WO2014049180A1 - Triacylglycérol synthases thermostables et leurs utilisations - Google Patents

Triacylglycérol synthases thermostables et leurs utilisations Download PDF

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WO2014049180A1
WO2014049180A1 PCT/ES2013/000211 ES2013000211W WO2014049180A1 WO 2014049180 A1 WO2014049180 A1 WO 2014049180A1 ES 2013000211 W ES2013000211 W ES 2013000211W WO 2014049180 A1 WO2014049180 A1 WO 2014049180A1
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tags
transformed
vector
polynucleotide
tdgat
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PCT/ES2013/000211
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Spanish (es)
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Gabriel MONCALIÁN MONTES
Fernando DE LA CRUZ CALAHORRA
Juan Antonio VILLA TORRECILLA
Beatriz LÁZARO PINTO
Matilde Andrea CABEZAS ISIDRO
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Universidad De Cantabria
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil

Definitions

  • the present invention generally relates to newly identified, isolated and optimized polynucleotides, to the proteins encoded by said polynucleotides, as well as to the methods for the production of said proteins and to the uses of said polynucleotides and proteins.
  • the present invention contemplates a polynucleotide isolated from the microorganism Thermomonospora curvata, which codes for a thermostable triacylglycerol synthase enzyme, the method for the expression of said enzyme and the use thereof for obtaining triglycerides from different substrates.
  • biodiesel the main renewable alternative to petroleum diesel. Every year, more than 7,000 million liters of biodiesel are consumed worldwide.
  • This biodiesel is composed of methyl and ethyl esters of fatty acids (FAMEs and FAEEs, respectively) produced by the chemical transesterification of animal and vegetable triglycerides (HUI, J, Nelson, E., Tilman, D., Polasky, S., and Tiffany, D. (2006).
  • FAMEs and FAEEs methyl and ethyl esters of fatty acids
  • biodiesel has obvious economic benefits such as security in the balance, reducing net emissions of C0 2 , or the development of rural areas, currently biodiesel called first generation does not meet many of these premises. Therefore, the development of the so-called second generation biofuels is required, whose main characteristic is that in their production they do not compete with vegetable crops for human consumption. The production of triglycerides by oleogenic microorganisms (whose greatest exponent to date are rhodococcus bacteria) for their transformation to biodiesel would therefore fall into this category of second generation biodiesel.
  • a solution to obtain generic oil or triglycerides (TAGs) for the production of biodiesel or enriched in omega-3 fatty acids for use in aquaculture is its production in single-celled organisms (SCO) single cell oils or unicellular oil ).
  • SCO single-celled organisms
  • the production of oils rich in omega-3 DHA fatty acids in unicellular algae and its commercialization as an additive for baby milks by the American company Martek has been successfully achieved.
  • these algae are difficult to modify genetically to produce other oils and grow relatively slowly with substrates. specific. That is why it has been a long time since we tried to produce unicellular oils in bacteria.
  • Escherichia coli is the most studied prokaryotic organism in the laboratory and with greater possibilities of transformation and modification, with a very short generation time (about 20 minutes) and a wide range of substrates that can be used as a culture medium.
  • the approach to produce TAGs in E. coli has been the expression of various genes that code for the WS / DGAT enzymes (wax synthase / diacylglycerol acyltransferase), responsible for the accumulation of neutral lipids in different microorganisms.
  • the expression of the WS / DGAT enzymes of Acinetobacter sp (Stóveken, T, Kalscheuer, R., Malkus, U., Reichelt, R., and Steinbüchel, A. (2005).
  • the wax ester synthase / acyl coenzyme A diacylglycerol acyltransferase from Acinetobacter sp.
  • strain ADP1 characterization of a novel type of acyltransferase. J. Bacteriol. 187, 1369-1376), Marinobacter aquaolei (Holtzapple, E., and Schmidt-Dannert, C. (2007). Biosynthesis of isoprenoid wax ester in Marinobacter hydrocarbonoclasticus DSM 8798: Identification and characterization of isoprenoid coenzyme A synthetase and wax ester synthases. J. Bacteriol.
  • Alcanivorax borkumensis ⁇ Kalscheuer, R., Stóveken, T, Malkus, U., Reichelt, R., Golyshin, PN, Sabirova, JS, Ferrer, M., Timmis, KN, and Steinbüchel, A. (2007). Analysis of storage lipid accumulation in Alcanivorax borkumensis: Evidence for alternative triacylglycerol biosynthesis routes in bacteria. J. Bacteriol. 189, 918-928) has not shown a remarkable accumulation of oil in E. coli.
  • the WS / DGAT enzyme of Acinetobacter calcoacéticus is collected in WO 03/074676 A2, for its ability to produce waxes and the WS / DGAT of M. aquaolei in document (US2009 / 01 17629 Al) for its ability to produce isoprenoid waxes.
  • a method has been described for producing ethyl esters of fatty acids directly in E. coli that could be used as biodiesel.
  • neither this method Karleuer, R., Stólting, T, and Steinbüchel, A. (2006). Microdiesel: Escherichia coli engineered for fuel production.
  • Microbiology Reading, Engl. 152, 2529-2536 or modifications thereof (Steen, EJ, Kang, Y., Bokinsky, G., Hu, Z, Schirmer, A., McClure, A., Del Cardayre, SB , and Keasling, JD (2010). Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 463, 559-562) has produced significant amounts of biodiesel using Acinetobacter calcoaceticus WS / DGAT enzyme. In both, a significant production of waxes (wax esters) but not triglycerides (TAGs) is observed.
  • the authors of the present invention after an important research work, have used an enzyme of the identified WS / DGAT family in a thermophilic organism such as Thermomonospora curvata (tDGAT), surprisingly observing that the expression of said enzyme in E. coli results in a large production of GADs compared to the use of other WS / DGAT proteobacteria enzymes.
  • tDGAT Thermomonospora curvata
  • the method developed based on the expression of this enzyme allows various substrates to be used to obtain oil in E.coli in a short period of time.
  • an isolated polynucleotide having at least 75% homology with the sequence SEQ ID NO 1, as well as the polypeptide encoded by said polynucleotide, which exhibits triacylglycerol synthase activity is the subject of the invention.
  • the object of the invention is also a vector comprising the polynucleotide of the invention.
  • a host cell transformed with the vector of the invention is the subject of the invention.
  • a method for obtaining TAGs based on the expression of the polypeptide encoded by the polynucleotide of the invention is also the subject of the invention. by culturing the transformed host cell of the invention in a medium comprising waste or industrial waste as a carbon source.
  • the use of the host cell of the invention for obtaining TAGs is an object of the invention.
  • FIG. 1 Analysis of the raw organic extracts obtained from E. coli strain C41.
  • Lane 1 oleic acid control; 2, triolein control; 3, pET 29c: mbDGATl (A1TX06) of M. aquaolei; 4 pET29c: mbDGAT2 (A1U572) of M. aquaolei; 5, pET29c: abDGATl (Q0VKV8) of A. borkumensis; 6 pET29c: tDGAT (YP 003301387) of T. curvata; 7, pET29c.
  • the white arrowhead indicates the migration distance of the TAGs.
  • FIG. 1 Relationship between the expressed tDGAT protein and the accumulation of TAG A. 10% SDS-PAGE gel with total lysates of the cells collected after the indicated induction periods.
  • the black arrowhead indicates the position of the tDGAT B protein.
  • Analysis of the raw organic extracts Calle 1, oleic acid control; 2, triolein control; 3, vector pET29c; 4, 5 and 6 pET29c: TDGAT (YP 003301387) of T. curvata induced 3, 6 and 24 hours respectively.
  • the white arrowhead indicates the migration distance of the TAGs.
  • FIG. 3 Microscopic location of the fluorescence produced by Nile red in E. Coli C41 cells at different wavelengths, red filter (543 nm), green filter (488 nm) and sum of the two.
  • red filter (543 nm)
  • green filter (488 nm)
  • sum of the two On the left panel (A) cells transformed with pET29, image with the two filters separately and the summation and zoom to the microorganism.
  • B cells transformed with pET29c: tDGAT, images with the two filters separately, the summation and zoom to a group of microorganisms.
  • the white arrowhead indicates lipid inclusions.
  • FIG. 4 Gas chromatography of FAMEs obtained from the extraction of TAGs from the culture of E. Coli strain C41 (DE3) transformed with (A) pET29c or (B) pET29c: tDGAT. In each of the graphs a series of peaks appear with the molecular species of fatty acid identified in the case of the majority.
  • FIG. 5 Production of TAG by C41 (DE3) pET29c: tDGAT with different substrates. Analysis of the raw organic extracts grown in 50 ml of minimum medium by varying the carbon source at a concentration of 40 g / 1. Lane 1, oleic acid control; 2, triolein control; 3, Glucose; 4, Gluconate; 5, Fructose; 6, Xilosa; 7, Lactose; 8, Glycerol; 9, Control pET29c. The white arrow indicates the migration distance of the TAGs.
  • Figure 6 TLC analysis of the raw organic extracts of the transformation with pBAD33: tDGAT from strains of E. coli DH5a.
  • the cells were cultured in LB with varying concentrations of arabinose and different induction times.
  • Lane 1 oleic acid control; 2, triolein control; 3, pBAD33: tDGAT 3 hours induction at 0 mM; 4, pBAD33: tDGAT 16 hours without induction; 5, pBAD33: tDGAT 3 hours of induction at 0.5 mM; 6, pBAD33: tDGAT 16 hours induction at 0.5 mM; 7, pBAD33: tDGAT 3 hours induction at 1 mM; 8, pBAD33: tDGAT 3 hours induction at 1 mM; 9, C41 pBAD33 16 hours without induction.
  • the white arrow indicates the migration distance of the TAGs.
  • the present invention relates to the identification, isolation and optimization of a gene encoding a WS / DGAT enzyme in Thermomonospora curvata (tDGAT).
  • the nucleotide sequence of the gene has been optimized for expression in gamma-proteobacteria, more specifically in Escherichia coli and is shown in SEQ ID NO 1 ( Figure 1).
  • the amino acid sequence of the encoded tDGAT enzyme has been identified by the gene and is shown in SEQ ID NO 2 (figure 2).
  • nucleotide sequence of SEQ ID NO 1 is particularly contemplated, any person skilled in the art understands that any other biologically functional equivalent form of said nucleotide sequence can be isolated using conventional DNA-DNA and DNA-RNA hybridization techniques.
  • the present invention also contemplates nucleotide sequences that hybridize with SEQ ID NO 1 and that encode proteins that exhibit the same or similar biological activity as the protein of SEQ ID NO 2.
  • the original nucleotide sequence of the gene is contemplated. , which has a 75% homology with SEQ ID NO 1 and which codes for the same polypeptide.
  • an isolated polynucleotide which codes for a WS / DGAT enzyme, which has at least 75% homology with the sequence SEQ ID NO 1, preferably at least 80%, more preferably is contemplated in a major aspect of the invention. at least 85%, even more preferably at least 90% and even more preferably at least 95% (polynucleotide of the invention).
  • an isolated polypeptide, with triacylglycerol synthase activity encoded by the polynucleotide of the invention, which has at least 75% homology with the sequence SEQ ID NO 2, preferably at least 80%, more preferably at least 85%, even more preferably at least 90% and even more preferably at least 95% (polypeptide of the invention).
  • the polynucleotides of the present invention can be used to produce proteins (WS / DGAT enzymes) by using recombinant expression vectors containing said polynucleotides.
  • a vector comprising the polynucleotide of the invention (vector of the invention) is contemplated.
  • vector of the invention a wide variety of existing vectors can be used for gene expression in bacteria, particularly in Escherichia coli, although in particular, said vector is selected from pET29c and pBAD33.
  • nucleotide sequence is inserted into the vector by means of conventional techniques, routinely used in the state of the art.
  • a host cell transformed with the vector of the invention is contemplated.
  • the host cell is a bacterium.
  • said bacterium is a gamma-proteobacterium. More preferably, said gamma-proteobacterium is Escherichia coli.
  • an Escherichia coli strain transformed with the pET29 vector comprising the sequence polynucleotide SEQ ID NO 1 is contemplated.
  • the present invention also contemplates methods for the production of triglycerides from the expression of the polypeptide of the invention, enzyme with WS / DGAT activity, from cells transformed with the polynucleotide sequence of the invention.
  • a method for obtaining TAGs which comprises the following steps: a) Obtaining the transformed host cell of the invention,
  • inducers are used for heterologous gene expression.
  • the gene expression is silenced until the inducer is added. This causes the cells to behave as if they were not transformed until they are induced. In this case, the cells grow without overexpressing the tDGAT gene until, preferably, IPTG (or arabinose) is added.
  • IPTG or arabinose
  • This also serves to verify that the observed phenotype (that is, the production of GAD), is directly related to the addition of inducer and, therefore, to the expression of the gene of interest.
  • a constitutive promoter could be used (which does not respond to inducer and is expressed at all times), but the production of GADs at will could not be controlled. It is also possible to use lactose instead of IPTG or other promoters that respond to other types of inductors (for example inductors cheaper than IPTG such as glucose or even light).
  • the method of the invention for obtaining TAGs comprises the following steps:
  • the tDGAT gene sequence (SEQ ID NO 1) is introduced into the clone vector pET29c, a vector that adds a tail of LEHHHHHH to the enzyme produced.
  • the IPTG inductor is used.
  • tDGAT tDGAT in E coli with IPTG
  • GADs a large production of GADs is surprisingly observed two hours after induction.
  • This production is not detected at all when other WS / DGAT proteobacteria enzymes were used ( Figure 1).
  • TAGs correlates perfectly with the amount of tDGAT produced, reaching a maximum of production three hours after induction ( Figure 2). In that short space of time the bacteria is capable of producing up to 50 mg of GAD per liter of culture.
  • the main advantage of the invention is the possibility of using various substrates to obtain oil in E coli in a short space of time.
  • the method of the present invention preferably employs those that are produced abundantly and cheaply or that are industrial wastes harmful to the environment.
  • glucose could be used (which can be obtained from the sugar cane sucrose, the starch of the cereals and even the cellulose of plants and trees), fructose (fruits and honey) or xylose ( obtained from the hemicellulose of plants and trees or as a residue from cellulose production industries), glycerol (byproduct of biodiesel production), lactose (byproduct of cheese production), molasses (byproduct of sugar production), waste organic, urban wastewater, cow manure, etc.
  • triglycerides obtained in the method of the invention, or a fraction thereof, as biofuel or as a starting material for obtaining biofuel is contemplated.
  • the use of the host cell of the invention for obtaining GADs is contemplated.
  • LB tryptone 10 g / L, yeast extract 5 g / L, was used for liquid culture
  • NaCl 5 g / L NaCl 5 g / L
  • LB Tryptone 10 g / L, yeast extract 5 g / L, was used for liquid culture
  • NaCl 5 g / L NaCl 5 g / L
  • w / v agar
  • kanamycin sulfate Sigma Aldrich, USA
  • chloramphenicol Sigma Aldrich, USA
  • a minimum medium (M9 salts, 2mM MgS0 4 and 0.1 mM CaCl 2 ) was used with the different carbon sources (glucose, gluconate, fructose, xylose, lactose and glycerol) at 40 g / 1 .
  • E. coli strains were conserved by centrifuging stationary phase cultures and resuspending them in a 0.75% (w / v) peptone solution and 50% glycerol. They were stored at -20 ° C for frequent use or -80 ° C for long-term preservation. Plasmid construction.
  • the plasmids used were constructed by digestion and cloning of fragments amplified by PCR in cloning vectors.
  • the PCR reactions were amplified with oligonucleotides with restriction targets at their ends, equal to those used in the vector to digest it.
  • a fragment containing SEQ ID NO 1 obtained by PCR was inserted with the primers suitable for adding at the ends the restriction sites Ndel and Xhol, respectively.
  • the construction was performed similarly using the restriction enzymes Kpnl and HindIII and adding a ribosome binding site.
  • the digestion of DNA with restriction enzymes was carried out with enzymes supplied by the companies Roche and Fermentas. In each case, the restriction conditions (buffer, time and temperature) and deactivation recommended by the commercial house were followed.
  • ligation reactions were performed in the cloning processes. using previously dephosphorylated vectors. The digested vector terminal phosphate groups were previously removed by treatment with alkaline phosphatase.
  • the reaction conditions were: 20 ⁇ of the restriction mixture, 1 ⁇ of 1 U / ⁇ alkaline phosphatase (Roche), 2.5 ⁇ of SAP lOx dephosphorylation buffer (Roche) and 1.5 ⁇ of milliQ water.
  • the mixture was incubated 1 h at 37 ° C.
  • Ligation reactions were performed with a 5: 1 molar ratio (insert: vector).
  • 1 ⁇ of phage ligase T4 5 U / ⁇ (Fermentas) and 2 ⁇ of the 10 X ligation buffer was used in a volume of 20 ⁇ . Incubation was performed overnight at 16 ° C. After the reaction, the ligase was inactivated 10 minutes at 65 ° C.
  • the constructions made were transformed by electroporation into a strain of E. coli, DH5a. The DNA sequences of all fragments cloned by PCR were verified by sequencing.
  • E. coli strains DH5a or C41 (DE3)
  • a procedure was used whereby they could be transformed with a frequency> 5 x 10 8 colonies / ⁇ g DNA.
  • the desired E. coli strain was grown at 37 ° C.
  • the isolated colonies were transferred to LB and grown under agitation at 37 ° C overnight.
  • the cells were diluted 1: 20 in LB and incubated with shaking to an OD at 600 nm of 0.6. They were then incubated on ice for 30 minutes and recovered by centrifugation at 4000 rpm for 10 minutes. They were finally washed with sterile water, recovered with 10% glycerol and frozen in aliquots at -80 0 C.
  • the DNA used to electroporate must be free of salts. Therefore, the ligation mixtures were microdialized before being mixed with the competent cells. They were placed on a Millipore GS nitrocellulose filter of 0.05 ⁇ pore size, in a Petri dish with sterile milliQ water and after 30 minutes, the filter drop was collected. For transformation, 5 ⁇ of the dialyzed ligation mixture was added to 50 ⁇ of competent cells thawed on ice. In the case of purified plasmid DNA, the amount used was 1 to 50 pg. This mixture was deposited in a 0.2 cm Gene Pulser electroporation cuvette (BioRad), cooled to 0 ° C.
  • BioRad Gene Pulser electroporation cuvette
  • the QIAprep Spin Miniprep kit (QIAGEN) was used for plasmid isolation.
  • GenElute Gel Extraction kit (Sigma-Aldrich) was used to obtain DNA fragments from bands extracted from agarose gels.
  • GenElute PCR Clean-Up kit (Sigma-Aldrich) was used. The concentration of the DNA samples was determined by measuring the absorbance at 260 nm with a Nano-Drop ND-1000 spectrophotometer.
  • ThermoPol 10X PCR buffer (BioLabs), which includes Mg 2 S0 4 at 20 mM, 5 ⁇ of total mO nucleotide mixture (2.5 mM of each nucleotide), 0.5 ⁇ of each oligonucleotide primer at 100 ⁇ , 0.5 ⁇ of Vent polymerase at 2 U / ⁇ , 10 ng of template DNA and milliQ water to a final volume of 50 ⁇ .
  • the PCRs used for clone testing were done with BIOTAQ polymerase (Bioline).
  • the reaction mixture with this polymerase was: 2.5 ⁇ of 10X N3 ⁇ 4 PCR buffer (Bioline), 2 ⁇ of total 10 mM nucleotide mixture, 2.5 ⁇ of each 10 ⁇ primer oligonucleotide, 0.2 ⁇ of 5 U / ⁇ polymerase, 1 ⁇ of MgCl 2 at 50 mM, the bacterial colony to be analyzed and milliQ water to a final volume of 25 ⁇ .
  • the PCRs were performed in a UNO II thermocycler (Biometra).
  • the program used consisted of an initial denaturing cycle of 5 minutes at 94 ° C, for later Perform 30 cycles of: 30 seconds at 94 ° C, 30 seconds at the DNA / primer hybridization temperature and elongation time (1 minute per Kb of amplified DNA) at 72 ° C. Finally, a 10-minute cycle was performed at 72 ° C to complete the amplification of the fragment, after which the reaction temperature was lowered to 4 ° C.
  • the synthetic ORFs were designed with codon dominance adapted to E. coli for the WS / DGATs of thermomonospora curvata and acquired from GenArt (currently Life Technologies).
  • GenArt currently Life Technologies
  • the oligonucleotides were supplied by Sigma-Genosys (Sigma-Aldrich, USA).
  • the different DNA molecules used were analyzed by agarose gel electrophoresis.
  • the agarose was dissolved in 0.5 X of TBE (45 mM Tris-HCl pH 8.2, 45 mM boric acid, 0.5 mM EDTA) at a concentration of 0.7-1% w / v, as necessary according to the size of the DNA fragments to solve.
  • SYBR Safe Invitrogene was used for DNA staining at a final concentration of 0.05 mg / mi.
  • the loading buffer or SAB (0.25% bromophenol blue (w / v), 30% glycerol (w / v) in 0.5 x TBE) was added to the DNA samples, in a 5: 1 ratio (DNA / SAB) to the volume of the sample.
  • HyperLadder I Biolabs
  • a horizontal BioRad electrophoresis system 80-120 volts was used to run the samples.
  • the agarose gels were visualized with a Gel Doc 2000 UV system and the images were analyzed with the Quantity One software (BioRad). Production and extraction of the TAGs fraction from a cell sample.
  • Cultures of 1 1 of C41 (DE3) or DH5D cells with the plasmid of interest were grown to an OD at 600 nm of 0.6 and induced with IPTG (0.1 or 1 mM final of IPTG) or Arabinosa (final 0.05 mM of arabinose) respectively.
  • Cells were collected after induction at different times by centrifugation at 4000 rpm for 12 minutes at 4 ° C.
  • the extraction of the lipid fraction was carried out by subjecting the pellet comprising the cells collected from a culture to 5 ml of a solution containing a 3: 2 mixture of hexane / 2- Isopropanol under stirring for 3 hours.
  • the mobile phase was composed of 200 ml of hexane, 50 ml of diethyl ether and 2.5 ml of acetic acid.
  • the samples were loaded in the form of small drops at the same point on a sheet of Polygram Silica Gel (Macherey-Nagel, Germany) (stationary matrix). When the front rose to the top of the sheet it was removed, allowed to dry and revealed with iodine vapor. This causes the separated TAGs in the matrix to be dyed an ocher color.
  • a sample (1 ⁇ ) of known lipid patterns (Sigma Aldrich) was run on one side to know how far they migrated. Obtaining FAMES.
  • fatty acids in the lipid fraction that accumulates E. coli transformed with tDGAT they had to be converted into methyl esters or FAMEs.
  • saponification reagent 45 g NaOH in 150 ml of methanol and 150 ml dH20
  • the sample was removed and stirred for 30 seconds and the sample was returned to the bath at 100 ° C for 25 minutes.
  • the tube was allowed to cool to room temperature and 2 ml of methylation reagent (325 ml 6 N HC1 with 275 ml of methanol) was added. It was stirred for 30 seconds and the sample was transferred to a bath at 80 ° C for 10 minutes. The sample was cooled on ice for five minutes and 1.25 ml of extraction reagent (200 ml hexane with 200 ml of methyl-t-butyl ether) was added, closing the tube and leaving it under stirring for 10 min. Two phases formed, discarding the lower one. 3 ml of wash reagent (10.8 g NaOH in 900 ml dH20) was added, allowed to stir gently for 5 minutes and the upper phase was discarded.
  • 2 ml of methylation reagent 325 ml 6 N HC1 with 275 ml of methanol
  • the fatty acids were analyzed by gas chromatography on a Shimadzu GC 2100 unit with FID detector and AOC 20i automatic injector. An HP Innowax 30 m x 0.32 mm, 0.5 Dm column was used. Calibration was performed with the FAME Mix RM-1 (Supelco) standard.
  • This lysate was ultracentrifuged at 40,000 rpm for 15 minutes at 4 ° C and the supernatant was loaded on a HisTrap HP GE Healthcare Nickel column (5 ml) previously equilibrated with an A buffer (1 M NaCl, 50 mM TrisHCl pH 7.5).
  • the protein was eluted with buffer B (same as buffer A but also contained 0.5 M imidazole), specifically 0.3 M imidazole.
  • the protein after elution by the HiTrap column was generally quite pure, it was concentrated and passed through a Superdex 75 GL10 30 column (Pharmacia) previously equilibrated in 150 mM NaCl buffer 50 mM TrisHCl pH 7.5 1 mM EDTA. The completely pure fractions were stored at -80 ° C at 10% in glycerol.
  • Electro foresis of proteins was carried out in polyacrylamide / SDS gels at 10% polyacrylamide.
  • the loading buffer 50 mM Tris-HCl pH 6.8, 4% SDS, 4% glycerol and 0.02% bromophenol blue
  • the samples were heated at 100 ° C for 5 minutes, before being loaded on the polyacrylamide gel.
  • the "Low Range" standard BioRad Laboratories
  • Electrophoresis was carried out in a 25 mM Tris-HCl buffer pH 8.3, 200 mM glycine and 1% SDS. Later, the gels were stained with a solution containing 0.1% Coomassie Brilliant Blue R-250 (Merck), 50% methanol and 10% glacial acetic acid at room temperature for 30 minutes.
  • the different oleogenic cells were prepared under different culture and growth conditions by incubating the samples for 30 min at 4 ° C in PBS containing 0.5 ⁇ g / ml Nile Red (stock solution 0.5 mg / mi in DMSO).
  • the cells were pelleted by centrifugation at 16000 g at 4 ° C, and resuspended in methanol for two minutes at -20 ° C.
  • the cells were then pelleted again by centrifugation, resuspended in PBS and mounted on sample holders with Vectashield (Vector Laboratories, Burlingame, CA, USA).
  • the samples were examined with a laser scanning LSM 510 microscope (Zeiss, Germany) with a 63 X oil immersion objective (1.4 NA).
  • the elution volume of the purified protein corresponds to a protein of about 53 KDa that co-indexes with the size of the tDGAT protein in a monomeric state.
  • E. coli C41 (DE3) transformed with pET29c: tDGAT was studied, using different carbon sources present in these wastes.
  • E. coli is an organism that can naturally take advantage of a large amount of carbohydrates and various types of alcohols. To corroborate it, this strain was grown in a M9 Minimum Medium (Materials and Methods), modifying only the carbon source. TAGs extracted from each of the crops were analyzed by TLC starting, in all cases, with the same amount of wet biomass. The accumulation of GADs in cultures with six carbon sources was induced different.
  • Glucose Hexose sugar of lignocellulose, starch, cane sugar, etc. Most abundant sugar of nature whose production from lignocellulose (the main component of biomass) is currently very developed.
  • Gluconate Main carbon source of other oleogenic microorganisms such as rhodococcus.
  • Fructose It is the main residue of many food industries that process, for example, molasses of plant origin.
  • Xilosa pentose lignocellulose sugar Not as abundant as glucose, but very abundant in the hemicellulose residue produced in the paper or textile cellulose producing industries.
  • Lactose Disaccharide formed by glucose and galactose discarded in food industries that usually work with dairy products.
  • the concentration of lactose sugar in whey is about 50 g 1.
  • Glycerol Major waste produced in the chemical esterification of biodiesel. Glycerol is produced as the main byproduct that is not used directly in the process.
  • TAGs are practically independent of the carbon source used. With xylose, lactose and, above all, glycerol, the microorganism grew more slowly than with glucose, gluconate, and fructose. Of all the sources used, it should be noted that TAGs were produced from pure lactose. This suggests that strain C41 (DE3) transformed with tDGAT could be used effectively to take advantage of industrial whey. EXAMPLE 7
  • tDGAT To express tDGAT in DH5 cells, the gene was cloned into plasmid pBAD33 whose promoter is recognized by E. coli RNA polymerase and is repressed in the absence of arabinose. Cloning of the pBAD33: tDGAT construct was performed as described in Materials and Methods and strains of E. coli DH5a were transformed. The cells were grown in the same manner as in the case of C41 pET: tDGAT, changing only inducer (arabinose instead of IPTG) and the antibiotic (chloramphenicol instead of kanamycin).
  • the pBAD33: tDGAT construct is functional and converts the DH5a bacteria into a bacterium that produces GADs.
  • the production of GADs by this strain was visible but was significantly lower in all treatments with respect to the case of C41 (DE3), although the levels of protein produced by plasmid pBAD33: tDGAT were similar to those produced by the pET system. This could be because C41 cells (DE3) have a larger membrane surface. If lipid inclusions begin to form in the membrane, as some models propose, this factor could be favoring the accumulation of GADs.

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Abstract

L'invention concerne un polynucléotide isolé qui présente une homologie d'au moins 75 % avec la séquence SEQ ID NO 1. L'invention concerne également un polypeptide codé par ledit polynucléotide, qui présente une activité triacylglycérol synthase. L'invention concerne également un vecteur qui comprend ledit polynucléotide. L'invention concerne également une cellule hôte transformée avec ledit vecteur. L'invention concerne également une méthode d'obtention de TAG fondée sur l'expression du polypeptide codé par ledit polynucléotide par culture de ladite cellule hôte transformée dans un milieu qui comprend des résidus ou des déchets industriels en tant que source de carbone. L'invention concerne également l'utilisation des TAG obtenus dans ladite méthode en tant que biocombustible ou que matériau de départ pour l'obtention de biocombustible.
PCT/ES2013/000211 2012-09-28 2013-09-23 Triacylglycérol synthases thermostables et leurs utilisations WO2014049180A1 (fr)

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Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
BAILLY-BECHET, M. ET AL.: "Codon usage domains over bacterial chromosomes, art e37", PLOS COMPUTATIONAL BIOLOGY, vol. 2, no. 4, 2006 *
DATABASE ENA 23 November 2011 (2011-11-23), "''Thermomonospora curvata DSM 43183 acyltransferase, WS/DGAT/MGAT'', Retrieved from ENA in EMBL-EBI Services: Codigo of acceso (ID)", accession no. CY99349.1 *
DATABASE UNIPROTKB 16 May 2012 (2012-05-16), CHERTKOV, O. ET AL.: "Complete genome sequence of Thermomonospora curvata type strain (B9).", accession no. 1AD40 *
DUAN, Y. ET AL.: "Of novo biosynthesis of biodiesel by Escherichia coli in optimized fed-batch cultivation, art e20265", PLOS ONE, vol. 6, no. 5, 2011 *
KALSCHEUER, R. ET AL.: "Microdiesel: Escherichia coli engineered for fuel production", MICROBIOLOGY, vol. 152, no. 9, 2006, pages 2529 - 2536 *
KALSCHEUER, R. ET AL.: "Synthesis of novel lipids in Saccharomyces cerevisiae by heterologous expression of an unspecific bacterial acyltransferase", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 70, no. 12, 2004, pages 7119 - 7125 *
STANDARDS IN GENOMIC SCIENCES, vol. 4, no. 1, February 2011 (2011-02-01), pages 13 - 22 *
STOLETZKI, N. ET AL.: "Synonymous codon usage in Escherichia coli: selection for translational accuracy", MOLECULAR BIOLOGY AND EVOLUTION, vol. 24, no. 2, 2007, pages 374 - 381 *

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