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A kind of Aspergillus oryzae that reduces the accumulation of by-products in the process of malic acid synthesis and its application

A technology of Aspergillus oryzae and malic acid, which is applied in the field of genetic engineering, can solve the problems such as no by-product regulation of malic acid, and achieve the effect of simple and efficient metabolic engineering methods and strategies.

Active Publication Date: 2020-09-04
HANGZHOU BIOKING BIOCHEM ENG
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, during the fermentation process, the by-products fumaric acid and succinic acid have been accumulated along with the synthesis of malic acid, especially succinic acid, and there is no report on the regulation of by-products in the process of malic acid synthesis

Method used

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  • A kind of Aspergillus oryzae that reduces the accumulation of by-products in the process of malic acid synthesis and its application
  • A kind of Aspergillus oryzae that reduces the accumulation of by-products in the process of malic acid synthesis and its application

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Experimental program
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Effect test

Embodiment 1

[0025] Construction of embodiment 1 overexpression vector

[0026] Primers were designed according to the gene sequences of FUM I, Ropyc and sfc1p published on NCBI:

[0027]

[0028] Target genes were amplified by PCR using the genomes of Saccharomyces cerevisiae and Rhizopus oryzae as templates. Among them, the extraction method of Saccharomyces cerevisiae genome is the method of Genome Extraction Kit for Rhizopus cerevisiae, and the method for extracting the genome of Rhizopus oryzae is the method of Genome Extraction Kit for Rhizopus oryzae Genome. The fusion product of the starting plasmid pMD-ETPBG and the target gene with the promoter and terminator derived from Aspergillus oryzae was cut with Not I and Asc I enzymes, and the target DNA was recovered and purified by 1% agarose gel electrophoresis. The purified digested products were ligated overnight with T4 DNA ligase, and transformed into competent Escherichia coli GM 109. The transformed bacterial suspension was...

Embodiment 2

[0029] Embodiment 2 Aspergillus oryzae protoplast transforms and the screening of transformant

[0030] The Aspergillus oryzae recombination method is the protoplast transformation method, and the protoplast preparation method refers to the literature Brown, S.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 Aspergillusoryzae NRRL3488 for increased production of L-malic acid. Appl Microb Biotech. 97, 8903-8912. For the protoplast transformation method, refer to the literature Blumhoff, M., Steiger, M.G., Marx, H., Mattanovich, D., Sauer, M., 2013. Six novel constitutive promoters formetabolic engineering of Aspergillusniger. Appl Microb Biotech.97, 259-267.

[0031] The host bacterium used for transformation is A.oryzaeGAA, and the transformation product is coated with a bleomycin-resistant plate. The genome of the regenerated protoplast strain was extracted for ...

Embodiment 3

[0032] Embodiment 3 Aspergillus oryzae fermentation produces L-malic acid

[0033] The spores of Aspergillus oryzae cultured at 34°C for 3-5 days were eluted with 0.05% Tween 80, and the mycelium was removed by filtration with mica cloth. The final concentration of spores in Aspergillus oryzae shake flask seed medium was 1×10 7 cells / mL, cultured at 30°C, 200rpm for 14h, transferred to the fermentation medium with a 10% inoculum size, and fermented at 30°C, 200rpm until the end of sugar consumption.

[0034] Heterologous expression of fumarase encoding gene FUM1 from Saccharomyces cerevisiae, Rhizopus oryzae pyruvate carboxylase encoding gene Ropyc and Saccharomyces cerevisiae succinate-fumarate mitochondrial transporter gene sfc1p in genetic engineering bacteria A.oryzaeGAA, The obtained recombinant strain GAAFPS was fermented by shake flask for 102h, and 130g cornstarch could be used to synthesize 105.3g malic acid, 0.21g fumaric acid and 3.8g succinic acid, compared with t...

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Abstract

The invention discloses aspergillus oryzae capable of reducing accumulation of a by-product in a synthesis process of aspergillus oryzae and an application of the aspergillus oryzae, and belongs to the field of genetic engineering. According to the invention, on the basis of A.oryzae GAA, over-expression of a fumarase coding gene derived from saccharomyces cerevisiae, a pyruvate carboxylase codinggene derived from rhizopus oryzae, a succinic acid-fumaric acid mitochondria transport protein coding gene derived from saccharomyces cerevisiae is implemented, so that conversion of fumaric acid andpyruvic acid in cytoplasm and mitochondria towards the malic acid and material exchange of cytoplasm and mitochondria are further enhanced. As for the finally obtained recombinant bacterium A.oryzaeCMPFS, a yield of L-malic acid undergoing shake-flask fermentation for 102h reaches 105.3g / L, concentration of succinic acid is 3.8g / L and concentration of fumaric acid is 0.21g / L; therefore, the concentration of the by-product is obviously reduced and product yield is improved; and a foundation is laid for production of the L-malic acid which is higher in purity through further metabolic transformation.

Description

technical field [0001] The invention relates to an aspergillus oryzae capable of reducing the accumulation of by-products in the synthesis process of malic acid and an application thereof, belonging to the field of genetic engineering. Background technique [0002] As one of the 12 priority bio-based platform compounds identified by the U.S. Department of Energy, malic acid has been widely used in food, health care products, chemicals, agriculture, cosmetics and medicine. The malic acid synthesized by the traditional chemical synthesis method is all DL-type. Since the L-type malic acid has higher nutritional value, the use of microbial fermentation to produce L-malic acid has attracted more and more researchers' attention in recent years. . [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 express...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/15C12P7/46C12R1/69
CPCC07K14/395C12N9/88C12N9/93C12P7/46C12Y402/01002C12Y604/01001
Inventor 刘龙陈坚刘晶晶堵国成李江华
Owner HANGZHOU BIOKING BIOCHEM ENG
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