to create a self-cloning strain that produces PaE under the control of the PaXYN1 promoter using uracil requirement. For this purpose, fusion PCR is carried out to produce a gene cassette in which the PaXYN1 promoter and PaCLE1 (PaE structural gene) are linked.

  First, P.I. The PaXYN1 promoter gene was amplified from chromosomal DNA of the Antactica GB-4 (0) strain using PaXYN1-609 and PaCLE1 + 25c-PaXYN1-1c as primers. Next, a gene fragment containing PaCLE1 and PaURA3 was amplified from the chromosomal DNA of PGB357 using PaURA3-300 and PaCLE1 + 1 as primers. These two gene fragments were connected by fusion PCR (FIG. 5). PaXYN1-609 was used as a primer on the PaXYN1 promoter side. On the other hand, as a primer on the PaURA3 side, not only PaURA3-300, but also the three types of genes having different promoter lengths using PaURA3-200 or PaURA3-100 whose promoter sequence to be amplified is shorter than PaURA3-300. Fragments were made (Figure 5). This is intended to create a situation where by cutting the promoter sequence of PaURA3 and weakening its expression, a sufficient amount of PaURA3 cannot be produced unless multiple gene fragments are introduced at one time, and cannot be grown on a selective medium. It is. By doing so, it is expected that a transformant having a plurality of gene fragments introduced therein, that is, a transformant capable of producing a large amount of PaE can be obtained. The prepared gene cassette was introduced into PGB045 strain (PaURA3 mutant strain obtained by ultraviolet irradiation) and PGB371 strain (PaURA3 natural mutant strain) to obtain a transformant (self-cloning strain).

(5) Evaluation of PaE activity of self-cloning strain The obtained transformant was applied to a plate medium (3 × FMM-PBSA emulsion / 8 mass% xylose) containing PBSA to evaluate the degradation of PBSA. Since this plate medium contains more PBSA emulsion than the conventional two-layer medium (3xFMM-PBSA emulsion / soybean oil multilayer plate medium; see also Example 7 described later if necessary), PBSA is decomposed. In order to form a transparent region (halo), more PBSA degrading enzyme is required. In addition, since xylose is used as a carbon source, expression of a gene under the control of the PaXYN1 promoter is strongly induced. When the above transformant was cultured at 30 ° C. on this medium, a transformant forming halo was observed on the third day, and halo was more clearly observed on the sixth day. For transformants containing the gene sequences of PaCLE1 and PaURA3 but introduced with any of the three types of gene fragments with different promoter lengths, 50 colonies were observed, and the PBSA emulsion was degraded to obtain a relatively clear The number of colonies in which halo formation was observed was counted. Table 5 shows the results of the transformants obtained by gene transfer into the PURA045 strain, which is a PaURA3 ultraviolet mutant.

  As shown in Table 5, there was a tendency that the acquisition rate of colonies forming large halos was higher when a fragment using a short promoter was introduced, as originally predicted. Subsequently, 2-50 strains introduced with fragments amplified using PaURA3-200 and 3-36 strains introduced with fragments using PaURA3-100 were selected, and these contained 30 mL of 3 × FMM-8 mass% xylose. A 300 mL Erlenmeyer flask was inoculated. And the PaE activity in a culture supernatant was measured after 24, 48, and 72 hours culture | cultivation.

The PaE activity was measured in the same manner as described in Example 3 (4). The results are shown in Table 6.

  As can be understood from Table 6, since the PaE activity was increased in the transgenic strain as compared with the parent strain, it is considered that the productivity of PaE was improved. In addition, for transformants obtained by gene transfer into the PURA371 strain, which is a natural mutant of PaURA3, by shortening the PaURA3 promoter, a strain with a high expression level of the introduced gene is highly likely to be obtained. Experimental results similar to those described above were obtained using as a parent strain.

(6) Preparation of another self-cloning strain (6-1) Preparation of plasmid The pAG32 plasmid (left in FIG. 6) provided by EUROSCARF was treated with SacI to be linearized. P.P. The gene fragment of PaCLE1 was amplified from the chromosomal DNA of the Antactica GB-4 (0) strain by PCR using SIu-PaCLE1-1575 and SIdc-PaCLE1 + 1426c as primers. This gene fragment was cloned into the linearized pAG32 to prepare plasmid pYT026 (in FIG. 6).

  And P.I. From the chromosomal DNA of the Antactica GB-4 (0) strain, the PaURA3 gene fragment was amplified by PCR using HindIII-PaURA3-651 and XbaI-PaURA3 + 1449c as primers. This gene fragment was cloned into pYT026 linearized with restriction enzymes HindIII and XbaI to prepare plasmid pYT108 (right in FIG. 6). In this pYT108, the drug resistance gene hphMX4 that was present in pYT026 It has been replaced with PaURA3 derived from Antactica.

(6-2) Preparation of donor strain of gene cassette A donor strain of PaCLE1-PaURA3c was prepared in order to efficiently use the gene cassette (PaCLE1-PaURA3c) in which PaCLE1 and PaURA3 were connected.
Specifically, pYT108 was linearized with the restriction enzyme EcoRV, and PaCLE1-PaURA3c was prepared by PCR using PaCLE1-1574 and PaURA3-651 as primers. This gene fragment was separated and extracted by agarose gel electrophoresis and introduced into PGB045 strain (PaURA3 mutant obtained by ultraviolet irradiation) to produce a transformant PGB459 strain. This PGB459 strain is used as a donor strain.

(6-3) Preparation of self-cloning strain The PaXYN1 promoter gene was amplified from the chromosomal DNA of the Antactica GB4- (0) strain using PaXYN1-609 and PaCLE1 + 25c-PaXYN1-1c as primers. Next, the PaCLE1-PaURA3c gene fragment was amplified from the chromosomal DNA of the PGB459 strain using PaCLE1 + 1 and PaURA3-651 as primers. Then, these two gene fragments were connected by fusion PCR using PaXYN1-609 and PaURA3-100 as primers. The gene cassette containing the PaXYN1 promoter and PaCLE1-PaURA3c thus prepared was introduced into the PGB371 strain (PaURA3 natural mutant strain) to obtain a transformant (self-cloning strain).

(6-4) Evaluation of PaE activity of self-cloning strain The obtained transformant was applied to a plate medium (3 × FMM-PBSA emulsion / 8 mass% xylose) containing PBSA to evaluate the degradation of PBSA. As a result, a plurality of transformants forming a halo were observed, and among these, the PGB469 strain showed a larger halo than the other transformants.

  S. As shown in other yeasts such as S. cerevisiae, in gene recombination using uracil requirement as an index, the marker gene such as URA3 used is deleted from the microbial cells, and the same marker gene is used repeatedly. Multiple expression cassettes can be introduced into one microbial cell. P. Even in the case of an antactica, it is expected that a strain having more expression cassettes can be produced by using this method.

[Example 6] P.I. Stabilization and concentration of biodegradable plastic-degrading enzyme PaE in an atactica culture solution (1) Purpose Even after obtaining a culture solution containing an enzyme such as PaE from Antactica, the target enzyme needs to be kept stable during storage and distribution in the state of the culture filtrate. Also, P. which is an enzyme-producing bacterium in the production process. If the anti-tactica is not completely removed or if germs are mixed, the obtained culture filtrate may be spoiled. Therefore, this example aims to establish a method for keeping the activity of PaE stable for a long time by a safe and simple method as much as possible. Another object of the present invention is to establish a method for sterilizing or completely sterilizing a culture solution (while maintaining the activity of PaE) from the viewpoint of preventing environmental contamination by recombinant organisms. In addition, to reduce product distribution costs, we will consider concentrating and compacting the PaE solution.

(2) Precipitation characteristics of PaE with ethanol At room temperature (about 20 ° C.), P.P. The culture solution of antactica and ethanol are mixed at a volume ratio of 100: 0 (0% ethanol), 75:25 (25% ethanol), 50:50 (50% ethanol), 30:70 (70% ethanol), or 10: Mixing with 90 (90% ethanol), it was examined whether a denatured precipitate was formed. As the culture solution, a filtrate obtained by filtering the culture supernatant of the L1-S12 strain with a filter and removing the cells was used. Then, precipitation did not occur with 0% ethanol and 25% ethanol, but with ethanol concentration of 50% or more, many precipitations occurred as the ethanol concentration was increased. This precipitate was collected by centrifugation at 10,000 × g for 10 minutes, redissolved with water in the same volume as the volume of the culture solution before mixing with ethanol, and then dissolved in 50% ethanol precipitate (sample A). ), A 70% ethanol precipitate redissolved solution (sample B), and a 90% ethanol precipitate redissolved solution (sample C). And the PaE activity and protein concentration of the said culture filtrate and sample AC were measured, and the recovery rate (activity recovery rate), protein precipitation rate, and specific activity of PaE activity were computed according to the following formula | equation.
Activity recovery rate = 100 × total PaE activity of redissolved solution / total PaE activity of culture solution Protein precipitation rate = 100 × total protein amount of redissolved solution / total protein amount of culture filtrate Specific activity = 100 × (resolved solution Total PaE activity / total protein amount of redissolved solution) / (total PaE activity of culture solution / total protein amount of culture filtrate)
= Activity recovery rate x total protein amount in culture filtrate / total protein amount in redissolved solution Note that PaE activity (U / mL) in this example means 25 mM Tris / HCl buffer (pH 9. 0) refers to the degradation activity of the PBSA emulsion, and the total PaE activity is obtained by multiplying the PaE activity by the volume (mL) of the sample. Table 7 shows the test results of precipitation characteristics of PaE with ethanol.

  As shown in Table 7, when the ethanol concentration was increased, both the activity recovery rate and the protein recovery rate increased, and 90% ethanol almost completely recovered the activity. This indicates that PaE is temporarily denatured and precipitated by high-concentration ethanol, but its activity can be recovered by redissolving the precipitate with an appropriate solvent.

(3) Ethanol tolerance of PaE At room temperature (about 20 ° C.), the culture filtrate of L1-S12 strain containing PaE and ethanol were mixed at a volume ratio of 100: 0 (0% ethanol), 90:10 (10% ethanol). ), 75:25 (25% ethanol), 50:50 (50% ethanol), 25:75 (75% ethanol), or 10:90 (90% ethanol). These mixed liquids were left as they were at room temperature, and the change with time of the PaE activity in the mixed liquid was examined. Samples with precipitation were sampled after being well agitated and homogenized with a touch mixer immediately before the activity measurement. The results are shown in Table 8.

  As shown in Table 8, even after leaving at room temperature for 70 days, when ethanol was added to the culture filtrate, the PaE activity in the mixed solution was remarkably stable compared to the case where no addition was made. It was kept in. And, especially during long-term storage, mild ethanol conditions such as 10 to 50% are considered preferable. In consideration of electrophoresis results of 50% saturated ammonium sulfate precipitate, 70% ethanol precipitate, and 90% ethanol precipitate, the fraction dissolved in 90% ethanol includes P.I. Although it was considered that a large amount of xylanase derived from the culture medium of antactica was contained, since xylanase activity was not actually detected from this fraction, the xylanase was inactivated in the presence of 90% ethanol. There is a possibility. Thus, it is considered that proteins other than PaE may have been irreversibly denatured or precipitated by ethanol treatment. Then, when ethanol treatment is performed, it is considered that PaE can be stabilized by inactivating an enzyme such as a protease that can cause PaE to become unstable or by suppressing the growth of microorganisms. It is done.

(4) Addition of ethanol after concentration by ammonium sulfate precipitation Ammonium sulfate was added to the culture filtrate of the L1-S12 strain containing PaE until 50% saturation was reached. The resulting precipitate (50% saturated ammonium sulfate precipitate) is recovered and redissolved with the same amount of water as the culture supernatant before addition of ammonium sulfate, and the 50% saturated ammonium sulfate precipitate redissolved solution (sample D) is obtained. Produced. PaE was recovered 95.0% in Sample D (Table 9). Further, the 50% saturated ammonium sulfate precipitate was suspended in 70% ethanol, insoluble matters were removed by centrifugation, and ethanol was further added to adjust the concentration to 90%. The resulting precipitate was recovered and redissolved with the same volume of water as the culture supernatant before addition of ammonium sulfate, and a 50% saturated ammonium sulfate precipitate / 70% ethanol soluble / 90% ethanol precipitate redissolved solution (sample E) was prepared. More than 90% of PaE was recovered in sample E (Table 9). And since specific activity rose nearly 3.5 times (Table 9), the refinement | purification degree of PaE was able to be raised by this method.

  In addition, by treating a 50% ammonium sulfate precipitate with 70% ethanol, an effective and powerful bactericidal action can be achieved. Therefore, the above-described method can prevent rot caused by microbial growth and prevent environmental pollution caused by recombinant organisms. It is considered effective for prevention. P. Considering only the purpose of recovering PaE from the culture filtrate of antactica, ethanol may be added directly to the culture filtrate until the concentration reaches 90%, but an ammonium sulfate precipitate is prepared to reduce the volume of the sample. The amount of ethanol used can be reduced by performing a precipitation operation with ethanol after the reduction.

[Example 7] Preparation of promoter evaluation method using uracil-requiring mutant and Selection of a new promoter of Antactica (1) Purpose In order to construct an efficient protein production system using genetic recombination of Pseudozyma sp., The gene of the target protein is expressed under the control of an appropriate promoter. There is a need. In this example, a system for evaluating the promoter activity of pseudozyma yeast is constructed. Specifically, a gene cassette for reporter assay is prepared by inserting a promoter sequence to be examined upstream of PaCLE1, which is a PaE gene. On the other hand, P.I. Using a PaURA3 mutant of Antactica GB-4 (0) as a parent strain, a strain in which the PaCLE1 gene is disrupted is prepared. The gene cassette is introduced into this strain using PaURA3 as a marker to construct an evaluation system using the biodegradable plastic degradation activity of PaE as an indicator of promoter strength.
For example, it is convenient if a protein can be produced under the control of a promoter that stably expresses a gene regardless of the components of the medium and the state of the cells. Such promoters are designated P.P. In order to search for the Antactica GB-4 (0) strain, the GB-4 (0) strain is cultured under a plurality of carbon source conditions, and a comprehensive gene expression analysis using the cells is performed to stably express the strain. Select genes. Then, using an evaluation system using PaE as a reporter, P.I. Attempts to select promoters that are stably expressed in antactica.

(2) Material The base sequences of the primers used in this example were as follows.
・ PvuII-PaCLE1 + 1968c
5'-GAACGCGGCCGCCAGTGTGTCTTGTCGTTGTACAC-3 '(SEQ ID NO: 57)
・ PvuII-PaCLE1 + 613
5'-TGAACGTTGGTGTTGTATCCCTGAAGCTTCGTACG-3 '(SEQ ID NO: 58)
・ EcoRV-PaCLE1 + 263c
5'-GAATTCATCGATGATGCCACGATGTTGGCAGTACCCT-3 '(SEQ ID NO: 59)
・ EcoRV-PaCLE1-1074
5'-ACTAGTGGATCTGATAACGTTGCCGATCCGCAGTCTC-3 '(SEQ ID NO: 60)
・ SalI-PaURA3-651
5'-TACGCTGCAGGTCGACGAGCTGCGACACCTCGACGTAGCG-3 '(SEQ ID NO: 61)
・ SacI-PaURA3 + 1449c
5'-TCATCGATGAATTCGGTGGCCATTGGCCGTGACAACAGTA-3 '(SEQ ID NO: 62)
・ EcoRI-PaCLE1-1574
5'-TATCATCGATGAATTCGTACGATATGCAGCCAATC-3 '(SEQ ID NO: 63)
・ EcoRI-PaCLE1 + 1426c
5'-AATGGCCACCGAATTCACTACCTGTTGCGTTCGTA-3 '(SEQ ID NO: 64)
PaCLE 1-1574 (as described in Example 5 (2))
PaURA3-300 (as described in Example 4 (2))
・ PaFRE1-903
5'-GCCGGAGTGATTCATGATTCGA-3 '(SEQ ID NO: 65)
PaXYN1-609 (as described in Example 5 (2))
PaCLE1 + 25c-PaXYN1-1c (as described in Example 5 (2))
・ PaCLE1 + 25c-PaFRE1-1c
5'-CGGCAAAGGTCGACTTGAACTGCATCTTGATCTATCGAGGCCCCGCACGA-3 '(SEQ ID NO: 66)
・ PaCLE1 + 25c-PaCTR1-1c
5'-CGGCAAAGGTCGACTTGAACTGCATGCCGGAGTGATTCATGATTCGAATC-3 '(SEQ ID NO: 67)
・ PaTDH3-1500
5'-CCATTGCAGGTCTCGACATCAC-3 '(SEQ ID NO: 68)
・ PaCLE1 + 25c-PaTDH3-1c
5'-CGGCAAAGGTCGACTTGAACTGCATTTTTGAGATGATGGATGGGGAGTGT-3 '(SEQ ID NO: 69)
・ PaCTR1-903
5'-CTTGATCTATCGAGGCCCCGCA-3 '(SEQ ID NO: 70)

The composition of the medium used in this example was as follows.
・ 3xFMM-PBSA emulsion ・ Soybean oil multilayer plate medium composition upper layer (carbon source and PBSA emulsion)
PBSA emulsion * 1.0% by mass
Soybean oil (Wako Pure Chemical Industries) 1.0 mass%
Agar 1.5% by mass
* Bionole emulsion EM-301 (PBSA) (Showa Denko)
Lower layer (3xFMM medium, composition excluding carbon source)
NaNO ₃ 0.2% by mass
KH ₂ PO ₄ 0.06% by mass
MgSO ₄ · 7H ₂ O 0.06% by mass
Yeast extract (Difco Laboratories) 0.3% by mass
(Production method)
The upper layer raw material was dissolved and sterilized in an Erlenmeyer flask containing a stirrer. About 15 mL of a lower layer plate medium was prepared in a 9 cm petri dish, and after solidifying, it was stored in an incubator at 45 ° C., and an upper layer of about 5 mL was overlaid. At this time, the upper layer was stirred with a stirrer so that the oil was uniformly mixed.

YPD medium for reporter assay Yeast extract (Difco Laboratories) 1% by mass
Peptone (Difco Laboratories) 2% by mass
Glucose 8% by mass
(Agar 2% by mass)
・ YPX medium for reporter assay Yeast extract (Difco Laboratories) 1% by mass
Peptone (Difco Laboratories) 2% by mass
8% by mass of xylose
(Agar 2% by mass)

(3) Production of PaE non-producing strain by gene disruption Endogenous PaCLE1 on the antitactic chromosome is disrupted (replaced) with natMX4 to produce a non-PaE producing strain. This natMX4 is S.I. It is a nourseotricin (cloneNAT) resistance gene that has recently been used in experiments using cerevisiae. As a construct for destroying PaCLE1, the chromosome of GB-4 (0) strain was obtained by PCR using KOD FX enzyme (Toyobo) to produce a gene sequence with natMX4 sandwiched between upstream and downstream sequences of PaCLE1. The sequence around PaCLE1 was amplified from DNA and cloned into a plasmid (pAG25: obtained for research from EUROSCARF) in which natMX4 was cloned using In-Fusion HD cloning kit.

  First, using PvuII-PaCLE1 + 1968c and PvuII-PaCLE1 + 613 as primers, the gene fragment was amplified by PCR using the chromosomal DNA of GB-4 (0) strain as a template, and the obtained gene fragment was cloned into pAG25 digested with PvuII. The resulting plasmid was named pYT007. Furthermore, pYT007 was digested with EcoRV, a gene fragment obtained by PCR using EcoRV-PaCLE1 + 263c and EcoRV-PaCLE1-1074 as primers was cloned, and the resulting plasmid was named pYT012. PYT012 digested with NotI was introduced into the PGB045 strain by the lithium method. From the obtained transformants, a strain that did not show PBSA degradation was selected on a 3 × FMM-PBSA emulsion / soybean oil multilayer plate medium and named PGB050 strain.

(4) Preparation of reporter assay gene sequence using PaE as an index First, in order to use PaURA3 as a marker for gene introduction, a plasmid in which natMX4 of pAG25 is substituted with PaURA3 is prepared. PaURA3 fragment for cloning was obtained by PCR using SalI-PaURA3-651 and SacI-PaURA3 + 1449c as primers. This was cloned into pAG25 digested with SacI and SalI, and the resulting plasmid was named pYT019.

  Next, a construct for introducing PaCLE1 is prepared. P. The PaCLE1 gene fragment was amplified from the chromosomal DNA of the Antactica GB-4 (0) strain by PCR using EcoRI-PaCLE1-1574 and EcoRI-PaCLE1 + 1426c as primers. This gene fragment was cloned into pYT019 digested with EcoRI to obtain a plasmid, and the resulting plasmid was named pYT040.

(5) Selection of homeostatic promoter of GB-4 (0) strain and evaluation of promoter by reporter assay using PaE 1) Selection of Candidate Promoter Sequences by Expression Analysis of Culture of Antactica GB-4 (0) Strain GB-4 (0) strain was added to 50 mL liquid medium (0.6% by weight NaNO containing 8% glucose or xylose). ₃ , after inoculating a 500 mL Erlenmeyer flask containing 0.06% by mass KH ₂ PO ₄ , 0.06% by mass MgSO ₄ .7H ₂ O, 0.3% by mass yeast extract (manufactured by Difco Laboratories)) The cells were cultured at 30 ° C. and 200 rpm for 24 hours and 168 hours. RNA was extracted from the cells and subjected to microarray analysis of GB-4 (0) strain.

  Based on the microarray analysis results under each culture condition, ORFs having a high RNA content were searched for stably under any condition. At this time, with reference to the annotation information of the GB-4 (0) strain genomic sequence, the ORF in which the function of the protein is presumed is encoded by an S. cerevisiae encoding a ferric reductase-like protein. PaFRE1 and S. cerevisiae having high homology with the cerevisiae FRE1 gene. PaCTR1 having high homology with the gene of CTR1 which is a copper transporter of cerevisiae was found, and attention was paid to these promoters. Each promoter sequence is designated P.P. As a result of extraction from the genomic sequence of the Antactica GB-4 (0) strain and comparison with each other, each promoter sequence is a common region on the genomic sequence of the GB-4 (0) strain and is used in opposite directions. I found out. S. The promoter of the gene for Tdh3, which is an isozyme of glyceraldehyde 3-phosphate dehydrogenase of S. cerevisiae, is S. cerevisiae. Since S. cerevisiae is often used as a stable promoter with high expression level, P. cerevisiae having high homology to TDH3. Focusing on the promoters of the antitactic gene (PaTDH3), we decided to investigate the functions of these promoters. For comparison, a PaCLE1 promoter and a highly expressed xylanase (PaXYN1) promoter known to be xylose-inducible were employed.

2) Construction of reporter assay system In order to obtain these focused gene fragments from the genome of GB-4 (0) strain, PCR using KOD plus was performed. Specifically, a gene fragment for evaluating the PaCLE1 promoter from pTY040 was prepared by PCR using PaCLE1-1574 and PaURA3-300 as primers (upper right of FIG. 7).

Gene fragments for evaluating PaXYN1, PaFRE1, PaCTR1, and PaTDH3 promoters were prepared by fusion PCR. Specifically, each promoter gene was amplified from the chromosomal DNA of the GB-4 (0) strain (upper left of FIG. 7). The primers used at this time were as shown in Table 10. The PaFRE1 promoter and the PaCTR1 promoter are promoters that are amplified with the same primer but have different directions.

Next, using PaCLE1 + 1 and PaURA3-300 as primers, a gene fragment of PaCLE1 that does not contain the PaCLE1 promoter was amplified from pTY040 (upper center in FIG. 7). This gene fragment was connected to each promoter gene by fusion PCR (FIG. 7). In addition, in order to produce a control gene fragment that does not contain PaCLE1 and contains only the marker gene, the gene fragment of PaURA3 is amplified by PCR from the chromosomal DNA of GB-4 (0) strain using PaURA3-300 and PaURA3 + 1449c as primers. did. Table 11 shows the primers used in this test example.

The sequence of the promoter region of each gene was as follows.
-Sequence of the promoter region of PaFRE1
(SEQ ID NO: 71)

-Sequence of the promoter region of PaCTR1
(SEQ ID NO: 72)

-PaTDH3 promoter region sequence
(SEQ ID NO: 73)

  Each of the gene fragments prepared as described above was introduced into the PGB050 strain, and a transformant showing degradation of PBSA was obtained on a 3 × FMM-PBSA emulsion / soybean oil multilayer plate medium. Using the obtained transformant, the function of each promoter sequence was evaluated. Specifically, (1) untransformed GB-4 (0) strain, (2) PaXYN1 promoter / PaE introduced PGB050 strain, (3) PaCLE1 promoter / PaE introduced PGB050 strain, (4) PaTDH3 promoter / PaE Introduced PGB050 strain, (5) PaCTR1 promoter / PaE introduced PGB050 strain, (6) PaFRE1 promoter / PaE introduced PGB050 strain, or (7) PGB050 strain into which only the marker gene was introduced, YPD medium for reporter assay (8% by mass) 2 mL of YPX medium for reporter assay (containing 8% by mass of xylose) and shaking culture in a test tube for 48 hours, and PaE activity of the culture supernatant was quantified. The result is shown in FIG. All promoters were confirmed to function in the presence of glucose or xylose. In particular, the PaFRE1 promoter (sample 6) exhibited a high PaE activity regardless of the sugar used, and thus may be a highly functional promoter. I was expecting.

Claims (14)

  Pseudozyma antarctica GB-4 (0) strain (Accession number NITE P-02392).   An auxotrophic mutant of Pseudozyma antactica GB-4 (0) strain (Accession No. NITE P-02392).   The auxotrophic mutant according to claim 2, which is lysine-requiring or uracil-requiring.   An auxotrophic mutant of the genus Pseudozyma having a mutation in at least a part of the base sequence of LYS2, LYS12, and / or LYS20, or a mutation in at least a part of the base sequence of URA3 and / or URA5 An auxotrophic mutant, characterized by comprising:   It has a deletion of at least a part of the gene encoding Met1-Glu195 of the LYS12 protein, or a substitution at position 113, substitution at position 867, and / or deletion at position 614 in the URA3 base sequence 5. An auxotrophic mutant according to claim 4 having a loss.   The auxotrophic mutant according to claim 4 or 5, wherein the pseudozyma genus is a strain of the genus Pseudozyma that does not form pseudohyphae. A transformant of Pseudozyma that expresses the protein of interest,
The transformant is a transformant of an auxotrophic mutant,
The transformant has at least one gene cassette;
The gene cassette contains a gene that encodes the protein of interest, a promoter of XYN1 or FRE1 of Pseudozyma sp., And a gene that complements the auxotrophy of the auxotrophic mutant, and encodes the protein of interest. A transformant, wherein the gene is located downstream of the promoter.   The transformant according to claim 7, wherein the target protein is a protein of the genus Pseudozyma or a protein of an organism other than the genus Pseudozyma.   The transformant according to claim 7 or 8, wherein a codon used in a gene encoding the protein of interest is optimized to a codon used by Pseudozyma sp.   Pseudozyma Antactica GB-4 (0) L1-S12 strain (Accession number NITE P-02393), GB-4 (0) PGB480 strain (Accession number NITE P-02638), GB-4 (0) PGB469 strain (Accession number) NITE P-02639) and GB-4 (0) PGB474 strain (Accession No. NITE P-02640), a transformant of Pseudozyma antactica. A method for producing a protein of interest comprising:
A process of preparing an auxotrophic mutant of pseudozaima,
Introducing at least one gene cassette into the auxotrophic mutant; and
Culturing the auxotrophic mutant introduced with the gene cassette to produce the protein of interest,
Including
The gene cassette contains a gene that encodes the protein of interest, a promoter of XYN1 or FRE1 of Pseudozyma sp., And a gene that complements the auxotrophy of the auxotrophic mutant, and encodes the protein of interest. A method wherein the gene is located downstream of said promoter. A method for obtaining a stabilized biodegradable plastic degrading enzyme,
Preparing a solution containing the biodegradable plastic-degrading enzyme;
Adding ethanol to the biodegradable plastic-degrading enzyme-containing solution to stabilize or precipitate the biodegradable plastic-degrading enzyme; and
Recovering the stabilized or precipitated biodegradable plastic-degrading enzyme;
A method comprising the steps of:   The method according to claim 12, further comprising a step of concentrating the biodegradable plastic degrading enzyme before the ethanol addition step.   14. The method of claim 12 or 13, further comprising the step of producing the biodegradable plastic degrading enzyme according to the method of claim 11.