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Application of MAL33 gene deletion in improving tolerance of saccharomyces cerevisiae to lignocellulose hydrolysate inhibitor

A technology of lignocellulose and Saccharomyces cerevisiae, applied in the direction of microorganism-based methods, applications, genetic engineering, etc., can solve the problem of low tolerance, achieve the effect of improving tolerance and shortening the lag period

Active Publication Date: 2022-01-28
QILU UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In the process of producing second-generation fuel ethanol by using lignocellulosic raw materials, there is a problem that Saccharomyces cerevisiae has low tolerance to inhibitors in lignocellulose hydrolyzate, and the present invention provides MAL3 Application of 3 gene deletion in improving the tolerance of Saccharomyces cerevisiae to lignocellulosic hydrolyzate inhibitors

Method used

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  • Application of MAL33 gene deletion in improving tolerance of saccharomyces cerevisiae to lignocellulose hydrolysate inhibitor
  • Application of MAL33 gene deletion in improving tolerance of saccharomyces cerevisiae to lignocellulose hydrolysate inhibitor
  • Application of MAL33 gene deletion in improving tolerance of saccharomyces cerevisiae to lignocellulose hydrolysate inhibitor

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Experimental program
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Effect test

Embodiment 1

[0038] Example 1: MAL33 Construction of gene-deleted yeast strains

[0039] 1. Extraction of Saccharomyces cerevisiae genome

[0040] Saccharomyces cerevisiae BY4741 strain was cultured overnight in 5mL YPD medium, the cells were collected, and the BY4741 genomic DNA was extracted.

[0041] 2. Amplification of Gene Knockout Fragments

[0042] (1) MAL33 Amplification of the upstream homology arm and the downstream homology arm: using the genomic DNA of Saccharomyces cerevisiae BY4741 as a template, using primers UPS- MAL33 -F (5'-AATGGTCACTCCAAGTAACGGTATTGTGATTTCAACAGAA-3') and UPS- MAL33 -R (5'- TATTAAGGGTTGTCGACCTG ATCTTGACAACTGAGCTCTTTCACAC-3’) (underlined with loxP- KanMX4-loxP Homologous sequences upstream of the fragment for use with loxP-KanMX4-loxP for fusion PCR), and DOS- MAL33 -F (5'- TGATATCAGATCCACTAGTG TAGGACCCCTCATCACAATGATT -3’) (the underlined part is the loxP-KanMX4-loxP Downstream homologous sequences for use with loxP-KanMX4-loxP fusi...

Embodiment 2

[0048] Example 2: MAL33 Gene deletion strains are resistant to acetic acid, other typical inhibitors and H 2 o 2 tolerance test

[0049] 1. MAL33 Evaluation of the fermentation of gene-deleted strains in YPD medium containing 3 g / L acetic acid in oxygen-limited shake flasks

[0050] to pick MAL33 Gene deletion strain BSPX051-3XI- mal33△ and control strain BSPX051-3XI were inoculated into 5 mL of YPD liquid medium, activated and cultivated in a shaker at 30°C at 200 rpm for 12 h to 24 h, and then transferred to 5 mL of fresh medium after the bacterial liquid was cloudy Carry out the second activation, the activation time is 12 h~24 h. Inoculate the activated seed solution into an oxygen-limited bottle filled with 30 mL YPD + 3 g / L acetic acid medium, and adjust the initial OD 600 0.2, at 30 ℃, 200 rpm shaker in the oxygen-limited shake flask fermentation, sampling every few hours, using a UV-visible spectrophotometer to measure the OD of the fermentation broth 600 ,...

Embodiment 3

[0054] Example 3: MAL33 Oxygen-limited shake flask evaluation of gene-deleted strains in mixed sugar medium containing 3.5 g / L acetic acid and mixed sugar medium containing mixed inhibitors

[0055] 1. MAL33 Evaluation of the oxygen-limited shake flask fermentation of gene-deleted strains in YPDX medium containing 3.5 g / L acetic acid

[0056] to pick MAL33 Gene deletion strain BSPX051-3XI- mal33△ and control strain BSPX051-3XI were inoculated into 5 mL of YPD liquid medium, activated and cultivated in a shaker at 30 °C at 200 rpm for 12 h to 24 h, and then transferred to 5 mL of fresh medium after the bacterial liquid was cloudy Carry out the second activation, the activation time is 12h~24h. Inoculate the activated seed solution into 30 mL of YPDX medium containing 3.5 g / L acetic acid, adjust the initial OD 600 0.2, at 30 ℃, 200 rpm shaker in the oxygen-limited shake flask fermentation, sampling every few hours, using a UV-visible spectrophotometer to measure the OD ...

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Abstract

The invention relates to application of MAL33 gene deletion in improving tolerance of saccharomyces cerevisiae to lignocellulose hydrolysate inhibitors, and belongs to the field of bioengineering. The invention provides an MAL33 gene deleted saccharomyces cerevisiae strain, and it is proved that compared with a control strain, the acetic acid tolerance of the deleted strain is greatly improved; the tolerance to other typical inhibitors and H2O2 in the lignocellulose hydrolysate is also improved; the delay phase in a glucose and xylose culture medium (YPDX) with 3.5 g / L acetic acid is shortened by 24 h, and the fermentation period of ethanol production by co-utilization of glucose and xylose is shortened by 20 h; the conditions of growth in a glucose and xylose culture medium (YPDX) containing a mixed inhibitor and ethanol production through co-fermentation of glucose and xylose are superior to those of a control strain; the invention provides an important method for improving the tolerance of the saccharomyces cerevisiae to the lignocellulose hydrolysate inhibitor, and provides a theoretical basis for overcoming the technical bottleneck in the production of second-generation fuel ethanol.

Description

technical field [0001] The present invention relates to MAL3 3 The application of gene deletion in improving the tolerance of Saccharomyces cerevisiae to lignocellulose hydrolyzate inhibitors belongs to the field of bioengineering. Background technique [0002] In recent years, the second-generation fuel ethanol produced by using abundant and cheap lignocellulosic raw materials (such as agricultural waste and forestry waste, etc.) is expected to replace non-renewable fossil energy, which can alleviate the current world energy crisis and reduce environmental pollution. wide attention from all over the world. Lignocellulosic raw materials undergo pretreatment, enzymatic hydrolysis and other processes to release sugars such as six-carbon sugars and five-carbon sugars that can be utilized by microorganisms such as Saccharomyces cerevisiae, and also produce various inhibitors, including weak acids, furans and phenolic compounds. [0003] Weak acids mainly include formic acid, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/31C12P7/06C12N1/19C12R1/865
CPCC12N15/81C07K14/395C12P7/06Y02E50/10C12N15/01C12N9/22C12N2500/32C12N2500/34C12N2500/74
Inventor 徐丽丽鲍晓明袁嫒夏天晴王赟韦方卿李在禄
Owner QILU UNIV OF TECH
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