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Antiphagin L-phenylalanine producing strain as well as breeding method and application thereof

A phenylalanine, anti-phage technology, applied in the field of bioengineering

Inactive Publication Date: 2011-04-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve the problem of breeding of anti-bacteriophage genetically engineered bacteria, the present invention also provides a method for breeding of anti-phage L-phenylalanine producing bacteria, which can be used for the host bacteria WSH-Z06 of recombinant E. (See reference for source: Enhanced L-phenylalanine biosynthesis by co-expression of pheA fbr andaro F wt .Haiyan Zhou, Xianyan Liao, Tianwen Wang, Guocheng Du, Jian Chen.Bioresource Technology) carried out mutagenesis treatment, screened to obtain the bacterial strain WSH-BR42 with phage resistance, and used the plasmid encoding the L-phenylalanine synthetase gene ( pAP-B03) was transformed into WSH-BR42 to obtain anti-phage L-phenylalanine producing bacteria WSH-BR42 (pAP-B03) (CCTCC M 2010008)

Method used

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  • Antiphagin L-phenylalanine producing strain as well as breeding method and application thereof
  • Antiphagin L-phenylalanine producing strain as well as breeding method and application thereof
  • Antiphagin L-phenylalanine producing strain as well as breeding method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Example 1 Isolation and purification of recombinant Escherichia coli WSH-Z06 (pAP-B03) susceptible phage BP-1

[0034] The source of the strain: the L-phenylalanine-producing strain recombinant Escherichia coli WSH-Z06 (pAP-B03) developed by our laboratory earlier, the strain preservation number is CCTCC M 2010009, which has been preserved in the China Center for Type Culture Collection. Date: January 14, 2010, Address: Wuhan University, Wuhan, China, taxonomic name: Escherichia coli ( Escherichia coli ).

[0035] Take 4mL of recombinant Escherichia coli WSH-Z06 (pAP-B03) fermentation broth infected by phage in a factory, centrifuge at 8000rpm for 10min, filter the supernatant through a 0.22μm microporous membrane, take out 100μL and put 6mL of the upper layer at 50℃ Add 100 μL of WSH-Z06 (pAP-B03) (CCTCC M2010009) bacterial suspension to the test tube of meat extract peptone medium at the same time, mix immediately, pour on the solidified bottom medium, spread evenly...

Embodiment 2

[0037] The breeding of embodiment 2 anti-phage Escherichia coli

[0038] A. Starting strain

[0039] The host strain WSH-Z06 of the recombinant Escherichia coli that has been industrialized for the production of L-phenylalanine.

[0040] Escherichia coli (Escherichia coli) WSH-Z06 (pAP-B03) (CCTCC M 2010009) is the starting strain for the selection of anti-phage recombinant bacteria in this laboratory.

[0041] B. Phage

[0042] The phage is BP-1, which was isolated through Example 1.

[0043] C. Medium

[0044] Seed medium: LB medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L); if the cells contain plasmid, add kanamycin sulfate 40μg / mL, pH 7.3, add 2% to the solid medium of agar.

[0045] Phage resistance detection medium: meat extract peptone medium (beef extract 5g / L, peptone 10g / L, glucose 10g / L, NaCl 5g / L), pH 7.0-7.2, agar 2%.

[0046] D. Mutagenesis and isolation screening of mutant strains

[0047] Put a ring of WSH-Z06 from a fresh slant into LB medium (25m...

Embodiment 3

[0050] Embodiment 3 Plasmid pAP-B03 transforms WSH-BR42 and WSH-BR130

[0051] Competent cells of Escherichia coli WSH-Z06 mutant strains WSH-B42 and WSH-BR130 were prepared by the method provided by the efficient preparation of competent cell kit (Shanghai Sangong). The prepared competent cells were placed on ice, and the plasmid pAP-B03 (constructed earlier, Enhanced L-phenylalanine biosynthesis by co-expression of pheA) was added to each tube (110 μL). fbr and aroF wt ) 2 μL, swirl gently to mix the contents, place on ice for 30 minutes, then place in a water bath at 42°C for 90 seconds, then quickly transfer to an ice bath to cool the cells for 1-2 minutes. Add 600 μL of liquid seed medium to each tube, and bathe in water at 37°C for 2 hours. Pipette 50 μL of transformed competent cells onto LB plates containing kanamycin sulfate, spread evenly, and culture overnight in a 37°C incubator. When a single colony grows, it is placed on an LB slant containing kanamycin sulfa...

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Abstract

The invention discloses an antiphagin L-phenylalanine producing strain as well as a breeding method and application thereof, belonging to the technical field of bioengineering. The breeding method comprises the steps of: inducing and recombining a host bacterium WSH-Z06 of escherichia coli with nitrosoguanidine (NTG), breeding a strain with phage (CCTCC M) resistance by using phage separated from WSH-Z06(pAP-B03)(CCTCC M2010009) fermentation broth as a sieve, and transforming a plasmid pAP-B03 containing a L-phenylalanine synthase gene to finally obtain the antiphagin L-phenylalanine producing strain WSH-BR42(pAP-B03)(CCTCC M 2010008). Compared with a starting strain, the antiphagin L-phenylalanine recombinant strain WSH-BR42(pAP-B03) has the resistance to the phage (CCTCC M) and suffers from no influence to the growth speed and acid forming capability to the strain body. In the invention, the problem that the L-phenylalanine fermentation production is constantly polluted by the phage is solved.

Description

Technical field: [0001] A phage-resistant L-phenylalanine high-yield bacterium, a breeding method and its application belong to the technical field of bioengineering, and in particular relate to a method for producing L-phenylalanine by a biological method. Background technique: [0002] L-phenylalanine (L-phenylalanine, L-Phe for short) is one of the eight kinds of amino acids that cannot be synthesized in humans and animals. People call it an essential amino acid. L-phenylalanine is the raw material for conversion into L-tyrosine in the living body, so L-phenylalanine becomes an essential component of some amino acid infusions and amino acid diets; moreover, L-phenylalanine is a synthetic drug An important raw material for ergotamine, antibiotics and vitamin B6; at the same time, L-phenylalanine is also an intermediate in the synthesis of some anticancer drugs; in the food industry, the main use of L-phenylalanine is to synthesize dipeptide sweeteners Flavoring agent aspa...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/01C12P13/22C12R1/19
Inventor 陈坚堵国成周海岩廖鲜艳薛晓驰王天文
Owner JIANGNAN UNIV
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