CN113462588A - Construction method of yarrowia lipolytica genetic engineering bacteria for producing citric acid or itaconic acid by using acetic acid - Google Patents

Construction method of yarrowia lipolytica genetic engineering bacteria for producing citric acid or itaconic acid by using acetic acid Download PDF

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CN113462588A
CN113462588A CN202110548580.8A CN202110548580A CN113462588A CN 113462588 A CN113462588 A CN 113462588A CN 202110548580 A CN202110548580 A CN 202110548580A CN 113462588 A CN113462588 A CN 113462588A
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yarrowia lipolytica
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citric acid
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邓利
萧琦
刘欢
刘军锋
王芳
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Beijing University of Chemical Technology
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Abstract

A construction method of yarrowia lipolytica genetic engineering bacteria for producing citric acid or itaconic acid by using acetic acid, belonging to the field of genetic engineering. The research discloses a yarrowia lipolytica genetic engineering bacterium for producing citric acid or itaconic acid, which is characterized in that a citrate synthase gene CAS, an acetyl coenzyme A synthase gene ACS, a mitochondrial carboxylic acid transporter gene MTT and a cis-aconitate decarboxylase gene CADA are introduced into leucine auxotrophic yarrowia lipolytica to construct yarrowia lipolytica genetic engineering bacterium YLA01 (containing genes CAS, ACS and MTT) and YLA02 (containing genes CAS, ACS, MTT and CADA) capable of metabolizing acetic acid. The yarrowia lipolytica genetic engineering bacteria constructed by the research can utilize acetic acid as a carbon source, efficiently produce citric acid or itaconic acid, and provide a good solution for acetic acid which is a waste in the fermentation industry.

Description

Construction method of yarrowia lipolytica genetic engineering bacteria for producing citric acid or itaconic acid by using acetic acid
Technical Field
The research belongs to the field of gene engineering, and particularly relates to yarrowia lipolytica gene engineering bacteria capable of producing citric acid or itaconic acid by using acetic acid and application thereof.
Background
Citric acid, which is a natural component of various citrus fruits, pineapples, pears, peaches and figs, is the most important food acidulant at present, and can improve the flavor of food, inhibit the growth of microorganisms and play a role in keeping food fresh, so the consumption of the citric acid in the food industry is always the highest among organic acids. Furthermore, citric acid may also be used as a chelating agent and a preferred additive for detergents, animal feeds, lubricants and plasticizers. Thus, the market for citric acid is in great demand and is on a continuing growth trend. Citric acid is mainly produced by chemical synthesis or fermentation of aspergillus niger, which generally uses glucose, starch and other food crops as fermentation substrates and acetic acid or acetate as a carbon source.
Itaconic acid, which contains a carbon-carbon double bond and two carboxyl groups with strong chemical activity in its molecule, can participate in numerous chemical reactions, has wide application in the polymer chemistry industry, and is used for synthesizing resins, plastics, rubbers, drugs, and the like. The market demand of itaconic acid is also huge, and fermentation of aspergillus terreus is a traditional itaconic acid production mode, but the production of itaconic acid by aspergillus terreus cannot be improved more than ever in decades, and with the development of molecular biology technology, the production of itaconic acid by means of metabolic engineering becomes the current mainstream research direction, and in the production of itaconic acid, the fermentation production process using acetic acid or acetate as a substrate is rarely involved.
Acetic acid, also known as acetic acid, is generally present as a byproduct in biosynthesis, is usually a metabolic waste, and contains a large amount of acetate in plant wastewater, so that the research on the utilization of acetic acid is a process of reusing waste in order to develop a new carbon source and follow the principle of green chemistry. In addition, during the process of converting acetic acid into citric acid, acetic acid can be easily converted into acetyl-coenzyme A, and then combined with oxaloacetate to form citric acid, and the carbon chain transfer is much shorter than that of the traditional carbon source such as glucose, so that the carbon source is favorable for producing citric acid.
Yarrowia lipolytica is a lipid yeast that converts most of the carbon atoms into fatty acids. It has been recognized as a safe (GRAS) organism for the food and health care industry to produce organic acids and natural products, with the ability to degrade a variety of substrates, including hexose/pentose sugars, glycerol, hydrocarbons, Volatile Fatty Acids (VFA), agricultural wastes, and even urea or urine, and can produce a variety of organic acids including citric acid, isocitric acid, alpha-ketoglutaric acid, and pyruvic acid, as a highly advantageous organic acid producing strain.
However, no report on the production of citric acid by yarrowia lipolytica using acetic acid as a carbon source has been found yet.
Disclosure of Invention
The research establishes the genetic engineering bacteria of the Yarrowia lipolytica and lays a foundation for further synthesizing the citric acid or the itaconic acid by utilizing the acetic acid or the acetate.
Therefore, the first objective of the present study is to overcome the deficiencies of the prior art and to provide a genetically engineered yarrowia lipolytica that can produce citric acid or itaconic acid using acetic acid.
The second purpose of the research is to provide a construction method of yarrowia lipolytica gene engineering bacteria for producing citric acid or itaconic acid by using acetic acid.
The third purpose of the research is to provide the application of yarrowia lipolytica gene engineering bacteria for producing citric acid or itaconic acid by using acetic acid.
In order to achieve the above purpose, the following technical solutions are adopted in the present research:
as the first aspect of the research, a yarrowia lipolytica genetic engineering bacterium for producing citric acid or itaconic acid, wherein a citrate synthase gene CAS, an acetyl coenzyme A synthase gene ACS and a mitochondrial carboxylic acid transporter gene MTT are introduced into leucine auxotrophic yarrowia lipolytica to construct yarrowia lipolytica genetic engineering bacterium YLA01 capable of metabolizing acetic acid; introducing citrate synthetase gene CAS, acetyl coenzyme A synthetase gene ACS, mitochondrial carboxylic acid transporter gene MTT and aconitic acid decarboxylase gene CADA into leucine auxotrophic yarrowia lipolytica to construct yarrowia lipolytica genetically engineered strain YLA02 capable of metabolizing acetic acid,
the nucleotide sequence of the citrate synthase gene CAS is shown as SEQ ID No.1, the nucleotide sequence of the acetyl coenzyme A synthase gene ACS is shown as SEQ ID No.2, the nucleotide sequence of the mitochondrial carboxylic acid transporter gene MTT is shown as SEQ ID No.3, and the nucleotide sequence of the aconitate decarboxylase gene CADA is shown as SEQ ID No. 4.
According to this study, the leucine auxotrophic yarrowia lipolytica is yarrowia lipolytica.
As a second aspect of the present study, a method for constructing yarrowia lipolytica genetically engineered bacterium producing citric acid and itaconic acid, comprising the steps of:
the method comprises the steps of firstly, connecting three genes of a citrate synthase gene CAS, an acetyl coenzyme A synthase gene ACS and a mitochondrial carboxylic acid transporter gene MTT to an expression plasmid PYLXP 'in sequence by means of homologous recombination and T4 connection to obtain a recombinant plasmid PYcam, and connecting the citrate synthase gene CAS, the acetyl coenzyme A synthase gene ACS, the mitochondrial carboxylic acid transporter gene MTT and an aconitate decarboxylase gene CADA to the expression plasmid PYLXP' by means of homologous recombination and T4 connection to obtain the recombinant plasmid PYcam. .
And step two, introducing the recombinant plasmid into yarrowia lipolytica by an acetic acid lithiation transformation method to construct strains YLA01(PYcam) and YLA02 (PYcam).
And (3) lithiation conversion method of acetic acid:
first, fresh yeast POIG was inoculated on YPD plates by plating and cultured at 30 ℃ for 24 hours for the purpose of activating the cells.
② single colony is selected, the plate is streaked into another clean YPD plate, and cultured for 20-22 h.
③ adding 90 mu L of PEG4000 with volume fraction of 50 percent, 5 mu L of salmon sperm single-stranded DNA and 5 mu L of lithium acetate (2mol/L) into a sterilized centrifuge tube in a super clean bench, and mixing evenly by vortex oscillation.
And fourthly, scraping the yeast cultured in the step 2 into a mixing system, wherein the amount of the yeast is not excessive, and the yeast can be uniformly mixed by shaking. Thus far it was thought that yeast competence was well established.
Fifthly, adding 800-1000ng recombinant plasmid into the cells, taking out the cells, uniformly mixing, placing the cells in warm water at 30 ℃ for 40min, taking out the cells every 600s, and carrying out vortex oscillation and uniform mixing.
Sixthly, the mixed transformation system is transferred to 39 ℃ and placed for 600 s.
Seventhly, taking out the mixture, adding 100 mu L of sterile water, and fully mixing.
And coating the mixed system on a leucine defect culture medium, and culturing for 3 days at the temperature of 30 ℃.
According to the research, the nucleotide sequence of the recombinant plasmid PYcam is shown as SEQ ID NO.5, and the nucleotide sequence of the recombinant plasmid PYcama is shown as SEQ ID NO. 6.
As a third aspect of the present study, the use of a yarrowia lipolytica genetically engineered bacterium capable of producing citric acid or itaconic acid as described above for producing citric acid and itaconic acid, wherein said genetically engineered bacterium is capable of producing citric acid or itaconic acid using acetic acid or acetate directly as a carbon source.
Further, the carbon source for culturing the yarrowia lipolytica genetic engineering bacterium capable of producing citric acid or itaconic acid is acetic acid, the concentration of the acetic acid is 5g/L-50g/L, and the concentration is the ratio of the mass of the acetic acid to the volume of a culture system. In the fermentation process, the pH of the fermentation liquor is adjusted once every 6 to 36 hours until the pH is between 3.0 and 9.0.
The construction method of the yarrowia lipolytica gene engineering bacterium for producing citric acid or itaconic acid has the following beneficial effects:
(1) according to the research, an acetic acid utilization pathway is firstly constructed in yarrowia lipolytica, so that the engineering strain can utilize the acetic acid to carry out biosynthesis of high value-added chemicals, and the process can provide a new carbon source pathway for production of citric acid, itaconic acid and related organic acids.
(2) The research provides a new, more environment-friendly and more economic utilization mode for acetic acid and acetate which are biological industrial byproducts or wastes.
Drawings
FIG. 1 shows the metabolic pathway of yarrowia lipolytica for the production of citric acid using acetate after the introduction of the three genes CAS, ACS, MTT.
FIG. 2 shows the metabolic pathway of yarrowia lipolytica for the production of citric acid from acetic acid after the introduction of the four genes CAS, ACS, MTT, CADA.
FIG. 3 is a graph showing the growth curve of strain YLA01 using acetic acid as a carbon source, the consumption curve of acetic acid, and the synthesis curve of citric acid.
FIG. 4 is a graph showing the growth curve of strain YLA01 using acetic acid as a carbon source, the consumption curve of acetic acid, and the synthesis curve of itaconic acid.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The study is further illustrated by the following specific examples, but the scope of the study is not limited thereto.
Although Yarrowia lipolytica was used as the starting strain in the present examples, a conventional uracil and leucine auxotrophic Yarrowia lipolytica (Yarrowia lipolytica) strain was used as the starting strain for transformation to perform the experiments in the examples.
Example 1 construction of genetically engineered yarrowia lipolytica for production of citric acid Using acetic acid as carbon Source
A. Construction of plasmid PYcam
(1) The citrate synthase CAS gene and the acetyl coenzyme A synthetase ACS gene are both from yarrowia lipolytica genome, and the acquisition mode is to design primers CAS-F, CAS-R and ACS-F, ACS-R, PCR amplifies corresponding citrate synthase CAS gene and acetyl coenzyme A synthetase ACS gene from the genome, and PCR amplifies corresponding citrate synthase CAS gene and acetyl coenzyme A synthetase gene ACS from Aspergillus terreus mitochondrial carboxylic acid transport protein gene MTT, and the primers are sequentially connected to a vector PYLXP' to obtain a recombinant plasmid, namely PYcam, the nucleotide sequence of which is shown as SEQ ID NO. 5.
Primer sequences used in Table 1
Figure BDA0003074509030000051
B. Yarrowia lipolytica transformation of plasmid PYcam
The transformation method of the plasmid is a lithium acetate yeast transformation method which is commonly used for yeast transformation.
The concrete mode is as follows:
first, fresh yeast POIG was inoculated on YPD plates by plating and cultured at 30 ℃ for 24 hours for the purpose of activating the cells.
② single colony is selected, the plate is streaked into another clean YPD plate, and cultured for 20-22 h.
③ adding 90 mu L of PEG4000 with volume fraction of 50 percent, 5 mu L of salmon sperm single-stranded DNA and 5 mu L of lithium acetate (2mol/L) into a sterilized centrifuge tube in a super clean bench, and mixing evenly by vortex oscillation.
And fourthly, scraping the yeast cultured in the step 2 into a mixing system, wherein the amount of the yeast is not excessive, and the yeast can be uniformly mixed by shaking. Thus far it was thought that yeast competence was well established.
Fifthly, adding 800-1000ng recombinant plasmid into the cells, taking out the cells, uniformly mixing, placing the cells in warm water at 30 ℃ for 40min, taking out the cells every 600s, and carrying out vortex oscillation and uniform mixing.
Sixthly, the mixed transformation system is transferred to 39 ℃ and placed for 600 s.
Seventhly, taking out the mixture, adding 100 mu L of sterile water, and fully mixing.
And coating the mixed system on a leucine defect culture medium, and culturing for 3 days at the temperature of 30 ℃.
Example 2 construction of yarrowia lipolytica Gene engineering bacteria for production of itaconic acid Using acetic acid as carbon Source
A. Construction of plasmid PYcama
(1) The citrate synthase CAS gene and the acetyl coenzyme A synthetase ACS gene are both from yarrowia lipolytica genomes, the acquisition mode is that primers CAS-F, CAS-R and ACS-F, ACS-R are designed, corresponding citrate synthase CAS gene and acetyl coenzyme A synthetase ACS gene are amplified from the genomes through PCR, the citrate synthase ACS gene is from Aspergillus terreus mitochondrial carboxylic acid transport protein gene MTT and aconitic acid decarboxylase gene CADA, the citrate synthase ACS gene and the ACS gene are sequentially connected to a vector PYLXP' to obtain a recombinant plasmid PYcama, the nucleotide sequence of the recombinant plasmid is shown as SEQ ID No.6, and the used sequences are shown as Table 1.
B. Yarrowia lipolytica transformation of plasmid PYcam
The transformation method of the plasmid is a lithium acetate yeast transformation method which is commonly used for yeast transformation.
The concrete mode is as follows:
first, fresh yeast POIG was inoculated on YPD plates by plating and cultured at 30 ℃ for 24 hours for the purpose of activating the cells.
② single colony is selected, the plate is streaked into another clean YPD plate, and cultured for 20-22 h.
③ adding 90 mu L of PEG4000 with volume fraction of 50 percent, 5 mu L of salmon sperm single-stranded DNA and 5 mu L of lithium acetate (2mol/L) into a sterilized centrifuge tube in a super clean bench, and mixing evenly by vortex oscillation.
And fourthly, scraping the yeast cultured in the step 2 into a mixing system, wherein the amount of the yeast is not excessive, and the yeast can be uniformly mixed by shaking. Thus far it was thought that yeast competence was well established.
Fifthly, adding 800-1000ng recombinant plasmid into the cells, taking out the cells, uniformly mixing, placing the cells in warm water at 30 ℃ for 40min, taking out the cells every 600s, and carrying out vortex oscillation and uniform mixing.
Sixthly, the mixed transformation system is transferred to 39 ℃ and placed for 600 s.
Seventhly, taking out the mixture, adding 100 mu L of sterile water, and fully mixing.
And coating the mixed system on a leucine defect culture medium, and culturing for 3 days at the temperature of 30 ℃.
Example 3 YLA01 Strain ability to ferment with acetic acid as carbon Source to produce citric acid
1. Experimental materials: strain YLA01
2. The experimental method comprises the following steps:
seed culture medium: leucine acetate-deficient medium, YNB1.5 g/L, acetic acid 23g/L, ammonium sulfate 4.6g/L, CSM-leu0.8 g/L. Sterilizing at 121 deg.C for 25 min.
Fermentation medium: leucine acetate deficient medium, YNB1.7 g/L, acetic acid 41g/L, ammonium sulfate 0.825g/L, CSM-leu 0.69 g/L. Sterilizing at 121 deg.C for 25 min.
Strain YLA01 was inoculated into 5mL of seed medium, cultured at 28 ℃ and 220rpm for 48 hours, 100. mu.L of seed solution was inoculated into 50mL of fermentation medium, cultured at 28 ℃ and 220rpm for 144 hours, and the concentration of citric acid and the residual amount of acetic acid in the fermentation broth were measured.
3. Method for detecting product
Citric acid is easily dissolved in water, so products are all enriched in fermentation liquor. And (5) detecting the concentration of the product in the fermentation liquor by using high performance liquid chromatography when the fermentation is finished.
4. Results of the experiment
After the strain YLA01 is fermented for 5 days, the yield and the biomass of the strain are plotted in figure 3, the biomass accumulation OD600 can reach 16, and the accumulation amount of citric acid can reach 12g/L, which shows that the expression genes ACS, ACS and MTT are helpful for the accumulation of citric acid on the basis of the expression CAS gene, and the growth of thalli cannot be obviously influenced.
Example 4YLA02 Strain ability to ferment with acetic acid as carbon Source to produce itaconic acid
1. Experimental materials: strain YLA02
2. The experimental method comprises the following steps:
seed culture medium: leucine acetate-deficient medium, YNB1.5 g/L, acetic acid 23g/L, ammonium sulfate 4.6g/L, CSM-leu0.8 g/L. Sterilizing at 121 deg.C for 25 min.
Fermentation medium: leucine acetate deficient medium, YNB1.7 g/L, acetic acid 41g/L, ammonium sulfate 0.825g/L, CSM-leu 0.69 g/L. Sterilizing at 121 deg.C for 25 min.
Strain YLA01 was inoculated into 5mL of seed medium, cultured at 28 ℃ and 220rpm for 48 hours, 100. mu.L of seed solution was inoculated into 50mL of fermentation medium, cultured at 28 ℃ and 220rpm for 144 hours, and the itaconic acid concentration and the residual amount of acetic acid in the fermentation broth were measured.
3. Method for detecting product
Itaconic acid can be dissolved in water, so the products are all enriched in the fermentation liquor. And (5) detecting the concentration of the product in the fermentation liquor by using high performance liquid chromatography when the fermentation is finished.
4. Results of the experiment
After the strain YLA02 is fermented for 5 days, the yield and the biomass are plotted in figure 4, the accumulated OD600 of the biomass can reach 16, and meanwhile, the accumulated amount of the itaconic acid can reach 1.2g/L, which shows that the production of the itaconic acid can be really carried out by utilizing the acetic acid.
The above description is only an embodiment of the present research, and it should be understood in advance that the above-mentioned modes are not to be taken as limitations for the implementation of the present research, and simple modifications and transformations for those skilled in the art are not complicated, so that the protection of the present research shall deviate from the content of the present statement and shall be protected by the present research under the principle of the present research.
Sequence listing
Beijing University of Chemical Technology
Construction method of yarrowia lipolytica genetic engineering bacteria for producing citric acid or itaconic acid by using acetic acid
SEQ ID NO.1
1709 bp
atgatttctg ctattcgtcc cgccgttcga tcttccgttc gtgttgccc ctatggcca acaccgcct tccgggcct actctaccc aggatgtga gtatttctttt ctttcatcaa ttggttgctgtgcgacggatttcgttgcgtcagcctgattgcaacagccttaggccccattttcgacctgttcttgcctcggcaaaagtttttccgaatgcatgtgacacgtcgaatgtggtgctttcaagcagcagcagcagcataaaatatggaatgtgttgtgtgcagaagtcgacattacataaccccgcggcaaccatacgagatggcagtcataacaattgcaattgagcaatacaaaccacactgcaacccactaaaaagaaacacgactaacaaatagggtcttaaggagcgattcgccgagctcatccccgagaacgtcgagaagatcaagaagctccgaaaggagaagggtaacaccgtcatcggcgaggtcatcctcgaccaggcttacggtggtatgcgaggtattaagggtctcgtctgggagggatccgtcctcgaccccgaggagggtatccgattccgaggtctgactatccccgacctccagaagcagctcccccacgcccctggcggaaaggagcctctccccgagggtcttttctggctcctgctcaccggcgagatccccactgatgctcaggtcaagggtctgtccgctgactgggcctctcgagccgagatccccaagcatgttgaggagctcatcgaccgatgcccccccaccctccaccccatggctcagctcggtattgccgtcaacgctctggagtccgagtctcagttcaccaaggcttacgagaagggtgttaacaagaaggagtactggcagtacacctacgaggattccatgaacctcattgccaagctccccgtcattgcttctcgaatctaccgaaaccttttcaaggacggaaagattgttggctccattgacaactctcttgactactctgctaacttcgcctctctgctcggctttggcgacaacaaggagttcattgagcttctgcgactctacctcaccatccacgctgaccacgagggaggtaacgtctctgcccacaccaccaagcttgttggttctgctctctcctctcccttcctctctctgtccgctggtctcaacggtcttgccggtcctctccacggccgagctaaccaggaggtccttgagtggattctcgagatgaagtccaagattggctctgatgtcaccaaggaggacattgagaagtacctctgggatacccttaaggccggtcgagtcgtccccggttacggacacgccgttctccgaaagaccgatcctcgatacaccgcccagcgagagttcgccctcgagcacatgcccgactacgacctcttccacctcgtttccaccatctacgaggttgcccccaaggttctcaccgagcacggcaagaccaagaacccctggcccaatgtggactcccactccggtgtcctcctccagtactacggtctcactgagcagtcttactacactgttctcttcggtgtttcccgagctatcggtgtcctgccccagctcatcatggaccgagcttacggtgctcccatcgagcgacccaagtccttctctaccgagaagtacgctgagctcgttggcctcaagctctaa
SEQ ID NO.2
1974 bp
atgtctgaagaccacccagccatccacccaccctccgagttcaaggacaaccacccccacttcggaggcccccacctcgactgtctgcaggactaccaccagctgcacaaggagtccattgaggaccccaaggccttctggaagaagatggccaacgagctcatctcctggtcaaccccctttgaaactgtgcgatctggcggcttcgagcacggcgacgtggcctggttccccgagggccagctcaacgcctcctacaactgtgtggatcgacacgcctttgccaaccccgacaagcccgccatcatttttgaggccgatgagccgggccagggccgaatcgtcacctacggcgaactgctgcgacaggtgtctcaggtcgcagccaccctgcgatccttcggcgtccagaagggcgatactgtggccgtctacctgcccatgatccccgaggccattgtcactctgctggccatcacccgaattggcgctgtccactcggtcatcttcgccggcttctcctccggttctctgcgagaccgaatcaacgacgccaagtccaaggttgtcgtcaccaccgacgcctccatgcgaggaggcaagaccatcgacaccaagaagattgtcgatgaagccttgcgagactgcccctctgttacccacaccctggtcttccgacgagcaggtgtcgagaacctggcctggactgagggccgggacttctggtggcacgaggaggtcgtcaagcaccgaccctaccttgcccccgtccccgttgcctccgaggaccccatcttcctgctttacacctctggatccaccggcacccccaagggtctggcccacgctaccggtggctacctgcttggtgctgccctgaccgccaagtacgtgtttgacatccacggagacgacaagctgttcaccgctggagacgttggctggatcaccggccacacctacgtgctctacggtcctctgatgctcggagccaccactgttgtgttcgagggaacccctgcctacccctccttctcgcgatactgggacattgtcgacgaccacaagatcacccacttctacgtggctcccaccgccctgcgtctcctgaagcgggccggcacccatcacattaagcacgacctgtcgtctctgcgaaccctcggctctgtgggtgagcccattgcccccgacgtgtggcagtggtacaacgacaacattggccgaggcaaggcccacatctgtgacacctactggcagaccgagactggctcgcatatcattgcccccatggccggcgtgacccccaccaagcccggttctgcttccctgcctgtctttggaattgatcccgttatcattgatcccgtgtctggcgaggagctcaagggtaacaacgttgagggtgttcttgccctgcgatctccctggccctccatggcccgaaccgtgtggaacacccacgagcgatacatggagacctacctgcggccctaccccggctactacttcaccggtgatggtgctgcccgagacaatgacggcttttactggatccgaggccgagtcgacgacgttgtcaacgtttctggccaccgtctttccaccgccgagattgaggctgctctcattgagcacgctcaggtgtctgagtctgccgttgttggtgtccatgacgatctgactggccaggccgtcaacgcctttgtggctctcaagaaccccgtcgaggatgtggacgctctgcgaaaggagcttgttgtgcaggtgcgaaagaccattggaccctttgctgctcccaagaatgtcatcatcgtggacgatctgcccaagactcggtctggcaagatcatgcgacgaattctgcgaaaggtgcttgctggcgaggaggaccagctcggagacatttccactcttgctaaccccgacgttgtccagaccatcattgaggttgttcactcgttgaaaaagtaa
SEQ ID NO.3
909 bp
Atggattccaagattcagaccaacgtccctctgcccaaggctcctctgactcagaaggcccgaggtaagcgaaccaaaggtattcccgctctggtggctggtgcttgtgctggtgccgtggagatctccatcacctacccctttgagtccgccaagacccgagcccagctgaagcgacgaaaccacgacgtcgctgctatccgacccggcattcgaggttggtacgctggttacggtgccactctggtcggcaccactctgaaggcctccgtccagttcgcctccttcaacatctatcgatccgccctcgccggtcccaacggtgaaatctctaccggcgcttctatgctggccggtttcggtgctggtgtcaccgaggctgtgctcgctgtcactcccgctgaggccatcaagaccaagatcatcgacgcccgaaaggtcggtaacgccgagctgtctaccaccttcggtgccatcgccggcattctccgagatcgaggccccctcggtttcttctctgccgtgggccccactattctccgacagtcctctaacgccgccgtcaagttcaccgtgtacaacgagctgatcggtctggcccgaaagtactcccacaacggtgaggacgtccatcctctggcctctactctggtgggttctgtcaccggtgtctgttgcgcttggtccacccagcctctcgacgtcatcaagacccgaatgcagtctctgcaagcccgacagctgtacggcaacacctttaactgcgtgaagactctgctccgatccgagggcatcggtgtgttctggtccggtgtctggttccgaaccggtagactgtctctgacctccgccatcatgttccccgtctacgagaaggtctacaagttcctcacccagcctaactaa
SEQ ID NO 4
1470 bp
acaaaacagagcgccgatagcaatgccaagagcggtgtgaccagcgaaatctgccactgggccagtaatctggccaccgacgatatccctagtgacgttctggagcgtgccaagtatctgattttagatggtattgcttgtgcttgggttggtgcccgtgtgccttggagcgaaaagtacgtgcaagctaccatgagctttgaaccgccgggcgcatgccgcgtgattggttatggtcagaaactgggcccggttgccgcagcaatgaccaacagcgccttcatccaagctaccgagctggatgactatcatagcgaagccccgctgcatagcgccagtattgttctgccggcagtgttcgcagccagcgaagttctggccgaacaaggtaaaaccatcagcggcattgatgttattctggccgccattgtgggcttcgaaagtggtccgcgcatcggtaaggcaatctatggcagcgatttactgaacaatggctggcactgtggtgcagtttacggtgcccccgctggtgccttagcaactggtaagctgctgggtttaacacccgatagcatggaagatgctttaggcatcgcatgcacccaagcatgtggtctgatgagcgcccagtacggtggcatggttaagcgcgttcagcatggtttcgccgcacgcaacggcttactgggtggcttactggcacatggtggttacgaggcaatgaagggcgttctggaacgcagctatggcggctttctgaaaatgttcaccaaaggcaatggccgcgaacctccgtataaagaagaagaggtggtggccggtctgggcagcttttggcacaccttcaccatccgcattaaactgtatgcttgttgtggtttagtgcatggtccggtggaagccatcgagaatttacaaggtcgctatccggagctgctgaaccgtgcaaatctgagtaacatccgccatgtgcacgtgcagctgagcacagccagcaatagccattgcggctggattcccgaagaacgtccgattagtagcattgctggtcagatgagcgttgcatatattttagccgtgcagctggtggatcagcagtgtttactgagccagttcagcgagtttgatgacaatttagaacgcccggaggtttgggacttagcccgtaaagtgacaagcagtcagagcgaggagtttgatcaagatggcaactgtctgagcgctggtcgtgttcgcattgagttcaacgatggcagcagcatcaccgaaagcgttgaaaagccgctgggcgttaaggaaccgatgccgaacgaacgcattttacataagtatcgcactttagccggcagcgttacagacgaaagccgcgtgaaggagattgaggatttagtgctgggcttagaccgtttaaccgatattagcccgctgttagagctgctgaattgcccggttaagagtccgctggtgtga
SEQ ID NO 5
14015 bp
cgatgcttttcgtagataatggaatacaaatggatatccagagtatacacatggatagtatacactgacacgacaattctgtatctctttatgttaactactgtgaggcgttaaatagagcttgatatataaaatgttacatttcacagtctgaacttttgcagattacctaatttggtaagatattaattatgaactgaaagttgatggcatccctaaatttgatgaaagcctagggacgacagagaccgggttggcggcgcatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgacccagtagcgggcccaaccccggcgagagcccccttctccccacatatcaaacctcccccggttcccacacttgccgttaagggcgtagggtactgcagtctggaatctacgcttgttcagactttgtacttgtttctttgtctggccatccgggtaacccatgccggacgcaaaatagactactgaaaatttttttgctttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccgaaatcgttaagcatttccttctgagtataagaatcattcaaatctagaatggtgagtttcagaggcagcagcaattgccacgggctttgagcacacggccgggtgtggtcccattcccatcgacacaagacgccacgtcatccgaccagcactttttgcagtactaaccgcagatttctgctattcgtcccgccgttcgatcttccgttcgtgttgcccctatggccaacaccgccttccgggcctactctacccaggatgtgagtatttcttttctttcatcaattggttgctgtgcgacggatttcgttgcgtcagcctgattgcaacagccttaggccccattttcgacctgttcttgcctcggcaaaagtttttccgaatgcatgtgacacgtcgaatgtggtgctttcaagcagcagcagcagcataaaatatggaatgtgttgtgtgcagaagtcgacattacataaccccgcggcaaccatacgagatggcagtcataacaattgcaattgagcaatacaaaccacactgcaacccactaaaaagaaacacgactaacaaatagggtcttaaggagcgattcgccgagctcatccccgagaacgtcgagaagatcaagaagctccgaaaggagaagggtaacaccgtcatcggcgaggtcatcctcgaccaggcttacggtggtatgcgaggtattaagggtctcgtctgggagggatccgtcctcgaccccgaggagggtatccgattccgaggtctgactatccccgacctccagaagcagctcccccacgcccctggcggaaaggagcctctccccgagggtcttttctggctcctgctcaccggcgagatccccactgatgctcaggtcaagggtctgtccgctgactgggcctctcgagccgagatccccaagcatgttgaggagctcatcgaccgatgcccccccaccctccaccccatggctcagctcggtattgccgtcaacgctctggagtccgagtctcagttcaccaaggcttacgagaagggtgttaacaagaaggagtactggcagtacacctacgaggattccatgaacctcattgccaagctccccgtcattgcttctcgaatctaccgaaaccttttcaaggacggaaagattgttggctccattgacaactctcttgactactctgctaacttcgcctctctgctcggctttggcgacaacaaggagttcattgagcttctgcgactctacctcaccatccacgctgaccacgagggaggtaacgtctctgcccacaccaccaagcttgttggttctgctctctcctctcccttcctctctctgtccgctggtctcaacggtcttgccggtcctctccacggccgagctaaccaggaggtccttgagtggattctcgagatgaagtccaagattggctctgatgtcaccaaggaggacattgagaagtacctctgggatacccttaaggccggtcgagtcgtccccggttacggacacgccgttctccgaaagaccgatcctcgatacaccgcccagcgagagttcgccctcgagcacatgcccgactacgacctcttccacctcgtttccaccatctacgaggttgcccccaaggttctcaccgagcacggcaagaccaagaacccctggcccaatgtggactcccactccggtgtcctcctccagtactacggtctcactgagcagtcttactacactgttctcttcggtgtttcccgagctatcggtgtcctgccccagctcatcatggaccgagcttacggtgctcccatcgagcgacccaagtccttctctaccgagaagtacgctgagctcgttggcctcaagctctaaggtaccgactagttccatggcctgtccccacgttgccggtcttgcctcctactacctgtccatcaatgacgaggttctcacccctgcccaggtcgaggctcttattactgagtccaacaccggtgttcttcccaccaccaacctcaagggctctcccaacgctgttgcctacaacggtgttggcatttaggcaattaacagatagtttgccggtgataattctcttaacctcccacactcctttgacataacgatttatgtaacgaaactgaaatttgaccagatattgttgtaaatagaaaatctggcttgtaggtggcaaaatgcggcgtctttgttcatcaattccctctgtgactactcgtcatccctttatgttcgactgtcgtatttcttattttccatacatatgcaagtgagatgcccgtgtccgttatcaaatctagttagctagggacgacagagaccgggttggcggcgcatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgacccagtagcgggcccaaccccggcgagagcccccttctccccacatatcaaacctcccccggttcccacacttgccgttaagggcgtagggtactgcagtctggaatctacgcttgttcagactttgtacttgtttctttgtctggccatccgggtaacccatgccggacgcaaaatagactactgaaaatttttttgctttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccgaaatcgttaagcatttccttctgagtataagaatcattcaaatctagaatggtgagtttcagaggcagcagcaattgccacgggctttgagcacacggccgggtgtggtcccattcccatcgacacaagacgccacgtcatccgaccagcactttttgcagtactaaccgcagtctgaagaccacccagccatccacccaccctccgagttcaaggacaaccacccccacttcggaggcccccacctcgactgtctgcaggactaccaccagctgcacaaggagtccattgaggaccccaaggccttctggaagaagatggccaacgagctcatctcctggtcaaccccctttgaaactgtgcgatctggcggcttcgagcacggcgacgtggcctggttccccgagggccagctcaacgcctcctacaactgtgtggatcgacacgcctttgccaaccccgacaagcccgccatcatttttgaggccgatgagccgggccagggccgaatcgtcacctacggcgaactgctgcgacaggtgtctcaggtcgcagccaccctgcgatccttcggcgtccagaagggcgatactgtggccgtctacctgcccatgatccccgaggccattgtcactctgctggccatcacccgaattggcgctgtccactcggtcatcttcgccggcttctcctccggttctctgcgagaccgaatcaacgacgccaagtccaaggttgtcgtcaccaccgacgcctccatgcgaggaggcaagaccatcgacaccaagaagattgtcgatgaagccttgcgagactgcccctctgttacccacaccctggtcttccgacgagcaggtgtcgagaacctggcctggactgagggccgggacttctggtggcacgaggaggtcgtcaagcaccgaccctaccttgcccccgtccccgttgcctccgaggaccccatcttcctgctttacacctctggatccaccggcacccccaagggtctggcccacgctaccggtggctacctgcttggtgctgccctgaccgccaagtacgtgtttgacatccacggagacgacaagctgttcaccgctggagacgttggctggatcaccggccacacctacgtgctctacggtcctctgatgctcggagccaccactgttgtgttcgagggaacccctgcctacccctccttctcgcgatactgggacattgtcgacgaccacaagatcacccacttctacgtggctcccaccgccctgcgtctcctgaagcgggccggcacccatcacattaagcacgacctgtcgtctctgcgaaccctcggctctgtgggtgagcccattgcccccgacgtgtggcagtggtacaacgacaacattggccgaggcaaggcccacatctgtgacacctactggcagaccgagactggctcgcatatcattgcccccatggccggcgtgacccccaccaagcccggttctgcttccctgcctgtctttggaattgatcccgttatcattgatcccgtgtctggcgaggagctcaagggtaacaacgttgagggtgttcttgccctgcgatctccctggccctccatggcccgaaccgtgtggaacacccacgagcgatacatggagacctacctgcggccctaccccggctactacttcaccggtgatggtgctgcccgagacaatgacggcttttactggatccgaggccgagtcgacgacgttgtcaacgtttctggccaccgtctttccaccgccgagattgaggctgctctcattgagcacgctcaggtgtctgagtctgccgttgttggtgtccatgacgatctgactggccaggccgtcaacgcctttgtggctctcaagaaccccgtcgaggatgtggacgctctgcgaaaggagcttgttgtgcaggtgcgaaagaccattggaccctttgctgctcccaagaatgtcatcatcgtggacgatctgcccaagactcggtctggcaagatcatgcgacgaattctgcgaaaggtgcttgctggcgaggaggaccagctcggagacatttccactcttgctaaccccgacgttgtccagaccatcattgaggttgttcactcgttgaaaaagtaaggtaccgactagttccatggcctgtccccacgttgccggtcttgcctcctactacctgtccatcaatgacgaggttctcacccctgcccaggtcgaggctcttattactgagtccaacaccggtgttcttcccaccaccaacctcaagggctctcccaacgctgttgcctacaacggtgttggcatttaggcaattaacagatagtttgccggtgataattctcttaacctcccacactcctttgacataacgatttatgtaacgaaactgaaatttgaccagatattgttgtaaatagaaaatctggcttgtaggtggcaaaatgcggcgtctttgttcatcaattccctctgtgactactcgtcatccctttatgttcgactgtcgtatttcttattttccatacatatgcaagtgagatgcccgtgtccgttatcaaatctagttagctagggacgacagagaccgggttggcggcgcatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgacccagtagcgggcccaaccccggcgagagcccccttctccccacatatcaaacctcccccggttcccacacttgccgttaagggcgtagggtactgcagtctggaatctacgcttgttcagactttgtacttgtttctttgtctggccatccgggtaacccatgccggacgcaaaatagactactgaaaatttttttgctttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccgaaatcgttaagcatttccttctgagtataagaatcattcaaatctagaatggtgagtttcagaggcagcagcaattgccacgggctttgagcacacggccgggtgtggtcccattcccatcgacacaagacgccacgtcatccgaccagcactttttgcagtactaaccgcaggattccaagattcagaccaacgtccctctgcccaaggctcctctgactcagaaggcccgaggtaagcgaaccaaaggtattcccgctctggtggctggtgcttgtgctggtgccgtggagatctccatcacctacccctttgagtccgccaagacccgagcccagctgaagcgacgaaaccacgacgtcgctgctatccgacccggcattcgaggttggtacgctggttacggtgccactctggtcggcaccactctgaaggcctccgtccagttcgcctccttcaacatctatcgatccgccctcgccggtcccaacggtgaaatctctaccggcgcttctatgctggccggtttcggtgctggtgtcaccgaggctgtgctcgctgtcactcccgctgaggccatcaagaccaagatcatcgacgcccgaaaggtcggtaacgccgagctgtctaccaccttcggtgccatcgccggcattctccgagatcgaggccccctcggtttcttctctgccgtgggccccactattctccgacagtcctctaacgccgccgtcaagttcaccgtgtacaacgagctgatcggtctggcccgaaagtactcccacaacggtgaggacgtccatcctctggcctctactctggtgggttctgtcaccggtgtctgttgcgcttggtccacccagcctctcgacgtcatcaagacccgaatgcagtctctgcaagcccgacagctgtacggcaacacctttaactgcgtgaagactctgctccgatccgagggcatcggtgtgttctggtccggtgtctggttccgaaccggtagactgtctctgacctccgccatcatgttccccgtctacgagaaggtctacaagttcctcacccagcctaactaaggtaccgactagttccatggcctgtccccacgttgccggtcttgcctcctactacctgtccatcaatgacgaggttctcacccctgcccaggtcgaggctcttattactgagtccaacaccggtgttcttcccaccaccaacctcaagggctctcccaacgctgttgcctacaacggtgttggcatttaggcaattaacagatagtttgccggtgataattctcttaacctcccacactcctttgacataacgatttatgtaacgaaactgaaatttgaccagatattgttgtaaatagaaaatctggcttgtaggtggcaaaatgcggcgtctttgttcatcaattccctctgtgactactcgtcatccctttatgttcgactgtcgtatttcttattttccatacatatgcaagtgagatgcccgtgtccgttatcaaatctagttagctagcgagacaataacggaggagtcgactatgtctgataaaaggatgtaacataggcaagctgctcgtgagtgttgagtacgaaccttagatccaaatcacccgcacccacggatatacttgcttgaatatacagtagtatgctcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctctgggtcattttcggcgaggaccgctttcgctggagcgcgacgatgatcggcctgtcgcttgcggtattcggaatcttgcacgccctcgctcaagccttcgtcactggtcccgccaccaaacgtttcggcgagaagcaggccattatcgccggcatggcggccgacgcgctgggctacgtcttgctggcgttcgcgacgcgaggctggatggccttccccattatgattcttctcgcttccggcggcatcgggatgcccgcgttgcaggccatgctgtccaggcaggtagatgacgaccatcagggacagcttcaaggatcgctcgcggctcttaccagcctaacttcgatcactggaccgctgatcgtcacggcgatttatgccgcctcggcgagcacatggaacgggttggcatggattgtaggcgccgccctataccttgtctgcctccccgcgttgcgtcgcggtgcatggagccgggccacctcgacctgaatggaagccggcggcacctcgctaacggattcaccactccaagaattggagccaatcaattcttgcggagaactgtgaatgcgcaaaccaacccttggcagaacatatccatcgcgtccgccatctccagcagccgcacgcggcgcatctcgggcagcgttgggtcctggccacgggtgcgcatgatcgtgctcctgtcgttgaggacccggctaggctggcggggttgccttactggttagcagaatgaatcaccgatacgcgagcgaacgtgaagcgactgctgctgcaaaacgtctgcgacctgagcaacaacatgaatggtcttcggtttccgtgtttcgtaaagtctggaaacgcggaagtcagcgccctgcaccattatgttccggatctgcatcgcaggatgctgctggctaccctgtggaacacctacatctgtattaacgaagcgctggcattgaccctgagtgatttttctctggtcccgccgcatccataccgccagttgtttaccctcacaacgttccagtaaccgggcatgttcatcatcagtaacccgtatcgtgagcatcctctctcgtttcatcggtatcattacccccatgaacagaaatcccccttacacggaggcatcagtgaccaaacaggaaaaaaccgcccttaacatggcccgctttatcagaagccagacattaacgcttctggagaaactcaacgagctggacgcggatgaacaggcagacatctgtgaatcgcttcacgaccacgctgatgagctttaccgcagcagatccgcggccgcataggccaatagtggatctgctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccga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SEQ ID NO 6
16483 bp
cgatgcttttcgtagataatggaatacaaatggatatccagagtatacacatggatagtatacactgacacgacaattctgtatctctttatgttaactactgtgaggcgttaaatagagcttgatatataaaatgttacatttcacagtctgaacttttgcagattacctaatttggtaagatattaattatgaactgaaagttgatggcatccctaaatttgatgaaagcctagggacgacagagaccgggttggcggcgcatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgacccagtagcgggcccaaccccggcgagagcccccttctccccacatatcaaacctcccccggttcccacacttgccgttaagggcgtagggtactgcagtctggaatctacgcttgttcagactttgtacttgtttctttgtctggccatccgggtaacccatgccggacgcaaaatagactactgaaaatttttttgctttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccgaaatcgttaagcatttccttctgagtataagaatcattcaaatctagaatggtgagtttcagaggcagcagcaattgccacgggctttgagcacacggccgggtgtggtcccattcccatcgacacaagacgccacgtcatccgaccagcactttttgcagtactaaccgcagatttctgctattcgtcccgccgttcgatcttccgttcgtgttgcccctatggccaacaccgccttccgggcctactctacccaggatgtgagtatttcttttctttcatcaattggttgctgtgcgacggatttcgttgcgtcagcctgattgcaacagccttaggccccattttcgacctgttcttgcctcggcaaaagtttttccgaatgcatgtgacacgtcgaatgtggtgctttcaagcagcagcagcagcataaaatatggaatgtgttgtgtgcagaagtcgacattacataaccccgcggcaaccatacgagatggcagtcataacaattgcaattgagcaatacaaaccacactgcaacccactaaaaagaaacacgactaacaaatagggtcttaaggagcgattcgccgagctcatccccgagaacgtcgagaagatcaagaagctccgaaaggagaagggtaacaccgtcatcggcgaggtcatcctcgaccaggcttacggtggtatgcgaggtattaagggtctcgtctgggagggatccgtcctcgaccccgaggagggtatccgattccgaggtctgactatccccgacctccagaagcagctcccccacgcccctggcggaaaggagcctctccccgagggtcttttctggctcctgctcaccggcgagatccccactgatgctcaggtcaagggtctgtccgctgactgggcctctcgagccgagatccccaagcatgttgaggagctcatcgaccgatgcccccccaccctccaccccatggctcagctcggtattgccgtcaacgctctggagtccgagtctcagttcaccaaggcttacgagaagggtgttaacaagaaggagtactggcagtacacctacgaggattccatgaacctcattgccaagctccccgtcattgcttctcgaatctaccgaaaccttttcaaggacggaaagattgttggctccattgacaactctcttgactactctgctaacttcgcctctctgctcggctttggcgacaacaaggagttcattgagcttctgcgactctacctcaccatccacgctgaccacgagggaggtaacgtctctgcccacaccaccaagcttgttggttctgctctctcctctcccttcctctctctgtccgctggtctcaacggtcttgccggtcctctccacggccgagctaaccaggaggtccttgagtggattctcgagatgaagtccaagattggctctgatgtcaccaaggaggacattgagaagtacctctgggatacccttaaggccggtcgagtcgtccccggttacggacacgccgttctccgaaagaccgatcctcgatacaccgcccagcgagagttcgccctcgagcacatgcccgactacgacctcttccacctcgtttccaccatctacgaggttgcccccaaggttctcaccgagcacggcaagaccaagaacccctggcccaatgtggactcccactccggtgtcctcctccagtactacggtctcactgagcagtcttactacactgttctcttcggtgtttcccgagctatcggtgtcctgccccagctcatcatggaccgagcttacggtgctcccatcgagcgacccaagtccttctctaccgagaagtacgctgagctcgttggcctcaagctctaaggtaccgactagttccatggcctgtccccacgttgccggtcttgcctcctactacctgtccatcaatgacgaggttctcacccctgcccaggtcgaggctcttattactgagtccaacaccggtgttcttcccaccaccaacctcaagggctctcccaacgctgttgcctacaacggtgttggcatttaggcaattaacagatagtttgccggtgataattctcttaacctcccacactcctttgacataacgatttatgtaacgaaactgaaatttgaccagatattgttgtaaatagaaaatctggcttgtaggtggcaaaatgcggcgtctttgttcatcaattccctctgtgactactcgtcatccctttatgttcgactgtcgtatttcttattttccatacatatgcaagtgagatgcccgtgtccgttatcaaatctagttagctagggacgacagagaccgggttggcggcgcatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgacccagtagcgggcccaaccccggcgagagcccccttctccccacatatcaaacctcccccggttcccacacttgccgttaagggcgtagggtactgcagtctggaatctacgcttgttcagactttgtacttgtttctttgtctggccatccgggtaacccatgccggacgcaaaatagactactgaaaatttttttgctttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccgaaatcgttaagcatttccttctgagtataagaatcattcaaatctagaatggtgagtttcagaggcagcagcaattgccacgggctttgagcacacggccgggtgtggtcccattcccatcgacacaagacgccacgtcatccgaccagcactttttgcagtactaaccgcagtctgaagaccacccagccatccacccaccctccgagttcaaggacaaccacccccacttcggaggcccccacctcgactgtctgcaggactaccaccagctgcacaaggagtccattgaggaccccaaggccttctggaagaagatggccaacgagctcatctcctggtcaaccccctttgaaactgtgcgatctggcggcttcgagcacggcgacgtggcctggttccccgagggccagctcaacgcctcctacaactgtgtggatcgacacgcctttgccaaccccgacaagcccgccatcatttttgaggccgatgagccgggccagggccgaatcgtcacctacggcgaactgctgcgacaggtgtctcaggtcgcagccaccctgcgatccttcggcgtccagaagggcgatactgtggccgtctacctgcccatgatccccgaggccattgtcactctgctggccatcacccgaattggcgctgtccactcggtcatcttcgccggcttctcctccggttctctgcgagaccgaatcaacgacgccaagtccaaggttgtcgtcaccaccgacgcctccatgcgaggaggcaagaccatcgacaccaagaagattgtcgatgaagccttgcgagactgcccctctgttacccacaccctggtcttccgacgagcaggtgtcgagaacctggcctggactgagggccgggacttctggtggcacgaggaggtcgtcaagcaccgaccctaccttgcccccgtccccgttgcctccgaggaccccatcttcctgctttacacctctggatccaccggcacccccaagggtctggcccacgctaccggtggctacctgcttggtgctgccctgaccgccaagtacgtgtttgacatccacggagacgacaagctgttcaccgctggagacgttggctggatcaccggccacacctacgtgctctacggtcctctgatgctcggagccaccactgttgtgttcgagggaacccctgcctacccctccttctcgcgatactgggacattgtcgacgaccacaagatcacccacttctacgtggctcccaccgccctgcgtctcctgaagcgggccggcacccatcacattaagcacgacctgtcgtctctgcgaaccctcggctctgtgggtgagcccattgcccccgacgtgtggcagtggtacaacgacaacattggccgaggcaaggcccacatctgtgacacctactggcagaccgagactggctcgcatatcattgcccccatggccggcgtgacccccaccaagcccggttctgcttccctgcctgtctttggaattgatcccgttatcattgatcccgtgtctggcgaggagctcaagggtaacaacgttgagggtgttcttgccctgcgatctccctggccctccatggcccgaaccgtgtggaacacccacgagcgatacatggagacctacctgcggccctaccccggctactacttcaccggtgatggtgctgcccgagacaatgacggcttttactggatccgaggccgagtcgacgacgttgtcaacgtttctggccaccgtctttccaccgccgagattgaggctgctctcattgagcacgctcaggtgtctgagtctgccgttgttggtgtccatgacgatctgactggccaggccgtcaacgcctttgtggctctcaagaaccccgtcgaggatgtggacgctctgcgaaaggagcttgttgtgcaggtgcgaaagaccattggaccctttgctgctcccaagaatgtcatcatcgtggacgatctgcccaagactcggtctggcaagatcatgcgacgaattctgcgaaaggtgcttgctggcgaggaggaccagctcggagacatttccactcttgctaaccccgacgttgtccagaccatcattgaggttgttcactcgttgaaaaagtaaggtaccgactagttccatggcctgtccccacgttgccggtcttgcctcctactacctgtccatcaatgacgaggttctcacccctgcccaggtcgaggctcttattactgagtccaacaccggtgttcttcccaccaccaacctcaagggctctcccaacgctgttgcctacaacggtgttggcatttaggcaattaacagatagtttgccggtgataattctcttaacctcccacactcctttgacataacgatttatgtaacgaaactgaaatttgaccagatattgttgtaaatagaaaatctggcttgtaggtggcaaaatgcggcgtctttgttcatcaattccctctgtgactactcgtcatccctttatgttcgactgtcgtatttcttattttccatacatatgcaagtgagatgcccgtgtccgttatcaaatctagttagctagggacgacagagaccgggttggcggcgcatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgacccagtagcgggcccaaccccggcgagagcccccttctccccacatatcaaacctcccccggttcccacacttgccgttaagggcgtagggtactgcagtctggaatctacgcttgttcagactttgtacttgtttctttgtctggccatccgggtaacccatgccggacgcaaaatagactactgaaaatttttttgctttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccgaaatcgttaagcatttccttctgagtataagaatcattcaaatctagaatggtgagtttcagaggcagcagcaattgccacgggctttgagcacacggccgggtgtggtcccattcccatcgacacaagacgccacgtcatccgaccagcactttttgcagtactaaccgcaggattccaagattcagaccaacgtccctctgcccaaggctcctctgactcagaaggcccgaggtaagcgaaccaaaggtattcccgctctggtggctggtgcttgtgctggtgccgtggagatctccatcacctacccctttgagtccgccaagacccgagcccagctgaagcgacgaaaccacgacgtcgctgctatccgacccggcattcgaggttggtacgctggttacggtgccactctggtcggcaccactctgaaggcctccgtccagttcgcctccttcaacatctatcgatccgccctcgccggtcccaacggtgaaatctctaccggcgcttctatgctggccggtttcggtgctggtgtcaccgaggctgtgctcgctgtcactcccgctgaggccatcaagaccaagatcatcgacgcccgaaaggtcggtaacgccgagctgtctaccaccttcggtgccatcgccggcattctccgagatcgaggccccctcggtttcttctctgccgtgggccccactattctccgacagtcctctaacgccgccgtcaagttcaccgtgtacaacgagctgatcggtctggcccgaaagtactcccacaacggtgaggacgtccatcctctggcctctactctggtgggttctgtcaccggtgtctgttgcgcttggtccacccagcctctcgacgtcatcaagacccgaatgcagtctctgcaagcccgacagctgtacggcaacacctttaactgcgtgaagactctgctccgatccgagggcatcggtgtgttctggtccggtgtctggttccgaaccggtagactgtctctgacctccgccatcatgttccccgtctacgagaaggtctacaagttcctcacccagcctaactaaggtaccgactagttccatggcctgtccccacgttgccggtcttgcctcctactacctgtccatcaatgacgaggttctcacccctgcccaggtcgaggctcttattactgagtccaacaccggtgttcttcccaccaccaacctcaagggctctcccaacgctgttgcctacaacggtgttggcatttaggcaattaacagatagtttgccggtgataattctcttaacctcccacactcctttgacataacgatttatgtaacgaaactgaaatttgaccagatattgttgtaaatagaaaatctggcttgtaggtggcaaaatgcggcgtctttgttcatcaattccctctgtgactactcgtcatccctttatgttcgactgtcgtatttcttattttccatacatatgcaagtgagatgcccgtgtccgttatcaaatctagttagctagggacgacagagaccgggttggcggcgcatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgacccagtagcgggcccaaccccggcgagagcccccttctccccacatatcaaacctcccccggttcccacacttgccgttaagggcgtagggtactgcagtctggaatctacgcttgttcagactttgtacttgtttctttgtctggccatccgggtaacccatgccggacgcaaaatagactactgaaaatttttttgctttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccgaaatcgttaagcatttccttctgagtataagaatcattcaaatctagaatggtgagtttcagaggcagcagcaattgccacgggctttgagcacacggccgggtgtggtcccattcccatcgacacaagacgccacgtcatccgaccagcactttttgcagtactaaccgcagacaaaacagagcgccgatagcaatgccaagagcggtgtgaccagcgaaatctgccactgggccagtaatctggccaccgacgatatccctagtgacgttctggagcgtgccaagtatctgattttagatggtattgcttgtgcttgggttggtgcccgtgtgccttggagcgaaaagtacgtgcaagctaccatgagctttgaaccgccgggcgcatgccgcgtgattggttatggtcagaaactgggcccggttgccgcagcaatgaccaacagcgccttcatccaagctaccgagctggatgactatcatagcgaagccccgctgcatagcgccagtattgttctgccggcagtgttcgcagccagcgaagttctggccgaacaaggtaaaaccatcagcggcattgatgttattctggccgccattgtgggcttcgaaagtggtccgcgcatcggtaaggcaatctatggcagcgatttactgaacaatggctggcactgtggtgcagtttacggtgcccccgctggtgccttagcaactggtaagctgctgggtttaacacccgatagcatggaagatgctttaggcatcgcatgcacccaagcatgtggtctgatgagcgcccagtacggtggcatggttaagcgcgttcagcatggtttcgccgcacgcaacggcttactgggtggcttactggcacatggtggttacgaggcaatgaagggcgttctggaacgcagctatggcggctttctgaaaatgttcaccaaaggcaatggccgcgaacctccgtataaagaagaagaggtggtggccggtctgggcagcttttggcacaccttcaccatccgcattaaactgtatgcttgttgtggtttagtgcatggtccggtggaagccatcgagaatttacaaggtcgctatccggagctgctgaaccgtgcaaatctgagtaacatccgccatgtgcacgtgcagctgagcacagccagcaatagccattgcggctggattcccgaagaacgtccgattagtagcattgctggtcagatgagcgttgcatatattttagccgtgcagctggtggatcagcagtgtttactgagccagttcagcgagtttgatgacaatttagaacgcccggaggtttgggacttagcccgtaaagtgacaagcagtcagagcgaggagtttgatcaagatggcaactgtctgagcgctggtcgtgttcgcattgagttcaacgatggcagcagcatcaccgaaagcgttgaaaagccgctgggcgttaaggaaccgatgccgaacgaacgcattttacataagtatcgcactttagccggcagcgttacagacgaaagccgcgtgaaggagattgaggatttagtgctgggcttagaccgtttaaccgatattagcccgctgttagagctgctgaattgcccggttaagagtccgctggtgtgaggtaccgactagttccatggcctgtccccacgttgccggtcttgcctcctactacctgtccatcaatgacgaggttctcacccctgcccaggtcgaggctcttattactgagtccaacaccggtgttcttcccaccaccaacctcaagggctctcccaacgctgttgcctacaacg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Claims (4)

1. A yarrowia lipolytica genetically engineered bacterium producing citric acid is characterized in that a citric acid synthase gene CAS, an acetyl coenzyme A synthase gene ACS and a mitochondrial carboxylic acid transporter gene MTT derived from Aspergillus terreus which are endogenous in yarrowia lipolytica are expressed in vivo to construct a yarrowia lipolytica genetically engineered bacterium YLA01 capable of metabolically producing citric acid,
the nucleotide sequence of the citrate synthase gene CAS is shown as SEQ ID NO.1, the nucleotide sequence of the acetyl coenzyme A synthase gene ACS is shown as SEQ ID NO.2, and the nucleotide sequence of the mitochondrial carboxylic acid transporter gene MTT is shown as SEQ ID NO. 3.
2. A yarrowia lipolytica genetically engineered bacterium producing itaconic acid, characterized in that a citric acid synthase gene CAS and an acetyl coenzyme A synthase gene ACS derived from yarrowia lipolytica endogenesis, a mitochondrial carboxylic acid transporter gene MTT and a aconitic acid decarboxylase gene CADA derived from aspergillus terreus are expressed in yarrowia lipolytica to construct a yarrowia lipolytica genetically engineered bacterium YLA02 capable of metabolically producing citric acid,
the nucleotide sequence of the citrate synthase gene CAS is shown as SEQ ID No.1, the nucleotide sequence of the acetyl coenzyme A synthase gene ACS is shown as SEQ ID No.2, the nucleotide sequence of the mitochondrial carboxylic acid transporter gene MTT is shown as SEQ ID No.3, and the nucleotide sequence of the aconitate decarboxylase gene CADA is shown as SEQ ID No. 4.
3. The method for constructing yarrowia lipolytica genetically engineered bacterium that produces citric acid or itaconic acid of claim 1 or 2, comprising the steps of:
step one, connecting three genes of a citrate synthase gene CAS, an acetyl coenzyme A synthase gene ACS and a mitochondrial carboxylic acid transporter gene MTT to an expression plasmid PYLXP 'in sequence by means of homologous recombination and T4 connection to obtain a recombinant plasmid PYcam, and connecting the citrate synthase gene CAS, the acetyl coenzyme A synthase gene ACS, the mitochondrial carboxylic acid transporter gene MTT and an aconitate decarboxylase gene CADA to the expression plasmid PYLXP' by means of homologous recombination and T4 connection to obtain a recombinant plasmid PYcama;
step two, introducing the recombinant plasmid into yarrowia lipolytica to construct strains YLA01(PYcam) and YLA02 (PYcam); the nucleotide sequence of the recombinant plasmid PYcam is shown as SEQ ID NO.5, and the nucleotide sequence of the recombinant plasmid PYcama is shown as SEQ ID NO. 6.
4. The use of yarrowia lipolytica genetically engineered strain producing citric acid or itaconic acid of claim 1 or 2 for the production of citric acid or itaconic acid using acetic acid or acetate directly as carbon source.
CN202110548580.8A 2021-05-20 2021-05-20 Construction method of yarrowia lipolytica genetic engineering bacteria for producing citric acid or itaconic acid by using acetic acid Pending CN113462588A (en)

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Application publication date: 20211001