CN108641970B - Aspergillus oryzae capable of reducing accumulation of byproducts in malic acid synthesis process and application thereof - Google Patents
Aspergillus oryzae capable of reducing accumulation of byproducts in malic acid synthesis process and application thereof Download PDFInfo
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Abstract
The invention discloses aspergillus oryzae capable of reducing accumulation of byproducts in a malic acid synthesis process and application thereof, and belongs to the field of genetic engineering. On the basis of A.oryzae GAA, the invention over-expresses a fumarase coding gene derived from saccharomyces cerevisiae, a pyruvate carboxylase coding gene derived from rhizopus oryzae and a succinic acid-fumaric acid mitochondrial transporter coding gene derived from saccharomyces cerevisiae, thereby further strengthening the conversion of fumaric acid and pyruvic acid to malic acid in cytoplasm and mitochondria and the material exchange of cytoplasm and mitochondria. The yield of the finally obtained recombinant bacterium A.oryzae CMPFS after 102h shake flask fermentation of L-malic acid reaches 105.3g/L, the concentration of succinic acid is 3.8g/L, and the concentration of fumaric acid is 0.21g/L, thereby realizing the obvious reduction of the concentration of byproducts and the improvement of the yield of products, and laying a foundation for further metabolic modification to produce L-malic acid with higher purity.
Description
Technical Field
The invention relates to aspergillus oryzae capable of reducing accumulation of byproducts in a malic acid synthesis process and application thereof, belonging to the field of genetic engineering.
Background
Malic acid is widely applied to the fields of food, health products, chemical industry, agriculture, cosmetics, medicines and the like as one of 12 preferentially developed and utilized bio-based platform compounds identified by the U.S. department of energy. The malic acid synthesized by the traditional chemical synthesis method is DL-type, and the L-malic acid has higher nutritional application value, so that the L-malic acid produced by the microbial fermentation method attracts more and more attention of researchers in recent years.
Aspergillus oryzae as a "genetically regarded as safe" (GRAS) strain has been applied to fermentation industries of food, feed, acid production, wine brewing and the like for over 1000 years ago, and is an ideal eukaryotic expression vector. Previous studies have demonstrated that aspergillus oryzae has a variety of amylase lines that enable more efficient use of soluble starch for the synthesis of L-malic acid by enhancing expression of key amylases. However, during the fermentation process, the byproducts fumaric acid and succinic acid are accumulated along with the synthesis of malic acid, especially succinic acid, and no report is provided for the regulation of the byproducts in the synthesis process of malic acid at present.
Disclosure of Invention
The invention aims to solve the technical problems of constructing a food safety strain which has improved L-malic acid production capacity and reduced by-products and is suitable for industrial production, and a method for producing L-malic acid and upstream and downstream products by using the strain.
The first purpose of the invention is to provide a recombinant aspergillus oryzae, which heterologously expresses saccharomyces cerevisiae fumarase, rhizopus oryzae pyruvate carboxylase and saccharomyces cerevisiae fumaric acid-succinic acid transport protein and can reduce the concentration of byproducts while realizing high yield of L-malic acid by using corn starch as a raw material.
In one embodiment of the invention, the heterologous expression is expression of the Saccharomyces cerevisiae fumarase gene FUM1, the Rhizopus oryzae pyruvate carboxylase encoding gene, Ropyc, and the Saccharomyces cerevisiae succinate-fumarate mitochondrial transporter gene, sfc1 p.
In one embodiment of the invention, the fumarase encoding Gene FUM1 is selected from the group consisting of NCBI-Gene ID: 855866; the Gene encoding pyruvate carboxylase, Ropyc, as NCBI-Gene ID: HM 130700.1; genes encoding the succinate-fumarate mitochondrial transporter sfc1p are as described in NCBI-Gene ID: NM _ 001181753.1.
In one embodiment of the invention, the recombinant aspergillus oryzae uses aspergillus oryzae (a.oryzae) GAA as a starting strain; the aspergillus oryzae GAA was disclosed in patent application publication No. CN106591158A at 26.4.2017.
The second purpose of the invention is to provide a construction method of the recombinant aspergillus oryzae, which takes aspergillus oryzae GAA as an initial strain to express a saccharomyces cerevisiae fumarase gene FUM1, a rhizopus oryzae pyruvate carboxylase encoding gene Ropyc and a saccharomyces cerevisiae succinic acid-fumaric acid mitochondrial transporter gene sfc1 p.
In one embodiment of the invention, the fumarase encoding Gene FUM1 is selected from the group consisting of NCBI-Gene ID: 855866; the Gene encoding pyruvate carboxylase, Ropyc, as NCBI-Gene ID: HM 130700.1; genes encoding the succinate-fumarate mitochondrial transporter sfc1p are as described in NCBI-Gene ID: NM _ 001181753.1.
In one embodiment of the invention, the plasmid for constructing the expression cassette of the target gene is constructed by connecting Aspergillus oryzae tyrosinase promoter Peno, TtryC and bleomycin resistance gene ble on the basis of pMD 19-Tvector.
The third purpose of the invention is to provide the application of the recombinant aspergillus oryzae.
In one embodiment of the invention, the application comprises the fermentative production of L-malic acid using corn starch as a substrate.
In one embodiment of the invention, the fermentation is carried out at 28-30 ℃ for 60-150 h.
The invention also provides application of the recombinant aspergillus oryzae in preparation of products containing L-malic acid or upstream and downstream products thereof.
Has the advantages that: on the basis of an engineering bacterium A.oryzae GAA over-expressing glucoamylase, a-glucosidase and a-amylase, the recombinant Aspergillus oryzae further improves the metabolic flow of byproducts fumaric acid, pyruvic acid and succinic acid to malic acid, the yield of L-malic acid of the obtained recombinant bacterium A.oryzae GAAFPS after 102h shake flask fermentation reaches 105.3g/L, the concentration of succinic acid is 3.8g/L, and the concentration of fumaric acid is 0.21g/L, and an excellent starting strain is provided for further metabolic modification and fermentation optimization. The metabolic engineering method and the strategy provided by the invention are simple, convenient and efficient, and are suitable for metabolic regulation of similar strains and fermentation production of related products.
Detailed Description
And (3) aspergillus oryzae seed culture and fermentation:
seed medium (g/L): glucose 30, peptone fish meal 3, KH2PO40.75，K2HPO40.75，NaH2PO40.56，K2HPO40.56。
Fermentation medium (g/L): corn starch 100, peptone fish meal 12, K2HPO40.15，KH2PO40.15，CaCO390。
The culture conditions are as follows:
the spore production condition of the Aspergillus oryzae plate is that the Aspergillus oryzae is cultivated for 3 to 5 days at a constant temperature of 34 ℃, and the final concentration of spores in a seed culture medium of the Aspergillus oryzae shake flask is 1 × 107Culturing at 30 deg.C and 200rpm for 14 hr, inoculating 10% of the culture medium, and fermenting at 30 deg.C and 200rpm until sugar consumption is over.
The method for measuring malic acid comprises the following steps:
high Performance Liquid Chromatography (HPLC) detection: agilent 1200, UV detector, C18 column (250 × 4.6mm, 5 μm), mobile phase: 10% methanol and 0.75mmol of dilute phosphoric acid. The flow rate is 0.7mL/min, the column temperature is 20 ℃, the wavelength is 210nm, and the injection volume is 10 mu L.
Sample preparation: to a known volume of fermentation broth, an equal volume of 2M HCl was added to dissolve the resulting calcium malate precipitate and residual CaCO3. Diluting several times after centrifuging, filtering with 0.22 μm filter membrane, and detecting the filtrate with liquid phase.
EXAMPLE 1 construction of overexpression vectors
Primers were designed based on the gene sequences of FUM I, Ropyc and sfc1p published on NCBI:
and respectively carrying out PCR amplification on target genes by taking saccharomyces cerevisiae and rhizopus oryzae genomes as templates. The method for extracting the saccharomyces cerevisiae genome is a rhizopus oryzae genome extraction kit method, and the method for extracting the rhizopus oryzae genome is a rhizopus oryzae plant genome extraction kit method. The target DNA is recovered and purified by 1% agarose gel electrophoresis using a fusion product of the fast-cutting enzyme Not I and Asc I double-enzyme digestion starting plasmid pMD-ETPBG and the target gene with a promoter and a terminator from Aspergillus oryzae. The purified cleavage products were ligated overnight using T4DNA ligase to transform E.coli GM 109 competent. And coating the transformed bacterial suspension on a screening plate containing an aminobenzyl antibiotic, and culturing for 12h to obtain a colony, and screening positive transformants by PCR. Recombinant plasmids are extracted by a biological medium particle extraction kit method. And (3) carrying out double digestion on the recombinant plasmid by using fast cutting enzymes Sal I and Asc I, and recovering a target fragment for protoplast transformation.
Example 2 Aspergillus oryzae protoplast transformation and selection of transformants
The Aspergillus oryzae recombinant method is a protoplast transformation method, and the protoplast preparation method is described in 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 Aspergillus oryzae NRRL3488for embedded production of L-bulk acid. applied MicrobBiotech.97, 8903-8912. Protoplast transformation methods are described in Blumhoff, M., Steiger, M.G., Marx, H., Mattanovich, D., Sauer, M.,2013.Six novel consistent microorganisms for metabolic engineering of Aspergillus niger. appl. Microb Biotech.97, 259-267.
The host strain used for transformation was a. oryzae gaa, and the transformation products were spread on resistant plates containing bleomycin. Extracting protoplast regeneration strain genome, performing PCR verification, and carrying out passage on the obtained positive transformant on a resistance plate for several times.
EXAMPLE 3 Aspergillus oryzae fermentation production of L-malic acid
The spores of Aspergillus oryzae cultured at 34 deg.C for 3-5 days were eluted with 0.05% Tween 80, and the mycelia were removed by filtration using mica cloth, and the final concentration of spores in seed medium of Aspergillus oryzae shake flask was 1 × 107Culturing at 30 deg.C and 200rpm for 14 hr, inoculating 10% of the culture medium, and fermenting at 30 deg.C and 200rpm until sugar consumption is over.
A fumarase encoding gene FUM1, a rhizopus oryzae pyruvate carboxylase encoding gene Ropyc and a saccharomyces cerevisiae succinic acid-fumaric acid mitochondrial transporter gene sfc1p derived from saccharomyces cerevisiae are heterologously expressed in a genetically engineered bacterium A.oryzae GAA, and after 102h shake flask fermentation, 105.3g of malic acid, 0.21g of fumaric acid and 3.8g of succinic acid can be synthesized by 130g of corn starch, compared with a starting strain A.oryzae GAA (80.8g of malic acid, 0.79g of fumaric acid and 9.2g of succinic acid), the conversion rate of relative corn starch is improved to 0.81g/g, and the production intensity of malic acid is 1.02 g/L/h. In addition, the yields of recombinant bacteria expressing FUMI gene separately on the basis of GAA of Aspergillus oryzae and co-expressing FUMI gene and Ropyc gene are shown in Table 1.
The invention takes the crude corn starch as the substrate, and the L-malic acid is produced with high yield by the aspergillus oryzae fermentation method, thereby laying a foundation for producing the high-purity L-malic acid with low cost and high efficiency.
TABLE 1 Effect of metabolic engineering of Aspergillus oryzae on L-malic acid production
Note: oryzaeGAAF expresses FUMI gene on the basis of GAA; GAAFP expresses the gene of copyc on the basis of GAAF; GAAFPS expresses sfc1p gene on the basis of GAAFP.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
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Claims (9)
1. A recombinant Aspergillus oryzae (Aspergillus oryzae) is characterized in that Aspergillus oryzae GAA is used as an initial strain, saccharomyces cerevisiae fumarase, rhizopus oryzae pyruvate carboxylase and saccharomyces cerevisiae succinic acid-fumaric acid mitochondrial transporter are expressed in a heterologous mode, and corn starch is used as a raw material to produce L-malic acid in a high yield and reduce the concentration of byproducts.
2. The recombinant Aspergillus oryzae of claim 1, wherein the heterologous expression is expression of the Saccharomyces cerevisiae fumarase gene FUM1, the Rhizopus oryzae pyruvate carboxylase encoding gene Ropyc, and the Saccharomyces cerevisiae succinate-fumarate mitochondrial transporter gene sfc1 p.
3. The recombinant Aspergillus oryzae according to claim 2, wherein the fumarase encoding Gene FUM1 is selected from the group consisting of NCBI-Gene ID: 855866; the Gene encoding pyruvate carboxylase, Ropyc, as NCBI-Gene ID: HM 130700.1; genes encoding the succinate-fumarate mitochondrial transporter sfc1p are as described in NCBI-Gene ID: NM _ 001181753.1.
4. A method for constructing the recombinant Aspergillus oryzae of any one of claims 1-3, which takes Aspergillus oryzae GAA as an initial strain to express a Saccharomyces cerevisiae fumarase gene FUM1, a Rhizopus oryzae pyruvate carboxylase encoding gene Ropyc and a Saccharomyces cerevisiae succinic acid-fumaric acid mitochondrial transporter gene sfc1 p.
5. The method of claim 4, wherein the plasmid used for constructing the expression cassette of the desired gene is constructed by linking the Aspergillus oryzae tyrosinase promoter Peno, TtryC and bleomycin resistance gene ble to pMD19-T vector.
6. Use of the recombinant aspergillus oryzae of any one of claims 1 to 3 in the preparation of food, pharmaceutical and health care products by fermentation.
7. A method for producing L-malic acid, which comprises using corn starch as a substrate and fermenting recombinant Aspergillus oryzae of any one of claims 1-3 to produce L-malic acid.
8. The method according to claim 7, wherein the fermentation is carried out at 28-30 ℃ for 60-150 h.
9. Use of a recombinant aspergillus oryzae as claimed in any one of claims 1 to 3 for the preparation of L-malic acid or upstream and downstream products thereof.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105754963A (en) * | 2016-05-19 | 2016-07-13 | 江南大学 | Method for improving yield of fumaric acid |
| CN106222099A (en) * | 2016-07-27 | 2016-12-14 | 江南大学 | A kind of method being effectively improved aspergillus oryzae L malic acid production intensity |
| CN106591158A (en) * | 2017-01-18 | 2017-04-26 | 江南大学 | Method for improving L-malic acid synthesis through fermentation of starch by using Aspergillus oryzae |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105754963A (en) * | 2016-05-19 | 2016-07-13 | 江南大学 | Method for improving yield of fumaric acid |
| CN106222099A (en) * | 2016-07-27 | 2016-12-14 | 江南大学 | A kind of method being effectively improved aspergillus oryzae L malic acid production intensity |
| CN106591158A (en) * | 2017-01-18 | 2017-04-26 | 江南大学 | Method for improving L-malic acid synthesis through fermentation of starch by using Aspergillus oryzae |
Non-Patent Citations (3)
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| Isolation and Cloning of Four Subunits of a Fission Yeast TFIIIC Complex That Includes an Ortholog of the Human Regulatory Protein TFIIICb;Ying Huang等;《THE JOURNAL OF BIOLOGICAL CHEMISTRY》;20000721;第275卷(第40期);参见全文 * |
| The Aspergillus nidulans acuL gene encodes a mitochondrial carrier required for the utilization of carbon sources that are metabolized via the TCA cycle;Michel Flipphi等;《Fungal Genetics and Biology》;20140514;参见全文 * |
| 基于代谢工程策略合成L-苹果酸研究进展;周丽等;《食品工业科技》;20141121;第36卷(第10期);参见全文 * |
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Effective date of registration: 20210909 Address after: 310000 Tangqi town (Industrial Zone), Yuhang District, Hangzhou City, Zhejiang Province Patentee after: HANGZHOU BIOKING BIOCHEMICAL ENGINEERING Co.,Ltd. Address before: No. 1800 Lihu Avenue, Wuxi City, Jiangsu Province Patentee before: Jiangnan University |