CN102643774B - Gene engineering bacterium for producing succinic acid and method for producing succinic acid by fermentation of gene engineering bacterium - Google Patents
Gene engineering bacterium for producing succinic acid and method for producing succinic acid by fermentation of gene engineering bacterium Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, and relates to a succinic acid genetic engineering bacterium and a method for producing succinic acid by fermentation. The succinic acid-producing genetic engineering strain is classified and named as Escherichia coli BA306, and the preservation number is CCTCC NO: and M2012103. The construction process mainly comprises inactivating or knocking out phosphoenolpyruvate carboxylase genes and ptsG genes in a phosphate transport system, and co-expressing phosphoenolpyruvate carboxylase and nicotinic acid phosphoribosyltransferase in excess, so that the recombinant escherichia coli can efficiently utilize monosaccharides such as glucose, xylose, arabinose and fructose, and simultaneously efficiently utilize mixed sugars and cellulose hydrolysate in various proportions to grow, and the synthesis efficiency of succinic acid is greatly improved. The fermentation method adopts a two-stage fermentation mode, biomass is improved in an aerobic stage, and acid is produced in an anaerobic stage.
Description
Technical field
The invention belongs to technical field of bioengineering, the method that relates to a strain succinic acid-producing genetic engineering bacterium and fermentation production of succinic acid thereof, specifically a plant height effect is utilized the monose such as glucose, wood sugar, pectinose and fructose, and efficiently utilizes various ratio mixing sugar and cellulosic hydrolysate growth succinic acid-producing recombinant bacterial strain and utilize this strain fermentation to produce the method for succinic acid.
Background technology
Succinic acid (succinic acid) claim again succsinic acid, be widely used in the industries such as medicine, agricultural chemicals, dyestuff, spices, paint, food and plastics, as C4 hardware and software platform compound, can be used for synthetic 1, organic chemicals and poly butylene succinate (PBS) the class Biodegradable materials such as 4-butyleneglycol, tetrahydrofuran (THF), gamma-butyrolactone, thought one of biorefinery product of following 12 kinds of most worthies by USDOE.
The production method of succinic acid mainly comprises chemical synthesis and microbe fermentation method, utilizes microbe fermentation method to transform renewable resources, because raw material sources are extensive and cheap, pollute little, environmental friendliness, and can absorb fixation of C O during the fermentation
2, can effectively alleviate Greenhouse effect, opened up the new way that greenhouse gases carbonic acid gas utilizes, become the focus of research this year.The production bacterial strain of succinic acid mainly concentrates on Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens, restructuring Corynebacterium glutamicum and recombination bacillus coli.Although wherein utilize wild strain to produce succinic acid, obtained higher production concentration, culturing process culture medium cost is higher, and the byproducts build-up such as formic acid, acetic acid is more, has hindered its process of industrialization.And recombination bacillus coli because genetic background is clear, easy to operate, easy-regulating, substratum require the simple and advantage such as rapid of growing, and is widely used in recent years research to obtain the outstanding bacterial strain of succinic acid-producing.
The structure thinking of existing succinic acid-producing recombination bacillus coli mainly comprises that inactivation by product generates the key enzyme (as pyruvate formate-lyase and serum lactic dehydrogenase) of approach, the activity that strengthens enzyme (as phosphoric acid enol pyruvic acid carboxylase) in succinic acid route of synthesis and external source importing and can guide the enzyme (as pyruvate carboxylase) of synthesizing succinic acid to improve it to the utilization ratio of glucose and throughput rate.Wherein, E.coli NZN111 due to while inactivation pyruvate formate-lyase and serum lactic dehydrogenase, NADH can not be regenerated as NAD in time
+, cause the imbalance (NADH/NAD of coenzyme NAD (H) in born of the same parents
+ratio surpasses 2), finally cause bacterial strain under anaerobic condition can not utilize glucose.Its spontaneous mutation strain E.coli AFP111 is due to the ptsG gene having suddenlyd change in glucose obligate movement system, reduced the generation speed of NADH in EMP Embden Meyerbof Parnas pathway, recovered NAD (H) balance, make bacterial strain under anaerobic can utilize glucose, and product is mainly succinic acid, at aerobic anaerobism two stage fermentations, cultivate in AFP11 1 process, succinic acid mass yield reaches 96%, and production intensity is 1.21 g L
-1h
-1.Therefore,, in high succinic acid-producing coli strain building process, guarantee that the balance of coenzyme NAD (H) in born of the same parents is one of key factor of the high succinic acid-producing of recombination bacillus coli.
The biosynthesizing of NAD in intestinal bacteria (H) and decomposition approach as shown in Figure 1, relate to its synthetic gene and mainly contain three (pncB, nadD, nadE), and relate to catabolic gene and mainly contain two (viaD, yrfE), and NAD
+reach more than 300 with NADH conversion reaction each other.Correlative study shows, utilizes DNA recombinant technology to transform the effective means that NAD (H) biosynthetic pathway is raising NAD (H) total amount.The people such as San (Metab Eng, 2002,4:238-247; Metab Eng, 2002,4:182-192), in research cofactor regulates and controls the influence process of Metabolism of E. coli flow point cloth, by overexpression nicotinic acid phosphoribosyl transferase, make NAD in born of the same parents (H) total amount improve 41.7%; The people such as Heuser (Eng Life Sci, 2007,7:343-353) by overexpression nicotinic acid phosphoribosyl transferase and NAD synthase, or express this two enzymes simultaneously, NAD (H) total amount in bacterial strain born of the same parents has been improved more than 2 times, and apply it in synthetic (R)-methyl-3-hydroxyl butylamine process of enzymatic conversion, make the amount of NAD (H) no longer become limiting factor, thereby improved the efficiency of enzymatic conversion.Numerous scientific practices also prove and utilize fermentation control means can effectively regulate NAD (H) total amount and NADH/NAD
+ratio, and then effectively improve utilization ratio and the product production level of substrate.Utilizing Saccharomyces cerevisiae TMB3001 (Biotechnol Bioeng, 2002,78:172-178) with Fusarium oxysporum (J Biosci Bioeng, 2004,97:299-304.Enzyme Micro Technol, 2005,36:100-106) in the process of xylose-fermenting production ethanol, add acetoin as external source electron acceptor(EA), effectively increased NAD in born of the same parents
+content, has improved the productive rate of ethanol; The people such as San (Metab Eng, 2002,4:182-192), utilizing intestinal bacteria to produce in 1,2-PD process, find to be 0.1 h in thinning ratio
-1in perseveranceization anaerobism culture systems, along with the increase of carbon source reductibility, NADH/NAD in born of the same parents
+ratio is increased to 0.75 (glucose) and 0.94 (sorbyl alcohol) from 0.51 (gluconic acid), and causes center metabolism apoblema ethanol (consuming 2 mol NADH) to be respectively 0.29,1 and 3.62 to the ratio of acetic acid (not consuming NADH).
PTS movement system is the main transporting mode of glucose transport in intestinal bacteria, but the existence of PTS movement system, make intestinal bacteria can not utilize glucose and other various monose metabolism growth simultaneously, and wild-type e. coli can utilize respectively under anaerobic metabolism growth of the monose such as glucose, wood sugar, fructose, pectinose, but succinic acid is not its main metabolites, for reducing by product, generate, intestinal bacteria knock out or inactivation serum lactic dehydrogenase (LDH) gene, and pyruvate formate-lyase (PFL) gene activity reduces the generation of by product.But when take glucose or fructose during as carbon source, owing to knocking out serum lactic dehydrogenase (LDH) gene and pyruvate formate-lyase (PFL) gene, cause bacterial strain coenzyme uneven, and a large amount of pyruvic acid have been accumulated, cause thalline can not utilize metabolism growth under glucose and fructose anaerobic condition, and produce succinic acid; And take wood sugar or pectinose during as carbon source, owing to knocking out serum lactic dehydrogenase (LDH) gene and pyruvate formate-lyase (PFL) gene, reduced the generation of pyruvic acid and acetic acid, thereby the ATP that follows pyruvic acid and acetic acid to generate is reduced, finally cause recombination bacillus coli can not utilize wood sugar and pectinose metabolism growth, and produce succinic acid.
Corn cob is waste more common in agriculture production, because its composition contains a large amount of Mierocrystalline celluloses, therefore its hydrolyzed solution is concerning microorganism fermentation, it is a kind of green carbon source of good sustainable use, but its dilute acid hydrolysis liquid contains high density wood sugar, so in prior art, most of succinic acid-producing intestinal bacteria can not utilize Corncob hydrolysate fermentation production of succinic acid.Jiang Min etc. press solid-liquid ratio 1:5 (mass volume ratio) preparation corn cob feed liquid, material particular diameter 250～380 μ m, H by corn cob
2sO
4consumption 3% (volume fraction), 126 ℃ of hydrolysis temperatures, reaction times 215 h, utilizes charcoal absorption and Ca (OH)
2the modes such as neutralization, carry out detoxification desalting treatment to corn cob polycomponent liquid glucose, and total Sugar concentration is 50 g/L, and wherein wood sugar accounts for more than 80%.
Rice straw is an important class renewable biomass resource.At present, except the utilization in paper-making industry industrial aspect, the overwhelming majority goes out of use, serious waste resource and polluted environment.Its main component is Mierocrystalline cellulose, hemicellulose and xylogen, therefore its hydrolyzed solution is concerning microorganism fermentation, it is a kind of green carbon source of good sustainable use, but its hydrolyzed solution contains high density wood sugar, therefore in prior art, most of succinic acid-producing intestinal bacteria can not utilize rice straw hydrolyzed solution fermentation production of succinic acid, rice straw 1 h is processed by 121 ℃ of dilute sulphuric acids in the inscription on pottery Yihe River etc., use the NaOH of 20 g/L in 121 ℃ of Treating straw 1h, the two total mass concentration of glucose and xylose reaches 50 g/L left and right again.
When being cane sugar manufacture, squeezes bagasse sugar remaining main component afterwards, therefore its hydrolyzed solution is concerning microorganism fermentation, it is a kind of green carbon source of good sustainable use, but its hydrolyzed solution contains high density wood sugar, therefore in prior art, most of succinic acid-producing intestinal bacteria can not utilize rice straw hydrolyzed solution fermentation production of succinic acid, approximately contain 50% Mierocrystalline cellulose by pulverize and the pre-treatment of alkali/oxidation style can to obtain total reducing sugar quality be 50 g/L, wherein wood sugar accounts for more than 80%.
Molasses are in industrial sugaring process, after crystallization of sucrose, remaining uncrystallizable, but still contain the liquid residue compared with polysaccharide.Yu Wei etc. mix molasses and water 1:1, with the vitriol oil (5 mol/L), its pH are adjusted to 2.0, in 100 ℃ of heating 20 min, and process with 15% lime slurry neutralization, and total reducing sugar quality is 600 g/L, and glucose and fructose respectively account for 50%.
In intestinal bacteria phosphoenolpyruvic acid, by phosphoric acid enol pyruvic acid carboxylase (PPC), generate oxaloacetic acid, in this process, there is no the generation of ATP, but in Bacillus subtilis, phosphoenolpyruvic acid generates oxaloacetic acid by phosphoenolpyruvic acid carboxylation kinases (PCK), in this process, there is the generation of ATP, and Millard etc. are overexpression E.coli ppc and pck in intestinal bacteria, research finds that overexpression ppc can make succsinic acid as the primary product of mixed acid fermentation, and output improves 3.5 times compared with starting strain, and overexpression pck is on not impact of fermentation results, but in ppc defect bacterial strain, the overexpression of pck can improve the output of succsinic acid, this is the K due to PPC
mtherefore less 100 times than PCK, only in ppc defect bacterial strain, the overexpression of pck can improve the output of succsinic acid.
Summary of the invention
Technical purpose of the present invention is to provide a plant height effect to utilize the monose such as glucose, wood sugar, pectinose and fructose, and efficiently utilize various ratio mixing sugar and cellulosic hydrolysate growth succinic acid-producing recombinant bacterial strain and construction process thereof, and utilize this bacterial strain anaerobically fermenting to produce succinic acid, the construction process that reaches bacterial strain is simple and convenient, the strain fermentation method simple possible that structure obtains, be easy to industrialization, the object that acid producing ability is strong, thereby greatly reduce production costs, increase economic efficiency.
For realizing technical purpose of the present invention, the present invention by the following technical solutions.
One, the invention provides a strain succinic acid-producing genetically engineered bacteria strain, its Classification And Nomenclature is colon bacillus (Escherichia coli) BA306, and its preserving number registration number is CCTCC NO:M2012103.
Two, the construction process of colon bacillus BA306 of the present invention, it is characterized in that lacking serum lactic dehydrogenase (LDH) gene, the bacterial strain intestinal bacteria of pyruvate formate-lyase (PFL) gene activity are starting strain, utilize homologous recombination technique to knock out the ptsG gene in phosphoric acid enol pyruvic acid carboxylase (PPC) gene and phosphoric acid movement system (PTS), and after excessive coexpression phosphoenolpyruvic acid carboxylation kinases and nicotinic acid phosphoribosyltransferase, obtain efficiently utilizing glucose, wood sugar, the monose such as pectinose and fructose, and efficiently utilize the colon bacillus BA306 of various ratio mixing sugar and cellulosic hydrolysate growth succinic acid-producing.
Further, described concrete construction step is as follows:
(1) to lack lactate dehydrogenase gene (ldhA), the active E.coli NZN111 bacterial strain of pyruvate formate-lyase gene (pflB) is starting strain, knock out the ptsG gene in wherein phosphoric acid enol pyruvic acid carboxylase (PPC) gene and PTS movement system, obtain lacking the competence bacterial strain of ldhA, pflB, ppc and ptsG simultaneously;
(2) purifying amplifies phosphoenolpyruvic acid carboxylation kinase gene (pck), builds and obtains the kinase whose expression plasmid of overexpression phosphoenolpyruvic acid carboxylation;
(3) purifying amplifies nicotinic acid phosphoribosyltransferase gene (pncB), is connected on the described recombinant plasmid of step (2), builds the expression plasmid that obtains excessive coexpression phosphoenolpyruvic acid carboxylation kinases and nicotinic acid phosphoribosyltransferase;
(4) plasmid step (3) Suo Shu is imported to the competence bacterial strain that step (1) obtains, obtain positive transformant;
(5) utilize the excessive coexpression phosphoenolpyruvic acid of positive transformant carboxylation kinases and the nicotinic acid phosphoribosyltransferase of step (4), recover its under anaerobic metabolism, obtain efficiently utilizing the monose such as glucose, wood sugar, pectinose and fructose, and efficiently utilize the colon bacillus BA306 of various ratio mixing sugar and cellulosic hydrolysate growth succinic acid-producing.
Three, utilize the method for colon bacillus BA306 fermentation production of succinic acid of the present invention, it is characterized in that adopting two stage fermentation modes, the aerobic stage is improved biomass, anaerobic stages fermentation and acid.
Further, concrete steps are as follows.
Colon bacillus BA306 is inoculated to aerobic in aerobic stage fermentation substratum by 1% (v/v) inoculum size and cultivate, when aerobic is cultivated thalline OD
600iPTG to 0.4～0.6 use 0.3 mM is induced to OD
600, by inoculum size 10% (v/v), be forwarded to anaerobically fermenting in anaerobic stages fermention medium at=3 o'clock.
Wherein said aerobic stage fermentation substratum is that in prior art, aerobic is cultivated the colibacillary conventional medium of succinic acid-producing; Carbon source in described anaerobic stages fermention medium includes but not limited to glucose, wood sugar, pectinose, fructose or its combination; Also comprise Corncob hydrolysate, rice straw hydrolyzed solution, molasses hydrolyzed solution or bagasse hydrolyzed solution.
Beneficial effect of the present invention is:
First, the present invention is to lack serum lactic dehydrogenase (LDH) gene, the bacterial strain intestinal bacteria NZN111 of pyruvate formate-lyase (PFL) gene activity is starting strain, utilize homologous recombination technique to knock out the ptsG gene in phosphoric acid enol pyruvic acid carboxylase (PPC) gene and PTS movement system, and excessive coexpression phosphoenolpyruvic acid carboxylation kinases and nicotinic acid phosphoribosyltransferase, make it efficiently utilize the monose such as glucose, wood sugar, pectinose and fructose, and efficiently utilize various ratio mixing sugar and cellulosic hydrolysate growth succinic acid-producing.Thisly by molecular biology method, improve strains A TP biosynthesis ability, improve NAD (H) generative capacity, change NADH/NAD in born of the same parents
+efficiently utilize the monose such as glucose, wood sugar, pectinose and fructose, make bacterial strain can efficiently utilize various ratio mixing sugar and cellulosic hydrolysate metabolism growth, a large amount of raising output of succinic acid and the method for throughput have no open, and this application will advance progress and the development of succinic acid industry greatly.
Secondly, utilize the fermentation results of bacterial strain colon bacillus BA306 of the present invention to show that the new recombination bacillus coli colon bacillus BA306 building can efficiently utilize the monose such as glucose, wood sugar, pectinose and fructose, and efficiently utilize various ratio mixing sugar and cellulosic hydrolysate fermentation, and accumulate in a large number succinic acid, compare starting strain, can not utilize the monose such as glucose, wood sugar, pectinose and fructose, various ratio mixing sugar and cellulosic hydrolysate fermentation, the feature that there is no succinic acid accumulation, it is huge that it produces sour changing features.
Finally, produce in a large number the by product to the toxic effect of bacterial strain such as acetic acid etc. in anaerobic fermentation process, therefore consider two stage fermentation modes, the aerobic stage is improved biomass, and anaerobic stages carries out acidogenic fermentation.Also can optionally adopt membrane separation technique, reach the object of separating thallus, then and then for anaerobically fermenting.
Accompanying drawing explanation
The biosynthesizing of NAD in Fig. 1 intestinal bacteria (H) and decomposition approach.
The electrophoresis of the linear DNA fragment of Fig. 2 embodiment 1 is identified figure.
The electrophoresis of the homologous recombination positive recombinant of Fig. 3 embodiment 1 is identified figure.
The electrophoresis of the linear DNA fragment of Fig. 4 embodiment 2 is identified figure.
The electrophoresis of the homologous recombination positive recombinant of Fig. 5 embodiment 2 is identified figure.
The double digestion electrophoresis of Fig. 6 plasmid pTrc99a-pck is identified figure.
The double digestion electrophoresis of Fig. 7 plasmid pTrc99a-pck-pncB is identified figure.
The Classification And Nomenclature of microorganism of the present invention is colon bacillus (Escherichia coli) BA306, its preservation date is on April 8th, 2012, depositary institution's full name is Chinese Typical Representative culture collection center, referred to as CCTCC, address is: China. Wuhan. and Wuhan University, deposit number: CCTCC NO:M 2012103.
Embodiment
The following examples elaborate to the present invention, but to not restriction of the present invention.
The explanation in the source of biomaterial of the present invention:
1, the source of apramycin resistance gene of the present invention is: pIJ773, the azure professor of Shao of Nanjing Normal University is so kind as to give.
2, the source of plasmid that can abduction delivering λ recombinase of the present invention is: pKD46, and purchased from Introvegen company.
3, the source of the plasmid that produces FLP recombinase of can inducing of the present invention is: pCP20, and purchased from Introvegen company.
4, the genomic source of Bacillus subtilis of the present invention is: purchased from Chinese Typical Representative culture collection center.
5, expression plasmid of the present invention with the source of pTrc99a is: purchased from Introvegen company.
There are two places in the source of the competence bacterial strain of 6, starting strain: E.coli NZN1 11:
（1）Biotechnol?Bioeng,2001,74:89～95。Applicant is first by finding the above-mentioned document source of this biomaterial, and to have contacted utterer be the David P.Clark professor of Univ Chicago USA, and its this biomaterial of gifting of mail requests, and freely obtained this biomaterial; And applicant guaranteed in 20 years, to the public, to provide this biomaterial from the application's day;
(2) this biomaterial also discloses and obtains the authorization in the patent documentation of Chinese patent (application number 96198547.X, applying date 1996.10.31 authorize day on January 1st, 2003, Granted publication CN1097632C).
7, the design of primer of the present invention and synthetic: designed, designed outer Si Rui covered with gold leaf biotech company are synthetic.
The present embodiment explanation utilizes homologous recombination technique to knock out phosphoric acid enol pyruvic acid carboxylase ppc gene in starting strain NZN111, the process of the apramycin resistant strain that is eliminated.
1, utilize LB substratum, under 37 ℃, aerobic conditions, cultivate intestinal bacteria NZN111 to OD
600=0.4～0.6, be prepared into electricity and turn competence.
2, plasmid pKD46 electricity is proceeded to competent intestinal bacteria NZN1 11.Electric shock condition is: 200 Ω, 25 μ F, electric shock voltage 2.3 kV, electric shock times 4～5 ms.After electric shock, rapidly thalline is added to the SOC substratum of precooling 1 mL, the LB culture medium flat plate of coating band penbritin (amp) after 150 r/min, 30 ℃ of cultivation 1 h filters out positive transformant intestinal bacteria NZN1 11 (pKD46).
3, the L-arabinose that adds 10 mM in LB substratum induces plasmid pKD46 to give expression to λ recombinase at 30 ℃, makes electricity and turns competence.
4, take both sides is template with the apramycin resistance gene in FRT site, utilizes High fidelity PCR amplification system, take plasmid pIJ773 as template, and designs two ends with the amplimer of PPC homologous fragment, amplifies linear DNA homologous fragment, and primer sequence is as follows:
Upstream band homology arm primer H1-P1, underscore is homologous fragment:
5’-
ATGAACGAACAATATTCCGCATTGCGTAGTAATGTCAGTATGCTC
GGCATTCCGGGGATCCGTCGACC-3’。
Downstream band homology arm primer H2-P2, underscore is homologous fragment:
5’-
AGCACGAGGGTTTGCAGAAGAGGAAGATTAGCCGGTATTACGCAT
ACCTGTAGGCTGGAGCTGCTTC-3’。
Reaction system: with each 0.5 μ L of upstream and downstream primer (100 pmol/ μ L) of homology arm; Template DNA (100ng/ μ L) 0.5 μ L; 10 * buffer, 5 μ L; Each 1 μ L of dNTPs (10 mM); DMSO (100%) 2.5 μ L; Pyrobest archaeal dna polymerase (2.5 U/ μ L) 1 μ L; ddH
2o 36/35.5 μ L; Cumulative volume 50 μ L.
Reaction conditions: 94 ℃, 2 min; (94 ℃ of 45 sec; 50 ℃ of 45 sec; 72 ℃ of 90 sec; 10 circulations); (94 ℃ of 45 sec; 50 ℃ of 45 sec; 72 ℃ of 90 sec; 15 circulations); 72 ℃, 5 min.
The evaluation of linear DNA fragment is as Fig. 2.
5, electricity turns linear DNA fragment intestinal bacteria NZN1 11 (pKD46) competence of abduction delivering λ recombinase extremely, and coats with the LB flat screen of apramycin and select positive recombinant, and has carried out PCR evaluation, and electrophorogram as shown in Figure 3.
6, positive recombinant make pour into after competence can abduction delivering FLP recombinase plasmid pCP20, after FLP recombinase is expressed in 42 ℃ of heat shocks, can eliminate apramycin resistance.Utilize pair of plates, carry out parallel point sample, can on non-resistant flat board, grow, but can not in resistant panel, grow all very knocked out the bacterial strain of resistance.
The bacterial strain that knocks out ppc gene that the present embodiment explanation utilizes case study on implementation 1 to obtain, utilizes homologous recombination technique to knock out ptsG gene in PTS movement system, the process of the apramycin resistant strain that is eliminated again.
1, utilize LB substratum, under 37 ℃, aerobic conditions, cultivate case study on implementation 1 has obtained the bacterial strain that knocks out ppc gene to OD
600=0.4～0.6, be prepared into electricity and turn competence.
2, plasmid pKD46 electricity is proceeded in this competent cell.Electric shock condition is: 200 Ω, 25 μ F, electric shock voltage 2.3 kV, electric shock times 4～5 ms.After electric shock, rapidly thalline is added to the SOC substratum of precooling 1 mL, the LB culture medium flat plate of coating band penbritin (amp) after 150 r/min, 30 ℃ of cultivation 1 h filters out positive transformant intestinal bacteria NZN1 11/ △ ppc (pKD46).
3, the L-arabinose that adds 10 mM in LB substratum induces plasmid pKD46 to give expression to λ recombinase at 30 ℃, makes electricity and turns competence.
4, take both sides is template with the apramycin resistance gene in FRT site, utilize High fidelity PCR amplification system, take plasmid pIJ773 as template, and design two ends with the amplimer of ptsG DNA homolog fragment, amplify linear DNA homologous fragment, primer sequence is as follows:
Upstream band homology arm primer H1-P1, underscore is homologous fragment:
5’-
ATGTTTAAGAATGCATTTGCTAACCTGCAAAAGGTCGGTAAATCGCTG
ATTCCGGGGATCCGTCGACC-3’。
Downstream band homology arm primer H2-P2, underscore is homologous fragment:
5’-
TTAGTGGTTACGGATGTACTCATCCATCTCGGTTTTCAGGTTATCGGA
TGTAGGCTGGAGCTGCTTC-3’。
Reaction system: with each 0.5 μ L of upstream and downstream primer (100 pmol/ μ L) of homology arm; Template DNA (100ng/ μ L) 0.5 μ L; 10 * buffer, 5 μ L; Each 1 μ L of dNTPs (10 mM); DMSO (100%) 2.5 μ L; Pyrobest archaeal dna polymerase (2.5 U/ μ L) 1 μ L; ddH
2o 36/35.5 μ L; Cumulative volume 50 μ L.
Reaction conditions: 94 ℃, 2 min; (94 ℃ of 45 sec; 50 ℃ of 45 sec; 72 ℃ of 90 sec; 10 circulations); (94 ℃ of 45 sec; 50 ℃ of 45 sec; 72 ℃ of 90 sec; 15 circulations); 72 ℃, 5 min.
The evaluation of linear DNA fragment is as Fig. 4.
5, electricity turns linear DNA fragment intestinal bacteria NZN111/ △ ppc (pKD46) competence of abduction delivering λ recombinase extremely, and coats with the LB flat screen of apramycin and select positive recombinant, and has carried out PCR evaluation, and electrophorogram as shown in Figure 5.
6, positive recombinant make pour into after competence can abduction delivering FLP recombinase plasmid pCP20, after FLP recombinase is expressed in 42 ℃ of heat shocks, can eliminate apramycin resistance.Utilize pair of plates, carry out parallel point sample, can on non-resistant flat board, grow, but can not in resistant panel, grow all very knocked out the bacterial strain of resistance.
The present embodiment explanation builds the kinase whose expression plasmid of overexpression phosphoenolpyruvic acid carboxylation.
1, build the kinase whose expression plasmid of overexpression phosphoenolpyruvic acid carboxylation, its process comprises:
(1) the synthetic primer with SacI and XbaI enzyme cutting site,
Upstream primer: 5 '-CGAGCTCATGAACTCAGTTGATTTGACCG-3 ';
Downstream primer: 5 '-GCTCTAGAGCATTCCGTCAATTAAAACAAG-3 '.
(2) take Bacillus subtilis genome is template, pcr amplification goal gene fragment, and reaction conditions is: 94 ℃, 5 min; (94 ℃ of 45 s, 53 ℃ of 45 s, 72 ℃ of 100 s, 35 circulations); 72 ℃, 10 min.After the pck gene that purifying amplifies, expression plasmid with pTrc99a respectively with SacI and XbaI double digestion, is connected acquisition recombinant plasmid pTrc99a-pck.The double digestion electrophoresis of plasmid pTrc99a-pck is identified as shown in Figure 6.
Embodiment 4
The present embodiment explanation builds the expression plasmid of excessive coexpression phosphoenolpyruvic acid carboxylation kinases and nicotinic acid phosphoribosyltransferase, make recombinant bacterium can efficiently utilize the monose such as glucose, wood sugar, pectinose and fructose, and efficiently utilize various ratio mixing sugar and cellulosic hydrolysate fermentation, and accumulate in a large number succinic acid, obtain the method for bacterial strain colon bacillus BA306.
1, the expression plasmid that builds excessive coexpression phosphoenolpyruvic acid carboxylation kinases and nicotinic acid phosphoribosyltransferase, its process comprises:
(1) synthetic upstream and downstream primer is all with the primer of HindIII restriction enzyme site,
Upstream primer: 5 '-CCCAAGCTTATGACACAATTCGCTTCTCCTG-3 '
Downstream primer: 5 '-CCCAAGCTTCACTTGTCCACCCGTAAATGG-3 '
(2) take e. coli k12 series as template, pcr amplification goal gene fragment, reaction conditions is: 94 ℃, 5 min; (94 ℃ of 45 s, 55 ℃ of 45 s, 72 ℃ of 1 min, 35 circulations); 72 ℃, 10 min.After the pncB gene that purifying amplifies, for plasmid pTrc99a-pck, HindIII single endonuclease digestion, connection obtain recombinant plasmid pTrc99a-pck-pncB.The double digestion electrophoresis of plasmid pTrc99a-pck-pncB is identified as shown in Figure 7.
2, plasmid pTrc99a-pck-pncB is imported to the competence bacterial strain that simultaneously lacks ldhA, pflB, ppc and ptsG in case study on implementation 2, the positive transformant of acquisition is new structure bacterial strain Escherichia coliBA306 of the present invention.
Embodiment 5
The contrast of the present embodiment explanation colon bacillus BA306 and starting strain xylose-fermenting acid producing ability.
Colon bacillus BA306 can efficiently utilize wood-sugar fermentation, and accumulates in a large number succinic acid, adopts two stage fermentation modes, it is characterized in that by 1% (v/v) inoculum size from cryopreservation tube access triangular flask, when aerobic is cultivated thalline OD
600to 0.4～0.6 left and right, with the IPTG of 0.3 mM, be induced to OD
600during=3 left and right, by inoculum size 10%, be forwarded to anaerobically fermenting in serum bottle, fermentation 48 h.
Aerobic stage substratum is: LB+Amp (penbritin 50 μ g/mL).
Anaerobic stages substratum is: LB+ wood sugar (35 g/L)+magnesium basic carbonate 0.6 g+Amp (penbritin 50 μ g/mL)+0.3 mM IPTG.
Fermentation results is in Table 1.
The result comparison of table 1 Escherichia coli BA206 and starting strain fermentation and acid
Note: ND represents not detect.
Embodiment 6
The contrast of the present embodiment explanation colon bacillus BA306 and starting strain glucose fermentation acid producing ability.
Colon bacillus BA306 can efficiently utilize glucose fermentation, and accumulates in a large number succinic acid, adopts two stage fermentation modes, it is characterized in that by 1% (v/v) inoculum size from cryopreservation tube access triangular flask, when aerobic is cultivated thalline OD
600to 0.4～0.6 left and right, with the IPTG of 0.3 mM, be induced to OD
600during=3 left and right, by inoculum size 10%, be forwarded to anaerobically fermenting in serum bottle, fermentation 48 h.
Aerobic stage substratum is: LB+Amp (penbritin 50 μ g/mL).
Anaerobic stages substratum is: LB+ glucose (35 g/L)+magnesium basic carbonate 0.6 g+Amp (penbritin 50 μ g/mL)+0.3 mM IPTG.
Fermentation results is in Table 2.
The result comparison of table 2Escherichia coli BA306 and starting strain fermentation and acid
Note: ND represents not detect.
Embodiment 7
The contrast of the present embodiment explanation colon bacillus BA306 and starting strain fermentation pectinose acid producing ability.
Colon bacillus BA306 can efficiently utilize pectinose fermentation, and accumulates in a large number succinic acid, adopts two stage fermentation modes, it is characterized in that by 1% (v/v) inoculum size from cryopreservation tube access triangular flask, when aerobic is cultivated thalline OD
600to 0.4～0.6 left and right, with the IPTG of 0.3 mM, be induced to OD
600during=3 left and right, by inoculum size 10%, be forwarded to anaerobically fermenting in serum bottle, fermentation 48 h.
Aerobic stage substratum is: LB+Amp (penbritin 50 μ g/mL).
Anaerobic stages substratum is: LB+ pectinose (35 g/L)+magnesium basic carbonate 0.6 g+Amp (penbritin 50 μ g/mL)+0.3 mM IPTG.
Fermentation results is in Table 3.
The result comparison of table 3Escherichia coli BA306 and starting strain fermentation and acid
Note: ND represents not detect.
Embodiment 8
The contrast of the present embodiment explanation colon bacillus BA306 and starting strain fermentation fructose acid producing ability.
Colon bacillus BA306 can efficiently utilize fructose fermentation, and accumulates in a large number succinic acid, adopts two stage fermentation modes, it is characterized in that by 1% (v/v) inoculum size from cryopreservation tube access triangular flask, when aerobic is cultivated thalline OD
600to 0.4～0.6 left and right, with the IPTG of 0.3 mM, be induced to OD
600during=3 left and right, by inoculum size 10%, be forwarded to anaerobically fermenting in serum bottle, fermentation 48 h.
Aerobic stage substratum is: LB+Amp (penbritin 50 μ g/mL).
Anaerobic stages substratum is: LB+ fructose (35 g/L)+magnesium basic carbonate 0.6 g+Amp (penbritin 50 μ g/mL)+0.3 mM IPTG.
Fermentation results is in Table 4.
The result comparison of table 4Escherichia coli BA306 and starting strain fermentation and acid
Note: ND represents not detect.
Embodiment 9
The contrast of the present embodiment explanation colon bacillus BA306 and starting strain fermentation total reducing sugar acid producing ability.Colon bacillus BA306 can efficiently utilize mass ratio for glucose: wood sugar: pectinose: the mixing sugar fermentation that fructose is 1:1:1:1, and accumulate in a large number succinic acid, adopt two stage fermentation modes, it is characterized in that by 1% (v/v) inoculum size from cryopreservation tube access triangular flask, when aerobic is cultivated thalline OD
600to 0.4～0.6 left and right, with the IPTG of 0.3 mM, be induced to OD
600during=3 left and right, by inoculum size 10%, be forwarded to anaerobically fermenting in serum bottle, fermentation 48 h.
Aerobic stage substratum is: LB+Amp (penbritin 50 μ g/mL).
Anaerobic stages substratum is: LB+ total reducing sugar (35 g/L)+magnesium basic carbonate 0.6 g+Amp (penbritin 50 μ g/mL)+0.3 mM IPTG.
Fermentation results is in Table 5.
The result comparison of table 5 Escherichia coli BA306 and starting strain fermentation and acid
Note: ND represents not detect.
Embodiment 10
The contrast of the present embodiment explanation colon bacillus BA306 and starting strain fermentation total reducing sugar acid producing ability.
Colon bacillus BA306 can efficiently utilize mass ratio for glucose: wood sugar: pectinose: the mixing sugar fermentation that fructose is 1:2:3:4, and accumulate in a large number succinic acid, adopt two stage fermentation modes, it is characterized in that by 1% (v/v) inoculum size from cryopreservation tube access triangular flask, when aerobic is cultivated thalline OD
600to 0.4～0.6 left and right, with the IPTG of 0.3 mM, be induced to OD
600during=3 left and right, by inoculum size 10%, be forwarded to anaerobically fermenting in serum bottle, fermentation 48 h.
Aerobic stage substratum is: LB+Amp (penbritin 50 μ g/mL).
Anaerobic stages substratum is: LB+ total reducing sugar (35 g/L)+magnesium basic carbonate 0.6 g+Amp (penbritin 50 μ g/mL)+0.3 mM IPTG.
Fermentation results is in Table 6.
The result comparison of table 6 Escherichia coli BA306 and starting strain fermentation and acid
Note: ND represents not detect.
Embodiment 11
The contrast of the present embodiment explanation colon bacillus BA306 and starting strain fermentation total reducing sugar acid producing ability.
Colon bacillus BA306 can efficiently utilize mass ratio for wood sugar: glucose: pectinose: the mixing sugar fermentation that fructose is 1:2:3:4, and accumulate in a large number succinic acid, adopt two stage fermentation modes, it is characterized in that by 1% (v/v) inoculum size from cryopreservation tube access triangular flask, when aerobic is cultivated thalline OD
600to 0.4～0.6 left and right, with the IPTG of 0.3 mM, be induced to OD
600during=3 left and right, by inoculum size 10%, be forwarded to anaerobically fermenting in serum bottle, fermentation 48 h.
Aerobic stage substratum is: LB+Amp (penbritin 50 μ g/mL).
Anaerobic stages substratum is: LB+ total reducing sugar (35 g/L)+magnesium basic carbonate 0.6 g+Amp (penbritin 50 μ g/mL)+0.3 mM IPTG.
Fermentation results is in Table 7.
The result comparison of table 7Escherichia coli BA306 and starting strain fermentation and acid
Note: ND represents not detect.
Embodiment 12
The contrast of the present embodiment explanation colon bacillus BA306 and starting strain fermentation total reducing sugar acid producing ability.
Colon bacillus BA306 can efficiently utilize mass ratio for pectinose: wood sugar: glucose: the mixing sugar fermentation that fructose is 1:2:3:4, and accumulate in a large number succinic acid, adopt two stage fermentation modes, it is characterized in that by 1% (v/v) inoculum size from cryopreservation tube access triangular flask, when aerobic is cultivated thalline OD
600to 0.4～0.6 left and right, with the IPTG of 0.3 mM, be induced to OD
600during=3 left and right, by inoculum size 10%, be forwarded to anaerobically fermenting in serum bottle, fermentation 48 h.
Aerobic stage substratum is: LB+Amp (penbritin 50 μ g/mL).
Anaerobic stages substratum is: LB+ total reducing sugar (35 g/L)+magnesium basic carbonate 0.6 g+Amp (penbritin 50 μ g/mL)+0.3 mM IPTG.
Fermentation results is in Table 8.
The result comparison of table 8 Escherichia coli BA306 and starting strain fermentation and acid
Note: ND represents not detect.
Embodiment 13
The contrast of the present embodiment explanation colon bacillus BA306 and starting strain fermentation total reducing sugar acid producing ability.
Colon bacillus BA306 can efficiently utilize mass ratio for fructose: pectinose: wood sugar: the mixing sugar fermentation that glucose is 1:2:3:4, and accumulate in a large number succinic acid, adopt two stage fermentation modes, it is characterized in that by 1% (v/v) inoculum size from cryopreservation tube access triangular flask, when aerobic is cultivated thalline OD
600to 0.4～0.6 left and right, with the IPTG of 0.3 mM, be induced to OD
600during=3 left and right, by inoculum size 10%, be forwarded to anaerobically fermenting in serum bottle, fermentation 48 h.
Aerobic stage substratum is: LB+Amp (penbritin 50 μ g/mL).
Anaerobic stages substratum is: LB+ total reducing sugar (35 g/L)+magnesium basic carbonate 0.6 g+Amp (penbritin 50 μ g/mL)+0.3 mM IPTG.
Fermentation results is in Table 9.
The result comparison of table 9Escherichia coli BA306 and starting strain fermentation and acid
Note: ND represents not detect.
Embodiment 14
The contrast of the present embodiment explanation colon bacillus BA306 and starting strain fermentation and acid ability.
Colon bacillus BA306 can efficiently utilize Corncob hydrolysate fermentation, and accumulates in a large number succinic acid, adopts two stage fermentation modes, it is characterized in that by 1% (v/v) inoculum size from cryopreservation tube access triangular flask, when aerobic is cultivated thalline OD
600to 0.4～0.6 left and right, with the IPTG of 0.3 mM, be induced to OD
600during=3 left and right, by inoculum size 10%, be forwarded to anaerobically fermenting in serum bottle, fermentation 48 h.
Aerobic stage substratum is: LB+Amp (penbritin 50 μ g/mL).
Anaerobic stages substratum is: LB+ Corncob hydrolysate (by total reducing sugar 30 g/L meterings)+magnesium basic carbonate 0.6g+Amp (penbritin 50 μ g/mL)+0.3 mM IPTG.
Fermentation results is in Table 10.
The result comparison of table 10 Escherichia coli BA306 and starting strain fermentation and acid
Note: ND represents not detect.
Embodiment 15
The contrast of the present embodiment explanation colon bacillus BA306 and starting strain fermentation and acid ability.
Colon bacillus BA306 can efficiently utilize the fermentation of rice straw hydrolyzed solution, and accumulates in a large number succinic acid, adopts two stage fermentation modes, it is characterized in that by 1% (v/v) inoculum size from cryopreservation tube access triangular flask, when aerobic is cultivated thalline OD
600to 0.4～0.6 left and right, with the IPTG of 0.3 mM, be induced to OD
600during=3 left and right, by inoculum size 10%, be forwarded to anaerobically fermenting in serum bottle, fermentation 48 h.
Aerobic stage substratum is: LB+Amp (penbritin 50 μ g/mL).
Anaerobic stages substratum is: LB+ rice straw hydrolyzed solution (by total reducing sugar 35 g/L meterings)+magnesium basic carbonate 0.6 g+Amp (penbritin 50 μ g/mL)+0.3 mM IPTG.
Fermentation results is in Table 11.
The ferment result comparison of main acid of table 11Escherichia coli BA306 and starting strain
Note: ND represents not detect.
Embodiment 16
The contrast of the present embodiment explanation colon bacillus BA306 and starting strain fermentation and acid ability.
Colon bacillus BA306 can efficiently utilize the fermentation of molasses hydrolyzed solution, and accumulates in a large number succinic acid, adopts two stage fermentation modes, it is characterized in that by 1% (v/v) inoculum size from cryopreservation tube access triangular flask, when aerobic is cultivated thalline OD
600to 0.4～0.6 left and right, with the IPTG of 0.3 mM, be induced to OD
600during=3 left and right, by inoculum size 10%, be forwarded to anaerobically fermenting in serum bottle, fermentation 48 h.
Aerobic stage substratum is: LB+Amp (penbritin 50 μ g/mL).
Anaerobic stages substratum is: LB+ molasses hydrolyzed solution (by total reducing sugar 35 g/L meterings)+magnesium basic carbonate 0.6g+Amp (penbritin 50 μ g/mL)+0.3 mM IPTG.
Fermentation results is in Table 12.
The result comparison of table 12Escherichia coli BA306 and starting strain fermentation and acid
Note: ND represents not detect.
Embodiment 17
The contrast of the present embodiment explanation colon bacillus BA306 and starting strain fermentation and acid ability.
Colon bacillus BA306 can efficiently utilize the fermentation of bagasse hydrolyzed solution, and accumulates in a large number succinic acid, adopts two stage fermentation modes, it is characterized in that by 1% (v/v) inoculum size from cryopreservation tube access triangular flask, when aerobic is cultivated thalline OD
600to 0.4～0.6 left and right, with the IPTG of 0.3 mM, be induced to OD
600during=3 left and right, by inoculum size 10%, be forwarded to anaerobically fermenting in serum bottle, fermentation 48 h.
Aerobic stage substratum is: LB+Amp (penbritin 50 μ g/mL).
Anaerobic stages substratum is: LB+ bagasse hydrolyzed solution (by total reducing sugar 35 g/L meterings)+magnesium basic carbonate 0.6g+Amp (penbritin 50 μ g/mL)+0.3 mM IPTG.
Fermentation results is in Table 13.
The result comparison of table 13 Escherichia coli BA306 and starting strain fermentation and acid
Note: ND represents not detect.
Claims (5)
1. a strain succinic acid-producing genetically engineered bacteria strain BA306, its Classification And Nomenclature be colon bacillus (
escherichia coli), its preserving number is numbered CCTCC NO:M2012103; To lack lactate dehydrogenase gene, the bacterial strain intestinal bacteria of pyruvate formate-lyase gene activity
e.colinZN111 is starting strain, utilizes homologous recombination technique to knock out phosphoric acid enol pyruvic acid carboxylase gene and phosphotransferase system
ptsGgene, and after excessive coexpression phosphoenolpyruvic acid carboxylation kinases and nicotinic acid phosphoribosyltransferase, obtain colon bacillus BA306; Concrete construction step is as follows:
(1) to lack lactate dehydrogenase gene, pyruvate formate-lyase gene activity
e.colinZN111 bacterial strain is starting strain, knocks out wherein phosphoric acid enol pyruvic acid carboxylase gene and phosphotransferase system
ptsGgene is lacked simultaneously
ldhA,
pflB,
ppcwith
ptsGcompetence bacterial strain;
(2) purifying amplifies phosphoenolpyruvic acid carboxylation kinase gene, builds and obtains the kinase whose expression plasmid of overexpression phosphoenolpyruvic acid carboxylation;
(3) purifying amplifies nicotinic acid phosphoribosyltransferase gene, is connected on the described expression plasmid of step (2), builds the expression plasmid that obtains excessive coexpression phosphoenolpyruvic acid carboxylation kinases and nicotinic acid phosphoribosyltransferase;
(4) plasmid step (3) Suo Shu is imported to the competence bacterial strain that step (1) obtains, obtain positive transformant;
(5) utilize the excessive coexpression phosphoenolpyruvic acid of positive transformant carboxylation kinases and the nicotinic acid phosphoribosyltransferase of step (4), recover its under anaerobic metabolism, obtain colon bacillus BA306.
2. the method for utilizing the colon bacillus BA306 fermentation production of succinic acid described in claim 1, is characterized in that adopting two stage fermentation modes, and the aerobic stage is improved biomass, anaerobic stages fermentation and acid.
3. method according to claim 2, it is characterized in that colon bacillus BA306 is inoculated to aerobic in aerobic stage fermentation substratum by 1% inoculum size to be cultivated, when the IPTG of aerobic cultivation thalline OD600 to 0.4～0.6 use 0.3 mM is induced to OD600=3, by inoculum size 10%, be forwarded to anaerobically fermenting in anaerobic stages fermention medium.
4. method according to claim 3, is characterized in that the carbon source of described anaerobic stages fermention medium is glucose, wood sugar, pectinose, fructose or its combination.
5. method according to claim 3, is characterized in that the carbon source of described anaerobic stages fermention medium is Corncob hydrolysate, rice straw hydrolyzed solution, molasses hydrolyzed solution or bagasse hydrolyzed solution.
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| Title |
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| 于丽等.过量表达Bacillus subtilis 磷酸烯醇式丙酮酸羧化激酶对大肠杆菌产琥珀酸的影响.《微生物学通报》.2010,第37卷(第3期),325－330. |
| 刘嵘明等.过量表达烟酸转磷酸核糖激酶对大肠杆菌NZN111产丁二酸的影响.《生物工程学报》.2011,第27卷(第10期),1438－1447. |
| 过量表达Bacillus subtilis 磷酸烯醇式丙酮酸羧化激酶对大肠杆菌产琥珀酸的影响;于丽等;《微生物学通报》;20100320;第37卷(第3期);325－330 * |
| 过量表达烟酸转磷酸核糖激酶对大肠杆菌NZN111产丁二酸的影响;刘嵘明等;《生物工程学报》;20111025;第27卷(第10期);1438－1447 * |
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