CN105567748B - A kind of method of fermentation production of succinic acid - Google Patents
A kind of method of fermentation production of succinic acid Download PDFInfo
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Abstract
The invention discloses a kind of method of fermentation production of succinic acid, step includes acid-producing bacteria strain building and recombinant bacterial strain fermentation production of succinic acid.Construct recombinant bacterial strain starting strain be Actinobacillus succinogenes (Actinobacillus succinogenes) NJ113(deposit number CGMCC No.1716).Succinic acid is produced using this method, the method for obtaining acid-producing bacteria strain is simple and convenient, fermentation process simple possible, and acid producing ability is stronger, can substantially reduce production cost, increase economic efficiency.Recombinant bacterial strain is in synthetic media without growth and high-yield succinic in the culture medium of external source addition glutamic acid, when using glucose as carbon source, fermentation 72h in anaerobism serum bottle, the glucose for consuming 27g/L produce the succinic acid of 19.53g/L, and succinic acid yield reaches 72.33%.
Description
Technical field
The present invention relates to a kind of methods of fermentation production of succinic acid to belong to industrial microorganism and fermentation technical field.
Background technique
Succinic acid also known as succinic acid are a kind of common natural organic acids, are widely present in human body, animal, plant and micro-
In biology.Succinic acid is as one of the TCA intermediate product recycled and the terminal reduzate of anaerobic metabolism, in biological metabolism mistake
It is played a very important role in journey.Result of study in recent years shows that succinic acid is also used as C4 platform chemicals synthesis 1,
The bulk chemicals such as 4- butanediol, tetrahydrofuran, gamma-butyrolacton and poly butylene succinate (PBS) class biodegradable
Polyester.In recent years, it is standby to prepare succinic acid using bioanalysis for getting worse with the increasingly depleted and environmental problem of fossil resource
It attracts attention, succinic acid is classified as one of the biorefinery product of following 12 kinds of most worthies by U.S. Department of Energy.
Relative to chemical synthesis, the method that biosynthesis prepares succinic acid is received more and more attention.Biosynthesis
Succinic acid is using bacterium, and the various microorganisms such as fungi send out using glucose or other various hydrolyzates as carbon source through microorganism
Ferment produces succinic acid, and relative to chemically synthesized method, the big advantage of one is that raw material are the hot spot that acid becomes Recent study.
Fermentation strain is one of the key point of succinic acid biosynthesis, and most bacteriums and fungi can generate succinic acid, but
It is the succinic acid that only part bacterial strain can produce high concentration, succinic acid industrial production bacterium bag includes some propionate production bacterium, typical case
Gastrointestinal bacteria and rumen bacteria.Currently, the research of succinic acid industrial fermentation bacterial strain is concentrated mainly on succinic acid-producing anaerobism spiral shell
Bacterium (Anaerobiospirillum succiniciproducens), succinic acid-producing Actinobacillus (Actinobacillus
Succinogenes), Corynebacterium glutamicum (Corynebacterium glutamicum) and Escherichia coli (Escherichia
Coli) etc..Wherein in the fermenting and producing of succinic acid, Actinobacillus succinogenes because its with resistance to high substrate,
The advantages that production concentration, high-yield succinic, carbon source is extensive have become at present most succinic acid industrialization production potential bacterial strain it
One.
McKinlay etc. has studied Actinobacillus succinogenes fermentating metabolism mistake using synthetic media
Journey finds that the essential amino acid of the bacterial strain is glutamic acid, cysteine and methionine, and analyzes the metabolism net of the bacterial strain
Network, it was demonstrated that the bacterial strain is glutamic acid deficiency.Then by detecting the extracellular TCA correlation enzyme activity discovery of its living body,
Actinobacillus succinogenes lacks key enzyme-citrate synthetase and isocitric acid dehydrogenation in TCA circulation
Enzyme, therefore its TCA circulation is not exclusively, can not accumulate the precursor α-ketoglutaric acid of glutamic acid synthesis, cause it that cannot independently synthesize paddy
Propylhomoserin (such as Fig. 1).Xi Yonglan is in the paper (machine towards the Actinobacillus succinogenes synthesizing succinic acid that cheap living resources utilize
System parsing and process adjustment) in point out: Actinobacillus succinogenes NJ113 does not add paddy in synthetic media
Propylhomoserin utilizes glucose fermentation succinic acid-producing, and experimental result is shown, initial sugar concentration is 10g/L, ferments after 12h, there are about 8g/L
Glucose it is remaining.And compare, glucose runs out of entirely substantially.This result is that is to say, bright Actinobacillus
Succinogenes NJ113 is also glutamic acid deficient strain.Without suitable Genetic tools so can not be to production amber because before
Sour Actinobacillus carries out genetic modification, therefore can only meet the growth demand of bacterial strain by optimization culture based formulas.
Summary of the invention
The purpose of technology of the invention is to be not easy to cultivate for existing succinic acid-producing bacterial strain, and the low problem of acid producing ability mentions
For a kind of method of fermentation production of succinic acid, succinic acid is produced using this method, the method for obtaining acid-producing bacteria strain is simple and convenient, hair
Fermenting process simple possible, and acid producing ability is stronger, can substantially reduce production cost, increase economic efficiency.
To achieve the object of the present invention, The technical solution adopted by the invention is as follows:
Step 1: independently screening Actinobacillus succinogenes NJ113 (the deposit number CGMCC of acquisition with laboratory
It No.1716) is starting strain, it is characterised in that recycling incomplete Actinobacillus succinogenes NJ113 with TCA is bacterium germination
Strain, after benefit co-expresses isocitric dehydrogenase and citric acid synthesized enzyme gene, obtaining one plant can be not necessarily in synthetic media
External source adds the recombinant type Actinobacillus succinogenes of the high-yield succinic of glutamic acid；
Further, told that specific step is as follows:
(1) using electrotransformation (as thallus OD660When value is between 0.3-0.7, it is centrifuged 10min under 4100r/min revolving speed,
It is washed 3 times with the sucrose cushion solution of 272mM, it is 2.5Kv, 25uF, 200 Ω that electricity, which turns condition, is carried out in 0.2cm electricity revolving cup)
Prepare Actinobacillus succinogenes competence bacterial strain；
(2) 5 ' end of synthesis a pair has the primer of restriction enzyme site, and using E.coli BL21 genomic DNA as template, purifying is expanded
After increasing icdh gene out, expression plasmid pLGZ920 with the consistent enzyme double digestion of restriction enzyme site designed by primer, connection obtains
Obtain recombinant plasmid pLGZ920-icdh；
(3) primer of 5 ' homologous recombination of the end with BamHI restriction enzyme site of synthesis a pair, with E.coli BL21 genome
DNA is template, after purifying the cs gene amplified, with the recombinant plasmid pLGZ920-icdh of building with enzyme designed by primer
The consistent enzyme single endonuclease digestion of enzyme site is the recombinant plasmid pLGZ920-icdh-cs that connection obtains with one-step cloning；
(4) it by the competence bacterial strain in recombinant plasmid pLGZ920-icdh-cs steps for importing (1) described in step (3), obtains
Obtain positive transformant；
(5) using the positive transformant coexpression isocitric dehydrogenase and citrate synthetase of step (4), restore its
The ability for synthesizing glutamic acid under anaerobic condition in synthetic media obtains cultivating using under glucose anaerobic condition in synthesis
The genetic engineering bacterium Actinobacillus succinogenes BD106 of middle synthesis glutamic acid and succinic acid-producing.
Utilize the method for Actinobacillus succinogenes of the present invention fermentation succinic acid-producing, it is characterised in that use molecule
Means carry out genetic modification to it, allow to independently synthesize glutamic acid on synthetic media and produce fourth two using glucose
Acid.
Further, the specific steps are as follows:
Seed culture: Actinobacillus succinogenes BD106 being activated in plate, 35-37 DEG C, is trained overnight in anaerobic box
It supports.The bacterial strain activated in plate is linked into serum bottle, 35-37 DEG C, 200r/min cultivates 10-12 hour, as one
Grade seed.First order seed is linked into serum bottle with 6%-10% inoculum concentration, 35-37 DEG C, 200r/min cultivates 10-12 small
When, as secondary seed.Seed culture medium is passed through CO in serum bottle22min, 35-37 DEG C of cultivation temperature, incubation time 10-
12h；
Fermentation succinic acid-producing: seed culture medium is inoculated into anaerobic fermentation in anaerobic fermentation culture medium, inoculum concentration volume ratio
6%-10% fills CO22min, 35-37 DEG C of fermentation temperature, fermentation time 24-72h.
Wherein the anaerobic stages fermentation medium is using glucose as the conjunction of the Actinobacillus succinogenes of carbon source
At culture medium.
In the present invention, the glutamic acid metabolism approach of NJ113 is constructed using expression vector pLGZ920 (being purchased from ATCC), it is different
Citrate synthetase and isocitric dehydrogenase are expressed in source so that the glutamic acid metabolism approach of NJ113 is covered so that its
It may not need external source addition glutamic acid in synthetic media and utilize glucose succinic acid-producing.Fourth two is produced using method of the invention
Acid, the method for obtaining acid-producing bacteria strain is simple and convenient, fermentation process simple possible, and acid producing ability is strong.
Detailed description of the invention
Fig. 1 is glutamic acid synthesis and decomposition approach in Actinobacillus succinogenes.
Fig. 2 is the building map of recombinant plasmid pLGZ920-icdh.
Fig. 3 is the building map of recombinant plasmid pLGZ920-icdh-cs.
Fig. 4 is the agarose gel electrophoresis qualification figure of PCR product icdh.
Fig. 5 is the agarose gel electrophoresis qualification figure of PCR product cs.
Fig. 6 is the bacterium colony PCR qualification figure of recombinant plasmid pLGZ920-icdh.
Fig. 7 is the bacterium colony PCR qualification figure of recombinant plasmid pLGZ920-icdh-cs.
Specific embodiment
It elaborates below with reference to embodiment and attached drawing explanation to the present invention, but there is no limit to the present invention.
Embodiment 1
This example demonstrates that the expression plasmid of building coexpression isocitric dehydrogenase, obtains recombinant type bacterial strain succinic acid-producing
Method.
It constructs pLGZ920-icdh plasmid (such as Fig. 2), process includes: (1) synthesis with SacI and BamHI restriction enzyme site
Primer:
Upstream primer: 5 '-CGGGATCCCAAACCAGTAGCGCTCGAAGGAGAG-3 '；
Downstream primer: 5 '-CGAGCTCCGCCCGTTGCAATATTAAACACATG-3 '；
(2) using E.coli BL21 genomic DNA as template, PCR amplification target gene fragment, reaction condition are as follows: 94 DEG C,
10min；(94 DEG C of 45s, 62 DEG C of 45s, 72 DEG C of 80s, 35 circulations)；72 DEG C, 10min.
After the icdh gene that purifying amplifies (such as Fig. 4), expression plasmid pLGZ920 SacI and BamHI double digestion, connection
It obtains recombinant plasmid pLGZ920-icdh (such as Fig. 6).
Embodiment 2
This example demonstrates that the expression plasmid of building coexpression isocitric dehydrogenase and citrate synthetase, restores wild
Type Actinobacillus succinogenes synthesize the ability of glutamic acid in synthetic media, obtain the method that recombinant type bacterial strain produces butyric acid.
1, the expression plasmid (such as Fig. 3) of building coexpression isocitric dehydrogenase and citrate synthetase, process include:
(1) synthesis upstream and downstream all has the primer of the homologous recombination of the one-step cloning of BamHI restriction enzyme site,
Upstream primer: 5 '-ATGAGGTGATCTAGAGGATCCCAGGTTGATGTGCGAAGGC-3 '；
Downstream primer: 5 '-GCGCTACTGGTTTGGGATCCTCTATTAAAGGCGGGTCCGGAAAG-3 '；
(2) using E. coli BL21 genomic DNA as template, PCR amplification target fragment, reaction condition are as follows: 94
DEG C, 10min；(94 DEG C of 45s, 55 DEG C of 45s, 72 DEG C of 100s, 35 circulations)；72 DEG C, 10min.Purify the cs (such as Fig. 5) amplified
After gene, recombinant plasmid pLGZ920-icdh- is obtained after plasmid pLGZ920-icdh BamHI single endonuclease digestion, one-step cloning recombination
Cs (such as Fig. 7).
2, plasmid pLGZ920-icdh-cs is imported to the competence bacterial strain of Actinobacillus succinogenes NJ113, the sun of acquisition
Property transformant is the recombinant type bacterial strain BD106 newly constructed of the invention.
Embodiment 3
This example demonstrates that recombination Actinobacillus succinogenes BD106 and starting strain NJ113 that overexpression newly constructs
The comparison of consumption sugar and acid producing ability of the glutamic acid in synthetic media is not added.
Actinobacillus succinogenes NJ113 co-expresses isocitric dehydrogenase after importing plasmid pLGZ920-icdh-cs
Its ability that glutamic acid is synthesized in synthetic media is restored with citrate synthetase.
Seed culture: Actinobacillus succinogenes BD106 is activated in plate, 37 DEG C, is incubated overnight in anaerobic box.It will
The bacterial strain activated in plate is linked into serum bottle, and 37 DEG C, 200r/min cultivates 12 hours, as first order seed.By one
Grade seed is linked into serum bottle with 6% inoculum concentration, and 37 DEG C, 200r/min cultivates 12 hours, as secondary seed.Serum bottle
Middle seed culture medium is passed through CO22min, 37 DEG C of cultivation temperature, incubation time 12h；
Fermentation succinic acid-producing: seed culture medium is inoculated into anaerobic fermentation in anaerobic fermentation culture medium, inoculum concentration volume ratio
6%, fill CO22min, 37 DEG C of fermentation temperature, fermentation time 72h.
The fermentation culture medium of anaerobism serum bottle are as follows: fermentation medium+glucose (30g/L)+basic magnesium carbonate 0.48g/L
+ Amp (30 μ g/mL of ampicillin).
Fermentation medium: CH3COONa 1.36g/L, NaCl 1g/L, MgCl20.2g/L, CaCl20.2g/L, Na2HPO4
0.31g/L, NaH2PO41.6g/L, K2HPO43g/L, NH4HCO31.57g/L, biotin (D-biotin) 0.01g/L, niacin
7.0,121 DEG C of sterilizing 15min of (L-nicotinic acid) 0.025g/L, methionine (L-methionine) 0.11g/L, pH.
The measurement result of various parameters is shown in Table 1 after anaerobism serum bottle culture.
The measurement result of each parameter after 1 anaerobism serum bottle culture of table
Fermentation 72h in anaerobism serum bottle, the glucose for consuming 27g/L produce the succinic acid of 19.53g/L, and succinic acid produces
Rate reaches 72.33%
Embodiment 4
This example demonstrates that recombination Actinobacillus succinogenes BD106 and starting strain NJ113 that overexpression newly constructs
The comparison of consumption sugar and acid producing ability of the glutamic acid in synthetic media is not added.
The building mode of bacterial strain is the same as embodiment 3.
Seed culture: Actinobacillus succinogenes BD106 is activated in plate, 35 DEG C, is incubated overnight in anaerobic box.It will
The bacterial strain activated in plate is linked into serum bottle, and 35 DEG C, 200r/min cultivates 11 hours, as first order seed.By one
Grade seed is linked into serum bottle with 8% inoculum concentration, and 35 DEG C, 200r/min cultivates 11 hours, as secondary seed.Serum bottle
Middle seed culture medium is passed through CO22min, 35 DEG C of cultivation temperature, incubation time 11h；
Fermentation succinic acid-producing: seed culture medium is inoculated into anaerobic fermentation in anaerobic fermentation culture medium, inoculum concentration volume ratio
8%, fill CO22min, 35 DEG C of fermentation temperature, fermentation time 48h.
The fermentation culture medium of anaerobism serum bottle are as follows: fermentation medium+glucose (30g/L)+basic magnesium carbonate 0.48g/L
+ Amp (30 μ g/mL of ampicillin).
Fermentation medium: CH3COONa 1.36g/L, NaCl 1g/L, MgCl20.2g/L, CaCl20.2g/L, Na2HPO4
0.31g/L, NaH2PO41.6g/L, K2HPO43g/L, NH4HCO31.57g/L, biotin (D-biotin) 0.01g/L, niacin
7.0,121 DEG C of sterilizing 15min of (L-nicotinic acid) 0.025g/L, methionine (L-methionine) 0.11g/L, pH.
The measurement result of various parameters is shown in Table 2 after anaerobism serum bottle culture.
The measurement result of each parameter after 2 anaerobism serum bottle culture of table
Therefore the fermentation 48h in anaerobism serum bottle, the glucose for consuming 20g/L produce the succinic acid of 11.35g/L, fourth two
Sour yield is 56.75%
Embodiment 5
This example demonstrates that recombination Actinobacillus succinogenes BD106 and starting strain NJ113 that coexpression newly constructs are not
Add the comparison of consumption sugar and acid producing ability of the glutamic acid in synthetic media.
The building mode of bacterial strain is the same as embodiment 3.
Seed culture: Actinobacillus succinogenes BD106 is activated in plate, 36 DEG C, is incubated overnight in anaerobic box.It will
The bacterial strain activated in plate is linked into serum bottle, and 36 DEG C, 200r/min cultivates 10 hours, as first order seed.By one
Grade seed is linked into serum bottle with 10% inoculum concentration, and 36 DEG C, 200r/min cultivates 10 hours, as secondary seed.Serum
Seed culture medium is passed through CO in bottle22min, 36 DEG C of cultivation temperature, incubation time 10h；
Fermentation succinic acid-producing: seed culture medium is inoculated into anaerobic fermentation in anaerobic fermentation culture medium, inoculum concentration volume ratio
10%, fill CO22min, 36 DEG C of fermentation temperature, fermentation time is for 24 hours.
The fermentation culture medium of anaerobism serum bottle are as follows: fermentation medium+glucose (30g/L)+basic magnesium carbonate 0.48g/L
+ Amp (30 μ g/mL of ampicillin).
Fermentation medium: CH3COONa 1.36g/L, NaCl 1g/L, MgCl20.2g/L, CaCl20.2g/L, Na2HPO4
0.31g/L, NaH2PO41.6g/L, K2HPO43g/L, NH4HCO31.57g/L, biotin (D-biotin) 0.01g/L, niacin
7.0,121 DEG C of sterilizing 15min of (L-nicotinic acid) 0.025g/L, methionine (L-methionine) 0.11g/L, pH.
The measurement result of various parameters is shown in Table 3 after anaerobism serum bottle culture.
The measurement result of each parameter after 3 anaerobism serum bottle culture of table
Therefore the fermentation in anaerobism serum bottle is for 24 hours, the glucose for consuming 12g/L produces the succinic acid of 5.76g/L, and succinic acid produces
Rate is 48%.
Claims (3)
1. a kind of method of fermentation production of succinic acid, which is characterized in that the method is carried out in two steps, specific as follows:
The building of first step recombinant bacterial strain: include the following steps:
(1) with the Actinobacillus succinogenes of deposit number CGMCC No.1716 (Actinobacillus succinogenes)
NJ113 is starting strain, prepares A.succinogenes competence bacterial strain by electrotransformation；As thallus OD660Value is in 0.3-
When between 0.7, it is centrifuged 10min under 4100r/min revolving speed, is washed 3 times with the sucrose cushion solution of 272mM；Electrotransformation condition
For 2.5Kv, 25uF, 200 Ω, carried out in 0.2cm electricity revolving cup；
(2) 5 ' end of synthesis a pair has the primer of restriction enzyme site, and using E.coli BL21 genomic DNA as template, purifying is amplified
Isocitric acid dehydrogenase gene icdh gene after, by expression plasmid pLGZ920 with consistent with restriction enzyme site designed by primer
Enzyme double digestion, connection obtain recombinant plasmid pLGZ920-icdh；
(3) 5 ' end of synthesis a pair has the primer of the homologous recombination of BamHI restriction enzyme site, is with E.coli BL21 genomic DNA
Template, purifying amplify citrate synthetase cs gene after, with the recombinant plasmid pLGZ920-icdh of building with set by primer
The consistent enzyme single endonuclease digestion of the restriction enzyme site of meter, the recombinant plasmid pLGZ920-icdh-cs that the connection of one-step cloning method obtains；
(4) by the competence bacterial strain that plasmid steps for importing (1) obtains described in step (3), positive transformant is obtained, it is as described
Recombinant bacterial strain；
Second step recombinant bacterial strain fermentation: recombinant bacterial strain is activated in plate, is incubated overnight in anaerobic box, after be forwarded to anaerobism hair
Anaerobic fermentation in ferment culture medium.
2. the method for fermentation production of succinic acid according to claim 1, which is characterized in that the specific step of recombinant bacterial strain fermentation
It is rapid as follows:
Seed culture: recombinant bacterial strain being activated in plate, 35-37 DEG C, is incubated overnight in anaerobic box；By what is activated in plate
Bacterial strain is linked into serum bottle, and 35-37 DEG C, 200r/min cultivates 10-12 hour, as first order seed；By first order seed with
6%-10% inoculum concentration is linked into serum bottle, and 35-37 DEG C, 200r/min cultivates 10-12 hour, as secondary seed；Blood
Seed culture medium is passed through CO in clear bottle2, 35-37 DEG C of cultivation temperature, incubation time 10-12h；
Fermentation succinic acid-producing: seed culture medium is inoculated into anaerobic fermentation in anaerobic fermentation culture medium, inoculum concentration volume ratio 6%-
10%, fill CO2, 35-37 DEG C of fermentation temperature, fermentation time 24-72h.
3. the method for fermentation production of succinic acid according to claim 1, which is characterized in that the anaerobic fermentation culture medium are as follows:
Fermentation medium+30 μ g/mL of glucose 30g/L+ basic magnesium carbonate 0.48g/L+ ampicillin；The fermentation medium is prepared
Are as follows: CH3COONa 1.36g/L, NaCl 1g/L, MgCl20.2g/L, CaCl20.2g/L, Na2HPO40.31g/L, NaH2PO4
1.6g/L, K2HPO43g/L, NH4HCO31.57g/L, biotin 0.01g/L, niacin 0.025g/L, methionine 0.11g/L, pH
7.0,121 DEG C of sterilizing 15min.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1884484A (en) * | 2006-06-14 | 2006-12-27 | 南京工业大学 | Succinic acid-producing strain and its screening method and uses |
| CN101712970A (en) * | 2009-12-29 | 2010-05-26 | 南京工业大学 | Method for preparing butanedioic acid through fermentation |
| CN102605009A (en) * | 2012-03-23 | 2012-07-25 | 南京工业大学 | Method for improving strength and concentration of butane diacid generated from anaerobic fermentation |
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2015
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1884484A (en) * | 2006-06-14 | 2006-12-27 | 南京工业大学 | Succinic acid-producing strain and its screening method and uses |
| CN101712970A (en) * | 2009-12-29 | 2010-05-26 | 南京工业大学 | Method for preparing butanedioic acid through fermentation |
| CN102605009A (en) * | 2012-03-23 | 2012-07-25 | 南京工业大学 | Method for improving strength and concentration of butane diacid generated from anaerobic fermentation |
Non-Patent Citations (3)
| Title |
|---|
| Insights into Actinobacillus succinogenes Fermentative Metabolism in a Chemically Defined Growth Medium;James B. McKinlay;《 APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;20051130;6651-6656 |
| 从原料到产品：生物基丁二酸研究进展;税宗霞等;《应用与环境学报》;20150225;13 |
| 发酵法生产丁二酸的化学合成培养基的筛选;马江锋等;《生物加工过程》;20130915;45-48 |
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