CN104830917A - Method for manufacturing 3-hydroxypropionic acid from microbial fermentation glycerol - Google Patents
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Abstract
The invention relates to a method for manufacturing 3-hydroxypropionic acid from microbial fermentation glycerol. The method is implemented by the aid of first microorganisms and second microorganisms. The first microorganisms have glycerol dehydration activity, and the glycerol can be converted into 3-hydroxy-propionaldehyde or 1, 3-propylene glycol by the first microorganisms. The second microorganisms have glycol dehydration activity, and the 3-hydroxy-propionaldehyde or the 1, 3-propylene glycol glycerol can be converted into the 3-hydroxypropionic acid by the second microorganisms. The first microorganisms can be selected from Klebsiella pneumoniae and the like and genetically engineered bacteria of the Klebsiella pneumoniae and the like; the second microorganisms are Gluconobacter oxydans and genetically engineered bacteria of the Gluconobacter oxydans. The method has the advantages that the glycerol is used as a substrate, the two types of microorganisms are subjected to sectioned mixed fermentation to manufacture the 3-hydroxypropionic acid, the concentration of final products can be improved as compared with an existing single-bacterium fermentation method mainly on the basis of genetically engineered bacteria, the rate of converting the glycerol into the 3-hydroxypropionic acid can be increased as compared with the existing single-bacterium fermentation method, lactic acid in fermentation liquor can be converted into acetic acid, accordingly, the concentration of the lactic acid of byproducts can be reduced, and separation and purification stress can be relieved; the concentration of the 3-hydroxypropionic acid can reach 75gL<-1> at least, and accordingly the method has an excellent industrial application prospect.
Description
Technical field
The present invention relates to technical field of bioengineering, particularly relate to field of microbial fermentation, specifically refer to a kind of method utilizing fermentable glycerol production 3-hydroxy-propionic acid.
Background technology
3-hydroxy-propionic acid (3-hydroxypropionic acid, be called for short 3-HP) be a kind of important platform chemicals, it can generate the multiple important chemical substances such as vinylformic acid, propanedioic acid, 1,3-PD by dehydration, redox, esterification etc.It also can as the eco-friendly macromolecular material-poly (hydroxyalkanoate) of monomer synthesize.In addition, it can also be used for the production of coating, glue paste, Water Treatment Chemicals and personal-care supplies as industrial chemicals, one of USDOE 12 kinds of Chemicals being once classified as most Development volue in 2004.
The preparation of current 3-hydroxy-propionic acid is mainly chemical method, as vinylformic acid hydration method, and 3-HPA oxidation style, 3-hydroxypropionitrile hydrolysis method and this base of thunder formal (Reformatsky) reaction etc.But chemical method raw material is many from oil, and exist high to equipment requirements, energy consumption is high, pollutes the problems such as large.By comparison, biological process has the advantages such as simple to operate, mild condition, environmental protection, therefore receives much attention and enjoys the favor of domestic and international researchist.
Production by biological 3-HP just has report as far back as the sixties in last century, but substrate price needed for naturally occurring bacterial strain is high and 3-HP productive rate is all lower, is therefore in laboratory stage for a long time.In recent years, along with the development of Protocols in Molecular Biology, large quantity research starts to concentrate on and carries out approach transformation to Natural strains, utilizes recombinant bacterium with the cheap carbon source such as glycerine or glucose for substrate produces 3-HP.It is substrate and glycerine substrate two kinds that the structure of genetic engineering bacterium is mainly divided into glucose according to the difference of substrate.Glucose is that the transformation routes of substrate is the most ripe with the research of U.S. Jia Ji (Cargill) company, the said firm constructs many paths from glucose to 3-HP, wherein the representational process of most is: glucose through diphosphate pathway to pyruvic acid, pyruvic acid reduction generates lactic acid, lactic acid generates lactoyl CoA under the effect of propionyl CoA transferring enzyme, the latter generates acryloyl CoA under the effect of lactoyl CoA dehydratase, and then under the effect of 3-hydroxyl propionyl dehydratase, generate 3-hydroxyl propionyl CoA, final generation 3-hydroxy-propionic acid.
Be that substrate generation 3-HP is made up of two-step reaction usually with glycerine: glycerine first generates 3-HPA through glycerol dehydratase dehydration, and 3-HPA generates 3-HP through aldehyde dehydrogenase dehydrogenation.Compared with glucose, glycerine is short to 3-HP generation pass, builds and regulates and controls more effectively simple.And Biodiesel development in recent years rapidly, produces a large amount of by-product glycerin in its production process, if be used appropriately, can reduce the production cost of biofuel, improve its market competitiveness.Therefore be that raw material product 3-HP has agreed with bioenergy and ecological requirements with glycerine, having good Social benefit and economic benefit, is the study hotspot that current biological method produces 3-HP.
Be that substrate structure genetic engineering bacterium mainly comprises again two large classes according to Host Strains difference with glycerine, one class introduces aldehyde dehydrogenase in the bacterial strain containing dha operon with glycerol dehydratase activity (as klebsiella pneumoniae, Klebsiella pneumoniae).Another kind of be express glycerol dehydratase and aldehyde dehydrogenase in the bacterial strain (conventional is intestinal bacteria, Escherich.coli) not containing glycerol dehydratase simultaneously.Be starting strain as the Kumar of Busan, Korea national university, Ashok etc. once report respectively with K.pneumoniae, introduce aldehyde dehydrogenase gene and build 3-HP route of synthesis.Jung etc. then express the production ways that glycerol dehydratase and aldehyde dehydrogenase construct 3-HP in E.coli simultaneously, and have carried out many-sided transformation to host, to improve productive rate and the transformation efficiency of 3-HP.The E.coli that Rathnasingh etc. build Raj has carried out further transformation, and after replacing aldehyde dehydrogenase ALDH with semialdehyde dehydrogenase KGSADH, produces 3-HP concentration to improve recombinant bacterium.The E.coli engineering bacteria that Kim also builds Kwak etc. has carried out further transformation, and replace aldehyde dehydrogenase ALDH with more efficient semialdehyde dehydrogenase PSALDH, recombinant bacterium 3-HP concentration reaches 57.3g/L.The Chu of Korea S screens highly active desaturase GabD4 from Cupriavidus necator, and this enzyme is after orthogenesis, and with glycerol dehydratase coexpression in E.coli, final recombinant bacterium 40h produces 3-hydroxy-propionic acid concentration and reaches 71.9g/L.This is that report take glycerine as the highest level that substrate produces 3-hydroxy-propionic acid at present.
Also there are similar work report in domestic East China University of Science, Nanjing University of Technology, Southern Yangtze University, Beijing University of Chemical Technology, wherein Beijing University of Chemical Technology Tian Pingfang professor research team has used the various new biology techniques means such as overall disturbance, sense-rna to regulate and control K.pneumoniae, study the most deep, but its 3-HP output does not have large breakthrough at present.The Huang Yanna of East China University of Science etc. express the aldehyde dehydrogenase ALDH of E.coli in K.pneumoniae, and through optimizing, under micro-oxygen conditions, 3-HP concentration reaches 49.8g/L.This is the highest level that the K.pneumoniae of report restructuring at present produces 3-HP.
Build the research that genetic engineering bacterium prepares 3-hydroxy-propionic acid up to now to make some progress, but also there is many problems: 1) approach transformation itself needs loaded down with trivial details molecular biology manipulations, later stage cultivates to be needed additionally to add microbiotic and inductor etc. toward contact, increases operation easier and strengthens production cost.2) select permeability of Host Strains during engineering bacteria is built.Take K.pneumoniae as host, produce a large amount of 1,3-PD in 3-HP production process, the latter is the natural end product of K.pneumoniae, and constructive ways is difficult to cut off completely, and this just makes 3-HP concentration and transformation efficiency all be difficult to improve.Take E.coli as host, 3-HP output is relatively high, but lives owing to himself not having glycerol dehydratase enzyme, and building process is more complicated.And E.coli self can not generate the coenzyme VB of glycerol dehydratase
12, need additionally to add in fermentation system, because it is expensive, thus fermentation costs increased.3) in recombinant bacterium, the equilibrium problem of glycerol dehydratase and aldehyde dehydrogenase also constrains the raising of 3-HP output.Only under anaerobism and micro-aerobic condition, just tool is active for the glycerol dehydratase of current discovery, and with this understanding NAD (P) H can not generate timely the cofactor NAD (P) of aldehyde dehydrogenase+, this not only makes 3-HP produce rate reduction, the 3-HPA of strong cytotoxicity also can be caused to accumulate, thus cause necrocytosis fermentation to stop.
In sum, current this area needs a kind of simple and effective technique can be 3-hydroxy-propionic acid by the carbohydrate-modifying of relative moderate, and can obtain comparatively high product concentration and productive rate, to adapt to the demand of suitability for industrialized production.
Summary of the invention
The object of the invention is the shortcoming overcoming above-mentioned prior art, provide a kind of can realize the glycerine of relative moderate is utilized two kinds of microorganism segmentations, the method utilizing fermentable glycerol production 3-hydroxy-propionic acid that mixed fungus fermentation is converted into 3-hydroxy-propionic acid, described method carries out segmentation, mixed fungus fermentation for utilizing two kinds of microorganisms;
Above-mentioned two kinds of microorganisms comprise:
Have dehydrating glycerin activity, glycerine converting generates the microorganism one of 3-HPA or 1,3-PD;
Transform the microorganism two of 3-HPA or 1,3-PD generation 3-hydroxy-propionic acid;
Leading portion fermentative process conditions is the substratum containing glycerine 20 ~ 120g/L, the seed culture fluid of access microorganism one, and inoculum size 3 ~ 10%, passes into air or nitrogen, 30 ~ 42 DEG C, and ferment 10 ~ 45 hours, pH is 6.0 ~ 8.0;
Back segment fermentative process conditions is seed liquor or the microorganism cells of microorganism two in leading portion nutrient solution, and seed liquor inoculum size is 10 ~ 20%, and cell inoculum size is 3 ~ 20g/L, passes into air or oxygen, 20 ~ 37 DEG C, and cultivate 10 ~ 40 hours, pH is 5.0 ~ 7.0;
It is embrane-associated protein that 1,3-PD or 3-HPA are transformed the relevant enzyme generating 3-hydroxy-propionic acid by described microorganism two.
Preferably, described microorganism one is selected from: Klebsiella pneumoniae (Klebsiella pneumoniae), citrobacter freundii (Citrobacter freundii), clostridium butyricum (Clostridia butyricum), Clostridium pasteurianum (Clostridia pastruianu), and their genetic engineering bacterium.
Preferably, described microorganism two is Gluconobacter oxvdans (Gluconobacter oxydans), and its genetic engineering bacterium.
Preferably, described embrane-associated protein with pyrroloquinoline quinone (Pyrroloquinoline quinonePQQ) for cofactor.
Preferably, the seed liquor of described microorganism one obtains by the following method:
By strain inoculation in seed culture medium, culture temperature 28 ~ 37 DEG C, passes into air or nitrogen and carries out aerobic or Anaerobic culturel 10 ~ 15 hours, namely obtain the seed liquor of microorganism one.
Preferably, the seed liquor of described microorganism two obtains by the following method:
Gluconobacter oxydans kind is inoculated in seed culture medium, culture temperature 25 ~ 32 DEG C, cultivates 20 ~ 30 hours, namely obtain the seed liquor of microorganism two.
Preferably, the microorganism cells of described microorganism two is prepared by the following method:
Gluconobacter oxydans kind is inoculated in seed culture medium, culture temperature 25 ~ 32 DEG C, cultivates 20 ~ 30 hours, low-temperature centrifugation, collect bacterial sediment, with phosphoric acid buffer washing, supernatant discarded, namely obtains microorganism cells.
The present invention utilizes two kinds of microorganism segmentations, mixed fungus fermentation glycerine produces 3-HP, and tool has the following advantages compared with current conventional genetic engineering bacterium glycerine converting:
1) the present invention two kinds of microorganisms used can be the natural microbials that nature exists, and eliminate loaded down with trivial details genetic manipulation step.
2) the present invention utilizes two kinds of microorganisms, segmentation, mixed fermentation, access microorganism titanium dioxide gluconobacter suboxydans in fermentation system, can by 3-HPA in system and 1, ammediol is all converted into 3-hydroxy-propionic acid, thus improves production concentration and the glycerine transformation efficiency to 3-HP.
3) 1 is oxidized in the present invention, ammediol and 3-HPA and desaturase be the membrane bound protein of microorganism two, produce 3-HP direct secretion in extracellular, thus avoid the material turnover energy consumption of cell and rate limiting, there is higher production intensity; Also make the tolerance of cell to 3-HP higher in addition, thus can have higher final product concentrations, 3-HP concentration of the present invention can reach 75g L
-1above, there is good prospects for commercial application.
4) time usually with the microorganisms producing 3-such as intestinal bacteria or klebsiella spp hydroxy-propionic acid, microorganism utilizes glycerine to produce 3-HP can produce a large amount of lactic acid simultaneously, lactic acid be the isomers of 3-hydroxy-propionic acid and physico-chemical property and 3-hydroxy-propionic acid very close to thus cause separation and purification difficulty, the present invention accesses microorganism titanium dioxide gluconobacter suboxydans, acetic acid can be generated by lactic acid in transformation fermentation liquid, thus reduce by product lactic acid concn and slow down separation and purification pressure.
Embodiment
In order to more clearly describe technology contents of the present invention, conduct further description below in conjunction with specific embodiment.
embodiment 1, Klebsiella pneumoniae (K.pneumoniae) glycerol dehydratase strengthening bacterial strain structure
Utilize round pcr from Klebsiella pneumoniae, amplify the gene dhaB (GenBankaccession No.U30903) of encoding glycerol dehydratase, be connected to expression vector pKM13 (Ping Zheng, Kirsten Wereath etc., ProcessBiochemistry 41 (2006) 2160 – 2169) on, obtain recombinant plasmid pKM13-dhaB, by recombinant plasmid transformed Klebsiella pneumoniae ATCC25955, obtain recombinant bacterium ATCC25955/pKM13-dhaB.
Concrete building process is:
(1) structure of expression plasmid:
Design two primers according to Klebsiella pneumoniae dhaT gene order, its sequence is as follows:
Primer 1:5 '-ccg
gaattcatgaaaagatcaaaacgatttgc-3 '
Primer 2: 5 '-tgc
tctagattaattcgcctgaccggccag-3 '
With Klebsiella pneumoniae genomic dna for template, pcr amplification goal gene
PCR reaction system (50 μ l): 10x GC Buffer 5 μ l, 2.5mmol/l DNTPs 4 μ l, template DNA 1 μ l, TaqDNAPolymerase1 μ l, primer 1 and each 0.1nM of primer 2, distilled water supplies 50 μ l.
Reaction conditions: 95 DEG C of sex change 5min; 95 DEG C of 50s, 55 DEG C of 90s, 72 DEG C of 5min, 35 circulations, 72 DEG C, 10min.
Obtain PCR primer electrophoretic analysis to confirm, after PCR primer purification kit (vast Tyke) purifying, EcoR I and Xba I double digestion, reclaim the fragment of wherein 4.4kb, connection carrier pKM13, obtains recombinant plasmid pKM13-dhaB.
(2) expression of gene:
Recombinant plasmid pKM13-dhaB electricity is transformed into Klebsiella pneumoniae ATCC25955, and coating is dull and stereotyped containing kantlex, picking positive transformant, obtains restructuring Klebsiella pneumoniae ATCC25955/pKM13-dhaB.
embodiment 2, Gluconobacter oxvdans (G.oxydans) ethanol dehydrogenase strengthening bacterial strain structure
Utilize round pcr from Gluconobacter oxvdans DSM2003, amplify the gene adh AB of coding film in conjunction with ethanol dehydration enzyme, expression vector pBBR1MCS-5 is connected to DNA ligase, obtain recombinant plasmid pBBR1MCS-5-adhAB, by recombinant plasmid transformed Gluconobacter oxvdans DSM2003, obtain recombinant bacterium DSM2003/pBBR1MCS-5-adhAB.
Concrete building process is:
(1) structure of expression plasmid:
Design two primers according to Gluconobacter oxvdans alcohol dehydrogenase gene sequence (G.oxydans 621H GGOX1068), its sequence is as follows:
Primer 1:5 '-AAA
gTCGACaTGAACGACACGGTGCCCGAGC-3 '
Primer 2: 5 '-TC
tCTAGAcAGGGGTGGGGACGCTTATTGTGCG-3 '
With Gluconobacter oxvdans DSM2003 genomic dna for template, pcr amplification goal gene.
PCR reaction system (50 μ l): 10x GC Buffer 5 μ l, 2.5mmol/l DNTPs 4 μ l, template DNA 1 μ l, TaqDNAPolymerase1 μ l, primer 1 and each 0.1nM of primer 2, distilled water supplies 50 μ l.
Reaction conditions: 94 DEG C of sex change 3min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 4min, 30 circulations, 72 DEG C, 10min.
Obtain PCR primer electrophoretic analysis to confirm, after PCR primer purification kit (vast Tyke) purifying, SalI and Xba I double digestion, reclaims the fragment of wherein 4.2kb, connection carrier pBBR1MCS-5, obtains recombinant plasmid pBBR1MCS-5-adhAB.
(2) expression of gene:
Transformed by recombinant plasmid pBBR1MCS-5-adhAB electricity and import Gluconobacter oxvdans DSM2003, coating is containing the plate screening transformant of gentamicin, and picking positive transformant, obtains recombinant bacterium DSM2003/pBBR1MCS-5-adhAB.
embodiment 3, microorganism one seed culture
Seed culture based formulas (g/L): peptone 10, yeast powder 5, NaCl 10, glycerine 2, adjusts pH to 7.0,121 DEG C of sterilizing 20min with 2mol/L KOH.
Get 250ml triangular flask, seed culture medium liquid amount 50ml, by shown in table 1, respectively access the bacterial classification of a ring slant preservation with transfering loop, culture temperature 28 ~ 37 DEG C, pass into air or nitrogen carries out aerobic or Anaerobic culturel 10 ~ 15h, shaking speed 150 ~ 200r/min.
Described bacterial classification is: Klebsiella pneumoniae (Klebsiella pneumoniae) ATCC25955, citrobacter freundii (Citrobacter freundii) DSM30040, clostridium butyricum (Clostridia butyricum) DSM5431, Clostridium pasteurianum (Clostridia pastruianu) DSM525.
Table 1, microorganism one seed culture condition
embodiment 4, microorganism two seed liquor cultivation
Seed culture based formulas (g/L): D-glucitol 40, yeast powder 10, KH
2pO
42, MgSO
47H
2o 0.5; Get 500ml triangular flask, seed culture medium liquid amount 30 ~ 100ml, access the Gluconobacter oxvdans of a ring slant preservation with transfering loop, culture temperature 25 ~ 32 DEG C, shaking speed 250r/min, cultivate 20 ~ 30h.
Table 2, microorganism two seed culture
embodiment 5, Gluconobacter oxvdans cell acquisition
The seed culture fluid that Example 4 obtains, in 4 ~ 10 DEG C, the centrifugal 15min of 8000rpm, supernatant discarded, the bacterial sediment of gained washs with the phosphate buffer solution of pH 7.00, and recentrifuge, abandons supernatant, namely obtains Gluconobacter oxvdans cell.
embodiment 6, the leading leading portion fermentation of microorganism one
Glycerin medium formula (g/L): KCl 0.75, NaH
2pO
42H
2o 1.5, (NH
4)
2sO
42.35, Na
2sO
40.28, MgSO
47H
2o 0.26, citric acid 0.62, corn steep liquor 4.0, glycerine 20 ~ 120, micro-0.3ml/L, adjusts pH to 7.0,121 DEG C of sterilizing 20min with the KOH of 2mol/L.
Wherein said trace element solution composition (g/L): ZnCl
234.2, FeCl
36H
2o 2.7, MnCl
24H
2o 10, CuCl
22H
2o 0.85, CoCl
26H
2o 23.8, H
3bO
30.31, Na
2moO
42H
2o 0.25.
Get 500ml triangular flask, substratum liquid amount 200ml, by condition shown in table 2, access the seed liquor that embodiment 3 obtains respectively, inoculum size 3 ~ 10%, culture temperature 30 ~ 42 DEG C, pass into air or nitrogen and carry out aerobic or Anaerobic culturel 10 ~ 45 hours, shaking speed 150r/min.
The leading portion fermentation that table 3, microorganism one are dominated
embodiment 7, the leading back segment fermentation of microorganism two
The 5# fermented liquid obtained in Example 6 is sub-packed in 1000ml triangular flask, liquid amount 100ml/ bottle, the nutrient solution that access embodiment 4 obtains, or the microorganism cells that access embodiment 5 obtains, 20 ~ 37 DEG C, pass into air or oxygen, shaking speed 250r/min, carries out aerobic and cultivates 10 ~ 40 hours, the centrifugal 5min of 12000rpm, get supernatant liquor and measure wherein 3-HP and lactic acid concn, the results are shown in Table 3.
The back segment fermentation that table 4, oxidation grape acidfast bacilli are leading
embodiment 8, the mensuration of 3-HP and lactic acid concn adopts high pressure liquid chromatography (HPLC) analytical procedure, and operational condition is as follows:
Chromatographic column: Zorbax SB-Aq, Agilent (4.6mm × 250mm, 5 μm)
Moving phase: methyl alcohol: 0.1% phosphoric acid solution, 10:90 ~ 30:70.
Flow velocity: 0.5-0.8mL/min.
Sample size: 3-10 μ l
Column temperature: 30 DEG C.
UV analyser, 210nm scans.
The present invention utilizes two kinds of microorganism segmentations, mixed fungus fermentation glycerine produces 3-HP, and tool has the following advantages compared with current conventional genetic engineering bacterium glycerine converting:
1) the present invention two kinds of microorganisms used can be the natural microbials that nature exists, and eliminate loaded down with trivial details genetic manipulation step.
2) the present invention utilizes two kinds of microorganisms, segmentation, mixed fermentation, access microorganism titanium dioxide gluconobacter suboxydans in fermentation system, can by 3-HPA in system and 1, ammediol is all converted into 3-hydroxy-propionic acid, thus improves production concentration and the glycerine transformation efficiency to 3-HP.
3) 1 is oxidized in the present invention, ammediol and 3-HPA and desaturase be the membrane bound protein of microorganism two, produce 3-HP direct secretion in extracellular, thus avoid the material turnover energy consumption of cell and rate limiting, there is higher production intensity; Also make the tolerance of cell to 3-HP higher in addition, thus can have higher final product concentrations, 3-HP concentration of the present invention can reach 75g L
-1above, there is good prospects for commercial application.
4) time usually with the microorganisms producing 3-such as intestinal bacteria or klebsiella spp hydroxy-propionic acid, microorganism utilizes glycerine to produce 3-HP can produce a large amount of lactic acid simultaneously, lactic acid be the isomers of 3-hydroxy-propionic acid and character and 3-hydroxy-propionic acid very close to thus cause separation and purification difficulty, the present invention accesses microorganism titanium dioxide gluconobacter suboxydans, acetic acid can be generated by lactic acid in transformation fermentation liquid, thus reduce by product lactic acid concn and slow down separation and purification pressure.
In this description, the present invention is described with reference to its specific embodiment.But, still can make various amendment and conversion obviously and not deviate from the spirit and scope of the present invention.Therefore, specification sheets is regarded in an illustrative, rather than a restrictive.
Claims (7)
1. utilize a method for fermentable glycerol production 3-hydroxy-propionic acid, it is characterized in that, described method carries out segmentation, mixed fungus fermentation for utilizing two kinds of microorganisms;
Above-mentioned two kinds of microorganisms comprise:
Have dehydrating glycerin activity, glycerine converting generates the microorganism one of 3-HPA or 1,3-PD;
Transform the microorganism two of 3-HPA or 1,3-PD generation 3-hydroxy-propionic acid;
Leading portion fermentative process conditions is the substratum containing glycerine 20 ~ 120g/L, the seed culture fluid of access microorganism one, and inoculum size 3 ~ 10%, passes into air or nitrogen, 30 ~ 42 DEG C, and ferment 10 ~ 45 hours, pH is 6.0 ~ 8.0;
Back segment fermentative process conditions is seed liquor or the microorganism cells of microorganism two in leading portion nutrient solution, seed liquor inoculum size be 10 ~ 20% or cell inoculum size be 3 ~ 20g/L, pass into air or oxygen, 20 ~ 37 DEG C, cultivate 10 ~ 40 hours, pH is 5.0 ~ 7.0;
It is embrane-associated protein that the conversion 1,3-PD of described microorganism two or 3-HPA generate 3-hydroxy-propionic acid relevant enzyme.
2. the method utilizing fermentable glycerol production 3-hydroxy-propionic acid according to claim 1, it is characterized in that, described microorganism one is selected from: Klebsiella pneumoniae (Klebsiella pneumoniae), citrobacter freundii (Citrobacterfreundii), clostridium butyricum (Clostridia butyricum), Clostridium pasteurianum (Clostridiapastruianu), and their genetic engineering bacterium.
3. the method utilizing fermentable glycerol production 3-hydroxy-propionic acid according to claim 1, is characterized in that, described microorganism two is Gluconobacter oxvdans (Gluconobacter oxydans), and its genetic engineering bacterium.
4. the method utilizing fermentable glycerol production 3-hydroxy-propionic acid according to claim 1, is characterized in that, described embrane-associated protein with pyrroloquinoline quinone (Pyrroloquinoline quinonePQQ) for cofactor.
5. the method utilizing fermentable glycerol production 3-hydroxy-propionic acid according to claim 1, is characterized in that, the seed liquor of described microorganism one obtains by the following method:
By strain inoculation in seed culture medium, culture temperature 28 ~ 37 DEG C, passes into air or nitrogen and carries out aerobic or Anaerobic culturel 10 ~ 15 hours, namely obtain the seed liquor of microorganism one.
6. the method utilizing fermentable glycerol production 3-hydroxy-propionic acid according to claim 1, is characterized in that, the seed liquor of described microorganism two obtains by the following method:
Gluconobacter oxydans kind is inoculated in seed culture medium, culture temperature 25 ~ 32 DEG C, cultivates 20 ~ 30 hours, namely obtain the seed liquor of microorganism two.
7. the method utilizing fermentable glycerol production 3-hydroxy-propionic acid according to claim 1, is characterized in that, the microorganism cells of described microorganism two is prepared by the following method:
Gluconobacter oxydans kind is inoculated in seed culture medium, culture temperature 25 ~ 32 DEG C, cultivates 20 ~ 30 hours, low-temperature centrifugation, collect bacterial sediment, with phosphoric acid buffer washing, supernatant discarded, namely obtains microorganism cells.
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| US20110125118A1 (en) * | 2009-11-20 | 2011-05-26 | Opx Biotechnologies, Inc. | Production of an Organic Acid and/or Related Chemicals |
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| CN114958928B (en) * | 2022-02-28 | 2024-05-14 | 江苏大学 | Method for co-producing 3-hydroxy propionic acid and 1, 3-propylene glycol by genetic engineering bacterium fermentation |
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