CN102869768B

CN102869768B - Produce fatty acid and the microbial project of derivative of fatty acid

Info

Publication number: CN102869768B
Application number: CN201180022036.4A
Authority: CN
Inventors: 格里戈里·斯特凡诺普洛斯; 赛义德·侯赛因·伊马姆·阿比迪
Original assignee: Massachusetts Institute of Technology
Current assignee: Massachusetts Institute of Technology
Priority date: 2010-03-02
Filing date: 2011-03-02
Publication date: 2016-11-30
Anticipated expiration: 2031-03-02

Abstract

Some aspects of the present invention relate to use engineered microorganism that carbon source is converted into bio-fuel or the method for bio-fuel precursor.Some aspects of the present invention relate to finding the key regulator of lipid metabolism in microorganism.Some aspects of the present invention relate to produce bio-fuel or the engineered microorganism of bio-fuel precursor.

Description

Produce fatty acid and the microbial project of derivative of fatty acid

Related application

According to 35U.S.C. § 119 (e), this application claims the U.S. Provisional Patent Application submitted on March 2nd, 2010 U.S.S.N.61/309, the priority of 782, entire contents is incorporated herein by.

Technical field

The present invention relates to using engineered cell or microorganism that carbohydrate source is converted into biology at least in part Fuel or the field of bio-fuel precursor (such as fatty acid or derivative of fatty acid, such as triacylglycerol).

Federal government subsidizes research

The research (at least partly) in terms of some of invention disclosed herein is caused to be subsidized 69106899 supports by Ministry of Energy. U.S. government has certain rights in the invention.

Background technology

The bio-fuel of sustainable production is the succedaneum of Fossil fuel, and can aid in and alleviate can be easily obtained The consumption of fossil fuel reserves, avoids the pollution relevant to Fossil fuel and greenhouse gas emission simultaneously, thus with continuable Mode meets the growing demand to the energy afforded.But, some shortcomings of existing production method hamper extensively General enforcement bio-fuel produces, and the plant and the cereal crops that such as produce bio-fuel compete the land area having agronomical value, Or use the industrial material of the most limited supply as carbon source.

Summary of the invention

The sustainability of Fossil fuel and the concern day by day increased of recyclability are made to develop separate sources many Plant substituting bio-fuel, including the lipid synthesized from Renewable resource by microorganism (such as antibacterial or yeast).Can be used as biology The lipid of fuel or bio-fuel precursor includes such as fatty acid and derivant (such as, triacylglycerol) thereof.

The bio-fuel of Microbe synthesis or the economic feasibility of bio-fuel precursor depend on use have include multiple The applicable microorganism of the phenotype of the combination of advantageous feature, described advantageous feature such as allow to be efficiently converted into carbon bio-fuel or The metabolism of bio-fuel precursor, high biomass formation rate, bio-fuel or the high yield of bio-fuel precursor, bio-fuel or The intracellular accumulation of high level of bio-fuel precursor or secretion, to raw material (carbon source and related substances) with synthesized product is (such as, Fatty acid or triacylglycerol) well tolerable and bio-fuel or the stability of bio-fuel precursor (such as dense in low-carbon-source Under degree).Conversion yield (grams of oil produced by every gram of substrate (such as glucose)) is particularly important.It is generally employed to produce biology The microorganism of fuel or bio-fuel precursor does not meets the required table enough allowing to produce the most economically bio-fuel Type.

Some aspects of the present invention relate to transforming in microorganism desirable characteristics to produce bio-fuel or bio-fuel before Body.Although from nineteen thirties and nineteen forties, having studied the lipid in microorganism and fatty acid Metabolism (see, e.g. Woodbine, M.1959, Microbial fat:Microorganisms as potential fat Producers.Prog.Ind.Microbiol.1:181), but, although at genetically modified microorganism or optimize production process Condition aspect has made many effort, and in microorganism, transformation produces the progress in terms of relevant expectation phenotype still to bio-fuel The least.Up to the present, genetic modification work is concentrated mainly on operation fatty acid synthesis circuit upstream or gene target therein Mark and optimization are fermented or condition of culture (such as, by supplementing growth medium with fatty acid).

One major obstacle of genetically modified microorganism is the absence of in target microorganism (such as oil-producing (oleaginous) yeast In) genomic information of Key Metabolic path regulatory factor and annotation.Therefore, in the microorganism for producing bio-fuel still So lack and control carbohydrate to the Function Identification of the key regulator of lipid Transfer and annotation.

Some aspects of the present invention relate to being accredited as giving birth to oleaginous yeast Yarrowia lipolytica (Y.lipolytica) Produce bio-fuel or the microorganism of bio-fuel precursor.Some aspects of the present invention relate to finding fatty acid metabolism in microorganism Key regulator.Some aspects of the present invention relate to finding that stearyl-coenzyme A desaturase (SCD) is as carbon water in microorganism Compound is to the key regulator of lipid Transfer.Some aspects of the present invention relate to the pass of fatty acid metabolism in encoding microbial The nucleic acid of the separation of key regulatory factor.Some aspects of the present invention provide the key of the fatty acid metabolism of coding oleaginous microorganism The nucleic acid of the separation of regulatory factor (such as, SCD gene outcome).

Some aspects of the present invention relate to operating the activity (such as, passing through genetic manipulation) of fatty acid metabolism regulatory factor and come Transformation is for producing the microorganism of bio-fuel.Some aspects of the present invention relate to transforming for producing bio-fuel or biological combustion The microorganism of the separation of material precursor.Some aspects of the present invention relate to for carbohydrate source is converted into bio-fuel or life The microorganism of the separation that thing fuel precursor is optimized, such as, comprises the oleaginous microorganism of the SCD gene outcome of activity increase. Some aspects of the present invention relate to transformation for producing bio-fuel or the culture of microorganism of bio-fuel precursor.The present invention's Some aspects are directed to use with transforming and carbohydrate source are converted into fatty acid or fat for producing the microorganism of bio-fuel The method of acid derivative.Some aspects of the present invention are directed to use with transformation for producing the microorganism of bio-fuel by carbon hydrate Thing is converted into the bioreactor of fatty acid or derivative of fatty acid.Some aspects of the present invention provide the engineered micro-life of use Carbohydrate source is converted into bio-fuel or the method for bio-fuel precursor by thing at least in part.

Some aspects of the present invention relate to separate oil-producing cell, its comprise raising selected from hemoglobin, cytochrome, GLUT, malate dehydrogenase (malic enzyme), ACC, SCD, FAA1, ACS, ACS2, FAT1, FAT2, PCS60, ACLY, FAS, acyl The genetic modification of the expression of the one or more of genes of base CoA synthase, pyruvate carboxylase and AMPK gene, and/or Reduce selected from JNK2 and the genetic modification of the expression of the gene of Δ-12 desaturase.In some embodiments, the oil-producing of separation Cell comprises nucleic acid construct, and described nucleic acid construct comprises: (a) is included under the control of suitable homology or allogeneic promoter The expression cassette of the nucleic acid of encoding gene product；B () is included in the interference controlling lower coding targeted gene product of allogeneic promoter The expression cassette of the nucleic acid of RNA；And/or (c) inserts the nucleic acid construct of cellular genome, described construct comprises raising or reduces The nucleotide sequence of gene product expression.In some embodiments, allogeneic promoter is induction type or constitutive promoter.One In a little embodiments, the natural regulation of the natural gene of nucleic acid construct suppression or destruction (disrupt) encoding gene product, lead Cause the process LAN of natural gene.In some embodiments, nucleic acid construct suppression or the expression of elimination natural gene.At some In embodiment, a part for the regulatory region or regulatory region that lack, destroy, suddenly change and/or replace regulator gene expression it is situated between Lead suppression or the destruction of the natural regulation of natural gene, or by lacking, destroy, suddenly change and/or replace the coding of natural gene Regulatory region or a part for regulatory region that sequence or regulation natural gene are expressed mediate suppression or the elimination that natural gene is expressed. In some embodiments, by composing type or inducible expression targeting JNK2 and/or Δ-12 delta 8 desaturase genes product and suppression The nucleic acid of described gene expression mediates JNK2 and/or the reduction of Δ-12 delta 8 desaturase genes expression.In some embodiments In, the nucleic acid of targeting JNK2 and/or Δ-12 delta 8 desaturase genes transcript is by the expression of RNAi approach suppression transcript.? In some embodiments, the nucleic acid of targeting JNK2 and/or Δ-12 desaturase transcript is siRNA, shRNA or micro RNA.In some embodiments, by knocking out wild type gene in microorganism (such as, by nucleic acid construct (such as targeting Property carrier) with genome JNK2 or the homologous recombination of Δ-12 delta 8 desaturase genes seat) thus destroy wild type gene expression come Reduce JNK2 and/or the expression of Δ-12 desaturase.In some embodiments, nucleic acid construct is inserted the gene of cell In group.In some embodiments, the raising of gene product expression or reduce give cell carbohydrate source is converted into The advantageous phenotypes of fatty acid, fatty acid derived and/or TAG.In some embodiments, advantageous phenotypes is the fatty acid changed Spectrum, the triacylglycerol spectrum changed, the fatty acid increased and/or triacylglycerol synthetic ratio, the conversion yield of increase, cell increase Triacylglycerol accumulation and strengthen the tolerance to osmotic pressure, the multiplication rate of increase, the cell volume of increase and/or cell pair Concentration be lethal and/or suppress its same type cell the propagation without modified cells material strengthen tolerance.At some In embodiment, with same cell type without compared with modified cells, the fatty acid profile of the change of cell or the trigalloyl of change Glycerol stave reveals the ratio of C18 fatty acid and C16 fatty acid and increases at least 2 times.In some embodiments, with identical carefully Comparing without modified cells of born of the same parents' type, the fatty acid profile of the change of cell or the triacylglycerol stave of change reveal C18 fat Acid increases at least 2.5 times with the ratio of C16 fatty acid.In some embodiments, the most modified with same cell type Cell is compared, and the fatty acid profile of the change of cell or the triacylglycerol stave of change reveal the ratio of C18 fatty acid and C16 fatty acid Rate increases at least 5 times.In some embodiments, with same cell type without compared with modified cells, the change of cell Fatty acid profile or the triacylglycerol stave of change reveal the ratio of C18 fatty acid and C16 fatty acid and increase at least 6.5 times. In some embodiments, cell is survived under the conditions of to the osmotic pressure lethal without modified cells.In some embodiments, Cell osmotic pressure level by tolerated without modified cells the 200% of top level under conditions of survive.Implement at some In scheme, cell osmotic pressure level by tolerated without modified cells the 300% of top level under conditions of survive.One In a little embodiments, cell osmotic pressure level by tolerated without modified cells the 400% of top level under conditions of deposit Live.In some embodiments, with same cell type without compared with modified cells, cell proliferation rate increases at least 5 Again, at least 10 times, at least 20 times, at least 25 times or at least 30 times.In some embodiments, with same cell type without Modified cells is compared, and the volume of cell increases at least 2 times.In some embodiments, cell tolerance concentration be lethal and/or The material without modified cells propagation of suppression same cell type.In some embodiments, described material is fermentable sugars, Described concentration is at least 80g/l, at least 100g/l, at least 150g/l, at least 200g/l, at least 300g/l.Some embodiment party In case, with same cell type without compared with modified cells, the fatty acid of cell or the synthetic ratio of triacylglycerol increase to Few 5 times or at least 10 times.In some embodiments, cell is with at least about 20g/g, at least about 25g/g or at least about 30g/g Carbohydrate source is converted into fatty acid or triacylglycerol by conversion ratio.In some embodiments, cell be prokaryotic cell or Eukaryotic cell.In some embodiments, cell is bacterial cell, alga cells, fungal cell or yeast cells.Real at some Executing in scheme, cell is oleaginous yeast cell.In some embodiments, cell is yarrowia lipolytica cell.

Some aspects of the present invention relate to culture, its oil-producing cell comprising separation and carbohydrate source, described product Oil cell comprise raising selected from hemoglobin, cytochrome, GLUT, malate dehydrogenase, ACC, SCD, FAA1, ACS, ACS2, FAT1, The one or more of genes of FAT2, PCS60, ACLY, FAS, acyl-CoA synthetase, pyruvate carboxylase and AMPK gene The genetic modification of expression, and/or reduce the genetic modification of the expression of JNK2 and/or Δ-12 delta 8 desaturase genes product.One In a little embodiments, the oil-producing cell of separation is engineered microorganism provided in this article.In some embodiments, described carbon Hydrate source is fermentable sugars.In some embodiments, described carbohydrate source is monosaccharide (monomeric sugar).In some embodiments, described carbohydrate source is glucose and glycerol.In some embodiments, described Carbohydrate source sterilized.In some embodiments, under described culture is maintained at non-sterilising conditions.Implement at some In scheme, described culture does not comprise the oil-producing cell selectively antibiotic separated or antiproliferative.Implement at some In scheme, described carbohydrate source is from plant or algal biomass.In some embodiments, described carbohydrate source From cellulose, hemicellulose, starch, glycerol or derivatives thereof.In some embodiments, described culture also comprises fibre Dimension element or hydrolysis of hemicellulose enzyme.In some embodiments, biomass or cellulose or hemicellulose at hot water or diluted acid or Pretreatment in the process of ammonia filament expansion, utilizes hydrolytic enzyme pretreatment, steam pre-treatment and/or Calx preconditioning.Implement at some In scheme, it is to the most modified wild type, identical not with the oil-producing cell type separated that described culture comprises concentration The material of modified cell-lethal.In some embodiments, described material is (such as acetic acid class at pretreatment carbohydrate source (acetate), furfural or aromatic compounds) period produce noxious substance.In some embodiments, described material is carbon water Compound source.In some embodiments, described material is fermentable sugars.In some embodiments, described material is monosaccharide. In some embodiments, described culture comprises concentration is at least 80g/l, at least 100g/l, at least 150g/l, at least The fermentable sugars of 200g/l, at least 250g/l or at least 300g/l.

Some aspects of the present invention relate to a kind of method, and it includes making carbohydrate source and connects with the oil-producing cell separated Touch, and under conditions of being suitable to make cell at least in part described carbohydrate source are converted into fatty acid or triacylglycerol Hatch and the carbohydrate source of described cells contacting, described cell comprise raising selected from hemoglobin, cytochrome, GLUT, Malate dehydrogenase, ACC, SCD, FAA1, ACS, ACS2, FAT1, FAT2, PCS60, ACLY, FAS, acyl-CoA synthetase, acetone The genetic modification of expression of the one or more of genes of acid carboxylase and AMPK gene outcome, and/or reduce JNK2 and/or The genetic modification that Δ-12 delta 8 desaturase genes is expressed.In some embodiments, the oil-producing cell of described separation is to be carried herein The engineered microorganism of confession.In some embodiments, described carbohydrate source be sugar (such as glucose, xylose etc.) or from Plant or the starch of algal biomass.In some embodiments, described carbohydrate source is from cellulose or hemicellulose. In some embodiments, in the presence of cellulose or hydrolysis of hemicellulose enzyme, make described carbohydrate source with described carefully Born of the same parents contact.In some embodiments, at 55 DEG C in the presence of every gram of biomass about 15IU cellulose or half fiber hydrolase, Make described carbohydrate source and described cells contacting 48 hours.In some embodiments, fine by hot water or dilute acid or ammonia Tie up expansion process and/or hydrolytic enzyme preprocessing biomass or cellulose or hemicellulose.In some embodiments, with separate The carbohydrate source of oil-producing cells contacting comprise concentration pair identical with the described oil-producing cell type separated without repairing Decorations cell is the material of lethasl concentration.In some embodiments, described material is in pretreatment carbohydrate source (such as second Acids) period produce noxious substance.In some embodiments, described material is described carbohydrate source.Real at some Executing in scheme, carbohydrate source is fermentable sugars, with oil-producing cells contacting after the concentration of described fermentable sugars be at least 80g/l, at least 100g/l, at least 200g/l or at least 300g/l.In some embodiments, under non-sterilising conditions, carbon water is made Compound source and the oil-producing cells contacting separated.In some embodiments, hatch under non-sterilizing condition and the oil-producing separated The carbohydrate source of cells contacting.In some embodiments, hatch in open reactor and connect with the oil-producing cell separated The carbohydrate source touched.In some embodiments, in fed batch process (fed batch process), carbon aquation is made Compound source and the oil-producing cells contacting separated also hatch described carbohydrate source is converted into fatty acid or triacylglycerol.? In some embodiments, continuous process makes carbohydrate source and the oil-producing cells contacting separated hatching with by described carbon Hydrate source is converted into fatty acid or triacylglycerol.In some embodiments, by solvent extraction from the oil-producing separated The carbohydrate source extraction fatty acid of cells contacting or triacylglycerol.In some embodiments, described solvent extraction is molten Agent hexane extracts.In some embodiments, by fatty acid or triacylglycerol from the carbon aquation with the oil-producing cells contacting separated Compound source separates, then by ester exchange reaction (transesterification) refine.

Some aspects of the present invention relate to a kind of method, it include by improve selected from hemoglobin, cytochrome, GLUT, malate dehydrogenase, ACC, SCD, FAA1, ACS, ACS2, FAT1, FAT2, PCS60, ACLY, FAS and AMPK gene outcome The expression of one or more of gene outcomes, and/or the expression reducing JNK2 and/or Δ-12 delta 8 desaturase genes changes fat Fat acid spectrum, triacylglycerol spectrum, fatty acid synthetic ratio (fatty acid synthesis rate), triacylglycerol synthetic ratio, cell The degree of middle derivative of fatty acid accumulation, secretion rate (the rate of fatty acid derivative of derivative of fatty acid Secretion) speed that, carbohydrate converts to fatty acid or derivative of fatty acid, carbohydrate are to fatty acid or fat Effective productivity that pipecolic acid derivative converts, tolerance, multiplication rate, cell volume or cell to osmotic pressure are to for carbon hydrate The tolerance of thing source noxious substance in the conversion of fatty acid or triacylglycerol.In some embodiments, described fat is changed In acid spectrum, described triacylglycerol spectrum, described fatty acid synthetic ratio, described triacylglycerol synthetic ratio, cell, derivative of fatty acid amasss The derivative of fatty acid secretion rate of tired degree or cell is to increase fatty acid, the fatty acid being synthesized by cell, accumulate or being secreted and spreads out Biology and/or the amount of triacylglycerol.In some embodiments, the carbohydrate changing cell spreads out to fatty acid or fatty acid The efficiency of bioconversion is that transformation efficiency increases at least 2 times, at least 3 times, at least 4 times or at least 5 times.Implement at some In scheme, derivative of fatty acid is triacylglycerol.In some embodiments, cell is changed to the tolerance of osmotic pressure or to poisonous The tolerance of material is to confer to resistance to without the osmotic pressure that modified cells is mortality levels or noxious substance to same cell type It is subject to.In some embodiments, change multiplication rate be described multiplication rate is increased at least 2 times, at least 5 times, at least 10 Again, at least 20 times or at least 30 times.In some embodiments, changing described cell volume is to be increased to by described cell volume At least 2 times.In some embodiments, described cell is yeast cells.In some embodiments, described yeast is oil-producing ferment Female.In some embodiments, described oleaginous yeast is Yarrowia lipolytica.

Some aspects of the present invention relate to the nucleic acid molecules separated, and it comprises: a) coding SEQ ID NO:1 (solves fat Ye Shi Yeast SCD) nucleotide sequence, or nucleotides sequence b) and a) shows the nucleotide sequence of homogeneity of at least 85%.One In a little embodiments, the nucleotide sequence of coding SEQ ID NO:1 is SEQ ID NO:2.In some embodiments, nucleotide The nucleotides sequence of sequence and SEQ ID NO:2 shows the homogeneity of at least 85%.In some embodiments, nucleotide sequence with The nucleotides sequence of SEQ ID NO:2 shows the homogeneity of at least 90%.In some embodiments, nucleotide sequence and SEQ ID The nucleotides sequence of NO:2 shows the homogeneity of at least 95%.In some embodiments, nucleotide sequence is with SEQ ID NO:2's Nucleotides sequence shows the homogeneity of at least 97.5%.In some embodiments, nucleotide sequence and the nucleoside of SEQ ID NO:2 Acid sequence has the homogeneity of at least 99%.In some embodiments, it is provided that nucleic acid construct, it comprises as described herein The nucleic acid molecules (nucleic acid molecules of separation the most described in this paragraph) separated and the promoter of the separation of allos.Some embodiment party In case, promoter is constitutive promoter or inducible promoter.In some embodiments, described constitutive promoter is to turn over Translate elongation factor (Translation Elongation Factor, TEF) promoter.In some embodiments, described induction Type promoter is drug induced promoter.In some embodiments, the nucleic acid molecules of separation comprises modified SCD startup Son.In some embodiments, modification is the wild type SCD promoter sequence that lacks wholly or in part and/or suddenly change, and causes correspondence Answer the destruction of the feedback suppression of the described SCD promoter of, derivative of fatty acid sour in high levels of fat and/or triacylglycerol.? In some embodiments, described modification is heterologous sequence to insert wild type SCD promoter region, optionally in combination with wholly or in part Disappearance and/or sudden change wild type SCD promoter sequence, cause responding to high levels of fat acid, derivative of fatty acid and/or three The destruction of the feedback suppression of the described SCD promoter of acyl glycerol.

Some aspects of the present invention relate to carrier, and it comprises expression cassette, any expression cassette the most mentioned herein.This Some aspects of invention relate at least one of cell comprising expression cassette as described herein or carrier as described herein.

In some cases, the theme of the application can relate to associated products, the alternative solution of particular problem and/ Or individual system or the multiple different purposes of goods.

The further advantage of the present invention, feature and purposes are by by the described below of non-limiting embodiments of the present invention and tie Close accompanying drawing and become obvious.In the case of this specification comprises conflicting disclosure with the file being incorporated by reference into, Should be as the criterion with this specification.

Accompanying drawing explanation

The fatty acid profile of Fig. 1: Yarrowia lipolytica (Yarrowia lipolytica).A) in shake flat experiment, gas is used Phase chromatograph-mass spectrometer coupling (GC-MS) measures total trip of the Yarrowia lipolytica logarithmic (log) phase culture being incubated in minimal medium From fatty acid (free fatty acid, FFA).B) during stable trophophase, measure under the same conditions in identical culture Total FFA.C) during stable phase, the TL (FFA and esterified fatty acid) in identical culture is measured.

Fig. 2: analyze the TL of Yarrowia lipolytica.A) in minimal medium, wildtyp Y. lipolytica bacterium is cultivated Strain is until 72 hours stable phase cultures, and uses GC-MS to measure TL in shake flat experiment.B) cultivation is measured to stable phase (72 hours) and half constitutive promoter control under process LAN SCD (natural Δ 9 desaturase) mutant strain in total fat Matter.C) with Nile red to cultivating the confocal microscopy dyeed to the wild-type strain of stable phase.D) Nile red is used The mutant strain cultivated to stable phase is dyeed and is analyzed with Laser Scanning Confocal Microscope.

Fig. 3: in shaking flask, Yarrowia lipolytica mutant-1 (overexpressing cell pigment B, hemoglobin, Glut1 and Δ 9-desaturase (SCD), (D9, ■)) and wild type (LS, ◆) glucose consumption to pure glucose.With wild type solution fat Family name's Yeast Phase ratio, Yarrowia lipolytica mutant-1 show faster glucose consumption characteristic and with wild type in observed by arrive Not exclusively consume the complete glucose consumption compared.

72 hour in Fig. 4: A), Yarrowia lipolytica mutant 1 (overexpressing cell in corn straw hydrolyzate (Hz) Pigment B, hemoglobin, Glut1 and Δ 9-desaturase (SCD)) and mutant 2 (overexpressing cell pigment B, hemoglobin and Glut1) sugar consumption.B) within a few hours, the generation of the oil of mutant 1 and mutant 2 in corn straw Hz.

Fig. 5: compare wild type and the growth characteristics of engineered microorganism.YL-eng: process LAN Δ 9-desaturase (SCD) Sudden change Yarrowia lipolytica.YL-wild: wildtyp Y. lipolytica.In the basic training containing the sugar that concentration is 250g/l Support and base is cultivated cell.Wild-type cell can not grow under these conditions, and mutant cell is resistant to high-caliber sugar also Well-grown, shows to obtain higher bio-fuel in the method using mutant strain or bio-fuel precursor produces Rate.Y-axis: OD value.X-axis: with the time hour represented.

Fig. 6: the sugar consumption of the Yarrowia lipolytica mutant of process LAN Δ 9-desaturase (SCD) and growth characteristics.? Culture medium containing 160g/l sugar is cultivated cell, the OD of monitoring culture and sugar consumption.Mutant cell was at 48 hours internal consumptions The sugar supplied, and after in batches supplementing sugar continued growth.This illustrates for fed-batch operation and semicontinuous biology One embodiment of biofuel production process.

Fig. 7: engineered Yarrowia lipolytica (process LAN Δ 9-desaturase (SCD), CYB and hemoglobin) Lipid produces.

Fig. 8: after cultivating 72 hours, mutant strain (process LAN Δ 9-desaturase (SCD), CYB and blood red egg In vain；The often post in left side in group) and the fatty acid profile of wildtyp Y. lipolytica bacterial strain (the often post on right side in group).

Fig. 9: the growth kinetics of the different sudden change Yarrowia lipolytica strains compared with wildtyp Y. lipolytica. CB: CYB process LAN body；D9:SCD process LAN body.

Figure 10: under different glucose levels, the different sudden changes compared with wildtyp Y. lipolytica solve fat Ye Shi ferment The growth kinetics of mother strains.Wild: wildtyp Y. lipolytica；C18: the sudden change of process LAN Δ 9-desaturase (SCD) Yarrowia lipolytica.

Figure 11: sudden change (process LAN Δ 9-desaturase (SCD)) and the growth of wildtyp Y. lipolytica and lipid are raw Produce kinetics.

Figure 12: pYLEX1, the expression vector (A) of transgene expression in Yarrowia lipolytica.For people in the art This carrier well known to Yuan can comprise selected marker or the deficiency URA3 labelling from Yarrowia lipolytica URA3 gene, its Allow complementary uracil auxotrophy, as by LE DALL etc., Curr.Genet., the URA3d described in 26,38-44 (1994) Labelling.Sequence such as activated promoter and terminator sequence in Ye Shi yeast for regulating and expressing.Some embodiment party In case, this carrier comprises induction type or constitutive promoter.In some embodiments, can be from pYLEX1 mistake in microorganism Expressing gene, such as, by being cloned into pYLEX1 by purpose construct (the SCD cDNA as under the control of promoter).Display CYB under TEF promoter controls and the exemplary clones (B, C) of hemoglobin cDNA.

Figure 13: cultivate engineered microorganism in algal biomass.Obtain the algae being dried and go forward side by side horizontal high voltage process to split Solve cell and make starch gelatinization.By α-amylase enzymatic treatment through the cell of HIGH PRESSURE TREATMENT to discharge glucose.With our contain There is the mutant yeast cell inoculation gained culture medium of Δ 9-desaturase and cytochrome, Glut1 and hemoglobin.Figure shows Go out Ye Shi yeast mutants Johnson & Johnson head in the fermentation medium not having any additive.Cell reached in 4-5 days OD43.This shows that the growth of mutant yeast is not suppressed.

The microscope of Figure 14: the yeast cells being incubated in algae hydrolyzate is observed.Cultivate under the conditions of described in Figure 13 Cell.Harvesting also dyes with Nile red to identify oil.Microdroplet in yeast cells represents oil.

The microscope of Figure 15: the yeast cells being incubated in crude glycerol is observed.Harvesting and with Nile red dyeing with Identify oil.Microdroplet in yeast cells represents oil.

Figure 16: for producing comprising Δ 12-delta 8 desaturase genes flank region and resisting of Δ 12-desaturase knock-out bacterial strain The Δ 12-desaturase of raw element resistance sequence knocks out the schematic construction of construct.

Figure 17: engineered microorganism growth in 3% acetic acid class (84 little add 2% glycerol constantly).

Describe in detail

Introduce

In view of the minimizing of fossil fuel resource, numerous studies work is devoted to develop reproducible succedaneum.Before one has The method of scape is that transformation microorganism is to produce bio-fuel (such as biodiesel or biodiesel precursor such as three from renewable carbon source Acyl glycerol), such as by using generation fatty acid or the microorganism Microalgae as a raw of derivative of fatty acid Material for biofuels production (Gouveia L, Oliveira Δ C.J Ind Microbiol Biotechnol.2009Feb；36 (2): 269-74).Although some aspects of this invention are directed to use with photosynthetic microorganism (such as algae Class) produce bio-fuel or bio-fuel precursor, but the use of photosynthetic microorganism creates a series of technological challenge (Cadoret JP, Bernard O.J Lipid biofuel production with microalgae:potential and challenges Soc Biol.2008；202 (3): 201-11).The focus of research work turns to transformation microorganism to send out dark During ferment by renewable carbon source (fermentable sugars of Tathagata authigenic material (such as glucose or from Semen Maydis or the sugar of Caulis Sacchari sinensis) or Person's not fermentable carbohydrate polymer (such as cellulose or hemicellulose)) be converted into bio-fuel or bio-fuel before Body.

Produce bio-fuel needs: (i) identifies suitable microorganism viable economicallyly, and (ii) transforms in microorganism Going out to need and/or desired phenotype, this phenotype can comprise multifrequency nature.This type of in this phenotype needs and/or desired spy Property example include but not limited to that rapidly and effectively biomass produce, beyond the growth vigor of undesirably microorganism, effectively, Ideally close to carbohydrate theoretically to the conversion ratio of oil, with to substrate and the high tolerance of end-product.In these characteristics Some are the prerequisite producing bio-fuel based on microorganism the most viable economicallyly.It is desirable that it is engineered Microorganism should show the combination of advantageous feature, and it gives and allows by can be by abundant in the way of scale, cost savings Carbon source is efficiently converted into bio-fuel or the phenotype of bio-fuel precursor.Bio-fuel or the micro-organisms of bio-fuel precursor

Some aspects of the present invention relate to bio-fuel or the production of bio-fuel precursor of microbe-mediated.Term is " raw Thing fuel " refer to the fuel of biological origin (such as living cells, microorganism, fungus or plant).This term includes the most direct The fuel (such as by traditional extraction, distillation or method of refining) that obtains from biogenetic derivation and by processing to obtain biological origin The fuel (such as by chemical modification such as ester exchange method) that bio-fuel precursor produces.The example of the bio-fuel that can directly obtain Son is alcohol (such as ethanol, propanol and butanol), fatty and oily.The life obtained by processing bio-fuel precursor (such as, lipid) The example of thing fuel is biodiesel (such as, being produced) by the ester exchange reaction of lipid and green diesel/modified oil fires Material (such as, is produced by the hydrogenation of oil).Biodiesel is also referred to as fatty acid methyl (or second) ester, and it is the most most important One of bio-fuel, and can be produced on an industrial scale by the ester exchange reaction of lipid, in ester exchange reaction, hydrogen-oxygen Change sodium and methanol (or ethanol) to react to produce biodiesel and glycerol with lipid (such as triacylglycerol).For on an industrial scale The raw material producing biodiesel includes Animal fat, vegetable oil, Petiolus Trachycarpi oil, Fructus Cannabis, Semen sojae atricolor, Semen Brassicae campestris, Caulis et Folium Lini, Helianthi and contains Oil algae.In other methods, microorganism converts biomass into bio-fuel precursor such as lipid, and then it is extracted and goes forward side by side One step is processed to produce bio-fuel.Term " biomass " refers to grown by living cells or biology (such as microorganism) and/or bred The material produced.Biomass can include cell, microorganism and/or intracellular inclusions (such as cell fatty acid and TAG) with And extracellular material.Extracellular material includes but not limited to fatty acid or the TAG such as secreted by the compound of emiocytosis.With In the important kind of biomass producing bio-fuel be algal biomass and the biomass from plant, such as corn straw and The wood fiber.In some embodiments, the biomass being used for producing bio-fuel or bio-fuel precursor can include from planting The sugar of thing, such as from Caulis Sacchari sinensis or the sugar of Semen Maydis.

It is raw that some aspects of the present invention relate to identifying, transform and be developed for viable economically ground commercial scale biodiesel The microbial source of the lipid produced, these be not the most all reported.Term " lipid " refers to fatty acid and derivant thereof.Cause This, the example of lipid includes fatty acid (FA, saturated and unsaturation)；Glyceride or glycerol lipid, also referred to as acylglycerol Such as monoglyceride (monoacylglycerol), diglyceride (diacylglycerol), triglyceride (triacylglycerol, TAG or natural fat)；Phosphoric acid Glyceride (phosphoglyceride)；(sphingolipid, sterin fat include terpene including cholesterol and steroid hormone, pregnant amylene alcohol ester to nonglyceride Class, fatty alcohol, wax and polyketone)；And complex lipid derivant (connecting sugared lipid or glycolipid and the lipid of connection protein). Lipid is living cells and the pith of microorganism plasma membrane.Some cells and microorganism also produce lipid to store energy, such as Form with the triacylglycerol in lipid microdroplet.

Some aspects of the present invention relate to the suitable lipid metabolism according to microorganism and identify for producing bio-fuel or life The microorganism of thing fuel precursor.Term " lipid metabolism " refers to producing or the molecular process of degraded of lipid.Fatty acid closes One-tenth, fatty acid oxidation, fatty acid desaturation, TAG synthesis, TAG storage and TAG degraded are the parts as cytolipin metabolism The example of process.Therefore, term " fatty acid " metabolism " refer to fatty acid synthesis, produce, convert or degrade all Cell or organic process.Fatty acid synthesis, fatty acid oxidation, TAG synthesis and TAG degraded are as cellular fatty acid metabolism The example of the process of part.

Term " triacylglycerol " (TAG, otherwise referred to as triglyceride) refers to such molecule: its comprise by ester bond with A part glycerol that three fatty acid molecules (aliphatic monocarboxylic acid) are covalently bound, each fatty acid is connected to glycerol molecule One of three hydroxyls (OH).Due to reduction, the anhydrous nature of triacylglycerol, so it is the highly enriched storage shape of metabolisable energy Formula, and be the raw material being suitable for production of biodiesel.

Many cells and the biological form with fatty acid and derivative of fatty acid (such as TAG) store metabolisable energy.Fatty acid And derivant (such as TAG) provides the ideal form storing metabolisable energy.It is contained in the energy in C-C key and can pass through β-oxygen Changing and effectively discharge, beta oxidation is the reaction of the reverse being in form equivalent to fatty acid biological synthesis, but it is by constituting difference The different enzymes mediation of molecular pathways and regulation.Microorganism can be supplied from outside, and endogenous conversion and de novo synthesis produce fat Acid.Some aspects of the present invention relate to being effectively synthesized also depot fat acid or fatty acid according to the carbon source that microorganism is externally supplied The ability of derivant is identified for producing bio-fuel or the microorganism of bio-fuel precursor.

A kind of microorganism produced for bio-fuel

Some aspects of the present invention relate to the general on an industrial scale identified for producing bio-fuel or bio-fuel precursor The carbohydrate-modifying applicable microorganism for lipid.The most not yet identify allow the most viable economicallyly from Carbohydrate source produces bio-fuel or the microorganism of bio-fuel precursor.Some aspects of the present invention relate to according to solving fat Oleaginous yeast Yarrowia lipolytica is accredited as production bio-fuel by the favourable alkali metabolism (base metabolism) of family name's yeast Or the biology of bio-fuel precursor.

Yarrowia lipolytica is non-pathogenic oleaginous yeast, and it can utilize several kinds of carbon source to include organic acid, hydrocarbon and multiple Fatty and oily.Term " oil-producing " refers to that the microorganism that can accumulate the lipid more than 20% of its dry cell weight (sees C.Ratledge etc., Microbial routes to lipids.Biochem Soc Trans.1989Dec；17 (6): 1139- 41).According to certain aspects of the invention, Yarrowia lipolytica is the micro-life for producing bio-fuel or bio-fuel precursor Thing, because Yarrowia lipolytica is the obligate need that can assimilate carbohydrate (such as glucose) or glycerol as sole carbon source Oxygen animalcule, and compared with other yeast strain, in Yarrowia lipolytica, glucose is to fatty acid and triacylglycerol (TAG) Convert higher and lipid storage ability is higher.See for example Beopoulos A, Cescut J, Haddouche R, Uribelarrea JL, Molina-Jouve C, Nicaud JM, Yarrowia lipolytica as a model for bio-oil production.Prog Lipid Res.2009Nov；48 (6): 375-87.Additionally, Yarrowia lipolytica is to grind Study carefully one of the most deep " unconventional " yeast species, and be recently completed the gene order-checking of Yarrowia lipolytica, including MtDNA sequencing.Kerscher S, Durstewitz G, Casaregola S, Gaillardin C, Brandt U., Th e complete mitoch ondrial genome of Yarrowia lipolytica.Comp Funct Genomics.2001；2 (2): 80-90.Even if not yet completing the functional annotation of genome sequence, genomic sequence data can The most more convenient genetic manipulation of property.See, e.g. Sokolova L, Wittig I, B arth HD, Schigger H, Brutschy B, Brandt U., LILBID-mass spectrometry of protein complexes from blue- Nativegels, a sensitive top-down proteomic approach.Proteomics. discloses 2010 on the net Feb 1, PMID:20127694.

In wildtyp Y. lipolytica, start the fatty acid from carbon source and TAG synthesis at stable trophophase, show to deposit In the tight regulatory mechanism controlling lipid metabolism.This regulatory mechanism controls the amount of the lipid that can be synthesized and store, and it is notable Limit the raw material conversion yield to lipid.Therefore, the metabolizing parameters of wildtyp Y. lipolytica is unsuitable for viable economically Ground produces bio-fuel or bio-fuel precursor on an industrial scale.

A kind of key regulator of microbial fatty acid metabolic

Some aspects of the present invention relate to it was unexpectedly found that: (i) satisfied fatty acid by feedback circuit suppression fat The de novo synthesis of acid and TAG store, and (ii) (such as solves fat in the microorganism being suitable to produce bio-fuel or bio-fuel precursor Ye Shi yeast) in process LAN SCD, Δ 9-desaturase be enough to restrain fatty acid synthesis and TAG stores this feedback suppression, cause Fatty acid and/or TAG synthesis, store dramatically increase.

Some aspects of the present invention relate to it has unexpectedly been discovered that, except make fatty acid and/or the synthesis of TAG and store increase In addition, in microorganism, process LAN SCD also gives microorganism (such as, Yarrowia lipolytica) and produces bio-fuel or bio-fuel The advantageous phenotypes of precursor, it includes but not limited to: (i) TAG stores the superactivation of approach, (ii) growth vigor, and (iii) continues Oil production, (iv) to carbohydrate source material in culture medium (such as, glucose and other sugar) tolerance strengthen, (v) Fatty acid profile changes, and such as, the beneficially ratio of the saturated and unsaturated fatty acid that bio-fuel or bio-fuel precursor produce becomes Change.

The SCD of the present invention be in oleaginous microorganism fatty acid metabolism and TAG synthesis key regulator discovery for The method being intended to renewable carbon source be changed into bio-fuel or bio-fuel precursor by engineered cell is significant. Some aspects based on the present invention, it now is possible to change the fatty acid of microorganism (such as oleaginous yeast such as Yarrowia lipolytica) And/or TAG spectrum, this change gives to be managed the carbohydrate-modifying pole for bio-fuel or bio-fuel precursor on an industrial scale Think phenotype, as dramatically increased fatty acid synthesis, TAG synthesis, fatty acid and TAG, yield of biomass and strengthening in culture medium The tolerance of high concentration substrate, product and/or toxin.

According to certain aspects of the invention, the lipid in microorganism or fatty acid generation are changed according to method provided herein Thank (such as by independent process LAN SCD or with other hereditary or non-genetic modification provided in this article combine) allow produce for The microorganism that bio-fuel or bio-fuel precursor production process are optimized.Some aspects of the present invention relate to engineered micro- Fatty acid metabolism in biology, causes fatty acid and derivative of fatty acid synthesis rate and accumulation in microorganism to increase.

Natural fat acid molecule is generally of branchiess aliphatic chain or the tail of 4 to 28 carbon atoms.If aliphatic All carbon atoms of chain are all connected by C--C single bond, then fatty acid is referred to as " saturated ", if or two or more carbon Atom is connected by C-C double bond, then fatty acid is referred to as " undersaturated ".Unsaturated fatty acid membrane fluidity, cytoactive, Metabolism, control genetic transcription nuclear incident regulation in play an important role.

In yeast, fatty acid profile is mainly by C16 and C18 fatty acid (such as Palmic acid (C16), palmitoleic acid (C16), tristearin Acid (C18) and oleic acid (C18)) composition.Palmic acid is the unbranched satisfied fatty acid (carbon of the aliphatic chain with 16 carbon atoms Atom/unsaturated bond: 16.0).Stearic acid is the unbranched satisfied fatty acid (18.0) of the aliphatic chain with 18 carbon atoms. Palmitoleic acid is the monounsaturated fatty acid (16.1) of the aliphatic chain of 16 carbon atoms.Oleic acid is the aliphatic of 18 carbon atoms The monounsaturated fatty acid (18.1) of chain.In yeast, minority fatty acid species includes C14 and C26 fatty acid, and they are at protein Modification has critical function or respectively as sphingolipid and the component of GPI anchor.

The de novo synthesis of fatty acid uses macrometabolic element, acetyl group coenzyme A, ATP and NADPH, thus with depend on this Other cell processes of a little compounds has competition.Needed for NADPH is two reduction steps in fatty acid prolonging circulation, this is by fat Fat acid synthesis associates with the metabolism state of cell, cause fatty acid synthesis be limited to cell have high-energy load condition (by The acetyl group coenzyme A deposit instruction of the ATP/AMP ratio, the reducing equivalent of rising and the rising that increase) under.In fatty acid metabolism Relate to nearly all subcellular organelle, demonstrate the need for being adjusted maintaining fatty acid stable state in multiple levels.

Most of biologies (including yeast) can be from several kinds of carbon source de novo synthesis fatty acid.In initial step, by by Acetyl-CoA carboxylase (ACC；Being encoded by ACC1 and HFA1 in yeast) add CO to malonyl coenzyme A₂Carry out carboxylation acetyl Coenzyme A.Biotin is the important cofactor in this reaction, and passes through biotin: apolipoprotein ligase (in yeast by BPL1/ACC2 encodes) covalently bound with ACC apolipoprotein (apoprotein).ACC is three functional enzymes, with biotin carboxyl Carrier protein (BCCP) domain, biotin carboxylase (BC) domain and carboxyl transferase (CT) domain.Most of thin In bacterium, these domains are expressed as single polypeptide and are assembled into different complex.On the contrary, eucaryon ACC (includes that mitochondrion ACC becomes Body (Hfa1 in yeast)) on single polypeptide with these functions.The malonyl coenzyme A produced by ACC is being closed by fatty acid As the donor of two carbon in enzyme, FAS and prolongation enzymatic circulation serial reaction.

In yeast, in the synthesis of endochylema fatty acid, involved independent function shows as one or two not homopolypeptide respectively Separated domains on chain.Yeast endochylema fatty acid synthase (FAS) is by two subunit Fas1 (β subunit) and Fas2 (α subunit) The complex constituted, it is organized as six poly-α 6 β 6 complex.Fas1 is with acetyltransferase, enoyl reductase, dehydratase With malonyl palmitoyl transferase activity.Fas2 comprises acyl carrier protein, chlC4,3-ketone group synthase and phosphoric acid Pantetheine transferase active.

Yeast mitochondrial fatty acid synthesis carried out by Type II FAS system, its on not homopolypeptide with individually Enzymatic activity: Acp1, carries phosphopantetheine group prothetic group (prosthetic phosphopantetheine Group) acyl carrier protein；Cem1, beta-keto acyl base-ACP synthase；Oar1,3-oxoacyl-[acyl carrier protein] is also Protoenzyme；Htd2,3-hydroxyl acyl group-thioesters dehydratase；Etr1, enoyl-ACP reductase.The function of Ppt2 is phosphopan tetheine mercapto The connection of base ethamine: protein transferase, its catalytic phosphatase pantetheine prothetic group and apoACP.

The direct product of fatty acid de novo synthesis is satisfied fatty acid.Known in eukaryote (including yeast), saturated Fatty acid is the precursor of unsaturated fatty acid.Unsaturated fatty acid is typically passed through saturated fat by enzyme-specific (referred to as desaturase) The desaturation of C--C single bond in fat acid and produce.The regulatory mechanism that control satisfied fatty acid is converted into unsaturated fatty acid is the completeest Full understanding.In eukaryote, unsaturated fatty acid is in the core thing of membrane fluidity, cytoactive, metabolism and control genetic transcription The regulation of part is played an important role.Generally, the yeast fat acid of about 80% is monounsaturated, it is intended that they are at its fat Race's chain comprises a unsaturated bond.

A vital step in monounsaturated fatty acid biosynthesis is to draw at Δ 9 (between carbon 9 and 10) Enter the first cis-double bonds.It is catalyzed this oxidation reaction by stearyl-coenzyme A desaturase (SCD, also referred to as Δ 9-desaturase).Though So the insertion of double bond betides several different acyl CoA substrate wherein with methylene, it is preferable that the end of SCD Thing is palmityl (16.0) coenzyme A and the stearoyl being separately converted to palmitoleoyl (16.1) coenzyme A and oleoyl (18.1) coenzyme A (18.0) coenzyme A (Ntambi, J.Lipid Res., 1999,40,1549-1558).

It is Ole1 by stearyl-coenzyme A desaturase gene identification in nineteen ninety in saccharomyces cerevisiae (S.cerevisiae) (Stukey JE etc., J Biol Chem., 1990,265 (33): 20144-9).In 1994, by separating from human fat tissue The Partial cDNA of 0.76kb partly characterize human stearoyl coa desaturase gene (Li etc., Int.J.Cancer, 1994, 57,50348-352).Characterized completely at this gene in 1999, and it was found that the alternative of polyadenylation site uses and produces Two kinds of transcripts (Zhang etc., Biochem.J., 1999,340,255-264) of 3.9kb and 5.2kb.In saccharomyces cerevisiae, by Endoplasmic reticulum (ER) resident and basic Δ 9-desaturase Olel catalysis fatty acid single desaturation (Martin CE, Oh CS, Jiang Y, Regulation of long chain unsaturated fatty acid syntnesis in yeast..Biochim Biophys Acta.2007Mar；1771 (3): 271-85.Epub 2006 Jul 13).

Some aspects (at least partly) of the present invention relate to identifying saccharomyces cerevisiae in Yarrowia lipolytica as herein described The homologue SCD of Ole1.

The non-limitative example of the representative series of Yarrowia lipolytica SCD described below:

＞ gi | 0548053 | ref | XP_501496.1 | YALl0C05951p [Yarrowia lipolytica]

MVKNVDQVDLSQVDTIASGRDVNYKVKYTSGVKMSQGAYDDKGRHISEQPFTWANWHQHINWLNFILV

IALPLSSFAAAPFVSFNWKTAAFAVGYYMCTGLGITAGYHRMWAHRAYKAALPVRIILALFGGGAVEG

SIRWWASSHRVHHRWTDSNKDpYDARKGFWFSHFGNMLLVpNpKNKGRTDISDLNNDWVVRLQHKYYV

YVLVFMAIVLpTLVCGFGWGDWKGGLVYAGIMRYTFVQQVTFCVNSLAHWIGEQpFDDRRTPRDHALT

ALVTFGEGYHNFHHEFpSDYRNALIWYQYDpTKWLIWTLKQVGLAWDLQTFSQNAIEQGLVQQRQKKL

DKWRNNLNWGIPIEQLpVIEFEEFQEQAKTRDLVLISGIVHDVSAFVEHHPGGKALIMSAVGKDGTAV

FNGGVYRHSNAGHNLLATMRVSVIRGGMEVEVWKTAQNEKKDQNIVSDESGNRIHRAGLQATRVENPG

MSGMAA (SEQ ID NO:1)

＞ gi | 50548052 | ref | XM_501496.1 | Yarrowia lipolytica YALI0C05951p (YALI0C05951g)

MRNA, complete cds

ATGGTGAAAAACGTGGACCAAGTGGATCTCTCGCAGGTCGACACCATTGCCTCCGGCCGAGATGTCAA

CTACAAGGTCAAGTACACCTCCGGCGTTAAGATGAGCCAGGGCGCCTACGACGACAAGGGCCGCCACA

TTTCCGAGCAGCCCTTCACCTGGGCCAACTGGCACCAGCACATCAACTGGCTCAACTTCATTCTGGTG

ATTGCGCTGCCTCTGTCGTCCTTTGCTGCCGCTCCCTTCGTCTCCTTCAACTGGAAGACCGCCGCGTT

TGCTGTCGGCTATTACATGTGCACCGGTCTCGGTATCACCGCCGGCTACCACCGAATGTGGGCCCATC

GAGCCTACAAGGCCGCTCTGCCCGTTCGAATCATCCTTGCTCTGTTTGGAGGAGGAGCTGTCGAGGGC

TCCATCCGATGGTGGGCCTCGTCTCACCGAGTCCACCACCGATGGACCGACTCCAACAAGGACCCTTA

CGACGCCCGAAAGGGATTCTGGTTCTCCCACTTTGGCTGGATGCTGCTTGTGCCCAACCCCAAGAACA

AGGGCCGAACTGACATTTCTGACCTCAACAACGACTGGGTTGTCCGACTCCAGCACAAGTACTACGTT

TACGTTCTCGTCTTCATGGCCATTGTTCTGCCCACCCTCGTCTGTGGCTTTGGCTGGGGCGACTGGAA

GGGAGGTCTTGTCTACGCCGGTATCATGCGATACACCTTTGTGCAGCAGGTGACTTTCTGTGTCAACT

CCCTTGCCCACTGGATTGGAGAGCAGCCCTTCGACGACCGACGAACTCCCCGAGACCACGCTCTTACC

GCCCTGGTCACCTTTGGAGAGGGCTACCACAACTTCCACCACGAGTTCCCCTCGGACTACCGAAACGC

CCTCATCTGGTACCAGTACGACCCCACCAAGTGGCTCATCTGGACCCTCAAGCAGGTTGGTCTCGCCT

GGGACCTCCAGACCTTCTCCCAGAACGCCATCGAGCAGGGTCTCGTGCAGCAGCGACAGAAGAAGCTG

GACAAGTGGCGAAACAACCTCAACTGGGGTATCCCCATTGAGCAGCTGCCTGTCATTGAGTTTGAGGA

GTTCCAAGAGCAGGCCAAGACCCGAGATCTGGTTCTCATTTCTGGCATTGTCCACGACGTGTCTGCCT

TTGTCGAGCACCACCCTGGTGGAAAGGCCCTCATTATGAGCGCCGTCGGCAAGGACGGTACCGCTGTC

TTCAACGGAGGTGTCTACCGACACTCCAACGCTGGCCACAACCTGCTTGCCACCATGCGAGTTTCGGT

CATTCGAGGCGGCATGGAGGTTGAGGTGTGGAAGACTGCCCAGAACGAAAAGAAGGACCAGAACATTG

TCTCCGATGAGAGTGGAAACCGAATCCACCGAGCTGGTCTCCAGGCCACCCGGGTCGAGAACCCCGGT

ATGTCTGGCATGGCTGCTTAG (SEQ ID NO:2)

Stearyl-coenzyme A desaturase or SCD introduce double bond at the A9-C of its fatty acid substrate being esterified by coenzyme A. This activity influence satisfied fatty acid and the ratio of unsaturated fatty acid (such as stearic acid and oleic acid).Stearic acid is the main of SCD Substrate, but other chain length fatty acid can also be processed by SCD.Think that the stearyl-coenzyme A desaturase in human body is fat Fat generates enzyme, is not only as its key player in the biosynthesis of monounsaturated fatty acid, due also to it passes through meals The pattern (Ntambi, Lipid Res., 1999,40,1549-1558) that food and insulin are adjusted.Therefore, stearoyl-coenzyme The regulation of A desaturase the most important and its active to changes in diet, hormonal imbalance, growth course, temperature become Change, metal, alcohol, Pexoxisome proliferator and phenolic compound sensitive (Ntambi, Lipid Res., 1999,40,1549- 1558)。

Animal model is non-in the research by polyunsaturated fatty acids (PUFA) regulation stearyl-coenzyme A desaturase Chang Youyong.Such as, observe, when two groups are all fed in the thin fatty tissue with fatty Zucker rats (Jones etc.) During the meals that PUFA content is high compared with control diet, stearyl-coenzyme A desaturase mRNA decrease 75% (Jones etc., Am.J.Physiol., 1996,271, E44-49).Similar result is obtained with tissue culture system.At Mus 3T3-L1 fat In cell line, arachidonic acid, linoleic acid, linolenic acid and eicosapentaenoic acid make stearoyl-coenzyme in dose-dependent mode The expression of A desaturase reduces (Sessler etc., J.Biol.Chem., 1996,271,29854-29858).

The molecular mechanism of PUFA regulation stearyl-coenzyme A desaturase gene expression in different tissues is still understood seldom. Understanding to regulatory mechanism at present relates to PUFA and combines with the PUFA associated proteins of presumption, then, by PUFA associated proteins Combination generation Transcription inhibition with cis acting PUFA response element (SREBP) of stearyl-coenzyme A desaturase gene (Ntambi, Lipid Res., 1999,40,1549-1558；Zhang etc., Biochem.J., 2001,357,183-193).

Although, in different biologies, have studied the regulation of SCD gene catalysis activity, but the expression of SCD gene and tune Save the meaning itself to lipid metabolism and do not become the object of further investigation yet.Have pointed out SCD and affect satisfied fatty acid and unsaturation The ratio of fatty acid (such as stearic acid and oleic acid).

Some aspects of the present invention relate to it was unexpectedly found that SCD is also as (such as Yarrowia lipolytica in microorganism In) key regulator of fatty acid and TAG metabolism plays a role.Some aspects of the present invention relate to it was unexpectedly found that single Solely process LAN SCD gene outcome not only changes the ratio of the satisfied fatty acid in impacted cell and unsaturated fatty acid, and Be enough to cause significantly and unexpectedly increasing of fatty acid and/or TAG synthesis rate and/or storage.Individually operated desaturation Expression of enzymes is to give very good by the carbohydrate-modifying microorganism for lipid (such as oleaginous yeast cell) on an industrial scale Phenotype should be it has unexpectedly been discovered that effectively produce bio-fuel from renewable carbon source to by the sweat of microbe-mediated Or bio-fuel precursor has far reaching significance.The downward that fatty acid synthesizes and stores is restrained by process LAN SCD in microorganism Not only give fatty acid synthetic ratio and accumulation that microorganism increases, also overcome FA/TAG synthesis in cultivation and be limited to the steady of microorganism Regularly limit.Unexpectedly, SCD in microorganism (such as producing bio-fuel or the microorganism of bio-fuel precursor) Process LAN also give microorganism to high concentration substrate (such as fermentable sugars) and noxious substance (the such as substrate relevant to substrate The by-product of preprocessing process) tolerance.The phenotype (the most above-mentioned tolerance improves phenotype) given by SCD process LAN allows inciting somebody to action (see Figure 11 overcomes FA by SCD process LAN to the lipid of the acquisition high concentration that sugar is converted in the industrial fermentation processes of lipid Synthesis negative regulator).

According to certain aspects of the invention, operate other gene to can help to be given birth on a large scale from carbon source by fermentable Produce bio-fuel or bio-fuel precursor.Such as, impact (such as sugar) containing carbon substrate turns to the gene that fatty acid synthesizes.Therefore, Some aspects of the present invention provide operation regulation carbon and flow in or out the expression of the involved gene of lipid synthesis approach with reality The method of the improvement of existing lipid production parameter.

Some aspects of the present invention provide the lipid of the microorganism that regulation produces bio-fuel or bio-fuel precursor The method that the expression of other gene outcome of metabolism and/or activity carry out operating.Present invention operation in terms of some is intended to increase Carbohydrate to the conversion of fatty acid and/or TAG with for from carbohydrate source large-scale production lipid to operated life Thing is optimized.Some aspects of the present invention provide operation such as specific gene product process LAN, knock out, strike low, activation and/ Or suppress alone or in combination, and/or to implement with other operative combination well known by persons skilled in the art.Term " operates " Refer to genetic manipulation (process LAN of such as specific gene product, knock out, strike low, activate and/or suppress) and non-genetic manipulation is (such as The operation of culture medium, substrate, substrate pretreatment, pH, temperature, method for transformation etc.) the two.

Gene expression manipulations (referred to herein as the regulation and control of gene expression) can be natural to given gene expression The destruction of regulation or suppression, process LAN, the suppression of expression or being completely eliminated of expressing.At native gene sequence (the most natural SCD Gene order) upstream insert allogeneic promoter or in promoter deleting regulatory sequences (such as mediation SCD gene pass through saturated fat The regulation sequence of the feedback suppression of fat acid) it is the example destroying or suppressing the natural regulation expressed.The strategy that controlling gene is expressed Hereditary change (such as by recombinant technique such as gene target or viral transduction) can be included, or non-hereditary change is (the most Know that the environment causing gene expression to raise or to lower changes, or medicine or tiny RNA are such as divided by the instantaneous delivery of regulatory factor Son is instantaneous is delivered to target cell).The method of hereditary and non-hereditary change microorganism is known to those skilled in the art and retouches It is set forth in such as: J.Sambrook and D.Russell, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press；3rd edition (January 15,2001)；David C.Amberg, Daniel J.Burke；With Jeffrey N.Strathern, Methods in Yeast Genetics:A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press (April 2005)；John N.Abelson, Melvin I.Simon, Christine Guthrie and Gerald R.Fink, Guide to Yeast Genetics and Molecular Biology, Part A, Volume 194 (Methods in Enzymology Series, 194), Academic Press (March 11,2004)；Christine Guthrie and Gerald R.Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part B, the 350th volume (Methods In Enzymology, volume 350), Academic Press；1st edition (July2,2002)；Christine Guthrie and Gerald R.Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part C, Volume 351, Academic Press；1st edition (July 9,2002)；Gregory N.Stephanopoulos, Aristos A.Aristidou and Jens Nielsen, Metabolic Engineering:Principles and Methodologies, Academic Press；1st edition (October 16,1998)；And Christina Smolke, The Metabolic Pathway Engineering Handbook:Fundamentals, CRC Press；1st edition (July28,2009), they are whole It is incorporated herein by.

Term as used herein " process LAN " refer to with reference to cell (wild-type cell of such as same cell type or Same cell type but lack the cell of specific modification such as genetic modification) compare, given cell, cell type or cell state In the increase of given gene expression dose.The satisfied fatty acid of SCD gene expression in wild-type cell can be suppressed dense showing In the yarrowia lipolytica cell of degree, continuous expression SCD gene is an example of gene overexpression forcibly.

Term as used herein " knocks out " the functional of expression referred to gene outcome (such as RNA or protein) and breaks Bad.It generally by causing gene outcome (the such as mRNA from recombination with targeting construct targeting each genome district Or protein) completely inhibiting of expressing, the specific part in described targeting construct and described genome district is recombinated and lacks The part in described district and/or insert HETEROLOGOUS NUCLEOTIDE or nucleotide sequence.In diploid, such homologous recombination events leads to The most only affect in two allele.Homozygosity can by multiple strategy obtain, such as by breeding heterozygote and Screening filial generation.In diplont (such as yeast), term " knock-out bacterial strain " generally refers to non-functional allele and isozygotys Bacterial strain.

Term as used herein " strikes " to refer to the expression of part suppressor gene product (such as mRNA or protein) low.This The known several genes in field strike low strategy can be used for inhibition of gene expression (such as suppression or resource transfers is gone out lipid synthesis approach The expression of gene, ACS2, FAT1, PCS60 and/or the AMPK as in oleaginous yeast such as Yarrowia lipolytica).Such as, may be used RNA interference (RNAi) and/or micro RNA (miRNA) approach is utilized (to include that siRNA (siRNA), bob press from both sides to use RNA (shRNA), double-stranded RNA (dsRNA), miRNA and other little disturbing molecule based on nucleic acid known in the art) gene Strike low strategy.In one embodiment, (such as shRNA or shRNA-mir expresses structure to use RNAi pattern based on carrier Body) reduce (such as in oleaginous yeast cell such as yarrowia lipolytica cell) gene in cell and (such as suppress or by resource transfers Go out the gene of lipid synthesis approach, such as ACS2, FAT1, PCS60 and/or AMPK) expression.According to the present invention in terms of some point From plasmid can comprise the promoter that is effectively connected of gene with coding small RNA (such as shRNA).Implement at some In scheme, it is possible to use the plasmid vector of separation produces can the virus of infection cell (such as yeast cells or bacterial cell) Grain (such as retrovirus or phage).Exemplary viral include adenovirus, retrovirus, slow virus, adeno-associated virus, Phage and other virus known in the art and disclosed herein.

Some aspects of the present invention provide and operate in the microorganism for producing bio-fuel or bio-fuel precursor The method of stearyl-coenzyme A desaturase (SCD) activity.SCD be with the interleaving of stearic C9 and C10 of coenzyme A coupling Enter Δ 9 desaturase of double bond, as other place in this article is described in detail, this inserting step be produce desat fatty acid and The committed step of its derivant.In some embodiments, this operation is process LAN.In some embodiments, operation is passed through Make the microorganism of production bio-fuel or bio-fuel precursor and be effectively connected allogeneic promoter (such as, composing type or induction type Promoter) the expression construct comprising SCD gene outcome (such as SCD albumen) code nucleic acid contact carry out.Real at some Executing in scheme, the nucleic acid of coding SCD gene outcome comprises the coded sequence of SEQ ID NO:2.In some embodiments, SCD It is Yarrowia lipolytica SCD, such as, comprises the Yarrowia lipolytica SCD of the aminoacid sequence of SEQ ID NO:1.Implement at some In scheme, microorganism is Yarrowia lipolytica.In some embodiments, carry out in microorganism to SCD activity operation with Give on a large scale by the carbohydrate-modifying advantageous phenotypes for lipid, the lipid synthesis rate such as increased, increase Carbohydrate is to the efficiency of lipid Transfer, the lipid storage of increase and the growth rate of increase, the carbon source raising concentration Or the tolerance of the enhancing of lipid products.Stearyl-coenzyme A desaturase gene and gene outcome sequence are those skilled in the art Well known to.Visible exemplary representative under GeneID:852825 entrance in ncbi database (www.ncbi.nlm.nih.gov) Property gene and gene outcome sequence.

Some aspects of the present invention provide and operate in the microorganism for producing bio-fuel or bio-fuel precursor The method of the activity of c-Jun N-terminal Kinase 2 (JNK2).JNK2 is positioned in Cytoplasm and is catalyzed fatty acid during hunger Decompose and be used for producing energy and carbon shielding (carbonblock).JNK2 is that energy homeostasis is required and responding to the low sucrose solution of cell Flat lipase activation plays key player.See Grimard V, Massier J, Richter D, Schwudke D, Kalaidzidis Y, Fava E, Hermetter A, Thiele C., siRNA screening revealsJNK2 as an evolutionary conserved regulator of triglyceride homeostasis.JLipid Res.2008Nov；49 (11): 2427-40.Epub 2008 Jul 8.In some embodiments, eliminate or reduce production life JNK2 activity in the microorganism of thing fuel or bio-fuel precursor is low by knocking out or striking the most respectively.Some embodiment party In case, reduce JNK2 activity in the microorganism producing bio-fuel or bio-fuel precursor with increase product stability and/or Reduce decomposition product metabolism.In some embodiments, employ conditionality suppression system, and at carbohydrate source (such as Fermentable sugars) suppress JNK2 activity during little production process.In some embodiments, to JNK2 gene in microorganism The activity of product carries out operating to give on a large scale by the carbohydrate-modifying advantageous phenotypes as lipid, such as increasing Lipid synthesis rate, increase carbohydrate to the efficiency of lipid Transfer, the lipid storage of increase and increase growth speed The tolerance that rate, the carbon source raising concentration or lipid products strengthen.JNK2 gene and gene outcome sequence are people in the art Well known to member.Visible exemplary representative under GeneID:5601 entrance in ncbi database (www.ncbi.nlm.nih.gov) Property gene and gene outcome sequence.

Some aspects of the present invention provide and operate in the microorganism for producing bio-fuel or bio-fuel precursor The method of the activity of Δ-12 delta 8 desaturase genes product.It is senior chain that Δ-12 desaturase participates in the lipid Transfer containing oleic acid Lipid.In some embodiments, such as in view of the cold flow characteristic of gained bio-fuel, it is desirable to avoid the life of long-chain fatty acid Produce or minimize it to produce bio-fuel.In some embodiments, production bio-fuel or biological combustion are eliminated or reduce The activity of Δ-12 desaturase in the microorganism of material precursor, the most respectively by (such as, knocking out) or partial gene deletion completely Or strike low.In some embodiments, the Δ-12 reduced in the microorganism for producing bio-fuel or bio-fuel precursor goes Saturated enzymatic activity is to increase product stability, obtain desired TAG spectrum and/or reduce the catabolism of product in microorganism. In some embodiments, conditionality suppression system suppression Δ-12 desaturase activity is used.In some embodiments, exist In microorganism, activity to Δ-12 delta 8 desaturase genes product operates and to give by carbohydrate-modifying is on a large scale The advantageous phenotypes of lipid, the lipid synthesis rate such as increased, the carbohydrate of increase is to the efficiency of lipid Transfer, the fat of increase Matter stores, and the content of the C18 fatty acid of increase, the total fatty acids of the microorganism of increase stores up the percent of middle C18 fatty acid, institute The cold flow characteristic of the increase of the lipid, oil or the TAG that produce, the growth rate of increase, the carbon source that concentration is raised or lipid products The tolerance strengthened.Δ-12 delta 8 desaturase genes and gene outcome sequence are known to those skilled in the art.Ncbi database (www.ncbi.nlm.nih.gov) visible exemplary representative gene and gene outcome under GeneID:2909806 entrance in Sequence.

Some aspects of the present invention provide and operate in the microorganism for producing bio-fuel or bio-fuel precursor The method of the activity of hemoglobin gene product.Hemoglobin gene product (is included for some embodiments of the invention Hemoglobin gene product) general introduction, see Frey AD, Kallio PT.Bacterial hemoglobins and Flavoh emoglobins:versatile proteinsand their impactonmicrobiology and biotechnology.FEMS MicrobiolRev.2003Oct；27 (4): 525-45.In some embodiments, the most logical Cross the nucleic acid of process LAN encoding haemoglobin protein to increase (the such as hemoglobin egg of hemoglobin gene product in microorganism White matter) activity.In some embodiments, in microorganism, the process LAN of hemoglobin causes oxygen transmission in microorganism to increase. In some embodiments, the oxygen owing to increasing flows into and needs the route of synthesis (such as fatty acid synthesis pathway) of hyperoxia, so The hemoglobin activity increased causes bio-fuel or the synthesis of bio-fuel precursor to increase.In some embodiments, in micro-life In thing, the activity of operation hemoglobin gene product is to give on a large scale by the carbohydrate-modifying advantageous phenotypes as lipid, The lipid synthesis rate that such as increases, the carbohydrate of increase are to the efficiency of lipid Transfer, the lipid storage of increase and increase Growth rate, to concentration raise carbon source or lipid products strengthen tolerance.Hemoglobin gene and gene outcome sequence are Known to one of skill in the art.GeneID:7738539 (Deide in ncbi database (www.ncbi.nlm.nih.gov) 12990) visible exemplary representative gene and gene outcome sequence under entrance.

Some aspects of the present invention provide and operate in the microorganism for producing bio-fuel or bio-fuel precursor The method of the activity of cytochrome genes product, described cytochrome genes product such as cytochrome b gene product, particularly Ground is cytochrome B5 gene outcome.In some embodiments, by the nucleic acid of such as process LAN Codocyte chromoprotein Increase the activity of cytochrome genes product in microorganism (such as cytochrome protein).In some embodiments, micro- In biology, overexpressing cell pigment causes the oxygen transmission in microorganism to increase.In some embodiments, due to the oxygen stream increased Enter to need the route of synthesis (such as fatty acid synthesis pathway) of hyperoxia, thus increase cytochrome activity cause bio-fuel or The synthesis of bio-fuel precursor increases.In some embodiments, in microorganism operate cytochrome genes product activity with Give on a large scale by the carbohydrate-modifying advantageous phenotypes for lipid, such as the lipid synthesis rate increased, the carbon water of increase Compound is to the efficiency of lipid Transfer, the lipid storage of increase and the growth rate of increase, the carbon source raising concentration or fat The tolerance of the enhancing of matter product.Cytochrome genes and gene outcome sequence are known to those skilled in the art.NCBI data Visible exemplary representative gene and gene outcome sequence under GeneID:1528 entrance in storehouse (www.ncbi.nlm.nih.gov) Row.

Some aspects of the present invention provide and operate in the microorganism for producing bio-fuel or bio-fuel precursor The method of the activity of glucose transporter (GLUT) gene outcome (such as Glut 1 gene outcome).In some embodiments In, increase (the such as GLUT albumen of GLUT gene outcome in microorganism by the nucleic acid of such as process LAN coding GLUT protein Matter) activity.In some embodiments, in microorganism, the nucleic acid of process LAN coding GLUT protein causes the Portugal of microorganism Grape Sugar intake increases.In some embodiments, owing to the picked-up of glucose increases, so the GLUT activity increased causes biology Fuel or the synthesis of bio-fuel precursor increase.In some embodiments, in microorganism, operate the activity of GLUT gene outcome To give on a large scale by the carbohydrate-modifying advantageous phenotypes as lipid, such as the lipid synthesis rate increased, the carbon of increase Hydrate to the efficiency of lipid Transfer, the lipid storage of increase and the growth rate of increase, to concentration raise carbon source or The tolerance that lipid products strengthens.GLUT gene and gene outcome sequence are known to those skilled in the art.Ncbi database (www.ncbi.nlm.nih.gov) visible exemplary representative gene and gene outcome sequence under GeneID:38109 entrance in Row.

Some aspects of the present invention provide and operate in the microorganism for producing bio-fuel or bio-fuel precursor The method of the activity of pyruvate carboxylase (PC) gene outcome.In some embodiments, PC egg is encoded by such as process LAN The nucleic acid of white matter increases the activity of PC gene outcome in microorganism (such as PC protein).In some embodiments, micro- In biology, the nucleic acid of process LAN coding PC protein causes the glucose uptake of microorganism to increase.In some embodiments, by Picked-up in glucose increases, so the PC activity increased causes bio-fuel or the synthesis of bio-fuel precursor to increase.At some In embodiment, in microorganism operate PC gene outcome activity with give on a large scale by carbohydrate-modifying as lipid Advantageous phenotypes, the lipid synthesis rate such as increased, the carbohydrate of increase to the efficiency of lipid Transfer, increase lipid store up The tolerance that the growth rate deposited and increase, the carbon source raising concentration or lipid products strengthen.PC gene and gene outcome sequence It is classified as known to one of skill in the art.In ncbi database (www.ncbi.nlm.nih.gov) under GeneID:5091 entrance Visible exemplary representative gene and gene outcome sequence.

Some aspects of the present invention provide and operate in the microorganism for producing bio-fuel or bio-fuel precursor The method of the activity of malate dehydrogenase (ME) gene outcome.ME catalysis (S)-malate oxidation decarboxylation becomes acetone acid, simultaneously with two The release of carbonoxide and the conversion of NADP+ to NADPH.In some embodiments, ME protein is encoded by such as process LAN Nucleic acid increase the activity of ME gene outcome in microorganism (such as ME protein).In some embodiments, in microorganism The nucleic acid of middle process LAN coding ME protein causes NADPH level in microorganism to increase, and causes the reductivemetabolism of adequate level Thing (such as NADPH) is used for increasing fatty acid synthesis.In some embodiments, owing to NADPH level increases, so increase ME activity causes bio-fuel or the synthesis of bio-fuel precursor to increase.In some embodiments, microorganism operates ME base Because the activity of product is to give on a large scale by the carbohydrate-modifying advantageous phenotypes as lipid, the such as lipid synthesis increased Rate, increase carbohydrate to the efficiency of lipid Transfer, the lipid storage of increase and the growth rate of increase, to concentration liter The tolerance that high carbon source or lipid products strengthen.ME gene and gene outcome sequence are known to those skilled in the art.NCBI Visible exemplary representative gene and gene under GeneID:17436 entrance in data base (www.ncbi.nlm.nih.gov) Product Sequence.

Some aspects of the present invention provide in the microorganism for producing bio-fuel or bio-fuel precursor (such as In Yarrowia lipolytica) operation acetyl-CoA carboxylase (ACC) gene outcome method.ACC gene outcome mediation acetylcoenzyme A (the main C2 precursor in fatty acid synthesis) is converted into malonyl coenzyme A, and this conversion is considered as first in fatty acid synthesis Committed step and also be considered as fatty acid synthesis in rate-limiting step (see Cao Y, Yang J, Xian M, Xu X, Liu W.Increasing unsaturated fatty acid contents in Esch erich ia coli by coexpression of three different genes.Appl Microbiol Biotechnol.2010).At some In embodiment, the operation of ACC activity is ACC process LAN.In some embodiments, in microorganism, ACC process LAN increases fat Acid synthetic ratio and/or give on a large scale by carbohydrate-modifying for bio-fuel or the advantageous phenotypes of bio-fuel precursor, The lipid synthesis rate that such as increases, the carbohydrate of increase are to the efficiency of lipid Transfer, the lipid storage of increase, the life of increase Long speed, tolerance to the enhancing of material (such as carbon source, bio-fuel or bio-fuel precursor or noxious substance) concentration.ACC Gene and gene outcome sequence are known to those skilled in the art.In ncbi database (www.ncbi.nlm.nih.gov) Visible exemplary representative gene and gene outcome sequence under GeneID:855750 entrance.

Some aspects of the present invention provide and operate in the microorganism for producing bio-fuel or bio-fuel precursor The method of the activity of acyl-CoA synthetase (ACS).ACS is that catalysis has the thioesterification of the fatty acid of coenzyme A to form activation The enzyme family of intermediate (sees Lu X, Vora H, Khosla C., Ov erproduction of free fatty acids In E.coli:implications for biodiesel production Metab Eng.2008 Nov；10 (6): 333- 9).These intermediate are phospholipid, fatty acid cholesterol ester or the precursor of alcohol fatty acid ester (such as TAG).Yarrowia lipolytica comprises With two kinds of acyl-CoA synthetase predicted known to two kinds.In some embodiments of the present invention, seviparous life is being produced In thing, process LAN ACS enzyme is to give on a large scale by the carbohydrate-modifying advantageous phenotypes as lipid, the such as lipid increased Synthetic ratio, increase carbohydrate to the efficiency of lipid Transfer, the lipid storage of increase and/or secretion, increase growth speed The tolerance that rate, the carbon source raising concentration or lipid products strengthen.ACS gene and gene outcome sequence are those skilled in the art Well known to.Visible exemplary representative under GeneID:851245 entrance in ncbi database (www.ncbi.nlm.nih.gov) Property gene and gene outcome sequence.

Some aspects of the present invention provide and operate in the microorganism for producing bio-fuel or bio-fuel precursor The method of the activity of acyl-CoA synthetase 2 (ACS2), enzyme that ACS2 is in peroxisome and participate in fatty acid Degraded.In some embodiments, suppression ACS2 stops or suppression fatty acid is degraded by yeast catabolism, and one In a little embodiments, this suppression supplements the increase of FAA1 gene product activity so that the fatty acid increased is secreted into culture medium In.Yarrowia lipolytica comprises ACS2 acetyl group CoA synthase and (sees Beopoulos A, Cescut J, Haddouche R, Uribelarrea JL, Molina-Jouve C, Nicaud JM., Yarrowia lipolytica as a model for bio-oil production.Prog Lipid Res.2009Nov；48 (6): 375-87).In some embodiments, micro- Biology knocks out, strikes low and/or suppression ACS2 gene product expression or activity to give on a large scale by carbohydrate-modifying For bio-fuel or the advantageous phenotypes of bio-fuel precursor, the lipid synthesis rate such as increased, the carbohydrate of increase are to fat Matter convert efficiency, the lipid storage of increase and the growth rate of increase, to material (such as carbon source, bio-fuel or biology Fuel precursor or noxious substance) tolerance of enhancing of concentration.ACS2 gene and gene outcome sequence are those skilled in the art institutes Known.Visible exemplary representativeness under GeneID:850846 entrance in ncbi database (www.ncbi.nlm.nih.gov) Gene and gene outcome sequence.

Some aspects of the present invention provide and operate in the microorganism for producing bio-fuel or bio-fuel precursor The method of the activity of FAA1 gene outcome.The catalysis of FAA1 gene outcome has the Cytoplasm thioesterification of the long-chain fatty acid of coenzyme A To produce the intermediate of activation.Yarrowia lipolytica FAA1 is saccharomyces cerevisiae P30624FAA1 long chain fatty acid coa ligase Homologue.This enzyme participates in generation and the secretion of fatty acid that free fatty stores up.In some embodiments, it is being used for In the microorganism of production bio-fuel or bio-fuel precursor, process LAN FAA1 gene outcome is to give on a large scale by carbon aquation Compound is converted into the advantageous phenotypes of lipid, and the lipid synthesis rate that such as increases, the carbohydrate of increase are to the effect of lipid Transfer The tolerance that rate, the lipid storage of increase and the growth rate of increase, the carbon source raising concentration or lipid products strengthen.FAA1 Gene and gene outcome sequence are known to those skilled in the art.In ncbi database (www.ncbi.nlm.nih.gov) Visible exemplary representative gene and gene outcome sequence under GeneID:854495 entrance.

Some aspects of the present invention provide and operate in the microorganism for producing bio-fuel or bio-fuel precursor The very method of long-chain fatty acid CoA synthase (FAT1) activity.Think that FAT1 controls the very long-chain fat with coenzyme A The fatty acid transport of acid and thioesterification.Yarrowia lipolytica comprises FAT1 very long-chain fatty acid CoA synthase.Real at some Execute in scheme, suppress FAT1 activity to stop synthesis and/or the increasing of very length fatty acids derivant by such as genetic manipulation Add storing up of free fatty.In some embodiments, knock out in microorganism, strike low and/or suppression FAT1 gene outcome Expression or activity with give on a large scale by carbohydrate-modifying as bio-fuel or the advantageous phenotypes of bio-fuel precursor, The lipid synthesis rate that such as increases, the carbohydrate of increase are to the efficiency of lipid Transfer, the lipid storage of increase and increase Growth rate, tolerance to the enhancing of material (such as carbon source, bio-fuel or bio-fuel precursor or noxious substance) concentration. FAT1 gene and gene outcome sequence are known to those skilled in the art.Ncbi database (www.ncbi.nlm.nih.gov) Visible exemplary representative gene and gene outcome sequence under middle GeneID:852329 entrance.

Some aspects of the present invention provide the method for operation PCS60, and PCS60 is also referred to as FAT2, AMP associated proteins acyl group CoA synthase or peroxisome CoA synthase, it is the peroxisome acyl having and not determining substrate specificity Base CoA synthase.Yarrowia lipolytica comprises saccharomyces cerevisiae PCS 60 homologue.Suppression PCS60 will stop very long fat The synthesis of acid derivative also increases storing up of free fatty.In some embodiments of the present invention, knock out in microorganism, Strike the low and/or expression of suppression PCS60 gene outcome or activity to fire carbohydrate-modifying for biological on a large scale to give Material or the advantageous phenotypes of bio-fuel precursor, the lipid synthesis rate such as increased, the carbohydrate of increase are to lipid Transfer Efficiency, the lipid storage of increase and the growth rate of increase, to material (such as carbon source, bio-fuel or bio-fuel precursor Or noxious substance) tolerance of enhancing of concentration.FAT2 gene and gene outcome sequence are known to those skilled in the art.NCBI Visible exemplary representative gene and base under GeneID:852523 entrance in data base (www.ncbi.nlm.nih.gov) Because of Product Sequence.

Some aspects of the present invention provide for large-scale production bio-fuel or the microorganism of bio-fuel precursor The method of process LAN ATP citrate-lyase (ACLY) in (such as Yarrowia lipolytica).Be suitable to produce on an industrial scale biology Some microorganisms of fuel or bio-fuel precursor generally produce a large amount of citric acid at (including Yarrowia lipolytica).ACLY mediates lemon Lemon acid is converted into coenzyme A, according to certain aspects of the invention, this reaction can by ACLY express promote (see Holz M,A, Mauersberger S, Barth G, Aconitase overexpression changes theproduct ratio of citric acidproduction by Yarrowia lipolytica.Appl Microbiol Biotechnol.2009Jan；81 (6): 1087-96).In some embodiments, ACLY process LAN reduces less desirable Fructus Citri Limoniae Acid produces and/or provides other S-acetyl-coenzyme-A originated for synthesising biological fuel or bio-fuel precursor.Some embodiment party In case, suppression citric acid in the microorganism (including Yarrowia lipolytica) for producing bio-fuel or bio-fuel precursor Excess produces.In some embodiments, in microorganism, (such as in Yarrowia lipolytica), ACLY process LAN increases fatty acid conjunction One-tenth rate and/or give on a large scale by carbohydrate-modifying for bio-fuel or the advantageous phenotypes of bio-fuel precursor, such as Increase lipid synthesis rate, increase carbohydrate to the efficiency of lipid Transfer, the lipid storage of increase and the life of increase Long speed, tolerance to the enhancing of material (such as carbon source, bio-fuel or bio-fuel precursor or noxious substance) concentration.Also join See Lasserre JP, Nicaud JM, Pagot Y, Joubert-Caron R, Caron M, Hardouin J.Talanta.First complexomic study of alkane-binding protein complexes in the yeast Yarrowia lipolytica. 2010 Feb 15；80 (4): 1576-85.ACLY gene and gene outcome sequence are Known to one of skill in the art.Can under GeneID:108728 entrance in ncbi database (www.ncbi.nlm.nih.gov) See exemplary representative gene and gene outcome sequence.

The method that some aspects of the present invention provide process LAN fatty acid synthase complex (FAS).Although ACC is probably Rate-limiting enzyme in fatty acid synthesis, but also have pointed out other step and this approach is controlled, most notably FAS (see Schweizer E,H, Regler R, Rottner G.J, Genetic control of Yarrowia lipolytica fatty acid synthetase biosynthesis and function.Basic Microbiol.1988；28 (5): 283-92).This complex is the strongest (most of substrate in whole lipid synthesis approach Substrate-intensive) multifunctional polypeptides of fatty acid chain is extended during.In some embodiments, microorganism In (such as in Yarrowia lipolytica) ACLY process LAN add fatty acid synthetic ratio and/or give on a large scale by carbon hydrate Thing is converted into bio-fuel or the advantageous phenotypes of bio-fuel precursor, the lipid synthesis rate that such as increases, the carbon hydrate of increase Thing to the efficiency of lipid Transfer, the lipid storage of increase and/or secretion, the growth rate of increase, to material (such as carbon source, life Thing fuel or bio-fuel precursor or noxious substance) tolerance that strengthens of concentration.FAS gene and gene outcome sequence are this area skill Well known to art personnel.GeneID:853653 and GeneID:855845 in ncbi database (www.ncbi.nlm.nih.gov) Visible exemplary representative gene and gene outcome sequence under entrance.

Some aspects of the present invention provide the method for suppression AMP activated protein kinase (AMPK).AMPK is by response In cell AMP:ADP ratio by phosphorylation regulate other oroteins activity regulatory enzyme (see Lee-Young RS, Palmer MJ, Linden KC, LePlastrier K, Canny BJ, Hargreaves M, Wadley GD, Kemp BE, McConell GK.Carbohydrate ingestion does not alter skeletal muscle AMPK signaling during exercise in humans.Am J Physiol Endocrinol Metab.2006Sep；291 (3): E566-73).In yeast, AMPK shows targeting ACC and INO 1, needed for INO1 is lipids, biological synthesis step in early days Gene.In AMPK knocks out mutant, lack ACC phosphorylation cause the ACC of superactivation and the excess generation of fatty acid.One In a little embodiments, AMPK is suppressed to cause the superactivation of lipid synthesis in microorganism.In some embodiments, by such as Knock out the AMPK activity that AMPK gene is completely eliminated in microorganism.In some embodiments, by the most hereditary or non-heredity The activity of AMPK in operation suppression microorganism.The suppression (different from being completely eliminated) of AMPK activity can be avoided being regulated by AMPK The negative interaction of other cell processes.In some embodiments, knock out in microorganism (such as Yarrowia lipolytica), strike low And/or the suppression expression of AMPK gene outcome or activity with give on a large scale by carbohydrate-modifying as bio-fuel or life The advantageous phenotypes of thing fuel precursor, the lipid synthesis rate such as increased, the carbohydrate of increase are to the efficiency of lipid Transfer, increasing The lipid storage added and/or secretion, the growth rate of increase, to material (such as carbon source, bio-fuel or bio-fuel precursor or have Poisonous substance matter) concentration strengthen tolerance.AMPK gene and gene outcome sequence are known to those skilled in the art.Ncbi database (www.ncbi.nlm.nih.gov) in, under GeneID:100145903 entrance, visible exemplary representative gene and gene produce Thing sequence.

The nucleic acid separated

Some aspects of the present invention provide to give into microorganism (such as Yarrowia lipolytica) and produce bio-fuel or life The needs of thing fuel precursor and/or the code nucleic acid of the gene outcome of expectation phenotype.In some embodiments, this nucleic acid comes From the nucleic acid of Yarrowia lipolytica.In some embodiments, this nucleic acid coding desaturase, such as Δ 9 desaturase.One In a little embodiments, this nucleic acid coding Yarrowia lipolytica Δ 9 desaturase.In some embodiments, this nucleic acid comprises SEQ ID NO:1.In some embodiments, this nucleic acid is SEQ ID NO:1.In some versions, this nucleic acid coding gene outcome, Such as by the protein of SEQ ID NO:1 coding.

Some aspects of the present invention provide gene outcome (such as protein), and it is that microorganism (such as solves fat Ye Shi ferment Female) give production bio-fuel or the needs of bio-fuel precursor and/or expectation phenotype.In some embodiments, this albumen Matter is the protein from Yarrowia lipolytica.In some embodiments, this protein is desaturase, and such as Δ 9 goes to satisfy And enzyme.In some embodiments, this protein is Yarrowia lipolytica Δ 9 desaturase.In some embodiments, this egg The aminoacid sequence of white matter is provided in the aminoacid sequence in SEQ ID NO:2.

Term " nucleic acid " refers to comprise the molecule of the nucleotide of multiple connection." nucleic acid " and " nucleic acid molecules " exchanges and uses also And mean oligoribonucleotide and oligodeoxyribonucleotide.This term is also contemplated by many nucleoside (i.e. polynucleotide removing phosphoric acid) With other organic base any comprising nucleic acid.Organic base includes adenine,uracil,guanine, thymus pyrimidine, cytosine and flesh Glycosides.Nucleic acid can be strand or double-strand.Nucleic acid can be natural or non-natural.Nucleic acid can obtain from natural origin or Nucleic acid synthesizer can be used to synthesize (i.e. synthesizing).The separation and the suitably method that carry out nucleic acid routinely in the art can See standard molecular biology textbook.(see, e.g. Maniatis ' Handbook of Molecular Biology.) such as this Literary composition is described, and nucleic acid can be DNA or RNA, such as genomic DNA, mitochondrial DNA, mRNA, cDNA, rRNA, miRNA, PNA or LNA Or a combination thereof.It is used as unnatural nucleic acids such as bacterial artificial chromosome (BAC) and yeast according to certain aspects of the invention Artificial chromosome (YAC).

Some aspects of the present invention are directed to use with nucleic acid derivative.As will be described herein, some nucleic acid is used to spread out Biology can increase its stability by preventing the digestion of nucleic acid of the present invention, particularly may comprise nucleic acid when they are exposed to During the biological specimen of enzyme.Nucleic acid derivative used herein is unnatural nucleic acids or its unit.Nucleic acid derivative can wrap Connect containing non-natural element such as non-natural nucleotides and non-natural backbone.Permissible according to present invention nucleic acid derivative in terms of some Comprise backbone modification such as but be not limited to thiophosphate connect, di-phosphate ester modify nucleic acid, di-phosphate ester and sulfur for phosphorus The combination of acid esters nucleic acid, methyl orthophosphoric acid, alkylphosphonate, phosphate ester, D2EHDTPA Arrcostab, phosphoramidate, carbamic acid Ester, carbonic ester, phosphotriester, acetamide ester (acetamidate), carboxymethyl ester, D2EHDTPA methyl ester, phosphorodithioate, To ethyoxyl and combinations thereof.The skeleton composition of nucleic acid can be homology or allos.

Nucleic acid derivative according to certain aspects of the invention can comprise the replacement in sugar and/or alkali or modification.Such as, Some nucleic acid derivatives can include the nucleic acid with skeleton sugar, and skeleton sugar is covalently bound in addition to the hydroxyl at 3 ' positions and removes 5 ' Low-molecular-weight organic group (such as 2 '-O-alkylated ribose group) outside phosphate at Wei.Nucleic acid derivative can include Non-ribose sugar arabinose.Nucleic acid derivative can comprise substituted purine and pyrimidine such as C-5 propine modified base, 5-methyl born of the same parents Pyrimidine, 2-aminopurine, 2-amido-6-chloropurine, 2,6-diaminopurine, hypoxanthine, 2-deracil and the different born of the same parents of vacation are phonetic Pyridine.

In some embodiments, nucleic acid can include peptide nucleic acid(PNA) (PNA), lock nucleic acid (LNA), DNA, RNA or above-mentioned Combined nucleic acid (co-nucleic acid) is such as DNA-LNA combined nucleic acid.

The nucleic acid that term as used herein " nucleic acid molecules of separation " refers to not in its natural surroundings, such as, (i) leads to Cross means known in the art to extract from cell or microorganism (such as antibacterial or yeast) and or the nucleic acid of/purification, described ability Known method such as alkaline bleach liquor cleavage host cell in territory is the most such as by silicon oxide purification via adsorption-based process nucleic acid；(ii) by the most poly- The nucleic acid of synthase chain reaction (PCR) amplification in vitro；(iii) produced by clone (such as nucleic acid clone being entered expression vector) restructuring Nucleic acid；(iv) digested by such as vitro enzyme or pass through to shear fragmentation and the nucleic acid of size separation that then glue separates；Or The nucleic acid that person (v) is synthesized by such as chemosynthesis.In some embodiments, by recombinant DNA skill well known in the art The nucleic acid of the easily operated separation of art.It is therefore contemplated that be cloned into the nucleic acid of carrier or be delivered to host cell and be integrated into host's base Because the nucleic acid of group is to separate, but under the native state in its natural host nucleic acid (such as in the genome of host) is then It is not.The nucleic acid separated can be basic purification, but need not so.Such as, the core of separation in clone or expression vector Acid is not pure, its can only comprise little percentage ratio its residing for material in cell.But it is this in term as used herein Nucleic acid is to separate.

Some aspects of the present invention relate to giving and produce that the microorganism of bio-fuel or bio-fuel precursor is required or expectation The code nucleic acid of the gene outcome of phenotype, it is connected with promoter or other transcriptional activation element.In some embodiments, Encoding gene product and the nucleic acid being connected with promoter are contained in expression vector or expression construct.Term as used herein " expression vector " or " expression construct " refers to a series of permission specific nucleic acids in host microorganism (such as oleaginous yeast) The nucleic acid construct that the specific nucleic acid element transcribed recombinantly or synthetically produces.In some embodiments, expression vector can be A part for plasmid, virus or nucleic acid fragment.In some embodiments, expression vector comprises treating of being effectively connected with promoter The code nucleic acid transcribed.Promoter is to promote to treat that transcribed nucleic acid carries out the nucleic acid elements transcribed.Promoter is usually located at same chain The upstream (or 5 ') of the nucleotide sequence transcribed is controlled with it.In some embodiments, expression vector comprises and allos startup The code nucleic acid to be transcribed that son effectively connects.The startup that allogeneic promoter right and wrong are effectively connected with given nucleotide sequence natively Son.Such as, the SCD gene in Yarrowia lipolytica is effectively connected with Yarrowia lipolytica SCD gene promoter natively.Cause This, any startup being effectively connected with SCD gene or its part in addition to wildtyp Y. lipolytica SCD gene promoter Son (such as in expression construct) is allogeneic promoter.

In some embodiments, expression vector comprises the code nucleic acid effectively connecting constitutive promoter, such as, encode The nucleic acid of SCD gene outcome.Term " constitutive promoter " refers to the promoter of the gene processive transcription allowing it to be associated.? In some embodiments, expression vector comprises the code nucleic acid effectively connecting inducible promoter, and such as, coding SCD gene produces The nucleic acid of thing.Replace the term " inducible promoter " used in this article to refer to only exist with term " conditional promoter " Or when there is not biology or abiotic component, allow the promoter of its genetic transcription that is associated.Induction type well known in the art starts The example of son is that (such as tetracycline/doxycycline inducible promoter, tamoxifen induction type start drug induced promoter Son) and depend on the promoter (restructuring in the loxP site of such as crc-mediation) that recombination event activates.

As well known to those skilled in the art expression vector or expression construct are delivered in microorganism (such as yeast cells) Method.Nucleic acid (including expression vector) can be delivered to former by multiple method known to biological various equivalent modifications Core microorganism or eukaryotic microorganisms.According to certain aspects of the invention the method in delivery of nucleic acids to microorganism is included but not Being limited to different chemistry, electrochemistry and biological methods, such as, heat-shock transformed, electroporation, transfection are (the most liposome-mediated Transfection, the transfection of DEAE-Dextran mediation or calcium phosphate transfection).In some embodiments, use for transfer thing Nucleic acid construct (such as SCD expression construct) is introduced in host microorganism by supporting agent or the carrier of matter.For by hereditary material Transfer to the carrier in microorganism known to those skilled in the art, including such as plasmid, artificial chromosome and viral vector. (include comprising composing type or the expression construct of induction type allogeneic promoter, knocking out or strike low structure for building nucleic acid construct Build body) method and be people in the art for method that nucleic acid or nucleic acid construct are delivered in microorganism and carrier Well known to member, and be described in such as following in: J.Sambrook and D.Russell, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor LaboratoryPress；3rd edition (January 15,2001)； David C.Amberg, Daniel J.Burke and Jeffrey N.Strathern, Methods in Yeast Genetics: ACold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press(April 2005)；John N.Abelson, Melvin I.Simon, Christine Guthrie and Gerald R.Fink, Guide to Yeast Genetics and Molecular Biology, Part A, the 194th volume (Methods In Enzymology Series, 194), Academic Press (March 11,2004)；Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part B, Volume 350 (Methods in Enzymology, volume 350), Academic Press；1st edition (July 2,2002)； Christine Guthrie and

Gerald R, Fink, Guide to Yeast Genetics and Molecular and Cell Biology, Part C, volume 351, Academic Press；1st edition (July 9,2002)；Gregory N.Stephanopoulos, Aristos A.Aristidou and Jens Nielsen, Metabolic Engineering: Principles and Methodologies, Academic Press；1st edition (October 16,1998)；And Christina Smolke, The Metabolic Pathway Engineering Handbook:Fundamentals, CRC Press；1st edition (July 28,2009), it is all incorporated herein by.

In some embodiments, to giving needed for microorganism or the coding of gene outcome of expectation phenotype in microorganism The natural promoter (the most natural SCD promoter) of gene carries out modifying to change the regulation of its transcriptional activity.Implement at some In scheme, compared with the most modified homologue, modified promoter shows the transcriptional activity of increase.Art used herein Language " modified promoter " refers to that its nucleotide sequence is by the promoter of manual change.The example of such manual change is single Only or the nucleotide deletion of combination, insert or suddenly change.Artificial promoters's change can be carried out in the way of targeting, such as, pass through Methods of homologous recombination, such as gene target, knocks out, strikes low, rite-directed mutagenesis or the most Zinc finger nuclease-mediated strategy.Or, can With by random or semi-random event as irradiated or non-targeted nucleotide incorporation is subsequently selected carries out such change.Typically Carry out promoter to modify to regulate and control the transcription activating characteristic of each promoter.Such as, destroy or lack mediation respond to intracellular fat Sour water is put down rising and is suppressed the regulating element of SCD promoter to be activated by the processive transcription causing SCD gene, even if at intracellular fat Also it is such under conditions of the flat rising of sour water.Similarly, the transcriptional activation element that composing type activates is inserted conditional promoter District can make each gene process LAN under the conditions of normal inhibitory.Targeting destruction in microorganism as well known to those skilled in the art The method of (targeting such as causing transcription rate to increase destroys) natural promoter (the most natural SCD promoter).

In some embodiments, it is provided that nucleic acid construct is for producing bio-fuel or micro-life of bio-fuel precursor Thing knocks out Δ-12 delta 8 desaturase genes.In some embodiments, knock out construct and comprise microorganism Δ-12 desaturase The genome sequence of gene, the nucleotides sequence of its flank destroys Δ-12 desaturase when being listed in insertion Δ-12 delta 8 desaturase genes The expression of gene outcome.In some embodiments, the nucleic acid destroying Δ-12 delta 8 desaturase genes Product Expression is that antibiotic resists Property label, such as phleomycin resistant gene.In some embodiments, Δ-12 desaturase knockout carrier comprises SEQ The sequence that IDNO:28 is provided.The method being delivered in microorganism by knockout carrier is known to those skilled in the art, micro- The method carrying out homologous recombination in biology (such as in yeast) is known to those skilled in the art.The present invention is not limited to the party Face.

Microorganism remodeling method

Some aspects of the present invention relate to engineered microorganism (such as Yarrowia lipolytica) to show with extensive raw Produce the required and/or expectation phenotype of bio-fuel or bio-fuel precursor.Some aspects of the present invention relate to Metabolically engineered SCD way Footpath is to obtain for producing the microorganism that bio-fuel is optimized.Some aspects of the present invention relate to Metabolically engineered regulation carbon Flow in or out the gene of fatty acid synthesis pathway to obtain for producing the microorganism that bio-fuel is optimized.

Some aspects of the present invention provide and are greatly increased solution fat by the natural grease metabolism of regulation Yarrowia lipolytica The carbon source of family name's yeast mediation is to the method for the transformation efficiency of lipid.Some aspects of the present invention relate to such discovery: process LAN The gene (such as SClD) increasing fatty acid or triacylglycerol accumulation not only causes lipid accumulation to increase, and causes lipid synthesis Rate, lipid content and/or growth rate increase.Significantly and unexpectedly, according to certain methods provided by the present invention Lipid metabolism regulation also gives other favorable property, such as to raw material (including high concentration substrate (such as glucose)) And/or the tolerance generally polluting the noxious substance of raw material (the most pretreated raw material) strengthens.The one of such polluter A little non-limitative examples are furfural, 5 hydroxymethyl furfural and acetic acid class.The raw material thing of the noxious substance of pollution is produced after pretreatment The non-limiting examples of matter is the raw material from timber, corn straw and bagasse.

Some aspects of the present invention relate in Yarrowia lipolytica by restraining the key regulator of lipid metabolism Transcription inhibition (such as by restraining the Transcription inhibition of SCD) transforms out required and/or expectation phenotype.Also contemplate and producing life Key regulator (the example of operation lipid metabolism in other microorganisms (such as yeast, antibacterial, fungus or algae) of thing fuel As, SCD).

In order to transform the biology (such as oleaginous yeast) for producing bio-fuel on an industrial scale, it is important that manage in detail Solve and each biology regulates and controls fatty acid and the molecular mechanism of lipid metabolism.Until the present invention, the generation oil produced for bio-fuel Microorganism (such as oleaginous yeast) in fatty acid and the qualification of lipid metabolism regulatory factor and functional annotation the most unresolved.This Some aspects of invention provide qualification and the function note of the key regulator gene SCD in oleaginous yeast Yarrowia lipolytica Release.Additionally provide SCD nucleic acid and the protein molecule of separation.

Some aspects of the present invention relate to being transformed out in microorganism for producing bio-fuel or life by genetic modification The expectation phenotype of thing fuel precursor.Some aspects of the present invention relate to operation in microorganism and participate in producing bio-fuel or biology The gene of fuel precursor (such as fatty acid or triacylglycerol).Some aspects of the present invention relate to operating abreast in microorganism Participate in producing bio-fuel or multiple genes of bio-fuel precursor.

In some embodiments, the method operation individual gene provided by some aspects of the present invention transforms micro-life Thing is used for producing bio-fuel or bio-fuel precursor, and described gene such as Δ 9 desaturase (such as, SCD), GLUT are (such as, Glut1), hemoglobin, cytochrome (such as, cytochrome B5), malate dehydrogenase, ACC, ACS, ACS2, FAA1, FAT1, FAT2, ACLY, FAS, AMPK, JNK2 or Δ-12 desaturase.In some embodiments, by some aspects of the present invention The method provided operates multiple genes and transforms microorganism for producing bio-fuel or bio-fuel precursor, and described gene is such as Δ 9 desaturase (such as, SCD), GLUT (such as, Glut1), hemoglobin, cytochrome (such as, cytochrome B5), Herba Marsileae Quadrifoliae In fruit acid enzyme, ACC, ACS, ACS2, FAA1, FAT1, FAT2, ACLY, EAS, JNK2, Δ-12 desaturase and/or AMPK two Individual or more combination in any.In some embodiments, transformation microorganism with comprise increase level SCD gene outcome and Other operation (such as genetic manipulation) of other gene product expression, described other gene outcome such as GLUT is (such as, Glut1), hemoglobin, cytochrome (such as, cytochrome B5), malate dehydrogenase, ACC, ACS, ACS2, FAA1, FAT1, FAT2, ACLY, FAS, JNK2, Δ-12 desaturase or AMPK gene outcome.In some embodiments, transformation microorganism with Comprise SCD gene outcome and the hemoglobin gene product of increase level.In some embodiments, transformation microorganism is to comprise The SCD gene outcome of increase level and GLUT gene outcome (such as Glut1 gene outcome).In some embodiments, transformation Microorganism is to comprise the SCD gene outcome of increase level, GLUT gene outcome (such as Glut1 gene outcome) and hemoglobin And/or cytochrome genes product.In some embodiments, transformation microorganism is to comprise the SCD gene outcome of increase level Knock out with Glut1, hemoglobin and cytochrome b5 and optional Δ 12 desaturase.In some embodiments, micro-life Thing is Yarrowia lipolytica.

For producing the engineered microorganism of bio-fuel

Some aspects of the present invention relate to for large-scale production bio-fuel or bio-fuel precursor carried out transformation and/ Or the microorganism optimized.In some embodiments, it is provided that by the method provided by some aspects of the present invention or make The engineered microorganism operated with the nucleic acid provided by some aspects of the present invention or protein.In some embodiments In, it is provided that process LAN gives for microorganism needed for producing bio-fuel or bio-fuel precursor according to certain aspects of the invention And/or the engineered microorganism of the gene outcome of the phenotype of expectation.In some embodiments, it is provided that comprise the SCD of increase The microorganism of gene product activity.In some embodiments, microorganism shows the fatty acid synthetic ratio of increase, increase TAG stores and/or other required or anticipant character.

In some embodiments, engineered microorganism is oleaginous yeast, such as Yarrowia lipolytica.Some embodiment party In case, engineered yeast provided by the present invention shows one or more very good and unexpected phenotypic characteristic, example As: the conversion (such as with the ratio close to theoretical value) of the carbon of increase to oil, Johnson & Johnson head, lasting oil production, significantly biology The tolerance that matter produces and strengthens carbon source and related substances.

In some embodiments, some aspects of the present invention engineered microorganism (such as, the engineered ferment provided Female) show about 0.02g/g (grams of the glucose of the grams of produced oil, lipid or TAG/consumed) to about 0.3g/g Carbon to oil conversion ratio.In some embodiments, some aspects of the present invention the engineered microorganism provided is (such as through changing Acrasis) show following carbon to oil conversion ratio: about 0.010g/g (glucose of the grams of produced TAG/consumed gram Number), about 0.02g/g, about 0.025g/g, about 0.03g/g, about 0.04g/g, about 0.05g/g, about 0.06g/g, about 0.07g/g, about 0.075g/g, about 0.08g/g, about 0.09g/g, about 0.1g/g, about 0.11g/g, about 0.12g/g, about 0.13g/g, about 0.14g/ G, about 0.15g/g, about 0.16g/g, about 0.17g/g, about 0.18g/g, about 0.19g/g, about 0.2g/g, about 0.21g/g, about 0.22g/g, about 0.23g/g, about 0.24g/g, about 0.25g/g, about 0.26g/g, about 0.27g/g, about 0.28g/g, about 0.29g/g Or about 0.3g/g or close to theoretical value.In some embodiments, some aspects of the present invention the engineered micro-life provided Thing (the most engineered yeast) shows following carbon to oil conversion ratio: at least about 0.010g/g (produced TAG grams/disappeared The glucose grams of consumption), at least about 0.02g/g, at least about 0.025g/g, at least about 0.03g/g, at least about 0.04g/g, at least About 0.05g/g, at least about 0.06g/g, at least about 0.07g/g, at least about 0.075g/g, at least about 0.08g/g, at least about 0.09g/g, at least about 0.1g/g, at least about 0.11g/g, at least about 0.12g/g, at least about 0.13g/g, at least about 0.14g/g, At least about 0.15g/g, at least about 0.16g/g, at least about 0.17g/g, at least about 0.18g/g, at least about 0.19g/g, at least about 0.2g/g, at least about 0.21g/g, at least about 0.22g/g, at least about 0.23g/g, at least about 0.24g/g, at least about 0.25g/g, At least about 0.26g/g, at least about 0.27g/g, at least about 0.28g/g, at least about 0.29g/g or at least about 0.3g/g or close reason Opinion value.

In some embodiments, compared with wild-type yeast, some aspects of the present invention the engineered yeast provided Show and increase to following biomass and produce: about 2 times, about 2.5 times, about 5 times, about 7.5 times, about 10 times, about 15 times, about 20 Again, about 25 times, about 30 times, about 32 times, about 35 times or about 40 times.In some embodiments, some aspects of the present invention carry The engineered yeast of confession show carbon source and/or related substances concentration are tolerated the most highly concentrated by tolerated by wild-type yeast Degree the most about 150%, the most about 175%, the most about 200%, the most about 225%, the most about 250%, the most about 275%, The most about 300%, the most about 325%, the most about 350%, the most about 375%, the most about 400% or the most about 500%.Carbon source The non-limitative example of related substances includes the noxious substance polluting carbon source, such as, produce during pretreatment carbon source or use Material (such as, acidic materials such as acetic acid class or ammonia).

Data shown herein determine a new rate-limiting step of lipid accumulation in oleaginous yeast, transform it and make The characteristic for producing bio-fuel from carbohydrate source (such as glucose) must be greatly improved through the microorganism of operation.Cause This, method provided by the present invention and production are to use microorganism (such as yeast) fermentation from renewable carbohydrate source to substitute Property ground generate bio-fuel major progress.

For producing the culture of microorganism of bio-fuel

Some aspects of the present invention relate to presented herein or that transform according to certain aspects of the invention or Comprise the nucleic acid of separation presented herein or the culture of microorganism of protein.

In some embodiments, culture comprises presented herein or transforms according to certain aspects of the invention Or the microorganism that comprises the nucleic acid from the separation listed herein or protein, and culture medium (such as liquid culture Base).

In some embodiments, culture comprises presented herein or transforms according to certain aspects of the invention Or the nucleic acid that comprises separation presented herein or the microorganism of protein, and carbohydrate source.

In some embodiments, culture comprises presented herein or transforms according to certain aspects of the invention Or the nucleic acid that comprises separation presented herein or the microorganism of protein, and set up comply with microorganism survival, Growth and or microorganism in the carbohydrate salinity, osmotic pressure and the pH condition that convert to bio-fuel or bio-fuel precursor Salt and/or buffer.

In some embodiments, culture comprises other component, such as additive.The limiting examples of additive is Nutrient, enzyme, aminoacid, albumin, somatomedin, enzyme inhibitor (such as protease inhibitor), fatty acid, lipid, hormone (such as dexamethasone and gibberellins), trace element, inorganic compound (such as reducing agent such as manganese), redox modulating agent (example Such as antioxidant), stabilizer (such as dimethyl sulfoxide), Polyethylene Glycol, polyvinylpyrrolidone (PVP), gelatin, antibiotic (example Such as brefeldin A), salt (such as NaCl), chelating agen (such as EDTA, EGTA) and enzyme (such as cellulase, Bacillus polymyxa Neutral proteinase (dispase), hyaluronidase or DNA enzymatic).In some embodiments, culture can comprise induction or suppression from condition The medicine that property or inducible promoter are transcribed, such as doxycycline, tetracycline, tamoxifen, IPTG, hormone or metal ion.

Although concrete condition of culture (concentration of such as carbon source) depends on engineered microorganism to be cultivated, but is used for producing Conventional method and the condition of culture of raw culture of microorganism are known to a person skilled in the art, and be described in such as following in: J.Sambrook and D.Russell, Molecular Cloning:ALaboratory Manual, Cold Spring Harbor Laboratory Press；3rd edition (January 15,2001)；David C.Amberg, Daniel J.Burke and Jeffrey N.Strathern, Methods in Yeast Genetics:A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press (April 2005)；John N.Abelson, Melvin I.Simon, Christine Guthrie and Gerald R.Fink, Guide to Yeast Genetics and Molecular Biology, Part A, volume 194 (Methods in Enzymology Series, 194), Academic Press (March 11,2004)；Christine Guthrie and Gerald R. Fink, Guide to Yeast Genetics and Molecular and CEll Biology, Part B, the 350th volume (Methods in Enzymology, Vol 350), Academic Press；1st plate (July 2,2002)；And Christine Guthrie and Gerald R.Fink, Guide to Yeast Genetics and Molecular and Cell Biology Part C, the 351st is apt to, Academic Press；1st edition (July 9,2002), it is all incorporated herein by.As it is known in the art, for Oil produces, and cultivates engineered culture of microorganism as herein described under conditions of being suitable to oil accumulation.

In some embodiments, it is provided that display is beyond the wild-type microorganisms of same type and/or beyond other is micro- Biology (is such as commonly found in carbon source micro-life of contaminating microorganisms culture in bio-fuel or bio-fuel precursor convert Thing) the engineered microorganism of growth vigor.Such as, in some embodiments, it is provided that micro-with the wild type of same type Biological or other microbial than show increase multiplication rate and/or to the wild-type microorganisms of identical type and/or Other microorganism is poisonous or the tolerance of enhancing under conditions of limiting growth or breeding or viability.In some embodiments, The growth of engineered microorganism provided by some aspects of the present invention and/or proliferative advantage change into use non-sterilizing to cultivate and Fermentation condition produces bio-fuel or the probability of bio-fuel precursor, because so alleviating or having fully phased out by polluting micro- The culture undue growth problem that biology causes.In some embodiments, cultivate by the one of the present invention under non-sterilising conditions The engineered microorganism that a little aspects provide is to produce bio-fuel or bio-fuel precursor.Such as, in some embodiments, make With non-sterilizing raw material, non-sterilising medium, non-sterilizing additive or non-sterile biological reactor (opening under the most non-sterilising conditions Put reactor) produce bio-fuel or bio-fuel precursor.

For producing the method/raw material/bioreactor of bio-fuel

Before some aspects of the present invention are directed to use with modified micro-organisms bio-fuel or the bio-fuel of the present invention The method of body.In some embodiments, it is provided that produce bio-fuel or the method for bio-fuel precursor on an industrial scale.

Use the method provided by some aspects of the present invention that several kinds of carbon source can be converted into bio-fuel or biological combustion Material precursor.Sugar, starch and fiber apply to the carbohydrate source of the method for transformation by some aspects offer of the present invention Non-limitative example.According to certain aspects of the invention, carbohydrate source can comprise refine and/or unrefined sugar, shallow lake Powder and/or fiber or its combination in any.The non-limitative example of sugar is fermentable sugars, such as glucose, fructose, sucrose, xylose And lactose.The non-limitative example of starch is amylase and amylopectin.The non-limitative example of fiber is Plant fiber, such as fibre Dimension element, hemicellulose and wood fibre.Some aspects of the present invention are directed to use with industry byproduct, intermediate product or waste product (example Such as thick plant extract, molasses, straw or sewage) as carbon source.In some embodiments, carbon source is from algae.At some In embodiment, specially produce algal biomass to use in producing at the bio-fuel of microbe-mediated or bio-fuel precursor Make carbon source.

In some embodiments, it is provided that for producing bio-fuel or the method for bio-fuel precursor, it includes making With carbon source raw material that is cheap, abundant and that can be easily obtained as carbon source.In some embodiments, cellulose or half fibre are used Dimension is as carbon source.In some embodiments, cellulose or hemicellulose are from industry byproduct or waste product.Some embodiment party In case, cellulose or hemicellulose are directly from plant or algal biomass.Plant or algal biomass are the abundantest former One of material, and include a large amount of not fermentable sugars and fiber, such as cellulose and hemicellulose.In some embodiments, in advance Process biomass material so that fermentable sugars or fiber fermentable sugars will do not changed into, so that it can be used for growth of microorganism and micro- Bio-fuel or the bio-fuel precursor of biological mediation produce.In some embodiments, the pretreatment of biomass material includes Use well known to a person skilled in the art that cellulose and/or hemi-cellulose components depolymerization are monosaccharide by preprocess method, described pre- Processing method such as diluted acid or ammonia filament expansion (AFEX) method (see, e.g. Yang B, Wyman CE.Dilute acid and autohydrolysis pretreatment.Methods Mol Biol.2009；581:103-14；Balan V, Bals B, Chundawat SP, Marshall D, Dale BE, Lignocellulosic biomasspretreatment using AFEXMethods Mol Biol.2009；581:61-77).For by other method that biomass polymers depolymerization is monosaccharide being As well known to those skilled in the art and consideration is used in some embodiments of the present invention.

In some embodiments, use the biomass material that diluted acid method pretreatment comprises not fermentable sugars sending out Ferment sugar depolymerization is for fermenting monosaccharide.In some embodiments, appropriateness gentleness at a temperature of with dilute sulfuric acid with the time determined Process biomass.Such as, in some embodiments, with about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5% or about The sulfuric acid treatment biomass of 6%.In some embodiments, about 30 DEG C, about 37 DEG C, about 40 DEG C, about 50 DEG C, about 60 DEG C, about 70 DEG C, about 80 DEG C, about 90 DEG C, about 100 DEG C, about 110 DEG C, about 120 DEG C, about 130 DEG C, about 140 DEG C, about 150 DEG C, about 175 DEG C, about 200 DEG C or more than processing biomass at about 200 DEG C.

In some embodiments, gained hydrolyzate comprises insoluble lignin and soluble cellulosic polymers and half Cellulosic polymer.Can be by well known to a person skilled in the art method (such as by with cellulase or other hydrolytic enzyme) Process the product processed further below to produce hexose and pentose such as glucose and xylose monomer.In some embodiments In, the by-product including cytotoxic compound is resulted in dilute acid pretreatment not fermentable sugars, according to certain aspects of the invention, Its suppression is not transformed the growth of microorganism, is reduced its vigor and/or suppress its bio-fuel or the production of bio-fuel precursor.? In some embodiments, wash pretreated raw material, supplement and support growth of microorganism and bio-fuel or bio-fuel precursor Produce culture medium and/or add superfluous lime with removing toxic substances.

In some embodiments, use the biomass material that AFEX method pretreatment comprises not fermentable sugars so that can not Sugar fermentation depolymerization is for fermenting monosaccharide.In some embodiments, at high temperature and pressure with liquefied ammonia with the time-triggered protocol determined Biomass.In some embodiments, biomass are processed about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 points Clock, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes or longer.In some embodiments, about 30 DEG C, about 37 DEG C, about 40 DEG C, about 50 DEG C, about 60 DEG C, about 70 DEG C, about 80 DEG C, about 90 DEG C, about 100 DEG C, about 110 DEG C, about 120 DEG C, about 130 DEG C, About 140 DEG C, about 150 DEG C, about 175 DEG C, about 200 DEG C or more than processing biomass at about 200 DEG C.In some embodiments, AFEX pretreatment cause the crystalline cellulose being contained in raw material be converted into amorphous can fermentation form.In some embodiments In, before the biomass material of AFEX pretreatment does not comprise a large amount of suppression growth of microorganism and/or bio-fuel or bio-fuel Without detoxifying for producing bio-fuel or bio-fuel precursor before the harmful by-products that body produces, and use.

In some embodiments, with hydrolysis or the enzyme (such as with cellulase or hemicellulase) of solution chitosan polymer Process preprocessed or the most pretreated biomass material.In some embodiments, raw material is made to contact in the liquid phase with enzyme, And make to hatch at a temperature of enzyme catalysis depolymerization or hydrolysis be enough to hydrolyze or significant quantity in depolymerization biomass material not Fermentable sugars or a period of time of fiber.In some embodiments, hatch after hydrolysis and depolymerization, will by being such as centrifuged The liquid phase of the raw material contacted with enzyme separates with the solid phase comprising not fermentable sugars and fiber (comprising solubility fermentable sugars fraction). In some embodiments, the liquid fraction of raw material is made subsequently and for being converted into micro-life of bio-fuel or bio-fuel precursor Thing (such as being provided by some aspects of the present invention) contact.In some embodiments, not fermentable sugars or the enzymatic of fiber Conversion betides in united bioprocess, such as with micro-to bio-fuel or bio-fuel precursor of produced fermentable sugars Bioconversion is in identical time and/or same reactor.In some embodiments, first carry out Enzymatic transformation, then make with The raw material of enzyme contact contacts with the microorganism producing bio-fuel or bio-fuel precursor.In some embodiments, identical Time and carry out enzymatic and microorganism in same reactor and convert.

In some embodiments, cultivate engineered microorganism provided herein using acetic acid class as primary carbon source, Such as process LAN SCD gene and optionally carry as herein described other modify Yarrowia lipolytica.Such as, implement at some In scheme, about 1%, about 2%, about 3%, about 4%, about 5%, about cultivating microorganism in the acetic acid class solution that concentration is following: 6%, about 7%, about 8%, about 9% or about 10%.In some embodiments, acetic acid class concentration is about 3%～10%.At some In embodiment, cultivate using the cell comprising engineered microorganism as herein provided that acetic acid class is cultivated as primary carbon source Thing contacts with glycerol or is impregnated in glycerol.In some embodiments, microorganism is made to contact off and on glycerol.Implement at some In scheme, microorganism is made continuously or semi-continuously to contact with glycerol.In some embodiments, microorganism is about with concentration 0.5%, the glycerol contact of about 1%, about 2%, about 3%, about 4% or about 5%.Make engineered microorganism provided in this article with Glycerol contact provides with greater need for metabolite for producing TAG, and produces the reduction part needed for fatty acid from carbohydrate. In some embodiments, the carbon source (any carbon source the most as herein described) in addition to acetic acid class is being used to carry out bio-fuel Or bio-fuel precursor production method mixes glycerol.

In some embodiments, the carbohydrate of large scale microbial mediation can to the sweat of lipid Transfer To carry out in bioreactor.Term " bioreactor " and " fermentor " of replacing use herein refer to wherein give birth to Thing and/or the closure (enclosure) of chemical reaction (it at least partly relates to the part of living organism or living organism) or portion Divide closure." Large Scale Biology reactor " or " commercial scale bioreactor " is for business or half commercial mass production The bioreactor of product (such as bio-fuel or bio-fuel precursor such as fatty acid and/or TAG).Large Scale Biology reacts The volume range of device is generally at several litres, Shuo Baisheng, thousands of liter or bigger.

Microorganism or culture of microorganism can be comprised according to present invention bioreactor in terms of some.Implement at some In scheme, bioreactor can comprise the spore of the microorganism arbitrarily separated such as provided by some aspects of the present invention And/or the rest cell type of any kind, such as in drying regime.In some embodiments, anti-to such biology Answer and device adds suitable carbohydrate source can cause the sprouting and the most extremely of the activation of rest cell, such as yeast spores Small part carbohydrate source is converted into bio-fuel or bio-fuel precursor.

Cell culture system, wherein microorganism and shifting can be comprised according to the present invention some bioreactors in terms of some Hydrodynamic body and/or bubble contact.Microorganism according to certain aspects of the invention or culture of microorganism can in suspension or Person is attached on solid phase carrier cultivate.The non-limitative example of carrier system includes that microcarrier (can be such as porous or non- The polymer spheres of porous, microballon and micro-dish), have specified chemical group (such as tertiary amine groups) crosslinking pearl (such as Portugal gather Sugar), the 2D microcarrier that comprises the cell being trapped in non-porous polymeric fibres, 3D microcarrier is (for example, it is possible to comprise porous fibre Carrier fibre, hollow fibre, many barrel reactors and semipermeable membrane), there is the microcarrier of the ion-exchange capacity of reduction, encapsulating Cell, capillary tube and aggregation.Carrier can be made up of material such as glucosan, gelatin, glass and cellulose.

Can carry out according to the present invention plant-scale carbon aquation in terms of some with continuous, semicontinuous or non-continuous mode Compound is to the conversion process of lipid.The non-limitative example of operations according to the instant invention pattern be in batches, fed-batch, extension divide Batch (extended batch), repeated batch, take out/fill out, rotate the operation of wall, revolving bottle and/or fill-up mode.

In some embodiments, it is possible to use allow continuously or semi-continuously to feed substrate raw material (example from bioreactor Such as carbohydrate source) and/or continuously or semi-continuously separate product (such as secrete lipid, the organic facies comprising lipid and/or Show the cell of required lipid content) bioreactor.

The non-limitative example of bioreactor of the present invention is stirred-tank fermenter, is stirred by rotary blender Bioreactor, chemostat, by shaking device be stirred bioreactor, airlift fermentation device, packed bed react Device, fixed bed reactors, fluidized bed aerosol generator, utilize the bioreactor that ripple induction type stirs, centrifugal biological respinse Device, roller bottle and hollow fibre bioreactor, roller arrangement (the most desk-top, vehicle-mounted and/automatically kind), vertical stacking Dish, rolling bottle, stir or shake bottle, oscillatory type porous plate, MD bottle, T bottle, Roux bottle, multi-surface tissue culture incubator, improvement Fermentor and coated pearl (such as, are coated to prevent cell adhesion with serum albumin, nitrocellulose or carboxymethyl cellulose Pearl).

Can optionally comprise sensor according to present invention bioreactor in terms of some and fermentor and/or control system Unite to measure and/or regulation response parameter.The non-limitative example of response parameter is: biological parameter such as growth rate, cell Size, cell number, cell density, cell type or cell state；At the bottom of chemical parameters such as pH, oxidation-reduction potential, reaction Thing and/or the concentration of product, the concentration such as oxygen concentration and CO of dissolved gas₂Concentration, nutrient concentrations, metabolite concentration, Portugal Grape sugar concentration, glutamine concentration, concentrations of pyruvate, apatite concentration, oligomeric peptide concentration, amino acid concentration, vitamine concentration, Hormone concentration, additive concentration, serum-concentration, ionic strength, ion concentration, relative humidity, molar concentration, osmotic pressure, other The concentration of chemicals (such as buffer agent, adjuvant or byproduct of reaction)；Physical/chemical parameter, such as density, conductivity, stirring Degree, pressure and flow velocity, shear stress, shear rate, viscosity, color, turbidity, light absorption, mixing rate, conversion ratio and heat Kinetic parameter, such as temperature, light intensity/light quality etc..

The sensor that can measure parameters is well known to mechanics and electricity those skilled in the relevant art.Base It is bioreactor engineering neck in the control system of parameter in bioreactor can be regulated from sensor as herein described input Well known to the technical staff in territory.

Multiple different microorganisms that some aspects of the present invention are provided can be cultivated with root in suitable bioreactor Carry out large-scale carbohydrate according to some aspects of the present invention to convert to bio-fuel or bio-fuel precursor, such as from Many primary yeasts (such as oleaginous yeast), antibacterial, the microorganism in algae and fungi source.

The non-limitative example of yeast cells is from following cell: Yarrowia lipolytica, multiple-shaped nuohan inferior yeast (Hansenula polymorpha), Pichia sp. (Pichia pastoris), saccharomyces cerevisiae (Saccharomyces Cerevisiae), saccharomyces bayanus (S.bayanus), Kluyveromyces lactis (S.K.lactis), Waltomyces Lipofer.Mortierella alpine, Mortierella isabellina, multiple-shaped nuohan inferior yeast (Hansenula Polymorpha), Zygosaccharomyces rouxii (Mucor rouxii), trichosporon cutaneum (Trichosporon cutaneu), viscous red ferment Female (Rhodotorula glutinis), amylase yeast (Saccharomyces diastasicus), perhaps prosperous yeast (Schwanniomyces occidentalis), saccharomyces cerevisiae (S.cerevisiae), pichia stipitis (Pichia And schizosaccharomyces pombe (Schizosaccharomyces pombe) stipitis).

The non-limitative example of antibacterial be bacillus subtilis (Bacillus subtilis), Salmonella (Salmonella), Escherichia coli (Escherichia coli), vibrio cholera (Vibrio cholerae), streptomycete (Streptomyces), glimmering Light pseudomonas (Pseudomonas fluorescens), pseudomonas putida (Pseudomonas putida), pseudomonas Belong to (Pseudomonas sp), Rhod (Rhodococcus sp), streptomyces (Streptomvces sp) and Alcaligenes Belong to (Alcaligenes sp.).

Can be from following species cultivation fungal cell: aspergillosis western meat Sa rice (Aspergillus Shirousamii), Aspergillus niger (Aspergillus niger) and trichoderma reesei (Trichoderma reesei).

The non-limitative example of alga cells is from following cell: rich oil new chlorella (Neochloris Oleoabundans), scenedesmus obliquus (Scenedesmus obliquus), microalgae are belonged to (Nannochloropsis sp), Du Family name algae (Dunaliella tertiolecta), chlorella (Chlorella vulgaris), swim chlorella (Chlorella And spirulina maxim (Spirulina maxima) emersonii).

The type of the carbohydrate source being converted into bio-fuel or bio-fuel precursor according to some aspects of the present invention takes Certainly in the specified microorganisms used.Some microorganisms provided by some aspects of the present invention may be used for effectively converting spy Determine carbohydrate source, and different carbohydrate sources may not efficiently be processed by identical microorganism or can not at all Process.According to certain aspects of the invention, oleaginous yeast Yarrowia lipolytica can effectively by sugar (such as glucose, fructose, sugarcane Sugar and/or lactose) and the higher carbohydrate source (such as molasses) of sugared content and Plant fiber be converted into fatty acid and spread out Biological.

In some embodiments, some aspects of the present invention (such as oleaginous yeast is as solved fat in the microorganism provided Family name's yeast) secrete bio-fuel or the bio-fuel precursor, such as fatty acid or trigalloyl being produced from carbon source raw material at least in part Glycerol.In some embodiments, make microorganism that some aspects of the present invention provide and carbohydrate source in the reactor In aqueous solution contact, and secreted bio-fuel or bio-fuel precursor formed can be separated from the water organic facies. Term as used herein organic facies refers to comprise nonpolar organic compound (such as fatty acid, TAG and/or other is nonpolar Lipid) liquid phase.The organic facies of the present invention can comprise microorganism, carbohydrate further or see in each bioreactor Other compound in other phase.Known to those skilled in the art for plant-scale separated method.At some In embodiment, organic facies is continuously or semi-continuously absorbed.In some embodiments, use and comprise continuously or half Extract the bioreactor of the separator of organic facies continuously.

In some embodiments, bio-fuel or bio-fuel precursor are according to long-pending in present invention cell in terms of some Tired.In some embodiments, continuously or semi-continuously separate from bioreactor by being such as centrifuged, settle or filter Have accumulated bio-fuel or the cell of bio-fuel precursor of desired amount.Can such as based on cell physical characteristic (as cell is big Little or density) change by well known to a person skilled in the art the cell separation that method is carried out further.It is then possible to use Standard extraction method as well known to those skilled in the art (such as solvent hexane extraction) extracts the biological combustion accumulated from each cell Material or bio-fuel precursor.In some embodiments, collect microbial cell and the hexane with cell volume collected by 3 times enters Row extraction.In some embodiments, further refine is extracted bio-fuel or bio-fuel precursor.Some embodiment party In case, use and well known to a person skilled in the art that method (such as ester exchange method) is by bio-fuel precursor (such as triacylglycerol) It is converted into bio-fuel (such as biodiesel).

The function of these and other embodiment of the present invention and advantage will become to be more fully understood from from the following example. Following example are intended to explain the benefit of the present invention, but do not represent the four corner of the present invention.

Embodiment

Material and method

Gene construct: by each gene (such as, GLUT1, hemoglobin, cytochrome, pyruvate carboxylase, SCD etc.) It is cloned between site Pm1I and Kpn of plasmid YLEX (Figure 12).The restriction site used is Pm1I and KpnI.To all CDNA carries out checking order and comparison in genome database (mapped).The cloned cDNA's of comparison is exemplary representational Sequence database entries includes GLUT1: gene I/D: 6513；Hemoglobin: excrement Vitreoscilla (Vitreoscilla Stercoraria) Vitreosicilla hemoglobin gene, ACCESSION L77863；Cytochrome: gene I/D: 1528, CYB5A cells Pigment b5A type；Pyruvate carboxylase: gene I/D: 5091；SCD stearyl-coenzyme A desaturase (SCD): gene I/D: 710155.

The representative series (such as coded sequence) that can be used for producing process LAN microorganism is, such as: hemoglobin is (thin Bacterium)

ATGTTAGACCAACAAACCGTAGACACCAGCAAAGCCACTGTTCCTGTATTGAAAGAGCATGGCGTGACC ATTACCACGACGTT

TTACCAAAATTTGTTTGCCAAACATCCTGAAGTACGACCTTTGTTTGACATGGGTCGCCAAGCATCTTT GGAACAGCCTAAGG

CTTTGGCGATGACGGTTGGGGCGGCGGCACAAAACATTGAAAATTTACCTGCAATTTTGCCTGCAGTAC AAAAAATTGCCGTC

AAACATTGTCAAGCAGGCGTGGCGGCACGACATTATCCGATTGTGGGTCAAGAATTGTTGGGTGCGATT AAAGAATTATTGGG

TGATGCGGCGACCGATGATATTTTGGATGCGTGGGGCAAGGCTTATGGCGTGATTGCCGATGTTTTTAT TCAAGTGGAAGCGG

ATTTGTACGCTCAAGACGCTGAATAA (SEQ ID NO:3)

CYB (Ye Shi yeast)

ATGATCATCAACGGCAAGGTCTACGACATCTCCAGCTTCGTTGACGAGCATCCCGGTGGAGAGGAGGTT CTTCTTGATGCCGG

TGGAACTGAGGCCACCAACGCTTTCGACGACGTTGGACACTCTGAGGACGCTTACGGCATCCTTAACGA CCTCTATGTCGGTG

AGGTTGACCCCAGCGAGGACGTTATCCGAAAGACTCACACTGTCAAGACTTCTTACGAGGACGGCGAGT CTGTTGGTGATGAC

CACGGATCTTCTTCCATGATCTTCCTCATTGTTGCTGCTGCTGTTGCCGCCGCTGCTTTCTTCTACCTCCAGGGTCA GAAATAA (SEQ ID NO:4)

GLUT (rat)

ATGGAGCCCAGCAGCAAGAAGGTGACGGGCCGCCTTATGTTGGCCGTGGGAGGGGCAGTGCTCGGATCC CTGCAGTTCGGCTA

TAACACCGGTGTCATCAACGCCCCCCAGAAGGTAATTGAGGAGTTCTACAATCAAACATGGAACCACCG CTATGGAGAGTCCA

TCCCATCCACCACACTCACCACACTCTGGTCTCTCTCCGTGGCCATCTTCTCTGTCGGGGGCATGATTG GTTCCTTCTCTGTG

GGCCTCTTTGTTAATCGCTTTGGCAGGCGGAACTCCATGCTGATGATGAACCTGTTGGCCTTTGTGTCT GCCGTGCTTATGGG

TTTCTCCAAACTGGGCAAGTCCTTTGAGATGCTGATCCTGGGCCGCTTCATCATTGGAGTGTACTGTGG CCTGACCACCGGCT

TTGTGCCCATGTATGTGGGGGAGGTGTCACCCACAGCTCTTCGTGGAGCCCTGGGCACCCTGCACCAGC TGGGCATCGTCGTT

GGGATCCTTATTGCCCAGGTGTTCGGCTTAGACTCCATCATGGGCAATGCAGACTTGTGGCCTCTACTG CTCAGTGTCATCTT

CATCCCAGCCCTGCTACAGTGTATCCTGTTGCCCTTCTGCCCTGAGAGCCCCCGCTTCCTGCTCATCAA TCGTAACGAGGAGA

ACCGGGCCAAGAGTGTGCTGAAAAAGCTTCGAGGGACAGCCGATGTGACCCGAGACCTGCAGGAGATGA AAGAAGAGGGTCGG

CAGATGATGCGGGAGAAGAAGGTCACCATCTTGGAGCTGTTCCGCTCACCCGCCTACCGCCAGCCCATC CTCATCGCCGTGGT

GCTGCAGCTGTCCCAGCAGCTGTCGGGCATCAATGCTGTGTTCTACTACTCAACGAGCATCTTCGAGAA GGCAGGTGTGCAGC

AGCCTGTGTATGCCACCATCGGCTCGGGTATCGTCAACACGGCCTTCACTGTGGTGTCGCTGTTCGTCG TGGAGCGAGCTGGC

CGTCGGACCCTGCACCTCATTGGTCTGGCTGGCATGGCGGGCTGTGCTGTGCTCATGACCATCGCCCTG GCCCTGCTGGAGCA

GCTGCCCTGGATGTCCTATCTGAGTATCGTGGCCATCTTTGGCTTTGTGGCCTTCTTTGAAGTAGGCCC TGGTCCTATTCCAT

GGTTCATTGTGGCCGAGCTGTTCAGCCAGGGGCCCCGACCTGCTGCTGTTGCTGTGGCTGGCTTCTCTA ACTGGACCTCAAAC

TTCATCGTGGGCATGTGCTTCCAATATGTGGAGCAACTGTGTGGCCCCTACGTCTTCATCATCTTCACG GTGCTGCTGGTACT

CTTCTTCATCTTCACCTACTTCAAAGTTCCTGAGACCAAAGGCCGGACCTTCGATGAGATCGCTTCCGG CTTCCGGCAGGGGG

GTGCCAGCCAGAGCGACAAGACACCTGAGGAGCTCTTCCACCCTCTGGGGGCTGACTCCCAAGTGTGA (SEQ ID NO:5)

Malate dehydrogenase (Ye Shi yeast)

ATGTTACGACTACGAACCATGCGACCCACACAGACCAGCGTCAGGGCGGCGCTTGGGCCCACCGCCGCG GCCCGAAACATGTC

CTCCTCCAGCCCCTCCAGCTTCGAATACTCGTCCTACGTCAAGGGCACGCGGGAAATCGGCCACCGAAA GGCGCCCACAACCC

GTCTGTCGGTTGAGGGCCCCATCTACGTGGGCTTCGACGGCATTCGTCTTCTCAACCTGCCGCATCTCA ACAAGGGCTCGGGA

TTCCCCCTCAACGAGCGACGGGAATTCAGACTCAGTGGTCTTCTGCCCTCTGCCGAAGCCACCCTGGAG GAACAGGTCGACCG

AGCATACCAACAATTCAAAAAGTGTGGCACTCCCTTAGCCAAAAACGGGTTCTGCACCTCGCTCAAGTT CCAAAACGAGGTGC

TCTACTACGCCCTGCTGCTCAAGCACGTTAAGGAGGTCTTCCCCATCATCTATACACCGACTCAGGGAG AAGCCATTGAACAG

TACTCGCGGCTGTTCCGGCGGCCCGAAGGCTGCTTCCTCGACATCACCAGTCCCTACGACGTGGAGGAG CGTCTGGGAGCGTT

TGGAGACCATGACGACATTGACTACATTGTCGTGACTGACTCCGAGGGTATTCTCGGAATTGGAGACCA AGGAGTGGGCGGTA

TTGGTATTTCCATCGCCAAGCTGGCTCTCATGACTCTATGTGCTGGAGTCAACCCCTCACGAGTCATTC CTGTGGTTCTGGAT

ACGGGAACCAACAACCAGGAGCTGCTGCACGACCCCCTGTATCTCGGCCGACGAATGCCCCGAGTGCGA GGAAAGCAGTACGA

CGACTTCATCGACAACTTTGTGCAGTCTGCCCGAAGGCTGTATCCCAAGGCGGTGATCCATTTCGAGGA CTTTGGGCTCGCTA

ACGCACACAAGATCCTCGACAAGTATCGACCGGAGATCCCCTGCTTCAACGACGACATCCAGGGCACTG GAGCCGTCACTTTG

GCCTCCATCACGGCCGCTCTCAAGGTGCTGGGCAAAAATATCACAGATACTCGAATTCTCGTGTACGGA GCTGGTTCGGCCGG

CATGGGTATTGCTGAACAGGTCTATGATAACCTGGTTGCCCAGGGTCTCGACGACAAGACTGCGCGACA AAACATCTTTCTCA

TGGACCGACCGGGTCTACTGACCACCGCACTTACCGACGAGCAGATGAGCGACGTGCAGAAGCCGTTTG CCAAGGACAAGGCC

AATTACGAGGGAGTGGACACCAAGACTCTGGAGCACGTGGTTGCTGCCGTCAAGCCCCATATTCTCATT GGATGTTCCACTCA

GCCCGGCGCCTTTAACGAGAAGGTCGTCAAGGAGATGCTCAAACACACCCCTCGACCCATCATTCTCCC TCTTTCCAACCCCA

CACGTCTTCATGAGGCTGTCCCTGCAGATCTGTACAAGTGGACCGACGGCAAGGCTCTGGTTGCCACCG GCTCGCCCTTTGAC

CCAGTCAACGGCAAGGAGACGTCTGAGAACAATAACTGCTTTGTTTTCCCCGGAATCGGGCTGGGAGCC ATTCTGTCTCGATC

AAAGCTCATCACCAACACCATGATTGCTGCTGCCATCGAGTGCCTCGCCGAACAGGCCCCCATTCTCAA GAACCACGACGAGG

GAGTACTTCCCGACGTAGCTCTCATCCAGATCATTTCGGCCCGGGTGGCCACTGCCGTGGTTCTTCAGG CCAAGGCTGAGGGC

CTAGCCACTGTCGAGGAAGAGCTCAAGCCCGGCACCAAGGAACATGTGCAGATTCCCGACAACTTTGAC GAGTGTCTCGCCTG

GGTCGAGACTCAGATGTGGCGGCCCGTCTACCGGCCTCTCATCCATGTGCGGGATTACGACTAG (SEQ ID NO:6)

Ye Shi yeast Δ (9) desaturase (stearyl-coenzyme A desaturase)

ATGGTGAAAAACGTGGACCAAGTGGATCTCTCGCAGGTCGACACCATTGCCTCCGGCCGAGATGTCAAC TACAAGGTCAAGTA

CACCTCCGGCGTTAAGATGAGCCAGGGCGCCTACGACGACAAGGGCCGCCACATTTCCGAGCAGCCCTT CACCTGGGCCAACT

GGCACCAGCACATCAACTGGCTCAACTTCATTCTGGTGATTGCGCTGCCTCTGTCGTCCTTTGCTGCCG CTCCCTTCGTCTCC

TTCAACTGGAAGACCGCCGCGTTTGCTGTCGGCTATTACATGTGCACCGGTCTCGGTATCACCGCCGGC TACCACCGAATGTG

GGCCCATCGAGCCTACAAGGCCGCTCTGCCCGTTCGAATCATCCTTGCTCTGTTTGGAGGAGGAGCTGT CGAGGGCTCCATCC

GATGGTGGGCCTCGTCTCACCGAGTCCACCACCGATGGACCGACTCCAACAAGGACCCTTACGACGCCC GAAAGGGATTCTGG

TTCTCCCACTTTGGCTGGATGCTGCTTGTGCCCAACCCCAAGAACAAGGGCCGAACTGACATTTCTGAC CTCAACAACGACTG

GGTTGTCCGACTCCAGCACAAGTACTACGTTTACGTTCTCGTCTTCATGGCCATTGTTCTGCCCACCCT CGTCTGTGGCTTTG

GCTGGGGCGACTGGAAGGGAGGTCTTGTCTACGCCGGTATCATGCGATACACCTTTGTGCAGCAGGTGA CTTTCTGTGTCAAC

TCCCTTGCCCACTGGATTGGAGAGCAGCCCTTCGACGACCGACGAACTCCCCGAGACCACGCTCTTACC GCCCTGGTCACCTT

TGGAGAGGGCTACCACAACTTCCACCACGAGTTCCCCTCGGACTACCGAAACGCCCTCATCTGGTACCA GTACGACCCCACCA

AGTGGCTCATCTGGACCCTCAAGCAGGTTGGTCTCGCCTGGGACCTCCAGACCTTCTCCCAGAACGCCA TCGAGCAGGGTCTC

GTGCAGCAGCGACAGAAGAAGCTGGACAAGTGGCGAAACAACCTCAACTGGGGTATCCCCATTGAGCAG CTGCCTGTCATTGA

GTTTGAGGAGTTCCAAGAGCAGGCCAAGACCCGAGATCTGGTTCTCATTTCTGGCATTGTCCACGACGT GTCTGCCTTTGTCG

AGCACCACCCTGGTGGAAAGGCCCTCATTATGAGCGCCGTCGGCAAGGACGGTACCGCTGTCTTCAACG GAGGTGTCTACCGA

CACTCCAACGCTGGCCACAACCTGCTTGCCACCATGCGAGTTTCGGTCATTCGAGGCGGCATGGAGGTT GAGGTGTGGAAGAC

TGCCCAGAACGAAAAGAAGGACCAGAACATTGTCTCCGATGAGAGTGGAAACCGAATCCACCGAGCTGG TCTCCAGGCCACCC

GGGTCGAGAACCCCGGTATGTCTGGCATGGCTGCTTAG (SEQ ID NO:7)

Pyruvate carboxylase (people)

ATGCTGAAGTTCCGAACAGTCCATGGGGGCCTGAGGCTCCTGGGAATCCGCCGAACCTCCACCGCCCCC GCTGCCTCCCCAAA

TGTCCGGCGCCTGGAGTATAAGCCCATCAAGAAAGTCATGGTGGCCAACAGAGGTGAGATTGCCATCCG TGTGTTCCGGGCCT

GCACGGAGCTGGGCATCCGCACCGTAGCCATCTACTCTGAGCAGGACACGGGCCAGATGCACCGGCAGA AAGCAGATGAAGCC

TATCTCATCGGCCGCGGCCTGGCCCCCGTGCAGGCCTACCTGCACATCCCAGACATCATCAAGGTGGCC AAGGAGAACAACGT

AGATGCAGTGCACCCTGGCTACGGGTTCCTCTCTGAGCGAGCGGACTTCGCCCAGGCCTGCCAGGATGC AGGGGTCCGGTTTA

TTGGGCCAAGCCCAGAAGTGGTCCGCAAGATGGGAGACAAGGTGGAGGCCCGGGCCATCGCCATTGCTG CGGGTGTTCCCGTT

GTCCCTGGCACAGATGCCCCCATCACGTCCCTGCATGAGGCCCACGAGTTCTCCAACACCTACGGCTTC CCCATCATCTTCAA

GGCGGCCTATGGGGGTGGAGGGCGTGGCATGAGGGTGGTGCACAGCTACGAGGAGCTGGAGGAGAATTA CACCCGGGCCTACT

CAGAGGCTCTGGCCGCCTTTGGGAATGGGGCGCTGTTTGTGGAGAAGTTCATCGAGAAGCCACGGCACA TCGAGGTGCAGATC

TTGGGGGACCAGTATGGGAACATCCTGCACCTGTACGAGCGAGACTGCTCCATCCAGCGGCGGCACCAG AAGGTGGTCGAGAT

TGCCCCCGCCGCCCACCTGGACCCGCAGCTTCGGACTCGGCTCACCAGCGACTCTGTGAAACTCGCTAA ACAGGTGGGCTACG

AGAACGCAGGCACCGTGGAGTTCCTGGTGGACAGGCACGGCAAGCACTACTTCATCGAGGTCAACTCCC GCCTGCAGGTGGAG

CACACGGTCACAGAGGAGATCACCGACGTAGACCTGGTCCATGCTCAGATCCACGTGGCTGAGGGCAGG AGCCTACCCGACCT

GGGCCTGCGGCAGGAGAACATCCGCATCAACGGGTGTGCCATCCAGTGCCGGGTCACCACCGAGGACCC CGCGCGCAGCTTCC

AGCCGGACACCGGCCGCATTGAGGTGTTCCGGAGCGGAGAGGGCATGGGCATCCGCCTGGATAATGCTT CCGCCTTCCAAGGA

GCCGTCATCTCGCCCCACTACGACTCCCTGCTGGTCAAAGTCATTGCCCACGGCAAAGACCACCCCACG GCCGCCACCAAGAT

GAGCAGGGCCCTTGCGGAGTTCCGCGTCCGAGGTGTGAAGACCAACATCGCCTTCCTGCAGAATGTGCT CAACAACCAGCAGT

TCCTGGCAGGCACTGTGGACACCCAGTTCATCGACGAGAACCCAGAGCTGTTCCAGCTGCGGCCTGCAC AGAACCGGGCCCAA

AAGCTGTTGCACTACCTCGGCCATGTCATGGTAAACGGTCCAACCACCCCGATTCCCGTCAAGGCCAGC CCCAGCCCCACGGA

CCCCGTTGTCCCTGCAGTGCCCATAGGCCCGCCCCCGGCTGGTTTCAGAGACATCCTGCTGCGAGAGGG GCCTGAGGGCTTTG

CTCGAGCTGTGCGGAACCACCCGGGGCTGCTGCTGATGGACACGACCTTCAGGGACGCCCACCAGTCAC TGCTGGCCACTCGT

GTGCGCACCCACGATCTCAAAAAGATCGCCCCCTATGTTGCCCACAACTTCAGCAAGCTCTTCAGCATG GAGAACTGGGGAGG

AGCCACGTTTGACGTCGCCATGCGCTTCCTGTATGAGTGCCCCTGGCGGCGGCTGCAGGAGCTCCGGGA GCTCATCCCCAACA

TCCCTTTCCAGATGCTGCTGCGGGGGGCCAATGCTGTGGGCTACACCAACTACCCAGACAACGTGGTCT TCAAGTTCTGTGAA

GTGGCCAAAGAGAATGGCATGGATGTCTTCCGTGTGTTTGACTCCCTCAACTACTTGCCCAACATGCTG CTGGGCATGGAGGC

GGCAGGAAGTGCCGGAGGCGTGGTGGAGGCTGCCATCTCATACACGGGCGACGTGGCCGACCCCAGCCG CACCAAGTACTCAC

TGCAGTACTACATGGGCTTGGCCGAAGAGCTGGTGCGAGCTGGCACCCACATCCTGTGCATCAAGGACA TGGCCGGGCTGCTG

AAGCCCACGGCCTGCACCATGCTGGTCAGCTCCCTCCGGGACCGCTTCCCCGACCTCCCACTGCACATC CACACCCACGACAC

GTCAGGGGCAGGCGTGGCAGCCATGCTGGCCTGTGCCCAGGCTGGAGCTGATGTGGTGGATGTGGCAGC TGATTCCATGTCTG

GGATGACTTCACAGCCCAGCATGGGGGCCCTGGTGGCCTGTACCAGAGGGACTCCCCTGGACACAGAGG TGCCCATGGAGCGC

GTGTTTGACTACAGTGAGTACTGGGAGGGGGCTCGGGGACTGTACGCGGCCTTCGACTGCACGGCCACC ATGAAGTCTGGCAA

CTCGGACGTGTATGAAAATGAGATCCCAGGGGGCCAGTACACCAACCTGCACTTCCAGGCCCACAGCAT GGGGCTTGGCTCCA

AGTTCAAGGAGGTCAAGAAGGCCTATGTGGAGGCCAACCAGATGCTGGGCGATCTCATCAAGGTGACGC CCTCCTCCAAGATC

GTGGGGGACCTGGCCCAGTTTATGGTGCAGAATGGATTGAGCCGGGCAGAGGCCGAAGCTCAGGCGGAA GAGCTGTCCTTTCC

CCGCTCCGTGGTGGAGTTCCTGCAGGGCTACATCGGTGTCCCCCATGGGGGGTTCCCCGAACCCTTTCG CTCTAAGGTACTGA

AGGACCTGCCAAGGGTGGAGGGGCGGCCTGGAGCCTCCCTCCCTCCCCTGGATCTGCAGGCACTGGAGA AGGAGCTGGTAGAC

CGGCATGGGGAGGAGGTGACGCCGGAAGATGTGCTCTCAGCAGCTATGTACCCCGATGTGTTTGCCCAC TTCAAGGACTTCAC

TGCCACCTTTGGCCCCCTGGATAGCCTGAATACTCGCCTCTTCCTGCAGGGACCCAAGATCGCAGAGGA GTTTGAGGTGGAGC

TGGAGCGGGGCAAGACGCTGCACATCAAAGCCCTGGCCGTGAGCGACCTGAACCGGGCCGGCCAGAGGC AGGTCTTCTTTGAG

CTCAATGGGCAGCTGCGGTCCATCTTGGTCAAGGACACCCAGGCCATGAAGGAGATGCACTTCCACCCC AAGGCCCTAAAGGA

CGTGAAGGGCCAGATCGGGGCGCCCATGCCTGGGAAGGTGATAGACATCAAAGTGGTGGCAGGGGCCAA GGTGGCCAAGGGCC

AGCCCCTGTGTGTGCTCAGTGCCATGAAGATGGAGACTGTGGTGACCTCACCCATGGAGGGTACTGTCC GCAAGGTTCATGTG

ACCAAGGACATGACACTGGAAGGTGACGACCTCATCCTGGAGATCGAGTGA (SEQ ID NO:8)

ACC (saccharomyces cerevisiae)

TTATTTCAAAGTCTTCAACAATTTTTCTTTATCATCGGTAGATAACATCTTGATAACTTCAGATAATCC ATCAATAGCATTGT

CATGGTCGCTTCTGATCTTTTTAGCTAAGTCTTGAGCGAATGACTCTAATTTCAAACCCTTTAGTTTAT CGTCCAAAGTTTTG

TAGTTTTCTTCAATCCATGTTGCGACTTGCCTATCATCTTCATGGTCCACTGAAGCAGGGTACCACGAT CTAATTCTTGCGAT

CTTTTCTAATCTTGATGCTTCGCCTACCTGATGGCTCAACCTTTTAATCAAATATTCTTCGTTCAATCT TCTTCTCAATCTCC

AGAAGAAGAAACGACGTGCCTCGGTCCATTCCAGTTCCTTAGAAATAACACCCTTGGCCACCATACGTG AAGACCTATCGTGC

AAATCAGCAAATTGAAGACTGATTTGTCCGTAAATTGGCAATAGTTCTCTCTCACGATCAGCTAATTGC TTGGATATTTGCTG

ATGTACTTCTGGAGCCAAACTCTTGTTGGATAATTGAGATCTCAATTCTCTGTACTTGTCATCCAATCT GTTCATGGTGTCCA

GCAATTTTTCTCTACGGAACTTGATACCAACCATACCTTGTGGTTCCAAAACACCAGCTCTAGCGTTGA CGTCGGCATACATT

TCCATTTGGTCAGCGTTGATAGTTGGATCGACAACAACCCATGAACCACCTCTTAGTTCACCGGTAGGT GGGATATAGATAAT

AATTGGTTGTTTGTAATCCACCAATGCGTCAACAATAAACGAACCATACTTCAAGACTTCGTTGAACAT ATCACGTTGACCAC

CAGAGAAACCTCTCCAGTTGGCCAAAATCATCATTGGCAATTGTTCACCGTTGTTAAAGTCATTGATAG CTTGAGCAGTCTTG

AAGGCGGAGTTTGGATGCCAAACTTGACCAGGTTCTTGAATTAATGTTTCAGCACTATTTGGATTAGCT GGATCAGCAGGAAT

CAAGTTCTCGACAGTTCTTGTTTCAACACCAATAACACCCAGTGGAATACCACCAAGACGGGCTCTACC AACGACAACACCTT

TGGCCCATCCTGACAAAGTTTCAAAGAAAGACCCTTTATCAAACAAACCATATTCAAATCCACTTTCAG TCTCACGACCTTCA

ATCATCCATCTTACATCGTAAGTTTCATCATTAGTTGGAGTGAAATCAACTGGTCTATCCCATGTGTCT TTAGTTTCCAAGAT

AGGAACTGGCATATTACGCTTGGCTGGAACATAAGACATCCATTCAACAATCTTCTCTACACCAGCTAA ATCGTCAACAGCAG

TCAAATGTGAAACACCGTTGTTATACATGATTTGAGTACCACCCAATTGTAAGTTAGAAGTATAAACTT CTCTACCCAGCATT

TTGTTGATTGCAGGAGCACCAGTTAAAATAATTGGCTGGCCTTCGACCTGAATAGCTCTTTGACCCAAA CGAACCAAATAAGC

ACCGATACCGACGGATCTACAAGTGACTAAGGTGATAGTGAAGATATCGTGGTAAGCCCTTGACGTTGC ACCAGCAATTAAAC

CAGATCCACGTAGACATTCGACACCTAACCCATCTTCAGAACCAATAATTGTCTTGATGACAAATCTTT CTTCACCGTTTATA

ACAGTACGTTCAGTGAGAACAGAATTTTCTTTGTCAAATTTCTTTAAAGTTTCCATACCTTCACTTGTT AAGTATAAGTATTG

GAAGCCCTTGTCCGGATTGGCAGCATCATTCCATGCAACTTGAAATAGTGGAACAATCTCTTCAGCCAT ACCAATTCTGGCAC

CTGAGTTTGCAGCCAAGTAAATTCTTGGGATACCACGCTTTCTAGCATATTCAGTAACCTTATTGAAGA ATTCGTCTTCTTGT

GGACCAAAGGAACCGATCTTGAATGTGATATCGTTAGCAACAACAACAAATTGACGGCCTCTTGGATAT TCAGGAGTCTTTAC

AGTAATCTTAAAGGCAACCATACCAATAGCGTTGGCACCAGGTTCTCTTTCCACCTCAGTTAATTCGCC GTTTTCATCTTCAA

TCAACTCGTTGGAAATAAAGAAATCATCTGTTAACTTAACATCTGCAGAGAAATTTTTCCATTGGGATG ACGATGCTTGGCGG

AATAATTCTGGGAAGTCATAGACATATGTGGTACCCATCAAGTGTGCCTTATAACGTTTTGGTTGCAAC CATTCCTTAACAGG

GTAAGGAGTAGCAATAGGTCTTAAATGCATGGATCCAGGTTTACCCAAAGACTTAAATACCCATTCACC TTTTGCGTTCTTGA

CTTCGGTGTACATTTCTGTTTTGATAACATAACCAGAAACGTTATTGATCAAGGCACGCAATGGTACTG GGGCACCTGTTTGA

GGATCTTTGATGATGATTCTAATTTCGGCAGAAGAAACACGCAATCTCAACAATCTCTTACCAAATCTT TCTAAGAAACCACC

GAAGGCGGCTTCGACATCTTCTGGAGAGATATCAAACACCGCAATGAAGTTGATGAAGATATGATTCAA ATCAGAATTTGAAG

TGTCGGTGACTTCTAAATTATCCAATATATCACTCATCAATCTGTTAGCTTCAGAAGTCAGATATTCTT GAATAGAAATGTCA

TCACGGATATGACCCGTTCTAATAATACCTCTTGTAAAGAATCTCTTATCCAATGGAGAAGTCTTACTA ACAGCTTCGTAGAC

ATGGATGTTTCTATTATCAGTGAAAATTGGTTTAATGTTGAAGTTGGACAATCTTCCTAATTCCAGTTG GAAGGCCAAAGCCG

GCTCAATGTGACGAATTGTTTCATTTTCGTTATAATTTGGACCGTTAAAAGTATAATACTTTGGATAAG ACCCATCTTTAAAA

CCGAACATAAATGTGATACGACGGATAGAAGCATTGATTAATTCCTGCTTATTCAAATCCAAAATTTCT CTCAACCTTACCAA

AATTTCCTCTTCAGATTCGAAACCTTCTGTAGAAGCAACACAAACATTAGCAACATTACTCAACGATGC GGAGCTACCAGAAC

GATCAGGAGCAGGTCCGTTAGAAGAAGATTGGTGACGAGGAATAACTTCCAAACTTTGTGACAAAATTT CATCAACATCATCT

AAATGATCCACAGCCATCAAAATACCTTCTCTTAACGGAGATGACTGACTGTTTGCAACATATGACAAA TCTGAAACAGAAAC

AGCCCTGTTCATACCCATTTTAGATTTAACAGTTGGAAAGGTGGAGAACGCAGCTGAAGGTAGTTGGAA TTTCCATTCAACAA

TTGGAACTGTGACACCTTCGTGAACTCTAATATCTCCTATGGTGTAAGCACGATAAGCACGACGAATAT AGACTTGAGCAGCT

GCAGCAGTCACAACTGGGTCTTGATGGGTTAGGAATTGAAGTAAAACATCGAACACAACGTAATTAGAA TCGATCAAGTCCTT

CAAGATATTCAAATCTGGTTCAGAGCGCTTTGGATTGGATGAGCCATAGGCAACCTTCACAACAGAGGA TTTTAAGATATGTT

CAATTTGTTCAGTTCTTTCCTTGACCGAAGGTAAAGCGCCTTGAATCAAAATTTCTCTTGCTTGTAGAG CGACCTTAGCGGTA

GCCTTAGATTCTAGTTCAACAATATGTTGTAGAGGAGTAGAGAAAATGGCAGAAACTTTAGAAGATAAC TTGCACAATGGTTG

ATAATGTTTCAAGATAGCTAGGATCAGGTTATTCTTCGCTGAAACTTTCGAATGAGACAAAACAGTTAG CGCAACTTTATCTA

GATCTTTAGGGTTTTCATCACGCAATTTCAGAATGATATTTTCCTCACGAACATTTGGACCATTGAATA ACTTTTCAACTTCG

TAATATTCTTCCAAGAAATGGACAAATATAGAATGTTCATGGGCTTCTAACCCGTTAGAGTACTTATGA GCAATATCCGCCAA

TGGTTCCACGACGGCGCCCAGCAATTTGTCGGGGTTGTATTCAGGATTCTTCACGGCCATATCAATCAA TTTACTTAATTGTC

TAGCTGGGAAAACAGCACCACGTCTCAAAGAACGTGCAACTAACTCTTCCATTTGTTCATCTAGCTTAG CAGGCAATCTTGAA

TGTAAAGCAGAGATGTGTAGTTTCCATTCTGAGTAAGGCAGTTTTGGATTTCTCAAAACCTCTATCAAT TGTTGCAAGGAAGC

GTTCATAATAACTTGGTTGTCATAACCCTTCAAAATGTTTTCCAAAGTAGACACTAATGACTTGAATTT ATAGGCAGGTTTGG

TTCCTTCGATAACTGGAGAACCAAAATCTGGCAGCATACCTTCAAATGGTAGAGCGTGCTTGACCTTGG ATGGATCGTCAAGA

GTCATAATAGCCATGATATCACCTGCAACAATGGTAGAACCAGGTTGCTTTAATAACTGGACGATACCA TTTTCTTGAGAAAC

CAAAGGCATTTGCATTTTCATAACTTCAATTTCTGCATATGGTTGGCCCTTGATAATGTGTTCACCATT TTCCACCAAGAATT

TAACCAATTTACCAGGGGATGGAGTACGCAACTGGGTTGGATCGTTTTCAACTTCCAACAAAGTAGTCA TAGAGTCAACGGAT

AATCTTGTAGCAGCAACTTCTTCTTTCCAATAGATGGTATGCGATTTACCGCCTATGGCAATCAAAAGA CCACCATCAGATAG

TTGACGCAGTATGATATCACATTTAGAACCATTGATAAATAATGTGTAACGGTCATTACCGGATTTAGC TACGGTGAACTTGT

ATCTTTTACCCTCATGGATAAAATCTACAGGGAACATAGTTTGCAGTAGGTCTTTAGATAGAACTTGTC CCTTTTGTAAGGAT

TCGATATACTTGTGGCGGGCTTCTTCAGATGCTAAGAAAGCCTTTGTAGCGGCACCGCAAATGACGGCA AGAGTTGGATCAGG

CTTTTCAGCGGTCATTTTATGAGTAATCAAATCGTCCAACCAACCGGTGGTAATAGTGTTATCCTCGAA ATCTTCAGTTTCCA

AAAGTTTGATCAAGTATTCCACAGTAGTTCTGAAATCACCCCTAATGGACAATTCCTTCAGGGCAACAA CCATGTGTTTCCTG

GAAGCTTGTCTATTTTCACCAAAAGCAAAAATATGGCCGAACTGAGAGTCCGAAAAGGAGTGAATATTA CCATTGTTACCCAC

GGAGAAGTAACCCCAAACATTAGAGGAAGAACGGAAGTTTAGTTCATGCAAAGTACCACCCGATGGCTT GAATCCATCGTTTG

GATCTTCTGATGTGATACGACAAGCGGTACAATGACCCTTTGGAATAGGTCTTCTTTGTTTCTTGGTGG CATCTTGAGTTTTG

AATTCGAAATCGATTTCTGAGGCAGAATGAGGATTCATACCATATAAAGTTCTAATGTCACTTATTCTA TGCATAGGGATACC

CATAGCGATTTGTAATTGAGCTGCAGGTAAGTTAACACCGGAGACCATTTCCGTTGTTGGATGCTCGAC TTGTAATCTTGGGT

TCAATTCTAAAAAGTAGAATTTTCCATCATCATGAGAATATAGATACTCCACGGTACCGGCAGAGACAT AACCGACTAGTTTC

CCCAGTCTGACGGCAGCCTTTTCCATCTCGTGAAATGTTTCAGCCTTGGCAATTGTAACTGGTGCTTCT TCGATAATTTTTTG

ATGACGTCTCTGAACGGAACAGTCTCTACCGAACAAGGAAATATTTGTACCGTACTGATCTGCTAGCAG TTGAACTTCCAAGT

GACGCGCTCTACCGGCCAACTTCATGATGAAAATGGGGGAGCCTGGAATTTCGTTGGCTGCCTGGTGGT ATAAAGCGATGAAA

TCTTCTTCACGTTCAACTTGTCTGATACCTTTACCACCACCACCTTCGGATGCCTTAATCATGACAGGA AAACCAATACGCTT

GGCCTTTTGTAAACCATCTTCAGGAGAGGTACAACAACCCTTTTGATAGATGTCATCGTCGACAGAGAC CAGACCGGTTTTCT

CGTCCACGTGAACGGTGTCAACACCGGTACCAGACCATGGAATACATGGGACTTTAGCACTTTGAGCGA CAATGGTAGAGGAG

ATTTTATCACCTAAAGACCTCATGGCGTTACCTGGAGGCCCAATAAAGATGACTTTCCTCTTAGACTGG GACAATTTTTCAGG

CAATAGTGGATTCTCGGAGGCGTGACCCCAGCCAGCCCATACGGCGTCTACGTCTGCTCTTTCGGCGAT GTCTACGATCAAGT

CTACGTTAGCGTAGTTGTTATTATTAGTACCACCTGGCACTTCAATGTATTGATCGGCCATACGGATAT ATTCTGCGTTGGCC

TCCAGATCTTCTGGGGTGGCCATGGCGACGAATTGGACGGTTCTGTCATCGCCGAACGTCTCGTATGCC CATTTTCTGACGGA

TCTAATTTCTTTCACGGCGGCAATACCATTATTTGCTATCAGGATCTTGGATATGACCGTGTGACCACC GTGACTCTTAACAA

AGTCCCTTAACGGGGACTCCTCTAGTTTATCTACTGTATTGAGGCCAATGAAATGACCTGGAAGTTCTG TATGTCTTTCTGAG

TAGTTTGTAATTTCGTACTCCATCTTCTGTGGAGAAGACTCGAATAAGCTTTCTTCGCTCAT (SEQ ID NO:9)

FAA (saccharomyces cerevisiae)

ATGGTTGCTCAATATACCGTTCCAGTTGGGAAAGCCGCCAATGAGCATGAAACTGCTCCAAGAAGAAAT TATCAATGCCGCGA

GAAGCCGCTCGTCAGACCGCCTAACACAAAGTGTTCCACTGTTTATGAGTTTGTTCTAGAGTGCTTTCA GAAGAACAAAAATT

CAAATGCTATGGGTTGGAGGGATGTTAAGGAAATTCATGAAGAATCCAAATCGGTTATGAAAAAAGTTG ATGGCAAGGAGACT

TCAGTGGAAAAGAAATGGATGTATTATGAACTATCGCATTATCATTATAATTCATTTGACCAATTGACC GATATCATGCATGA

AATTGGTCGTGGGTTGGTGAAAATAGGATTAAAGCCTAATGATGATGACAAATTACATCTTTACGCAGC CACTTCTCACAAGT

GGATGAAGATGTTCTTAGGAGCGCAGTCTCAAGGTATTCCTGTCGTCACTGCCTACGATACTTTGGGAG AGAAAGGGCTAATT

CATTCTTTGGTGCAAACGGGGTCTAAGGCCATTTTTACCGATAACTCTTTATTACCATCCTTGATCAAA CCAGTGCAAGCCGC

TCAAGACGTAAAATACATAATTCATTTCGATTCCATCAGTTCTGAGGACAGGAGGCAAAGTGGTAAGAT CTATCAATCTGCTC

ATGATGCCATCAACAGAATTAAAGAAGTTAGACCTGATATCAAGACCTTTAGCTTTGACGACATCTTGA AGCTAGGTAAAGAA

TCCTGTAACGAAATCGATGTTCATCCACCTGGCAAGGATGATCTTTGTTGCATCATGTATACGTCTGGT TCTACAGGTGAGCC

AAAGGGTGTTGTCTTGAAACATTCAAATGTTGTCGCAGGTGTTGGTGGTGCAAGTTTGAATGTTTTGAA GTTTGTGGGCAATA

CCGACCGTGTTATCTGTTTTTTGCCACTAGCTCATATTTTTGAATTGGTTTTCGAACTATTGTCCTTTT ATTGGGGGGCCTGC

ATTGGTTATGCCACCGTAAAAACTTTAACTAGCAGCTCTGTGAGAAATTGTCAAGGTGATTTGCAAGAA TTCAAGCCCACAAT

CATGGTTGGTGTCGCCGCTGTTTGGGAAACAGTGAGAAAAGGGATCTTAAACCAAATTGATAATTTGCC CTTCCTCACCAAGA

AAATCTTCTGGACCGCGTATAATACCAAGTTGAACATGCAACGTCTCCACATCCCTGGTGGCGGCGCCT TAGGAAACTTGGTT

TTCAAAAAAATCAGAACTGCCACAGGTGGCCAATTAAGATATTTGTTAAACGGTGGTTCTCCAATCAGT CGGGATGCTCAGGA

ATTCATCACAAATTTAATCTGCCCTATGCTTATTGGTTACGGTTTAACCGAGACATGCGCTAGTACCAC CATCTTGGATCCTG

CTAATTTTGAACTCGGCGTCGCTGGTGACCTAACAGGTTGTGTTACCGTCAAACTAGTTGATGTTGAAG AATTAGGTTATTTT

GCTAAAAACAACCAAGGTGAAGTTTGGATCACAGGTGCCAATGTCACGCCTGAATATTATAAGAATGAG GAAGAAACTTCTCA

AGCTTTAACAAGCGATGGTTGGTTCAAGACCGGTGACATCGGTGAATGGGAAGCAAATGGCCATTTGAA AATAATTGACAGGA

AGAAAAACTTGGTCAAAACAATGAACGGTGAATATATCGCACTCGAGAAATTAGAGTCCGTTTACAGAT CTAACGAATATGTT

GCTAACATTTGTGTTTATGCCGACCAATCTAAGACTAAGCCAGTTGGTATTATTGTACCAAATCATGCT CCATTAACGAAGCT

TGCTAAAAAGTTGGGAATTATGGAACAAAAAGACAGTTCAATTAATATCGAAAATTATTTGGAGGATGC AAAATTGATTAAAG

CTGTTTATTCTGATCTTTTGAAGACAGGTAAAGACCAAGGTTTGGTTGGCATTGAATTACTAGCAGGCA TAGTGTTCTTTGAC

GGCGAATGGACTCCACAAAACGGTTTTGTTACGTCCGCTCAGAAATTGAAAAGAAAAGACATTTTGAAT GCTGTCAAAGATAA

AGTTGACGCCGTTTATAGTTCGTCTTAA (SEQID NO:10)

Acyl-CoA synthetase

ATGACTGTTACCCCACAGCACCAAGTCGTCCACGAGGCCAACGGTGTCACCCCAAGACCCACTCCTAAG GAGTTTTTTGACAA

ACAGCCCCGTCCTGGCCATATCACCTCCATCGAACAGTACCAGGAATTATACCAGAAGTCCATCGCCGA CCCTGAAGGATTCT

TTGGTCCTATGGCCAAGGAGTTGTTGTCGTGGGACAGAGACTTCGACAAGGTCAAGTCCGGTTCTTTGA AGGACGGTGACGTT

GCCTGGTTCATTGGCGGCCAGTTGAACGCTTCCTACAACTGTGTAGACAGATGGGCCTATGCGACTCCA GACAAGACTGCCAT

CATCTACGAAGCTGACGAAGAAAAGGACTCGTACAAGTTGACCTACGCCGAGTTGTTGAGAGAAGTCTC CAAGGTAGCTGGTG

TGTTGAAGAGCTGGGGCATCAAAAAGGGTGATACTGTTGCTATCTACTTGCCAATGACTCCTCAAGCTG TTATTGCTATGCTC

GCTGTAGCCAGATTAGGTGCCATCCACTCGGTTATCTTTGCAGGTTTCTCTTCTGGTTCCATCAGAGAC AGAGTCAACGATGC

TTCTTGTAAGGCTCTTATTACCTGTGACGAAGGTAGAAGAGGTGGTAAGACCGTTAACATCAAGAAATT GTGCGACGAAGCCT

TGAAGAGCTGTCCTACTGTAGAAAAGGTGCTTGTTTTCAAGAGAACCGGAAACGAAAATATTGAATTGG AAGAGGGTAGAGAT

TTCTGGTGGGATGAAGAAACCGCCAAGTTCTCGGGTTACTTGCCACCTGTTCCAGTCAATTCTGAAGAC CCATTGTTCTTGTT

GTATACATCTGGTTCCACTGGTACTCCTAAGGGTGTTGTCCACACCACTGGGGGCTACCTCTTAGGTGC TGCCATGACCACCA

AGTACATTTTCGACGTCCACCCAGAAGACATCTTGTTCACTGCCGGTGATGTCGGTTGGATTACTGGTC ACACCTATGCTTTG

TACGGACCTTTGGCTCTCGGTATCCCAACAATCGTTTTTGAAGGTACTCCAGCCTACCCAGACTTTGGT AGATTCTGGCAAAT

TGTCGAAAAGCACAAGGCTACCCACTTCTACGTAGCTCCTACTGCCCTCAGATTGTTGAGAAAGAGTGG CGAGCAAGAGATTC

CAAAGTACGACTTGTCTTCTTTGAGAACATTGGGCTCTGTTGGTGAACCTATCTCCCCTGATATCTGGG AATGGTACAACGAG

CACGTTGGACAAGGCAGATGCCACATCTCCGACACCTACTGGCAAACTGAGTCTGGTTCTCACTTCATT GCTCCAATTGCCGG

TGTCACTCCAAACAAACCTGGTTCAGCCTCTTTGCCATTCTTTGGTATCGAGACCGCTCTTATTGATCC AGTTTCCGGCCACG

AACTCGAAGGTAACGACATCGAAGGTGTTCTTGCCATCAAGAGCACCTGGCCATCTATGGCTAGATCTG TCTGGAACAACCAC

ACCAAGTACATGGACACATACTTGAACCCATACCCAGGCTACTACTTTACCGGCGACGGTGCTGCCAGA GATCACGACGGCTA

CTACTGGATTAGAGGTAGAGTCGATGATGTCGTCAATGTGTCTGGTCACAGATTGTCTACTGCTGAAAT AGAAGCTGCCCTCA

TCGAACACAACGGTGTTTCTGAAGCTGCTGTGGTTGGTATTACCGACGACTTAACTGGTCAAGCCGTAG TTGCCTACGTTGCT

CTCAAGAACGAATACGTCGACAAGATCGCCGGCAAGGAAACCAGCGACGAAGCCTTTGCCTTGAGAAAG GAATTGATCATGAC

CGTCAGAAAGGAAATCGGACCTTTCGCAGCTCCAAAGAGCGTCATCATTGTCGCCGACTTGCCAAAGAC CAGATCTGGTAAGA

TCATGAGAAGAATCTTGAGAAAGATCTCTGCCAACGAAGCAGACCAATTGGGTGACATCACCACTTTGT CCAACCCTCAGTCT

GTCGTTGGTATAATCGACTCCTTTGCTGCTCAATTTGCTAAGAAATAA (SEQ ID NO:11)

FAT

ATGGGGAGACACTTGGCCTTGCTTCTGCTTCTGCTCTTCTTCCTCCAGCATTTTGGAGATGGTGATGGA AGCCAAAGACTTGA

ACCGACCCCTTCCCTCCAGTTTACACACGTCCAGTACAATGTCACTGTGCACGAAAACTCGGCCGCAAA GACCTATGTCGGCC

ACCCTAGAAAAATGGGCATCTACATCTTAGACCCCTCGTGGGAAATAAGGTACAAAATCATCTCAGGAG ACAACGAAAACCTA

TTCAAAGCGGAAGAGTATGTTCTCGGAGACTTTTGCTTTCTAAGGATAAGAACCAAGGGAGGGAATACT GCCATCCTGAACCG

AGAAGTGAGAGACCATTACACACTGGTAATCAAAGCAGTTGAAAAAGTCACAGATGCCGAGGCCCGAGC CAAGGTCAGGGTGC

AAGTGCTGGATACAAACGACTTACGGCCGTTGTTCTCACCCACGTCCTACAGCGTTTCTCTGCCGGAAA ACACAGCCATAAGG

ACCAGTATCGCAAGAGTCAGTGCCACGGATGCGGACATTGGAACCAACGGCGAATTTTACTACAGCTTT AAAGACAGAACGGA

CATGTTTGCCATCCACCCAACCAGTGGTGTGGTTGTTTTGACTGGCAGGCTTGATGTCCTGGAGACCCA GCGCTATGAGCTGG

AGATCTTGGCTGTGGACCGGGGAATGAAGCTGTACGGTAGCAGTGGGGTCAGCAGTCTGGCCAAGCTGA CGGTTCACGTGGAG

CAGGCTAACGAGTGTGCACCCGGGATAACCGCCGTGACGTTATCACCATCTGAGCTGGACAAGGACCCA ACGTACGCCATTAT

CACTGTGGAGGACTGCGATCAGGGTGCCAACGGGGAGATAGCATCTTTGAGCATTGTGGCTGGCGACCT CCTTCAGCAGTTTA

AAACGGTGAGGTCTTTCCCAGGGAGTAAAGCATTCAAAGTGAAAGCCGTCGGGGGCGTCGACTGGGACA GCCATCCTTATGGC

TACAACCTGACAGTGCAGGCTAAAGACAAAGGAACTCCTCCGCAGTTTTCCCCTGTGAAAGTCATTCAC GTCATTTCTCCTCA

GTTCAGAGCTGGCCCGGTCAAGTTTGAAATGGATGTTTACAGAGCTGAGATCAGTGAGTTTGCCCCTCC ACATACACCCGTGG

TCCTGGTCAAGGCTATTCCTAGTTATTCCCATTTGAGGTACGTTTTTAAAAGCACTCCTGGAAAACCCA AATTCGGTTTAAAT

CACAACACGGGTCTCATTTCCATTTTAGAACCAATTAAAAGGCAGCACACATCCCATTTTGAGCTTGAG GTGACAACAAGTGA

CAGACGAGCCTCCACCAAAGTCGTGGTCAAAGTTGTAGGTACAAACAGCAACCCCCCGGAGTTTACACA GACCTCGTACAAAG

CATCCTTTGATGAGAATGCACCCGTCGGTACCCCGGTCATGAGGGTGAGCGCGGTTGACCCTGACGAGG GGGAGAATGGCTAC

GTGACTTACAGTATTGCAAACTTAAATCACGTGCCATTTGTCATCGACCACTTTACGGGTGCTGTGAGT ACCTCTGAGAATCT

GGACTATGAACTGATGCCTCGAGTCTACACGCTGAGGATTCGTGCTTCCGACTGGGGCTTACCGTACCG CCGGGAAGTTGAAG

TCCTTGCCACAATTACTCTGAATAACCTGAATGACAACACCCCCCTGTTTGAGAAGACAAACTGTGAAG GGACAATTCCCCGA

GACCTGGGTGTAGGGGAGCAGATAACCACGGTTTCTGCCATTGACGCTGATGAGCTGCAGTTGGTCCGG TACCAGATTGAAGC

TGGAAATGAGTTGGATTTGTTTGGCTTAAACCCCAGCTCTGGTGTGCTGTCATTGAAGCACTCGCTCAT GGACGGCTTGGGTG

CAAAGGTTTCCTTTCACAGCTTGAGAATCACAGCTACAGACGGAGAAAATTTTGCCACACCATTATATA TCAACCTAACGGTG

GCTGCCAGTCGCAAGCCAGTAAACTTGCGGTGTGAGGAGACCGGTGTTGCCAAAATGCTGGCAGAGAAA CTCCTGCAGGCGAA

TAAATTACACCATCAGGGGGACGCGGAGGATATTTTCTTTGATTCTCACTCCGTCAACGCCCATGCCCC ACAGTTTAGGGGTT

CTCTTCCAACAGGAATTGAGGTAAAGGAGGACCTCCCAGTGGGCGCCAGTATACTATTCATGAATGCTA CTGACCTTGACTCT

GGCTTCAATGGGAAACTGGTCTATGCTATCTCTGGAGGGAATGATGACAGTTGCTTTACTGTTGACATG GAAACAGGAATGCT

GAAAGTCCTCTCTCCACTTGACCGAGAAGTAACGGACAAATACACACTGAACATTACCGTGTATGACCT TGGTATACCCCAGA

GGGCTGCCTGGCGCCTTCTGGATGTCACCGTCCTGGATGCCAATGACAACGCGCCCGAGTTTTTACAGG AGAGCTATTTTGTC

GAAGTGAGCGAAGACAAGGAGATAAACAGTGAAATCATCCAGGTAGAGGCCACCGATAAAGACCTGGGC CCCAGCGGACACGT

GACATACGCCATCCTCACGGACACAGAGAAGTTTGCGATCGACAGGGTGACCGGTGTGGTGAAAATTAT CCAGCCTTTGGATC

GTGAAGTGCAGCGTGTACATTACCTGAAGATCGAGGCCAGGGACCAAGCCACAGAGGAACCCTGGCTGT CCTCCACTGTGCTT

CTGAAAGTGTCACTCGATGATGTTAATGACAACCCACCTAGGTTCATTCCACCCAGTTACTCCGTGAAG GTTCGAGAAGACCT

ACCGGAAGGAACCATCATCATGTGGTTAGAAGCCCATGACCCTGATGTAGGTCAGTCCAGTCAGGTGAG ATACAGCCTCCTGG

ACCACGGAGAAGGCCACTTCGATGTGGATAAACTCAGCGGGGCAGTGAGAATTGTCCAGCAGCTGGACT TTGAGAAGAAGCAA

CTGTATAATCTCACCGTGAGGGCCAAAGACAAAGGGAAGCCGGCGTCTCTGTCTTCCACTGGCTACGTG GAAGTGGAGGTCGT

GGACGTGAATGAGAACTTACACGCGCCAGTGTTCTCCAGCTTCGTGGAGAAGGGCACAGTGAAAGAAGA CGTCCCTATGGGCT

CATCAGTAATGACCGTGTCAGCTCACGATGAGGACACCGGGAGAGATGGAGAGATCCGGTATTCCATCA GAGATGGCTCTGGT

GTTGGTGTTTTCAGGATAGATGAAGAAACAGGTGTCATAGAGACCTCAGATCGACTGGACCGAGAGTCG ACTTCCCACTACTG

GCTCACCGTCTACGCCACAGATCAGGGTGTGGTGCCTCTGTCATCCTTCATAGAGGTCTACATAGAGGT TGAGGATGTCAATG

ACAACGCACCACAGACATCAGAGCCTGTGTATTATCCTGAAATAATGGAGAATTCACCCAAGGATGTAT CTGTGGTCCAGATT

GAGGCATTTGACCCGGATTCCAGCTCCAGTGACAAGCTGACGTACAGAATTACAAGTGGAAATCCCCAA GGGTTCTTCTCAAT

ACACCCTAAAACAGGTCTCATCACAACCACATCGAGGAAGCTGGACCGAGAGCAGCAGGATGAACACAT TCTGGAAGTTACTG

TGACAGACAATGGTGTACCTCCCAGATCCACCATTGCCAGGGTCATTGTGAAAATCCTGGATGAGAACG ACAACAGGCCTCAG

TTCCTTCAGAAGTTTTATAAAATCAGGCTCCCGGAGCGAGAAAAAGCTGATGGAGACCGGAGCGCGAAG CGCGAGCCTCTCTA

CCGAGTCATAGCCGCAGATAAGGATGAAGGGCCCAATGCCGAGCTCTCCTACAGCATCGAGGAAGGGAA CGAGCACGGCCGGT

TTTCCATTGAACCCAAGACAGGAGTGGTCTCATCCAAAAAGTTCTCTGCGGCTGGAGAATACGACATTC TTTCTATTAAGGCA

ATTGACAATGGGCGCCCCCAGAAGTCATCGACCACCAGACTCCATATTGAATGGATCTCCAAACCCAAG CCGTCCTTGGAGCC

GATTTCGTTTGAGGAATCGGTTTTCTCGTTTACTGTAATGGAGAGTGATCCGGTGGCTCACATGATCGG CGTGATCTCCGTTG

AGCCTCCTGGCATGCCTCTGTGGTTTGACATCATCGGGGGCAACTATGACAGTCACTTTGATGTGGACA AGGGCACTGGAACC

ATCATTGTGGCCAAGCCCCTTGACGCAGAGCAGAAGTCCAGCTATAACCTCACAGTGGAGGCGACAGAC GGGACCTCCACTAT

CCTCACCCAGGTACTCATCAAAGTAATAGATACCAATGACCACCGCCCTCAGTTTTCTACCTCGAAATA CGAAGTCTCTGTTC

CCGAAGACACAGAGCCAGAAACAGAGATTCTGCAAATCAGCGCCGTAGACAGGGACGAGAAAAACAAAC TGATCTACACCCTC

CAGAGCAGCATAGATCCAGCAAGTCTCAAGAAATTCCGCCTCGATCCTGCAACAGGCGCTCTCTACACA TCTGAGAAGCTCGA

TCACGAAGCCATTCACCAGCACGTCCTCACAGTCATGGTCCGGGATCAGGATGTCCCTGTGAAACGCAA CTTTGCCAGAATCA

TTGTGAATGTCAGTGACATGAATGACCACTCTCCGTGGTTCACCAGTTCGTCCTATGAAGGGCGGGTTT ATGAGTCGGCAGCC

GTGGGCTCGGTCGTGCTACAGGTTACAGCTCTGGACAGAGACAAAGGGAGAAATGCTGAAGTGCTCTAC TCCATCGAGTCAGG

AAACATTGGAAATTCCTTTACAATCGACCCCATCTTGGGCTCTATAAAAACTGCCAGAGAATTGGATCG AAGTCACCAAGTAG

ACTATGATTTAATGGTAAAAGCTACAGACAAAGGGGAGCCACCAATGAGCGAAATGACCTCCGTGCGGA TCTCTGTCACCGTC

GCCGACAATGCCTCTCCTAAGTTCACATCCAAGGAGTACTCGGCTGAGATTAGTGAAGCCATCAGGATT GGGAGTTTTGTTGG

AATGGTCTCTGCTCACAGTCAGTCATCAGTGATGTATGAAGTAAAAGATGGAAATATAGGCGATGCATT TAATATCAATCCAC

ATTCAGGAAGCATCGTCACTCAGAGAGCCTTGGATTTTGAGACACTGCCCATTTATACATTGACAGTAC AAGGGACCAACATG

GCCGGCTTGTCCACCAATACAACGGTGGTAGTGCACATACAGGATGAGAATGACAACCCTCCAGCTTTC ACACGGGCGGAATA

TTCAGGATTCATTAGTGAATCAGCCTCAGTCAACAGCGTGGTGCTAACGGATAAGAATGTTCCGCTCGT GATCCGAGCCACCG

ACGCTGATCGGGAATCCAATGCTCTGCTCGTCTATCAAATTGTCGAGCCATCTGTGCACAACTATTTTG CCATTGATCCCACC

ACCGGTGCCATCCATACCGTACTGAGTCTGGACTATGAAGAGACACGTGTCTTTCACTTCACCGTCCAA GTGCATGACATGGG

GACGCCTCGTCTGTTTGCTGAGTATGCAGCAAATGTGACCGTGCATGTGATTGACATCAATGACTGCCC CCCTGTCTTCTCTA

AGTCACTGTACGAAGCATCCCTCCTATTGCCGACGTACAAAGGCGTGAACGTCATCACAGTGAATGCCA CAGATGCCGACTCC

AGGGCGTTCTCCCAGTTAATATACTCCATCACCAAAGGCAACATTGGGGAGAAGTTCTCCATGGACCAC AAGACTGGCACCAT

AGCAATTCAGAACACAACCCAGTTACGGAGCCGCTATGAGCTGACCGTCCGCGCCTCCGATGGCCGGTT TACAAGCGTGGCCT

CCGTGAGAATCAACGTGAAGGAAAGCAGAGAGAGTCCTCTCAAGTTTACCCAAGATGCCTACTCTGCGG TGGTGAAGGAGAAC

TCCACCGAAGCCAAAACCTTAGCTGTCATTACCGCGATAGGGAACCCGATTAACGAGCCTTTGTTTTAC CGTATCCTCAACCC

AGACCGCAGATTTAAAATCAGCCACACCTCAGGCGTGTTGTCAACCACTGGGATACCATTTGATCGGGA GCAACAGGAGACGT

TTGTTGTGGTGGTAGAGGTGACTAAAGAACGGGAGCCGTCGGCCGTGGCCCACGTTGTGGTGAAGGTCA CCGTGGAAGACCAG

AATGATAATGCACCCGTGTTTGTCAACCTTCCCTACTATGCTGTGGTGAAGGTGGATGCTGAGGTGGGC CATGTCATCCGCCA

CGTCACTGCCATTGACAGAGACAGTGGCAGAAACGGTGACGTTCACTACTACCTTAAGGAGCATCATGA CCACTTTGAGATTG

GACCCTCTGGTGACATTTCTCTGAAAAAGCAATTTGAGCACGACACCTTGAATAAAGAATACCTTGTCA CAGTGGTTGCGAAG

GACGGGGGGAACCCAGCTTTCTCCGCAGAAGTTCTAGTTCCCATCACCGTCATGAACAAAGCCATGCCC GTGTTTGAAAAGGC

TTTCTACAGTGCAGAGATTCCCGAGAACGTCCAGACGCACAGCCCAGTGGTCCACGTCCAAGCCAACAG CCCAGAAGGGTTGA

AAGTGTTCTACAGTATCACAGACGGGGACCCTTTTAGTCAGTTTACTATCAACTTCAACACTGGGGTGA TAAACGTCATCGCA

CCGCTGGACTTTGAGTCCCACCCAGCCTATAAGCTAAGCATACGGGCCACTGACTCCCTGACTGGCGCC CACGCTGAAGTGTT

TGTTGACATCGTAGTAGAAGACATCAATGACAACCCTCCCGTGTTTGTGCAACAGTCTTACTCGACAAC CCTGTCTGAAGCAT

CTGTCATCGGAGCGCCTATCCTTCAAGTTAGAGCCACCGACTCTGACTCGGAACCAAATAGAGGGATTT CCTACCAGCTGATT

GGAAATCACAGCAAAAGCCACGATCACTTTCACATAGATAGTCACACTGGGCTGATTTCACTGGTGAGG GCTTTGGATTACGA

ACAGTTCCAGCAGCACAAGCTGCTCGTAAGGGCTGTTGATGGAGGAATGCCGCCACTGAGCAGCGATGT GGTCGTCACTGTGG

ATGTCACCGACCTCAACGATAACCCGCCTCTGTTTGAACAACAGGTTTACGAAGCTAGGATCAGTGAGC ACGCTGCCCACGGG

CATTTTGTGATGTGCGTAAAGGCCTGTGATGCAGATCGCTCAGACCTAGACAGGCTGGAGTACTCCATT CTGTCCGGCAATGA

TCACAAGAGCTTTGTCATTGACGGGGAGACAGGAATCATCACGCTCTCCAACCCGCGCCGCCACACCTT GAAGCCGTTCTATA

GTCTCAACGTTTCTGTGTCTGATGGGGTTTTCCGAAGCTCGGCTCGGGTGAATGTCACCGTGATGGGAG GGAATTTGCACAGC

CCTGTCTTTCACCAGAATGAGTATGAGGTAGAGCTGGCTGAAAACGCCCCCTTGCACACCCTGGTGGTC CAAGTGAAGGCTAC

TGACAGAGATTCCGGTATCTACGGCCACCTGACTTACCACCTTGTAAATGACTTTGCCAAAGACAGGTT TTACGTGAACGACG

GAGGGCAGGTCTTCACTCTGGAGAGACTTGATCGAGAGGCTCCAGCAGAGAAAGTGATCTCAGTCCGTT TAATGGCTAAGGAT

GCTGGGGGGAAGGTCGCCTTCTGCACTGTCAACGTCATCCTCACGGACGACAATGACAACGCACCACAG TTTCGCTCAACCAA

GTACGAGGTGAACGTGGGGTCCAGCGCCGCCAAAGGGACGTCGGTCGTCAAGGTCTTCGCGAGTGATGC CGATGAGGGGTCGA

ATGCTGACGTCACCTACGCCATCGAGGCAGATTCGGAAAGTGTCGAGGAGAACTTGGAAATCAACCAAC TGACCGGCCTCATT

ACTACAAAGGAAAGCTTAATAGGTTTAGAGAATGAATTCTTCACTTTCTTCGTTAGAGCTGTGGATAAC GGGTCTCCGCCCAA

AGAGTCTGTTGTTCCTGTCTATGTTAAAATACTTCCCCCGGAAGTGCAGCTTCCTAGGTTCTCAGAGCC CTTTTATACCTATT

CCATTTCAGAAGACATGCCTATTGGCACAGAGATTGACCTCATCCGGGTAGAGCATAGCGGGACTGTTC TCTACACCCTGGTC

AAAGGCAATACTCCCGAGAGTAACAGGGACGAGTTCTTTGTGATTGACCGGCAGAGTGGGAGACTGAAG CTGGAGAAGAGCCT

TGACCACGAGACCACTAAGTGGTATCAGTTTTCCATCCTGGCCAGGTGTACTCTGGATGACTACGAGGT GGTGGCTTCTATAG

ATGTCAGTATCCAGGTGAAAGACGCTAATGATAACAGCCCAGTTTTGGAGTCCAATCCATACGAGGCAT TTATTGTCGAAAAC

CTGCCAGCAGGGAGTAGGGTCATCCAGGTCAGAGCATCTGACCTAGACTCAGGAGTCAACGGCCAAGTC ATGTACAGTCTAGA

TCAGTCCCAAGATGCAGACATCATCGAGTCTTTTGCCATTAACATGGAAACAGGCTGGATTACAACCCT CAAGGAGCTTGACC

ATGAAGAGAGAGCCAGTTACCAGATTAAAGTGGTTGCCTCAGACCATGGTGAAAAGGTGCAGCTGTCTT CCACCGCCATTGTG

GATGTCACCGTCACTGACGTCAACGACAGCCCGCCTCGATTCACAGCTGAGATTTATAAAGGGACAGTG AGTGAGGATGACCC

CCCAGGGGGTGTGATCGCCATCTTGAGCACCACTGACGCCGACTCTGAAGAGATTAACCGACAAGTGTC GTACTTCATAACAG

GAGGGGATGCATTGGGACAGTTTGCTGTGGAAAATATGCAGAATGACTGGAGGGTGTACGTGAAGAAAC CTCTCGACAGGGAA

CAAAAGGACAGTTACCTTCTGACCGTCACTGCAACAGATGGGACCTTCTCTTCCAAAGCTAGAGTTGAA GTCAAGGTTCTCGA

TGCCAATGATAACAGTCCAGTGTGTGAGAGGACCGCATATTCTGATGCCATTCCCGAAGACGCTCTTCC GGGGAAGCTGGTCA

TGCAGGTCTCTGCCACAGATGCAGATATCCGGTCCAACGCGGAGATCACTTACACTTTATTTGGCTCAG GTGCAGAAAAGTTT

AAACTGAATCCAGACACAGGTGAACTGAGAACATTAGCCCTCCTTGATCGTGAGGAGCAAGCAGTTTAT CATCTTCTGGTCAA

GGCCACAGACGGAGGGGGCAGATCCTGTCAGGCAACTATTGTGCTCACGTTAGAAGATGTAAATGACAA CACCCCCGAGTTCA

CCGCGGATCCATACGCCATCACGGTATTTGAAAACACAGAGCCTGGGACACCGTTGACCAGAGTGCAGG CCACCGATGCAGAC

GCAGGGTTGAATCGGAAGATTTCCTACTCACTGCTTGACTCTGCTGACGGGCAGTTCTCCATTAACGAG CAGTCCGGAATTCT

TCAGTTGGAAAAGCATTTGGACAGGGAACTACAGGCAGTCTATACTCTCACTTTGAAAGCAGCGGACCA AGGATTGCCAAGGA

AATTGACAGCCACTGGCACGGTGGTTGTGTCTGTTTTGGATATAAATGACAACCCACCTGTGTTTGAGT ACCGTGAATATGGT

GCCACCGTGTCAGAGGACATTGTCATCGGGACCGAAGTTCTCCAGGTGTACGCAGCCAGTCGGGATATC GAGGCGAATGCAGA

AATCACATACGCAATCATAAGTGGGAACGAACACGGAAAATTCAGCATCGATTCTAAGACAGGGGCCAT ATTTATCATTGAGA

ACCTGGATTATGAAAGCTCCCATGGCTATTACCTGACTGTGGAAGCCACTGATGGAGGCACGCCCTCGT TGAGTGACGTGGCG

ACCGTGAACATCAACATCACAGATATTAACGATAACAGCCCAGTGTTCAGCCAGGACAGCTACACCACA GTGGTCAGCGAAGA

CGCGGCCCTGGAGCAGCCCGTCATTACAATTATGGCTGATGATGCTGATGGCCCTTCAAACAGCCACAT CCTCTACTCCATTA

TAGAGGGTAACCAAGGAAGTCCATTCACAATCGACCCTGTCAGAGGAGAAATCAAAGTAACGAAGCCCC TAGACCGCGAAACG

ATCTCAGGTTATACGCTCACGGTGCAGGCTGCCGACAACGGCAATCCACCCAGAGTCAACACCACCACA GTGAACATCGATGT

CTCCGATGTCAACGACAATGCTCCCCTCTTCTCCAGAGACAACTACAGTGTCATCATCCAGGAAAACAA GCCCGTGGGTTTCA

GCGTCCTGAAGCTAGTAGTGACAGACAAGGACTCGTCCCACAACGGCCCCCCTTTCTCCTTTGCTATTG TGAGTGGAAATGAT

GACAACATGTTTGAGGTGAACCAGCACGGGGTCCTCCTGACAGCGGCAACAGTCAAGAGGAAAGTGAAG GACCATTACCTTCT

GCACGTTAAGGTGGCTGACAATGGAAAGCCTCAGCTGTCTTCGTTGACACACATTGACATCAGGGTTAT TGAGGAGAGCATCC

ACCCTCCTGCCATTTTGCCACTGGAGATTTTCATCACTGCTTCTGGAGAGGAATACTCAGGCGGGGTCA TAGGAAAGATCCAT

GCCACAGACCAGGATGTGTATGACACCTTGACGTACAGTCTGGATCCCCACATGGATGGCCTGTTCTCT GTTTCCAGCACGGG

GGGTAAACTGATTGCACACAGAAAGCTGGATATAGGCCAGTACCTTCTTAATGTCAGCGTGACAGACGG GAAGTTTACAACGG

TGGCTGACATCACCGTGCACATCCAGCAAGTGACCCAGGAGATGCTGAACCACACCATCGCTATCCGAT TTGCAAATCTCACC

CCGGAAGAGTTTGTCGGCGACTACTGGCGCAACTTCCAGCGAGCTTTACGCAACATCCTGGGCATCCGG AAGAACGACATACA

GATTGTCAGCTTGCAGCCCTCCGAACCCCACTCCCACCTTGACGTCTTACTCTTTGTAGAGAAATCAGG GGGCACCCAGATCT

CAACGAAACAACTTCTGCACAAGATCAATTCTTCCGTCACGGACATCGAGGAAATCATTGGCGTGAGGA TACTGGATGTGTTC

CAGAAACTCTGTGCAGGGCTGGATTGCCCGTGGAAATTCTGTGATGAGAAGGTTTCTGTGGATGAAAAC ATTATGTCAACTCA

TAGCACAGCCAGACTGAGTTTTGTGACTCCCCGGCACCATAGAACAGCCGTGTGTCTCTGCAAAGATGG GACATGCCCGCCTG

TCCACCAAGGGTGCGAAGATAACCCCTGTCCTGCAGGATCCGAATGTGTCGCTGATCCCCGAGAAGAGA AGTACAGCTGTGTG

TGTCCTGGTGGCGGGTTCGCCAAATGTCCAGGGAGTTCATCCATAACTTTTACCGGCAGCAGCTTTGTG AAATATCGTCTGAT

GGAAAATGAAAACCGACTGGAGATGAAGTTGACCATGCGCCTGAGAACCTACTCTTCCCACGCGGTTGT GATGTACGCTCGAG

GAACTGACTACAGTATCCTGGAGATTCATACTGGGAGACTGCAGTACAAATTTGACTGTGGAAGTGGCC CTGGGATCGTCTCT

GTTCAGAGCATTCAAGTCAACGATGGGCAGTGGCATGCAGTGTCCCTGGAAGTGGAGGGGAATTATGCA AAATTGGTTCTAGA

TGAAGTCCACACTGCCTCGGGCACAGCCCCAGGAGCTCTGAAAACCCTCAACCTGGATAACTACGTAAT TTTTGGTGGCCACC

TCCGCCAGCAAGGGACAAAACATGGACGAAACACCCAGGTGGCCAATGGTTTCAGGGGCTGCATGGACT CTATTTATTTGAAT

GGGCAGGAGCTACCTTTGAACAACAAACCAAGAGCCTATGCACACATCGAAGAATGGGTGGACCTAGCT CATGGGTGCTTGTT

AACTGCCACCGAAGACTGTTCCAGCAACCCTTGTCAGAATGGAGGCGTCTGCAATCCCTCGCCCACTGG AGGTTATTACTGCA

AGTGCAGTGCATTGCACGCAGGGACGTACTGTGAGGTGAGCGTCAACCCGTGCTCCTCCAACCCCTGCC TCTACGGAGGAACG

TGCATGGTAGACAACGGAGGTTTTGTTTGCCAGTGCAGGGGGCTGTACACTGGCCAGAGATGTCAGCTT AGTCCGTACTGCAA

AGATGAACCCTGTAAAAATGGTGGAACGTGTTTTGACAGTTTGGATGGTGCTGTCTGTCAGTGTGACTC AGGCTTTAGGGGAG

AAAGATGTCAGAGTGACATTGACGAGTGTGCTGGGAACCCCTGTCGGAACGGGGCCCTTTGCGAGAACA CGCATGGCTCCTAT

CACTGTAACTGCAGCCAGGAGTACAGAGGGAAGCACTGTGAGGATGCCACTCCCAACCACTACGTGTCC ACCCCGTGGAACAT

CGGACTGGCCGAAGGAATCGGAATTATTGTGTTTATAGCCGGGATATTCTTACTGGTGGTGGTGTTTGT CCTCTGCCGAAAGA

TGATCAGTCGGAAGAAGAAACACCAGGCGGAACCTGAAGACAAGCGTTTGGGGCCAACCACGGCTTTCT TACAGAGACCTTAC

TTTGATTCCAAGCCGAGCAAGAACATTTACTCTGACATCCCGCCCCAGGTGCCCGTGCGTCCCATTTCC TACACTCCGAGCAT

TCCCAGTGACTCTAGAAACAATCTGGACCGGAACTCGTTTGAAGGCTCGGCAATCCCAGAGCACCCAGA ATTCAGCACTTTTA

ACCCCGAGTCTATGCACGGACATCGGAAAGCCGTGGCTGTGTGCAGCGTGGCTCCAAACTTGCCTCCCC CACCCCCTTCCAAC

TCTCCCTCAGACAGCGACTCCATTCAGAAGCCCAGCTGGGACTTCGACTACGACGCTAAAGTGGTGGAT CTTGACCCTTGTCT

TTCCAAGAAGCCCCTGGAGGAAAAACCCTCTCAGCCATACAGTGCCCGGGAGAGCCTGTCCGAGGTGCA GTCCCTTAGCTCCT

TCCAGTCAGAGTCCTGTGATGACAATGGGTACCACTGGGATACATCAGACTGGATGCCCAGTGTTCCTC TGCCAGACATACAA

GAGTTCCCCAATTACGAGGTTATCGATGAGCACACGCCCCTCTACTCAGCTGATCCAAATGCCATCGAC ACTGACTATTACCC

TGGGGGTTATGACATTGAAAGTGACTTTCCACCCCCACCAGAGGACTTCCCTGCACCCGATGAACTGCC ACCATTGCCTCCAG

AATTCAGCGACCAGTTCGAGTCCATACACCCACCCAGAGACATGCCCGCAGCAGGTAGCTTGGGGTCTT CCTCCAGGAATCGT

CAGAGGTTCAACCTGAATCAGTACCTGCCCAATTTCTACCCCGTCGATATGTCTGAACCTCAGAAACAA GGCGCTGGTGAGAA

CAGTACCTGTAGAGAACCCTACACTCCCTACCCTCCAGGGTATCAAAGAAACTTCGAGGCGCCCACCAT AGAAAACATGCCCA

TGTCTGTGTACACCTCTACGGCTTCCTGCTCCGATGTGTCAGCGTGCTGCGAAGTGGAGTCTGAGGTCA TGATGAGTGACTAC

GAGAGCGGGGACGACGGCCACTTTGAAGAGGTGACCATTCCCCCGCTAGATTCCCAGCAGCATACGGAAGTGTGA (SEQ IDNO:12)

FAT

ATGAAGATTAAAAAATATGTAACTCCTGTAAAAAGAAAAGCTTTCACCATACTCCAATGGATTTCACTA CTGTGTAGTCTATG

GTTGATCCCCACTGTACAAAGCAAGGCCGATGAGAAGCACACGGCGACCCTGGAGTATAGACTAGAGAA CCAACTGCAAGATC

TATATAGGTTTAGCCATAGTGTATATAATGTTACCATACCAGAAAATAGTCTGGGCAAGACTTACGCCA AGGGAGTATTGCAT

GAAAGACTGGCCGGCCTGAGAGTTGGCTTGAACGCAGAGGTTAAGTATAGGATAATTAGTGGCGATAAG GAGAAGCTATTTAA

GGCCGAGGAGAAACTGGTCGGAGATTTTGCCTTCTTAGCGATTCGAACGCGGACAAATAACGTTGTGCT AAACAGAGAAAAAA

CTGAGGAATACGTTATAAGAGTGAAGGCACATGTACATTTGCACGACCGAAATGTATCAAGCTATGAAA CGGAGGCGAATATC

CACATCAAAGTACTGGATCGCAATGACCTGAGTCCGCTGTTTTATCCGACCCAGTACACCGTTGTTATT CCGGAGGACACGCC

CAAATATCAAAGTATTTTAAAGGTCACAGCTGACGATGCTGACCTCGGCATCAATGGGGAAATCTACTA CAGCCTCCTGATGG

ATAGTGAATACTTTGCTATCCATCCAACAACTGGCGAAATTACTCTCCTGCAGCAGCTTCAGTATGCGG AGAACTCGCACTTC

GAGCTCACGGTGGTGGCCTACGATCGGGGATCATGGGTGAACCATCAGAACCACCAGGCCAGCAAGACG AAGGTTAGTATTTC

GGTGAAACAGGTTAACTTTTACGCTCCAGAGATTTTCACGAAAACCTTCTCGAGCGTGACGCCAACATC AAACCCTTTGATTT

ATGGAATTGTACGAGTAAACGACAAAGACACTGGGATAAATGGCAACATAGGGCGATTGGAAATCGTCG ATGGAAATCCGGAT

GGCACGTTTCTTCTGAAGGCGGCGGAGACCAAAGACGAGTACTACATCGAATTGAATCAGTTTGCCCAT CTTAACCAGCAACA

TTTCATTTACAACTTAACCCTACTGGCGGAGGACCTCGGAACTCCCCGTCGATTCGCCTACAAATCCGT TCCGATTCAAATCA

AGCCCGAGAGCAAAAATATACCCATATTCACACAGGAGATTTACGAAGTATCCATTCCAGAAACGGCAC CCATTAACATGCCT

GTGATAAGGCTCAAAGTAAGCGATCCAGATTTGGGCAAAAATGCATTGGTCTACTTGGAAATCGTGGGT GGAAATGAGGGCGA

CGAGTTCCGAATTAATCCCGATTCGGGAATGTTGTACACAGCAAAGCAACTGGATGCCGAAAAGAAGTC AAGTTATACCTTAA

CAGTCTCCGCCATTGATCAGGCAAATGTTGGGTCGCGGAAACAATCTTCAGCCAAGGTGAAAATCAGCG TACAGGATATGAAC

GACAATGATCCCATTTTTGAGAATGTCAATAAGGTCATTAGTATCAATGAGAACAACTTGGCTGGCTCG TTTGTTGTGAAGCT

TACTGCCAAGGACAGGGATTCTGGTGAAAATTCATACATATCGTATAGTATTGCCAATCTAAATGCGGT TCCATTTGAAATCG

ATCACTTTAGCGGTATAGTTAAGACCACATCACTGCTTGACTTTGAAACAATGAAGCGTAACTATGAGC TGATAATCCGTGCA

TCCGATTGGGGATTGCCGTACAGAAGACAGACGGAAATCAAACTGTCCATCGTCGTCAAGGATATCAAC GATAATCGGCCGCA

GTTTGAACGTGTGAACTGCTATGGCAAAGTGACCAAATCGGCGCCGATGGGCACCGAGGTATTCGTTAC CTCAGCCATTGACT

TTGATGCAGGCGATATAATATCCTATAGGTTGAGCGACGGCAACGAGGATGGCTGCTTTAACTTGGACC CCACATCGGGTTCC

CTGTCTATTTCCTGCGACCTGAAGAAAACAACCTTAACAAACCGTATTCTCAAAGTTTCCGCCACGGAC GGCACCCACTTTTC

CGATGACTTGATCATCAATGTACACCTAATGCCCGAAGATTTGGGTGGAGATTCCAGTATTCTACATGG TTTTGGATCCTTTG

AGTGCCGGGAAACCGGCGTGGCCAGGAGATTGGCGGAAACATTATCGTTGGCCGAAAAAAACAATGTAA AGAGTGCATCGCCA

TCCGTTTTCAGTGACTTGTCTCTAACACCCAGTCGATATGGCCAAAATGTGCATAGACCAGAGTTCGTG AACTTCCCTCAGGA

GCTGTCCATTAACGAAAGTGTCCAATTGGGCGAAACAGTTGCTTGGATAGAGGCCAAAGATCGCGATTT GGGCTACAATGGAA

AGCTGGTATTTGCAATTTCAGACGGGGACTACGATTCGGTTTTTCGTATTGATCCAGACCGCGGTGAAC TGCAGATTATTGGA

TATTTGGATAGAGAGCGTCAAAATGAATATGTTCTCAACATCACCGTCTACGATCTGGGTAACCCGACC AAATCGACGTCAAA

AATGTTGCCAATAACGATCCTCGACGTGAACGATAATCGCCCGGTTATTCAGAAGACGTTGGCCACCTT CCGGCTGACTGAGA

GCGCCAGGATAGGAACTGTGGTACACTGCCTTCATGCCACGGATGCGGATTCTGGAATCAATGCTCAGG TGACATATGCCCTG

TCGGTTGAGTGCAGCGATTTCACAGTAAATGCTACTACGGGATGTCTTCGTCTGAACAAACCACTGGAT CGCGAGAAGCAGGA

TAACTACGCTCTTCACATAACTGCCAAGGATGGTGGCAGTCCCGTGCTATCCTCGGAGGCATTGGTTTA CGTCCTGGTCGACG

ATGTCAACGACAACGCGCCCGTTTTCGGAGTGCAAGAGTACATATTTAAGGTGCGCGAAGATCTGCCCC GTGGAACAGTGTTG

GCCGTAATCGAGGCGGTGGACGAAGATATTGGACCCAATGCCGAGATCCAATTCTCTTTGAAAGAGGAG ACCCAGGATGAGGA

ACTATTCAGAATCGATAAGCACACGGGTGCAATTAGGACTCAAGGATATCTGGACTATGAGAACAAACA AGTGCACAACCTTA

TTGTCAGTGCCATCGATGGCGGAGATCCCTCTCTAACTTCGGACATGTCCATCGTAATAATGATCATCG ACGTCAACGAGAAC

CGATTTGCGCCCGAATTCGACGACTTTGTGTACGAGGGAAAGGTAAAGGAGAACAAGCCGAAGGGAACG TTCGTAATGAATGT

CACAGCACGGGATATGGACACGGTGGACCTGAACTCCAAGATCACGTACTCAATAACAGGTGGCGATGG ACTGGGAATTTTTG

CGGTTAACGACCAAGGTTCAATAACTTCCTTGTCGCAACTCGATGCGGAGACGAAAAACTTTTACTGGC TGACGCTCTGTGCA

CAGGATTGCGCAATAGTTCCCCTCAGCAATTGTGTGGAAGTTTACATACAAGTCGAAAACGAAAACGAT AACATTCCTCTTAC

GGACAAACCAGTGTACTACGTTAATGTCACGGAAGCCAGTGTGGAAAATGTGGAGATCATTACCCTAAA GGCTTTCGATCCCG

ATATAGATCCCACTCAGACTATAACATATAACATAGTTTCCGGAAATCTTGTCGGGTACTTTGAAATTG ATTCGAAAACAGGA

GTGATTAAGACGACAGAACGCAAATTGGATAGAGAAAATCAAGCGGAACATATTTTGGAGGTGGCTATA TCAGATAACGGATC

TCCAGTACTATCTTCTACATCGCGAATCGTTGTGTCAGTACTGGATATTAACGATAACAGCCCCGAGTT TGACCAAAGGGTCT

ACAAGGTGCAAGTTCCGTCTTCAGCCACAGTCAATCAATCTATTTTTCAGGTTCACGCTATCGACAGCG ACAGTGGCGAAAAT

GGTCGAATTACCTACTCAATTAAGTCCGGAAAGGGTAAGAATAAATTTCGCATCGATAGCCAAAGGGGC CATATACATATAGC

AAAACCATTGGACTCCGACAATGAGTTTGAGATTCACATCAAGGCTGAGGACAACGGAATTCCTAAAAA GAGTCAAACTGCTA

GAGTTAATATTGTTGTAGTTCCTGTAAATCCTAATTCCCAAAATGCACCGTTGATAGTCAGAAAGACAT CCGAAAATGTCGTT

GATCTTACGGAAAATGACAAGCCTGGATTTTTGGTCACTCAAATTTTAGCTGTCGATGATGACAACGAC CAGCTGTGGTACAA

CATTTCCAATGGCAATGACGACAATACCTTTTACATTGGCCAAGACAACGGAAACATACTGCTTTCAAA ATATTTGGACTACG

AGACCCAACAGTCCTATAATCTGACTATCAGCGTCACTGATGGCACATTCACAGCGTTTACTAATCTTT TGGTTCAAGTGATC

GATATTAATGACAACCCCCCTCAGTTCGCTAAAGATGTGTATCATGTCAATATATCCGAAAATATTGAA GAGGAATCAGTTAT

AATGCAACTCCACGCCACTGACAGAGATGAGGACAAGAAGCTATTCTATCACCTGCACGCAACTCAGGA TCCGTCGTCGCTGG

CATTGTTCCGAATCGATTCCATAAGTGGAAATGTCATTGTCACTCAGAGATTGGATTTTGAAAAGACTG CGCAGCATATACTC

ATCGTTTTTGTTAAGGATCAAGGAGCGCCTGGAAAAAGAAACTATGCCAAGATAATTGTAAACGTGCAT GACCACAACGACCA

TCATCCAGAATTTACTGCTAAAATAATTCAAAGTAAGGTTCCCGAAAGCGCAGCTATTGGCTCTAAGTT AGCCGAAGTGAGGG

CCATAGATAGAGATAGTGGTCACAATGCCGAGATCCAGTACTCGATTATCACGGGTAACGTGGGTAGTG TGTTTGAGATTGAT

CCGACTTTCGGTATAATCACATTGGCTGGCAACTTGAATATCAACAAGATCCAGGAGTACATGCTTCAA GTGAAGGCCGTAGA

TCTGGGAAATCCACCGCTGTCATCGCAGATTCCGGTACACATCATTGTCACCATGTCCGAGAACGATCC TCCGAAGTTCCCAA

CCAACAACATTGCCATTGAAATATTCGAAAACCTGCCCATCGGAACATTTGTTACTCAAGTCACCGCTC GGTCGTCGTCATCC

ATATTCTTCAATATTATTTCCGGCAACATCAACGAAAGCTTCCGCATTAACCCATCTACTGGAGTTATT GTTATCAATGGAAA

TATCGACTATGAATCCATCAAAGTATTCAACCTTACGGTTAAAGGAACCAATATGGCAGCCGAGTCATC CTGCCAAAATATAA

TTATACATATCCTAGATGCTAACGATAATATTCCGTATTTCGTTCAAAATGAATATGTTGGAGCATTAC CCGAATCCGCCGCT

ATTGGATCTTACGTACTGAAAGTACACGACTCATCAAAAGATCATTTAACATTACAAGTTAAGGATGCG GATGTCGGAGTAAA

CGGAATGGTTGAATACCACATAGTTGACGATCTGGCAAAAAACTTTTTTAAAATAGATTCGACAACTGG CGCTATTGAACTGT

TACGACAATTGGACTATGAAACAAACGCTGGTTATACCTTTGACGTTACGGTTAGTGATATGGGAAAGC CCAAACTACATTCC

ACTACAACTGCACATGTGACGATTCGTGTCATAAATGTTAACGATTGTCCTCCAGTATTTAATGAGCGT GAACTCAATGTAAC

TTTGTTCCTTCCAACTTTTGAGAATGTGTTTGTAAGACAAGTTAGCGCAAAGGATGCTGATAACGATAC CTTAAGGTTTGATA

TTGTGGATGGAAACACCAACGAATGTTTCCAGATCGAAAAATACACCGGAATAATTACAACACGAAATT TTGAAATACTAAAT

AACGAAAATGATCGGGACTATGCCTTGCACGTCCGTGCCTCCGACGGAATTTTCTCTGCAATTTTAATA GTTAAAATTAAGGT

TTTGTCCGCCATCGATTCGAATTTCGCATTCCAACGTGAATCGTACAGATTTTCTGCATTTGAAAATAA CACAAAGGTAGCTA

CCATTGGATTGGTGAACGTAATAGGAAACACACTGGACGAAAACGTTGAGTATCGCATCCTGAACCCAA CACAATTGTTTGAT

ATTGGAATCAGTTCGGGAGCCCTAAAAACCACTGGAGTTATTTTCGATCGCGAAGTAAAGGATTTGTAC AGACTCTTCGTGGA

AGCAAAGTCAATGCTATACGACGGCATGAATTCAAATGTTCGCAGAGCAGTAACGTCCATAGATATATC CGTCTTGGATGTGA

ACGACAATTGCCCCTTGTTTGTCAATATGCCCTATTATGCCACAGTCTCTATTGACGATCCAAAAGGAA CGATTATTATGCAG

GTCAAGGCCATTGACTTGGACAGTGCAGAAAACGGCGAAGTTCGGTACGAACTTAAGAAGGGCAATGGG GAGTTGTTCAAACT

GGACCGCAAATCTGGGGAGTTATCCATAAAGCAGCATGTCGAAGGTCATAACCGAAACTATGAATTGAC AGTGGCTGCCTATG

ATGGCGCCATAACACCATGCTCCTCGGAAGCTCCTCTGCAGGTTAAGGTTATAGATCGTTCGATGCCCG TTTTTGAAAAGCAG

TTTTATACTGTTAGCGTCAAGGAAGACGTGGAAATGTACTCAGCCCTTTCCGTATCCATTGAAGCAGAA AGTCCCCTGGGAAG

GAGTTTAATTTACACAATATCTTCCGAGAGTCAATCGTTTGAAATTGATTACAACACGGGATCAATTTT TGTCGTAAATGAAT

TGGATTACGAGAAAATAAGCTCACACGATGTTTCCATTCGAGCGACTGACAGTCTTTCTGGTGTTTATG CTGAAGTCGTTTTA

TCTGTTTCCATTATGGATGTCAATGACTGCTATCCAGAAATTGAGAGTGATATATACAACCTAACCATT CCGGAAAATGCATC

GTTTGGAACACAAATTCTGAAGATTAATGCAACTGATAACGACTCGGGAGCAAATGCAAAACTTTCCTA TTACATTGAGTCCA

TTAATGGGCAAAATAATTCAGAACTGTTTTACATTGACGTCACAGACGGAAATCTGTATTTAAAGACTC CATTGGACTATGAA

CAAATCAAGTATCATCATATAGTCGTTAACGTAAAGGACCATGGATCGCCATCATTAAGTTCCCGATCA AACGTATTTATAAC

AGGTAGAATTCTATGTCGCTTTATCTCTTACAAACTAATTTATGATTCTATTATTCCAGTTAAAGACTT AAACGACAACGCTC

CATGTTTCGTTGAGCCGTCGTACTTCACCAAAGTGTCAGTGGCAGCTGTTCGTGGACAATTTGTTGCTT TACCTAAAGCATAC

GATAAGGATATTTCCGATACCGATTCTCTGGAATACAAAATTGTTTACGGAAATGAATTGCAAACCTAT AGTATTGATAAGCT

AACAGGAGTGATTTCCCTTCAAAATATGTTAAATTTCACTGATAAAAGTAGCACAGTCTTGAATATTTC CGTCTCCGATGGAG

TTCATACGGCATATGCCCGGCTCAAAATATCCTTATTGCCAGAAAACGTTTACAGTCCACTGTTTGATC AAAGTACTTATGAG

GCTCAAGTACCTGAAAACTTGCTACACGGTCATAATATAATCACGGTAAAAGCATCGGATGGAGACTTT GGCACCTACGCCAA

TCTTTACTACGAAATAGTTTCGGAGGAAATGAAAAAAATCTTTCTCATCGACCAAACGACGGGTGTAAT AACCTCAAAAGTAA

CTTTCGACCGTGAAAAAAAGGATGAGTACGTGGTGCTACTGAAGGTGTCCGACGGTGGCGGAAAATTCG GATTTGCCTCTCTC

AAGGTCATAGTCGTCGACGTGAACGATAACGTTCCTTACTTCCTATTGAAGGAATACAAAATGGTTGTT AGCACAACAGTGGA

AGCAAACCAAACTATCCTGACGGTCAAAGCCAAAGACGACGATATTGTTGATAATGGATCGGTGCATTT CCAAATTGTTCAAA

AATCCAACGATAAGGCAGTAAAGGATGTAATCGAAATCAACGAGAAAACTGGGGATATTGTGTTTAAAA GCAAGGCGGAATCT

TACGGAGTGAACTCATATCAGTTTTTCGTTCGCGCTTCCGATCGCGGTGAACCTCAATTTCATTCGGAA GTTCCAGTGTCAAT

CGAAATAATCGAGACTGATGCCAATATTCCCACTTTTGAGAAATCGTCAGTTCTACTAAAGATCATAGA GTCAACGCCACCAG

GAACCGTGCTAACGAAGCTACATATGATTGGAAACTATACGTTCAAATTCTCAATAGCAGCGGATCAGG ATCACTTCATGATA

TCCGATAGTGGTGAACTGATCCTTCAGCAGACATTGGACAGGGAGCAGCAAGAGTCGCACAATTTGATT GTAGTGGCGGAAAC

TTCCACGGTTCCCGTTTTTTTCGCCTACGCTGATGTTTTGATTGACGTTAGGGACGAAAATGATAACTA TCCCAAGTTTGACA

ACACATTCTACAGTGCCAGTGTTGCGGAAAACAGTGAAAAGGTGATATCCTTGGTGAAAGTATCGGCCA CAGATGCGGACACT

GGGCCAAATGGCGACATTCGCTACTACTTGGAAAGTGATACTGAAAACATTCAAAATATTTTTGACATT GACATTTACTCTGG

CTGGATCACCTTGCTAACCTCCTTGGACAGAGAAGTTCAGTCCGAGTACAATTTCAAAGTAATTGCTGC CGATAATGGCCACC

CAAAGCATGATGCAAAAGTACCTGTAACTATCAAAATCGTAGACTATAATGATAACGCACCAGTATTTA AGTTGCCTATCGAA

GGGCTTTCTGTTTTCGAAAACGCGCTGCCTGGCACGGTTTTAATCAACTTACTCCTAATTGATCCCGAT ATCGAGAAACAGGA

AATGGATTTCTTTATCGTTTCTGGGGACAAGCAAGCCCAGTTTCAGATCGGTAAGAGCGGAGAGTTATT TATTGCCAAACCAT

TAGATCGCGAACAACTCATGTTCTACAACTTAAGCATAATAGCCACTGATGGAAAATTCACTGCCAAAG CCAATGTGGAAATA

GATGTAAAAGACATAAACGACAATACGCCTTACTGCCTAAAACCCCGCTATCATATCTCCACTAATGAA TCAATCTCGATTGG

AACTACACTCGTTGAGGTCAAGGCGATTGACTTTGATTTTCAAAGCAAACTGCGCTTCTATCTTTCGGG CAAAGGTGCGGACG

ACTTCAGTATAGGAAAGGAAAGTGGCATCCTGAAGGTGGCAAGCGCACTGGATCGGGAGACAACCCCCA AGTACAAATTGGTC

GCACATGTACAGGATGGCAAGGACTTTACGCAAGAGTGTTTCTCGGAAATAATCATCACGGTCAATGAC ATAAATGACAATAT

GCCCATTTTCTCAATGGCTCAATATAGAGTGAGTGTACCCGAGGATGCACAACTGAACACATTGATCAC GAAAGTGCACGCGA

TGGATAAGGATTTCGGGGTAAATAGACAAATCAAATACTCGCTAATGGGTGAAAACCATGATTATTTCA AAATATCAAAATCG

ACTGGTATCATAAGGCTGCACAAAAGTCTCGATCGTGAAACAATTTCATTGTTTAATCTCACTGTGAAG GCGGAGGACTGTGG

CGTTCCAAAACTACACTCCATTGCAACAGTTGCTGTGAACATATTGGACATTAATGACAATCCACCCGA GTTCAGTATGCGTC

AGTATTCGTGCAAAATTCTGGAAAACGCCACACACGGCACAGAAGTGTGCAAAGTTTATGCCACTTCGA TAGATATTGGGGTA

AATGCGGATATTCACTACTTCATAATGAGTGGCAACGAGCAGGGGAAGTTCAAAATGGATTCCACGACG GGCGACTTGGTGCT

AAATGCAACCTTGGACTATGAAATGTCCAAGTTTTACTTCTTGACCATTCAAGCAATCGATGGCGGCAC TCCACCGCTTAGCA

ACAATGCATATGTGAACATCTCTATTCTGGACATTAATGACAACAGTCCCACGTTTCTGCAAAACCTGT ACCGCATTAATGTC

AATGAAGATATTTTCGTGGGCTCCAAGATTCTGGACGTCAAAGCCACGGACGAAGATTCAGATGTAAAT GGTCTTGTAACTTA

CAACATTGAAAGAGGCGACAATATAGGCCAGTTTTCAATAGATCCGAAAAACGGAACAATTAGCGTTTC GAGGCCATTAGATC

GTGAGACTATTTCGCACTACACTCTTGAAATTCAAGCCTGTGATCAGGGAGATCCTCAGAGATGCAACA GTGTTCCAATCAAT

ATAAACATTTTGGACACTAACGATAATGCACCCATATTTTCCAGCTCTAACTACAGTGTAGTACTTCAA GAAAACCGACTTCT

GGGCTATGTATTCCTTACCTTCAAGATATCAGACGCAGACGAAACACCCAATACCACGCCATACACCTT CGATATTAGGTCTG

GAAATGAGGGTGGGCTTTTCCGGCTGGAGCAAGATGGTTCCTTGAGAACGGCCTCGCGATTTAATCACA ATCTGCAGGACGAA

TTCGTGATTCAAGTTCGAGTTTTCGACAACGGCACACCTCCATTATATTCCGATGCCTGGGTGGTTGTG AAAATAATTGAAGA

AAGCCAATACCCGCCCATTGTCACACCCCTAGAAGTAACCATAAATTCATTCGAGGACGATTTTTCGGG CGCATTCATTGGCA

AAGTTCATGCCTCGGATCAGGACAAGTATGATGAATTGAACTTTAGTTTGGTGTCCGGTCCCGATGACA TGTATCAGAGCTCG

AAGCTGTTCAACATTTCCAACAACACGGGAAAGATCTATGCCATATCCAACCTGGATATTGGTCTGTAC AAGCTAAATGTGTC

CGTTTCGGATGGTAAATTTCATGTGTTCTCCATTGTCAAAATCAACGTGGAACTGGTAACCAATGATAT GCTAAAAGAGTCGG

TTGTCATTCGATTCAGAAGGATTTCAGCATCTGAGTTTCTGCTGAGTCACAGGAAAACCTTTATGCGCT CCATTCGCAATATA

ATGCGATGTCGCCAAAAGGATGTAATTCTCATCACCCTTCAATCGGATTATCAAAAAGCATCACAACAT GCTGTGGGTAATCG

ACGAGCCAGGTCCATTGACTCCGATTTGAACGTGGTGTTTGCAGTGCGAAAGCAGCAAATAATACCCGA TTCCGATGAATTCT

TCACAAGTGATGAAATTCGGCAGACACTGATAGACAAGAAGAACGAGATTGAAAACGAAACCAACCTGG TGGTGGAGGATGTA

CTACCATCCACCTGTCAAAGCAACAAAAACGACTGCGTTCACGGGGAATGCAAACAGATATTACAGATC CTGAAGAACAACGT

TACCACCACCTTTACGGATGTGATTAGTTTTGCTGCTCCATCTTACATTCCGGTGAATACGTGTGTCTG TCGACCAGGATTCG

ATGGAAAGCACTGCAAAGAGACTGTGAATGCCTGCTCCACGGATCCATGTTCCCCGCAGAGGATCTGCA TGCCGTCTGGCTCG

GCTTTGGGTTACCAATGTGTGTGTCCCAAGGGATTTTCAGGAACCTACTGCGAGCGGAAGTCTTCGAAG TGCAGCAATGAGTC

CTGTGACATGGGTCTATTCACTGCGGTGTCCTTTGGCGGAAAGAGCTATGCCCACTACAAGATCAACAA GGTGAAGGCGAAGT

TCACGCTGGAAAACGGGTTTTCCTACTCCCTGCAGATAAGAACTGTGCAACAAACTGGGACTCTGCTGT ATGCCAGCGGCAAG

GTGGACTACAACATCCTGGAGATCATAAACGGAGCTGTTCAGTACAGATTCGATTTGGGCTCGGGCGAG GGAGTCATCAGTGT

GTCCAGCATTAACATCTCTGACGGCGAGTGGCATCAAATCAGCCTAGAGCGGTCCCTCAATAGTGCCAA AGTGATGGTGGACA

ACAAGCACGTCTCCCATGGCAGTGCTCCGGGTGTGAATGGCATCCTGAACATCCAGTCGAACGATATCT TTGTAGGCGCCGAG

GTTCGTCCGCATCCATCGATAATTGGCTACGAGGATATTCAGCGTGGCTTCATCGGTTGCATGGCAAAC ATCAAAATAGCCAA

AGAGTCGCTGCCATTGTACATTTCCGGTGGGAGTACCATTGCTGCCTTGAAACGTTTTACGAATGTCGA GTTCAAGTGCGATC

CGTCGAATGTTCTGGTGCGCCTGGGCATTTGCGCATCTCAGCCGTGTGCCAATAGTGGAATCTGCAAGG AACTCGATACGGAC

GTGTTTGAATGCGCCTGTCAGCCCCGATATTCCGGCAAGCATTGCGAGATTGATTTGGACCCTTGCTCA TCGGGACCCTGCTT

GTTTGGCGGCAGGTGCGACTACCACGGACCGAACAACTACAGCTGCACGTGTCCCATCCACTTATCCGG AAAGAGGTGTGAGT

ACGGCAAGTTCTGCACGCCGAACCCGTGCAAAAACGGTGGCATTTGCGAGGAAGGCGATGGAATATCGC ACTGCATGTGCCGC

GGCTACACGGGACCCACTTGTGAGATCGATGTGGATGAGTGCGAGAACCAGCCGTGCGGCAATGGAGCG ACCTGCATCAATGA

ACCCGGAAGTTTCCGTTGCATTTGTCCATCTTATCTCACAGGAeCCAGCTGCGGCGATCCCCTGTATTCGAACTCTA TTTCTA

CAAAGCTGAAGAACTTTTCTATAGAGCACATTAGCGGGATCATTTCCGGCGTGGCCGTGGTACTGGTCA TCATCAGTTGTGTC

CTGTGTTGeGTGGTGTTGAAGAGGAGTTCCTCTTCAAAGCGAAGGAACCGACTAGAAAAGGACAAGAACAAGTCGTC GTACAA

GGAGGCGAACTTGAACTCACTGGTGGACAAGGACAATTACTGCAAACCAAACGTAAAGTTGAGTAACTT GGAGGTTAACCAGC

GTCCAATTAGCTACACAGCAGTTCCAAATGACAACCTAGTCCTGAGCAATAGGAATTTTGTAAATAACT TAGACATCTTGCGT

AGCTACGGTTCGGCCGGCGATGAACTGGAAAATGTGCCATTCGAGTACCAGAAGGTTAATCGAAACAAA CAGCATGTGAACAT

AAACTCCTGCCATTCAACCGATGCAGATAATGCCTACAAACAAGAATGGTGCGAGCAAATGCATTTAAG AACCTTCAGTGAAA

ATAAACTGAACAATGAACTTAAACGGGATTTCGGACCATCTGTGAGTCGCTTTTCAACTGGGAAACTAA TCCAAGTTGAAATG

CCCAACGTGTGCCACTCTTCCAGTGCGAATTTCGTTGATTATTCAGCTCTTGCCAATGGTCAGTATCAT TGGGACTGTTCCGA

CTGGGTTCGCAAAAGCCATAATCCCTTGCCAGATATAACCGAAGTTCCTGGAGCAGAAATAGCTGATTC GTCGAGCTTACACA

GCAACGATAGCAACGAGTCCAAGTCGAAGAAAGCCTTTTTCGTGCACAGGGAAGACGGAGATGTTGATC CGACGAGGGATATA

GCCGCGTTGAATGAGGATATCGGATCGGAGTATTTGGACTCGGAGGCAGAGAGCTGCTTGGAGCCGTTT ATGTTGCCAAGATC

AAGTAATCAGCCACTTTCAAGACTGAGTTCTTTTAATAATATCGAGAATGAAGACTATAAATCAAATAC AGGCAAAGTATATT

TAAGACATCeTGATTCGTATTTACCGACGATGCATTTTCCAAGTGAGACCGATGGGGAAAGCTCTATGACCGAGGGG CCGATT

TCTAGGATGGAAATAAAAACCAGGAGGACGATAAGTGAAAATTCAGAGGAGGCATACCTATTTCCATGeACTGTCGG AGAAAT

TGGATCCAACAGCAACATTTCGGTTCGACTGTGTGAAATTGAAGATTCTGAGTTGGAGGAGTTTTTACC ACAACAACAAACAA

ACAATTAA (SEQID NO:13)

PCS (saccharomyces cerevisiae)

CTACAACTTACTCTTATTTCTGCTGCTCTTAGCAAAAGTTTCTGCGATAACTCTTCTCTGGATTTTACC TGTAGCGGTTTTTG

GTAGCTTATCAACAAAGTACACCTTGGTTGGAATTTTGAAAGAGGCTAGGTGCTTCTTTAAGAAGTTCA CCAGTTCTTCGTAG

GTCATTTTTTCTCCCTTCTTCAAAACAATGGCGGCTTGAACTACTTGGCCGTACATATCGTCGGGAACA CCAAATGCAACGGC

TTCATCGATCTTTGGATGCGATAGCATAATGCCGTeGAGCTCAATGGGTGAAATCTTTTCACCACCCCTGTTGATAA GCTCTT

TGATTCTGCCTGTAAGGACCAAAAACCCCTCAGGGTCGAAATAACCTTGGTCACCGGTTCTGAAATAGT TCTCTCTCTTGGTG

AAGTTCTCCTTGTTAGCTTTTGGATTATTAGCATACCCCAAAGTGACGTTTTCGCCTCTGATGGAAACT TCGCCGACTTTGCC

CGGGGGCAAGACATTGTCATTGTCATCTAGAATGACGACGGTGACTCCTTGTGGCTGGCCCACAGTACC AGGCTTTCTCTTTC

CTGGAGGCAGATTGTTTGAGGTCATTTGATGTGATGCTTCGGTCATCGCATAGGCCTCCAAGACAGGTG CATTGAATTCCTTC

TCCAGCTTATGGAACGTTGCTGGAGCCAAAGCAGAAGAACACGATCTGATGAATCTAATGTGTGGGAAA GGGTTTGGTTTGGG

CATGTTCAGCATAATCATGCTTATTGTGGGAACGCAACTGAACCAATTACAGTTGTACTTAAeAAATTGGTCCCAGA ATAACT

TTGGATGGAATCCATCGGGAACCACAACAGAACCCTGAGTTCTAAAAGTGGAAAGTAAAACACCAATTA ACCCATGGACGTGG

AAAAGAGGCATCACGACATAAGATCTGTCCAAGGGCGTTAGCTTGTAAGTGTTAGCAATGTTCAACGTG CTTCTCACAATGTT

eAAATGTAACAAAGGCACCGTTTTTGGAGTGGAGGTGGTACCACTGGTATGCAAAATCAGGGCAACGTCACTGGAAC GGGCAA

ACCCAGGGAATTTAACGGGATTTGTGTTGACAAATTTGGCGTTGTTCAAAGACCGGTAAATAACCCTTT TGTAGTTGTCCTCT

GGAGAGTATATATCATACTCTACCCTAAACCTGGTCGCATCGAAGGCCAGCTCTACGATAAAACATCCA AACGTGGAGGCAGA

TTTTAGAATTTCAGAACTCTGTAACTTTGTGGTACCCTTTGGGACGCAAATCGCCTTAGATTTCAGGTC ATTCAAATAAAAAT

TGAACTCCTTTTCCTTATAATTGGGATTCAAGGGCGCGCCAATTTTAGCGTCCATAGTAGCACCGAGGA AAGCGACGATAAAT

TCCAGCCCATTACGCATGGATATCGCCACTGTATCTTGTCTGAAAACAGCTCCGTACAATGGAGAATTA GGATTTGTGAACAT

GGTCTGGAAGTGACCCACCATGTGGGATAGATCCCTGTAGGTCACCTGAGTGTCCGTTTCAGGAACAAT AACGGCGACATTAT

CGGATACGCTAAAAGTATCGTTGAACGAAGCAGTAACAGTAGCGGCACTTGTCAT (SEQ ID NO: 14)

ACLY (people (Homo sapiens))

GCGAGCCGATGGGGGCGGGGAAAAGTCCGGCTGGGCCGGGACAAAAGCCGGATCCCGGGAAGCTACCGG CTGCTGGGGTGCTC

CGGATTTTGCGGGGTTCGTCGGGCCTGTGGAAGAAGCTGCCGCGCACGGACTTCGGCAGAGGTAGAGCA GGTCTCTCTGCAGC

CATGTCGGCCAAGGCAATTTCAGAGCAGACGGGCAAAGAACTCCTTTACAAGTTCATCTGTACCACCTC AGCCATCCAGAATC

GGTTCAAGTATGCTCGGGTCACTCCTGACACAGACTGGGCCCGCTTGCTGCAGGACCACCCCTGGCTGC TCAGCCAGAACTTG

GTAGTCAAGCCAGACCAGCTGATCAAACGTCGTGGAAAACTTGGTCTCGTTGGGGTCAACeTCACTCTGGATGGGGT CAAGTC

CTGGCTGAAGCCACGGCTGGGACAGGAAGCCACAGTTGGCAAGGCCACAGGCTTCCTCAAGAACTTTCT GATCGAGCCCTTCG

TCCCCCACAGTCAGGCTGAGGAGTTCTATGTCTGCATCTATGCCACCCGAGAAGGGGACTACGTCCTGT TCCACCACGAGGGG

GGTGTGGACGTGGGTGATGTGGACGCCAAGGCCCAGAAGCTGCTTGTTGGCGTGGATGAGAAACTGAAT CCTGAGGACATCAA

AAAACACCTGTTGGTCCACGCCCCTGAAGACAAGAAAGAAATTCTGGCCAGTTTTATCTCCGGCCTCTT CAATTTCTACGAGG

ACTTGTACTTCACCTACCTCGAGATCAATCCCCTTGTAGTGACCAAAGATGGAGTCTATGTCCTTGACT TGGCGGCCAAGGTG

GACGCCACTGCCGACTACATCTGCAAAGTCAAGTGGGGTGACATCGAGTTCCCTCCCCCCTTCGGGCGG GAGGCATATCCAGA

GGAAGCCTACATTGCAGACCTCGATGCCAAAAGTGGGGCAAGCCTGAAGCTGACCTTGCTGAACCCCAA AGGGAGGATCTGGA

CCATGGTGGCCGGGGGTGGCGCCTCTGTCGTGTACAGCGATACCATCTGTGATCTAGGGGGTGTCAACG AGCTGGCAAACTAT

GGGGAGTACTCAGGCGCCCCCAGCGAGCAGCAGACCTATGACTATGCCAAGACTATCCTCTCCCTCATG ACCCGAGAGAAGCA

CCCAGATGGCAAGATCCTCATCATTGGAGGCAGCATCGCAAACTTCACCAACGTGGCTGCCACGTTCAA GGGCATCGTGAGAG

CAATTCGAGATTACCAGGGCCCCCTGAAGGAGCACGAAGTCACAATCTTTGTCCGAAGAGGTGGCCCCA ACTATCAGGAGGGC

TTACGGGTGATGGGAGAAGTCGGGAAGACCACTGGGATCCCCATCCATGTCTTTGGCACAGAGACTCAC ATGACGGCCATTGT

GGGCATGGCCCTGGGCCACCGGCCCATCCCCAACCAGCCACCCACAGCGGCCCACACTGCAAACTTCCT CCTCAACGCCAGCG

GGAGCACATCGACGCCAGCCCCCAGCAGGACAGCATCTTTTTCTGAGTCCAGGGCCGATGAGGTGGCGC CTGCAAAGAAGGCC

AAGCCTGCCATGCCACAAGATTCAGTCCCAAGTCCAAGATCCCTGCAAGGAAAGAGCACCACCCTCTTC AGCCGCCACACCAA

GGCCATTGTGTGGGGCATGCAGACCCGGGCCGTGCAAGGCATGCTGGACTTTGACTATGTCTGCTCCCG AGACGAGCCCTCAG

TGGCTGCCATGGTCTACCCTTTCACTGGGGACCACAAGCAGAAGTTTTACTGGGGGCACAAAGAGATCC TGATCCCTGTCTTC

AAGAACATGGCTGATGCCATGAGGAAGCACCCGGAGGTAGATGTGCTCATCAACTTTGCCTeTCTeCGCTCTGCCTA TGACAG

CACCATGGAGACCATGAACTATGCCCAGATCCGGACCATCGCCATCATAGCTGAAGGCATCCCTGAGGC CCTCACGAGAAAGC

TGATCAAGAAGGCGGACCAGAAGGGAGTGACCATCATCGGACCTGCCACTGTTGGAGGCATCAAGCCTG GGTGCTTTAAGATT

GGCAACACAGGTGGGATGCTGGACAACATCCTGGCCTCCAAACTGTACCGCCCAGGCAGCGTGGCCTAT GTCTCACGTTCCGG

AGGCATGTeCAACGAGCTCAACAATATCATCTCTCGGACCACGGATGGCGTCTATGAGGGCGTGGCCATTGGTGGGG ACAGGT

ACCCGGGCTCCACATTCATGGATCATGTGTTACGCTATCAGGACACTCCAGGAGTCAAAATGATTGTGG TTCTTGGAGAGATT

GGGGGCACTGAGGAATATAAGATTTGCCGGGGCATCAAGGAGGGCCGCCTCACTAAGCCCATCGTCTGC TGGTGCATCGGGAC

GTGTGCCACCATGTTCTCCTCTGAGGTCCAGTTTGGCCATGCTGGAGCTTGTGCCAACCAGGCTTCTGA AACTGCAGTAGCCA

AGAACCAGGCTTTGAAGGAAGCAGGAGTGTTTGTGeCCCGGAGCTTTGATGAGCTTGGAGAGATCATCCAGTCTGTA TACGAA

GATCTCGTGGCCAATGGAGTCATTGTACCTGCCCAGGAGGTGCCGCCCCCAACCGTGCCCATGGACTAC TCCTGGGCCAGGGA

GCTTGGTTTGATCCGCAAACCTGCCTCGTTCATGACCAGCATCTGCGATGAGCGAGGACAGGAGCTCAT CTACGCGGGCATGC

CCATCACTGAGGTCTTCAAGGAAGAGATGGGCATTGGCGGGGTCCTCGGCCTCCTCTGGTTCCAGAAAA GGTTGCCTAAGTAC

TCTTGCCAGTTCATTGAGATGTGTCTGATGGTGACAGCTGATCACGGGCCAGCCGTCTCTGGAGCCCAC AACACCATCATTTG

TGCGCGAGCTGGGAAAGACCTGGTCTCCAGCCTCACCTCGGGGCTGCTCACCATCGGGGATeGGTTTGGGGGTGCCT TGGATG

CAGCAGCCAAGATGTTCAGTAAAGCCTTTGACAGTGGCATTATCCCCATGGAGTTTGTGAAeAAGATGAAGAAGGAA GGGAAG

CTGATCATGGGCATTGGTCACCGAGTGAAGTCGATAAACAACCCAGACATGCGAGTGCAGATCCTCAAA GATTACGTCAGGCA

GCACTTCCCTGCCACTCCTCTGCTCGATTATGCACTGGAAGTAGAGAAGATTACCACCTCGAAGAAGCC AAATCTTATCCTGA

ATGTAGATGGTCTCATCGGAGTCGCATTTGTAGACATGCTTAGAAACTGTGGGTCCTTTACTCGGGAGG AAGCTGATGAATAT

ATTGACATTGGAGCCCTCAATGGCATCTTTGTGCTGGGAAGGAGTATGGGGTTCATTGGACACTATCTT GATCAGAAGAGGCT

GAAGCAGGGGCTGTATCGTCATCCGTGGGATGATATTTCATATGTTCTTCCGGAACACATGAGeATGTAA (SEQ ID NO:15)

FAS (cow mycobacteria subspecies, Mycobacterium bovid subsp.bovis):

ATGAGTCAGACGGTGCGCGGTGTGATCGCACGACAAAAGGGCGAACCCGTTGAGCTGGTGAACATTGTC GTCCCGGATCCCGG

ACCCGGCGAGGCCGTGGTCGACGTCACeGCCTGCGGGGTATGCCATACCGACCTGACCTACCGCGAGGGCGGCATCA ACGACG

AATACCCTTTTCTGCTCGGACACGAGGCCGCGGGCATCATCGAGGCCGTCGGGCCGGGTGTAACCGCAG TCGAGCCCGGCGAC

TTCGTGATCCTGAACTGGCGTGCCGTGTGCGGCCAGTGCCGGGCCTGCAAACGCGGACGGCCCCGCTAC TGCTTCGACACCTT

TAACGCCGAACAGAAGATGACGCTGACCGACGGCACCGAGCTCACTGCGGCGTTGGGCATCGGGGCCTT TGCCGATAAGACGC

TGGTGCACTCTGGCCAGTGCACGAAGGTCGATCCGGCTGCCGATCCCGCGGTGGCCGGCCTGCTGGGTT GCGGGGTCATGGCC

GGCCTGGGCGCCGCGATCAACACCGGCGGGGTAACCCGCGACGACACCGTCGCGGTGATCGGCTGCGGC GGCGTTGGCGATGC

CGCGATCGCCGGTGCCGCGCTGGTCGGCGCeAAACGGATCATCGCGGTCGACACCGATGACACGAAGCTTGACTGGG CCCGeA

CeTTCGGCGCCACCCACACCGTCAACGCCCGCGAAGTCGACGTCGTCCAGGCCATCGGCGGCCTCACGGATGGATTC GGCGCG

GACGTGGTGATCGACGCCGTCGGCCGACCGGAAACCTACCAGCAGGCCTTCTACGCCCGCGATCTCGCC GGAACCGTTGTGCT

GGTGGGTGTTCCGACGCCCGACATGCGCCTGGACATGCCGCTGGTCGACTTCTTCTCTCACGGCGGTGC GCTGAAGTCGTCGT

GGTACGGCGATTGCCTGCCCGAAAGCGACTTCCCCACGCTGATCGACCTTTACCTGCAGGGCCGGCTGC CGCTGCAGCGGTTC

GTTTCCGAACGCATCGGGCTCGAAGACGTCGAGGAGGCGTTCCACAAGATGCATGGCGGCAAGGTATTG CGTTCGGTGGTGAT

GTTGTGA (SEQ ID NO:1G)

AMPK (people)

AGTTCCTGGAGAAAGATGGCGACAGCCGAGAAGCAGAAACACGACGGGCGGGTGAAGATCGGCCACTAC ATTCTGGGTGACAC

GCTGGGGGTCGGCACCTTCGGCAAAGTGAAGGTTGGCAAACATGAATTGACTGGGCATAAAGTAGCTGT GAAGATACTCAATC

GACAGAAGATTCGGAGCCTTGATGTGGTAGGAAAAATCCGCAGAGAAATTCAGAACCTCAAGCTTTTCA GGCATCCTCATATA

ATTAAACTGCACCAGGTCATCAGTACACCATCTGATATTTTCATGGTGATGGAATATGTCTCAGGAGGA GAGCTATTTGATTA

TATCTGTAAGAATGGAAGGAAATCTGATGTACCTGGAGTAGTAAAAACAGGCTCCACGAAGGAGCTGGA TGAAAAAGAAAGTC

GGCGTeTGTTCCAACAGATCCTTTCTGGTGTGGATTATTGTCACAGGCATATGGTGGTCCATAGAGATTTGAAACCT GAAAAT

GTCCTGCTTGATGCACACATGAATGCAAAGATAGCTGATTTTGGTCTTTCAAACATGATGTCAGATGGT GAATTTTTAAGAAC

AAGTTGTGGCTCACCCAACTATGCTGCACeAGAAGTAATTTCAGGAAGATTGTATGCAGGCCCAGAGGTAGATATAT GGAGCA

GTGGGGTTATTCTCTATGCTTTATTATGTGGAACCCTTCCATTTGATGATGACCATGTGCCAACTCTTT TTAAGAAGATATGT

GATGGGATCTTCTATACCCCTCAATATTTAAATCCTTCTGTGATTAGCCTTTTGAAACATATGCTGCAG GTGGATCCCATGAA

GAGGGCCACAATCAAAGATATCAGGGAACATGAATGGTTTAAACAGGACCTTCCAAAATATCTCTTTCC TGAGGATCCATCAT

ATAGTTCAACCATGATTGATGATGAAGCCTTAAAAGAAGTATGTGAAAAGTTTGAGTGCTCAGAAGAGG AAGTTCTCAGCTGT

CTTTACAACAGAAATCACCAGGATCCTTTGGCAGTTGCCTACCATCTCATAATAGATAACAGGAGAATA ATGAATGAAGCCAA

AGATTTCTATTTGGCGACAAGCCCACCTGATTCTTTTCTTGATGATCATCACCTGACTCGGCCCCATCC TGAAAGAGTACCAT

TCTTGGTTGCTGAAACACCAAGGGCACGCCATAeCCTTGATGAATTAAATCCACAGAAATCCAAACACCAAGGTGTA AGGAAA

GCAAAATGGCATTTAGGAATTAGAAGTCAAAGTCGACCAAATGATATTATGGCAGAAGTATGTAGAGCA ATCAAACAATTGGA

TTATGAATGGAAGGTTGTAAACCCATATTATTTGCGTGTACGAAGGAAGAATCCTGTGACAAGCACTTA CTCCAAAATGAGTC

TACAGTTATACCAAGTGGATAGTAGAACTTATCTACTGGATTTCCGTAGTATTGATGATGAAATTACAG AAGCCAAATCAGGG

ACTGCTACTCCACAGAGATCGGGATCAGTTAGCAACTATCGATCTTGCCAAAGGAGTGATTCAGATGCT GAGGCTCAAGGAAA

ATCCTCAGAAGTTTCTCTTACCTCATCTGTGACCTCAeTTGACTCTTCTCCTGTTGACCTAACTCCAAGACCTGGAA GTCACA

CAATAGAATTTTTTGAGATGTGTGCAAATCTAATTAAAATTeTTGCACAATAA (SEQ ID NO:17)

Conversion carrier selection on leucine auxotrophy type agar plate in Po 1g Yarrowia lipolytica strain.Use PCR The bacterium colony that screening is correctly inserted in genome.

Δ 9-FW AATGGTGAAAAACGTGGACCAAGTGGA (SEQ ID NO:18)

Δ 9-REV ATGGATCCCTAAGCAGCCATGCCAGACATAC (SEQ ID NO:19)

GLUT1-FW AATGGAGCCCAGCAGCAAGAAGGTGA (SEQ ID NO:20)

GLUT1-REV AATGGGTACCTCACACTTGGGAGTCAGeC (SEQ ID NO:21)

Hemoglobin F W AGAGACCGGGTTGGCGGCGCA (SEQ ID NO:22)

Hemoglobin REV CAGCGTCTTGAGCGTACAAA (SEQ ID NO:23)

Cytochrome FW AATGATCATCAACGGCAAGGTCT (SEQ ID NO:24)

Cytochrome REV TTATTTCTGACCCTGGAGGTAGAAG (SEQ ID NO:25)

AATGCTGAAGTT DEG C of CGAACAGT of pyruvate carboxylase FW (SEQ ID NO:26)

Pyruvate carboxylase CGATGGTACCTCACTCGATCTCCAGGATG (SEQ ID NO:27)

(substantially cultivate in YPD culture medium (complete medium: yeast extract, peptone, glucose) and YNB culture medium Base, comprises all nutrients, but without aminoacid, and the most nitrogenous source or carbon source) in cultivate gained bacterium colony.When using YNB training When foster base is cultivated, providing nitrogen with ammonium sulfate and provide carbon with glucose, carbon-nitrogen ratio is 150.It is long-pending that this C/N is compared to start oil Tired is necessary.After consuming nitrogen in yeast, excessive glucocorticoid is converted into oil accumulation.

Results oil: cultivate cell in the constrained culture medium of nitrogen.Harvesting be dried 2 at 60 DEG C after 72 hours My god.At 90 DEG C, directly process cell 24 hours with 1% sulphuric acid and methanol.Oil is converted into FAME (fatty acid methyl ester) and passes through Hexane extracts.Hexane extraction is repeated twice to reclaim the FAME of 95%.Evaporation of hexane fraction is the most resuspended in 5ml hexane.Will 10 μ l fraction inject GC-MS to quantify FAME.

(Voelker and Davies, 1994) as described by early stage, gathers in the crops and prepares cell culture and divide for fatty acid Analysis.The quadrupole MS of list with electron bombardment ionization source is used to be quantified the content of fatty acid of each sample by GC-MS.GC post is ID For 0.25mm and HP-5MS (5% the phenyl)-methyl polysiloxane of 30m length that film thickness is 25 μm.GC elution requirement is as follows: 100 DEG C as initial temperature (5 minutes), within 15 minutes, rise to 250 DEG C, at 250 DEG C keep 10 minutes.

Embodiment 1

GC-MS detection is used to be grown on logarithm and stablize total free fatty in trophophase Yarrowia lipolytica culture (FFA) the qualitative spectrum (Figure 1A-C) stored up.Main FFA stores up and comprises saturated Palmic acid and stearic acid and unsaturated oleic acid. The FFA spectrum of relatively two trophophases demonstrates that stable phase does not has oleic acid, and observes the class of stearic acid and oleic acid in logarithmic (log) phase As peak density (Figure 1A, B).The analysis of the oleic acid that the TL (FFA+ lipid) in stable phase has reclaimed partial amount shows, oil Acid enters TAG forming feature (Fig. 1 C).The residue of oleic acid is stored up for downstream polyunsaturated fatty acids and therefore can not be helped Rescue.Therefore, oleic acid imports TAG with instantaneous versions during stable trophophase and is formed, its store with intracellular TAG pathway activation time Between meet.This shows that can there is monitoring point mechanism carrys out monitor and detection oleic levels with regulation oil accumulation.

Embodiment 2

Because in mice, SCD is necessary (to see, e.g. Regulation of stearoyl for lipogenesis Coenzyme A desaturases and role in metabolism.Prog Lipid Res.2004Mar；43 (2): 91- 104), and be in the news in most of biologies synthesize unsaturated fatty acid be important, so we test understanding fat Family name yeast SCD in TAG accumulates as the role of rate-limiting step.The saccharomyces cerevisiae OLE1 gene of coding SCD and solution fat Ye Shi ferment The protein sequence analysis display protein of parent protein sequence has the homogeneity of 51%.Yarrowia lipolytica desaturase bag Containing typical 3 histidine boxes and cytochrome b5 domain for other stearic acid coa desaturase.Because going to satisfy (see for example, Regulation of stearovl coenzyme A desaturase by gene transcription level with enzyme polyunsaturated fatty acids and cholesterol. James M.Ntambi.Journal of Lipid Research, volume 40,1549-1558, September 1999) and (see Mol at logarithm and the stable phase of cell growth Cell Biol Res Commun.1999Apr；1 (1): 36-43) by altitude mixture control, so we use half constitutive promoter Express to natural Yarrowia lipolytica delta 8 desaturase genes.The modified stable gene of single copy is integrated into base Because of in group.The GC-MS spectrum of saltant type and wild-type strain shows in terms of unsaturation with satisfied fatty acid ratio and dramatically increases (Fig. 2 A, B).Show unsaturated fatty acid with the confocal microscopy of the intracellular lipid of Nile red dyeing to increase and TAG Mutual relation (Fig. 2 C: wild type, Fig. 2 D:SCD process LAN type) between excess accumulation.In most of the cases, SCD process LAN The whole cell volume of cell is completely filled with TAG (Fig. 2 D).These evidences finding to provide key regulatory genes, described gene Be enough to the process LAN by changing unsaturated fatty acid and ratio induction intracellular TAG of satisfied fatty acid unexpectedly.

The Confocal Images of trophophase and stable phase cell demonstrates dramatically different in oil accumulation pattern.We test Intracellular TAG entering back into the stable phase rich oil mutant cell after cell division cycle moves.We are with containing high concentration sugar (300g/l) minimal medium feeds stable phase cell in batches.Cell enters back into logarithmic (log) phase effectively, and then fast-growth is also The biomass carrying out consuming all sugar in 96 hours produce.Even if it is interesting that graphical analysis illustrates during logarithmic (log) phase also The mutant strain of the intracellular oil of accumulation excess, this is atypical for oleaginous yeast.Although wild-type cell can not be at height Sugar concentration grows, but even if under the sugared concentration of favourable growth, logarithmic (log) phase does not the most exist lasting oil and produces and yeast Sample sign.

In a word, these results use the sugar of high concentration to establish continuous print fed batch process, show the yeast of transformation Strain can accumulate oil at exponential phase during stablizing trophophase continuously.

Embodiment 3

Create two kinds of mutant yeast, the following gene of its process LAN: mutant 1:SCD, hemoglobin, Glut1, Cytochrome；Mutant 2: hemoglobin, Glut1, cytochrome.Each gene is cloned into the site PmlI of plasmid YLEX with Between Kpn.Select with vector Po 1g Yarrowia lipolytica strain and on leucine auxotrophy agar plate.Use PCR screening The bacterium colony being correctly inserted in genome.Gained bacterium is cultivated in the YPD culture medium that carbon-nitrogen ratio (C/N) is 150 and YNB culture medium Fall.This C/N comparison causes oil accumulation to be necessary.After consuming nitrogen, the sugar of excess enters the oil accumulation in yeast.

In order to measure maximum oil accumulation, nitrogen restricted type culture medium is cultivated cell.After 72 hours, harvesting and It is dried 2 days at 60 DEG C.At 90 DEG C, cell is directly processed 24 hours with 1% sulphuric acid and methanol.Oil is converted into FAME (fat Acid methyl ester) and extracted by hexane.Hexane extraction is repeated twice to reclaim the FAME of 95%.Evaporation of hexane fraction at 5ml In hexane resuspended.10 μ l fraction are injected GC-MS to quantify FAME.Maximum oil accumulation in mutant strain is 80g/l.

Compare mutant 1 (" D9 ") and the glucose uptake kinetics of wild-type yeast (" LS ").Fig. 3 illustrates 72 hours Rear mutant 1 consumes the whole sugar provided, and wild-type yeast only consumes about the 70% of provided sugar.It was additionally observed that Even if in a period of time extended, wild-type strain does not consumes all sugar yet.

It is then determined that whether mutant strain can use biomass hydrolysate as carbon source.2L bioreactor is arranged For comprising corn straw hydrolyzate (Hz) in the presence of 1% yeast extract.Hz comprises 20g glucose.We add (benefit Material is in batches) 180g glucose extremely final concentration of 200g/l.We determined that wild type can not grow in hazard biological matter Hz.Shaking Cultivating mutant 1 and mutant 2 cell in Ping to the final OD in 50ml is 3.Overnight culture is added each bioreactor And carry out fermenting 72 hours at 30 DEG C.Running two reactors under the same conditions, one of them has mutant 1, another Individual have mutant 2.Stirring is set to 5.5 for 800rpm, pH.

Due to the restriction of some nutrient factors in culture medium, so in 72 hours, two kinds of bacterial strains all consume and carried About 50% (Fig. 4 A, it is shown that mutant 1 bacterial strain) for glucose.The reason of 50% sugar consumption of two kinds of bacterial strains is to there is Glut1, Glucose transport is entered cell by known Glut1.Mutant 1 consumes 123 grams of glucoses and mutant 2 consumes 105 grams of sugar.Should Result shows compared with wild type, mutant cell can consumption sugar almost 50% and toxicity robust against Hz, in early days In test, wild type growth is bad and consumes less than 10g sugar.Mutant strain illustrates Johnson & Johnson head consumption sugar well.Due to battalion Support some consumption of the factor, so not consuming remaining sugar (seeing Fig. 5 and 6).

Compared with mutant 2, mutant 1 (having the combination of process LAN gene) illustrates the oil synthesis of increase.Every liter of sudden change Body 1 produces 26g oil (Fig. 4 B), and every liter of mutant 2 produces 14g oil.This shows that the combination of process LAN gene not only causes is provided The consuming of sugar increases and produce the bio-fuel precursor of more oil-useful with causing increase.

Embodiment 4

Then, we measure transformation bacterial strain and the growth vigor of wild-type strain, TL life in 2 liters of rounds Produce, carbohydrate substrates tolerates to transformation efficiency and the substrate of lipid Transfer.

GC-MS is used to measure the total amount (Fig. 7) of lipid.Transformation bacterial strain is observed than wild-type strain 10 times Lipid production (80g/l), is represented as other people (S.Papanikolaou I.Chevalot, M.Komaitis, I.Marc G.Aggelis, Single cell oil production by Yarrowia lipolytica growing on an industrial derivative of animal fat in batch cultures Appl Microbiol Biotechnol.2002Mar；58 (3): 308-12) increase of the Yarrowia lipolytica strain described in 20 times.Monounsaturated fatty acid The main species of acid is oleic acid, and it increases to 8.5 times (g/l) (Fig. 8) compared with control strain.Total unsaturated fatty acid is with saturated The ratio of fatty acid significantly increases, and total unsaturated fatty acid does not increases to exceed satisfied fatty acid, but wherein minority is c18.1 Situation (seeing Fig. 7 and 8).The sugar of mutant strain-oil conversion ratio is defined as 0.28g/g, when in view of being used for producing biomass Sugared time, it is close to theoretical value.

Notable and unexpected 32 times of growth vigors (figure is observed between engineered bacterial strain and wild-type strain 9).The growth characteristics of mutant strain keep identical (Figure 10) under the sugared concentration lethal to wild-type strain permeability.Higher Sugar tolerance is particularly important for the high gravity being generally used for industrial bio fuel production ferments.Before, fat Ye Shi ferment is being solved Female culture is observed negative correlation that higher biomass produce between lipid accumulation (Papanikolaou S, Chevalot I, Komaitis M, Marc I, Aggelis G.Single cell oil production by Yarrowia lipolytica growing on an industrial derivative of animal fat in batch cultures.Appl Microbiol Biotechnol.2002Mar；58 (3): 308-12.).Therefore, the bacterium of our transformation The contact that biomass higher in strain produce between the lipid accumulation of excess is unexpected.Owing to fat stores is mainly used In synthesis film and activity (the FEBS J.2008 Nov that sprouts；275 (22): 5552-63), a probability of logarithmic (log) phase cell is Guide excess lipid again to flow to film by activating cell division approach to synthesize and/or by Lipid Secretion to extracellular matrix.This The excessive lipid production after lipid within endothelial cells accumulation in early days will be entered after stable phase cell cycle in, equilibrium.It is true that it is higher Biomass produce relevant to growth Lipid Secretion in early days.

Figure 11 illustrates sudden change and the growth of wildtyp Y. lipolytica and lipid production kinetics.With wild type (comparison) Bacterial strain is compared, and mutant strain not only shows strong growth vigor, and it also produces the fatty acid of significantly higher amount.

In a word, these results show on the basis of glucose is as single carbon source, effective Metabolically engineered oleaginous yeast table Reveal very good multiple phenotypes.

Embodiment 5

Detect the SCD regulatory mechanism causing the multiple phenotypic characteristic of mutant strain.In view of Yarrowia lipolytica desaturation The low sequence of the similar functions gene in enzyme gene and nematode C. elegans (Caenorhabditis elegans) and mice Row homogeneity, tests the fatty acid specificity across species SCD cloned in Yarrowia lipolytica.Ye Shi natural with process LAN The mutant of yeast genes is similar to, and Caenorhabditis elegans has similar specificity with the SCD in mice to stearic acid, shows Higher yield of biomass.During co-focusing imaging confirms stable trophophase, the oily of excess accumulates.These results show oil The desaturase activity of acid is relevant to the excess accumulation of TAG.Owing to the SCD in known bakery yeast (baker ' s yeast) exists Transcribe regulated with post-transcriptional level (see Tabor DE, Kim JB, Spiegelman BM, Edwards PA, Identification of conserved cis-elements and transcription factors required For sterol-regulated transcription of stearovl coenzyme A desaturase.J Biol Chem.1999 Jul 16；274 (29): 20603-10；Shimano H, Sterol regulatoryelement-binding protein family as global regulators of lipid synthetic genes in energy metabolism.Vitam Horm.2002；65:167-94), have studied the oleic acid feedback suppression conduct to delta 8 desaturase genes Possible regulation mechanism.We stably incorporate the natural delta 8 desaturase genes of single copy and the 1kb upstream comprising promoter region Sequence.Mutant strain have accumulated excessive oil and has growth vigor and sugar tolerance as mutant before.This shows and bread Yeast is different, and oil accumulation is not driven the promoter sequence regulation and control that desaturase is expressed.This means desaturation in Ye Shi yeast The negative regulator of enzyme gene is to be non-dependent on transcribing and is likely to occur in metaboilic level.These data provide for the first time In oleaginous yeast, the inhibitory action by overcoming oleic acid carries out the mechanism that oil regulates.

Embodiment 6

The engineered microorganism that the present invention provides can be cultivated in multiple substrate.Figure 13 illustrates using algae bio as carbon The Johnson & Johnson head of sudden change Yarrowia lipolytica strain during hydrate source.Figure 14 illustrates be incubated in algal biomass engineered micro- Oil accumulation in biological cell.Figure 15 illustrates the oil accumulation in the engineered cell being incubated in crude glycerine.

Embodiment 7

It is senior chain lipid that Δ-12 desaturase is responsible for the lipid Transfer comprising oleic acid.In order to produce bio-fuel, examine Consider the cold flow characteristic to diesel fuel, preferably C18 chain fatty acid such as stearic acid and oleic acid.Therefore, in some embodiments, the phase Hope that blocking or suppress C18 convert fatty acids is longer chain fatty acid.

It can complete (example by the expression of wild type Δ-12 delta 8 desaturase genes in suppression or blocking-up host microorganism Microorganism such as process LAN Δ 9 desaturase (SCD)).To this end, create nucleic acid construct to knock out in Yarrowia lipolytica Wild type Δ-12 desaturase.The schematic structure knocking out construct is shown in Figure 16.It is phleomycin that this carrier comprises flank (such as, Zeocin^TM) genome sequence of Δ-12 delta 8 desaturase genes of resistant gene.The sequence of this construct is shown in following.

Δ-12 desaturase knockout carrier sequence

CCAACAGACCGACCATAGAAATGGATTCGACCACGCAGACCAACACCGGCACCGGCAAGGTGGCCGTGC AGCCCCCCACGGCC

TTCATTAAGCCCATTGAGAAGGTGTCCGAGCCCGTCTACGACACCTTTGGCAACGAGTTCACTCCTCCA GACTACTCTATCAA

GGATATTCTGGATGCCATTCCCCAGGAGTGCTACAAGCGGTCCTACGTTAAGTCCTACTCGTACGTGGC CCGAGACTGCTTCT

TTATCGCCGTTTTTGCCTACATGGCCTACGCGTACCTGCCTCTTATTCCCTCGGCTTCCGGCCGAGCTG TGGCCTGGGCCATG

TACTCCATTGTCCAGGGTCTGTTTGGCACCGGTCTGTGGGTTCTTGCCCACGAGTGTGGCCACTCTGCT TTCTCCGACTCTAA

CACCGAGAGACCGGGTTGGCGGCGCATTTGTGTCCCAAAAAACAGCCCCAATTGCCCCAATTGACCCCA AATTGACCCAGTAG

CGGGCCCAACCCCGGCGAGAGCCCCCTTCACCCCACATATCAAACCTCCCCCGGTTCCCACACTTGCCG TTAAGGGCGTAGGG

TACTGCAGTCTGGAATCTACGCTTGTTCAGACTTTGTACTAGTTTCTTTGTCTGGCCATCCGGGTAACC CATGCCGGACGCAA

AATAGACTACTGAAAATTTTTTTGCTTTGTGGTTGGGACTTTAGCCAAGGGTATAAAAGACCACCGTCC CCGAATTACCTTTC

CTCTTCTTTTCTCTCTCTCCTTGTCAACTCACACCCGAAATCGTTAAGCATTTCCTTCTGAGTATAAGA ATCATTCAAAATGG

CCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCG ACCGGCTCGGGTTC

TCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCG GTCCAGGACCAGGT

GGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAGTGGTCGGA GGTCGTGTCCACGA

ACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCC TGCGCGACCCGGCC

GGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGATCCATGGCCTGTCCCCACGTTGCCGGTCTT GCCTCCTACTACCT

GTCCATCAATGACGAGGTTCTCACCCCTGCCCAGGTCGAGGCTCTTATTACTGAGTCCAACACCGGTGT TCTTCCCACCACCA

ACCTCAAGGGCTCTCCCAACGCTGTTGCCTACAACGGTGTTGGCATTTAGGCAATTAACAGATAGTTTG CCGGTGATAATTCT

CTTAACCTCCCACACTCCTTTGACATAACGATTTATGTAACGAAACTGAAATTTGACCAGATATTGTTG TAAATAGAAAATCT

GGCTTGTAGGTGGCAAAATGCGGCGTCTTTGTTCATCAATTCCCTCTGTGACTACTCGTCATCCCTTTA TGTTCGACTGTCGT

ATTTCTTATTTTCCATACATATGCAAGTGAGATGCCCGTGTCCTGGCCATCACCTACCTGCAGCACACC GACCCCACTCTGCC

CCACTACCACGCCGACCAGTGGAACTTCACCCGAGGAGCCGCCGCCACCATCGACCGAGAGTTTGGCTT CATCGGCTCCTTCT

GCTTCCATGACATCATCGAGACCCACGTTCTGCACCACTACGTGTCTCGAATTCCCTTCTACAACGCCC GAATCGCCACTGAG

AAGATCAAGAAGGTCATGGGCAAGCACTACCGACACGACGACACCAACTTCATCAAGTCTCTTTACACT GTCGCCCGAACCTG

CCAGTTTGTTGAAGGTAAGGAAGGCATTCAGATGTTTAGAAACGTCAATGGAGTCGGAGTTGCTCCTGA CGGCCTGCCTTCTA

AAAAGTAGAGCTAGAAATGTTATTTGATTGTGTTTTAACTGAACAGCA (SEQ ID NO:28)

A-12 desaturase at Yarrowia lipolytica knocks out process LAN series of genes in cell and (includes A9 desaturation Enzyme, Glut1, hemoglobin and cytochrome b5) flow into the sugar of cell with further increasing and increase oil content.Observe cell Dramatically increasing of size, wherein the cell the most up to 95% is filled with oil.

Embodiment 8

The Yarrowia lipolytica 148 hours (Figure 17) of process LAN SCD is cultivated in 3% acetic acid class solution.At about 84 hours Time to cell culture add 2% glycerol with provide glycerol to drive fatty acid to produce.The latter is to use acetic acid class as raw material Produce the bottleneck of oil.Observed by confocal laser microscope and use the acetic acid class culture medium mixing glycerol to dramatically increase Oil produces, and is the more economical new method effectively producing oil.

Although being described herein as and illustrated several embodiments of the present invention, but those skilled in the art being readily able to Envision other method multiple and/or structure is used for realizing function as herein described and/or obtaining result as herein described and/or Individual or more advantages, and think that these each changing and/or changing are also within the scope of the invention.More generally, Those skilled in the art will be apparent from all parameters as herein described, yardstick, material and structure and are intended to exemplary, and Actual parameter, yardstick, material and/or structure will depend upon which the concrete application that present invention teach that used.People in the art Member is it will be appreciated that maybe can use not more than routine experiment to determine many equivalents of invention as described herein specific embodiments Variant.It is therefore understood that previous embodiment scheme is only meant as exemplary, and in appended claims and its equivalent In the range of variant, can implement with the differently present invention being specifically described and claim.Present invention is generally directed to institute herein Each feature, system, goods, material, test kit and/or the method stated.As long as additionally, these features, system, goods, material, Test kit and/or method are the most conflicting, then two or more these features, system, goods, material, test kit and/or The combination in any of method is also contained in the scope of the present invention.

Defined and used herein being defined should be understood to control dictionary definition, in the file that is incorporated by reference into Definition and/or the conventional sense of defined term.

Contradiction unless clearly indicated, the indefinite article used the most in the specification and claims is to be understood that For meaning " at least one ".

The phrase "and/or" used in the specification and claims should be understood to united element " or Or the two ", i.e. in some cases, element exists with being combined, and in other cases, element is separately present.Unless it is clear Show and contradict, otherwise relevant or uncorrelated to those elements specifically determined, except concrete by phrase "and/or" Other element beyond element that ground determines can be optionally present.Accordingly, as non-limitative example, with reference to " A and/or B ", When being used in combination with open language such as " comprise/include ", in one embodiment, can refer to that A does not has B (optionally Element including in addition to B)；In another embodiment, can refer to that B does not has A (optionally comprising the element in addition to A)； In yet another embodiment, A and B both (optionally comprising other element) etc. can be referred to.

The "or" used in the specification and claims should be understood to have with "and/or" as defined above Identical implication.Such as, when the project in list being separated, "or" or "and/or" should be understood to include, i.e. include At least one in some elements or element list, also includes more than one, and optionally other unlisted project.The most clear Point out to Chu the term of contradiction, such as " only one " or " one exactly ", or when in claims, " consist of " refers to the element accurately comprising in some elements or element list.Typically, when being above exclusiveness art When language such as "or", " one in ... " " only one in ... " or " one accurately in ... ", it should will be made herein Term "or" be only understood to mean that exclusive alternative (that is, " and one or another but not the two ").When for right Time in claim, " substantially by ... composition " should have as in Patent Law field use its conventional sense.

The phrase " at least one " referring to one or more kinds of element list used in the specification and claims Should be understood at least one element of any one in element list or more element, but unnecessarily include tool At least one of each element being listed in element list body, and it is not excluded for the combination in any of element in element list.Nothing Opinion is relevant or uncorrelated to specifically defined those elements, this definition also allow for being optionally present except in element list specifically The element beyond element limited.Accordingly, as a non-limitative example, " at least one of A and B " (or, equally, " at least one of A or B, " or equally " at least one of A and/or B ") at least one can be referred in one embodiment Individual, optionally include more than one, A and there is not B (optionally including the element in addition to B)；In another embodiment, may be used To refer at least one, optionally include more than one, B and there is not A (optionally including the element in addition to A)；In another reality Execute in scheme, can be referred at least one, optionally include more than one, A and at least one, optionally include more than one, B (with optionally including other element)；Deng.

It is also understood that contradiction unless clearly indicated, otherwise include required by this paper of more than one step including Any means in, the order of the step of method is unnecessarily limited by the order of cited method step.

REFERENCES

1.J.Sambrook and D, Russell, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press；3rd edition (January 15,2001), 978-0879695774

2.David C.Amberg, Daniel J.Burke；And Jeffrey N.Strathern, Methods in Yeast Genetics:A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Laboratory Press (April 2005), 978-0879697280

3.John N.Abelson, Melvin I.Simon, Christine Guthrie, and Gerald R.Fink, Guide to Yeast Genetics and Molecular Biology, Part A, Volume 194 (Methods in Enzymology Series, 194), Academic Press (March 11,2004), 978-0121827786

4.Christine Guthrie and Gerald R.Fink, Guide to Yeast Geneties and Molecular and Cell Biology, Part B, Volume 350 (Methods in Enzymology, Vol 350), Academic Press；1st edition (July 2,2002), 978-0123106711

5.Christine Guthrie and Gerald R.Fink, Guide to Yeast Geneties and Molecular and Cell Biology, PartC, Volume 351, Academic Press；1st edition(July 9,2002), 978-0123106728

6.Gregory N.Stephanopoulos, Aristos A.Aristidou and Jens Nielset, Metabolic Engineering:Principles and Methodologies, Academic Press；1edition (October 16,1998), 978-0126662603

7, Christina Smolke, The Metabolic Pathway Engineering Handbook: Fundamentals, CRC Press；1edition (July 28,2009), 978-1439802960

All publications, patent and sequence library entrance (including being listed in those above entries) mentioned herein Full content be incorporated herein by, be equal to represent that each single application or patent are specifically and individually by drawing With being expressly incorporated herein.In the case of conflicting, it is as the criterion with the application (including the most arbitrarily defining).

Claims

1. the oil-producing fungal cell separated, it comprises restructuring stearyl-coenzyme A desaturase (SCD) gene improving SCD expression And/or improve what SCD expressed, and relative to same type unmodified cell, at least one extra SCD gene copy, and

Reduce the genetic modification that Δ-12 desaturase is expressed.

2. the oil-producing fungal cell of the separation described in claim 1, wherein reduces the something lost of Δ-12 delta 8 desaturase genes Product Expression Biography is modified to convert with nucleic acid construct, and described nucleic acid construct comprises:

A () comprises the expression cassette of suitable homology or allogeneic promoter,

B () comprises the expression cassette of the nucleic acid of the RNA interfering of coding targeting Δ-12 delta 8 desaturase genes product, and/or

C () reduces the nucleotide sequence of the expression of Δ-12 delta 8 desaturase genes product when inserting the genome of described cell.

3. the oil-producing fungal cell of the separation described in claim 1, wherein:

Described cell comprises restructuring stearyl-coenzyme A desaturase (SCD) gene；

Described restructuring SCD gene comprises the endogenous nucleotide sequences in described cellular genome；

Described endogenous nucleotide sequences encodes the SCD albumen of described cellular endogenous；

Described restructuring SCD gene is that the described endogenous nucleotide sequences that is because of restructuring has passed through mutation or passed through recombination event It is changed；And

Described mutation or recombination event improve the expression of natural SCD albumen.

4. the oil-producing fungal cell of the separation described in claim 2, wherein said nucleic acid construct suppresses or eliminates described Δ-12 The expression of delta 8 desaturase genes product.

5. the oil-producing fungal cell of the separation described in claim 4,

Wherein by lacking, destroy, suddenly change and/or replace the coded sequence of natural Δ-12 delta 8 desaturase genes or regulating natural Regulatory region or a part for regulatory region that Δ-12 delta 8 desaturase genes is expressed mediate Δ-12 delta 8 desaturase genes product table The suppression reached or elimination.

6. the oil-producing fungal cell of the separation described in claim 2,4 or 5, wherein said nucleic acid construct inserts described cell In genome.

7. the oil-producing fungal cell of the separation according to any one of claim 1 to 6, the raising that wherein SCD expresses gives described Carbohydrate source is converted into fatty acid, derivative of fatty acid and/or the advantageous phenotypes of triacylglycerol (TAG) by cell.

8. the oil-producing fungal cell of the separation according to any one of claim 1 to 7, wherein said cell is that oleaginous yeast is thin Born of the same parents.

9. the oil-producing fungal cell of the separation described in claim 8, wherein said cell is Yarrowia lipolytica (Y.lipolytica) cell.

10. culture, it comprises the oil-producing fungal cell according to any one of claim 1 to 9.

The culture of 11. claim 10, it also comprises carbohydrate source.

Culture described in 12. claim 11, wherein said carbohydrate source is fermentable sugars.

Culture described in 13. claim 11 or 12, wherein said carbohydrate source is monosaccharide.

Culture described in 14. claim 11 or 12, wherein said carbohydrate source is glucose and/or glycerol.

Culture according to any one of 15. claim 11 to 14, the non-sterilizing of wherein said carbohydrate source.

Culture according to any one of 16. claim 11 to 15, wherein said culture is maintained under non-sterilising conditions.

Culture according to any one of 17. claim 11 to 16, wherein said culture does not comprise described oil-producing fungus Cell selectively antibiotic or antiproliferative.

Culture according to any one of 18. claim 11 to 17, wherein said carbohydrate source is from plant or algae Biomass.

Culture according to any one of 19. claim 11 to 18, wherein said carbohydrate source is from cellulose, half fibre Dimension element, starch or glycerol.

Culture described in 20. claim 18 or 19, it also comprises cellulolytic enzyme or hydrolysis of hemicellulose enzyme.

Culture according to any one of 21. claim 18 to 20, wherein said biomass or described cellulose or half fiber Element pretreatment in hot water or diluted acid or the process of ammonia filament expansion, uses hydrolytic enzyme pretreatment, by steam pre-treatment and/or Calx Pretreatment.

22. 1 kinds are used for the method producing fatty acid or triacylglycerol, and it includes

Carbohydrate source is made to contact with the oil-producing fungal cell of the separation any one of claim 1-9；With

Be suitable to be converted into by described carbohydrate source at least in part the condition of fatty acid or triacylglycerol by described cell Under hatch the described carbohydrate source with described cells contacting.

Method described in 23. claim 22, wherein said carbohydrate source is fermentable sugars or raw from plant or algae The carbohydrate starch of material.

Method described in 24. claim 22 or 23, wherein said carbohydrate source is from cellulose or hemicellulose.

Method according to any one of 25. claim 22 to 24, wherein at cellulolytic enzyme or hydrolysis of hemicellulose enzyme In the presence of, make described carbohydrate source and described cells contacting.

Method according to any one of 26. claim 22 to 25, wherein at every gram of biomass 15IU cellulolytic enzyme or half In the presence of fiber hydrolase, described carbohydrate source is made to contact 48 hours with described cell in 55 DEG C.

Method according to any one of 27. claim 22 to 26, wherein process at hot water or diluted acid or ammonia filament expansion neutralize/ Or with biomass described in hydrolytic enzyme pretreatment or described cellulose or hemicellulose.

28. 1 kinds are used for the method producing restructuring oil-producing fungal cell, and it includes with the first nucleic acid and the second nuclear transformation fungus Cell, wherein:

Described first nucleic acid is arranged to natural stearyl-coenzyme A desaturase (SCD) gene recombinaton with described cell, or institute State the first nucleic acid and comprise the SCD gene of at least one copy；

Described first nucleic acid improves the expression of SCD；With

Described second nucleic acid reduces the expression of Δ-12 desaturase.

Method described in 29. claim 28, wherein said fungal cell is yeast cells.

Method described in 30. claim 29, wherein said yeast cells is Yarrowia lipolytica.

The oil-producing fungal cell of the separation of 31. claim 1, wherein said SCD gene is

The nucleotide sequence of coding SEQ ID NO:1.

The oil-producing fungal cell of the separation described in 32. claim 31, wherein said nucleotide sequence is SEQ ID NO:2.

The oil-producing fungal cell of the separation according to any one of 33. claim 31 to 32, it also comprises the promoter of allos.

The oil-producing fungal cell of the separation described in 34. claim 33, wherein said allogeneic promoter be constitutive promoter or Inducible promoter.

The oil-producing fungal cell of the separation described in 35. claim 33, wherein said allogeneic promoter is translation elongation factor (TEF) promoter.

The oil-producing fungal cell of the separation described in 36. claim 33, wherein said allogeneic promoter is drug induced startup Son.

The oil-producing fungal cell of the separation described in 37. claim 33, it also comprises modified SCD promoter.

The oil-producing fungal cell of the separation described in 38. claim 1, the restructuring that wherein said cell comprises raising SCD expression is hard Acyl coenzyme A desaturase (SCD) gene.

The oil-producing fungal cell of the separation described in 39. claim 1, wherein said cell comprises what raising SCD expressed, relative to Same type unmodified cell, at least one extra SCD gene copy.

The oil-producing fungal cell of the separation described in 40. claim 8, wherein said cell is selected from: Yarrowia lipolytica, the multiform Chinese Inferior yeast (Hansenula polymorpha), Pichia sp. (Pichia pastoris), saccharomyces bayanus (S.bayanus), Crewe Family name's saccharomyces lactis (K.lactis), Waltomyces lipofer, Mortierella alpine, Mortierella Isabellina, Zygosaccharomyces rouxii (Mucor rouxii), trichosporon cutaneum (Trichosporon cutaneu), rhodotorula glutinis (Rhodotorula glutinis), amylase yeast (Saccharomyces diastasicus), perhaps prosperous yeast (Schwanniomyces occidentalis), pichia stipitis (Pichia stipitis) and schizosaccharomyces pombe (Schizosaccharomyces pombe)。

The oil-producing fungal cell of the separation described in 41. any one of claim 1 to 7, wherein said cell is selected from: the western meat of aspergillosis Sa rice (Aspergillus shirousamii), Aspergillus niger (Aspergillus niger) and trichoderma reesei (Trichoderma reesei)。