Clostridium modified by kinase gene with phosphorylation function and application of clostridium

A technology of genetic modification and phosphorylation, applied in the field of microbial engineering, can solve problems such as affecting the synthesis of Clostridium metabolites

Active Publication Date: 2018-06-29
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These kinases with phosphorylation function can transfer phosphate groups in Clostridium and activate downstream proteins, so they may regulate the physiological metabolism of Clostridia, and may also affect

Method used

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  • Clostridium modified by kinase gene with phosphorylation function and application of clostridium
  • Clostridium modified by kinase gene with phosphorylation function and application of clostridium
  • Clostridium modified by kinase gene with phosphorylation function and application of clostridium

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Experimental program
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Embodiment 1

[0026] This embodiment includes the following steps:

[0027] (1) Vector construction

[0028] Gene knockout: use the pSY6 plasmid containing class II intron as the vector, such as figure 1 A. The intron fragment is modified by overlap extension PCR to make it have the function of specifically recognizing the target gene, and insert the target site to block the expression of the target gene, as shown in the illustration figure 1 b. Overlap extension PCR primers were designed through the "Clostron" website (www.clostron.com), and the insertion site in the gene to be knocked out was predicted according to the algorithm published in the literature (Journal of Molecular Biology, 2004, 336:421-439). The sequence is shown in Table 1. In the first round of PCR, primers IBS and EBSuniversal were used to amplify intron fragment 1, and primers EBS1d and EBS2 were used to amplify intron fragment 2. The reaction system and reaction conditions were shown in Table 2. In the second round ...

Embodiment 2

[0048] Fermentative production of butanol by a kinase-inactive strain with phosphorylation function

[0049] This example mainly includes the following steps: the seed medium used is CGM medium, and the CGM medium is deoxygenated by nitrogen gas for 20 minutes before use, then sterilized at 121°C for 15 minutes, cooled to room temperature, and inserted into 2 mL of -80°C refrigerator. Histidine kinase inactivated bacteria. After culturing at 37°C for 16-20 hours, insert the P2 fermentation medium and carry out anaerobic fermentation in the fermenter. After the liquid pH is lower than 5.0, the fermentation pH is controlled to be maintained above 5.0 by automatically feeding 50% (v / v) ammonia water. Samples were taken regularly to detect the bacterial concentration and solvent content.

[0050] CGM medium: every liter of medium contains glucose 20g, yeast powder 2g, tryptone 4g, potassium dihydrogen phosphate 0.5g, dipotassium hydrogen phosphate 0.5g, ammonium acetate 2.2g and...

Embodiment 3

[0055] Cell Morphology Detection of Kinase-inactive Strain with Phosphorylation Function

[0056] The cell morphological changes of the starting strain ATCC55025, cac3319 knockout bacteria and cac0437 knockout bacteria were observed by scanning electron microscope. The specific steps include: taking materials: during the acidogenic period (8h), the acidogenic to solventogenic transition period (16h), the solventogenic period (32h) and the decay period (72h), take 4mL of fermentation broth and centrifuge at 8000rpm for 5min, discard the supernatant , wash with 0.1mol / L PBS for 10min, repeat 3 times; fix: after washing, centrifuge and discard the supernatant, immediately add 2.5% glutaraldehyde to fix for more than 2h; wash: wash with 0.1mol / L PBS for 10min, repeat this step 3 times Dehydration: use 50%, 70%, 90%, 95% tert-butanol to process the sample step by step for each 10min and then use pure tert-butanol to process the sample for 10min, this step is repeated 3 times; Put ...

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Abstract

The invention discloses clostridium modified by a kinase gene with a phosphorylation function and application of the clostridium. A method is a method for modifying by the kinase gene with the phosphorylation function to improve the yield of butanol; specifically, the phosphorylation function in the clostridium is knocked out to obtain the kinase (including histidine kinase, serine kinase and threonine kinase) gene, so that downstream gene expression is regulated and controlled. After the kinase gene with the phosphorylation function is knocked out or overexpressed, the yield of the butanol inmutant strains is remarkably changed when being compared with that of wild strains; some kinases participate in spore formation and cell morphological changes, so that clostridium cells keep a high-activity state and the butanol synthesis capability of the cells is improved. The clostridium modified by the kinase gene with the phosphorylation function has a good application prospect in the aspectof production of the butanol.

Description

technical field [0001] The invention belongs to the technical field of microbial engineering, and in particular relates to a Clostridium modified with a phosphorylated kinase gene and a method for increasing butanol production through the gene modified phosphorylated kinase. Background technique [0002] Butanol is an important chemical raw material and a new type of bioenergy. Compared with ethanol, butanol has the advantages of high energy density, low vapor pressure, low corrosion, easy pipeline transportation, and can be mixed with gasoline in any ratio without modification of vehicles (Biotechnology Advances, 2013, 31:1575- 1587). Butanol is produced by Clostridium fermentation, and the butanol concentration at the end of the fermentation usually does not exceed 2.0% (w / v) because the fermentation product butanol has a strong toxicity inhibitory effect on the strain. With the rapid development of molecular biology technology, the molecular transformation of butanol-pr...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P7/16C12R1/145
CPCC12N9/1205C12P7/16Y02E50/10
Inventor 薛闯杜广庆张萌
Owner DALIAN UNIV OF TECH
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