Metabolic pathway reconstruction method for reducing lignin content and use thereof

A pathway, phenethyl alcohol technology, applied in the field of genetic engineering, can solve the problem of 2-phenylethyl alcohol synthesis and other problems, achieve great research value and application potential, and improve the effect of glucose efficiency

Inactive Publication Date: 2014-12-17
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Metabolic pathway reconstruction method for reducing lignin content and use thereof
  • Metabolic pathway reconstruction method for reducing lignin content and use thereof
  • Metabolic pathway reconstruction method for reducing lignin content and use thereof

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Embodiment 1

[0025] The construction of the plant expression vector of embodiment 1 2-phenylethanol synthesis pathway:

[0026] 1. Yeast 2-phenylethanol synthesis pathway genes: ARO9 , ARO10 with ADH2 gene cloning

[0027] In order to construct the 2-phenylethanol synthesis pathway in plants, according to the report of the 2-phenylethanol synthesis pathway in Saccharomyces cerevisiae, select ARO9 (NM_001179267), ARO10 (NM_001180688) and ADH2 (NM_001182812) Construction of 2-phenylethanol synthesis pathway. Take Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) extracted their genomic DNA, searched GenBank to obtain the full-length cDNA sequences of the two, respectively designed gene-specific primers, and used PCR technology to amplify the full-length cDNA of the three genes; finally, the two were connected into pMD19-T for sequencing. Amplification conditions were: 95°C for 5 min for pre-denaturation; 94 for 30 s, 55°C for 40 s, 72°C for 1 min, 35 cycles; 72°C for 8 min ...

Embodiment 2

[0036] Example 2 Application of constructing 2-phenylethanol synthesis pathway to reduce plant lignin content:

[0037] 1. Agrobacterium-mediated transformation of Arabidopsis

[0038] Will ARO9 with ARO10 The co-overexpressed plant expression vectors were transformed into Agrobacterium, and positive clones were picked and cultured overnight until OD=2.0. Centrifuge 300ml of overnight culture at room temperature to collect bacteria, and resuspend with 150ml transformation buffer (transformation buffer: containing 0.5% sucrose solution and 0.02% Silwet). Put the inflorescences of Arabidopsis thaliana upside down and immerse them in the bacterial solution. Try to immerse all the inflorescences in the bacterial solution for 5-6 times. Then put the Arabidopsis thaliana upside down in the water receiving tray, cover it with plastic wrap, and place it in the culture room. (22°C) overnight, and then cultured normally. After one week of transformation, it can be retransformed ag...

Embodiment 3

[0052] Example 3 Construction of 2-phenylethanol synthesis pathway to improve the efficiency of enzymatic hydrolysis and saccharification of plant cell walls

[0053] Efficiency analysis of cell wall extracts from Arabidopsis thaliana stems hydrolyzed by cellulase. The specific steps are:

[0054] (1) Divide the cell wall extracts into two treatment groups. The first group was directly treated with cellulose and β-glucosidase. Take 100 mg of the cell wall extracts from the control and transgenic plants, respectively, at 50 C. Treat for 24 hours under the condition of pH4.8, take 500 μl of the enzymatic hydrolyzate at 1, 3, 6, and 24 hours respectively, and measure the glucose content in the hydrolyzate.

[0055] (2) The second group took the cell wall extract and treated it with hot water (121°C) for 1 hour, then treated it with cellulase and glucosidase at 1, 3, 6, and 24 hours to measure the glucose content in the enzymatic hydrolysis product . Follow the instructions of...

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Abstract

The invention relates to the field of gene engineering and discloses a metabolic pathway reconstruction method for reducing lignin content and a use thereof. The metabolic pathway reconstruction method can construct a 2-phenylethyl alcohol synthesis pathway in the plant so that lignin synthesis is reduced and thus fiber biomass conversion efficiency is improved. The 2-phenylethyl alcohol synthesis pathway is from yeast and comprises three key genes of amino acid transaminase gene ARO9, pyruvate decarboxylase gene ARO10 and ethanol dehydrogenase gene ADH2. The 2-phenylethyl alcohol synthesis pathway is constructed in arabidopsis thaliana, and through competition between 2-phenylethyl alcohol synthesis and lignin synthesis in initial substrate phenylalanine use, carbon metabolism shunting is caused and synthesis of lignin in the transgenic plant is reduced. The method provided by the invention can reduce fiber biomass pretreatment difficulty by reducing plant lignin synthesis. At present, the method can reduce lignin synthesis in arabidopsis thaliana and can improve glucose release efficiency of stem cell wall extract enzymolysis.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method and application for reducing lignin synthesis by constructing a 2-phenylethanol synthesis pathway in transgenic plants. Background technique [0002] Lignocellulosic biomass (biomass) is the most abundant renewable resource on earth. Its conversion products are abundant and can be converted into various products such as biofuels and bio-based chemicals. With the increasing shortage of mineral resources, as well as the environmental pollution and climate deterioration caused by the use of mineral resources such as petroleum, the development and utilization of fiber biomass resources has attracted widespread attention worldwide. Lignin is one of the main components of lignocellulosic biomass, and its existence has always been the main barrier limiting the efficient conversion and utilization of cellulosic biomass. Previous research results have proved that changing the...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81A01H5/00C12N15/82
Inventor 周功克祁广柴国华孔英珍王殿唐贤丰胡瑞波
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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