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Genetically engineered bacterium for high producing beta-alanine and co-culture preparation of D-pantothenic acid

A technology of genetically engineered bacteria and alanine, applied in genetic engineering, plant genetic improvement, bacteria, etc., can solve problems such as complex regulation and suboptimal biological production performance

Active Publication Date: 2021-04-09
ZHEJIANG UNIV OF TECH
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AI Technical Summary

Problems solved by technology

In order to economically and effectively use microorganisms to produce D-pantothenic acid, the production of pantothenic acid and β-alanine can be regulated in the bacteria, and then D-pantothenic acid can be synthesized; but this regulation is very complicated, because the precursor of the two - phosphoenol The interconversion and important role of pyruvate and pyruvate require fine molecular elements to regulate, and the balance of pantoate and β-alanine pathways needs to be achieved by adjusting the relative expression intensity of pathway genes in the two modules
This requires optimization of gene copy number, expression promoter strength, and ribosome binding sites, etc., which are limited by existing bioengineering tools and often result in suboptimal bioproduction performance

Method used

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  • Genetically engineered bacterium for high producing beta-alanine and co-culture preparation of D-pantothenic acid
  • Genetically engineered bacterium for high producing beta-alanine and co-culture preparation of D-pantothenic acid
  • Genetically engineered bacterium for high producing beta-alanine and co-culture preparation of D-pantothenic acid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1: Determination of D-pantothenic acid content in fermented liquid by HPLC method

[0077] The detection method is as follows:

[0078] Sample treatment: Take 1ml of fermentation broth and centrifuge to get the supernatant, dilute the supernatant with ultrapure water to keep the D-pantothenic acid content between 0.05g / L and 0.40g / L;

[0079] Chromatographic conditions: C 18 Column (250×4.6mm, particle size 5μm, Agilent Technologies Co., Santa Clara, CA, USA), detection wavelength: 200nm, column temperature: 30°C, flow rate: 0.9ml / min;

[0080] Mobile phase: acetonitrile / water / phosphoric acid: (50 / 949 / 1);

[0081] Data collection time: 23min.

Embodiment 2

[0082] Embodiment 2: HPLC method measures beta-alanine content in fermented liquid

[0083] The detection method is as follows:

[0084] Sample treatment: Take 1ml of fermentation broth and centrifuge to get the supernatant, dilute the supernatant with ultrapure water to keep the content of β-alanine between 0.05g / L and 0.50g / L; take 100μL sample and add 1.5mL In the EP tube, add 100 μL of derivatization reagent (2,4-dinitrofluorobenzene) and 100 μL of derivatization buffer (0.5M NaHCO 3 ), then 300 μL of the system was reacted in a 60°C metal bath for 1h15min (400rpm); after the reaction, 700μL of 0.2M PB buffer was added to the system, mixed well, and centrifuged for 12-13min, when precipitation appeared, take 200μL of the supernatant In the lining tube, spare.

[0085] Chromatographic conditions: C 18 Column (250×4.6mm, particle size 5μm, Agilent Technologies Co., Santa Clara, CA, USA), detection wavelength: 360nm, column temperature: 40°C, flow rate: 1ml / min;

[0086] ...

Embodiment 3

[0089] Embodiment 3: Construction and shake flask fermentation of the bacterial strain ALA1 of overexpression panD gene

[0090] Using genetically engineered bacteria E.coli W3110(DE3) (CCTCC NO: M 2018914) as the starting strain, using CRISPR-Cas9-mediated gene editing technology such as figure 1 (Yu Jiang et al.2015Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System.Applied Environmental Microbiology.81:2506-2514), with the Trc promoter derived from pTrc99A (nucleotide sequence shown in SEQ ID No.1 ), to replace the natural promoter of the panD gene in the genome to enhance the expression intensity of the panD gene.

[0091] (1) Construction of pTarget-panD plasmid: use pTarget F plasmid (Addgene Plasmid#62226) as a template, use pT-trc-panD F / pT-trc-panD R as primers for PCR amplification, and the obtained PCR products are subjected to Dpn I in Incubate and digest at 37°C for 3 hours, then transform into E.coli DH5α-transformation-competent medium, ...

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Abstract

The invention relates to a genetically engineered bacterium for high producing beta-alanine, co-culture preparation of D-pantothenic acid, a construction method of the genetically engineered bacterium and application of the genetically engineered bacterium to co-culture preparation of the D-pantothenic acid. According to the invention, original promoters of panD, aspC and ppc genes on a genome are replaced with a pTrc99A-derived Trc promoter and a ribosomebinding site (RBS) sequence so as to enhance synthesis of the beta-alanine, and genes pykA and pykF are knocked out to block consumption of phosphoenolpyruvate (PEP) and modify a glucose uptake path of escherichia coli, and thus, a non-phosphotransferase system (non-PTS) transport system is enhanced to block a PTS transport system, and synthesized precursor PEP is accumulated; on the basis, heterologous aspartate decarboxylase genes panD and aspC of E.coli W3110 are introduced to enhance the enzyme activity of key enzymes, so that supply and conversion of beta-alanine precursors are enhanced; gdhA genes of E.coli W3110 are introduced to enhance cyclic regeneration of coenzymes NADP / DNAPH, and finally, the titer of the beta-alanine is increased from 0 to 2.48g / L. The strain and a previous D-pantothenic acid producing strain DPA 21 / pBCST3 undergo construction of a primary co-culture system; the inoculation ratio is optimized; and when the inoculation ratio of the two strains is 1: 1, the co-culture strain can produce 3.08 g / L of D-pantothenic acid in a same fermentation medium.

Description

[0001] (1) Technical field [0002] The invention relates to a genetically engineered bacterium for high-yield beta-alanine, co-cultivation for preparing D-pantothenic acid and its construction method, and its application in co-culturing for preparing D-pantothenic acid. [0003] (2) Background technology [0004] Vitamin B5 is also known as pantothenic acid because it is acidic in nature and widely found in a variety of foods. There are two configurations of pantothenic acid - D-form and L-form, and only the D-form has biological activity in the living body. D - Pantothenic acid and its derivatives (calcium D-pantothenate and D-panthenol) are widely used in food, medicine, feed, cosmetics and other fields. Because it is unstable under conditions such as acid, alkali, light and heat, a more stable form-calcium pantothenate is often used in storage, transportation, commodity trading and use. [0005] In 1933, D-pantothenic acid was extracted from the liver for the first time ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/60C12N15/54C12P39/00C12P13/06C12P13/02C12R1/19
CPCC12N9/88C12N9/1096C12N9/1205C12N15/70C12P39/00C12P13/06C12P13/02C12Y401/01015C12Y206/01021C12Y401/01031C12Y207/0104C12Y402/03012C12Y207/01002
Inventor 柳志强陈力张博李波王培郑裕国
Owner ZHEJIANG UNIV OF TECH
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