CN106591209A - Recombinant strain and preparation method thereof and method for producing L-threonine - Google Patents

Recombinant strain and preparation method thereof and method for producing L-threonine Download PDF

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CN106591209A
CN106591209A CN201611248603.9A CN201611248603A CN106591209A CN 106591209 A CN106591209 A CN 106591209A CN 201611248603 A CN201611248603 A CN 201611248603A CN 106591209 A CN106591209 A CN 106591209A
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bacterial strain
threonine
recombinant bacterial
pntab
strain
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程江红
张捧
康培
刁刘洋
毛贤军
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Langfang Meihua Bio Technology Development Co Ltd
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Abstract

The invention relates to the field of microbial technology and particularly relates to a recombinant strain and a preparation method thereof and a method for producing L-threonine. The recombinant strain is transformed by taking escherichia coli as an original strain, wherein the transformation comprises reinforcement of pntAB gene and knockout of iclR gene. Through fermentation culture of the recombinant strain provided by the invention, the L-threonine yield can reach 15.3g/L, the sugar acid conversion rate can reach 18.5%, plasmid burden is avoided, and the construction method is simple.

Description

The method of recombinant bacterial strain and preparation method thereof and production L-Threonine
Technical field
The present invention relates to microbial technology field, more particularly to recombinant bacterial strain and preparation method thereof and production L-Threonine Method.
Background technology
L-Threonine (Thr) is one of necessary 8 kinds of aminoacid of humans and animals growth, and it is widely used in feedstuff, food Product add and prepared by medicine auxiliary material etc..Threonine is a kind of important nutrition enhancer, can be with intensified cereal, cake, breast Product, there is alleviation human-body fatigue, the effect of enhancing development as tryptophan.Pharmaceutically, due in the structure of threonine Containing hydroxyl, there is water holding ability to human body skin, combined with oligonucleotide chain, to protecting cell membrane to play an important role, in vivo can Promote phospholipid synthesis and fatty acid oxidation.Its preparation has the medicinal efficiency of promotion human development's anti-fatty liver, is aminoacids complex A composition in transfusion.Meanwhile, threonine is again the raw material of the antibiotic-monobactam for manufacturing the efficient low allergy of a class.
At present L-Threonine is mainly produced by fermentable, and various bacteria can be used for L-Threonine production, such as large intestine The mutant that the wild type induction of bacillus, corynebacterium, Serratia etc. is obtained is used as production bacterial strain.Instantiation includes (Japanese patent application is public for various auxotrophs such as mutants resistant to amino acid analogues or methionine, lysine, isoleucine The number of opening 224684/83；Korean Patent Publication No 8022/87).However, classic mutagenesis breeding causes bacterium due to random mutation Strain poor growth and the more by-product of generation, are difficult to obtain superior strain.
With being continuously increased for global threonine requirement amount, the structure of high yield L-threonine-producing strain and transform particularly important. In the Chinese patent CN03811059.8 of CJ Co., Ltd. of Korea application in 2003, using escherichia coli, by lacking threonine The 39bp sequences of the -56th to -18 of operon sequence, strengthen threonine synthesis key gene thrABC expression, threonine life Force of labor improves 22%.In the Chinese patent 201610119758.6 of Flos Mume group application in 2016, by strengthening thrA*BC, striking Except tdh obtains MHZ-0213-3 bacterial strains, the bacterial strain production amount of threonine is 4.2g/L, conversion ratio is about 8.9%, and plasmid-free is negative Load.
The content of the invention
In view of this, the invention provides the method for recombinant bacterial strain and preparation method thereof and production L-Threonine.The present invention The fermented culture of recombinant bacterial strain of offer, produces the amount of threonine up to 15.3g/L, and saccharic acid conversion ratio is up to 18.5%.
In order to realize foregoing invention purpose, the present invention provides technical scheme below：
The invention provides a kind of recombinant bacterial strain, is transformed by starting strain of escherichia coli, its transformation includes：Reinforcing PntAB genes and knockout iclR genes.
Preferably, reinforcing pntAB genes are：The natural promoter of pntAB genes is replaced by into Ptac promoteres.
In the embodiment that the present invention is provided, starting strain is escherichia coli MHZ-0213-3.
In the embodiment that the present invention is provided, the deposit number of recombinant bacterial strain is CGMCC No.13402.
Present invention also offers a kind of construction method of recombinant bacterial strain, including：Changed by starting strain of escherichia coli Make, its transformation includes：Reinforcing pntAB genes and knockout iclR genes.
Preferably, reinforcing pntAB genes are：The natural promoter of pntAB genes is replaced by into Ptac promoteres.
In the embodiment that the present invention is provided, starting strain is escherichia coli MHZ-0213-3.
Preferably, transformation is carried out using CRISPR-Cas9 gene editings technology.
Present invention also offers a kind of method for producing L-Threonine, is built using the recombinant bacterial strain or the present invention of the present invention It is fermentation strain that method builds the recombinant bacterial strain for obtaining.
Preferably, the method for production L-Threonine is：Recombinant bacterial strain is activated, being inoculated into seed culture medium is carried out Seed culture, being then seeded into fermentation medium carries out fermentation culture.
Preferably, seed culture medium includes：
Preferably, fermentation medium includes：
Preferably, the temperature of activation is 37 DEG C, the time is 18～24h.
Preferably, the temperature of seed culture is 37 DEG C, rotating speed is 90rpm, and the time is 4.5～5.5h, OD650Control exists 2。
Preferably, the temperature of fermentation culture is 37 DEG C.
The invention provides the method for recombinant bacterial strain and preparation method thereof and production L-Threonine.The recombinant bacterial strain is with large intestine Bacillus is transformed for starting strain, and its transformation includes：Reinforcing pntAB genes and knockout iclR genes.The present invention at least has One of following beneficial effect：
1st, the natural promoter of pntAB genes is replaced by Ptac promoteres by the present invention, knocks out iclR genes, is recombinated Bacterial strain, fermented culture produces the amount of threonine up to 15.3g/L, and saccharic acid conversion ratio is up to 18.5%.
2nd, the recombinant bacterial strain plasmid-free burden that the present invention is obtained, construction method is easy.
3rd, the present invention builds the recombinant bacterial strain for obtaining no coupling product acetic acid during fermentation culture and is formed.
Biological deposits explanation
Classification And Nomenclature：Colon bacillus, Escherichia coli were deposited in Chinese micro- life on November 30th, 2016 Thing culture presevation administration committee common micro-organisms center (CGMCC), collection address is BeiChen West Road, Chaoyang District, BeiJing City 1 Number institute 3, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.13402.
Specific embodiment
The invention discloses the method for recombinant bacterial strain and preparation method thereof and production L-Threonine, those skilled in the art can To use for reference present disclosure, technological parameter realization is suitably modified.Specifically, all similar replacements and change are to this It is it will be apparent that they are considered as being included in the present invention for art personnel.The method of the present invention and application are It is described by preferred embodiment, related personnel substantially can be in without departing from present invention, spirit and scope to herein Described methods and applications are modified or suitably the technology of the present invention is realized and applied to change with combining.
It is of the invention with wild type W3110 as starting strain according to the metabolic pathway of L-Threonine in escherichia coli, in its base Because carrying out related transformation in group, key gene in the metabolic pathway of threonine end is modified, such as：Release aspartokinase Feedback suppression, reinforcing thrA*BC enhance L-Threonine end route of synthesis, knock out tdh blocked L-Threonine degraded produce The formation of thing glycine, the engineering strain of acquisition is named as MHZ-0213-3, and it can effectively accumulate threonine, and need not add Plus aminoacid and plasmid-free burden.
The present invention carries out related retrofit work on the basis of MHZ-0213-3 bacterial strains.
The biosynthesiss of many industrial useful compounds are required for cofactor NADPH, such as escherichia coli (E.coli) Biosynthesiss lycopene (Alper, H. etc., (2005) Metab.Eng.7,155-164), Corynebacterium glutamicum (Corynebacterium glutamicum) high yield lysine (Ikeda.J Ind Microbiol Biotechnol.2006, 33:610-615).Metabolite accumulation of the threonine in escherichia coli needs also exist for enough reducing power supplies.
The pyridine nucleotide transhydrogenase of known pntAB coding using electro chemical proton gradient as driving force, by general NADH is oxidized to NAD+And NADP+It is reduced to NADPH, it can be understood as can improve the NADPH of intracellular by strengthening pntAB Level.
IclR genes in glyoxylate pathway, its encoding transcription regulatory protein IclR can negative regulator encoding isocitrate lyase With the expression of the aceBA genes of malate synthase.
The construction work of escherichia coli W3110 mutant strains, make use of CRISPR- in the document of the reports such as Jiang Y Cas9 methods (Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Jiang Y, Chen B, et al.Appl.Environ Microbiol, 2015) are carried out on genome of E.coli Genetic manipulation.
In following examples, the kanamycin final concentration of 50 μ g/ml in the medium, the spectinomycin is in training Final concentration of 50 μ g/ml in foster base.
In following examples, agents useful for same can be buied by market.The high conversion threonine production bacterium that the present invention is provided The parental strain planted is W3110, belongs to Escherichia (Escherichia).Primer sequence see the table below used in embodiment.
The primer sequence of table 1
Title SEQ ID No. Sequence
gRNApntABup-f1 1 aatactagtgaagggaatatcatgcgaatgttttagagctagaaatagc
gRNApntABdn-r1 2 cgccccggcacgaatcactattcaaaaaaagcaccgactcgg
pntABup-f1 3 tagtgattcgtgccggggcg
Ptac-pntABup-r1 4 tgattaattgtcaacagctcgatattcccttccatcggtttta
Ptac-pntABdn-f1 5 gagctgttgacaattaatcatcggctcgtataatgtgtggaattgtgagcggataacaa
Ptac-pntABdn-f2 6 attgtgagcggataacaatttcacacaggaaacagcgatgcgaattggcataccaagag
pntABdn-r1 7 aactgcagtcgccatcgagcttagtgcg
pntAB-up 8 tatcacattccttaagccaa
pntAB-dn 9 cccatactttgaacttgttc
gRNAiclRup-f1 10 aatactagtccattagattgcgcaggatagttttagagctagaaatagc
gRNAiclRdn-r1 11 tttaaacatcaaaccacttaattatttcaaaaaaagcaccgactcgg
iclRup-f1 12 ataattaagtggtttgatgttt
iclRup-r1 13 gactgtcatggtcgcacccgcagcgtgtattttcgatgag
iclRdn-f1 14 ctcatcgaaaatacacgctgcgggtgcgaccatgacagtc
iclRdn-r1 15 aactgcagtcgataactctggatcatgg
iclR-up 16 aatttaatatgattacaact
iclR-dn 17 cgatcacttccggtttactg
The Gene Name being related in the present invention is explained as follows：
thrA：Aspartokinase and I-homoserine dehydrogenase；
thrB：Homoserine kinase；
thrC：Threonine synthetase；
tdh：L-Threonine dehydrogenase；
pntAB：Pyridine nucleotide transhydrogenase；
iclR：Isocitrate lyase/malate synthase negative growth factor；
SgRNA (single guide RNA are referred to as)：It is that CRISPR gene knockouts knock in ingredient important in system, The previous guide RNA for finding, by two parts-tracRNA and crRNA is constituted, and after two parts amalgamation and expression, i.e., sgRNA also can The function of guide is exercised well, and with Cas9 protein binding, guiding Cas9 enzyme target gene groups DNA are sheared；
Trc (Ptac) promoter：It is the split promoter of trp promoteres and lac promoteres, equally has higher than trp Transcriptional efficiency and by lacI repressor proteins regulate and control strong promoter characteristic.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1：Prepare the bacterial strain MHZ-0213-3 (Ptac-pntAB) of reinforcing pntAB genes
(1) pTargetT-Ptac-pntAB plasmids are built
Primer pair gRNApntABup-f1/gRNApntABdn-r1 goes out pntAB's with pTargetT plasmids as template amplification SgRNA fragments, primer pair pntABup-f1/Ptac-pntABup-r1 goes out Ptac-pntAB with W3110 genomes as template amplification Left half section, it is right that primer pair Ptac-pntABdn-f1/pntABdn-r1 goes out Ptac-pntAB with W3110 genomes as template amplification Half section 1, primer pair Ptac-pntABdn-f2/pntABdn-r1 goes out Ptac- for right half section 1 with Ptac-pntAB as template amplification PntAB is right half section 2,3 fragments is carried out with OE-PCR amplifications and obtains gRNA-Ptac-pntAB fragments (total length 0.9kb), with SpeI/PstI carries out enzyme action, in being inserted into the identical restriction enzyme site of plasmid pTargetT, obtains pTargetT-Ptac-pntAB Plasmid.
(2) prepared by competent cell and electricity turns pTargetT-Ptac-pntAB plasmids
PCas plasmids are transformed into in MHZ-0213-3 competent cells (method for transformation and the equal reference of competence preparation method 《Molecular cloning III》), picking MHZ-0213-3 (pCas) single bacterium colonies are Arabic containing kanamycin and final concentration of 10mM in 5ml In the LB test tubes of sugar, 30 DEG C of 200r/min are cultivated to OD650Turn competent cell (competence preparation method is joined to prepare electricity after 0.4 According to《Molecular cloning III》).
PTargetT-Ptac-pntAB plasmid electricity is proceeded to into (electricity conversion bar in MHZ-0213-3 (pCas) competent cell Part：The μ F of 2.5kV, 200 Ω, 25), to coat on the LB flat boards containing spectinomycin and kanamycin, 30 DEG C of quiescent cultures are to single bacterium Fall visible.
(3) recombination checking：(positive fragment is about to carry out bacterium colony PCR checkings using pntAB-up/pntAB-dn primers 0.9kb), correct laggard step sequence verification is verified.
(4) build related plasmids to lose：It is dense containing kanamycin and end that the correct single bacterium colony of picking sequence verification is inoculated in 5ml In spending the LB test tubes for 0.5mMIPTG, line after 30 DEG C of incubated overnight on the LB flat boards containing kanamycin, 30 DEG C of incubated overnight Afterwards picking single bacterium colony is to putting on the LB flat boards containing kanamycin, spectinomycin LB flat boards and containing only kanamycin, if containing card Can not grow on that mycin, the LB flat boards of spectinomycin, grow on the LB flat boards of kanamycin, show pTargetT-Ptac- PntAB plasmids are lost.The positive bacterium colony of picking pTargetT-Ptac-pntAB plasmid loss, in being inoculated in nonreactive LB test tube, Line on LB flat boards after 42 DEG C of culture 8h, 37 DEG C of incubated overnight；Next day picking single bacterium colony is to putting in the flat boards of LB containing kanamycin On nonreactive LB flat board；If can not grow on the LB flat boards containing kanamycin, grow on nonreactive LB flat board, show pCas matter Grain is lost, and obtains MHZ-0213-3 (Ptac-pntAB) bacterial strain.
Embodiment 2：Prepare the bacterial strain MHZ-0213-3 (Δ iclR) for knocking out iclR genes
(1) pTargetT-iclR plasmids are built
Primer pair gRNA-iclRup-f1/gRNA-iclRdn-r1 goes out iclR's with pTargetT plasmids as template amplification SgRNA fragments；Primer pair iclRup-f1/iclRup-r1 goes out iclR and knocks out half section of a left side with W3110 genomes as template amplification；Draw Thing goes out iclR and knocks out half section of the right side with W3110 genomes to iclRdn-f1/iclRdn-r1 as template amplification；To three fragments OE- PCR amplifications obtain gRNA-iclR and knock out fragment (total length 1.0kb), and with speI/PstI enzyme action is carried out, and are inserted into plasmid In the identical restriction enzyme site of pTargetT, pTargetT-iclR plasmids are obtained.
(2) prepared by competent cell and electricity turns pTargetT-iclR plasmids
PCas plasmids are transformed into in MHZ-0213-3 competent cells (method for transformation and the equal reference of competence preparation method 《Molecular cloning III》), picking MHZ-0213-3 (pCas) single bacterium colonies are Arabic containing kanamycin and final concentration of 10mM in 5ml In the LB test tubes of sugar, 30 DEG C of 200r/min are cultivated to OD650Turn competent cell (competence preparation method is joined to prepare electricity after 0.4 According to《Molecular cloning III》).
PTargetT-iclR plasmid electricity is proceeded to into (electric conversion condition in MHZ-0213-3 (pCas) competent cell： The μ F of 2.5kV, 200 Ω, 25), to coat on the LB flat boards containing spectinomycin and kanamycin, 30 DEG C of quiescent cultures can to single bacterium colony See.
(3) recombination checking：Bacterium colony PCR checkings (positive fragment about 1.0kb) are carried out using iclR-up/iclR-dn primers, The correct laggard step sequence verification of checking.
(4) build related plasmids to lose：PTargetT-iclR, pCas plasmid loss method obtains MHZ- with embodiment 1 0213-3 (Δ iclR) bacterial strain.
Embodiment 3：Prepare the bacterial strain MHZ-0213-3 (Ptac-pntAB, Δ iclR) for knocking out iclR genes
MHZ-0213-4 (pCas) bacterial strain obtained in embodiment 1 is prepared into into electricity and turns competent cell, then electricity consumption turns The plasmid pTargetT-iclR that change method obtains embodiment 2 proceeds to competent cell (electric conversion condition：2.5kV, 200 Ω, 25 μ F), authentication method obtains MHZ-0213-3 (Ptac-pntAB, Δ iclR) bacterial strain with embodiment 2.
The product threonine genetic modification bacterial strain that embodiment 1-3 is obtained is as shown in table 2 below.
The genetic engineering bacterium that the present invention of table 2 builds
Embodiment 4：Produce the checking of L-Threonine genetic engineering bacterium shake flask fermentation
Bacterial strain MHZ-0213-3, MHZ-0213-4, MHZ-0214-1, MHZ-0214-2 are taken from cryopreservation tube, totally 4 plants, The flat lining out activation of LB, 37 DEG C of culture 18-24h；Thalline is scraped into next ring from flat board, is inoculated into equipped with 50mL seed culture In the shaking flask of base (being shown in Table 2), 37 DEG C, rotating speed 90rpm is cultivated about 5 hours, OD650Control is 2；2mL seed liquor is transferred to and is contained In the shaking flask of 20mL fermentation medium (being shown in Table 3), 37 DEG C of reciprocal shaker, 100rpm fermentation culture exhausts up to residual sugar, and fermentation is eventually Test sample product OD650, and L-Threonine content is determined with the method for HPLC, and the sugared situation of consumption is determined with bio-sensing instrument method, produce acid And conversion results are shown in Table 4.
The seed culture medium of table 3 (g/L)
Composition Concentration
Glucose 25
Semen Maydis pulp 25
Soybean meal hydrolysate 7.7
Yeast extract 2.5
KH2PO4 1.4
Magnesium sulfate heptahydrate 0.5
FeSO4、MnSO4 20mg/L
pH 7.0
The fermentation medium of table 4 (g/L)
Composition Concentration
Glycerol 30
Semen Maydis pulp 6
Soybean meal hydrolysate 7.7
Magnesium sulfate heptahydrate 0.5
KH2PO4 1.0
Aspartic acid 10
FeSO4、MnSO4 30mg/L
Biotin 50μg
Thiamine 500μg
pH 7.2
Table 5 produces the threonine genetic engineering bacterium productivity and compares
As shown in Table 5, the bacterium MHZ-0213-3 conversion ratios that set out are 8.8%, and strengthen the transformation bacterium MHZ-0213-4 of pntAB Conversion ratio 9.5%, improves 0.7 percentage point；Knock out conversion ratio after the iclR genes of the bacterium that sets out to improve to 12.2%, improve 2.7 percentage point；For the transformation bacterial strain MHZ-0214-2 that reinforcing pntAB knocks out iclR produces threonine 15.3g/L, conversion simultaneously Rate 18.5%, conversion ratio improves 9.5 percentage points, with significant effect；Illustrating to strengthen while reducing power is supplied strengthens There is glyoxylate pathway collaboration to strengthen the effect that threonine conversion ratio is lifted.Escherichia coli of the present invention, plasmid-free is born Load, construction method is easy and L-Threonine yield is higher than control strain, shows that it can apply to L-Threonine and ferments and follow-up Research.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.
SEQUENCE LISTING
<110>Langfang Flos Mume biotechnology development corporation, Ltd.
<120>The method of recombinant bacterial strain and preparation method thereof and production L-Threonine
<130> MP1623788
<160> 17
<170> PatentIn version 3.3
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<211> 22
<212> DNA
<213>Artificial sequence
<400> 12
ataattaagt ggtttgatgt tt 22
<210> 13
<211> 40
<212> DNA
<213>Artificial sequence
<400> 13
gactgtcatg gtcgcacccg cagcgtgtat tttcgatgag 40
<210> 14
<211> 40
<212> DNA
<213>Artificial sequence
<400> 14
ctcatcgaaa atacacgctg cgggtgcgac catgacagtc 40
<210> 15
<211> 28
<212> DNA
<213>Artificial sequence
<400> 15
aactgcagtc gataactctg gatcatgg 28
<210> 16
<211> 20
<212> DNA
<213>Artificial sequence
<400> 16
aatttaatat gattacaact 20
<210> 17
<211> 20
<212> DNA
<213>Artificial sequence
<400> 17
cgatcacttc cggtttactg 20

Claims (10)

1. a kind of recombinant bacterial strain, it is characterised in that transformed by starting strain of escherichia coli, its transformation includes：Reinforcing PntAB genes and knockout iclR genes.
2. recombinant bacterial strain according to claim 1, it is characterised in that the reinforcing pntAB genes are：By pntAB genes Natural promoter be replaced by Ptac promoteres.
3. recombinant bacterial strain according to claim 1 and 2, it is characterised in that the starting strain is escherichia coli MHZ- 0213-3。
4. recombinant bacterial strain according to any one of claim 1 to 3, it is characterised in that the preservation of the recombinant bacterial strain is compiled Number be CGMCC No.13402.
5. a kind of construction method of recombinant bacterial strain, it is characterised in that include：Transformed by starting strain of escherichia coli, its Transformation includes：Reinforcing pntAB genes and knockout iclR genes.
6. construction method according to claim 5, it is characterised in that the transformation adopts CRISPR-Cas9 gene editings Technology is carried out.
7. a kind of method for producing L-Threonine, it is characterised in that using the restructuring as any one of Claims 1-4 It is fermentation strain that bacterial strain or the construction method as described in claim 5 or 6 build the recombinant bacterial strain for obtaining.
8. method according to claim 7, it is characterised in that the method for the production L-Threonine is：By recombinant bacterial strain Activated, being inoculated into seed culture medium carries out seed culture, being then seeded into fermentation medium carries out fermentation culture.
9. method according to claim 8, it is characterised in that the seed culture medium includes：
The fermentation medium includes：
10. method according to claim 8 or claim 9, it is characterised in that the temperature of the activation is 37 DEG C, the time is 18～ 24h；
The temperature of the seed culture is 37 DEG C, and rotating speed is 90rpm, and the time is 4.5～5.5h, OD650Control is 2；
The temperature of the fermentation culture is 37 DEG C.
CN201611248603.9A 2016-12-29 2016-12-29 Recombinant strain and preparation method thereof and method for producing L-threonine Pending CN106591209A (en)

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