CN101448950B - Process for production of panthenol - Google Patents
Process for production of panthenol Download PDFInfo
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- CN101448950B CN101448950B CN200780017876.5A CN200780017876A CN101448950B CN 101448950 B CN101448950 B CN 101448950B CN 200780017876 A CN200780017876 A CN 200780017876A CN 101448950 B CN101448950 B CN 101448950B
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- panthenol
- acid salt
- aminopropanol
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
Abstract
The present invention provides a process for the production of panthenol by culturing a microorganism capable of overexpressing at least one enzyme selected from the group consisting of the enzymes in the pantoate biosynthesis and PanC under suitable culturing conditions, with co-feeding of 3-aminopropanol or a suitable derivative thereof and optionally recovering the panthenol from the cell culturing medium.
Description
The present invention relates to the technique for the production of panthenol.More properly, the present invention relates to the technique of fermentative production panthenol, described technique is by under suitable fermentation condition, in suitable fermention medium, with the common feed of 3-aminopropanol (AMP) or derivatives thereof, carry out culturing micro-organisms, and, if necessary, reclaiming panthenol realizes.
Panthenol is 2,4-dihydroxyl-N-(3-hydroxypropyl)-3,3-amide dimethyl butyrate or pantothenylalcohol, and it is the alcohol corresponding to pantothenic acid.Due to its film protection feature, panthenol is widely used in cosmetic industry.The D (+) of panthenol related to the present invention-or R-form be preferred form, it has vitamin activity, so its purposes as preventive in medicine and food tonic field is well-known, its new application is also in continuing exploitation.
The conventional manufacturing process of panthenol is synthetic R-pantolactone (Alpha-hydroxy-β, beta-dimethyl-gamma-butyrolactone alpha-hydroxy-beta) (see for example Schnider with the chemical condensation of 3-aminopropanol, O.:Synthesis of panthenoland its transformation into pantothenic acid.Jubilee Vol.Emil Barell 1946,85-91).
Do not describe up to now and use microorganism by biotechnical processes, to produce the method for panthenol.
CN 1367253 has described and has used the Fusariummonoliforme that produces D-pantoyl lactone lytic enzyme be hydrolyzed DL-pantoic acid (pantoic acid) lactone and made the Pantothenic acid obtaining react to produce D-panthenol with 3-aminopropanol by microorganism enzymatic.
The be produced into biosynthesizing originally panthenol carried out wholly or in part lower with the chemical technology than known remains the attractive technical object that people want realization.
The be produced into biosynthesizing originally panthenol carried out wholly or in part lower with the chemical technology than known remains the attractive technical object that people want realization.
The technique of the claimed production D-VB5 of EP 859 848 B1 (BASF AG), described technique is included in and while there is Beta-alanine, cultivates transformant E.coli.The claimed method of producing pantothenate or pantoic acid salt (pantoate) by cultivating genetically modified following microorganism of WO 01/21772 (OmnigeneBioproducts/BASF AG); described microorganism is the genus of Bacillus; B.subtilis particularly, at least one enzyme that is wherein selected from PanB (ketopantoic acid salt (ketopantoate) hydroxymethyl transferases), PanC (pantothenate synthetic enzyme), PanD (aspartic acid-α-decarboxylase) and PanE (ketopantoic acid salt reductase enzyme) is crossed is expressed.
Although known PanC can be in microorganism the condensation of catalysis Beta-alanine and pantoic acid salt form pantothenate, shockingly find that at present PanC can also catalysis 3-aminopropanol and pantoic acid salt condensation formation panthenol.
Therefore, the present invention relates to a kind of technique of producing panthenol, it is characterized in that: by cultivating following microorganism and optionally reclaim panthenol realize from cell culture medium with 3-aminopropanol or the common feed of its suitable derivative under suitable culture condition, described microorganism can cross at least one enzyme of expressing the group that the enzyme that is selected from the biosynthesizing of pantoic acid salt and PanC form.
The invention still further relates to according to the panthenol of this class explained hereafter, and PanC or its purposes the technique of producing panthenol from 3-aminopropanol or derivatives thereof and pantoic acid salt of mutant of catalytic activity with raising.
Microorganism of the present invention can be eucaryon or protokaryon.Preferably, this microorganism is protokaryon.Prokaryotic micro-organisms can be gram-positive or gram-negative.Gram-positive microorganism includes but not limited to belong to the microorganism of one of Bacillus, Corynebacterium, Lactobacillus, Lactococus and Streptomyces genus.Preferably, this microorganism belongs to Bacillus genus.Example is Bacillus licheniformis, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus puntis, Bacillus halodurans etc.Most preferably, this microorganism is Bacillussubtilis.
The enzyme of pantoic acid salt biosynthetic pathway and their DNA sequence dna of encoding are well known by persons skilled in the art, and it includes but not limited to the enzyme of PanB, Pan E, IIvB, IIvC, IIvD, IIvN, GlyA, SerA and SerC and glycine cutting approach.
In preferred embodiments more of the present invention, at least one enzyme in PanB, PanC, PanE and IIvD is crossed expresses.In another specific embodiment of the present invention, by partially or completely lacking corresponding PanD gene, make catalysis aspartic acid α-decarboxylation form the PanD inactivation of Beta-alanine.
Many microorganisms through specificity genetic manipulation and conversion have been described in document, it can cross one or more enzymes of expressing pantoic acid salt biosynthetic pathway, or the microorganisms producing of raising pantoic acid salt, so it can be used to (optionally after introducing other favourable change) in the present invention.In patent documentation, the open example of this quasi-microorganism is:
WO 97/10340, EP 1 001 027, EP 1 006 189, WO 01/21772, WO01/92556, EP 1 167 520, WO 02/24936, WO 02/29020, WO 02/055711, WO 02/057474, WO 02/057476, WO 02/061108, WO 02/064806, WO02/072838, WO 02/072840, WO 02/072854, WO 02/072855, EP 1 247868, WO 03/004672, WO 03/006664, DE 102 01 540 A1, WO03/029476, WO 2004/005525 and WO 2004/005527.
The various part of speech forms that term " is crossed and expressed " represent that gene product is to modify before and the horizontal expression of the higher level of expressing in the suitable microorganism of unmodified than microorganism.In one embodiment, microorganism of the present invention crosses one or more genes of expressing the group that is selected from following gene formation: panB, panC, panE, ilvB, ilvC, ilvD, ilvN, glyA, serA and serC, and relate to the gcv gene that glycine cuts approach; And mutant, described mutant causes the synthetic enzyme being encoded with improved catalysis characteristics.
Term " de-adjusting " or " de-adjusting " represent change or the modification of gene in microorganism, make the level of gene product in microorganism or activity be changed or modify.Preferably, at least one gene is changed or modifies, make gene product be enhanced/increase or decay/reduce.
Can carry out crossing of gene in microorganism according to any method as known in the art expresses or de-adjusting.In one embodiment, microorganism can be by genetic manipulation, for example, by genetic engineering procedure.Genetic manipulation can include but not limited to: adjusting sequence or site that change or modification are relevant to the expression of concrete gene, for example, by adding strong promoter, inducible promoter or multiple promoter, thereby or by removals, regulate sequence to make expression become composing type; Modify the chromosomal localization of concrete gene, change the nucleotide sequence adjacent with concrete gene as ribosome bind site or transcription terminator, improve the copy number of concrete gene, modification relates to the protein (for example regulate albumen, suppress son, enhanser, transcription activator etc.) of concrete gene transformation and/or concrete gene product translation, or any other conventional means of the common concrete genetic expression of de-adjusting in this area (include but not limited to use antisense nucleic acid molecule, for example, seal and suppress sub-protein expression).The example of suitable promotor is, but is not limited to P
veg, P
15and P
26(Lee et al., 1980, Mol.Gen.Genet.180:57-65and Moran etal., 1982, Mol.Gen.Genet.186:339-46).
Or microorganism can be operated by physics (physically) or environment (environmentally), with before operation microorganism or the level of the higher level expressed in not operated suitable microorganism cross expressing gene product.For example, can be with the agent treated microorganism of the concrete genetic transcription of raising known or under a cloud and/or the translation of concrete gene product, or when there is described reagent culturing micro-organisms, thereby strengthen or improve and transcribe and/or translate.In addition, can be at selected temperature culturing micro-organisms, make to transcribe and/or translate and be enhanced or improve, described temperature can improve the translation of transcribing of concrete gene and/or concrete gene product.
Term " culturing micro-organisms under suitable culture condition " refers to well known in the art maintaining and/or the life method of microorganism of the present invention of growing.Can be in liquid, solid or semisolid medium culturing micro-organisms.Preferably, cultivate microorganism of the present invention in comprising the liquid nutrient medium of nutrient substance, described nutrient substance is critical or useful for maintaining and/or growing of microorganism.This class nutrient substance includes but not limited to, carbon source or carbon substrate, if alcohol, sugar, sugar alcohol, complex carbohydrates are as starch, hydrocarbon, lipid acid, other organic acid, oil, fat; Nitrogenous source, for example, from vegetable-protein, yeast extract, peptone, peptide and the amino acid of cereal, beans and tubers, or from animal-origin as meat or breast, meat extract and caseic hydrolysate; Inorganic nitrogen-sourced as urea, ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate; Phosphorus source, for example phosphoric acid and sodium salt thereof or sylvite; Trace element, for example magnesium, iron, manganese, calcium, copper, zinc, boron, molybdenum and/or cobalt salt; And somatomedin is as VITAMIN, growth promotor etc.
Preferably, culturing micro-organisms under controlled pH.In one embodiment, under the pH between 6.0 and 8.5, culturing micro-organisms under approximately 7 pH more preferably.Can maintain by any method known to those skilled in the art the pH of expectation.
Preferably, culturing micro-organisms at controlled ventilation and controlled temperature also.In one embodiment, controlled temperature comprises temperature between 15 and 70 ℃, the preferably temperature between 20 and 55 ℃, the temperature between 30 and 45 ℃ or between 30 and 50 ℃ more preferably.
Can cultivate or fermentation by cellar culture method culturing micro-organisms continuously, semi-continuously or in batches in liquid medium within as static cultivation, test tube cultivation, wave and culture, ventilation turn.Preferably, culturing micro-organisms in fermentor tank.Fermenting process of the present invention comprises in batches, fed-batch and continuous fermentation process.Developed multiple this class technique, they are known in the field.
Cultivate the time that conventionally continues enough to produce the panthenol amount of expectation.
According to the present invention, the key feature of culturing micro-organisms is the common feed of 3-aminopropanol (AMP) or its suitable derivative.Suitable AMP derivative is those derivatives that are converted into AMP under culture condition, as 3-alkoxy propyl amine, is preferably 3 methoxypropyl amine or 3-ethoxy propylamine, 3-halogen propylamine, for example 3-chlorine propylamine or 3-amino glycerol.
Although microbial culture was carried out during 72 hours, for shaking flask and fermenting experiment, for the two, care should be used to improves the pattern of the common feed of AMP, to adapt to the panthenol productive experiment of every type.In shaking flask, in when inoculation with add AMP with the concentration of 40g/L in growth medium.During fed-batch fermentation, first carry out first cultivation stage of 24 hours without the common charging of AMP, then during 24 hours with 14g/L to AMP charging.This AMP charging that increases for last 24 hours of then cultivating.In both cases, by with in HCl and to maintain pH constant be critical (pH of shake flat experiment is 7.2, and the pH of stirring tank fermentation is 6.8).
In the specific embodiment of the present invention, can, by the precursor of pantoic acid salt in common feed pantoic acid simultaneously or discontinuously, suitable pantoic acid derivative or biosynthetic pathway, improve the productive rate of the panthenol of microorganisms producing.Under Incubation Condition, the optimal dose of the common feed of this class can easily be measured by normal experiment.
The panthenol obtaining in fermented liquid according to the present invention can not reclaim and use or use after recovery.Term " recovery " comprises compound separation, extraction from substratum, collects, separates or purifying.Can carry out separating compound according to any conventional isolated or purified method known in the art, described method includes but not limited to by conventional plastic resin treatment, by conventional sorbent treatment, changes pH, solvent extraction, dialysis, filtration, concentrated, crystallization, recrystallization, adjusting pH etc.For example, can be by first microorganism being removed and reclaim panthenol compound from substratum from culture.Then by solution by or without Zeo-karb to remove undesired positively charged ion, then by or without anionite-exchange resin to remove undesired inorganic anion and organic acid.
By below the description of general method and non-limiting specific embodiment further being set forth to the present invention.
general method
Bacterial strain and plasmid.Bacillus subtilis bacterial strain of the present invention comes from bacterial strain 1A747 (Bacillus Genetic Stock Center, The Ohio State University, Columbus, Ohio43210 USA), it is the prototroph derivative of B.subtilis 168 (trpC2) (GenBank AL009126).Chloramphenicol resistance gene (cat) box derives from plasmid pC194 (GenBank M19465, Cat#1E17 Bacillus Genetic Stock Center, The Ohio State University, Columbus, Ohio 43210 USA).S.aureus erythromycin resistance gene (GenBank V01278) increases from plasmid pDG646 (Gu é rout-Fleury et al., 1995).S.aureus spectinomycin resistant gene (XO3216) increases from plasmid pDG1726 (Gu é rout-Fleury et al., 1995).The P of B.subtilis phage SPO1
15and P
26promotor (Lee et al., 1980, Mol.Gen.Genet.
180: the derivative (H ü mbelin et al., 1999, the J.Ind.Microbiol.Biotech. that 57-65) derive from plasmid pX12
22: 1-7), described plasmid contains from RB50::[pRF69]:: the SPO1-15 of [pRF93] and SPO1-26 promotor (Perkins et al., 1999, J.Ind.Microbiol.Biotech.22:8-18).
Substratum.Standard minimal medium (MM) for B.subtilis contains 1x Spizizen salt, 0.04% Sodium Glutamate and 0.5% glucose.Standard solid perfect medium is Tryptones blood agar culture-medium (Tryptone Blood Agar Broth) (TBAB, Difco).Standardized liquid perfect medium be veal fusion-yeast extract medium (Veal Infusion-Yeast Extract broth) (VY).The composition of these substratum is as described below:
TBAB substratum: 33g Difco Tryptones BAB (Catalog # 0232), 1L water.Autoclaving.
VY substratum: 25g Difco veal merges substratum (Catalog # 0344), 5g Difco yeast extract (Catalog #0127), 1L water.Autoclaving.
Minimal medium (MM): 100ml 10x Spizizen salt; 10ml50% glucose; 1ml40% Sodium Glutamate, water is supplied 1L (qsp 1L water).
10X Spizizen salt: 140g K
2hPO
4; 20g (NH
4)
2sO
4; 60g KH
2pO
4; 10g Trisodium Citrate 2H
2o; 2g MgSO
47H
2o; Water is supplied 1L.
10X VFB minimal medium (10X VFB MM): 2.5g Sodium Glutamate; 15.7gKH
2pO
4; 15.7g K
2hPO
4; 27.4g Na
2hPO
4.12H
2o; 40g NH
4cl; 1g citric acid; 68g (NH
4)
25O
4; Water is supplied 1L.
Trace element solution: 1.4g MnSO
4h
2o; 0.4g CoCl
26H
2o; 0.15g (NH
4) 6Mo
7o
244H
2o; 0.1g AlCl
36H
2o; 0.075g CuCl
22H
2o; Water is supplied 200ml.
Fe solution: 0.21g FeSO
47H
2o; Water is supplied 10ml.
CaCl2 solution: 15.6g CaCl
2.2H
2o; Water is supplied 500ml.
Mg/Zn solution: 100gMg SO
4.7H
2o; 0.4g ZnSO
4.7H
2o; Water is supplied 200ml.
VFB MM substratum: 100ml 10X VFB MM; 10ml 50% glucose; 2ml trace element solution; 2ml Fe solution; 2ml CaCl
2solution; 2ml Mg/Zn solution; 882ml aseptic deionized water.
VFB MMGT substratum: 100ml 10X VFB MM; 100ml 0.5 M Tris (pH6.8); 44ml 50% glucose; 2ml trace element solution; 2ml Fe solution; 2ml CaCl
2solution; 2ml Mg/Zn solution; 748ml aseptic deionized water.
VF fermentation batch culture base: suitably sterilizing in solution: 0.75g Sodium Glutamate; 4.71gKH
2pO
4; 4.71g K
2hPO
4; 8.23g Na
2hPO
412H
2o; 0.23g NH
4cl; 1.41g (NH
4)
2sO
4; 11.77g yeast extract (Merck); 0.2ml Basildon defoamer; Supply 1L.
As adding in fermentor tank through autoclaved solution: 27.3g glucose H
2o; Supply 1L.
Solution as sterilizing after filtration adds in fermentor tank: 2ml trace element solution; 2mlCaCl
2-solution; 2ml Mg/Zn-solution; 2ml Fe-solution; Supply 1L.
VF fermentation charging substratum: 660g glucose H
2o; Supply 1L.Autoclaving.Add 2gMgSO
47H
2o; 14.6mg MnSO
4h
2o; 4mg ZnSO
4h
2o; Supply 1L (autoclaving).
VYS substratum (g/l): calf is merged substratum, 30; Yeast extract, 5; Sorbyl alcohol 10; K
2hPO
42.5.This substratum was used in the first stage of inoculum.
Common charging substratum: the storage solutions of preparing pantoic acid salt and 3-aminopropanol is respectively to the final concentration of 415g/l and 980g/l.
molecule and genetic technique.The heredity of standard and Protocols in Molecular Biology are generally known in the art, and have previously been described.DNA transforms, the B.subtilis genetic technique of PBS1 generalized transduction and other standard is also generally known in the art, and has previously been described (Harwoodand Cutting, 1992).
fermentation.For example, in stirred tank fermentor (the New Brunswick20 riser that contains at first the 6 liters of fermentation of the VF containing dextrose/saline solution batch culture bases), cultivate bacterial strain.By NBSBiocommand 32 business softwares (New Brunswick Scientific Co., Inc., Edison, NJ, USA), carry out computer control; Use Lucullus software (Biospecktra AG, Schlieren, Switzerland) for data gathering and control glucose charging.
In order to prepare the inoculum of the use of fermenting, use two inoculum (two-seed) culture scheme.In the first stage, by previously preparing and being stored in the 2ml stock culture of-25 ℃, inoculated in the 25ml VYS substratum in 100ml Erlenmeyer flask, then it is hatched 3 hours on rotary shaker at 200rpm39 ℃.In subordinate phase, this culture of 0.1ml is transferred in 300ml productive culture base.This second is cultivated in advance in 2L Erlenmeyer flask and carries out, and again hatches 21 hours at 39 ℃, in this time, is issued to OD>12.For the last stage, be transferred in stirred-tank reactor (fermentor tank) the content of this class flask is aseptic, to provide the inoculum concentration of approximately 5% w/w.Prepare as mentioned below freezing bacterium original seed: in VY substratum by microbial culture to exponential phase (OD
600=0.8-1.0), add the final concentration of aseptic glycerine to 20%, freezing 1ml sample on dry ice then, and freezing bacterium is stored in to-80 ℃.
During fermentation, the pH6.8 that solution of ammonium hydroxide and the 3M HCl by automatic interpolation 27% maintains in reactor is constant.Leavening temperature is 39 ℃.Automatic classification (automaticcascading) (scope is from 400rpm to 1000rpm) by agitator and higher than the air-flow of 1vvm level, reaches the Cmin of 15% dissolved oxygen (pO2).Manually add as required defoamer (Basildon).
Fermentation can be batchwise process, but preferably carbohydrate is restricted, the process of fed-batch.Therefore, after original glucose consumption, to reactor, provide definite VF fermented feed solution (seeing above), this is the situation after 6-8 hour process period normally.Now, start to add feedstock solution with the constant rate of 84g/h.
measure panthenol, pantothenate, pantoic acid salt, aminopropanol and THMP (2,3,4 trihydroxyies, 3 methyl
pentane)
By
1h NMR spectrography, according to the amount of hereinafter described measuring panthenol, pantothenate, pantoic acid salt, aminopropanol and THMP: add the standardized solution that 500 μ l accurately contain known quantity toxilic acid (5.607g/l) in 500 μ l supernatant liquors.Freeze-drying and be heavily dissolved in 650 μ l D
2after in O, on BrukerAvance600 spectro-metre, under 300 K 600 MHz, measure
1h NMR spectrum.Relaxation delay (relaxation delay) is adjusted to 30 seconds, with guarantee scanning between completely lax.Total has been measured 16 scanning.According to the ratio between the integration (integral) from described compound methyl resonance, calculate the accurate amount of these components that exist.
embodiment 1
This embodiment has described the structure to the bacterial strain PA12 of excessive production pantothenate.
With polymerase chain reaction (PCR), in the promoter region of the panBCD operon of B.subtilis prototroph bacterial strain 1A747, produce deletion mutantion, wherein the Nucleotide district of the 215bp length between birA and panB is replaced by chlorampenicol resistant (cat) box from Staphylococcus aureus (GeneBank M58515).For this reason, first with and the orientation of the transcriptional orientation opposite of panB cat box is introduced between the NheI and ClaI site of pBR322 plasmid (GeneBank J01749).Then produce two PCR fragments " arm ": as described in manufacturers (Expand High fidelity PCR System-RocheApplied Science), in 50 μ l reaction volumes, in 0.1 μ g 1A747 chromosomal DNA, add the primer panB/up2/for/R1 of 0.2 μ l and the 100mM solution of panB/up2/rev/ClaI or primer panB/down2/for/NheI and panB/down2/rev/Bam (table 1), the 10X damping fluid of 40mM dNTP ' s, the 5 μ l that contain 1 μ l in described 50 μ l reaction volumes and 0.75 μ l PCR enzyme (Taq and Tgo).30 circulations are carried out in PCR reaction, use the annealing temperature of 58 ℃ and the extension time of 60 seconds.The fragment that purifying obtains (being called respectively F1 and F2), sequentially inserts respectively between the EcoRI of pBR322 and ClaI site (F1) and between NheI and BamHI site (F2) subsequently.The DNA of connection is transformed in E.coli TOP10 cell (Invitrogen), under the concentration of 100 μ g/ml, selects Ampicillin Trihydrate resistance.This produces E.coli plasmid pPA5.Then by DNA, transform in the karyomit(e) of B.subtilis1A747 and introduce Δ panB
p:: cat lacks box, uses standard conditions to select chlorampenicol resistant (Cm on the TBAB agar plate that contains 5 μ g/ml paraxin (Cm)
r).The single Cm that contains disappearance in separated panB promoter region
rbacterium colony, and called after PA1 (Δ panB
p:: cat).The same as expected, PA1 is also the auxotrophic (Pan of pantothenate
-), it is growth needs pantothenate in minimal medium.Use panB/up2/for/R1 and panB/down2/rev/Bam primer (table 1), reuse standard reaction condition by diagnostic PCR checking deletion mutantion.
Table 1. is for generation of containing Δ panB
p:: the primer of the B.subtilis bacterial strain of cat deletion mutantion.
Next step is strong constitutive promoter to be introduced to the upstream of panB gene.This class promotor is described in the literature, comprises the P from B.subtilis SP01 phage
15and P
26(Lee et al., 1980).Use long flank homology polymerase chain reaction (LFH-PCR) (Wach, 1996) to produce DNA fragmentation, P is contained in its ribosome bind site at panB (RBS) upstream
15.For this reason, first produce two PCR " arm ": as described in manufacturers (Expand High fidelity PCR System-RocheApplied Science), in 50 μ l reaction volumes, in 0.1 μ g 1A747 chromosomal DNA, add the primer P1panBCD of 0.2 μ l and the 100mM solution of P2panBCD or primer P3panBCD and P4panBCD (table 2), the 10X damping fluid of 40mM dNTP ' s, the 5 μ l that contain 1 μ l in described 50 μ l reaction volumes and 0.75 μ l PCR enzyme (Taq and Tgo).30 circulations are carried out in PCR reaction, use the annealing temperature of 55.7 ℃ and the extension time of 45 seconds.The fragment obtaining is called respectively F3 and F4, is purified and is used as primer in second takes turns PCR.F3 and F4 fragment are diluted to 50 times, and every kind of 1 μ l is added in the linearizing plasmid pXI23roDTD-SPO1-15 of 0.1 μ g in 50 μ l reaction volumes (containing P15 promotor).In 10 initial circulations, use the annealing temperature of 63 ℃ and the extension time of 6 minutes.In ensuing 20 circulations, after each circulation, will extend time lengthening 20 seconds.Then in third round PCR, use the product obtaining as template.By 50 times of PCR product dilutions, and the 100mM solution of 1 μ l and 0.2 μ l primer P1panBCD and P4panBCD is combined in 50 μ l reaction volumes, described 50 μ l reaction volumes contain dNTP ' s, damping fluid and enzyme as mentioned above.PCR reaction parameter with in second takes turns PCR, use identical.Then by DNA, transform the PCR fragment completing is converted in the bacterial strain PA1 of panB promoter deletion, use standard conditions on minimal medium agar plate, to select pantothenate prototroph (Pan
+).These Pan
+bacterium colony is also (the Cm of paraxin sensitivity
s), confirmed the insertion of promotor box.Separated single Pan
+cm
sbacterium colony called after PA12 (P
15panBCD), this bacterium colony contains from P
15the panBCD operon of promoter expression.By diagnosis PCR, use P15seq and P4panBCD primer (table 2), reuse standard reaction condition Verification panB upstream P
15the existence of promotor.
Table 2. is for generation of the primer of the B.subtilis bacterial strain containing P15panBCD expression cassette.
embodiment 2
This embodiment has described the structure to the bacterial strain PA49 of excessive production pantothenate.
In order to build the bacterial strain that also contains the strong constitutive promoter of panE upstream region of gene (ylbQ), first build deletion mutantion.The inspection of PanE gene has been disclosed to two possible starting points: starting point 1 (5 '-AAATTGGGTG-3 ' (RBS)-7nt-ATG) is overlapping and be positioned at starting point 2 upstream 33bp with BspHI site, starting point 2 (5 '-GGAGG-3 ' (RBS)-5nt-TTG) is overlapping with BsaXI site.Therefore, use S.aureus erythromycin resistance (Em
r) gene (GenBank V01278) 219bp that builds panE/ylbQ promoter region by LFH-PCR lacks.For this reason, first produce two PCR fragments " arm ": to the 100mM solution that adds 0.2 μ l primer P1panE and P2panE/Er or primer P3panE/Er/2 and P4panE (table 3) in 0.1 μ g 1A747 chromosomal DNA in 50 μ l reaction volumes, described in the 10X damping fluid of 40mM dNTP ' s, 5 μ l that described 50 μ l reaction volumes contain 1 μ l and 0.75 μ l PCR enzyme (Taq and Tgo) ，Ru manufacturers (Expand High fidelityPCR System-Roche Applied Science).30 circulations are carried out in PCR reaction, use the annealing temperature of 55.7 ℃ and the extension time of 45 seconds.The fragment obtaining is known as respectively F1 and F2, is purified and is used as primer in second takes turns PCR.F1 and F2 fragment are diluted to 50 times, and every kind of 1 μ l is added into the linearizing plasmid pDG646 of 0.1 μ g in 50 μ l reaction volumes (containing erm box; Gu é rout-Fleury et al., 1995) in.In 10 initial circulations, use the annealing temperature of 63 ℃ and the extension time of 6 minutes.In ensuing 20 circulations, after each circulation, will extend time lengthening 20 seconds.Then in third round PCR, use the product obtaining as template.By 50 times of PCR product dilutions, and the 100mM solution of 1 μ l and 0.2 μ l primer P1panE and P4panE is combined in 50 μ l reaction volumes, described 50 μ l reaction volumes contain dNTP ' s, damping fluid and enzyme as mentioned above.PCR reaction parameter with in second takes turns PCR, use identical.Then the PCR fragment completing is transformed into PA4 (Trp
+bacterium colony is by transforming and derive from B.subtilis CU550 trpC2 ilvC4 leu-124 with 1A747 chromosomal DNA) in, the auxotrophic Em of pantothenate obtained
rbacterium colony.These bacterium colonies are known as PA5 (Δ panE
p:: erm ilvC leuC).Use the structure of diagnosis PCR checking disappearance.Under non-congression concentration (non-congressional concentration), by PA1 chromosomal DNA, transform panB promoter deletion is caused in PA5 subsequently, produce PA6 (ilvC leuC Δ panE
p:: cat Δ panE
p:: erm).
Table 3. is for generation of containing Δ panE
p:: the primer of the B.subtilis bacterial strain of ermt deletion mutantion
Next step is to introduce strong composing type P at panB and panE upstream simultaneously
15promotor.Reuse LFH-PCR and produce following DNA fragmentation, described DNA fragmentation contains P in the opening code-reading frame upstream of panB
15and P is contained in the opening code-reading frame upstream at panE
15.For this reason, use primer P1panB and P2panB/P15 (F1) and primer P3panB/P15 and P4panB (F2) (table 1 and table 2, P
15panB builds) and P1panE and P2panE/P15 (F1) and P3panE/P15 and P4panE (F2) (table 3 and table 4, P
15panE builds) for panB and panE, produce two PCR fragments " arm ", use for building the identical PCR scheme (seeing above) of PA12.
Table 4. is for generation of containing P
15the primer of the B.subtilis bacterial strain of panE expression cassette
Then by DNA, transform the P completing
15panB and P
15panE PCR fragment is transformed into bacterial strain PA6 (the ilvC leuC Δ panB of panB and panE promoter deletion together
p:: cat Δ panEp::erm), use standard conditions on minimal medium agar plate, to select pantothenate prototroph (Pan
+).The Pan reclaiming
+bacterium colony is also Cm
s(Em with erythromycin-sensitive
s), this has confirmed the insertion of promotor box.Separated single Pan
+cm
sem
sbacterium colony, and be called PA32, described bacterium colony contains from P
15the panBCD operon of promoter expression and panE gene.By diagnosis PCR, use P15seq and P4panB primer (table 1 and 2), reuse standard reaction condition Verification panB upstream region of gene P
15the existence of promotor.Use P15seq and P4panE primer (table 3 and 4) on panE gene, to carry out same contrast.Yet, P before panE subsequently
15the order-checking of promotor has disclosed P
15the excalation of promotor.
For the structure with correct is replaced the P of excalation
15panE gene, is used chromosomal DNA (the Δ panE from PA5
p:: erm ilvC leuC) by DNA, transform Δ panE
p:: erm sudden change is introduced in PA32 again, and selects erythromycin resistance.This producing bacterial strain PA41 (P
15panBCD Δ panE
p:: erm ilvC leuC).Then by the P producing by LFH-PCR as previously mentioned
15panEDNA fragment is transformed in PA41, selects Pan
+prototroph.This producing bacterial strain PA43 (ilvCleuC P
15panBCD P
15panE).Then use standard step, use the PBS1 phage lysate of preparing on wild-type B.subtilis1A747 by this Ilv
-leu
-auxotrophic strain transduction is Ilv
+leu
+prototroph.This producing bacterial strain PA49 (P
15panBCD P
15panE).
embodiment 3
This embodiment has described the structure to bacterial strain PA112, and it is the PA49 derivative that contains panC disappearance.
Use long flank homology polymerase chain reaction (LFH-PCR) to produce deletion mutantion in the coding region of the panC of panBCD operon opening code-reading frame, wherein the 487bp-longer nucleotide region of panC is replaced by paraxin (cat) the resistance box (GenBankM58515) from Staphylococcus aureus.For this reason, first produce two PCR fragments " arm ": to the 100mM solution that adds 0.2 μ l primer P1panC and P2panC-cat or primer P3panC and P4panC-cat (table 5) in 0.1 μ g 1A747 chromosomal DNA in 50 μ l reaction volumes, described in the 40mM dNTP ' s that described 50ul reaction volume contains 1 μ l, the 10X damping fluid of 5 μ l and 0.75 μ l PCR enzyme (Taq and Tgo) ，Ru manufacturers (Expand High fidelity PCR System-Roche AppliedScience).30 circulations are carried out in PCR reaction, use the annealing temperature of 55.7 ℃ and the extension time of 45 seconds.The fragment obtaining is called respectively F1 and F2, is purified and is used as primer in second takes turns PCR.F1 and F2 fragment are diluted to 50 times, and every kind of 1 μ l is added in the linearizing plasmid pPA4 of 0.1 μ g in 50 μ l reaction volumes (containing cat box).In 10 initial circulations, use the annealing temperature of 63 ℃ and the extension time of 3 minutes.In ensuing 20 circulations, after each circulation, will extend time lengthening 20 seconds.Then in third round PCR, use the product obtaining as template.By 50 times of PCR product dilutions, and the 100mM solution of 1 μ l and 0.2 μ l primer P1panC and P4panC is combined in 50 μ l reaction volumes, described 50 μ l reaction volumes contain dNTP ' s, damping fluid and enzyme as mentioned above.PCR reaction parameter with in second takes turns PCR, use identical.Then the PCR fragment completing is transformed in the bacterial strain PA49 of overexpression pantothenate, on TBAB substratum, selects chlorampenicol resistant (Cm
r).Separated single Cm
rbacterium colony, and called after PA112 (P
wtpanB Δ C::catD).Use P1panC and P4panC, reuse standard reaction condition by the existence of diagnostic PCR checking cat box.With regard to phenotype, PA112 can not be supplemented with 1mM pantoic acid salt or 1mM pantoic acid salt and adding the upper growth of minimal medium (MM) of 1mM Beta-alanine, but on the MM that contains 1mM pantothenate normal growth.In shake-flask culture, PA112 upgrowth situation when having 1 μ M or 10 μ M pantothenate is not good, but when pantothenate is supplementary while being increased to 100 μ M or 1mM normal growth.
Table 5. is for the production of the primer of Δ panC::cat deletion mutantion
Underscore sequence and panC district homology.
embodiment 4
This embodiment has described the structure of bacterial strain PA121, and it is the PA49 derivative that contains panCD disappearance
Use long flank homology polymerase chain reaction (LFH-PCR) to produce deletion mutantion, it comprises the coding region of panC and the panD opening code-reading frame of panBCD operon, and wherein the 874bp-longer nucleotide region of panC and panD is replaced by paraxin (cat) the resistance box (GenBank M58515) from Staphylococcus aureus.For this reason, first produce two PCR fragments " arm ": to the 100mM solution that adds 0.2 μ l primer P1panC and P2panC-cat or primer P3panD-cat and P4panD (table 6) in 0.1 μ g 1A747 chromosomal DNA in 50 μ l reaction volumes, described in the 10X damping fluid of 40mM dNTP ' s, 5 μ l that described 50 μ l reaction volumes contain 1 μ l and 0.75 μ l PCR enzyme (Taq and Tgo) ，Ru manufacturers (Expand High fidelity PCR System-RocheApplied Science).30 circulations are carried out in PCR reaction, use the annealing temperature of 55.7 ℃ and the extension time of 45 seconds.The fragment obtaining is called respectively F1 ' and F2 ', is purified and follows in second takes turns PCR to be used as primer.F1 ' and F2 ' fragment are diluted to 50 times, and every kind of 1 μ l is added in the linearizing plasmid pPA4 of 0.1 μ g in 50 μ l reaction volumes (containing cat box).In 10 initial circulations, use the annealing temperature of 63 ℃ and the extension time of 5 minutes.In ensuing 20 circulations, after each circulation, will extend time lengthening 20 seconds.Then in third round PCR, use the product obtaining as template.By 50 times of PCR product dilutions, and the 100mM solution of 1 μ l and 0.2 μ l primer P1panC and P4panD is combined in 50 μ l reaction volumes, described 50 μ l reaction volumes contain dNTP ' s, damping fluid and enzyme as mentioned above.PCR reaction parameter with in second takes turns PCR, use identical.Then the PCR fragment completing is transformed in the bacterial strain PA49 of overexpression pantothenate, on TBAB substratum, selects chlorampenicol resistant (Cm
r).The single Cm that separated panCD is lacked
rbacterium colony, and called after PA121 (P
wtpanB Δ CD::cat).Use PlpanC and P4panD, reuse standard reaction condition by the existence of diagnostic PCR checking cat box.As bacterial strain PA112, bacterial strain PA121 can not be supplemented with 1mM pantoic acid salt or 1mM pantoic acid salt and adding the upper growth of minimal medium (MM) of 1mM Beta-alanine in phenotype, but on the MM that contains 1mM pantothenate normal growth.In shake-flask culture, PA121 upgrowth situation when having 1 μ M or 10 μ M pantothenate is not good, but when pantothenate is supplementary while being increased to 100 μ M or 1mM normal growth.
Table 6. is for generation of the primer of Δ panCD::cat deletion mutantion
Underlined sequence and panCD district homology.
embodiment 5
This embodiment has described the structure to the bacterial strain PA73 of excessive production pantothenate.
Use LFH-PCR to produce ilvD upstream and contain P
26the DNA fragmentation of promotor.First use primer P1/ilvD/for and P2/ilvD/f/26 (F1 arm) and primer P3/ilvD/r/26 and P4/ilvD/rev (F2 arm) (table 7) two the PCR fragments " arm " that increase.Template is the chromosomal DNA of 1A747.Using linearization plasmid pUC18SP01-26 (containing P
26second taking turns in PCR the F1 obtaining and F2 arm as primer promotor).The F1-P obtaining with primer P1/ilvD/for and P4/ilvD/rev (table 7) amplification in third round PCR
26-F2LFH-PCR product.To contain P
26the F1-P of ilvD
26-F2LFH-PCR fragment is transformed in the bacterial strain PA24 (Δ ilvD::spec) of ilvD promoter deletion, obtains PA27 (P
26ilvD).Use the PBS1 lysate from PA27 to pass through transduction by P
26ilvD introduces the bacterial strain PA60 (P of ilvD promoter deletion
15panBCD P
15panE Δ ilvD::spec) in.On minimum agar plate, select the responsive (Spec of ilvD prototroph and spectinomycin
s) bacterium colony.Bacterial strain PA62 (the P obtaining
15panBCD P
15panE P
26ilvD
g320D) in ilvD coding region, with a single point, suddenly change, it causes the amino acid change of the arrive-Asp of Gly-of residue 320.Then use LFH-PCR, by first removing the interior zone of the ilvD gene that contains this sudden change, this ilvD encoding sequence is reverted to wild-type.By primer Plc/ilvD/for and P2c/ilvD/cat (F1 arm) and primer P3c/ilvD/cat and P4c/ilvD/rev (F2 arm) (table 7) two the PCR fragments " arm " that increase.Template DNA is the chromosomal DNA of 1A747.Using linearization plasmid pTH5 (containing cat box; Second taking turns in PCR the F1 obtaining and F2 arm as primer GenBank M58515).F1-cat-F2 LFH-PCR fragment is transformed in PA62, and selects Cm
rand IlvD
-auxotrophic strain PA64 (P
15panBCD P
15panE P26 Δ ilvD::cat).Use the structure of diagnosis PCR checking disappearance.Then use the chromosomal DNA of wild type strain 1A747 that bacterial strain PA64 is converted into prototroph.Bacterial strain PA73 (the P obtaining
15panBCD P
15panEP
26ilvD), in, by diagnosis PCR, use P26seq and P4/ilvD/rev primer (table 7) checking ilvD upstream P
26not not existing of the existence of promotor and cat gene.
The ilvD that table 7. is set forth sudden change and P26-driving for the production of DilvD::spec crosses the primer of expression
Be added with sequence and the ilvD district homology of underscore.
embodiment 6
This embodiment has described the structure to PA207, and it is the PA12 derivative that contains panD genetically deficient.
Build and there is P in two steps
15the bacterial strain of panBC Δ panD::spec operon.First panD is lacked to box and be transformed in bacterial strain PA1, the disappearance that described PA1 contains panB leader.Use long flank homology polymerase chain reaction (LFH-PCR) to produce deletion mutantion in the coding region of the panD of panBCD operon opening code-reading frame, wherein the Nucleotide region of panD is replaced by the VITAMIN B4 transferring enzyme (accession number XO3216) of giving spectinomycin resistance from Staphylococcus aureus.For this reason, first produce two PCR fragments " arm ": to the 100mM solution that adds 0.2 μ l primer P1panD and P2panD-spec or primer P3panD-spec and P4panDb (table 8) in 0.1 μ g 1A747 chromosomal DNA in 50 μ l reaction volumes, described in the 10X damping fluid of 40mM dNTP ' s, 5 μ l that described 50 μ l reaction volumes contain 1 μ l and 0.75 μ l PCR enzyme (Taq and Tgo) ，Ru manufacturers (Expand High fidelity PCR System-Roche AppliedScience).30 circulations are carried out in PCR reaction, use the annealing temperature of 55.7 ℃ and the extension time of 45 seconds.The fragment obtaining is called respectively F1 and F2, is purified and is used as primer in second takes turns PCR.F1 and F2 fragment are diluted to 50 times, and every kind of 1 μ l is added in the linearizing plasmid pDG1726 of 0.1 μ g in 50 μ l reaction volumes (containing spec box).In 10 initial circulations, use the annealing temperature of 63 ℃ and the extension time of 3 minutes.In ensuing 20 circulations, after each circulation, will extend time lengthening 20 seconds.Then in third round PCR, use the product obtaining as template.By 50 times of PCR product dilutions, and the 100mM solution of 1 μ l and 0.2 μ l primer P1panD and P4panD is combined in 50 μ l reaction volumes, described 50 μ l reaction volumes contain dNTP ' s, damping fluid and enzyme as mentioned above.PCR reaction parameter with in second takes turns PCR, use identical.Then the PCR fragment completing is transformed in bacterial strain PA1, on TBAB substratum, selects spectinomycin resistance (Sp
r).The single Sp that separated panD is lacked
rbacterium colony, and called after PA203.Then will contain P
15the DNA fragmentation of promotor (seeing the structure of PA12) is transformed in bacterial strain PA203, and rebuild is through the P of engineering operation
15panBC Δ D operon.By recovering Pan
+prototroph and forfeiture chlorampenicol resistant are selected the cell that contains this operon.This producing bacterial strain PA207.Phenotype analytical points out that PA207 is Beta-alanine trophism slowly (bradytrophic).Reuse standard reaction condition, by diagnosis PCR, use the existence of P1panD and P4panDb checking spec box.
Table 8. is for the production of the primer of Δ panD::spec deletion mutantion
embodiment 7
This embodiment has set forth by producing panthenol to bacterial strain PA49, PA73, PA112, PA121 and PA207 feed aminopropanol.
By the overnight culture of PA112, PA121 and PA207,37 ℃ of cultivations, and 1:100 dilutes in the SMG substratum of the fresh preparation of 200ml.Then minute styles such as three parts of 40ml of every kind of substratum are transferred in the 250ml flask containing polystyrene foamed plastics stopper (styroform stoppers).To adding the final concentration to 40g/l through the 3-aminopropanol solution (pH7.2) of HCl neutralization in each flask.At 37 ℃, continue to cultivate 37 ° of C, now by centrifugal removal cell and by supernatant liquor sterile filtration.In acellular substratum, add pantoic acid salt and 3-aminopropanol and hatch 72 hours in contrast at 37 ℃.By NMR, measure panthenol, pantoic acid salt and pantothenate level, the results are shown in Table 9.In bacterial strain PA49, PA73 and PA207, clearly detect the production of panthenol.The bacterial strain PA112 and the PA121 that contain panC disappearance do not produce panthenol.When pantoic acid salt and 3-aminopropanol are hatched 72 hours in substratum (acellular), can't detect spontaneous panthenol and form.These results are pointed out conventionally and the panC enzyme of pantoic acid salt and Beta-alanine coupling formation pantothenate also can coupling pantoic acid salt and 3-aminopropanol formation panthenol.Obviously, can use the method for well known to a person skilled in the art (for example PCR mutagenesis, protein evolution) significantly to strengthen the enzyme characteristic that panC catalysis pantoic acid salt and 3-aminopropanol are converted into panthenol.
The multiple pantothenate that table 9. is cultivated when existing 40g/ to rise aminopropanol is produced in bacterial strain and is produced panthenol.
athe average of three samples.
bin culture, add 1mM pantothenate, because PA112 and PA121 are pantothenate auxotroph.
embodiment 8
This embodiment has set forth and in stirred-tank reactor, has used fed-batch operation mode by bacterial strain PA207 feed aminopropanol and pantoic acid salt are produced to panthenol.
In the limited fed-batch fermentation of standard glucose, bacterial strain PA207 is cultivated 71 hours.Batch phase initial～after 6 hours, use 80% glucose of feed within the scope of 84 to 95g/h to start the batch feed stage.After the fermentation time of 25 hours, the mean rate with 14g/h in 22 hours provides 98% 3-aminopropanol solution.Start after 3-aminopropanol feed ten hours, with the mean rate of 26g/h, add 41.5% pantoic acid salts solution, approximately add 16 hours.
As shown in table 10, when to ferment common feed pantoic acid salt and 3-aminopropanol the two time, by NMR, clearly detect panthenol.
Table 10. is produced panthenol in the pantoic acid salt of bacterial strain PA207 (PA49 Δ panD::spec) and the common feed fermentation of 3-aminopropanol.
athree samples average.
Claims (8)
1. produce the method for panthenol, described method by cultivating following Bacillus subtilis microorganism and optionally reclaim panthenol and realize from cell culture medium with 3-aminopropanol or the common feed of its suitable derivative under suitable culture condition, described Bacillus subtilis microorganism can cross at least one enzyme and the PanC expressing in the biosynthesizing of pantoic acid salt, at least one enzyme in the biosynthesizing of wherein said pantoic acid salt selects free ketopantoic acid salt hydroxymethyl transferases (PanB), the group that ketopantoic acid salt reductase enzyme (PanE) and IlvD form, wherein said 3-aminopropanol derivative selects free 3-alkoxy propyl amine, the group that 3-halogen propylamine and 3-amino glycerol form.
2. method claimed in claim 1, wherein PanE is crossed and is expressed.
3. method claimed in claim 1, wherein PanB is crossed and is expressed.
4. method claimed in claim 1, wherein PanC is crossed and is expressed.
5. method claimed in claim 1, wherein PanD is by inactivation.
6. the purposes of the PanC of catalytic activity with raising the method for preparing panthenol from 3-aminopropanol or derivatives thereof and pantoic acid salt, described derivative is selected from 3-alkoxy propyl amine, 3-halogen propylamine and 3-amino glycerol.
7. purposes according to claim 6, wherein said 3-alkoxy propyl amine is 3 methoxypropyl amine or 3-ethoxy propylamine.
8. purposes according to claim 6, wherein said 3-halogen propylamine is 3-chlorine propylamine.
Applications Claiming Priority (3)
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|---|---|---|---|
| EP06010054.2 | 2006-05-16 | ||
| EP06010054 | 2006-05-16 | ||
| PCT/EP2007/004249 WO2007131750A1 (en) | 2006-05-16 | 2007-05-15 | Process for the production of panthenol |
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| EP2468712A1 (en) * | 2010-12-14 | 2012-06-27 | Basf Se | Method for producing and purifying 3-aminopropanol |
| EP2723878B1 (en) | 2011-06-21 | 2020-05-20 | DSM IP Assets B.V. | Vitamin b5 production |
| WO2012175574A1 (en) | 2011-06-21 | 2012-12-27 | Dsm Ip Assets B.V. | Increased pantothenate (vitamin b5) production |
| CN111893083A (en) * | 2019-05-05 | 2020-11-06 | 中国科学院上海高等研究院 | Modified Klebsiella bacteria and application thereof in producing 2, 3-dihydroxy isovaleric acid |
| CN113317512A (en) * | 2021-05-19 | 2021-08-31 | 安徽华恒生物科技股份有限公司 | Preparation method and application of D-panthenol aqueous solution with high stability |
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