CN102433312A - D-3-phosphoglycerate-dehydrogenase as well as coding gene and construction method thereof - Google Patents

Abstract

The invention relates to D-3-phosphoglycerate-dehydrogenase and a coding gene thereof. The amino acid sequence of the D-3-phosphoglycerate-dehydrogenase is shown as a sequence 1; the inhibition constant Ki of L-serine of the D-3-phosphoglycerate-dehydrogenase is greater than 250mM; and the sequence of the coding gene is shown as a sequence 2. The difference between the amino acid sequence of the D-3-phosphoglycerate-dehydrogenase (PGDH) provided by the invention and the amino acid sequence (sequence 3) of the escherichia coli wild type PGDH lies in that amino acid at 344th site is not histidine but lactamine and amino acid at 346th site is not asparagines but lactamine and the PGDH generated by mutation is not inhibited by the L-serine. In addition, the enzyme activity of a PGDH variant disclosed by the invention has no change compared with the wild type PGDH variant.

Description

A kind of D-3-phosphoglycerate dehydrogenase and encoding sox and construction process

Technical field

The invention belongs to the genetically engineered field, especially a kind of D-3-phosphoglycerate dehydrogenase and encoding sox and construction process.

Background technology

PGDH (D-3-phosphoglycerate-dehydrogenase; PGDH), i.e. D-3-phosphoglycerate dehydrogenase [EC1.1.1.95], this enzyme is a L-Serine synthetic key enzyme; 3-phoshoglyceric acid is converted into phosphohydroxy pyruvic acid, is the synthetic the first step of L-Serine.

Biological method is produced Serine and is mainly contained precursor methods and direct fermentation at present.Precursor methods mainly adopts serine hydroxymethylase (SHMT) that glycocoll is converted into the L-Serine; This legal system is equipped with the L-Serine and has yield height, advantage such as with short production cycle, but how from enzyme reaction solution separation and Extraction L-Serine be a difficult problem; Direct fermentation promptly is to utilize the direct fermentation of saccharic utilization of carbon source engineering bacteria, reaches the purpose of accumulation Serine, and this method can significantly reduce the production cost of Serine.

But directly utilize saccharic carbon source through fermentation L-Serine to exist bottleneck: there is feedback inhibition in the L-Serine to the D-3-phosphoglycerate dehydrogenase; Promptly when the L-serine concentration is higher, can make the structural modification of D-3-phosphoglycerate dehydrogenase; Cause reducing enzyme and live, this has just limited the resultant quantity of L-Serine.Therefore, how to guarantee that the feedback inhibition of removing this enzyme under the prerequisite that enzyme is lived just seems significant.

Peters-Wendisch etc. (2002; Appl.Microbiol.Biotechnol.60:437-441) the C-terminal deletion of Corynebacterium glutamicum (Corynebacterium glutamicum) PGDH has been described; Realized the elimination of L-Serine feedback inhibition, lost but caused the part enzyme to be lived simultaneously.

Patented claim CN200410063527.5 has described in the 349th glycocoll of intestinal bacteria PGDH or the replacement of the 372nd Threonine; The PGDH variant of gained has reduced the sensitivity to the L-Serine; Obtained effect preferably, but do not removed the feedback inhibition of L-Serine fully PGDH.

Summary of the invention

The object of the present invention is to provide a kind of D-3-phosphoglycerate dehydrogenase and encoding sox and construction process, it compares this desaturase with intestinal bacteria wild-type PGDH, does not receive L-Serine feedback inhibition, and enzyme is lived quite.Use the intestinal bacteria system; Gene to expressing PGDH is cloned, sequential analysis and rite-directed mutagenesis; Utilize recombinant gene to make up the recombinant expression vector that contains mutator gene then; Change recombinant expression vector over to corresponding host strain and express, and then obtain this D-3-phosphoglycerate dehydrogenase.

The objective of the invention is to realize through following technical scheme:

A kind of D-3-phosphoglycerate dehydrogenase, aminoacid sequence is seen sequence 1.

And, the inhibition constant K i＞250mM of its L-Serine.

A kind of encoding sox of D-3-phosphoglycerate dehydrogenase is characterized in that: gene order is seen sequence 2.

A kind of method that makes up the D-3-phosphoglycerate dehydrogenase, its construction process comprises following step:

(1) gene amplification: according to the gene order of reporting among the Genbank; The design primer; From e. coli k12 MG1655 bacterial strain clone obtain D-3-phosphoglycerate dehydrogenase expressing gene serA, this gene is connected to cloning vector pUC19 after, the sequence of goal gene is analyzed and is compared;

(2) rite-directed mutagenesis of gene: with the recombinant cloning vector in the said step 1 is template, utilizes overlapping pcr, and the 344th of gene and 346 bit codons are carried out rite-directed mutagenesis;

(3) recombinant expression vector makes up: the mutator gene muserA that is obtained is connected with expression vector pET28a (+); Carry out the double digestion checking; The correct expression vector of checking is transformed into E.coli BL21 (DE3), expresses the D-3-phosphoglycerate dehydrogenase after suddenling change.

Advantage of the present invention and positively effect are following:

The present invention is the basis with intestinal bacteria, utilizes genetic engineering technique that D-3-phosphoglycerate dehydrogenase (PGDH) expressing gene is carried out rite-directed mutagenesis, has obtained a kind of PGDH variant; This variant has been removed the feedback inhibition of L-Serine to it; And it is suitable with wild PGDH that enzyme is lived, even this has just guaranteed that PGDH still has activity in L-serine concentration conditions of higher following time; The synthetic of L-Serine can be continued, for industrial mass production L-Serine provides a strong instrument.

Description of drawings:

Fig. 1 is serA gene amplification result of the present invention and pUC19-serA double digestion qualification result: wherein: M:DNA Marker; 1:serA gene PCR product; 2: the recombinant cloning vector enzyme is cut the result.

Fig. 2 is mutator gene hypomere of the present invention and epimere amplification: wherein: M:DNAMarker; 1: following fragment gene; 2: go up fragment gene.

Embodiment

Below in conjunction with embodiment, the present invention is further specified, following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.

A kind of rite-directed mutagenesis method of D-3-phosphoglycerate dehydrogenase is following:

1, clone serA gene

Total DNA with intestinal bacteria MG1655 bacterial strain is a template, designs following primer and is used to clone D-3-phosphoglycerate dehydrogenase expressing gene serA:

Upstream primer serA-F:5 '-AGC GAG CTCATG GCAAAG GTATCG CTG GAG-3 ' (underscore is the SacI restriction enzyme site).

Downstream primer serA-R:5 '-ACG C GT CGA CTT AGT ACA GCA GAC GGG CGC-3 ' (underscore is the SalI restriction enzyme site).

Amplification system (50 μ L):

ddH ₂O 34.5μL，

10×buffer 5.0μL，

dNTP(2.5mmol/L) 5.0μL，

Upstream primer serA-F 2.0 μ L,

Downstream primer serA-R 2.0 μ L,

Dna profiling 1.0 μ L,

Pyrobest high-fidelity enzyme 0.5 μ L.

Amplification program:

95 ℃ of 5min, 1 circulation;

94 ℃ of 45s, 61 ℃ of 45s, 72 ℃ of 80s, 30 circulations;

72 ℃ of 10min, 1 circulation.

Carry out double digestion with restriction enzyme SacI and SalI respectively with cloning vector pUC19 behind the dna fragmentation purifying that obtains of amplification, then electrophoresis, cut cloning vector and PCR product after glue recovery enzyme is cut.The double digestion condition is 37 ℃, 4h, and system is (50 μ L) as follows:

SacI 2μL，

SalI 2μL，

10×Buffer?T 5μL，

BSA 5μL，

PUC19 or serA gene 20 μ L,

Sterilized water 16 μ L.

Carrier pUC19 behind the purifying utilizes Takara test kit (Takara Ligation Kit ver2.0) to be connected with serA Gene Double endonuclease bamhi, and condition of contact is 37 ℃, 16h, and system is (10 μ L) as follows:

Solution?I 5μL，

pUC19 1μL，

SerA gene 4 μ L,

Connect mixture transformed into escherichia coli DH5 α competent cell, corresponding transformant screens with ammonia benzyl mycin.Goal gene amplification and recombinant cloning vector pUC19-serA double digestion qualification result are seen Fig. 1.

2, the rite-directed mutagenesis of serA gene

Utilize overlapping PCR to carry out the codon 344 of serA gene and 346 rite-directed mutagenesis.Use the described carrier pUC19-serA of step 1 as template.

(1) carries out PCR one time with upstream primer serA-F and epimere mutant primer serA-muR:5 '-CAC GCC CGG ACG GGC TTCGGC GAT GTG CAT CA-3 ', amplification mutator gene epimere sequence.

Amplification system (50 μ L):

ddH ₂O 34.5μL，

10×buffer 5.0μL，

dNTP(2.5mmol/L) 5.0μL，

Upstream primer serA-F 2.0 μ L,

Epimere mutant primer serA-muR 2.0 μ L,

pUC19-serA 1.0μL，

Pyrobest high-fidelity enzyme 0.5 μ L.

Amplification program:

95 ℃ of 5min, 1 circulation;

94 ℃ of 45s, 55 ℃ of 45s, 72 ℃ of 30s, 30 circulations;

72 ℃ of 10min, 1 circulation.

(2) with hypomere mutant primer serA-muF:5 '-TGATGC ACATCG CCGAAG CCC GTC CGG GCGTG-3 ' and downstream primer serA-R: carry out PCR one time, amplification mutator gene hypomere sequence.

Amplification system (50 μ L):

ddH ₂O 34.5μL，

10×buffer 5.0μL，

dNTP(2.5mmol/L) 5.0μL，

Last primer serA-muF 2.0 μ L,

Following primer serA-R 2.0 μ L,

pUC19-serA 1.0μL，

Pyrobest high-fidelity enzyme 0.5 μ L.

Amplification program:

95 ℃ of 5min, 1 circulation;

94 ℃ of 45s, 50 ℃ of 45s, 72 ℃ of 60s, 30 circulations;

72 ℃ of 10min, 1 circulation.

Two kinds of PCR products are carried out the sepharose purifying respectively obtain two segment DNA fragments, the about 1kb of mutator gene epimere, the about 250bp of mutator gene hypomere sees Fig. 2.Then with this two segment DNAs fragment template each other; Use upstream primer serA-F and downstream primer serA-R to carry out PCR according to method described in the step 1; Product carries out the sepharose purifying, and the dna fragmentation that obtains is the serA mutator gene, after confirming through order-checking; Mutator gene with serA described in step 1 connects into expression vector pET28a (+), carrier called after pET28a (+)-muserA of generation.Among the transformed into escherichia coli BL21 (DE3), corresponding transformant screens with kantlex.

3, confirm PGDH activity and suppressor factor constant K i.

For confirming that PGDH enzymic activity and L-Serine are to its active influence; The LB substratum (Tryptones of 10g/L at the penbritin that contains 100mg/L of 100mL volume; 5g/L yeast extract, the NaCl of 10g/L) in, inoculation 2mL incubated overnight pET28a (+)-muserA/BL21 (DE3) bacterium liquid; And at 30 ℃, shaking table is cultivated under the 200r/min.When cell concentration reaches OD _600nmBe about at 0.8 o'clock, in nutrient solution, adding final concentration is sec.-propyl-β-thiogalactoside (IPTG) inducible gene expression of 1.0mmol/L, continues to cultivate 3h.Centrifugal collecting cell, washing resuspending are in 2mL damping fluid (100mM potassiumphosphate, pH7.0 then; 10mM MgCl ₂The 1mM WR 34678) in.Cell uses Ultrasonic Cell Disruptor to carry out fragmentation, and 20000g is centrifugal so that crude extract is clarified, and (1980, J.Bacteriol.141:235-245) mensuration PGDH is active to use McKitrick and Pizer test method(s).Activity is seen table 1.Suppressing constant K i expressed enzyme activity is the active 50% o'clock inhibition concentration of under the unrestraint agent, measuring.The PGDH variant has the enzyme identical with wild-type PGDH enzyme lives, and anti-inhibition ability greatly improves.Have only when the L-serine concentration is very high (＞250mM), enzymic activity just receives part and suppresses.Therefore PGDH variant of the present invention has been removed the feedback inhibition of L-Serine.

Table 1:PGDH enzyme is lived and Ki

Allelotrope	Sudden change	Enzyme [unit/mg] alive	Ki[mM]
				serA	Wild-type	0.05	＜0.01
muserA	H344A/N346A	0.05	＞250

Sequence one: the aminoacid sequence of D-3-phosphoglycerate dehydrogenase among the present invention (the underscore place is a mutating acid)

MAKVSLEKDKIKFLLVEGVHQKALESLRAAGYTNIEFHKGALDDEQLKESIRDAHFIGLRSRTHLTEDVINAAEKLVAIGCFCIGTNQVDLDAAAKRGIPVFNAPFSNTRSVAELVIGELLLLLRGVPEANAKAHRGVWNKLAAGSFEARGKKLGIIGYGHIGTQLGILAESLGMYVYFYDIENKLPLGNATQVQHLSDLLNMSDVVSLHVPENPSTKNMMGAKEISLMKPGSLLINASRGTVVDIPALCDALASKHLAGAAIDVFPTEPATNSDPFTSPLCEFDNVLLTPHIGGSTQEAQENIGLEVAGKLIKYSDNGSTLSAVNFPEVSLPLHGGRRLMHI AE ARPGVLTALNKIFAEQGVNIAAQYLQTSAQMGYVVIDIEADEDVAEKALQAMKAIPGTIRARLLY

Sequence two: the gene order of D-3-phosphoglycerate dehydrogenase among the present invention (the underscore place is a mutating alkali yl)

ATGGCAAAGGTATCGCTGGAGAAAGACAAGATTAAGTTTCTGCTGGTAGAAGGCGTGCACCAAAAGGCGCTGGAAAGCCTTCGTGCAGCTGGTTACACCAACATCGAATTTCACAAAGGCGCGCTGGATGATGAACAATTAAAAGAATCCATCCGCGATGCCCACTTCATCGGCCTGCGATCCCGTACCCATCTGACTGAAGACGTGATCAACGCCGCAGAAAAACTGGTCGCTATTGGCTGTTTCTGTATCGGAACAAACCAGGTTGATCTGGATGCGGCGGCAAAGCGCGGGATCCCGGTATTTAACGCACCGTTCTCAAATACGCGCTCTGTTGCGGAGCTGGTGATTGGCGAACTGCTGCTGCTATTGCGCGGCGTGCCGGAAGCCAATGCTAAAGCGCACCGTGGCGTGTGGAACAAACTGGCGGCGGGTTCTTTTGAAGCGCGCGGCAAAAAGCTGGGTATCATCGGCTACGGTCATATTGGTACGCAATTGGGCATTCTGGCTGAATCGCTGGGAATGTATGTTTACTTTTATGATATTGAAAATAAACTGCCGCTGGGCAACGCCACTCAGGTACAGCATCTTTCTGACCTGCTGAATATGAGCGATGTGGTGAGTCTGCATGTACCAGAGAATCCGTCCACCAAAAATATGATGGGCGCGAAAGAAATTTCACTAATGAAGCCCGGCTCGCTGCTGATTAATGCTTCGCGCGGTACTGTGGTGGATATTCCGGCGCTGTGTGATGCGCTGGCGAGCAAACATCTGGCGGGGGCGGCAATCGACGTATTCCCGACGGAACCGGCGACCAATAGCGATCCATTTACCTCTCCGCTGTGTGAATTCGACAACGTCCTTCTGACGCCACACATTGGCGGTTCGACTCAGGAAGCGCAGGAGAATATCGGCCTGGAAGTTGCGGGTAAATTGATCAAGTATTCTGACAATGGCTCAACGCTCTCTGCGGTGAACTTCCCGGAAGTCTCGCTGCCACTGCACGGTGGGCGTCGTCTGATGCACATC GCCGAA GCCCGTCCGGGCGTGCTAACTGCGCTGAACAAAATCTTCGCCGAGCAGGGCGTCAACATCGCCGCGCAATATCTGCAAACTTCCGCCCAGATGGGTTATGTGGTTATTGATATTGAAGCCGACGAAGACGTTGCCGAAAAAGCGCTGCAGGCAATGAAAGCTATTCCGGGTACCATTCGCGCCCGTCTGCTGTACTAA

Sequence three: the wild D-3-phosphoglycerate dehydrogenase of intestinal bacteria aminoacid sequence (the underscore place is for treating catastrophe point)

MAKVSLEKDKIKFLLVEGVHQKALESLRAAGYTNIEFHKGALDDEQLKESIRDAHFIGLRSRTHLTEDVINAAEKLVAIGCFCIGTNQVDLDAAAKRGIPVFNAPFSNTRSVAELVIGELLLLLRGVPEANAKAHRGVWNKLAAGSFEARGKKLGIIGYGHIGTQLGILAESLGMYVYFYDIENKLPLGNATQVQHLSDLLNMSDVVSLHVPENPSTKNMMGAKEISLMKPGSLLINASRGTVVDIPALCDALASKHLAGAAIDVFPTEPATNSDPFTSPLCEFDNVLLTPHIGGSTQEAQENIGLEVAGKLIKYSDNGSTLSAVNFPEVSLPLHGGRRLMHI HE NRPGVLTALNKIFAEQGVNIAAQYLQT?SAQMGYVVIDIEADEDVAEKALQAMKAIPGTIRARLLY。

Claims (4)

1. D-3-phosphoglycerate dehydrogenase, it is characterized in that: aminoacid sequence is seen sequence 1.

2. D-3-phosphoglycerate dehydrogenase according to claim 1 is characterized in that: the inhibition constant K i＞250mM of its L-Serine.

3. the encoding sox of a D-3-phosphoglycerate dehydrogenase as claimed in claim 1, it is characterized in that: gene order is seen sequence 2.

4. method that makes up D-3-phosphoglycerate dehydrogenase as claimed in claim 1, it is characterized in that: its construction process comprises following step:

(1) gene amplification: according to the gene order of reporting among the Genbank; The design primer; From e. coli k12 MG1655 bacterial strain clone obtain D-3-phosphoglycerate dehydrogenase expressing gene serA, this gene is connected to cloning vector pUC19 after, the sequence of goal gene is analyzed and is compared;

(2) rite-directed mutagenesis of gene: with the recombinant cloning vector in the said step 1 is template, utilizes overlapping pcr, and the 344th of gene and 346 bit codons are carried out rite-directed mutagenesis;

(3) recombinant expression vector makes up: the mutator gene muserA that is obtained is connected with expression vector pET28a (+); Carry out the double digestion checking; The correct expression vector of checking is transformed into E.coli BL21 (DE3), expresses the D-3-phosphoglycerate dehydrogenase after suddenling change.

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