CN113755352B - Construction and application of saccharomyces cerevisiae genetic engineering bacteria for producing heme by fermentation method - Google Patents
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Abstract
The invention discloses construction and application of saccharomyces cerevisiae genetic engineering bacteria for producing heme by a fermentation method. The invention provides a Saccharomyces cerevisiae (Saccharomyces cerevisiae) TIB H1 with a preservation registration number of CGMCC No.21000. The invention also protects the application of the saccharomyces cerevisiae TIB H1 in the production of heme. The invention also provides a method for producing the heme, which comprises the following steps: and fermenting and culturing the saccharomyces cerevisiae TIB H1. The invention lays a certain foundation for the efficient production of the heme by the saccharomyces cerevisiae and has industrial value for the industrial production of the heme.
Description
Technical Field
The invention belongs to the technical field of metabolic engineering, and relates to construction and application of saccharomyces cerevisiae genetic engineering bacteria for producing heme by a fermentation method, in particular to application of the genetic engineering bacteria for producing heme.
Background
Heme is a small molecule substance containing protoporphyrin and a ferrous ion coordinated in the ring, and is present in most organisms, such as animals, plants, and microorganisms. The heme is an important prosthetic group of myoglobin and hemoglobin in prokaryotic cells and eukaryotic cells and is composed of Fe 2+ And protoporphyrin, play an important role in the aspects of oxygen transfer, active oxygen removal, electron transfer and energy generation. At present, heme is widely applied to the health care industry as a biological iron supplement; in the food industry, hemoglobin and myoglobin which are rich in heme and stable in structure are added to endow the artificial meat with true color. However, the method of obtaining heme and hemoglobin from animal blood and plant tissues by organic extraction or enzymatic hydrolysis is time-consuming, labor-consuming, low in yield, and unfavorable for ecological environment, and cannot be applied to commercial production of artificial meat on a large scale. Therefore, the construction of cell factories to produce bio-based heme has become a hot topic of research.
The saccharomyces cerevisiae, as a common eukaryotic model organism in modern molecular and cell biology, is not only an important industrial microorganism, but also one of the most common underpan cells in synthetic biology, is used for research and development of diversified products such as biofuels, bulk chemicals, raw materials of medicines and health care products, and has huge market potential and social and economic benefits. Saccharomyces cerevisiae has many advantages as a cell factory: firstly, saccharomyces cerevisiae can avoid the potential risk of human or animal origin pathogens, with GRAS (general regulated as safe) certification; yeast are capable of performing post-translational modifications, including protein end modifications, folding, and subunit assembly; in addition, a large amount of research is carried out on saccharomyces cerevisiae as an eukaryotic model organism, the genome and physiological and biochemical backgrounds are quite clear, the genetic manipulation technology is mature, and the exogenous adaptability is strong.
Disclosure of Invention
The invention aims to provide construction and application of saccharomyces cerevisiae gene engineering bacteria for producing heme by a fermentation method.
The invention provides a Saccharomyces cerevisiae (Saccharomyces cerevisiae) TIB H1 which is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 of Beijing Corp West Lu No. 1 of the morning-Yangxi district, china academy of sciences) in 11 months and 04 days of 2020, and the preservation registration number is CGMCC No.21000.
The invention also protects the application of the saccharomyces cerevisiae TIB H1 in the production of heme.
The invention also provides a method for producing the heme, which comprises the following steps: and fermenting and culturing the saccharomyces cerevisiae TIB H1.
The culture medium adopted by the fermentation culture comprises the following components: uracil, feSO 4 Glutamic acid, glucose.
The culture medium adopted by the fermentation culture comprises the following components: 1-3g/L uracil, 80-120. Mu.M FeSO 4 0.5-1.5g/L glutamic acid and 40-60g/L glucose.
The culture medium adopted by the fermentation culture comprises the following components: 2g/L uracil, 100. Mu.M FeSO 4 1g/L glutamic acid, 50g/L glucose.
The culture medium adopted by the fermentation culture comprises the following components: (NH) 4 ) 2 SO 4 、KH 2 PO 4 、MgSO 4 Uracil, feSO 4 Glutamic acid, glucose.
The culture medium adopted by the fermentation culture consists of the following components: (NH) 4 ) 2 SO 4 、KH 2 PO 4 、MgSO 4 Uracil, feSO 4 Glutamic acid, glucose and the balance of water.
The culture medium adopted by the fermentation culture consists of the following components: 5-10g/L (NH) 4 ) 2 SO 4 、10-20g/L KH 2 PO 4 、0.1-1g/L MgSO 4 ·7H 2 O, 1-3g/L uracil, 80-120. Mu.M FeSO 4 0.5-1.5g/L glutamic acid, 40-60g/L glucose and the balance of water.
The culture medium adopted by the fermentation culture consists of the following components: 7.5g/L (NH) 4 ) 2 SO 4 、14.4g/L KH 2 PO 4 、0.5g/L MgSO 4 ·7H 2 O, 2g/L uracil, 100. Mu.M FeSO 4 1g/L glutamic acid, 50g/L glucose and the balance of water.
The culture medium adopted by the fermentation culture is called fermentation culture medium for short.
The fermentation culture conditions specifically include: culturing at 25-35 deg.C under shaking at 200-300rpm for 36-60h.
The fermentation culture conditions specifically include: shaking and culturing at 30 deg.C and 250rpm for 48h.
Initial OD of the fermentation culture 600nm Is 0.4-0.6.
Initial OD of the fermentation culture 600nm Is 0.5.
In the fermentation culture, the seed solution is inoculated into a fermentation culture medium, and then the culture is started.
The preparation method of the seed liquid comprises the following specific steps: saccharomyces cerevisiae TIB H1 was inoculated into YPD seed medium, and shake-cultured at 30 ℃ and 250rpm for 24 hours.
The invention also protects a recombinant saccharomyces cerevisiae, which is obtained by introducing a coding gene of glutamyl-t RNA synthetase (gltX), a coding gene of glutamyl tRNA reductase (hemA), a coding gene of glutamyl-1-semialdehyde transaminase (hemL) and a coding gene of porphobilinogen deaminase (HEM 3) into starting saccharomyces cerevisiae.
The gene encoding glutamyl-t RNA synthetase is also called the gltX gene.
The gene encoding glutamyl tRNA reductase is also known as the hemA gene.
The gene encoding glutamyl-1-semialdehyde transaminase is also known as the hemL gene.
The coding gene of the porcupine deaminase is also called as HEM3 gene.
In the recombinant saccharomyces cerevisiae, a gltX gene, a hemA gene and a hemL gene are integrated into a saccharomyces cerevisiae genome DNA.
The gltX gene is integrated between the 93963 and 93964 sites of chromosome XI of the s.cerevisiae genome.
The hemA gene integrates between the 195461 and 195462 sites of the X chromosome of the s.cerevisiae genome.
The hemL gene is integrated between positions 796317 and 796318 of chromosome XII of the s.cerevisiae genome.
The construction method of the recombinant saccharomyces cerevisiae comprises the following steps:
(1) pMEL10 plasmid and DNA molecule Up1-P ADH1 -gltX-T CYC1 Introducing Down1 into Saccharomyces cerevisiae, culturing to obtain genome DNA integrated with DNA molecule Up1-P ADH1 -gltX-T CYC1 -a recombinant yeast strain of Down 1;
(2) pROS10 substanceParticle and DNA molecule Up2-P TEF1 -hemA-T TEF1 Down2 and DNA molecule Up3-P ENO2 -hemL-T ADH1 Introducing Down3 into the recombinant yeast strain obtained in the step (1), and culturing to obtain a DNA molecule Up2-P integrated in the genome DNA TEF1 -hemA-T TEF1 Down2 and DNA molecule Up3-P ENO2 -hemL-T ADH1 -a recombinant yeast strain of Down 3;
(3) And (3) introducing the pRS426-hem3 plasmid into the recombinant yeast strain obtained in the step (2) to obtain the target recombinant saccharomyces cerevisiae.
The saccharomyces cerevisiae can be specifically saccharomyces cerevisiae TIB H.
The invention also protects the application of the recombinant saccharomyces cerevisiae in the production of heme.
The invention also provides a method for producing the heme, which comprises the following steps: and (3) fermenting and culturing the recombinant saccharomyces cerevisiae.
The culture medium adopted by the fermentation culture comprises the following components: uracil, feSO 4 Glutamic acid, glucose.
The culture medium adopted by the fermentation culture comprises the following components: 1-3g/L uracil, 80-120. Mu.M FeSO 4 0.5-1.5g/L glutamic acid and 40-60g/L glucose.
The culture medium adopted by the fermentation culture comprises the following components: 2g/L uracil, 100. Mu.M FeSO 4 1g/L glutamic acid, 50g/L glucose.
The culture medium adopted by the fermentation culture comprises the following components: (NH) 4 ) 2 SO 4 、KH 2 PO 4 、MgSO 4 Uracil, feSO 4 Glutamic acid, glucose.
The culture medium adopted by the fermentation culture consists of the following components: (NH) 4 ) 2 SO 4 、KH 2 PO 4 、MgSO 4 Uracil, feSO 4 Glutamic acid, glucose and the balance of water.
The culture medium adopted by the fermentation culture consists of the following components: 5-10g/L (NH) 4 ) 2 SO 4 、10-20g/L KH 2 PO 4 、0.1-1g/L MgSO 4 ·7H 2 O, 1-3g/L uracil, 80-120. Mu.M FeSO 4 0.5-1.5g/L glutamic acid, 40-60g/L glucose and the balance of water.
The culture medium adopted by the fermentation culture consists of the following components: 7.5g/L (NH) 4 ) 2 SO 4 、14.4g/L KH 2 PO 4 、0.5g/L MgSO 4 ·7H 2 O, 2g/L uracil, 100. Mu.M FeSO 4 1g/L glutamic acid, 50g/L glucose and the balance of water.
The culture medium adopted by the fermentation culture is called fermentation culture medium for short.
The conditions of the fermentation culture can be specifically as follows: culturing at 25-35 deg.C under shaking at 200-300rpm for 36-60h.
The fermentation culture conditions specifically include: shaking and culturing at 30 deg.C and 250rpm for 48h.
Initial OD of the fermentation culture 600nm Is 0.4-0.6.
Initial OD of the fermentation culture 600nm Is 0.5.
In the fermentation culture, the seed liquid is inoculated into a fermentation culture medium, and then the culture is started.
The preparation method of the seed liquid comprises the following specific steps: the recombinant saccharomyces cerevisiae is inoculated to YPD seed culture medium and is subjected to shaking culture at 30 ℃ and 250rpm for 24 hours.
The gltX gene is shown as 625-2040 th nucleotides in sequence 1 of the sequence table.
The hemA gene is composed of 587 th-1843 th nucleotides in a sequence 2 of a sequence table.
The hemL gene is composed of nucleotides 805-2085 in a sequence 3 of a sequence table.
The HEM3 gene is shown as 2694-3677 th nucleotide in a sequence 4 of a sequence table.
DNA molecule Up1-P ADH1 -gltX-T CYC1 -Down1, which is a double-stranded DNA molecule, as shown in sequence 1 of the sequence listing.
DNA molecule Up2-P TEF1 -hemA-T TEF1 -Down2, which is a double-stranded DNA molecule, as shown in sequence 2 of the sequence listing.
DNA molecule Up3-P ENO2 -hemL-T ADH1 -Down3, double strandedThe DNA molecule is shown as a sequence 3 in a sequence table.
The pRS426-hem3 plasmid is a circular plasmid (double-stranded DNA circular plasmid) and is shown as a sequence 4 in a sequence table.
The pMEL10 plasmid is a circular plasmid (double-stranded DNA circular plasmid) and is shown as a sequence 5 in a sequence table.
The pROS10 plasmid is a circular plasmid (double-stranded DNA circular plasmid) and is shown as a sequence 6 in a sequence table.
The invention constructs saccharomyces cerevisiae engineering bacteria TIB H1 by integrating glutamyl-t RNA synthetase (gltX), glutamyl tRNA reductase (hemA) and glutamyl-1-semialdehyde transaminase (hemL) into a saccharomyces cerevisiae genome and overexpressing porchogen deaminase (HEM 3). Furthermore, the inventor deposits the saccharomyces cerevisiae engineering bacteria TIB H1 by a patent program. When the saccharomyces cerevisiae engineering bacteria TIB H1 are used for shake flask fermentation for 48 hours, the heme yield reaches 0.596mg/L/OD. The invention lays a certain foundation for the efficient production of the heme by the saccharomyces cerevisiae and has industrial value for the industrial production of the heme.
Drawings
FIG. 1 shows the results of the hemoglobin production in example 3.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way. The experimental procedures in the following examples, unless otherwise specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Unless otherwise stated, the quantitative tests in the following examples were carried out in triplicate, and the results were averaged. The saccharomyces cerevisiae TIB H has the genotype MATa ura3-52 can1 delta and cas9-natNT2 TRP1 LEU2 HIS3.SD synthetic medium: beijing Pankeno, catalog No. YGM003A-3.SD medium: taking 8g of SD synthetic medium and 20g of glucose, adding distilled water to a constant volume of 1L, sterilizing at 121 ℃ for 15min, and adding uracil to a concentration of 2g/L after sterilization. URA3 deficiency screening medium: taking 8g SD synthetic culture medium, 20g glucose and 20g agar, adding distilled water to constant volume to 1L, sterilizing at 121 deg.C for 15min.5-FOA screening culture medium: taking 8g of SD synthetic culture medium, 20g of glucose, 1g of 5-FOA and 20g of agar, adding distilled water to fix the volume to 1L, sterilizing at 121 ℃ for 15min, and adding uracil to make the concentration of uracil be 2g/L after sterilization.
Example 1 construction of DNA molecules and recombinant plasmids
DNA molecule Up1-P ADH1 -gltX-T CYC1 -Down1, which is a double-stranded DNA molecule, as shown in sequence 1 of the sequence listing. In the sequence 1, the 1 st to 207 th nucleotides form Up1 (207 bp), and the 208 th to 624 th nucleotides form a promoter P ADH1 (417 bp), the 625-2040 th nucleotide constitutes the gltX gene (1416 bp), and the 2041-2295 th nucleotide constitutes the terminator T CYC1 (255 bp), the 2296-2515 th nucleotide constitutes Down1 (220 bp). Up1 is the upstream homology arm, down1 is the downstream homology arm.
DNA molecule Up2-P TEF1 -hemA-T TEF1 -Down2, which is a double-stranded DNA molecule, as shown in sequence 2 of the sequence listing. In the sequence 2, the 1 st to 243 nd nucleotides form Up2 (243 bp), and the 244 th to 586 th nucleotides form a promoter P TEF1 (343 bp), nucleotides 587-1843 constitute hemA gene (1257 bp), nucleotides 1844-2041 constitute terminator T TEF1 (198 bp), 2042-2277 is the nucleotide composition Down2 (236 bp). Up2 is the upstream homology arm, down2 is the downstream homology arm.
DNA molecule Up3-P ENO2 -hemL-T ADH1 -Down3, which is a double-stranded DNA molecule, as shown in sequence 3 of the sequence listing. In the sequence 3, the 1 st to 255 th nucleotides form Up3 (255 bp), and the 256 th to 804 th nucleotides form a promoter P ENO2 (549 bp), the 805-2085 th nucleotide constitutes hemL gene (1281 bp), the 2086-2250 th nucleotide constitutes terminator T ADH1 (165 bp), nucleotides 2251-2499 constitute Down3 (249 bp). Up3 is the upstream homology arm, down3 is the downstream homology arm.
The pRS426-hem3 plasmid is a circular plasmid (double-stranded DNA circular plasmid) and is shown as a sequence 4 in a sequence table. In the sequence 4, nucleotides 2013-2693 form a promoter TDH3 (681 bp), nucleotides 2694-3677 form an HEM3 gene (984 bp), and nucleotides 3678-3995 form a terminator CYC1 (318 bp).
The pMEL10 plasmid is a circular plasmid (double-stranded DNA circular plasmid) and is shown as a sequence 5 in a sequence table. In the sequence 5, the 3587 th-3685 th nucleotides encode sgRNA (the nucleotides encoding the binding region of the target sequence are 'CTAATGTGTCCGCGTTTCTA').
The pROS10 plasmid is a circular plasmid (double-stranded DNA circular plasmid) and is shown as a sequence 6 in a sequence table. In the sequence 6, the 3730 th to 3828 th nucleotides encode one sgRNA (the nucleotides encoding the binding region of the target sequence are "CTCTCTCGAAGTGGTCACGTGC") and the 5218 th to 5120 th nucleotides encode the other sgRNA (the nucleotides encoding the binding region of the target sequence are "GGTATGTGCAGTTGATTCAC").
Helper plasmids (both the pMEL10 plasmid and the pres 10 plasmid) have a gene encoding orotidine-5' -monophosphate decarboxylase (OMP decarboxylase), which converts 5-Fluoroorotic Acid (5-fluorogenic Acid, 5-FOA) into a form toxic to the yeast cells, thereby causing the yeast cells to die. Thus, after obtaining a recombinant yeast having a foreign DNA molecule, 5-FOA can be used for further screening, and only a recombinant yeast that integrates the foreign DNA molecule into the genomic DNA and does not carry a helper plasmid can grow normally.
The gltX gene, hemA gene, and hemL gene are all derived from e.
Up1、P ADH1 、T CYC1 、Down1、Up2、P TEF1 、T TEF1 、Down2、Up3、P ENO2 、T ADH1 The Down3, the promoter TDH3, the HEM3 gene and the terminator CYC1 are all derived from saccharomyces cerevisiae.
Example 2 construction and preservation of recombinant Saccharomyces cerevisiae TIB H1 engineered Strain
Using the Frozen-EZ Yeast Transformation II Kit TM The kit (Zymo company, catalog T2001) is transformed to construct recombinant Saccharomyces cerevisiae. EZ1 reagent, EZ2 reagent and EZ3 reagent are reagent kit components.
1. Construction of recombinant Saccharomyces cerevisiae TIB Ha engineering strain
1. A single colony of Saccharomyces cerevisiae TIB H was inoculated into 2-3mL of SD medium, and cultured at 30 ℃ for 24 hours with shaking at 250 rpm.
2. After completion of step 1, the bacterial solution was inoculated into 5mL of SD medium (initial OD of system) 600nm Value ≈ 0.2), shaking culture at 30 deg.C and 250rpm to OD 600nm Value =0.8-1.0.
3. After step 2, centrifuging at 3000rpm for 5min, and discarding the supernatant; resuspending the precipitate with EZ1 reagent preheated at 30 ℃, transferring to a 2mL EP tube, centrifuging at 3000rpm for 5min, and discarding the supernatant; the pellet was resuspended with EZ2 reagent after preheating at 30 ℃.
4. 50. Mu.L of the suspension obtained in step 3, 1. Mu.g of the pMEL10 plasmid and 1. Mu.g of the DNA molecule Up1-P ADH1 -gltX-T CYC1 -Down1 was gently mixed, then 500. Mu.L of EZ3 reagent was added thereto, and mixed well by a vortex shaker, shaken for 20 seconds, and then cultured at 30 ℃ for 2 to 3 hours with shaking at 100 to 150 rpm.
5. After step 4, mix well with vortex oscillator, take 100 μ L bacterial liquid to coat the plate (for keeping the bacterial), the residual bacterial liquid 3000rmp centrifugate for 5min, collect the precipitate.
6. Resuspending the pellet obtained in step 5 with 100. Mu.L sterile water, coating URA3 defect screening medium plate, standing and culturing at 30 ℃ for 48h, and obtaining the introduced DNA molecule Up1-P by PCR amplification and sequencing verification ADH1 -gltX-T CYC1 -recombinant yeast strain of Down 1.
7. And (3) streaking and inoculating the recombinant yeast strain obtained in the step (6) in a 5-FOA screening culture medium plate, and statically culturing at 30 ℃ for 48h to obtain a strain capable of normally growing, namely a recombinant saccharomyces cerevisiae TIB Ha genetic engineering strain, which is referred to as saccharomyces cerevisiae TIB Ha for short.
2. Construction of recombinant saccharomyces cerevisiae TIB Hb engineering strain
1. A single colony of Saccharomyces cerevisiae TIB Ha is inoculated into 2-3mL SD culture medium, and is subjected to shaking culture at 30 ℃ and 250rpm for 24h.
2. The same as step one, 2.
3. The same as step one, 3.
4. 50. Mu.L of the suspension obtained in step 3 and 1. Mu.g of pROS10 substance were addedParticle, 1. Mu.g DNA molecule Up2-P TEF1 -hemA-T TEF1 Down2 and 1. Mu.g of the DNA molecule Up3-P ENO2 -hemL-T ADH1 -Down3 was gently mixed, then 500. Mu.L of EZ3 reagent was added thereto, and mixed well by a vortex shaker, shaken for 20 seconds, and then cultured at 30 ℃ for 2 to 3 hours with shaking at 100 to 150 rpm.
5. The same as step one, 5.
6. Resuspending the pellet obtained in step 5 with 100. Mu.L sterile water, coating URA3 defect screening medium plate, standing at 30 deg.C for 48h, and PCR amplification and sequencing verification to obtain introduced DNA molecule Up2-P TEF1 -hemA-T TEF1 Down2 and DNA molecule Up3-P ENO2 -hemL-T ADH1 -recombinant yeast strain of Down 3.
7. And (3) streaking the recombinant yeast strain obtained in the step (6) in a 5-FOA screening culture medium plate, and statically culturing at 30 ℃ for 48h to obtain a strain capable of normally growing, namely a recombinant saccharomyces cerevisiae TIB Hb genetic engineering strain, namely saccharomyces cerevisiae TIB Hb for short.
3. Construction of recombinant saccharomyces cerevisiae TIB H1 engineering strain
1. The single colony of Saccharomyces cerevisiae TIB Hb was inoculated into 2-3mL SD medium, and cultured at 30 ℃ for 24h with shaking at 250 rpm.
2. The same as step one, 2.
3. The same as step one, 3.
4. And (3) gently mixing 50 mu L of the suspension obtained in the step (3) with 1 mu g of pRS426-hem3 plasmid, adding 500 mu L of EZ3 reagent, fully mixing by using a vortex oscillator, shaking for 20 seconds, and then culturing at 30 ℃ and 100-150rpm for 2-3h.
5. The same as step one, 5.
6. And (3) resuspending the precipitate obtained in the step (5) by using 100 mu L of sterile water, coating a URA3 defect screening culture medium plate, standing and culturing for 48 hours at 30 ℃, then selecting a recombinant strain with normal growth, and then obtaining a recombinant yeast strain with an HEM3 gene through PCR amplification and sequencing verification, namely the recombinant saccharomyces cerevisiae TIB H1 genetic engineering strain, namely saccharomyces cerevisiae TIB H1 for short.
4. Preservation of Saccharomyces cerevisiae TIB H1
Saccharomyces cerevisiae TIB H1 has been deposited in China general microbiological culture Collection center (CGMCC, no. 3 of the institute of microbiology, china academy of sciences) at 04.11.2020 of China, and has been deposited with the registration number of CGMCC No.21000.
Example 3 fermentation production of heme by recombinant Saccharomyces cerevisiae
The test bacteria are respectively as follows: saccharomyces cerevisiae TIB H, saccharomyces cerevisiae TIB Ha, saccharomyces cerevisiae TIB Hb or Saccharomyces cerevisiae TIB H1.
1. Single colonies of the test bacteria were inoculated into YPD seed medium and cultured at 30 ℃ for 24 hours under shaking at 250 rpm.
YPD seed culture Medium: dissolving 20g of peptone and 10g of yeast extract with appropriate amount of distilled water, sterilizing at 121 deg.C for 15min, and adding sterile glucose aqueous solution to 1000ml; the concentration of glucose in the medium was 20g/L.
2. Inoculating the bacterial liquid obtained in the step 1 into a fermentation medium (initial OD of a system) 600nm 0.5), 30 ℃ and 250rpm for 48h.
Fermentation medium: containing 7.5g/L (NH) 4 ) 2 SO 4 、14.4g/L KH 2 PO 4 、0.5g/L MgSO 4 ·7H 2 O, 2g/L uracil, 100. Mu.M FeSO 4 1g/L glutamic acid, 50g/L glucose and the balance of water.
3. And (3) after the step 2 is finished, taking an n ml system, and detecting the yield of heme produced by each OD bacterial cell.
A heme detection method comprises the following steps: collecting n ml system, centrifuging, and collecting cell precipitate to obtain 8OD cell 600nm 4ml of the system is taken if the OD value of the system is 2, and then the cell sediment is collected by centrifugation, namely 8OD 600nm Cell amount of (b), resuspended with 500. Mu.L of 20mM oxalic acid aqueous solution, and then left to stand at 4 ℃ for 16 hours; then adding 500 μ l of 70 deg.C preheated 2M oxalic acid aqueous solution, mixing, adding half volume of the mixture into amber tube, heating at 95 deg.C for 30min, and keeping the other half volume of the mixture at room temperature for 30min; two mixtures of 200. Mu.l each supernatant were transferred to black 96-well plates at λ =400nm excitation wavelength and λ =600nm emission wavelengthAnd (4) detecting the fluorescence value. The total porphyrin level (bound heme and free porphyrin) corresponds to the heated sample and the free porphyrin level corresponds to the unheated sample. Standard curve equation between porphyrin concentration and fluorescence value: fluorescence =71.223 × porphyrin concentration (mg/L) +58.049. Heme level = total porphyrin level-free porphyrin level. The heme production was calculated as the heme level, i.e., the amount of heme (mg) per liter of system that completed step 2 divided by the amount of cells (OD) in that liter of system.
The results of heme production are shown in FIG. 1. The heme yields of the saccharomyces cerevisiae TIB H, the saccharomyces cerevisiae TIB Ha, the saccharomyces cerevisiae TIB Hb or the saccharomyces cerevisiae TIB H1 are 0.011mg/L/OD, 0.044mg/L/OD, 0.096mg/L/OD and 0.596mg/L/OD in sequence, and the heme yield of the saccharomyces cerevisiae TIB H1 is the highest and is improved by 52 times compared with the saccharomyces cerevisiae TIB H.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
Construction and application of saccharomyces cerevisiae gene engineering bacteria for producing heme by fermentation method
<130> GNCYX210740
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2515
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
agccatctaa ccatcatacc cccatacggt aacaaaacct cttctaagaa aagaagtctc 60
tgctcctccg ccatcttatt tttattcgct gcgcgcgttt attgtcgcat cgctagccag 120
caaaaagttg gttgcctttt tttacctaaa aaagacacat ctaactgatt agttttccgt 180
tttaggatat tgacgccaag cgtgcgtata ataggcgcat gcaacttctt ttcttttttt 240
ttcttttctc tctcccccgt tgttgtctca ccatatccgc aatgacaaaa aaatgatgga 300
agacactaaa ggaaaaaatt aacgacaaag acagcaccaa cagatgtcgt tgttccagag 360
ctgatgaggg gtatctcgaa gcacacgaaa ctttttcctt ccttcattca cgcacactac 420
tctctaatga gcaacggtat acggccttcc ttccagttac ttgaatttga aataaaaaaa 480
agtttgctgt cttgctatca agtataaata gacctgcaat tattaatctt ttgtttcctc 540
gtcattgttc tcgttccctt tcttccttgt ttctttttct gcacaatatt tcaagctata 600
ccaagcatac aatcaactat ctcaatgaaa atcaaaactc gcttcgcgcc aagcccaaca 660
ggctatctgc acgttggcgg cgcgcgtact gctctttact cctggctttt tgcacgtaac 720
cacggcggtg agttcgtgct gcgtattgaa gacaccgatc ttgagcgttc cacgccggaa 780
gctatcgaag ccattatgga tggcatgaac tggctgagcc tggagtggga tgaaggtccg 840
tactaccaga ccaaacgttt tgatcgctac aacgcggtga tcgatcagat gctggaagag 900
ggcactgctt ataaatgcta ttgctctaaa gagcgcctgg aagcgctgcg cgaagagcaa 960
atggcgaaag gtgagaagcc gcgttatgac ggtcgctgcc gccacagcca tgagcatcat 1020
gctgatgatg aaccgtgtgt tgtacgtttt gctaacccgc aggaaggttc tgttgttttt 1080
gacgatcaga tccgtggtcc gatcgagttc agcaaccagg aactggacga tcttattatc 1140
cgccgtaccg atggttcccc aacctataac ttctgtgtgg ttgtcgatga ctgggatatg 1200
gaaatcaccc acgttatccg tggcgaagac catatcaaca acacgccacg ccagatcaac 1260
attcttaagg ccctgaaagc gccggtgccg gtttacgcgc acgtttctat gatcaatggc 1320
gatgacggta aaaaactgtc caaacgtcac ggggcagtca gcgtaatgca gtatcgtgat 1380
gacggttatt tgccagaagc actgctgaac tatctggtgc gtctgggctg gtcccacggc 1440
gatcaggaaa tcttcactcg tgaagagatg atcaaatact tcactttgaa tgccgtcagc 1500
aaatctgcca gtgcgttcaa caccgacaag ctgctgtggc tgaaccatca ctacattaac 1560
gcgctgccgc cggagtatgt tgctactcac ttacagtggc acattgagca ggaaaatatc 1620
gatacccgta acggcccgca gctggctgat ctggtgaaac tgctgggcga acgctgcaag 1680
acgctgaaag agatggcaca gagctgccgt tatttctacg aagattttgc tgagttcgat 1740
gccgacgccg cgaaaaaaca tctgcgtccg gtagcgcgtc agccgctgga agtggttcgt 1800
gacaaactgg ccgcgattac tgactggacc gctgaaaacg ttcatcacgc tattcaggcg 1860
acggcggatg agctggaagt gggtatgggt aaagttggta tgccgctgcg tgtcgccgta 1920
accggtgcgg ggcagtctcc agcactggat gttaccgttc acgcaattgg taagacccgc 1980
agtatcgagc gtatcaacaa agcgctggat tttattgctg aacgcgaaaa tcagcagtaa 2040
gtcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc 2100
gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg 2160
ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg 2220
tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgataggc 2280
tttaatttgc aagctgcaga acttgcaaac ataccaaaat cctttattct tgttcactca 2340
ttttacatca aaaaataata tttcagttat taaggaaaat aaaaaaatag attagagaag 2400
cattttgaag aaatagtata ttcttttatt gaacctaaga gcgtgatatt tttactcgaa 2460
ataaaatacg aaaaatctat acactcatct ttccgactac tattggctcc tgctc 2515
<210> 2
<211> 2277
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cataattcta ccaccttaca ctcaacttac tctttaactc ctatagtata atatcgccac 60
tgaccccata ttaaaaaatt tttttgctcg atcttctatc ctctttaggt taattgtcgc 120
tgttattgtc tagatttttt ctcggagatg gcgcatctat ttgccgtcaa aagatcctct 180
cataccatat taagtaaatt gcctccattt ctttttcctc gggcagagaa actcgcaggc 240
aacgacatgg aggcccagaa taccctcctt gacagtcttg acgtgcgcag ctcaggggca 300
tgatgtgact gtcgcccgta catttagccc atacatcccc atgtataatc atttgcatcc 360
atacattttg atggccgcac ggcgcgaagc aaaaattacg gctcctcgct gcagacctgc 420
gagcagggaa acgctcccct cacagacgcg ttgaattgtc cccacgccgc gcccctgtag 480
agaaatataa aaggttagga tttgccactg aggttcttct ttcatatact tccttttaaa 540
atcttgctag gatacagttc tcacatcaca tccgaacata aacaacatga cccttttagc 600
actcggtatc aaccataaaa cggcacctgt atcgctgcga gaacgtgtat cgttttcgcc 660
ggataagctc gatcaggcgc ttgacagcct gcttgcgcag ccgatggtgc agggcggcgt 720
ggtgctgtcg acgtgcaacc gcacggaact ttatcttagc gttgaagagc aggataacct 780
gcaagaggcg ttaatccgct ggctttgcga ttatcacaat cttaatgaag aagatctgcg 840
taaaagcctc tactggcatc aggataacga cgcggttagc catttaatgc gtgttgccag 900
cggcctggat tcattggttc ttggggagcc gcagatcctc ggtcaggtta aaaaagcgtt 960
tgccgattcg caaaaaggcc atatgaaggc cagcgaactg gaacgcatgt tccagaaatc 1020
tttctctgta gcgaaacgcg ttcgcactga aacagatatc ggtgccagcg ctgtgtctgt 1080
cgcttttgcg gcttgtacgc tggcgcggca gatctttgaa tcgctctcta cggtcacagt 1140
gttgctggta ggcgcgggcg aaaccatcga gctggtagcg cgtcatctgc gcgaacataa 1200
agtacagaag atgattatcg ccaaccgcac tcgcgaacgt gcccaaatac tggcagatga 1260
agttggcgcg gaagtgattg ccctgagtga gatcgacgaa cgtctgcgcg aagccgatat 1320
catcatcagt tccaccgcca gcccgttacc gattatcggg aaaggcatgg tggagcgcgc 1380
attaaaaagc cgtcgcaacc aaccaatgct gttggtggat attgccgttc cgcgcgatgt 1440
tgagccggaa gttggcaaac tggcgaatgc ttatctttat agcgtggacg atctgcaaag 1500
catcatttcg cacaacctgg cgcagcgtaa agccgcagcg gttgaggcgg aaactattgt 1560
cgctcaggaa accagcgaat ttatggcgtg gctgcgagca caaagcgcca gcgaaaccat 1620
tcgcgagtat cgcagccagg cagagcaagt tcgcgatgag ttaaccgcca aagcgttagc 1680
ggcccttgag cagggcggcg acgcgcaagc cattatgcag gatctggcat ggaaactgac 1740
taaccgcttg atccatgcgc caacgaaatc acttcaacag gccgcccgtg acggggataa 1800
cgaacgcctg aatattctgc gcgacagcct cgggctggag tagactgaca ataaaaagat 1860
tcttgttttc aagaacttgt catttgtata gtttttttat attgtagttg ttctatttta 1920
atcaaatgtt agcgtgattt atattttttt tcgcctcgac atcatctgcc cagatgcgaa 1980
gttaagtgcg cagaaagtaa tatcatgcgt caatcgtatg tgaatgctgg tcgctatact 2040
gattggggtt tccgtgtagc cttcccctga atagtgtggg acgttttatg agaagccgta 2100
agaaataggc aaattgagtt atgacaagta gacatgatgc cgcagccttg cctgacttta 2160
cgtctccttc atgaataagt ttttctatcg agttcttttc cttttttcgc cttaattagc 2220
tcaattaagc ctgtcctcac tacttttctt tttcttatcg gctttgtgcc acaccta 2277
<210> 3
<211> 2499
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
tggcctgcag actaaactgt atggtggtct tggaatgata aagatctgtt taatagattt 60
agtagataca atagcacatc tcattaccca gttatgattg acgtcattct gagttacaat 120
gatcttagcg acctgtgctc attatttgct ccactaattc taattttcct cgcctttcat 180
atttcgtatc tttattctat atcctaaaat ttttttggca aatcccagat ttggctttga 240
ttttggcatc ggttccgctc agcatctgct tcttcccaaa gatgaacgcg gcgttatgtc 300
actaacgacg tgcaccaact tgcggaaagt ggaatcccgt tccaaaactg gcatccctaa 360
ttgatacatc tacacaccgc acgccttttt tctgaagccc actttcgtgg actttgccat 420
atgcaaaatt catgaagtgt gataccaagt cagcatacac ctcactaggg tagtttcttt 480
ggttgtattg atcatttggt tcatcgtggt tcattaattt tttttctcca ttgctttctg 540
gctttgatct tactatcatt tggatttttg tcgaaggttg tagaattgta tgtgacaagt 600
ggcaccaagc atatataaaa aaaaaaagca ttatcttcct accagagttg attgttaaaa 660
acgtatttat agcaaacgca attgtaatta attcttattt tgtatctttt cttcccttgt 720
ctcaatcttt tatttttatt ttatttttct tttcttagtt tctttcataa caccaagcaa 780
ctaatactat aacatacaat aataatgagt aagtctgaaa atctttacag cgcagcgcgc 840
gagctgatcc ctggcggtgt gaactcccct gttcgcgcct ttactggcgt gggcggcact 900
ccactgttta tcgaaaaagc ggacggcgct tatctgtacg atgttgatgg caaagcctat 960
atcgattatg tcggttcctg ggggccgatg gtgctgggcc ataaccatcc ggcaatccgc 1020
aatgccgtga ttgaagccgc cgagcgtggt ttaagctttg gtgcaccaac cgaaatggaa 1080
gtgaaaatgg cgcaactggt gaccgaactg gtcccgacca tggatatggt gcgcatggtg 1140
aactccggca ccgaggcgac gatgagcgcc atccgtctgg cccgtggttt taccggtcgc 1200
gacaaaatta ttaaatttga aggatgctac cacggtcacg ctgactgcct gctggtgaaa 1260
gccggttctg gcgcactcac gttaggtcag ccaaactcgc cgggcgttcc ggcagatttc 1320
gccaaacata ccttaacctg tacttataac gatctggctt ctgtacgcgc cgcatttgag 1380
caatacccgc aagagattgc ctgtattatc gtcgagccgg tggcaggcaa tatgaactgt 1440
gttccaccgc tgccagactt cctgccaggt ctgcgcgcgc tgtgcgacga atttggcgcg 1500
ttgctgatca tcgatgaagt gatgaccggt ttccgcgtag cgctagctgg cgcacaggat 1560
tattacggcg tagtgccaga tttaacctgc ctcggcaaaa tcatcggcgg tggaatgccg 1620
gtaggcgcat tcggtggtcg tcgtgatgta atggatgcgc tggccccgac gggtccggtc 1680
tatcaggcgg gtacgctttc cggtaacccg attgcgatgg cagcgggttt cgcctgtctg 1740
aatgaagtcg cgcagccggg cgttcacgaa acgctggatg agctgacaac acgtctggca 1800
gaaggtctgc tggaagcggc agaagaagcc ggaattccgc tggtcgttaa ccacgttggc 1860
ggcatgttcg gtattttctt taccgacgcc gagtccgtga cgtgctatca ggatgtgatg 1920
gcctgtgacg tggaacgctt taagcgtttc ttccatatga tgctggacga aggtgtttac 1980
ctggcaccgt cagcgtttga agcgggcttt atgtccgtgg cgcacagcat ggaagatatc 2040
aataacacca tcgatgctgc acgtcgggtg tttgcgaagt tgtgagagcg acctcatgct 2100
atactgagaa agcaacctga cctacaggaa agagttactc aagaataaga attttcgttt 2160
taaaacctaa gagtcacttt aaaatttgta tacacttatt ttttttataa cttatttaat 2220
aataaaaatc ataaatcata agaaattcgc caagccactt ctcatgacat atattggtaa 2280
gtaacttcat caatactaat tagtctttgc cggttaccca tctggcccct gacttgcgat 2340
gcttaggaag ttccatactc gcggctcttc ccaacagtag cacatccgtg aaacttctgg 2400
cgctattcat tatgcagtac caggacaaga agttaaaaaa aaagctctgt tacaagttca 2460
atggtggtgc aaggattgaa gttattatcc aggaggcac 2499
<210> 4
<211> 7709
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240
ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300
agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360
cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt 420
cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat 480
ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca 540
aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg 600
tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg 660
ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca 720
aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac 780
acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa 840
aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg 900
gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct 960
ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac 1020
ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg 1080
atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa 1140
gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa 1200
gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac 1260
aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac 1320
agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat 1380
tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa 1440
tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca 1500
agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 1560
gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta 1620
aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg 1680
cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 1740
gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg 1800
gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 1860
cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg 1920
taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat 1980
acgactcact atagggcgaa ttgggtaccg ggtcagttcg agtttatcat tatcaatact 2040
gccatttcaa agaatacgta aataattaat agtagtgatt ttcctaactt tatttagtca 2100
aaaaattagc cttttaattc tgctgtaacc cgtacatgcc caaaataggg ggcgggttac 2160
acagaatata taacatcgta ggtgtctggg tgaacagttt attcctggca tccactaaat 2220
ataatggagc ccgcttttta agctggcatc cagaaaaaaa aagaatccca gcaccaaaat 2280
attgttttct tcaccaacca tcagttcata ggtccattct cttagcgcaa ctacagagaa 2340
caggggcaca aacaggcaaa aaacgggcac aacctcaatg gagtgatgca acctgcctgg 2400
agtaaatgat gacacaaggc aattgaccca cgcatgtatc tatctcattt tcttacacct 2460
tctattacct tctgctctct ctgatttgga aaaagctgaa aaaaaaggtt gaaaccagtt 2520
ccctgaaatt attcccctac ttgactaata agtatataaa gacggtaggt attgattgta 2580
attctgtaaa tctatttctt aaacttctta aattctactt ttatagttag tctttttttt 2640
agttttaaaa caccaagaac ttagtttcga ataaacacac ataaacaaac aaaatgggcc 2700
ctgaaactct acatattggt gggagaaaat cgaaattggc ggtaatacaa tccaaccatg 2760
ttttaaaact gatcgaagaa aagtatccgg actacgactg caaggttttc actttgcaaa 2820
ctcttggtga ccagattcaa ttcaaacctt tgtactcatt tggcggtaaa gctttatgga 2880
caaaggagtt ggaagaccat ctttaccatg acgatccctc aaagaagctt gacttgatcg 2940
ttcattctct gaaggacatg cccactttac taccagaggg tttcgagctg gggggtatca 3000
ctaagcgggt cgatccaaca gattgtcttg tcatgccctt ttactctgct tataagtctc 3060
tggatgacct tccagacggg gggattgtgg gaacctcatc cgtgagaaga tctgctcagc 3120
taaaaagaaa atacccacat ttgaaatttg aaagtgtcag aggaaatata caaactagat 3180
tacaaaaact agacgaccca aaatctccgt accaatgcat catcttggcg tctgctgggt 3240
tgatgcgtat ggggttggaa aacagaatta cgcagcgatt ccattcggat acaatgtacc 3300
atgcagttgg acaaggcgcc ctgggtatag aaattagaaa gggtgacacc aagatgatga 3360
agattcttga cgaaatttgc gatctaaatg caactatatg ttgcctttcg gagcgtgctt 3420
tgatgagaac tttagagggg ggttgttccg ttcctattgg tgtggaatct aaatacaatg 3480
aagagactaa aaaattacta ttaaaggcca ttgtagttga cgttgaaggc acagaagcag 3540
tagaagacga aattgaaatg ctaatagaaa atgttaaaga agattccatg gcgtgtggta 3600
agatactagc tgaaagaatg attgccgatg gcgcaaagaa aattctggat gaaattaatt 3660
tagacagaat caaatgacac gtccgacggc ggcccacggg tcccaggcct cggagatccg 3720
tccccctttt cctttgtcga tatcatgtaa ttagttatgt cacgcttaca ttcacgccct 3780
ccccccacat ccgctctaac cgaaaaggaa ggagttagac aacctgaagt ctaggtccct 3840
atttattttt ttatagttat gttagtatta agaacgttat ttatatttca aatttttctt 3900
ttttttctgt acagacgcgt gtacgcatgt aacattatac tgaaaacctt gcttgagaag 3960
gttttgggac gctcgaaggc tttaatttgc aagctccccc cctcgaggtc gacggtatcg 4020
ataagcttga tatcgaattc ctgcagcccg ggggatccac tagttctaga gcggccgcca 4080
ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa 4140
tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 4200
ggagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgaggta actcacatta 4260
attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 4320
tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 4380
ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 4440
gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 4500
ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 4560
cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 4620
ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 4680
accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 4740
catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 4800
gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 4860
tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 4920
agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 4980
actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 5040
gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 5100
aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 5160
gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 5220
aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 5280
atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 5340
gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 5400
atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 5460
ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 5520
cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 5580
agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 5640
cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 5700
tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 5760
agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 5820
gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 5880
gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 5940
ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 6000
tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 6060
tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 6120
gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 6180
caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 6240
atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgaa 6300
cgaagcatct gtgcttcatt ttgtagaaca aaaatgcaac gcgagagcgc taatttttca 6360
aacaaagaat ctgagctgca tttttacaga acagaaatgc aacgcgaaag cgctatttta 6420
ccaacgaaga atctgtgctt catttttgta aaacaaaaat gcaacgcgag agcgctaatt 6480
tttcaaacaa agaatctgag ctgcattttt acagaacaga aatgcaacgc gagagcgcta 6540
ttttaccaac aaagaatcta tacttctttt ttgttctaca aaaatgcatc ccgagagcgc 6600
tatttttcta acaaagcatc ttagattact ttttttctcc tttgtgcgct ctataatgca 6660
gtctcttgat aactttttgc actgtaggtc cgttaaggtt agaagaaggc tactttggtg 6720
tctattttct cttccataaa aaaagcctga ctccacttcc cgcgtttact gattactagc 6780
gaagctgcgg gtgcattttt tcaagataaa ggcatccccg attatattct ataccgatgt 6840
ggattgcgca tactttgtga acagaaagtg atagcgttga tgattcttca ttggtcagaa 6900
aattatgaac ggtttcttct attttgtctc tatatactac gtataggaaa tgtttacatt 6960
ttcgtattgt tttcgattca ctctatgaat agttcttact acaatttttt tgtctaaaga 7020
gtaatactag agataaacat aaaaaatgta gaggtcgagt ttagatgcaa gttcaaggag 7080
cgaaaggtgg atgggtaggt tatataggga tatagcacag agatatatag caaagagata 7140
cttttgagca atgtttgtgg aagcggtatt cgcaatattt tagtagctcg ttacagtccg 7200
gtgcgttttt ggttttttga aagtgcgtct tcagagcgct tttggttttc aaaagcgctc 7260
tgaagttcct atactttcta gagaatagga acttcggaat aggaacttca aagcgtttcc 7320
gaaaacgagc gcttccgaaa atgcaacgcg agctgcgcac atacagctca ctgttcacgt 7380
cgcacctata tctgcgtgtt gcctgtatat atatatacat gagaagaacg gcatagtgcg 7440
tgtttatgct taaatgcgta cttatatgcg tctatttatg taggatgaaa ggtagtctag 7500
tacctcctgt gatattatcc cattccatgc ggggtatcgt atgcttcctt cagcactacc 7560
ctttagctgt tctatatgct gccactcctc aattggatta gtctcatcct tcaatgctat 7620
catttccttt gatattggat catactaaga aaccattatt atcatgacat taacctataa 7680
aaataggcgt atcacgaggc cctttcgtc 7709
<210> 5
<211> 6109
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
acaggcaaca cgcagatata ggtgcgacgt gaacagtgag ctgtatgtgc gcagctcgcg 60
ttgcattttc ggaagcgctc gttttcggaa acgctttgaa gttcctattc cgaagttcct 120
attctctaga aagtatagga acttcagagc gcttttgaaa accaaaagcg ctctgaagac 180
gcactttcaa aaaaccaaaa acgcaccgga ctgtaacgag ctactaaaat attgcgaata 240
ccgcttccac aaacattgct caaaagtatc tctttgctat atatctctgt gctatatccc 300
tatataacct acccatccac ctttcgctcc ttgaacttgc atctaaactc gacctctaca 360
ttttttatgt ttatctctag tattactctt tagacaaaaa aattgtagta agaactattc 420
atagagtgaa tcgaaaacaa tacgaaaatg taaacatttc ctatacgtag tatatagaga 480
caaaatagaa gaaaccgttc ataattttct gaccaatgaa gaatcatcaa cgctatcact 540
ttctgttcac aaagtatgcg caatccacat cggtatagaa tataatcggg gatgccttta 600
tcttgaaaaa atgcacccgc agcttcgcta gtaatcagta aacgcgggaa gtggagtcag 660
gcttttttta tggaagagaa aatagacacc aaagtagcct tcttctaacc ttaacggacc 720
tacagtgcaa aaagttatca agagactgca ttatagagcg cacaaaggag aaaaaaagta 780
atctaagatg ctttgttaga aaaatagcgc tctcgggatg catttttgta gaacaaaaaa 840
gaagtataga ttctttgttg gtaaaatagc gctctcgcgt tgcatttctg ttctgtaaaa 900
atgcagctca gattctttgt ttgaaaaatt agcgctctcg cgttgcattt ttgttttaca 960
aaaatgaagc acagattctt cgttggtaaa atagcgcttt cgcgttgcat ttctgttctg 1020
taaaaatgca gctcagattc tttgtttgaa aaattagcgc tctcgcgttg catttttgtt 1080
ctacaaaatg aagcacagat gcttcgttca ggtggcactt ttcggggaaa tgtgcgcgga 1140
acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 1200
ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt 1260
gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg 1320
ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg 1380
gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg 1440
agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag 1500
caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca 1560
gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg 1620
agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc 1680
gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg 1740
aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg 1800
ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac 1860
tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg 1920
tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg 1980
gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact 2040
atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa 2100
ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 2160
aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 2220
ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 2280
ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 2340
tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 2400
cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 2460
gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 2520
gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 2580
tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 2640
ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 2700
gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 2760
ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 2820
tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 2880
ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 2940
gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga 3000
acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg 3060
cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg 3120
aaagcgggca gtgagcgcaa cgcaattaat gtgagttacc tcactcatta ggcaccccag 3180
gctttacact ttatgcttcc ggctcctatg ttgtgtggaa ttgtgagcgg ataacaattt 3240
cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg 3300
gaacaaaagc tggagcttct ttgaaaagat aatgtatgat tatgctttca ctcatattta 3360
tacagaaact tgatgttttc tttcgagtat atacaaggtg attacatgta cgtttgaagt 3420
acaactctag attttgtagt gccctcttgg gctagcggta aaggtgcgca ttttttcaca 3480
ccctacaatg ttctgttcaa aagattttgg tcaaacgctg tagaagtgaa agttggtgcg 3540
catgtttcgg cgttcgaaac ttctccgcag tgaaagataa atgatcctaa tgtgtccgcg 3600
tttctagttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga 3660
aaaagtggca ccgagtcggt ggtgcttttt ttgtttttta tgtcttcgag tcatgtaatt 3720
agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg aaaaggaagg 3780
agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag 3840
aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa 3900
cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt taatttgcgg 3960
ccggtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta 4020
caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 4080
cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 4140
cgcagcctga atggcgaatg gcgcgacgcg ccctgtagcg gcgcattaag cgcggcgggt 4200
gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 4260
gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 4320
gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 4380
tagggtgatg gttcacgtag tgggccgttg aacattctta ggctggtcga atcatttaga 4440
cacgggcatc gtcctctcga aaggtggcat aggccactag tggatctgat atcacctaat 4500
aacttcgtat agcatacatt atacgaagtt atattaaggg ttctcgagag ctcgttttat 4560
ttaggttcta tcgaggagaa aaagcgacaa gaagagatag accatggata aactgattat 4620
gttctaaaca ctcctcagaa gctcatcgaa ctgtcatcct gcgtgaagat taaaatccaa 4680
cttagaaatt tcgagcttac ggagacaatc atatgggaga agcaattgga agatagaaaa 4740
aaggtactcg gtacataaat atatgtgatt ctgggtagaa gatcggtctg cattggatgg 4800
tggtaacgca tttttttaca cacattactt gcctcgagca tcaaatggtg gttattcgtg 4860
gatctatatc acgtgatttg cttaagaatt gtcgttcatg gtgacacttt tagctttgac 4920
atgattaagc tcatctcaat tgatgttatc taaagtcatt tcaactatct aagatgtggt 4980
tgtgattggg ccattttgtg aaagccagta cgccagcgtc aatacactcc cgtcaattag 5040
ttgcaccatg tccacaaaat catataccag tagagctgag actcatgcaa gtccggttgc 5100
atcgaaactt ttacgtttaa tggatgaaaa gaagaccaat ttgtgtgctt ctcttgacgt 5160
tcgttcgact gatgagctat tgaaacttgt tgaaacgttg ggtccataca tttgcctttt 5220
gaaaacacac gttgatatct tggatgattt cagttatgag ggtactgtcg ttccattgaa 5280
agcattggca gagaaataca agttcttgat atttgaggac agaaaattcg ccgatatcgg 5340
taacacagtc aaattacaat atacatcggg cgtttaccgt atcgcagaat ggtctgatat 5400
caccaacgcc cacggggtta ctggtgctgg tattgttgct ggcttgaaac aaggtgcgca 5460
agaggtcacc aaagaaccaa ggggattatt gatgcttgct gaattgtctt ccaagggttc 5520
tctagcacac ggtgaatata ctaagggtac cgttgatatt gcaaagagtg ataaagattt 5580
cgttattggg ttcattgctc agaacgatat gggaggaaga gaagaagggt ttgattggct 5640
aatcatgacc ccaggtgtag gtttagacga caaaggcgat gcattgggtc agcagtacag 5700
aaccgtcgac gaagttgtaa gtggtggatc agatatcatc attgttggca gaggactttt 5760
cgccaagggt agagatccta aggttgaagg tgaaagatac agaaatgctg gatgggaagc 5820
gtaccaaaag agaatcagcg ctccccatta attatacagg aaacttaata gaacaaatca 5880
catatttaat ctaatagcca cctgcattgg cacggtgcaa cactcacttc aacttcatct 5940
tacaaaagat cacgtgatct gttgtattgg gatctctaga cctaataact tcgtatagca 6000
tacattatac gaagttatat taagggttgt cgacctgcag cgtacgaagg tgcctattga 6060
tgatctggcg gaatgtctgc cgtgccatag ccatgccttc acatatagt 6109
<210> 6
<211> 5577
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tctagagaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct 60
gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 120
atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 180
caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 240
gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 300
ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 360
cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 420
taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 480
cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 540
acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 600
aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt 660
atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 720
atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 780
gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 840
gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 900
ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 960
ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt 1020
tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt 1080
accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt 1140
atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc 1200
cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa 1260
tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg 1320
tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt 1380
gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc 1440
agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt 1500
aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg 1560
gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac 1620
tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc 1680
gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt 1740
tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg 1800
aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag 1860
catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa 1920
acaaataggg gttccgcgca ctcgagacta tatgtgaagg catggctatg gcacggcaga 1980
cattccgcca gatcatcaat aggcacgcgg ccgccctgat gcggtatttt ctccttacgc 2040
atctgtgcgg tatttcacac cgcatagggt aataactgat ataattaaat tgaagctcta 2100
atttgtgagt ttagtataca tgcatttact tataatacag ttttttagtt ttgctggccg 2160
catcttctca aatatgcttc ccagcctgct tttctgtaac gttcaccctc taccttagca 2220
tcccttccct ttgcaaatag tcctcttcca acaataataa tgtcagatcc tgtagagacc 2280
acatcatcca cggttctata ctgttgaccc aatgcgtctc ccttgtcatc taaacccaca 2340
ccgggtgtca taatcaacca atcgtaacct tcatctcttc cacccatgtc tctttgagca 2400
ataaagccga taacaaaatc tttgtcgctc ttcgcaatgt caacagtacc cttagtatat 2460
tctccagtag atagggagcc cttgcatgac aattctgcta acatcaaaag gcctctaggt 2520
tcctttgtta cttcttctgc cgcctgcttc aaaccgctaa caatacctgg gcccaccaca 2580
ccgtgtgcat tcgtaatgtc tgcccattct gctattctgt atacacccgc agagtactgc 2640
aatttgactg tattaccaat gtcagcaaat tttctgtctt cgaagagtaa aaaattgtac 2700
ttggcggata atgcctttag cggcttaact gtgccctcca tggaaaaatc agtcaagata 2760
tccacatgtg tttttagtaa acaaattttg ggacctaatg cttcaactaa ctccagtaat 2820
tccttggtgg tacgaacatc caatgaagca cacaagtttg tttgcttttc gtgcatgata 2880
ttaaatagct tggcagcaac aggactagga tgagtagcag cacgttcctt atatgtagct 2940
ttcgacatga tttatcttcg tttcctgcag gtttttgttc tgtgcagttg ggttaagaat 3000
actgggcaat ttcatgtttc ttcaacacta catatgcgta tatataccaa tctaagtctg 3060
tgctccttcc ttcgttcttc cttctgttcg gagattaccg aatcaaaaaa atttcaaaga 3120
aaccgaaatc aaaaaaaaga ataaaaaaaa aatgatgaat tgaattgaaa agctgtggta 3180
tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg 3240
ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 3300
gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 3360
gcgacagctg cacctttcga gaggacgatg cccgtgtcta aatgattcga ccagcctaag 3420
aatgttcaac ccctcactaa agggaacaaa agctggagct tctttgaaaa gataatgtat 3480
gattatgctt tcactcatat ttatacagaa acttgatgtt ttctttcgag tatatacaag 3540
gtgattacat gtacgtttga agtacaactc tagattttgt agtgccctct tgggctagcg 3600
gtaaaggtgc gcattttttc acaccctaca atgttctgtt caaaagattt tggtcaaacg 3660
ctgtagaagt gaaagttggt gcgcatgttt cggcgttcga aacttctccg cagtgaaaga 3720
taaatgatcc tctcgaagtg gtcacgtgcg ttttagagct agaaatagca agttaaaata 3780
aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtggtgctt tttttgtttt 3840
ttatgtcttc gagtcatgta attagttatg tcacgcttac gttcacgccc tccacgcatt 3900
taagcataaa cacgcactat gccgttcttc tcatgtatat atatatacag gcaacacgca 3960
gatataggtg cgacgtgaac agtgagctgt atgtgcgcag ctcgcgttgc attttcggaa 4020
gcgctcgttt tcggaaacgc tttgaagttc ctattccgaa gttcctattc tgtagaaagt 4080
ataggaactt cagagcgctt ttgaaaacca aaagcgctct gaagacgcac tttcaaaaaa 4140
ccaaaaacgc accggactgt aacgagctac taaaatattg cgaataccgc ttccacaaac 4200
attgctcaaa agtatctctt tgctatatat ctctgtgcta tatccctata taacctaccc 4260
atccaccttt cgctccttga acttgcatct aaactcgacc tctacatttt ttatgtttat 4320
ctctagtatt actctttaga caaaaaaatt gtagtaagaa ctattcatag agtgaatcga 4380
aaacaatacg aaaatgtaaa catttcctat acgtagtata tagagacaaa atagaagaaa 4440
ccgttcataa ttttctgacc aatgaagaat catcaacgct atcactttct gttcacaaag 4500
tatgcgcaat ccacatcggt atagaatata atcggggatg cctttatctt gaaaaaatgc 4560
acccgcagct tcgctagtaa tcagtaaacg cgggaagtgg agtcaggctt tttttatgga 4620
agagaaaata gacaccaaag tagccttctt ctaaccttaa cggacctaca gtgcaaaaag 4680
ttatcaagag actgcattat agagcgcaca aaggagaaaa aaagtaatct aagatgcttt 4740
gttagaaaaa tagcgctctc gggatgcatt tttgtagaac aaaaaagaag tatagattct 4800
ttgttggtaa aatagcgctc tcgcgttgca tttctgttct gtaaaaatgc agctcagatt 4860
ctttgtttga aaaattagcg ctctcgcgtt gcatttttgt tttacaaaaa tgaagcacag 4920
attcttcgtt ggtaaaatag cgctttcgcg ttgcatttct gttctgtaaa aatgcagctc 4980
agattctttg tttgaaaaat tagcgctctc gcgttgcatt tttgttctac aaaatgaagc 5040
acagatgctt cgttggaggg cgtgaacgta agcgtgacat aactaattac atgactcgaa 5100
gacataaaaa acaaaaaaag caccaccgac tcggtgccac tttttcaagt tgataacgga 5160
ctagccttat tttaacttgc tatttctagc tctaaaacgt gaatcaactg cacataccga 5220
tcatttatct ttcactgcgg agaagtttcg aacgccgaaa catgcgcacc aactttcact 5280
tctacagcgt ttgaccaaaa tcttttgaac agaacattgt agggtgtgaa aaaatgcgca 5340
cctttaccgc tagcccaaga gggcactaca aaatctagag ttgtacttca aacgtacatg 5400
taatcacctt gtatatactc gaaagaaaac atcaagtttc tgtataaata tgagtgaaag 5460
cataatcata cattatcttt tcaaagaagc tccagctttt gttcccttta gtgagggtat 5520
tcacgtagac ggataggtat agccagacat cagcagcata cttcgggaac cgtaggc 5577
Claims (7)
1. Saccharomyces cerevisiae TIB H1 with preservation registration number CGMCC No.21000.
2. Use of the saccharomyces cerevisiae TIB H1 as defined in claim 1 for the production of heme.
3. A method for producing hemoglobin, comprising the steps of: fermenting and culturing the saccharomyces cerevisiae TIB H1 of claim 1.
4. The recombinant Saccharomyces cerevisiae is obtained by introducing glutamyl-t RNA synthetase encoding gene, glutamyl tRNA reductase encoding gene, glutamyl-1-semialdehyde transaminase encoding gene and porcupro-deaminase encoding gene into starting Saccharomyces cerevisiae.
5. The recombinant Saccharomyces cerevisiae yeast according to claim 4, wherein: the gene coding for glutamyl-t RNA synthetase, the gene coding for glutamyl tRNA reductase and the gene coding for glutamyl-1-semialdehyde transaminase are incorporated into the Saccharomyces cerevisiae genome.
6. Use of the recombinant Saccharomyces cerevisiae described in claims 4 or 5 for the production of heme.
7. A method of producing heme comprising the steps of: and (3) carrying out fermentation culture on the recombinant saccharomyces cerevisiae of claim 4 or 5.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004701A (en) * | 2014-06-18 | 2014-08-27 | 江南大学 | Method for building high-yield 5-aminolevulinic acid escherichia coli engineering strains |
| CN105745332A (en) * | 2013-09-11 | 2016-07-06 | 非凡食品有限公司 | Secretion of heme-containing polypeptides |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105745332A (en) * | 2013-09-11 | 2016-07-06 | 非凡食品有限公司 | Secretion of heme-containing polypeptides |
| CN104004701A (en) * | 2014-06-18 | 2014-08-27 | 江南大学 | Method for building high-yield 5-aminolevulinic acid escherichia coli engineering strains |
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| 李德茂 ; 曾艳 ; 周桔 ; 王钦宏 ; 孙际宾 ; 江会锋 ; 马延和 ; .生物制造食品原料市场准入政策比较及对我国的建议.中国科学院院刊.(第08期),全文. * |
| 陈丹园 ; 沈云杰 ; 杨燕 ; 唐蕾 ; .关键酶基因的过表达与环境因素对大肠杆菌血红素合成的调控.食品与发酵工业.(第11期),全文. * |
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