A method of improvement L-arginine producing strains
阅读说明：本技术 一种改良l-精氨酸产生菌的方法 (A method of improvement L-arginine producing strains ) 是由 杨晟 蒋宇 杨俊杰 董枫 刘映淼 于 2018-05-22 设计创作，主要内容包括：本发明公开了一种改良L-精氨酸产生菌的方法,包括下述步骤：敲除谷氨酸棒杆菌ATCC13032基因组中的argR基因,获得基因敲除菌株ATCC13032(ΔargR)；对基因敲除菌株的基因组中argB基因进行A26V和M31V突变,获得基因突变菌株ATCC13032(ΔargR,argBmut(A26V M31V))；对基因突变菌株的基因组中ABC型糖通透酶基因NCgl2374起始密码子上游调控区的碱基序列进行突变,获得基因工程菌株ATCC13032(ΔargR,argBmut(A26V M31V),NCgl2374mut4)。本发明的方法能够将菌株的L-精氨酸生产能力提高14倍。(The invention discloses a kind of method for improveing L-arginine producing strains, includes the following steps: to knock out the argR gene in Corynebacterium glutamicum ATCC13032 genome, obtain gene knock-out bacterial strain ATCC13032 (Δ argR)；A26V and M31V mutation is carried out to argB gene in the genome of gene knock-out bacterial strain, is obtained gene mutation strains A TCC13032 (Δ argR, argBmut (A26V M31V))；The base sequence of ABC type sugar permease gene NCgl2374 upstream from start codon control region in the genome of gene mutation bacterial strain is mutated, obtain engineering strain ATCC13032 (Δ argR, argBmut (A26V M31V), NCgl2374mut4).The L-arginine production capacity of bacterial strain can be improved 14 times by method of the invention.)
1. a kind of method for improveing L-arginine producing strains, comprising the following steps:
A. the argR gene in Corynebacterium glutamicum ATCC13032 genome is knocked out, gene knock-out bacterial strain ATCC13032 is obtained (ΔargR)；
B. in the genome of gene knock-out bacterial strain ATCC13032 (Δ argR) described in step A argB gene carry out A26V and M31V mutation, obtains gene mutation strains A TCC13032 (Δ argR, argBmut (A26V M31V))；
C. in the genome of gene mutation strains A TCC13032 described in step B (Δ argR, argBmut (A26V M31V)) The base sequence of ABC type sugar permease gene NCgl2374 upstream from start codon control region is mutated, by SEQ ID NO: 13 sport SEQ ID NO:14, and acquisition L-arginine production capacity is than the engineering strain that the gene mutation bacterial strain improves ATCC13032(ΔargR,argBmut(A26V M31V),NCgl2374mut4)。
2. the method as described in claim 1, which is characterized in that gene knock-out bacterial strain ATCC13032 (Δ described in step A ArgR it) is prepared by following methods:
A1. using ATCC13032 genome as template, it is using the primer argR-aL-F and sequence that sequence is SEQ ID NO:1 The primer argR-aL-R of SEQ ID NO:2 carries out PCR amplification, obtains the argR-aL segment of about 1kb；
A2. using ATCC13032 genome as template, it is using the primer argR-aR-F and sequence that sequence is SEQ ID NO:3 The primer argR-aR-R of SEQ ID NO:4 carries out PCR amplification, obtains the argR-aR segment of about 1kb；
A3. the plasmid pK18mobsacB for the use of HindIII and EcoRI digestion GenBank accession number being FJ437239.1, glue return Receipts obtain 5.7kb carrier segments；
The above-mentioned argR-aL segment of A4.Gibson connection, argR-aR segment and carrier segments convert DH5 α competent cell, apply Cloth kanamycins LB plate, is incubated overnight；
A5. PCR amplification is carried out using primer argR-aL-F and argR-aR-R and verify transformant, obtain plasmid pK18mobsacB- argR；
A6. corynebacterium glutamicum ATCC13032 competent cell is prepared；
A7. plasmid pK18mobsacB-argR is transformed into ATCC13032 competent cell；
A8. SacB sucrose counter-selection is carried out, carries out PCR amplification using primer argR-aL-F and argR-aR-R, verifying is flat in BHIS Plate is grown in the transformant that the BHIS plate containing kanamycins cannot be grown, and obtains strains A TCC13032 (Δ argR).
3. the method as described in claim 1, which is characterized in that gene mutation bacterial strain described in step B passes through following methods system It is standby:
B1. using ATCC13032 genome as template, it is using the primer argB-aL-F and sequence that sequence is SEQ ID NO:5 The primer argB-aL-R of SEQ ID NO:6 carries out PCR amplification, obtains the argB-aL segment of about 1kb；
B2. using ATCC13032 genome as template, it is using the primer argB-aR-F and sequence that sequence is SEQ ID NO:7 The primer argB-aR-R of SEQ ID NO:8 carries out PCR amplification, obtains the argB-aR segment of about 1kb；
B3. the plasmid pK18mobsacB for the use of HindIII and EcoRI digestion GenBank accession number being FJ437239.1, glue return Receipts obtain 5.7kb carrier segments；
The above-mentioned argB-aL segment of B4.Gibson connection, argB-aR segment and carrier segments convert DH5 α competent cell, apply Cloth kanamycins LB plate, is incubated overnight；
B5. PCR amplification is carried out using primer argB-aL-F and argB-aR-R and verify transformant, obtain plasmid pK18mobsacB- argBmut；
B6. corynebacterium glutamicum ATCC13032 (Δ argR) competent cell is prepared；
B7. plasmid pK18mobsacB-argBmut is transformed into ATCC13032 (Δ argR) competent cell；
B8. SacB sucrose counter-selection is carried out, carries out PCR amplification using primer argB-aL-F and argB-aR-R, verifying is flat in BHIS Plate growth and in the transformant that the BHIS plate containing kanamycins cannot be grown, obtain strains A TCC13032 (Δ argR, argBmut(A26V M31V))。
4. the method as described in claim 1, which is characterized in that engineering strain described in step C passes through following methods system It is standby:
C1. using ATCC13032 genome as template, using sequence be SEQ ID NO:9 primer NCgl2374mut4-aL-F and The primer NCgl2374mut4-aL-R that sequence is SEQ ID NO:10 carries out PCR amplification, obtains about 0.5kb's NCgl2374mut4-aL segment；
C2. using ATCC13032 genome as template, the primer NCgl2374mut4-aR-F for the use of sequence being SEQ ID NO:11 Primer NCgl2374mut4-aR-R progress PCR amplification with sequence is SEQ ID NO:12, obtains about 0.5kb's NCgl2374mut4-aR segment；
C3. the plasmid pK18mobsacB for the use of HindIII and EcoRI digestion GenBank accession number being FJ437239.1, glue return Receipts obtain 5.7kb carrier segments；
The above-mentioned NCgl2374mut4-aL segment of C4.Gibson connection, NCgl2374mut4-aR segment and carrier segments, conversion DH5 α competent cell is coated with kanamycins LB plate, is incubated overnight；
C5. PCR amplification is carried out using primer NCgl2374mut4-aL-F and NCgl2374mut4-aR-R and verify transformant, obtain Plasmid pK18mobsacB-NCgl2374mut4；
C6. corynebacterium glutamicum ATCC13032 (Δ argR, argBmut (A26V M31V)) competent cell is prepared；
C7. plasmid pK18mobsacB-NCgl2374mut4 is transformed into ATCC13032 (Δ argR, argBmut (A26V M31V)) competent cell；
C8. SacB sucrose counter-selection is carried out, carries out PCR expansion using primer NCgl2374mut4-aL-F and NCgl2374mut4-aR-R Increase verifying in BHIS plated growth and in the transformant that the BHIS plate containing kanamycins cannot be grown, obtains bacterial strain ATCC13032(ΔargR,argBmut(A26V M31V),NCgl2374mut4)。
5. the method as described in any one of claim 2-4, which is characterized in that step A7, be converted into chlorine described in B7 and C7 Change calcium conversion method or electrotransformation.
6. a kind of genetic engineering bacterium ATCC13032 (Δ argR, argBmut (A26V M31V), NCgl2374mut4), feature It is, is constructed according to method as described in claim 1.
7. the application that genetic engineering bacterium as claimed in claim 6 is used to produce L-arginine.
8. the use as claimed in claim 7, which is characterized in that produce L- essence ammonia by the fermentation of the genetic engineering bacterium Acid.
9. the use as claimed in claim 7, which is characterized in that fermentation medium composition is as follows: 60g/L glucose, 5g/L are beautiful Rice & peanut milk, 30g/L (NH4)2SO4, 8g/L KCl, 2g/L urea, 0.5g/L KH2PO4, 0.5g/L K2HPO4, 1g/L MgSO4· 7H2O, 1g/L NaCl, 20mg/L FeSO4·7H2O, 10mg/L MnSO4·5H2O, 20mg/L niacin, 20mg/L β-the third ammonia Acid, 10mg/L VB1,0.2mg/L biotin, 30g/L CaCO3, KOH tune PH=7.7.
10. the use as claimed in claim 7, which is characterized in that seed culture medium composition is as follows: 3g/L NaCl, 5g/L yeast Extract, 7g/L beef extract, 10g/L peptone, 10g/L glucose.
Technical field
The invention belongs to genetic engineering fields, specifically, being related to a kind of method for improveing L-arginine producing strains, especially It is related to a kind of method that L-arginine producing strains production capacity is improved by genetic engineering.
Background technique
L-arginine (L-argnine, abbreviation L-Arg) is one of half required basic amino acid needed in human body, as one Basic amino acid of the kind containing guanidine radicals, is a kind of important intermediate metabolites of organism urea cycle, has a variety of unique lifes Reason and pharmacological action for treatment physiological function, cardiovascular disease, excitation immune system, the nutrient balance for maintaining baby, promote Human body removing toxic substances etc. all has good curative effect, is known as that the important carrier of amino acid is transported and stored in body by expert, in flesh It is particularly important in intracellular metabolite.It is the essential amino acid for synthesizing plasmosin and nucleoprotein；Flesh is participated in as unique ammonia source The synthesis of acid；As the important intermediate of urea cycle, the role for excluding extra ammonia is play in liver, prevents ammonia excessively product Tire out and causes to be poisoned；It also has the function of adjusting body immunity, can inhibit tumour growth, promote wounded tissue healing etc.. Also, arginine is the direct precursor of nitric oxide, urea, ornithine and flesh butylamine, is the important essence of synthesis muscle element, and It is used as the synthesis of polyamine, citrulling and glutamine.Therefore, L-arginine has in terms of medicine, food and chemical field It is important and be widely applied.For example, clinically, in addition to one of main component as Hausmam Amin 20, L-arginine And its esters is also widely used in all kinds of hepatic coma for the treatment of and avoids with sodium glutamate person and viral liver class glutamic-pyruvic transaminase exception person, it is right Virus hepatitis is significant in efficacy.There is therapeutic effect to diseases such as enteron aisle ulcer, thrombosis and neurasthenia.In addition, L- essence ammonia Acid is the important component of sport nutrition drink formula, and a kind of important feed addictive, is also widely used in height Hold aquaculture.According to statistics, L-arginine demand in the whole world is at 15000 tons or more at present, and demand is with annual 12%- 15% speed increases.
There are two types of the production methods of L-arginine: first is that protein hydrolyzes extraction method, second is that microbe fermentation method.Hydrolyze method There are when operating cost, yield and low output, it is at high cost the problems such as, and be not suitable for large-scale raw there are serious pollution It produces.It is relatively easy and environmentally friendly that fermentation method produces L-arginine technique, therefore has very big development potentiality, becomes state One important trend of inside and outside amino acids industry.In the world the such as Japanese aginomoto of famous amino acid company, consonance fermentation and German Digao is husky mainly to carry out L-arginine production using biofermentation and technique for gene engineering.But domestic microorganism hair The acid yield that ferment produces L-arginine is generally lower, and higher cost, production level and yield are far from satisfying domestic demand, Therefore the research for improving L-arginine fermentation level has great importance.
What the fermenting microbe of L-arginine was studied more mainly has Corynebacterium glutamicum (Corynebacterium Glutamicum), brevibacterium flavum (Brevibacterium flavum), Corynebacterium crenatum (Corynebacterium Crenatum), Escherichia coli (Escherichia coli), bacillus subtilis (Bacillus subtilis) etc., but at present It is mainly Corynebacterium glutamicum and Corynebacterium crenatum for producing the microbial strains of L-arginine.Technique for gene engineering is for essence Propylhomoserin Producing Strain breeding has important impetus, is a kind of efficient using genetic engineering building L-arginine superior strain The breeding technique of rate, rationalization, but building filters out that be suitable for the high yield recombinant bacterium of commercial scale be a kind of urgent always Demand.
Summary of the invention
For the defect for overcoming existing L-arginine-producing bacteria strain fermentation level low, the present invention using technique for gene engineering come Corynebacterium glutamicum is transformed, it, can be by bacterial strain by enhancing gene relevant to L-arginine generation, reduction branched metabolic pathway L-arginine production capacity increase substantially.
In order to achieve the above object, the present invention adopts the following technical scheme:
A method of improvement L-arginine producing strains, comprising the following steps:
A. the argR gene in Corynebacterium glutamicum ATCC13032 genome is knocked out, gene knock-out bacterial strain is obtained ATCC13032(ΔargR)；
B. argB gene in the genome of gene knock-out bacterial strain ATCC13032 (Δ argR) described in step A is carried out A26V and M31V mutation, obtains gene mutation strains A TCC13032 (Δ argR, argBmut (A26V M31V))；
C. to the gene of gene mutation strains A TCC13032 described in step B (Δ argR, argBmut (A26V M31V)) The base sequence of ABC type sugar permease gene NCgl2374 upstream from start codon control region is mutated in group, by SEQ ID NO:13 sports SEQ ID NO:14, and acquisition L-arginine production capacity is than the gene that Corynebacterium glutamicum ATCC13032 is improved Engineered strain ATCC13032 (Δ argR, argBmut (A26V M31V), NCgl2374mut4).
In one embodiment, under gene knock-out bacterial strain ATCC13032 (Δ argR) described in above-mentioned steps A passes through State method preparation:
A1. using ATCC13032 genome as template, the primer argR-aL-F and sequence that the use of sequence are SEQ ID NO:1 PCR amplification is carried out for the primer argR-aL-R of SEQ ID NO:2, obtains the argR-aL segment of about 1kb；
A2. using ATCC13032 genome as template, the primer argR-aR-F and sequence that the use of sequence are SEQ ID NO:3 PCR amplification is carried out for the primer argR-aR-R of SEQ ID NO:4, obtains the argR-aR segment of about 1kb；
A3. the plasmid pK18mobsacB for the use of HindIII and EcoRI digestion GenBank accession number being FJ437239.1, Glue recycles to obtain 5.7kb carrier segments；
The above-mentioned argR-aL segment of A4.Gibson connection, argR-aR segment and carrier segments, conversion DH5 α competence are thin Born of the same parents are coated with kanamycins LB plate, are incubated overnight；
A5. PCR amplification is carried out using primer argR-aL-F and argR-aR-R and verify transformant, obtain plasmid pK18mobsacB-argR；
A6. corynebacterium glutamicum ATCC13032 competent cell is prepared；
A7. plasmid pK18mobsacB-argR is transformed into ATCC13032 competent cell；
A8. SacB sucrose counter-selection is carried out, carries out PCR amplification using primer argR-aL-F and argR-aR-R, verifying exists BHIS plated growth and in the transformant that the BHIS plate containing kanamycins cannot be grown, obtain strains A TCC13032 (Δ argR)。
In one embodiment, gene mutation bacterial strain described in above-mentioned steps B is prepared by following methods:
B1. using ATCC13032 genome as template, the primer argB-aL-F and sequence that the use of sequence are SEQ ID NO:5 PCR amplification is carried out for the primer argB-aL-R of SEQ ID NO:6, obtains the argB-aL segment of about 1kb；
B2. using ATCC13032 genome as template, the primer argB-aR-F and sequence that the use of sequence are SEQ ID NO:7 PCR amplification is carried out for the primer argB-aR-R of SEQ ID NO:8, obtains the argB-aR segment of about 1kb；
B3. the plasmid pK18mobsacB for the use of HindIII and EcoRI digestion GenBank accession number being FJ437239.1, Glue recycles to obtain 5.7kb carrier segments；
The above-mentioned argB-aL segment of B4.Gibson connection, argB-aR segment and carrier segments, conversion DH5 α competence are thin Born of the same parents are coated with kanamycins LB plate, are incubated overnight；
B5. PCR amplification is carried out using primer argB-aL-F and argB-aR-R and verify transformant, obtain plasmid pK18mobsacB-argBmut；
B6. corynebacterium glutamicum ATCC13032 (Δ argR) competent cell is prepared；
B7. plasmid pK18mobsacB-argBmut is transformed into ATCC13032 (Δ argR) competent cell；
B8. SacB sucrose counter-selection is carried out, carries out PCR amplification using primer argB-aL-F and argB-aR-R, verifying exists BHIS plated growth and in the transformant that the BHIS plate containing kanamycins cannot be grown, obtain strains A TCC13032 (Δ argR,argBmut(A26V M31V))。
In one embodiment, engineering strain described in above-mentioned steps C is prepared by following methods:
C1. using ATCC13032 genome as template, the primer NCgl2374mut4- for the use of sequence being SEQ ID NO:9 The primer NCgl2374mut4-aL-R that aL-F and sequence are SEQ ID NO:10 carries out PCR amplification, obtains about 0.5kb's NCgl2374mut4-aL segment；
C2. using ATCC13032 genome as template, the primer NCgl2374mut4- for the use of sequence being SEQ ID NO:11 The primer NCgl2374mut4-aR-R that aR-F and sequence are SEQ ID NO:12 carries out PCR amplification, obtains about 0.5kb's NCgl2374mut4-aR segment；
C3. the plasmid pK18mobsacB for the use of HindIII and EcoRI digestion GenBank accession number being FJ437239.1, Glue recycles to obtain 5.7kb carrier segments；
The above-mentioned NCgl2374mut4-aL segment of C4.Gibson connection, NCgl2374mut4-aR segment and carrier segments turn Change DH5 α competent cell, is coated with kanamycins LB plate, is incubated overnight；
C5. PCR amplification is carried out using primer NCgl2374mut4-aL-F and NCgl2374mut4-aR-R verify transformant, Obtain plasmid pK18mobsacB-NCgl2374mut4；
C6. corynebacterium glutamicum ATCC13032 (Δ argR, argBmut (A26V M31V)) competent cell is prepared；
C7. plasmid pK18mobsacB-NCgl2374mut4 is transformed into ATCC13032 (Δ argR, argBmut (A26V M31V)) competent cell；
C8. SacB sucrose counter-selection is carried out, is carried out using primer NCgl2374mut4-aL-F and NCgl2374mut4-aR-R PCR amplification is verified in BHIS plated growth and in the transformant that the BHIS plate containing kanamycins cannot be grown, and bacterial strain is obtained ATCC13032(ΔargR,argBmut(A26V M31V),NCgl2374mut4)。
Calcium chloride transformation or electrotransformation, preferably electrotransformation are converted into described in above-mentioned steps A7, B7 and C7.
According to the second aspect of the invention, a kind of genetic engineering bacterium ATCC13032 (Δ argR, argBmut are provided (A26V M31V), NCgl2374mut4), it is constructed according to above-mentioned method.
According to the third aspect of the present invention, application of the said gene engineering bacteria in the production of L-arginine is provided.
The genetic engineering bacterium both can produce L-arginine by fermenting directly as fermentation strain, can also be used as out Bacterium germination strain does further improvement to filter out the new production bacterium that L-arginine production capacity further increases.
When by being fermented said gene engineering bacteria to produce L-arginine, when fermentation, culture medium used can be with It is any culture medium suitable for Corynebacterium glutamicum growth fermentation.
Preferred embodiment in accordance with the present invention, fermentation medium composition are as follows: 60g/L glucose, 5g/L corn pulp, 30g/ L(NH4)2SO4, 8g/L KCl, 2g/L urea, 0.5g/L KH2PO4, 0.5g/L K2HPO4, 1g/L MgSO4·7H2O, 1g/L NaCl, 20mg/L FeSO4·7H2O, 10mg/L MnSO4·5H2O, 20mg/L niacin, 20mg/L Beta-alanine, 10mg/L VB1,0.2mg/L biotin, 30g/L CaCO3, KOH tune PH=7.7.
In a preferred embodiment, include when above-mentioned L-arginine-producing bacteria is fermented seed growth phase and Thallus fermentation stage.The two stages use seed culture medium and fermentation medium respectively, and fermentation medium can be trained with seed It is same to support base phase, it can not also be identical.
Preferably, when fermentation medium and seed culture medium be not identical, seed culture medium composition is as follows: 3g/L NaCl, 5g/L yeast extract, 7g/L beef extract, 10g/L peptone, 10g/L glucose.
Method of the invention can make the original strain Corynebacterium glutamicum for producing L-arginine The L-arginine production capacity of ATCC13032 improves 14 times, has and popularizes value.
Detailed description of the invention
Fig. 1 is the schematic diagram of plasmid pK18mobsacB, which is presented by the river Institute of Microorganism, Academia Sinica Liu Shuan It gives.GenBank:FJ437239.1.Specifying information referring tohttps://www.ncbi.nlm.nih.gov/nuccore/ 215434894。
Fig. 2 is the schematic diagram for the recombinant plasmid pK18mobsacB-argR that the present invention constructs.
Fig. 3 is the schematic diagram for the recombinant plasmid pK18mobsacB-argBmut that the present invention constructs.
Fig. 4 is the schematic diagram for the recombinant plasmid pK18mobsacB-NCgl2374mut4 that the present invention constructs.
Specific embodiment
The present invention is described in further details below in conjunction with specific embodiment.It should be understood that following embodiment is only used for The bright present invention is not for limiting the scope of the invention.
Additive amount, content and the concentration of many kinds of substance is referred to herein, wherein the percentage composition, except special instruction Outside, all refer to mass percentage.
Herein, term " Corynebacterium glutamicum ATCC13032 ", " strains A TCC13032 ", " ATCC13032 " are indicated Identical meaning all refers to the original strain ATCC13032 as genetic modification object, is a kind of L-arginine producing strains, purchase It buys from Shanghai Industrial institute of microbiology.
Herein, term " clpp gene degerming (strain) " and " ATCC13032 (Δ argR) " indicate identical meaning, are all Refer to the bacterial strain that argR gene is knocked in ATCC13032 genome.
Similarly, term " gene mutation bacterium (strain) ", " ATCC13032 (Δ argRargBmut (A26V M31V) ", " ATCC13032 (Δ argR, argBmut (A26V M31V) " and the identical meaning of " CIBTS1512 " expression, all refer to clpp gene ArgBmut gene mutation is the bacterial strain of argBmut (A26V M31V) gene in degerming ATCC13032 (Δ argR) genome.
Term " genetic engineering bacterium (strain) ", " ATCC13032 (Δ argRargBmut (A26V M31V) NCgl2374mut4)”、“ATCC13032(ΔargR,argBmut(A26V M31V),NCgl2374mut4)”、“CIBTS1512 (NCgl2374mut4) " and " CIBTS3008 " indicates identical meaning, all refer to gene mutation bacterium ATCC13032 (Δ argR, ArgBmut (A26V M31V)) be further by NCgl2374 gene mutation in genome NCgl2374mut4 gene bacterial strain, The mutation refers to the base sequence SEQ ID NO of ABC type sugar permease gene NCgl2374 upstream from start codon control region: 13 sport SEQ ID NO:14.
Gene argR is that a generally existing arginine operon in bacterium adjusts gene, is had in different bacteriums Different functions, inventor the study found that by knock out Corynebacterium glutamicum gene group in this negative regulator gene, can be certain Its inhibition to arginine synthesis is released in degree.
N-acetylglutamat kinases (N-acetylglutamatekinase) is completed arginine for Corynebacterium glutamicum and is closed Have catalysis without the feedback inhibition by final product L-arginine by glutamic acid to acetylglutamate at the approach first step.Hair 26th alanine (A) is sported valine the study found that by being mutated to N-acetylglutamat kinases by bright people (V), the 31st methionine (M) sports valine (V), can effectively improve L- in Corynebacterium glutamicum ATCC13032 Arginic synthesis capability.Herein, the mutated gene of N-acetylglutamat kinase gene argB is abbreviated as argBmut Or argBmut (A26V M31V).
The base sequence SEQ ID NO:13 of ABC type sugar permease gene NCgl2374 upstream from start codon control region becomes Changing, which can cause the change of arginine synthesis capability to be one, has been surprisingly found that.Being sported SEQ ID NO:14 can be certain The L-arginine yield of Corynebacterium glutamicum is improved in degree.
Experiment discovery, the L-arginine production capacity of gene mutation bacterial strain CIBTS1512 are mentioned than original strain ATCC13032 It is about 13 times high；The L-arginine production capacity of engineering strain CIBTS3008 improves 11.4% than CIBTS1512 again, That is CIBTS3008 improves at least 14 times than original strain ATCC13032, demonstrates genetic engineering design philosophy of the invention Correctness.