CN101735971A - Method for screening high-yield glutamic acid strains in high flux - Google Patents
Method for screening high-yield glutamic acid strains in high flux Download PDFInfo
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Abstract
The invention relates to a method for screening high-yield glutamic acid strains in high flux and belongs to the technical field of fermentation engineering. The screening of high-yield strains is always the bottleneck of selecting seeds of the high-yield glutamic acid strains. A traditional screening method comprises the following steps: selecting a single colony from a slab; performing fermentation and preliminary screening on the colony through a test tube or a shake flask; and performing fermentation and second screening in the shake flask. The method improves the traditional screening, and comprises the following steps: selecting the single colony on the slab, and performing primary screening by a microporous cell cultivation plate and fermentation and second screening in the shake flask according to the characteristics of the high-yield glutamic acid strains. The method effectively increases the number of the colonies in each screening, reduces the intensity of screening work, and improves the efficiency of the whole screening work.
Description
Technical field
The present invention relates to a kind of based on dull and stereotyped, micropore Tissue Culture Plate fast, the method for screening high-yield glutamic acid strains in high flux, belong to the fermentation engineering field.
Background technology
L-glutamic acid is traditional large leavened prod, is the amino acid of large scale fermentation production the earliest, also is the present amino acid of turnout maximum in the world.L-glutamic acid has at aspects such as food, medicine, industry, agriculturals widely to be used, and its single sodium salt-Sodium Glutamate (trade name monosodium glutamate) is important seasonings.China's glutamic acid yield was about 1,600,000 tons in 2008, accounted for about 75% of global total amount.Yet China mainly with L-glutamic acid (monosodium glutamate) as pure seasonings (ratio is 85%), other countries only are 52% with L-glutamic acid as the ratio of pure seasonings, and with its ratio that is used as chemical industry, medicine and protective foods intermediate up to 48% (Japanese aginomoto company food cause report, 2002 years).Estimate 5-20 from now on, China will improve the ratio that L-glutamic acid is used as chemical industry, medicine and protective foods intermediate greatly.Estimate that the year two thousand twenty whole world glutamic acid yield can reach 3,200,000 tons, China accounts for 70% of total share, will reach 2,240,000 tons.
China is that the big producing country of L-glutamic acid consumes big country.But there is the bottleneck problem of some serious restriction industry developments in China's L-glutamic acid industry at present, most importantly: lack the high yield strain excellent that the modern biotechnology method is transformed, the key technical indexes of fermentative production is starkly lower than the level of external advanced producer, cause the too high (Food science of production and operation cost, 2009,30 (4): 40-43).
Good yield glutamic acid strains in high is the key of fermentative production L-glutamic acid.China is the sixties in last century, and successively isolation and selection has obtained Corynebacterium Pekinese AS1.299, corynebacterium crenatum AS1.452 and Tianjin tyrothricin T6-13.Subsequently by UV,
60Methods such as mutafacient system such as Co radiation, NTG, DES and protoplastis fusion, seed selection be fit to industrial glutamate-producing strain kind.Is starting strain cloudlike meeting continuous heavy rain etc. with T6-13, adopts UV and methods such as NTG mutagenesis and protoplastis mutagenesis, by mutagenic treatment repeatedly, selects the S9114 bacterial strain.This bacterial strain has anti-high sugar (25%-30%), anti-high glutamic acid (20%), does not utilize the characteristics of L-glutamic acid.Shake flask test is sugar 15.5% just, produces acid 8.56%, transformation efficiency 55.15%.This bacterial strain is applied (the scientific and technological communication of fermenting, 1993,22 (1): 3-7) in how tame Gourmet Powder Factory.But production practice are found, produce the L-glutamic acid bacterial classification in process is gone down to posterity in preservation, production performance can fail gradually, and producing acid and transformation efficiency can constantly descend, therefore, must regularly carry out rejuvenation, promptly from the bacterial strain of decline, filter out the not bacterial strain of decline by separation, purifying, the highly yielding ability that could keep bacterial strain like this, constantly satisfy and produce needs (the scientific and technological communication of fermenting, 1993,22 (2): 38-34).
So it is essential that the seed selection of high yield corynebacterium glutamicum strain is operated in the fermentative production of L-glutamic acid.No matter be, use of the transformation of modern genetic engineering means existing yield glutamic acid strains in high with traditional mutagenesis, still the rejuvenation of superior strain, all need from numerous single strains, to pick out superior strain, and screening method commonly used at present, all be to select single bacterium colony from specific flat board earlier, produce sour primary dcreening operation by test tube or little shake flask fermentation then, produce the multiple sieve of acid with shake flask fermentation again, select superior strain.Because in the L-glutamic acid shake flask fermentation process, need keep fermented liquid pH7.0 by repeatedly adding urea or ammoniacal liquor, complex operation, workload is big, and simultaneously personal errors is also big, and with test tube or shake bottle for a short time, the limited amount of each fermentation test, screening operation speed is slow, therefore, becomes a bottleneck of superior strain seed selection.
The present invention is according to the characteristics of high yield corynebacterium glutamicum strain, adopt and select flat board, as the plate screening limiting factor be: high sugar, high glutamic acid, L-glutamic acid sole carbon source, resistance (Sulphaguanidine, ketone group propanedioic acid, propanedioic acid, tonka bean camphor etc.), succsinic acid responsive type, auxotroph (Isoleucine, methionine(Met), phenylalanine defective type) etc., or low pH tolerance, or high pH tolerance.
Picking is at the bacterium colony of selecting dull and stereotyped growth; With micropore Tissue Culture Plate replacing test tubes or shake bottle and produce sour primary dcreening operation, a micropore Tissue Culture Plate just can carry out 24 to 384 samples while shaker fermentations, can place a plurality of well plates on the shaking table, has increased the colony counts of each screening effectively.And the present invention adds acid base indicator in fermention medium, has effectively pointed out the time of adding urea, thereby reduces personal errors, reduces screening operation intensity, has improved the efficient of whole screening work.Therefore, the present invention has positive effect for the seed selection of high yield glutamate-producing strain strain.
Summary of the invention
The purpose of this invention is to provide a kind of based on selecting dull and stereotyped, micropore Tissue Culture Plate culture technique, foundation fast, the method for screening high-yield glutamic acid strains in high flux.
Technical scheme of the present invention:
(1) will be applied to dull and stereotyped 28-35 ℃ and cultivate 48-72h from nature separate microorganism sample or by engineered product L-glutamic acid mutant bacteria sample.The plate screening limiting factor is: high sugar, high glutamic acid, L-glutamic acid sole carbon source, resistance (Sulphaguanidine, ketone group propanedioic acid, propanedioic acid, tonka bean camphor etc.), succsinic acid responsive type, auxotroph (defective typies such as Isoleucine, methionine(Met), phenylalanine), low pH tolerance, high pH tolerance etc.
Form by following compositions as the resistant panel substratum: glucose 5-10g, peptone 5-10g, yeast extract paste 1-5g, extractum carnis 3-10g, NaCl 2-5g, agar 15-20g, and contain Sulphaguanidine 5-20g, ketone group propanedioic acid 0.5-2.5g, propanedioic acid 5-20g, tonka bean camphor 1-5g, distilled water is settled to 1L, pH7.0.
Form by following compositions as the sugared plate culture medium of height: glucose 250-300g, peptone 5-10g, yeast extract paste 1-5g, extractum carnis 3-10g, NaCl 2-5g, agar 15-20g, distilled water is settled to 1L, pH7.0.
Form by following compositions as the high glutamic acid substratum: Sodium Glutamate 150-200g, peptone 5-10g, yeast extract paste 1-5g, extractum carnis 3-10g, NaCl 2-5g, agar 15-20g, distilled water is settled to 1L, pH7.0.
Form by following compositions as L-glutamic acid sole carbon source substratum: paddy atmosphere acid sodium 5-10g, (NH
4)
2SO
40.5-2.5g, K
2HPO
40.5-2.5g, KH
2PO
40.1-0.8g, MgSO
40.5-5g, FeSO
41-10mg, MnSO
41-10mg, VitB1 0.05-0.2mg, vitamin H 0.01-0.1mg, washing agar 15-20g, distilled water is settled to 1L, and NaOH transfers pH7.0.
Form by following compositions as the used substratum of seed selection succsinic acid responsive type bacterial strain: glucose 5-10g, peptone 5-10g, yeast extract paste 1-5g, extractum carnis 3-10g, NaCl 2-5g, succsinic acid 0.1-3g, agar 15-20g, distilled water is settled to 1L, pH7.0.
Form by following compositions as low pH gradient plate: glucose 1-10g, peptone 5-10g, yeast extract paste 1-5g, extractum carnis 3-10g, NaCl 2-5g, agar 15-20g, distilled water is settled to 1L, and pH 4.0-5.0 is regulated with L-glutamic acid in the sterilization back.
Form by following compositions as high pH gradient plate: glucose 100-150g, K
2HPO
40.5-1.5g, MgSO
40.5-5g, FeSO
41-10mg, MnSO
41-10mg, VitB1 0.01-0.1mg, urea 3-10g, corn steep liquor 1-10g, bromine thymol blue 0.05-0.5g, agar 15-20g, distilled water is settled to 1L, and pH 10.0-12.0 is transferred with NaOH in the sterilization back.
(2) well plates primary dcreening operation: select single bacterium colony in the flat board growth, be inoculated in every hole and contain the well plates of 0.8-1.2mL seed culture medium, after 28-35 ℃ of shaking table cultivated 12-24h, press the 5%-10% inoculum size, be forwarded to the well plates that the 0.8-1.2mL fermention medium is contained in every hole, 30-35 ℃ of shaking table cultivated 32-48h, shaking speed is 200-300rpm, and when fermentation 12-24h, add the urea 50-100 μ L of mass concentration 10%-20%, continue fermentation 20-36h.Fermented liquid in the well plates detects glutamic acid yield by SBA-40C bio-sensing analyser, chooses the high bacterial strain of output and shakes the multiple sieve of bottle.
The well plates that adopts is the square deep-well plates in 24 holes or 48 holes or 96 holes or 384 holes.
Seed culture medium is made up of following compositions: glucose 15-25g, K
2HPO
40.5-2.5g, MgSO
40.3-6g, FeSO
41-10mg, MnSO
41-15mg, urea 1-3g, corn steep liquor 20-35g are settled to 1L, and pH 7.0.
Fermention medium is made up of following compositions: glucose 100-150g, K
2HPO
40.5-1.5g, MgSO
40.1-6g, FeSO
41-5mg, MnSO
41-5mg, VitB1 0.01-0.1mg, urea 3-10g, corn steep liquor 1-10g are settled to 1L, and pH 7.0.
(3) shake the multiple sieve of bottle: the inoculation that above-mentioned product L-glutamic acid content is high is in the 250mL triangular flask that contains the 10-30mL seed culture medium, after 28-35 ℃ of shaking table cultivated 8-12h, be forwarded in the 500mL triangular flask that contains the 10-50mL fermention medium by 2.5%-10%, in fermention medium, add the acid base indicator of 0.01-0.1g/L simultaneously.Cultivation is carried out on the reciprocating type shaking table of the rotary shaking table of 200-250rpm or 90-150rpm, according to indicator colour-change in the fermented liquid, add urea 3-8 time of mass concentration 10%-40%, each dosage 100-800 μ L, fermentation 40-50h, detect glutamic acid yield by SBA-40C bio-sensing analyser, select superior strain.
Glucose and L-glutamic acid content detect with SBA-40C bio-sensing analyser in the fermented liquid: 10-100 times of fermented liquid dilution, sample feeding amount 25 μ L multiply by extension rate according to the instrument display data and are L-glutamic acid and glucose content.
Shake that thalli growth detects with 752 type ultraviolet-visible pectrophotometers in the bottle: shake in the bottle bacterium liquid suitably after the dilution, detect OD in spectrophotometer
660Thalli growth is measured with microplate reader in the well plates: draw bacterium liquid in the 200 μ L microwell plates to the new microwell plate, the lid lid utilizes microplate reader to measure OD after being placed on microplate reader concussion 30s
660
Beneficial effect of the present invention: traditional screening method is a picking list bacterium colony from the flat board, by test tube or little shake flask fermentation primary dcreening operation, carries out shake flask fermentation again and sieves again then.The present invention improves traditional screening, and according to the characteristics of high yield corynebacterium glutamicum strain, picking list bacterium colony carries out primary dcreening operation with the micropore Tissue Culture Plate on flat board, and shake flask fermentation sieves again.The inventive method has increased the colony counts of each screening effectively, reduces screening operation intensity, has improved the efficient of whole screening work.
Embodiment
Embodiment 1 well plates is used to ferment and produces the feasibility of L-glutamic acid
Glutamate producing bacterium SW07-1 with the laboratory preservation, this bacterial strain the sixth of the twelve Earthly Branches is at article " seed selection of temperature tolerance L-glutamic acid fermentation bacterial classification " (biological processing, 2009,7 (6): open 74-78), and use from the inclined-plane and rule to the dull and stereotyped activation of LB 24h, inoculating needle is chosen a ring and is shaken bottle to the 250mL that contains the 10mL seed culture medium, 30 ℃, 220rpm cultivates 12h, when seed is transferred to fermention medium, at 4 angles of 96 hole micropore Tissue Culture Plates and edge one row and middle portion reserve some holes at random and contrast as aseptic blank sample, and choose the parallel sample of 11 representative positions inoculations as fermentation culture, inoculum size with 8% is seeded to this 11 holes, and the 1mL fermention medium is contained in every hole.Sterile gauze places 30 ℃ of shaking tables on the inoculation bonnet, 220rpm cultivates, 10% urea, 80 μ L are all added in all holes (comprising blank sample) behind the 24h, continue to detect each hole L-glutamic acid content of (comprising blank sample) respectively behind the fermentation 24h, utilize microplate reader to measure the OD of each sample simultaneously
660, investigate the microbiological contamination situation that whether occurs.The OD of the blank sample of result
660All less than 0.1, the OD of parallel sample
660All greater than 0.8, so can think there is not the microbiological contamination phenomenon.
The used medium component of this example is as follows: seed culture medium: glucose 25g, K
2HPO
41.5g, MgSO
40.6g, FeSO
45mg, MnSO
45mg, urea 2.5g, corn steep liquor 30g are settled to 1L, and pH 7.0.
Fermention medium: glucose 140g, K
2HPO
41g, MgSO
46g, FeSO
45mg, MnSO
45mg, VitB1 0.05mg, urea 7g, corn steep liquor 3g are settled to 1L, and pH 7.0.
Directly measure the L-glutamic acid content of fermented liquid in the orifice plate with SBA-40C bio-sensing analyser, the result shows that the L-glutamic acid content of all blank samples is lower than 10
-2G/L, and the result of 11 parallel samples (unit: 10
-2G/L) as follows: 55.7,55.1,59.2,59.1,57.7,57.6,61.9,59.4,58.4,56.7,57.4, mean value is 58.1, variance s
2=3.60, standard deviation sigma=1.81, according to the principle of work and the instrument characteristic of SBA-40C type bio-sensing analyser, its relative error is about 1%, and is not more than 2%.And the relative error between each hole is 2.48% in this experiment, has only 0.48% after the eliminating instrumental error, shows the product acid situation that reflects bacterial classification that 96 well culture plates can be more stable.
Embodiment 2 usefulness resistant panel-well plates to X ray mutagenesis after the screening of bacterial strain
Select dull and stereotyped the composition: glucose 10g, peptone 10g, yeast extract paste 5g, extractum carnis 10g, NaCl 5g, Sulphaguanidine 20g, ketone group propanedioic acid 2.5g, propanedioic acid 16g, tonka bean camphor 2g, agar 20g are settled to 1L, pH7.0.
Seed culture medium: glucose 25g, K
2HPO
41.5g, MgSO
40.6g, FeSO
45mg, MnSO
45mg, urea 2.5g, corn steep liquor 30g are settled to 1L, and pH 7.0.
Fermention medium: glucose 140g, K
2HPO
41g, MgSO
46g, FeSO
45mg, MnSO
45mg, VitB1 0.05mg, urea 7g, corn steep liquor 3g are settled to 1L, and pH 7.0.
With the glutamate producing bacterium strain SW07-1 that preserve in the laboratory, 40min obtains the mutant bacteria storehouse with X ray mutagenesis.It is dull and stereotyped that bacteria suspension after the mutagenesis is applied to selection, cultivated 3 days for 30 ℃, choose totally 89 of the bacterium colonies that grow on the flat board, be seeded to respectively (its mesopore A1, B1 are original bacterium contrast) in 96 holes of the micropore Tissue Culture Plate that contains the 1mL seed culture medium, 30 ℃, after 220rpm cultivates 24h, be forwarded in the 96 hole micropore Tissue Culture Plates that every hole contains the 1mL fermention medium with 8% inoculum size, 30 ℃, after 220rpm cultivates 24h, 10% urea 80 μ L are added in every hole, continue 32 ℃, and 250rpm cultivates the 24h fermentation ends.SBA-40C bio-sensing analyser detects the L-glutamic acid content (unit: 10 of fermented liquid in each hole
-2G/L), wherein 21 plant heights are in contrasting data, result such as table 1.
Resistant mutant strain 96 hole micropore Tissue Culture Plate results after the mutagenesis of table 1X ray
Embodiment 3-4 with pH tolerate flat board-well plates to mutagenesis after the screening of bacterial strain
With the glutamate producing bacterium strain SW07-1 that preserve in the laboratory, 40min obtains the mutant bacteria storehouse with X ray mutagenesis.Bacteria suspension separate application after the mutagenesis as for low pH gradient plate and high pH gradient plate, was cultivated 3 days for 30 ℃.
Wherein, low pH gradient plate is made up of following compositions: glucose 10g, peptone 10g, yeast extract paste 5g, extractum carnis 10g, NaCl 5g, agar 20g, be settled to 1L, and pH4.6-5.0 is regulated with L-glutamic acid in the sterilization back.
High pH gradient plate is made up of following compositions: glucose 140g, K
2HPO
41g, MgSO
46g, FeSO
45mg, MnSO
45mg, VitB1 0.05mg, urea 7g, corn steep liquor 3g, bromine thymol blue 0.1g, agar 20g are settled to 1L, and it is 10.5-11.2 that pH is transferred with NaOH in the sterilization back.
Select on low pH gradient plate 126 of the single bacterium colonies of growth, 280 of the single bacterium colonies of the dull and stereotyped growth of high pH.With these 406 bacterium colonies, be inoculated into 96 hole micropore Tissue Culture Plates by the method for embodiment 2 and carry out primary dcreening operation, obtain 9 strains and the 41 strain glutamic acid yield bacterial strain high respectively than control strain.
The bottle that shakes of 5 pairs of micropore Tissue Culture Plates of embodiment primary dcreening operation bacterial strain sieves again
The bacterial strain 71 strain bacterial strains that micropore Tissue Culture Plate primary dcreening operation obtains behind the dull and stereotyped cultivation of LB 24h, carry out the shake flask fermentation experiment.Inoculation is shaken in the bottle to the 250mL that contains 10mL seed culture medium (seed culture medium, fermentation culture based component are with embodiment 2), and fermentation shake flask has 2 parallel samples, 30 ℃, 220rpm cultivates 8h, 8% inoculum size is forwarded to the fermention medium that contains 0.01% toluylene red, thalline produces acid after producing alkali earlier in the fermenting process, fermented liquid becomes redness again by initial red yellowing, when reddening once more, fermented liquid (ferments 8～10h) approximately, every 2h adds an amount of urea and keeps about pH7.0, (ferments 40～48h) approximately until residual sugar less than 1g/L.
After diluting 100 times, shake flask fermentation liquid detects L-glutamic acid and glucose content by SBA-40C bio-sensing analyser.The result has the product acid amount and the glucose acid invert ratio of 13 plant mutant strains to be higher than control strain.
This 13 strain bacterium carries out aforesaid shake flask fermentation test after continuous 6 generations of transferring again.The results are shown in Table 2 (data are the mean value of two parallel samples in the table).The output that wherein is numbered D-2-2, D-2-3, D-2-17, G-6-13, G-6-32, G-7-2, X-43 bacterial strain still is higher than control strain.
The product L-glutamic acid of bacterial strain after table 2 continuous passage
The bottle that shakes of low yield acid bacterial strain is checked in the embodiment 6 micropore Tissue Culture Plates
Produce acid and be less than in the negative mutant strain that contrasts strain from the 96 hole micropore Tissue Culture Plates of embodiment 2-4, picked at random 15 strains are numbered-1 ,-2 ,-3, arrive-15.Carry out the shake flask fermentation test by the method for embodiment 5, the results are shown in Table 3 (data are the mean value of two parallel samples in the table), 15 strain bacterium output of negative sudden change group all are less than former bacterium, illustrate to utilize the method for microwell plate fermentation screening L-glutamic acid high yield bacterium the possible little of sieve to occur leaking.
The shake flask fermentation result of the negative mutant strain of table 3
| Bacterium number | SW07-1 (contrast) | ??-1 | ??-2 | ??-3 | ??-4 | ??-5 | ??-6 | ??-7 |
| Glutamic acid yield (g/L) | ??64.25 | ??27.30 | ??15.15 | ??38.55 | ??1.30 | ??15.65 | ??4.80 | ??15.60 |
| Bacterium number | ??-8 | ??-9 | ??-10 | ??-11 | ??-12 | ??-13 | ??-14 | ??-15 |
| Glutamic acid yield (g/L) | ??11.60 | ??1.80 | ??7.45 | ??16.75 | ??21.10 | ??6.95 | ??0.55 | ??0.40 |
Claims (7)
1. the method for a screening high-yield glutamic acid strains in high flux is characterized in that:
(1) from the nature separation or by engineered glutamate-producing strain sample, be applied to and select flat board, cultivate 48-72h for 28-35 ℃;
(2) well plates primary dcreening operation: select single bacterium colony in the flat board growth, be inoculated into the well plates that contains seed culture medium, 28-35 ℃ of shaking table cultivated 12-24h, is forwarded to the well plates that contains fermention medium, 28-35 ℃ of shaking table cultivated 32-48h, measures the content of L-glutamic acid in the liquid;
(3) shake the multiple sieve of bottle: the inoculation that above-mentioned product L-glutamic acid content is high is to containing the shaking in the bottle of seed culture medium, after 28-35 ℃ of shaking table cultivated 8-12h, be forwarded to the shake flask fermentation 40-50h that contains fermention medium, detect fermented liquid two-story valley histidine content, select superior strain.
2. method according to claim 1, it is characterized in that described selection is dull and stereotyped, plate culture medium is the selectivity flat board that contains limiting factor, the screening limiting factor is: high sugar, or high glutamic acid, or L-glutamic acid sole carbon source or resistance: Sulphaguanidine, ketone group propanedioic acid, propanedioic acid, tonka bean camphor resistance, or succsinic acid responsive type, or auxotroph: Isoleucine, methionine(Met), phenylalanine defective type, or low pH tolerance, or high pH tolerance.
3. method according to claim 1 is characterized in that described seed culture medium is made up of following compositions: glucose 15-25g, K
2HPO
40.5-2.5g, MgSO
40.3-6g, FeSO
41-10mg, MnSO
41-15mg, urea 1-3g, corn steep liquor 20-35g are settled to 1L, and pH 7.0.
4. method according to claim 1 is characterized in that described fermention medium is made up of following compositions: glucose 100-150g, K
2HPO
40.5-1.5g, MgSO
40.1-6g, FeSO
41-5mg, MnSO
41-5mg, VitB1 0.01-0.1mg, urea 3-10g, corn steep liquor 1-10g are settled to 1L, and pH 7.0.
5. method according to claim 1, when it is characterized in that with the well plates primary dcreening operation, the well plates that adopts is the square deep-well plates in 24 holes or 48 holes or 96 holes or 384 holes, liquid amount is 0.8-1.2mL, the inoculum size of fermentation culture is 5%-10%, and shaking speed is 200-300rpm, and when fermentation culture 12-24h, add the urea 50-100 μ L of mass concentration 10%-20%, continue fermentation 20-36h.
6. method according to claim 1 is characterized in that shaking bottle when sieving again, and seed culture adopts 250mL triangular flask, liquid amount 10-30mL; Fermentation culture adopts the 500mL triangular flask, liquid amount 10-50mL, inoculum size 2.5%-10%, cultivation is carried out on the reciprocating type shaking table of the rotary shaking table of 200-250rpm or 90-150rpm, according to indicator colour-change in the fermented liquid, add urea 3-8 time of mass concentration 10%-40%, each dosage 100-800 μ L, behind the fermentation 40-50h, detect fermented liquid two-story valley histidine content.
7. method according to claim 4 is characterized in that described fermention medium, when shake flask fermentation, adds the acid base indicator of 0.01-0.1g/L in the fermention medium, and that acid base indicator is selected for use is phenol red, toluylene red, o-cresolsulfonphthalein or purpurum bromocresolis.
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| CN111394291A (en) * | 2020-03-27 | 2020-07-10 | 江南大学 | Method for producing L-glutamic acid by utilizing recombinant corynebacterium glutamicum fermentation |
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