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A method for knocking out arca to improve the production of 1,3-propanediol in Klebsiella

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

Active Publication Date: 2019-10-18
JIANGNAN UNIV
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  • 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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  • A method for knocking out arca to improve the production of 1,3-propanediol in Klebsiella
  • A method for knocking out arca to improve the production of 1,3-propanediol in Klebsiella
  • A method for knocking out arca to improve the production of 1,3-propanediol in Klebsiella

Examples

Experimental program
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Effect test

example 1

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

[0029] 1 K.pneumoniae was inoculated into liquid LB medium and cultivated to OD at 37℃ 600 =0.6, preparation of electrotransformation competent.

[0030] 2 Introduce the pKD46 plasmid containing chloramphenicol resistance into K.pneumoniae competence, incubate at 30°C for 12h, and transfer to liquid LB medium (containing chloramphenicol 30μg·L -1 ), cultivate to OD at 30℃ 600 =0.25, 30mM arabinose was added, the pKD46 plasmid was induced to express the three proteins of Exo, Bet and Gam at 37°C, and the K. pneumonia (pKD46) competence was prepared again.

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

[0032] The upstream primer sequence is:

[0033] GGACTTTGGTACTTCCTGTTTCGATTTAGTTGGCAATT...

example 2

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

[0041] 1 K.pneumoniae total RNA extraction

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

[0043] (2) Add 1 mL of TRIzon to fully lyse the bacteria, leave it at room temperature for 5 minutes, and separate the protein and nucleic acid complex.

[0044] (3) Add 0.2 mL of chloroform to the solution in the previous step, close the cap, shake vigorously for 15 seconds, and place at room temperature for 3 minutes.

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

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

[0047] (6) 4℃, 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℃, 12000...

example 3

[0068] Example 3. Using the constructed K. pneumoniaeΔarcA to ferment to produce 1,3-propanediol.

[0069] 1 Prepare seed culture 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, KOH adjusts the pH to about 8.5.

[0070] 2 Inoculate K.pneumonia and the mutant strain obtained above in liquid LB medium, 37℃, 100r·min -1 Shake culture for 16h, transfer seed medium at 0.5% (v / v) inoculum, 37℃, 100r·min -1 Cultivate to OD 600 =3, transfer the seed liquid to 50mL fermentation medium according to 4% inoculum amou...

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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, which belongs to the technical field of metabolic engineering. Background technique [0002] 1,3-Propanediol (PDO) is an important chemical raw material, and its main purpose 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. Due to the shortcomings of chemical synthesis, such as high pollution, high energy consumption, and high cost, people turn their attention to biological methods with green production advantages. Among them, Klebsiella (Klebsiella) fermented glycerol to synthesize PDO under microaerobic conditions has the advantages of high substrate conversion rate, development value and application prospects. However, the supply of reducing power in cells has been the main bottleneck restricting the production of 1,3-propaned...

Claims

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Application Information

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Patent Type & Authority Patents(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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