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Construction and application of C.glutamicum subspecies lactofermentum with high L-methionine yield

A Corynebacterium glutamicum and methionine technology, applied in the field of bioengineering, can solve the problems of no industrial application of methionine, unfavorable food safety fermentation, unfavorable strain metabolic engineering transformation, etc.

Inactive Publication Date: 2014-10-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] At present, the fermentation method to synthesize methionine mainly uses Escherichia coli or Corynebacterium glutamicum as the production strain, but because Escherichia coli itself produces endotoxin, it is not conducive to food safety fermentation
At the same time, the methionine metabolic pathway in Escherichia coli is more complicated than that in Corynebacterium glutamicum, which is not conducive to the metabolic engineering of strains
Corynebacterium glutamicum, as a food-safe production strain, has a high production capacity of glutamic acid and aspartic acid family amino acids, and is also used to produce L-methionine. It has been widely used in In the industrial production of lysine, histidine, isoleucine and other amino acids, but there is no example of industrial application of methionine

Method used

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  • Construction and application of C.glutamicum subspecies lactofermentum with high L-methionine yield

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1, the construction of recombinant Corynebacterium glutamicum

[0020] In this example, plasmids for knocking out and expressing related genes in Corynebacterium glutamicum were first constructed, and then the constructed plasmids were transformed into corresponding Corynebacterium glutamicum respectively, so as to realize the expression of related genes. knockout and overexpression.

[0021] 1.1 Gene encoding homoserine kinase thr B and the gene encoding the repressor protein McbR wxya Construction of knockout plasmids

[0022] 1.1.1 Knockout the upstream and downstream homology arms mcbRU, mcbRD, thrBU, thrBD and loxp - can - Amplification of loxp fragments

[0023] In this example, primers for amplification were first designed, and the genome and plasmids used for amplifying the template were extracted, and then relevant gene fragments were amplified by PCR. details as follows:

[0024] According to Genebank report thr B , wxya For the gene sequ...

Embodiment 2

[0104] Embodiment 2, recombinant Corynebacterium glutamicum shake flask level production methionine

[0105] The medium formula used in this example is as follows:

[0106] Seed medium: 25 g / L glucose, 20 g / L corn steep liquor, 1 g / L KH 2 PO 4 , 0.5 g / L MgSO 4 , 1.25 g / L urea.

[0107] Fermentation medium: 100 g / L glucose, 20 g / L corn steep liquor, 20 g / L (NH 4 ) 2 SO 4 , 1 g / L KH 2 PO 4 , 0.5 g / L MgSO 4 , 0.01 g / L MnSO 4 , 0.01 g / L FeSO 4 , 1 mg / L VB1, 6 mg / L VB6, 0.1 mg / L VH, 0.2 g / L VB12.

[0108] Pick activated single colony in 30mL / 250mL seed medium, culture at 30°C, 200 rpm for 18 h. Then press initial OD 562 1 was transferred to 50mL / 500mL fermentation medium, wherein 20 g / L calcium carbonate was added to the fermentation medium to balance the pH. Incubate at 30°C, 200 rpm for 72 h. Sampling was carried out to determine the relevant amino acid production.

[0109] In the present embodiment, the amino acid content of the fermented liquid adopts HPLC to me...

Embodiment 3

[0112] Example 3, the best mutant strain WTQ102 / pJYW-4- hom m - lysC m - brnFE Production of methionine at fermenter level

[0113] The seed medium and fermentation medium used in this example were the same as those in Example 2.

[0114] Pick activated single colony in 50mL / 500mL seed medium, culture at 30°C, 200 rpm for 18 h. Then press initial OD 562Transfer to 1.2L / 3L fermenter for 1. Adjust the pH to 7.0 by feeding ammonia water, control the dissolved oxygen to 30% by correlating the stirring speed and aeration rate, and control the residual sugar above 20g / L by adding 50% glucose. Samples were taken every 4 h to determine the bacterial concentration, residual sugar and amino acid content. Wherein the amino acid detection method is exactly the same as that in Example 2. The test results showed that the methionine yield reached the highest level at 48 h of fermentation, and the highest yield was 3.1 g / L.

[0115] SEQ ID NO.1

[0116] According to genebank rep...

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Abstract

Belonging to the field of bioengineering, the invention relates to construction and application of C.glutamicum subspecies lactofermentum with high L-methionine yield. The methionine high-yielding recombinant strain C.glutamicum subspecies lactofermentum QW102 / pJYW-4-hom<m>-lysC<m>-brnFE provided by the invention is preserved in China Center for Type Culture Collection with a preservation number of CCTCC M2014232. The recombinant strain has the genetic characteristics of: expression of transport protein BrnFE, expression of aspartokinase fragment lysC and homoserine dehydrogenase fragment hom, and deletion of thrB and mcbR genes on a genome at the same time. According to the invention, by a genetic engineering method, a recombinant strain with high methionine yield is obtained. The invention also provides a method for high yield of L-methionine by the recombinant strain.

Description

technical field [0001] The invention relates to the construction and application of a high-yield L-methionine Corynebacterium glutamicum, which belongs to the field of bioengineering, and specifically relates to a method for high-yield L-methionine. Background technique [0002] L-methionine is one of the sulfur-containing α-sulfur amino acids. The synthesis methods are mainly chemical synthesis and fermentation. At present, the main application of industrial production is the chemical synthesis of methionine, but there is still no industrial application example of the fermentation method. L-methionine is widely used in the feed industry. Because feed production does not require high-purity L-methionine, chemical synthesis is still the main method. However, due to the large amount of harmful substances produced by chemical synthesis, the production of methionine by fermentation has attracted more and more attention. However, due to the high energy consumption of the methi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P13/12C12R1/15
Inventor 王小元秦天宇胡晓清李烨李颜颜
Owner JIANGNAN UNIV
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