CN105861341B - The method that the inferior Dbaly yeast bacterial strain of one plant of Chinese and its fermentation prepare 3- hydracrylic acid - Google Patents
The method that the inferior Dbaly yeast bacterial strain of one plant of Chinese and its fermentation prepare 3- hydracrylic acid Download PDFInfo
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Abstract
The invention discloses the inferior Dbaly yeast bacterial strain of one plant of Chinese, the inferior Dbaly yeast bacterial strain of the Chinese be the inferior Dbaly yeast bacterium of the Chinese (Debaryomyces hansenii.) IS451, deposit number are as follows: CGMCC No.11893.The method that the strain fermentation prepares 3- hydracrylic acid, is prepared as follows: IS451 strain inoculated being activated in slant medium, then accesses seed culture medium, is cultivated in constant temperature oscillator, IS451 bacterial strain seed liquor is made in culture to logarithmic phase；Cultured IS451 bacterial strain seed liquor is inoculated in fermentation medium according to volume ratio for 10%, pH to 6.0 is adjusted；It is cultivated in constant temperature oscillator, fermentation time 5 days, fermentation liquid is made；It detects 3- hydracrylic acid in fermentation liquid and contains yield up to 48.96g/L, be higher by more than 3 times, be with a wide range of applications than document report microbial fermentation production 3- hydroxypropionic acid content.
Description
Technical field
The present invention relates to the methods that the inferior Dbaly yeast bacterial strain of one plant of Chinese and its fermentation prepare 3- hydracrylic acid, belong to biology
Chemical industry and field of fermentation engineering.
Background technique
It is influenced by world petroleum resource, price, environmental protection and Global climate change etc., exploitation bio-based source has become perhaps
Multinational is improved energy resource safety, GHG emissions mitigation, the important measures for coping with climate change.3- hydracrylic acid (3-hp)
It is the important chemical intermediate of one kind of rising in recent years, 3-hp has hydroxyl and carboxyl Liang Zhong functional group, is that many optics are living
Property substance precursor, be a kind of important platform chemicals, yield directly affects the production of many high valuable chemicals.
3-hp can be used as production malonic acid, 1,3-PD (PDO), succinic acid, and the raw material of specialty polyesters and acrylic acid can also be made
Standby a variety of important fine chemical products.Currently, being classified as one of the chemical products of most potentiality to be exploited by U.S. Department of Energy.Tradition system
Standby 3-hp is chemical synthesis, and such as adjacent halohydrin and potassium cyanide act on the 3- hydroxyl nitrile generated, passes through hydrolysis or Reformatsky
Reaction is made.The technique for being catalyzed 3 monohydroxy propionic aldehyde oxidation production 3-hp and its salt by platinum.Use solid acid catalyst ZSM5
Zeolite propene hydrate acid produces 3-hp.It is produced using chemical synthesis, difficulty is larger, and separation and purification of products is more complex, raw
It produces at high cost.
In contrast, these unfavorable factors can be effectively avoided in biological method, have the spies such as at low cost, mild condition
Point.Therefore, the modern research hotspot most gazed in the world is had become with biological method preparation 3-hp, especially genetic engineering bacterium method
One of.Cargill-Dow company, which develops, expresses alanine 2,3, aminomutase using genetic engineering bacterium, with carbon hydrate
The technique of object production 3-hp.South Korea scholar in recent years studies bioanalysis production 3-hp: the table in recombination bacillus coli
Up to malonyl-CoA reductase, acetyl-CoA decarboxylase, biotin enzyme and transhydrogenase gene, the genetic engineering bacterium of building can
Using the 3-hp for generating 1.2 mmol/L as substrate using glucose；The recombination bacillus coli using glycerol as substrate is constructed, is expressed
Glycerol dehydratase and acetaldehyde dehydrogenase gene, 3-hp yield are 0.58 g/L；Additionally by knockout glycerol dehydrogenase and mistake
Acetaldehyde dehydrogenase gene is expressed, the 3-hp yield of recombinant bacterium is 2.07 g/L.Domestic Southern Yangtze University is constructed using similar thinking
Recombination bacillus coli, using glycerol as substrate, 3-hp yield is 4.92 g/L, the report of other related bioanalysis production 3-hp
Also seldom.
Since building genetic engineering bacterium is related to several genes, and stability of these genes in host cell, enzyme
Activity expression and the faces enormous challenge in actual production such as cell metabolism flow control, still have there are many technical problem
It is to be solved.That reports at present can be less using the natural bacterium of glycerol production 3-hp, and yield is lower.It is known can fermenting and producing 3-
The wild strain of hp mainly has:Han-senuamiso、Fusarium merismoides、Candida rugosa、 Byssochlamys sp.、Rhodococcus eryt-hropolisLG12 andKlebsiella terrigenaDeng.Wherein,
Li Bing and Pei Jiang is gloomy to screen the metabolizable microbial strains Klebsiella terrigena for generating 3-hp from natural environment
(Klebsiella terrigena) can produce 3-hp by sole carbon source of glycerol, and acid producing ability is 1% (10 g/L), for the first time
Realize that wild-type strain is directly converted into 3-hp from glycerol.
Fan Junying breeding obtains one plant of 3-HP and efficiently synthesizes bacterial strain, they are research material with the soil of acquisition and fecal specimens
Material, therefrom
Screening obtains one plant can be using the yeast Y-11 of propionic fermentation production 3-HP, through Physiology and biochemistry identification and 18S
The analysis of rDNA sequence, primarily determines that the bacterial strain of screening isCandida sp.(Candida).Again using Y-11 as starting strain,
Through ultraviolet-nitrosoguanidine -60Co γ complex mutation, further screening has obtained mutant character stabilization and heritable superior strain
The yield of 5-13B, bacterial strain 3-HP are 11.78 g/L, are 2.46 times of starting strain.This result produces microbial fermentation
The research of 3-HP is increased to new level.In such microbial resources library abundant, due to natural environment and other physical and chemical items
The difference of part still has the microorganism that can much generate 3- hydracrylic acid undiscovered, and what is be separated to now is also only a drop in the ocean.
Therefore, the microbial strains for producing 3- hydracrylic acid also need further to find and study.
Flourishing country has been realized in the industrialization of biology base conversion 3-hp in the world, and produces as biological based platform
Other important fine chemical products have been widely used in medicine, biomaterial, functional material, bio-fuel and other fine
The industries such as chemicals become indispensable important raw material in national economy production.In the world, using microorganism conversion and
Engineering bacteria synthesis 3-hp has become mainstream technology, effectively chemical synthesis can be avoided to be brought using microbial fermentation production 3-hp
Unfavorable factor, and there is with short production cycle, abundant raw material, environmentally protective, the advantages such as production cost is low can be with production economy valence
It is worth higher chemical intermediate.The improvement and innovation for constantly carrying out the prior art solve the environmental protection in production, reduce life
Production cost and expansion industrialized scale etc. have become the research emphasis in the current technical field.
New 3- hydracrylic acid producing strains are screened, the type that biological fermentation process prepares 3- hydracrylic acid bacterial strain can be enriched, be
The research and application of biochemical industry and biofermentation provide foundation, provide new resource for building 3-hp high efficiency engineering bacterium, benefit
The problems such as mankind, solution energy security, all has very important significance.
Summary of the invention
The present invention provides new microorganism for building 3- hydracrylic acid (3-hp) high efficiency engineering bacterium, specifically provides one plant of Chinese
Inferior Dbaly yeast bacterial strain, the inferior Dbaly yeast bacterial strain of the Chinese be the inferior Dbaly yeast bacterium of the Chinese (Debaryomyces hansenii.) IS451, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center；Address: north
No. 3 Institute of Microorganism, Academia Sinica, institute of the Chaoyang District Jing Shi North Star West Road 1；Preservation date: on December 18th, 2015；Preservation
Number are as follows: CGMCC No.11893.
A kind of method that the inferior Dbaly yeast strain fermentation of the Chinese as described above prepares 3- hydracrylic acid, as follows
Preparation:
Step 1) by the inferior Dbaly yeast bacterium of the Chinese (Debaryomyces hansenii.) IS451 strain inoculated is in inclined-plane
It is activated in culture medium, 30 DEG C of constant temperature incubations for 24 hours, take 2 ring inclined-plane IS451 bacterial strains, i.e., connect inclined-plane bacterium with 2 points of oese
Strain；
Each component and its dosage in the slant medium are as follows: 20.00 g/L of glucose, 10.00 g/L of yeast extract,
20.00 g/L of peptone, 20.00 g/L of agar powder.
Seed culture medium is accessed again, and culture medium liquid amount is 50mL/250mL, is cultivated in constant temperature oscillator, 180 r/ of revolving speed
Min, cultivates 48h at 30 DEG C, and IS451 bacterial strain seed liquor is made in culture to logarithmic phase；
Each component and its dosage in the seed culture medium are as follows: glucose, 20.00 g/L；Yeast extract, 10.00 g/
L；(NH4)2SO4, 10.00 g/L；KH2PO4 , 5.00 g/L； K2HPO4, 2.00 g/L；MgSO4·7 H2O, 1.00 g/L；
FeSO4·7 H2O, 0.03 g/L；TES 5 mL；
The cultured IS451 bacterial strain seed liquor of step 1) is inoculated in fermentation medium according to volume ratio for 10% by step 2
In, adjust pH to 6.0；Culture medium liquid amount is 100mL/250mL, is cultivated in constant temperature oscillator, revolving speed 180 r/min, 30 DEG C,
Fermentation time 5 days, fermentation liquid is made；
Each component and its dosage in the fermentation medium are as follows: 35.00 g/L of glucose 25.00-；Yeast extract 10.00
g/L；KH2PO4, 5.00 g/L；K2HPO4, 2.00 g/L；(NH4)2SO4, 10.00-25.00 g/L；MgSO4·7H2O,
1.00 g/L；FeSO4·7 H2O, 0.03 g/L；Propionic acid, 10.00-25.00 g/L；Glycerol, 5.00-15.00 g/L；TES, 5
mL；
Step 3) detecting step 2) made from 3- hydroxypropionic acid content in fermentation liquid；
In above-mentioned steps, each component and its dosage in the TES are as follows: HCl, 3.65 g/L；H3BO4, 0.30 g/L；
CuCl2·6H2O, 0.20 g/L；ZnSO4·7H2O, 0.10 g/L；MnSO4 , 0.03 g/L；NaMoO4·2KH2PO4, 0.03
g/L；NiCl2·6H2O, 0.02 g/L；CuSO4·5H2O, 0.01 g/L.
Each component and its dosage advanced optimize in fermentation medium in the step 2) are as follows: 30.00 g/L of glucose；
10.00 g/L of yeast extract；KH2PO4, 5.00 g/L；K2HPO4, 2.00 g/L；(NH4)2SO4, 15.00 g/L；MgSO4·7H2O,
1.00 g/L；FeSO4·7 H2O, 0.03 g/L；Propionic acid, 15.00 g/L；Glycerol, 10.00 g/L；TES, 5 mL；
Each component and its dosage in the TES are as follows: HCl, 3.65 g/L；H3BO4, 0.30 g/L；CuCl2·6H2O,
0.20 g/L；ZnSO4·7H2O, 0.10 g/L；MnSO4 , 0.03 g/L；NaMoO4·2KH2PO4, 0.03 g/L；NiCl2·
6H2O, 0.02 g/L；CuSO4·5H2O, 0.01 g/L.Technical solution of the present invention is summarized as follows:
The invention has the advantages that:
The present invention filters out the inferior Dbaly yeast of the Chinese that a plant height produces 3- hydracrylic acid from orchard soil and human feces
Bacterium (Debaryomyces hansenii.) IS451, the bacterial strain adaptability is stronger, and breeding is fast, with short production cycle；With hair
Ferment culture abundant raw material multiplicity, also environmental protection while inexpensive, while the advantages such as can continuously be mass produced；In optimal culture side
Under case, the amount which produces 3- hydracrylic acid is up to 48.96 g/L, produces 3- hydroxyl third than document report microbial fermentation
Acid content is higher by more than 3 times, which is with a wide range of applications.
Detailed description of the invention
Fig. 1 be the inferior Dbaly yeast bacterium of the Chinese (Debaryomyces hansenii.) IS451 bacterial strain cellular morphology figure；
Fig. 2 is the inferior Dbaly yeast bacterium of the Chinese (Debaryomyces hansenii.) IS451 bacterial strain colonial morphology；
Fig. 3 is the PCR amplification result of the inferior Dbaly yeast bacterium of the Chinese (Debaryomyces hansenii.) IS451 bacterial strain；
Fig. 4 is the Phylogenetic of the inferior Dbaly yeast bacterium of the Chinese (Debaryomyces hansenii.) IS451 bacterial strain
Analysis；
Fig. 5 is 3- hydracrylic acid mark product HPLC figure；
Fig. 6 is the inferior Dbaly yeast bacterium of the Chinese (Debaryomyces hansenii.) under 2 fermentation condition of the present embodiment
IS451 produces the HPLC figure of 3- hydroxypropionic acid content；
Fig. 7 is the inferior Dbaly yeast bacterium of the Chinese (Debaryomyces hansenii.) under 3 fermentation condition of the present embodiment
IS451 produces 3- hydroxypropionic acid content HPLC figure.
Specific embodiment
Method and effect of the invention are described further below with reference to embodiment.
Embodiment 1: the inferior Dbaly yeast bacterium of the Chinese (Debaryomyces hansenii.) IS451 bacterial strain separation screening
With purifying
The inferior Dbaly yeast bacterium of the Chinese (Debaryomyces hansenii.) IS451 bacterial strain sample acquisition: main source
It is isolated in the orchard acquisition soil returned and the human feces sample of acquisition.
It screens isolated method are as follows: by a small amount of sample of human feces of soil and acquisition from orchard acquisition back
It is soaked in sterile saline respectively, stands 12 h；Bacteria suspension is subjected to gradient dilution after 12 h, gradient dilution concentration according to
Secondary is 10-4, 10-5, 10-6, 10-7, 10-8, 10-9；Bacteria suspension after dilution, it is to be transferred；
Preparing propionic acid content (V/V) is 0.4%, 0.8%, 1.2%, 1.6%, 2.0% screening and culturing medium, and is adjusted with pH meter
PH value is 6.0, and after sterilizing, inverted plate, the amount control of each plate screening culture medium is screening flat board after cooling in 10 mL；
Bacterial suspension inoculation after drawing 0.2 mL dilution respectively with liquid-transfering gun is in the screening flat board cooled down, and with sterilized
The spreading rod of bacterium is gently coated on screening flat board surface so that sample distribution is uniform, and each bacteria suspension dilution gradient is coated on difference
In the screening flat board of propionic acid content；
The screening flat board for having accessed strain dilution is placed in 30 DEG C of constant incubators and is inverted culture 48h, observes micro- life
Object growing state；Further to obtain purpose bacterial strain, need to take isolated method of crossing.The plate that constant temperature is inverted culture is taken
Out, in an aseptic environment, it is fallen with oese picking single bacterium therein and is crossed in above-mentioned screening flat board, then proceed to cultivate, weight
This multiple operation 3 to 5 times, cultivates 2 d or so, until there is single bacterium colony every time.
The screening and culturing medium (g/L): glucose 20.00, yeast extract 10.00, (NH4)2SO410.00 KH2PO4
5.00 K2HPO4 2.00 MgSO4·7 H2O 1.00, FeSO4·7 H25 mL of O 0.03, TES.
The slant medium (g/L): glucose 20.00, yeast extract 10.00, peptone 20.00, agar powder
20.00。
TES(trace element solution g/L): HCl 3.65, H3BO4 0.30, CuCl2·6H2O 0.20, ZnSO4·7H2O
0.10, MnSO4 0.03, NaMoO4·2 KH2PO40.03, NiCl2·6H2O 0.02, CuSO4·5H2O 0.01。
Visible bacterial strain thallus is spherical under the bacterial strain microscope, sees Fig. 1；It is greyish white that bacterium colony presentation is cultivated on base plate
Color is that protrusion is spherical, surface is soft and wet, is easy to provoke, neat in edge is shown in Fig. 2.
Its physiological and biochemical property are as follows: carbon assimilation glucose, sucrose, maltose, lactose, soluble starch；Nitrogen source assimilation
Ammonium sulfate, potassium nitrate, ammonium nitrate；Urea cannot be assimilated；The pH range for being suitble to this Yeast Growth is 4.0-6.0, the most suitable growth
Temperature is 30 DEG C.
Bacterial strain 18S rDNA sequence is obtained after testing, as shown in SEQ ID No.1 in sequence table.
It is analyzed by 18S rDNA sequence, PCR amplification figure is shown in Fig. 3, morphologic observation and Physiology and biochemistry identification, determines
IS451 bacterium be the inferior Dbaly yeast bacterium of the Chinese (Debaryomyces hansenii.), Fig. 4 is bacterial strainDebaryomyces hansenii.The phylogenetic tree analysis on Position of IS451.
Embodiment 2: under optimal fermentation condition,Debaryomyces hansenii.IS451 bacterial strain high yield 3- hydroxyl third
Sour situation
The method that the inferior Dbaly yeast strain fermentation of the Chinese prepares 3- hydracrylic acid, is prepared as follows:
Step 1) by the inferior Dbaly yeast bacterium of the Chinese (Debaryomyces hansenii.) IS451 strain inoculated is in inclined-plane
It is activated in culture medium, 30 DEG C of constant temperature incubations for 24 hours, take 2 ring inclined-plane IS451 bacterial strains, i.e., connect inclined-plane bacterium with 2 points of oese
Strain；
Each component and its dosage in the slant medium are as follows: 20.00 g/L of glucose, 10.00 g/L of yeast extract, egg
White 20.00 g/L of peptone, 20.00 g/L of agar powder.
Seed culture medium is accessed again, and culture medium liquid amount is 50mL/250mL, is cultivated in constant temperature oscillator, 180 r/ of revolving speed
Min, cultivates 48h at 30 DEG C, and IS451 bacterial strain seed liquor is made in culture to logarithmic phase；
Each component and its dosage in the seed culture medium are as follows: glucose, 20.00 g/L；Yeast extract, 10.00 g/
L；(NH4)2SO4, 10.00 g/L；KH2PO4 , 5.00 g/L； K2HPO4, 2.00 g/L；MgSO4·7 H2O, 1.00 g/L；
FeSO4·7 H2O, 0.03 g/L；TES 5 mL；
Each component and its dosage in the TES are as follows: HCl, 3.65 g/L；H3BO4, 0.30 g/L；CuCl2·6H2O,
0.20 g/L；ZnSO4·7H2O, 0.10 g/L；MnSO4 , 0.03 g/L；NaMoO4·2KH2PO4, 0.03 g/L；NiCl2·
6H2O, 0.02 g/L；CuSO4·5H2O, 0.01 g/L.
The cultured IS451 bacterial strain seed liquor of step 1) is inoculated in fermentation medium according to volume ratio for 10% by step 2
In, adjust pH to 6.0；Culture medium liquid amount is 100mL/250mL, is cultivated in constant temperature oscillator, revolving speed 180 r/min, 30 DEG C,
Fermentation time 5 days, fermentation liquid is made；
Each component and its dosage in the fermentation medium are as follows: 30.00 g/L of glucose；10.00 g/L of yeast extract；
KH2PO4, 5.00 g/L；K2HPO4, 2.00 g/L；(NH4)2SO4, 15.00 g/L；MgSO4·7H2O, 1.00 g/L；FeSO4·
7 H2O, 0.03 g/L；Propionic acid, 15.00 g/L；Glycerol, 10.00 g/L；TES, 5 mL；
Each component and its dosage in the TES are as follows: HCl, 3.65 g/L；H3BO4, 0.30 g/L；CuCl2·6H2O,
0.20 g/L；ZnSO4·7H2O, 0.10 g/L；MnSO4 , 0.03 g/L；NaMoO4·2KH2PO4, 0.03 g/L；NiCl2·
6H2O, 0.02 g/L；CuSO4·5H2O, 0.01 g/L.
Step 3) detecting step 2) made from 3- hydroxypropionic acid content in fermentation liquid；With high performance liquid chromatography (chromatographic column:
Ecosile C18 column (250 mm × 4.6 mm, 5 μm)；Mobile phase: 3% methanol uses H3PO4Adjust pH to 2.0；Testing conditions:
210 nm of UV detector, 35 DEG C of column temperature, 0.8 mL/ min of flow velocity, 20 μ L of sample volume) the 3- hydroxyl in fermentation liquid of detecting step 2
The content of base propionic acid.At this point,Debaryomyces hansenii.The content of 3- hydracrylic acid reaches after IS451 strain fermentation
48.96 g/L.3- hydracrylic acid mark product HPLC figure is shown in that Fig. 5,3- hydroxypropionic acid content manufactured in the present embodiment, HPLC figure are shown in
Fig. 6.
Embodiment 3: under general fermentation condition,Debaryomyces hansenii.IS451 bacterial strain produces 3- hydracrylic acid
Situation
The method that the inferior Dbaly yeast strain fermentation of the Chinese prepares 3- hydracrylic acid, is prepared as follows:
Step 1) by the inferior Dbaly yeast bacterium of the Chinese (Debaryomyces hansenii.) IS451 strain inoculated is in inclined-plane
It is activated in culture medium, 30 DEG C of constant temperature incubations for 24 hours, take 2 ring inclined-plane IS451 bacterial strains, i.e., connect inclined-plane bacterium with 2 points of oese
Strain；
Each component and its dosage in the slant medium are as follows: 20.00 g/L of glucose, 10.00 g/L of yeast extract, egg
White 20.00 g/L of peptone, 20.00 g/L of agar powder.
Seed culture medium is accessed again, and culture medium liquid amount is 50mL/250mL, is cultivated in constant temperature oscillator, 180 r/ of revolving speed
Min, cultivates 48h at 30 DEG C, and IS451 bacterial strain seed liquor is made in culture to logarithmic phase；
Each component and its dosage in the seed culture medium are as follows: glucose, 20.00 g/L；Yeast extract, 10.00 g/
L；(NH4)2SO4, 10.00 g/L；KH2PO4 , 5.00 g/L； K2HPO4, 2.00 g/L；MgSO4·7 H2O, 1.00 g/L；
FeSO4·7 H2O, 0.03 g/L；TES 5 mL.
The cultured IS451 bacterial strain seed liquor of step 1) is inoculated in fermentation medium according to volume ratio for 10% by step 2
In, adjust pH to 6.0；Culture medium liquid amount is 100mL/250mL, is cultivated in constant temperature oscillator, revolving speed 180 r/min, 30 DEG C,
Fermentation time 5 days, fermentation liquid is made；
Each component and its dosage in the fermentation medium are as follows: 35.00 g/L of glucose；10.00 g/L of yeast extract；
KH2PO4, 5.00 g/L；K2HPO4, 2.00 g/L；(NH4)2SO4, 10.00 g/L；MgSO4·7H2O, 1.00 g/L；
FeSO4·7 H2O, 0.03 g/L；Propionic acid, 10.00 g/L；Glycerol, 5.00 g/L；TES, 5 mL；
Each component and its dosage in the TES are as follows: HCl, 3.65 g/L；H3BO4, 0.30 g/L；CuCl2·6H2O,
0.20 g/L；ZnSO4·7H2O, 0.10 g/L；MnSO4 , 0.03 g/L；NaMoO4·2KH2PO4, 0.03 g/L；NiCl2·
6H2O, 0.02 g/L；CuSO4·5H2O, 0.01 g/L.
Step 3) detecting step 2) made from 3- hydroxypropionic acid content in fermentation liquid；With high performance liquid chromatography (chromatographic column:
Ecosile C18 column (250 mm × 4.6 mm, 5 μm)；Mobile phase: 3% methanol uses H3PO4Adjust pH to 2.0；Testing conditions:
210 nm of UV detector, 35 DEG C of column temperature, 0.8 mL/ min of flow velocity, 20 μ L of sample volume) the 3- hydroxyl in fermentation liquid of detecting step 2
The content of base propionic acid.At this point,Debaryomyces hansenii.The content of 3- hydracrylic acid reaches after IS451 strain fermentation
36.39 g/L；3- hydracrylic acid mark product HPLC figure is shown in Fig. 5；3- hydroxypropionic acid content manufactured in the present embodiment, HPLC figure, is shown in
Fig. 7.
<110>Xuzhou Engineering Institute
The method that the inferior Dbaly yeast bacterial strain of<120>one plants of Chinese and its fermentation prepare 3- hydracrylic acid
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 597
<212> DNA
<213>artificial sequence
<400> 1
AAAGAAACCA ACAGGGATTG CCTTAGTAAC GGCGAGTGAA GCGGCAAAAG CTCAAATTTG 60
AAATCTGGCG CCTTCGGTGT CCGAGTTGTA ATTTGAAGAA GGTAACTTTG GAGTTGGCTC 120
TTGTCTATGT TCCTTGGAAC AGGACGTCAC AGAGGGTGAG AATCCCGTGC GATGAGATGC 180
CCAATTCTAT GTAAAGTGCT TTCGAAGAGT CGAGTTGTTT GGGAATGCAG CTCTAAGTGG 240
GTGGTAAATT CCATCTAAAG CTAAATATTG GCGAGAGACC GATAGCGAAC AAGTACAGTG 300
ATGGAAAGAT GAAAAGAACT TTGAAAAGAG AGTGAAAAAG TACGTGAAAT TGTTGAAAGG 360
GAAGGGCTTG AGATCAGACT TGGTATTTTG CGATCCTTTC CTTCTTGGTT GGGTTCTCCG 420
CAGCTTACTG GGCCAGCATC GGTTTGGATG GTAGGATAAT GATTAAGGAA TGTGGCTCTA 480
CTTCGGTGGA GTGTTATAGC CTTGGTTGAT ACTGCCTGTC TAGACCGAGG ACTGCGTCTT 540
TGACTAGGAT GCTGGCATAA TGATCTTAAG CCACCCGTC 579
Claims (2)
1. the inferior Dbaly yeast bacterial strain of one plant of Chinese, it is characterised in that: the inferior Dbaly yeast bacterial strain of the Chinese is Han Xunde Bali ferment
Female bacterium (Debaryomyces hansenii.) IS451, deposit number are as follows: CGMCC No.11893；The bacterial strain 18S rDNA sequence
Column are as shown in SEQ ID No.1 in sequence table.
2. a kind of method that the inferior Dbaly yeast strain fermentation of the Chinese as described in claim 1 prepares 3- hydracrylic acid, feature
It is, is prepared as follows:
Step 1) is by the inferior Dbaly yeast bacterium of the Chinese (Debaryomyces hansenii.) IS451 strain inoculated in slant medium
In activated, 30 DEG C of constant temperature incubations for 24 hours, take 2 ring inclined-plane IS451 bacterial strains, i.e., connect inclined-plane bacterial strain with 2 points of oese；
Seed culture medium is accessed again, culture medium liquid amount is 50mL/250mL, it is cultivated in constant temperature oscillator, revolving speed 180r/min,
48h is cultivated at 30 DEG C, IS451 bacterial strain seed liquor is made in culture to logarithmic phase；
The cultured IS451 bacterial strain seed liquor of step 1) is inoculated in fermentation medium according to volume ratio for 10% by step 2),
Adjust pH to 6.0；Culture medium liquid amount is 100mL/250mL, is cultivated in constant temperature oscillator, and revolving speed 180r/min, ferments by 30 DEG C
Time 5 days, fermentation liquid is made；
Step 3) detecting step 2) made from 3- hydroxypropionic acid content in fermentation liquid；
Each component and its dosage in the slant medium are as follows: glucose 20.00g/L, yeast extract 10.00g/L, peptone
20.00g/L agar powder 20.00g/L；
Each component and its dosage in the seed culture medium are as follows: glucose, 20.00g/L；Yeast extract, 10.00g/L；(NH4)2SO4, 10.00g/L；KH2PO4, 5.00g/L；K2HPO4, 2.00g/L；MgSO4·7H2O, 1.00g/L；FeSO4·7H2O,
0.03g/L；TES, 5mL；
Each component and its dosage in the fermentation medium are as follows: glucose 30.00g/L；Yeast extract 10.00g/L；KH2PO4,
5.00g/L；K2HPO4, 2.00g/L；(NH4)2SO4, 15.00g/L；MgSO4·7H2O, 1.00g/L；FeSO4·7H2O, 0.03g/
L；Propionic acid, 15.00g/L；Glycerol, 10.00g/L；TES, 5mL；
Each component and its dosage in the TES are as follows: HCl, 3.65g/L；H3BO4, 0.30g/L；CuCl2·6H2O, 0.20g/L；
ZnSO4·7H2O, 0.10g/L；MnSO4, 0.03g/L；NaMoO4·2KH2PO4, 0.03g/L；NiCl2·6H2O, 0.02g/L；
CuSO4·5H2O, 0.01g/L.
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