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L-tryptophan production genetically engineered bacterium and construction method and application thereof

A technology of genetically engineered bacteria and construction methods, applied in biochemical equipment and methods, bacteria, introduction of foreign genetic material using vectors, etc., can solve problems such as low efficiency, and achieve the effect of avoiding consumption, improving level, and improving ability

Inactive Publication Date: 2016-05-04
MEIHUA BIOTECH LANGFANG CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] PTS system-deficient Escherichia coli can transport glucose into the cell through the synergistic action of D-Galactose / H+symporter (GalP) and glucokinase (Glk) ( figure 1 ), with ATP as the phosphate group donor (1molGlucose+1molATP→1molGlucose-6-P+1molADP), but the efficiency of phosphorylation and transport of glucose by GalP and Glk is low (NeidhardtFC, PostmaPW, LengelerJW, etal.EscherichiacoliandSalmonella. 2nded.WashingtonD.C.:ASMPress,1996:1824–1866.;Hernández-MontalvoV,MartínezA,Hernández-ChavezG,etal.ExpressionofgalPandglkinaEscherichiacoliPTSmutantrestoresglucosetransportandincreasesglycolyticfluxtofermentationproducts.BiotechnolBioeng,2003,83(6):687–694.;GossetG.ImprovementofEscherichiacoliproductionstrainsbymodificationofthephosphoenolpyruvate:sugarphosphotransferasesystem. Microb Cell Fact, 2005, 4(1): 14.)

Method used

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  • L-tryptophan production genetically engineered bacterium and construction method and application thereof
  • L-tryptophan production genetically engineered bacterium and construction method and application thereof
  • L-tryptophan production genetically engineered bacterium and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Preparation of bacterial strain SA01△ptsHIcrr with knockout of ptsH, ptsI and crr genes

[0062] 1. Preparation of mutant strain SA01△ptsHIcrr::kan

[0063] Using the plasmid pKD4 (CGSCNO.7632) as a template, use primers pts1 and pts2 to amplify the ptsHIcrr mutant gene fragment by PCR. The primer sequences are as follows:

[0064] Forward primer pts1:

[0065] 5'-CTAGACTTTAGTTCCCACAACACTAAACCTATAAGTTGGGGAAATACAATGGTGTAGGCTGGAGCTGCTTC-3' (SEQ ID NO.1)

[0066] Reverse primer pts2:

[0067] 5'-AAATGGCGCCGATGGGCGCCATTTTTCACTGCGGCAAGAATTACTTCTTGCATATGAATATCCTCCTTAG-3' (SEQ ID NO.2)

[0068] The PCR amplification reaction system is: HF buffer (without MgSO 4 ) 10 μL, dNTP (10 mM) 1 μL, High-Fidelity DNA polymerase 0.5 μL, forward primer (10 μM) 2.5 μL, reverse primer (10 μM) 2.5 μL, template 0.5 μL, add ddH 2 0 to 50 μL (the high-fidelity PCR reaction reagents used in the present invention are all purchased from NEB Company).

[0069] The PCR amplificat...

Embodiment 2

[0083] Embodiment 2: Construction of recombinant plasmid pMG56

[0084] 1. Construction of pET28-glfglk

[0085] Using plasmid pSC6.090B (ATCCNo.PTA-2578) as a template, use primers glfglk1 and glfglk2 to amplify about 2.3kb of FK1 fragment (upstream sequence of glf-glk gene), and use primers glfglk3 and glfglk4 to amplify about 1.0kb of FK2 Fragment (downstream sequence of glf-glk gene). The above PCR products were subjected to agarose gel electrophoresis and gel recovery to obtain DNA fragments of FK1 and FK2. Using the DNA fragments of FK1 and FK2 as templates, the glf-glk gene fragment of about 3.3 kb was amplified with primers lfglk1 and glfglk4. XbaI and SphI restriction enzyme sites were introduced at the 5' ends of primers glfglk1 and glfglk4, respectively, and the primer sequences were as follows:

[0086] Forward primer glfglk1:

[0087] 5′-GC TCTAGA CGACATCATAACGGTTCTG-3' (SEQ ID NO. 6)

[0088] Reverse primer glfglk2:

[0089] 5'-ACGCGCATGGGTTCCACCGATGTCAAT...

Embodiment 3

[0103] Example 3: Obtaining of L-tryptophan-producing recombinant strains

[0104] The Escherichia coli mutant SA01△ptsHIcrr obtained in Example 1 to knock out the ptsHIcrr gene was made into competent cells (for the preparation and transformation methods of competent cells, refer to page 96, chapter 1 of "Molecular Cloning Experiment Guide III"), and the 2. The obtained recombinant plasmid pMG56 was transformed into the host strain SA01ΔptsHIcrr, and the genetically engineered strain MHZ-0811 (pMG56 / SA01ΔptsHIcrr, preservation number: 11940) was obtained for the fermentation and production of L-tryptophan.

[0105] The genetically engineered strain MHZ-0811 is biologically deposited, and its relevant information is as follows:

[0106] Strain MHZ-0811: Classification and name: Escherichia coli, Escherichiacoli. It was preserved in the General Microorganism Center of China Committee for the Collection of Microorganisms on December 25, 2015, and the address is No. 1, Beichen We...

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Abstract

The invention relates to the field of microorganisms, and discloses an L-tryptophan production genetically engineered bacterium and a construction method and application thereof. A common pathway of an Escherichia coli central carbon metabolic pathway is modified through a genetic engineering method, a phosphoric enol pyruvic acid-phosphotransferase system with phosphoric enol pyruvic acid as a substrate of escherichia coli is knocked out, a glucose transfer system with non-PEP as a substrate is enhanced, a glucose transfer endocytosis pathway is changed, the L-tryptophan production genetically engineered bacterium is obtained, and consumption of L-tryptophan synthesis precursor phosphoric enol pyruvic acid in the glucose transferring process is avoided, so that the intracellular PEP level is improved, and then the capability of L-tryptophan produced through strain fermentation is improved. An experiment shows that the constructed L-tryptophan production genetically engineered bacterium is an escherichia coli L-tryptophan high-producing strain, L-tryptophan can be effectively accumulated, the yield of the L-tryptophan is improved, and a foundation is laid for industrial production of the L-tryptophan.

Description

technical field [0001] The invention belongs to the field of microorganisms, and relates to a genetic engineering bacterium producing L-tryptophan and its construction method and application. Background technique [0002] Tryptophan, scientific name β-indolylalanine (β-indolylalanine), is one of the essential amino acids in human and animal life activities, and plays an important role in the growth, development and metabolism of humans and animals. The second essential amino acid is widely used in medicine, food and feed. In the field of medicine, tryptophan is an important component of amino acid infusion and an important pharmaceutical intermediate. In the field of food applications, tryptophan can be used to strengthen food, improve flavor, and can also be used in bread to promote fermentation. In the field of feed addition, when lysine and methionine are satisfied, tryptophan becomes an important limiting amino acid in diets. Supplementing exogenous tryptophan can incr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P13/22
CPCC12N9/1223C12P13/227C12Y207/03009
Inventor 吴涛毛贤军赵津津王小霞
Owner MEIHUA BIOTECH LANGFANG CO LTD
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