CN113788881B - Cysteine transporter mutants and their use in the production of L-cysteine - Google Patents
Cysteine transporter mutants and their use in the production of L-cysteine Download PDFInfo
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Abstract
本发明综合利用随机突变、饱和突变和组合突变方法，得到了多种L‑半胱氨酸外排能力增强的转运蛋白EamB突变体，相对于野生的EamB转运蛋白，存在I83M、G156A、N157M、G156S、N157S中一个或多个突变。在具体实施中，分别过表达上述EamB突变体，底盘菌株的L‑半胱氨酸发酵产量可提升约30%~70%，并且不会对菌体正常生长造成影响。因此，本发明提供的有益突变体可以为工业化高效生产L‑半胱氨酸及下游代谢产物奠定了良好的基础。
The invention comprehensively utilizes random mutation, saturation mutation and combined mutation methods to obtain a variety of transporter EamB mutants with enhanced L-cysteine efflux capacity. Compared with the wild EamB transporter, there are I83M, G156A, N157M, One or more mutations in G156S, N157S. In a specific implementation, the above-mentioned EamB mutants were respectively overexpressed, the L-cysteine fermentation yield of the chassis strain could be increased by about 30%-70%, and the normal growth of the bacterial cells would not be affected. Therefore, the beneficial mutants provided by the present invention can lay a good foundation for the efficient industrial production of L-cysteine and downstream metabolites.
Description
Technical Field
The invention belongs to the field of bioengineering, and particularly relates to an L-cysteine transporter mutant with enhanced efflux capability and application thereof in preparation of L-cysteine.
Background
L-cysteine is an important sulfur-containing amino acid and has wide application in the fields of food, medicine, cosmetics, feed industry and the like, wherein the application of the food additive accounts for about six components of the yield of cysteine. In recent years, researchers have developed many new cysteine products, such as bread fermentation modifiers, cysteine oral tablets, acetylcysteine eye drops, health chewing gums, and the like. With the continuous development of the terminal application of cysteine, the global cysteine market demand is rapidly increased, and the market prospect is very bright. The cysteine yield of China accounts for about 2/3 of the global total yield, the annual output is kept above 80%, and the cysteine production plays an important role in the international market with intense competition.
At present, most of the L-cysteine is obtained by industrial hydrolysis of human hair and poultry feathers, but is often associated with low yields and serious environmental and safety problems. In recent years, the microbial fermentation production of L-cysteine is receiving more and more attention, and compared with the current proteolysis preparation method, the microbial fermentation method provides a substitute production strategy which is safe in food, high in economic benefit and environment-friendly. The development of systemic metabolic engineering and synthetic biology has provided various tools and techniques for engineering microbial strains to efficiently produce bio-based chemicals. In the strategies, the transfer capacity of the optimized substrate, metabolic intermediate or metabolite is modified, so that good opportunities are provided for the strain to enhance substrate absorption, overcome metabolic inhibition and protect cells from toxic compounds, and the method is an effective method for improving the robustness and production efficiency of a microbial cell factory. Unfortunately, most natural transporters have inherent limitations such as low substrate transport activity, poor substrate affinity, and unexpected substrate/product inhibitory effects that prevent their widespread use in industrial biotechnology.
The construction of efficient microbial cell factories for the industrial fermentative production of L-cysteine is challenging due to the cytotoxic and complex feedback effects of high concentrations of L-cysteine. To mitigate their cytotoxicity, microorganisms have evolved strategies to maintain low levels of intracellular L-cysteine, for example, by reducing intracellular L-cysteine concentrations through both degradation and efflux pathways. Therefore, it is necessary to promote the L-cysteine efflux system for the fermentative production of L-cysteine by microorganisms. In E.coli, several transporters have been shown to be closely related to the export of L-cysteine, including the inner membrane transporters EamB (YfiK), Eama (Yded), Bcr, CydDC and TolC. As a main L-cysteine efflux protein, the overexpression of EamB can promote the growth of Escherichia coli cellsOThe secretion process of the-acetylserine and the L-cysteine has important significance in improving the fermentation production of the L-cysteine by the engineering strain. However, the lack of research on the structure and transport mechanism of EamB transporter increases the difficulty of improving its transport efficiency through design and modification.
Disclosure of Invention
In view of the above requirements, the primary object of the present invention is to provide an L-cysteine transporter mutant, so as to improve the substrate transport capacity thereof, and facilitate the microbial fermentation production of metabolites such as L-cysteine.
The invention provides a polypeptide derived fromEscherichia coliEamB mutant of L-cysteine transporter MG1655, characterized in that, relative to the amino acid sequence shown in SEQ ID No.2, there are only mutations at one or more of position 83 from isoleucine I to methionine M, position 156 from glycine G to alanine A or serine S, and position 157 from asparagine N to methionine M or serine S.
In a preferred embodiment, only one of the positions 83, 156, 157, 83, N, to M, is mutated from isoleucine I to methionine M, glycine G to serine S, and asparagine N to serine S; only combined mutations with isoleucine I to methionine M at position 83 and glycine G to serine S at position 156 were present; only combined mutations with isoleucine I to methionine M at position 83 and asparagine N to serine S at position 157 exist; only combined mutations with glycine G to serine S at position 156 and asparagine N to serine S at position 157 were present; there are only combined mutations with isoleucine I to methionine M at position 83, glycine G to serine S at position 156 and asparagine N to serine S at position 157.
More preferably, there is only a combined mutation of glycine G to serine S at position 156 and asparagine N to serine S at position 157, most preferably the amino acid sequence thereof is shown in SEQ ID No. 3.
The invention further provides a coding gene of the EamB mutant of the L-cysteine transporter.
In a preferred embodiment, the nucleotide sequence of the gene encoding the EamB mutant of the L-cysteine transporter is obtained by mutation based on the nucleotide sequence shown in SEQ ID No. 1. In a more preferred embodiment, the nucleotide sequence of the gene encoding the EamB mutant of the L-cysteine transporter is shown as SEQ ID No.4 (encoding I83M-G156S), SEQ ID No.5 (encoding I83M-N157S), SEQ ID No.6 (encoding G156S-N157S) and SEQ ID No.7 (encoding I83M-G156S-N157S).
The invention also provides an expression vector and a host cell containing the EamB mutant encoding gene of the L-cysteine transporter.
The invention also provides the application of the EamB mutant of the L-cysteine transporter or the coding gene thereof or the recombinant host cell in the preparation of L-cysteine.
In a specific embodiment, the L-cysteine is produced by culturing a microbial cell into which an expression vector containing a gene encoding the EamB mutant of the L-cysteine transporter has been introduced, and further comprising the steps of collecting and purifying the L-cysteine.
In a more specific embodiment, the expression vector is a low copy plasmid pMW118, which is used for fermentative production of metabolites such as L-cysteine by constructing a recombinant plasmid containing a gene encoding an EamB mutant and introducing it into microbial cells. In a more preferred embodiment, the microbial cell is Escherichia coli or Corynebacterium glutamicum.
The invention further provides a recombinant bacterium for enhancing the L-cysteine production capacity, which is characterized in that the encoding gene is over-expressed in the original strain. Preferably, L-cysteine degradation pathway genes are knocked out from the starting straintanAAndyhaMsimultaneous overexpression of L-cysteine synthetic pathway genescysE. In a specific embodiment, the starting strain is Escherichia coli, Corynebacterium glutamicum, Pantoea ananatis, Bacillus subtilis, preferably Escherichia coli, Corynebacterium glutamicum.
The invention is realized by the following technical idea that Escherichia coli is usedE. coliMG1655 genome is template, EamB coding gene mutation library is obtained by error-prone PCR random mutation method, One-Step high-efficiency seamless Cloning technology (Clonexpress II One Step Cloning Kit, Vazyme Biote)ch, China) to construct it on an expression vector to obtain a recombinant plasmid library containing the EamB mutant encoding gene of the L-cysteine transporter. Introduction of the recombinant plasmid library intoE. coli MG 1655L-cysteine Transporter deleted background bacteria: (∆eamA ∆eamB ∆bcr) Through solid plate primary screening and 96-well plate secondary screening, beneficial mutants capable of improving the resistance of a background strain to L-cysteine are searched, and finally three mutants with improved transport activity are obtained through screening, wherein the mutation sites of the mutants are I83M, G156A and N157M respectively. Two mutants, G156S and N157S, with further improved transport activity were obtained by high throughput screening, followed by saturation mutagenesis of each amino acid residue site. Finally, the beneficial mutants I83M, G156S and N157S obtained are subjected to combined mutation to obtain the optimal mutant combination G156S-N157S, and the mutant can endow Chassis bacteria with the highest L-cysteine tolerance and productivity.
The invention has the beneficial effects that through mutation and screening of EamB encoding gene of L-cysteine transporter from escherichia coli, the mutant which can obviously endow the strain with enhanced L-cysteine tolerance and production capacity is obtained, and the L-cysteine fermentation yield of the chassis strain can be improved by about 30-70%. Therefore, the EamB mutant of the cysteine transporter provided by the invention can lay a good foundation for producing L-cysteine and downstream products by high-efficiency fermentation.
Drawings
FIG. 1 resistant growth test of L-cysteine transporter EamB mutants.
FIG. 2 fermentation analysis of L-cysteine transporter EamB mutants.
Detailed Description
The invention will be described in further detail with reference to specific embodiments and drawings for better understanding of the objects, technical solutions and advantages thereof, but the invention should not be construed as being limited thereto. The experimental methods used in the examples are all conventional methods unless otherwise specified. Materials, reagents and the like used in examples are commercially available unless otherwise specified.
Example 1 construction of L-cysteine Transporter EamB mutant library
The invention adopts EasyTaq DNA polymerase with low fidelity toE. coliMG1655 genome as template, and primer P1 (5'-TATGACCATGGTGACACCGACCCTTTTAAGTGCTTTTTGGACTTAC-3') and P2 (5'-GGGCGACCTCTTAATAGAAAATGCGTACCGCGCAATAGACCAGCA-3') were used to obtain by error-prone PCR methodeamBEncoding a library of gene mutations. By adding magnesium ions and manganese ions with certain concentration into a PCR reaction system, the fidelity in the PCR amplification process is further reduced, and the obtained product is controlledeamBThe coding gene contains 2-3 point mutations. The error-prone PCR system adopted by the invention is as follows: 5 μ L10 × EasyTaq buffer, 0.2 μ M upstream primer P1, 0.2 μ M downstream primer P2, 200 μ M dNTPs, 0.8 mM MnCl2、6 mM MgSO 450 ng of template DNA and 1. mu.L of EasyTaq DNA polymerase (TransGen Biotech, China) were added with sterile water to prepare a 50. mu.L system. The PCR reaction program is: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30 s; annealing at 58 ℃ for 30 s; extending for 2 min at 72 ℃ and circulating for 35 times; extending for 5min at 72 ℃, and storing at 4 ℃.
The invention adopts Phusion High-Fidelity DNA polymerase with High Fidelity, takes pMW118 plasmid as a template, and utilizes primers P3 (5'-TTTCTATTAAGAGGTCGCCCTCTTCCGCTTAGTAACTTGCTACTTAAG-3') and P4 (5'-TCGGTGTCACCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCC-3') to obtain the pMW118 plasmid skeleton by a common PCR method. In order to ensure the sequence correctness in the plasmid backbone amplification, the PCR reaction is carried out by adopting the high-efficiency super-fidelity DNA polymerase which is commercially used at present, and the system is as follows: mu.L of 5 XPHUSION HF buffer, 0.5. mu.M forward primer P3, 0.5. mu.M reverse primer P4, 200. mu.M dNTPs, 3% DMSO, 50 ng template DNA, 0.5. mu.L Phusion High-Fidelity DNA polymerase (Thermo Scientific, USA), sterile water was added to make up the 50. mu.L system. The PCR reaction program is: pre-denaturation at 98 ℃ for 1 min; denaturation at 98 ℃ for 10 s; annealing at 62 ℃ for 20 s; extending for 3 min at 72 ℃ and circulating for 35 times; extending for 5min at 72 ℃, and storing at 4 ℃.
P was cloned based on Clonexpress One-Step directed Cloning seamless Cloning kit (Clonexpress II One Step Cloning kit, Vazyme Biotech, China)Obtained by CReamBConnecting the coding gene mutation library with pMW118 plasmid skeleton to obtain a plasmid containingeamBRecombinant expression plasmid pMW118-eamB of coding gene different mutantmut. The connecting system is as follows: 4 mu L of 5 XCE II buffer solution, 50-200 ng of gene mutation fragment, 50-200 ng of plasmid skeleton and 2 mu L of Exnase II. After the system configuration is finished, the reaction is carried out for 30 min at 37 ℃, and after the reaction is finished, the reaction product is placed in an ice water bath for cooling for 5 min. The plasmid library obtained above was then introduced into E.coli DH 5. alpha. competent cells according to a conventional E.coli heat shock transformation method.
Example 2 screening of L-cysteine Transporter EamB mutant library
The basic basis of the high-throughput screening process is that high-concentration L-cysteine has obvious cytotoxicity, the recombinant plasmid mutation library in the embodiment 1 is introduced into a background bacterium with deletion of an Escherichia coli L-cysteine transporter, a strain is subjected to L-cysteine tolerance screening to obtain a beneficial mutant, the mutant with stronger transport capacity can discharge excessive L-cysteine to the outside of the cell, and the strain has enhanced tolerance capacity under the pressure of high-concentration L-cysteine. The Escherichia coli L-cysteine transporter deletion bacterium is characterized inE. coliThree potential L-cysteine transporters EamA, EamB and Bcr are knocked out in MG1655, and the genotype of the strain is as follows:∆eamA ∆eamB ∆bcr. The EamB mutant library is screened on the basis of the Escherichia coli L-cysteine transporter deletion bacterium, so that the problems of false positive and the like in the screening process can be effectively reduced.
The specific screening steps are as follows: due to the cytotoxic effect of L-cysteine, EamB mutants with enhanced efflux capacity will confer stronger L-cysteine resistance to the strain. Coli L-cysteine transporter deletion background bacteria containing the library of recombinant plasmid mutations described in example 1 were plated on pressure agar plates of selective antibiotics and L-cysteine. Randomly picking colonies from selective pressure agar plates, inoculating the colonies into 96-well microtiter plates containing 200. mu.L LB medium per well, and culturing overnight at 37 ℃ and 800 rpm; mu.L of overnight culture was then transferred to 96-well microtiter plates containing 200. mu.L of fresh M9-glucose medium (to which 1 mM L-cysteine had been added) and incubated at 37 ℃ for 14 hours with shaking at 800 rpm. The pMW118-eamB recombinant plasmid containing the natural EamB transporter is introduced into an Escherichia coli L-cysteine transporter deletion background bacterium to be used as a control of a screening experiment. And selecting clones with better growth state than that of the control bacteria under the condition of L-cysteine screening pressure, and re-screening by using higher L-cysteine pressure. After two rounds of screening procedures, three different EamB beneficial mutants were finally obtained, i.e., I83M, G156A and N157M, as determined by Sanger sequencing.
The LB culture medium comprises the following components: 1% yeast extract, 2% tryptone, 1% NaCl. The M9-glucose medium comprises the following components: 0.8% glucose, 12.8 g/L Na2HPO4·7H2O，3 g/L KH2PO4，0.5 g/L NaCl，1 g/L NH4Cl，2 mM MgSO4，0.1 mM CaCl2。
Example 3 site-directed saturation mutagenesis of L-cysteine Transporter EamB mutants
Aiming at the three EamB beneficial mutants (I83M, G156A and N157M) obtained in example 2, random degenerate primers NNK (wherein N represents A, C, G or T, and K represents G or T) are designed at the amino acid residue positions 83, 156 and 157 by adopting a strategy of site-directed saturation mutation, and a recombinant plasmid library containing the site-directed saturation mutation is obtained by a PCR method. By adopting the screening strategy and method in example 2, the recombinant plasmid library containing the saturation mutation at the specific site was introduced into E.coli L-cysteine transporter-deficient bacteria, and screening was performed under the L-cysteine pressure condition. Through Sanger sequencing, the optimal mutation types are finally obtained at the 83 th, 156 th and 157 th amino acid residue positions, namely the 83 th combined mutation of isoleucine I to methionine M, the 156 th combined mutation of glycine G to serine S and the 157 th combined mutation of asparagine N to serine S.
The degenerate primer NNK is respectively as follows:
I83-Sat-F：5'-TTTTGAGTTGGGCGGGGGCGGCATATNNKGTCTGGCTG-3'
I83-Sat-R：5'-CCCGCCCAACTCAAAAGGTGTACCGCT-3'
G156-Sat-F：5'-TGGCGATGATTGGGACGTTTNNKAATGTGTGCTGG-3'
G156-Sat-R：5'-CGTCCCAATCATCGCCAGCAAAACGCTGAC-3'
G157-Sat-F：5'-TGGCGATGATTGGGACGTTTGGCNNKGTGTGCTGG-3'
G157-Sat-R：5'-CGTCCCAATCATCGCCAGCAAAACGCTGAC-3'
the influence of EamB and different single mutants thereof on the growth of the strain under the condition of L-cysteine pressure is tested. As shown in figure 1, the results show that all EamB mutants can remarkably endow the strains with enhanced L-cysteine tolerance, and the biomass of the recombinant strains under the pressure of 1 mM L-cysteine is improved by about 1.7-2.4 times compared with the wild type natural L-cysteine transporter EamB.
Example 4 combinatorial mutations at beneficial mutation sites further enhance Transporter Activity
In order to further improve the efflux capability of the transporter, the optimal mutation sites obtained in the above, I83M, G156S and N157S, are subjected to combined mutation. The Site-Directed Mutagenesis related to the invention is completed by adopting a QuikChange Site-Directed Mutagenesis Kit (QuikChange Site-Directed Mutagenesis Kit), and related primers are as follows:
I83M-F：5'-TTTTGAGTTGGGCGGGGGCGGCATATATGGTCTGGCTG-3'
I83M-R：5'-CCCGCCCAACTCAAAAGGTGTACCGCT-3'
G156S-F：5'-TGGCGATGATTGGGACGTTTAGCAATGTGTGCTGG-3'
G156S-R：5'-CGTCCCAATCATCGCCAGCAAAACGCTGAC-3'
G157S-F：5'-TGGCGATGATTGGGACGTTTGGCAGTGTGTGCTGG-3'
G157S-R：5'-CGTCCCAATCATCGCCAGCAAAACGCTGAC-3'
the QuikChange site-directed mutant is: mu.L of 5 XPHUSION HF buffer, 0.5. mu.M forward primer, 0.5. mu.M reverse primer, 200. mu.M dNTPs, 3% DMSO, 50 ng template DNA, 0.5. mu.L Phusion DNA polymerase (Thermo Scientific, USA) was added and the 50. mu.L system was replenished with sterile water. The PCR reaction program is: pre-denaturation at 98 ℃ for 1 min; denaturation at 98 ℃ for 10 s; annealing at 62 ℃ for 20 s; extending for 3 min at 72 ℃ and circulating for 35 times; extending for 5min at 72 ℃, and storing at 4 ℃.
Single colonies were picked for sequencing verification and finally 3 double mutation combinations (I83M-G156S, I83M-N157S, G156S-N157S) and 1 triple mutation combination (I83M-G156S-N157S) were constructed. At the same time, the effect of the combination of mutations described above on the growth of the strain under L-cysteine stress conditions was tested. The results showed that the biomass of the recombinant strains containing the four combinatorial mutants was increased by about 2.1-fold, 2.6-fold, 2.9-fold and 2.4-fold at 1 mM L-cysteine pressure, respectively, relative to the wild-type native L-cysteine transporter EamB. The results show that the combination of the two mutations G156S-N157S is the optimal combination, and the mutant can endow the strain with the strongest L-cysteine tolerance.
Example 5 fermentation test of L-cysteine Transporter EamB mutants
The beneficial mutants I83M, G156S, N157S, I83M-G156S, I83M-N157S, G156S-N157S and I83M-G156S-N157S obtained in the above examples are introduced into an Escherichia coli basic engineering bacterium E.coli-CYS with certain cysteine production capacity according to a conventional Escherichia coli heat shock transformation method for fermentation test. coli-CYS is inE. coliKnocking out two L-cysteine degradation pathway genes in W3110tanAAndyhaMsimultaneously express an L-cysteine synthetic pathway genecysEThe strain has certain L-cysteine production capacity under the fermentation condition.
The strain obtained above was inoculated into LB medium overnight, and after overnight culture, it was inoculated into a fresh fermentation medium (50 g/L glucose, 12 g/L K) in an inoculum size of 10%2HPO4，3 g/L KH2PO4，0.1 g/L NaCl，5 g/L (NH4)2SO4，0.3 g/L MgSO4·7H2O，0.015 g/L CaCl2·2H2O，0.002 g/L FeSO4·7H2O, 1 g/L sodium citrate, 5 mg/L microorganism B1 and 1 mL/L trace element solution) were subjected to shake flask fermentation. Fermentation of bacterial strainsAfter 42 h of culture, samples were taken for determination of the L-cysteine content.
As shown in FIG. 2, the beneficial mutants obtained in the above examples can improve the L-cysteine production capacity of the basic engineering bacteria to different degrees, wherein the optimum mutation combination G156S-N157S can improve the L-cysteine fermentation content of the basic engineering bacteria by about 70%. Therefore, the EamB mutants of the L-cysteine transporter disclosed by the invention have important application values in the efficient production of L-cysteine by microbial Chassis bacteria such as escherichia coli and the like.
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> cysteine transporter mutant and application thereof in production of L-cysteine
<130>
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cgcgtgctggcagggatgagtctgggatttttgattgtgatgttactgtgtgcgggcatt
tcattttcactggcagtgattgacccggcagcggtacaccttttgagttgggcgggggcg
gcatatattgtctggctggcgtggaaaatcgccaccagcccaacaaaggaagacggactt
caggcaaaaccaatcagcttttgggccagctttgctttgcagtttgtgaacgtcaaaatc
attttgtacggtgttacggcactgtcgacgtttgttctgccgcaaacacaggcgttaagc
tgggtagttggcgtcagcgttttgctggcgatgattgggacgtttagcagtgtgtgctgg
gcgctggcggggcatctgtttcagcgattgtttcgccagtatggtcgccagttaaatatc
gtgcttgccctgttgctggtctattgcgcggtacgcattttctattaa 588
<210> 7
<211> 588
<212> DNA
<213> Escherichia coli MG1655
<400> 7
gtgacaccgacccttttaagtgctttttggacttacaccctgattaccgctatgacgcca
ggaccgaacaatattctcgcccttagctctgctacgtcgcatggatttcgtcaaagtacc
cgcgtgctggcagggatgagtctgggatttttgattgtgatgttactgtgtgcgggcatt
tcattttcactggcagtgattgacccggcagcggtacaccttttgagttgggcgggggcg
gcatatatggtctggctggcgtggaaaatcgccaccagcccaacaaaggaagacggactt
caggcaaaaccaatcagcttttgggccagctttgctttgcagtttgtgaacgtcaaaatc
attttgtacggtgttacggcactgtcgacgtttgttctgccgcaaacacaggcgttaagc
tgggtagttggcgtcagcgttttgctggcgatgattgggacgtttagcagtgtgtgctgg
gcgctggcggggcatctgtttcagcgattgtttcgccagtatggtcgccagttaaatatc
gtgcttgccctgttgctggtctattgcgcggtacgcattttctattaa 588
<210> 8
<211> 46
<212> DNA
<213> Artificial sequence
<400> 8
TATGACCATGGTGACACCGACCCTTTTAAGTGCTTTTTGGACTTAC 46
<210> 9
<211> 45
<212> DNA
<213> Artificial sequence
<400> 9
GGGCGACCTCTTAATAGAAAATGCGTACCGCGCAATAGACCAGCA 45
<210> 10
<211> 48
<212> DNA
<213> Artificial sequence
<400> 10
TTTCTATTAAGAGGTCGCCCTCTTCCGCTTAGTAACTTGCTACTTAAG 48
<210> 11
<211> 47
<212> DNA
<213> Artificial sequence
<400> 11
TCGGTGTCACCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCC 47
<210> 12
<211> 38
<212> DNA
<213> Artificial sequence
<400> 12
TTTTGAGTTGGGCGGGGGCGGCATATNNKGTCTGGCTG 38
<210> 13
<211> 27
<212> DNA
<213> Artificial sequence
<400> 13
CCCGCCCAACTCAAAAGGTGTACCGCT 27
<210> 14
<211> 35
<212> DNA
<213> Artificial sequence
<400> 14
TGGCGATGATTGGGACGTTTNNKAATGTGTGCTGG 35
<210> 15
<211> 30
<212> DNA
<213> Artificial sequence
<400> 15
CGTCCCAATCATCGCCAGCAAAACGCTGAC 30
<210> 16
<211> 35
<212> DNA
<213> Artificial sequence
<400> 16
TGGCGATGATTGGGACGTTTGGCNNKGTGTGCTGG 35
<210> 17
<211> 30
<212> DNA
<213> Artificial sequence
<400> 17
CGTCCCAATCATCGCCAGCAAAACGCTGAC 30
<210> 18
<211> 38
<212> DNA
<213> Artificial sequence
<400> 18
TTTTGAGTTGGGCGGGGGCGGCATATATGGTCTGGCTG 38
<210> 19
<211> 27
<212> DNA
<213> Artificial sequence
<400> 19
CCCGCCCAACTCAAAAGGTGTACCGCT 27
<210> 20
<211> 35
<212> DNA
<213> Artificial sequence
<400> 20
TGGCGATGATTGGGACGTTTAGCAATGTGTGCTGG 35
<210> 21
<211> 30
<212> DNA
<213> Artificial sequence
<400> 21
CGTCCCAATCATCGCCAGCAAAACGCTGAC 30
<210> 22
<211> 35
<212> DNA
<213> Artificial sequence
<400> 22
TGGCGATGATTGGGACGTTTGGCAGTGTGTGCTGG 35
<210> 23
<211> 30
<212> DNA
<213> Artificial sequence
<400> 23
CGTCCCAATCATCGCCAGCAAAACGCTGAC 30
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| WO2013148559A1 (en) * | 2012-03-24 | 2013-10-03 | Carnegie Institution Of Washington | Pathogen resistant plant cells and methods of making |
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