CN103642766A - Protein, DNA molecule, conversion host containing DNA and method for production of L-valine by utilization of conversion host - Google Patents
Protein, DNA molecule, conversion host containing DNA and method for production of L-valine by utilization of conversion host Download PDFInfo
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Abstract
The invention relates to the biotechnology field, and especially relates to a protein, a DNA molecule, a conversion host containing the DNA and a method for production of L-valine by utilization of the conversion host. A coding gene leuDH (the nucleotide sequence is shown in SEQ ID No.2) of a leucine dehydrogenase with an auxiliary factor of NADH is converted and introduced into corynebacteria, and genetically engineered bacterium with changed redox equilibrium in cells are obtained. The bacterial strain is cultured and can be used for production of L-valine, and the yield of L-valine is raised remarkably.
Description
Technical field
The present invention relates to biological technical field, particularly the DNA molecular of albumen, this albumen of encoding, contain this DNA molecular this conversion of conversion host host for the production of the method for Valine.
Background technology
Valine (L-valine), chemical name is L-valine, molecular formula is C
5h
11nO
2, relative molecular mass is 117.15.Be white in color crystallization or crystalline powder, odorless, bitter, solubleness in water: 25 ℃ is 88.5g/L, and 50 ℃ is 96.2g/L, is insoluble to cold ethanol, ether, acetone.Iso-electric point is 5.96,315 ℃ of fusing points.Valine (L-Val) is one of eight kinds of indispensable amino acids of human body, again one of three kinds of branched-chain amino acids (comprising α-amino-isovaleric acid, leucine, Isoleucine), because of its special structure and function, in human life's metabolism, there is the status of particularly important.Can be widely used in medicine industry, foodstuffs industry and fodder industry etc.In medicine industry, the main component of amino acid transfusion, comprehensive amino acid preparation can be made, liver failure, central nervous system function disorder can be treated.As lacked, can cause neurological disorder, stasi, weight loss, anaemia etc.In foodstuffs industry, can be used as foodstuff additive, liquid and flavour agent etc. are augmented in nutrition.In metric system cake, add α-amino-isovaleric acid (1g/kg), product has sesame fragrant, for bread, also can improve local flavor.Valine also can be used as amino acid energy beverage and sports drink, has the muscle of formation, strengthens liver function, alleviates the effects such as muscle fatigue.In fodder industry, the mammary tissue lactation of animal is had to important promoter action.Use it in chicken feed, can improve the immunological competence of chick to newcastle disease virus.And Valine is a kind of limiting amino acid in animal-feed, so Valine can be used as the deficiency that fodder additives improves aminoacids content in animal diets.
The production method of Valine has three kinds: extraction method, chemical synthesis, microbe fermentation method.Extraction method and chemical synthesis, because raw material sources are restricted, production cost is high, contaminate environment, are difficult to realize suitability for industrialized production.Production by Microorganism Fermentation Valine has that raw materials cost is low, reaction conditions is gentle, easily realize the advantages such as scale operation, is to produce at present the topmost method of Valine.By the seed selection of bacterial strain, to remove the feedback inhibition in metabolism adjusting and to check, reach the object of excess accumulation Valine, be microbe fermentation method industrial application means the most widely.
Rod bacillus is for the production of L-amino acid whose representative microorganism, particularly Corynebacterium glutamicum (Corynebacterium glutamicum), Beijing corynebacterium (Corynebacterium pekinense) and brevibacterium flavum (Breviabacterium flavum).Yet the output that existing strain fermentation is produced Valine is lower, cannot meet the need of market.
In order to improve the Valine throughput of microorganism, can to producing bacterial strain, constantly transform by methods such as traditional mutagenesis and metabolic engineerings.Tradition selection by mutation, refer to by specific starting strain being carried out to physics, chemistry or the two merging ground mutagenic treatment, select again auxotroph and/or Amino acid analogue resistant mutant strain, to remove the feedback inhibition in metabolism adjusting or to check effect, thereby reach certain amino acid whose object of excess accumulation.Metabolic engineering breeding adopts genetic engineering technique engineered cells metabolic system to improve efficiency of pcr product or to improve cell performance exactly on to the basis of metabolism network systems analysis.Mainly comprise following means: to the structure of the transformation of original pathways metabolism, novel metabolic pathways, the metabolism critical path of group scale or the identification of target spot.Metabolic engineering by rationality builds amino acid preparation strain, becomes just gradually the main policies of amino acid breeding.
Summary of the invention
In view of this, the invention provides a kind of albumen, this albumen of encoding DNA molecular, contain this DNA molecular this conversion of conversion host host for the production of the method for Valine.Encoding gene leuDH by the leucine dehydrogenase that is NADH by cofactor transforms in introduction rod bacillus, obtain the genetic engineering bacterium that in born of the same parents, redox equilibrium changes, cultivate this bacterial strain and can be used in production Valine, the utmost point has significantly improved the output of Valine.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a kind of leucine dehydrogenase or its function equivalent with the aminoacid sequence as shown in SEQ ID No.1.
The metabolic pathway of synthesizing of α-amino-isovaleric acid, is from dimolecular pyruvic acid, through three intermediate products such as acetylactis, dihydroxyl isovaleric acid and ketone group isovaleric acid, finally forms α-amino-isovaleric acid.And these mesostates respectively catalytic activity by acetohydroxy acid synthetase, Acetohydroxy acid isomeroreductase, dihydroxyacid dehydratase and transaminase produce, as shown in Figure 1.
Acetohydroxy acid synthetase (AHAS) is that first the shared enzyme on Valine route of synthesis is also key enzyme, and 1 molecule acetylactis, the i.e. precursor of α-amino-isovaleric acid are synthesized in catalysis 2 molecule pyruvic acid decarboxylations.AHAS is subject to any feedback inhibition in three kinds of branched-chain amino acids.
Dihydroxy acid reduction isomerase (AHAIR) is second enzyme on Valine route of synthesis, is a tetramer enzyme, and the subunit of the same race that is 53kDa by size forms, and catalysis acetylactis generates 2,3-dihydroxyl isovaleric acid.Reaction comprises isomery and the reduction of alkyl; There is this reaction and also need Mg
2+(activator) and NADPH (hydrogen donor).
Dihydroxyacid dehydratase (DHAD) is the 3rd enzyme on Valine route of synthesis, and the dimer enzyme being comprised of two subunits can catalysis 2, and 3-dihydroxyl isovaleric acid generates 2-ketone group isovaleric acid.
Transaminase (TA) B is last enzyme in α-amino-isovaleric acid building-up process.Transaminase B, Transaminase C and aromatic aminotransferase (gene is respectively ilvE, avtA and tyrB) all have catalytic activity in branched-chain amino acid synthetic, but mainly by three kinds of synthetic final steps of branched-chain amino acid of Transaminase B catalysis, are reacted.
Leucine dehydrogenase (Leucine Dehydrogenase, LDH, EC1.4.1.9) is a kind of NAD
+the oxydo-reductase of dependent form, it is catalysis L-Leu and corresponding ketone acid and the analogue thereof of some branched-chain l-amino acids reaction generation reversibly, as follows:
The leucine dehydrogenase that derives from bacillus megaterium (Bacillus megaterium) ATCC14945 has good thermostability, chemical stability and operational stability.
NADH and NADPH are important coenzyme in cell, most redox reactions of involved in sugar, fat, protein three class substance metabolisms, and NAD/NADH and NADP/NADPH are respectively its corresponding redox couples.The leucine dehydrogenase encoding gene leuDH that derives from bacillus megaterium is imported in excellent bacillus or Flavobacterium, and its coded product be take NADH as coenzyme.The expression of this enzyme can supplement and take the function of the transaminase that NADPH is coenzyme originally as coenzyme with NADH, thereby reduce the consumption to NADPH, reach the effect of the balance that regulates the interior NADH of born of the same parents and NADPH, the result that transforms and express is to make also corresponding being changed of the interior redox equilibrium of born of the same parents.
As preferably, the function equivalent with the leucine dehydrogenase of the aminoacid sequence as shown in SEQ ID No.1 provided by the invention obtains by replacing in the aminoacid sequence shown in SEQ ID No.1, lacking or add one or more amino-acid residues.
The present invention also provides leucine dehydrogenase or its function equivalent with the aminoacid sequence as shown in SEQ ID No.1 for catalysis 2-ketone group isovaleric acid, to generate the application of α-amino-isovaleric acid.
The present invention also provides coding to have the leucine dehydrogenase of aminoacid sequence or the DNA molecular of its function equivalent as shown in SEQ ID No.1.
In some embodiments of the invention, coding provided by the invention has the leucine dehydrogenase of the aminoacid sequence as shown in SEQ ID No.1 or the DNA molecular nucleotide sequence of its function equivalent as shown in SEQ ID No.2.
The present invention also provides coding to have the leucine dehydrogenase of aminoacid sequence or the DNA molecular of its function equivalent as shown in SEQ ID No.1, and the homology of the nucleotide sequence shown in its nucleotide sequence and SEQ ID No.2 is greater than more than 95%.
The present invention also provides coding to have the leucine dehydrogenase of aminoacid sequence as shown in SEQ ID No.1 or the carrier of the DNA molecular of its function equivalent; Coding has the leucine dehydrogenase of aminoacid sequence as shown in SEQ ID No.1 or the DNA molecular of its function equivalent can be greater than more than 95% DNA molecular for the DNA molecular of nucleotide sequence as shown in SEQ ID No.2 or the homology of the nucleotide sequence shown in nucleotide sequence and SEQ ID No.2.
In some embodiments of the invention, coding has the leucine dehydrogenase of aminoacid sequence as shown in SEQ ID No.1 or the carrier of the DNA molecular of its function equivalent is pMX119.
The present invention also provides coding to have the leucine dehydrogenase of aminoacid sequence as shown in SEQ ID No.1 or the conversion host of the DNA molecular of its function equivalent; Coding has the leucine dehydrogenase of aminoacid sequence as shown in SEQ ID No.1 or the DNA molecular of its function equivalent can be greater than more than 95% DNA molecular for the DNA molecular of nucleotide sequence as shown in SEQ ID No.2 or the homology of the nucleotide sequence shown in nucleotide sequence and SEQ ID No.2.
In some embodiments of the invention, coding has the DNA molecular that the leucine dehydrogenase of aminoacid sequence as shown in SEQ ID No.1 or the DNA molecular of its function equivalent can be greater than more than 95% for the homology of the DNA molecular of nucleotide sequence as shown in SEQ ID No.2 or the nucleotide sequence shown in nucleotide sequence and SEQ ID No.2 and imports by the conversion method that shocks by electricity.
In some embodiments of the invention, coding has the DNA molecular that the leucine dehydrogenase of aminoacid sequence as shown in SEQ ID No.1 or the DNA molecular of its function equivalent can be greater than more than 95% for the homology of the DNA molecular of nucleotide sequence as shown in SEQ ID No.2 or the nucleotide sequence shown in nucleotide sequence and SEQ ID No.2 and expresses enhancing.
As preferably, express the mode that strengthens and can be greater than for the homology of the DNA molecular of nucleotide sequence as shown in SEQ ID No.2 or the nucleotide sequence shown in nucleotide sequence and SEQ ID No.2 more than 95% DNA molecular as under the control of strong promoter or increase its copy number for coding has the leucine dehydrogenase of aminoacid sequence as shown in SEQ ID No.1 or the DNA molecular of its function equivalent.
In some embodiments of the invention, transforming host is excellent bacillus or tyrothricin.
In other embodiment of the present invention, transforming host is excellent bacillus, its deposit number for its deposit number be CGMCC No.7041.
The present invention also provides above-mentioned conversion host the purposes for fermentative production Valine.
The present invention also provides the method for above-mentioned conversion host fermentative production Valine, it is characterized in that, gets after described conversion host increases in seed culture medium, is placed in fermention medium and cultivates 50～120h in 28 ℃～37 ℃, obtains.
The invention provides a kind of albumen, the DNA molecular of this albumen of encoding, contain this DNA molecular this conversion of conversion host host for the production of the method for Valine.Encoding gene leuDH by the leucine dehydrogenase that is NADH by cofactor transforms in introduction rod bacillus, obtains the genetic engineering bacterium that in born of the same parents, redox equilibrium changes, and cultivates this bacterial strain and can be used in production Valine.In the fermented liquid of shake flask fermentation, the Valine output of starting strain CGMCC1.299 is 1.2g/L, and the Valine output of excellent bacillus provided by the invention is 3.8g/L, than starting strain output, improves 216%.In the fermented liquid of 50L tank, the Valine output of starting strain CGMCC1.299 is 2.9g/L, and the Valine output of excellent bacillus provided by the invention is 9.1g/L, than starting strain output, improve 213.7%, the utmost point has significantly improved Valine output (P < 0.01).Comprehensive above-mentioned fermentation test result, the excellent bacillus that deposit number provided by the invention the is CGMCCNo.7041 all utmost point has significantly improved Valine output (P < 0.01).
Biological preservation explanation
Classification And Nomenclature: Beijing corynebacterium Corynebacterium pekinense is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 26th, 2012, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number for its deposit number be CGMCC No.7041.
accompanying drawing explanation
Fig. 1 shows Valine biosynthesizing route map;
Fig. 2 shows the schematic diagram of recombinant plasmid pXMJ19-leuDH.
Embodiment
The invention discloses a kind of albumen, the DNA molecular of this albumen of encoding, contain this DNA molecular this conversion of conversion host host for the production of the method for Valine.Those skilled in the art can use for reference content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can change methods and applications as herein described or suitably change and combination within not departing from content of the present invention, spirit and scope, realizes and apply the technology of the present invention.
The DNA molecular of albumen provided by the invention, this albumen of encoding, this conversion of conversion host host of containing this DNA molecular all can be buied by market for the production of agents useful for same in the method for Valine.Wherein, the deposit number of starting strain is CGMCC1.299.
Below in conjunction with embodiment, further set forth the present invention:
The structure of embodiment 1 expression plasmid pXMJ19-leuDH
1. the genomic dna of bacillus megaterium Bacillus megaterium ATCC14945 of take is template, with the primer pair that leuF and leuR form, carries out pcr amplification, obtains pcr amplification product:
LeuF:5-' G
aAGCTTaTGAAAATTTTTGACTAT-3 ' (underscore is that HindIII enzyme is cut recognition site)
LeuR:5-' C
gAATTCtTAGCGCGTATGACGTCTAC-3 ' (underscore is that EcoRI enzyme is cut recognition site)
2. the PCR product of recycling step 1 link carrier pEASY
tM-T5 (purchased from Beijing Quanshijin Biotechnology Co., Ltd), obtains recombinant plasmid pT5-leuDH, and order-checking obtains leuDH gene order, as shown in SEQ ID No.1;
3. with two kinds of restriction enzymes (HindIII and EcoRI) double digestion plasmid pT5-leuDH and carrier pXMJ19, cut after glue reclaims and obtain respectively leuDH fragment (about 1100bp) and pXMJ19 carrier framework (about 6600bp);
4. two double digestion products of step 3 gained are connected, transform intestinal bacteria, LB culture medium flat plate screening through containing 17 μ g/mL paraxin obtains positive colony, extracts recombinant plasmid pXMJ19-leuDH after amplification cultivation, and its structural representation as shown in Figure 2.
The structure of embodiment 2 engineering bacteria CGMCC1.299/pXMJ19-leuDH
1. prepare the electric shock transformed competence colibacillus cell of excellent bacillus;
2. electric shock transforms: the pXMJ19-leuDH plasmid of 3-8 μ L is joined in competent cell, place 10min on ice; Proceed to 1mm electric shock cup, 1.8 or 2.1kV electric shock 5-7ms;
3. add LB substratum 1mL, 33 ℃, 150rpm are cultivated 1h; After concentrated, coat containing on the brain heart culture medium flat plate of 25 μ g/mL kantlex 33 ℃ of constant temperature culture 36-48h;
4. screening positive clone, carries out PCR evaluation (specific band of the 1.1kb that has an appointment positive) by the primer pair with leuF/leuR forms, and obtains recombinant bacterium.
By recombinant bacterium called after Beijing corynebacterium (Corynebacterium pekinense) MHZ-1010, this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on December 26th, 2012, and deposit number is CGMCC No.7041.
Embodiment 3 deposit number provided by the invention is that the excellent bacillus fermentation of CGMCC No.7041 is produced Valine
1. substratum
Seed activation substratum: yeast extract 1%, peptone 1%, sodium-chlor 0.5%, glucose 0.5%, agar 2%, pH7.2;
Seed culture medium: corn steep liquor 2.5%, glucose 1.0%, ammonium sulfate 0.4%, magnesium sulfate 0.04%, potassium primary phosphate 0.1%, urea 0.1%, CaCO
30.5%, pH7.2;
Fermention medium: corn steep liquor 0.5%, glucose 12.0%, ammonium sulfate 4.0%, magnesium sulfate 0.04%, potassium primary phosphate 0.1%, CaCO
34%, V
h50ug/L, V
b1hCl100 μ g/L, pH7.2;
2. shake flask fermentation is produced Valine
(1) seed culture: picking excellent bacillus provided by the invention inclined-plane seed 1 articulating is to being equipped with in the 500mL triangular flask of 20mL seed culture medium, 33 ℃, 220r/min shaking culture 16-22h;
(2) fermentation culture: 2mL seed liquor is seeded in the 500mL triangular flask that 20mL fermention medium is housed to 33 ℃, 220r/min shaking culture 72h; During 24h, adding IPTG(final concentration is 10 μ mol/L) induction;
(3) get 1mL fermented liquid centrifugal (12000rpm, 2min), collect supernatant liquor, with HPLC test bar bacillus and the Valine content contrasting in fermented liquid, its concentration is as shown in table 1.
The comparative result of Valine content in the fermented liquid of table 1 shake flask fermentation
As shown in data in table 1, the Valine output of starting strain CGMCC1.299 is 1.2g/L, and the Valine output of excellent bacillus provided by the invention is 3.8g/L, than starting strain output, improve 216%, the utmost point has significantly improved Valine output (P < 0.01).
3.50L tank fermentative production Valine
(1) secondary seed is cultivated: picking rod bacillus inclined-plane seed 2-3 articulating is to being equipped with in the 2L triangular flask of 100mL seed culture medium, 33 ℃, the about 12-16h of 220r/min shaking culture; Proceeded to again in the 5L triangular flask containing 1L nutrient solution 33 ℃, 220r/min continuation cultivation 16-20h;
(2) 50L fermentation: 3L seed liquor is seeded in the 50L fermentor tank that 22L fermention medium is housed, cultivates 72h for 31 ℃; In process, with ammoniacal liquor, regulate pH7.0, with the glucose of 80% (m/v), maintaining remaining sugar concentration is 2-3%, and dissolved oxygen is controlled between 10-20% relatively, and it is 10 μ mo l/L that 24h adds IPTG(final concentration constantly) induction;
(3) get 1mL fermented liquid centrifugal (12000rpm, 2min), collect supernatant liquor, HPLC test bar bacillus and the Valine content contrasting in fermented liquid, its concentration is as shown in table 2.
The comparative result of Valine content in the fermented liquid of table 250L tank
As shown in table 2, the Valine output of starting strain CGMCC1.299 is 2.9g/L, and the Valine output of excellent bacillus provided by the invention is 9.1g/L, than starting strain output, improve 213.7%, the utmost point has significantly improved Valine output (P < 0.01).
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (16)
1. leucine dehydrogenase or its function equivalent with the aminoacid sequence as shown in SEQ ID No.1.
2. function equivalent according to claim 1 obtains by replacing in the aminoacid sequence shown in SEQ ID No.1, lacking or add one or more amino-acid residues.
3. leucine dehydrogenase according to claim 1 and 2 or its function equivalent generate the application of α-amino-isovaleric acid for catalysis 2-ketone group isovaleric acid.
4. the DNA molecular of coding leucine dehydrogenase as claimed in claim 1 or 2 or its function equivalent.
5. DNA molecular according to claim 4, is characterized in that, its nucleotide sequence is as shown in SEQ ID No.2.
6. the DNA molecular of coding leucine dehydrogenase as claimed in claim 1 or 2 or its function equivalent, is characterized in that, the homology of the nucleotide sequence shown in its nucleotide sequence and SEQ ID No.2 is greater than more than 95%.
7. the carrier with the DNA molecular as described in claim 5 or 6 any one.
8. carrier according to claim 7, is characterized in that, it is pMX119.
9. the conversion host with the DNA molecular as described in claim 5 or 6 any one.
10. conversion host according to claim 9, is characterized in that, the DNA molecular as described in claim 5 or 6 any one imports by electric shock conversion method.
11. conversion hosts according to claim 9, is characterized in that, the DNA molecular as described in claim 5 or 6 any one is expressed and strengthened.
12. conversion hosts according to claim 11, it is characterized in that, the mode that described expression strengthens be DNA molecular as described in claim 5 or 6 any one as under the control of strong promoter or the copy number of the DNA molecular of increase as described in claim 5 or 6 any one.
13. conversion hosts according to claim 9, is characterized in that, it is excellent bacillus or tyrothricin.
14. conversion hosts according to claim 9, is characterized in that, its deposit number is CGMCC No.7041.
15. according to the conversion host described in claim 9 to 14 any one for the purposes of fermentative production Valine.
16. 1 kinds of excellent bacillus fermentations as described in claim 9 to 14 any one are produced the methods of Valines, it is characterized in that, get described conversions host in seed culture medium, expand numerous after, be placed in fermention medium in 28 ℃～37 ℃ cultivation 50～120h, obtain.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113278641A (en) * | 2020-05-27 | 2021-08-20 | 安徽华恒生物科技股份有限公司 | Recombinant escherichia coli for producing L-valine, construction method and application thereof |
| CN113278655A (en) * | 2020-05-13 | 2021-08-20 | 安徽华恒生物科技股份有限公司 | Recombinant microorganism for producing L-valine and construction method and application thereof |
| WO2021238184A1 (en) * | 2020-05-27 | 2021-12-02 | 安徽华恒生物科技股份有限公司 | Recombinant microorganism for producing l-valine, construction method therefor and use thereof |
| CN113913478A (en) * | 2021-11-26 | 2022-01-11 | 江南大学 | A kind of method of fermenting Brevibacterium flavum to produce L-valine |
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| WO2021238183A1 (en) * | 2020-05-27 | 2021-12-02 | 安徽华恒生物科技股份有限公司 | Recombinant microorganism for producing l-valine, a construction method therefor, and application thereof |
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| CN113913478A (en) * | 2021-11-26 | 2022-01-11 | 江南大学 | A kind of method of fermenting Brevibacterium flavum to produce L-valine |
| CN113913478B (en) * | 2021-11-26 | 2023-06-02 | 江南大学 | Method for producing L-valine by fermenting Brevibacterium flavum |
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