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Error-prone whole-genome shuffling approach for Zymomonas mobilis and furfural-tolerant Zymomonas mobilis

A Zymomonas and sports technology, applied in the field of bioengineering, to achieve the effect of strong furfural tolerance, safe and environmentally friendly reagents, and solving energy crisis

Active Publication Date: 2018-04-06
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional error-prone PCR technology can only effectively mutate specific genes, while the whole genome shuffling technology needs to combine physical and chemical mutagenesis to obtain excellent strains, and then realize the shuffling of the whole gene of each strain through protoplast fusion, so as to screen out tolerance traits Improved bacteria [7]

Method used

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  • Error-prone whole-genome shuffling approach for Zymomonas mobilis and furfural-tolerant Zymomonas mobilis
  • Error-prone whole-genome shuffling approach for Zymomonas mobilis and furfural-tolerant Zymomonas mobilis
  • Error-prone whole-genome shuffling approach for Zymomonas mobilis and furfural-tolerant Zymomonas mobilis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Genomic DNA Extraction:

[0035] Extract the genomic DNA of starting bacterial strain Zymomonas mobilis CP4, ( figure 1 ), the specific operation is as follows: the above-mentioned bacterial solution in the glycerol tube was transferred to a test tube containing 5 mL of RM liquid medium, and cultured at 30°C overnight to make OD600=2-4. Transfer the culture to a centrifuge tube, centrifuge at 13,000 rpm at 4°C for 1 minute, discard the supernatant, resuspend the pellet with 30 μL of sterile water, and discard the supernatant by centrifugation. Add 120 μL of bacteriostasis buffer, 60 μL of Tris saturated phenol, 60 μL of chloroform, 120 μL of TE buffer, and 120 μL of quartz sand into the centrifuge tube. After fully shaking for 4 min, centrifuge at 13000 rpm / 5 min at 4 °C to obtain the supernatant. Add 1mL of absolute ethanol to the supernatant, mix it upside down, place it at ‐20°C for 30min, centrifuge at 13000rpm / 25min at 4°C, discard the supernatant, dry t...

Embodiment 2

[0041] Example 2 Error-prone PCR amplification of the whole genome:

[0042] Take 5 μL of dNTPS stock solution, 16.6 μL of 100 uM 10-17 base random primers, 3 μL of 15 mM MnCl 2 , 5 μL of 10× mutation buffer, 20ng of genomic DNA obtained in step (1), 2.5 μL of 2U / μL Taq DNA polymerase, supplemented with sterile water to 50 μL; the dNTPS stock solution contains 10mM dCTP and dTTP, 2mM dATP and dGTP ; The 10× mutation buffer contains 1mM Tris-HCl pH8.3, 0.7mM MgCl 2 , 5mM KCl, 1g / 100mL glycerol;

[0043] Error-prone PCR reaction conditions: 94°C pre-denaturation for 5 minutes; 92°C for 1 minute, then annealing at 37°C for 1 minute, then rising from 37°C to 55°C at a rate of 0.1°C per second, and extending at 55°C for 4 minutes. 50 cycles, supplementary extension at 55°C for 10 min; amplification products such as figure 1 shown.

[0044] 16.6μL of 100uM any primer with 10-17 bases can amplify error-prone PCR products.

[0045] 16.6μL of 100uM10-17 bases are:

[0046] 8.3 μL...

Embodiment 3

[0060] Example 3 Whole genome shuffling:

[0061] The various PCR products obtained in Example 2 were mixed and concentrated by ethanol precipitation 10 times for electrotransformation;

[0062] Inoculate the starting strain Zymomonas mobilis CP4 into 3ml RM liquid medium and grow it to the late logarithmic period, inoculate its culture into 50ml RM liquid medium with 0.5% inoculum amount, and culture it statically at 30°C until OD 600 =0.4-0.5 (also can be any value of 0.4-0.5); 6000rpm centrifuges 5min, collects thallus, washes 2 times with 10% glycerol aqueous solution with the volume percentage content of ice bath, with 2ml volume percentage content is 10% aqueous solution of glycerol was suspended and mixed to obtain competent cells;

[0063] Take 4 parts of competent cells, 50 μl each, add 3 μL, 4 μL, 5 μL of 10-fold concentrated PCR products, and 5 μL of sterile ultrapure water to 50 μl of competent cells, and transfer them to sterile pre-cooled 1mm electroporation In...

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Abstract

The invention discloses an error-prone whole-genome shuffling method of zymomonas mobilis and furfural-tolerant zymomonas mobilis. The furfural-tolerant zymomonas mobilis is classified and named as zymomonas mobilis F2.5-4 with the preservation number CCTCC NO: M2015366. The furfural-tolerant zymomonas mobilis obtained by the method is capable of growing in an RM culture medium with 3g / L furfural efficiently. Compared with a traditional whole-genome shuffling technology, the error-prone whole-genome shuffling method has the advantages that since the error-prone PCR (polymerase chain reaction) technology is applied effectively, the furfural-tolerant zymomonas mobilis can be obtained rapidly and effectively without physical and chemical mutagenesis and protoplast fusion. Growth of a starting zymomonas mobilis strain in the culture medium with the 3g / L furfural is inhibited severely, and the furfural-tolerant zymomonas mobilis obtained by the method is stronger than the starting zymomonas mobilis strain in furfural tolerance.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a method for improving the furfural tolerance of Zymomonas mobilis by using the error-prone whole genome reorganization technology and the furfural-tolerant Zymomonas mobilis. Background technique [0002] With the depletion of fossil energy and the gradual emergence of the greenhouse effect, clean alternative energy has become a global research hotspot, and the research and development of fuel ethanol produced by fermentation of lignocellulose as raw material has attracted more attention. Renewable lignocellulose resources are extremely abundant in nature, and its main components are cellulose, hemicellulose and lignin. Saccharomyces cerevisiae and Zymomonas mobilis have attracted much attention as ethanol-producing strains. Zymomonas mobilis is currently the only microorganism that anaerobically ferments glucose through the ED pathway. Compared with Saccharomyces cerevisiae, it has...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/10C12R1/01
Inventor 马媛媛张敏华洪解放邹少兰
Owner TIANJIN UNIV
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