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Application of gene SNG1 deletion in improving vanillic aldehyde resistance of saccharomyces cerevisiae

A technology of Saccharomyces cerevisiae and gene deletion, applied in the application, genetic engineering, plant genetic improvement and other directions, to achieve the effect of shortening the fermentation cycle, improving resistance and saving production costs

Active Publication Date: 2020-08-18
QILU UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The search found that there are few reports on the application of Saccharomyces cerevisiae SNG1 gene deletion in improving vanillin resistance

Method used

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  • Application of gene SNG1 deletion in improving vanillic aldehyde resistance of saccharomyces cerevisiae
  • Application of gene SNG1 deletion in improving vanillic aldehyde resistance of saccharomyces cerevisiae
  • Application of gene SNG1 deletion in improving vanillic aldehyde resistance of saccharomyces cerevisiae

Examples

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Embodiment 1

[0045] Example 1: Construction of sng1Δ mutant

[0046] (1) Extraction of Saccharomyces cerevisiae BY4741 genome

[0047] Inoculate Saccharomyces cerevisiae BY4741 in 5mL YEPD medium, cultivate overnight at 30°C, 200rp, collect the bacteria by centrifugation, wash once with 1mL of sterile water, add 200μL of lysis buffer to suspend the cells, and transfer to a cell containing 0.4g of glass beads. Add 200 μL of phenol / chloroform / isoamyl alcohol (25:24:1) to the screw-top broken cell tube, vortex for 3 minutes, add 200 μL of TE solution to mix, centrifuge at 13000 r / min for 10 minutes at 4 °C, transfer to Clear to a new Eppendorf tube, add 1ml of -20°C absolute ethanol to precipitate DNA, then centrifuge at 13000r / min for 10min at 4°C, and discard the supernatant. After vacuum drying, add 400 μL of TE solution to dissolve DNA, then add 10 μL of 10 mg mL -1 RNase A, after incubation at 37°C for 15min, add 10μL of 4mol L -1 After mixing the ammonium acetate and 1mL of absolute ...

Embodiment 2

[0089] Example 2: Application of sng1Δ mutant in improving vanillin resistance (vanillin tolerance)

[0090] (1) Vanillin tolerance experiment of sng1Δ mutant

[0091] The obtained sng1Δ mutant was mixed with 6mmol L -1 Vanillin gradient growth experiments were performed on YEPD plates. The specific operation is as follows:

[0092] Inoculate the sng1Δ mutant in 1-2mL of YEPD medium, culture overnight at 30°C until the logarithmic growth phase, and the culture product was measured at the initial OD 600 Transfer 0.2 to 5mL fresh culture medium, collect the cells by centrifugation after cultivating overnight, wash the suspended cells with 1mL sterile water for 3 times, centrifuge at 8000r / min, suspend the cells with sterile water again, put them in Placed at 30°C for 9h to prepare resting cells. During the preparation of the culture medium, pour it on the plate and let it dry naturally. Adjust the concentration of bacterial suspension to OD 600 1.0, followed by 10-fold ser...

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Abstract

The invention discloses an application of saccharomyces cerevisiae SNG1 gene deletion in improving vanillic aldehyde resistance. The gene sequence of the related SNG1 gene deletion mutant is composedof -18 to +203 bp nucleotide fragments of a saccharomyces cerevisiae SNG1 gene, nucleotide fragments of loxp-KanMX4-loxp and +1446 to +1644 bp nucleotide fragments of the saccharomyces cerevisiae SNG1gene, and the nucleotide sequence of the mutant is as shown in SEQ ID No.1. Experiments prove that under no stress, the wild type is not different from the growth of the sng1 delta mutant disclosed by the invention; however, under the stress of vanillic aldehyde, the maximum specific growth rate of the sng1 delta mutant is 70% higher than that of a control strain, and the specific consumption rate of vanillic aldehyde is 52% higher than that of vanillic aldehyde. It is prompted that the mutant strain is suitable for vanillin / alcohol production or construction of a high-vanillin-tolerance saccharomyces cerevisiae strain for producing second-generation fuel ethanol or other high-value compounds by taking lignocellulose as a raw material.

Description

technical field [0001] The invention relates to the application of SNG1 deletion of Saccharomyces cerevisiae gene in improving vanillin resistance, and belongs to the field of biotechnology. Background technique [0002] Saccharomyces cerevisiae is a traditional ethanol production strain. It has high hexose utilization, high ethanol and low pH tolerance robustness. At the same time, Saccharomyces cerevisiae is a model fungus, and people have a better understanding of its molecular genetic manipulation. It is abundant and has many operating tools and means, so it has become the main cell factory for the production of ethanol and high-value compounds by fermentation (Liu et al., 2009). [0003] Lignocellulose is the most abundant renewable biomass resource on earth. The production of bioethanol or other high-value compounds from lignocellulose has been widely concerned by scientists around the world. Vanillin is a major phenolic inhibitor in lignocellulose hydrolyzate, and i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N1/19C12N15/31C12R1/865
CPCC12N15/905C07K14/395C12N15/81C12R2001/865C12N1/36
Inventor 鲍晓明王心凝曹文妍赵伟全李在禄
Owner QILU UNIV OF TECH
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