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A method for synthesizing xylitol by Aspergillus oryzae engineering bacteria with enhanced saccharification ability of hemicellulose

A technology of hemicellulose and Aspergillus oryzae, applied in the field of xylitol synthesis by Aspergillus oryzae engineering bacteria, can solve the problem of low saccharification ability of hemicellulose

Active Publication Date: 2022-07-29
HUAQIAO UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the hemicellulose saccharification ability of the mutant strain C3-1 is still low, so enhancing the hemicellulose saccharification ability of Aspergillus oryzae is the key to further improving the yield of xylitol synthesized by CBP method

Method used

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  • A method for synthesizing xylitol by Aspergillus oryzae engineering bacteria with enhanced saccharification ability of hemicellulose
  • A method for synthesizing xylitol by Aspergillus oryzae engineering bacteria with enhanced saccharification ability of hemicellulose
  • A method for synthesizing xylitol by Aspergillus oryzae engineering bacteria with enhanced saccharification ability of hemicellulose

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Effect test

Embodiment 1

[0042] 1. Construction of pyrG knockout targeting vector pMD19-pyrGAB

[0043] The construction process of the pyrG knockout targeting vector pMD19-pyrGAB is as follows figure 1 (A). Using the genome of A.oryzae CICC2012 as a template, the upstream and downstream homologous recombination fragments 1.0kb PA and 1.5 of pyrG (GenBank accession number: GQ496621) were amplified by PCR with primers PA-F / PA-R and PB-F / PB-R, respectively. kb PB, the upstream and downstream fragments were spliced ​​by overlap extension PCR with primers PA-F / PB-R, and the 2.5kb spliced ​​fragment was ligated with the 2.7kb linear vector pMD19-T to obtain a 5.2kb pyrG knockout targeting vector pMD19-pyrGAB, and Enzyme cleavage verification.

[0044] 2. In the pyrG knockout box pMD19-pyrGAB, both ends of the fusion fragment PA and PB contain Sac I and BamH I restriction sites, and the fusion fragment also contains a BamH I restriction site at about 0.6kb, so pyrG After the knockout cassette pMD-pyrGAB ...

Embodiment 2

[0052] 1. Construction of xdh seamless knockout targeting vector pMD19-pyrG-xdhABC

[0053] The construction process of the xdh seamless knockout targeting vector pMD19-pyrG-xdhABC is as follows figure 1 (B). For the positions of the upstream fragments xdh-A, xdh-B and the downstream fragment xdh-C of the Aspergillus oryzae xdh (GenBank accession number: GQ222265) gene, see Image 6 . PCR amplification of xdh-A, xdh-B, xdh-C and Four fragments of pyrG. The pyrG fragment was ligated with pMD19-T to obtain the recombinant plasmid pYRG. The recombinant plasmid pYRG-B was obtained by double digestion with restriction enzymes SphI / HindIII and ligated with the xdh-B fragment. It was digested with SacI / SmaI and ligated with the xdh-A fragment to obtain the recombinant plasmid pYRG-AB. The plasmid was digested with BamH I, and then smoothed and dephosphorylated with T4 DNAPolymerase and Alkaline Phosphatase, respectively, and blunt-end ligated with the xdh-C fragment treated wit...

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Abstract

The invention discloses a method for synthesizing xylitol by Aspergillus oryzae engineering bacteria with enhanced hemicellulose saccharification ability, comprising the following steps: 1) obtaining Aspergillus oryzae strain; 2) using gene homologous recombination technology to delete whey as a screening marker Nucleotide-5'-phosphate decarboxylase gene (pyrG), to construct a uridine auxotrophic homologous transformation system based on the deletion of pyrG to provide uridine auxotrophic host bacteria for subsequent genetic modification; 3) The use of selectable markers can be recycled The gene deletion technology used is a traceless deletion of the xdh gene to obtain a xylitol CBP engineered bacterium with enhanced hemicellulose saccharification ability.

Description

technical field [0001] The invention relates to a method for synthesizing xylitol by Aspergillus oryzae engineering bacteria with enhanced saccharification ability of hemicellulose. Background technique [0002] Xylitol is xylitol, which is ubiquitous in nature, such as fruits, vegetables and grains, but the content is extremely low. The production methods of xylitol mainly include extraction method, chemical synthesis method and biotransformation method. Among them, the extraction method cannot meet the market demand due to low yield and high production cost. In industrial production, the traditional chemical hydrogenation method is mainly used to produce xylitol. Although the technology is mature, the complex process, high energy consumption and poor safety performance are not conducive to the popularization and production of xylitol. Biotransformation is a new and efficient production method of xylitol. It mainly treats the lignocellulose contained in forestry and anima...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/80C12N15/66C12P7/18C12R1/69
CPCC12P7/18C12N15/80C12N15/66
Inventor 陈宏文杨春发谢桂贞杜钰刘薇
Owner HUAQIAO UNIVERSITY
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