CN113755518B - Method for constructing recombinant yarrowia lipolytica and application thereof - Google Patents
Method for constructing recombinant yarrowia lipolytica and application thereof Download PDFInfo
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Abstract
The application discloses a method for improving the temperature tolerance of yarrowia lipolytica or improving the erythritol yield of yarrowia lipolytica under high temperature conditions, which comprises the following steps: over-expressing the high temperature tolerance gene in yarrowia lipolytica of the recipient to obtain yarrowia lipolytica of interest which is higher in high temperature tolerance or erythritol yield than the yarrowia lipolytica of the recipient. According to the application, the heat-resistant gene information of Saccharomyces cerevisiae and thermophilic microorganisms is excavated and arranged through NCBI, PDB and other databases, genes with different functions are used as candidate elements, a plasmid expression vector is constructed after codon optimization, the plasmid expression vector is transformed into yarrowia lipolytica industrial strains, recombinant expression strains are screened, and through fermentation evaluation, the heat-resistant genes Yap1, hsp12, vma2, hul5, tsa1 and Groes with different functions, which can improve the erythritol yield under the condition of 35 ℃, are obtained.
Description
Technical Field
The application relates to a method for constructing recombinant yarrowia lipolytica and application thereof in the field of biotechnology.
Background
Erythritol (1, 2,3, 4-butanetetrachol) is a four carbon functional sugar alcohol that is widely distributed in fruits, fermented foods and animals. Because of low metabolic energy in the organism, the compound can prevent and treat dental caries, has low insulin metabolic response and the like, is a low-calorie sweetener, and is widely applied to the fields of food, medicine, chemical industry and the like. The erythritol has huge market development space and very broad application prospect. Although foods such as fruits and kelp widely contain erythritol, the production of erythritol by fermentation with hypertonic or hypertonic-resistant yeast organisms is a major production method. At present, yarrowia lipolytica (yarrowia lipolytica) has realized large-scale erythritol fermentation production, and has achieved better economic benefits. However, the fermentation temperature for producing erythritol by using the strain is generally 30 ℃, and the fermentation temperature needs to be subjected to circulating cooling treatment during the production in seasons with higher temperature such as summer, thereby increasing the production cost, even leading to the shutdown of factories, and seriously affecting the production operation and economic benefits of enterprises. Therefore, the temperature tolerance of yarrowia lipolytica is improved, the production period is increased, and the economic benefit is improved.
Traditional mutagenesis is time-consuming and labor-consuming, has poor genetic stability of the strain, and is generally not suitable for constructing industrial microbial species at the expense of reduced product yield. Along with the elucidation of the response and defense mechanism of the saccharomyces cerevisiae under high temperature stress and the characterization of the thermophilic bacteria source heat-resistant element, a new target is provided for rational breeding of the high temperature resistant yeast. For example, researchers have found that overexpression of ubiquitin ligase Rsp5 and ubiquitin binding enzyme results in improved multiple stress tolerance at higher temperatures (39 ℃); heat stress also causes ROS damage, and yeast heat resistance can be improved by knocking out the glycosyl phosphatidylinositol-immobilized membrane protein encoding gene DFG 5. Liu et al have significantly higher cell viability at 42℃than the control strain by introducing a thermoelement from Thermoanaerobactertengcondensis. Zhou et al greatly enhanced the high temperature growth of E.coli and Saccharomyces cerevisiae (2.6 fold increase in biomass at 36 ℃) by heterologous expression of the cold shock protein cspL from Bacillus coagulans 2-6. Wang et al maintained the high erythritol production properties at 33℃by over-expressing the Rsp5 gene alone in yarrowia lipolytica.
Under high temperature stress, the functional proteins in the yarrowia lipolytica cells can be subjected to thermal denaturation, misfolding, aggregation and the like; intracellular pH homeostasis may be imbalanced; intracellular structures may be subject to oxidative damage; the nucleic acid may be degraded or mutated. Therefore, aiming at the influence of high temperature stress on the yeast, the element with the corresponding protection function is mined, and the method has very important significance for improving the capacity of yarrowia lipolytica for high-yield erythritol at high temperature.
Disclosure of Invention
The technical problem to be solved by the application is how to improve the temperature tolerance of yarrowia lipolytica and how to improve the erythritol yield of yarrowia lipolytica under high temperature conditions.
In order to solve the above technical problems, a first object of the present application is to provide a method for constructing recombinant yarrowia lipolytica, comprising introducing a high temperature tolerance gene into yarrowia lipolytica to obtain the recombinant yarrowia lipolytica; the high temperature tolerance gene is at least one of the following genes: groes gene, hsp12 gene, tsa1 gene, hul gene, vma2 gene, and Yap1 gene;
the Groes gene encodes any one of the following proteins A1) to A3):
a1 Amino acid sequence is protein of sequence 12 in the sequence table,
a2 Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of A1), has more than 80 percent of identity with the protein shown in A1) and has high temperature tolerance activity,
a3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of A1) or A2);
the Hsp12 gene encodes any one of the following proteins B1) -B3):
b1 Amino acid sequence is protein of sequence 8 in the sequence table,
b2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of B1), has more than 80 percent of identity with the protein shown in A1) and has high temperature tolerance activity,
b3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of B1) or B2);
the Tsa1 gene encodes any one of the following C1) -C3) proteins:
c1 Amino acid sequence is protein of sequence 11 in the sequence table,
c2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of C1), has more than 80 percent of identity with the protein shown in C1) and has high temperature tolerance activity,
c3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of C1) or C2);
the Hul gene encodes any one of the following proteins D1) -D3):
d1 Amino acid sequence is protein of sequence 10 in the sequence table,
d2 Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of D1), has more than 80 percent of identity with the protein shown in D1) and has high temperature tolerance activity,
d3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of D1) or D2);
the Vma2 gene encodes any one of the following proteins E1) -E3):
e1 Amino acid sequence is protein of sequence 9 in the sequence table,
e2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of E1), has more than 80 percent of identity with the protein shown in E1) and has high temperature tolerance activity,
e3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of E1) or E2);
the Yap1 gene encodes any one of the following proteins F1) -F3):
f1 Amino acid sequence is protein of sequence 7 in the sequence table,
f2 Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of F1), has more than 80 percent of identity with the protein shown in F1) and has high temperature tolerance activity,
f3 Fusion proteins obtained by ligating protein tags to the N-terminal or/and C-terminal of F1) or F2).
The high temperature is 31-40 ℃, and can be 35 ℃ in particular.
Wherein, the sequence 7 in the sequence table consists of 650 amino acid residues, the sequence 8 in the sequence table consists of 109 amino acid residues, the sequence 9 in the sequence table consists of 517 amino acid residues, the sequence 10 in the sequence table consists of 910 amino acid residues, the sequence 11 in the sequence table consists of 196 amino acid residues, and the sequence 12 in the sequence table consists of 101 amino acid residues.
In the method, the protein can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing.
In the above method, the protein may be derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) or Thermophilus (Thermus thermophilus).
In the above method, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with the target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.
In the above method, the identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.
In the above method, the 80% identity or more may be at least 80%, 85%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.
In the above method, the Groes gene is a gene represented by a 1) or a 2) as follows:
a1 Coding sequence (CDS) of the coding strand is a cDNA molecule or a DNA molecule of sequence 6 in the sequence table,
a2 The nucleotide of the coding chain is a cDNA molecule or a DNA molecule of a sequence 6 in a sequence table;
the Hsp12 gene is a gene shown in the following b 1) or b 2):
b1 The coding sequence (CDS) of the coding strand is a cDNA molecule or a DNA molecule of a sequence 2 in a sequence table,
b2 The nucleotide of the coding chain is a cDNA molecule or a DNA molecule of a sequence 2 in a sequence table;
the Tsa1 gene is a gene shown in the following c 1) or c 2):
c1 Coding sequence (CDS) of the coding strand is a cDNA molecule or a DNA molecule of sequence 5 in the sequence listing,
c2 The nucleotide of the coding chain is a cDNA molecule or a DNA molecule of a sequence 5 in a sequence table;
the Hul gene is a gene shown in the following d 1) or d 2):
d1 Coding sequence (CDS) of the coding strand is a cDNA molecule or a DNA molecule of sequence 4 in the sequence table,
d2 The nucleotide of the coding chain is a cDNA molecule or a DNA molecule of a sequence 4 in a sequence table;
the Vma2 gene is a gene shown in the following e 1) or e 2):
e1 Coding sequence (CDS) of the coding strand is cDNA molecule or DNA molecule of sequence 3 in the sequence table,
e2 The nucleotide of the coding chain is a cDNA molecule or a DNA molecule of a sequence 3 in a sequence table;
the Yap1 gene is a gene shown in the following f 1) or f 2):
f1 Coding sequence (CDS) of the coding strand is cDNA molecule or DNA molecule of sequence 1 in the sequence table,
f2 The nucleotide of the coding chain is a cDNA molecule or a DNA molecule of a sequence 1 in a sequence table.
Wherein, the sequence 1 in the sequence table consists of 1953 nucleotides, and codes the protein shown in the sequence 7 in the sequence table; sequence 2 in the sequence table consists of 330 nucleotides and codes protein shown in sequence 8 in the sequence table; sequence 3 in the sequence table consists of 1554 nucleotides and codes protein shown in sequence 9 in the sequence table; sequence 4 in the sequence table consists of 2733 nucleotides and codes protein shown in sequence 10 in the sequence table; sequence 5 in the sequence table consists of 591 nucleotides and codes protein shown in sequence 11 in the sequence table; sequence 6 in the sequence table consists of 306 nucleotides and codes for a protein shown in sequence 12 in the sequence table.
In the above method, the recombinant yarrowia lipolytica has a higher erythritol yield than the yarrowia lipolytica under high temperature conditions. The high temperature is 31-40 ℃, and can be 35 ℃ in particular.
A second object of the present application is to protect recombinant yarrowia lipolytica constructed in the above-described manner.
A third object of the present application is the use of a high temperature tolerance gene for increasing erythritol production by yarrowia lipolytica under high temperature conditions or for increasing high temperature tolerance by yarrowia lipolytica, said high temperature tolerance gene being at least one of the following genes: groes gene, hsp12 gene, tsa1 gene, hul gene, vma2 gene, and Yap1 gene;
the Groes gene encodes any one of the following proteins A1) to A3):
a1 Amino acid sequence is protein of sequence 12 in the sequence table,
a2 Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of A1), has more than 80 percent of identity with the protein shown in A1) and has high temperature tolerance activity,
a3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of A1) or A2);
the Hsp12 gene encodes any one of the following proteins B1) -B3):
b1 Amino acid sequence is protein of sequence 8 in the sequence table,
b2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of B1), has more than 80 percent of identity with the protein shown in A1) and has high temperature tolerance activity,
b3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of B1) or B2);
the Tsa1 gene encodes any one of the following C1) -C3) proteins:
c1 Amino acid sequence is protein of sequence 11 in the sequence table,
c2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of C1), has more than 80 percent of identity with the protein shown in C1) and has high temperature tolerance activity,
c3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of C1) or C2);
the Hul gene encodes any one of the following proteins D1) -D3):
d1 Amino acid sequence is protein of sequence 10 in the sequence table,
d2 Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of D1), has more than 80 percent of identity with the protein shown in D1) and has high temperature tolerance activity,
d3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of D1) or D2);
the Vma2 gene encodes any one of the following proteins E1) -E3):
e1 Amino acid sequence is protein of sequence 9 in the sequence table,
e2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of E1), has more than 80 percent of identity with the protein shown in E1) and has high temperature tolerance activity,
e3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of E1) or E2);
the Yap1 gene encodes any one of the following proteins F1) -F3):
f1 Amino acid sequence is protein of sequence 7 in the sequence table,
f2 Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of F1), has more than 80 percent of identity with the protein shown in F1) and has high temperature tolerance activity,
f3 Fusion proteins obtained by ligating protein tags to the N-terminal or/and C-terminal of F1) or F2).
The application also provides application of the high-temperature tolerance gene in constructing high-temperature resistant recombinant yarrowia lipolytica, wherein the high-temperature tolerance gene comprises at least one of the following genes: groes gene, hsp12 gene, tsa1 gene, hul gene, vma2 gene, and Yap1 gene;
the Groes gene encodes any one of the following proteins A1) to A3):
a1 Amino acid sequence is protein of sequence 12 in the sequence table,
a2 Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of A1), has more than 80 percent of identity with the protein shown in A1) and has high temperature tolerance activity,
a3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of A1) or A2);
the Hsp12 gene encodes any one of the following proteins B1) -B3):
b1 Amino acid sequence is protein of sequence 8 in the sequence table,
b2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of B1), has more than 80 percent of identity with the protein shown in A1) and has high temperature tolerance activity,
b3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of B1) or B2);
the Tsa1 gene encodes any one of the following C1) -C3) proteins:
c1 Amino acid sequence is protein of sequence 11 in the sequence table,
c2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of C1), has more than 80 percent of identity with the protein shown in C1) and has high temperature tolerance activity,
c3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of C1) or C2);
the Hul gene encodes any one of the following proteins D1) -D3):
d1 Amino acid sequence is protein of sequence 10 in the sequence table,
d2 Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of D1), has more than 80 percent of identity with the protein shown in D1) and has high temperature tolerance activity,
d3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of D1) or D2);
the Vma2 gene encodes any one of the following proteins E1) -E3):
e1 Amino acid sequence is protein of sequence 9 in the sequence table,
e2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of E1), has more than 80 percent of identity with the protein shown in E1) and has high temperature tolerance activity,
e3 Fusion proteins obtained by ligating protein tags at the N-terminal or/and C-terminal of E1) or E2);
the Yap1 gene encodes any one of the following proteins F1) -F3):
f1 Amino acid sequence is protein of sequence 7 in the sequence table,
f2 Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the protein of F1), has more than 80 percent of identity with the protein shown in F1) and has high temperature tolerance activity,
f3 Fusion proteins obtained by ligating protein tags to the N-terminal or/and C-terminal of F1) or F2).
In the above application, the elevated temperature is 31 ℃ to 40 ℃, and may be specifically 35 ℃.
In order to solve the technical problems, the application also provides the application of the method or the recombinant yarrowia lipolytica in erythritol production. The erythritol is produced by producing erythritol at a high temperature of 31-40 ℃, and specifically can be 35 ℃.
The agent provided by the application contains a substance for promoting the expression of the high temperature tolerance gene in yarrowia lipolytica.
The active ingredient of the above-mentioned agent may be a substance that promotes the expression of a high temperature tolerance gene in yarrowia lipolytica, the active ingredient of the above-mentioned agent may further contain other biological components or/and non-biological components, and the other active ingredient of the above-mentioned agent may be determined by one skilled in the art based on the effect of improving the high temperature tolerance or erythritol production of yarrowia lipolytica.
The application also protects the application of the method or the reagent in erythritol production.
According to the application, the sequenced Saccharomyces cerevisiae, thermophilic microorganisms and heat-resistant gene information thereof are mined and sorted through NCBI, PDB and other databases, and genes with different functions and obvious reported effects in the existing literature are combined as candidate elements. Constructing a plasmid expression vector after codon optimization, transforming into yarrowia lipolytica strains, and screening recombinant expression strains. Through fermentation evaluation, different functional heat-resistant genes Yap1, hsp12, vma2, hul5, tsa1 and Groes which can improve the erythritol yield under the condition of 35 ℃ are obtained. Compared with a control strain transformed with an empty plasmid, the growth of the strain expressing the heat-resistant genes Yap1, hsp12, vma2, hul5, tsa1 or Groes under the condition of 35 ℃ is improved, and the erythritol yield is also obviously increased.
Preservation description
Strain name: yarrowia lipolytica
Latin name: yarrowia lipolytica
Strain number: ERY01
Preservation mechanism: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
The preservation organization is abbreviated as: CGMCC
Address: beijing city, chaoyang area, north Chenxi Lu No. 1 and 3
Preservation date: 2021, 7, 27
Accession numbers of the preservation center: CGMCC No.22966
Drawings
FIG. 1 shows the growth of each strain at 35℃for 72 hours in example 1 of the present application.
FIG. 2 shows erythritol yields of the respective strains of example 1 of the present application when fermented at 35℃for 72 hours.
Detailed Description
The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.
The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the examples described below are all conventional biochemical reagents, unless otherwise specified, and are commercially available.
Strain 1
Saccharomyces cerevisiae BY4741 in the examples described below is described in non-patent documents "Zhang G, lin Y, qi X, li L, wang Q, ma Y.TALENs-assisted multiplex editing for accelerated genome evolution to improve yeast phenolates.ACS Synth biol.2015;4 (10): 1101-11", available to the public from the institute of Tianjin Industrial biotechnology, national academy of sciences, to repeat the experiments of the present application, are not useful for other purposes.
Yarrowialipolyticastrain W29 (CLIB 89) in the examples described below is ARS Culture Collection (NRRL) product.
Yarrowia lipolytica industrial strain eri 01 in the following examples was deposited with the China general microbiological culture Collection center, accession number: CGMCC No.22966, classification name: yarrowia lipolytica (Yarrowia lipolytica), date of preservation: 2021, 7, 27, deposit unit address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.
2 Medium
The seed liquid culture medium consists of a solute and a solvent, wherein the solvent is water, and the concentration of the solute is as follows: glucose 20% (w/v: g/mL), yeast extract 1%, potassium dihydrogen phosphate 0.1%, ammonium citrate 0.5%, magnesium sulfate heptahydrate 0.05%, and North fungus 800mg/L.
The fermentation medium consists of a solute and a solvent, wherein the solvent is water, and the concentration of the solute is as follows: 337.5g/L of food-grade glucose (one crystal water), 8.6g/L of yeast extract, 6.1g/L of corn steep liquor, 0.173g/L of magnesium sulfate (seven crystal water), 3.07g/L of diammonium hydrogen phosphate, VB10.0086 g/L, 0.013g/L of manganese sulfate (one crystal water), 0.002g/L of copper sulfate (anhydrous), 0.0173g/L of sodium glutamate and 800mg/L of Noralserin.
In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA, and the last position is the 3' terminal nucleotide of the corresponding DNA.
Example 1
The technical route for achieving the purpose of the application is as follows:
(1) And searching the heat-resistant genes of Saccharomyces cerevisiae origin and thermophilic microorganism origin, which have gene sequences in the biological information database and are reported in the prior literature, so as to obtain a heat-resistant element library.
The sequenced Saccharomyces cerevisiae, thermophilic microorganisms and heat-resistant gene information thereof are excavated and sorted through NCBI, PDB and other databases, and genes with different functions and obvious effects in the prior literature report are combined as candidate elements to form a heat-resistant element library.
(2) Genes in the heat-resistant element library are classified according to sources and functions, and heat-resistant genes with different functions from different species are screened as candidate genes.
Classifying genes in the heat-resistant element library in the step (1) according to functions to obtain heat-resistant gene types, wherein the specific types of the heat-resistant genes are shown in Table 1:
TABLE 1 Heat resistance Gene information for different functions
(3) Performing PCR amplification on candidate genes derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) BY using a Saccharomyces cerevisiae BY4741 genome as a template; candidate genes derived from thermophilic bacteria (Thermusthermophilus) were synthesized codon optimized according to yarrowia lipolytica.
The candidate genes Yap1, hsp12, vma2, hul and Tsa1 derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) were amplified BY PCR using the genome of Saccharomyces cerevisiae BY4741 as a template, and the primers used were as follows:
amplifying the Yap1 gene fragment (the nucleotide sequence is shown as the 1 st to 1953 rd positions of the sequence 1 in the sequence table) by using a primer pair Yap1-F/Yap 1-R:
Yap1-F:5′-GACATATCTACAGCAATGAGTGTGTCTACCGCCAA-3' (underlined nucleotides are homologous sequences that match perfectly with plasmid pTIB);
Yap1-R:5′-ACCGGCAACGTGGGGTTAGTTCATATGCTTATTCA-3' (underlined nucleotides are homologous sequences that match perfectly with plasmid pTIB).
The Hsp12 gene fragment is amplified by a primer pair Hsp12-F/Hsp12-R (the nucleotide sequence is shown as the 1 st to 330 th positions of the sequence 2 in the sequence table):
Hsp12-F:5′-GACATATCTACAGCAATGTCTGACGCAGGTAGAAA-3' (underlined nucleotides are homologous sequences that match perfectly with plasmid pTIB);
Hsp12-R:5′-ACCGGCAACGTGGGGTTACTTCTTGGTTGGGTCTT-3' (underlined nucleotides are homologous sequences that match perfectly with plasmid pTIB).
Amplifying a Vma2 gene fragment (the nucleotide sequence is shown as 1 st to 1554 th positions of sequence 3 in a sequence table) by using a primer pair Vma2-F/Vma 2-R:
Vma2-F:5′-GACATATCTACAGCAATGGTTTTGTCTGATAAGGA-3' (underlined nucleotides are homologous sequences that match perfectly with plasmid pTIB);
Vma2-R:5′-ACCGGCAACGTGGGGTTAGATTAGAGATTCTTCTT-3' (underlined nucleotides are homologous sequences that match perfectly with plasmid pTIB).
Amplifying Hul gene fragment (nucleotide sequence is shown in 1 st to 2733 th positions of sequence 4 of a sequence table) by using a primer pair Hul5-F/Hul 5-R:
Hul5-F:5′-GACATATCTACAGCAATGTTAAACTTCACCGGTCA-3' (underlined nucleotides are homologous sequences that match perfectly with plasmid pTIB);
Hul5-R:5′-ACCGGCAACGTGGGGTTATGATAAGTCAAACCTGG-3' (underlined nucleotides are homologous sequences that match perfectly with plasmid pTIB).
Amplifying Tsa1 gene fragment (the nucleotide sequence is shown as the 1 st to 591 st positions of sequence 5 in a sequence table) by using a primer pair Tsa1-F/Tsa 1-R:
Tsa1-F:5′-GACATATCTACAGCAATGGTCGCTCAAGTTCAAAA-3' (underlined nucleotides are homologous sequences that match perfectly with plasmid pTIB);
Tsa1-R:5′-ACCGGCAACGTGGGGTTATTTGTTGGCAGCTTCGA-3' (underlined nucleotides are homologous sequences that match perfectly with plasmid pTIB).
The candidate gene Groes of thermophilic bacteria (Thermusthermophilus) is delivered to a company to carry out codon optimization synthesis according to yarrowia lipolytica genome, so that Groes gene fragments are synthesized, and the nucleotide sequence of the Groes gene fragments is shown as the 1 st to 306 th positions of sequence 6 in a sequence table.
(4) Construction of the overexpression vector pTIB:
the commercial plasmid pUC57 is used as a template, and a primer17/primer18 is used for amplifying a prokaryotic replication initiation site and an ampicillin antibiotic gene expression cassette fragment, which is named ori-Amp:
Primer 17:ATCAATGGGCCTTGGTTTCCATAGGCTCCGCCCCC (underlined nucleotides are homologous sequences that match exactly to ChrE 1-DOWN)
Primer 18:CGCCCAAAATGCCAGCGCGGAACCCCTATTTGTTT (underlined nucleotides are homologous sequences which match perfectly with pXPR 2)
The promoter of gene XPR2 was amplified using primer1/primer2 using Yarrowialipolyticastrain W (CLIB 89) genome as template and designated pXPR2:
Primer 1:AATAGGGGTTCCGCGCTGGCATTTTGGGCGTTTTC (underlined nucleotides are homologous sequences that match perfectly with ori-Amp)
Primer 2:CAAAGTAGTACCCATTGTTGGATTGGAGGATTGGA (underlined nucleotides are homologous sequences that match exactly to Nat)
The amino acid sequence of the North-si-resistance gene (from Cloning vectorpNZ) was searched for at NCBI, and was synthesized by Jin Weizhi company after optimization according to the codon of yarrowia lipolytica, and the North-si-resistance gene was amplified using primer3/primer4 and designated as Nat:
Primer 3:TCCTCCAATCCAACAATGGGTACTACTTTGGATGA (underlined nucleotides are homologous sequences which match perfectly with pXPR 2)
Primer 4:GATTCCGAACAGAAGTCATGGACATGGCATAGACA (underlined nucleotides are homologous sequences which match perfectly with Tlip 2)
ERY01 genome is used as a template, and a primer5/primer6 is used for amplifying a terminator of gene lip2, which is named Tlip2: primer 5:ATGCCATGTCCATGACTTCTGTTCGGAATCAACCT (underlined nucleotides are homologous sequences that match exactly to Nat)
Primer 6:TCCATTATCTACGAACAGATGCATTCTTGGGCGGT (underlined nucleotides are homologous sequences which match exactly to CEN 1)
The eukaryotic replication initiation site was amplified using the Yarrowia lipolytica strain W (CLIB 89) genome as template, using primer7/primer8, designated CEN1:
Primer 7:CCAAGAATGCATCTGTTCGTAGATAATGGAATACA (underlined nucleotides are homologous sequences which match perfectly with Tlip 2)
Primer 8:TTCGGGGTCTGCCAGCATCAAATTTAGGGATGCCA (underlined nucleotides are homologous sequences that match exactly with ChrE 1-UP)
The ERY01 genome is used as a template, and a primer9/primer10 is used for amplifying an upstream homology arm 500bp sequence fragment of a gene insertion site ChrE1, which is named ChrE1-UP:
Primer 9:TCCCTAAATTTGATGCTGGCAGACCCCGAAACCTA (underlined nucleotides are homologous sequences which match exactly to CEN 1)
Primer 10:GGCGCCAAACTCCTTACAACAGCCAGTCCTCCACG (underlined nucleotides are homologous sequences that match exactly to pExp 1)
The promoter pExp1 fragment was amplified using the Yarrowia lipolytica strain W (CLIB 89) genome as template and primer11/primer12, designated pExp1:
Primer 11:AGGACTGGCTGTTGTAAGGAGTTTGGCGCCCGTTT (underlined nucleotides are homologous sequences that match exactly with ChrE 1-UP)
Primer 12:ACCGGCAACGTGGGGGCGATCGCTGCTGTAGATATGTCTTGTG (underlined nucleotides are homologous sequences that match exactly to TTef)
The terminator TTef fragment was amplified using the Yarrowia lipolytica strain W (CLIB 89) genome as template, primer13/primer14, named TTef:
Primer 13:TACAGCAGCGATCGCCCCCACGTTGCCGGTCTTGC (underlined nucleotides are homologous sequences that match exactly to pExp 1)
Primer 14:GGTGTCCGAGCGTCGGAATTCGGACACGGGCATCT (underlined nucleotides are homologous sequences that match exactly to ChrE 1-DOWN)
The ERY01 genome is used as a template, and a primer15/primer16 is used for amplifying a 500bp sequence fragment of a downstream homology arm of a gene insertion site ChrE1, which is named ChrE1-DOWN:
Primer 15:CCCGTGTCCGAATTCCGACGCTCGGACACCTCTGG (underlined nucleotides are homologous sequences that match exactly to TTef)
Primer 16:CGGAGCCTATGGAAACCAAGGCCCATTGATTGAGA (underlined nucleotides are homologous sequences that match perfectly with ori-Amp)
Fusing the amplified ori-Amp and pXPR2 through fusion PCR to obtain fragment ori-Amp-pXPR2; the Tlip2 and CEN1 are fused by fusion PCR to obtain a fragment Tlip2-CEN1; fusing the Nat and the Tlip2-CEN1 by fusion PCR to obtain a fragment Nat-Tlip2-CEN1; fusing ori-Amp-pXPR2 and Nat-Tlip2-CEN1 through fusion PCR to obtain fragment ori-Amp-pXPR2-Nat-Tlip2-CEN1; fusion of the Chu E1-UP and the pExp1 is carried out through fusion PCR, so as to obtain a fragment Chu E1-UP-pExp1; fusion of TTef and ChrE1-DOWN is carried out through fusion PCR, so that fragments TTef-ChrE1-DOWN are obtained; the fragments ori-Amp-pXPR2-Nat-Tlip2-CEN1, chrE1-UP-pExp1 and TTef-ChrE1-DOWN were assembled using a Trelief SoSoo Cloning Kit Ver.2 kit, and sequenced to obtain the correct overexpressing vector pTIB (nucleotide sequence is shown as sequence 13 in the sequence Listing, wherein positions 1-1752 are pXPR2 promoter sequence, positions 1753-2325 are Nat sequence, positions 2326-3262 are Tlip2 sequence, positions 3263-3481 are CEN1 sequence, positions 3482-3966 are ChrE1-UP sequence, positions 3967-4967 are pExp1 sequence, positions 4968-4975 are AsiSI enzyme recognition site, positions 4976-5385 are TTef sequence, positions 5386-5885 are ChrE1-DOWN sequence, and positions 5886-7610 are ori-Amp sequence).
(5) And (3) connecting the heat-resistant gene fragment with an over-expression vector pTIB to obtain a recombinant expression vector.
The heat-resistant gene fragments obtained by PCR amplification in the step (3) are connected to a vector pTIB cut by restriction enzyme AsiSI, and the correct recombinant vector is obtained by sequencing, specifically as follows:
the Yap1 gene fragment (the nucleotide sequence is shown as the 1 st to 1953 rd positions of the sequence 1 in the sequence table) is connected to a body pTIB which is cut by restriction enzyme AsiSI to obtain a recombinant expression vector of Yap1 protein, which is named pTIB-Yap1.pTIB-Yap1 contains a Yap1 gene whose coding sequence (CDS) is shown in the 1 st to 1953 rd positions of sequence 1 in the sequence table, and transcription of the Yap1 gene is initiated by the promoter pExp 1.
The Hsp12 gene fragment (the nucleotide sequence is shown as the 1 st to 330 th positions of the sequence 2 in the sequence table) is connected to the body pTIB which is cut by restriction enzyme AsiSI to obtain a recombinant expression vector of the Hsp12 protein, which is named pTIB-Hsp12.pTIB-Hsp12 contains Hsp12 genes whose coding sequences (CDS) are shown in the 1 st to 330 th positions of sequence 2 in a sequence table, and the transcription of the Hsp12 genes is started by a promoter pExp 1.
The Vma2 gene fragment (the nucleotide sequence is shown as the 1 st to 1554 th positions of the sequence 3 in the sequence table) is connected to the body pTIB which is cut by restriction enzyme AsiSI to obtain a recombinant expression vector of the Vma2 protein, which is named pTIB-Vma2.pTIB-Vma2 contains a coding sequence (CDS) which is the Vma2 gene shown in the 1 st to 1554 th positions of the sequence 3 in the sequence table, and the transcription of the Vma2 gene is started by the promoter pExp 1.
The Hul gene fragment (the nucleotide sequence is shown as the 1 st to 2733 th positions of the sequence 4 in the sequence table) is connected to a body pTIB which is cut by restriction enzyme AsiSI to obtain a recombinant expression vector of Hul protein, which is named pTIB-Hul5.pTIB-Hul contains Hul gene whose coding sequence (CDS) is shown in sequence 4 of the sequence table at positions 1-2733, and transcription of Hul gene is initiated by promoter pExp 1.
The Tsa1 gene fragment (the nucleotide sequence is shown as the 1 st to 591 st positions of the sequence 5 in the sequence table) is connected to a body pTIB which is cut by restriction enzyme AsiSI to obtain a recombinant expression vector of Tsa1 protein, which is named pTIB-Tsa1.pTIB-Tsa1 contains a coding sequence (CDS) which is a Tsa1 gene shown in the 1 st to 591 st positions of a sequence 5 in a sequence table, and transcription of the Tsa1 gene is started by a promoter pExp 1.
And (3) directly connecting the Groes gene fragment synthesized in the step (3) to an AsiSI enzyme cutting site of pTIB, keeping other sequences of the pTIB vector unchanged, and obtaining a recombinant expression vector of Groes protein, which is named pTIB-Groes. pTIB-Groes contains the Groes gene whose coding sequence (CDS) is shown in positions 1 to 306 of sequence 6 in the sequence listing, and transcription of the Groes gene is initiated by the promoter pExp 1.
(5) The recombinant vector is transformed into yarrowia lipolytica industrial strain ERY01 with high erythritol yield to obtain recombinant strain.
The recombinant vectors (pTIB-Yap 1, pTIB-Hsp12, pTIB-Vma2, pTIB-Hul, pTIB-Tsa1 and pTIB-Groes, with empty plasmid pTIB as a control) obtained in the step (4) are respectively transformed into a yarrowia lipolytica industrial strain ERY01 with high erythritol yield, and the correctly transformed recombinant strains are obtained by sequencing, wherein the following specific operation steps are performed:
preparation of competent cells:
the original strain ERY01 is placed in a 30 ℃ incubator for culturing 24 hours after being streaked on a YPD flat plate; the cultured strain is streaked on YPD plates again and then is placed in a 30 ℃ incubator for culturing for 24 hours; scraping off the cultured cells, re-suspending the cells in 1mL of sterile water, centrifuging for 5min at 3000g, and removing the supernatant, wherein the steps are repeated twice; cells were resuspended with 1mL of sterile water and OD was determined 600 The method comprises the steps of carrying out a first treatment on the surface of the The bacteria with OD value of 9.2 are taken out and placed in a new sterile centrifuge tube, and the supernatant is removed by centrifugation at 3000g for 5min, so as to obtain ERY01 competent cells for one-time transformation.
The transformation process comprises the following steps:
1) 500ng of plasmids (pTIB-Yap 1, pTIB-Hsp12, pTIB-Vma2, pTIB-Hul5, pTIB-Tsa1, pTIB-Groes and empty plasmid pTIB, respectively) were added to ERY01 competent cells;
2) Resuspension of cells with the transformation mixture:
TABLE 2
Composition of the components | Content of |
PEG(Stock50%;sterile-filtrated;end43.8%) | 87.5μl |
LiAc(Stock2M;sterile-filtrated;end0.1M) | 5.0μl |
ssDNA(Stock10mg/ml;end0.25g/l) | 2.5μl |
DTT(stock2M;sterile-filtrated;end100mM) | 5.0μl |
3) Incubating at 39 ℃ for 1h;
4) Centrifuging 3000g for 5min, removing supernatant, adding 500 μl of YPD medium, suspending, and shake culturing at 30deg.C for 2 hr;
5) After centrifugation at 3000g for 5min, the supernatant was removed, resuspended in 100. Mu.l of sterile water and plated onto YPD plates containing Noralserin (800 mg/L) and incubated at 30 ℃.
According to the above operation steps, the recombinant vector pTIB-Yap1 is transformed into a yarrowia lipolytica industrial strain ERY01 with high erythritol yield, and the recombinant yarrowia lipolytica strain containing pTIB-Yap1 is obtained by sequencing and named ERY01pTIB-Yap1.
According to the above operation steps, the recombinant vector pTIB-Hsp12 is transformed into a yarrowia lipolytica industrial strain ERY01 with high erythritol yield, and the recombinant yarrowia lipolytica strain containing pTIB-Hsp12 is obtained through sequencing and named ERY01pTIB-Hsp12.
According to the above operation steps, the recombinant vector pTIB-Vma2 is transformed into a yarrowia lipolytica industrial strain ERY01 with high erythritol yield, and the recombinant yarrowia lipolytica strain containing pTIB-Vma2 is obtained through sequencing and named ERY01pTIB-Vma2.
According to the above procedure, the recombinant vector pTIB-Hul5 was transformed into the yarrowia lipolytica industrial strain ERY01 with high erythritol yield, and the recombinant yarrowia lipolytica strain containing pTIB-Hul5 was obtained by sequencing and named ERY01 pTIB-Hul.
According to the above operation steps, the recombinant vector pTIB-Tsa1 is transformed into a yarrowia lipolytica industrial strain ERY01 with high erythritol yield, and the recombinant yarrowia lipolytica strain containing pTIB-Tsa1 is obtained through sequencing and named ERY01pTIB-Tsa1.
According to the above procedure, the recombinant vector pTIB-Groes was transformed into the industrial strain ERY01 of yarrowia lipolytica producing erythritol at high yield, and the recombinant strain of yarrowia lipolytica containing pTIB-Groes was obtained by sequencing and named ERY01pTIB-Groes.
According to the above procedure, the empty plasmid pTIB was transformed into the industrial strain ERY01 of yarrowia lipolytica which was highly erythritol-producing, and the recombinant yarrowia lipolytica strain containing pTIB was obtained by sequencing and designated ERY01pTIB.
(6) Fermenting and culturing each recombinant strain at 35 ℃, and measuring the growth condition of cells by a spectrophotometer; the yield of erythritol was determined by HPLC.
YPD plates containing Norrissin were streaked on the above recombinant yarrowia lipolytica strains (ERY 01pTIB-Yap1, ERY01pTIB-Hsp12, ERY01pTIB-Vma2, ERY01pTIB-Hul5, ERY01pTIB-Tsa1, ERY01pTIB-Groes, ERY01 pTIB) respectively, and after growing, the single clone was selected to a seed liquid medium for cultivation. The culture temperature is 30 ℃, and the seed liquid is obtained after the culture for 36 hours.
Transferring the seed liquid to the fermentation medium to an initial OD 600 The culture was carried out at 35℃for 72 hours at 0.1, and the fermentation broth was collected. 3 replicates were run for each strain.
Diluting the fermentation broth, and performing OD 600 Determination of erythritol content was performed simultaneously by HPLC. In HPLC, erythritol (product CAS number 149-32-6, beijing Soy Co., ltd.) was used as a standard substance and quantitative analysis of erythritol was performed by a standard curve method (external standard method) based on the retention time of the standard substance. HPLC detection conditions: HPLC (HPX-87H chromatography column, 300 mm. Times.7.8 mm, bio-Rad, hercules, calif., USA) at 55℃with a mobile phase of 0.008M sulfuric acid solution, flow rate of 0.6M L/min and sample loading of 5. Mu.L.
Data were processed using Excel software and experimental results were expressed as mean ± standard deviation.
The measurement results are shown in fig. 1 and 2. Compared with the control strain ERY01pTIB transferred into empty plasmid pTIB, the growth of all recombinant plasmid-type strains (ERY 01pTIB-Yap1, ERY01pTIB-Hsp12, ERY01pTIB-Vma2, ERY01pTIB-Hul5, ERY01pTIB-Tsa1 and ERY01 pTIB-Groes) expressing heat-resistant genes under the condition of 35 ℃ is improved, and the erythritol yield is also obviously increased. Wherein, the yield of erythritol of ERY01pTIB-Groes is highest and is 31.6+/-0.6 g erythritol/L fermentation liquor; the erythritol yield of ERY01pTIB-Yap1 is 14.2+ -0.3 g erythritol/L fermentation broth; the erythritol yield of ERY01pTIB-Hsp12 is 22+ -1.4 g erythritol/L fermentation broth; the erythritol yield of ERY01pTIB-Vma2 is 19.8+ -0.7 g erythritol/L fermentation broth; the erythritol yield of ERY01pTIB-Hul5 is 20.3+ -1.5 g erythritol/L broth; the erythritol yield of ERY01pTIB-Tsa1 is 21.3+ -1.1 g erythritol/L fermentation broth; the erythritol yield of ERY01pTIB was 11.+ -. 0.9g erythritol/L broth.
(7) Screening to obtain heat-resistant genes with different functions, which can enable industrial strains of yarrowia lipolytica to grow well under the condition of 35 ℃ and produce erythritol in high yield.
According to the result of the step (6), different heat-resistant genes Yap1, hsp12, vma2, hul5, tsa1 and Groes which can enable the yarrowia lipolytica industrial strain to grow well under the condition of 35 ℃ and produce erythritol with high yield are obtained through screening.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Sequence listing
<110> institute of Tianjin Industrial biotechnology, national academy of sciences
<120> a method for constructing recombinant yarrowia lipolytica and use thereof
<130> GNCSY212191
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1953
<212> DNA
<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 1
atgagtgtgt ctaccgccaa gaggtcgctg gatgtcgttt ctccgggttc attagcggag 60
tttgagggtt caaaatctcg tcacgatgaa atagaaaatg aacatagacg tactggtaca 120
cgtgatggcg aggatagcga gcaaccgaag aagaagggta gcaaaactag caaaaagcaa 180
gatttggatc ctgaaactaa gcagaagagg actgcccaaa atcgggccgc tcaaagagct 240
tttagggaac gtaaggagag gaagatgaag gaattggaga agaaggtaca aagtttagag 300
agtattcagc agcaaaatga agtggaagct acttttttga gggaccagtt aatcactctg 360
gtgaatgagt taaaaaaata tagaccagag acaagaaatg actcaaaagt gctggaatat 420
ttagcaaggc gagatcctaa tttgcatttt tcaaaaaata acgttaacca cagcaatagc 480
gagccaattg acacacccaa tgatgacata caagaaaatg ttaaacaaaa gatgaatttc 540
acgtttcaat atccgcttga taacgacaac gacaacgaca acagtaaaaa tgtggggaaa 600
caattacctt caccaaatga tccaagtcat tcggctccta tgcctataaa tcagacacaa 660
aagaaattaa gtgacgctac agattcctcc agcgctactt tggattccct ttcaaatagt 720
aacgatgttc ttaataacac accaaactcc tccacttcga tggattggtt agataatgta 780
atatatacta acaggtttgt gtcaggtgat gatggcagca atagtaaaac taagaattta 840
gacagtaata tgttttctaa tgactttaat tttgaaaacc aatttgatga acaagtttcg 900
gagttttgtt cgaaaatgaa ccaggtatgt ggaacaaggc aatgtcccat tcccaagaaa 960
cccatctcgg ctcttgataa agaagttttc gcgtcatctt ctatactaag ttcaaattct 1020
cctgctttaa caaatacttg ggaatcacat tctaatatta cagataatac tcctgctaat 1080
gtcattgcta ctgatgctac taaatatgaa aattccttct ccggttttgg ccgacttggt 1140
ttcgatatga gtgccaatca ttacgtcgtg aatgataata gcactggtag cactgatagc 1200
actggtagca ctggcaataa gaacaaaaag aacaataata atagcgatga tgtactccca 1260
ttcatatccg agtcaccgtt tgatatgaac caagttacta atttttttag tccgggatct 1320
accggcatcg gcaataatgc tgcctctaac accaatccca gcctactgca aagcagcaaa 1380
gaggatatac cttttatcaa cgcaaatctg gctttcccag acgacaattc aactaatatt 1440
caattacaac ctttctctga atctcaatct caaaataagt ttgactacga catgtttttt 1500
agagattcat cgaaggaagg taacaattta tttggagagt ttttagagga tgacgatgat 1560
gacaaaaaag ccgctaatat gtcagacgat gagtcaagtt taatcaagaa ccagttaatt 1620
aacgaagaac cagagcttcc gaaacaatat ctacaatcgg taccaggaaa tgaaagcgaa 1680
atctcacaaa aaaatggcag tagtttacag aatgctgaca aaatcaataa tggcaatgat 1740
aacgataatg ataatgatgt cgttccatct aaggaaggct ctttactaag gtgttcggaa 1800
atttgggata gaataacaac acatccgaaa tactcagata ttgatgtcga tggtttatgt 1860
tccgagctaa tggcaaaggc aaaatgttca gaaagagggg ttgtcatcaa tgcagaagac 1920
gttcaattag ctttgaataa gcatatgaac taa 1953
<210> 2
<211> 330
<212> DNA
<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 2
atgtctgacg caggtagaaa aggattcggt gaaaaagctt ctgaagcttt gaagccagac 60
tctcaaaagt catacgctga acaaggtaag gaatacatca ctgacaaggc cgacaaggtc 120
gctggtaagg ttcaaccaga agacaacaag ggtgtcttcc aaggtgtcca cgactctgcc 180
gaaaaaggca aggataacgc tgaaggtcaa ggtgaatctt tggcagacca agctagagat 240
tacatgggag ccgccaagtc caagttgaac gatgccgtcg aatatgtttc cggtcgtgtc 300
cacggtgaag aagacccaac caagaagtaa 330
<210> 3
<211> 1554
<212> DNA
<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 3
atggttttgt ctgataagga gttgtttgcc ataaataaga aagccgtcga acaaggtttc 60
aatgtgaagc ctagattgaa ctataatacg gtcagtggtg tgaacggtcc attagtcatt 120
ttggaaaagg tcaagttccc acgttacaac gaaattgtta atttgacatt gccagatgga 180
accgtgagac aaggtcaagt tttggaaatt agaggagata gagccattgt gcaagtgttt 240
gaaggtacat ctggtattga tgtcaagaag actaccgtgg aattcactgg tgagagtttg 300
agaattcctg tgtctgaaga catgttgggt agaatttttg acggttctgg tagacccatt 360
gacaacggtc ctaaagtttt cgcagaggat tacttggaca ttaacggttc tcctatcaac 420
ccatatgctc gtatttatcc agaagaaatg atttctactg gtgtttctgc tattgacaca 480
atgaactcca ttgccagagg tcaaaagatc ccaattttct ccgcatcagg tttaccacac 540
aacgaaattg cagcacaaat ttgtagacag gctggtttgg tgagacctac caaggatgtt 600
catgatggtc atgaagaaaa tttctccatc gtttttgctg ccatgggtgt caacttggaa 660
accgctagat ttttcaaaca ggatttcgaa gaaaatgggt ctttggaaag aacttcatta 720
tttttgaact tggctaatga ccctaccatt gaaagaatta tcactccaag attggccttg 780
accaccgctg aataccttgc ttaccaaacg gaacgtcatg tgttgaccat cttgaccgat 840
atgtcatcgt atgctgatgc tcttagagaa gtttccgctg ctagagaaga agttccaggt 900
agaagaggtt atcctggtta catgtataca gatttgtcca caatttatga aagagcaggt 960
agagtagagg gtcgtaacgg gtccatcact caaataccta tcttgacaat gcctaacgat 1020
gatattacgc atccaattcc ggatttgacc ggttatatta ccgagggtca aatcttcgtt 1080
gaccgtcaat tacataacaa gggtatctac ccaccaatca acgtcttgcc ttcgttgagt 1140
agattgatga aatctgccat cggtgaaggt atgaccagaa aggaccacgg tgacgtttct 1200
aaccaattgt atgccaagta cgccatcggt aaggacgctg ctgctatgaa ggccgttgtc 1260
ggtgaagagg cgttatccat cgaagataag ttatctttgg aatttttgga aaaattcgaa 1320
aagaccttta tcacacaagg cgcctacgag gacagaaccg ttttcgaaag tttggaccag 1380
gcatggagtt tgctaagaat ctaccctaag gagatgttga atagaatctc cccaaagatt 1440
cttgatgaat tttacgatag agccagagac gatgccgacg aagatgaaga agatcccgac 1500
acaagaagct ccggtaagaa gaaggacgcc agccaagaag aatctctaat ctaa 1554
<210> 4
<211> 2733
<212> DNA
<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 4
atgttaaact tcaccggtca aacaaggaga agaaatgtca atttagggaa taggactcga 60
aattcaaaga aggatttact ggaaaaggcc aaaagggaac gtgaaaggag agcacaagat 120
aagctcaaag aagacgccag taaaaccatt caaaaaagca tcagaagaca tttttcaaat 180
gtgagactct tcaaaaacac atttactagt tcgcaacttg ttcatatgat accagcttac 240
gggggcaaat taatctatta catttctcaa tatgatctgc agcaactgct aaaattatct 300
cataattttt tgagttctta tcctaattct ttaggcaaca gacagctatt gagcttgttg 360
aagctatatc aagatgatgc actggtggct gaaactctaa gcgatcttaa catggactgc 420
cctacagttg acgaattttt agatagtcta tccgtttatc tctgtcgggc ttcctccttg 480
agttattcct cagcttctaa gctagctgat gtcatagaag catgggaagt aatgcatagc 540
agtgcctcca ttagtatttt ttcgatatcg ataggatctt acgaaaaacg gccatttgca 600
ctacagtttt attgcatact tgccgaaaga aaccttttgc cccaacttat caacacaaat 660
ccgatattat gggataacat ggcaaagaca tattcacact gtagtaaagg tggccaaaaa 720
aatatcgcca agctactcat accaaacttc aacaatcata ttgctccatc agtcttgcgt 780
agtgataacg actatgtctt gaaattttat gaaaaggcat ttatagacga agttattgct 840
actactgcaa attacgtttc tgatgaagat cacgtgaaga atttgatgtg ctacattgca 900
agctctccca atcaaagctg taagaattct gtccttatta ccttactgtc taacaaagac 960
ttcgtgagaa ggctctcatg ggaattcttt cacactaaat tcaacgccag caagactgag 1020
gctcaccccc tgttttcagt tttggcacag cttatcgaca tgcacctttt aatatcgaca 1080
gatcgggagt tattagacta caactctgtg atacctatcg aagaactaaa gaaatttaca 1140
tccacgttaa aagattttac tttccgacaa tattgggaac tacctaaaag tgaaaggaac 1200
cctatgttaa aggaagcagt accacttttg agcaaagtct acgaaaggga ctcaagattg 1260
cactttctat ccacggagaa taatccaacc tattgggaaa actctgaaaa gcaattttta 1320
aatttgagat tttacgaaga attacaggag tacgaagatc tgtatagaga acacttagaa 1380
gaggaaagtg atgaggatat ggaaaaggaa atagatcttg ataaagaaag gcctcctttg 1440
aagtctttat tattgaacaa aatgaaaaag agattaaagt catcattacg tttccgaaag 1500
ttggaaatac ttttggaatt accatttttt attccttttg aagaaagggt ggacttattt 1560
tatatgttca tcgcactgga caagaagcga ttatctttag atgacgatca caacttgatc 1620
aatatgttta ccccctgggc ctccaccggt atgaggaagc aatccgctat catctctagg 1680
gataatgtct tagaagatgc tttcaacgca tttaactcta taggagaaag gttcaaagct 1740
tcattagatg ttacttttat taatgaattt ggtgaagaag ctggtattga tgggggcggc 1800
attaccaagg aatttttaac tactgtgtct gatgaaggat ttaaagatcc aaagcacgag 1860
ttatttcgga cgaatgatcg ctacgaatta tatccttctg ttgtttatga cgctacgaaa 1920
cttaagtata tatggtttct gggaaaggtt gtaggcaaat gtctatatga gcatgttttg 1980
atagatgtat cttttgctga tttctttttg aaaaaattat tgaattactc gaacgggttt 2040
ttatcttctt tttctgatct gggaagctat gattcggtgt tgtataataa tttgatcaaa 2100
ttattgaaca tgaccactga cgagatcaag tctttagatt taacctttga aatagatgag 2160
cctgaaagtt ccgcaaaagt tgttgactta attccgaatg gttcaaaaac gtacgtgacg 2220
aaggataatg tgttgctcta cgttactaaa gtaacggatt acaaattaaa caaaagatgc 2280
ttcaaaccag tttcggcata ccatggaggg ctcagtgtta tcattgcccc acattggatg 2340
gagatgttta actctataga actacaaatg ttaatatcag gtgagagaga taatatcgat 2400
ttagacgatc tgaaatctaa cacagaatac gggggttata aagaagaaga tcagacaatt 2460
gttgattttt gggaggtttt gaatgagttt aagtttgaag agaaattgaa ttttttgaaa 2520
tttgtcacgt ccgttccgca agctcctctg caaggcttca aggcattaga tccaaaattt 2580
ggtatcagaa atgcagggac agagaaatac aggctgccta cggcatctac ctgtgttaat 2640
ttattaaaat tgccagatta tagaaacaaa acaattttga gagagaaatt attatatgca 2700
ataaactcag gcgccaggtt tgacttatca taa 2733
<210> 5
<211> 591
<212> DNA
<213> Saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 5
atggtcgctc aagttcaaaa gcaagctcca acttttaaga aaactgccgt cgtcgacggt 60
gtctttgacg aagtctcctt ggacaaatac aagggtaagt acgttgtcct agcctttatt 120
ccattggcct tcactttcgt ctgtccaacc gaaatcattg ctttctcaga agctgctaag 180
aaattcgaag aacaaggcgc tcaagttctt ttcgcctcca ctgactccga atactccctt 240
ttggcatgga ccaatatccc aagaaaggaa ggtggtttgg gcccaatcaa cattccattg 300
ttggctgaca ccaaccactc tttgtccaga gactatggtg tcttgatcga agaagaaggt 360
gtcgccttga gaggtttgtt catcatcgac ccaaagggtg tcattagaca catcaccatt 420
aacgatttgc cagtcggtag aaacgttgac gaagccttga gattggttga agccttccaa 480
tggaccgaca agaacggtac tgtcttgcca tgtaactgga ctccaggtgc tgctaccatc 540
aagccaaccg ttgaagactc caaggaatac ttcgaagctg ccaacaaata a 591
<210> 6
<211> 306
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
atggccgccg aggtgaagac cgtgatcaag cccctgggcg accgagtggt cgtgaagcga 60
atcgaggaag agcccaagac caagggcggc atcgtgctgc ccgacaccgc caaggagaag 120
cctcagaagg gcaaggtgat cgccgtgggc accggccgag tgctggagaa cggacagcga 180
gtgcccctgg aggtgaagga gggcgacatc gtggtgttcg ccaagtacgg cggcaccgag 240
atcgagatcg acggcgagga gtacgtgatc ctgtctgagc gagacctgct ggccgtgctg 300
cagtaa 306
<210> 7
<211> 650
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 7
Met Ser Val Ser Thr Ala Lys Arg Ser Leu Asp Val Val Ser Pro Gly
1 5 10 15
Ser Leu Ala Glu Phe Glu Gly Ser Lys Ser Arg His Asp Glu Ile Glu
20 25 30
Asn Glu His Arg Arg Thr Gly Thr Arg Asp Gly Glu Asp Ser Glu Gln
35 40 45
Pro Lys Lys Lys Gly Ser Lys Thr Ser Lys Lys Gln Asp Leu Asp Pro
50 55 60
Glu Thr Lys Gln Lys Arg Thr Ala Gln Asn Arg Ala Ala Gln Arg Ala
65 70 75 80
Phe Arg Glu Arg Lys Glu Arg Lys Met Lys Glu Leu Glu Lys Lys Val
85 90 95
Gln Ser Leu Glu Ser Ile Gln Gln Gln Asn Glu Val Glu Ala Thr Phe
100 105 110
Leu Arg Asp Gln Leu Ile Thr Leu Val Asn Glu Leu Lys Lys Tyr Arg
115 120 125
Pro Glu Thr Arg Asn Asp Ser Lys Val Leu Glu Tyr Leu Ala Arg Arg
130 135 140
Asp Pro Asn Leu His Phe Ser Lys Asn Asn Val Asn His Ser Asn Ser
145 150 155 160
Glu Pro Ile Asp Thr Pro Asn Asp Asp Ile Gln Glu Asn Val Lys Gln
165 170 175
Lys Met Asn Phe Thr Phe Gln Tyr Pro Leu Asp Asn Asp Asn Asp Asn
180 185 190
Asp Asn Ser Lys Asn Val Gly Lys Gln Leu Pro Ser Pro Asn Asp Pro
195 200 205
Ser His Ser Ala Pro Met Pro Ile Asn Gln Thr Gln Lys Lys Leu Ser
210 215 220
Asp Ala Thr Asp Ser Ser Ser Ala Thr Leu Asp Ser Leu Ser Asn Ser
225 230 235 240
Asn Asp Val Leu Asn Asn Thr Pro Asn Ser Ser Thr Ser Met Asp Trp
245 250 255
Leu Asp Asn Val Ile Tyr Thr Asn Arg Phe Val Ser Gly Asp Asp Gly
260 265 270
Ser Asn Ser Lys Thr Lys Asn Leu Asp Ser Asn Met Phe Ser Asn Asp
275 280 285
Phe Asn Phe Glu Asn Gln Phe Asp Glu Gln Val Ser Glu Phe Cys Ser
290 295 300
Lys Met Asn Gln Val Cys Gly Thr Arg Gln Cys Pro Ile Pro Lys Lys
305 310 315 320
Pro Ile Ser Ala Leu Asp Lys Glu Val Phe Ala Ser Ser Ser Ile Leu
325 330 335
Ser Ser Asn Ser Pro Ala Leu Thr Asn Thr Trp Glu Ser His Ser Asn
340 345 350
Ile Thr Asp Asn Thr Pro Ala Asn Val Ile Ala Thr Asp Ala Thr Lys
355 360 365
Tyr Glu Asn Ser Phe Ser Gly Phe Gly Arg Leu Gly Phe Asp Met Ser
370 375 380
Ala Asn His Tyr Val Val Asn Asp Asn Ser Thr Gly Ser Thr Asp Ser
385 390 395 400
Thr Gly Ser Thr Gly Asn Lys Asn Lys Lys Asn Asn Asn Asn Ser Asp
405 410 415
Asp Val Leu Pro Phe Ile Ser Glu Ser Pro Phe Asp Met Asn Gln Val
420 425 430
Thr Asn Phe Phe Ser Pro Gly Ser Thr Gly Ile Gly Asn Asn Ala Ala
435 440 445
Ser Asn Thr Asn Pro Ser Leu Leu Gln Ser Ser Lys Glu Asp Ile Pro
450 455 460
Phe Ile Asn Ala Asn Leu Ala Phe Pro Asp Asp Asn Ser Thr Asn Ile
465 470 475 480
Gln Leu Gln Pro Phe Ser Glu Ser Gln Ser Gln Asn Lys Phe Asp Tyr
485 490 495
Asp Met Phe Phe Arg Asp Ser Ser Lys Glu Gly Asn Asn Leu Phe Gly
500 505 510
Glu Phe Leu Glu Asp Asp Asp Asp Asp Lys Lys Ala Ala Asn Met Ser
515 520 525
Asp Asp Glu Ser Ser Leu Ile Lys Asn Gln Leu Ile Asn Glu Glu Pro
530 535 540
Glu Leu Pro Lys Gln Tyr Leu Gln Ser Val Pro Gly Asn Glu Ser Glu
545 550 555 560
Ile Ser Gln Lys Asn Gly Ser Ser Leu Gln Asn Ala Asp Lys Ile Asn
565 570 575
Asn Gly Asn Asp Asn Asp Asn Asp Asn Asp Val Val Pro Ser Lys Glu
580 585 590
Gly Ser Leu Leu Arg Cys Ser Glu Ile Trp Asp Arg Ile Thr Thr His
595 600 605
Pro Lys Tyr Ser Asp Ile Asp Val Asp Gly Leu Cys Ser Glu Leu Met
610 615 620
Ala Lys Ala Lys Cys Ser Glu Arg Gly Val Val Ile Asn Ala Glu Asp
625 630 635 640
Val Gln Leu Ala Leu Asn Lys His Met Asn
645 650
<210> 8
<211> 109
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 8
Met Ser Asp Ala Gly Arg Lys Gly Phe Gly Glu Lys Ala Ser Glu Ala
1 5 10 15
Leu Lys Pro Asp Ser Gln Lys Ser Tyr Ala Glu Gln Gly Lys Glu Tyr
20 25 30
Ile Thr Asp Lys Ala Asp Lys Val Ala Gly Lys Val Gln Pro Glu Asp
35 40 45
Asn Lys Gly Val Phe Gln Gly Val His Asp Ser Ala Glu Lys Gly Lys
50 55 60
Asp Asn Ala Glu Gly Gln Gly Glu Ser Leu Ala Asp Gln Ala Arg Asp
65 70 75 80
Tyr Met Gly Ala Ala Lys Ser Lys Leu Asn Asp Ala Val Glu Tyr Val
85 90 95
Ser Gly Arg Val His Gly Glu Glu Asp Pro Thr Lys Lys
100 105
<210> 9
<211> 517
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 9
Met Val Leu Ser Asp Lys Glu Leu Phe Ala Ile Asn Lys Lys Ala Val
1 5 10 15
Glu Gln Gly Phe Asn Val Lys Pro Arg Leu Asn Tyr Asn Thr Val Ser
20 25 30
Gly Val Asn Gly Pro Leu Val Ile Leu Glu Lys Val Lys Phe Pro Arg
35 40 45
Tyr Asn Glu Ile Val Asn Leu Thr Leu Pro Asp Gly Thr Val Arg Gln
50 55 60
Gly Gln Val Leu Glu Ile Arg Gly Asp Arg Ala Ile Val Gln Val Phe
65 70 75 80
Glu Gly Thr Ser Gly Ile Asp Val Lys Lys Thr Thr Val Glu Phe Thr
85 90 95
Gly Glu Ser Leu Arg Ile Pro Val Ser Glu Asp Met Leu Gly Arg Ile
100 105 110
Phe Asp Gly Ser Gly Arg Pro Ile Asp Asn Gly Pro Lys Val Phe Ala
115 120 125
Glu Asp Tyr Leu Asp Ile Asn Gly Ser Pro Ile Asn Pro Tyr Ala Arg
130 135 140
Ile Tyr Pro Glu Glu Met Ile Ser Thr Gly Val Ser Ala Ile Asp Thr
145 150 155 160
Met Asn Ser Ile Ala Arg Gly Gln Lys Ile Pro Ile Phe Ser Ala Ser
165 170 175
Gly Leu Pro His Asn Glu Ile Ala Ala Gln Ile Cys Arg Gln Ala Gly
180 185 190
Leu Val Arg Pro Thr Lys Asp Val His Asp Gly His Glu Glu Asn Phe
195 200 205
Ser Ile Val Phe Ala Ala Met Gly Val Asn Leu Glu Thr Ala Arg Phe
210 215 220
Phe Lys Gln Asp Phe Glu Glu Asn Gly Ser Leu Glu Arg Thr Ser Leu
225 230 235 240
Phe Leu Asn Leu Ala Asn Asp Pro Thr Ile Glu Arg Ile Ile Thr Pro
245 250 255
Arg Leu Ala Leu Thr Thr Ala Glu Tyr Leu Ala Tyr Gln Thr Glu Arg
260 265 270
His Val Leu Thr Ile Leu Thr Asp Met Ser Ser Tyr Ala Asp Ala Leu
275 280 285
Arg Glu Val Ser Ala Ala Arg Glu Glu Val Pro Gly Arg Arg Gly Tyr
290 295 300
Pro Gly Tyr Met Tyr Thr Asp Leu Ser Thr Ile Tyr Glu Arg Ala Gly
305 310 315 320
Arg Val Glu Gly Arg Asn Gly Ser Ile Thr Gln Ile Pro Ile Leu Thr
325 330 335
Met Pro Asn Asp Asp Ile Thr His Pro Ile Pro Asp Leu Thr Gly Tyr
340 345 350
Ile Thr Glu Gly Gln Ile Phe Val Asp Arg Gln Leu His Asn Lys Gly
355 360 365
Ile Tyr Pro Pro Ile Asn Val Leu Pro Ser Leu Ser Arg Leu Met Lys
370 375 380
Ser Ala Ile Gly Glu Gly Met Thr Arg Lys Asp His Gly Asp Val Ser
385 390 395 400
Asn Gln Leu Tyr Ala Lys Tyr Ala Ile Gly Lys Asp Ala Ala Ala Met
405 410 415
Lys Ala Val Val Gly Glu Glu Ala Leu Ser Ile Glu Asp Lys Leu Ser
420 425 430
Leu Glu Phe Leu Glu Lys Phe Glu Lys Thr Phe Ile Thr Gln Gly Ala
435 440 445
Tyr Glu Asp Arg Thr Val Phe Glu Ser Leu Asp Gln Ala Trp Ser Leu
450 455 460
Leu Arg Ile Tyr Pro Lys Glu Met Leu Asn Arg Ile Ser Pro Lys Ile
465 470 475 480
Leu Asp Glu Phe Tyr Asp Arg Ala Arg Asp Asp Ala Asp Glu Asp Glu
485 490 495
Glu Asp Pro Asp Thr Arg Ser Ser Gly Lys Lys Lys Asp Ala Ser Gln
500 505 510
Glu Glu Ser Leu Ile
515
<210> 10
<211> 910
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 10
Met Leu Asn Phe Thr Gly Gln Thr Arg Arg Arg Asn Val Asn Leu Gly
1 5 10 15
Asn Arg Thr Arg Asn Ser Lys Lys Asp Leu Leu Glu Lys Ala Lys Arg
20 25 30
Glu Arg Glu Arg Arg Ala Gln Asp Lys Leu Lys Glu Asp Ala Ser Lys
35 40 45
Thr Ile Gln Lys Ser Ile Arg Arg His Phe Ser Asn Val Arg Leu Phe
50 55 60
Lys Asn Thr Phe Thr Ser Ser Gln Leu Val His Met Ile Pro Ala Tyr
65 70 75 80
Gly Gly Lys Leu Ile Tyr Tyr Ile Ser Gln Tyr Asp Leu Gln Gln Leu
85 90 95
Leu Lys Leu Ser His Asn Phe Leu Ser Ser Tyr Pro Asn Ser Leu Gly
100 105 110
Asn Arg Gln Leu Leu Ser Leu Leu Lys Leu Tyr Gln Asp Asp Ala Leu
115 120 125
Val Ala Glu Thr Leu Ser Asp Leu Asn Met Asp Cys Pro Thr Val Asp
130 135 140
Glu Phe Leu Asp Ser Leu Ser Val Tyr Leu Cys Arg Ala Ser Ser Leu
145 150 155 160
Ser Tyr Ser Ser Ala Ser Lys Leu Ala Asp Val Ile Glu Ala Trp Glu
165 170 175
Val Met His Ser Ser Ala Ser Ile Ser Ile Phe Ser Ile Ser Ile Gly
180 185 190
Ser Tyr Glu Lys Arg Pro Phe Ala Leu Gln Phe Tyr Cys Ile Leu Ala
195 200 205
Glu Arg Asn Leu Leu Pro Gln Leu Ile Asn Thr Asn Pro Ile Leu Trp
210 215 220
Asp Asn Met Ala Lys Thr Tyr Ser His Cys Ser Lys Gly Gly Gln Lys
225 230 235 240
Asn Ile Ala Lys Leu Leu Ile Pro Asn Phe Asn Asn His Ile Ala Pro
245 250 255
Ser Val Leu Arg Ser Asp Asn Asp Tyr Val Leu Lys Phe Tyr Glu Lys
260 265 270
Ala Phe Ile Asp Glu Val Ile Ala Thr Thr Ala Asn Tyr Val Ser Asp
275 280 285
Glu Asp His Val Lys Asn Leu Met Cys Tyr Ile Ala Ser Ser Pro Asn
290 295 300
Gln Ser Cys Lys Asn Ser Val Leu Ile Thr Leu Leu Ser Asn Lys Asp
305 310 315 320
Phe Val Arg Arg Leu Ser Trp Glu Phe Phe His Thr Lys Phe Asn Ala
325 330 335
Ser Lys Thr Glu Ala His Pro Leu Phe Ser Val Leu Ala Gln Leu Ile
340 345 350
Asp Met His Leu Leu Ile Ser Thr Asp Arg Glu Leu Leu Asp Tyr Asn
355 360 365
Ser Val Ile Pro Ile Glu Glu Leu Lys Lys Phe Thr Ser Thr Leu Lys
370 375 380
Asp Phe Thr Phe Arg Gln Tyr Trp Glu Leu Pro Lys Ser Glu Arg Asn
385 390 395 400
Pro Met Leu Lys Glu Ala Val Pro Leu Leu Ser Lys Val Tyr Glu Arg
405 410 415
Asp Ser Arg Leu His Phe Leu Ser Thr Glu Asn Asn Pro Thr Tyr Trp
420 425 430
Glu Asn Ser Glu Lys Gln Phe Leu Asn Leu Arg Phe Tyr Glu Glu Leu
435 440 445
Gln Glu Tyr Glu Asp Leu Tyr Arg Glu His Leu Glu Glu Glu Ser Asp
450 455 460
Glu Asp Met Glu Lys Glu Ile Asp Leu Asp Lys Glu Arg Pro Pro Leu
465 470 475 480
Lys Ser Leu Leu Leu Asn Lys Met Lys Lys Arg Leu Lys Ser Ser Leu
485 490 495
Arg Phe Arg Lys Leu Glu Ile Leu Leu Glu Leu Pro Phe Phe Ile Pro
500 505 510
Phe Glu Glu Arg Val Asp Leu Phe Tyr Met Phe Ile Ala Leu Asp Lys
515 520 525
Lys Arg Leu Ser Leu Asp Asp Asp His Asn Leu Ile Asn Met Phe Thr
530 535 540
Pro Trp Ala Ser Thr Gly Met Arg Lys Gln Ser Ala Ile Ile Ser Arg
545 550 555 560
Asp Asn Val Leu Glu Asp Ala Phe Asn Ala Phe Asn Ser Ile Gly Glu
565 570 575
Arg Phe Lys Ala Ser Leu Asp Val Thr Phe Ile Asn Glu Phe Gly Glu
580 585 590
Glu Ala Gly Ile Asp Gly Gly Gly Ile Thr Lys Glu Phe Leu Thr Thr
595 600 605
Val Ser Asp Glu Gly Phe Lys Asp Pro Lys His Glu Leu Phe Arg Thr
610 615 620
Asn Asp Arg Tyr Glu Leu Tyr Pro Ser Val Val Tyr Asp Ala Thr Lys
625 630 635 640
Leu Lys Tyr Ile Trp Phe Leu Gly Lys Val Val Gly Lys Cys Leu Tyr
645 650 655
Glu His Val Leu Ile Asp Val Ser Phe Ala Asp Phe Phe Leu Lys Lys
660 665 670
Leu Leu Asn Tyr Ser Asn Gly Phe Leu Ser Ser Phe Ser Asp Leu Gly
675 680 685
Ser Tyr Asp Ser Val Leu Tyr Asn Asn Leu Ile Lys Leu Leu Asn Met
690 695 700
Thr Thr Asp Glu Ile Lys Ser Leu Asp Leu Thr Phe Glu Ile Asp Glu
705 710 715 720
Pro Glu Ser Ser Ala Lys Val Val Asp Leu Ile Pro Asn Gly Ser Lys
725 730 735
Thr Tyr Val Thr Lys Asp Asn Val Leu Leu Tyr Val Thr Lys Val Thr
740 745 750
Asp Tyr Lys Leu Asn Lys Arg Cys Phe Lys Pro Val Ser Ala Tyr His
755 760 765
Gly Gly Leu Ser Val Ile Ile Ala Pro His Trp Met Glu Met Phe Asn
770 775 780
Ser Ile Glu Leu Gln Met Leu Ile Ser Gly Glu Arg Asp Asn Ile Asp
785 790 795 800
Leu Asp Asp Leu Lys Ser Asn Thr Glu Tyr Gly Gly Tyr Lys Glu Glu
805 810 815
Asp Gln Thr Ile Val Asp Phe Trp Glu Val Leu Asn Glu Phe Lys Phe
820 825 830
Glu Glu Lys Leu Asn Phe Leu Lys Phe Val Thr Ser Val Pro Gln Ala
835 840 845
Pro Leu Gln Gly Phe Lys Ala Leu Asp Pro Lys Phe Gly Ile Arg Asn
850 855 860
Ala Gly Thr Glu Lys Tyr Arg Leu Pro Thr Ala Ser Thr Cys Val Asn
865 870 875 880
Leu Leu Lys Leu Pro Asp Tyr Arg Asn Lys Thr Ile Leu Arg Glu Lys
885 890 895
Leu Leu Tyr Ala Ile Asn Ser Gly Ala Arg Phe Asp Leu Ser
900 905 910
<210> 11
<211> 196
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 11
Met Val Ala Gln Val Gln Lys Gln Ala Pro Thr Phe Lys Lys Thr Ala
1 5 10 15
Val Val Asp Gly Val Phe Asp Glu Val Ser Leu Asp Lys Tyr Lys Gly
20 25 30
Lys Tyr Val Val Leu Ala Phe Ile Pro Leu Ala Phe Thr Phe Val Cys
35 40 45
Pro Thr Glu Ile Ile Ala Phe Ser Glu Ala Ala Lys Lys Phe Glu Glu
50 55 60
Gln Gly Ala Gln Val Leu Phe Ala Ser Thr Asp Ser Glu Tyr Ser Leu
65 70 75 80
Leu Ala Trp Thr Asn Ile Pro Arg Lys Glu Gly Gly Leu Gly Pro Ile
85 90 95
Asn Ile Pro Leu Leu Ala Asp Thr Asn His Ser Leu Ser Arg Asp Tyr
100 105 110
Gly Val Leu Ile Glu Glu Glu Gly Val Ala Leu Arg Gly Leu Phe Ile
115 120 125
Ile Asp Pro Lys Gly Val Ile Arg His Ile Thr Ile Asn Asp Leu Pro
130 135 140
Val Gly Arg Asn Val Asp Glu Ala Leu Arg Leu Val Glu Ala Phe Gln
145 150 155 160
Trp Thr Asp Lys Asn Gly Thr Val Leu Pro Cys Asn Trp Thr Pro Gly
165 170 175
Ala Ala Thr Ile Lys Pro Thr Val Glu Asp Ser Lys Glu Tyr Phe Glu
180 185 190
Ala Ala Asn Lys
195
<210> 12
<211> 101
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 12
Met Ala Ala Glu Val Lys Thr Val Ile Lys Pro Leu Gly Asp Arg Val
1 5 10 15
Val Val Lys Arg Ile Glu Glu Glu Pro Lys Thr Lys Gly Gly Ile Val
20 25 30
Leu Pro Asp Thr Ala Lys Glu Lys Pro Gln Lys Gly Lys Val Ile Ala
35 40 45
Val Gly Thr Gly Arg Val Leu Glu Asn Gly Gln Arg Val Pro Leu Glu
50 55 60
Val Lys Glu Gly Asp Ile Val Val Phe Ala Lys Tyr Gly Gly Thr Glu
65 70 75 80
Ile Glu Ile Asp Gly Glu Glu Tyr Val Ile Leu Ser Glu Arg Asp Leu
85 90 95
Leu Ala Val Leu Gln
100
<210> 13
<211> 7610
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
ctggcatttt gggcgttttc ggccttcatg gagcgcatgg agcgaaacta cctccgggac 60
cagagtggca tgagaaacca gcttctttgt ctggaccatt tggtccaatt tatgcttccc 120
tcactgtaca agcaccttga gaagaccgag tcaaccaatc tgtttttctt cttcagaatg 180
ctgctggtgt ggttcaagcg agagttgctc tgggatgacg ttttgcgtct gtgggaggtg 240
ttgtggacag attacctgtc gtcccaattt gttctatttg tgtgcctggc tatcctcgat 300
aagcacaagg acgtcatgat tgaccatctg gctgggtttg atgagattct gaagtacatg 360
aacgagctgt ccatgaccat cgatttggac gagcttcttg ttcgtgccga gctcttgttc 420
taccgattca gacgtacggt cgagcttatt gaccgaaaga acgaggacag acgcaactca 480
gcggacggct ccgagcctgt ttccatcaca gaggacctgc gggaattgtt atctcggaaa 540
gtcattgttg tgcgtgaggg tgagcgtcct gaaggcgtaa tgggtgggta ggtaatgcag 600
tttgcatgca tgaagacact aaacaagcca accatacagc agaagtatgt agccttgcac 660
atgatttatt gacaggccac ccaaacaggc gtatgtatag tactgtacct tcagtagact 720
attgtagcta acatgtcgtt gcgtggcgta tgtaccaagc cacagaaatt atgtcagaga 780
taaggtcgcg acagttagag cagcaacgcg tggagagttt gggttttggg ttacgtacgt 840
agagccgttt gatagatggt acatccaccg gctagcggaa cacagtgtca agacaagcct 900
gcaacacagt cataatattt gcgatattca ggcgtatcag gtacaatctg aggtgtctca 960
caagtgccgt gcagtcccgc ccccacttgc ttctctttgt gtgtagtgta cgtacattat 1020
cgagaccgtt gttcccgccc acctcgatcc ggggtcctat gcatccctga aacattgatt 1080
ggaaattaac atatgagctg cgtgcttttt gcattcaagg gcgcagctta tcttgtatcc 1140
ttaattacac atgacctctt gagcgccacg gtacattcct ggcgtcagtt cggtggagcg 1200
gacacttttc tctcctttgt ctgacatgtt ggttaagttg tagtccaggg acacaagggg 1260
ttccaacggc agtggcagcc taccccacgc tacccaccac tggccctggt ctaacttcga 1320
cgatcggcat cagggttcat ggataggcgg tgtgatttac gatgtgatgg acaatgttag 1380
agagatccca ctacttgtag tcaggccatc ttttacgtac gcactgtacc atgatgtcaa 1440
tggagtatga tgaaccgact ttgagagact cacatctgca caacaccatg tttcagcgga 1500
atccgacttc caacccaaac ccaagcccct gtcagatatc gtgagaaggc acggcaccaa 1560
ctaatgcaca cactccacct gtattgcacc aagataatga gggcatcgtc ttggcgcgtc 1620
ttggcgagag ccgtgtttcg tgacgcaatc agagcagttt ctggatagta tcttgtccag 1680
aaacacgata taaaccccat cgacgggccc gttgaagagc accaacccac tatccaatcc 1740
tccaatccaa caatgggtac tactttggat gatactgctt acagatacag aacttctgtt 1800
ccaggtgatg ctgaagctat tgaagctttg gatggttctt tcactactga cactgttttc 1860
agagttactg ctactggtga tggtttcact ttgagagaag ttccagttga tccaccattg 1920
actaaggttt ttccagatga tgaatccgat gacgaatctg atgatggtga agatggtgat 1980
ccagattcta gaacttttgt tgcttatggt gatgacggtg acttggctgg ttttgttgtt 2040
gtttcttatt ctggttggaa tagaagattg accgtcgaag atattgaagt tgctccagaa 2100
catagaggtc atggtgttgg tagagctttg atgggtttgg ctactgaatt tgctagagaa 2160
agaggtgctg gtcatttgtg gttggaagtt actaatgtta acgctccagc tattcatgcc 2220
tatagaagaa tgggttttac cttgtgtggt ttggatactg cattatacga tggtactgca 2280
tcagatggtg aacaagcctt gtatatgtct atgccatgtc catgacttct gttcggaatc 2340
aacctcaagg ttaacggcca cgatcccctc gttgttactc ttggtcagcc cattgtcggt 2400
aacgctggct ttgctaactg ggtcgataaa ctcttctttg gccaggagaa ccccgatgtc 2460
tccaaggtgt ccaaagaccg aaagctctac cgaatcaccc accgaggaga tatcgtccct 2520
caagtgccct tctgggacgg ttaccagcac tgctctggtg aggtctttat tgactggccc 2580
ctgatccacc ctcctctctc caacgttgtc atgtgccagg gccagagcaa taaacagtgc 2640
tctgccggta acactctgct ccagcaggtc aatgtgattg gaaaccatct gcagtacttc 2700
gtcaccgagg gtgtctgtgg tatctaagct atttatcact ctttacaact tctacctcaa 2760
ctatctactt taataaatga atatcgttta ttctctatga ttactgtata tgcgttcctc 2820
taagacaaat cgaaaccagc atgcgatcga atggcataca aaagtttctt ccgaagttga 2880
tcaatgtcct gatagtcagg cagcttgaga agattgacac aggtggaggc cgtagggaac 2940
cgatcaacct gtctaccagc gttacgaatg gcaaatgacg ggttcaaagc cttgaatcct 3000
tgcaatggtg ccttggatac tgatgtcaca aacttaagaa gcagccgctt gtcctcttcc 3060
tcgaaactct caaacacagt ccagaagtcc tttatagttt gatctgtatc cagatagcct 3120
ccgtaattgg tgtgtgtctt caaatcccag acgtccacat tggcatgtcc tccactgata 3180
agcatttgaa gttcatctgc gttgaacatt gagacccacg aagggtcaat gagctggtat 3240
agaccgccca agaatgcatc tgttcgtaga taatggaata caaatggata tccagagtat 3300
acacatggat agtatacact gacacgacaa ttctgtatct ctttatgtta actactgtga 3360
ggcgttaaat agagcttgat atataaaatg ttacatttca cagtctgaac ttttgcagat 3420
tacctaattt ggtaagatat taattatgaa ctgaaagttg atggcatccc taaatttgat 3480
gctggcagac cccgaaacct atgtgggggg gggttagaag caattggaga agaaacgttc 3540
agtaacttca aaacgagttt tcctctgtca aattttggat cagatcaagt gtcctatcag 3600
tctcaccctt gtgtttgcac ggtctgccaa gttctctcag atgatctacg ttcaactaat 3660
gagacagaaa gagcacgcag gcttgaacgc aggcttgaac gcatttggtt gagtcattgt 3720
acggcaactg gaagggctga gttgatctat ctgtcttctt cttcctcttc ttcttcaaat 3780
gacatcagca ctggagattc tctcctagct catggactga gactactatg ttctatggag 3840
ttgtgctctc aatccaacca tctttcacaa catcaaactc aagtccactg agaacatctc 3900
gctcgacttg tctctatctc aacacccagg gctatctgag ctggatcgtg gaggactggc 3960
tgttgtaagg agtttggcgc ccgttttttc gagccccaca cgtttcggtg agtatgagcg 4020
gcggcagatt cgagcgtttc cggtttccgc ggcgggacga gagcccatga tgggggctcc 4080
caccaccagc aatcagggcc ctgattacac acccacctgt aatgtcatgc tgttcatcgt 4140
ggttaatgct gctgtgtgct gtgtgtgtgt gttgtttggc gctcattgtt gcgttatgca 4200
gcgtacacca caatattgga agcttattag cctttctatt ttttcgtttg caaggcttaa 4260
caacattgct gtggagaggg atggggatat ggaggccgct ggagggagtc ggagaggcgt 4320
tttggagcgg cttggcctgg cgcccactcg cgaaacgcac ctaggaccct ttggcacgcc 4380
gaaatgtgcc acttttcagt ctagtaacgc cttacctacg tcattccatg catgcatgtt 4440
tgcgcctttt ttcccttgcc cttgatcgcc acacagtaca gtgcactgta cagtggaggt 4500
tttggggggg tcttagatgg gagctaaaag cggcctagcg gtacactagt gggattgtat 4560
ggagtggcat ggagcctggg tggagcctga caggacgcac gaccggctag cccgtgacag 4620
acgatgggtg gctcctgttg tccaccgcgt acaaatgttt gggccaaagt cttgtcagcc 4680
ttgcttgcga acctaattcc caattttgtc acttcgcacc cccattgatc gagccctaac 4740
ccctgcccat caggcaatcc aattaagctc gcattgtctg ccttgtttag tttggctcct 4800
gcccgtttcg gcgtccactt gcacaaacac aaacaagcat tatatataag gctcgtctct 4860
ccctcccaac cacactcact tttttgcccg tcttcccttg ctaacacaaa agtcaagaac 4920
acaaacaacc accccaaccc ccttacacac aagacatatc tacagcagcg atcgccccca 4980
cgttgccggt cttgcctcct actacctgtc catcaatgac gaggttctca cccctgccca 5040
ggtcgaggct cttattactg agtccaacac cggtgttctt cccaccacca acctcaaggg 5100
ctctcccaac gctgttgcct acaacggtgt tggcatttag gcaattaaca gatagtttgc 5160
cggtgataat tctcttaacc tcccacactc ctttgacata acgatttatg taacgaaact 5220
gaaatttgac cagatattgt tgtaaataga aaatctggct tgtaggtggc aaaatcccgt 5280
ctttgttcat caattccctc tgtgactact cgtcatccct ttatgttcga ctgtcgtatt 5340
tttattttcc atacatacgc aagtgagatg cccgtgtccg aattccgacg ctcggacacc 5400
tctggacaca agcactatcc tctgctgcga cgaactacac gcgaaagtcc actaatggct 5460
tttctattgc cagcgttctg ctagatacag gaggagggtt tctgtcaatg ggacagctgg 5520
ttttcgacgg tatcttcgac cacgacaggt cagggtatct acaagaatgt gcccaagttt 5580
ttgctttcct gggtcaccat cttcttcgac gtggttctca tgctcccgcg cgactggttc 5640
ccaggagcta cagtacccac atccaagatg gtcaagatgt gagtgaagag cctgtgtata 5700
aggcaagggg agcactggcg tattttgaat agattgagta gcatgaagag catagacggg 5760
ttggcccagt ttctctcgtt agccttctgt tgcaagtgaa cagcgcacga tcttccatgt 5820
tccggtgcac aagtagacac cctagacacg ccgaaaatat ctccgtctca atcaatgggc 5880
cttggtttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 5940
aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 6000
gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 6060
ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 6120
cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 6180
ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 6240
actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 6300
tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 6360
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 6420
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 6480
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 6540
ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt 6600
tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc 6660
agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc 6720
gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata 6780
ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg 6840
gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc 6900
cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct 6960
acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa 7020
cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt 7080
cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca 7140
ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac 7200
tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca 7260
atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt 7320
tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc 7380
actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca 7440
aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata 7500
ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc 7560
ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg 7610
Claims (10)
1. A method of constructing a recombinant yarrowia lipolytica comprising: the method comprises introducing a high temperature tolerance gene into yarrowia lipolytica to obtain the recombinant yarrowia lipolytica; the high temperature tolerance gene is Groes gene;
the Groes gene codes the protein of which the amino acid sequence is the sequence 12 in the sequence table.
2. The method according to claim 1, characterized in that: the Groes gene is a gene shown in the following a 1) or a 2):
a1 Coding sequence (CDS) of the coding strand is a cDNA molecule or a DNA molecule of sequence 6 in the sequence table,
a2 The nucleotide of the coding strand is a cDNA molecule or a DNA molecule of sequence 6 in the sequence table.
3. The method according to claim 1 or 2, characterized in that: the recombinant yarrowia lipolytica has higher erythritol yield than the yarrowia lipolytica under high temperature conditions.
4. A method according to claim 3, characterized in that: the high temperature was 35 ℃.
5. Recombinant yarrowia lipolytica constructed by the method of any one of claims 1-4.
6. Use of a high temperature tolerance gene, said high temperature tolerance gene being a Groes gene, in increasing erythritol production by yarrowia lipolytica under high temperature conditions or in increasing high temperature tolerance by yarrowia lipolytica;
the Groes gene codes the protein of which the amino acid sequence is the sequence 12 in the sequence table.
7. The application of a high-temperature tolerance gene in constructing high-temperature resistant recombinant yarrowia lipolytica, wherein the high-temperature tolerance gene is Groes gene;
the Groes gene codes the protein of which the amino acid sequence is the sequence 12 in the sequence table.
8. Use according to claim 6 or 7, characterized in that: the high temperature was 35 ℃.
9. Use of the method of any one of claims 1-4 or the recombinant yarrowia lipolytica of claim 5 in erythritol production.
10. The use according to claim 9, characterized in that: the erythritol is produced by producing erythritol at high temperature.
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CN107257851A (en) * | 2015-01-16 | 2017-10-17 | 法国农业科学研究院 | Positive influences are natural or combination of bacterial chaperonin of physiology of eukaryotic of engineering |
CN109971663A (en) * | 2019-04-28 | 2019-07-05 | 江南大学 | One plant of heat-resisting sub- sieve solution rouge yeast and its application |
CN113151341A (en) * | 2020-12-21 | 2021-07-23 | 江南大学 | Method for improving heat resistance of erythritol lipolysis lipolytica |
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CN107257851A (en) * | 2015-01-16 | 2017-10-17 | 法国农业科学研究院 | Positive influences are natural or combination of bacterial chaperonin of physiology of eukaryotic of engineering |
CN109971663A (en) * | 2019-04-28 | 2019-07-05 | 江南大学 | One plant of heat-resisting sub- sieve solution rouge yeast and its application |
CN113151341A (en) * | 2020-12-21 | 2021-07-23 | 江南大学 | Method for improving heat resistance of erythritol lipolysis lipolytica |
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