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Method for synthesizing xylitol by aspergillus oryzae engineering bacteria with enhanced hemicellulose saccharification capacity

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: 2020-04-10
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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  • Method for synthesizing xylitol by aspergillus oryzae engineering bacteria with enhanced hemicellulose saccharification capacity
  • Method for synthesizing xylitol by aspergillus oryzae engineering bacteria with enhanced hemicellulose saccharification capacity
  • Method for synthesizing xylitol by aspergillus oryzae engineering bacteria with enhanced hemicellulose saccharification capacity

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Embodiment 1

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

[0043] The construction process of pyrG knockout targeting vector pMD19-pyrGAB is as follows: figure 1 (A) shown. Using the genome of A. oryzae CICC2012 as a template, primers PA-F / PA-R and PB-F / PB-R were used to PCR amplify the upstream and downstream homologous recombination fragments 1.0 kb PA and 1.5 kb of pyrG (GenBank accession number: GQ496621) kb PB, using primers PA-F / PB-R to splice the upstream and downstream fragments by overlap extension PCR, the 2.5kb spliced ​​fragment was ligated with the 2.7kb linear vector pMD19-T to obtain the 5.2kb pyrG knockout targeting vector pMD19-pyrGAB, and Enzyme digestion 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 about 0.6kb of the fusion fragment also contains a BamH I restriction site, so pyrG After the knockout cassette pMD-pyrGAB was digested w...

Embodiment 2

[0052] 1. Construction of xdh traceless 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) shown. The positions of the upstream fragments xdh-A, xdh-B and the downstream fragment xdh-C of Aspergillus oryzae xdh (GenBank accession number: GQ222265) gene are shown in Image 6 . Using primers xdh-A1 / xdh-A2, xdh-B1 / xdh-B2, xdh-C1 / xdh-C2 and pyrG-F / pyrG-R respectively, PCR amplified xdh-A, xdh-B, xdh-C and four fragments of pyrG. The pyrG fragment was connected with pMD19-T to obtain the recombinant plasmid pYRG. Digested with restriction endonuclease SphI / HindIII and ligated with the xdh-B fragment to obtain the recombinant plasmid pYRG-B. Digested with SacI / SmaI and ligated with the xdh-A fragment to obtain the recombinant plasmid pYRG-AB. The plasmid was single-digested with BamH I, then smoothed and dephosphorylated with T4 DNA Polymerase and Alkaline Phos...

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Abstract

The invention discloses a method for synthesizing xylitol by aspergillus oryzae engineering bacteria with enhanced hemicellulose saccharification capacity. The method comprises the steps of 1) obtaining an aspergillus oryzae strain; 2) deleting an orotic acid nucleoside-5'-phosphate decarboxylase gene (pyrG) as a selection marker by utilizing a gene homologous recombination technology, constructing an uridine auxotroph homologous transformation system based on pyrG deletion, and providing uridine auxotroph host bacteria for subsequent gene modification; and 3) carrying out traceless deletion on the xdh gene by using a gene deletion technology of which the selection marker can be recycled to obtain the xylitol CBP engineering bacteria with enhanced hemicellulose saccharification capacity.

Description

technical field [0001] The invention relates to a method for synthesizing xylitol by aspergillus oryzae engineering bacteria with enhanced hemicellulose saccharification ability. Background technique [0002] Xylitol, also known as xylitol, is ubiquitous in nature, such as in 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 needs of the market because the yield is very low and the production cost is high. In industrial production, xylitol is mainly produced by the traditional chemical hydrogenation method. Although the technology is mature, the process is complicated, the energy consumption is high, and the safety performance is poor, which is not conducive to the popularization and production of xylitol. Biotransformation is a new and efficient way of producing xylitol. It is mainly to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/18C12N15/80C12N15/66C12R1/69
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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