CN107418962A

CN107418962A - A kind of thiolase and encoding gene and its preparation and application

Info

Publication number: CN107418962A
Application number: CN201610348088.5A
Authority: CN
Inventors: 尹恒; 王江; 谭海东; 李悝悝
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Panjin Industrial Technology Research Institute Co ltd Of Dalian Institute Of Chemical Materials Chinese Academy Of Sciences
Priority date: 2016-05-24
Filing date: 2016-05-24
Publication date: 2017-12-01

Abstract

The invention discloses one kind, from Methylobacter, (Methylobacterium sp. are in the resource number of Chinese Bei Na DSMZs：BNCC195932 gene order of thiolase (PhaA)) and its preparation method and application, i.e. design primer amplifies the nucleotide sequence of the gene from the bacterium, again by the gene cloning of the enzyme to coli expression carrier, obtain can the heterogenous expression enzyme E. coli recombinant stain, the thiolase prepared with the bacterial strain heterogenous expression, can be used for synthesizing polyhydroxyalkanoateby (PHA) and hydroxy fatty acid.

Description

A kind of thiolase and encoding gene and its preparation and application

Technical field

The present invention relates to a kind of gene order of thiolase and preparation method thereof.The invention provides The recombinant plasmid and recombination engineered strain of the thiolase and its synthesis poly-hydroxy fatty acid Application in terms of ester (PHA) and hydroxy fatty acid.Thiolase provided by the invention can extensive use In fields such as agricultural, food, medicine and environmental protection.

Background technology

Polyhydroxyalkanoate (polyhydroxyalkanoates, being abbreviated as PHAs) is micro- life A kind polyester (PHB is PHA one kind) for thing synthesis, because it has advantageous property, such as Biodegradability, biocompatibility and optical property etc., there is very extensive purposes.Biology can Degradability and hot-workability cause it to make polybag, agricultural mulching and agricultural herbicide, kill Worm agent, the carrier of fertilizer, improve their utilization ratio and polluted without generation environment.Biology Compatibility causes it to can be used as medical embedded material, pharmaceutical carrier, can adjust the release speed of medicine Rate.Chiral drug：Its chiral monomer (R types) can be used as chiral drug or chiral drug The intermediate of synthesis.Good gas phase causes it to can be used as the packaging material of food etc. every property. In addition PHA can also be used to produce bioenergy, have combustibility, after esterification is handled, Can directly as bio-fuel, only lower than the combustion heat of diesel oil 15% or so.Because it has this A little good characteristics, so having attracted the broad interest of scientific and technological circle and industrial quarters.

Hydroxy fatty acid is a kind of common medicine and the synthetic intermediate of other many compounds, Many medicines and compound can be synthesized based on this.

Beta-keto thiolase (PhaA) is a key enzyme in PHA metabolic pathway of synthesizing, This enzyme research in Ralstonia eutropha is more detailed, determines enzyme activity and power Learn characteristic, it is determined that activated centre and the work such as rite-directed mutagenesis were done, by the enzyme and other two Individual enzyme constructs PHA metabolic pathways, yield in Escherichia coli or Pichia pastoris together Compare high, also change PHA component characteristic by way of adding different substrates to produce tool There is the PHA of some properties, in Ralstonia eutropha, substantially related aspect All studied, but studied in other kinds it is seldom, it is also very shallow, Simply confirm do not have in the Pseudomonas there is this enzyme in Methylobacterium Pseudomonas Heterogenous expression, also without detailed survey enzyme activity and dynamics.Due to organic reagent pair Many enzymes have stronger inhibitory action, the higher structure of enzyme is changed and is denatured mistake It is living, and Methylobacterium can grow in methanol nutrient solution, so deriving from The enzyme in Methylobacterium should be to having certain tolerance to machine reagent, this It is the characteristic that this enzyme in other sources may not have.We have studied The vigor size and dynamics of the enzyme, preferably recognize in Methylobacterium sp. With make use of the enzyme.

The content of the invention

First purpose of the present invention is to provide a kind of new thiolase PhaA and its coding base Cause.

Second object of the present invention is to provide a kind of method for preparing new thiolase PhaA.

Third object of the present invention is to provide containing described thiolase PhaA genetic recombination tables Up to plasmid and recombination engineered strain.

Fourth object of the present invention is to provide in a kind of new thiolase PhaA synthesis PHA Using.

The 5th purpose of the present invention is to provide a kind of new thiolase PhaA in production hydroxyl fat The application of sour aspect.

Thiolase PhaA provided by the present invention, the Methylobacter in soil (Methylobacterium sp. are in the resource number of Chinese Bei Na DSMZs： BNCC195932), the thiolase PhaA encoding genes (being named as phaA) therefrom amplified, With one of following nucleotide sequence feature：

1) in sequence table SEQ ID NO.1 DNA (DNA) sequence；

2) in polynucleotide SEQ ID NO.2 amino acid sequences DNA (DNA) Sequence；

3) homology with SEQ ID NO.1 DNA (DNA) sequences limited reaches 80% and more than, and can coding for thiolase reactive protein DNA (DNA) sequence.

4) in sequence table SEQ ID NO.1 DNA (DNA) sequence carry out one or Several nucleotides substitutions, missing or nucleotides of the coding with thiolysis enzymatic activity obtained from addition Sequence.

Present invention also offers thiolase PhaA amino acid sequence, has one of following feature：

1) 1-396s of the SEQ ID NO.2 since aminoterminal in sequence table is active Thiolysis PhaA amino acid sequence, 397-402 are His-Tag amino acid sequence.

2) by 1-396 amino acids of the SEQ ID NO.2 since aminoterminal in sequence table Residue carries out one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, missing or additions and formed with thiolysis enzymatic activity Constant amino acid sequence.

The thiolase PhaA of present invention amino acid sequence and its nucleotide coding sequence can also According to the PhaA of prediction amino acid sequence and its artificial synthesized acquisition of nucleotide coding sequence.

Prepare restructuring enzyme PhaA method, it is that encoding gene PhaA is cloned into recombination expression Carrier, host cell is imported, obtain the thiolase of recombination expression.

Described recombination expression thiolase PhaA expression vector can be that Bacillus coli expression carries Body, Yeast expression carrier, hay bacillus expression vector, lactic acid bacteria expression vectors, streptomycete table Up to carrier, phage vector, filamentous fungi expression vector, plant expression vector, insect expression Carrier or mammalian cell expression vector etc..

Can be big for recombinantly expressing thiolase PhaA recombinant bacterium or transgenic cell line Enterobacteria host cell (such as Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH5 α etc.), yeast host cells (such as Saccharomyces Cerevisiae, Pichia pastoris, Kluyveromyces lactis etc.), hay bacillus host Cell (such as Bacillus subtilis R25, Bacillus subtilis 9920), lactic acid bacteria host Cell (such as Lactic acid bacteria COCC101), actinomyces host cell are (such as Streptomyces spp. etc.), filamentous fungal host cell (such as Trichoderma viride, Trichoderma reesei, Aspergillus niger, Aspergillus nidulans etc.), insect Cell (such as Bombyx mori, Antharaea eucalypti etc.) or mammalian cell is (such as Chinese hamster ovary cell CHO, baby hamster kidney cell BHK, CHL cells CHL etc.).

The thiolase PhaA of present invention gene order is from Methylobacter by round pcr Clone obtains in (Methylobacterium sp.).Gene code head of district 1185bp, category In belonging to the thiolase in cond-enzymes superfamilies.

The thiolase PhaA that the present invention is obtained from Recombinant protein expression, it is auxiliary with acetoacetyl When enzyme A is substrate, it is 0.5-1000U/mg to be measured under conditions of 28 DEG C, pH8.1 than work.

The present invention thiolase PhaA can extensively using agricultural, packaging material for food, medicine and The fields such as environmental protection.

Brief description of the drawings

Fig. 1：Thiolysis gene phaA agarose gel electrophoresis detects.

Fig. 2：The SDS-PAGE figures of thiolysis PhaA purifying.The sample that each swimming lane adds is respectively： Swimming lane 1- pre-dyed Protein Markers；Swimming lane 5-PhaA purifying proteins.

Fig. 3：Vapor detection PHB

Embodiment

The thiolase full-length gene of embodiment 1 is cloned

Reference gene group DNA purification kits (Thermo companies) operating procedure is extracted (Methylobacterium sp. receive in culture presevation Methylobacterium sp. in Chinese north The resource number of the heart is：BNCC195932 genomic DNA).To The National Center Thiolysis gene order is carried out more in for Biotechnology Information (NCBI) database After sequence alignment analysis, primer phaa-F is designed:5’ATGGCAGCCAGCGAAGATATC-3’； phaa-R:5 '-TCAGACGCGCTCGACGCACA-3 ', with the Methylobacterium of extraction Sp. genomic DNA is template, and the gene order of amplification coding thiolase maturation protein (is not wrapped Include signal peptide gene).PCR reaction conditions are：98 DEG C of 3min, 1 circulation；98 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 1min, 30 circulations；72 DEG C of 3min, 1 circulation.PCR primer Enter after row agarose gel electrophoresis analysis (see Fig. 1), gel extraction carried out to purpose fragment, It is sequenced after the method for double digestion is connected on prokaryotic expression carrier pET28a.

The thiolase gene sequence analysis of embodiment 2

The result of sequencing uses the Basic Local Alignment in GenBank databases Search Tool (BLAST) are analyzed, and the softwares of Vector NTI Suite 8.0 carry out Multiple Sequence Alignment, Analytical sequence information.

Thiolase gene (being named as phaA) the coding head of district 1191bp of acquisition, its nucleotides Sequence is as shown in SEQ ID NO 1.PhaA encodes 396 amino acid and a terminator codon, For its amino acid sequence as shown in SEQ ID NO 2, protein theoretical molecular is 40928Da, It is 6.29 to predict isoelectric point, powered in amino acid to have 99, accounts for 25.13%, acid amino Acid has 45, accounts for 11.42%, and basic amino acid has 43, accounts for 10.91%, Polar Amides Acid has 68, accounts for 17.26%, hydrophobic amino acid has 163, accounts for 41.37%.Ala has 67, Gly has 44, and Glu has 21.

(1) SEQ ID No.1 information (referring to sequence table)

(a) sequence signature

* length：1209 base-pairs

* type：Nucleic acid

* chain：Double-strand

* topological structure：Linearly

(b) molecule type：cDNA

(c) assume：It is no

(d) antisense：It is no

(e) initial source：Methylobacterium sp. bacterial strains.

Sequence table

SEQ ID NO.1

ATGGCAGCCAGCGAAGATATCGTCATTGTCGGGGCGGCGCGCACGCCGGTGG GTTCGTTCGCCGGCGCCTTCGGCTCGGTGCCCGCGCACGAACTCGGCGCGGTG GCGATCAAGGCGGCTTTGGAGCGGGCGGGCGTTTCGCCGGACGACGTGGAC GAGGTGATCTTCGGCCAGGTGCTCACGGCGGCCGCTGGCCAGAACCCGGCGC GCCAAGCCGCCATCGCCGCCGGCATCCCGGAGAAGGCCACCGCCTGGGGCCT CAACCAGGTCTGCGGCTCTGGTCTGCGCACGGTCGCCGTCGGCATGCAGCAGA TCGCCAGCGGCGACGCCAAGGTGATCGTGGCGGGCGGCCAGGAATCGATGTC GCTTTCCCCCCACGCTCAATACCTGCGCGGCGGCCAGAAGATGGGTGACCTCA AGCTCGTCGACACGATGATCAAGGACGGCCTGTGGGATGCCTTCAACGGCTAC CACATGGGCCAGACCGCCGAGAACGTCGCGCAGGCCTTCCAGCTCACCCGCG AGCAGCAGGACCAGTTCGCGGTCCGCTCGCAGAACAAGGCCGAGGCCGCCCG CAAGGAGGGCCGGTTCAAGGAGGAGATCGTCCCCGTCACCGTGAAGGGCCG GAAGGGCGACACGGTCGTCGATACCGACG

AGTACATCCGCGACGGCGCCACCGTTGAAGCGATGGCCAAGCTCAAGCCCGCC TTCACCAAGGACGGCACCGTGACCGCCGCCAACGCCTCGGGCCTCAACGACG GTGCCGCCGCCCTCGTGCTGATGTCGGCCTCTGAGGCCGAGCGCCGGGGCATC AAGCCGCTGGCCAAGATCGTCTCCTGGGCGACCGCGGGCGTCGATCCCAAGGT GATGGGCACCGGCCCGATTCCGGCCTCGCGCAAGGCGCTCGACAAGGCCGGC TGGAAGCCTGCCGACCTCGACCTGATCGAGGCGAACGAGGCCTTCGCCGCCCA GGCTCTGGCCGTGAACAAGGACATGGGCTGGGAC

GACGAGAAGGTGAACGTCAATGGCGGCGCCATCGCCATCGGCCACCCGATCG GCGCCTCGGGCGCCCGCGTCCTCATCACCCTGCTGCACGAGCTGAAGCGCCGC GATGCCAGACGAGGCCTTGCCACCCTCTGCATCGGCGGTGGCATGGTCGTCGC GATGTGCGTCGAGCGCGTCCTCGAGCACCACCACCACCACCACTGA

SEQ ID No.2 information

(a) sequence signature

* length：402 amino acid residues

* type：Amino acid

* chain：It is single-stranded

* topological structure：Linearly

(b) molecule type：Albumen

SEQ ID NO.2

MAASEDIVIVGAARTPVGSFAGAFGSVPAHELGAVAIKAALERAGVSPDDVDEVIF GQVLTAAAGQNPARQAAIAAGIPEKATAWGLNQVCGSGLRTVAVGMQQIASGD AKVIVAGGQESMSLSPHAQYLRGGQKMGDLKLVDTMIKDGLWDAFNGYHMGQ TAENVAQAFQLTREQQDQFAVRSQNKAEAARKEGRFKEEIVPVTVKGRKGDTVV DTDEYIRDGATVEAMAKLKPAFTKDGTVTAANASGLNDGAAALVLMSASEAERR GIKPLAKIVSWATAGVDPKVMGTGPIPASRKALDKAGWKPADLDLIEANEAFAAQ ALAVNKDMGWDDEKVNVNGGAIAIGHPIGASGARVLITLLHELKRRDARRGLAT LCIGGGMVVAMCVERVLEHHHHHH

Recombination expression and purifying of the phaA genes of embodiment 3 in Escherichia coli

For the ease of the recombination expression of gene, divide in the upstream and downstream primer of the embodiment 1 of design Yin Ru not Ncol and Xhol restriction enzyme sites.Pcr amplification product and expression vector pET28a difference Double digestion is carried out with Ncol and Xhol, digestion products, will after being separated by electrophoresis and gel reclaims By pET28a carrier T of the PCR primer of double digestion with also passing through double digestion₄DNA connects Connect enzyme connection.Connection product Transformed E .coli TOP10 competent cells are coated on containing 50 On the solid Luria-Bertani culture mediums of μ g/mL ampicillins, 37 DEG C of culture 12-16h. Monoclonal is accessed in the liquid Luria-Bertani culture mediums containing 50 μ g/mL ampicillins Culture, extract plasmid；Double digestion is carried out to the plasmid of extraction using restriction endonuclease Ncol and Xhol, In electrophoresis detection, the correct recombinant plasmid of clip size send Hua Da gene sequencing, the results showed that, Inserted between pET28a Ncol and Xhol restriction enzyme sites shown in SEQ ID NO 1 PhaC genes, and direction of insertion is correct, it was demonstrated that the recombinant plasmid of structure is correct, and this is recombinated Plasmid is named as pET28a-PhaC.

By pET28a-PhaC Transformed E .coli BL21 (DE3), cultivated in liquid LB nutrient solutions, , will when when OD values reach 0.4-0.6, addition 0.01mm IPTG induced expressions are three small Bacterium solution centrifuges, sonicated cells, then detects PHA synthase with polyacrylamide gel electrophoresis PhaC expression and purifying situation, as a result as shown in Fig. 2 PHA synthase PhaC after purification It is in single band on running gel, and position matches with the molecular weight predicted.

The thiolase PhaA of embodiment 4 enzyme activity determination

(1) concentration (BCA RNA isolation kits) of albumen is determined

A. according to sample size, 1 volume BCA reagents B (50 is added by 50 volume BCA reagent As:1) Appropriate BCA working solutions are prepared, are fully mixed.BCA working solutions room temperature is stablized in 24 hours.

B. protein standard substance is completely dissolved, takes 10 μ l to be diluted to 100 μ l, makes final concentration of 0.5mg/ml.For protein sample in what solution, standard items also preferably use any solution dilution.But It is for simplicity 0.9%NaCl or PBS dilution standard product can also be used.

C. standard items are pressed (can determine to reduce front or behind according to the content of actual albumen Two points) 0,1,2,4,8,12,16,20 μ l (protein content represented respectively as：0、0.5、 1st, 2,4,6,8,10ug albumen) it is added in the standard sample wells of 96 orifice plates, add for dilute The solution for releasing standard items is supplied to 20 μ l.

D. in the sample well for taking 5,10,15 μ l samples to 96 orifice plates, add for dilution standard The solution of product is to 20 μ l.

E. each hole adds 200 μ l BCA working solutions, and 37 DEG C are placed 30 minutes.

F. A562 is determined, protein concentration is calculated according to standard curve.

(2) measure of thiolase PhaA vigor

A, prepare and include 62.4mM Tris-Cl pH 8.1,50mM MgCl₂Buffer solution.Weigh Tris 0.755g, MgCl₂·6H₂O 1.0165g are dissolved in 100mL deionized waters, are adjusted with hydrochloric acid PH value sterilizes to 8.1.

B, using the acetoacetyl-CoA and CoA of buffer 140uM concentration：Weigh Acetoacetyl-CoA 1mg, add 8.387mL buffer solution, and CoA 1mg add buffer solution 9.306mL.It is fitted into sterilized 10mL centrifuge tubes, -20 degree preserve.

C, standard curve is done：Prepare 0 respectively with mother liquor and buffer solution, 10,20,30,40, 50th, 60, the acetoacetyl-CoA and CoA of 70uM concentration.Do three it is parallel, on ice Operation, acetoacetyl-CoA and CoA mother liquors respectively take 1mL to take 0 after mixing, 14.28,28.57, 42.86th, 57.12,71.43,85.72,100 μ l, buffer solution takes 100 respectively, 85.72,71.43, 57.12nd, 42.86,28.57,14.28,0 μ l, 10min is placed in 28 degrees Celsius of incubator, Absorbance is determined at 304nm, draws the song of absorption value and acetoacetyl-CoA concentration Line.

D, 178.29 μ l acetoacetyl-CoA and CoA mother liquors are taken, do three it is parallel, take 10 or 20 μ l enzyme liquids and inactivation compare, then mend the μ l of buffer solution 21.71 or 11.71,28 5min is placed in the incubator of degree, absorbance is determined at 304nm.

E, according to the acetoacetyl-CoA of survey decrement and protein content, enzyme activity is calculated. The ratio work for measuring PhaA enzymes in this way is 0.5-1000U/mg.

The enzyme of embodiment 5 builds PHA metabolism way together with PhaB, PhaC in Escherichia coli Footpath

(1), Ncol is chained in the gene order of the enzyme and PhaB, PhaC gene order With Xhol digestions position.

(2) after, three genetic fragments are cut away with the two restriction endonucleases, then connected with T4DNA Connect enzyme three gene orders are connected to above expression vector pET28a, be transferred in Escherichia coli.

(3), with the LB nutrient solutions culture Escherichia coli 3 days to produce PHA.

(4), thalline is centrifuged lyophilized.

(5), gas chromatographic detection PHA content, the PHA contents detected are dry cell weight 10%-90%.

Such as Fig. 3.

Claims

1. one kind is from Methylobacter, (Methylobacterium sp. receive bacterium in Chinese north The resource number of kind of collection is：BNCC195932) the gene (phaA) of thiolase, its Nucleotide sequence has one of following feature：

1) there is the 1-1188 of SEQ ID NO.1 DNA (DNA) sequence in sequence table Or 1-1191 or 1-1209DNA sequences；

2) 1-396 positions or 1-402 position ammonia of the SEQ ID NO.2 since aminoterminal are encoded DNA (DNA) sequence of base acid sequence；

3) to the 1-1188 of SEQ ID NO.1 DNA (DNA) sequence in sequence table Or 1-1191 or 1-1209DNA sequences carry out one or more than two nucleotides substitutions, missings Or nucleotides of the coding with thiolysis enzymatic activity obtained from the one or two or more kinds in addition Sequence.

2. a kind of thiolase of the thiolase gene coding described in claim 1, its amino acid Sequence has one of following feature：

1) there is in sequence table 1-396 positions of the SEQ ID NO.2 since aminoterminal or the 1-402 amino acids residue sequences；

2) to the amino acid sequence in sequence table shown in SEQ ID NO.2 since aminoterminal 1-396 positions or 1-402 positions carry out one or more than two 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors, missing or addition In one or two or more kinds and formed the amino acid sequences with thiolysis enzymatic activity.

A kind of 3. preparation method of the thiolase described in claim 2, it is characterised in that：Will Thiolase gene described in claim 1 is cloned into recombinant expression carrier, imports host cell, Obtain the thiolase of recombination expression.

4. according to the preparation method described in claim 3, it is characterised in that：Described restructuring table Up to the expression vector of thiolase, refer to coli expression carrier, Yeast expression carrier, withered grass Bacillus expression vector, lactic acid bacteria expression vectors, streptomyces expression vector, phage vector, silk Shape fungus expression vector, plant expression vector, insect expression vector or mammalian cell table Up to carrier.

5. according to the preparation method described in claim 3, it is characterised in that：The host cell It is the recombinant bacterium or transgenic cell line for recombinantly expressing thiolase, refers to escherichia coli host Cell (such as Escherichia coli BL21, Escherichia coli JM109, Escherichia coli DH5 α etc.), yeast host cells (such as Saccharomyces cerevisiae, Pichia Pastoris, Kluyveromyces lactis etc.), hay bacillus host cell (such as Bacillus Subtilis R25, Bacillus subtilis 9920 etc.), lactic acid bacteria host cell (such as Lactic acid Bacteria COCC101 etc.), actinomyces host cell (such as Streptomyces spp.), silk Shape fungal host cells (such as Trichoderma viride, Trichoderma reesei, Aspergillus Niger, Aspergillus nidulans etc.), insect cell (such as Bombyx mori, Antharaea Eucalypti etc.), mammalian cell (such as Chinese hamster ovary cell CHO, baby hamster Kidney cell BHK, CHL cells CHL etc.) in one kind.

A kind of 6. application of PHA synthase as claimed in claim 2, it is characterised in that：

The enzyme can catalyze and synthesize polyhydroxyalkanoate (PHA).

7. according to the application of the PHA synthase described in claim 6, it is characterised in that：

For the raw material that is used when synthesizing polyhydroxyalkanoateby (PHA) for acetyl coenzyme A.