CN102477407A - Black-pigmented engineered escherichia coli 9F2 and 4HPPD enzyme generated by the same - Google Patents

Abstract

The invention discloses a black-pigmented engineered escherichia coli 9F2 and 4HPPD enzyme generated by the same. The engineered bacteria can produce a black pigment. The engineered bacteria grows under a culture condition of a temperature of 37 DEG C, and the growth is in a good condition. The bacteria contains 4HPPD gene(a length of over 1083 bases pairs ) and encodes 4HPPD enzyme (361 amino acids), and the molecular weight is 40.4 Kda.

Description

The 4HPPD enzyme that one strain is produced melanic colibacillus engineering 9F2 and produced

Technical field

The present invention relates to the intestinal bacteria 9F2 that a strain has transformed the 4HPPD enzyme that the clone obtains from the grand genomic library in deep-sea; Engineering bacillus is expressed the 4HPPD enzyme; The 4-HPPA generates homogentisic acid under the effect of this enzyme, homogentisic acid generates melanochrome through oxidation and multimerization.

Background technology

Melanochrome comprises two big types, and one big type is tyrosine, polyphenol and their related compound metabolism final products; Melanochrome belongs to pattern former times class and apparent black pigment for other one big type, mainly is distributed in the plant.First kind melanochrome is complicated black superpolymer, and occurring in nature can be divided three classes basically: i.e. eumelanin, phaeomelanin and different melanocyte.Eumelanin is nitrogenous, sulfur atom-containing not, and color is dark-brown or the nitrogenous and sulphur atom of black phaeomelanin, color is that reddish brown or yellow different melanocyte is brown or black, mainly is present in the plant.According to alkaline hydrolysis or oxidative breakdown product, can melanochrome be divided into two kinds of indoles framework and catechol frameworks.

Melanochrome can be as UV light absorber, inhibitor and new type natural pharmaceutical carrier, in order to treat some relevant nervous system disorders with short of melanin, like xeroderma pitmentosum, Parkinson's disease, senile dementia, prosperous Ting Shi tarantism etc.Black have very big application potential, as aspect makeup or the hair dye decoration function, remove radical, the protection of biotic pesticide light etc., some solubility melanochrome have remarkable restraining effect external to HIV virus, resisiting influenza virus, cell death inducing.

Can produce melanochrome in a lot of moulds, bacterium, actinomycetes etc., like black mold, it is embraced a son in generation and secretes melanochrome simultaneously.It is generally acknowledged that melanochrome is not that microorganism growth, metabolism, breeding institute are necessary, poison but melanochrome can improve the mikrobe preventing from heavy metal, uvioresistant and phototoxis, anti-oxidant, thus strengthen its viability.

Because the special national conditions of melanochrome characteristics of product and China, China melanochrome market have the characteristic that is different from foreign market, different other pigment product markets.China's melanochrome number of the enterprise is less, and is small, so total size is little.In recent years, along with developing rapidly of foodstuff additive industry, the foreign market increases melanic demand just year by year.China's people's living standard improves constantly in recent years, and heath food is paid attention to more, and especially natural black pigment will impel melanic demand to increase considerably in Application in Food, and its market outlook are very good.Domestic industry looked up in 2009, and melanic demand is taken a turn for the better, and annual melanochrome industry market scale has reached 21,921 ten thousand yuan.Constantly high along with the increase of demand and product price from now on, its market scale will further increase, and expect melanochrome market scale in 2015 and will reach 40,880 ten thousand yuan.At present, the melanic size of capacity of China has surpassed 2000 tons.According to a preliminary estimate, Chinese melanic throughput in 2010 will reach 2150 tons above 2100 tons.

China's melanochrome industrial production raw material is mainly black soya bean and Semen Sesami Nigrum at present, and raw material supply receives weather, seasonal effect easily.And microorganisms producing melanochrome possesses and do not receive region, season limit, is easy to industrialized characteristics, in following melanochrome industry development, has very high value.

Summary of the invention

The object of the invention is to provide a strain esherichia coli engineering bacteria DH5 α 9F2 (Esherichia coli DH5 α 9F2); Engineering bacillus is expressed the 4HPPD enzyme; The 4-HPPA generates homogentisic acid under the effect of this enzyme, homogentisic acid generates melanochrome through oxidation and multimerization.This intestinal bacteria 5C11 culture presevation number is CCTCC NO:M 2010244, and preservation date is on September 21st, 2010, and the preservation place is a Wuhan City, Hubei Province Wuhan University, and preservation mechanism is Chinese typical culture collection center.

The pelagic deposit matter sample carries out enrichment from the Eastern Pacific for we, extracts enriched sample DNA and sets up the Fosimid library, and (Fosmid inserts fragment 40～45kb) to plasmid vector, and the host is intestinal bacteria (Escherichia coli), obtains about 3000 clone's.Through screening, obtain clone's.This is cloned in the LB cultivation and presents red-brown after 36 hours.

This clone finds that through analyzing the 4HPPD enzyme gene on the Pseudomonasstutzeri A1501 (GenBank:CP000304.1) among this sequence and the GENEBANK has very high similarity after inserting the order-checking of fragment total length.According to the sequences Design primer, as template, pcr amplification goes out the fragment of a treaty 1.5Kb with full length sequence, and gene order is as shown in Figure 2.This gene clone is transformed into the DH5a competent cell to the T carrier, obtains engineering bacteria 9F2.

Clone's 4HPPD enzyme gene, 1083 base pairs of total length, the 4HPPD enzyme of having encoded (361 amino acid), molecular weight is 40.4Kda.Through experimental verification; Phenylalanine(Phe) in the substratum and tyrosine can generate 4-hydroxy benzo ketone acid p-hydroxyphenylpyruvate (PHPP) under the effect of escherichia coli host tyrosine oxidase; PHPP generates homogentisic acid under the effect of this enzyme, homogentisic acid generates melanochrome through oxidation and multimerization.

Description of drawings:

Fig. 1: represented is the upgrowth situation that engineering bacteria 9F2 cultivates with LB, and 9F2 cultivated in LB 24 hours.

Fig. 2: represented is that HGA detects in the 9F2 nutrient solution.

Embodiment

Below in conjunction with embodiment the present invention is done further description, but do not constitute any restriction of the present invention.

Embodiment 1

The acquisition of engineering bacteria of the present invention

Engineering bacteria provided by the invention is to adopt following method to obtain:

1. abyssal sediment sample enrichment: take out Eastern Pacific's pelagic deposit matter sample 10g of 4 ℃ of preservations, in artificial seawater actinomycetes enrichment 500ml substratum, carry out enrichment culture, culture condition is 28 °, 200rpm, 10 days.

2. abyssal sediment enriched sample DNA extraction: enriched product 30ml, centrifugal 15 minutes of 10000g removes supernatant; Add 13.5ml DNA extraction buffer (100mM Tris-HCl, 100mM EDTA-Na, 100mM sodium phosphate buffer; 1.5M Nacl; CTAB pH 8.0), behind the mixing, add N,O-Diacetylmuramidase (10mg/ml); 37 degree levels vibration 30 minutes; Add 1.5ml 20%SDS, it as for water-bath in the 65 degree water-bath whirlpools 2 hours, was accompanied by the gentle mixing of putting upside down in every 15-20 minute simultaneously; With centrifuge tube 6000g centrifugal ten minutes, remove supernatant, deposition repeat to wash twice (4.5ml buffer+0.5mlSDS, 65 degree, 10min, centrifugal 6000g, 10min) this step can omit; Concentrate three resulting supernatants, add isopyknic phenol chloroform, the centrifugal 15min of 15000g; Water intaking phase solution adds the Virahol of 0.6 times of volume, precipitation at room temperature 1 hour again in another centrifuge tube; The centrifugal 20min of 16000g obtains the DNA crude extract; Use 70% washing with alcohol, be positioned over operator's console after centrifugal to dry up, use deionized water dissolving at last.

3. the structure of the grand genomic library of abyssal sediment enriched sample:

A.DNA shears

Extract the DNA of environmental sample or institute's enrichment thalline, detect with agarose gel electrophoresis

B.DNA selects

1. downcut the DNA of the about 40K to 45K of fragment,, at 70.C colloidal sol 10min, be that 1 μ l calculates by 1mg solid gums dissolving back volume, add GELase 50XBuffer, per 100 μ l glue add 1U GELase Enzyme, 45.C water-bath 1 hour.

2.70.C water-bath 10min, deactivation GELase Enzyme

3. the agar that centrifugal removal is not degraded.

4. add 1/10 volume sodium-acetate, gentle mixing.

5. the absolute ethyl alcohol that adds 2.5 times of volumes, gentle mixing.

6. precipitation at room temperature 10min.

7.13000rpm/min centrifugal 20min carefully absorbs supernatant

8. will precipitate with 70% ethanol of precooling and wash twice

9. after drying up DNA is dissolved in TE Buffer.

The C.DNA end-filling

1. mend flat system:

X μ l sterilized water

8μl 10X?End-Repair?Buffer

8μl 2.5mM?dNTP?Mix

8μl 10mM?ATP

Up to 20 μ g sheared insert DNA (about 0.5 μ g/ μ l)

4μl End-Repaie?Enzyme?Mix

80 μ l total reaction volume

2. room temperature is placed 45min.

3. add the phenol-chloroform extracting

4. get supernatant, add 1/10 volume sodium-acetate, gentle mixing.

5. the absolute ethyl alcohol that adds 2.5 times of volumes, gentle mixing.

The deposition 6.-20.C spend the night.

7.13000rpm/min centrifugal 20min carefully absorbs supernatant

8. will precipitate with 70% ethanol of precooling and wash twice

9. after drying up DNA is dissolved in TE Buffer.

D. ligation

1. linked system:

X μ l sterilized water

1μl 10X?Fast-Link?Ligation?Buffer

1μl 10mM?ATP

1μl CopyControl?pCC1FOS?Vecter

Xμl concertrated?insert?DNA

1μl Fast-Link?DNA?Ligase

10 μ l total reaction volume

2. room temperature is placed and is connected 2 hours

3. reaction system places 70 ℃, and 10 minutes, deactivation DNA Ligase.

E. clone's preparation

1. EPI300 is changed in the LB liquid nutrient medium that adds 10mM MgSO4, be cultured to OD and reach 0.8-1.0,4 degree are preserved bacterium liquid and can be preserved 72 hours.

2. put and melt a tube packaging albumen on ice, when melting in l to new centrifuge tube of rapid sucking-off 25 μ.25 remaining μ l still place-the subsequent use of 70.C.

3.25 add 10 μ l ligation products in the μ l packaging protein.

4.30.C water-bath 90min.

5. add other 25 μ l packaging proteins, 30.C water-bath 90min.

6. add Phage dilution buffer 940 μ l, make TV reach 1ml.

F.Titering?the?Packaged?CopyControl?Fosmid?Clones

1. add packaged solution of 10 μ l and 100 μ l EPI300-T1R host cell mixings.

2.37.C water-bath 20min.

3. be applied on the LC flat board that contains 12.5 μ g/ml paraxin incubated overnight

4. calculate clone's subnumber order.

G. clone's is confirmed.

1. extract the sub-plasmid of clone, agarose gel electrophoresis detects clip size.

2. plasmid enzyme restriction atlas analysis

The final library that makes up successfully about 3000 clone's.

4.4HPPD gene clone transforms

Find that one is cloned in LB and cultivates after 36 hours and present red-brown, it contains the 4HPPD gene through total length order-checking back discovery, and this gene clone is transformed into the DH5a competent cell to the T carrier, competence activation under 37 ℃, 120RPM condition.Get 50 microlitre coated plates, obtain engineering bacteria 9F2 containing on the LB flat board of penbritin after 10 hours.After 24 hours, visible engineering bacillus is produced pigment on flat board.

The checking of engineering bacteria melanochrome that 9F2 produces:

4HPPD enzyme in existing report mikrobe or the grand genomic library can and produce melanochrome at expression in escherichia coli.Phenylalanine(Phe) in the substratum and tyrosine can generate 4-hydroxy benzo ketone acid p-hydroxyphenylpyruvate (PHPP) under the effect of escherichia coli host tyrosine oxidase; PHPP generates homogentisic acid under the effect of 4HPPD enzyme, homogentisic acid generates melanochrome through oxidation and multimerization.We detect the melanuric acid in the engineering bacteria 9F2 LB substratum through HPLC, see Fig. 1.

Embodiment 2

The clone of enzyme gene and order-checking:

Through fosmid is cloned the full length sequence analysis of son and the interpretation of result of the BLAST in Gene Bank; This full length sequence and Pseudomonas stutzeri A1501 (GenBank:CP000304.1) similarity are the highest; Wherein the 4HPPD dna homolog property of fragment and Pseudomonas stutzeri A1501 is the highest in the full length sequence, is 98%.According to sequence and analytical results design amplification 4HPPD gene primer, primer is 9F2-PF

5′-TCAGGTCCATCCCATAGCAG-3′；9F2-PR?5′-GTGATACCGCGTGATCTCAAT-3′。

The following 95 ℃ of preparatory sex change of PCR step 5 minutes; 95 ℃ of sex change 1 minute, 50 ℃ of renaturation 45s, 68 ℃ were extended 2 minutes: 30 circulations were extended 4 ℃ of insulations 10 minutes for back 68 ℃.And with the fragment cloning of gained to the T carrier, and check order.

According to recording sequence, show that through DNA and albumen contrast the fragment that obtains of cloning is exactly the sequence of needed 4HPPD enzyme gene and 200 base left and right sides of upstream and downstream length thereof.

The recognition site that the fragment of being cloned is set according to primer carries out corresponding enzyme and cuts processing, and links to each other with carrier that same enzyme is handled well, finally is transformed in the E.Coli EPI300 competent cell and carries out abduction delivering.Substratum is LB, and rotating speed 180rpm cultivated 24 hours for 37 ℃.

Claims (7)

1. a strain colibacillus engineering DH5 α 9F2 (Esherichia coli DH5 α 9F2), preserving number is: CCTCC NO:M2010244.

2. a kind of colibacillus engineering as claimed in claim 1 is characterized in that, engineering bacillus is to change intestinal bacteria over to by the gene that contains the 4HPPD enzyme to obtain.

3. colibacillus engineering 9F2 as claimed in claim 1 (Esherichia coli DH5 α 9F2) is characterized in that synthesizing a kind of melanochrome.

4. colibacillus engineering 9F2 as claimed in claim 1 (Esherichia coli DH5 α 9F2); It is characterized in that engineering bacillus can express the 4HPPD enzyme; Phenylalanine(Phe) in the substratum and tyrosine can generate 4-hydroxy benzo ketone acid p-hydroxyphenylpyruvate (PHPP) under the effect of escherichia coli host tyrosine oxidase; PHPP generates homogentisic acid under the effect of this enzyme, homogentisic acid generates melanochrome through oxidation and multimerization.

5. a 4HPPD enzyme gene is characterized in that, has the base sequence shown in the SEQ ID NO.1 and function equivalent or varient.

6. a 4HPPD enzyme is characterized in that, has the described aminoacid sequence of SEQ ID NO.2.

7. produce melanic method for one kind; It is characterized in that: the aminoacid sequence that adopts the described intestinal bacteria 9F2 of claim 1 or its 4HPPD enzyme gene or 4HPPD enzyme; Obtain the 4HPPD enzyme; Generate homogentisic acid with this enzymatic conversion 4-hydroxy benzo ketone acid, homogentisic acid generates melanochrome through oxidation and multimerization.

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