CN106591158A - Method for improving L-malic acid synthesis through fermentation of starch by using Aspergillus oryzae - Google Patents
Method for improving L-malic acid synthesis through fermentation of starch by using Aspergillus oryzae Download PDFInfo
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- CN106591158A CN106591158A CN201710036762.0A CN201710036762A CN106591158A CN 106591158 A CN106591158 A CN 106591158A CN 201710036762 A CN201710036762 A CN 201710036762A CN 106591158 A CN106591158 A CN 106591158A
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- C12P7/44—Polycarboxylic acids
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Abstract
The invention discloses a method for improving L-malic acid synthesis through fermentation of starch by using Aspergillus oryzae, and belongs to the field of gene engineering. According to the present invention, on the basis of genetic engineering bacteria A. Oryzae PN5, glucosidase coding gene agdA and amylase coding gene amyB are simultaneously subjected to overexpression, and the obtained recombinant strain A. Oryzae GAA is subjected to shake flask fermentation for 96 h, such that 100 g/L of starch can be converted into 72 g/L of L-malic acid, 6.4 g/L of succinic acid and 0.18 g/L of fumaric acid, wherein the conversion rate (g/g) of the malic acid relative to the starch is 0.72, and the production intensity is 0.75. With the method of the present invention, the foundation is established for the further metabolic transformation of Aspergillus oryzae in the starch fermentation production of L-malic acid.
Description
Technical field
The present invention relates to a kind of method for improving aspergillus oryzae fermentation Starch synthesis L MALIC ACID, belongs to genetic engineering field.
Background technology
Malic acid be mainly used as food, beverage as a kind of four important carbon dicarboxylic acids in acidic flavoring agent, metal object it is clear
Lotion, and agricultural, cosmetics and medicine and other fields.2004, USDOE by malic acid and fumaric acid, succinic acid this three
Kind of four carbon dicarboxylic acids as can by microorganism using renewable raw materials fermentation large-scale production 12 kinds of platform chemicals it
One.
At present fermentative Production malic acid mainly passes through antibacterial or filamentous fungis glucose fermentation, or High Density Cultivation
Ustilago trichophora fermentation glycerols etc..Antibacterial can produce the by-products such as acetic acid, lactic acid during carbohydrate metabolism；Silk
The carbohydrate metabolism of shape funguses is then along with the generation of higher citric acid, succinic acid and fumaric acid etc..Glycerol fermentation produce acid need compared with
The thalline of high concentration and longer fermentation period, are unfavorable for industrialized production, and using glucose as fermentation raw material, with industry shallow lake
Powder is compared, and cost is higher by 1.2～1.5 times.
Starchy material source is wide, cheap, is preferable carbon source in commercial production.It is well known that aspergillus oryzae conduct
" generally regarded as safe " (GRAS) bacterial strain, was applied to food, feedstuff, product before more than 1000 years
The fermentation industries such as acid, wine brewing, with extremely strong amylase secretion ability.Based on prior art with regard to the Starch Production malic acid that ferments
Research, aspergillus oryzae fermentation Starch Production malic acid become possibility, but still suffered from very big room for promotion.Because solubility is formed sediment
Powder is dissolved in after hot water into pasty state, and extremely low mobility can affect mass transfer and thalli growth, therefore, improve utilization of the thalline to starch
Ability plays an important role for the fermentation level for improving malic acid.
The content of the invention
First purpose of the present invention is to provide a kind of genetic engineering bacterium, is the coexpression coding with aspergillus oryzae as host
The gene agdA of diastatic gene amyB and coding beta-glucosaccharase
In one embodiment of the invention, the gene amyB is the gene that NCBI Gene ID are 5996200, institute
State the gene that gene agdA is that NCBI Gene ID are 5992274.
In one embodiment of the invention, with pBg32 as carrier, the pBg32 is in Shen for the genetic engineering bacterium
Please number for 201610603538.0 patent of invention disclosed in, the plasmid with pMD19 as the plasmid that sets out, by connection aspergillus oryzae phosphorus
Acid glycerol kinase promoter (Ppgk), benomyl resistance gene (e) of aspergillus nidulanses and glucoamylase terminator
(Tgla) it is built-up.
In one embodiment of the invention, with starch inducible strong promoter PamyB expressing gene amyB, with
PagdA expressing gene agdA；The PamyB sequences are as shown in SEQ ID NO.1；The PagdA sequences such as SEQ ID NO.2 institutes
Show.
In one embodiment of the invention, the genetic engineering bacterium is glucose amylase gene with overexpression
The engineering bacteria A.oryzae PN5 of glaA are host；The A.oryzae PN5 are with plasmid pMD19 as carrier, with SEQ ID
The strong promoter mediation of the starch induction type shown in NO.7, overexpression NCBI Gene ID are 5999830 glaA genes
Restructuring aspergillus oryzae.
Second object of the present invention is to provide the construction method of the genetic engineering bacterium, and methods described is by Gene ID
Gene amyB, Gene ID for 5996200 is that 5992274 gene agdA is connected with carrier, is converted into host cell.
Third object of the present invention is to provide a kind of method for producing L MALIC ACID as substrate with starch, and methods described is
The genetic engineering bacterium is seeded in fermentation medium, 28～30 DEG C, 200～250rpm, 48～96h of fermentation.
In one embodiment of the invention, the fermentation medium contains per L：Soluble starch 100g, Trypsin
Peptone 12g, K2HPO40.15g, KH2PO40.15g, MgSO4.7H20 0.1g, CaCl2.H20 0.1g, CaCO390,0.005g
NaCl, 0.005g FeSO4.7H20。
In one embodiment of the invention, the fermentation is two-step fermentation, and the first step is to train 32～34 DEG C of constant temperature
The spore of foster 3-7 days is connected in seed culture medium so that spore final concentration of 1.5 × 106Individual/mL, 30～34 DEG C, 200rpm trainings
Support 12～16h；Second step is that cultured seed liquor is connected in fermentation medium with 5～10% inoculum concentration, 30～34 DEG C,
200rpm cultivates 48～96h.
Fourth object of the present invention is to provide the genetic engineering bacterium and produces answering in organic acid production in fermentation arts
With.
Beneficial effect：The restructuring aspergillus oryzae that the present invention is provided improves the conversion ratio and production intensity that fermentation starch produces acid, and
By-product (heteroacid) content in fermented product is few.The genetic engineering bacterium A.oryzae GAA that the present invention builds are through 96h shaking flasks
The yield of fermentation L MALIC ACID reaches 72g/L, and heteroacid succinic acid concentration is less than 6.4g/L, and fumaric acid content is less than 0.18g/L, is
Further metabolic engineering aspergillus oryzae production malic acid is had laid a good foundation.It is Metabolically engineered thread true that the present invention is provided
The method of bacterium production organic acid is simple, is easy to use, with application prospect well.
Specific embodiment
Seed culture medium (g/L)：Glucose 30, tryptone 3, KH2PO40.75, K2HPO40.75, NaH2PO4
0.56, K2HPO4 0.56,MgSO4.7H20 0.075, CaCl2.H20 0.075,0.75mL micro- nutritional solution (5g NaCl, 5g
FeSO4.7H20th, 1g citric acids).
Fermentation medium (g/L)：Soluble starch 100, tryptone 12, K2HPO40.15, KH2PO40.15,
MgSO4.7H20 0.1, CaCl2.H20 0.1,0.005g NaCl, 0.005g FeSO4.7H20, CaCO3 90。
Condition of culture：Aspergillus oryzae flat board Sporulation condition coats PDA plate, 34 DEG C of constant temperature culture 3-7 for appropriate spore liquid
My god.Aspergillus oryzae shake-flask seed culture medium spore final concentration of 1.5 × 106Individual/mL, 30 DEG C, 200rpm culture 14h, with 10%
Inoculum concentration switching fermentation medium, 30 DEG C, 200rpm fermentation 96h.
The assay method of malic acid：Agilent 1200, UV detector, C18 posts (250 × 4.6mm, 5 μm), mobile phase：
10% methanol and 0.75mmol phosphoric acid,diluteds.Flow velocity 0.7mL/min, 20 DEG C of column temperature, wavelength 210nm, sampling volume is 10 μ L.
Sample preparation：The calcium malate that isopyknic 2M HCl are added in the fermentation liquid of known volume to dissolve generation sinks
The CaCO for forming sediment and remaining3.Dilute 5 times after centrifugation again, Jing after 0.22 μm of membrane filtration, filtrate is used for Liquid Detection.
The structure of the over-express vector of embodiment 1
According to the upper of the Amylase EC encoding gene amyB and glucoside enzyme coding gene agdA announced on NCBI
Downstream sequence, designs primer：
With aspergillus oryzae genome as template, by PCR amplifying target genes.The extracting method of the genome of aspergillus oryzae is day
Root Plant Genome extracts kit method.Original plasmid for construction of expression vector is with ammonia benzyl and benomyl resistance
PBg32 (application numbers：201610603538.0).AmyB gene expressions are obtained using fast enzyme cutting Hind III and Asc I double digestions
Purpose fragment, using fast enzyme cutting by the purpose fragment of Sal I and Asc I double digestion agdA gene expressions, 1% agarose coagulates
Gel electrophoresis recovery purifying target DNA.
The screening of the aspergillus oryzae protoplast transformation of embodiment 2 and transformant
Aspergillus oryzae recombination method is protoplast transformation, and method for preparing protoplast is referring to paper disclosed in 2013
《Metabolic engineering of Aspergillus oryzae NRRL3488 for increased
production of L-malic acid》；Protoplast transformation method is referring to paper disclosed in 2013《Six novel
constitutive promoters for metabolic engineering of Aspergillus niger.》。
As above described in the content of the invention, the Host Strains for conversion are for the preparation of aspergillus oryzae protoplast and method for transformation
A.oryzae PN5, converted product resistant panel of the coating containing benomyl.Extract Protoplast regeneration strains Genomic PCR to test
Card, gained positive transformant is passed on for several times in resistant panel.
The aspergillus oryzae fermenting and producing L MALIC ACID of embodiment 3
By the spore of 34 DEG C of cultures aspergillus oryzae of 3-7 days with 0.05% Tween 80 eluting, it is filtered to remove using mica cloth
Mycelium.Aspergillus oryzae shake-flask seed culture medium spore final concentration of 1.5 × 106Individual/mL, 30 DEG C, 200rpm culture 14h, with
10% inoculum concentration switching fermentation medium, 30 DEG C, 200rpm fermentation 96h.
A.oryzae PN5 with non-overexpression amyB and agdA are (with plasmid pMD19 as carrier, with SEQ ID NO.7 institutes
The strong promoter mediation of the starch induction type for showing, overexpression NCBI Gene ID are the restructuring rice of 5999830 glaA genes
Aspergillosis), and (it is respectively designated as A.oryzae by the genetic engineering bacterium for only expressing amyB or agdA respectively of above-mentioned construction of strategy
GA1 and A.oryzae GA2) compare, culture under the same conditions, fermentation determine the content of malic acid in fermentation liquid, as a result
Show, Jing 96h shake flask fermentations, A.oryzae PN5 yield be 63g/L, the malic acid of A.oryzae GA1 and A.oryzae GA2
Yield is respectively 66g/L and 65g/L.As simultaneously overexpression amyB and agdA, recombinant bacterium A.oryzae GAA Fructus Mali pumilae acid yields are carried
Up to more than 72g/L, compared to control A.oryzae PN5 14.3% is improve.Malic acid turning relative to starch in the present invention
Rate is 0.72g/g, and production intensity is 0.75.Meanwhile, the genetic engineering bacterium provided in the present invention is accompanied by a small amount of by-product
The generation of thing, succinic acid content is less than 6.4g/L, and fumaric acid content is less than 0.18g/L.The achievement in research of the present invention is further demonstrate,proved
Real aspergillus oryzae ferments the potentiality of Starch synthesis malic acid, and for further metabolic regulation excellent starting strain is provided,
Industrialized production for malic acid provides cheap fermentation raw material.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention
Enclosing should be by being defined that claims are defined.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>A kind of method for improving aspergillus oryzae fermentation Starch synthesis L MALIC ACID
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<170> PatentIn version 3.3
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Claims (10)
1. a kind of genetic engineering bacterium, it is characterised in that with aspergillus oryzae as host, coexpression encodes diastatic gene amyB and volume
The gene agdA of code glucosidase.
2. genetic engineering bacterium according to claim 1, it is characterised in that the gene amyB is that NCBI Gene ID are
5996200 gene, the gene agdA is the gene that NCBI Gene ID are 5992274.
3. genetic engineering bacterium according to claim 1 and 2, it is characterised in that with pBg32 as expression vector, lured with starch
Conductivity type strong promoter PamyB expressing gene amyB, with PagdA expressing gene agdA.
4. genetic engineering bacterium according to claim 3, it is characterised in that the glucose amylase gene glaA with overexpression
Aspergillus oryzae be host.
5. the method for building genetic engineering bacterium described in claim 2, it is characterised in that by the gene that Gene ID are 5996200
AmyB, Gene ID is that 5992274 gene agdA is connected with carrier, is converted into Aspergillus oryzae cell.
6. method according to claim 5, it is characterised in that the carrier is pBg32.
7. it is a kind of with starch as substrate produce L MALIC ACID method, methods described is that the genetic engineering bacterium is seeded to into fermentation
In culture medium, 30～34 DEG C, 200～250rpm, 48～96h of fermentation.
8. method according to claim 7, it is characterised in that be first connected in the spore of 32～34 DEG C of constant temperature culture 3-7 days
In seed culture medium so that spore final concentration >=1.5 × 106Individual/mL, 30～34 DEG C, 200～250rpm cultivates 12～16h, then
It is forwarded in fermentation medium by the inoculum concentration of volume 5～10%, 30～34 DEG C, 200rpm cultivates 48～96h.
9. method according to claim 8, it is characterised in that the fermentation medium contains per L：Soluble starch
100g, tryptone 12g, K2HPO40.15g, KH2PO40.15g, MgSO4.7H200.1g, CaCl2 .H200.1g, CaCO3
90,0.005g NaCl, 0.005g FeSO4 .7H20。
10. application of the arbitrary genetic engineering bacterium of claim 1-2,4 in terms of fermentation arts prepare organic acid.
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| CN108641970A (en) * | 2018-04-16 | 2018-10-12 | 江南大学 | It is a kind of to reduce the aspergillus oryzae of byproducts build-up and its application in malic acid building-up process |
| CN112195110A (en) * | 2020-09-22 | 2021-01-08 | 天津科技大学 | Recombinant aspergillus oryzae strain and kojic acid fermentation method and application thereof |
| CN112939238A (en) * | 2021-02-10 | 2021-06-11 | 杭州楠大环保科技有限公司 | Microecological preparation for efficiently removing COD (chemical oxygen demand) in domestic sewage |
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| CN108641970A (en) * | 2018-04-16 | 2018-10-12 | 江南大学 | It is a kind of to reduce the aspergillus oryzae of byproducts build-up and its application in malic acid building-up process |
| CN108641970B (en) * | 2018-04-16 | 2020-09-04 | 江南大学 | Aspergillus oryzae capable of reducing accumulation of byproducts in malic acid synthesis process and application thereof |
| CN112195110A (en) * | 2020-09-22 | 2021-01-08 | 天津科技大学 | Recombinant aspergillus oryzae strain and kojic acid fermentation method and application thereof |
| CN112195110B (en) * | 2020-09-22 | 2022-09-09 | 天津科技大学 | Recombinant aspergillus oryzae strain and kojic acid fermentation method and application thereof |
| CN112939238A (en) * | 2021-02-10 | 2021-06-11 | 杭州楠大环保科技有限公司 | Microecological preparation for efficiently removing COD (chemical oxygen demand) in domestic sewage |
| CN112939238B (en) * | 2021-02-10 | 2022-05-24 | 杭州楠大环保科技有限公司 | Microecological preparation for efficiently removing COD (chemical oxygen demand) in domestic sewage |
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