Construction method and application of recombinant mold capable of increasing yield of citric acid

A technology for increasing production and constructing methods, which is applied in the field of bioengineering to achieve the effects of increasing production, shortening fermentation cycle, and increasing citric acid production

Active Publication Date: 2017-05-31
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Aspergillus niger is an important industrial production bacterium. The current research on the metabolic transformation of Aspergillus niger focuses on the transformation of the central metabolic pathway and respiratory chain, including the single gene expression and gene coordination of key enzymes in the glycolysis pathway, TCA cycle, and rTCA cycle. expression, as well as overexpression and knockout of alternative oxidases, but these modifications had little effect on citrate production. Among the above methods, only the enhancement of the rTCA cycle pathway promoted the increase of citrate production

Method used

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  • Construction method and application of recombinant mold capable of increasing yield of citric acid
  • Construction method and application of recombinant mold capable of increasing yield of citric acid
  • Construction method and application of recombinant mold capable of increasing yield of citric acid

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Example 1 Extraction of Aspergillus niger RNA

[0044] Inoculate Aspergillus niger spores into citric acid fermentation medium, culture at 250r / min at 35°C for 48 hours, collect the balls with mirocloth filter cloth, wash them with sterile ultrapure water for 3 times, drain the water and quickly place them in liquid nitrogen freezing. The tissue was thoroughly ground by grinding with liquid nitrogen, and the total RNA of Aspergillus niger was extracted using the QIAGEN RNeasy Plant Mini Kit kit. The RNA was reverse-transcribed into cDNA with TAKARA PrimeScript RT reagent Kit with gDNA Eraser.

Embodiment 2

[0045] Example 2 Extraction of Aspergillus niger Genomic DNA

[0046] Aspergillus niger spores were inoculated into ME liquid medium (3% malt extract, 0.5% tryptone), cultured at 250r / min at 35°C for 48h, the balls were collected with a mirocloth filter cloth, and washed with sterile ultrapure water for 3 After drying, the water was quickly frozen in liquid nitrogen. The tissue was thoroughly ground by grinding with liquid nitrogen, and the genome of filamentous fungi was extracted using DNeasy Plant MiniKit from QIAGEN.

Embodiment 3

[0047] Example 3 Construction of HGT1 protein expression cassette and hygromycin resistance expression cassette

[0048] Use primers trp-F (sequence shown in SEQ ID NO.6) and trp-R (sequence shown in SEQ ID NO.7) to amplify the trp terminator with pAN7-1 as a template, and the upstream and downstream sequences contain Pst I and The Hin dIII digestion site was connected to pMD19 for sequencing, and then digested with these two restriction endonucleases, and the sequence was connected to the same digested pUC19 to obtain pUC19-trp.

[0049]Use primers Pgpd-F (sequence shown in SEQ ID NO.8) and Pgpd-R (sequence shown in SEQ ID NO.9) to amplify the PgpdA promoter from pAN7-1, and the sequence contains Eco RI and Kpn I at both ends Restriction site, after digestion, the sequence was connected to pUC19-trp that was also digested to obtain pUC-PgpdA-trp.

[0050] Utilize primer HGT1-F (sequence as shown in SEQ ID NO.10) and HGT1-R (sequence as shown in SEQ ID NO.11) to amplify HGT1 ...

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Abstract

The invention relates to a construction method of a recombinant mold capable of increasing the yield of citric acid. The construction method comprises the following steps that an HGT1 protein expression cassette is constructed, wherein the HGT1 protein expression cassette contains a constitutive promoter, an HGT1 protein coding gene connected with the constitutive promoter and a terminator connected with the HGT1 protein coding gene; a resistant gene expression cassette is constructed, wherein the resistant gene expression cassette contains a constitutive promoter, an hph gene connected with the constitutive promoter and a terminator connected with the hph gene; the HGT1 protein expression cassette and the resistant gene expression cassette are converted into a mold, and the recombinant mold is obtained. The invention further provides the HGT1 protein expression cassette and the recombinant mold which are constructed through the method and application of the recombinant mold constructed through the method in fermentation production of citric acid. According to the recombinant mold, by starting high-affinity glucose transporter expression, the yield of citric acid in the mold is increased, and the fermentation time is greatly shortened.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a construction method and application of a recombinant mold with improved citric acid production. Background technique [0002] Aspergillus niger is an important industrial production bacterium. The current research on the metabolic transformation of Aspergillus niger focuses on the transformation of the central metabolic pathway and respiratory chain, including the single gene expression and gene coordination of key enzymes in the glycolysis pathway, TCA cycle, and rTCA cycle. expression, as well as overexpression and knockout of alternative oxidases, but these modifications had little effect on citric acid production. Among the above methods, only the enhancement of the rTCA cycle pathway promoted the increase of citric acid production. [0003] In the industrial production of citric acid, the raw material is starch, which is liquefied by amylase and then filtered to obt...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12P7/48C12R1/685C12R1/69C12R1/66
CPCC07K14/38C12P7/48
Inventor 刘龙殷娴陈坚堵国成李江华
Owner JIANGNAN UNIV
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