CDT (carbohydrate deficient transferrin)-2 new use and method using CDT-2 to promote microbial cells to transport xylo-oligosaccharides and application thereof

A CDT-2, 1. CDT-2 technology, applied in the new use of CDT-2 and the use of it to promote microbial cell transport of xylooligosaccharides and its application fields, to achieve cost-saving effects

Active Publication Date: 2014-11-12
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, as an ideal production strain for bioproducts and

Method used

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  • CDT (carbohydrate deficient transferrin)-2 new use and method using CDT-2 to promote microbial cells to transport xylo-oligosaccharides and application thereof
  • CDT (carbohydrate deficient transferrin)-2 new use and method using CDT-2 to promote microbial cells to transport xylo-oligosaccharides and application thereof
  • CDT (carbohydrate deficient transferrin)-2 new use and method using CDT-2 to promote microbial cells to transport xylo-oligosaccharides and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1. Determination of expression of cdt-2 and cdt-1 genes in wild-type strains under different carbon source conditions and determination of growth of Neurospora crassa with knockout of cdt-2 gene on various carbon sources

[0054] 1. cdt-2 and cdt-1 double gene knockout strain

[0055] 1. Source of cdt-1 and cdt-2 single gene knockout strains

[0056] Neurospora crassa strains with cdt-1 and cdt-2 single-gene knockouts were purchased from Fungal Genetics Stock Center (FGSC) fungal strain preservation bank in the United States and obtained from commercial channels. The FGSC numbers are FGSC16575 (Δcdt-1), FGSC17868 (Δcdt-2), respectively.

[0057] In the present invention, the cdt-2 gene sequence of the used FGSC17868 (Δcdt-2) strain is shown in sequence 2 in the sequence listing, and the amino acid residue sequence of the CDT-2 protein encoded by it is shown in sequence 1 in the sequence listing.

[0058] 2. Construction of cdt-2 and cdt-1 double gene knockout ...

Embodiment 2

[0071] Example 2. Xylooligosaccharide (xylobiose or xylotriose) transport experiment of Neurospora crassa with knockout of cdt-2 gene

[0072] Mycelia culture: all strains (wild-type Neurospora crassa: WT, cdt-2 knockout strain: Δcdt-2, cdt-2 complementation strain: Pc-cdt-2, that is, to retransform the cdt-2 gene into crassa sporozoa cdt-2 mutant strain Δcdt-2 to see if it restores the traits lost after cdt-2 knockout) were inoculated respectively (the inoculum size was 10 6 spores / mL) in 100 mL of Vogel salt medium supplemented with 2% sucrose, and cultured at 25 °C for 16 h. After the cultivation, the mycelia were collected by centrifugation and washed three times with 1×Vogel salt.

[0073] Induced expression of transporter: Divide mycelia into 2 parts, transfer them to Vogel salt medium supplemented with 0.5% xylan or 0.5% sucrose (as a control), and culture at 25°C for 4 hours to induce the expression of transporter. After the culture is over, the protein expression cu...

Embodiment 3

[0078] Example 3, Cloning of gh43-2 gene, expression in Saccharomyces cerevisiae and determination of ability to hydrolyze xylooligosaccharides

[0079] The gh43-2 gene is predicted to be a xylosidase or arabinosidase gene. The purpose of this example is to determine the function of the gh43-2 encoded protein GH43-2 by cloning the gh43-2 gene and expressing it in Saccharomyces cerevisiae.

[0080] 1. Construction of a recombinant Saccharomyces cerevisiae strain carrying the gh43-2 gene

[0081] gh43-2 was PCR amplified from cDNA of Neurospora crassa using primers 1900-F (sequence: 5'-GCATACTAGTAAAAATGTACACCGCCGACCCCCTCCGC-3') and 1900-R (sequence: 5'-ATGAATTCTTAATGATGATGATGATGATGCTTCCCAGCCGGCTGCTTTTCC-3' with a His tag) For the coding reading frame of the gene, the PCR reaction system is: 5×phusion HF buffer 10 μl, 10 mM dNTPs 1 μl, 1900-F 2.5 μl, 1900-R 2.5 μl, cDNA 1 μl, Phusion DNA polymerase 0.5 μl, water 32.5 μl. The PCR reaction conditions were as follows: first 98°C fo...

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Abstract

The invention discloses a method for promoting microbial cells to transport and / or hydrolyze xylo oligosaccharides and application thereof. Experiments prove that cello-oligosaccharide transport protein CDT-2 also has xylo-oligosaccharide transport capacity, beta xylosidase GH43-2 has hydrolysis effect on the xylo-oligosaccharides, according to the method, cdt-2 gene and / or gh43-2 gene are/ is introduced into a microbial strain to obtain a recombinant microbial strain, and compared with a starting strain, the recombinant microbial strain acquires or improves the capability for transporting the xylo-oligosaccharides from extracellular to intracellular and / or hydrolyzing the xylo-oligosaccharides into xylose. By discovery of the xylo-oligosaccharide transport protein CDT-2 and application of the beta xylosidase GH43-2, an idea using modified yeast to directly use the xylo-oligosaccharides derived from hemicellulose degradation for producing biological chemicals is provided, co-fermentation of glucose, xylose, cello-oligosaccharide and xylo-oligosaccharide mixed sugar can be realized by co expression of cdt-2 and gh43-2 and cdt-1 and gh43-1 in brewer's yeast containing xylose metabolic pathway to produce the biological chemicals, and the production cost is greatly saved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a new application of gene cdt-2 in affecting the transport of xylooligosaccharides by cells, that is, by expressing the cdt-2 gene, microbial strains can obtain or improve the ability of transporting xylooligosaccharides, and using the gene After being co-introduced into the microbial strain together with the β-xylosidase coding gene gh43-2, the microbial strain can obtain or improve the ability of xylooligosaccharide transport and hydrolysis of xylooligosaccharide. Background technique [0002] Biomass degradation is mainly through lignocellulose degrading enzymes secreted by some lignocellulosic degrading strains, such as Neurospora crassa, to decompose biomass into different monosaccharides and polysaccharides for use by the bacteria. Biomass sugars produced include monosaccharides such as glucose, xylose, and arabinose, and oligosaccharides such as cellooligosaccharides and xylooli...

Claims

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Application Information

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IPC IPC(8): C12N15/81C12N1/19C12P7/06C12P7/10C12R1/865
CPCY02E50/10
Inventor 田朝光蔡鹏丽顾芮萌王邦李金根万里马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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