CN113788881A - Cysteine transporter mutant and application thereof in production of L-cysteine - Google Patents

Cysteine transporter mutant and application thereof in production of L-cysteine Download PDF

Info

Publication number
CN113788881A
CN113788881A CN202111344079.6A CN202111344079A CN113788881A CN 113788881 A CN113788881 A CN 113788881A CN 202111344079 A CN202111344079 A CN 202111344079A CN 113788881 A CN113788881 A CN 113788881A
Authority
CN
China
Prior art keywords
cysteine
eamb
serine
mutant
asparagine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202111344079.6A
Other languages
Chinese (zh)
Other versions
CN113788881B (en
Inventor
刘君
刘光辉
徐宁
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tianjin National Synthetic Biotechnology Innovation Center Co ltd
Original Assignee
Tianjin Institute of Industrial Biotechnology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tianjin Institute of Industrial Biotechnology of CAS filed Critical Tianjin Institute of Industrial Biotechnology of CAS
Priority to CN202111344079.6A priority Critical patent/CN113788881B/en
Publication of CN113788881A publication Critical patent/CN113788881A/en
Application granted granted Critical
Publication of CN113788881B publication Critical patent/CN113788881B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/12—Methionine; Cysteine; Cystine

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention comprehensively utilizes random mutation, saturation mutation and combined mutation methods to obtain various EamB mutants of the transporter with enhanced L-cysteine efflux capability, and compared with the wild EamB transporter, the EamB transporter has one or more mutations in I83M, G156A, N157M, G156S and N157S. In the specific implementation, the EamB mutants are respectively overexpressed, the fermentation yield of the L-cysteine of the chassis strain can be improved by about 30-70%, and the normal growth of thalli cannot be influenced. Therefore, the beneficial mutant provided by the invention can lay a good foundation for the industrial high-efficiency production of L-cysteine and downstream metabolites.

Description

Cysteine transporter mutant and application thereof in production of L-cysteine
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.
Due to the high concentration of L-moietyCystine has cytotoxicity and complex feedback effect, and the construction of a high-efficiency microbial cell factory for industrial fermentation production of L-cysteine has a great challenge. 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 the template, utilize error-prone PCR random mutation method to obtain EamB coding gene mutation library, adopt One-Step method high-efficient seamless Cloning technology (Clonexpress II One Step Cloning Kit, Vazyme Biotech, China), construct it on expression vector, obtain the recombinant plasmid library containing L-cysteine transport protein EamB mutant coding gene. 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; extension at 72 deg.C for 5min, 4 deg.CAnd (5) storing.
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 ℃.
PCR-obtained products were cloned into a seamless Cloning kit (Clonexpress II One Step Cloning kit, Vazyme Biotech, China) by One-Step methodeamBConnecting 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 Escherichia coli L-cysteine transporter deletion, a beneficial mutant is obtained by screening the strain for L-cysteine tolerance, the mutant with stronger transport capability can discharge excessive L-cysteine out of cells, and the strain is subjected to high-concentration L-cysteine-increased tolerability under cysteine stress. 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 obtained in the above examples, I83M, G156S, N157S, I83M-G156S, I83M-N157S, G156S-N157S and I83M-G156S-N157S, were transformed by heat shock according to conventional E.coliThe method comprises introducing Escherichia coli basic engineering bacteria E.coli-CYS with certain cysteine production capacity, and performing 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. After the strain is fermented and cultured for 42 hours, sampling is carried out to determine the content of the L-cysteine.
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>
<160> 23
<170> PatentIn version 3.5
<210> 1
<211> 588
<212> DNA
<213> Escherichia coli MG1655
<400> 1
gtgacaccgacccttttaagtgctttttggacttacaccctgattaccgctatgacgcca
ggaccgaacaatattctcgcccttagctctgctacgtcgcatggatttcgtcaaagtacc
cgcgtgctggcagggatgagtctgggatttttgattgtgatgttactgtgtgcgggcatt
tcattttcactggcagtgattgacccggcagcggtacaccttttgagttgggcgggggcg
gcatatattgtctggctggcgtggaaaatcgccaccagcccaacaaaggaagacggactt
caggcaaaaccaatcagcttttgggccagctttgctttgcagtttgtgaacgtcaaaatc
attttgtacggtgttacggcactgtcgacgtttgttctgccgcaaacacaggcgttaagc
tgggtagttggcgtcagcgttttgctggcgatgattgggacgtttggcaatgtgtgctgg
gcgctggcggggcatctgtttcagcgattgtttcgccagtatggtcgccagttaaatatc
gtgcttgccctgttgctggtctattgcgcggtacgcattttctattaa 588
<210> 2
<211> 195
<212> PRT
<213> Escherichia coli MG1655
<400> 2
MTPTLLSAFWTYTLITAMTPGPNNILALSSATSHGFRQSTRVLAGMSLGFLIVMLLCAGI
SFSLAVIDPAAVHLLSWAGAAYIVWLAWKIATSPTKEDGLQAKPISFWASFALQFVNVKI
ILYGVTALSTFVLPQTQALSWVVGVSVLLAMIGTFGNVCWALAGHLFQRLFRQYGRQLNI
VLALLLVYCAVRIFY 195
<210> 3
<211> 195
<212> PRT
<213> Escherichia coli MG1655
<400> 3
MTPTLLSAFWTYTLITAMTPGPNNILALSSATSHGFRQSTRVLAGMSLGFLIVMLLCAGI
SFSLAVIDPAAVHLLSWAGAAYIVWLAWKIATSPTKEDGLQAKPISFWASFALQFVNVKI
ILYGVTALSTFVLPQTQALSWVVGVSVLLAMIGTFSSVCWALAGHLFQRLFRQYGRQLNI
VLALLLVYCAVRIFY 195
<210> 4
<211> 588
<212> DNA
<213> Escherichia coli MG1655
<400> 4
gtgacaccgacccttttaagtgctttttggacttacaccctgattaccgctatgacgcca
ggaccgaacaatattctcgcccttagctctgctacgtcgcatggatttcgtcaaagtacc
cgcgtgctggcagggatgagtctgggatttttgattgtgatgttactgtgtgcgggcatt
tcattttcactggcagtgattgacccggcagcggtacaccttttgagttgggcgggggcg
gcatatatggtctggctggcgtggaaaatcgccaccagcccaacaaaggaagacggactt
caggcaaaaccaatcagcttttgggccagctttgctttgcagtttgtgaacgtcaaaatc
attttgtacggtgttacggcactgtcgacgtttgttctgccgcaaacacaggcgttaagc
tgggtagttggcgtcagcgttttgctggcgatgattgggacgtttagcaatgtgtgctgg
gcgctggcggggcatctgtttcagcgattgtttcgccagtatggtcgccagttaaatatc
gtgcttgccctgttgctggtctattgcgcggtacgcattttctattaa 588
<210> 5
<211> 588
<212> DNA
<213> Escherichia coli MG1655
<400> 5
gtgacaccgacccttttaagtgctttttggacttacaccctgattaccgctatgacgcca
ggaccgaacaatattctcgcccttagctctgctacgtcgcatggatttcgtcaaagtacc
cgcgtgctggcagggatgagtctgggatttttgattgtgatgttactgtgtgcgggcatt
tcattttcactggcagtgattgacccggcagcggtacaccttttgagttgggcgggggcg
gcatatatggtctggctggcgtggaaaatcgccaccagcccaacaaaggaagacggactt
caggcaaaaccaatcagcttttgggccagctttgctttgcagtttgtgaacgtcaaaatc
attttgtacggtgttacggcactgtcgacgtttgttctgccgcaaacacaggcgttaagc
tgggtagttggcgtcagcgttttgctggcgatgattgggacgtttggcagtgtgtgctgg
gcgctggcggggcatctgtttcagcgattgtttcgccagtatggtcgccagttaaatatc
gtgcttgccctgttgctggtctattgcgcggtacgcattttctattaa 588
<210> 6
<211> 588
<212> DNA
<213> Escherichia coli MG1655
<400> 6
gtgacaccgacccttttaagtgctttttggacttacaccctgattaccgctatgacgcca
ggaccgaacaatattctcgcccttagctctgctacgtcgcatggatttcgtcaaagtacc
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

Claims (13)

1. Is derived fromEscherichia coliThe L-cysteine transporter EamB mutant of MG1655 is characterized in that, relative to the amino acid sequence shown in SEQ ID No.2, only one or more site mutations of isoleucine I to methionine M at position 83, glycine G to alanine A at position 156, asparagine N to methionine M at position 157, and asparagine N to serine S at position 157 exist.
2. The EamB mutant as claimed in claim 1, wherein only the 83 th position is mutated from isoleucine I to methionine M, the 156 th position is mutated from glycine G to alanine A, the 157 th position is mutated from asparagine N to methionine M, the 157 th position is mutated from asparagine N to serine S; or only a combined mutation of isoleucine I to methionine M at position 83 and glycine G to serine S at position 156; or there is only a combined mutation of isoleucine I to methionine M at position 83 and asparagine N to serine S at position 157; or only a combined mutation of glycine G to serine S at position 156 and asparagine N to serine S at position 157; or there is only a combination of mutations from 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.
3. The EamB mutant according to claim 1, characterized in that its amino acid sequence is shown in SEQ ID No. 3.
4. The EamB mutant of any one of claims 1-3 encoding gene.
5. The encoding gene according to claim 4, wherein the nucleotide sequence is obtained by mutation based on the nucleotide sequence shown in SEQ ID No. 1.
6. The coding gene of claim 5, wherein the nucleotide sequence is selected from the group consisting of SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No. 7.
7. An expression vector comprising the coding gene according to any one of claims 4 to 6.
8. A recombinant host cell comprising the gene encoding any one of claims 4 to 6.
9. The recombinant host cell of claim 8, wherein the recombinant host cell is escherichia coli, corynebacterium glutamicum, pantoea ananatis, or bacillus subtilis.
10. Use of an EamB mutant according to any one of claims 1 to 3, or a gene encoding according to any one of claims 4 to 6, or a recombinant host cell according to any one of claims 8 to 9 for the production of L-cysteine or a downstream metabolite thereof.
11. A recombinant bacterium having an enhanced L-cysteine producing ability, characterized in that the encoding gene according to any one of claims 4 to 6 is overexpressed in the starting strain.
12. The recombinant bacterium of claim 11, wherein a L-cysteine degradation pathway gene is knocked out in the starting straintanAAndyhaMsimultaneous overexpression of L-cysteine synthetic pathway genescysE。
13. The recombinant bacterium according to claim 11 or 12, wherein the starting strain is escherichia coli, corynebacterium glutamicum, pantoea ananatis, or bacillus subtilis.
CN202111344079.6A 2021-11-15 2021-11-15 Cysteine transporter mutant and application thereof in production of L-cysteine Active CN113788881B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111344079.6A CN113788881B (en) 2021-11-15 2021-11-15 Cysteine transporter mutant and application thereof in production of L-cysteine

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111344079.6A CN113788881B (en) 2021-11-15 2021-11-15 Cysteine transporter mutant and application thereof in production of L-cysteine

Publications (2)

Publication Number Publication Date
CN113788881A true CN113788881A (en) 2021-12-14
CN113788881B CN113788881B (en) 2022-02-11

Family

ID=78955294

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111344079.6A Active CN113788881B (en) 2021-11-15 2021-11-15 Cysteine transporter mutant and application thereof in production of L-cysteine

Country Status (1)

Country Link
CN (1) CN113788881B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115029371A (en) * 2022-06-14 2022-09-09 大连工业大学 Efficient separation method of natural active product produced by microorganism

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1261626A (en) * 1998-12-30 2000-08-02 味之素株式会社 Method for producing L-amino acid
WO2013148559A1 (en) * 2012-03-24 2013-10-03 Carnegie Institution Of Washington Pathogen resistant plant cells and methods of making
CN111040979A (en) * 2018-10-11 2020-04-21 中国科学院天津工业生物技术研究所 Method for high-yield production of L-cysteine by corynebacterium glutamicum through metabolic engineering transformation
WO2020204179A1 (en) * 2019-04-05 2020-10-08 Ajinomoto Co., Inc. Method of producing l-amino acids
CN113549633A (en) * 2021-06-08 2021-10-26 天津科技大学 L-cysteine transporter mutant and application thereof in production of L-cysteine

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1261626A (en) * 1998-12-30 2000-08-02 味之素株式会社 Method for producing L-amino acid
WO2013148559A1 (en) * 2012-03-24 2013-10-03 Carnegie Institution Of Washington Pathogen resistant plant cells and methods of making
CN111040979A (en) * 2018-10-11 2020-04-21 中国科学院天津工业生物技术研究所 Method for high-yield production of L-cysteine by corynebacterium glutamicum through metabolic engineering transformation
WO2020204179A1 (en) * 2019-04-05 2020-10-08 Ajinomoto Co., Inc. Method of producing l-amino acids
CN113549633A (en) * 2021-06-08 2021-10-26 天津科技大学 L-cysteine transporter mutant and application thereof in production of L-cysteine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PITTMAN, MS等: "Cysteine is exported from the Escherichia coli cytoplasm by CydDC, an ATP-binding cassette-type transporter required for cytochrome assembly", 《JOURNAL OF BIOLOGICAL CHEMISTRY 》 *
沈观宇等: "氨基酸生产菌的氨基酸外向转运蛋白研究进展", 《高校化学工程学报》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115029371A (en) * 2022-06-14 2022-09-09 大连工业大学 Efficient separation method of natural active product produced by microorganism
CN115029371B (en) * 2022-06-14 2024-01-02 大连工业大学 Efficient separation method of natural active product produced by microorganisms

Also Published As

Publication number Publication date
CN113788881B (en) 2022-02-11

Similar Documents

Publication Publication Date Title
KR102013873B1 (en) A microorganism of the genus Corynebacterium producing purine nucleotide and method for producing purine nucleotide using the same
JP7373661B2 (en) Genetically modified bacterium producing L-arginine, its construction method and use
CN109055289B (en) Recombinant escherichia coli for high yield of L-methionine and application thereof
CN102080062B (en) An l-cysteine-producing bacterium and a method for producing l-cysteine
WO2018152918A1 (en) L-homoserine-producing bacterial strain, construction method for same, and applications thereof
JP2016529901A (en) Modified microorganisms for improving alanine production
CN112210519A (en) Genetically engineered bacterium for secreting acetaldehyde dehydrogenase by using edible fungi
CN107227283B (en) Corynebacterium glutamicum and construction method and application thereof
CN113788881B (en) Cysteine transporter mutant and application thereof in production of L-cysteine
US20230024648A1 (en) Ean B Mutants and Their Uses
CN112375726B (en) Genetically engineered bacterium for producing L-homoserine and application thereof
CN112725251B (en) Engineering bacterium for producing spermidine
CN110564659B (en) Escherichia coli resistant to sodium acetate, sodium chloride and isobutanol and construction method thereof
CN110846333B (en) Recombinant strain modified by deoB gene and construction method and application thereof
CN109055417B (en) Recombinant microorganism, preparation method thereof and application thereof in production of coenzyme Q10
CN114181288B (en) Process for producing L-valine, gene used therefor and protein encoded by the gene
CN116121160A (en) Genetically engineered bacterium for over-expressing pyrB gene and method for producing L-arginine by using genetically engineered bacterium
RU2694041C1 (en) Embodiment of o-acetyl-homoserine-sulfhydrylase and method of producing l-methionine using this embodiment
CN114134127A (en) Diaminobutyrate acetyltransferase mutants for the synthesis of ectoin
KR102605543B1 (en) Methionine-producing yeast
WO2021035793A1 (en) Mvin protein mutant, expression vector and host cell including said mutant, and application thereof
CN110872595B (en) Acid-resistant expression cassette and application thereof in fermentation production of organic acid
CN108866017B (en) Method for preparing β -hydroxy- β -methylbutyric acid by enzyme method
CN110964682B (en) L-lysine-tolerant bacteria, producing bacteria and use thereof
CN117625665A (en) Method for constructing L-lysine-producing engineering bacteria based on intracellular multienzyme assembly of fiber small scaffold protein and application of engineering bacteria

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
TR01 Transfer of patent right

Effective date of registration: 20240423

Address after: No. 15 West 8th Road, Airport Economic Zone, Binhai New Area, Tianjin, 300308

Patentee after: Tianjin National Synthetic Biotechnology Innovation Center Co.,Ltd.

Country or region after: China

Address before: No.32, Xiqi Road, Airport Economic Zone, Binhai New Area, Tianjin 300308

Patentee before: TIANJIN INSTITUTE OF INDUSTRIAL BIOTECHNOLOGY, CHINESE ACADEMY OF SCIENCES

Country or region before: China

TR01 Transfer of patent right