CN106635939A - Genetically engineered bacterium for producing multiple L-amino acids and application - Google Patents

Genetically engineered bacterium for producing multiple L-amino acids and application Download PDF

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CN106635939A
CN106635939A CN201610853074.9A CN201610853074A CN106635939A CN 106635939 A CN106635939 A CN 106635939A CN 201610853074 A CN201610853074 A CN 201610853074A CN 106635939 A CN106635939 A CN 106635939A
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genetic engineering
ncgl2773
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engineering bacterium
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王小元
高云飞
胡晓清
李颜颜
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Jiangnan University
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Abstract

The invention discloses a genetically engineered bacterium for producing multiple L-amino acids and application and belongs to the field of genetic engineering. A knockout carrier of a gene NCgl2773 is established, a host bacterium C.glutamincum ATCC 13869 is converted by adopting an electronic transformation method, and the genetically engineered bacterium WGYF001 is obtained by homologous recombination. Compared with an original strain, a mycolic acid layer disappears and becomes thin and loose, both amino acid synthesis and secretion are improved, and the L-amino acid yield is increased remarkably. The yield of L-glutamic acid, L-aspartic acid and L-threonine can be remarkably improved by adopting the established genetically engineered bacterium WGYF001 and shake flask fermentation and are respectively improved by 34.67 times, 13.05 times and 37.28 times compared with the original strain, meanwhile the yield of L-histidine, L-tyrosine, L-valine, L-methionine, L-phenylalanine, L-isoleucine and L-leucine is efectilvey improved, and yield increases are all 20% or above compared with the original strain.

Description

A kind of genetic engineering bacterium for producing various l-amino acids and application
Technical field
The present invention relates to a kind of genetic engineering bacterium for producing various l-amino acids and application, belong to genetic engineering field.
Background technology
Amino acid refers to the common name of the class organic compound containing amino and carboxyl, feed addictive, food additives, The fields such as medicine, cosmetics and surfactant are widely used.According to statistics, the amino acid starting material production in the whole world in 2006 2,800,000 tons are reached, wherein for about 400,000 tons of the fields such as medicine, health care and cosmetics, accounting for 14.5%；Add for feed Plus about 600,000 tons of agent, account for 21.5%；For about 1,800,000 tons of flavor enhancement and food additives, 64% is accounted for.To 2010, amino acid Yield exceed tens million of tons.
At present the industrial mode of production of amino acid mainly passes through microbe fermentation method, i.e., using microorganism in specific ring Under the conditions of border, add the nutriments such as carbon source, nitrogen source, make microbial growth and obtain purpose amino acid in a large number, it is conventional The microorganism of production amino acid is Escherichia coli, bacillus subtilis, Corynebacterium glutamicum, saccharomycete, mould etc., but above-mentioned Microorganism often can only one to two kind of specific amino acids of high yield, often yield is relatively low or even can not produce for remaining amino acid.
The content of the invention
First purpose of the present invention is to provide a kind of genetic engineering bacterium of product several amino acids, and the genetic engineering bacterium is The Corynebacterium glutamicum of coding polyketide synthase gene NCgl2773 is knocked out.
In one embodiment of the invention, the genetic engineering bacterium for producing several amino acids, its Classification And Nomenclature is paddy Propylhomoserin bar bacterium (Corynebacterium glutamicum) WGYF001, is preserved in Chinese Typical Representative on 22nd in August in 2016 Culture collection, deposit number CCTCC NO:M 2016425, preservation address is Wuhan, China Wuhan University.
The method that second object of the present invention is to provide the genetic engineering bacterium, methods described is will to have knocked out coding to gather The carrier of ketone synthase gene NCgl2773 is proceeded in Corynebacterium glutamicum (C.glutamicum), is obtained by homologous recombination NCgl2773 defective gene engineering bacterias.
In one embodiment of the invention, the carrier nucleosides for having knocked out coding polyketide synthase gene NCgl2773 Acid sequence is as shown in SEQ ID NO.1-NO.4.
In one embodiment of the invention, the carrier for having knocked out coding polyketide synthase gene NCgl2773 is root Build according to following method：(1) with C.glutamicum ATCC13869 genomes as template, amplification NCgl2773 genes are upper and lower Trip fragment, respectively as shown in SEQ ID NO.1 and SEQ ID NO.3, (2) are with pDTW202 as template amplification Kan resistances for its sequence Genetic fragment, (3) are connected said gene fragment with carrier pBlueScript II SK (+)；The pBlueScript II SK (+) sequence is as shown in SEQ ID NO.4.
In one embodiment of the invention, methods described is mainly comprised the following steps：
(1) with C.glutamicum ATCC13869 genomes as template, it is with 2773U-F/R and 2773D-F/R respectively Primer expands NCgl2773 gene upstream and downstream fragments 2773-U and 2773-D of each 1000bp；With pDTW202 (Jinyu Hu, Yanzhen Tan, Yanyan Li, Xiaoqing Hu, Daqing Xu, Xiaoyuan Wang, 2013.Construction and application ofan efficient multiple-gene-deletion system in Corynebacterium glutamicum.) it is template, Kan-lox-F/R is that primer expands Kan resistance gene fragments；2773- , with XhoI, BamHI digestion, with EcoRI, XbaI enzyme cutting, with BamHI, EcoRI digestion, three fragments are together for Kan fragments for 2773-D for U PBlueScript II SK (+) with XhoI and XbaI enzyme cutting is connected into, the plasmid i.e. NCgl2773 knockout carriers for completing are built, It is named as pYFW-1 (Fig. 1)；
(2) NCgl2773 knockout carriers pYFW-1 is converted into Host Strains, NCgl2773 genes is obtained by homologous recombination and is lacked Swaged expressive host bacterium.
Third object of the present invention is to provide the method that the genetic engineering bacterium produces l-amino acid, methods described be with Brain heart infusion broth culture medium, 30 DEG C, 200～250r/min cultivates 10～90h.
In one embodiment of the invention, the brain heart infusion broth culture medium contains 37g/L brain heart infusions.
Fourth object of the present invention is to provide application of the genetic engineering bacterium in the product containing amino acid is prepared.
In one embodiment of the invention, the application includes preparing feed addictive, food additives, health care The fields such as product, pharmaceutical preparation, cosmetics, surfactant.
Beneficial effect：The genetic engineering bacterium of the present invention does not need external source addition biotin and surfactant in fermentation, Production cost is saved, beneficial to industrialized production.Compared with the bacterium that sets out, mycomycete acid layer disappears, and becomes thin and loose, l-amino acid Synthesis and secretion are improved, and significantly improve l-amino acid yield.The genetic engineering bacterium WGYF001 that the present invention builds passes through Shake flask fermentation is remarkably improved the yield of Pidolidone, L-Aspartic acid, L-threonine, compared with the bacterium that sets out, is respectively increased 34.67 times, 13.05 times, 37.28 times, at the same effectively increase L-Histidine, TYR, Valine, METHIONINE, The yield of L-phenylalanine, ILE and L-Leu, compared with the bacterium that sets out, yield amplification is more than 20%.
Biomaterial preservation
Corynebacterium glutamicum (Corynebacterium glutamicum) WGYF001, in the preservation on the 22nd of August in 2016 In China typical culture collection center, deposit number CCTCC NO:M 2016425, preservation address is that Wuhan, China Wuhan is big Learn.
Description of the drawings
Fig. 1 is the structure of NCgl2773 gene knockout carrier pYFW-1；
Fig. 2 is the genetic engineering bacterium WGYF001 shake flask fermentation generation of L-glutamic acid contents of the present invention；
Fig. 3 is that the genetic engineering bacterium WGYF001 shake flask fermentations of the present invention produce L-Aspartic acid content；
Fig. 4 is that the genetic engineering bacterium WGYF001 shake flask fermentations of the present invention produce L-threonine content；
Fig. 5 is that the genetic engineering bacterium WGYF001 shake flask fermentations of the present invention produce L-Histidine content；
Fig. 6 is that the genetic engineering bacterium WGYF001 shake flask fermentations of the present invention produce TYR content；
Fig. 7 is that the genetic engineering bacterium WGYF001 shake flask fermentations of the present invention produce Valine content；
Fig. 8 is that the genetic engineering bacterium WGYF001 shake flask fermentations of the present invention produce METHIONINE content；
Fig. 9 is that the genetic engineering bacterium WGYF001 shake flask fermentations of the present invention produce L-phenylalanine content；
Figure 10 is that the genetic engineering bacterium WGYF001 shake flask fermentations of the present invention produce ILE content；
Figure 11 is that the genetic engineering bacterium WGYF001 shake flask fermentations of the present invention produce the bright content of glutamic acid of L-；
Figure 12 is the genetic engineering bacterium WGYF001 intracellular L-Aspartic acid contents of the present invention；
Figure 13 is the genetic engineering bacterium WGYF001 intracellular Pidolidone contents of the present invention.
Specific embodiment
Primer
Primer according to the present invention is as shown in table 1.
Table 1 is the present invention relates to primer
Culture medium
Electricity turns competence culture medium (g/L)：Peptone 10, yeast extract 5, NaCl 10, glycine 30, Tween801, Deionized water is prepared, natural pH；
Electricity turns recovery media (g/L)：Peptone 5, yeast extract 2.5, NaCl 5, D-glucitol 91, brain heart infusion 18.5, deionized water is prepared, natural pH；
Brain heart infusion broth culture medium (BHIS culture mediums) is (g/L)：Brain heart infusion 37, D-glucitol 91, deionized water is matched somebody with somebody System, natural pH, solid-state adds 15 agar powders；
Brain heart infusion broth fermentation medium (BHI culture mediums) is (g/L)：Brain heart infusion 37, deionized water is prepared, natural pH。
HPLC detects amino acid
Sample pretreatment：The amino acid in zymotic fluid is detected using HPLC OPA (OPA) pre-column derivatization method. Zymotic fluid is centrifuged into 5min in 12000r/min, is suitably diluted with 10% trichloroacetic acid, make the final concentration of 100- of l-amino acid 500mg/L, 4 DEG C of placement 12h, 12000r/min centrifugation 20min, 0.22 μm of water phase pin type filter is filtered.
Testing conditions：Chromatographic column：Thermo Hypersil ODS-24.6*250mm (5 μm), column temperature：40℃.Mobile phase： Water phase A：Then plus the μ L of triethylamine 200 anhydrous sodium acetate 3.01g, is first dissolved in water, and, tetrahydrochysene is barked the 5mL that mutters, ddH2O is settled to 1L, 5% acetic acid is adjusted to pH7.2；Organic phase B：Anhydrous sodium acetate 3.01g, ddH2O constant volumes be 200mL, 5% acetic acid adjust to PH7.2, then adds again 400mL methyl alcohol (chromatographically pure) and 400mL second eyeballs (chromatographically pure), and flow velocity is 1mL/min.Sample is run before pillar Column front derivation need to be carried out, the derivative sample for completing carries out gradient elution into chromatographic column, and elution program is as follows：
The structure of the NCgl2773 gene knockout carrier pYFW-1 of embodiment 1
With C.glutamicum ATCC13869 genomes as template, respectively with 2773U-F/R and 2773D-F/R as primer Expand NCgl2773 gene upstream and downstream fragments 2773-U and 2773-D of each 1000bp, its sequence respectively such as SEQ ID NO.1 and Shown in SEQ ID NO.2；With pDTW-202 (Jinyu Hu, Yanzhen Tan, Yanyan Li, Xiaoqing Hu, Daqing Xu, Xiaoyuan Wang, 2013.Construction and application of an efficient multiple- Build in gene-deletion system in Corynebacterium glutamicum) it is template, Kan-lox-F/R is Primer expands Kan resistance gene fragments, and its sequence is as shown in SEQ ID NO.3；2773-U is with XhoI, BamHI digestion, 2773-D With EcoRI, XbaI enzyme cutting, with BamHI, EcoRI digestion, three fragments are connected into together the sequence with XhoI and XbaI enzyme cutting to Kan fragments PBlueScript II SK (+) of the row as shown in SEQ ID NO.4, builds the plasmid i.e. NCgl2773 knockout carriers for completing, life Entitled pYFW-1 (Fig. 1)
The structure of the NCgl2773 gene defection type expressive host bacterium of embodiment 2
By pYFW-1 electricity conversion Corynebacterium glutamicums, by homologous recombination, Kan resistance screenings, NCgl2773 genes are obtained Transformant is knocked out, then is isolated and purified by transformant, picking positive transformant, carry out bacterium colony PCR and gene sequencing checking, finally Obtain deficiency expressive host bacterium.
The conversion ratio of embodiment 3
The competent cell of ATCC13869 and WGYF001 is prepared respectively, while electricity conversion plasmid pDXW-10 (Daqin Xu, Yanzhen Tan, Feng Shi, Xiaoyuan Wang, 2010.An improved constitutive shuttle vector constructed for metabolic engineering research in Corynebacterium Build in glutamicum), shock by electricity condition 1.80KV, 6ms；1.62KV, 6ms；1.44KV, 6ms, conversion fluid coating BHIS Kan Flat board, after 30 DEG C of culture 50h, counts transformant number, the results are shown in Table 1.Can be seen according to result, after knocking out NCgl2773, WGYF001 cell permeabilities change, and in the case where voltage is reduced, conversion quantum count significantly increases, and conversion ratio compares open country Raw bacterium is remarkably reinforced.
Table 1 converts sub-count
The minimal inhibitory concentration of embodiment 4 (MIC)
30 DEG C, 200rpm incubated overnights ATCC13869 and WGYF001 are diluted to respectively new BHI culture mediums, make OD562= 1.96 new orifice plates are taken, from the beginning of A1 holes, 90ul bacterium solutions is added per hole, be then separately added into from left to right dilute through continuous two times The antibiotic solution 10ul for releasing, covers, 30 DEG C, after 200rpm incubated overnights, measures OD562.Positive controls are set：90ul Bacterium solution+10ul PBSs；Negative control group：90ul BHI culture medium+10ul PBSs；Blank control group：100ul PBS.Defining minimal inhibitory concentration (MIC) is：Just meet | ODExperimental group-ODPositive control|≤0.05*ODPositive controlWhen, institute is right The antibiotic concentration in the hole answered, schematic diagram such as table 2, the results are shown in Table 3.Can be seen according to result, WGYF001 drug resistances compare open country Raw bacterium has and declines by a relatively large margin, and chloramphenicol and gentamicin are to act on bacterium rRNA, need, through cell membrane, thus may be used Infer that WGYF001 cell membranes compare wild mushroom more thin and loose.
2 two times of continuity methods of table are illustrated
1 2 3 4 5 6 7 8 9 10 11 12
20 21 22 23 24 25 26 27 28 29 210 211
212 213 214 215 216 217 218 219 220 221 222 223
It is positive It is negative It is blank
Numerical value is the antibiotic extension rate in the hole
The drug resistance of the bacterial strain of table 3
The shake flask fermentation of embodiment 5
Draw from the glycerol tube of preservation strain WGYF001 and take a ring bacterium solution and rule on solid-state BHIS culture medium, 30 DEG C are trained Foster 48h.A ring lawn is scraped from the flat board for having activated using oese and is transferred to the 250mL tri- equipped with 50mL seed culture mediums In the bottle of angle, 30 DEG C, 200r/min culture 10h.Draw certain bacterium solution from cultured seed liquor to be seeded to equipped with 100mL fermentations In the 500mL triangular flasks of culture medium, initial OD=0.1 is made, 30 DEG C, 200rpm is cultivated, until fermentation ends (t=78h).
Intracellular Contents of Amino Acids, before taking remaining thalline is centrifuged, and PBS is washed twice, final concentration 20mg/mL 37 DEG C of process 1h of lysozyme, carry out ultrasonication, crush two-wheeled, determine intracellular amino acid content.Compared with the bacterium that sets out, WGYF001 intracellulars Pidolidone and L-Aspartic acid are only detected in 14h, are not detected at afterwards, and the L- of two plants of bacterium Threonine is not detected in all four time point, illustrates that intracellular threonine almost all is secreted into extracellular.
Contents of Amino Acids result shows in cell free fermentation liquid, and compared with the bacterium that sets out, WGYF001 can by shake flask fermentation Significantly improve the yield of Pidolidone, L-Aspartic acid, L-threonine, compared with the bacterium that sets out, be respectively increased 34.67 times, 13.05 Again, 37.28 times, while effectively increasing L-Histidine, TYR, Valine, METHIONINE, L-phenylalanine, L- The yield of isoleucine and L-Leu, compared with the bacterium that sets out, yield amplification is more than 20%.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention Enclosing should be by being defined that claims are defined.

Claims (8)

1. it is a kind of produce several amino acids genetic engineering bacterium, it is characterised in that the genetic engineering bacterium is to have knocked out coding polyketone The Corynebacterium glutamicum of synthase gene NCgl2773.
2. genetic engineering bacterium according to claim 1, it is characterised in that Classification And Nomenclature is Corynebacterium glutamicum (Corynebacterium glutamicum) WGYF001, is preserved in China typical culture collection on 22nd in August in 2016 Center, deposit number CCTCC NO:M 2016425, preservation address is Wuhan, China Wuhan University.
3. the construction method of genetic engineering bacterium described in claim 1 or 2, it is characterised in that knock out in Corynebacterium glutamicum and encode The gene NCgl2773 of polyketide synthase.
4. method according to claim 3, it is characterised in that described to have knocked out coding polyketide synthase gene NCgl2773's Vector nucleotide sequence is as shown in SEQ ID NO.4.
5. method according to claim 3, it is characterised in that described to have knocked out coding polyketide synthase gene NCgl2773's Carrier is built according to following method：(1) with C.glutamicum ATCC13869 genomes as template, NCgl2773 is expanded Gene upstream and downstream fragment, its sequence respectively as shown in SEQ ID NO.1 and SEQ ID NO.2, by template of pDTW202 expand by (2) Increase Kan resistance gene fragments, its sequence as shown in SEQ ID NO.3 (3) by said gene fragment and carrier pBlueScript II SK (+) connection；PBlueScript II SK (+) sequences are as shown in SEQ ID NO.4.
6. the method that genetic engineering bacterium described in a kind of application claim 1 produces l-amino acid, methods described is with brain heart infusion Broth bouillon, 30 DEG C, 200～250r/min cultivates 10～90h.
7. method according to claim 6, it is characterised in that the brain heart infusion broth culture medium contains brain heart infusion 37g/L。
8. application of the genetic engineering bacterium described in claim 1 in the product containing amino acid is prepared.
CN201610853074.9A 2016-09-26 2016-09-26 Genetically engineered bacterium for producing multiple L-amino acids and application Pending CN106635939A (en)

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CN108330095A (en) * 2018-03-01 2018-07-27 江南大学 It is a kind of accumulation N-acetyl-neuraminate recombination Corynebacterium glutamicum and its application
KR102266232B1 (en) * 2021-01-15 2021-06-17 씨제이제일제당 주식회사 Novel polyketide synthase variant and a method for producing XMP or GMP using the same

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ROLAND GANDE等: "Acyl-CoA Carboxylases (accD2 and accD3), Together with a Unique Polyketide Synthase (Cg-pks), Are Key to Mycolic Acid Biosynthesis in Corynebacterianeae Such as Corynebacterium glutamicum and Mycobacterium tuberculosis", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108330095A (en) * 2018-03-01 2018-07-27 江南大学 It is a kind of accumulation N-acetyl-neuraminate recombination Corynebacterium glutamicum and its application
KR102266232B1 (en) * 2021-01-15 2021-06-17 씨제이제일제당 주식회사 Novel polyketide synthase variant and a method for producing XMP or GMP using the same
WO2022154188A1 (en) * 2021-01-15 2022-07-21 씨제이제일제당 (주) Novel polyketide synthase variant and method for producing xmp or gmp using same

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Application publication date: 20170510