GENE CASSETTE FOR HOMOLOGOUS RECOMBINATION KNOCK-OUT IN YEAST CELLS
20190153475 · 2019-05-23
Assignee
Inventors
- Xianzhong Chen (Wuxi, CN)
- Lihua Zhang (Xinyang, CN)
- Zheng Xiang (Wuxi, CN)
- Wei Shen (Wuxi, CN)
- Li Li (Shanghai, CN)
- Markus Pötter (Münster, DE)
Cpc classification
C12N1/00
CHEMISTRY; METALLURGY
International classification
C12N15/90
CHEMISTRY; METALLURGY
Abstract
There is provided a gene cassette for disruption of at least one target gene in a yeast cell, wherein the gene cassette comprises: (d) a URA3 gene capable of being used as a marker gene; (e) at least one gene disruption auxiliary (gda) sequence; and (f) an upstream and a downstream sequences of the target gene,
wherein the gda sequence is at least 300 to 600 bp in length and selected from within the nucleotide sequence of SEQ ID NO:39.
Claims
1. A gene cassette for disruption of at least one target gene in a yeast cell, wherein the gene cassette comprises: (a) a URA3 gene capable of being used as a marker gene; (b) at least one gene disruption auxiliary (gda) sequence; and (c) an upstream and a downstream sequences of the target gene, wherein the gda sequence is from 300 to 600 bp in length and selected from within the nucleotide sequence of SEQ ID NO:39 and variants thereof.
2. The gene cassette according to claim 1, wherein (b) the gda sequence is from 300 to 500 bp in length.
3. The gene cassette according to claim 2, wherein (b) the gda sequence is selected from within the nucleotide sequence of SEQ ID NO:40.
4. The gene cassette according to claim 2, wherein (b) the gda sequence is selected from within the nucleotide sequence of SEQ ID NO:41.
5. The gene cassette according to claim 2, wherein (b) the gda sequence is selected from within the nucleotide sequence of SEQ ID NO: 42.
6. The gene cassette according to claim 2, wherein (b) the gda sequence is selected from within the nucleotide sequence of SEQ ID NO:43.
7. The gene cassette according to claim 1, wherein (b) the gda sequence is at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 16, 14 18 21 and 24.
8. The gene cassette according to claim 1, wherein the yeast cell is selected from the group consisting of Candida albicans, Candida tropicalis, Candida parapsilopsis, Candida krusei, Cryptococcus neoformans, Hansenular polymorpha, Issatchenkia orientalis, Kluyverei lactis, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Yarrowia lipolytica.
9. The gene cassette according to claim 1, wherein the yeast cell is uracil auxotrophic C. tropicalis.
10. The gene cassette according to claim 1, wherein (a) the URA3 gene comprises the nucleotide sequence of SEQ ID NO:3.
11. The gene cassette according to claim 1, wherein (c) the upstream and downstream sequences of the target gene are each 50 bp in length.
12. A method of disrupting the expression of at least one target gene in at least one yeast cell, the method comprises transforming the yeast cell with at least one vector comprising the gene cassette according to claim 1.
13. The method of claim 12, wherein the yeast cell is uracil auxotrophic C. tropicalis.
14. The method according to claim 12, wherein the gda sequence is at least one nucleotide sequence selected from the group consisting of SEQ ID NOs: 16, 14, 18, 21 and 24.
15. A genetically modified yeast cell comprising a gene cassette according to claim 1.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
[0081] The foregoing describes preferred embodiments, which, as will be understood by those skilled in the art, may be subject to variations or modifications in design, construction or operation without departing from the scope of the claims. These variations, for instance, are intended to be covered by the scope of the claims.
Methods and Materials
[0082] Uracil auxotroph strain C. tropicalis XZX was used as a target strain for gene disruption. The uracil auxotroph strain was derived by screening of C. tropicalis ATTC 20336 after physical or chemical mutagenesis, and the open reading frame of the URA3 gene of the mutant strain comprised a missense mutation which altered the amino acid sequence.
[0083] The specific method is as follows: C. tropicalis ATTC 20336 as the starting strain was subjected to mutagenesis 11 times and screened with FOA selection medium, and a total of 127 colonies grew from the FOA selection medium (SM+5-fluoroorotic acid 2 g/L). The grown colonies were separately cultured on a SM plate and a MM plate. Finally, 13 URA3/URA3 mutant strains were identified; 3 of the 13 strains were selected, and designated as C. tropicalis XZW, C. tropicalis XZX, and C. tropicalis XZB respectively. DNA sequencing analysis showed that the common mutation in the URA3 gene sequence was the mutation of base G to A happening at the base pair at position +608. This mutation in base, which incurred, changed the protein sequence, and was the main cause of the functional defect of the URA3 gene (see Zheng Xiang, Xianzhong Chen et al. 2014).
[0084] The following culture media and compositions were used in the examples of the present invention: MM (yeast nitrogen base without amino acids & ammonium sulfate, YNB 6.7 g/L; glucose 20 g/L; (NH4)2SO4 10 g/L); SM (MM+uracil 60 mg/L); and FOA culture medium (SM+5-fluoroorotic acid 2 g/L).
[0085] The recombination efficiency calculated in all the examples and the results shown in table 1 was calculated according to the following formula:
[0086] The marker gene pop-out efficiency calculated in all the examples and the results shown in the table 2 was calculated according to the following formula:
Example 1
[0087] Disruption of First CAT Gene in C. tropicalis XZX by Transformation of the Gene Disruption Cassette CAT1-Gda488-URA3-CAT1 [0088] 1. Culturing of C. tropicalis ATTC 20336 strain. [0089] C. tropicalis ATTC 20336 was inoculated in SM or MM medium, and cultured in a shake flask at 30 C., 200 rpm until the desired microbial concentration was reached to extract chromosomal DNA. [0090] 2. Isolation of C. tropicalis ATTC 20336 chromosomal DNA. [0091] (1) Centrifugation was carried out to obtain the cells; (2) a suitable amount of sorbitol-Na.sub.2EDTA buffer solution (sorbitol 1 mol/L, Na.sub.2EDTA 0.1 mol/L, pH 7.5) was added to form a microbial suspension, a suitable amount of Snailase solution (50 mg/mL) was next added, and after mixing until uniform, digestion was carried out at 37 C. for 4 h in order to remove the yeast cell walls; (3) centrifugation was carried out to collect the cells, the supernatant was discarded, a suitable amount of Tris-HCl-Na.sub.2EDTA solution (Tris 50 mmol/L, Na.sub.2EDTA 20 mmol/L, pH 7.4) was used to gently suspend the cells, a suitable amount of SDS solution (SDS 100 g/L) was added, and the mixture was stirred until uniform and incubated at 65 C. for 30 min; (4) after the microbial suspension became clear, 200 L of potassium acetate solution (potassium acetate 5 mol/L) was added, the mixture was stirred until uniform, and it was placed for 1 h in an ice bath; (5) centrifugation was carried out at 12000 rpm for 5 min. The supernatant was transferred to a fresh EP tube, an equivalent volume of isopropanol was added, and the mixture was stirred until uniform and then allowed to stand at room temperature for 15 min; (6) centrifugation was carried out at 12000 rpm for 5 min, the supernatant was discarded, the precipitation were washed with 200 L of 70% ethanol solution. The ethanol solution was discarded, the precipitate was allowed to dry naturally, 33 L of sterile water was added to dissolve the precipitation, 2 L of RNaseA was added, the mixture was stirred until uniform, and it was incubated at 37 C. for 1 h to digest the RNA; (7) after incubation was completed, the C. tropicalis ATTC 20336 chromosomal DNA was obtained, which could be applied directly as a PCR template or stored at 20 C. [0092] 3. Preparation of URA3 gene fragment and Tm-URA3 vector [0093] C. tropicalis ATTC 20336 chromosomal DNA was used as a template, the URA3 gene upstream primer URAU: 5-tactctaacgacgggtacaac-3 (SEQ ID NO: 1), and the downstream primer URAR: 5-acccgatttcaaaagtgcaga-3 (SEQ ID NO: 2) were designed according to the URA3 gene of C. tropicalis in NCBI (GenBank Accession No. AB006207), PCR amplification was conducted to produce a URA3 gene fragment (SEQ ID NO: 3) with a size of 1581 bp. The URA3 gene fragment was ligated to a commercial vector pMD18-T Vector (Takara Biotechnology (Dalian) Co., Ltd, Dalian, China) to obtain a recombinant plasmid, which was then introduced into E. coli JM109 for amplification. The recombinant plasmid was designated as Tm-URA3. [0094] 4. Preparation of gda488 sequence (URA3 gene fragment from +671 to +1158). [0095] C. tropicalis ATTC 20336 chromosomal DNA was used as a template, and the synthetic gda488 sequence formed using the upstream primer Ugda488 5-aactgcagttctgactggtaccgat-3 (SEQ ID NO: 4) and the downstream primer Dgda 5-gcgtcgacacccgatttcaaaagtgcaga-3 (SEQ ID NO: 5) were used in PCR. PCR amplification was conducted to produce a gda488 sequence (SEQ ID NO. 6). [0096] 5. PstI and SalI were used to double digest the above gda488 fragment and the recombinant vector Tm-URA3. Then ligating to form a recombinant plasmid, which was introduced into E. coli JM109 for amplification. This new recombinant plasmid was designated as Tm-gda488-URA3. [0097] 6. C. tropicalis ATTC 20336 chromosomal DNA was used as a template, CAT gene upstream primer CATU 5-gtttaactttaagttgtcgc-3 (SEQ ID NO: 7), and the downstream primer CATR: 5-tacaacttaggcttagcatca-3 (SEQ ID NO: 8) were used in PCR. PCR amplification was conducted to produce a CAT1 gene (SEQ ID NO: 9) with a size of 1881 bp, after which it was ligated to a pMD18-T Simple Vector (Takara Biotechnology (Dalian) Co., Ltd, Dalian, China) commercial vector to obtain a recombinant plasmid, which was introduced into E. coli JM109 for amplification. The recombinant plasmid was designated as Ts-CAT1. [0098] 7. Recombinant plasmid Ts-CAT1 was used as a template, inverse PCR primer rCATU: 5-aactgcagccaaaattcagccaaccagt-3 (SEQ ID NO: 10), and rCATR: 5-gctctagaagatgattcaaccaggcgaac-3 (SEQ ID NO: 11) were used to amplify by inverse PCR and to obtain a fragment with upstream and downstream CAT gene homology arms, which was designated as CAT1-Ts-CAT1. [0099] 8. Restriction endonuclease PstI and XbaI were used to double digest vector Tm-gda488-URA3. The gda488-URA3 fragment was recovered and ligated with a PstI and XbaI double digested CAT1-Ts-CAT1 fragment to form a recombinant plasmid. Then the plasmid was introduced into E. coli JM109 for amplification. The recombinant plasmid was designated as Ts-CAT1-gda488-URA3. [0100] 9. Using recombinant plasmid Ts-CAT1-gda488-URA3 as a template and CAT gene up/downstream primer CATU/CATR, PCR amplification was carried out to obtain a first CAT allele gene disruption cassette, designated as CAT1-gda488-URA3-CAT1. [0101] 10. The fully-constructed gene disruption cassette CAT1-gda488-URA3-CAT1 was transformed using the lithium chloride transformation method into uracil auxotroph C. tropicalis XZX and then applied onto a MM plate. After growth of the transformants was completed, chromosomal DNA isolated according to method of steps 1 and 2 was used in PCR identification, and the strain that was identified as correct transformants was designated as strain 01-1. The PCR identification primers were CATU and CATR. The total number of transformants on MM plate was 28, and the number of transformants identified was 24, the number of transformants identified as correct transformation was 6, the recombination efficiency was 1 transformant/ng DNA (Table 1). PCR identification results are shown in
Example 2
[0105] Disruption of First CAT Gene in C. tropicalis XZX by Transformation of the Gene Disruption Cassette CAT1-Gda324-URA3-CAT1 [0106] 1. Using C. tropicalis ATTC 20336 chromosomal DNA as a template, the synthetic gda324 sequence upstream primer Ugda324 5-aactgcagactaagcttctaggacgtcat-3 (SEQ ID NO. 13), and the downstream primer Dgda(SEQ ID NO:5) as primers, PCR amplification was conducted to produce a gda324 sequence (URA3 gene fragment from +835 to +1158) (SEQ ID NO. 14). [0107] 2. PstI and SalI were used to double digest the above gda324 fragment and the recombinant vector Tm-URA3, the fragments were ligated to form a new recombinant plasmid, which was introduced into E. coli JM109 for amplification. This plasmid was designated as Tm-gda324-URA3. [0108] 3. PstI and XbaI were used to double digest vector Tm-gda324-URA3. The gda324-URA3 fragment was recovered, and ligated to the PstI and XbaI double digested CAT1-Ts-CAT1 fragment to form a recombinant plasmid, which was introduced into E. coli JM109 for amplification. This plasmid was designated as Ts-CAT1-gda324-URA3. [0109] 4. Using recombinant plasmid Ts-CAT1-gda324-URA3 as a template, PCR amplification was carried out according to the method of step 9 of Example 1 to obtain a first CAT allele disruption cassette CAT1-gda324-URA3-CAT1. [0110] 5. The XZX strain was transformed according to step 10 of Example 1 and PCR identification was carried out. PCR identification results are shown in
Example 3
[0112] Disruption of the First CAT Gene in C. tropicalis XZX by Transformation of the Gene Disruption Cassette CAT1-Gda245-URA3-CAT1 [0113] 1. Using C. tropicalis ATTC 20336 chromosomal DNA as a template, the synthetic gda245 sequence upstream primer Ugda 5-aactgcagaatggatgtagcagggatggt-3 (SEQ ID NO: 15) and the downstream primer Dgda(SEQ ID NO:5) as primers, PCR amplification was conducted to produce a gda245 sequence (URA3 gene fragment from +914 to +1158) (SEQ ID NO: 16). [0114] 2. PstI and SalI were used in a double digest the above gda245 fragment and the recombinant vector Tm-URA3. The fragments were ligated to form a recombinant plasmid, which was introduced into E. coli JM109 for amplification. This plasmid was designated as Tm-gda245-URA3. [0115] 3. Vector Tm-gda245-URA3 was double digested with PstI and XbaI. The gda245-URA3 fragment was recovered and ligated to the PstI and XbaI double digested CAT1-Ts-CAT1 fragment to form a recombinant plasmid, which was introduced into E. coli JM109 for amplification. This plasmid was designated as Ts-CAT1-gda245-URA3. [0116] 4. Using recombinant plasmid Ts-CAT1-gda245-URA3 as a template, PCR amplification was carried out according to the method of step 9 of Example 1 to obtain a first CAT allele disruption cassette CAT1-gda245-URA3-CAT1. [0117] 5. The XZX strain was transformed according to the method of step 10 of Example 1 and PCR identification was carried out with the primers URAU/CATR. The identification results as shown in
Example 4
[0119] Disruption of the First CAT Gene in C. tropicalis XZX by Transformation of the Gene Disruption Cassette CAT1-Gda143-URA3-CAT1 [0120] 1. Using C. tropicalis ATTC 20336 chromosomal DNA as a template, the synthetic gda143 sequence upstream primer Ugda143 5-aactgcagtgcttgaaggtattcacgta-3 (SEQ ID NO: 17), and the downstream primer Dgda(SEQ ID NO: 5), as primers, PCR amplification was conducted to produce a gda143 sequence (URA3 gene fragment from +1016 to +1158) (SEQ ID NO. 18). [0121] 2. PstI and SalI were used to double digest the above gda143 fragment and the recombinant vector Tm-URA3. The fragments were ligated to form a recombinant plasmid, which was introduced into E. coli JM109 for amplification. This plasmid was designated as Tm-gda143-URA3. [0122] 3. Vector Tm-gda143-URA3 was double digested by PstI and XbaI. The gda143-URA3 fragment was recovered and ligated to the PstI and XbaI double digested CAT1-Ts-CAT1 fragment to form a recombinant plasmid, which was introduced into E. coli JM109 for amplification. This plasmid was designated as Ts-CAT1-gda143-URA3. [0123] 4. Using recombinant plasmid Ts-CAT1-gda143-URA3 as a template, PCR amplification was carried out according to the method of step 9 of Example 1 to obtain a first CAT allele disruption cassette CAT1-gda143-URA3-CAT1. [0124] 5. The XZX strain was transformed according to the method of step 10 of Example 1 and PCR identification was carried out and the results showed that the total number of transformants on MM plate was 31, number of transformants identified was 24, number of transformants identified as correctly transformed was 11, and the recombination efficiency was 1.95 transformants/g DNA (see Table 1). PCR identification results are shown in
Example 5
[0126] Disruption of the first CAT gene in C. tropicalis XZX by transformation of the gene disruption cassette CAT1-gda325-URA3-CAT1 1. Using C. tropicalis ATTC 20336 chromosomal DNA as a template, the synthetic gda325 sequence upstream primer Ugda325 5-aactgcagtcgtgattgggttcatcgc-3 (SEQ ID NO. 19), and the downstream primer Dgda325 5-gcgtcgaccaatgacgtcctagaagc-3 (SEQ ID NO. 20) as primers, PCR amplification was conducted to produce a gda325 sequence (URA3 gene fragment from +533 to +857) (SEQ ID NO. 21). [0127] 2. PstI and SalI were used to double digest the above gda325 fragment and the recombinant vector Tm-URA3. The fragments were ligated to form a recombinant plasmid, which was introduced into E. coli JM109 for amplification. This plasmid was designated as Tm-gda325-URA3. [0128] 3. Vector Tm-gda325-URA3 was double digested with PstI and XbaI. The gda325-URA3 fragment was recovered and ligated to the PstI and XbaI double digested CAT1-Ts-CAT1 fragment to form a recombinant plasmid, which was introduced into E. coli JM109 for amplification. This plasmid was designated as Ts-CAT1-gda325-URA3. [0129] 4. Using recombinant plasmid Ts-CAT1-gda325-URA3 as a template, PCR amplification was carried out according to the method of step 9 of Example 1 to obtain a first CAT allele disruption cassette CAT1-gda325-URA3-CAT1. [0130] 5. The XZX strain was transformed according to the method of step 10 of Example 1 and PCR identification was carried out (results shown in
Example 6
[0132] Disruption of the First CAT Gene in C. tropicalis XZX by Transformation of the Gene Disruption Cassette CAT1-URA3-Gda305-CAT1 [0133] 1. Using C. tropicalis ATTC 20336 chromosomal DNA as a template, the synthetic gda305 sequence upstream primer Ugda305 5-gctctagatctaacgacgggtacaacga-3 (SEQ ID NO: 22), and the downstream primer Dgda305 5-cggaattcacgtgactagtatggcaat-3 (SEQ ID NO: 23) as primers, PCR amplification was conducted to produce a gda305 sequence (URA3 gene fragment from 420 to 116) (SEQ ID NO: 24). [0134] 2. XbaI and EcoRI were used to double digest the above gda305 fragment and the recombinant vector Tm-URA3. The fragments were ligated to form a recombinant plasmid, which was introduced into E. coli JM109 for amplification. This plasmid was designated as Tm-URA3-gda305. [0135] 3. Vector Tm-URA3-gda305 was double digested by PstI and EcoRI. The URA3-gda305 fragment was recovered. PstI and XbaI were used to double digest CAT1-Ts-CAT1. pfu DNA polymerase was then used to fill in the sticky ends of the CAT1-Ts-CAT1 and dp1305-URA3 fragments in order to carry out blunt end ligation and obtain a recombinant plasmid, which was then introduced into E. coli JM109 for amplification. This plasmid was designated as Ts-CAT1-URA3-gda305. [0136] 4. Using recombinant plasmid Ts-CAT1-URA3-gda305 as a template, PCR amplification was carried out according to the method of step 9 of Example 1 to obtain a first CAT allele disruption cassette CAT1-URA3-gda305-CAT1. [0137] 5. The XZX strain was transformed according to the method of step 10 of Example 1 and PCR identification was carried out, and the identification results showed the disruption of the first CAT allele in C. tropicalis XZX by transformation of the gene disruption cassettes CAT1-gda325-URA3-CAT1 (Example 5) and CAT1-URA3-gda305-CAT1. The successful transformants (true positives) were selected for the next step. PCR identification results are shown in
Example 7
[0139] Disruption of the First CAT Gene in C. tropicalis XZX by Transformation of the Gene Disruption Cassette CAT1-URA3-Gda302-CAT1 [0140] 1. Using C. tropicalis ATTC 20336 chromosomal DNA as a template, the synthetic gda302 sequence upstream primer Ugda302 5-gctctagacatacacagaaagggcatc-3 (SEQ ID NO: 25), and the downstream primer Dgda302 5-cggaattcgtactgcaacatcacgg-3 (SEQ ID NO: 26) as primers, PCR amplification was conducted to produce a gda302 sequence (URA3 gene fragment from +17 to +318) (SEQ ID NO: 27). [0141] 2. XbaI and EcoRI were used to double digest the above gda302 fragment and the recombinant vector Tm-URA3. The fragments were ligated to form a recombinant plasmid, which was introduced into E. coli JM109 for amplification. This plasmid was designated as Tm-URA3-gda302. [0142] 3. Vector Tm-URA3-gda302 were double digested with PstI and EcoRI. The URA3-gda302 fragment was recovered. PstI and XbaI were used to double digest CAT1-Ts-CAT1. pfu DNA polymerase was then used to fill in the sticky ends of the CAT1-Ts-CAT1 and URA3-gda302 fragments in order to carry out blunt end ligation and obtain a recombinant plasmid, which was then introduced into E. coli JM109 for amplification. This plasmid was designated as Ts-CAT1-URA3-gda302. [0143] 4. Using recombinant plasmid Ts-CAT1-URA3-gda302 as a template, PCR amplification was carried out according to the method of step 9 of Example 1 to obtain a first CAT allele disruption cassette CAT1-URA3-gda302-CAT1. [0144] 5. The XZX strain was transformed according to the method of step 10 of Example 1, PCR identification was carried out, where the identification results showed the success of disrupting the first CAT allele in C. tropicalis XZX by transformation of the gene disruption cassette CAT1-URA3-gda302-CAT1. The band of false-positive transformants (CAT1 original gene) had a size of 1881 bp, and the positive transformants or the disruption cassette integrated transformants (CAT1-URA3-gda302-CAT1) had a band size of 2521 bp. The PCR primers were CATU/CATR. PCR identification results are shown in
Comparative Example 1
[0146] Disruption of the First CAT Gene in C. tropicalis XZX by Transformation of the Gene Disruption Cassette CAT1-hisG-URA3-hisG-CAT1 [0147] 1. Isolation of hisG fragment: PCR amplification was carried out using the two pairs of primers hisG-F1 5-ccggaattcttccagtggtgcatgaacgc-3 (SEQ ID NO: 28) and hisG-R1 5-cgcggattcgctgttccagtcaatcagggt-3 (SEQ ID NO: 29) as well as hisG-F2 5-acgcgtcgacttccagtggtgcatgaacgc-3 (SEQ ID NO: 30) and hisG-R2 5-aactgcaggctgttccagtcaatcagggt-3 (SEQ ID NO: 31). PCR was carried out as taught in Ko et al. (2006), using plasmid pCUB6 as a template to obtain two 1.1 kb hisG fragments. These were designated: [0148] hisG1 (SEQ ID NO: 32) where the two ends had EcoRI and Bam HI restriction enzyme loci; and [0149] hisG2 (SEQ ID NO: 33) where the two ends had SalI and PstI restriction enzyme loci. [0150] 2. The restriction enzymes EcoRI and Bam HI were used to digest the hisG1 fragment, then the digested fragment was inserted into a Tm-URA3 plasmid that had been digested with the same enzymes to obtain the recombinant plasmid Tm-hisG1-URA3. [0151] 3. The restriction enzymes PstI and SalI were used to digest the hisG2 fragment, then the digested fragment was inserted into a Tm-hisG1-URA3 plasmid that had been digested with the same enzymes to obtain the recombinant plasmid Tm-hisG1-URA3-hisG2, abbreviated as Tm-HUH. [0152] 4. PstI and EcoRI were used to double digest the recombinant plasmid Tm-HUH, and gel recycling was used to obtain a hisG1-URA3-hisG2 fragment; PstI and XbaI were used to double digest CAT1-Ts-CAT1; pfu DNA polymerase was then used to fill in the sticky ends of the CAT1-Ts-CAT1 and hisG1-URA3-hisG2 fragment in order to carry out blunt end ligation to obtain the recombinant plasmid Ts-CAT1-hisG1-URA3-hisG2. [0153] 5. Using recombinant plasmid Ts-CAT1-hisG1-URA3-hisG2 as a template, PCR amplification was carried out according to the method of step 9 of Example 1 to obtain the first CAT allele disruption cassette CAT1-hisG1-URA3-hisG2-CAT1. [0154] 6. The XZX strain was transformed according to the method of step 10 of Example 1, PCR identification was carried out, where the identification results showed the disruption of the first CAT allele disrupted in C. tropicalis XZX by transformation of the gene disruption cassette CAT1-hisG-URA3-hisG-CAT1. The PCR amplification band of positive transformants or the gene disruption cassette integrated transformants (hisG1) had a size of 1149 bp. The PCR primers used were His-F1 and His-R1. PCR identification results are shown in
Example 8
[0156] Disruption of the Second CAT Allele in C. tropicalis02 (URA3/URA3, cat::gda324/CAT) by transformation of the gene disruption cassette CAT2-gda324-URA3-CAT2 [0157] 1. Using C. tropicalis ATTC 20336 chromosomal DNA as a template, the CAT2 upstream primer CAT2ndU 5-ctgaaggctccgacatcacc-3 (SEQ ID NO; 34), and the CAT2 downstream primer CAT2ndR: 5-caaccttgtcggcgctgcta-3 (SEQ ID NO: 35) as primers, PCR amplification was conducted to produce a CAT2 fragment (SEQ ID NO: 36), after which it was linked to a commercial vector pMD18-T Simple Vector to obtain a recombinant plasmid, which was introduced into E. coli JM109 for amplification. The recombinant plasmid was designated as Ts-CAT2. [0158] 2. Using recombinant plasmid Ts-CAT2 as a template, the inverse PCR upstream primer rCAR2ndU: 5-aactgcagatctgttttgaccgtccccgtg-3 (SEQ ID NO: 37), and the downstream primer rCAT2ndR: 5-aactgcagatctgttttgaccgtccccgtg-3 (SEQ ID NO: 38) as primers, inverse PCR amplification was carried out to obtain a fragment having upstream and downstream CAT2 gene homology arms, which was designated as CAT2-Ts-CAT2. [0159] 3. PstI and XbaI were used to double digest vector Tm-gda324-URA3. The gda324-URA3 fragment was recovered and ligated to the PstI and XbaI double digested CAT2-Ts-CAT2 fragment to form a recombinant plasmid, which was designated as Ts-CAT2-gda324-URA3. The plasmid was introduced into E. coli JM109 for amplification. [0160] 4. Using recombinant plasmid Ts-CAT2-gda324-URA3 as a template and the CAT2 gene fragment upstream and the downstream primers CAT2ndU and CAT2ndR as primers, PCR amplification was carried out to obtain a second CAT allele disruption cassette designated as CAT2-gda324-URA3-CAT2. [0161] 5. Strain 02 from Example 1 was transformed according to the method of step 10 of Example 1, PCR identification was carried out using CAT2ndU and CATR as primers to identify the successful strains with disruption of the second CAT allele in C. tropicalis 02 (URA3/URA3, cat::gda324/CAT) by transformation of the gene disruption cassette CAT2-gda324-URA3-CAT2. The disruption cassette integrated transformant had a PCR amplification band (CAT2-gda324-URA3-CAT2-fragment from downstream homology arm of CAT2 to downstream homology arm of CAT1-CAT1) size of 3027 bp. The control used was PCR products with C. tropicalis XZX chromosomal DNA as a template (CAT gene fragment from CAT2 gene upstream homology arm to CAT1 downstream homology arm), which has a size of 1312 bp. The PCR primers were CAT2ndU/CATR. PCR identification results are shown in
[0163] Sequencing of the PCR product CAT1-gda324-CAT1-CATLD at the CAT gene locus after the marker gene was popped out according to Example 1, revealed that there was fragment loss between the two CAT1 homology arms of a single CAT allele, and the lost fragment was substituted by a gda sequence. This was confirmed by carrying out a sequence comparison. Thus, it was verified at the molecular level that this single copy of the CAT sequence was disrupted, and it was also verified that in the process of pop-out of the marker gene, only the URA3 gene fragment between the two gda sequences having the same direction was popped out(in the two gda sequences, one gda sequence exists in the URA3 gene, the other one gda sequence is from the gene disruption cassette, the two gda sequences are exactly the same). Sequencing of the PCR product CAT2-gda324-CAT2-CAT1 at the CAT gene locus after the marker gene was popped out according to Example 8 showed that the sequence of the PCR product conformed with the sequence according to theoretical prediction (identity of the two sequences was 97.05%) and only the fragment between the two CAT2 homology arms was replaced by a gda fragment. Thus two-copy CAT allele disruption was further verified at the molecular level, showing that the gene disruption cassette of the used may be suitable for two-copy and multiple gene disruption of C. tropicalis.
TABLE-US-00001 TABLE 1 Comparison of recombination efficiency of gene disruption cassette of the present invention and conventional gene disruption cassette Number of transformants Recombination Total Total no. No. of identified as efficiency Gene disruption DNA of transformants correct (transformants/ cassette wt. (g) transformants identified transformation g DNA) CAT1-gda143- 7.27 31 24 11 1.95 URA3-CAT1 CAT1-gda245- 7.04 21 12 9 2.24 URA3-CAT1 CAT1-gda324- 4.98 17 11 4 1.24 URA3-CAT1 CAT1-gda488- 7.01 28 24 6 1.00 URA3-CAT1 CAT1-His-URA3- 15.51 49 48 2 0.13 His-CAT1
[0164] It can be seen from the statistical results shown in Table 1 that the transformation/recombination efficiency of the gene disruption cassette with gda143, gda245, gda324, gda488 used was greater by an order of magnitude than that of the gene disruption cassette of prior art (hisG-URA3-hisG).
TABLE-US-00002 TABLE 2 Effect of gda sequence length on URA3 gene pop-out efficiency No. No. of Of identified No. of Total no. iden- detection colonies of cells tified marker gda on applied to trans- gene Ex. size FOA FOA form- trans- Detection no. (bp) plate plate ants formants efficiency 4 143 0, 1, 3 2.715 10.sup.9 4 3 3.7 10.sup.10 3 245 0, 0, 2 2.86 10.sup.9 2 2 2.33 10.sup.10 2 324 92, 99, 2.055 10.sup.9 8 8 5.58 10.sup.8 132 1 488 73, 82, 5.395 10.sup.9 6 6 1.65 10.sup.8 112 7 302 61, 72, 5.16 10.sup.9 12 12 1.42 10.sup.8 87 6 305 62, 99, 5.84 10.sup.9 12 12 1.73 10.sup.8 145 5 325 145, 146, 2.33 10.sup.9 11 9 5.61 10.sup.8 188 comp. hisG 744, 711, 9.43 10.sup.9 12 12 7.5 10.sup.8 ex. 1 657
[0165] It can be seen from the statistical results shown in Table 2 that when the gda fragment length of the gene disruption cassette was 143 bp, the URA3 gene was efficiently popped out. When the gda fragment was longer than 300 bp, URA3 gene pop-out efficiency was markedly higher and was comparable to that of conventional HisG disruption cassettes, which further improved the overall efficiency of C. tropicalis gene disruption.
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