CN102337303B - Method for directly synthesizing fatty alcohol in heterotrophic microbes - Google Patents

Abstract

The invention discloses a method for synthesizing fatty alcohol in microbes. The method comprises the following steps of: cloning acyl-CoA reductase gene to a corresponding expression vector; and transforming constructed plasmid into a corresponding host bacterium, and modifying a fatty acid metabolism pathway in heterotrophic microbes through inducible expression of acyl-CoA reductase to generate the fatty alcohol. By the method, the fatty alcohol is synthesized in the microbes by a biosynthesis method; and the technology is renewable and low in energy consumption; and the environment-friendly production mode can reduce the consumption of scarce resources of petroleum, and has great application prospect in the industry, and the fatty alcohol can be directly produced without chemical modification.

Description

A kind of in the heterotrophic microorganism body the direct method of synthetic fatty alcohol

Technical field

The invention belongs to renewable energy source and biomass energy and industrial chemicals production field, thereby be specifically related to transform the method that heterotrophic microorganism own fat acid metabolic approach is produced fatty alcohol.

Background technology

Fatty alcohol (fatty alcohol) is one of raw material of detergent use tensio-active agent, and a large amount of utilizations are arranged in washing composition, skin care product, makeup, medicine.Fatty alcohol is produced by spermaceti the earliest, the mixed fatty alcohol of gained through in sulfonation and after become vitriol, be a kind of anionic detergent the earliest.Thereafter developing source is raw material than more rich Oleum Cocois, plam oil and butter, and hydrolysis gained lipid acid restores as alcohol, is referred to as natural fatty alcohol.After the petrochemical industry development, take petroleum products as raw material, the fatty alcohol of production is called synthetic fatty alcohol.But rely on the fatty alcohol that petroleum products is raw material also must rely on the margin of oil, day by day rare today in resource, this development pattern will be eliminated gradually, therefore, needs a kind of environmental protection more, the reproducible mode of production.If can be by heterogenous expression fatty alcohol synthetic enzyme in improved fatty acid metabolism path, acyl-CoA reductase, can ferment and produce a large amount of fatty alcohol by microorganism.

The people such as Michael at purifying in 2000 the fatty acyl CoA reductase of jojoba, and prove that this gene---jojoba far can become fatty alcohol by the lipid acid reduction, this work is published in " Plant Physiology " (James G. Metz, Michael R. Pollard 1, Lana Anderson, Thomas R. Hayes, and Michael W. Lassner. 2000.Purification of a Jojoba Embryo Fatty Acyl-Coenzyme A Reductase and Expression of Its cDNA in High Erucic Acid Rapeseed. Vol. 122:635 – 644).Tan etc. have expressed respectively and have come from jojoba in the cyanobacteria Syn-LY2 bacterial strain that lacks the synthetic fatty alcohol ability, mouse, the acyl-CoA reductase FAR of Arabidopis thaliana etc., find heterogenous expression jojoba and wherein the cyanobacteria of a kind of Arabidopis thaliana FAR can synthetic fatty alcohol, and other the cyanobacteria of several FAR of heterogenous expression can not produce fatty alcohol.This work is published in " Metabolic Engineering " upper (Tan X, Yao L, Lu X. et al.2010.Photosynthesis driven conversion of carbon dioxide to fatty alcohols and hydrocarbons in cyanobacteria. Metabolic Engineering 13 (2011) 169 – 176).The people such as professor Liu Tiangang have developed " cell free " system, rate-limiting step in the fatty acid metabolism approach is studied, instruct the fatty acid content how improved in microbe, this work is published in " Metabolic Engineering " (Tiangang Liu, Harmit Vora, 12: 378 – 386 of Chaitan Khosla.2010.Quantitative analysis and engineering of fatty acid biosynthesis in E. coli. Metabolic Engineering).The aliphatic alcohol reductase gene is convenient to be optimized, the microbe species that can carry out at present genetic manipulation is various, the Fatty acid biosynthesis metabolism approach is again the requirement of every kind of microorganism existence, and current research is comparatively deep to this approach understanding, its rate-limiting step is substantially clear, is beneficial to and is transformed.Only there is at present the scholar to utilize jojoba far genes produce fatty alcohol in can carrying out photoautotrophic cyanobacteria, as everyone knows, because the heterotrophic microorganism growth is rapid and need external source extra-nutrition material, this facilitates by supplemental medium or increases some nutritive ingredient and improves its throughput, makes it in the industrial large-scale industrialized production that realizes.And, because it must rely on artificial recharge nutriment, so can strictly control the stages of its production, prevent that it from growing wild and causing uncontrollable situation.At present industrial had manyly utilize heterotrophic microorganism to produce the examples of many successful that fermentation obtains product, utilize the aliphatic alcohol reductase gene to produce fatty alcohol very large application prospect is arranged by transforming himself pathways metabolism in the heterotrophic microorganism body.

Summary of the invention

The object of the present invention is to provide a kind of method of utilizing heterotrophic microorganism to carry out the biosynthesizing fatty alcohol, in the heterotrophic microorganism body, its fatty acid metabolism approach of transformation and expression foreign protein are to produce important industrial chemicals---fatty alcohol.

For achieving the above object, provided by the invention in the heterotrophic microorganism body method of synthetic fatty alcohol, it is that the acyl-CoA reductase gene is imported in the heterotrophic microorganism body, and by this gene clone on expression vector, import in corresponding heterotrophic microorganism body, and abduction delivering.

Described acyl-CoA reductase gene is preferably Jojoba farGene, the protein shown in its coding SEQ ID No.2.The aminoacid sequence shown in SEQ ID No.2 of perhaps encoding is substituted, replaces and/or increases one or several amino acid, have jojoba far albumen with isoreactivity by the derivative protein obtained of jojoba far.

Heterotrophic microorganism of the present invention can be eucaryon heterotrophic microorganism or protokaryon heterotrophic microorganism, comprises bacterium, fungi, actinomycetes etc.

In addition, the inventive method also comprises by transformation heterotrophic microorganism pathways metabolism or according to the preferences of microorganism codon, the aliphatic alcohol reductase gene is optimized, to improve the output of fatty alcohol.

In addition, the inventive method also comprises carries out tolerance screening to transforming aliphatic alcohol reductase gene heterotrophic microorganism, obtains dominant strain, thereby improves the output of fatty alcohol.

Wherein, the heterotrophic microorganism pathways metabolism of described transformation is the fatty acid metabolism approach in this microbe, comprise: improve lipid acid output or reduce lipid acid output in order to produce the approach of fatty alcohol, for example: import the pMSD8 plasmid in intestinal bacteria, overexpression fatty acetyl-CoA carboxylase, improve in order to produce the fatty acid content of fatty alcohol; For example change this microorganism body fat acid metabolic approach intermediate, in order to produce the approach of fatty alcohol: knock out fadE gene in intestinal bacteria to accumulate the more acyl CoA of fattiness acid metabolic approach intermediate, thereby obtain more fatty alcohol; Change this microorganism body fat acid metabolic approach derivative in order to produce the approach of fatty alcohol.

The present invention also provides a kind of heterotrophic microorganism, and it is the heterotrophic microorganism that transforms the acyl-CoA reductase gene.In addition, also comprise as mentioned above the heterotrophic microorganism of its pathways metabolism being transformed to the fatty alcohol raising obtained.

Advantage of the present invention is to introduce himself fatty acid metabolism approach of foreign gene transformation to realize the purpose of manufacture feedstock fat alcohol in heterotrophic microorganism, do not need by too much chemosynthesis reaction, reduce the pollution of environment, also reduced the consumption to prospective oil.Simultaneously, due to most of heterotrophic microorganism fast growth, be convenient to carry out genetic manipulation, its antifouling property excellence, and manually can regulate its speed of growth and prevent its welding that grows wild, all factors from showing to transform heterotrophic microorganism that to carry out industrial production be practicable.

The accompanying drawing explanation

Fig. 1 synthesizes for design Jojoba farGene structure figure.

The pRL104 expression vector schematic diagram that Fig. 2 is design construction.

The schematic diagram that Fig. 3 is plasmid pRL105, Jojoba farGene passes through its two ends BamThe H I and XhoThe I restriction endonuclease sites is cloned on the pRL104 carrier.

Schematic diagram that Fig. 4 is plasmid pRL106, pcr amplification goes out Jojoba farGene passes through its two ends NdeI and XhoThe I restriction endonuclease sites also is cloned on the pET28 carrier.

Fig. 5 for the yeast of pRL104 after semi-lactosi is induced, its culture carries out GC-MS after fatty alcohol extraction figure as a result.C15:0 means pentadecanol.

Fig. 6 for the yeast of pRL105 after semi-lactosi is induced, its culture carries out GC-MS after fatty alcohol extraction figure as a result.C12:0 means dodecanol; C14:0 means tetradecanol; C15:0 means pentadecanol; C16:0 means hexadecanol; C18:0 means octadecanol.

Fig. 7 be intestinal bacteria RL1 after IPTG induces, its culture carries out GC-MS after fatty alcohol extraction figure as a result.C15:0 means pentadecanol.

Fig. 8 be intestinal bacteria RL2 after IPTG induces, its culture carries out GC-MS after fatty alcohol extraction figure as a result.C12:0 means dodecanol; C14:0 means tetradecanol; C15:0 means pentadecanol; C16:0 means hexadecanol; C16:1 means Δ 9-hexadecanol; C18:1 means Δ 9-octadecanol.

Fig. 9 be intestinal bacteria RL3 after IPTG induces, its culture carries out GC-MS after fatty alcohol extraction figure as a result.C12:0 means dodecanol; C14:0 means tetradecanol; C15:0 means pentadecanol; C16:0 means hexadecanol; C16:1 means Δ 9-hexadecanol; C18:1 means Δ 9-octadecanol.

Figure 10 be intestinal bacteria RL4 after IPTG induces, its culture carries out GC-MS after fatty alcohol extraction figure as a result.C12:0 means dodecanol; C14:0 means tetradecanol; C15:0 means pentadecanol; C16:0 means hexadecanol; C16:1 means Δ 9-hexadecanol; C18:1 means Δ 9-octadecanol.

Figure 11 be intestinal bacteria RL5 after IPTG induces, its culture carries out GC-MS after fatty alcohol extraction figure as a result.C12:0 means dodecanol; C14:0 means tetradecanol; C15:0 means pentadecanol; C16:0 means hexadecanol; C16:1 means Δ 9-hexadecanol; C18:1 means Δ 9-octadecanol.

Embodiment

Purpose of the present invention reaches by following measures:

Introduce foreign gene in the heterotrophic microorganism body---acyl-CoA reductase, thus the reduction of the lipid acid of catalysis self forms fatty alcohol.

The acyl-CoA reductase reductase enzyme of introducing derives from plant exactly Simmondsia chinensisThe far gene, this gene is called again jojoba far gene.

A kind of as in heterotrophic microorganism of yeast cell, it is beneficial to cultivation, contamination resistance is strong, be convenient to carry out genetic manipulation, in embodiment, select a kind of yeast saccharomyces cerevisiae YPH499 (ATCC:76625) as producing bacterial strain, and built expression plasmid pRL104, and transform it as can express jojoba far gene pRL105 carrier.Intestinal bacteria are also clearly heterotrophic microorganisms of a kind of genetic manipulation background, its fast growth, the culture condition gentleness, it is one of first-selected bacterial strain carried out fermentative production, in embodiment, select a kind of e. coli bl21 (DE3) as producing bacterial strain, choose its expression plasmid pET28, and transform it as can express jojoba far gene pRL106 carrier.

Following examples are used for further illustrating the present invention, but should not be construed as limitation of the present invention.

Embodiment 1

Design a fragment gene sequence, it not only comprises Jojoba farThe homing sequence of gene and pRL104 plasmid, also added other restriction enzyme site at two ends, after being convenient to the replacing of carrier to optimize the expression of this fragment gene, added one section His-tag label at its N end simultaneously and be beneficial to whether identify protein expression.

Optionally be optimized (SEQ ID No.3, synthetic by the Jin Sirui bio tech ltd) according to yeast preference codon, and pass through BamThe H I and XhoTwo restriction enzyme sites of I are cloned in it on pRL104 carrier, and the plasmid built is named as pRL105.

Yeast saccharomyces cerevisiae YPH499 is the uridylic defective type, and on the pRL104 plasmid with the uridylic expressing gene, the plasmid pRL105 that builds and pRL104 are transformed respectively and enter in yeast saccharomyces cerevisiae YPH499, utilize SC-ura substratum (glucose that carbon source is 1%) to be screened.

In a little SC-ura substratum that is cloned in 5ml successfully transformed (carbon source is 1% glucose) of picking, tentatively cultivate for 30 ℃, the middle continuation of SC-ura substratum (carbon source is 1% glucose) that after 12h, it is proceeded to 50ml cultivated the 7h left and right at 30 ℃, surveys its OD.

If now OD is a, according to 300*0.4/a=120/a, calculate, get the centrifugal 5min of culture 5000rpm of 120/a ml, remove substratum and collect thalline, use again the centrifugal 5min of 10ml sterilized water 5000rpm, add about 5ml sterilized water suspension cell after removing supernatant, it is proceeded to the middle enlarged culturing of SC-ura substratum (raffinose that carbon source is 1%) of 270ml, owing to there being thalline to run off in the process at aseptic washing thalline, now the OD in substratum will be less than 0.4, after now continuing to cultivate about 1h, be about at 0.4 o'clock until its OD, add the semi-lactosi of 30ml 20% to carry out the semi-lactosi abduction delivering, culture temperature is still 30 ℃.

After semi-lactosi abduction delivering 24h, the culture of getting 100ml carries out the fatty alcohol extraction.

Extracting method is: in the 100ml culture, add the pentadecanol of 100ul 10mg/ml as internal reference, the solution A (hexane:isopranol=3:2) that adds again 200ml, after violent extraction 10min, standing 10min, after removing lower aqueous solution, then add the solution B (12g sodium sulfate is dissolved in 180ml water) of 180ml, continue violent extraction 10min, in standing 10min, remove lower aqueous solution again, the round-bottomed flask that the upper strata organic layer is proceeded to 500ml is rotated evaporation.Condition is: 40 ℃ of water-baths, pressure is 250psi.After organic liquid has been evaporated, 3 hexane with 3ml dissolve the product of round-bottomed flask inwall respectively, then this 9ml liquid rotating is moved on in the round-bottomed flask of a 25ml, under similarity condition, are rotated evaporation.After organic layer evaporates, use the hexane of 300ul by the product stripping in round-bottomed flask, it is transferred in a sample bottle with built-in pipe.

The sample of handling well is carried out to GC-MS(chromatography of gases-GC-MS) detect.The 5975C/7890A system that GC-MS is Agilent, the use pillar is HP-INNOWax, helium flow velocity 1ml/min, sample size 1ul, splitting ratio is 10:1, program temperature is: 50 ℃ of 2min, per minute raises 10 ℃ to 240 ℃, keeps 10min.

Experimental result: fatty alcohol detected in the yeast saccharomyces cerevisiae with pRL105, and only in the yeast saccharomyces cerevisiae with pRL104, fatty alcohol do not detected, prove pRL105 that the present invention builds can be in the yeast body synthetic fatty alcohol.By the reference internal reference, calculate, the fatty alcohol output of this kind of method production is about 3mg/L., from result, see simultaneously, utilize this method can access C12, C14, the fatty alcohol of C16 and C18.

Embodiment 2

Jojoba far gene on pRL105 is passed through to PCR method (primer sequence: SEQ ID No.4 and 5) add at its two ends NdeI and XhoTwo restriction enzyme sites of I, and it is upper to utilize these two restriction enzyme sites that it is cloned in to pET28, constructed plasmid is called pRL106.

By BL21(DE3) in fatty acyl-CoA dehydrogenase fadE gene knockout, can reduce the consumption of acyl CoA, thereby accumulate more acyl CoA for the production of fatty alcohol.To knock out e. coli bl21 (DE3) the called after TL101 of acyl CoA in this example.

The plasmid pMSD8 that John professor Cronan once built can be in intestinal bacteria overexpression fatty acetyl-CoA carboxylase(Mark S. Davis, Jose ' Solbiati, and John E. Cronan, Jr. 2000.Overproduction of Acetyl-CoA Carboxylase Activity Increases the Rate of Fatty Acid Biosynthesis in Escherichia coli. THE JOURNAL OF BIOLOGICAL CHEMISTRY .275 (37): 28593 – 28598.) in this example, the present invention uses this plasmid to see the output that whether can improve fatty alcohol.

With the tetradecyl alcohol (tetradecanol) of the lauryl alcohol that includes 0.4g/L (dodecanol) and 0.4g/L and the hexadecanol (hexadecanol) of 0.4g/L, TL101 is carried out to the fatty alcohol tolerance screening, the fatty alcohol that is 1.2g/L by above-mentioned concentration total concn (fatty alcohol) carries out the screening of a week continuously to TL101, by the cell conservation of finally surviving, and called after TL101*.

PET28 is transformed and enters BL21 (DE3), and the transformant that utilizes the kantlex screening to succeed, by its called after RL1.

PRL106 is transformed and enters BL21 (DE3), and the transformant that utilizes the kantlex screening to succeed, by its called after RL2.

PRL106 is transformed and enters TL101, and the transformant that utilizes the kantlex screening to succeed, by its called after RL3.

PMSD8 and pRL106 cotransformation are entered to TL101, and the transformant that utilizes kantlex and Pyocianil screening to succeed, by its called after RL4.

PMSD8 and pRL106 cotransformation are entered to TL101*, and the transformant that utilizes kantlex and Pyocianil screening to succeed, by its called after RL5.

Respectively RL1, RL2, RL3, RL4, five kinds of bacterium of RL5 are inoculated in respectively in the 5mlLB substratum 30 ℃ cultivate about 12h after, by its proceed in the 300mlLB substratum 30 ℃ continue to cultivate about 90min after, reach at 0.6 o'clock until its OD, add the IPTG of 0.25mM to carry out abduction delivering, culture temperature is still 30 ℃.

After abduction delivering 18h, get the 100ml culture and carry out fatty alcohol extraction.

Extracting method is: in the 100ml culture, add the pentadecanol of 100ul 10mg/ml as internal reference, the solution A (hexane:isopranol=3:2) that adds again 200ml, after violent extraction 10min, standing 10min, after removing lower aqueous solution, then add the solution B (12g sodium sulfate is dissolved in 180ml water) of 180ml, continue violent extraction 10min, in standing 10min, remove lower aqueous solution again, the round-bottomed flask that the upper strata organic layer is proceeded to 500ml is rotated evaporation.Condition is: 40 ℃ of water-baths, pressure is 250psi.After organic liquid has been evaporated, 3 hexane with 3ml dissolve the product of round-bottomed flask inwall respectively, then this 9ml liquid rotating is moved on in the round-bottomed flask of a 25ml, under similarity condition, are rotated evaporation.After organic layer evaporates, use the hexane of 300ul by the product stripping in round-bottomed flask, it is transferred in a sample bottle with built-in pipe.

The sample of handling well is carried out to GC-MS(chromatography of gases-GC-MS) detect.The 5975C/7890A system that GC-MS is Agilent, the use pillar is HP-INNOWax, helium flow velocity 1ml/min, sample size 1ul, splitting ratio is 10:1, program temperature is: 50 ℃ of 2min, per minute raises 10 ℃ to 240 ℃, keeps 10min.

Experimental result: except RL1, fatty alcohol all detected at all the other four bacterial classifications, the fatty alcohol output of RL2 is about 9.7mg/L, RL3 fatty alcohol output is about 10mg/L, RL4 fatty alcohol output is about 10.7mg/L, RL5 fatty alcohol output is about 12.3mg/L, proved that jojoba far gene can transform into fatty alcohol by the interior lipid acid of catalysis heterotrophic microorganism body really, and proof can obtain the fatty alcohol of higher output yield by the fatty acid metabolism approach in transformation heterotrophic microorganism body.Simultaneously, figure shows can obtain C12 in intestinal bacteria as a result, C14, C16, Δ 9-C16:1, the fatty alcohol of Δ 9-C18:1.

The sequence table explanation: SEQ ID No.1 and 2 is respectively Jojoba farGene order and protein sequence; SEQ ID No.3 is for pressing the yeast expression preference after codon optimized Jojoba farGene order.

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ttggataata aggctatctt ggttaccggt gccactggtt ccttggctaa gatttttgtt 180

gaaaaggtat tgagaagtca acctaacgtt aaaaagttgt atttgttgtt gagagctaca 240

gatgacgaaa ccgctgcatt gagattgcaa aacgaagttt tcggtaaaga attgtttaaa 300

gtattgaagc aaaatttggg tgcaaacttt tactctttcg tctcagaaaa ggttacagtt 360

gttccaggtg acattaccgg tgaagacttg tgtttgaagg atgttaattt gaaggaagaa 420

atgtggagag aaatagatgt cgttgtaaac ttagccgcta caattaattt catcgaaaga 480

tacgacgtct cattgttgat taacacctac ggtgctaagt acgttttgga tttcgctaag 540

aaatgcaata agttgaagat atttgtacat gtctctactg cttacgtttc aggtgaaaag 600

aatggtttga tcttagaaaa gccttattac atgggtgaat ctttgaacgg tagattgggt 660

ttggatataa acgtagaaaa gaaattggtt gaagcaaaga ttaatgaatt gcaagcagcc 720

ggtgccactg aaaaatccat taagagtaca atgaaggata tgggtataga aagagctaga 780

cactggggtt ggccaaacgt ttacgttttt actaaggcat tgggtgaaat gttgttgatg 840

caatacaagg gtgacattcc tttgactata atcagaccaa caatcataac ttccacattc 900

aaagaaccat ttcctggttg ggtcgaaggt gttagaacaa ttgataacgt ccctgtttat 960

tacggtaaag gtagattgag atgtatgtta tgcggtcctt ctaccataat cgacttaatc 1020

ccagctgata tggtcgttaa cgctactatt gtagcaatgg tcgcacatgc caatcaaaga 1080

tatgttgaac cagtaaccta ccacgttggt tcttcagctg caaatcctat gaaattatct 1140

gcattgccag aaatggccca tagatacttc acaaagaatc catggataaa ccctgataga 1200

aatccagtac atgtcggtag agccatggta ttttccagtt tctcaacctt ccacttgtat 1260

ttgactttga actttttatt gccattgaag gttttggaaa tcgcaaacac tattttctgt 1320

caatggttca agggtaaata catggacttg aagagaaaga caagattgtt gttgagattg 1380

gttgatatct ataaacctta cttatttttc caaggtatct tcgatgacat gaacacagaa 1440

aagttgagaa tagccgctaa ggaatctatc gttgaagccg acatgtttta tttcgatcca 1500

agagctatta attgggaaga ctactttttg aagactcatt ttcctggtgt cgttgaacat 1560

gtattgaact aaagatctag atctgtcgac ctcgag 1596

<210> 4

<211> 28

<212> DNA

<213 > artificial sequence

<400> 4

gcatcatatg gaagaaatgg gttcaatc 28

<210> 5

<211> 34

<212> DNA

<213 > artificial sequence

<400> 5

atatgcgctc ttagttcaat acatgttcaa cgac 34

Claims (7)

1. the method for a synthetic fatty alcohol in the heterotrophic microorganism body, it is that the acyl-CoA reductase gene is imported in the heterotrophic microorganism body, and makes this gene at this microorganism expression in vivo, wherein, described heterotrophic microorganism is yeast saccharomyces cerevisiae, and described acyl-CoA reductase gene is Jojoba farGene, its nucleotide sequence is as shown in SEQ ID No.3.

2. method according to claim 1, is characterized in that, the acyl-CoA reductase gene clone, on expression vector, is imported in corresponding heterotrophic microorganism body, and abduction delivering.

3. method according to claim 1 and 2, is characterized in that, also comprises the pathways metabolism of and degraded synthetic by transformation microorganism lipid acid, to improve the output of fatty alcohol.

4. method according to claim 3, is characterized in that, the heterotrophic microorganism pathways metabolism of transformation is to improve lipid acid output in this microbe in order to produce the approach of fatty alcohol.

5. method according to claim 3, is characterized in that, the heterotrophic microorganism pathways metabolism of transformation is to change this heterotrophic microorganism body fat acid metabolic approach intermediate in order to produce the approach of fatty alcohol.

6. method according to claim 3, is characterized in that, the heterotrophic microorganism pathways metabolism of transformation is to change this heterotrophic microorganism body fat acid metabolic approach derivative in order to produce the approach of fatty alcohol.

7. a heterotrophic microorganism, it is the heterotrophic microorganism that turns the acyl-CoA reductase gene, and wherein, described heterotrophic microorganism is yeast saccharomyces cerevisiae, and described acyl-CoA reductase gene is Jojoba farGene, its nucleotide sequence is as shown in SEQ ID No.3.

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