Construction of threonine producing strain and method for producing threonine
阅读说明：本技术 苏氨酸生产菌种的构建及生产苏氨酸的方法 (Construction of threonine producing strain and method for producing threonine ) 是由 刘慧敏 康培 尹春筱 于 2020-06-28 设计创作，主要内容包括：本发明涉及微生物领域,特别涉及高转化率苏氨酸生产菌种的构建及生产苏氨酸的方法。本发明所述新型大肠杆菌L-苏氨酸产量均高于各自的对照菌株,其中mqo表达活性增强的菌株摇瓶转化率在24-30％之间,较出发菌提高6-12个转化率；cyoABCDE表达活性增强的菌株摇瓶转化率在26-30％之间,同样较出发菌株提高8-12个转化率。有这两个位点改造的摇瓶结果可以得出,将苹果酸:醌氧化还原酶基因mqo的表达加强或者增强呼吸链末端氧化酶的活性均可以明显提高苏氨酸的生产能力。(The invention relates to the field of microorganisms, in particular to a method for constructing a threonine production strain with high conversion rate and producing threonine. The yield of the novel escherichia coli L-threonine is higher than that of respective control strains, wherein the shake flask conversion rate of the strain with enhanced mqo expression activity is 24-30%, and is improved by 6-12 conversion rates compared with the strain; the shake flask conversion rate of the strain with enhanced cyoABCDE expression activity is 26-30%, and is also increased by 8-12 compared with the strain. The shake flask result modified by the two sites can be obtained, and the threonine production capacity can be obviously improved by enhancing the expression of the malic acid quinone oxidoreductase gene mqo or enhancing the activity of the respiratory chain terminal oxidase.)
1. Use of enhancing the expression of an mqo gene and/or enhancing the expression of a cyoabccde gene in the production of L-threonine.
2. The use of claim 1, wherein the enhancement comprises a combination of one or more of promoter enhancement, RBS sequence enhancement, or increased copy number.
3. Strain characterized in that the expression of the mqo gene is enhanced and/or the expression of the cyoABCDE gene is enhanced.
4. The strain of claim 3, wherein the enhancement comprises a combination of one or more of promoter enhancement, RBS sequence enhancement, or increased copy number.
5. Use of the strain according to claim 3 or 4 for the production of L-threonine.
6. Method for constructing a strain according to claim 3 or 4, wherein the expression of the mqo gene and/or the expression of the cyoABCDE gene is enhanced.
7. The method of construction of claim 6, wherein the enhancement comprises a combination of one or more of promoter enhancement, RBS sequence enhancement, or increased copy number.
8. A process for producing L-threonine, characterized in that the strain according to claim 3 or 4 is a fermentation strain.
Technical Field
The invention relates to the field of microorganisms, in particular to a method for constructing a threonine production strain with high conversion rate and producing threonine.
Background
L-threonine is one of 8 amino acids essential for human and animal growth, and is widely used in feed, food additives, preparation of auxiliary materials for medicines, and the like. Currently, L-threonine is mainly produced by fermentation of microorganisms, and various bacteria are available for L-threonine production, such as mutant strains induced by wild-type strains of Escherichia coli, Corynebacterium, Serratia, and the like, as production strains. Specific examples include amino acid analogue resistant mutants or various auxotrophs such as methionine, lysine, isoleucine and the like. However, in the conventional mutation breeding, the strain grows slowly and generates more byproducts due to random mutation, so that a high-yield strain is not easy to obtain.
With the increasing demand of threonine in the world, the construction and modification of high-yield threonine strains are particularly important. In the prior art, Escherichia coli is used, the 39bp sequence from-56 th to-18 th of a threonine operon sequence is deleted, so that the thrABC expression of a threonine synthesis key gene is enhanced, and the threonine productivity is improved by 22%. KwangHoLee et al utilize the strategy of systematic metabolic engineering, relieve the feedback inhibition of products by mutating the genes thrA and lysC for coding aspartokinase I and III, remove the byproducts glycine and isoleucine by knocking out tdh and weakening ilvA, provide more precursors for threonine synthesis by inactivating competitive pathway genes metA and lysA, and the like, and the finally obtained TH28C (pBRThrABCR3) strain can produce 82.4g/L acid after 50h fermentation, and the conversion rate of saccharic acid is 39.3%. In the Chinese patent 201611248603.9 filed by the plum blossom group in 2016, MHZ-0214-2 strain was obtained by strengthening pntAB and knocking out iclR, and the strain had threonine yield of 15.3g/L, conversion rate of about 18.5% and no plasmid load.
Disclosure of Invention
In view of this, the present invention provides a method for constructing a threonine-producing strain that further improves the conversion rate of sugar acid and a method for producing threonine.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of enhancing the expression of an mqo gene and/or enhancing the expression of a cyoABCDE gene in the production of L-threonine.
In some embodiments of the invention, the enhancement comprises a combination of one or more of promoter enhancement, RBS sequence enhancement, or increased copy number. The promoter enhancement comprises selecting a strong promoter; the strong promoter includes the tac promoter. The RBS sequence is enhanced to introduce an RBS sequence.
The invention also provides strains that enhance the expression of the mqo gene and/or enhance the expression of the cyoABCDE gene.
In some embodiments of the invention, the enhancement comprises a combination of one or more of promoter enhancement, RBS sequence enhancement, or increased copy number. The enhancement includes a combination of one or more of promoter enhancement, RBS sequence enhancement, or increased copy number. The promoter enhancement comprises selecting a strong promoter; the strong promoter includes the tac promoter. The RBS sequence is enhanced to introduce an RBS sequence.
In some embodiments of the invention, the starting strain of the strain is MHZ-0214-2, which is described in detail in the patent application publication No. 106591209A.
The invention also provides application of the strain in producing L-threonine.
The invention also provides a construction method of the strain, and the construction method can strengthen the expression of the mqo gene and/or strengthen the expression of the cyoABCDE gene.
In some embodiments of the invention, the enhancement comprises a combination of one or more of promoter enhancement, RBS sequence enhancement, or increased copy number. The enhancement includes a combination of one or more of promoter enhancement, RBS sequence enhancement, or increased copy number. The promoter enhancement comprises selecting a strong promoter; the strong promoter includes the tac promoter. The RBS sequence is enhanced to introduce an RBS sequence. The invention also provides a method for producing L-threonine by taking the strain as a fermentation strain.
The yield of the novel escherichia coli L-threonine is higher than that of respective control strains, wherein the shake flask conversion rate of the strain with enhanced mqo expression activity is 24-30%, and is improved by 6-12 conversion rates compared with the strain; the shake flask conversion rate of the strain with enhanced cyoABCDE expression activity is 26-30%, and is also increased by 8-12 compared with the strain. The shake flask result modified by the two sites can be obtained, and the threonine production capacity can be obviously improved by enhancing the expression of the malic acid quinone oxidoreductase gene mqo or enhancing the activity of the respiratory chain terminal oxidase.
The shake flask conversion rate of the strain simultaneously transformed by the two sites is 35-52%, and the conversion rate of threonine is further improved. The yield of the strain MHZ-0216-5 with the best performance is 44.6g/l, the conversion rate is 52.4%, the yield of the starting strain MHZ-0214-2 threonine is 15.3g/l, the yield is 18.0%, the yield is increased by 191.50% compared with the starting strain, and the conversion rate is increased by 191.35%, so that the threonine production capacity of the modified strain is obviously better than that of the MHZ-0214-2.
Detailed Description
The invention discloses a construction method of threonine production strains with high conversion rate and a method for producing threonine, and a person skilled in the art can realize the method by properly improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
According to the metabolic pathway of L-threonine in Escherichia coli, MHZ-0214-2 is taken as an initial strain (plum blossom group patent application publication No. 106591209A), related transformation is carried out on the genome of the initial strain, and corresponding enhancement and overexpression are carried out on key genes in the metabolic pathway of threonine, such as: enhancing the expression of the malate quinone oxidoreductase gene mqo, including replacing the original promoter with a strong promoter, or enhancing the strength of RBS and increasing the copy number of mqo on the genome; increasing the expression strength of the respiratory chain end oxidase cyoABCDE, including replacing the original promoter with a strong promoter, or increasing the strength of the RBS, and increasing the copy number of cyoABCDE on the genome; in addition, both are overexpressed simultaneously, increasing the supply of precursors and energy, to improve the ability of the strain to produce threonine.
The CRISPR-Cas9 gene Editing technology (Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System, Jiang Y, Chen B, et al. appl. environ Microbiol,2015) reported by JianngY et al is mainly used for reference in Genome Editing of Escherichia coli.
In the following examples, the final concentration of Kanamycin (Kanamycin) in the medium was 50. mu.g/mL, and the final concentration of spectinomycin (spectinomycin) in the medium was 50. mu.g/mL.
In the following examples, all reagents used are commercially available. The parent strain of the threonine producing strain with high conversion rate provided by the invention is MHZ-0214-2, belonging to W3110 (Escherichia)). The primer sequences used in the examples are shown in Table 1.
TABLE 1
The names of the genes involved in the present invention are explained as follows:
thrA: aspartokinase and I-homoserine dehydrogenase;
thrB: homoserine kinase;
thrC: a threonine synthase;
mqo: malic acid, quinone oxidoreductase;
tdcC: serine/threonine H+A symporter protein;
cyoABCDE: cytochrome bo3 ubiquitin oxidase;
pyc: a pyruvate carboxylase;
the raw materials and reagents used in the construction of the threonine production strain with high conversion rate and the method for producing threonine provided by the invention can be purchased from the market.
The invention is further illustrated by the following examples:
example 1: preparation of Mqo Gene-enhanced Strain MHZ-0214-3 (promoter-enhanced)
(1) pTarget-tac-mqo plasmid construction
Step1: using pTargetT plasmid as template (from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System, JiangY, Chen B, et al. appl. environ Microbiol,2015), selecting sgRNAmqotac-F/sgRNA-R primer pair, amplifying sgRNA fragment I with N20; step2: using a W3110 genome as a template, selecting an mqotac-UF/mqotac-UR primer pair, and amplifying an upstream homology arm II containing a tac promoter; step3: amplifying a downstream homology arm (c) containing a tac promoter by using a W3110 genome as a template and selecting an mqotac-DF/mqotac-DR primer pair; step4, using the first, second and third as templates, selecting sgRNA mqotac-F/mqotac-DR primer pair to amplify sgRNA-up-Ptac-down segment, also called Donor DNA; step5 double SpeI and PstI digestion of Donor DNA and ligation with the vector fragment (double digested and dephosphorylated) in the appropriate ratio using T4 ligase at 22 ℃. Subsequently, Trans1-T1 competent cells were transformed to obtain pTarget-Ptac-mqo, and enzyme digestion was performed for identification. (2) Competent cell preparation and electrotransformation of pTarget-Ptac-mqo plasmid
Step1: electrically transferring pCas plasmid (derived from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System, JiangY, ChenB, et al. appl. environ Microbiol,2015) into MHZ-0214-2 competent cells (the transformation method and the competence preparation method refer to molecular clone III); step2: a single MHZ-0214-2(pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ and 200r/min to OD650After 0.4, electroporation competent cells were prepared (see molecular clone III). Step3: the pTarget-Ptac-mqo plasmid was electroporated into MHZ-0214-2(pCas) competent cells (electroporation conditions: 2.5kV, 200. omega., 25. mu.F), spread on a medium containing spectinomycin and a cardOn LB plate of natamycin, static culture was carried out at 30 ℃ until single colony was visible.
(3) Recombination verification
And (3) amplifying the target fragment by using a primer pair mqotac-F/mqotac-R, and sequencing the amplified product to verify the integrity of the sequence.
(4) Construction of related plasmid losses
Step1: selecting a single colony with correct sequencing verification, inoculating the single colony into a 5mL LB test tube containing kanamycin and having a final concentration of 0.5mMIPTG, performing overnight culture at 30 ℃, and streaking the single colony on an LB flat plate containing kanamycin; step2: picking a single colony point on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, and culturing overnight at 30 ℃, wherein if the single colony point cannot grow on the LB plate containing kanamycin and spectinomycin and grows on the LB plate containing kanamycin, the pTarget-Ptac-mqo plasmid is lost; step3: selecting positive colonies lost by pTarget-Ptac-mqo plasmid, inoculating into an anti-LB-free test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; step4: single colonies were picked and spotted on both kanamycin-containing LB plates and non-resistant LB plates, and if they could not grow on kanamycin-containing LB plates, they grew on non-resistant LB plates, indicating that pCas plasmid was lost, resulting in MHZ-0214-3(Ptac-mqo) strain.
Example 2: preparation of RBS-enhanced Mqo Gene Strain MHZ-0214-4(RBS-mqo)
(1) pTarget-RBS3-mqo plasmid construction
Step1: using pTargetT plasmid as template (from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System, JiangY, Chen B, et al. appl. environ Microbiol,2015), selecting sgRNAmqoRBS-F/sgRNA-R primer pair, amplifying sgRNA fragment I with N20; step2: using a W3110 genome as a template, selecting an mqoRBS-UF/mqoRBS-UR primer pair, and amplifying an upstream homology arm (II) containing RBS; step3: selecting an mqoRBS-UF/mqoRBS-RBSR primer pair by taking the fragment II as a template to amplify an upstream homology arm III containing RBS; step4: using a W3110 genome as a template, selecting an mqoRBS-DF/mqoRBS-DR primer pair, and amplifying a downstream homology arm (IV) containing RBS; step5, using the first, the third and the fourth as templates, selecting sgRNA-mqoRBS-F/mqoRBS-DR primer pairs, amplifying sgRNA-up-RBS-down fragments, also called DonorDNA; step6 SpeI and BglII double digestion of the DonORDNA fragment and ligation of the vector fragment (double digestion and dephosphorylation) in the appropriate ratio with T4 ligase at 22 ℃. Subsequently, Trans1-T1 competent cells were transformed to obtain pTarget-RBS-mqo, and enzyme cleavage identification was performed.
(2) Competent cell preparation and electrotransformation of pTarget-RBS-mqo plasmid
Step1: the pCas plasmid (derived from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System, Jiang Y, Chen B, et al. appl. environ Microbiol,2015) was electroporated into MHZ-0214-2 competent cells (the transformation method and the competent preparation method are referred to molecular clone III); step2: a single MHZ-0214-2(pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ and 200r/min to OD650After 0.4, electroporation competent cells were prepared (see molecular clone III). Step3: the pTarget-RBS-mqo plasmid was electroporated into MHZ-0214-2(pCas) competent cells (electrotransformation conditions: 2.5kV, 200. omega., 25. mu.F), spread on LB plates containing spectinomycin and kanamycin, and cultured at 30 ℃ until a single colony was visible.
(3) Recombination verification
Step1: carrying out colony PCR verification on the single colony by using a primer pair mqoRBS-RBSR/mqoRBS-UF; step2: the strains which are identified by PCR to be correct are amplified by a primer pair mqoRBS-F/mqoRBS-R, and the amplified products are sequenced to verify the integrity of the sequences.
(4) Construction of related plasmid losses
Step1: selecting a single colony with correct sequencing verification, inoculating the single colony into a 5mL LB test tube containing kanamycin and 0.5mM IPTG (isopropyl-beta-thiogalactoside) with final concentration, culturing overnight at 30 ℃, and streaking on an LB plate containing kanamycin; step2: picking a single colony point on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, and culturing overnight at 30 ℃, wherein if the single colony point cannot grow on the LB plate containing kanamycin and spectinomycin and grows on the LB plate containing kanamycin, the pTarget-RBS-mqo plasmid is lost; step3: selecting positive colonies lost by pTarget-RBS-mqo plasmid, inoculating the positive colonies in an anti-LB-free test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; step4: single colonies were picked and spotted on both kanamycin-containing LB plates and non-resistant LB plates, and if they could not grow on kanamycin-containing LB plates, they grew on non-resistant LB plates, indicating that pCas plasmid was lost, resulting in MHZ-0214 (RBS-mqo) strain.
Example 3: preparation of a Strain with an enhanced mqo Gene MHZ-0214-5 (multicopy)
(1) Construction of plasmid pTarget-zwf mqo
Step1: using pTargetT plasmid as template (from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System, JiangY, Chen B, et al. appl. environ Microbiol,2015), selecting sgRNAmqo2-F/sgRNA-R primer pair, amplifying sgRNA fragment I with N20; step2: using a W3110 genome as a template, and selecting an mqo2-UF/mqo2-UR primer pair to amplify an upstream homology arm II; step3, selecting an mqo-F/mqo-R primer pair by taking a W3110 genome as a template to amplify an mqo fragment c; step4: using a W3110 genome as a template, selecting an mqo2-DF/mqo2-DR primer pair, and amplifying a downstream homology arm (r) containing RBS; step5, selecting a primer pair of sgRNA mqo2-F/mqo2-DR by taking the first, the second, the third and the fourth as templates, and amplifying an sgRNA-up-mqo-down fragment, which is also called DonORDNA; step6 double SpeI and PstI digestion of the DonORDNA and ligation with the vector fragment (double digested and dephosphorylated) in the appropriate ratio using T4 ligase at 22 ℃. Subsequently, Trans1-T1 competent cells were transformed to obtain pTarget-zwf:: mqo, and enzyme digestion was performed.
(2) Competent cell preparation and electrotransformation of pTarget-RBS3-mqo plasmid
Step1: (ii) electrotransfering a pCas plasmid (derived from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System, JiangY, Chen B, et. appl. environ Microbiol,2015) into MHZ-0214-2 competent cells (the transformation method and the competent preparation method refer to molecular clone III); step2: a single MHZ-0214-2(pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ and 200r/min to OD650After 0.4, electroporation competent cells were prepared (see molecular clone III). Step3: the plasmid pTarget-zwf:: mqo was electrotransferred into MHZ-0214-2(pCas) competent cells (electrotransformation conditions: 2.5kV, 200. omega., 25. mu.F), spread on LB plate containing spectinomycin and kanamycin, and incubated at 30 ℃ until a single colony was visible.
(3) Recombination verification
Step1: the single colony was amplified using primer pair mqo2-F/mqo2-R and the amplified products were sequenced to verify sequence integrity.
(4) Construction of related plasmid losses
Step1: selecting a single colony with correct sequencing verification, inoculating the single colony into a 5mL LB test tube containing kanamycin and having a final concentration of 0.5mMIPTG, performing overnight culture at 30 ℃, and streaking the single colony on an LB flat plate containing kanamycin; step2: picking a single colony point on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, culturing overnight at 30 ℃, if the single colony point cannot grow on the LB plate containing kanamycin and spectinomycin, and growing on the LB plate containing kanamycin, indicating that pTarget-zwf:: mqo plasmid is lost; step3: selecting positive colonies with pTarget-zwf that the mqo plasmid is lost, inoculating the positive colonies in an anti-LB-free test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; step4: single colonies were picked and spotted on both kanamycin-containing LB plates and non-resistant LB plates, and if they could not grow on kanamycin-containing LB plates, they grew on non-resistant LB plates, indicating that pCas plasmid was lost, yielding MHZ-0214-5(zwf:: mqo) strain.
Example 4: preparation of CyoABCDE Gene-enhanced Strain MHZ-0214-6 (promoter-enhanced)
(1) pTarget-tac-cyoABCDE plasmid construction
Step1: selecting a sgRNA-F/sgRNA-R primer pair to amplify a sgRNA fragment (i) with N20 by using pTargetT plasmid as a template (derived from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System, JiangY, ChenB, et al, apple. environ Microbiol, 2015); step2: using W3110 genome as template, selecting cyo-UF/cyo-UR primer pair to amplify upstream homology arm; step3: amplifying a downstream homology arm (c) containing a tac promoter by using a W3110 genome as a template and selecting a cyotac-F/cyo-DR primer pair; step4: taking the third step as a template, selecting a cyotac-DF/cyo-DR primer pair, and amplifying a downstream homology arm (fourth step) containing a tac promoter; step5, selecting the sgRNA cyo-F/cyo-DR primer pair with the first, the second and the fourth as templates to amplify an sgRNA-up-Ptac-down fragment, also called DonORDNA; step6 double SpeI and PstI digestion of Donor DNA and ligation with the vector fragment (double digested and dephosphorylated) in the appropriate ratio using T4 ligase at 22 ℃. Subsequently, Trans1-T1 competent cells were transformed to obtain pTarget-Ptac-cyoABCDE, and enzyme digestion was performed.
(2) Preparation of competent cells and electrotransformation of pTarget-Ptac-cyoABCDE plasmid
Step1: the pCas plasmid (derived from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System, JiangY, ChenB, et al. appl. environ Microbiol,2015) was electroporated into MHZ-0214-2 competent cells (the transformation method and the competent preparation method are referred to molecular clone III); step2: a single MHZ-0214-2(pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ and 200r/min to OD650After 0.4, electroporation competent cells were prepared (see molecular clone III). Step3: the pTarget-Ptac-cyoABCDE plasmid was electroporated into MHZ-0214-2(pCas) competent cells (electroporation conditions: 2.5kV, 200. omega., 25. mu.F), spread on LB plates containing spectinomycin and kanamycin, and cultured by standing at 30 ℃ until a single colony was visible.
(3) Recombination verification
The primers were used to amplify the target fragment from cyo-F/cyo-R and the amplified product was sequenced to verify the integrity of the sequence.
(4) Construction of related plasmid losses
Step1: selecting a single colony with correct sequencing verification, inoculating the single colony into a 5mL LB test tube containing kanamycin and having a final concentration of 0.5mMIPTG, performing overnight culture at 30 ℃, and streaking the single colony on an LB flat plate containing kanamycin; step2: picking a single colony point on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, and culturing overnight at 30 ℃, wherein if the single colony point cannot grow on the LB plate containing kanamycin and spectinomycin and grows on the LB plate containing kanamycin, the pTarget-Ptac-cyoABCDE plasmid is lost; step3: selecting a positive colony lost by pTarget-Ptac-cyoABCDE plasmid, inoculating the positive colony in an anti-LB-free test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; step4: single colonies were picked on both kanamycin-containing LB plates and non-resistant LB plates, and if they could not grow on kanamycin-containing LB plates, they grew on non-resistant LB plates, indicating that the pCas plasmid was lost, resulting in MHZ-0214-6(Ptac-cyoABCDE) strain.
Example 5: preparation of RBS-enhanced Strain of cyoABCDE Gene
MHZ-0214-7(RBS::cyoABCDE)
(1) pTarget-RBS-cyoABCDE plasmid construction
Step1: using pTargetT plasmid as template (from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System, JiangY, Chen B, et al. appl. environ Microbiol,2015), selecting sgRNAcy-F/sgRNA-R primer pair, amplifying to obtain sgRNA fragment I with N20; step2: using W3110 genome as template, selecting cyo-UF/cyoRBS-UR primer pair, amplifying upstream homology arm containing RBS; step3, using W3110 genome as template, selecting cyoRBS-DF/cyo-DR primer pair to amplify upstream homology arm (c) containing RBS; step4, using the first, second and third as templates, selecting sgRNA cyo-F/cyo-DR primer pair to amplify sgRNA-up-RBS-down fragment, also called DonORDNA; step5 double SpeI and PstI digestion of Donor DNA and ligation with the vector fragment (double digested and dephosphorylated) in the appropriate ratio using T4 ligase at 22 ℃. Subsequently, Trans1-T1 competent cells were transformed to obtain pTarget-RBS-cyoABCDE, and enzyme digestion was performed.
(2) Competent cell preparation and electrotransformation of pTarget-RBS-cyoABCDE plasmid
Step1: (ii) electrotransfering a pCas plasmid (derived from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System, JiangY, Chen B, et. appl. environ Microbiol,2015) into MHZ-0214-2 competent cells (the transformation method and the competent preparation method refer to molecular clone III); step2: a single MHZ-0214-2(pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ and 200r/min to OD6500.4 later electroporation competent cells (competent)The morphological preparation method is referred to molecular cloning III. Step3: pTarget-RBS-cyoABCDE plasmid was electroporated into MHZ-0214-2(pCas) competent cells (electroporation conditions: 2.5kV, 200. omega., 25. mu.F), spread on LB plate containing spectinomycin and kanamycin, and cultured by standing at 30 ℃ until a single colony was visible.
(3) Recombination verification
The primer pair cyo-F/cyo-R was used for amplification and the amplified product was sent for sequencing to verify the integrity of the sequence.
(4) Construction of related plasmid losses
Step1: selecting a single colony with correct sequencing verification, inoculating the single colony into a 5mL LB test tube containing kanamycin and having a final concentration of 0.5mMIPTG, performing overnight culture at 30 ℃, and streaking the single colony on an LB flat plate containing kanamycin; step2: picking a single colony point on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, and culturing overnight at 30 ℃, wherein if the single colony point cannot grow on the LB plate containing kanamycin and spectinomycin, the single colony point grows on the LB plate containing kanamycin, and the pTarget-RBS-cyoABCDE plasmid is lost; step3: selecting positive colonies lost by pTarget-RBS-cyoABCDE plasmid, inoculating into an anti-LB-free test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; step4: single colonies were spotted on both kanamycin-containing LB plates and non-resistant LB plates, and if they could not grow on kanamycin-containing LB plates, they grew on non-resistant LB plates, indicating that pCas plasmid was lost, yielding MHZ-0214-7(RBS:: cyoABCDE) strain.
Example 6: preparation of CyoABCDE Gene-introduced Strain MHZ-0214-8 (multicopy)
(1) pTarget-cyoABCDE plasmid construction
Step1: using pTargetT plasmid as template (from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System, JiangY, Chen B, et al. appl. environ Microbiol,2015), selecting sgRNAcy 2-F/sgRNA-R primer pair, amplifying sgRNA fragment I with N20; step2, amplifying an upstream homologous arm fragment II by taking a W3110 genome as a template and selecting a cyo2-UF/cyo2-UR primer pair; step3, using W3110 genome as template, selecting cyo2-DF/cyo2-DR primer pair to amplify downstream homology arm; step4, using W3110 genome as template, selecting cyo-F/cyo-R primer pair to amplify cyoABCDE fragment; step5, carrying out fusion PCR amplification on the first, second, third and fourth to obtain a complete fragment sgRNA-Up arm-cyoABCDE-Down arm; step6, carrying out SpeI and PstI double digestion on Donor DNA, connecting the DNA and a vector fragment (double digestion and dephosphorylation) according to a proper ratio by using T4 ligase at 23 ℃, and transforming Trans1-T1 competent cells to obtain pTargetT-cyoABCDE.
(2) Competent cell preparation and electrotransformation of pTarget-cyoABCDE plasmid
Step1: the pCas plasmid (derived from Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System, Jiang Y, Chen B, et al. appl. environ Microbiol,2015) was electroporated into MHZ-0214-2 competent cells (the transformation method and the competent preparation method are referred to molecular clone III); step2: a single MHZ-0214-2(pCas) colony was picked up and cultured in 5mL LB tube containing kanamycin and arabinose at a final concentration of 10mM at 30 ℃ and 200r/min to OD650After 0.4, electroporation competent cells were prepared (see molecular clone III). Step3: the pTargetT-cyoABCDE plasmid was electroporated into MHZ-0214-2(pCas) competent cells (electroporation conditions: 2.5kV, 200. omega., 25. mu.F), spread on LB plates containing spectinomycin and kanamycin, and cultured by standing at 30 ℃ until a single colony was visible.
(3) Recombination verification
Step1: the correct strain identified by PCR was amplified with the primer pair cyo2-F/cyo2-R and the amplification product was sequenced to verify sequence integrity.
(4) Construction of related plasmid losses
Step1: selecting a single colony with correct sequencing verification, inoculating the single colony into a 5mL LB test tube containing kanamycin and having a final concentration of 0.5mMIPTG, performing overnight culture at 30 ℃, and streaking the single colony on an LB flat plate containing kanamycin; step2: picking a single colony point on an LB plate containing kanamycin and spectinomycin and an LB plate only containing kanamycin, culturing overnight at 30 ℃, if the colony cannot grow on the LB plate containing kanamycin and spectinomycin, growing on the LB plate containing kanamycin, and indicating that the pTargetT-cyoABCDE plasmid is lost; step3: selecting a positive colony lost by the pTargetT-cyoABCDE plasmid, inoculating the positive colony in an anti-LB-free test tube, culturing at 42 ℃ for 8h, streaking on an LB plate, and culturing at 37 ℃ overnight; step4: picking single colony to point on an LB plate containing kanamycin and an LB plate without resistance, if the colony can not grow on the LB plate containing kanamycin and grows on the LB plate without resistance, showing that pCas plasmid is lost, obtaining the plasmid
MHZ-0214-8(tdcC:: cyoABCDE) strain.
Example 7: preparation of Strain in which mqo Gene was enhanced and CyoABCDE Gene was simultaneously introduced
According to the method of the examples 1-6, the mqo and cyoABCDE sites are overlapped in different modification modes to obtain 9 modified bacteria.
The threonine-producing genetically modified strains obtained in examples 1 to 7 are shown in Table 2.
TABLE 2 genetically engineered bacteria constructed according to the present invention
Example 8: shake flask fermentation verification of L-threonine producing genetically engineered bacteria
Step1 taking 16 MHZ-0214-2, MHZ-0214-3, MHZ-0214-4, MHZ-0214-5, MHZ-0214-6, MHZ-0214-7, MHZ-0214-8, MHZ-0216-0, MHZ-0216-1, MHZ-0216-2, MHZ-0216-3, MHZ-0216-4, MHZ-0216-5, MHZ-0216-6, MHZ-0216-7 and MHZ-0216-8 from the frozen tube, marking and activating on an LB plate, and culturing at 37 ℃ for 18-24 h; step2 the cells were scraped from the plate and inoculated into a shake flask containing 50mL of seed medium (see Table 3) and cultured at 37 ℃ and 90rpm for about 5 hours to OD650Controlling the content within 2; step3 transferring 2mL of seed solution into a shake flask containing 20mL of fermentation medium (see Table 4), performing fermentation culture at 37 ℃ and 100rpm in a reciprocating shaking table until residual sugar is exhausted, and measuring OD of a sample after fermentation is finished650And the content of L-threonine was measured by HPLC, and the amount of residual sugar was measured by biosensing. To ensure the realityThe reliability of the test, 3 times of repeated experiments on the shake flask, the average value of the results of acid production and conversion rate is shown in table 5.
TABLE 3 seed culture Medium (g/L)
Composition (I)
Concentration of
Glucose
25
Corn steep liquor
25
Soybean meal hydrolysate
7.7
Yeast cream
2.5
KH2PO4
1.4
Magnesium sulfate heptahydrate
0.5
FeSO4、MnSO4
20mg/L
pH
7.0
TABLE 4 fermentation Medium (g/L)
Composition (I)
Concentration of
Glucose
85
Corn steep liquor
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 comparison of productivity of threonine-producing genetically engineered bacteria
Note: denotes P value <0.01, indicating a clear difference from the control
As can be seen from Table 5, the L-threonine yields of the novel escherichia coli are higher than those of respective control strains, wherein the shake flask conversion rate of the strain with enhanced mqo expression activity is 24-30%, and is improved by 6-12 conversion rates compared with that of the strain; the shake flask conversion rate of the strain with enhanced cyoABCDE expression activity is 26-30%, and is also increased by 8-12 compared with the strain. The shake flask result modified by the two sites can be obtained, and the threonine production capacity can be obviously improved by enhancing the expression of the malic acid quinone oxidoreductase gene mqo or enhancing the activity of the respiratory chain terminal oxidase.
The shake flask conversion rate of the strain simultaneously transformed by the two sites is 35-52%, and the conversion rate of threonine is further improved. The yield of the strain MHZ-0216-5 with the best performance is 44.6g/l, the conversion rate is 52.4%, the yield of the starting strain MHZ-0214-2 threonine is 15.3g/l, the yield is 18.0%, the yield is increased by 191.50% compared with the starting strain, and the conversion rate is increased by 191.35%, so that the threonine production capacity of the modified strain is obviously better than that of the MHZ-0214-2.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Gallery plum blossom Biotechnology development Co., Ltd
<120> construction of threonine producing strain and method for producing threonine
<130> MP1832873
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<212> DNA
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gcaccgagtc ggtgcttttt tcgatgtctg gctgtttgat c 41
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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cacacattat acgagccgat gattaattgt caacagctca gaaatgaagg gatttttag 59
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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atcggctcgt ataatgtgtg gtcacacagg aaacagaatt ctatgaaaaa agtgactgcc 60
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gcttctgcag cataccgcga aacagcgagc 30
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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acgctggcga tgttgttgac 20
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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ggactagtca ataaagcgac taaaagtagt tttagagcta gaaatagcaa gt 52
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gcaccgagtc ggtgcttttt ttgaaggcag aagaacgcga tattatcctg c 51
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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ccaagttgcg ggcggggggg gttgattaat tatagggtta 40
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
cccccccgcc cgcaacttgg aggtaaaacc atgaaaaaag tgactgccat gctc 54
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gaagatctaa gcttctgcag gatgctgcca tcggcgtttt gcggg 45
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<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ggttttacct ccaagttgcg ggcggggggg 30
<210> 15
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
actggttgag aagtggctgg 20
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
atgcggaacg gtattgataa 20
<210> 17
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
atactagtcg ggaaaagcgc gcaaagtggt tttagagcta gaaatagc 48
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<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gcaccgagtc ggtgcttttt tggcaaagta gttaatggtg 40
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<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
cactttgcgc gcttttcccg 20
<210> 20
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
cgggaaaagc gcgcaaagtg ccggacggca taatattacg 40
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
cgatgatttt tttatcagtt ttgccgttac aacgcaatat ccgccac 47
<210> 22
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
cggcaaaact gataaaaaaa tcatcg 26
<210> 23
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gcttctgcag attgcggact caaatatttt c 31
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
ctgcgaggtc gccagcgacg 20
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
agaacttatt catcgcatcg 20
<210> 26
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
atactagtga ggtcgttaaa tgagactcgt tttagagcta gaaatagc 48
<210> 27
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
gcaccgagtc ggtgcttttt ttgctggatt atctggcgct ac 42
<210> 28
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
cacattatac gagccgatga ttaattgtca acagctcttt ggctgccagg tcacatatg 59
<210> 29
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
tcggctcgta taatgtgtgg tcacacagga aacagaattc tatgagactc agaaaatac 59
<210> 30
<211> 41
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
ctatgagact cagaaaatac aataaaagtt tgggatggtt g 41
<210> 31
<211> 30
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
gcttctgcag gaggtcactt tgaagtacac 30
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
acgcgctgga tttgacgcac 20
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
ccgggttcgt tggcgatcag 20
<210> 34
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
atcctgtcta gcttttttga ttaacgacct caattccac 39
<210> 35
<211> 59
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
tcaaaaaagc tagacaggat aatatttatg agactcagaa aatacaataa aagtttggg 59
<210> 36
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
ggactagtac agtcgctggc atcgagcagt tttagagcta gaaatagcaa g 51
<210> 37
<211> 52
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
aagtggcacc gagtcggtgc tttttttgaa agattgacgc gccattgcca ag 52
<210> 38
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
tccgtgctcg gcatgctcta gagaagtatt aatgtcaggt attgc 45
<210> 39
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
taatacttct ctagagcatg ccgagcacgg aaaacttaat cgaag 45
<210> 40
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
aactgcagca gtgctttctt atctggcta 29
<210> 41
<211> 31
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
gctctagaca aatccaagta acaggaattt a 31
<210> 42
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
catgcatgca gagaggtttt gttaccacac agc 33
<210> 43
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
cgagttaata ccgtcggcaa t 21
<210> 44
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gaacatcgta tacaaactgt 20
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