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Construction of a high-yielding l-serine recombinant Corynebacterium glutamicum and its fermentation method

A technology of Corynebacterium glutamicum and serine, applied in the biological field, can solve the problems of slow growth of bacteria, decreased production efficiency, decreased production rate, etc., and achieves improved yield and sugar-acid conversion rate, increased growth rate, and increased production intensity. Effect

Active Publication Date: 2017-06-20
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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  • Construction of a high-yielding l-serine recombinant Corynebacterium glutamicum and its fermentation method
  • Construction of a high-yielding l-serine recombinant Corynebacterium glutamicum and its fermentation method
  • Construction of a high-yielding l-serine recombinant Corynebacterium glutamicum and its fermentation method

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1: Construction of recombinant bacteria Corynebacterium glutamicum 33a ΔSS ΔavtA

[0024] Based on the genome sequence information of Corynebacterium glutamicum ATCC13032, primers for constructing a recombinant knockout plasmid for avtA knockout were designed. The primer sequences are as follows:

[0025] avtA-1:5'-GAAAAGCTTAAGGCCCTGCAGTAGTG-3'(HindIII)

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

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

[0028] avtA-4:5'-ATTCTAGACCTGCGCTGCCACGTTGT-3'(XbaI)

[0029] Use primers avtA-1 and avtA-2 to amplify the upstream fragments respectively, use primers avtA-3 and avtA-4 to 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 and downstream fragments are used as templates respectively, and the upstream and downstream fusion fragments are ampl...

Embodiment 2

[0030] Example 2: Construction of recombinant bacteria Corynebacterium glutamicum 33a ΔSS ΔavtAΔC-T ilvN

[0031] Using the recombinant strain constructed in Example 1 as the starting strain, the 249bp at the C-terminal of the coding regulatory gene ilvN of the acetohydroxyacid synthase (AHAS) that catalyzes the generation of α-acetolactate from pyruvate is precisely deleted to make acetohydroxyacid Synthase activity was reduced. The following primers were designed and synthesized for the construction of recombinant plasmids for knocking out ilvN:

[0032] ilvN-1:5'-CCCAAGCTTGCTGTTTCCAGATGACCAACC-3'(HindIII)

[0033] ilvN-2:5'-GGCGATAGTGGTCTCTTCATCAAGTCGCACGACTTTGAGC-3'

[0034] ilvN-3:5'-GAAGAGACCACTATCGCCACAGCAATTAATCTGATTGC-3'

[0035] ilvN-4:5'-CGCGGATCCCGTTCAGGTTTGGCTCGATG-3'(BamHI)

[0036] For specific experimental steps, refer to Example 1. For the construction diagram of the knockout plasmid pK18mobsacBΔC-T ilvN, see Figure 4 . The verification PCR primers of r...

Embodiment 3

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

[0041] Reaction system: 100mM potassium phosphate buffer (pH 7.3), 50mM sodium pyruvate, 10mM MgCl 2 and 100 μM flavin adenine dinucleotide (FAD). Take the Corynebacterium glutamicum cells in the logarithmic phase, centrifuge to remove the supernatant, wash 3 times with 0.2% ice KCl, 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 37°C for 20 min 2 SO 4 Stop the reaction. After incubating at 37° C. for 30 minutes, acetoin was produced from α-acetolactic acid, and the content of acetoin in the reaction liquid 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 enzyme activity a...

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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 Patents(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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