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Method for knocking out arcA to increase yield of Klebsiella 1,3-propylene glycol

A Klebsiella, propylene glycol technology, applied in the field of metabolic engineering, can solve problems such as limiting TCA cycle activity and affecting the regeneration of intracellular reducing power.

Active Publication Date: 2017-02-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the intracellular supply of reducing power has been the main bottleneck limiting the production of 1,3-propanediol
[0003] In microbial cells, the TCA cycle is an important source of intracellular reducing power, but low dissolved oxygen conditions will limit the activity of the TCA cycle and affect the regeneration of intracellular reducing power

Method used

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  • Method for knocking out arcA to increase yield of Klebsiella 1,3-propylene glycol
  • Method for knocking out arcA to increase yield of Klebsiella 1,3-propylene glycol
  • Method for knocking out arcA to increase yield of Klebsiella 1,3-propylene glycol

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0028] Example 1 This example illustrates the method for knocking out the arcA gene in K.pneumoniae using Red recombination technology

[0029] 1 K.pneumoniae was inoculated in liquid LB medium and cultured at 37°C until OD 600 =0.6, preparation of electrotransformation competent state.

[0030] 2 Introduce the pKD46 plasmid containing chloramphenicol resistance into K.pneumoniae competent, culture at 30°C for 12 hours, and transfer to liquid LB medium (containing chloramphenicol 30 μg L -1 ), cultured at 30℃ to OD 600 =0.25, add 30 mM arabinose, induce pKD46 plasmid to express Exo, Bet and Gam three proteins at 37°C, and prepare K.pneumonia (pKD46) competent again.

[0031] 3 Using the kanamycin-resistant pKD13 plasmid as a template, design amplification primers with arcA homologous fragments at both ends, and amplify to obtain linear DNA fragments. The primer sequences are as follows:

[0032] The upstream primer sequence is:

[0033] GGACTTTGGTACTTCCTGTTTCGATTTAGTTGGCA...

example 2

[0040] Example 2 Changes in transcription levels of key genes in the TCA cycle under microaerobic conditions after arcA gene knockout.

[0041] 1 Extraction of K.pneumoniae total RNA

[0042] (1) Grind the collected fresh bacteria immediately in liquid nitrogen.

[0043] (2) Add 1mL TRIzon to fully lyse the cells, and place at room temperature for 5 minutes to separate protein-nucleic acid complexes.

[0044] (3) Add 0.2 mL of chloroform to the solution in the upward step, cover the tube cap, shake vigorously for 15 s, and place at room temperature for 3 min.

[0045] (4) 4°C, 12000r·min -1 Centrifuge for 15 min, and transfer the upper aqueous phase to a new RNase-free centrifuge tube.

[0046] (5) Add an equal volume of isopropanol, pipette evenly, and place at room temperature for 10 minutes.

[0047] (6) 4°C, 12000r·min -1 Centrifuge for 10 min and discard the supernatant.

[0048] (7) Add 1 mL of RNase-free 75% ethanol to wash the precipitate.

[0049] (8) 4°C, 1200...

example 3

[0068] Example 3. Fermentation of 1,3-propanediol using constructed K.pneumoniaeΔarcA.

[0069] 1 Preparation of seed medium: yeast powder 10g L -1 , peptone 5g·L -1 , sodium chloride 10g·L -1 , the balance is water; fermentation medium: glycerol 40g L -1 , yeast powder 6g·L -1 , glucose 2g·L -1 , KH 2 PO 4 7.5g·L -1 , MgSO 4 2g·L -1 , (NH 4 ) 2 SO 4 2g·L -1 , FeSO 4 ·7H 2 O 0.005g·L -1 , VB 12 0.015g·L -1 , trace element solution (MnCl 2 4H 2 O 0.1g L -1 , Na 2 MoO 4 2H 2 O 0.035g·L -1 , CoCl 2 ·6H 2 O 0.2g·L -1 , ZnCl 2 0.07g·L -1 , CuCl 2 0.02g·L -1 , NiCl 2 ·6H 2 O 0.025g·L -1 , H 3 BO 3 0.06g·L -1 )100μL·L -1 , the balance is water, and KOH is used to adjust the pH to about 8.5.

[0070] 2 Inoculate K.pneumonia and the mutant strain obtained above in liquid LB medium, 37°C, 100r min -1 Shake culture for 16 hours, transfer to the seed medium according to the inoculum size of 0.5% (v / v), 37°C, 100r·min -1 Grow to OD 600 =3, tr...

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Abstract

The invention discloses establishment and application of a Klebsiella arcA gene-deleted strain. In the invention, the key gene arcA inhibiting TCA cycle of 1,3-propylene glycol producing Klebsiella is knocked out by a homologous recombination method, the TCA cycle activity of cells in a micro-aerobic condition is increased, the reducing power regeneration is enhanced, and the yield of 1,3-propylene glycol is increased. The yield of 1,3-propylene glycol synthesized by fermenting glycerin with knockout bacteria K.pneumoniae (delta)arcA under micro-aerobic conditions is increased by 12.57% to 17.64g / L, indicating that the yield of 1,3-propylene glycol can be effectively increased by knocking out the arcA gene participating in TCA cycle transcription regulation.

Description

technical field [0001] The invention provides a method for knocking out the arcA gene to increase the production of Klebsiella 1,3-propanediol, and belongs to the technical field of metabolic engineering. Background technique [0002] 1,3-Propanediol (PDO for short) is an important chemical raw material, and its main use is as a monomer for the synthesis of polyester and polyurethane materials. At present, the production method of 1,3-propanediol is mainly chemical synthesis. Because the chemical synthesis method has the disadvantages of high pollution, high energy consumption, and high cost, people turn their attention to the biological method with the advantages of green production. Among them, Klebsiella (Klebsiella) ferments glycerol to synthesize PDO under microaerobic conditions, which has the advantages of high substrate conversion rate, development value and application prospect. However, the intracellular supply of reducing power has been the main bottleneck limit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12P7/18C12R1/22
Inventor 诸葛斌陆竞争陆信曜宗红宋健任顺利
Owner JIANGNAN UNIV
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