CN102660484B - Rhodobacter azotoformans and culture method and application thereof - Google Patents

Rhodobacter azotoformans and culture method and application thereof Download PDF

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CN102660484B
CN102660484B CN201210156757.0A CN201210156757A CN102660484B CN 102660484 B CN102660484 B CN 102660484B CN 201210156757 A CN201210156757 A CN 201210156757A CN 102660484 B CN102660484 B CN 102660484B
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neurosporene
rhodobacter azotoformans
fixed nitrogen
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didehydrogenated
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肖敏
张金华
卢丽丽
钱新民
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Shandong University
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Abstract

The invention relates to Rhodobacter azotoformans Y6 and a culture method and application thereof. The strain has the function of producing carotenoid C-3,4 dehydrogenase which can catalyze neurosporene and lycopene for C-3,4 dehydrogenation reaction to obtain 3,4-didehydrogenated neurosporene and 3,4-didehydrogenated lycopene. In addition, the invention also provides the strain and the characteristics of the Rhodobacter azotoformans, the culture method for the Rhodobacter azotoformans, and a catalytic property of the carotenoid C-3,4 dehydrogenase of the Rhodobacter azotoformans. Recombinant enzyme which is subjected to recombinant expression by a crtD gene derived from the stain in Escherichia coli can catalyze the neurosporene or the lycopene to be dehydrogenated to form the 3,4-didehydrogenated neurosporene or the 3,4-didehydrogenated lycopene respectively, so that a novel enzyme source is provided for the microbial fermentation production of the 3,4-didehydrogenated neurosporene and the 3,4-didehydrogenated lycopene.

Description

The red bacterium of one strain fixed nitrogen and cultural method and application
Technical field
The present invention relates to the carotenoid C-3 that a strain produces catalysis neurosporene and Lyeopene dehydrogenation, the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 and cultural method and the application of 4 desaturases, belong to microbial technology field.
Background technology
Carotenoid is a kind of multiclass isoprenoid, is extensively present in microorganism, plant, animal body.Carotenoid comprises special long-chain polyenoid conjugated structure, makes it have special physiologically active, comprises photoabsorption, anti-oxidant, cancellation free radical etc.Carotenoid is produced the main plant extract of leaning at present, and output is limited, can not satisfy the demands, and microorganism fermentative production becomes study hotspot.Can produce different types of carotenoid by express different carotenoid synthases in engineering strain, seek the new microbe-derived carotenoid synthase that can obtain different catalytic characteristics, for the production of different sorts carotenoid provides new way.
Carotenoid C-3,4 desaturases (CrtD) are desaturases in carotenogenesis approach, conventionally catalysis 1-hydroxy kind carotene and 1 '-hydroxy kind carotene carry out C-3, and 4 and C-3 ', 4 ' dehydrogenation reaction.Product oxygen photosynthetic bacterium---in cyanobacteria, there are bibliographical information CrtD catalysis neurosporene and Lyeopene to carry out C-3, the reaction of 4 dehydrogenations, but in Anoxygenic photosynthetic bacteria, not yet find that CrtD possesses this catalysis characteristics.
Through retrieval, there is no in the world at present the red Production by Bacteria carotenoid of fixed nitrogen C-3, the bibliographical information of 4 desaturases.
Summary of the invention
The present invention is directed to existing microorganism carotenoid synthase kind limited, particularly Anoxygenic photosynthetic bacteria carotenoid C-3, the blank of 4 desaturase catalysis neurosporenes and Lyeopene dehydrogenation research, the carotenoid C-3 that provides a strain to produce catalysis neurosporene and Lyeopene dehydrogenation, the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 and cultural method and the application of 4 desaturases.
Summary of the invention
The red bacterium of fixed nitrogen of the present invention (Rhodobacter azotoformans) Y6, has the carotenoid of producing C-3,4 desaturases, can catalysis neurosporene and Lyeopene carry out C-3,4 dehydrogenation reactions, generate 3, the function of 4-bis-dehydrogenation neurosporenes and 3,4-, bis-dehydrogenation Lyeopenes.In addition the present invention also provides feature, cultural method and the application of this fixed nitrogen Rhodobacter strain.
Detailed Description Of The Invention
The red bacterium of one strain fixed nitrogen (Rhodobacter azotoformans) Y6 bacterial strain, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 8th, 2012, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, deposit number is CGMCC No.6086.
The cultural method of the red bacterium of above-mentioned fixed nitrogen (Rhodobacter azotoformans) Y6, step is as follows:
Red fixed nitrogen bacterium (Rhodobacter azotoformans) Y6 is inoculated in RCVBN substratum by 8 ~ 12wt% inoculum size, is that under the condition of 25 ~ 35 DEG C, constant temperature illumination leaves standstill and cultivates 48 ~ 72 hours in temperature;
Above-mentioned RCVBN nutrient media components is as follows:
MgSO 47H 2o 0.2g/L, CaCl 20.056g/L, FeSO 47H 2o 0.012g/L, Na 2eDTA 0.02g/L, H 3bO 32.8 × 10 -3g/L, MnSO 41.6 × 10 -3g/L, ZnSO 47H 2o 2.4 × 10 -4g/L, Na 2moO 42H 2o 7.52 × 10 -4g/L, Ca (NO3) 23H 2o 4 × 10 -5g/L, vitamin H 1.5 × 10 -5g/L, nicotinic acid 1 × 10 -3g/L, para-amino benzoic acid 1 × 10 -3g/L, vitamin 1 × 10 -3g/L, KH 2pO 40.6g/L, K 2hPO 43H 2o 0.9g/L, DL-oxysuccinic acid 4g/L, (NH 4) 2.SO 41g/L, pH6.8,115 DEG C of sterilizings 30 minutes.
Preferred according to the present invention, described culture temperature is 30 DEG C.
CrtD energy catalysis neurosporene and Lyeopene that the red bacterium Y6 of fixed nitrogen of the present invention bacterial strain produces carry out dehydrogenation reaction.
The application of the red bacterium of above-mentioned fixed nitrogen (Rhodobacter azotoformans) Y6 in neurosporene or Lyeopene dehydrogenation reaction.
Above-mentioned application, step is as follows:
Utilize round pcr taking the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 strain gene group DNA as template, adopt upstream primer D-F:TGCCATATGATGAGACAGATTGTGCCGAA(SEQ ID NO.3) and downstream primer D-R:TCGAAGCTTCCTCGATCGCGAAGCAAGGT(SEQ ID NO.4), amplification carotenoid crtD gene, this gene is expressed in e. coli bl21 (DE3) by expression vector pET-22b, collect recombinant Bacillus coli cells supersonic wave wall breaking and prepare recombinant C rtD crude enzyme liquid, recycling recombinant C rtD crude enzyme liquid carries out external dehydrogenation reaction to neurosporene or Lyeopene, generate 3, 4-bis-dehydrogenation neurosporenes or 3, 4-bis-dehydrogenation Lyeopenes.
Above operation steps, experiment condition and reagent if no special instructions, all adopt this area routine operation and common agents.
Beneficial effect
In the red bacterium of fixed nitrogen of the present invention (Rhodobacter azotoformans) Y6, contain carotenoid C-3,4-desaturase, this enzyme energy catalysis neurosporene or Lyeopene carry out dehydrogenation reaction and generate respectively 3,4-, bis-dehydrogenation neurosporenes or 3,4-, bis-dehydrogenation Lyeopenes, this feature is discovery first in Anoxygenic photosynthetic bacteria, the microorganism fermentative production that is 3,4-, bis-dehydrogenation neurosporenes or 3,4-, bis-dehydrogenation Lyeopenes provides new enzyme source.
Brief description of the drawings
Fig. 1 is the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 recombinant C rtD crude enzyme liquid catalysis neurosporene dehydrogenation product HPLC figure;
Wherein, 1,3,4-bis-dehydrogenation neurosporenes; 2, neurosporene;
Fig. 2 is the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 recombinant C rtD crude enzyme liquid catalysis Lyeopene dehydrogenation product HPLC figure;
Wherein, 3,3,4-bis-dehydrogenation Lyeopenes; 4, Lyeopene;
Fig. 3 is the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 recombinant C rtD crude enzyme liquid catalysis neurosporene dehydrogenation product 3,4-bis-dehydrogenation neurosporene optical absorption maps;
Fig. 4 is the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 recombinant C rtD crude enzyme liquid catalysis Lyeopene dehydrogenation product 3,4-bis-dehydrogenation Lyeopene optical absorption maps;
Fig. 5 is the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 recombinant C rtD crude enzyme liquid catalysis neurosporene dehydrogenation product 3,4-bis-dehydrogenation neurosporene mass-spectrograms;
Fig. 6 is the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 recombinant C rtD crude enzyme liquid catalysis Lyeopene dehydrogenation product 3,4-bis-dehydrogenation Lyeopene mass-spectrograms.
Embodiment
Below in conjunction with embodiment, the present invention is further elaborated, but institute of the present invention protection domain is not limited only to this.
The red bacterium of fixed nitrogen described in embodiment (Rhodobacter azotoformans) Y6, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences) on May 8th, 2012, deposit number is CGMCC No.6086.
Na 2eDTA is purchased from Amresco company of the U.S.; Na 2moO 42H 2o is purchased from Shanghai gelatinizing factory; Para-amino benzoic acid is purchased from Solution on Chemical Reagents in Shanghai purchasing and supply station; DL-oxysuccinic acid is purchased from Tianjin great Mao chemical reagent factory; Vitamin H, nicotinic acid, vitamin are purchased from China Medicine (Group) Shanghai Chemical Reagent Co.; Peptone and yeast powder are purchased from Oxoid company of Britain.
High performance liquid chromatograph and chromatographic column TC-C18 are all purchased from Agilent company of the U.S..
Embodiment 1:
The red bacterium of one strain fixed nitrogen (Rhodobacter azotoformans) separates and obtains in the Xiaoqinghe River sanitary sewage of Jinan City, Shandong Province, has following biological property:
(1) Morphological observation
Gram-negative, cell ellipse, thalline diameter is 0.6 μ m ~ 1.2 μ m; Under illumination anaerobism, dark aerobic and dark anaerobic condition, all can grow.Under illumination anaerobic condition, culture is brown color; Under illumination half anaerobic condition, culture is red.
(2) physiological and biochemical property is observed
Under illumination anaerobic condition, can utilize melampyrum to grow for sole carbon source, can not utilize soluble tartrate; Under dark anaerobism and saltpetre condition, can utilize fructose or wood sugar to grow for sole carbon source; Other features refer to table 1.
Table 1 fixed nitrogen red bacterium Y6 carbon source and electron donor utilization
Figure BDA00001658087100031
Note: +++, vigorous growth (OD660>1.0); ++, common growth (1.0>OD 660>0.2); +, faint growth (OD 660<0.2);-, do not grow
(3) 16S rDNA sequencing and homology analysis
Adopt bacterial genomes to extract test kit (purchased from BioFlux company) and extract the red bacterium Y6 of fixed nitrogen genomic dna, adopt the general upstream primer 27f:AGAGTTTGATCMTGGCTCAG(SEQ of bacterial 16 S rDNA ID NO.1) and downstream primer 1525r:AAGGAGGTGATCCAGCC(SEQ ID NO.2) taking genomic dna as template pcr amplification 16S rDNA sequence.
PCR reaction system (50 μ L):
TaKaRa taq archaeal dna polymerase (2.5U), 10 × TaKaRataq polysaccharase buffer(5 μ L), MgCl 2(1.5mM), genomic dna (10ng), dNTP(200 μ M), primer (0.4 μ M).
Reaction conditions is:
94 DEG C of denaturation 2min; 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 90s, 30 circulations; 72 DEG C are extended 10min.
The amplified production obtaining carries out sequencing by Shanghai Sheng Gong biotechnology company limited, size is 1459bp, this sequence is carried out to BLAST Nucleotide compare of analysis in NCBI website, 16S rDNA sequence (the GenBank number of including HM536622 with the red bacterium of 7 strain fixed nitrogen, GU990617, GU990616, NR_029215 etc.) homology is 99%.
The GenBank number of including of the 16S rDNA of above-mentioned bacterial strains is JF738027, the most approaching with the red bacterium of fixed nitrogen on evolutionary relationship, therefore by the red bacterium of this bacterial strain called after fixed nitrogen (Rhodobacter azotoformans) Y6 bacterial strain, and being deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences) on May 8th, 2012, deposit number is CGMCC No.6086.
Embodiment 2:
The cultural method of the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 described in embodiment 1, step is as follows:
Red fixed nitrogen bacterium (Rhodobacter azotoformans) Y6 is inoculated in RCVBN substratum by the inoculum size of 10wt%, is that under the condition of 30 DEG C, constant temperature illumination leaves standstill and cultivates 48 ~ 72 hours in temperature;
Above-mentioned RCVBN nutrient media components is as follows:
MgSO 47H 2o 0.2g/L, CaCl 20.056g/L, FeSO 47H 2o 0.012g/L, Na 2eDTA 0.02g/L, H 3bO 32.8 × 10 -3g/L, MnSO 41.6 × 10 -3g/L, ZnSO 47H 2o 2.4 × 10 -4g/L, Na 2moO 42H 2o 7.52 × 10 -4g/L, Ca (NO3) 23H 2o 4 × 10 -5g/L, vitamin H 1.5 × 10 -5g/L, nicotinic acid 1 × 10 -3g/L, para-amino benzoic acid 1 × 10 -3g/L, vitamin 1 × 10 -3g/L, KH 2pO 40.6g/L, K 2hPO 43H 2o 0.9g/L, DL-oxysuccinic acid 4g/L, (NH 4) 2.SO 41g/L; Adjust pH to 6.8,115 DEG C of sterilizings 30 minutes.
Embodiment 3:
The application of the red bacterium of above-mentioned fixed nitrogen (Rhodobacter azotoformans) Y6 in neurosporene or Lyeopene dehydrogenation reaction, concrete steps are as follows:
(a) adopt bacterial genomes to extract the genomic dna of the red bacterium Y6 of fixed nitrogen of test kit (BioFlux) extraction embodiment 2 cultivation acquisitions, adopt crtD upstream region of gene primer D-F:TGC cATATGaTGAGACAGATTGTGCCGAA(SEQ ID NO.3), underscore is NdeI restriction enzyme site) and downstream primer D-R:TCG aAGCTTcCTCGATCGCGAAGCAAGGT(SEQ ID NO.4), underscore is HindIIII restriction enzyme site), taking genomic dna as template pcr amplification crtD gene order,
PCR reaction system (50 μ L):
TaKaRa LAtaq archaeal dna polymerase (2.5U), 2 × TaKaRa GC damping fluid (25 μ L), MgCl 2(1.5mM), genomic dna (10ng), dNTP(200 μ M), primer (0.4 μ M).
94 DEG C of denaturation 2min; 94 DEG C of 30s, 61 DEG C of 30s, 72 DEG C of 90s, 30 circulations; 72 DEG C are extended 10min.
The final crtD gene that obtains, empirical tests, the GenBank number of including of the gene cluster at the crtD gene place of the red bacterium of fixed nitrogen (Rhodobacter azotoformans) Y6 is JF723980.
(b) amplified production that step (a) obtained adds restriction enzyme NdeI, HindIII(NEB, USA after reclaiming) cut 1 hour in 37 DEG C of enzymes, carry out DNA purifying, the gene fragment after making enzyme and cutting;
Adopt same enzyme blanking method to process pET-22b plasmid vector, the pET-22b plasmid vector after making enzyme and cutting;
PET-22b plasmid vector after gene fragment after enzyme is cut is cut with enzyme in molar ratio 4:1 mixes, and adopts T4 ligase enzyme (NEB, USA) in 16 DEG C of connections 10 hours, then adopts CaCl 2conversion method transforms Host Strains e. coli bl21 (DE3), the bacterium liquid coating amicillin resistance LB flat board after conversion, 37 DEG C of incubated overnight.The well-grown single bacterium colony of picking in LB nutrient solution 37 DEG C cultivate 10 hours, extract plasmid, by enzyme cutting method and PCR method checking positive transformant, obtain expressing the red bacterial carotenoid C-3 of fixed nitrogen, the intestinal bacteria recombinant bacterium of 4 desaturases;
The dull and stereotyped composition of above-mentioned LB: peptone 10g/L, yeast powder 5g/L, NaCl 7g/L, agar 15g/L, pH7.0～7.5,121 DEG C of sterilizings 20 minutes.
Above-mentioned LB nutrient solution composition: peptone 10g/L, yeast powder 5g/L, NaCl 7g/L, pH7.0～7.5,121 DEG C of sterilizings 20 minutes.
(c) will in step (b), express the red bacterial carotenoid C-3 of fixed nitrogen, the intestinal bacteria recombinant bacterium of 4 desaturases is inoculated in 200mL LB nutrient solution, and 37 DEG C are cultured to OD 600reach 0.5～0.9, add IPTG(sec.-propyl-β-D-sulfo-galactopyranoside) induction, IPTG final concentration is 0.5mmol/L, 25 DEG C of inducing culture 30 hours.Centrifugal collection somatic cells, with the resuspended rear ultrasonic disruption of 100mM phosphoric acid buffer (pH 8.0), obtains recombinant C rtD crude enzyme liquid;
Above-mentioned LB nutrient solution composition: peptone 10g/L, yeast powder 5g/L, NaCl 7g/L, pH7.0～7.5,121 DEG C of sterilizings 20 minutes.
(d) get the recombinant C rtD crude enzyme liquid 400 μ L that obtain in step (c), add 10 μ M neurosporenes or Lyeopene (purchased from CaroteNature company, Switzerland), reaction system is settled to 500 μ L with damping fluid; Mix 30 seconds (22kHz, 100W) with the ultrasonic disruption instrument of Sonics company of the U.S.; 30 DEG C of airtight oscillatory reactions of lucifuge 5 hours, obtain mixed solution;
(e) in the mixed solution making to step (d), add 30 μ L 2mol/LNaOH and 30 μ L 10wt%SDS solution, mix; Add 300 μ L 3mol/L NaCH 3cOOH(pH4.8) solution, vibrates 5 seconds; Centrifugal 1 minute of 12000rpm, discards supernatant, obtains protein-based precipitation;
(f) in the protein precipitation making to step (e), add 200 μ L acetone extraction carotenoid, stir 2 minutes, make to precipitate dispersed, obtain the acetone soln containing carotenoid;
(g) acetone soln containing carotenoid step (f) being made centrifugal 1 minute through 12000rpm; get acetone soln; after 0.22 μ m membrane filtration, HPLC(high performance liquid chromatography) method detection reaction product, chromatographic column is that TC-C18(is purchased from Agilent company; USA); mobile phase methanol/acetonitrile (volume ratio is 4/6), flow velocity 1mL/ minute, 30 DEG C of column temperatures; detect wavelength 474nm, detected result is as shown in Figure 1.
(h) collect the carotenoid component that step (g) HPLC detects, carry out the analyses of photoabsorption instrument (purchased from Thermo company, USA) and mass spectrograph (purchased from Kyoto company, Japan).Photoabsorption scanning wavelength scope is 350 ~ 600nm, solvent methanol/acetonitrile (volume ratio is 4/6); Mass ion source is ESI, positive ion mode, and moving phase is 98wt% acetonitrile (containing 0.05wt% formic acid).
Fig. 1 and Fig. 2 show that the red bacterium CrtD of above-mentioned restructuring fixed nitrogen catalysis neurosporene (component 2) or Lyeopene (component 4) have generated new carotenoid (component 1 and 3).Component 1 has three main absorption peaks, and wavelength is respectively 444nm, 472nm and 504nm; Molecular weight is 536.4, and this carotenoid is 3,4-, bis-dehydrogenation neurosporenes (Fig. 3, Fig. 5).Component 3 has three main absorption peaks, and wavelength is respectively 469nm, 496nm and 528nm; Molecular weight is 534.4, and this carotenoid is 3,4-, bis-dehydrogenation Lyeopenes (Fig. 4, Fig. 6).

Claims (2)

  1. The red bacterium of one strain fixed nitrogen ( rhodobacter azotoformans) Y6 bacterial strain, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address on May 8th, 2012: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, deposit number is CGMCC No.6086.
  2. The red bacterium of fixed nitrogen described in claim 1 ( rhodobacter azotoformans) application of Y6 in neurosporene or Lyeopene dehydrogenation reaction.
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