Genetically engineered bacterium of high-yield threonine and application method thereof

A technology of genetically engineered bacteria and threonine, which is applied in the fields of genetic engineering and fermentation engineering, can solve the problems of short fermentation period, low conversion rate and low conversion rate of Escherichia coli, reduce the synthesis of by-product acetic acid, increase production, Enhance the effect of metabolic balance

Active Publication Date: 2019-10-11
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] Since Escherichia coli is currently the high-yield threonine-producing bacteria, although Escherichia coli grows rapidly and the fermentation period is short, the conversion rate is not high
The starting strain of this industrial production currently has the best economic benefit of using glucose to produce threonine, but the conversion rate is low. When the shake flask is used as a control, the conversion rate is 40%.

Method used

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  • Genetically engineered bacterium of high-yield threonine and application method thereof
  • Genetically engineered bacterium of high-yield threonine and application method thereof
  • Genetically engineered bacterium of high-yield threonine and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of Recombinant Bacteria TWF001 / pFW01-phaCAB

[0033] (1) Taking Eutropha rosenbergii genome NC_008313.1 as a template, primers phaCAB-F / phaCAB-R were used to amplify the phaCAB gene cluster, and the sequence of the primer phaCAB-F / phaCAB-R for amplifying the phaCAB gene cluster was

[0034] phaCAB-F: 5'-CTGCTCGAGAGAAGGAGAATCAAATCATGGCTACCGG-3'

[0035] phaCAB-R: 5'-CCG GAATTC AGGTCAGCCCATATGCAGG-3'

[0036](2) Digest the PCR product with restriction endonuclease XhoI and EcoRI, vector pFW01 (see the article "Increasing l-threonine production in Escherichia coli by engineering the glyoxylate shunt and the l-threonine biosynthesis pathway" for the construction method of vector pFW01) with EcoRI After digesting with XhoI and purifying the digested product, use T4 ligase to treat overnight at 22°C, transform into E. coli DH5α, and screen the correct transformants. For the obtained correct transformants, extract the plasmid and use 1.8kV, 5ms for el...

Embodiment 2

[0038] Example 2 Application of Escherichia coli Genetic Engineering Bacteria TWF001 / pFW01-phaCAB Shake Flask Fermentation to Produce PHB and L-Threonine (1) Shake Flask Fermentation of Recombinant Genetic Engineering Bacteria

[0039] Seed medium I: LB medium: 10g / L peptone, 5g / L yeast powder, 10g / L NaCl;

[0040] Shake flask fermentation medium I: 30g / L glucose, 25g / L (NH 4 ) 2 SO 4 , 7.5g / L KH 2 PO 4 , 2g / L yeast extract, 2g / L citric acid, 1g / L MgSO 4 ·7H 2 O, 5mg / L FeSO 4 ·7H 2 O, 5mg / L MnSO 4 4H 2 O, 20g / L CaCO 3 , pH 6.8 or pH 7.0;

[0041] Culture method: 37°C, 200rpm, culture for 36h.

[0042] After the fermentation, under the condition of pH 6.8, more threonine was synthesized. The genetically engineered bacteria TWF001 / pFW01-phaCAB synthesized threonine 17.0g / L, the conversion coefficient of glucose was 0.57g / g, and the synthesis of acetic acid was 2.83g / L ; while the control bacteria TWF001 / pFW01 synthesized 9.72g / L threonine, the conversion coefficient...

Embodiment 4

[0061] (1) Determination of intracellular acetyl-CoA in recombinant bacteria TWF001 / pFW01-phaCAB and TWF001 / pFW01

[0062] Collect the OD prepared by step (1) in 2mL embodiment 2 600= 9.0 or so fermentation broth, put it on ice, and use the kit Acetyl-CoA Assay Kit (Solarbio, Beijing, China) to extract and measure acetyl-CoA, measure the light absorption value at 340nm, and the synthesis amount of acetyl-CoA By the formula Acetyl-CoA (nmol / g wetcell)=(1640*ΔA+0.012) / (OD 600 *1.7*0.002); ΔA=A 80s -A 20s Calculated, where A 80s OD of 80s after adding working solution 340nm , where A 20s OD 20s after adding working solution 340n .

[0063] The amount of acetyl-CoA synthesized by the genetically engineered bacteria TWF001 / pFW01-phaCAB per gram of wet cells was 4.51 μmol / g, while the amount of acetyl-CoA synthesized by the control bacteria TWF001 / pFW01 per gram of wet cells was 1.43 μmol / g;

[0064] (2) Determination of intracellular malic acid in recombinant bacteria TWF00...

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Abstract

The invention discloses a genetically engineered bacterium of high-yield threonine and an application method thereof, and belongs to the fields of genetic engineering and fermentation engineering. Thegenetically engineered bacterium is TWF001 / pFW01-phalpha CAB, phaCAB is over-expressed on a triclosan expression plasmid pFW01, threonine is synthesized extracellularly, and PHB is synthesized intracellularly. The synthetic amount of the constructed bacterium threonine is greatly increased, the shake flask fermentation level yield is 17.0 g / L, the synthetic threonine yield is 96.4 g / L on the 3-Ltank fermentation level, and the synthetic threonine yield is 133.5 g / L on the 10-L tank fermentation level. The genetically engineered bacterium is TWF001 / pFW01-phalpha CAB, the growth condition is good, no exogenous resistance gene sequence is introduced, and large-scale industrial production is better facilitated.

Description

technical field [0001] The invention relates to a genetically engineered bacterium with high threonine production and an application method thereof, belonging to the fields of genetic engineering and fermentation engineering. [0002] technical background [0003] As one of the eight essential amino acids, L-threonine is the second limiting amino acid in pig feed and the third limiting amino acid in poultry feed. Using the low-protein formula feed added with threonine as the poultry diet not only reduces the feeding cost, but also facilitates the poultry's absorption and promotes the growth of the poultry. In addition to alleviating the lack of natural protein, a reasonable nutritional ratio of poultry feed can also effectively reduce animal ammonia emissions, thereby reducing environmental pollution and benefiting the sustainable development of society. In addition, the amount of L-threonine used in food, medicine and cosmetics also shows a long-term steady growth trend. I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P13/08C12P7/62C12R1/19
CPCC12N9/0006C12N9/1029C12N15/52C12N15/70C12P7/625C12P13/08C12Y101/01036C12Y203/01009
Inventor 王小元王建莉马文渐方宇赵磊董笑飞詹依
Owner JIANGNAN UNIV
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