CN105602966B - It is a kind of encode 6-phosphogluconate dehydrogenase gene and its application - Google Patents
It is a kind of encode 6-phosphogluconate dehydrogenase gene and its application Download PDFInfo
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Abstract
The present invention provide it is a kind of encode 6-phosphogluconate dehydrogenase gene and its application, nucleotide sequence is as shown in SEQ ID NO:1.6-phosphogluconate dehydrogenase gene pgdhD2 derives from methylotrophic bacteria Methylobacterium sp.MB200, has Serine depression effect.Using methylotrophic bacteria Methylobacterium sp.MB200 as starting strain, pgdh deletion mutant bacterium DMB is constructed in homologous double-crossover method, it obtains and produces the Serine enhanced variant that Serine ability is much stronger than starting strain Methylobacterium sp.MB200, tolerable D-Ser concentration is much higher than starting strain.Pgdh deletion mutant bacterium provided by the invention has a good application prospect in fermentation method production Serine production.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of gene for encoding 6-phosphogluconate dehydrogenase
And its application.
Background technique
Serine has important physiological function and application value, is widely used in medicine, food and chemical field.Mesh
Before, the production method of Serine mainly has chemical synthesis, albumen hydrolysis, enzymatic conversion method and microbe fermentation method.Micro- life
Object fermentation method production Serine is a kind of green, environmental protection production method, but since there are feedback inhibition adjustment effect, micro- lifes
Object is difficult largely to accumulate Serine, therefore the yield of Fermentation of L-Serine is relatively low, limits this method
Application.
Release Serine feedback inhibition adjust, improve fermentation method production Serine yield method, first is that by pair
Crucial enzyme gene carries out evolution transformation in Serine biosynthesis pathway, and obtaining, there is anti-Serine feedback inhibition to adjust
Key enzyme, release the feedback inhibition of Serine, construct Serine superior strain；Second is that transformation non-synthetic approach such as EMP
The key enzyme of approach, HMP approach, Serine path for transformation, transporting pathway etc. can also reduce or release Serine feedback
Inhibit, constructs associated gene mutation type Serine superior strain, release the feedback inhibition of Serine.
Releasing Serine is the key that building Serine superior strain, base to the feedback inhibition of synthesis pathway key enzyme
Because transformation of evolving is the main means that metabolic pathway key enzyme and its gene expression regulating and controlling sequence is transformed.Have raw using molecule
The sensibility that object technology carries out correlative study to reduce key enzyme in route of synthesis to Serine, so that engineered strain has
Anti- Serine feedback inhibition is adjusted to improve Serine accumulation ability, improves the yield of biosynthesis Serine.As in
It is that state patent application (publication number 103436504A), which discloses with Serine producing bacterial strain Corynebacterium glutamicum SYPS-062,
Bacterium germination strain, using genome project means to be catalyzed its biosynthesis pathway first step reaction 3-phosphoglyceric acid dehydroenase into
Travelingization transformation realizes that the composing type of the 3-phosphoglyceric acid dehydroenase mutant of anti-Serine feedback inhibition stablizes expression,
The 3-phosphoglyceric acid dehydroenase mutant stablized and express anti-Serine feedback inhibition is obtained, Serine Producing Strain is constructed
Strain.Chinese patent (publication number 1609208) is disclosed the 349th of Escherichia coli 3-phosphoglyceric acid dehydroenase (3-PGDH)
The mutant of the substitution of glycine or the 372nd threonine, acquisition reduces the sensitivity of Serine, obtains preferable effect.
Chinese patent application (publication number 102433312A) equally uses rite-directed mutagenesis, by 3-phosphoglyceric acid dehydroenase (3-PGDH) the
344 hyte Histidine mutations are alanine and the 346th asparagine mutation is alanine, and mutant enzyme activity is not pressed down by Serine
It makes and enzyme activity can largely retain.
Methylotrophic bacteria Methylobacterium sp.MB200 is that one isolated is screened from the residue in methane-generating pit
Strain produces Serine bacterial strain, since it is using the low-carbon compound such as methanol-grown of non-C-C key, so have any different in other productions
The metabolic pathway of serine microorganism such as Escherichia coli, Corynebacterium glutamicum.The study found that methylotrophic bacteria such as
In Methylobacterium extorquens AM1, there is complicated metabolism network as shown in Figure 1.As shown in Figure 1, exist
In methylotrophic bacteria, methanol initial oxidation is methylene tetrahydrofolate, then acts on glycine and generates Serine.Methyl assimilation
Mainly realized by C1 approach and serine circulation, and C1 approach, serine circulation further through different carbon compounds,
Coenzyme, ATP etc. are contacted with EMP Embden Meyerbof Parnas pathway, HMP approach, TCA circulation, are formed one and are influenced each other, are implicative of each other and had
The metabolism network of sequence regulation.
The Serine biosynthesis of methylotrophic bacteria Methylobacterium sp.MB200 is in such metabolism net
In network, it is similarly subjected to stringent regulation, product Serine can in feedback inhibition metabolism network certain enzymes activity, make L-
Serine biosynthesis is in holddown.To release feedback regulation of the product to key enzyme in route of synthesis, it is usually taken
Strategy is breeding antimetabolic Regulatory mutant, and breeding product structure analog resistant mutant strain is that the Breeding Strategies are most common
Method.Mutant library is constructed using plasmid transposons insertion technology, being screened out from it D-Ser, (Serine structure is similar
Object) resistant mutants, and insertion sequence is cloned, it can analyze to obtain the gene being mutated, the enzyme of gene coding is possible
There are Serine feedback regulation effects.Pass through the relationship of analysis enzymatic activity and its function and Serine, it may be verified that the enzyme is
It is no that there is this kind of feedback inhibition.If the enzyme of the coded by said gene obtained has Serine feedback inhibition effect, to this
Gene carries out evolution transformation, constructs mutant strain, can establish Serine superior strain.In addition, the metabolism way of methylotrophic bacteria
Diameter is unique, and metabolism network is complicated, and EMP, HMP, TCA, C1 approach are all tied in a hundred and one ways with serine circulation, by above-mentioned
Method may clone to obtain the gene different from the presently found enzyme (such as 3-PGDH) with Serine feedback inhibition effect.
By building recombinant bacterium and enzyme activity experiment, related gene function and its relationship with production Serine are analyzed, related gene is constructed
Saltant releases methylotrophic bacteria Serine feedback inhibition and adjusts, obtains Serine superior strain, will be building L-
Serine superior strain provides new approaches, new way.
Summary of the invention
Fermenting for methylotrophic bacteria Methylobacterium sp.MB200, there are feedback inhibition tune for production Serine
Section, the problems such as yield is relatively low, the present invention construct methylotrophic bacteria using plasmid transposons insertion technology
The mutant library of Methylobacterium sp.MB200 filters out a kind of mutant with D-Ser resistance, clones
6-phosphogluconate dehydrogenase (6-PGDH) gene with Serine depression effect, and construct pgdh gene defection type
Serine superior strain, release methylotrophic bacteria metabolic process in 6-PGDH to produce Serine inhibiting effect, to mention
The yield of high Serine.
Technical solution provided by the invention are as follows:
A kind of gene pgdhD2 encoding 6-phosphogluconate dehydrogenase, nucleotide sequence such as SEQ ID NO:1 institute
Show.
In technical solution of the present invention, the gene pgdhD2 of the coding 6-phosphogluconate dehydrogenase, nucleosides
DNA in acid sequence SEQ ID NO:1 since 5 ' the 1st nucleotide in end initiation codon ATG to the 993rd nucleotide with end
Only codon TGA terminates, and there are a complete open reading frame (ORF), the 1-3 nucleotide from 5 ' ends are pgdhD2 base
The initiation codon ATG of cause, the 991-993 nucleotide from 5 ' ends are the terminator codon TGA of pgdhD2 gene.
In technical solution of the present invention, the gene pgdhD2 of the coding 6-phosphogluconate dehydrogenase, nucleosides
Open reading frame (ORF) in acid sequence SEQ ID NO:1, shares 993 nucleotide, and codified is made of 330 amino acid
Protein.
The present invention also provides a kind of gene pgdhD2 codings by the coding 6-phosphogluconate dehydrogenase
Protein, amino acid sequence is as shown in SEQ ID NO:2.
A kind of expression vector comprising encoding 6-phosphogluconate dehydrogenase gene pgdhD2 sequence.
A kind of methylotrophic bacteria of missing coding 6-phosphogluconate dehydrogenase gene pgdhD2 sequence
Methylobacterium sp.MB200 mutant cells system.
The present invention also provides the gene pgdhD2 of the coding 6-phosphogluconate dehydrogenase to compile in building missing
The application of code 6-phosphogluconate dehydrogenase gene pgdhD2 produced in Serine saltant type strain.
The method concrete operations that the present invention uses are as follows:
(1) methylotrophic bacteria Methylobacterium sp.MB200 be resistant to D-Ser mutant gene clone with
Analysis method: the mutation that technology constructs methylotrophic bacteria Methylobacterium sp.MB200 is inserted by plasmid transposons
Body library is screened out from it the mutant with D-Ser resistance.Total DNA is extracted to mutant, clone is connected to after digestion
On carrier pMD-18T, it imported into competent escherichia coli cell DH5 α, using the Double picking transformant on plate, and
Plasmid is extracted, the laggard pacing sequence of digestion verification carries out Blast sequence alignment and analysis in NCBI, show that mutant is inserted into
Segment is the 6-phosphogluconate dehydrogenase base that methylotrophic bacteria Methylobacterium sp.MB200 is resistant to D-Ser
Cause.
(2) clone of 6-phosphogluconate dehydrogenase gene, expression and enzyme activity analysis: according to Methylobacterium
The 6-phosphogluconate dehydrogenase gene order design primer of sp.MB200 is carried out by template of the total DNA of M.sp.MB200
PCR obtains pgdh genetic fragment (having I restriction enzyme site of EcoR I and Pst).By the pgdh segment and carrier pETBlue-2 of acquisition
Respectively to purify after I double digestion of EcoR I and Pst, connection is converted to the competent cell of e. coli bl21 (DE3) pLysS
In, to obtain recombinant bacterium BL21 (DE3) pLysS/pETBlue-2-pgdh.Recombinant protein PGDH is expressed under optimum conditions
Afterwards, and with nickel column purify.
(3) recombinant protein PGDH enzyme activity determination and functional verification: the measurement of recombinant protein PGDH enzyme activity.Including 100mM
Tris-HCl(pH8.0)、10mM MgCl2、3mM NADP+, in 3mM 6-PG reaction system, be added appropriate diluted enzyme solution, 40
Under DEG C reaction condition, measure and calculate the variation of NADPH concentration, enzyme activity be defined as 1min catalysis and generate the enzyme amount of 1mM NADPH be
One enzyme activity unit U.
The Serine of various concentration is added in enzyme activity determination system, measures the PGDH enzyme under different Serine concentration
Situation living analyzes influence of the Serine to recombinant protein PGDH enzyme activity, verifying 6-phosphogluconate dehydrogenase gene
Serine depression effect.
(4) methylotrophic bacteria Methylobacterium sp.MB2006- phosphogluconate dehydrogenase gene delection is prominent
Become the building of bacterium: using methylotrophic bacteria Methylobacterium sp.MB200 as starting strain, with homologous double-crossover method structure
Pgdh deletion mutant bacterium DMB is built, the pgdh deletion mutant bacterial strain of high yield Serine ability is obtained.Pass through analysis
Pgdh deletion mutant bacterium DMB produces Serine, resistance to D-Ser situation and enzyme activity determination, and verifying 6-phosphogluconic acid is de-
The function of hydrogenase gene.
Compared with prior art, the invention has the following beneficial effects:
1. the present invention constructs methylotrophic bacteria Methylobacterium sp.MB200 using plasmid transposons insertion technology
Mutant library, be screened out from it the mutant with D-Ser resistance, by sequencing analysis and the method for clone, compiled
The pgdh gene of code 6-phosphogluconate dehydrogenase (PGDH), and by building recombinant bacterium and enzyme activity experiment, show L- ammonia
Acid has depression effect to PGDH enzyme activity.The present invention is bacterium germination with methylotrophic bacteria Methylobacterium sp.MB200
Strain constructs pgdh deletion mutant bacterium DMB in homologous double-crossover method, and acquisition production Serine ability, which is much stronger than, sets out
The high yield Serine mutant strain DMB of bacterial strain Methylobacterium sp.MB200, can tolerate D-Ser is 5mg/
ML is much higher than the ability that starting strain tolerance D-Ser concentration is no more than 1mg/mL.
2. the present invention releases methylotrophic bacteria by building pgdh deletion mutant bacterial strain DMB
The Serine feedback inhibition of Methylobacterium sp.MB200 is adjusted, and obtains Serine superior strain, for building
Serine superior strain provides new thinking and approach.
Detailed description of the invention
Attached drawing 1 is the metabolism network of methylotrophic bacteria such as Methylobacterium extorquens AM1；
Attached drawing 2 is methylotrophic bacteria Methylobacterium sp.MB200, pgdh deletion mutant body, covers bacterium
The growing state of AMB and each bacterial strain of overexpression bacterium EMB in D-Ser gradient plate；
Attached drawing 3 is methylotrophic bacteria Methylobacterium sp.MB200, pgdh deletion mutant body, covers bacterium
Each bacterial strain fermentation liquor of AMB and overexpression bacterium EMB produces the DNS thin-layer chromatogram of Serine；
Explanation in relation to appended drawing reference:
A-M.sp.MB200；B- deletion mutant DMB；C- covers bacterium AMB；D- overexpression bacterium EMB；The sweet ammonia of 1- standard
Acid；2- standard serine；3-M.sp.MB200；4- deletion mutant DMB；5- covers bacterium AMB；6- overexpression bacterium EMB.
Specific embodiment
Below with reference to embodiment, specific embodiments of the present invention will be described in detail, it is to be understood that guarantor of the invention
Shield range is not limited by the specific implementation.
Unless otherwise explicitly stated, otherwise in entire disclosure and claims, term " includes " or its change
Changing such as "comprising" or " including " etc. will be understood to comprise stated element or component, and not exclude other members
Part or other component parts.
Embodiment 1:
1.1 methylotrophic bacteria Methylobacterium sp.MB200 be resistant to D-Ser mutant gene clone with
Analysis
1.1.1 technology is inserted by plasmid transposons and constructs methylotrophic bacteria Methylobacterium sp.MB200's
Mutant library is screened out from it the mutant D-2 with D-Ser resistance.
1.1.2 total DNA is extracted to mutant D-2, is connected to after digestion on cloning vector pMD-18T, imported into large intestine
In bacillus competent cell DH5 α, using the Double picking transformant on plate, and plasmid is extracted, after digestion verification further
Sequencing carries out Blast sequence alignment and analysis in NCBI.As a result obtain DNA fragmentation be 1875bp, the sequence with
The 6-phosphogluconate dehydrogenase gene of Methylobacterium populi BJ001 has highest similarity, consistency
Reach 89%, see Table 1 for details.
The mutant DNA fragments that table 1 is cloned into
The DNA fragmentation bioinformatic analysis of 1.2 mutant strain D-2 insertion
The DNA fragmentation obtained from mutant strain D-2 is 1875bp, searches mutant D-2 using ORF Finder on NCBI
The ORF of DNA fragmentation, wherein the ORF containing a 993bp.The ORF sequence and Methylobacterium populi BJ001,
The 6- of Methylobacterium extorquens AM1, Methylobacterium chloromethanicum CM4
Phosphogluconate dehydrogenase on amino acid levels up to 97%, thus may be used consistency up to 98% by similitude
Know, the sequential coding be M.sp.MB200 6-phosphogluconate dehydrogenase.
The predicted albumen is 330 containing total number of atnino acid, and wherein the biggish several amino acid of content and its content are:
Gly12.4%, Ala10.9%, Arg9.4%, Leu8.8%, Asp7.6%, Glue7.6%.Molecular composition is
C1572H2479N463O481S11.Molecular weight is 35913.4, theoretical pI=5.22.
Without obvious hydrophilic and hydrophobic, no transmembrane structure includes a pfam NAD binding structural domain.
1.36- phosphogluconate dehydrogenase gene cloning and expression
1.3.16- the clone of phosphogluconate dehydrogenase gene
The primers of foundation Methylobacterium sp.MB2006- phosphogluconate dehydrogenase gene,
It carries out PCR by template of the total DNA of M.sp.MB200, obtains pgdh genetic fragment (there is I restriction enzyme site of EcoR I and Pst).It will
The pgdh segment and carrier pETBlue-2 of acquisition after I double digestion of EcoR I and Pst respectively to purify, connection, conversion to large intestine bar
In the competent cell of bacterium BL21 (DE3) pLysS, to obtain recombinant bacterium BL21 (DE3) pLysS/pETBlue-2-pgdh.
1.3.2 the expression and purifying of recombinant protein PGDH
After recombinant protein is expressed under optimum conditions, and purified with nickel column.
1.4 recombinant protein PGDH enzymatic properties
1.4.1 the measurement of enzyme activity
Reaction system 300uL, wherein Tris-HCl containing 100mM (pH8.0), 10mM MgCl2, 3mM NADP+, 3mM 6-
PG is added appropriate diluted enzyme solution 50uL, supplies water, is uniformly mixed.40 DEG C, OD value is measured under 340nm, every 10S is recorded 1 time,
Total 10min.The variation of NADPH concentration can be calculated by the changing value of OD.
Enzyme activity definition: under optimum condition, the enzyme amount that 1min catalysis generates 1mM NADPH is an enzyme activity unit U.
1.4.2 influence of the L-Ser concentration to recombinant protein enzyme activity
The Serine of various concentration is added in enzyme activity determination system, measures the PGDH enzyme under different Serine concentration
Situation living.The result shows that the addition of Serine can be such that enzyme activity is suppressed, and depression effect is with the increase of Serine concentration
And reinforce, when concentration reaches 500mM, enzyme activity is reduced to 30% or so.
1.5 in Methylobacterium sp.MB200 verify 6-phosphogluconate dehydrogenase gene function and
Its Serine depression effect
1.5.1 the building of pgdh deletion mutant bacterium, covering bacterium, overexpression bacterium
Construct pgdh deletion mutant bacterium DMB in homologous double-crossover method, based on this by carrier pCM80 with
The overexpression bacterium EMB of method building covering bacterium AMB and the pgdh gene of three parents engagement.
1.5.2 pgdh deletion mutant bacterium, covering bacterium, 6-phosphogluconate dehydrogenase gene in overexpression bacterium
The verifying of function
With pgdh deletion mutant bacterium DMB, covering bacterium AMB, overexpression bacterium EMB and starting strain M.sp.MB200
6- is verified in above-mentioned each bacterial strain by producing Serine, resistance to D-Ser situation and enzyme activity determination as main study subject
The function of phosphogluconate dehydrogenase gene.
1.5.2.1 the case where each bacterial strain tolerance D-Ser
The case where each bacterial strain tolerance D-Ser of experimental study, as a result as shown in Figure 2.As known in the figure starting strain,
Overexpression bacterium is resistant to D-Ser and is no more than 1mg/mL, and it is bacterium germination out that it is 5mg/mL that deletion mutation strain, which can tolerate D-Ser,
5 times or more of strain, it is 1.2mg/mL that covering strain, which can tolerate D-Ser, more slightly higher than starting strain.
1.5.2.2 each bacterial strain produces the ability of Serine
Each bacterial strain carries out the energy that DNS chromatography semiqualitative analysis produces Serine after resting cell system fermented and cultured
Power, DNS tomographic map are as shown in Figure 3.The figure the result shows that: bacterial strain produce Serine ability be deletion mutation strain DMB it is most strong, return
Benefit strain AMB and overexpressing strain EMB is essentially identical, and M.sp.MB200 is most weak.
The aforementioned description to specific exemplary embodiment of the invention is in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can much be changed
And variation.The purpose of selecting and describing the exemplary embodiment is that explaining specific principle of the invention and its actually answering
With so that those skilled in the art can be realized and utilize a variety of different exemplary implementation schemes of the invention and
Various chooses and changes.The scope of the present invention is intended to be limited by claims and its equivalents.
Claims (3)
1. a kind of gene for encoding 6-phosphogluconate dehydrogenasepgdhD2 lacks coding 6-phosphogluconic acid in building and takes off
Hydrogenase genepgdhThe application of D2 produced in Serine strain, the strain that sets out of the strain areMethylobacteriumSp. MB200, it is characterised in that: the genepgdhD2 nucleotide sequence such as SEQ ID NO:1 institute
Show.
2. the gene of coding 6-phosphogluconate dehydrogenase according to claim 1pgdhD2 lacks coding 6- in building
Phosphogluconate dehydrogenase genepgdhThe application of D2 produced in Serine strain, the strain that sets out of the strain areMethylobacteriumSp. MB200, it is characterised in that: the genepgdhIn D2 nucleotide sequence SEQ ID NO:1
DNA initiation codon ATG to the 993rd nucleotide since 5 ' the 1st nucleotide in end is terminated with terminator codon TGA, is existed
One complete open reading frame (ORF), the 1-3 nucleotide from 5 ' ends arepgdhThe initiation codon ATG of D2 gene, from
5 ' end 991-993 nucleotide bepgdhThe terminator codon TGA of D2 gene.
3. the gene of coding 6-phosphogluconate dehydrogenase according to claim 1pgdhD2 lacks coding 6- in building
Phosphogluconate dehydrogenase genepgdhThe application of D2 produced in Serine strain, the strain that sets out of the strain areMethylobacteriumSp. MB200, it is characterised in that: the genepgdhIn D2 nucleotide sequence SEQ ID NO:1
Open reading frame (ORF) shares 993 nucleotide, the protein that codified is made of 330 amino acid.
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