WO2003000895A1

WO2003000895A1 - Yeast transformation vector system comprising multicopy paromomycin-resistance gene

Info

Publication number: WO2003000895A1
Application number: PCT/KR2002/001180
Authority: WO
Inventors: Jae Mahn Song
Original assignee: Jae Mahn Song
Priority date: 2001-06-21
Filing date: 2002-06-20
Publication date: 2003-01-03
Also published as: KR100422384B1; KR20020096576A

Abstract

A yeast transformation vector system using a paramomycin-resistance gene. The yeast transformation vector system is useful in producing paromomycin-resistant recombinants by isolating a multicopy paromomycin-resistance MPA1 gene, constructing pCABIOM101 vector (accession number: KCTC 1014BP) containing the MPA1 gene and pCABIOM111 derived therefrom, and transforming yeasts Saccharomyces cerevisiae and Pichia pastoris with the pCABIOM101 and pCABIOM111 respectively, and therefore plays an important role as a tool for basic studies of yeasts, as well as for use in biological industries.

Description

YEAST TRANSFORMATION VECTOR SYSTEM COMPRISING MULTICOPY PAROMOMYCIN-RESISTANCE GENE

Technical Field

The present invention relates to a yeast transformation vector system using a paromomycin-resistance gene. More particularly, the present invention relates to construction of a yeast transformation vector system, comprising isolating a multicopy paromomycin-resistance gene, MPAl, and constructing a pCABIOMlOl vector containing the MPAl gene and a pCABIOMlll vector derived from the pCABIOMlOl vector.

Background Art

Yeasts are safe for human consumption and have been used in bread, beer, distilled liquor, wine or clear strained rice wine production from the beginning of human history. They are now regarded as a GRAS (Generally Regarded As Safe) organism. Various studies of yeasts have been steadily carried out for developing industrial and commercially useful substances using yeasts and improving existing industrial yeasts. However, most industrial yeasts are diploid or polyploid organisms and thus difficult to mutate. Furthermore, they are sterile and thus spore formation and mating with other yeasts do not readily occur. For these reasons, satisfactory results have not been obtained in most yeast studies, unlike those using industrial bacteria. Recently, genetic recombination techniques using transformation systems for yeasts to overcome such limitations have been applied, thereby allowing development of yeasts having improved efficiency and productivity. The transformation systems for industrial yeasts, Pichia pastoris and Hansenula polymorpha have been developed and commercialized by Phillips Petroleum (Bartlesville, OK, USA) and Rhein Biotech (Dusseldorf, Germany) respectively. Recently, the transformation system for

Saccharomyces cerevisiae using an Aureobasidin A-resistance gene has been commercialized by Takara Shuzo (Shiga, Japan). As well, various yeast transformation vectors using wild-type genes, or antibiotic- or chemical-resistance genes have widely been used in Saccharomyces cerevisiae. On the other hand, with respect to industrial yeasts, yeast transformation vectors using antibiotic- or chemical- resistance genes (aureobasidin A, chloramphenicol, G418/geneticin, zeocin, copper, methatrexate, methylglyoxal, sulfometuron, and glyphosphate-resistance genes) have mainly been used. Therefore, in order to diversify the transformation vector systems for yeasts required for basic studies of yeasts or for industrial purposes, there is a need for development of antibiotic- or chemical-resistance genes that are different from the existing genes. Mutations associated with aminoglycoside antibiotics are useful in understanding the regulation mechanism of protein synthesis in both prokaryotes and eukaryotes. Nonsense suppressors designated as sup35 and sup45 that result in the suppression of nonsense mutations were isolated from yeasts. Most mutants that carried such nonsense suppressor genes grew slowly and exhibited various phenotypes (temperature sensitivity, osmotic sensitivity, drug sensitivity, etc.) (Song, J.M. and

Liebman, S.W., Genetics 115, 451-460 (1987)). Meanwhile, various genes that impact on actions of such nonsense suppressors were isolated from yeasts. Particularly, asu9 acts as an antisuppressor, reducing the effect of the sup45 suppressor.

The asu9 and sup45 induce sensitivity to the aminoglycoside antibiotic, paromomycin in yeasts. Attempts have been made to clone a wild-type gene ASU9⁺ that is complementary to the translational mutation, asu9-l, to facilitate the genetic analysis of the asu9. To this end, a yeast strain containing the asu9 mutation was transformed with YEp24 library into which partial fragments of Sau3A on yeast chromosomal DNA were inserted. The transformation event yielded two plasmids that were complementary to the paromomycin sensitivity of the asu9-l. The two plasmids, pJSl and pJS2 were complementary to the paromomycin sensitivity of the asu9-l and also manifested strong paromomycin-resistance in the ASU9⁺ yeast strains. The tetrad analysis has shown that such expression of the paromomycin-resistance is not caused by the yeast chromosomal ASU9⁺ gene but by other genes contained in the insertion DNA of the pJSl and ρJS2 (Song, J.M., Ph.D. Thesis, University of Illinois, Illinois, 1987).

However, no examinations have been carried out characterizing the gene exhibiting paromomycin-resistance when present at multicopy numbers in yeasts.

Disclosure of the Invention

Therefore, the present inventor had completed the invention by carrying out a molecular genetic analysis of genes inserted into plasmid pJS2, and isolating and analyzing the gene which is involved in resistance to paromomycin among them.

An object of the present invention is to construct pCABIOMlOl vector containing paromomycin-resistance gene, MPAl after isolating and analyzing the MPAl gene from the insertion genes in plasmid ρJS2. Another object of the present invention is to provide the pCABIOMlOl vector and another plasmid vector derived therefrom as a yeast transformation vector system.

In accordance with the present invention, the above and other objects can be accomplished by construction of pCABIOMlOl vector and pCABIOMlll vector derived therefrom, comprising isolating a multicopy paromomycin-resistance gene, MPAl and constructing the pCABIOMlOl vector containing the MPAl gene. Yeasts that are transformed with the pCABIOMlOl vector and the pCABIOMlll vector respectively exhibit strong resistance to paromomycin. Such transformation vector systems of yeasts are useful for basic studies of yeasts or for industrial purposes.

Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: Fig. 1 is a view showing deletions of plasmid pJS2;

Fig. 2 is a restriction map of pCABIOMlOl vector containing a multicopy paromomycin-resistance gene, MPAl; and

Fig. 3 is a restriction map of pCABIOMlll vector containing the ORF of a multicopy paromomycin-resistance gene, MPAl.

Best Mode for Carrying Out the Invention

Hereinafter, the constitutional elements and biological effects of the present invention will be described in more detail.

The present invention is accomplished by carrying out the following steps: deleting plasmid pJS2 to isolate a paromomycin-resistance gene from the insertion DNA of the plasmid pJS2; isolating and analyzing the paromomycin-resistance gene,

MPAl by DNA sequencing; constructing pCABIOMlOl vector containing the MPAl gene; analyzing a degree of resistance to paromomycin depending on copy number of the MPAl gene; selecting the paromomycin-resistant transformants obtained after transforming Saccharomyces cerevisiae with the pCABIOMlOl; and selecting the paromomycin-resistant transformants obtained after transforming Pichia pastoris with pCABIOMlll vector derived from the pCABIOMlOl vector.

Plasmid pJS2 used in the present invention is that disclosed in the present inventor's doctoral thesis (Song, J.M., Ph.D. Thesis, University of Illinois, Illinois, 1987).

The present invention will hereinafter be described more specifically by illustrative examples. It is, however, to be borne in mind that the present invention is by no means limited to or by them.

Example 1: Isolation of multicopy paromomycin-resistance MPAl gene Step 1: Deletions of the insertion DNA in plasmid pJS2

Plasmid pJS2 (Song, J.M., Ph.D. Thesis, University of Illinois, Illinois, 1987) is a recombinant plasmid constructed by inserting a 7.9 kb fragment of yeast chromosomal DNA into YEp24 vector that replicates at multicopy numbers by the replication determinant of the yeast 2μ in yeasts. The present inventor assumed in previous studies that a paromomycin-resistance gene among genes contained in the insertion DNA of pJS2 is positioned on one end of the 7.9 kb fragment where Sail and Nhel restriction enzyme sites are present. Based on the assumption, deletions of pJS2 were carried out for the paromomycin-resistance gene identification according to the following method. The 7.9 kb insertion fragment in the pJS2 has a Sail and a Nhel restriction enzyme site respectively. After the restriction enzyme sites were digested, two pJS2 deletions were obtained as below. As shown in Fig.l, one deletion called pJS2-l was obtained after Sail restriction enzyme digestion in the pJS2, resulting in a 5.8 kb Sail fragment of pJS2 being deleted. The other deletion called pJS2-l-l was obtained after Nhel restriction enzyme digestion in the pJS2-l, resulting in a 1.2 kb Nhel fragment of pJS2-l being deleted.

The pJS2 deletions, pJS2-l and pJS2-l-l were introduced into a yeast strain, SL680-7A (α asu9-l ade3-26 leu2-l met8-l trpl his5-2 ura3-52) to obtain respective transformants. The phenotypes of the transformants were investigated. The respective pJS2 deletions obtained by deleting the Sail and Nhel DNA fragments respectively from the pJS2 were expressed at multicopy numbers by 2μ present in the YEp24 vector when inserted into the yeast. The transformants contained the URA3 selectable marker genes, allowing Ura^~ cells to grow on uracil-deficient media. Therefore, the transformants were selected by growth on the uracil-deficient media. In order to investigate whether the introduced plasmid-derived gene exhibits paromomycin-resistance, the selected transformants were inoculated in 5 mg ml paromomycin-containing YPD complete media. The transformants containing the respective plasmids inoculated in the paromomycin-containing media exhibited the following results. YEp24 is a transformation vector carrying the URA3 selectable marker and transformants containing the YEp24 did not grow in the presence of paromomycin. Transformants containing pJS2 exhibited strong resistance to paromomycin, like pJS2-l and pJS2-l-l (see Table 1).

Table 1 Phenotypes of transformants

Growth** on:

Strain (plasmid)* YPD YPD+5mg ml SC SC-Ura paromomycin

SL680-7A (None) + - + -

(YEp24) + - + 4-

(pJS2) + + + +

(pJS2-l) + + + +

(pJS2-l-l) + + + +

(pCABIOMlOl) + + + +

(pCAB!OM102) + - + +

* Yeast strain was SL680-7A (α asu9-l ade3-26 his5-2 leu2-l met8-l trpl ura3-52), and plasmid pCABIOM102 was inserted after treated with the restriction enzyme, Ncol.

** Yeast suspensions were inoculated by spotting in yeast complete media (YPD), paromomycin-containing media (YPD+5mg/ml paromomycin), synthetic complete media (SC) and uracil deficient media (SC-Ura) respectively, Followed by comparison of growth characteristics. The evaluation of yeast growth is as follows: +, good growth by 4 days; -, no sign of growth by 7 days.

The above results show that it is not necessary to have entire sequences of genes contained in the pJS2 for expression of paromomycin-resistance. They also show that paromomycin-resistance is manifested when genes contained in the pJS2-l and pJS2-l-l fragments are expressed at multicopy numbers in yeasts.

Step 2: Isolation and analysis of the paromomycin-resistance MPAl gene

It could be seen from the step 1 that the paromomycin-resistance gene among the insertion DNA in plasmid pJS2 is present in a 2.3 kb SaH-BamHL DNA fragment and a 1.2 kb Saϊl-Nhel DNA fragment respectively. Therefore, these sites were DNA sequenced. It was found that 1,952 bp Bamtt site of the 2.3 kb SaU-BamHl DNA fragment and 770 bp Nhel site of the 1.2 kb SaR-Nhel DNA fragment correspond to N- terminal site of the YDR407C gene on yeast chromosome IV (Goffeau et al, Science 274, 546-567 (1997)). The above result shows that the N-terminal site (770 bp; 19.9% ORF) of the YDR407C gene (total 3,871 bp ORF) can be involved in the manifestation of paromomycin-resistance. The functions of the YDR407C gene and its mechanism have not been found, with the exception of its DNA base sequence. It was finally found in this step that the YDR407C gene is a multicopy paromomycin-resistance gene, and therefore the gene and its N-terminal site were called MPAl (multicopy paromomycin-resistance) gene.

Step 3: Construction of pCABIOMlOl vector containing the MPAl gene

It was re-confirmed in this step that the MPAl gene of the step 2 is contained in the 1.2 kb SaR-Nhel DNA fragment of the plasmid pJS2-l-l obtained in the step 1. To this end, first, the plasmid pJS2 was treated with Sail and Nhel restriction enzymes thereby to obtain 1.2 kb SaR-Nhel DNA fragment. Then, the DNA fragment so obtained was inserted into the Sail and Nhel restriction enzyme sites of YEp24 vector that replicates at multicopy numbers in yeasts, resulting in pCABIOMlOl vector (see Fig.2). Transformants obtained by inserting the pCABIOMlOl vector into yeast strain

SL680-7A exhibited resistance to paromomycin (Table 1). Based on this fact, it can be seen that the pCABIOMlOl vector contains a multicopy paromomycin-resistance gene, MPAl.

The pCABIOMlOl vector constructed by inserting the paromomycin- resistance gene MPAl into YEP24 vector was deposited at Genetic Resources Center,

Korea Research Institute of Bioscience and Biotechnology attached to Korea Institute of Science and Technology on May 28, 2001 under KCTC 1014BP.

Step 4: Investigation of degree of resistance to paromomycin depending on copy number of the MPAl gene A 2.2 kb EcøRI DNA. fragment which is the 2μ replication origin of YEp24, was deleted from the pCABIOMlOl vector constructed in the step 3, resulting in pCABIOM102 vector. The pCABIOM102 so obtained was inserted into yeast strain SL680-7A after being linearized by digestion of the Ncol restriction enzyme site of URA3 therein. The inserted pCABIOM102 were expressed at single-copy number, not at multicopy numbers after insertion into ura3-52 site in the yeast. The obtained transformants contained the URA3 selectable marker gene of the plasmid vector, allowing Ura^' cells to grow on uracil-deficient media. Therefore, the transformants were selected by growth on the uracil-deficient media. Where the selected transformants were inoculated into 5 mg/ml paromomycin-containing YPD complete media, there was no sign of growth (see Table 1). Judging from the fact that transformants containing pCABIOMlOl exhibited strong resistance to paromomycin but transformants containing pCABIOM102 of same MPAl gene exhibited sensitivity to paromomycin, it can be seen that resistance to paromomycin is accomplished only when the MPAl gene is expressed at multicopy numbers.

Example 2: Transformation of S. cerevisiae with the pCABIOMlOl vector

Phenotypes of transformants obtained by inserting the pCABIOMlOl vector into ASU9⁺ yeast (S. cerevisiae) strains, S73 (α serl-171 ura3-52) and JS243-7D (a leu2 trpl-Δ ura3-52) respectively were evaluated in this example. The obtained transformants contained the URA3 selectable marker gene of the plasmid vector, allowing Ura^~ cells to grow on uracil-deficient media. Therefore, the transformants were selected by growth on the uracil-deficient media. The selected transformants were inoculated into 10 mg/ml paromomycin-containing SC-Ura solid media in order to investigate whether the introduced plasmid-derived gene exhibits paromomycin- resistance. Each transformant inoculated into paromomycin-containing media exhibited the following results. The transformants containing transformation vector YEp24 with URA3 selectable marker did not grow in the presence of paromomycin. On the contrary, the transformants containing the pCABIOMlOl exhibited strong resistance to paromomycin (see Table 2). It can be seen from the above results that when the MPAl gene contained in the pCABIOMlOl vector is expressed at multicopy numbers in ASU9⁺ yeasts, paromomycin-resistance is manifested in the ASU9⁺ yeasts transformed with the pCABIOMlOl vector.

Table 2 Phenotypes of laboratory yeast transformants

Strain (plasmid)* Growth** on:

SC SC-Ura SC-Ura+lOmg/ml paromomycin

S73 (None) +

(YEp24) +

(pCABIOMlOl) +

143-7D (None) +

(YEp24) +

(pCABIOMlOl) + * Yeast strains were S73 (α ser 1-171 ura3~52) and JS143-7D (α leu2 trpl-Δ urα3-52). ** Yeast suspensions were inoculated by spotting in synthetic complete media (SC), uracil deficient media (SC- Ura) and paromomycin-containing media (SC-Ura+lOmg/ml paromomycin) respectively, followed by comparison of growth characteristics. The evaluation of yeast growth is as follows: +, good growth by 4 days; -, no sign of growth by 7 days.

Example 3: Transformation of yeast Pichia pastoris with pCABIOMlll vector

The present inventor investigated in this example whether the MPAl gene which is derived from yeast S. cerevisiae is expressed when being introduced into yeast Pichia pastoris. In order to do so, first, two primers, 5'-

GGAGACAAAAACCATGGATATTCTTAAGCA-3' (forward primer: SEQ ID NO: 4) and 5'-GTAGCTAGCTTTAGAGAAGCG-3' (reverse primer: SEQ ID NO: 5) based on base sequence of the MPAl gene in the pCABIOMlOl vector were constructed. The primers were designed to allow subsequent amplified DNA to contain the entire ORF of the MPAl gene in the pCABIOMlOl vector, an Ncol restriction enzyme site at the 5'- end, and an Nhel restriction enzyme site at the 3'-end. Exceptionally, the second codon in the MPAl ORF, AAT was changed to GAT, resulting in an Asn2→ Asp_2 amino acid substitution. Amplified DΝA by PCR amplification using the two primers was digested with Ncol and Nhel restriction enzymes, thereby to obtain a 771 bp Ncol- Nhel DΝA fragment. The obtained DΝA fragment was treated with Klenow enzyme, leaving blunt ends. At the same time, Pichia pastoris expression vector pHIL-D2 was digested with EcoRI restriction enzyme and then Klenow enzyme, thereby producing blunt-ends. Then, the 771 bp blunt-ended DΝA fragment was inserted into the blunt- ended vector thereby to give pCABIOMlll vector. The pCABIOMlll vector so obtained was digested with SaR restriction enzyme thereby to construct His⁺Mut⁺ transformants by transforming auxotroph, Pichia pastoris GS115 which has a nonfunctional histidinol dehydrogenase gene (his4) with the pCABIOMlll vector. In this case, the linearized DΝA of the pCABIOMlll vector obtained by digestion of SaR restriction enzyme was inserted into HIS4 site of the Pichia pastoris GS115 thereby producing His⁺ Mut⁺ Pichia transformants.

In order to investigate presence or absence of expression of the introduced plasmid-derived gene, selected transformants were inoculated into 10 mg ml paromomycin-containing minimal methanol (MM) media. Transformants containing respective plasmids exhibited the following results. The transformants containing pHIL-D2, which is a transformation vector containing HIS4 selectable marker, did not grow in the presence of paromomycin. On the contrary, the transformants containing the pCABIOMlll vector exhibited resistance to paromomycin (see Table 3). The above result shows that paromomycin-resistance can be expressed in the yeast Pichia pastoris transformed with the pCABIOMlll vector. Specifically, this indicates that when the MPAl gene in the pCABIOMlll vector is expressed in yeast Pichia pastoris, although it is not inserted at multicopy numbers, the AOX1 promoter of Pichia pastoris is induced by methanol and thus is involved in resistance to paromomycin. It was confirmed by PCR that the MPAl gene is introduced into the chromosomal DNA of Pichia pastoris. Specifically, PCR was carried out using 5' AOX1 primer and 3' AOX1 primer. When the amplified DNA was subjected to gel electrophoresis, a 2.2 kb wild-type AOX1 gene band and a 0.8 kb MPAl gene band were detected.

Table 3 Phenotypes of Pichia pastoris transformants

Strain [plasmid]* Growth** on:

MMH MM MM+10 mg ml paromomycin

GS115 [None]

[pHIS-D2] [pCABIOMlll]

* Yeast strain was Pichia pastoris GS115 Qιis4), and plasmids pHIS-D2 and pCABIOMlll were inserted after treated with the restriction enzyme, Sad.

** Yeast suspensions were inoculated by spotting in methanol minimal media supplemented with histidine (MMH), methanol minimal media (MM) and paromomycin-containing media (MM+lOmg/ml paromomycin) respectively, followed by comparison of growth characteristics. The evaluation of yeast growth is as follows: +, good growth by 4 days; ± , some growth by 4 days; -, no sign of growth by 7 days.

Industrial Applicability

As apparent from the above description, the present invention provides pCABIOMlOl vector (accession number: KCTC 1014BP) and pCABIOMlll vector derived therefrom as the transformation vector system of yeasts, obtained by isolating a multicopy paromomycin-resistance MPAl gene and constructing the pCABIOMlOl vector containing the MPAl gene. Therefore, paromomycin-resistant transformants can be obtained by transforming Saccharomyces cerevisiae and Pichia pastoris with the pCABIOMlOl vector and the pCABIOMlll vector respectively. Consequently, the present invention is excellent in that the transformation vector system for yeasts can be utilized for basic studies of yeasts or for industrial biological purposes.

Although the preferred embodiments of the present invention/ have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

Claims:

1. A paromomycin-resistance MPAl gene set forth in SEQ ID NO: 1.

2. An amino acid sequence set forth in SEQ ID NO: 2 encoded by the paromomycin-resistance MPAl gene as set forth in claim 1.

3. A pCABIOMlOl recombinant vector constructed by inserting the paromomycin-resistance MPAl gene as set forth in claim 1 into YEP24 vector (accession number: KCTC 1014BP).

4. A pCABIOMlll recombinant vector constructed by inserting MPAl ORF sequence set forth in SEQ ID NO: 3 into pHTL-D2 vector.

5. A yeast transformant, Saccharomyces cerevisiae pCABIOMlOl constructed by transforming yeast Saccharomyces cerevisiae with the pCABIOMlOl recombinant vector as set forth in claim 3.

6. A yeast transformant, Pichia pastoris pCABIOMlll constructed by transforming yeast Pichia pastoris with, the pCABIOMlll recombinant vector as set forth in claim 4.