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Methods for constructing and fermenting L-serine high-yielding recombinant corynebacterium glutamicum

A Corynebacterium glutamicum, construction method technology, applied in the direction of microorganism-based methods, fermentation, recombinant DNA technology, etc., can solve the problems of slow bacterial growth, reduced production efficiency, reduced production rate, etc., to achieve increased yield and sugar Effects of acid conversion, increased growth rate, and increased production intensity

Active Publication Date: 2015-04-29
JIANGNAN UNIV +1
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Problems solved by technology

At the same time, there is competition for metabolic flow in the pathway of L-serine synthesis, excessively changing the metabolic flow to the L-serine pathway will lead to a decrease in the metabolic flow to the pyruvate pathway and the tricarboxylic acid cycle, which will significantly reduce Growth rate of Corynebacterium glutamicum
Although the sugar-acid conversion rate of L-serine can be effectively improved by means of metabolic engineering, due to the reduction of metabolic flow to the tricarboxylic acid cycle, the production rate of Corynebacterium glutamicum will be reduced, which directly affects the production efficiency of L-serine. It is not conducive to the industrial production of L-serine, which has become an urgent problem to be solved in the production of L-serine by microbial fermentation
In order to solve the problem that the excessive flow of metabolic flow to the L-serine pathway causes the production efficiency of L-serine to decrease, this patent proposes a strategy of combining metabolic engineering transformation and fermentation technology. It flows to the L-serine synthesis pathway, which improves the sugar-acid conversion efficiency of L-serine; on the other hand, by adding nutrients to meet the nutritional needs of Corynebacterium glutamicum caused by metabolic transformation, it solves the problem of slow growth of bacteria. Increased production intensity of L-serine

Method used

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  • Methods for constructing and fermenting L-serine high-yielding recombinant corynebacterium glutamicum
  • Methods for constructing and fermenting L-serine high-yielding recombinant corynebacterium glutamicum
  • Methods for constructing and fermenting L-serine high-yielding recombinant corynebacterium glutamicum

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1: Recombinant bacteria Corynebacterium glutamicum SYPS-062-33a SS avtA build

[0024] Based on the genome sequence information of Corynebacterium glutamicum ATCC13032, it was designed to construct avtA The primers of the knockout recombinant knockout plasmid, the primer sequence is as follows:

[0025] avtA -1: 5’-GAAAAGCTTAAGGCCCTGCAGTAGTG-3’ ( Hin dIII)

[0026] avtA -2: 5’- CCCATCTGTTAAACTTAAACCAAACGGCTGAACATTGCTT-3’

[0027] avtA -3: 5’- GGTTTAAGTTTAACAGATGGGGGTGTGCGCAAAATCGG-3’

[0028] avtA -4: 5’- ATTCTAGACCTGCGCTGCCACGTTGT-3’ ( Xba I)

[0029] primers avtA -1 and avtA -2 to amplify the upstream fragment with primers avtA -3 and avtA -4 Amplify the downstream fragments, respectively purify the upstream and downstream fragments by agarose gel electrophoresis, see figure 2 , the lengths are 507bp and 548bp respectively; the upstream fragment and the downstream fragment are used as templates respectively, and the primers avt...

Embodiment 2

[0030] Embodiment 2: Recombinant bacteria Corynebacterium glutamicum SYPS-062-33a SS avtA C-T wxya build

[0031] Using the recombinant strain constructed in Example 1 as the starting strain, the coding regulatory gene of acetohydroxyacid synthase (AHAS) that catalyzes pyruvate to generate α-acetolactate wxya The 249bp of the C-terminus of the C-terminus was precisely deleted, so that the enzyme activity of acetohydroxyacid synthase was reduced. Design and synthesize the following primers for knockout wxya The recombinant plasmid:

[0032] wxya -1: 5’-CCCAAGCTTGCTGTTTCCAGATGACCAACC-3’ ( Hin dIII)

[0033] wxya -2: 5’-GGCGATAGTGGTCTCTTCATCAAGTCGCACGACTTTGAGC-3’

[0034] wxya -3: 5’-GAAGAGACCACTATCGCCACAGCAATTAATCTGATTGC-3’

[0035] wxya -4: 5’-CGCGGATCCCGTTCAGGTTTGGCTCGATG-3’ ( Bam HI)

[0036] Specific experimental steps refer to Example 1, knockout plasmid pK18mob sacB C-T wxya The construction diagram see Figure 4 . Recombinant bacteria Coryneba...

Embodiment 3

[0040] Embodiment 3: the enzyme activity assay method of acetohydroxyacid synthase (AHAS)

[0041] Reaction system: 100 mM potassium phosphate buffer (pH 7.3), 50 mM sodium pyruvate, 10 mM MgCl 2 and 100 μM flavin adenine dinucleotide (FAD). Take the Corynebacterium glutamicum cells in the logarithmic phase, centrifuge to remove the supernatant, wash with 0.2% ice KCl for 3 times, ultrasonically break and centrifuge to obtain the supernatant, take a certain amount of supernatant and add it to the reaction system, Add 100 μL of 50 % H after incubation at 37oC for 20 minutes 2 SO 4 Stop the reaction. After 30 minutes of incubation at 37oC, acetoin was produced from α-acetolactic acid, and the content of acetoin in the reaction solution was determined by gas chromatography. One unit of enzyme activity is set as: 1 nmol of α-acetolactate produced per milligram of protein per minute. Protein concentration was determined by the Bradford method. Acetohydroxyacid synthase enzym...

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Abstract

The invention provides a strategy combining metabolic engineering reconstruction with a fermentation process. On the one hand, a metabolic flow is changed to flow from a glycolysis route to an L-serine synthetic route by virtue of a metabolic engineering means, so that the sugar-acid conversion efficiency of L-serine is improved; and on the other hand, nutritional ingredients are added to meet requirements of corynebacterium glutamicum on nutrient substances after metabolic reconstruction, so that the problem of slow growth of thalli is solved, and the production strength of L-serine is improved.

Description

technical field [0001] The invention relates to the construction of high-yield L-serine strain Corynebacterium glutamicum and its fermentation process. The invention relates to a method for producing L-serine by using carbohydrate raw materials by microorganisms, and belongs to the field of biotechnology. Background technique [0002] L-serine is a non-essential amino acid, which has important physiological functions and application value, and is widely used in the fields of medicine, food and chemical industry. At present, the production of L-serine mainly relies on proteolysis, enzymatic conversion and microbial precursor fermentation. However, these methods have problems such as small yield, high production cost, and large environmental pollution, and are not suitable for large-scale industrial production of L-serine. Serine. The direct metabolism of renewable sugary raw materials to produce L-serine by microbial fermentation is a green and sustainable production method,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/77C12N1/21C12P13/14C12R1/15
Inventor 张晓梅许正宏史劲松朱勤健郭恒华张冬竹
Owner JIANGNAN UNIV
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