CN105018362A - Bacterial strain for producing long-chain dicarboxylic acids and preparation method and application of bacterial strain - Google Patents
Bacterial strain for producing long-chain dicarboxylic acids and preparation method and application of bacterial strain Download PDFInfo
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Abstract
The invention relates to a bacterial strain for producing long-chain dicarboxylic acids and a preparation method and application of the bacterial strain. The technical problem that the transformation effect of an existing long-chain dicarboxylic acid bacterial strain is not remarkable is solved through the bacterial strain. The classification and the name of the bacterial strain are candida mycoderma bacteria (Candida sp.) TDTC027. The preservation number of the bacterial strain is CGMCC No.8931. The bacterial strain can be widely used for the field of preparation of the long-chain dicarboxylic acids.
Description
Technical field
The present invention relates to a kind of bacterial classification and its preparation method and application, is that a kind of long-chain biatomic acid produces bacterial strain and its preparation method and application specifically.
Background technology
Long-chain biatomic acid is important industrial chemicals, has purposes extremely widely, can synthetic perfume, extraordinary nylon, a series of high added value of advanced lubrication wet goods chemical.Long-chain biatomic acid can be applicable to the top coat and oil pipe etc. of military domain, the coating of aerospacecraft, pipeline, automobile; At civil area, can be applicable to more than ten the high and new technology industries such as automobile, daily use chemicals spices, engineering plastics, nylon industry, more downstream industry can be developed, form emerging industrial chain.
In the past, long-chain biatomic acid adopted chemical synthesis to produce, and its patented technology is had by external.Chemical synthesis produces long-chain biatomic acid, and not only product category is single, and synthesis technique is complicated, cost is high, pollution is large.China uniquely can adopt microbial fermentation technology to realize the country of multiple long-chain biatomic acid large-scale industrial production in the world.Before this, the improvement of China's diprotic acid production bacterial classification all educates method realization by tradition such as different modes mutagenesis.Traditional breeding way has very large randomness, and screening is complicated.The performance improving bacterial strain further has been difficult to by the method for traditional breeding method.Still have many bottleneck problems in current long-chain biatomic acid commercial process, as substrate conversion efficiency have much room for improvement, production energy consumption is very huge etc.
Metabolic engineering technology can carry out bacterial classification molecular modification targetedly on gene level, obtains the new strains that performance is more excellent.As shown in Figure 1 and Figure 2, diprotic acid pathways metabolism mainly comprises omega oxidation approach and β-oxidation approach, and wherein the former is diprotic acid route of synthesis, and the latter relates to diprotic acid degradation pathway.The object of metabolic engineering improves omega oxidation activity by molecular modification means, and reduce β-oxidation activity.In the world, there is patent report (US005254466A) in Henkel company (Cognis company afterwards), optimizes diprotic acid produce bacterial strain, successively by 4 POX gene knockouts by gene knockout mode, reach and block β-oxidation completely, make substrate conversion efficiency rise to 100%.On this basis, the said firm, further by metabolic engineering means coexpression CYP monooxygenase and reductase enzyme, strengthens the object of omega oxidation, output increased 30% (World PatentWO/91/06660) to reach.
But utilize this bacterial strain to carry out batch fermentation experiment, its technique still cannot be competed with other diprotic acid production technique at that time, and does not finally carry out large-scale production.It is uracil auxotrophy that Henkel company carries out to bacterial classification the selection markers that molecular modification uses.The shortcoming of Henkel company patent of invention is: 1, starting strain is not suitability for industrialized production superior strain used; 2, the candiyeast that Production of Long-chain Dicarboxylic Acids by Fermentation Methods is used is diploid, and namely each cell has two cover karyomit(e)s, and each gene has corresponding allelotrope, and catalysis often walks the enzyme of biochemical reaction in body often by multiple genes encoding.Therefore, strengthened or weakened the activity of certain individual interior biochemical reaction by metabolic engineering means, needing to carry out molecular modification to the key gene of this enzyme of coding just has unusual effect.Otherwise effect also can not be remarkable after transformation.
Summary of the invention
The present invention produces the inapparent technical problem of long-chain biatomic acid effect to solve existing bacterial strain, and the long-chain biatomic acid providing a kind of production efficiency high produces bacterial strain and its preparation method and application.
For this reason, the invention provides a kind of long-chain biatomic acid and produce bacterial strain, its Classification And Nomenclature is candidiasis (Candida sp.) TDTC027, described bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 18th, 2014, be called for short CGMCC, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Its deposit number is: CGMCC No.8931.
In the present invention, the promotor that long-chain biatomic acid produces one of bacterial strain catalase allele C andidaA05093 and CandidaA09561 is replaced by strong promoter, the allelic base sequence of described catalase as shown in the sequence 8 in sequence table and sequence 9, catalase gene be encode in this strain gene group this enzyme multiple genes in the most key one is produced for long-chain biatomic acid.
The present invention provides a kind of long-chain biatomic acid to produce the preparation method of bacterial strain simultaneously, and it comprises the steps: (1) prepares primer Pro1, Pro2, Pro3 and Pro-4; (2) prepare long-chain biatomic acid and produce bacterium competence cell; (3) primer in use step (1) carries out increasing and obtains selection markers Sat1 gene fragment (primer Pro1 and Pro2) and strong promoter fragment (primer Pro3 and Pro4) respectively, take turns over-lap PCR through one again two fragments are coupled together, the product increased by this over-lap PCR again replaces the allelic original promoter of catalase by homologous recombination, reach the object strengthening catalase gene and express, the allelic base sequence of described catalase is as shown in the sequence 8 in sequence table and sequence 9; (4) turn by pcr amplification, purifying, electricity, screen, identify, obtain long-chain biatomic acid of the present invention and produce bacterial strain.
Preferably, in the present invention, long-chain biatomic acid produces the preparation method of bacterial strain, and the selection markers used in screening step is clonNAT resistance.
Preferably, long-chain biatomic acid produces the preparation method of bacterial strain, and the long-chain biatomic acid in step (2) produces bacterial strain candiyeast (Candida sp.) DC12.
The present invention provides long-chain biatomic acid to produce bacterial strain simultaneously and is producing the application in long-chain biatomic acid.
Preferably, after fermentation ends, fermented liquid is heated to 70 ~ 80 DEG C; Again pH is adjusted to 9 ~
9.5, removing bacterial sediment, retains supernatant; Decolouring, temperature remains on 70 ~ 90 DEG C, obtains cleaner liquid, is acidified to pH2.5 with acid, 70 ~ 90 DEG C of insulations, cooling, centrifugal or press filtration, washing, and it is dry that the precipitation after cleaning takes out final vacuum, obtains long-chain biatomic acid.
Long-chain biatomic acid in the present invention refers to the unbranched dicarboxylic acid containing more than ten carbon atoms, a kind of important fine chemistry industry intermediate raw material, particularly SL-AH (DC12), DC14 (DC14), 16-dicarboxylic acid (DC16) and DC18 (DC18).
The invention has the beneficial effects as follows, in order to break through the bottleneck producing bacterial classification genetic modification, the present invention is by resolving the genomics and transcription group feature of producing bacterial strain, analyze omega oxidation and β-oxidation pathways metabolism, genome global level establishes some critical target points that diprotic acid metabolism is relevant, by metabolic engineering means, molecular modification is carried out to these sites again, and experimental verification by fermentation, obtain the bacterial strain that performance is more excellent.TDTC027 bacterial strain of the present invention (preserving number: CGMCC No.8931), compares DC12 bacterial strain, and fermentation Laurate methyl produces acid amount and significantly improves, for large-scale production brings beneficial effect.
Accompanying drawing explanation
Fig. 1 is the relevant omega oxidation pathways metabolism of long-chain biatomic acid synthesis that the present invention relates to;
Fig. 2 is the relevant β-oxidation pathways metabolism of long-chain biatomic acid degraded that the present invention relates to;
Fig. 3 is that the promotor that the present invention relates to replaces schema;
Fig. 4 is the long-chain biatomic acid that HPLC analyzes DC12 fermentative production;
Fig. 5 is the long-chain biatomic acid that HPLC analyzes TDTC027 fermentative production.
Long-chain biatomic acid of the present invention produces bacterial strain, and its Classification And Nomenclature is candidiasis (Candidasp.) TDTC027; Its preservation mechanism is China Committee for Culture Collection of Microorganisms's common micro-organisms center, and be called for short CGMCC, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date is on March 18th, 2014, and preserving number is numbered: CGMCC No.8931.
Embodiment
In sequence table of the present invention, the title of sequence is as follows: sequence 1:Pro1; Sequence 2:Pro2; Sequence 3:Pro3; Sequence 4:Pro4; Sequence 5:Pro-U; Sequence 6:Pro-D; Sequence 7:CYPpromoter; Sequence 8:CandidaA05093; Sequence 9:CandidaA09561.
The bacterium colony observed in following examples and thalli morphology are summarized as follows:
On solid medium flat board, bacterium colony cheese shape, smooth surface, oyster white, full projection, colony diameter is about 2mm.Yeast sample is unicellular, and size is about 10x6 μm.Under most cases, with the unicellular form of yeast sample, have pseudomycelium simultaneously and formed, under stimulating at some growth phase or certain ambient conditions, pseudohypha increase more remarkable in regular meeting.
Substratum listed below employing in following examples:
1, YPD substratum, its formula is: the yeast extract paste of 1%, 2% peptone, and 2% glucose, if solid medium processed, adds 2% agar powder.Above-mentioned per-cent is quality volume percent, i.e. the grams of every 100 milliliters of substratum this component required.To add microbiotic, liquid nutrient medium is added to corresponding final concentration in use.When solid medium is cooled to 50 degrees centigrade after autoclaving, add microbiotic to corresponding final concentration, fall at once as in aseptic culture dish after mixing, rear inversion to be solidified, is put in 4 degrees Celsius of refrigerators, uses in two weeks.
2, the formula of seed culture medium: yeast extract paste 1 ~ 8g/L, corn steep liquor 1 ~ 8g/L, sucrose 5 ~ 25g/L, KH
2pO
44 ~ 12g/L, urea 0.5 ~ 4g/L, heavy wax 40 ~ 70g/L, 121 DEG C of sterilizings 30 minutes.Wherein, sucrose and urea separate independent 110 DEG C of sterilizings 20 minutes, remerge mixing after sterilizing.
3, fermentor cultivation based formulas: yeast extract paste 1 ~ 8g/L, corn steep liquor 1 ~ 8g/L, sucrose 5 ~ 30g/L, KH
2pO
44 ~ 15g/L, urea 0.5 ~ 4g/L, KNO
35 ~ 15g/L, NaCl0.5 ~ 2.5g/L, 121 DEG C of sterilizings 30 minutes.Wherein, the separately independent sterilizing of sucrose and urea, 110 DEG C of sterilizings 20 minutes, remerge mixing after sterilizing.Prepare 75% glucose solution in addition, 105 DEG C of sterilizings 20 minutes, the fermentation initial stage carries out stream and adds.
Yeast culture listed below employing in following examples and fermentation liquor treatment mode:
1, slat chain conveyor: be inverted in 30 DEG C of incubators after the flat lining out of YPD or coating, namely can be observed large and full oyster white bacterium colony for 2-3 days and is formed.
2, shaking table is cultivated: flat board is inoculated single bacterium colony in liquid nutrient medium, or comes from liquid nutrient medium switching, and 30 DEG C of shaking tables are cultivated, and rotating speed remains on 220 revs/min.
3, fermentor cultivation: accurately can control fermentation condition relatively in real time, as control of additive raw material, pH control, dissolved oxygen control and aeration-agitation intensity control etc.First stage, fermented liquid pH controls 5 ~ 6.8, and stream adds glucose solution simultaneously, is the thalli growth stage; Subordinate phase, fermented liquid pH controls 7.0 ~ 7.8, simultaneously current adding substrate (alkane, lipid acid or derivative of fatty acid, as methyl esters or ethyl ester etc.), based on fermentation and acid, also growth part thalline; Phase III, only produce acid, do not produce thalline, according to fermentation situation continued current adding substrate (alkane, lipid acid or derivative of fatty acid, as methyl esters or ethyl ester etc.).Whole fermenting process 10M NaOH solution auto-feeding control pH, makes dissolved oxygen remain on 20 ~ 40% by the adjustment of rotating speed simultaneously.
4, fermentation liquor treatment: after fermentation ends, is heated to 70 ~ 80 DEG C by fermented liquid, and maintains 60 minutes; Add 10M NaOH again and pH is adjusted to 9 ~ 9.5, with tubular-bowl centrifuge or press filtration removing bacterial sediment, retain supernatant; Add proper amount of active carbon decolouring, temperature remains on 70 ~ 90 DEG C, and soaking time is 60 minutes; After removing gac, obtain cleaner liquid, with concentrated hydrochloric acid or vitriol oil continuously acidizing to pH2.5,70 ~ 90 DEG C of insulations 2 hours, be cooled to 30 DEG C, centrifugal or press filtration, clear water washes one time, and it is dry that the precipitation after cleaning takes out final vacuum, obtains white diprotic acid.
Embodiment 1: bacterial strain metabolic engineering
1) gene order-checking
Utilize the genome of precious biotechnology company limited to extract test kit (Yeast DNAiso Kit) and prepare long-chain biatomic acid production bacterial strain candiyeast genomic dna, concrete operations are carried out according to test kit specification sheets.Genomic dna is again through steps such as library construction, new-generation sequencing technology sequencing analysis and data assemblings.By genome annotation and data analysis, obtain and produce bacterial strain and produce relevant gene information at diprotic acid, excavated the encoding sequence of this bacterium at omega oxidation and β-oxidation pathways metabolism genes involved.For yeast, β-oxidation is mainly sent out in peroxysome, particularly the β-oxidation of longer chain fatty acid, only betides in this organoid of peroxysome.In omega oxidation pathways metabolism, alkane is through CYP monooxygenase, fatty alcohol oxydase and alkanoic desaturase three step enzymic catalytic reaction, and an end of alkane is oxidized to carboxyl, generates lipid acid.Lipid acid takes turns omega oxidation process (three same step enzymic catalytic reactions) through one again, and two ends of alkane are all oxidized to carboxyl, generates diprotic acid.First two steps reaction in omega oxidation three-step reaction process all produces this by product of hydrogen peroxide.And if hydrogen peroxide is piled up in vivo, be virose for thalline.In order to ensure that omega oxidation continues to keep high reactivity, must effectively remove by product hydrogen peroxide.A more effective side is exactly slightly strengthen catalatic expression, removes hydrogen peroxide in time.And strengthen hydrogen peroxide expression of enzymes can by strengthen promoter activity realize.Sequential analysis finds, diprotic acid is produced strain gene group and is contained two catalase (catalase) genes, be respectively CandidaA05093 and CandidaA09561, coding region length is 1458bp, sequence similarity is up to 98%, upstream and downstream regional sequence is also highly similar, thus can judge it is pair of alleles.
2) transcript profile order-checking
Utilize the RNA of German Kai Jie company to extract test kit (RNeasy Mini Kit) and prepare the total serum IgE that long-chain biatomic acid produces bacterial strain candiyeast, concrete operations are carried out according to test kit specification sheets.The total serum IgE obtained is again through library construction, transcript profile order-checking and expression analysis.By transcript profile data analysis, CandidaA05093 and Candida09561 this 5125.7 and 5591.3RPKM are respectively to catalase allelotrope transcriptional level.And analyze and show, the transcriptional level of CandidaA06129 (coding CYP monooxygenase) is 20503.5RPKM, is the highest one of transcriptional level in full gene.The promotor of catalase gene is replaced with the promotor of CandidaA06129 by the present invention, to reach the object strengthening catalase gene and express.Here, RPKM is method of calculation of gene expression amount, represents Reads Per Kb Per Million Reads.Its calculation formula is
If the expression amount that RPKM (A) is Gene A, then C is the reads number of unique comparison to Gene A, and N is the total reads number of unique comparison to reference gene, and L is the base number of Gene A coding region.RPKM method can eliminate mrna length and order-checking amount difference to the impact calculating genetic expression, and the gene expression amount calculated can be directly used in icp gene differential expression.
Because omega oxidation is the pathways metabolism that long-chain biatomic acid synthesis is relevant, the object of bacterial classification transformation strengthens omega oxidation activity, to improve the ability of bacterial strain synthesis diprotic acid.Because omega oxidation produces a large amount of hydrogen peroxide, hydrogen peroxide is to the toxic effect of thalline.Catalase can hydrolyzed hydrogen oxide, generates water and oxygen.The present invention replaces the expression level strengthening hydrogen oxide enzyme by strong promoter, reach the object effectively removing hydrogen peroxide, ensures that omega oxidation process keeps efficiently carrying out, thus reaches the effect of more effectively synthesizing diprotic acid.
3) promotor replaces experiment flow as shown in Figure 3.Design of primers principle, homology arm and detect the design of primer and all select identical region in the comparison of CandidaA05093 and Candida09561 upstream of coding region promoter sequence and initiation region, coding region, thus reach two allelotrope promotors and replace and detect there is equal probability.Use Pro1 and Pro2 is primer, carries out pcr amplification and obtains Sat1 gene fragment, comprise promotor and terminator.Use Pro3 and Pro4 is primer, and it is masterplate that diprotic acid produces strain gene group DNA, carries out promotor (strong promoter) fragment that pcr amplification obtains CandidaA06129.The Sat1 gene fragment arrived with above-mentioned amplification again and strong promoter fragment are for masterplate, with Pro1 and Pro4 for primer, take turns over-lap PCR through one two fragments are coupled together, then the product that this over-lap PCR increases is incorporated in uracil auxotrophy strain gene group.Wherein 5 ' the end of primer Pro1 and Pro4 is homology arm sequence, corresponding to the upstream and downstream sequence of catalase gene promoter.Like this, the DNA fragmentation obtained after over-lap PCR amplification, upstream and downstream homology arm is with at two respectively, middle Sat1 gene and strong promoter.
Carry out electricity after the DNA fragmentation of amplification is purified to transform and import in somatic cells, the target site generation double exchange on the upstream and downstream homology arm of DNA fragmentation and thalline karyomit(e), reaches the object of promotor replacement.Because this double exchange is small probability event, need establishment method to screen.Here used resistant gene is Sat1, coding Knowles rhzomorph Transacetylase.Diprotic acid produces bacterial strain to Knowles rhzomorph (being clonNAT or NTC again) this medicaments insensitive, can not grow, and after expressing this enzyme, thalline can decompose Knowles rhzomorph, avoids lethal effect containing on the flat board of Knowles rhzomorph.Resistance screening mark is only incorporated into karyomit(e) and gets on and just can be expressed, and plays a role, and thalline can grown containing on the culture medium flat plate of resistance, and form bacterium colony.Simultaneously to the bacterium colony with resistance, carry out purifying and checking.
During checking, use a pair detection primer Pro-U and Pro-D, respectively with the upstream and downstream regions pair of target site.If do not have homologous recombination to occur, be the fragment that masterplate increases out by this genomic dna be the same in length, a band can only be observed in agarose electrophoresis.If the fragment of over-lap PCR amplification is incorporated into target site by homologous recombination, so just changes through the fragment length obtained that increases, two bands can be observed in agarose electrophoresis.Through the bacterial strain of checking, then in diprotic acid production, whether there is the advantage in performance by fermenting experiment checking metabolic engineering acquisition new strains.
4) pcr amplification
Wherein, plasmid pSFS2, to be the accession number of Gene (2004) 341:119 – 127, GeneBank database be reference: AY524979, derives from Microbe Inst., Chinese Academy of Sciences.Use Pro1 and Pro2 to be primer 1 and primer 2, plasmid pSFS2 is masterplate, carries out pcr amplification and obtains Sat1 gene, comprise promotor and terminator.Use Pro3 and Pro4 to be primer 1 and primer 2, it is masterplate that diprotic acid produces strain gene group DNA, carries out the strong promoter that pcr amplification obtains CYP monooxygenase gene (CandidaA06129).The Sat1 gene arrived with above-mentioned amplification again and strong promoter fragment, for masterplate, with Pro1 and Pro4 for primer, are taken turns over-lap PCR through one and two fragments are coupled together.The Pyrobest archaeal dna polymerase of archaeal dna polymerase Shi Bao biotechnology company limited, or the Phusion archaeal dna polymerase of NEB company, activity unit is 5U/ μ l.
PCR cycling condition is:
94 degree of 2 minutes (denaturation stages)
94 degree 20 seconds, 58 degree 20 seconds, 72 degree 1 ~ 6 minute (30 cyclic amplification stages, 1kb/ minute)
72 degree 10 minutes (finally extending the stage)
5) DNA purifying
After over-lap PCR reaction terminates, get the above-mentioned PCR sample of 5 μ l and carry out agarose electrophoresis detection, prove the amplification DNA fragmentation size of gained and the same of expectation, and assorted band, carrying out two-step purifying with concentrated, is conversion prepare dna sample below.
The first step utilizes PCR purification kit (purchased from Omega company), carries out to specifications.Be generally 50 μ l systems, do 4 pipes, cumulative volume is totally 200 μ l, the final step 50 or 100 μ l TE buffer solution elution pillars of PCR purifying.DNA after purifying, survey concentration with NanoDrop instrument, the DNA total amount calculated is about 20 μ g.
Second step is precipitated with ethanol/sodium-acetate/glycogen process by the DNA of purifying again.Specific practice is: the dehydrated alcohol adding 1 μ l glycogen (20mg/ml), 100 μ l sodium-acetate (3M, pH5.2) and 1ml precooling toward the DNA solution of the above-mentioned TE of being dissolved in damping fluid; Place-80 degrees Celsius of refrigerators and cool 30 minutes; 4 degree of whizzers, under the rotating speed of 14000 revs/min, centrifugal 10 minutes, stay precipitation; Precipitation washes twice with the ethanol of 75% precooling again; Air-dry in super clean bench; 4 degrees Celsius of refrigerator storage, it is for subsequent use that electricity is resuspended in 10 μ l ultrapure waters before turning.
6) competence preparation and electricity turn
Starting strain (Candida sp.DC12 is chosen from the flat board of fresh activation, see " microorganism journal " 20 (1): 88-93,1980, normal alkane fermentative production long-chain mixed dicarboxylic acid, can buy from Institute of Microorganism, Academia Sinica) single bacterium colony, 30 DEG C of shaking table overnight incubation, rotating speed is 220 revs/min; 2% transfers in 20ml YPD, and 30 DEG C of shaking tables are cultivated, and 220 revs/min, reach 1.8 to OD600; Be placed in by bacterium liquid and leave standstill 15 minutes on ice, make it stop growing, 4000 revs/min, 4 DEG C centrifugal 3 minutes, stays bacterial sediment; With the aseptic washing of 4ml precooling once; 4000 revs/min, 4 DEG C centrifugal 3 minutes, stays bacterial sediment; Add 4ml TE/0.1M LiOAc, 150 revs/min, vibration 90 minutes in the shaking table of 30 DEG C; Add 0.1ml1M DTT, vibration 30 minutes in the shaking table case continuing 150 revs/min, 30 DEG C; 4000 revs/min, 4 DEG C centrifugal 3 minutes, stays bacterial sediment; Add 4ml precooling sterilized water, wash 3 times; Add 2ml1M sorbyl alcohol, wash 1 time, 4000 revs/min, 4 DEG C centrifugal 3 minutes; Abandon supernatant, add 120 μ l sorbyl alcohols and cell is hanged; The cell suspension taking out 40ul, in 1.5ml centrifuge tube, is resuspended in the pcr amplification product (about 10 μ g) of sterilized water after adding the above-mentioned purifying of 5 μ l, mixing, is placed in 5 minutes on ice; Proceed in the electric revolving cup of precooling, dry electric revolving cup, carry out electricity and turn (electricity turns condition: the electric revolving cup of 2mm slit, and voltage is 1800 volts, and the electric shock time is 5 milliseconds); Electricity adds 1ml sorbyl alcohol after turning at once, and after mixing, sucking-off is put in the centrifuge tube of 1.5ml; 4000 revs/min centrifugal 3 minutes, abandons supernatant, adds 1ml YPD substratum, cultivates 2 hours in the shaking table of 37 DEG C; 4000 revs/min centrifugal 3 minutes, abandons supernatant, adds the resuspended thalline of 100 μ l YPD, spread plate (YPD+clonNAT), is put in 30 DEG C of incubators and is cultured to the appearance of single bacterium colony.
7) resistant clones is screened
Be inoculated into respectively in the centrifuge tube of 1ml YPD substratum by single bacterium colony that above-mentioned resistant panel grows, adding clonNAT to final concentration is 100 μ g/ml, 220 revs/min, and 30 DEG C of shaking tables are cultivated.Occur growth, illustrate with resistance, proves that resistant gene to be incorporated in phage gene group and to have played a role, this part bacterium colony is used for next step and verifies.Do not occur the bacterium colony grown, explanation is false positive, is equal to starting strain, stops process.
8) identify
Occur the resistant clones grown above, inoculation YPD liquid nutrient medium overnight incubation, draws 500 μ l bacterium liquid, and utilize the genome of precious biotechnology company limited to extract test kit and prepare genomic dna, concrete operations are carried out according to test kit specification sheets.With the genomic dna obtained for masterplate, Pro-U and Pro-D is that primer carries out pcr amplification, and concrete reaction system and reaction conditions are with reference to the 4th of the present embodiment 1) article.If the promotor of catalase gene is not replaced, the DNA fragmentation size increasing out from two allelosomals is the same, and agarose electrophoresis shows as a band.If the promotor of one of them catalase gene (CandidaA05093 or Candida09561) is replaced by strong promoter, the DNA fragmentation increasing out from two allelosomals is in different size, and agarose electrophoresis shows as two bands (see Fig. 3).Be accredited as the bacterium liquid that the positive and promotor of resistance is replaced, more further through the qualification of bacterium colony purifying and same procedure, obtain long-chain biatomic acid of the present invention and produce bacterial strain TDTC027.
Embodiment 2: the fermentative production of long-chain biatomic acid
Bacterial classification is by after conventional slant culture, and access 50ml first order seed cultivates 16 hours, and then being cultivated by first order seed transfers cultivates 16 hours into 500ml secondary seed.
The formula of seed culture medium: yeast extract paste 1 ~ 8g/L, corn steep liquor 1 ~ 8g/L, sucrose 5 ~ 25g/L, KH
2pO
44 ~ 12g/L, urea 0.5 ~ 4g/L, heavy wax 40 ~ 70g/L, 121 DEG C of sterilizings 30 minutes.Wherein, sucrose and urea separate independent 110 DEG C of sterilizings 20 minutes, remerge mixing after sterilizing.
After secondary seed has fermented, transfer into 5L fermentor tank.Fermentor cultivation based formulas: yeast extract paste 1 ~ 8g/L, corn steep liquor 1 ~ 8g/L, sucrose 5 ~ 30g/L, KH
2pO
44 ~ 15g/L, urea 0.5 ~ 4g/L, KNO
35 ~ 15g/L, NaCl0.5 ~ 2.5g/L, 121 DEG C of sterilizings 30 minutes.Wherein, the separately independent sterilizing of sucrose and urea, 110 DEG C of sterilizings 20 minutes, remerge mixing after sterilizing.Prepare 75% glucose solution in addition, 105 DEG C of sterilizings 20 minutes, the fermentation initial stage carries out stream and adds.Basic medium is 4L.At 30 DEG C with the ventilation volume of 1:0.5, control pH5.5 ~ 6.5, started to add 12 carbon straight-chain paraffins with the speed stream of 50ml/h after the 16th hour, fermentation time is 144-156 hour.Whole fermenting process 10M NaOH solution auto-feeding control pH, makes dissolved oxygen remain on 30% by the adjustment of rotating speed simultaneously.
As shown in Figure 4 and Figure 5, HPLC analyzes and shows starting strain DC12 and transform the ferment long-chain biatomic acid product that obtains of bacterial strain TDTC027 consistent, and purity is respectively 98.7% and 99.2%.
As can be seen from following table, improved bacterial strain, transformation efficiency is also improved.
Embodiment 3
According to the method for embodiment 2, just substrate changes Laurate methyl into from 12 carbon straight-chain paraffins.
Embodiment 4
According to the method for embodiment 2, just substrate changes Laurate ethyl into from 12 carbon straight-chain paraffins.
Claims (8)
1. long-chain biatomic acid produces a bacterial strain, and its Classification And Nomenclature is candidiasis (Candidasp.) TDTC027, and its deposit number is: CGMCC No.8931.
2. long-chain biatomic acid according to claim 1 produces bacterial strain, it is characterized in that the promotor of one of catalase allelotrope in described candidiasis genome is replaced by strong promoter.
3. produce bacterial strain according to claim 2 long-chain biatomic acid, it is characterized in that the described allelic base sequence of hydrogen peroxide hydrogen enzyme is as shown in the sequence 8 in sequence table and sequence 9.
4. long-chain biatomic acid as claimed in claim 1 produces the preparation method of bacterial strain, it is characterized in that comprising the steps:
(1) primer Pro1, Pro2, Pro3 and Pro4 is prepared;
(2) prepare long-chain biatomic acid and produce bacterium competence cell;
(3) primer in use step (1) carries out increasing and obtains selection markers Sat1 gene fragment primer Pro1, Pro2 and strong promoter fragment primer Pro3, Pro4 respectively, take turns over-lap PCR through one again two fragments are coupled together, the product increased by this over-lap PCR again replaces the allelic original promoter of catalase by homologous recombination, and the allelic base sequence of described catalase is as shown in the sequence 8 in sequence table and sequence 9;
(4) turn by pcr amplification, purifying, electricity, screen, identify, obtain long-chain biatomic acid and produce bacterial strain.
5. long-chain biatomic acid according to claim 4 produces the preparation method of bacterial strain, it is characterized in that the selection markers used in described screening step is clonNAT resistance.
6. long-chain biatomic acid according to claim 4 produces the preparation method of bacterial strain, it is characterized in that the long-chain biatomic acid production bacterial strain in described step (2) is candiyeast (Candida sp.) DC12.
7. long-chain biatomic acid as claimed in claim 1 is produced bacterial strain and is being produced the application in long-chain biatomic acid.
8. long-chain biatomic acid as claimed in claim 7 produces bacterial strain producing the application in long-chain biatomic acid, after it is characterized in that fermentation ends, fermented liquid is heated to 70 ~ 80 DEG C; Again pH is adjusted to 9 ~ 9.5, removing bacterial sediment, retains supernatant; Decolouring, temperature remains on 70 ~ 90 DEG C, obtains cleaner liquid, is acidified to pH2.5 with acid, 70 ~ 90 DEG C of insulations, cooling, centrifugal or press filtration, washing, and it is dry that the precipitation after cleaning takes out final vacuum, obtains long-chain biatomic acid.
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| CN111334444A (en) * | 2018-12-19 | 2020-06-26 | 中国科学院微生物研究所 | Long-chain dicarboxylic acid producing strain and preparation method and application thereof |
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| WO2004013336A2 (en) * | 2002-08-05 | 2004-02-12 | Cognis Corporation | Use of pox4 promoter to increase gene expression in candida tropicalis |
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| WO2004013336A2 (en) * | 2002-08-05 | 2004-02-12 | Cognis Corporation | Use of pox4 promoter to increase gene expression in candida tropicalis |
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| HIROFUMI OKADA,ET AL: "Catalase gene of the yeast Candida tropicalis", 《EUR. J. BIOCHEM.》 * |
| 高弘: "生物催化生产十三碳二元酸中β-氧化途径的代谢调控", 《中国博士学位论文全文数据库，工程科技I辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111334444A (en) * | 2018-12-19 | 2020-06-26 | 中国科学院微生物研究所 | Long-chain dicarboxylic acid producing strain and preparation method and application thereof |
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