CN110591989A - High-yield L-tryptophan engineering strain and application thereof - Google Patents
High-yield L-tryptophan engineering strain and application thereof Download PDFInfo
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Abstract
The invention discloses an L-tryptophan genetic engineering bacterium and a construction method thereof, wherein the preservation number is as follows: CGMCC NO. 15765. Related genes of an L-tryptophan catabolism pathway are deleted by means of genetic engineering, and meanwhile, the related genes of the L-tryptophan synthesis pathway are over-expressed by a proper expression vector to construct an L-tryptophan production underpan cell TD 0. On the basis, an L-tryptophan high-producing strain TD is screened out by a flow cytometer by combining with an L-tryptophan biosensor.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an L-tryptophan engineering bacterium and a method for producing L-tryptophan by using the same.
Background
L-tryptophan is one of amino acids essential for life activities of human bodies and animals, plays an important physiological role in growth, development and metabolism of the human bodies and the animals, and is widely applied to the fields of feeds, medicines, foods and the like. In the field of medicine, tryptophan is an important component and an important medical intermediate of amino acid infusion, and tryptophan is taken as a precursor to synthesize various metabolites with medical value, such as 5-hydroxytryptophan, nicotinic acid, alkaloid, coenzyme, pigment and the like. The L-tryptophan is added into the feed, so that the content of the tryptophan in the daily ration of livestock, poultry and fish can be effectively improved, the value and the utilization rate of protein in the feed are obviously improved, and the growth of animals is promoted. In the field of food applications, tryptophan can be used for fortifying food products, for example as a flavouring agent to be added to western-style pastries, and also for bread to promote fermentation. Due to the wide use of L-tryptophan, the demand for L-tryptophan is increasing at home and abroad. However, the production of L-tryptophan has been difficult and expensive for a long time, so that L-tryptophan cannot be widely popularized and applied in many fields.
L-tryptophan fermentation studies began in the early 60 s of the twentieth century. Although this method has been studied relatively early, it has not been able to meet the requirement of industrial production for a long period of development. The main reason is that the biosynthesis from glucose to L-tryptophan needs twenty-multiple steps of reactions, the pathway is long, the metabolic flow is weak, and on the other hand, the L-tryptophan biosynthesis pathway contains a complex regulation mechanism, thus increasing the difficulty of strain modification. Starting from the uptake of glucose by bacteria, it is necessary to pass through the Phosphate Transport System (PTS) on the cell membrane, the glycolysis Pathway (Embden Meyerhof Parnas, EMP) and the pentose phosphate Pathway (HMP), synthesize the precursors Phosphoenolpyruvate (PEP) and erythrose-4-phosphate (D-erythrose-4-phosphate, E4P), respectively, reach chorismic acid via the common shikimic acid Pathway, and then synthesize the three aromatic amino acids L-phenylalanine, L-tyrosine and L-tryptophan at the branch.
In the L-tryptophan metabolism process, the biosynthesis pathway from glucose to L-tryptophan is long, a plurality of precursors from different metabolic flow paths (such as PEP, E4P, L-serine, glutamine, PRPP and the like) are required, particularly PEP and L-serine are derived from an EMP pathway, E4P and PRPP are derived from an HMP pathway, and the metabolic balance is not easy to realize while the metabolic flow path is strengthened. In addition, metabolic regulation mechanisms in the L-tryptophan biosynthetic pathway are also complex, and various feedback inhibition and feedback repression exist. Berry A highly expresses aroG and trpECBA genes which remove feedback inhibition in Escherichia coli, ferments for 50 hours, produces tryptophan of 40-45g/L, and has a process conversion rate to glucose of more than 22% (Barry A, Improving production of aromatic compounds in Escherichia coli by metabolic engineering. trends Biotechnology, 14:250-256, 1996).
In 1979 Tribe and Pitard introduced trpE into E.coli (E.coli) for the first time by DNA recombination technology, and the yield of L-tryptophan reached 1g/L (Tribe D E et al, Applied and environmental microbiology,1979,38(2): 181-190.). Since then, with the wide application of recombinant DNA technology in microbial breeding, significant breakthroughs were made in the screening of L-tryptophan-producing bacteria and acid-producing strains. For example, in 1999, Ikeda et al highly expressed tktA gene in L-tryptophan-producing Corynebacterium glutamicum (Corynebacterium glutamicum) pIK9960 and enhanced the level of L-tryptophan synthesis precursor E4P, thereby increasing the L-tryptophan synthesis efficiency and achieving 58g/L of L-tryptophan yield at 80 hours of fermentation (Ikeda M et al, Applied and environmental microbiology,1999,65(6): 2497-2502.). In 2017, Zeng et al obtained a high-yield L-tryptophan strain S028 by a genetic engineering pure transformation method, and the L-tryptophan yield reached 40.3g/L by fermentation for 61h (Chen L et al, applied microbiology and biotechnology,2017,101(2): 559-568)
Although the modification method improves the L-tryptophan synthesis capacity of the strain to a certain degree, the traditional modification strategy cannot achieve the expectation due to the limitation of understanding the physiological and genetic backgrounds of the microorganisms. Therefore, a need exists for further development of engineering strategies for high-yield L-tryptophan engineering bacteria so as to obtain L-tryptophan engineering bacteria meeting the industrial requirements.
Disclosure of Invention
In view of the above, the invention aims to provide an engineering strain for producing L-tryptophan with high yield and a method for producing L-tryptophan by fermentation thereof, so as to meet the requirement of large-scale industrial production of L-tryptophan.
In order to achieve the above object, in a first aspect, the present invention provides a method for constructing a high-yield L-tryptophan engineering bacterium, comprising the following steps:
(1) knocking out tnaA and mlc genes in an original strain, and overexpressing aroG gene and trpeDCBA operon to obtain an L-tryptophan-producing chassis cell;
(2) constructing a biosensor for detecting the content of L-tryptophan,
(3) introducing the biosensor constructed in the step (2) into the chassis cells in the step (1);
(4) carrying out mutagenesis treatment on the underpan cells with the biosensor in the step (3);
(5) screening to obtain the mutant strain with the yield of L-tryptophan being improved compared with that of the Chassis cells.
Wherein the starting strain in the step (1) is bacteria, more preferably Escherichia coli, and further preferably MG 1655;
preferably, the core element of the biosensor in the step (2) is a tan c gene, and the protein TnaC expressed by the tan c gene is used as an input display depending on the concentration of intracellular tryptophan, and the concentration of intracellular tryptophan is converted into a GFP fluorescent protein signal for detection;
the mutagenesis method in the step (4) is chemical mutagenesis or physical mutagenesis, preferably physical mutagenesis, and more preferably ARTP mutagenesis;
preferably, the screening method of step (5) is high-throughput screening, preferably using a flow cytometer.
In a second aspect, the invention provides a high-yield L-tryptophan engineering bacterium, which is identified as Escherichia coli (Escherichia coli), and is preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3, Ministry of microbiology research, the postal code: 100101, of the Beijing city, Chaoyang district, North Cheng Wen Lu No. 1) in 16 days 5 and 2018, and the preservation number is CGMCC NO. 15765.
In a third aspect, the present invention provides a method for producing L-tryptophan, comprising the steps of:
(1) culturing the mutagenized strain having an increased L-tryptophan yield obtained in the first aspect or the strain of the second aspect under temperature conditions;
(2) transferring the strain in the step (1) to a fermentation medium according to 2% -10% of transfer amount and culturing for 30-60 hours;
(3) and (3) centrifuging and collecting the fermentation liquor in the step (2), and detecting by HPLC.
Wherein the culture temperature in the step (1) is 30-40 ℃, and preferably 37 ℃;
preferably, the amount of inoculation in step (2) is 5%.
In a fourth aspect, the present invention provides the use of a high L-tryptophan-producing strain for the production of L-tryptophan.
In a fifth aspect, the invention provides application of the high-yield L-tryptophan strain in feed, medicines and foods.
Drawings
FIG. 1 depicts a plasmid map of Ptrc99 a-trp.
FIG. 2. results of L-tryptophan biosensor response to extracellular alanine-tryptophan addition.
FIG. 3 is a bar graph of fermentation of high throughput screening strains.
FIG. 4 results of shake flask fermentation.
Detailed Description
Example 1: construction of L-Tryptophan-producing Chassis cells
Cas9-tnaA plasmid construction: using MG1655 as template, tanA-UP fragment with linker was amplified with primer tnaA-UP-F, tnaA-UP-R, and tnaA-Down fragment with linker was amplified with primer tnaA-Down-F, tnaA-Down-R. the tnaA-UP fragment and the tnaA-Down fragment were assembled into the tnaA-UD fragment. Using cas9 plasmid as template, using primer tnaA-n20-F, tnaA-ver-R to amplify plasmid skeleton tnaA-ver1 fragment, using primer tnaA-ver-F, tnaA-n20-R to amplify plasmid skeleton tnaA-ver2 fragment. The plasmid frameworks tnaA-ver1 and tnaA-ver2 and the fragment tnaA-UD are subjected to Gibsonassemmy (Gibson analysis method is that a plurality of DNA fragments are subjected to intermolecular ligation in 1 reaction by Gibson and the like) to obtain cas9-tnaA plasmid.
Cas9-mlc plasmid construction: the ligated mlc-UP fragment was amplified with the primer mlc-UP-F, mlc-UP-R and the ligated mlc-Down fragment was amplified with the primer mlc-Down-F, mlc-Down-R using MG1655 as a template. The mlc-UP fragment and the mlc-Down fragment were assembled into the mlc-UD fragment. The cas9 plasmid is used as a template, a plasmid skeleton mlc-ver1 fragment is amplified by using a primer mlc-n20-F, mlc-ver-R, and a plasmid skeleton mlc-ver2 fragment is amplified by using a primer mlc-ver-F, mlc-n 20-R. Plasmid frameworks mlc-ver1, mlc-ver2 and the above fragment mlc-UD gave cas9-mlc plasmid by Gibson assembly.
Construction of the plasmid pH5 a-aroG-trpeDCBA: the aroG fragment with the adaptor was amplified using the primer aroG-F, aroG-R, using MG1655 as a template. The adaptor-carrying pH5a-M fragment was amplified using the plasmid pH5a as template, pH5a-M-F, pH5 a-M-R. The aroG-M-Gibson fragment for Gibson assembly was obtained by amplifying the aroG-F, pH5a-M-R primer using the aroG fragment and the pH5a-M fragment as templates. A ligated trpeDCBA-Gibson fragment was amplified using the primer trpeDCBA-F, trpEDCBA-R, using MG1655 as a template. The plasmid backbone with the linker was amplified at pH5a-ver-F, pH5a-ver-R, and the above aroG-M-Gibson fragment and trpeDCBA-Gibson fragment were used to obtain the plasmid with pH5a-aroG-trpeDCBA by Gibson analysis.
The primers used in this section were as follows:
TABLE 1 construction of primers used for L-tryptophan-producing underpan cells
Plasmid cas9-tnaA was introduced into strain MG1655 to construct strain MG1655-cas 9-tnaA. Arabinose is added into a culture medium for induction knockout, and finally a strain MG1655 delta tnaA with the tnaA gene knocked out is constructed.
Plasmid cas9-mlc was introduced into strain MG 1655. delta. tnaA to construct strain MG 1655. delta. tnaA-ptrc99 a-mlc. Arabinose is added into a culture medium for induction knockout, and a strain MG1655 delta tnaA delta mlc with a mlc gene knocked out is finally constructed.
Strain TD0 was constructed by transformation of plasmid pH5 a-aroG-trpECBA into strain MG 1655. delta. tnaA. delta. mlc. The strains and plasmids used in this section were as follows:
TABLE 2 construction of strains and plasmids for L-tryptophan-producing Chassis cells
Example 2: construction of L-tryptophan biosensor
Construction of Ptrc99a-trp plasmid: the tnaC-tnaA (first 24bp of tnaC to 30bp after the tnaA promoter) fragment with the linker was amplified using MG1655 as a template and using a primer tnaC-F, tanC-R. The ligated GFP fragment was amplified with the primer GFP-F, GFP-R using the pMA5 plasmid as a template. The tnaC-GFP-Gibson fragment for Gibson assembly was amplified using the primers tanC-F, GFP-R, using the tanC-tnaA fragment and the GFP fragment as templates. The plasmid ptrc99a is used as a template, a primer ptrc99a-ver-F, ptrc99a-ver-R is used for amplification to obtain a ptrc99a plasmid skeleton, and the ptrc99a-trp plasmid is obtained by Gibson assembly with the tnAC-GFP-Gibson fragment, and the plasmid map is shown in figure 1. TnaC is used as an input display depending on the concentration of intracellular tryptophan, and the concentration of intracellular tryptophan is converted into a GFP fluorescent protein signal for detection.
The primers used in this section were as follows:
TABLE 3 primers used for construction of L-Tryptophan biosensors
TABLE 4 construction of L-Tryptophan biosensors Using strains and plasmids
To verify the effect of ptrc99a-trp, we introduced plasmid ptrc99a-trp into strain TD0 to obtain strain TD 0-trp. Selecting single colony to culture in LB overnight, transferring to M9 culture medium at a ratio of 1:100 after 12h, adding alanine-tryptophan dipeptide with different concentrations into the culture solution to test when OD is 0.4-0.5, culturing for 8h, and performing fluorescence quantification (excitation 483nm and emission 525nm) with enzyme labeling instrument. As shown in FIG. 2, the intracellular concentration of L-tryptophan has a linear relationship with the fluorescence intensity.
Example 3: high throughput screening
To obtain a relatively broad library of mutations, we performed plasma (ARTP) mutagenesis of the underplate cells. After the mutagenized bacteria are activated, the mixed bacteria liquid is screened by a two-wheel flow cytometer. In the first round of screening, the mutagenized cells are cultured overnight to grow to about 1.0 OD, flow screening is carried out, the cells with the fluorescence value reaching the first 0.3% are sorted out, and the cells are cultured in an enlarged way to reach about 1.0 OD. And in the second round of screening, after the cells sorted out in the first round are cultured, sorting the cells with the fluorescence value of up to the first 0.3 percent into a 96-well plate containing 500 mu L of culture medium by a flow cytometer overnight for culture and coating the cells in a solid culture medium to obtain single colonies.
Example 4: bacterial strain fermentation verification
The strains were separated by flow cytometry, and 96 single colonies (including one control strain) were picked and cultured overnight in a 96-well plate containing 500. mu.L of LB, then inoculated into a 96-well plate containing 500. mu.L of fermentation medium (three plates in parallel) at an inoculum size of 5% (v/v), and cultured at 37 ℃ for 38 hours at 200 rpm/min.
Example 5: high Performance Liquid Chromatography (HPLC) detection of fermentation strains
Centrifuging the fermentation liquid in a refrigerated centrifuge at 5500rpm/min for 15-20min, collecting supernatant, filtering the supernatant with 0.22 μm filter membrane, and performing HPLC detection. The fermentation result is shown in FIG. 3, the T20 strain has the highest L-tryptophan yield, and the tryptophan yield reaches 1.91g/L, which is 6.8 times of that of the Chassis strain. T20 strain was designated as strain TD after discarding the plasmid ptrc99 a-trp.
The HPLC conditions were as follows: the column was a ZORBAX Eclipse AAA (amino acid analysis) column, mobile phase A: 40mM Na2HPO4pH 7.8, flowAnd (3) moving phase B: methanol: acetonitrile: water 45:45:10, v/v/v. The elution gradient is 0-1min, 100% A; 9.8min, 43% A + 57% B; 100% B for 10 min; 100% B for 12 min; 12.5min, 100% A. The flow rate is 2.0mL/min, the RID and VWD detectors are connected in series, the temperature of the detection pool is controlled at 40 ℃, the sample injection amount is 10 mu L, the analysis time is 26min, and the ultraviolet detection wavelength is 338 nm.
Example 6: l-tryptophan engineering strain TD shake flask fermentation verification
Single colonies of the genetic engineering strain TD and the control strain TD0 were picked respectively and inoculated into a test tube containing 5ml of LB medium, cultured overnight at 37 ℃, then inoculated into a 250ml shake flask containing 30ml of fermentation medium according to the inoculum concentration of 5% for fermentation at 37 ℃ for 38 hours.
Centrifuging the fermentation liquid in a refrigerated centrifuge at 5500rpm/min for 15-20min, collecting supernatant, filtering the supernatant with 0.22 μm filter membrane, and performing HPLC detection. The fermentation result is shown in figure 4, the yield of the TD strain L-tryptophan is 1.88g/L, which is improved by 507% compared with the yield of the contrast strain.
Claims (10)
1. A construction method of high-yield L-tryptophan engineering bacteria comprises the following steps:
(1) knocking out tnaA and mlc genes in an original strain, and overexpressing aroG gene and trpeDCBA operon to obtain an L-tryptophan-producing chassis cell;
(2) constructing a biosensor for detecting the content of L-tryptophan;
(3) introducing the biosensor constructed in the step (2) into the chassis cells in the step (1);
(4) carrying out mutagenesis treatment on the underpan cells with the biosensor in the step (3);
(5) screening to obtain the mutant strain with the yield of L-tryptophan being improved compared with that of the Chassis cells.
2. The method according to claim 1, wherein the starting strain in step (1) is a bacterium, more preferably Escherichia coli, and still more preferably MG 1655.
3. The method of claim 1 or 2, wherein the core element of the biosensor in the step (2) is a tan c gene.
4. The method of construction according to any one of claims 1 to 3, wherein the mutagenesis in step (4) is chemical mutagenesis or physical mutagenesis, preferably physical mutagenesis, more preferably ARTP mutagenesis.
5. The method of any one of claims 1 to 4, wherein the screening method in step (5) is a high throughput screening, preferably a flow cytometry screening.
6. A mutant strain having an increased L-tryptophan production, which is selected by the method according to any one of claims 1 to 5.
7. The high-yield L-tryptophan engineering bacterium is characterized in that the engineering bacterium is Escherichia coli (Escherichia coli), and the preservation number is CGMCC NO. 15765.
8. A method for preparing L-tryptophan comprising the steps of:
(1) culturing a mutagenic strain with improved L-tryptophan yield obtained by screening according to claims 1 to 5 or the engineering bacteria according to claim 7 under certain temperature conditions;
(2) transferring the strain in the step (1) to a fermentation medium according to 2% -10% of transfer amount and culturing for 30-60 hours;
(3) and (3) centrifugally collecting the fermentation liquor obtained in the step (2), and detecting the yield of the L-tryptophan.
9. The method of claim 8, wherein the culturing temperature in step (1) is 30-40 ℃, preferably 37 ℃; the inoculation amount in the step (2) is 5 percent.
10. The mutant strain with high L-tryptophan yield obtained by screening according to claims 1 to 6 and the application of the engineering bacterium with high L-tryptophan yield to the production of L-tryptophan according to claim 7.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111235136A (en) * | 2020-01-19 | 2020-06-05 | 中国科学院天津工业生物技术研究所 | Isocitrate lyase mutant and application thereof in preparation of aromatic amino acid |
| CN113549588A (en) * | 2021-06-25 | 2021-10-26 | 天津科技大学 | Genetically engineered bacterium for producing 5-hydroxytryptophan and construction method and application thereof |
| WO2022007205A1 (en) * | 2020-07-08 | 2022-01-13 | 江南大学 | Method for increasing content of intracellular heme in escherichia coli |
| CN115820633A (en) * | 2022-08-15 | 2023-03-21 | 中国科学院天津工业生物技术研究所 | Biosensor for positively responding glucose absorption rate and application thereof |
| CN115895976A (en) * | 2022-12-20 | 2023-04-04 | 黑龙江金象生化有限责任公司 | Escherichia coli for producing L-tryptophan and application of escherichia coli for producing L-tryptophan |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101307301A (en) * | 2007-05-17 | 2008-11-19 | 浙江升华拜克生物股份有限公司 | L-tryptophan genetic engineering bacterium and process for producing L-tryptophan |
| EP2098597A1 (en) * | 2008-03-04 | 2009-09-09 | Evonik Degussa GmbH | Method for manufacturing L-amino acids using improved strains of the enterobacteriaceae family |
| CN102140431A (en) * | 2010-12-21 | 2011-08-03 | 大成生化科技(松原)有限公司 | L-tryptophan gene engineering bacterium, method for constructing same and method for fermenting and producing L-tryptophan by using same |
| CN102453691A (en) * | 2011-12-02 | 2012-05-16 | 山东鲁抗生物制造有限公司 | Escherichia coli engineering bacteria capable of realizing high yield of L-tryptophan |
| CN104388330A (en) * | 2014-09-26 | 2015-03-04 | 廊坊梅花生物技术开发有限公司 | L-tryptophan fermentation strain and method for fermentation production of L-tryptophan by using L-tryptophan fermentation strain |
| CN105543154A (en) * | 2016-01-13 | 2016-05-04 | 廊坊梅花生物技术开发有限公司 | L-tryptophan production genetically engineered bacterium and construction method and application thereof |
-
2019
- 2019-01-30 CN CN201910090483.1A patent/CN110591989A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101307301A (en) * | 2007-05-17 | 2008-11-19 | 浙江升华拜克生物股份有限公司 | L-tryptophan genetic engineering bacterium and process for producing L-tryptophan |
| EP2098597A1 (en) * | 2008-03-04 | 2009-09-09 | Evonik Degussa GmbH | Method for manufacturing L-amino acids using improved strains of the enterobacteriaceae family |
| CN102140431A (en) * | 2010-12-21 | 2011-08-03 | 大成生化科技(松原)有限公司 | L-tryptophan gene engineering bacterium, method for constructing same and method for fermenting and producing L-tryptophan by using same |
| CN102453691A (en) * | 2011-12-02 | 2012-05-16 | 山东鲁抗生物制造有限公司 | Escherichia coli engineering bacteria capable of realizing high yield of L-tryptophan |
| CN104388330A (en) * | 2014-09-26 | 2015-03-04 | 廊坊梅花生物技术开发有限公司 | L-tryptophan fermentation strain and method for fermentation production of L-tryptophan by using L-tryptophan fermentation strain |
| CN105543154A (en) * | 2016-01-13 | 2016-05-04 | 廊坊梅花生物技术开发有限公司 | L-tryptophan production genetically engineered bacterium and construction method and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| JACQUELINE PLUMBRIDGE: "Regulation of gene expression in the PTS in Escherichia coli: the role and interactions of Mlc", 《CURRENT OPINION IN MICROBIOLOGY》 * |
| MICHAEL BOTT ET AL.: "Need for speed - finding productive mutations using transcription factor-based biosensors, fluorescence-activated cell sorting and recombineering", 《MICROBIAL BIOTECHNOLOGY》 * |
| SUSY BEATRIZ CARMONA ET AL.: "Inactivation of the PTS as a Strategy to Engineer the Production of Aromatic Metabolites in Escherichia coli", 《J MOL MICROBIOL BIOTECHNOL》 * |
| 左祖桢: "大肠杆菌色氨酸高产菌株的构建", 《中国优秀博硕士学位论文全文数据库(硕士) 工程科技Ⅰ辑》 * |
Cited By (8)
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|---|---|---|---|---|
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| CN111235136B (en) * | 2020-01-19 | 2021-10-22 | 中国科学院天津工业生物技术研究所 | Isocitrate lyase mutant and application thereof in preparation of aromatic amino acid |
| WO2022007205A1 (en) * | 2020-07-08 | 2022-01-13 | 江南大学 | Method for increasing content of intracellular heme in escherichia coli |
| CN113549588A (en) * | 2021-06-25 | 2021-10-26 | 天津科技大学 | Genetically engineered bacterium for producing 5-hydroxytryptophan and construction method and application thereof |
| CN113549588B (en) * | 2021-06-25 | 2023-02-03 | 天津科技大学 | Genetically engineered bacterium for producing 5-hydroxytryptophan and construction method and application thereof |
| CN115820633A (en) * | 2022-08-15 | 2023-03-21 | 中国科学院天津工业生物技术研究所 | Biosensor for positively responding glucose absorption rate and application thereof |
| CN115895976A (en) * | 2022-12-20 | 2023-04-04 | 黑龙江金象生化有限责任公司 | Escherichia coli for producing L-tryptophan and application of escherichia coli for producing L-tryptophan |
| CN115895976B (en) * | 2022-12-20 | 2024-02-06 | 哈尔滨象柏生物科技有限公司 | Escherichia coli for producing L-tryptophan and application thereof in producing L-tryptophan |
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Application publication date: 20191220 |