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Method for effectively improving L-malic acid production intensity of Aspergillus oryzae pp25

A technology of Aspergillus oryzae and malic acid, which is applied in the field of genetic engineering, can solve problems such as incapability of large-scale production and application, and achieve the effects of being convenient for wide application, improving consumption rate and reducing accumulation of intracellular products

Inactive Publication Date: 2016-12-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Aspergillus flavus was the original malic acid producing strain, but due to its aflatoxin production it cannot be used for large-scale production, especially in the food industry

Method used

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  • Method for effectively improving L-malic acid production intensity of Aspergillus oryzae pp25
  • Method for effectively improving L-malic acid production intensity of Aspergillus oryzae pp25
  • Method for effectively improving L-malic acid production intensity of Aspergillus oryzae pp25

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 Aspergillus oryzae protoplast transforms and the screening of transformant

[0042] Aspergillus oryzae protoplast preparation and transformation method are as follows:

[0043] For the preparation method of protoplasts, refer to the literature Brown, S.

[0044] H., Bashkirova, L., Berka, R., Chandler, T., Doty, T., McCall, K., McCulloch, M., McFarland, S., Thompson, S., Yaver, D., Berry, A., 2013. Metabolic engineering of Aspergillus oryzae NRRL3488 for increased production of L-malic acid. Applied microbiology and biotechnology. 97, 8903-12.

[0045] For the protoplast transformation method, see the literature Blumhoff, M., Steiger, M.G., Marx, H., Mattanovich, D., Sauer, M., 2013. Six novel constitutive promoters for metabolic engineering of Aspergillus niger. Applied microbiology and biotechnology.97, 259-67.

[0046] The screened positive transformants were passed several times on the resistance plate to obtain a homozygous Aspergillus oryzae recombi...

Embodiment 2

[0047] The construction of embodiment 2 Aspergillus oryzae pp25

[0048] According to the upstream and downstream sequences of Aspergillus oryzae mdh and pyc genes published on NCBI, primers were designed to construct expression vectors of malate dehydrogenase mdh and pyruvate carboxylase pyc. At the same time, according to the upstream and downstream sequences of Escherichia coli pck and ppc published on NCBI, primers were designed to construct pck and ppc gene heterologous expression vectors.

[0049] Table 1 Primers for amplifying mdh and pyc genes

[0050] name

Sequence (5'-3')

serial number

mdh3-1

CCAATGCATGCCACCATGGTCAAAGCTGGTGAGTTAG

SEQ ID NO: 1

mdh3-2

GCTCTAGATTACTTTGGTGGTGGGTTCT

SEQ ID NO: 2

pyc-1

CCAATGCATGCCACCATGGCGGCTCCGTTTCGTCA

SEQ ID NO: 3

pyc-2

GCTCTAGATTACGCTTTGACGATCTTGCAG

SEQ ID NO: 4

ppc-1

CCTTAATTAAATGAACGAACAATATTCCGCATTGC

SEQ ID NO: 5

ppc-2

GGGGTACCTTAG...

Embodiment 3

[0055] Example 3 Overexpression of the four-carbon dicarboxylic acid transporter gene C4T318

[0056] Primers were designed according to the upstream and downstream sequences of Aspergillus oryzae C4T318 gene published on NCBI.

[0057] Table 2 Amplifies the primers of Aspergillus oryzae C4T318 gene

[0058] name

Sequence (5'-3')

serial number

C4T318-1

CCAATGCATGCCACCATGTTCAATAACGAACACCA

SEQ ID NO: 9

C4T318-2

GCTCTAGACTAATCAGATACATCCTCAT

SEQ ID NO: 10

[0059] Using the genome of Aspergillus oryzae as a template, related genes were amplified by PCR. The genome extraction method of Aspergillus is the method of Genome Extraction Kit of Tiangen Plant. Both the amplified target gene and the above vector plasmid pAg12 were double-digested with fast cutting enzymes Nsi I and Kpn I, and the target gene was ligated with the vector to obtain the expression plasmid pCAg22 of the C4T318 gene. Then use fast cutting enzyme AscI and Not...

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Abstract

The invention discloses a method for effectively improving L-malic acid production intensity of Aspergillus oryzae pp25, belonging to the field of genetic engineering. According to the invention, Aspergillus oryzae pp25 is used as a starting strain, a C4-dicarboxylate transport protein gene C4T318 of the Aspergillus oryzae pp25 and a malic acid transport protein gene maeI of Schizosaccharomyces pombe are expressed to obviously reduce the intracellular accumulation of malic acid, and finally, the shake flask yield of the L-malic acid is up to 93.17+ / -2.10g / L and is increased by 47.4%; the concentration of succinic acid is 5.17+ / -0.49g / L and is reduced by 41.8%; and the conversion rate of the malic acid relative to glucose is 0.85g / g and is increased by 49.1%. Meanwhile, the fermentation period is shortened from 90 hours to 80 hours, and the production intensity is up to 1.16g / L.h and is increased by 65.7%.

Description

technical field [0001] The invention relates to a method for effectively improving the production intensity of Aspergillus oryzae L-malic acid, which belongs to the field of genetic engineering. Background technique [0002] As an important four-carbon dicarboxylic acid, malic acid is mainly used as a sour agent in food and beverages, as a cleaning agent for metal objects, and in fields such as agriculture, cosmetics and medicine. In 2004, the U.S. Department of Energy listed malic acid, fumaric acid, and succinic acid as one of the 12 platform compounds that can be produced on a large scale by microbial fermentation using renewable raw materials. [0003] Aspergillus oryzae, as a "generally regarded as safe" (GRAS) safe strain, has been used in food, feed, acid production, winemaking and other fermentation industries as early as 1,000 years ago, and is an ideal eukaryotic expression vector. Aspergillus flavus was the original malic acid producing strain, but it cannot be u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12P7/46C12R1/69
CPCC07K14/38C07K14/39C12P7/46
Inventor 刘龙陈坚刘晶晶堵国成李江华房峻
Owner JIANGNAN UNIV
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