Method for increasing carbon source utilization rate in aspergillus oryzae L-malic acid synthesizing process

A technology of Aspergillus oryzae and malic acid, applied in the field of genetic engineering, can solve the problem of less metabolic transformation of Aspergillus oryzae, and achieve the effects of simple method, improved conversion rate and reduced production cost

Inactive Publication Date: 2016-09-28
JIANGNAN UNIV
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  • Application Information

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Problems solved by technology

[0005] There are relatively few reports on th

Method used

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  • Method for increasing carbon source utilization rate in aspergillus oryzae L-malic acid synthesizing process
  • Method for increasing carbon source utilization rate in aspergillus oryzae L-malic acid synthesizing process
  • Method for increasing carbon source utilization rate in aspergillus oryzae L-malic acid synthesizing process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Protoplast transformation of Aspergillus oryzae and selection of transformants

[0040] The preparation and transformation methods of Aspergillus oryzae protoplasts are as follows:

[0041] For the preparation method of protoplasts, refer to the literature Brown, S.

[0042] H., Bashkirova, L., Berka, R., Chandler, T., Doty, T., McCall, K., McCulloch, M., McFarland, S., Thompson, S., Yaver, D., Berry, A.,2013.Metabolic engineering ofAspergillus oryzae NRRL3488for increased production of L-malic acid.Applied microbiology and biotechnology.97,8903-12.

[0043] For the method of protoplast transformation, refer to 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.

[0044] The selected positive transformants were passed through the resistance plate several times to obtain a homozygous Aspergillus oryzae r...

Embodiment 2

[0045] Example 2 Overexpression of malate dehydrogenase mdh and pyruvate carboxylase pyc

[0046] According to the upstream and downstream sequences of Aspergillus oryzae mdh and pyc genes published on NCBI, primers were designed to construct malate dehydrogenase MDH and pyruvate carboxylase PYC expression vectors.

[0047] Table 1 Primers for amplifying mdh and pyc genes

[0048] 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

[0049] Using Aspergillus oryzae genome as template, related genes were amplified by PCR. The method for extracting the genome of Aspergillus oryzae is the sky root plant genome extraction kit method. The amplified target gene and the above-mentioned vector plasmid pBg32 are both digested with fast digestion enzymes Nsi I and KpnI, and the ...

Embodiment 3

[0051] Example 3 Construction of pck and ppc gene heterologous expression vector

[0052] According to the upstream and downstream sequences of E. coli pck and ppc published on NCBI, primers were designed.

[0053] Table 2 Primers for amplification of pck and ppc genes

[0054] name

Sequence (5’-3’)

Serial number

ppc-1

CCTTAATTAAATGAACGAACAATATTCCGCATTGC

SEQ ID NO: 5

ppc-2

GGGGTACCTTAGCCGGTATTACGCATACCTGCC

SEQ ID NO: 6

pck-1

TGCATGCATATGCGCGTTAACAATGGTTTGACCC

SEQ ID NO: 7

pck-2

GGGGTACCTTACAGTTTCGGACCAGCCGCTACC

SEQ ID NO: 8

[0055] Using the genome of E. coli BL21 as a template, the relevant genes were amplified by PCR using the above primers. The fast-cutting enzymes pac I and Kpn I were used to double-cut the target gene and the vector pHg11, and the target gene was connected to the vector to obtain the expression plasmid pPHg2 of the ppc gene and the expression plasmid pPCg32 of the pyc gene. Then, the target fragment was obtained by double digestion with ...

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Abstract

The invention discloses a method for increasing the carbon source utilization rate in the aspergillus oryzae L-malic acid synthesizing process and belongs to the field of genetic engineering. According to the method, Aspergillus oryzae NRRL 3488 serves as an original strain, metabolic flux for synthesizing malic acid from oxaloacetic acid is enhanced by expressing pyruvic carboxylase and malic dehydrogenase in aspergillus oryzae cytoplasm, and the shake flask yield of L-malic acid is increased to 50.5+/-1.06 g/L and is increased by 38.4%; then, the synthesizing efficiency of oxaloacetic acid from phosphoenolpyruvic acid in aspergillus oryzae cytoplasm is improved by expressing pyruvic carboxylase and pyruvate carboxykinase of Escherichia coli in the converter Aspergillus oryzae p16, finally, the yield of L-malic acid reaches 63.2+/-1.32 g/L, the concentration of succinic acid is 8.88+/-0.45 g/L, the production intensity is also improved to 0.7 g/(L.h) from original 0.24 g/(L.h) of Aspergillus oryzae NRRL 3488, and a foundation is laid for further metabolic engineering reform of Aspergillus oryzae for producing L-malic acid.

Description

Technical field [0001] The invention relates to a method for improving the utilization of carbon sources in the synthesis process of Aspergillus oryzae L-malic acid, and 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 foods and beverages, as a cleaning agent for metals, as well as in agriculture, cosmetics and medicine. In 2004, the U.S. Department of Energy identified malic acid, fumaric acid, and succinic acid as one of the 12 platform compounds that can be produced on a large scale through the fermentation of renewable raw materials by microorganisms. [0003] As a "generally regarded as safe" (GRAS) safe strain, Aspergillus oryzae has been used in food, feed, acid production, winemaking and other fermentation industries more than 1,000 years ago, and is an ideal eukaryotic expression vector. Aspergillus flavus is the original malic acid producing strain, but i...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N1/15C12P7/46C12R1/69
CPCC12N15/80C12N9/0006C12N9/88C12N9/93C12P7/46C12Y101/01037C12Y401/01C12Y401/01031C12Y604/01001
Inventor 刘龙陈坚刘晶晶堵国成李江华房峻
Owner JIANGNAN UNIV
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