A genetically engineered bacterium that weakens the accumulation of acetic acid in the fermentation process to enhance the production of l-tryptophan and its construction method

A fermentation process, tryptophan technology, applied in the fields of metabolic engineering and genetic engineering, can solve the problems of increasing costs, difficult to achieve dissolved oxygen conditions, etc., and achieve the effects of reducing specific activity, shortening transformation time, and increasing production.

Active Publication Date: 2017-09-26
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the acetic acid concentration exceeds 2g / L, it will affect the growth of the bacterial strain and the synthesis of L-Trp. In order to reduce the accumulation of acetic acid in the fermentation process as much as possible to ensure the normal growth of the bacteria, sufficient dissolved oxygen must be ensured in the fermentation process. However, This not only increases the cost in industrial production, but also makes it difficult to achieve the dissolved oxygen conditions provided by the laboratory.

Method used

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  • A genetically engineered bacterium that weakens the accumulation of acetic acid in the fermentation process to enhance the production of l-tryptophan and its construction method
  • A genetically engineered bacterium that weakens the accumulation of acetic acid in the fermentation process to enhance the production of l-tryptophan and its construction method
  • A genetically engineered bacterium that weakens the accumulation of acetic acid in the fermentation process to enhance the production of l-tryptophan and its construction method

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Experimental program
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Effect test

Embodiment 1

[0022] Example 1: Cloning of wild-type phosphate acetyltransferase gene and construction of recombinant strains

[0023] Using the gene sequence encoding phosphate acetyltransferase (Genbank accession number NC_000913.3: 2414747-2416891) as a template, the amplification primers P1 and P2 were designed to amplify the phosphoacetyltransferase gene pta by PCR and cloned into the pMD18 vector ( commercial tool vector), the ligation product was transformed into Escherichia coli JM109, and the transformation product was coated on an LB plate containing 100 mg / L ampicillin. After culturing overnight at 37°C, colonies were selected and inserted into LB liquid medium. After 8-10 hours, the plasmid was extracted and named pta / pMD18, and the plasmid was sequenced. The results showed that the inserted fragment was a 2145bp DNA fragment encoding a protein containing 704 amino acids. The pET24a(+) plasmid and the T vector containing the pta gene were subjected to double enzyme digestion wi...

Embodiment 2

[0028] Embodiment 2: the preparation of mutant

[0029] 1) Construction of mutants

[0030]Simulated the protein structure of phosphate acetyltransferase derived from the L-tryptophan producing strain (recombinant E. An amino acid site Pro69 in the acetyltransferase molecule has a potential effect on the stability of the hexamer of the enzyme. The 69th proline (Pro) in the phosphoacetyltransferase was mutated into leucine (Leu), named P69L. According to the gene sequence encoding phosphate acetyltransferase (Genbank accession number NC_000913.3: 2414747-2416891), primers R1 and R2 introducing the P69L mutation were designed and synthesized, and the plasmid pta / pET24a(+) was used as a template to transfer phosphoacetyl Site-directed mutagenesis of the enzyme gene. The PCR product was digested with Dpn I (Fermentas Company), and transformed into Escherichia coli JM109 competent cells. After the competent cells were cultured overnight in LB solid medium (containing 30 μg / mL ka...

Embodiment 3

[0038] Embodiment 3: DTNB chromogenic method measures the enzyme activity of phosphoacetyltransferase

[0039] 1) Enzyme activity assay method

[0040] The enzyme activity of phosphate acetyltransferase was determined by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) colorimetric method. At 25°C, phosphoacetyltransferase catalyzes the substrate acetyl coenzyme A to produce coenzyme A, and coenzyme A reacts with DTNB to produce a yellow substance with an absorbance value at a wavelength of 412nm. The amount of coenzyme A is directly proportional, so colorimetry can be performed at a wavelength of 412nm to calculate the enzyme activity. Definition of enzyme activity unit: under the above conditions, the amount of enzyme that catalyzes the production of 1 μmol of coenzyme A per minute is regarded as an activity unit.

[0041] Enzyme activity assay steps: at 25°C, add 850ml 0.1mol / L pH=7.4 phosphate buffer, 50ml 1.6mmol / L DTNB, 50ml 4mmol / L acetyl-CoA (DTNB solution) to 1ml cuvette ...

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Abstract

The invention discloses a genetically engineered bacterium that weakens the accumulation of acetic acid in the fermentation process to enhance the production of L-tryptophan and a construction method thereof, belonging to the fields of metabolic engineering and genetic engineering. The present invention takes L-tryptophan production strain (recombinant Escherichia coli) FB-04 (referred to as TS) phosphate acetyltransferase (Pta) as the parent, and uses molecular biology techniques to perform site-directed mutation on it. Under this modification, the specific activity of Pta was 60.4% of that before the mutation. Using Red recombination technology, the pta gene on the TS genome was mutated by a two-step traceless gene replacement method to construct an engineering bacterium TS‑PtaP69L. This strategy significantly reduces the specific activity of Pta, and the engineering bacteria TS-PtaP69L is compared with TS in the fermentation process: 1) The content of tryptophan in shake flask fermentation increases by 80%, and the content of acetic acid decreases by 15.5%; 2) Tryptophan in 3L fermenter fermentation The acid content increased by 12.6%, and the acetic acid content decreased by 73%. The tryptophan mutant strain has better industrial fermentation performance. This genomic site-directed mutagenesis strategy can also be used to improve the fermentation performance of E. coli producing other amino acids.

Description

technical field [0001] The present invention relates to a genetically engineered bacterium that weakens the accumulation of acetic acid in the fermentation process to enhance the production of L-tryptophan and its construction method, especially the technology of using the site-directed mutation method of protein engineering to reduce the specific activity and the use of Red recombination technology, through The invention relates to a technique for performing gene replacement on the genome by a two-step traceless replacement method, which belongs to the fields of metabolic engineering and genetic engineering. Background technique [0002] As an essential amino acid in the human body, L-tryptophan (L-Trp) is widely used in medicine, food, feed and other industries. In recent years, a number of high-yield L-Trp production strains have been gradually cultivated abroad by means of metabolic engineering, but there are still problems in the fermentation process, such as a signific...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/75C12N15/63C12P13/22
Inventor 吴敬刘莉娜段绪果
Owner JIANGNAN UNIV
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