Patent Application
20080044878
The following examples illustrate the present invention more specifically. However, these examples are by no means limitative of the scope of the invention.
The chromosomal DNAs of Saccharomyces cerevisiae and Candida maltosa were prepared using E.Z.N.A. Yeast DNA Kits (products of OMEGA BIOTEK). The preparation procedure was as described in the protocol attached to the kits.
Fragments of the Saccharomyces cerevisiae ACT1, GAP3, PMA1 and TEF1 genes the base sequences of which are already known were amplified as follows. The fragments were amplified by PCR using synthetic DNAs specified under SEQ ID NOs: 1 and 2 (for ACT1), SEQ ID NOs: 3 and 4 (for GAP3), SEQ ID NOs: 5 and 6 (for PMA1) and SEQ ID NOs: 7 and 8 (for TEF1) as primers and the Saccharomyces cerevisiae chromosomal DNA prepared in Example 1 as a template together with TaKaRa Ex Taq polymerase (product of Takara Shuzo). The conditions employed were as described in the protocol attached to the kit. More specifically, water was added, to make 0.1 ml, to a mixture of 1 μg of the template DNA, two primers each in an amount to give a final concentration of 1 μM, 2.5 U of Ex Taq polymerase, 0.01 ml of the attached buffer and 0.008 ml of the attached dNTP mixture. This was subjected to 25 cycles of PCR (each cycle comprising: 15 seconds at 98° C., 1 minute at 55° C., and 1 minute at 72° C.) to thereby amplify an ACT1 gene fragment of about 400 bp, or a GAP3 gene fragment of about 1 kb, or a PMA1 gene fragment of about 2.8 kb, or a TEF1 gene fragment of about 1.5 kb, of Saccharomyces cerevisiae.
The respective probe fragments amplified in the above manner were labeled with alkaline phosphatase using Amersham-Pharmacia' s Gene images AlkPhos kit according to the protocol attached thereto.
The hybridization was carried out overnight at 55° C. using Amersham-Pharmacia's Gene images AlkPhos kit according to the protocol attached thereto.
The washing was carried out at 55° C. and at room temperature using Amersham-Pharmacia' s Gene images AlkPhos kit according to the protocol attached thereto.
Positive clones were detected using Amersham-Pharmacia's CDP-Star kit according to the protocol attached thereto.
The chromosomal DNA of Candida maltosa as prepared in Example 1 was cleaved with the restriction enzyme BglII and fragments of about 1 kb to 3 kb were extracted from the gel by following agarose gel electrophoresis. These fragments were joined to pUC19, which was treated with the restriction enzyme BamHI, and the resulting recombined plasmids were used to transform the Escherichia coli DH5α strain. About 3,000 transformed colonies were screened to hybridization with the alkaline phosphatase-labeled Saccharomyces cerevisiae ACT1 gene fragment as a probe. As a result, 6 colonies of positive clone were obtained and, upon partial base sequence determination of the inserted fragment, the insert was found to be a gene containing the ACT1 promoter region of Candida maltosa. The partial sequence of the cloned fragment is shown under SEQ ID NO: 9.
The chromosomal DNA of Candida maltosa as prepared in Example 1 was cleaved with the restriction enzyme EcoRI and fragments of about 7 kb to 9 kb were extracted from the gel by following agarose gel electrophoresis. These fragments were joined to pUC19, which was treated with the same restriction enzyme, and the resulting recombined plasmids were used to transform the E. coli DH5α strain. About 2,000 transformed colonies were screened to hybridization with the alkaline phosphatase-labeled Saccharomyces cerevisiae GAP3 gene fragment as a probe. As a result, 2 colonies of positive clone were obtained and, upon partial base sequence determination of the inserted fragment, the insert was found to be a gene containing the GAP3 promoter region of Candida maltosa. The partial sequence of the cloned fragment is shown under SEQ ID NO: 10.
The chromosomal DNA of Candida maltosa as prepared in Example 1 was cleaved with the restriction enzyme XbaI and fragments of about 2 kb to 4 kb were extracted from the gel by following agarose gel electrophoresis. These fragments were joined to pUC19, which was treated with the same restriction enzyme, and the resulting recombined plasmids were used to transform the E. coli strain DH5α. About 5,000 transformed colonies were screened to hybridization with the alkaline phosphatase-labeled Saccharomyces cerevisiae PMA1 gene fragment as a probe. As a result, 5 colonies of positive clone were obtained and, upon base sequence determination of the inserted fragment, the insert was found to be a gene containing the PMA1 promoter region of Candida maltosa, as shown under SEQ ID NO: 11.
The chromosomal DNA of Candida maltosa as prepared in Example 1 was cleaved with the restriction enzymes EcoRI and PstI and fragments of about 2 kb to 4 kb were extracted from the gel by following agarose gel electrophoresis. These fragments were joined to pUC19 treated with the same restriction enzymes, and the resulting recombined plasmids were used to transform the E. coli DH5α strain. About 400 transformed colonies were screened to hybridization with the alkaline phosphatase-labeled Saccharomyces cerevisiae TEF1 gene fragment as a probe. As a result, 7 colonies of positive clone were obtained and, upon base sequence determination of the inserted fragment, the insert was found to be a gene containing the TEF1 promoter region of Candida maltosa, as shown under SEQ ID NO: 12.
An enzyme gene involved in polyester synthesis was synthesized based on the amino acid sequence of the Aeromonas caviae-derived polyhydroxyalkanoate (PHA) synthetase (T. Fukui, et al., FEMS Microbiology Letters, vol. 170, 69 (1999)). Since Candida maltosa is an yeast, which translates the CTG codon into serine, not into leucine, CTG was not assigned to the leucine codon. Those codons, which are frequently used in Candida maltosa, were preferentially selected as the codons corresponding to the respective amino acids. As for the frequency of the codon, the monograph “Nonconventional Yeasts in Biotechnology” written by Klaus Wolf (published by Springer) was consulted. The PHA-synthesizing enzyme gene (hereinafter referred to as “ORF2S” for short; SEQ ID NO: 13) was thus designed and the ORF2S gene portion was totally synthesized.
For the expression of the above ORF2S in Candida maltosa, the promoter ALK1p (SEQ ID NO: 15) of the Candida maltosa Alk1 gene was joined to the 5′ upstream thereof, and the terminator ALK1t (SEQ ID NO: 14) of the Candida maltosa Alk1 gene to the 3′ downstream thereof. The restriction enzyme sites for joining the promoter and terminator to the structural gene were produced by utilizing the PCR method. For the promoter portion, PCR was carried out using the DNAs shown under SEQ ID NO: 16 and SEQ ID NO: 17 with the promoter specified under SEQ ID NO: 15 as the template, and an ALK1p fragment having a PvuII site at the 5′ terminus and an EcoRI site at the 3′ terminus was prepared. For the terminator portion, PCR was carried out using the DNAs shown under SEQ ID NO: 18 and SEQ ID NO: 19 with the terminator specified under SEQ ID NO: 14 as the template, and an ALK1t fragment having a HindIII site at the 5′ terminus and an EcoRV site at the 3′ terminus was prepared. The ALK1p fragment was joined to pUCNT (described in WO 94/03613) at the PvuII-EcoRI site thereof, and the ALK1t fragment was joined to pUCNT at the HindIII-SspI site thereof, whereby pUAL1 was constructed. Then, the ORF2S fragment was joined to pUAL1 at the NdeI-PstI site thereof, whereby the plasmid pUAL-ORF2S (
Furthermore, pUTA1 (
A fragment containing the promoter, ORF2S and terminator was prepared from pSTV-ALK1-ORF2S using EcoT22I and inserted into pUTA1 at the PstI site thereof, whereby pUTA-ALK1-ORF2S shown in
An ORF2S expression vector was constructed using the Candida maltosa ACT1 promoter region cloned in Example 4, as follows. A fragment having a restriction enzyme EcoT22I site at the 5′-side terminus and a restriction enzyme NdeI site at the 3′-side terminus was obtained by carrying out PCR using the synthetic DNAs shown under SEQ ID NOs: 20 and 21 as the primers and the Candida maltosa ACT1 promoter region-containing fragment (SEQ ID NO: 9) as the template, and this fragment was treated with EcoT22I and NdeI. pSTV-ALK1-ORF2S was treated in the same manner with EcoT22I and NdeI to prepare a fragment free of the ALK promoter. The two fragments were joined together to construct pSTV-ACT1-ORF2S. By treating this plasmid with the restriction enzyme EcoT22I, ACT1-ORF2S fragment was prepared. This fragment was inserted into the PstI site of the pUTA1 obtained in Example 9, whereby an expression vector, pUTA-ACT1-ORF2S, was constructed as shown in
An ORF2S expression vector was constructed using the Candida maltosa GAP3 promoter region cloned in Example 5, as follows. A fragment having a restriction enzyme XhoI site at the 5′-side terminus and a restriction enzyme NdeI site at the 3′-side terminus was obtained by carrying out PCR using the synthetic DNAs shown under SEQ ID NOs: 22 and 23 as the primers and the Candida maltosa GAP3 promoter region-containing fragment (SEQ ID NO: 10) as the template, and this fragment was treated with XhoI and NdeI. The pSTV-ALK1-ORF2S plasmid was treated in the same manner with XhoI and NdeI to prepare a fragment free of the ALK promoter. The two fragments obtained were joined together to construct pSTV-GAP3-ORF2S. This plasmid was treated with the restriction enzymes XhoI and SalI to prepare a GAP3-ORF2S fragment. The pUTA1 plasmid was treated with SalI. The two fragments thus obtained were joined together, whereby an expression vector, pUTA-GAP3-ORF2S, was constructed as shown in
An ORF2S expression vector was constructed using the Candida maltosa PMA1 promoter region cloned in Example 6, as follows. A fragment having a restriction enzyme XhoI site at the 5′-side terminus and a restriction enzyme NdeI site at the 3′-side terminus was obtained by carrying out PCR using the synthetic DNAs shown under SEQ ID NOs: 24 and 25 as primers and the Candida maltosa PMA1 promoter region-containing fragment (SEQ ID NO: 11) as the template, and this fragment was treated with XhoI and NdeI. The pSTV-ALK1-ORF2S plasmid was treated in the same manner with XhoI and NdeI to prepare a fragment free of the ALK promoter. The two fragments obtained were joined together to construct pSTV-PMA1-ORF2S. This plasmid was treated with the restriction enzymes XhoI and SalI to prepare a PMA1-ORF2S fragment. The pUTA1 plasmid obtained in Example 9 was treated with SalI. Both the fragments obtained were joined together, whereby an expression vector, pUTA-PMA1-ORF2S, was constructed as shown in
An ORF2S expression vector was constructed using the Candida maltosa TEF1 promoter region cloned in Example 7, as follows. A fragment having a restriction enzyme XhoI site at the 5′-side terminus and a restriction enzyme NdeI site at the 3′-side terminus was obtained by carrying out PCR using the synthetic DNAs shown under SEQ ID NOs: 26 and 27 as the primers and the Candida maltosa TEF1 promoter region-containing fragment (SEQ ID NO: 12) as the template, and this fragment was treated with XhoI and NdeI. The pSTV-ALK1-ORF2S plasmid obtained in Example 9 was treated in the same manner with XhoI and NdeI to prepare a fragment free of the ALK promoter. The two fragments obtained were joined together to construct pSTV-TEF1-ORF2S. This plasmid was treated with the restriction enzymes XhoI and SalI to prepare a TEF1-ORF2S fragment. The pUTA1 plasmid obtained in Example 9 was treated with SalI. The two fragments thus obtained were joined together, whereby an expression vector, pUTA-TEF1-ORF2S, was constructed as shown in
The Candida maltosa AC16 strain (Deposit based on Budapest Treaty, International depository authority: National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (1-1-3 Higashi, Tsukuba, Ibaraki, Japan), deposited on Nov. 15, 2000, accession No. FERM BP-7366) was transformed by the electroporation method. The organism was precultured overnight on YPD medium at 30° C. One milliliter of the culture fluid was inoculated into 100 ml of the same medium placed in a 500-ml Sakaguchi flask and cultured at 30° C. for about 7 hours. After 10 minutes of centrifugation at 3,000 rpm and at room temperature, cells were washed with three portions (each about 50 ml) of an ice-cooled 1 M sorbitol solution. The cells were suspended in 3 ml of the same solution, and the suspension was divided into 0.1-ml portions and stored at −80° C. until use as cells to be transformed. For transformation, ECM 600 M (product of BTX) was used. Specifically, 0.1 ml of the cells to be transformed and about 1 μg of any one of the expression vector DNAs constructed in Examples 9 to 13 were placed in a cuvette having a gap width of 2 mm, electric pulses were applied thereto under the condition: mode 2.5 kV, voltage 1.9 kV, and resistance 246Ω. Immediately thereafter, the cuvette was ice-cooled, 0.5 ml of 1 M sorbitol was added, and the mixture was maintained at room temperature for 1 hour and then cultured on a YNB selection plate at 30° C. The selection plate used was composed of 0.67 w/v % Yeast Nitrogen Base without amino acid (product of Difco), 2 w/v % glucose, and 2 w/v % Bacto Agar (product of Difco).
By the above operations, 5 kinds of transformants, each containing any one of the expression vector constructed in Example 9 to 13, were obtained.
Each transformant obtained in Example 14 was precultured overnight on 0.67 w/v % Yeast Nitrogen Base without amino acid (product of Difco) plus 2 w/v % glucose. Then, 2.5 ml of the preculture was inoculated into 50 ml of the same medium placed in a 500-ml Sakaguchi flask and cultured at 30° C. for 24 hours. Only in a comparative example, namely in a culture of an ALK1 promoter-containing expression vector, 2 w/v % of n-dodecane was used as the carbon source. A portion, corresponding to about 10 ml, of the culture fluid was centrifuged at 3,000 rpm at room temperature for 10 minutes, and cells were washed with physiological saline and again centrifuged under the same conditions. The cell bodies were suspended in 1 ml of a 0.5 M potassium phosphate solution (pH 7.2), the suspension was mixed with the same volume of glass beads for fracturing yeast cells (0.45 mm; product of Biospec Products), and the cells were fractured by five times (each for 1 minute) of treatment on Mini Bead Beater (product of Biospec Products), followed by 10 seconds of centrifugation at 3,000 g on a centrifuge. The supernatant was recovered and used as an ORF2S activity assay sample. With the sample, the protein concentration was measured using Protein Assay Kit (product of BioRad) and the enzyme activity was determined as described in Valentin, H. E., et al., Appl. Microbiol. Biotechnol., vol. 40, 699 (1994).
Specifically, 0.01213 ml of aqueous solution containing 0.01 ml of cell fractured fluid and 7 mg/ml of (R)-3-hydroxybutylyl-CoA (product of Sigma), and 3.8 mg/ml of DTNB (product of Sigma) dissolved in 0.5 M potassium phosphate solution (pH 7.2) were added into 0.338 ml of water, and mixed. Then, absorbance variance at 412 nm of thus-obtained mixture was measured at room temperature, for 5 minutes. The measurement of the absorbance was carried out using a spectrophotometer produced by Shimadzu. The activity was calculated by the following formula.
Activity (U/ml)=ΔA412/min×103×VT/ε412×VE
In the formula, ΔA412/min represents an increase of absorbance per minute, VT represents an amount of reaction mixture, ε412 is 15.6×103 (m−1·cm−1), and VE represents an amount of an enzyme fluid.
The enzyme activity is expressed in terms of specific activity (U/mg) as in the above-cited document and the following formula.
Specific activity (U/mg)
=Activity(U/ml)/Protein Concentration (mg/ml)
The results thus obtained are shown in Table 1. From these results, it was revealed that the ACT1, GAP3, PMA1 and TEF1 promoter regions cloned in accordance with the present invention are promoters capable of functioning in Candida maltosa cells and are at least comparable in efficiency of the function to the ALK promoter.
Thus, the present invention has made it possible to carry out highly efficient expression of useful genes in yeasts of the genus Candida, in particular in Candida maltosa.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2002-105240 | Apr 2002 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP03/04426 | 4/8/2003 | WO | 00 | 4/20/2005 |
