CN101553558B

CN101553558B - Comprise fuel composition and the preparation and application thereof of farnesane and farnesane derivative

Info

Publication number: CN101553558B
Application number: CN200780045575.3A
Authority: CN
Inventors: 尼尔·斯蒂芬·伦宁格; 德里克·J·麦克菲
Original assignee: Amyris Inc
Current assignee: Amyris Inc
Priority date: 2006-10-10
Filing date: 2007-10-10
Publication date: 2015-08-26
Anticipated expiration: 2027-10-10
Also published as: ZA200902205B; CN101553558A

Abstract

Comprise farnesane (farnesane) and/or farnesane derivative and be selected from the fuel composition of conventional oil component of diesel oil fuel, rocket engine fuel, coal Water Oil Or Gas.This farnesane or farnesane derivative can be used as fuel element or fuel dope in this fuel composition.This fuel composition can comprise conventional oil additive further.Further disclose the preparation and application of this fuel composition.

Description

Comprise fuel composition and the preparation and application thereof of farnesane and farnesane derivative

At first related application

The application advocates the U.S. Provisional Patent Application submitted on October 10th, 2006 number 60/860 according to 35U.S.C. § 119 (e), the right of 881, with the U.S. Provisional Patent Application number 60/860 submitted on November 21st, 2006, the right of 854, it is all incorporated to herein at this in full by reference.

Technical field

Inter alia, the present invention includes the fuel composition such as diesel oil fuel and rocket engine fuel.Particularly, the present invention includes the fuel composition comprising farnesane, and prepare and use the method for this kind of fuel composition.In certain embodiments, the present invention includes stable fuel composition, it comprises that generate simply and effectively, at least part of farnesane from microorganism.In certain embodiments, the present invention includes the fuel composition comprising high-concentration biological through engineering approaches farnesane.

Background technology

In the past in decades, owing to rising steadily to oil price, the worry that rises of urgent supply restriction and global carbon dioxide quantity discharged, the fuel (" biofuel ") of biological production receives many concerns.The natural energy source non-renewable from such as oil and coal etc. is different, and biofuel comes from reproducible natural origin, is generally organism alive and metabolic by-prods thereof.

Up to now, the biofuel being applicable to the oil engines such as diesel motor comes from vegetables oil usually.The so-called first-generation " biofuel " is generally the C formed by vegetables oil transesterification ₁₆-C ₁₈fatty acid methyl ester.Recently, produce the s-generation " biofuel " by the such as novel process such as NExBTL technique (being disclosed in WO2006/075057), its by hydrogenated vegetable oil or animal tallow to obtain corresponding alkane or paraffin.Due to the character of this starting raw material, two kinds of methods all generate may with the uneven product mixture of the complexity of batch variation.The variability of this product makes to produce the fuel with specific specifications or requirement and becomes more complicated.Therefore, need fuel dope and fuel stack to assign to prepare fuel composition, and needs can be used for can simply preparing of such as diesel motor and jet engine and have the fuel element of recyclability.

Summary of the invention

The invention provides the fuel composition, fuel element or the fuel dope that comprise isoprenoid or derivatives thereof, and preparation and application.The embodiment of these compositions is believed to meet the demand.More particularly, isoprenoid and derivative thereof can be used as the fuel element in this fuel composition.In certain embodiments, this isoprenoid or derivatives thereof can be used as this fuel composition itself, the main ingredient of this fuel composition or the accessory constituent of this fuel composition.Isoprenoid and derivative thereof can prepare from microorganism (comprising Bioengineered microorganism).Fuel composition disclosed in this invention can be used as oil engine, as the fuel of petrol engine, diesel motor and jet engine.

In certain embodiments, the present invention includes the diesel oil fuel containing one or more Bioengineered fuel elements.In certain embodiments, the present invention includes the rocket engine fuel containing one or more Bioengineered fuel elements.In these embodiments, this Bioengineered fuel element can by the microorganism that can generate arbitrarily this Bioengineered fuel element, and its bacterial strain of such as genetically engineered microorganism, wild-type microorganisms or selection generates.In certain embodiments, this Bioengineered fuel element is isoprenoid or derivatives thereof disclosed in this invention.

In certain embodiments, described Bioengineered fuel element can from easy obtain, reproducible material.Impressively, the present invention thus provide easily obtain, the reproducible energy, and use them to carry out the method for energy generation.In certain embodiments, this Bioengineered fuel element can from sugar, such as monose or disaccharides.

In some other embodiment, described Bioengineered fuel element can from the non-fermented carbon source easily obtained, such as acetate or glycerine.

Accompanying drawing explanation

Fig. 1 is the schematic diagram of mevalonic acid (" the MEV ") approach generating isopentenyl pyrophosphate (" IPP ").

Fig. 2 is the schematic diagram of the DXP approach generating IPP and DPP (" DMAPP ").Dxs is DX-5-phosphate synthase; Dxr is DX-5-phosphoric acid reduction isomerase (also referred to as IspC); IspD is 4-CDP base-2C-methyl D-erythritol synthetic enzyme; IspE is 4-CDP base-2C-methyl D-erythritol synthetic enzyme; IspF is 2C-methyl D-erythritol 2,4-ring diphosphate synthase; IspG is 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (IspG); IspH is isopentene group/dimethylallyl diphosphate synthetic enzyme.

Fig. 3 shows the collection of illustrative plates of expression plasmid pAM97.

Fig. 4 shows the collection of illustrative plates of expression plasmid pAM408.

Fig. 5 shows the collection of illustrative plates of expression plasmid pAM424.

Fig. 6 A-E shows the ERG20-P of carrier pAM489 _gALthe ERG13-P of-tHMGR inset, carrier pAM491 _gALthe IDII-P of-tHMGR inset, carrier pAM493 _gALthe ERG10-P of-tHMGR inset, carrier pAM495 _gALthe ERG8-P of-ERG12 inset and carrier pAM497 _gALthe collection of illustrative plates of-ERG19 inset.

Fig. 7 shows the collection of illustrative plates of expression plasmid pAM373 and pAM342.

Fig. 8 shows the collection of illustrative plates of expression plasmid pAM404.

Fig. 9 shows the ASTM D975 test data from No. 2 diesel oil of BP Whiting refinery and the mixture of 5%, 20% and 50% farnesane (AMD-200) and this fuel.

Figure 10 shows ASTM D 975 test data meeting diesel oil fuel (CARB fuel) that California Air resource board of management (Caliornia Air Resources Board) requires and 5%, 20%, 50% and 65% farnesane mixture (AMD-200 and this fuel) from BP Carson refinery.This concrete sample of CARB fuel does not comprise the lubrication reinforcing agent be usually present in CARB fuel.

Figure 11 A-B shows the distillation curve of No. 2 diesel oil and the CARB diesel oil mixed from different amount farnesane (AMD-200).

Definition

By ASTM D 975 specification of ASTM international publication, some minimum acceptance requirements are proposed to the different grades diesel oil fuel used in the U.S..Such as, 2-D level ultra low sulfur diesel fuel should have the sulphur content (testing according to ASTM D2622) of by weight maximum 0.05%, the ash oontent (testing according to ASTM D 482) of maximum 0.01% by weight, minimum be 40 cetane value (testing according to ASTM D6079), viscosity (testing according to ASTM D 445) from 1.9cSt to 2.4cSt at 40 DEG C, and minimum be the flash-point of 52 DEG C.Japan and Europe have the diesel fuel oil specifications similar with the U.S. for the diesel oil fuel that grade is suitable.Such as, Japan JIS K2204 in, 2 grades of diesel oil fuels should have the MV minium viscosity of 2.0cSt at 40 DEG C, have the sulphur content of by weight maximum 0.05%, and minimum be 45 cetane value.Correspondingly, in the CEN 590 in Europe, A-F level diesel oil fuel should have the viscosity of 2.0cSt to 4.5cSt at 40 DEG C, has the sulphur content of by weight maximum 0.05%, and minimum be 49 cetane value.In some embodiments, fuel composition disclosed in this invention meet above-mentioned attribute at least one or all.

By the ASTMD1655 specification of ASTM international publication, some minimum acceptance requirements are proposed to Jet A.

" ash oontent " refers to the quantity of diesel oil fuel residue of remaining after combustion under the condition described in ASTM D 482.

" biofuel " refer to various come the diesel oil fuel of biological origin (such as vegetables oil or animal tallow).Biofuel mainly refers to the mixture of alkyl ester, comprises by the fatty acid methyl ester obtained after oil and methyl alcohol transesterification.Although soybean oil is the largest source of biofuel, the oil from other plant or animal tallow also can become raw material.

" Bioengineered fuel element " refers to, at least partly by host cell, comprise the fuel element that arbitrary archeobacteria, bacterium or eukaryotic cell generate.

" biofuel " refers to any fuel coming from biomass (biomass) (organism of namely surviving or its metabolic by-prods, the fertilizer of such as ox).As different in oil, coal and nuclear fuel from other natural origin, it is a kind of renewable energy source.

" C ₁₅isoprenoid starting raw material " compound that refers to farnesyl pyrophosphate (" FPP ") or can be derived by FPP.

" cetane value " refers to that under the condition described in ASTM D 613 fuel takes fire the easness of (spontaneous combustion).Have high hexadecane value fuel can injection cylinder after short period of time combustion, it has short time lag of inflammation.On the contrary, have the not easily spontaneous combustion of low-cetane fuel, it has longer time lag of inflammation.

The temperature of the brilliant opactitas of wax is there is in fuel sample first when " cloud point " refers to cool under the condition described in ASTM D 2500.

" cold filter clogging temperature " (CFPP) refers to that fuel first cannot by about instruction of temperature during wire netting in setting-up time section.The fuel cooled in ASTM D6371 test simulation fuel system is by the flowing of strainer.Therefore, CFPP is measuring of the kinetics cold flow attribute of fuel.

" diesel oil fuel " refers to the fuel being applicable to diesel motor, ignites in diesel motor under this fuel high pressure through air heating.The classification of diesel oil fuel comprises the hydro carbons with larger molecular weight scope.In some embodiments, diesel oil fuel of the present invention comprises the hydrocarbon comprising at least 15 carbon.In other embodiments, diesel oil fuel of the present invention comprises the hydrocarbon comprising at least 15 carbon, the alcohol comprising at least 3 carbon, comprises the fatty acid ester of at least 10 carbon, and composition thereof.The type of diesel oil fuel includes but not limited to petroleum diesel, biofuel, Bioengineered diesel oil or its mixture.Diesel oil fuel also can from synthol, such as shale oil or Fischer-Tropsch fuel, as obtained by the derivative fuel of synthetic gas and gelatin liquefaction thing.

" farnesane " refers to the compound with formula (III):

Or its steric isomer.In some embodiments, farnesane comprises substantially pure farnesane steric isomer.In other embodiments, farnesane comprises the mixture of the steric isomer (such as enantiomer and diastereomer) of farnesane.In further embodiment, the amount of often kind of steric isomer in farnesane mixture accounts for the about 0.1wt.% of this farnesane mixture total weight independently to about 99.9wt.%, about 0.5wt.% to about 99.5wt.%, about 1wt.% to about 99wt.%, about 5wt.% to about 95wt.%, about 10wt.% to about 90wt.%, about 20wt.% to about 80wt.%.

" α-farnesene " refers to the compound with following formula:

Or its steric isomer.In some embodiments, α-farnesene comprises substantially pure α-farnesene steric isomer.In other embodiments, α-farnesene comprises the mixture of steric isomer (such as, cis-trans isomers).In further embodiment, the amount of often kind of steric isomer in α-farnesene mixture accounts for the about 0.1wt.% of this α-farnesene mixture total weight independently to about 99.9wt.%, about 0.5wt.% to about 99.5wt.%, about 1wt.% to about 99wt.%, about 5wt.% to about 95wt.%, about 10wt.% to about 90wt.%, about 20wt.% to about 80wt.%.

" β-farnesene " refers to the compound with following formula: "

Or its steric isomer.In some embodiments, β-farnesene comprises substantially pure β-farnesene steric isomer.In other embodiments, β-farnesene comprises the mixture of steric isomer (such as, cis-trans isomers).In further embodiment, the amount of often kind of steric isomer in β-farnesene mixture accounts for the about 0.1wt.% of this β-farnesene mixture total weight independently to about 99.9wt.%, about 0.5wt.% to about 99.5wt.%, about 1wt.% to about 99wt.%, about 5wt.% to about 95wt.%, about 10wt.% to about 90wt.%, about 20wt.% to about 80wt.%.

" flash-point " refers to apply under the condition described in ASTM D93 the minimum temperature that burning things which may cause a fire disaster makes the steam on diesel oil fuel burn.

" fuel " refers to one or more hydrocarbon, one or more alcohol, one or more fatty acid esters or its mixture.Preferably, liquid hydrocarbon can be used.Fuel can be used for providing power to oil engines such as reciprocating engine (such as, petrol engine and diesel motor), Wankel engine, jet engine, some rocket engine, missile propulsive plant and gas turbine engines.In some embodiments, fuel comprises the mixture of the hydro carbons such as alkane, naphthenic hydrocarbon and aromatic hydrocarbon usually.In some embodiments, fuel comprises one or more C disclosed in this invention ₁₅isoprenoid compounds.

" fuel dope " points in fuel the emblem amount fuel element adding the character (such as, improving motor performance, fuel treatment, fuel stability or Environmental capacity) being changed this fuel, as chemical composition.The type of additive includes but not limited to antioxidant, thermal stability improvers, cetane number improver, stablizer, low temperature flow promoter, ignition dope, defoamer, anti-hazing additive, inhibiter, improver for lubricating performance, deicing agent, injector cleaning additive, smoke suppressant, drag reducing additive, metal passivator, dispersion agent, sanitising agent, demulsifying compound, dyestuff, marker, antistatic agent, sterilant and combination thereof.Term " conventional additives " refers to except isoprenoid compounds of the present invention, fuel dope well known by persons skilled in the art, such as above-mentioned fuel dope.

" fuel composition " refers to the fuel comprising at least two kinds of fuel elements.

" fuel element " refers to the mixture of any compound for preparation of fuels composition or compound.Exist " primary fuel component " and " secondary fuel components ".Main fuel element at least accounts for the volume of 50% in fuel composition; And the ratio of secondary fuel components in fuel composition is less than 50%.Fuel dope is secondary fuel components.Isoprenoid disclosed in this invention himself or can be used as main ingredient or accessory constituent with the mixture of other fuel element.

" isoprenoid " and " isoprenoid compounds " is interchangeable at this, and refers to by the derivative compound of isopentenyl pyrophosphate (" IPP ").

" initial boiling point " and " full boiling point " refers to the point in distillation curve, and it is relevant to the part that the temperature raised gradually by heating sample in sample removes.Initial boiling point is the boiling temperature of the first drop of liquid when leaving condenser, and the boiling temperature that full boiling point is last drop of liquid when leaving condenser.When sample is made up of single component, this is first identical with full boiling point, and is called as " boiling point ".ASTMM standard D86 is the general procedure measuring distillation of fuel curve.

" rocket engine fuel " refers to the fuel being adapted at using in jet engine.

" kerosene " refers to the specific distillation component of the oil (also referred to as " crude oil ") usually under atmospheric pressure between 150 DEG C and 275 DEG C.Crude oil forms primarily of paraffinic, cycloalkanes and aromatic hydrocarbon.

" oilness " refers to that diesel oil fuel carries out providing measuring of the ability of more effective abrasion protection to engine components in the intermetallic contact process of high pressure rolling point cantact under the condition described in ASTM D6079.

" petroleum diesel " refers to the specific distillation component of the oil usually under atmospheric pressure between 120 DEG C and 380 DEG C.In another embodiment, petroleum diesel is the distillation component of the oil between lower 150 DEG C and 370 DEG C of 1 normal atmosphere.

" pour point " refers to that fuel can inject container or shift out or the general instruction of minimum temperature by Flows from container, and pour point can measure under the condition described in ASTM D 97.Pour point is one of feature determining fuel availability in cold climates and suitability.

The composition of " substantially pure " compound refers to substantially not containing the composition of one or more other compounds, namely based on cumulative volume or the gross weight of said composition, this compound that said composition comprises and is greater than 80%, be greater than 90%, be greater than 95%, be greater than 96%, be greater than 97%, be greater than 98%, be greater than 99%, be greater than 99.5%, be greater than 99.6%, be greater than 99.7%, be greater than 99.8%, be greater than 99.9%, or comprise one or more other compounds being less than 0.01%.

The composition of " substantially not containing " a kind of compound refers to cumulative volume based on said composition or gross weight, comprise and be less than 20%, be less than 10%, be less than 5%, be less than 4%, be less than 3%, be less than 2%, be less than 1%, be less than 0.5%, be less than 0.1% or be less than 0.01% the composition of this compound.

Except above-mentioned definition, some compound of the present invention has one or more double bond, and it can exist as one or more steric isomers (such as cis-isomeride, trans-isomer(ide), E isomer and Z isomer).In certain embodiments, these compounds as independent steric isomer are not substantially containing other steric isomer.In some other embodiment, these compounds are the mixture of various steric isomer.

" Tx " refers to according to ASTM D-86 (being incorporated to by reference herein at this), the distillation temperature when x% of the initial volume of fuel composition is distilled.Such as, " T10 ", " T50 " and " T90 " refer to according to ASTMD 86, distillation temperature when 10%, 50% and 90% of the initial volume of this fuel composition is distilled respectively." T10 ", " T50 " and " T90 " are also called as 10vol.% temperature, 50vol.% temperature and 90vol% temperature respectively.

In the following description, all numerical value disclosed in this invention is about value, no matter whether it is connected to the word such as " approximately " or " about ".Numerical value may have 1%, 2%, 5% or be the change of 10-20% sometimes.When disclosed numerical range has lower limit R ^lwith upper limit R ^utime, then any number within the scope of this is all by open especially.Particularly, the following numerical value within the scope of this is by open especially: R=R ^l+ k* (R ^u-R ^l), wherein k is the variable increased progressively with 1% in 1% to 100% scope, namely k be 1%, 2%, 3%, 4%, 5%, 5% ..., 50%, 51%, 52 ..., 95%, 96%, 97%, 98%, 99 or 100%.In addition, the mentioned above any number scope defined by two R values is also disclosed especially.

Specific embodiments

Embodiment of the present invention provide one or more C comprised as main or secondary fuel components ₁₅the fuel composition of isoprenoid compounds.The present invention can use any C ₁₅isoprenoid compounds.In some embodiments, this C ₁₅each in isoprenoid has one of formula (I) and (II):

Wherein Z is H, O-R or O-C (=O) R; R is H, alkyl, cycloalkyl, aryl, alkaryl or aralkyl.In some embodiments, Z is O-R or O-C (=O) R; R is C ₁-C ₆alkyl.In other embodiments, Z is O-R or O-C (=O) R, and wherein R is methyl.In other embodiments, Z is O-R or O-C (=O) R, and wherein R is ethyl.In other embodiments, this C ₁₅isoprenoid compounds is farnesane, and namely the Z of formula (I) or (II) is H.

In one group of embodiment, described isoprenoid compounds is:

Wherein Z is as defined above.

In another group embodiment, described isoprenoid compounds is:

Wherein Z is as defined above.

In another group embodiment, described isoprenoid compounds is one or more compounds with following formula:

Wherein Z is as defined above.Formula (I-a), (I-b), (I-c) and (I-d) are the possible steric isomers of four kinds of formula (I), and formula (II) is formula (II-a), (II-b), (II-c) and (II-d) four kinds possible steric isomers.

In another group embodiment, described isoprenoid compounds is:

Or its steric isomer.

In another group embodiment, described isoprenoid compounds is:

Or its steric isomer, wherein R adopts aforementioned definitions.In another group embodiment, R is C ₁-C ₃alkyl.In another group embodiment, R is methyl.In another group embodiment, R is ethyl.

In another group embodiment, described isoprenoid compounds is:

In another group embodiment, described isoprenoid compounds has formula (III), (IV) or (V):

Wherein R is alkyl, such as methyl, ethyl, propyl group, sec.-propyl, butyl, isobutyl-, the tertiary butyl and linear or branched amyl group, hexyl, heptyl, octyl group, nonyl, decyl, dodecyl, four decyls, six decyls, eight decyls, eicosyl, docosyl etc.In other embodiments, the mixture of this isoprenoid compounds contained (III), (IV) or (V).

In another group embodiment, described isoprenoid compounds comprises and has formula (III), (IV) or (V) at least two kinds different compounds:

Or its steric isomer, wherein R is C ₁-C ₅alkyl, the amount that these two kinds of compounds exist at least accounts for about 5% of this fuel composition gross weight or cumulative volume respectively.

In another group embodiment, described isoprenoid compounds be as follows one of them:

Wherein R is as defined above.Formula (III-a), (III-b), (III-c) and (III-d) are the possible steric isomers of four kinds of formula (III).Formula (IV-a), (IV-b), (IV-c) and (IV-d) are the possible steric isomers of four kinds of formula (IV).Formula (V-a), (V-b), (V-c) and (V-d) are the possible steric isomers of four kinds of formula (V).

Each isoprenoid compounds in described fuel composition all as fuel element, can release energy when carrying out chemical reaction with oxygenants such as oxygen; Or can be used as fuel dope, change performance or the attribute of this fuel element.In some embodiments, this isoprenoid compounds accounts for the gross weight of this fuel composition or cumulative volume at least about 2%, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%.In other embodiments, this isoprenoid compounds accounts for gross weight or the cumulative volume maximum about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, maximum about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50%, maximum about 60%, at most about 70%, at most about 80% or at most about 90% of this fuel composition.In further embodiment, this isoprenoid compounds accounts for the gross weight of this fuel composition or cumulative volume about 2% to about 99%, about 2.5% to about 95%, about 5% to about 90%, about 7.5% to about 85%, about 10% to about 80%, about 15% to about 80%, about 20% to about 75% or about 25% to about 75%.

In some embodiments, described C ₁₅isoprenoid compounds is derived from Bioengineered C ₁₅isoprenoid starting raw material.In certain embodiments, this Bioengineered C ₁₅carbon source is converted into C by host cell by isoprenoid starting raw material ₁₅isoprenoid starting raw material prepares.

In other embodiments, described carbon source is sugar, such as monose, disaccharides or its a kind of or multiple combination.In certain embodiments, this sugar is the monose can supporting one or more Growth of Cells provided by the present invention.This monose is any monose well known by persons skilled in the art.The non-limiting example of the monose that part is suitable for comprises glucose, semi-lactosi, seminose, fructose, ribose and combination thereof.Some the non-limiting examples of the disaccharides be suitable for comprise sucrose, lactose, maltose, trehalose, cellobiose and combination thereof.

In other embodiments, described carbon source is polysaccharide.Some the non-limiting examples of the polysaccharide be suitable for comprise starch, glycogen, Mierocrystalline cellulose, chitin and combination thereof.

In other embodiments, described carbon source is not fermentable carbon source.Some non-limiting examples of suitable not fermentable carbon source comprise acetic ester or salt and glycerine.

In other embodiments, described fuel composition can comprise further come from oil, coal, timber or arbitrarily other hydro carbons source conventional oil component.The exemplary example of conventional oil component comprises diesel oil fuel, rocket engine fuel, kerosene, gasoline and Fischer-Tropsch derivatived fuel.In some embodiments, this conventional oil component is from oil or kerosene.In specific embodiments, this fuel element is diesel oil fuel, rocket engine fuel, kerosene, gasoline or its combination, or comprises diesel oil fuel, rocket engine fuel, kerosene, gasoline or its combination.In other embodiments, this fuel element is for distilling diesel oil fuel or comprising distillation diesel oil fuel.In further embodiment, the amount of this fuel element accounts for the gross weight of this fuel composition or at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of cumulative volume.In further embodiment, the amount of this fuel element accounts for gross weight or the cumulative volume maximum 10%, maximum 20%, maximum 30%, maximum 40%, maximum 50%, maximum 60%, maximum 70%, maximum 80% or maximum 90% of this fuel composition.

In some embodiments, described fuel composition can comprise conventional oil additive further.The character of these one or more additives and amount depend on the intended applications of final fuel composition.

In certain embodiments, described fuel composition expection is used for diesel motor.The U.S. detects and diesel oil fuel is divided into three large group with materialogy meeting (ASTM).The demand of classifying to these fuel comes from the different purposes of diesel motor, and this diesel motor is designed to available wherein a kind of standard diesel fuels and effectively operates.

No. 1-D is lighting end, similar with kerosene, can be used for the engine (such as, truck, tractor engine) needing frequent variations load and speed.This fuel has the flash-point being greater than 38 DEG C, and maximum cetane value is 40.This fuel is specially adapted to the running under cold snap.

No. 2-D is middle distillate fuel, has the volatility lower than No. 1-D and the density of Geng Gao.This fuel can be used for heavier engine, and such as, railroad engines, its comparable No. 1-D in use obtains at the uniform velocity but the running of more high loading.Its flash-point is greater than 52 DEG C, and minimum cetane value is 40.

No. 4-D is heavy fraction fuel, and it has the highest density and minimum volatility in three kinds of diesel oil fuels.It can be used for the low and medium-speed engine that can operate under sustained load, such as ship engine and generator.Its flash-point is greater than 55 DEG C, and minimum cetane number is 30

The diesel oil fuel that senior diesel oil fuel defines for the senior diesel oil met or exceeded one of national weights and measures trade council (NCWM) or Engine Manufacturers Association (EMA).

Generally speaking, diesel oil fuel is the complex mixture of thousands of kinds of individuation compounds.In these compounds, majority is C ₁₀-C ₂₂hydro carbons, and be generally paraffinic, cycloalkanes (ring paraffinic) and aromatic hydrocarbon based.Common paraffin refers to the alkane (being made up of hydrogen and carbon) with normal carbon chain.

Diesel oil fuel has the proportion in the distillation range of 390 to 715 °F (200 to 380 DEG C) and 0.760 to 0.935 scope at 1 atmosphere pressure usually.Except these character, diesel oil fuel should contain sulphur, the ash content of < 0.1wt.%, the water and sediment of < 0.5vol.% of < 1wt.%, and has the flash-point being greater than 55 DEG C.

Diesel oil fuel quality can characterize according to cetane value (usually in 30 to 60 scopes).High hexadecane value represents that this engine is easy to start and the potentiality of smooth operation.This cetane value and motor car engine cetane value similar, wherein n-Hexadecane (n-hexadecane, C ₁₆h ₃₄) there is designated value 100.At the other end of this grade, the isomer heptamethylnonane of n-Hexadecane has designated value 0.The cetane value of diesel oil fuel measures by comparing with the mixture of n-Hexadecane and heptamethylnonane.It is corresponding to the value of the volume parts of n-Hexadecane in the n-Hexadecane-heptamethylnonane mixture with the identical ignition quality of this fuel.

Generally speaking, ordinary diesel oil fuel has the aromatic content higher than 20wt.%, the sulphur content of hundreds of ppm or higher.They can comprise extra oxygen and/or nitrogen impurity further.For obtaining required diesel oil fuel, ordinary diesel oil fuel needs to transform usually, and the aromatic hydrocarbon wherein in this ordinary diesel oil fuel is converted into non-aromatic hydrocarbon, such as, and ring paraffin.This is achieved by ordinary diesel oil fuel being carried out in the presence of a hydrogenation catalyst hydrogenation usually.Also other method for transformation can be adopted.

Typically, " straight run " diesel oil fuel that crude oil generates through simple distillation has lower aromatic hydrocarbon content.But, generate the rising that can cause aromatic content by catalytic cracking Residual oil raising gasoline and diesel oil.Typical distillation diesel oil can comprise the aromatic hydrocarbon of 20 to 25% volumes, and the diesel oil being mixed with the raw material of catalytic cracking can have the aromatic hydrocarbon of 40 to 50%.The cumulative volume that the aromatic hydrocarbon content of fuel composition disclosed in this invention accounts for this fuel composition is less than about 50vol.%, about 45vol.%, about 40vol.%, about 35vol.%, about 30vol.%, about 25vol.% or about 20vol.%.In some embodiments, the cumulative volume that the aromatic hydrocarbon content of this fuel composition accounts for this fuel composition is less than about 15vol.%, is less than about 10vol.%, is less than about 5vol.%, is less than about 2.5vol.% or is less than about 1vol.%.In other embodiments, this fuel composition is not substantially containing aromatic hydrocarbons.

Aromatic hydrocarbon has poor spontaneous combustion quality, and the diesel oil fuel therefore containing aromatic hydrocarbon at high proportion has lower cetane value usually.The typical cetane value of straight-run diesel oil is in 50 to 55 scopes, and the cetane value of high aromatic hydrocarbon diesel oil fuel, even may be lower usually in 40 to 45 scopes.This may cause the difficulty of cold start-up due to long ignition delay, and improves combustion noise.

In order to reduce the sulphur content of fuel composition of the present invention, sulfur removal technology can be adopted to reduce the diesel fuel constituents in this fuel composition and/or higher isoprenoid compounds amount can be adopted.Any sulfur method embodiment used in the present invention.Also can use the step of other deoxidation and/or nitrogen, to obtain required diesel oil fuel.U.S. Patent number 5,611,912,5,068,025,4,746,420 and 4,675,102 disclose hydrogenation in embodiment used in the present invention and/or sulfur removal technology.The disclosure content of all aforementioned patent is incorporated to herein in full by reference at this.Fuel composition disclosed in this invention can have the sulphur content that maybe can be prepared into and have and account for this fuel composition gross weight and be less than about 500ppm, about 100ppm, about 50ppm, about 30ppm, about 20ppm or about 15ppm.In other embodiments, the sulphur content of this fuel composition is less than about 10ppm.In other embodiments, this fuel composition not sulfur-bearing substantially.

In certain embodiments, described fuel composition expection is used for jet engine.Modal rocket engine fuel is kerosene/paraffin fuel oil, and it is classified as Jet A-1, and its production meets International Standard.A kind of Jet A-1 type being called Jet A is only additionally used in the U.S..Another kind of rocket engine fuel conventional in civil aviaton is called as Jet B.Jet B be naphtha-kerosene (naptha-kerosene) field a kind of because of its cold weather performance by the lighter for ignition fuel oil used.The distillation range of Jet B is usually at 140 to 460 °F (50 to 250 DEG C).Jet A, Jet A-1 and Jet B ASTM specification D.1655-68 in illustrate.Alternatively, rocket engine fuel can adopt JP numbering system to carry out military classification in the whole world.Almost object civilian with it is consistent for part, only distinguishes to some extent in the amount of a small amount of additive.Such as, Jet A-1 and JP-8 is similar, and Jet B and JP-4 is similar.Or rocket engine fuel also can be classified as kerosene type or naphtha type.Some non-limiting examples of kerosene type jet fuel comprise Jet A, Jet A1, JP-5 and JP-8.Some non-limiting examples of naphtha type rocket engine fuel comprise Jet B and JP-4.In other embodiments, the Jet B according to ASTM specification D1655-68 is not comprised when fuel composition contained (III) or formula (I) or formula (II) (wherein Z is H).

Jet A is the standard jet type that the U.S. has used since nineteen fifty.Except the higher congealing point of-40 DEG C, JetA and Jet-A1 is similar.Similar with Jet A-1, Jet A has the higher flash point of minimum 38 DEG C, and spontaneous ignition temperature is 210 DEG C.

In certain embodiments, described fuel composition comprises at least one conventional oil additive.Some non-limiting examples of conventional oil additive comprise antioxidant, thermal stability improvers, cetane number improver, stablizer, low temperature flow promoter, ignition dope, defoamer, anti-hazing additive, inhibiter, improver for lubricating performance, deicing agent, injector cleaning additive, smoke suppressant, drag reducing additive, metal passivator, dispersion agent, sanitising agent, demulsifying compound, dyestuff, marker, antistatic agent, sterilant and combination thereof.The total amount of the fuel dope in this fuel composition can account for 0.001 to the 10wt% of this fuel composition gross weight, is 0.01 to 5wt% in one embodiment.

The people such as the visible Chunsham Song of description of some conventional oil additives, " Chemistry ofdisel fuel, " Taylor & Francis, London, chapter 1,32-36 page (2000), it is incorporated to herein by reference at this.In addition, the various additive that can be used for embodiment of the present invention disclosed in U.S. Patent as follows: 6,054,420,6,051,039,5,997,593,5,997,592,5,993,498,5,968,211,5,958,089,5,931,977,5,891,203,5,882,364,5,880,075,5,880,072,5,855,629,5,853,436,5,743,922,5,630,852,5,529,706,5,505,867,5,492,544,5,490,864,5,484,462,5,321,172 and 5,284,492.The disclosure content of all aforementioned patent is incorporated to herein in full by reference at this.

In some other embodiment, described fuel composition comprises the fuel dope as improver for lubricating performance or toughener.In some embodiments, one or more improver for lubricating performance mix mutually with this diesel oil fuel.The about 1ppm that the concentration of this improver for lubricating performance in this fuel accounts for the gross weight of this fuel composition usually to about 50,000ppm, about 10ppm to about 20,000ppm, 25ppm is to about 10,000ppm or 50ppm to about 1000ppm.Some non-limiting examples of proper lubrication improvement in performance agent comprise the ester of lipid acid, such as glyceryl monooleate and diiso decyl adipic acid ester; Based on the additive of acid amides, the additive (as LZ 539C) that such as can obtain from Lubrizol Chemical Company; Linoleic acid dimer; Aminoalkyl morpholine; Carbophenothion acid diesters-glycol; And there is the alkyl aromatic compound of at least one carbonyl group.Some improver for lubricating performance be suitable for or toughener are described in patent documentation, such as WO95/33805; WO94/17160; WO98/01516 and U.S. Patent number 5,484,462 and 5,490,864, and Danping Wei and H.A.Spikes, " TheLubricity of Diesel Fuels ", Wear, III (1986) 217235, all these documents are all incorporated to herein by reference at this.Some non-limiting examples of commercial improver for lubricating performance comprise OLI9000 (from Octel Corporation, Manchester, UK), PARADYNE ^tM655 and VEKTRON ^tM6010 (from Infineum, Linden, NJ), and HITEC ^tMe58O (from EthylCorporation, Richmond, VA).

In some other embodiment, described fuel composition comprises the fuel dope as sanitising agent.Generally speaking, the amount of this detergent additive accounts for being less than 10,00ppm, being less than 1000ppm, being less than 100ppm or being less than 10ppm of the gross weight of this fuel composition.Some non-limiting examples of suitable sanitising agent comprise the succimide of polyolefine replacement or the succinamide of polyamine, such as polyisobutene succinimide or PIBA succinamide, fatty amine, Mannich alkali or amine, and polyolefine (such as, polyisobutene) maleic anhydride.Some succimide sanitising agents be suitable for are described in GB960493, EP0147240, EP0482253, EP0613938, EP0557561 and WO98/42808 (being all incorporated to by reference herein at this).In some embodiments, this sanitising agent is the succimide that polyolefine replaces, such as polyisobutene succinimide.Some non-limiting examples of commercial detergent additive comprise F7661 and F7685 (from Infineum, Linden, NJ) and OMA4130D (from Octel Corporation, Manchester, UK).

In some other embodiment, described fuel composition comprises the fuel dope as cetane number improver.Some non-limiting examples of cetane number improver comprise superoxide, nitrate, nitrite, azo-compound etc.N-octyl nitrate, the 2-methyl-2-nitropropyl nitric ether of alkyl nitrate ester selected such as amyl nitrate, the own ester of nitric acid and mixing can be used, and the own ester of 2-acetyl nitrate.In some embodiments, this hexadecyl promotor is the own ester of 2-acetyl nitrate, and it can be buied from Associated OctelCompany Limited, and trade mark is C1-0801.The concentration of this hexadecyl promotor can be about 0.001 to the 5wt% of the gross weight of this fuel composition, in one embodiment from 0.01 to 2.5wt%.

In some other embodiment, described fuel composition comprises the fuel dope as stablizer.Some non-limiting examples of stablizer comprise primary t-alkyl-amine.Many stablizers also can be used as inhibiter.The concentration of stablizer in this fuel composition can be about 0.001 to the 2wt% of this fuel composition gross weight, 0.01 to 1wt% in one embodiment.

In some other embodiment, described fuel composition comprises the fuel dope as ignition dope.Some non-limiting examples of ignition dope comprise ferrocene (two (cyclopentadiene) iron), iron-based ignition dope (such as, TURBOTECT ^tMeR-18, come from Turbotect (USA) Inc., Tomball, Texas), barium base ignition dope, cerium ignition dope and iron and magnesium base ignition dope (such as, TURBOTECT ^tM703, from Turbotect (USA) Inc., Tomball, Texas).The concentration of ignition dope in this fuel composition can be about 0.001 to the 1wt% of this fuel composition gross weight, 0.01 to 1wt% in one embodiment.

On the other hand, provide and comprise following composition:

A () has the isoprenoid compounds of formula (I) or (II):

(b) conventional oil component; And

(c) fuel dope

Wherein Z is H, O-R or O-C (=O) R; R is H, alkyl, cycloalkyl, aryl, alkaryl or aralkyl; Based on the cumulative volume of this fuel composition, the amount of this isoprenoid is at least about 1vol.%, and the amount of this conventional oil component is at least about 5vol.%; And wherein this fuel composition has the flash-point being equal to or greater than 38 DEG C, and there is the initial boiling point between about 100 DEG C and about 200 DEG C.

In some embodiments, the amount of described isoprenoid compounds in fuel composition disclosed in this invention is at least 2vol.%, 3vol.% or 4vol.% of the cumulative volume of this fuel composition.In other embodiments, the amount of this isoprenoid compounds is that the about 1vol.% of the cumulative volume of this fuel composition is to about 90vol.%, about 2vol.% to about 90vol.%, about 3vol.% to about 90vol.% or about 4vol.% to about 90vol.%.

On the other hand, provide and comprise following composition:

A () has the isoprenoid compounds of formula (I) or (II):

(b) conventional oil component; And

(c) fuel dope

Wherein Z is H, O-R or O-C (=O) R; R is H, alkyl, cycloalkyl, aryl, alkaryl or aralkyl; Based on the cumulative volume of this fuel composition, the amount of this isoprenoid is at least about 5vol.%, and the amount of this conventional oil component is at least about 5vol.%; And wherein this fuel composition has the flash-point being equal to or greater than 38 DEG C, and there is the initial boiling point between about 100 DEG C and about 200 DEG C.

In some embodiments, the amount of described isoprenoid compounds in fuel composition disclosed in this invention is the about 5vol.% of the cumulative volume of this fuel composition to about 90vol.%.In other embodiments, the cumulative volume that the amount of this isoprenoid compounds accounts for this fuel composition is less than about 75vol.%, is less than about 65vol.%, is less than about 50vol.% or is less than about 45vol.%.In other embodiments, the amount of this isoprenoid compounds is about 5vol.% to about 10vol.%.In other embodiments, the amount of this isoprenoid compounds is about 15vol.% to about 25vol.%.In other embodiments, the amount of this isoprenoid compounds is about 45vol.% to about 55vol.%.

In other embodiments, based on the cumulative volume of described fuel composition, described in fuel composition disclosed in this invention, the amount of conventional oil component is at least about 20%, and the amount of described isoprenoid compounds is about 5% to about 75%.In certain embodiments, based on the cumulative volume of this fuel composition, the amount of this conventional oil component is at least about 30%, and the amount of this isoprenoid compounds is about 5% to about 65%.In certain embodiments, based on the cumulative volume of this fuel composition, the amount of this conventional oil is at least about 40%, and the amount of this isoprenoid compounds is about 5% to about 50%.In certain embodiments, based on the cumulative volume of this fuel composition, the amount of this conventional oil is at least about 50%, and the amount of this isoprenoid compounds is about 5% to about 45%.

In some embodiments, described conventional oil component is the fuel based on coal.In other embodiments, this conventional oil component is petroleum diesel.In other embodiments, this conventional oil component is kerosene.

In some embodiments, fuel composition disclosed in this invention has and is greater than about 100 DEG C, is greater than about 110 DEG C, is greater than about 120 DEG C, is greater than about 130 DEG C or be greater than the initial boiling point of about 140 DEG C.In other embodiments, this initial boiling point is about 100 DEG C to about 150 DEG C.

In some embodiments, fuel composition disclosed in this invention has the full boiling point being greater than about 200 DEG C.In other embodiments, this full boiling point is greater than about 225 DEG C, is greater than about 250 DEG C, is greater than about 275 DEG C, is greater than about 300 DEG C or be greater than about 325 DEG C.In further embodiment, this full boiling point is for being greater than about 350 DEG C.In certain embodiments, this full boiling point is for being greater than about 375 DEG C.

In other embodiments, fuel composition disclosed in this invention has the initial boiling point of about 100 DEG C to about 200 DEG C and is greater than the full boiling point of about 300 DEG C.In another embodiment, this fuel composition has the initial boiling point of about 110 DEG C to about 140 DEG C and is greater than the full boiling point of about 350 DEG C.In another embodiment, this fuel composition has the initial boiling point of about 110 DEG C to about 140 DEG C and is greater than the full boiling point of about 375 DEG C.

In some embodiments, fuel composition disclosed in this invention has the T90 distillation temperature of about 270 DEG C to about 350 DEG C.In other embodiments, this T90 distillation temperature is about 282 DEG C to about 338 DEG C.

In other embodiments, fuel composition disclosed in this invention has from about 175 DEG C to about 375 DEG C, from about 200 DEG C to about 350 DEG C, from about 225 DEG C to about 325 DEG C or from the T50 distillation temperature of about 250 DEG C to about 300 DEG C.

In other embodiments, fuel composition disclosed in this invention has from about 150 DEG C to about 350 DEG C, from about 175 DEG C to about 325 DEG C, from about 200 DEG C to about 300 DEG C or from the T10 distillation temperature of about 225 DEG C to about 275 DEG C.

In some embodiments, fuel composition disclosed in this invention have at least about 40, at least about 45, at least about 50, at least about 55, at least about 60 or at least about 65 cetane value.In other embodiments, this fuel composition has the cetane value at least about 70.In certain embodiments, this fuel composition has the cetane value of about 40 to 90, about 45 to 80 or about 50 to 70.

In some embodiments, fuel composition disclosed in this invention has the cloud point being equal to or less than 0 DEG C.In another group embodiment, this fuel composition has the cloud point being equal to or less than-5 DEG C.In another group embodiment, this fuel composition has the cloud point being equal to or less than-10 DEG C.In another group embodiment, this fuel composition has the cloud point being equal to or less than-15 DEG C.In another group embodiment, this fuel composition has the cloud point being equal to or less than-20 DEG C.In another group embodiment, this fuel composition has the cloud point being equal to or less than-25 DEG C.

In some embodiments, fuel composition disclosed in this invention has low sulfur content.In other embodiment, the sulphur content of this fuel composition compares the gross weight of this fuel composition lower than 500ppm.In other embodiments, this sulphur content compares the gross weight of this fuel composition lower than 250ppm, lower than 150ppm, lower than 100ppm, lower than 50ppm, lower than 25ppm, lower than 20ppm, lower than 1ppm or lower than 50ppm.In certain embodiments, this fuel composition does not have the sulphur content that can survey.

In some embodiments, fuel composition disclosed in this invention meets the regulation of ASTM D 975 for No. 2 diesel oil.

On the other hand, provide and comprise following composition:

A () amount is at least about the C of 1vol.% ₂₀hydrocarbon; With

B () has the isoprenoid compounds of formula (I) or (II)

Its amount is at least about 1vol.%, and wherein various amount is all based on the cumulative volume of this fuel composition, and Z is H, O-R or O-C (=O) R; R is H or C ₁-C ₆alkyl.In some embodiments, the amount of this isoprenoid compounds is at least about 2vol.%, 3vol.%, or 4vol.%.In some embodiments, this fuel composition comprises (c) further and accounts for the C of this fuel composition cumulative volume at least about 1vol.% ₁₀hydro carbons.

On the other hand, provide and comprise following composition:

A () amount is at least about the C of 1vol.% ₂₀hydrocarbon; With

B () has the isoprenoid compounds of formula (I) or (II)

Its amount is at least about 5vol.%, and wherein various amount is all based on the cumulative volume of this fuel composition, and Z is H, O-R or O-C (=O) R; R is H or C ₁-C ₆alkyl.In some embodiments, this fuel composition comprises (c) further and accounts for the C of this fuel composition cumulative volume at least about 1vol.% ₁₀hydro carbons.

In some embodiments, described C ₁₀the amount of hydro carbons is at least about 2vol.%, 3vol.%, 4vol.% or 5vol.%.In other embodiment, this C ₂₀the amount of hydro carbons is at least about 2vol.%, 3vol.%, 4vol.% or 5vol.%.

In some embodiments, described fuel composition comprises C further ₁₁-C ₁₉hydro carbons, wherein each group C ₁₁, C ₁₂, C ₁₃, C ₁₄, C ₁₅, C ₁₆, C ₁₇, C ₁₈, and C ₁₉the amount of hydro carbons be the cumulative volume of this fuel composition at least about 1vol%.

Fuel composition disclosed in this invention can be used for for the arbitrary equipment (such as reserve generator or oil engine) of needs fuel (such as, diesel oil fuel or rocket engine fuel) provides power.In certain embodiments, the emergency back-up fuel comprising one or more above-mentioned fuel compositions is provided.In certain embodiments, the invention provides the purposes of above-mentioned fuel composition as emergency back-up fuel.Term " emergency back-up fuel " refers to usually be stored in the fuel in the container beyond vehicle gas tank.This fuel should keep stable in the longer time period (such as, six to ten two months).When this motor vehicle fuel is used up, this emergency back-up fuel is added into the gas tank of this vehicle, and provides fuel to vehicle.Because the flash-point of diesel oil fuel prepared according to embodiment of the present invention is usually more than 140 °F, therefore can being stored in its safety in the luggage of diesel vehicle.This fuel composition also can be used as alternative fuel, as U.S. Patent number 6, and 096, described in 103, it is incorporated to herein by reference at this in full.

On the other hand, the fuel system of the fuel container comprised containing fuel composition provided by the present invention is provided.This fuel system comprises the engine-cooling system with recirculation engine coolant alternatively further, the fuel tube connecting this fuel container and oil engine and/or the fuel filter be arranged on fuel tube.Some non-limiting examples of oil engine comprise reciprocating engine (such as, petrol engine and diesel motor), Wankel engine, jet engine, some rocket engine and gas turbine engine.

In some embodiments, described fuel container and described cooling system are set together, thus allow described recirculation engine coolant to the heat trnasfer of the fuel composition in this fuel container.In other embodiments, this fuel system comprise further containing for the second fuel of diesel motor the second fuel container and be connected the second fuel tube of this second fuel container and this oil engine.Alternatively, this first and second fuel tube can be equipped with electromagnetic control valve, and this valve can open independently of one another or synchronously or cut out.In other embodiments, this second fuel is petroleum diesel.

On the other hand, provide engine installation, it comprises oil engine, comprises the fuel container of fuel composition disclosed in this invention, connects the fuel tube of this fuel container and oil engine.Alternatively, this engine installation comprises fuel filter further and/or comprises the engine-cooling system of recirculation engine coolant.In some embodiments, this oil engine is diesel motor.In other embodiments, this oil engine is jet engine.

When using fuel composition disclosed in this invention, need the particulate removed before being injected into this engine in this fuel composition.Therefore, need to select suitable fuel filter in fuel system disclosed in this invention.Water in automotive fuel, even if amount is few, can cause great harm to this engine.Therefore, the moisture removed before fuel composition injection engine is wherein needed.In some embodiments, by adopting the fuel filter of turbine centrifugal machine to remove water and particulate, the degree that wherein water and particulate are separated from this fuel composition makes the fuel composition after filtering can inject this engine and not bring the danger of this engine of damage.Also the fuel filter of other type can be adopted to make a return journey dewater and/or particulate.

On the other hand, provide the vehicles, it comprises oil engine, comprises the fuel container of fuel composition disclosed in this invention, connects the fuel tube of this fuel container and oil engine.Alternatively, these vehicles comprise fuel filter further and/or comprise the engine-cooling system of recirculation engine coolant.Some non-limiting examples of the vehicles comprise automobile, motorcycle, train, steamer and flyer.

On the other hand, the method for the isoprenoid compounds of preparation formula (I) or (II) is provided

Wherein Z is H, O-R or O-C (=O) R; R is H, alkyl, cycloalkyl, aryl, alkaryl or aralkyl.The method comprises:

A) C is obtained from biogenetic derivation ₁₅isoprenoid starting raw material, and

B) by this C ₁₅isoprenoid starting raw material is converted into described compound by chemosynthesis.

On the other hand, isoprenoid compounds (I) or (II) is provided

Wherein Z is H, O-R or O-C (=O) R; R is H, alkyl, cycloalkyl, aryl, alkaryl or aralkyl, and wherein this compound is prepared by the following method:

On the other hand, the biofuel generated by the following method is provided:

B) by this C ₁₅isoprenoid starting raw material is converted into the isoprenoid compounds of formula (I) or (II) by chemosynthesis:

Wherein Z is H, O-R or O-C (=O) R; R is H, alkyl, cycloalkyl, aryl, alkaryl or aralkyl.

In one group of embodiment, described C ₁₅isoprenoid compounds starting raw material is

It generates through over hydrogenation

Or its steric isomer.

In another group embodiment, described C ₁₅isoprenoid compounds starting raw material is

It generates through over hydrogenation and esterification

Or its steric isomer, wherein R is alkyl.

It generates through over hydrogenation and esterification

Or its steric isomer, wherein R is alkyl.

On the other hand, provide the method preparing fuel composition, it comprises:

A) be suitable for generating described C ₁₅under the condition of isoprenoid starting raw material, this C can be generated ₁₅the cell of isoprenoid starting raw material contacts with monose;

B) this C of hydrogenation ₁₅isoprenoid starting raw material, to form the C of hydrogenation ₁₅isoprenoid compounds; With

C) by the C of this hydrogenation ₁₅isoprenoid compounds mixes to prepare this fuel composition with one or more fuel elements or fuel dope mutually.

On the other hand, provide the method preparing fuel composition, it comprises:

A) be suitable for generating described C ₁₅under the condition of isoprenoid starting raw material, this C can be generated ₁₅the cell of isoprenoid starting raw material contacts with non-fermented carbon source;

On the other hand, the device for the production of fuel of the present invention, Bioengineered fuel element or Bioengineered fuel dope is provided.In certain embodiments, this device can produce described C with biological method ₁₅starting raw material.In certain embodiments, this device can prepare isoprenoid fuel dope or fuel element from this starting raw material further.

Described device can comprise microorganism can be adopted to prepare described C ₁₅the arbitrary structures of starting raw material.In some embodiments, this biological device comprises one or more cells disclosed in this invention.In some embodiments, this biological device comprises cell culture, and this cell culture at least comprises and accounts for its gross weight at least about 1wt.%, at least about 5wt.%, at least about 10wt.%, at least about 20wt.% or the C at least about 30wt.% ₁₅starting raw material.In other embodiments, this biological device comprises the fermentor tank containing one or more cells of the present invention.

The present invention can use any fermentor tank that can provide the stable and control environment being suitable for cell or microorganism growth or breeding.In some embodiments, this fermentor tank comprises the culture with one or more cells disclosed in this invention.In other embodiments, this fermentor tank comprises the cell culture that can generate farnesyl pyrophosphate (FPP) in biology mode.In other embodiments, this fermentor tank comprises the cell culture that can generate isopentenyl pyrophosphate (IPP) in biology mode.In certain embodiments, this fermentor tank comprises cell culture, and this cell culture at least comprises and accounts for its gross weight at least about 1wt.%, at least about 5wt.%, at least about 10wt.%, at least about 20wt.% or the C at least about 30wt.% ₁₅starting raw material.

Described device can comprise further can by described C ₁₅starting raw material produces the arbitrary structures of described fuel element or fuel dope.This structure can comprise for this C of hydrogenation ₁₅the hydrogenator of starting raw material.The present invention can use can by any hydrogenator that C=C reduction is C-C singly-bound under condition well known by persons skilled in the art.This hydrogenator can comprise hydrogenation catalyst disclosed in this invention.In some embodiments, this structure comprises the mixture of mixing tank, container and the hydrogenated products from step of hydrogenation in this embodiment and conventional oil additive further.

host cell

C ₁₅isoprenoid starting raw material is prepared by any means well known by persons skilled in the art, comprises biological method, chemosynthesis (not using the raw material of biological origin) and adopts the blending means of biology and chemical means simultaneously.As this C ₁₅when isoprenoid starting raw material is prepared by biological method, a kind of method comprises the host cell using and can generate target product through transformation.Similar with all isoprenoids, C ₁₅isoprenoid starting raw material is prepared through biochemical method by common intermediate isopentenyl pyrophosphate (" IPP ").

Described host cell can grow according to any technology well known by persons skilled in the art.Particularly, this host cell can grow in the substratum being applicable to this host cell.In favourable embodiment, this substratum comprises the reproducible component easily obtained.Therefore, the invention provides the reproducible energy easily obtained, use it for the method generating fuel composition.In certain embodiments, this host cell is being suitable for its growth and is generating C ₁₅undertaken growing or cultivating by contacting with monose under the condition of isoprenoid.In certain embodiments, this host cell by with glucose, semi-lactosi, seminose, fructose, ribose or its combine to contact and carry out growing or cultivating.Therefore, the invention provides from monose, the fuel composition of such as glucose, semi-lactosi, seminose, fructose, ribose and combination thereof, and generated the method for this fuel composition by this monose.

The host cell of any appropriate all can be used for implementing the present invention.In one embodiment, this host cell is genetically engineered host microorganism, and its nucleic acid molecule (namely suddenlys change through inserting, deleting or change; Such as by the insertion of Nucleotide, deletion, replacement and/or inversion) to generate required isoprenoid or isoprenoid derivative, or the output of this isoprenoid needed for improving or isoprenoid derivative.In another embodiment, this host cell can grow in liquid growth media.

The example of the host cell be suitable for comprises archeabacterial cell, bacterial cell and eukaryotic cell.The archeabacterial cell that some non-limiting examples of archeabacterial cell comprise and belong to gas fire Pseudomonas (Aeropyrum), Archaeglobus, Halobacterium (Halobacterium), methanococcus (Melhanococcus), Methanobacterium (Methanobacterium), hot-bulb Pseudomonas (Pyrococcus), sulfolobus solfataricus belong to (Sulfolobus) and thermophilic mycoplasma (Thermoplasma).Some non-limiting examples of archeobacteria bacterial strain comprise thermophile bacteria Aeropyrum pernix, ancient green-ball bacterium of glimmering, Methanococcus jannaschii, addicted to hot autotrophic methane bacteria, Pyrococcus abyssi, Pyrococcus honkoshii, thermoplasma acidophilum and Thermoplasmavolcamum etc.

Some non-limiting examples of bacterial cell comprise and belong to Agrobacterium, acidocaldarius belongs to, Anabaena, cyanobacteria belongs to group capsule, genus arthrobacter, azotobacter, bacillus, brevibacterium sp, Chromatium, Clostridium, corynebacterium, enterobacter, erwinia, Colibacter, lactobacillus genus, lactococcus, Bradyrhizobium, Methylobacter, emblem Bacillaceae, seat cyanobacteria belongs to, Rhodopseudomonas, red bacterium belongs to, Rhodopseudomonas, Crimson rhodospirillum, Rhod, salmonella, Scenedesmun, serratia, Shigella, Staphylococcus, streptomyces, the bacterial cell of Synnecoccus and zymomonas.

Some non-limiting examples of bacterial isolates comprise subtilis, medical science ring-type bacillus, Brevibacterium ammoniagenes, Brevibacterium immariophilum, Bai Jilinsiji (family name) clostridium, Enterobactersakazaku, intestinal bacteria, Lactococcus lactis, Autoinducer, pseudomonas aeruginosa, Pseudomonas mevalonii, Pseudomonas pudica, Rhodobacter capsulatus, Spherical red antibacterial, Crimson rhodospirillum, enteritis sramana (family name) bacterium, Salmonellas Corynebacterium diphtheriae, mouse typhus sramana (family name) bacterium, dysentery bacterium, Fu Shi will congratulates (family name) bacterium, shigella sonnei, streptococcus aureus etc.

Generally speaking, if use bacterial host cell, preferably avirulence bacterial strain is adopted.Some non-limiting examples of avirulence bacterial strain comprise subtilis, intestinal bacteria, Lactibacillusacidophilus, Switzerland's breast (acid) bacillus, Pseudomonas aeruginosa, Pseudomonas mevalonii, pseudomonas putida, Spherical red antibacterial, photosynthetic bacteria, Crimson rhodospirillum etc.

More eukaryotic non-limiting examples comprise fungal cell.Some non-limiting examples of fungal cell comprise the fungal cell belonging to Aspergillus, Candida, Chrysosporium, Cryotococcus, Fusarium, genus kluyveromyces, endogenetic fungus genus, neurospora, Penicillium, Pichia, Saccharomycodes and trichoderma.

Some non-limiting examples of eukaryotic cell bacterial strain comprise Aspergillus nidulans, aspergillus niger, aspergillus oryzae, Candida albicans, Chryosporium lucknowense, Fusarium graminearum, fusarium, Kluyveromyces lactis, Neuraspora crassa, Angus pichia spp, lucky illiteracy moral (family name) pichia spp, Pichia kudriavezii, Pichia membranaefaciens, pichia methanolica, silent yeast difficult to understand, pichia pastoris phaff, Pichiapijperi, Pichia quercuum, Pichia salictaria, Pichia thermotolerans, Pichiatrehalophila, pichia stipitis, actinomyces antibioticus, streptomyces aureus, chain streptococcus aureus, Saccaromyces bayanus, cloth Laplace yeast, yeast saccharomyces cerevisiae, kabicidin streptomycete, ash streptomyces chromogenes, streptomyces griseus, plumbous blue or green streptomycete, streptomyces olivochromogenes, branch streptomycete, field is without streptomycete, wine red streptomyces, and Trichodermareesei.

Generally speaking, when using eukaryotic cell, preferred avirulence bacterial strain.Some non-limiting examples of avirulence bacterial strain comprise Fusarium graminearum, fusarium, Pichia pastoris, cloth Laplace yeast and yeast saccharomyces cerevisiae.

In addition, some bacterial strain has been appointed as GRAS through Food and Drug Admistraton or has been it has been generally acknowledged that safety.Some non-limiting examples of these bacterial strains comprise subtilis, Lactibacillus acidophilus, Switzerland's breast (acid) bacillus and yeast saccharomyces cerevisiae.

iPP approach

There is the biosynthetic pathway of two kinds of known synthesis IPP and isomer DPP (" DMAPP ") thereof.Be different from plant, eukaryote exclusively uses mevalonic acid-dependency (" MEV ") Isoprenoid pathway that acetyl-CoA (" acetyl-CoA ") is converted into IPP, and IPP is isomerized to DMAPP subsequently.Prokaryotic organism (there is a few exceptions) use mevalonic acid-dependent/non-dependent or deoxy-D-xylulose sugar 5-phosphoric acid (" DXP ") approach to generate IPP and DMAPP respectively by a tapping point.Generally speaking, plant uses MEV and DXP approach to carry out IPP synthesis simultaneously.

mEV approach

Fig. 1 shows the schematic diagram of MEV approach.Generally speaking, this approach comprises six steps.

In the first step, two acetyl-CoA molecules merge formation acetoacetyl-CoA through enzyme catalysis.The enzyme of known this step of catalysis is, such as, and acetyl-CoA thiolase.Some the non-limiting examples of nucleotide sequence of this kind of enzyme of encoding comprise following GenBank accession number and can obtain the organism of this sequence: (NC_000913 REGION:2324131..2325315; Intestinal bacteria), (D49362; Micrococcus denitrificans) and (L20428; Yeast saccharomyces cerevisiae).

At the second step of MEV approach, acetoacetyl-CoA and another acetyl-CoA molecule condensation form 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA).The enzyme of known this step of catalysis is, such as, and HMG CoA synthase.Some the non-limiting examples of nucleotide sequence of this kind of enzyme of encoding comprise (NC_001145. complement 19061..20536; Yeast saccharomyces cerevisiae), (X96617; Yeast saccharomyces cerevisiae), (X83882; Arabidopis thaliana), (AB037907; Kitasatospora griseola), (BT007302; Homo sapiens) and (NC_002758, Locus tag SAV2546, GeneID 1122571; Streptococcus aureus).

In the 3rd step, the enzyme catalysis of HMG coenzyme A is converted into mevalonic acid.The enzyme of known this step of catalysis is, such as, and HMG CoA-reductase.Some the non-limiting examples of nucleotide sequence of this kind of enzyme of encoding comprise (NM_206548; Fruit bat), (NC_002758, Locus tag SAV2545, GeneID1122570; Streptococcus aureus), (NM_204485; Gallus gallus), (AB015627; Streptomyces KO3988), (AF542543; Gradually narrow leaf tobacco), (AB037907; Kitasatosporagriseola), (AX 128213, provides the sequence of coding brachymemma HMGR; Yeast saccharomyces cerevisiae) and (NC_001145: complement (115734..118898; Yeast saccharomyces cerevisiae).

In the 4th step, mevalonic acid is formed mevalonic acid 5-phosphoric acid by enzyme catalysis phosphorylation.The enzyme of known this step of catalysis is, such as, and Mevalonic kinase.Some the non-limiting examples of nucleotide sequence of this kind of enzyme of encoding comprise (L77688; Arabidopis thaliana) and (X55875; Yeast saccharomyces cerevisiae).

In the 5th step, add another phosphate group to described mevalonic acid 5-phosphatase catalytic, thus form mevalonic acid 5-tetra-sodium fat.The enzyme of known this step of catalysis is, such as, and Phosphomevalonic kinase.Some the non-limiting examples of nucleotide sequence of this kind of enzyme of encoding comprise (AF429385; Para rubber tree), (NM_006556; Homo sapiens) and (N_C001145. complement 712315..713670; Yeast saccharomyces cerevisiae).

In the 6th step, mevalonic acid 5-tetra-sodium fat is converted into IPP by enzyme catalysis, and the enzyme of known this step of catalysis is, such as, and mevalonate pyrophosphate fat decarboxylase.Some the non-limiting examples of nucleotide sequence of this kind of enzyme of encoding comprise (X97557; Yeast saccharomyces cerevisiae), (AF290095; Faecium) and (U49260; Homo sapiens).

As IPP being converted into DMAPP, then need to carry out the 7th step.The enzyme of known this step of catalysis is, such as, and IPP isomerase.Some the non-limiting examples of nucleotide sequence of this kind of enzyme of encoding comprise (NC_000913,3031087..3031635; Intestinal bacteria) and (AF082326; Haematocoocus Pluvialls).

dXP approach

Fig. 2 shows the schematic diagram of DXP approach.Generally speaking, DXP approach comprises seven steps.

In the first step, pyruvate salt and the condensation of D-glyceraldehyde 3 phosphate are formed DX-5-phosphoric acid.The enzyme of known this step of catalysis is, such as, and DX-5-phosphate synthase.Some the non-limiting examples of nucleotide sequence of this kind of enzyme of encoding comprise (AF035440; Intestinal bacteria), (NC_002947, locus tag PP0527; Pseudomonas putida KT2440), (CP000026, locustag SPA2301; Enteritis salmonella paratyphi, sees ATCC 9150), (NC_007493, locus tagRSP_0254; Spherical red antibacterial 2.4.1), (NC_005296, locus tag RPA0952; Rhodopseudomonas palustris CGA009), (NC_004556, locus tag PD 1293; Xylella fastidiosa Temeculal) and (NC_003076, locus tag AT5G11380; Arabidopis thaliana).

At second step, DX-5-phosphoric acid is converted into 2C-methyl D-erythritol-4-phosphoric acid.The enzyme of known this step of catalysis is, such as, and DX-5-phosphoric acid reduction isomerase.Some the non-limiting examples of nucleotide sequence of this kind of enzyme of encoding comprise (AB013300; Intestinal bacteria), (AF 148852; Arabidopis thaliana), (NC_002947, locus tag PP1597; Pseudomonas putida KT2440), (AL939124, locus tag SCO5694; Streptomyces coelicolor A3 (2)), (NC_007493, locus tag RSP_2709; Spherical red antibacterial 2.4.1) and (NC_007492, locus tagPf1_1107; Pseudomonas fluorescens PfO-1).

In the 3rd step, 2C-methyl D-erythritol-4-phosphoric acid is converted into 4-CDP base-2C-methyl D-erythritol.The enzyme of known this step of catalysis is, such as, and 4-CDP base-2C-methyl D-erythritol synthetic enzyme.Some the non-limiting examples of nucleotide sequence of this kind of enzyme of encoding comprise (AF230736; Intestinal bacteria), (NC_007493, locus_tag RSP_2835; Spherical red antibacterial 2.4.1), (NC_003071, locus_tag AT2G02500; Arabidopis thaliana) and (NC_002947, locus_tag PP1614; Pseudomonas putida KT2440).

In the 4th step, 4-CDP base-2C-methyl D-erythritol is converted into 4-CDP base-2C-methyl D-erythritol-2-phosphoric acid.The enzyme of known this step of catalysis is, such as, and 4-CDP base-2C-methyl D-erythritol kinases.Some the non-limiting examples of nucleotide sequence of this kind of enzyme of encoding comprise (AF216300; Intestinal bacteria) and (NC_007493, locus_tag RSP_779; Spherical red antibacterial 2.4.1).

In the 5th step, 4-CDP base-2C-methyl D-erythritol-2-phosphoric acid is converted into 2C-methyl D-erythritol 2,4-ring bisphosphate.The enzyme of known this step of catalysis is, such as, and 2C-methyl D-erythritol 2,4-ring diphosphate synthase.Some the non-limiting examples of nucleotide sequence of this kind of enzyme of encoding comprise (AF230738; Intestinal bacteria), (NC_007493, locus_tag RSP_6071; Spherical red antibacterial 2.4.1) and (NC_002947, locus_tag PP1618; Pseudomonas putida K.T2440).

In the 6th step, 2C-methyl D-erythritol 2,4-ring bisphosphate is converted into 1-hydroxy-2-methyl-2-(E)-butenyl-4-bisphosphate.The enzyme of known this step of catalysis is, such as, and 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase.Some the non-limiting examples of nucleotide sequence of this kind of enzyme of encoding comprise (AY033515; Intestinal bacteria), (NC_002947, locus_tag PP0853; Pseudomonas putida KT2440) and (NC_007493, locus_tag RSP_2982; Spherical red antibacterial 2.4.1).

In the 7th step, 1-hydroxy-2-methyl-2-(E)-butenyl-4-bisphosphate is converted into IPP or its isomer, i.e. DMAPP.The enzyme of known this step of catalysis is, such as, and isopentyl/dimethylallyl diphosphate synthetic enzyme.Some the non-limiting examples of nucleotide sequence of this kind of enzyme of encoding comprise (AY062212; Intestinal bacteria) and (NC_002947, locus_tagPP0606; Pseudomonas putida KT2440).

In some embodiments, " cross-talk " (or the interference) between the process that generates of the metabolic process of host cell self and the IPP of relating to provided by the invention is minimized or eliminates completely.Such as, when this host microorganism relies on DXP approach synthesis IPP specially, and when importing MEV approach to provide extra IPP, cross-talk is minimized or eliminates completely.This kind of host microorganism can not be endowed the ability of the expression changing this MEV path enzyme or be endowed the ability of the process intermediate relevant to this MEV approach.Special or the main organism relying on DXP approach comprises, such as, and intestinal bacteria.

In some embodiments, described host cell is specially by MEV approach or combine with DXP approach and generate IPP.In other embodiments, host DXP approach loses function, thus makes host cell generate IPP by the outside MEV approach introduced specially.Genetic expression or functionally inactive is stopped to make this DXP approach lose function by making one or more DXP path enzymes.

c ₁₅ isoprenoid starting raw material

Similar IPP, farnesyl pyrophosphate (" FPP ") is also prepared by biological method.Generally speaking, the DMAPP condensation of bimolecular IPP and a part can be formed FPP.In some embodiments, this reaction can by enzyme (such as, farnesyl pyrophosphoric acid synthetase) the institute catalysis of known this step of catalysis.

Some non-limiting examples of the nucleotide sequence of coding farnesyl pyrophosphoric acid synthetase comprise (ATU80605; Arabidopis thaliana), (ATHFPS2R; Arabidopis thaliana), (AAU36376; Artemisinin), (AF461050; Ox), (D00694; E. coli k-12), (AE009951, Locus AAL95523; Fusobacterium nucleatum subspecies nucleatum ATCC25586), (GFFPPSGEN; Rise storehouse gibberella), (CP000009, Locus AAW60034; Gluconobacter oxydans 621H), (AF019892; Sunflower Receptacle), (HUMFAPS; Homo sapiens), (KLPFPSQCR; Kluyveromyces lactis), (LAU15777; Lupinus albus), (LAU20771; Lupinus albus), (AF309508; House mouse), (NCFPPSGEN; Neurospora crassa), (PAFPS1; Guayule), (PAFPS2; Guayule), (RATFAPS; Brown rat), (YSCFPP; Yeast saccharomyces cerevisiae), (D89104; Schizosaccharomyces pombe), (CP000003, LocusAAT87386; Micrococcus scarlatinae), (CP000017, Locus AAZ51849; Micrococcus scarlatinae), (NC_008022, Locus YP_598856; Micrococcus scarlatinae MGAS 10270), (NC_008023, Locus YP_600845; Micrococcus scarlatinae MGAS2096), (NC_008024, LocusYP_602832; Micrococcus scarlatinae MGAS 10750) and (MZEFPS; Corn).

The method of biological IPP and FPP of generation is existing simultaneously describes, can with reference to comprising WO2006/014837 and U.S. Patent Publication No. 2003/0148479,2004/0005678 and 2006/0079476.Embodiment 1 and 2 additionally provides the embodiment of these compounds of preparation.

FPP can be converted into multiple C subsequently ₁₅isoprenoid.Generally speaking, can the C of non-ring type (band side chain or linear) and ring type (with or without side chain) ₁₅isoprenoid is as starting raw material.But, in enforcement of the present invention, by non-ring type C ₁₅isoprenoid generates target compound needs less chemical step.The C be suitable for ₁₅some non-limiting examples of isoprenoid starting raw material include but not limited to:

α-farnesene

α-farnesene, its structure is

Discovery is present in multiple biogenetic derivation, includes but not limited to, the coating of the Dufours gland of ant and apple and pears skin.From biological chemistry angle, α-farnesene can be generated by α-farnesene synthetic enzyme by FPP.Some the non-limiting examples of applicable nucleotide sequence of this kind of enzyme of encoding comprise (DQ30934; Pyruscommunis cultivar d ' Anjou) and (AY182241; Malus domestica).See people such as Pechouus, Planta219 (1): 84-94 (2004).

β-farnesene

β-farnesene, its structure is

Discovery is present in multiple biogenetic derivation, includes but not limited to, aphid and essential oil (as spearmint oil).In some plants (as wild potato), β-farnesene is synthesized as natural insect repellent.From biological chemistry angle, β-farnesene can be generated by β-farnesene synthetic enzyme by FPP.Some the non-limiting examples of applicable nucleotide sequence of this kind of enzyme of encoding comprise (AF024615; Mentha x pipperita) and (AY835398; Artemisia annua).See people such as Picaud, Phytochemistry 66 (9): 961-967 (2005).

farnesol

Farnesol, its structure is

Discovery is present in multiple biogenetic derivation, comprises insect and the essential oil from Vetiveria zizanoides (cintronella), orange flower oil, Cyclamen persicum, lemongrass, Tuberose and rose.From biological chemistry angle, farnesol can be generated by hydroxylases such as farnesol synthetic enzyme by FPP.Some the non-limiting examples of applicable nucleotide sequence of this kind of enzyme of encoding comprise (AF529266; Corn) and (YDR481C; Yeast saccharomyces cerevisiae).See Song, L., Applied Biochemistry and Biotechnology 128:149-158 (2006).

nerolidol

Nerolidol, its structure is

Also referred to as peruviol, its discovery is present in multiple biogenetic derivation, comprises the essential oil from orange flower oil, ginger, jasmine, lavandula angustifolia, tea tree and lemongrass.From biological chemistry angle, nerolidol can be generated by hydroxylases such as nerolidol synthetic enzyme by FPP.Some the non-limiting examples of applicable nucleotide sequence of this kind of enzyme of encoding comprise the AF529266 (Zea mays from corn; Gene tpsl).

In some embodiments, described isoprenoid starting raw material can obtain from naturally occurring terpenes or prepare, this terpenes can by various plants (such as, Copaifera langsdorfii, softwood tree, with root of Beijing euphorbia tree), insect (such as, dovetail butterfly, chrysomelid, termite and pine sawfoy) and marine organisms (such as, marine alga, sponge, coral, mollusk and fish) generate.

Copaifera langsdorfii or Copaifera sets also referred to as diesel oil tree and kerosene tree.It has multiple title in local language, comprise kupa ' y, cabismo and .Copaifera tree can generate a large amount of terpene hydrocarbons in its timber and leaf.Generally speaking, a Copaifera sets the terpenes oil that can generate about 30 to about 40 liters every year.

Terpenes oil also can obtain from softwood tree and root of Beijing euphorbia tree.Softwood tree belongs to the Coniferae (Pinophyta or Coniferae) in plant, and is generally the plant of carrying seed with pine nut with vascular tissue.In softwood tree, major part is tree, but some softwood tree may be shrub.Suitable more acerose non-limiting examples comprise cdear, cypress, Douglas fir, fir, needle juniper, shell pine, tamarack, pine tree, Chinese larch, dragon spruce and Japanese yew.The root of Beijing euphorbia is set, and also referred to as Euphorbia, is the plant extensively distributed in the whole world, belongs to root of Beijing euphorbia family (Euphorbiaceae).Root of Beijing euphorbia tree, by about 2160 kinds, is one of maximum species of vegetable kingdom.

Described C ₁₅isoprenoid starting raw material is sesquiterpene, and it is a part for the larger classification compound being called terpenes.As classification larger in hydro carbons, terpenes comprises half terpenes, monoterpene, sesquiterpene, two terpenes, sesterterpene, triterpene, tetraterpene and polyterpene.Therefore, the C be suitable for ₁₅the terpenes that isoprenoid starting raw material can adopt from the present invention is separating of oil.

chemical conversion

Fuel composition disclosed in this invention can comprise

Wherein Z is as defined above.Formula (I) or (II) prepare by any means known in the art, comprise biological method and chemosynthesis (not using the raw material of biogenetic derivation).In one embodiment, described C ₁₅isoprenoid starting raw material can obtain from the separation of naturally occurring source.Such as, farnesol can be separated with rose from Vetiveria zizanoides, enroli, Cyclamen persicum, lemongrass, Tuberose and to obtain.In another embodiment, this C ₁₅isoprenoid starting raw material prepares by the host cell through transforming the output generating this compound or improve this naturally occurring compound.

No matter why it originates, each C ₁₅isoprenoid starting raw material is all chemically converted to fuel element or fuel dope by any known reduction reaction (such as hydrogenation, or the combination of reduction reaction and esterification).In some embodiments, this C ₁₅isoprenoid starting raw material adopts catalyzer (such as Pd, Pd/C, Pt, PtO ₂, Ru (PPh ₃) ₂cl ₂, Raney Ni or its combination) be hydrogenated reduction.In one embodiment, this catalyzer is Pd catalyzer.In another embodiment, this catalyzer is 5%Pd/C.In further embodiment, this catalyzer is the 10%Pd/C in high-pressure reaction vessel, and this reaction allows to have proceeded to.Generally speaking, in the completed, this reaction mixture can be washed, concentrated, and drying is to obtain corresponding hydrogenated products.Alternatively, any reductive agent that C=C key can be reduced to C-C key can also be used.Such as, this C ₁₅isoprenoid starting raw material is by O ₂under, under catalyzer (such as, 5-ethyl-3-methyl lumiflavin perchloric acid ester) exists, obtain corresponding hydrogenated products with hydrazine process hydrogenation.This kind reduction reaction that hydrazine carries out is disclosed in the people such as Imada, J.Am.Chem.Soc.127,14544-14545 (2005) (being incorporated to by reference herein at this).

In some embodiments, described C ₁₅c=C key in isoprenoid starting raw material is at room temperature hydrogenated and is reduced to corresponding C-C key under the existence of catalyzer and hydrogen.In further embodiment, this catalyzer is the hereafter 10%Pd/C shown in flow process 1.

Flow process 1

According to the complete saturated C that above-mentioned flow process 1 obtains ₁₅alcohol is modified to generate corresponding saturated C further by any known esterifying agent (such as carboxylic acid, carboxylic acid halide's (such as fluorochemical, muriate, bromide and iodide) and carboxylic acid anhydride) ₁₅ester.This esterification can be carried out under the condition approved by technician arbitrarily.In some embodiments, this C ₁₅alcohol starting raw material by under the existence of acid or alkaline catalysts, or reacts esterified with objective carboxylic acid under Fischer or Steglich enzymatic synthesis condition.In other embodiments, this C ₁₅alcohol starting raw material can in the existence of alkaline catalysts (such as, amine or pyridine compounds) or disappearance be lower and objective carboxylic acid halide reaction is esterified.In other embodiments, as shown in hereafter flow process 2, this C ₁₅alcohol starting raw material can in the existence of alkaline catalysts (such as, amine compound (e.g., triethylamine)) or disappearance be lower and objective carboxylic acid anhydride reactant is esterified.The reaction mixture completed can be concentrated, wash the also corresponding ester of dry generation.

Flow process 2

Alternatively, described saturated C ₁₅ester can from described saturated C ₁₅alcohol obtains, and obtains required ester by the hereafter transesterification reaction shown in flow process 3.This transesterification reaction can carry out under the condition approved by technician arbitrarily.In some embodiments, this transesterification reaction is alkali catalyzed agent (such as basic metal (as Li, Na, K, Rb and Cs) and alkalescence (such as Mg, Ca, Sr and Ba) oxyhydroxide, carbonate or acetate or its combination) institute's catalysis.

Flow process 3

In some embodiments, described complete saturated C ₁₅alcohol is generated corresponding ester by any known alkylating agent as R-X modifies further, and wherein R is alkyl, and X is good leavings group, such as halogen, alkylsulfonyl, sulfate groups etc.Some non-limiting examples of alkylating agent comprise alkyl halide, alkyl sulfonic ester and alkyl sulfuric ester etc.Generally speaking, this C ₁₅first alcohol can be converted into C by alkali ₁₅alkoxide, then this C ₁₅alkoxide can react to R-X and form corresponding ester as shown in hereafter flow process 4, and wherein X is Cl, Br or I.In some embodiments, this alkali can be active metal, such as sodium Metal 99.5 or metal hydroxides, as sodium hydroxide, lithium aluminum hydride and sodium borohydride.

Flow process 4

Alternatively, C ₁₅enol can be partially alkylated or alkylated or esterification first as described above, and then as being hereafter hydrogenated as described in flow process 5, wherein R ' is R or C (=O) R, and R is H or alkyl.

Flow process 5

Reference hereafter flow process 6, described esterification is by carrying out with mode identical as mentioned above.Follow-up hydrogenation can be carried out in the same manner as described above.Alternatively, the hydrogenation catalyst not affecting arbitrarily described-O-C (=O) R group can be adopted to carry out the subsequent selective hydrogenation of double bond.In some embodiments, this hydrogenation catalyst is Pd/C, take diphenyl sulfide as catalyzer poison, thus selective reduction alkene luminous energy group, and can not this O-C of hydrogenation (=O) R group, see people such as Mori, Org.Lett.8,3279-3281 (2006), is incorporated to herein by reference at this.In other embodiments, PEG and Adams catalyzer (that is, PtO can be used ₂) as solvent, thus at 1 atmosphere pressure by hydrogen selective hydrogenation double bond.The use of Adams catalyzer is disclosed in the people such as Chandrasekhar, J.Org.Chem.71,2196-2199 (2006), and it is incorporated to herein by reference at this.

Flow process 6

Described C ₁₅the hydrogenation of isoprenoid starting raw material can be deposited and carry out in case at asymmetric hydrogenation catalyzer (such as rhodium-chiral diphosphine mixture), thus forms the stereospecificity hydrogenated products substantially not conforming to other steric isomer.The non-limiting example of this asymmetric hydrogenation catalyzer comprises rhodium-DIPAMP catalyzer.This rhodium-DIPAMP catalyzer and other asymmetric hydrogenation catalyzer are disclosed in the people such as Vineyard, J.Am.Chem.Soc.1977,99, (18), 5946; Ryoji Noyori, " AsymmetricCatalysis In Organic Synthesis, " John Wiley & Sons Inc., New York, the 2nd chapter, pp.16-94 (1994); With people such as Blaser, " Asymmetric Catalysis on Industrial Scale:Challenges, Approaches and Solutions; " Wiley-VCH, Weinheim, pp.23-52 (2004), it is all incorporated to herein at this in full by reference.

In some embodiments, α-farnesene and β-farnesene can asymmetric hydrogenation catalyzer deposit hydrogenation in case formed hereafter shown in one of the possible steric isomer of four kinds of farnesane, that is, compound (III-a), (IH-b), one of (III-c) and (III-d).

Similarly, farnesol can be deposited hydrogenation in case at asymmetric hydrogenation catalyzer and form one of possible steric isomer of four kinds of 3,7,11-trimethyldodecane-1-alcohol as follows.

Similarly, nerolidol can be deposited hydrogenation in case at asymmetric hydrogenation catalyzer and form one of possible steric isomer of four kinds of 3,7,11-trimethyldodecane-3-alcohol as follows.

Similarly, C ₁₅enol or it is alkylating, esterification, Sulfated, phosphorylation, Sulfonated or phosphorated product can be hydrogenated and form corresponding stereospecificity hydrogenated products under the existence of asymmetric hydrogenation catalyzer.

In the method that another is substituting, described C ₁₅the alkylation of enol, esterification, sulfation, sulfonated, phosphorylation or phosphoratedly can to carry out simultaneously.

fuel composition

Fuel composition disclosed in this invention is prepared by having cost-benefit and eco-friendly mode.Advantageously, isoprenoid compounds provided by the present invention generates by one or more microorganisms.Thus, these isoprenoid compounds can provide and can be used for diesel oil or rocket engine fuel, particularly the renewable energy source of fuel composition provided by the present invention.In addition, these isoprenoid compounds can reduce the dependency to non-renewable fuel, fuel element and/or fuel dope source.In certain embodiments, the present invention includes the fuel composition comprising Bioengineered farnesane.

As described above, embodiment of the present invention provide the various fuel compositions that can be used as diesel oil fuel or rocket engine fuel especially.The biodiesel fuel derivative with current diesel oil and methyl esterification of fatty acid is compared, and fuel composition disclosed in this invention can resistance to oxidative degradation better, thus has higher preservation period.Correspondingly, in some embodiments, this fuel composition have at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, at least about 10 years, at least about 15 years, at least about Two decades years or at least about the preservation period of 25 years.In other embodiments, this fuel composition has the preservation period at least about ISUZU company.In other embodiments, this fuel composition has the preservation period exceeding ISUZU company.

Although the present invention is described by limited amount embodiment, the special characteristic of an embodiment should owing to other embodiment of the present invention.The independent embodiment of neither one can represent all aspects of the present invention.In some embodiments, described composition or method can comprise the NM compound of this present invention multiple or step.In other embodiments, said composition or method do not comprise, or substantially do not comprise, any compound that the present invention does not enumerate or step.There is variant and variation in embodiment of the present invention.Such as, described diesel oil fuel needs not be the mixture of common paraffin and branched paraffins.It can comprise the hydrocarbon of any type, as long as the aromatic hydrocarbon content in this diesel oil fuel is less than 10% by weight, sulphur content is less than 100ppm.Although preferably this diesel oil fuel has the aromatic hydrocarbon content being less than 10% by weight and the sulphur content being less than 100ppm, for some object, the diesel oil fuel containing being greater than the aromatic hydrocarbon content of 10% and the sulphur content higher than 100ppm by weight can be accepted.It should be noted that the application of this diesel oil fuel is not limited to diesel motor; It can be used for any equipment needing diesel oil fuel, such as reserve generator.Although all diesel oil fuel of laws and regulations requirement has the cetane value of at least 40, and this laws and regulations requirement of diesel oil fuel demand fulfillment of not all embodiment of the present invention.In other words, can accept cetane value lower than 40 diesel oil fuel.It should be noted that described preparation and use the method for this diesel oil fuel to relate to multiple step.In some embodiments, these steps can be implemented in random order.In some embodiments, can omit or merge one or more step, but still realize substantially identical result.Additional claims are intended to cover all this kind of variant and variations falling into the scope of the invention.

All publications involved in this specification sheets and patent application are incorporated to herein all by reference, its quote degree as each independent publication and patent application specifically separately through quote be incorporated to the same herein.Although aforementioned invention is described in detail by the diagram and example of explaining object, those of ordinary skill in the art should understand easily can carry out certain change and adjustment when not departing from the spirit and scope of accessory claim to it according to instruction of the present invention.

Embodiment

Unless otherwise specified, enforcement of the present invention can adopt the routine techniques in the industries such as biosynthesizing within the scope of art technology.For this kind of technology that the present invention does not describe completely, people can find the abundant reference had about this in scientific and technical literature.

In following embodiment, we endeavour to ensure the accuracy of numeral used (such as amount, temperature etc.), but change and deviation should be allowed, and when there is the clerical error of this kind of numerical value herein, those skilled in the art should be able to according to of the present invention all the other openly derive correct value.Unless otherwise specified, temperature is with degree Celsius to represent, pressure equals or close to the normal atmosphere of sea level.Unless otherwise specified, all reagent obtains by chemical process.Following examples are intended to purposes of illustration, the scope do not limited the present invention in any way.

embodiment 1

The preparation method of the expression plasmid of the codase (comprising the enzyme of the yeast saccharomyces cerevisiae MEV approach regulated from operon) that present embodiment describes.

Expression plasmid pMevT generates after MevT operon is inserted pBAD33 carrier.Ubiquitous precursor acetyl-CoA can be converted into the enzyme of (R)-mevalonic acid, i.e. acetoacetyl coenzyme A thiolase, HMG CoA synthase and HMG CoA-reductase by this MevT operon coding MEV approach one group jointly.Obtain from genome of E.coli DNA encoding sequence (GenBank accession number NC_00913 REGION 2,324,131 2325315) (the encoding acetyl acetyl-CoA thiolase) of atoB gene, encoding sequence (the GenBank accession number X96617 of ERG13 gene is obtained from wine brewing ferment genomic dna, REGION 2201695) (coding HMG CoA synthase), and a fragment (GenBank accession number M22002 of the coding region of HMG1 gene is obtained from genes of brewing yeast group DNA, REGION 16603165) (coding brachymemma HMG CoA-reductase (tHMGR)), this MevT operon is generated by pcr amplification.Upstream PCR primer for HMG1 gene fragment amplification comprises artificial initiator codon.By overlap-extension PCR (SOEing) by the fragment assembly of amplification together, in this process, rrna bond site is imported to after atoB and ERG13 encoding sequence.After adding 3 ' A suspension, this MevT operon is connected to TA cloning vector pCR4 (Invitrogen, Carlsbad, CA).Subsequently, this MevT operon is connected to the XmaI PstI restriction site (people (1995) J Bacteriol 177 (14) 4121-4130 such as Guzman) of carrier pBAD33.P is subject to for making this operon _locthe control of promotor, replaces the araC-P of pBAD33 with the Nsil-XmaI fragment of pBBRI MCS _bADnsil-XmaI fragment, thus obtain expression vector pMevT (see United States Patent (USP) 7,192,751).

Expression plasmid pAM36-MevT66 generates by MevT66 operon is inserted pAM36 carrier.PAM36 carrier by the oligonucleotide box containing AscI-SfiI-AsiSI-XhoI-PacI-FsIl-Pmel restriction site is inserted pACYC 184 carrier (GenBank accession number XO6403), and to remove in pACYC184 tetramycin tolerance and obtains gene (conferring gene) and obtain afterwards.This MevT66 operon by with SEQ ID NO1 for templated synthesis obtains, this SEQ ID NO1 contains from colibacillary atoB gene (GenBank accession number NC_000913 REGION2324131..2325315), from ERG13 gene (the GenBank accession number X96617 of yeast saccharomyces cerevisiae, REGION 220..1695), and from truncation type HMG1 gene (the GenBank accession number M22002 of yeast saccharomyces cerevisiae, REGION 1777..3285), these three kinds of sequences all by codon optimized with at e. coli expression.The MevT66 operon that this synthesis generates by 5 ' EcoRl restriction site and 3 ' Hind III restriction site sandwich, therefore can be cloned the Compatible restriction sites entering cloning vector (such as standard pUC or pACYC source carrier).This MevT66 operon is by pcr amplification, and with SfiI and the AsiSI restriction site of flank, use SfiI and AsiSI restriction enzyme by the digestion of the DNA fragmentation of amplification completely, by gel electrophoresis separate reacted mixture, 4.2kb DNA fragmentation is about by gel purification kit (Qiagen, Valencia, CA) gel extraction, and the DNA fragmentation of separation is connected to the SfiI AsiSI restriction site of pAM36 carrier, thus obtain expression plasmid pAM36-MevT66.

Expression plasmid pAM25 generates by MevT66 operon is inserted pAM29 carrier.This pAM29 carrier is by assembling produce obtaining lacUV5 promotor that gene (Lutz and Bujard (1997) Nucl Acids Res.25:1203-1210) and oligonucleotide generate from the p15A ori of pZS24-MCS1 and kantlex tolerance.The DNA using EcoRI and Hind III restriction enzyme will comprise MevT66 operon (description see above-mentioned pAM36-MevT66) synthesizes construct digestion completely, by gel electrophoresis separate reacted mixture, gel extraction is about 4.2kbDNA fragment, and the DNA fragmentation of separation is connected to the EcoRI HindIII restriction site of pAM296, thus obtain expression plasmid pAM25.

Expression plasmid pMevB-Cm generates after MevB operon is inserted pBBR1MCS-1 carrier.This MevB operon is encoded one group and jointly (R)-mevalonic acid can be turned to the enzyme of IPP, i.e. Mevalonic kinase, Phosphomevalonic kinase and mevalonate pyrophosphate fat carboxylase.This MevB operon is by pcr amplification ERG12 gene (GenBank accession number X55875 from genes of brewing yeast group DNA, REGION:580..1911) (coding Mevalonic kinase), ERG8 gene (GenBank accession number Z49939, REGION:3363..4718) (coding Phosphomevalonic kinase), and MVDI gene (GenBank accession number X97557, REGION:544..1734) encoding sequence of (coding mevalonate pyrophosphate fat carboxylase), and obtain after PCR fragment being stitched together by overlap-extension PCR (SOEing).By selecting suitable primer sequence, in amplification procedure, the terminator codon of ERG12 and ERG8 is become TAG from TAA, to introduce ribosome bind site.After adding 3 ' A suspension, MevB operon is connected to TA cloning vector pCR4 (Invitrogen, Carlsbad, CA).By with this cloned construct of PstI restriction enzyme complete digestion to excise this MevB operon, by gel electrophoresis separate reacted mixture, gel extraction is about 4.2kb DNA fragmentation, and the DNA fragmentation of separation is connected to the PstI restriction site (people such as Kovach of pBBR1MCS-1 carrier, Gene 166 (1): 175-176 (1995)), thus obtain expression plasmid pMevB-Cm.

Expression plasmid pMBI generates by MBI operon is inserted pBBR1MCS-3 carrier.Except the enzyme of this MevB operon, this MBI operon also encoding isopentenyl Pyrophosphate phosphohydrolase isomerase, IPP is catalytically conveted to DMAP by it.This MBI operon is by such as under type acquisition: to comprise the encoding sequence of primer pcr amplification idi gene (GenBank accession number AF119715) from genome of E.coli DNA of XmaI restriction site at 5 ' end, use XmaI restriction enzyme by the digestion of the DNA fragmentation of amplification completely, by gel electrophoresis separate reacted mixture, gel extraction is about 0.5kb fragment, and the DNA fragmentation of separation is connected to the XmaI restriction site of pMevB-Cm expression plasmid, thus idi is placed in 3 ' end of MevB operon.This MBI operon subclone is entered the SalI SacI restriction site (people such as Kovach of carrier pBBR1MCS-3, Gene 166 (1): 175-176 (1995)), thus obtain expression vector pMBI (see United States Patent (USP) 7,192,751).

Expression plasmid pMBIS is generated by ispA gene is inserted pMBI.This ispA genes encoding farnesyl pyrophosphoric acid synthetase, two molecule I PP and a part DMAPP catalyzing and condensing are generated FPP by this enzyme.Adopt the encoding sequence of the forward primer with SacII restriction site and reverse primer this ispA gene of pcr amplification (GenBank accession number D00694, REGION:484..1383) from genome of E.coli DNA with SacI restriction site.Use SacII and ScI restriction enzyme by complete for the DNA product digestion of amplification, by gel electrophoresis separate reacted mixture, gel extraction is about 0.9kb fragment, and the DNA fragmentation of separation is connected to the SacII SacI restriction site of pMBI expression plasmid, thus this ispA gene is placed in 3 ' end of idi and MevB operon, and obtain expression plasmid pMBIS (see U.S. Patent number 7,192,751).

By described MBIS operon is inserted pAM36-MevT66, and before MBIS and MevT66 operon, add lacUV5 promotor to form expression plasmid pAM45.By comprising primer this MBIS operon of pcr amplification from pMBIS of 5 ' XhoI restriction site and 3 ' PacI restriction site, use XhoI and PacI restriction enzyme by the digestion of the PCR primer of amplification completely, by gel electrophoresis separate reacted mixture, gel extraction is about 5.4kb DNA fragmentation, and the DNA fragmentation of separation is connected to the XhoI PaI restriction site of pAM36-MevT66, obtain expression plasmid pAM43.The DNA fragmentation comprising the nucleotide sequence of coding lacUV5 promotor can be obtained by oligonucleotide synthesis, and subclone enters AscI SfiI and the AsiSI XhoI restriction site of pAM43, thus obtains expression plasmid pAM45.

embodiment 2

The preparation method of the expression vector of the codase (comprising the enzyme of the streptococcus aureus MEV approach regulated from operon) that present embodiment describes.

The encoding sequence that encoding sequence by the HMG1 gene (coding brachymemma yeast saccharomyces cerevisiae HMG CoA-reductase) by expression plasmid pAM25 replaces with mvaA gene (coding streptococcus aureus HMG CoA-reductase (GenBank accession number BA000017, REGION:2688925.-2687648)) obtains expression plasmid pAM41.Use the encoding sequence of primer 4-49mvaA SpeI (SEQ ID NO:13) and 4-49mvaAR XbaI (SEQ ID NO:14) pcr amplification mvaA gene from streptococcus aureus (ATCC 70069) genomic dna, use SpeI restriction enzyme by the digestion of the DNA fragmentation of amplification completely, by gel electrophoresis separate reacted mixture, gel extraction is about 1.3kb DN A fragment.By using HindIII restriction enzyme by pAM25 digestion completely, from this plasmid removing HMG1 encoding sequence.The end of T4DNA polysaccharase brachymemma gained linear DNA fragment is adopted to hang.Then adopt SpeI restriction enzyme this DNA fragmentation partial digested, by gel electrophoresis separate reacted mixture, gel extraction is about 4.8kb DNA fragmentation, the DNA fragmentation of separation is connected to the mvaA PCR primer of SpeI-digestion, to obtain expression plasmid pAM41.

The encoding sequence that encoding sequence by the ERG13 gene (encoding Saccharomyces cerevisiae HMG CoA synthase) by expression plasmid pAM41 replaces with mvaS gene (streptococcus aureus HMG CoA synthase (GenBank accession number BA000017, REGION:2689180..2690346)) obtains expression plasmid pAM52.Use the encoding sequence of primer HMGS 5 ' Sa mvaS-S (SEQ ID NO:15) and HMGS3 ' SamvaS-AS (SEQ ID NO:16) pcr amplification mvaS gene from streptococcus aureus (ATCC 70069) genomic dna, the DNA fragmentation of this amplification can be used as PCR primer, to replace the encoding sequence of the HMG1 gene in pAM41 according to the method for the people such as Geiser (BioTechniques 31:88-92 (2001)), thus obtain expression plasmid pAM52.The atoB (opt) comprised in pAM52: the nucleotides sequence of mvaS:mvaA operon is classified as SEQ ID NO:2.

(atoB (opt): mvaS:mvaA) operon by the MevT66 operon of expression plasmid pAM45 being replaced with pAM52 obtains expression plasmid pAM97.Use AsiSI and SfiI restriction enzyme to be digested completely by expression plasmid pAM45, by gel electrophoresis separate reacted mixture, gel extraction lacks the about 8.3kb DNA fragmentation of this MevT66 operon.Use (atoB (opt): mvaS:mvaA) operon of primer 19-25 atoBSfiI-S (SEQ ID NO:17) and 19-25mvaA-AsiSI-AS (SEQ ID NO:18) pcr amplification pAM52, use SfiI and AsiSI Methylation product digestion complete, by gel electrophoresis separate reacted mixture, gel extraction is about 3.8kb DNA fragmentation, the DNA fragmentation of separation is connected to the AsiSI SfiI restriction site of expression plasmid pAM45, obtains expression plasmid pAM97 (plasmid map see Fig. 3).

embodiment 3

Present embodiment describes the preparation method of the expression plasmid of codase (comprising the enzyme of the intestinal bacteria DXP approach regulated from operon).

Expression plasmid pAM408 is by generating after the gene insertion pAM29 carrier of the enzyme by " top " DXP approach of encoding.The enzyme of " top " DXP approach comprises this ketose of 1-deoxidation-D--5-phosphate synthase (by colibacillary dxs genes encoding), DX-5-phosphoric acid reduction isomerase (by colibacillary dxr genes encoding), 4-CDP base-2C-methyl D-erythritol synthetic enzyme (by colibacillary ispD genes encoding) and 4-CDP base-2C-methyl D-erythritol synthetic enzyme (by colibacillary ispE genes encoding), pyruvate salt and D-glyceraldehyde-3-phosphate are converted into 4-CDP base-2C-methyl D-erythritol-2-phosphoric acid by these enzymes jointly.The DNA fragmentation comprising the nucleotide sequence of the enzyme of coding " top " DXP approach by use the PCR primer pcr amplification shown in SEQ ID NO:19-26 from dxs (GenBank accession number U00096 REGION:437539..439401), the dxr (GenBank accession number U00096 REGION:193521..194717) of coli strain DH1 (ATCC#33849), ispD (GenBank accession number U00096 REION:2869803..2870512) and ispE (GenBank accession number U00096 REGION1261249..1262100) gene coded sequence and add best Shine Dalgarno sequence and 5 ' and 3 ' restriction site generate.Gel electrophoresis is separated PCR primer, gel extraction, and (XhoI and KpnI is for comprising the PCR primer of dxs gene to use suitable restriction enzyme; KpnI and ApaI is for comprising the PCR primer of dxr gene; ApaI and NdeI is for comprising the PCR primer of ispD gene; NdeI and MluI is for comprising the PCR primer of ispE gene) digest to complete, and use PCR purification kit (Qiagen, Valencia, CA) to carry out purifying.Then about equimolar each PCR primer is added into ligation, to allocate individual gene assembling into operon.The reaction mixture of this ligation of 1ul is used to the independent box gene of pcr amplification two, i.e. dxs-dxr and ispD-ispE box gene.Use primer 67-1A-C (SEQ ID NO:19) and this dxs-dxr box gene of 67-1D-C (SEQ ID NO:22) pcr amplification, and use primer 67-1E-C (SEQ ID NO:23) and this ispD-ispE box gene of 67-1H-C (SEQ ID NO:26) pcr amplification.Two kinds of PCR primer are separated by gel electrophoresis, and gel extraction.Use XhoI and ApaI restriction enzyme to comprise the PCR primer digestion of this dxs-dxr box gene completely, use ApaI and MluI restriction enzyme to comprise the PCR primer digestion of this isD-ispE box gene completely.These two kinds of PCR primer of purifying, and the DNA fragmentation of purifying is connected to the SalI MluI restriction site of pAM29 carrier, thus obtain expression plasmid pAM408 (plasmid map see Fig. 4).

Expression plasmid pAM409 is by generating after the gene insertion pAM369 carrier of the enzyme by " bottom " DXP approach of encoding.The enzyme of " bottom " DXP approach comprises 2C-methyl D-erythritol 2,4-ring diphosphate synthase (by colibacillary ispF genes encoding), 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (by colibacillary ispG genes encoding) and isopentene group/dimethylallyl diphosphate synthetic enzyme (by colibacillary ispH genes encoding), 4-CDP base-2C-methyl D-erythritol-2-phposphate is IPP and DMAPP by these enzymes jointly.IPP is also DMAPP by the active Transforming of isopentyl diphosphate isomerase (by colibacillary idi genes encoding).DMAPP is also further converted to FPP by the activity of Farnesyl pyrophosphate synthetase (such as by colibacillary ispA genes encoding).The coding enzyme of " bottom " DXP approach and the operon of isopentyl diphosphate isomerase and Farnesyl pyrophosphate synthetase are by using the PCR primer pcr amplification shown in SEQ ID NO:27-36 from the ispF (GenBank accession number U00096 REGION:2869323..2869802) of coli strain DH1 (ATCC#33849), ispG (GenBank accession number U00096REGION:2638708..2639826), ispH (GenBank accession number U00096 REGION:26277..27227), idi (GenBank accession number AF119715) and ispA (GenBank accession number D00694 REGION:484..1383) gene and add best Shine Dalgarno sequence and 5 ' and 3 ' restriction site generate.Gel electrophoresis is separated PCR primer, gel extraction, and (BamHI and ApaI is for comprising the PCR primer of ispF gene to use suitable restriction enzyme; KpnI and ApaI is for comprising the PCR primer of ispG gene; SalI and KpnI is for comprising the PCR primer of ispH gene; SalI and the HindIII PCR primer of attached bag containing idi gene; HindIII and NcoI is for comprising the PCR primer of ispA gene) digestion is to complete, and purifying.Then about equimolar each PCR primer is added into ligation, so that individual gene is assembled into operon.The reaction mixture of this ligation of 1ul is used to the independent box gene of pcr amplification two, i.e. ispF-ispG and ispH-idi-ispA box gene.Use primer 67-2A-C (SEQ ID NO:27) and this ispF-ispG box gene of 67-2D-C (SEQID NO:30) pcr amplification, and use primer 67-2E-C (SEQ ID NO:31) and this ispH-idi-ispA box gene of 67-2J-C (SEQ ID NO:36) pcr amplification.Two kinds of PCR primer are separated by gel electrophoresis, and gel extraction.Use BamHI and KpnI restriction enzyme to comprise the PCR primer digestion of this ispF-ispG box gene completely, use KpnI and NcoI restriction enzyme to comprise the PCR primer digestion of this ispH-idi-ispA box gene completely.Purifying two kinds of PCR primer.Carrier is by by the p15A ori from pAM29, and the lacUV5 promotor generated for beta lactamase gene (Lutz and Bujard (1997) Nucl Acids Res.25:1203-1210) and the oligonucleotide of Ampicillin Trihydrate tolerance from pZE12-1uc is assembled and generated afterwards.Two kinds that will comprise " bottom " DXP approach operon are separated the BamHINcoI restriction site that PCR primer is connected to pAM369 carrier, to obtain expression plasmid pAM409.

Expression plasmid PAM424, namely comprises the derivative of the expression plasmid pAM409 of the RK.2 ori of broad host range, generates by the ispFGH-idi-ispA operon of lacUV5 promotor and pAM409 being transferred to pAM257 carrier.Carrier pAM257 generates by such as under type: adopt primer 9-156A (SEQ ID NO:37) and 9-156B (SEQ ID NO:38) from RK2 plasmid DNA amplification RK2par locus people (1975) Science 190:1226-1228 such as () Meyer, use AatII and XhoI restriction enzyme by this 2.6kb PCR primer complete digestion, and this DNA fragmentation is connected to containing p15 ori and the plasmid (Lutz and Bujard (1997) Nucl Acids Res.25:1203-1210) obtaining gene from the paraxin tolerance of carrier pZA31-uc, to obtain plasmid pAM37-par, use restriction enzyme SacI and HindIII by pAM37-par digestion completely, this reaction mixture is separated by gel electrophoresis, and gel extraction comprises the DNA fragmentation of par locus and paraxin tolerance acquisition gene, the DNA fragmentation of separation is connected to SacI HindIII site people (1990) J Bacteriol. 172:6204-6216 such as () Roberts of emblem-RK.2 replicon pRR10, obtains carrier pAM133, use BglII and HindIII restriction enzyme by pAM133 digestion completely, this reaction mixture is separated by gel electrophoresis, and gel extraction lacks this Ampicillin Trihydrate genes conferring resistance and the oriT about 6.4kb DNA fragmentation in conjunction with initiator, the DNA fragmentation of separation is connected to the DNA fragmentation comprising the multiple clone site comprising PciI and XhoI restriction site that synthesis generates, obtains carrier pAM257.Use XhoI and PciI restriction enzyme by pAM409 digestion completely, this reaction mixture is separated by gel electrophoresis, and gel extraction is about 4.4kb DNA fragmentation, the DNA fragmentation of separation is connected to the XhoI PciI restriction site of pAM257 carrier, thus obtains expression plasmid pAM424 (plasmid map see Fig. 5).

embodiment 4

To present embodiment describes the nucleic acid directional integration of codase (comprising the enzyme of MEV approach) to the specific chromosome position of yeast saccharomyces cerevisiae to prepare the method for carrier.

Genomic dna is separated home-brewed wine yeast strain Y002 (CEN.PK2 background; MATA; Ura3-52; Trp1-289; Leu2-3,112; His3 Δ 1; MAL2-8C; SUC2), Y007 (S228C background MATA trp1 Δ 63), Y051 (S228C background; MAT α his3 Δ 1 leu2 Δ 0 lys2 Δ 0ura3 Δ 0 P _gAL1-HMG1 ^1586-3323p _gAL1.upc2-1 erg9::P _mET3-ERG9::HIS3P _gAL1-ERG20 P _gAL1-HMG1 ^1586-3323) and EG 123 (MATA ura3; Trp1; Leu2; His4can1).This bacterial strain is in the middle grow overnight of the liquid nutrient medium (YPD substratum) containing 1% yeast extract, 2% bacto peptone and 2% dextrose.By centrifugal for 10mL liquid culture 3,100rpm, with 10mL milli-Q water cell mass, recentrifuge is with isolated cell.Use Y-DER cerevisiae dna extraction agent box (Pierce Biotechnologies, Rockford, IL) according to the proposed projects extracting genomic dna of manufacturer.The genomic dna of extracting is resuspended to middle 100uL 10mMTris-Cl (pH8.5), ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE) reads OD _260/280reading, to determine genomic dna concentration and purity.

At Applied Biosystems 2720 Thermocycler (Applied Biosystems Inc, Foster City, CA) in, adopt Phusion high-fidelity DNA polymerase system (Finnzymes OY, Espoo, Finland), and the proposed projects of reference manufacturer, by polymerase chain reaction (PCR) DNA amplification.Be inserted into TOPOTA pCR2.1 cloning vector (Invitrogen completing, Carlsbad, after the pcr amplification of DNA fragmentation CA), by adding the QiagenTaq polysaccharase (Qiagen of 1uL to this reaction mixture, Valencia, CA), extra operation 10 minutes 72 DEG C of PCR stretch steps, then be cooled to 4 DEG C, hang with product nucleus thuja acid.After pcr amplification completes, 50% glycerine solution of 8uL is added into reaction mixture, and 1%TBE (0.89M Tris, 0.89M boric acid, the 0.02MEDTA sodium salt) sepharose complete mixture is loaded on containing 0.5ug/mL bromination ethidium.

Under 120V, 400mA, carry out agarose gel electrophoresis 30 minutes, and adopt ultraviolet to show DNA band.Use sterile razor blade from gel, excise DNA band, and the DNA of proposed projects to excision adopting Zymoclean gel DNA to reclaim test kit (Zymo Research, Orange, CA) reference manufacturer carry out gel-purified.The DNA wash-out of purifying is entered 10uL ultrapure water, ND-1000 spectrophotometer reads OD _260/280reading, to determine DNA concentration and purity.

Use the purified pcr product of 100-500ug and the T4DNA ligase enzyme (NewEngland Biolabs, Ipswich, MA) of high density, the proposed projects with reference to manufacturer connects.For carrying out plasmid propagation, the proposed projects of the construct of connection reference manufacturer is transformed into bacillus coli DH 5 alpha Competent cell (Invitrogen, Carlsbad, CA).Positive transformant is being selected containing on 1.5%Bacto agar, 1% Tryptones, 0.5% yeast extract, the suitable antibiotic solid medium of 1%NaCl and 50ug/mL.The transformant of separation is made at 37 DEG C, to grow 16 hours in the LB liquid medium containing 50ug/mL Pyocianil or kantlex, use QIAprep SpinMiniprep test kit (Qiagen, Valencia, CA) with reference to the proposed projects abstraction and purification plasmid of manufacturer.By diagnostic digestion with restriction enzyme checking construct, and be separated on sepharose and display DNA fragmentation.Also by the construct that DNA sequencing checking is selected, this work is completed by ElimBiopharmaceuticals Inc. (Hayward, CA).

By the ERG20-P by carrier pAM471 _gAL-tHMGR inset insertion vector pAM466 generates plasmid pAM489.By by DNA fragmentation ERG20-P _gAL-tHMGR inserts TOPOZeroBluntII cloning vector (Invitrogen, Carlsbad, CA) and generates carrier pAM471, this DNA fragmentation ERG20-P _gAL-tHMGR comprises open reading frame (ORF) (the ERG20 nucleotide position 1 to 1208 of ERG20; The A of ATG initiator codon is Nucleotide 1) (ERG20), comprise genomic gene seat (GAL1 nucleotide position-1 to-the 668) (P of divergent evolution GAL1 and GAL10 promotor _gAL), and the brachymemma ORF of HMG1 (HMG1 nucleotide position 1586 to 3323) (tHMGR).By by DNA fragmentation TRP1- ^{856 to+548}insert TOPO TA pCR2.1 cloning vector (Invitrogen, Carlsbad, CA) and generate carrier pAM466, this DNA fragmentation TRP1- ^{856 to+548}comprise the yeast saccharomyces cerevisiae wild-type TRP1 locus extending to position 548 from nucleotide position-856, and with non-natural inside XmaI restriction site between base-226 and-225.General introduction with reference to table 1 generates DNA fragmentation ERG20-P by pcr amplification _gAL-tHMGR and TRP1- ^{856 to+548}.For building pAM489, XmaI restriction enzyme (New England Biolabs, Ipswich, MA) is used to digest completely by the pAM466 of pAM471 and 100ng of 400ng, will corresponding to ERG20-P _gALthe DNA fragmentation gel-purified of-tHMGR inset and linearizing pAM466 carrier, and this purifying inset of 4 molar equivalents is connected with this kind of purified linear carrier of 1 molar equivalent, acquisition pAM489 (see the collection of illustrative plates of Fig. 6 A, and ERG20-P _gALthe nucleotide sequence SEQ ID NO:3 of-tHMGR inset).

By the ERG13-P by carrier pAM472 _gAL-tHMGR inset insertion vector pAM467 generates plasmid pAM491.By by DNA fragmentation ERG13-P _gALthe XmaI restriction site that-tHMGR inserts TOPO ZeroBlunt II cloning vector generates carrier pAM472, this DNA fragmentation ERG13-P _gAL-tHMGR comprises the ORF (ERG13 nucleotide position 1 to 1626) (ERG13) of ERG13, comprises genomic gene seat (GAL1 nucleotide position-1 to-the 668) (P of divergent evolution GAL1 and GAL10 promotor _gAL), and the brachymemma RF of HMG1 (HMG1 nucleotide position 1586 to 3323) (tHMGR).By by DNA fragmentation URA3 ^{-723 to 701}insert TOPO TA pCR2.1 cloning vector and generate carrier pAM467, this DNA fragmentation URA3 ^{-723 to 701}comprise the yeast saccharomyces cerevisiae wild-type URA3 locus extending to position-224 from nucleotide position-723, and with non-natural inside XmaI restriction site between base-224 and-223.General introduction with reference to table 2 generates DNA fragmentation ERG13-P by pcr amplification _gAL-tHMGR and URA3 ^{-723 to 701}.For building pAM491, XmaI restriction enzyme is used to digest completely by the pAM467 of pAM472 and 100ng of 400ng, will corresponding to ERG13-P _gALthe DNA fragmentation gel-purified of-tHMGR inset and linearizing pAM467 carrier, and this purifying inset of 4 molar equivalents is connected with this kind of purified linear carrier of 1 molar equivalent, acquisition pAM491 (see the collection of illustrative plates of Fig. 6 B, and ERG13-P _gALthe nucleotide sequence SEQ ID NO:4 of-tHMGR inset).

By the IDI1-P by carrier pAM473 _gAL-tHMGR inset insertion vector pAM468 generates plasmid pAM493.By by DNA fragmentation IDI1-P _gAL-tHMGR inserts TOPO Zero BluntII cloning vector and generates carrier pAM473, this DNA fragmentation IDI1-P _gAL-tHMGR comprises the ORF (IDI1 nucleotide position 1 to 1017) (IDI1) of IDI1, comprises genomic gene seat (GAL1 nucleotide position-1 to-the 668) (P of divergent evolution GAL1 and GAL10 promotor _gAL), and the brachymemma ORF of HMG1 (HMG1 nucleotide position 1586 to 3323) (tHMGR).By by DNA fragmentation ADE1 ^{-825 to 653}insert TOPO TA pCR2.1 cloning vector and generate carrier pAM468, this DNA fragmentation ADE1 ^{-825 to 653}comprise the yeast saccharomyces cerevisiae wild-type ADE1 locus extending to position 653 from nucleotide position-225, and with non-natural inside XmaI restriction site between base-226 and-225.General introduction with reference to table 3 generates DNA fragmentation IDI1-P by pcr amplification _gAL-tHMGR and ADE1 ^-825 ^{to 653}.For building pAM493, XmaI restriction enzyme is used to digest completely by the pAM468 of pAM473 and 100ng of 400ng, will corresponding to IDI1-P _gALthe DNA fragmentation gel-purified of-tHMGR inset and linearizing pAM468 carrier, and this purifying inset of 4 molar equivalents is connected with this kind of purified linear carrier of 1 molar equivalent, acquisition pAM493 (see the collection of illustrative plates of Fig. 6 C, and IDI1-P _gALthe nucleotide sequence SEQ ID NO:5 of-tHMGR inset).

By the ERG10-P by carrier pAM474 _gAL-ERG12 inset insertion vector pAM469 generates plasmid pAM495.By by DNA fragmentation ERG10-P _gAL-ERG12 inserts TOPO ZeroBlunt II cloning vector and generates carrier pAM474, this DNA fragmentation ERG10-P _gAL-ERG12 comprises the ORF (ERG10 nucleotide position 1 to 1347) (ERG10) of ERG10, comprises genomic gene seat (GAL1 nucleotide position-1 to-the 668) (P of divergent evolution GAL1 and GAL10 promotor _gAL), and the ORF of ERG12 (ERG12 nucleotide position 1 to 1482) (ERG12).By by DNA fragmentation HIS3 ^{-32 to-1000}-HISMX-HIS3 ^{504 to-1103}insert TOPO TA pCR2.1 cloning vector and generate carrier pAM469, this DNA fragmentation HIS3 ^{-32 to-1000}-HISMX-HIS3 ^{504 to-1103}comprise from nucleotide position-32 extend to position-1000 yeast saccharomyces cerevisiae wild-type HIS locus, HISMX mark and at HIS3 ^{504 to-1103}non-natural XmaI restriction site between sequence and this HISMX mark.General introduction with reference to table 4 generates DNA fragmentation ERG10-P by pcr amplification _gAL-ERG12 and HIS3 ^-32 ^to-1000-HISMX-HIS3 ^{504 to-1103}.For building pAM495, XmaI restriction enzyme is used to digest completely by the pAM469 of pAM474 and 100ng of 400ng, will corresponding to ERG10-P _gALthe DNA fragmentation gel-purified of-ERG12 inset and linearizing pAM469 carrier, and this purifying inset of 4 molar equivalents is connected with this kind of purified linear carrier of 1 molar equivalent, acquisition pAM495 (see the collection of illustrative plates of Fig. 6 D, and ERG10-P _gALthe nucleotide sequence SEQ ID NO:6 of-ERG12 inset).

By the ERG8-P by carrier pAM470 _gAL-ERG19 inset insertion vector pAM470 generates plasmid pAM497.By by DNA fragmentation ERG8-P _gAL-ERG19 inserts TOPO Zero BluntII cloning vector and generates carrier pAM475, this DNA fragmentation ERG8-P _gAL-ERG19 comprises the ORF (ERG8 nucleotide position 1 to 1512) (ERG8) of ERG8, comprises genomic gene seat (GAL1 nucleotide position-1 to-the 668) (P of divergent evolution GAL1 and GAL10 promotor _gAL), and the ORF of ERG19 (ERG19 nucleotide position 1 to 1341) (ERG19).By by DNA fragmentation LEU2 ^{-100 to} ⁴⁵⁰-HISMX-LEU2 ^{1096 to 1770}insert TOPO TA pCR2.1 cloning vector and generate carrier pAM470, this DNA fragmentation LEU2 ^{-100 to 450}-HISMX-LEU2 ^{1096 to 1770}comprise and extend to position 450 and nucleotide position 1096 from nucleotide position-100 and extend to two yeast saccharomyces cerevisiae wild-type LEU2 locus segments of position 1770, HISMX mark and LEU2 ^{1096 to 1770}non-natural XmaI restriction site between sequence and this HISMX mark.General introduction with reference to table 5 generates DNA fragmentation ERG8-P by pcr amplification _gAL-ERG19 and LEU2 ^{-100 to 450}-HISMX-LEU2 ^{1096 to 1770}.For building pAM497, XmaI restriction enzyme is used to digest completely by the pAM4709 of pAM475 and 100ng of 400ng, will corresponding to ERG8-P _gALthe DNA fragmentation gel-purified of-ERG19 inset and linearizing pAM470 carrier, and this purifying inset of 4 molar equivalents is connected with this kind of purified linear carrier of 1 molar equivalent, acquisition pAM497 (see the collection of illustrative plates of Fig. 6 E, and ERG8-P _gALthe nucleotide sequence SEQ ID NO:7 of-ERG19 inset).

embodiment 5

Present embodiment describes the preparation method of the expression plasmid of the enzyme of encoded translated FPP.

Expression plasmid pAM373 is by generating for after the nucleotide sequence insertion vector pTrc99A carrying out codon optimized coding Artemisinin β-farnesene synthetic enzyme (GenBank accession number AY835398) at expression in escherichia coli.Coding β-farnesene synthetic enzyme nucleotide sequence by with SEQ IDNO:8 for templated synthesis obtains, and adopt primer A (SEQ ID NO:86) and primer B (SEQ ID NO:87) for primer from DNA synthesis construct increased by PCR.In order to create leading NcoI restriction site in the PCR primer comprising this β-farnesene synthetic enzyme, replace in original peptide sequence with the codon of codes for aspartate (GAC) in 5 ' PCR primer the second amino acid whose codon (TCG encoding serine) of encoding.Use the PCR primer of the partial digested gained of NcoI restriction enzyme, and use SacI digestion with restriction enzyme complete, this reaction mixture is separated by gel electrophoresis, gel extraction comprises the about 1.7kb DNA fragmentation of this β-farnesene synthetic enzyme encoding sequence, the DNA fragmentation of separation is connected to the NcoI SacI restriction site of pTrc99A carrier, thus obtains expression plasmid pAM373 (plasmid map see Fig. 7).

Expression plasmid pAM342 is by generating for after the nucleotide sequence insertion vector pTrc99A carrying out codon optimized coding Picea excelsa α-farnesene synthetic enzyme (GenBank accession number AY473627, REGION:24..1766) at expression in escherichia coli.The nucleotide sequence of coding for alpha-farnesene synthetic enzyme by with SEQ ID NO:9 for templated synthesis obtains, and adopt primer C (SEQ IDNO:88) and primer D (SEQ ID NO:89) to be increased by PCR for primer synthesizes construct from DNA.Use the PCR primer of NcoI and SacI restriction enzyme complete digestion gained, this reaction mixture is separated by gel electrophoresis, gel extraction comprises the about 1.7kbDNA fragment of this α-farnesene synthetic enzyme encoding sequence, the DNA fragmentation of separation is connected to the NcoI SacI restriction site of pTrc99A carrier, thus obtains expression plasmid pAM342 (plasmid map see Fig. 7).

Expression plasmid pAM341 and pAM353 is by respectively by generating after the nucleotide sequence insertion vector pRS425-Gal1 of coding for alpha-farnesene synthetic enzyme or β-farnesene synthetic enzyme people such as (, (1994) Nucl.Acids.Res.22 (25): 5767-5768) MumbERG.This nucleotide sequence inset can adopt through all carrying out codon optimized Picea excelsa α-farnesene synthase gene (GenBank accession number AY473627, REGION:24..1766) encoding sequence or Artemisinin β-farnesene synthase gene (GenBank accession number AY835398) encoding sequence (being respectively SEQ ID NO:11 and 10) obtains as templated synthesis for expressing in yeast saccharomyces cerevisiae.The nucleotide sequence that this synthesis obtains is sandwiched by 5 ' BamHI and 3 ' XhoI restriction site, therefore can be cloned the Compatible restriction sites entering cloning vector (such as standard pUC or pACYC derived vectors).The nucleotide sequence of often kind of synthesis generation can adopt BamHI with XhoI restriction enzyme to obtain being separated after the digestion completely of this DNA synthesis construct.By gel electrophoresis separate reacted mixture, gel extraction comprises the about 1.7kb DNA fragmentation of this α-farnesene synthetic enzyme or β-farnesene synthetic enzyme encoding sequence, the DNA fragmentation of separation is connected to the BamHI XhoI restriction site of pRS425-Gall carrier, thus obtains expression plasmid pAM341 or pAM353 respectively.

Expression plasmid pAM404 is by generating after in yeast saccharomyces cerevisiae, the nucleotide sequence insertion vector pAM178 of codon optimized coding Artemisinin β-farnesene synthetic enzyme (GenBank accession number AY835398) is carried out in expression.Adopt primer GW-52-84pAM326 BamHI (SEQ ID NO:90) and GW-52-84pAM326 NheI (SEQ ID NO:91) from the nucleotide sequence of pAM353PCR amplification coding β-farnesene synthetic enzyme.Use the PCR primer of BamHI and NheI restriction enzyme complete digestion gained, this reaction mixture is separated by gel electrophoresis, gel extraction comprises the about 1.7kb DNA fragment of this β-farnesene synthetic enzyme encoding sequence, the DNA fragmentation of separation is connected to the BamHI NheI restriction site of pAM178 carrier, thus obtains expression plasmid pAM404 (plasmid map see Fig. 8).

embodiment 6

Present embodiment describes the generation of e. coli host bacteria strain used in the present invention.

As described in Table 6, Competent intestinal bacteria parental cell is transformed generation host strain by one or more expression plasmids by embodiment 1 to 3 and embodiment 5.

Host cell transformant is being selected containing on antibiotic Luria Bertoni (LB) agar.Mono-clonal is transferred to LB liquid nutrient medium containing 5mL and antibiotic culture tube from LB agar.The gyrate shaker of 250rpm hatches B526, B552 and B592 host cell transformant at 37 DEG C, until grow to stationary phase.The gyrate shaker of 250rpm hatches B650, B651, B652 and B653 host cell transformant 30 hours at 30 DEG C.By cell continuous passage 4 to 5 in containing 0.8% glucose and antibiotic M9-MOPS the substratum component of the M9-MOPS substratum (can see table 7) being taken turns, make cell adapted minimum substratum.By in cryovial at-80 DEG C of the cell storage that is contained in the 1mL storage liquid that is made up of aseptic 50% glycerine of 400uL and 600uL liquid nutrient medium.

embodiment 7

Present embodiment describes the generation of Wine brewing yeast strain used in the present invention.

In order to prepare Wine brewing yeast strain Y141 and Y140, remove expression plasmid (people (2006) the Nature 440:940-943 such as Ro by cultivating in rich medium from Wine brewing yeast strain EPY224; PCT patent disclosure WO2007/005604), obtain bacterial strain EPY300.Then transform bacterial strain EPY300 with expression plasmid pAM341 or pAM353, obtain host strain Y141 or Y140 respectively.Determination component substratum (SM-glu) containing 2% glucose and all amino acid whose synthesis except leucine selects host cell transformant.Single clone is transferred to and lacks in the culturing bottle of leucic liquid SM-glu containing 5mL, and at 30 DEG C this culture of oscillation incubation until reach stationary phase.By in cryovial at-80 DEG C of the cell storage that is contained in the 1mL storage liquid that is made up of aseptic 50% glycerine of 400uL and 600uL liquid nutrient medium.

For preparing Wine brewing yeast strain Y258, by replacing ERG9 promotor with yeast saccharomyces cerevisiae MET3 promotor, and replace ADE1ORF with Candida glabrata LEU2 gene (CgLEU2) and prepare Wine brewing yeast strain CEN.PK2-1C (Y002) (MATA; Ura3-52; Trp1-289; Leu2-3,112; His3 Δ 1; MAL2-8C; And CEN.PK2-1D (Y003) (MAT α SUC2); Ura3-52; Trpl-289; Leu2-3,112; His3 Δ 1; MAL2-8C; SUC2), for the derivable MEV pathway gene of importing (van Dijken etc. people (2000) Enzyme Microb.Technol.26 (9-10): 706-714).This realizes by following steps: the KanMX-PMET3 district (it comprises and 45 of natural promoter homology base pairs) using primer 50-56-pw 100-G (SEQ ID NO:93) and 50-56-pw101-G (SEQ ID NO:94) pcr amplification carrier pAM328, use 40%w/w PEG3350 (Sigma-Aldrich, St.Louis, MO), 100mM Lithium Acetate (Sigma-Aldrich, St.Louis, and 10ug salmon sperm dna (Invitrogen Corp. MO), Carlsbad, CA) PCR primer of 10ug gained is transformed Y002 and the Y003 cell entering exponential growth, this cell to be hatched at 30 DEG C after 30 minutes thermal shocking 30 minutes (Schiestl and Gietz. (1989) Curr.Genet.16 at 42 DEG C, 339-346).Positive recombinants by it containing 0.5ug/mL Geneticin (Geneticin) (Invitrogen Corp., Carlsbad, CA) ability that rich medium grows is identified, and confirms selected clone by diagnostic PCR.Gained clone designation is Y93 (M ATA) and Y94 (MAT α).Then use primer 61-67-CPK066-G (SEQ ID NO:95) and 61-67-CPK067-G (SEQ ID NO:96) from Candida glabrata genomic dna (ATCC, Manassas, VA) increase this 3.5kb CgLEU2 genomic gene seat (it comprises 50 base pairs with ADEI ORF homology), the PCR primer of 10ug gained is transformed Y93 and the Y94 cell entering exponential growth, select the positive recombinants of growth under not having leucine to add, and the clone selected by being confirmed by diagnosis PCR.Gained clone designation is Y 176 (MAT A) and Y 177 (MAT α).

Then pAM491 and the pAM495 plasmid DNA by using PmeI restriction enzyme (New England Biolabs, Beverly, MA) to digest 2ug, and the Y176 cell DNA inset of this purifying being imported exponential growth obtains bacterial strain Y188.The positive recombinants that selection can grow on the substratum lacking uridylic and Histidine, and confirm to be integrated into correct genomic gene seat by diagnostic PCR.

Then by using pAM489 and the pAM497 plasmid DNA of PmeI digestion with restriction enzyme 2ug, and Y 177 cell DNA inset of this purifying being imported exponential growth obtains bacterial strain Y 189.The positive recombinants that selection can grow on the substratum lacking uridylic and Histidine, and confirm to be integrated into correct genomic gene seat by diagnostic PCR.

In order to generate bacterial strain Y238, then by from about 1 × 10 of bacterial strain Y 188 and Y 189 ⁷cell on YPD culture plate under room temperature mixing within 6 hours, engage, then the cell culture of mixing is coated in lack Histidine, uridylic and tryptophane substratum on growth select diploid cell.Then with by this diploid cell of pAM493 plasmid DNA transformation of the 2ug of PmeI restriction enzyme complete digestion, and the DNA inset of purifying is imported the diploid cell of exponential growth.Selection can lack the positive recombinants that the substratum of VITAMIN B4 grows, and confirms to be integrated into correct genomic gene seat by diagnostic PCR.

Haploid strains Y211 (MAT α) is by such as under type generation: in 2% Potassium ethanoate and 0.02% raffinose liquid nutrient medium, make bacterial strain Y238 form spore, adopt Singer InstrumentsMSM300 series micrurgy instrument (Singer Instrument LTD, Somerset, UK) about 200 hereditary tetrads are separated, by lacking VITAMIN B4, Histidine, the ability qualification grown in the substratum of uridylic and tryptophane comprises the independent inheritance isolate of the suitable complement of the genetic material of induction, and the integration of the DNA of all inductions is confirmed by diagnostic PCR.

Finally, by obtaining host strain Y258 with pAM404 plasmid DNA transformation bacterial strain Y211.Determination component substratum (SM-glu) containing 2% glucose and all amino acid whose synthesis except leucine selects host cell transformant.Single clone is transferred to and lacks in the culturing bottle of leucic liquid SM-glu containing 5mL, and at 30 DEG C this culture of oscillation incubation until reach stationary phase.By in cryovial at-80 DEG C of the cell storage that is contained in the 1mL storage liquid that is made up of aseptic 50% glycerine of 400uL and 600uL liquid nutrient medium.

embodiment 8

Present embodiment describes and generate α-farnesene and β-farnesene by MEV approach in e. coli host bacteria strain.

The decile liquid storage of host strain B552 and B592 is added into separately independent containing in the antibiotic 125mL flask described in 25mLM9-MOPS, 0.8% glucose, 0.5% yeast extract and table 6, and grow overnight, to form the inoculum culture of each host strain.This inoculum culture can be used for the initial OD of about 0.05 ₆₀₀inoculate independent containing 40mL M9-MOPS, 2% glucose, 0.5% yeast extract and antibiotic 250mL flask.The gyrate shaker of 250rpm hatches culture until it reaches the OD of about 0.2 at 30 DEG C ₆₀₀, now by adding the generation of the farnesene in the 1M IPTG inducing host cell of 40uL to substratum.When inducing, culture covers the organic mat (organic overlay) of 8mL to catch farnesene.Every 24 hours by this organic mat of 2-10uL is transferred to containing 1mL ethyl acetate, and with the addition of transcaryophyllene as in interior target cleaning glass bottle to complete sampling.

Is being furnished with Agilent 5975 mass spectrographic Agilent 6890N gas chromatograph (GC/MS) (AgilentTechnologies Inc., Palo Alto, CA) on analyze this ethyl acetate sample with full ion scan pattern (50-500m/z).At employing HP-5MS post (Agilent Technologies, Inc., Palo Alto, CA), helium carrier gas and following temperature program(me) are separated the compound in the various samples of 1uL: 150 DEG C maintain 3 minutes, the 200 DEG C of temperature be warming up to 25 DEG C/min, be warming up to 300 DEG C with 60 DEG C/min, and maintain 1 minute at 300 DEG C.Use the program, show the retention time that β-farnesene has 4.33 minutes.Farnesene titre by by the peak area of generation be contained in add transcaryophyllene ethyl acetate in pure β-farnesene (Sigma-Aldrich Chemical Company, St.Louis, MO) quantitative correction curve compare after calculate.

Host strain B592 generated α-farnesene (mean value of 3 independent clonings of about 400mg/L at 120 hours; Induce at time point 0), and there is about 46mg/L/OD ₆₀₀maximum unit productivity (1 representative clone).Host strain B552 generated the β-farnesene (mean values of 3 independent clonings) of about 1.1g/L at 120 hours, and had about 96mg/L/OD ₆₀₀maximum unit productivity (1 representative clone).

embodiment 9

Present embodiment describes and generate β-farnesene by DXP approach in e. coli host bacteria strain.

The decile liquid storage of host strain B650, B651, B652 and B653 is added into separately independent containing in the antibiotic 125mL flask described in 25mLM9-MOPS, 0.8% glucose, 0.5% yeast extract and table 6, and grow overnight, to form the inoculum culture of each bacterial strain.This inoculum culture can be used for the initial OD of about 0.05 ₆₀₀inoculate independent containing 40mL M9-MOPS, 45ug/mL VitB1, micro-nutrients, 1.00E-5mol/LFeSO ₄, 0.1M MOPS, 2% glucose, 0.5% yeast extract and antibiotic 250mL flask.The moist incubation oscillator of 250rpm hatches culture until it reaches the OD of about 0.2 to 0.3 at 30 DEG C ₆₀₀, now by adding the generation of the β-farnesene in the 1M IPTG inducing host cell of 40uL to substratum.When inducing, culture covers the organic mat (organic overlay) of 8mL to catch β-farnesene.By the 100uL sample of upper strata organic mat is transferred in clean pipe to obtain the sample of different time points.This organic mat sample of 10uL, to be separated arbitrarily residual cell or substratum, is transferred in cleaning glass bottle in 500uL ethyl acetate by this pipe centrifugal, using β or transcaryophyllene as interior mark.By mixture vortex 30 seconds, then analyze with reference to described in embodiment 8.

Host strain B653 generates the β-farnesene (DCW refers to " dry cell weight ") of about 7mg/gDCW.

embodiment 10

Present embodiment describes and generate α-farnesene and β-farnesene in Saccharomyces cerevisiae host bacterial strain.

The decile liquid storage of host strain Y141, Y140 and Y258 is added into separately in the independent 125mL flask containing the leucic 25mL SM-gl of shortage, and grow overnight, to form the inoculum culture of each bacterial strain.This inoculum culture can be used for the initial OD of about 0.05 ₆₀₀inoculate independent 250mL and be with baffled flask, this flask comprises containing 0.2% glucose and 1.8% semi-lactosi, lacks the clear and definite substratum of component that leucic 40mL synthesizes.The gyrate shaker of 200rpm hatches culture at 30 DEG C.Y141 and Y140 culture is covered with the dodecane of 8mL; Y258 culture is covered with the isopropyl myristate of 8mL.Within every 24 hours, gather the sample of Y141 and Y140 culture, until 120 hours, and the sample of Y258 culture gathers by within latter 72 hours, being transferred to by the organic mat of 2uL to 10uL in the cleaning glass bottle of 500uL ethyl acetate in induction, using β or transcaryophyllene as interior mark.Y141 and Y140 sample can refer to described in embodiment 8 and analyzes, and this Y258 sample can refer to described in embodiment 11 and analyzes.

Host strain Y141 generated the α-farnesene (mean values of 3 independent clonings) of about 9.8mg/L at 120 hours, and had about 3mg/L/OD ₆₀₀maximum unit productivity (1 representative clone).Host strain Y140 generated the β-farnesene (mean values of 3 independent clonings) of about 56mg/L at 120 hours, and had about 20mg/L/OD ₆₀₀maximum unit productivity (1 representative clone).Host strain Y258 generated the β-farnesene (mean values of 3 independent clonings) of about 762mg/L at 72 hours, and had about 145mg/L/OD ₆₀₀maximum unit productivity (1 representative clone).

embodiment 11

Present embodiment describes and in e. coli host bacteria strain, generate β-farnesene under aerobic, nitrogen restriction, fed-batch condition.

The decile liquid storage of host strain B526 is added into containing in 50mL M9-MOPS substratum and antibiotic 250mL flask, and on the gyrate shaker of 250rpm, at 37 DEG C, hatch this culture spend the night, to form the inoculum culture of the host strain B526 for fermenting.This inoculum culture can be used for the initial OD of about 1 ₆₀₀inoculation is containing 40mL M9-MOPS substratum and antibiotic 250mL flask.The gyrate shaker of 250rpm again hatches this culture at 37 DEG C, until it reaches the OD of about 3 to 5 ₆₀₀.

Table 8 shows the final substratum composition of batch fermentation 070522-1 (nitrogen is excessive) and 070522-5 (nitrogen restriction).Substratum by the gross in two bio-reactors (2L Applikon Bioconsole ADI 1025, with ADI1010 controller, Applikon Biotechnology, Foster City, CA) is heated sterilizing in 30 minutes respectively at 121 DEG C.Additive (PSA) after sterilizing and microbiotic (Pyocianil of final concentration 100ug/L and the paraxin of final concentration 34ug/L) sterile filtration as storing solution, and are injected each bio-reactor by top board.All trace-metals are merged and pre-prepared is concentrated solution (table 9), is added into this PSA or the substratum that feeds intake.The initial volume of each batch fermentation is 1L.All inoculate by injecting 50mL inoculum culture (5% (v/v)) through top board for all batches.

Start when initial glucose group (15g) is consumed and there is the exponential phase glucose of 6 hour doubling time feed intake, and inject dissolved oxygen.Under the prerequisite being 31g/hr to the maximum, fermentor tank software (BioXpert, Applikon Biotechnology, Foster City, CA) is programmed to reference to following Equation for Calculating delivery rate:

m_{s} (t) = S_{0} μ e^{μ (t - t_{0})}

μ＝0.12hr ^-1

S ₀＝15g，

Wherein m _sbe substrate mass velocity (g/hr), μ is specific growth rate, t ₀initial glucose batch of material depleted time, S ₀it is initial substrate concentration.When reaching maximum rate, glucose feed is reduced to the speed of 11.7g/hr, and keeps this constant rate within the remaining time of this batch fermentation.

Ferment at the reduction temperature of 30 DEG C; Air flow set in bio-reactor is 1vvm; Initial stirring is 700rpm; Foam is controlled by Biospumex defoamer 200K; Height is used to fall to stirring (700-1,200rpm) and oxygen coalescence by dissolved oxygen tension control 40%; Use 9.9N NH ₄oH (2 parts of concentrated NH ₄oH, 1 part of H ₂o) pH is maintained 7.Ammonia is detected in the upper explanation with reference to manufacturer of NOVA Bioprofile 300 analyser (Nova Biomedical Corp., Waltham, MA).

At the OD of about 30 ₆₀₀under generate β-farnesene by the 1M IPTG inducing host cell adding 1mL to described substratum.β-the farnesene of volatilization is caught through the air purge instrument containing 200mL enanthol by making expellant gas.Then by this enanthol solution dilution to ethyl acetate (dilution factor 100 ×).Solubility β-farnesene extracts from fermented liquid by such as under type: by 50uL fermented liquid and 950uLHPLC level methanol mixed, at Fisher VortexGenie 2 ^tMmixing tank (Scientific Industries, Inc., Bohemia, NY) to vibrate this sample about 30 minutes with top speed on, 14, centrifugal 10 minutes of 000 × g from this sample acquisition cell debris group, and by this acetonitrile solution in glass HPLC bottle with the dilution of 990uL HPLC level ethyl acetate.

Be equipped with this ethyl acetate sample of the upper analysis of Agilent 6890N Network chromatography of gases system (Agilent Technologies, Inc., Palo Alto, CA) of flame ionization detector (GCFID).Each sample of injection 1uL decile, adopts DB1-MS post (30m × 250um × 0.25um; AgilentTechnologies, Inc., Palo Alto, CA), helium carrier gas and following temperature program(me) are separated the compound contained in this sample: 200 DEG C maintain 1 minute, are warming up to 230 DEG C with 10 DEG C/min, be warming up to 300 DEG C with 40 DEG C/min, and maintain 1 minute at 300 DEG C.Use the program, show the retention time that β-farnesene has 4.33 minutes.Farnesene titre by using the peak area of generation be contained in the addition of transcaryophyllene ethyl acetate (as interior mark) in pure β-farnesene (Sigma-AldrichChemical Company, St.Louis, MO) quantitative correction curve compare after calculate.

Batch fermentation 070522-5 (nitrogen restriction) shows the β-farnesene titre of lower cell culture density and Geng Gao than batch 070522-1 (nitrogen is excessive).The ammonium that batch fermentation 070522-5 (nitrogen restriction) is all in fermention medium at 50 hours internal consumptions, and batch 070522-1 (nitrogen is excessive) at the time point of all samplings all containing excessive ammonium.As shown in table 10, in nutrient solution, all contain most of β-farnesene of generation in two batch fermentation.

embodiment 12

Present embodiment describes the mensuration of the β-farnesene distribution in e. coli host bacteria strain culture.

Melt cultivating through 65.5 hours the freezing full cell culture fluid (WCB) obtained after (see embodiment 11) by batch fermentation 070522-1 at ambient temperature.The WCB of 1.4mL is placed in 2mL press cover pipe with a scale (snap-cap tube), and on outstanding cup type whizzer (swinging cup rotor) with 10,600RCF centrifugal 10 minutes.After centrifugal, three different layers can be seen in pipe: cell mass, supernatant liquor and organic solid (light solid) layer.When being tilted by pipe, another liquid (light liquid) layer (supernatant liquor of this light solid may be through) can be seen above this organic solid.Light liquid is drawn to independent pipe; Use pipette tip by light solid transfer to independent pipe weighing; Supernatant liquor is poured into independent pipe also again centrifugal to remove all cell debriss; And cell mass is resuspended in deionized water, gained volume is 1.4mL.HPLC level methyl alcohol is adopted to extract, to carry out GCFID analysis with reference to described in embodiment 11 to every one deck.

The about 50% β-farnesene generated in cultivation is present in this light solid.The about 32% β-farnesene generated is arranged in each layer, cannot determine, this may be because micro-scale volume operational difficulty caused.

embodiment 13

Present embodiment describes and obtain farnesane by α-farnesene hydrogenation.α-farnesene (204g, 1mole, 255mL) is added in the 500mL Parr high pressure vessel containing 10%Pd/C (5% of 5g, α-farnesene weight).By reaction vessel seal, vacuumize 5 minutes by suction cleaner, then at 25 DEG C with H ₂this reaction mixture is pressurized to 35psi.Vibrate this reaction mixture, until do not observe H ₂the further decline (about 16 hours) of pressure.By suction cleaner by unnecessary H ₂gas removes, and is disposed to N ₂in air.Thin-layer chromatography (" TLC ", Rf=0.95, hexane, p-anisaldehyde dyeing or iodine) shows described reactant completely dissolve.Through silica gel ( from Aldrich) pad this reactant composition of vacuum filtration, then wash from silica gel with hexane (2L).This filtrate is concentrated with Rotary Evaporators.The product that further vacuum-drying is separated, to remove residual hexane, obtains the farnesane (195g, 244mL, 95%) of colorless liquid. ¹h-NMR (CDCl ₃, 500MHz): δ 1.56-1.11 (m, 17H), 0.88-0.79 (overlapping t & d, 15H).

embodiment 14

Present embodiment describes 3,7,11-trimethyldodecane-2,6,10-triolefin-1-alcohol or farnesol hydrogenation is 3,7,11-trimethyldodecane-1-alcohol.

Farnesol (572g, 2.58 moles, 650mL) is added into the 1000mL Parr high pressure vessel containing 10%Pd/C (23g, 4% of farnesol weight).Reaction vessel is sealed, vacuumizes 5 minutes, then with H by suction cleaner ₂this reaction mixture is pressurized to 1000psi.At 25 DEG C, stir this reaction mixture, and judge to react completely by thin-layer chromatography (" TLC ", Rf=0.32,90: 10 hexanes: ethyl acetate) after about 12 hours.Reaction vessel is reduced pressure under vacuo, then passes through N ₂gas.Through silica gel ( , from Aldrich) and pad this reactant composition of vacuum filtration, then wash from silica gel with ethyl acetate (" EtOAc ", 3L).This filtrate is concentrated with Rotary Evaporators.The product of separation is dry to remove EtOAc residual arbitrarily further under a high vacuum, 3,7,11-trimethyldodecane-1-alcohol of pale yellow viscous liquid shape are provided. ¹h-NMR (CDCl ₃, 500MHz): δ 3.71 (m, 2H), 1.65-1.05 (m, 17H), 0.89-0.83 (overlapping t & d, 12H).

embodiment 15

Present embodiment describes from 3,7,11-trimethyldodecane-1-alcohol synthesis, 3,7,11-trimethyldodecane ethyl ester.

To the CH of 3,7, the 11-trimethyldodecane-1-alcohol (542g, 2.38 moles) in stirring at 25 DEG C ₂cl ₂solution (1500mL) adds diacetyl oxide (267g, 2.63mol, 247mL), then adds triethylamine (360g, 3.57mol, 497mL), to generate colourless solution.Continue stir about 12 hours at ambient temperature, then generate the solution of dark rust.TLC (Rf=0.32,96: 4 hexanes: ethyl acetate) analyzes judgement and reacts completely.Termination reaction as follows, and process.Rotary evaporation concentration response component is to remove CH ₂cl ₂, and dilute with EtOAc (2L).With H ₂o (3 ×, 1L) wash organic layer, then pour Erlenmeyer flask into.Add decolourising charcoal (20g), stir 15 minutes, filter through Celite bed, generate light yellow filtrate with EtOAc (2L) washing.Rotary evaporation concentrating filtrate, and further drying obtains 3,7,11-trimethyldodecane ethyl esters of pale yellow viscous liquid shape under vacuo. ¹h-NMR (CDCl ₃, 500MHz): δ 4.11 (t, 2H), 2.04 (s, 3H), 1.62-1.09 (m, 17H) 0.91-0.83 (overlapping t & d, 12H).

embodiment 16

Present embodiment describes microbe-derived β-farnesene hydrogenation is farnesane.β-farnesene (KJF-41-120-05 and KJF-41-120-06 of 5.014g) is loaded glass pressure bottle, in bottle, adds 101mg 10% carbon carry palladium (Sigma-Aldrich#205699-50G).Flask is vacuumized 10 minutes, then under vibration, be pressurized to 55psi with hydrogen (Airgas UHP).After 8 minutes, get rid of hydrogen, with hydrogen by vessel pressurization to 53psi, then in 16 minutes get rid of.Stop oscillation, then flask is open into 4L hydrogen gas cylinder more than 48 hours under 53psi.Use Fene-Fane-Split100 method to carry out analysis display with GC/MS to react completely, then flask is pressurized to 52psi, and shaken overnight.Within a couple of days subsequently, when pressure drop is to below 48psi, reaction is pressurized to 48psi again.When GC/MS analysis display reaction is still complete, the same carbon of adding other 101mg carries palladium, again reaction is pressurized to 48psi.After 17 minutes, hydrogen consumption is complete, then is pressurized to 48psi.Within a couple of days subsequently, when pressure drop is to below 48psi, reaction is pressurized to 48psi again, reacts completely until GC/MS analyzes display.How empty funnel uses silica gel filter this catalyzer, obtain 1.47g water white oil.GC/FID assay products display product purity is used to be 99.42%.

embodiment 17

Present embodiment describes the extensive hydrogenation of β-farnesene and generate farnesane.10wt.%Pd/C (doing) catalyzer of 4kg (4.65L=1.23 gallon) farnesene liquid and 24g is added to the reactors of 2 gallons.Obtain the primary catalyst loading capacity of 5.16g/L thus.By container sealing, with nitrogen purge, then vacuumize.Start to stir, and add compression hydrogen continuously with 100psig.Reactor is heated to 80 DEG C.After 23 hours, collecting sample is analyzed.Detecting this farnesane concentration through GC-FID is 45.87%.After other 4 hours, gather second sample and analyze.Detecting this farnesane concentration through GC-FID is 47%.Cool this reactor, open, add 10g 10wt.%Pd/C (doing) catalyzer (amounting to 34g).Reactor is made to return above-mentioned reaction conditions.After 24 hours, gather the 3rd sample and analyze.Detecting this farnesane concentration through GC-FID is 67.86%.Cool this reactor, open, add 24g10wt.%Pd/C (doing) catalyzer (amounting to 58g).Reactor is made to return above-mentioned reaction conditions.After 24 hours, gather the 4th sample and analyze.Detecting this farnesane concentration through GC-FID is 97.27%.Cool this reactor, open, add 10g 10wt.%Pd/C (doing) catalyzer (amounting to 68g).Reactor is made to return above-mentioned reaction conditions.After 24 hours, gather the 5th sample and final sample and analyze.Detecting final farnesane concentration through GC-FID is 99.71%.Cool this reactor, ventilate, open.Then use 0.5 Mm filter cylinder to filter this reaction mixture, collect with 1 gal glass bottle.Total reaction time is about 96 hours.

Based on the experience of previous batch, this flow process is modified, to carry out follow-up batch.10wt.%Pd/C (doing) catalyzer of 4kg (4.65L=1.23 gallon) farnesene liquid and 75g is added to the reactors of 2 gallons.Obtain the primary catalyst loading capacity of 16.13g/L thus.By container sealing, with nitrogen purge, then vacuumize.Start to stir, and add compression hydrogen continuously with 100psig.Reactor is heated to 80 DEG C.Total reaction time is about 48 hours.Detecting final farnesane concentration through GC-FID is 99.76%.Cool this reactor, ventilate, open.Then use 0.5 Mm filter cylinder to filter this reaction mixture, collect with 1 gal glass bottle.

If needed, this product is further purified by distillation.Provide exemplary 1L as follows and distill scheme.The farnesane of about 1L is loaded 2L round-bottomed flask, the Vigreaux post this flask having water-cooled still head and be connected with this joint.Stirred liq, and be evacuated to 14Torr.Now, by this liquid heat to 155 DEG C, flask is wrapped in and has in the glass wool of aluminium foil.When heating, liquid is become light yellow from clarification.Start to occur steam at 120 DEG C.The clarification farnesane of about 950mL is have collected before distillation stops.

embodiment 18

Present embodiment describes the ultra-low-sulphur diesel (No. 2 diesel oil reach ASTM D 975 standard) of 90% and the attribute comprising the mixing prod of the mixture of 3,7,11-trimethyldodecane base ethyl ester and farnesane of 10%.This kind of mixture mainly comprises 3,7,11-trimethyldodecane base ethyl ester, there is a small amount of farnesane.

Table 12

	ASTM testing method	90%ULSD and 10% farnesane and 3,7,11-trimethyldodecane yl acetate
			Cetane value	D613	50.4
Cold filter clogging temperature (DEG C)	D6371	＜-22
			Cloud point (DEG C)	D2500	＜-22
Pour point (DEG C)	D97	＜-24
			Viscosity at 40 DEG C	D445	3.594

embodiment 19

Present embodiment describes the detection of the farnesane of different amount the ultra-low-sulphur diesel obtained from the BP refinery of Whitting (Indiana) or the BP refinery of Carson (California).Diesel oil from BP Carson refinery is CARB fuel, and it meets California Air resource board of management (Caliornia AirResources Board) requirement.Although often add lubricant to CARB fuel in oil refining process, this CARB fuel sample obtained before any lubricant of interpolation.Fig. 9 and 10 shows the test data of the different amount farnesanes mixed from the diesel oil fuel from this refinery.Figure 11 A-B shows the various fuel of test and the distillation curve of mixture.

embodiment 20

Present embodiment describes the mensuration of the amount of the farnesane of natural discovery in petroleum diesel (there is the complex mixture of thousands of kinds of individually oriented compounds).In these compounds, majority is C ₁₀-C ₂₂hydro carbons, and be generally paraffinic, cycloalkanes and aromatic hydrocarbon based.

Diluted in hexane by diesel oil sample, then with reference to people such as Zielinska, J.Air & WasteManage.Assoc.54:1138-1150 (2004) is described to be measured with GC-MS.The result unit that table 13 shows is ug/mL, wt.% and vol.%.

Table 13

Except the most latter two sample of table 13, all diesel oil samples are the diesel oil fuel bought from service station.These No. 2 diesel oil come from BPWhiting refinery.This CARB diesel oil comes from BP Carson refinery, and not containing improver for lubricating performance.

embodiment 21

Present embodiment describes to farnesane with from BP Whitng refinery diesel oil or come from BPCarson refinery CARB diesel oil mixture in add improver for lubricating performance.

The InfiniumR696 improver for lubricating performance (being once called as ECD-1) of 200ppm should be comprised from the diesel oil fuel of BP Whiting refinery.Add 100ppm again to this basic fuel, and test the lubricity of the farnesane of 5vol.%, 20vol.% and 50vol% and the mixture of this basic fuel with reference to ASTM D 6079.The lubricity (HFRR@60 DEG C) of the mixture of the farnesane of 5vol.%, 20vl.% and 50vol% and this basic fuel is respectively 300 μm, 240 μm and 450 μm.

Come from the CARB diesel oil of BPCarson refinery not containing oiliness additive.Add the Infinium R696 of 300ppm to this basic fuel, and test the lubricity of the farnesane of 5vol.%, 20vol.%, 50vol% and 65vol% and the mixture of this basic fuel with reference to ASTM D6079.The lubricity (HFRR@60 DEG C) of the mixture of 5vol.%, 20vol.%, 50vol% and 65vol% is respectively 200 μm, 240 μm, 280 μm and 240 μm.

Sequence table

SEQ ID NO：1

MevT66 operon (5 ' to 3 ')

GAATTCAAAGGAGGAAAATAAAATGAAGAACTGTGTGATTGTTTCTGCGGTCCGCACGGCGATC

GGCAGCTTTAACGGCTCTTTAGCGAGCACCTCTGCAATCGATCTGGGTGCGACGGTCATTAAGGC

CGCCATTGAACGCGCCAAAATCGACAGCCAGCACGTTGATGAGGTGATCATGGGCAATGTGTTA

CAAGCCGGCCTGGGTCAAAACCCAGCGCGTCAAGCACTGTTAAAATCTGGTCTGGCCGAGACCG

TGTGTGGCTTCACCGTCAATAAGGTTTGCGGCTCTGGCCTGAAGAGCGTGGCCCTGGCAGCACAA

GCGATTCAAGCCGGTCAGGCACAAAGCATCGTTGCGGGTGGCATGGAGAACATGTCTCTGGCGC

CGTACTTATTAGATGCCAAAGCCCGCAGCGGTTATCGCCTGGGCGATGGTCAGGTGTACGACGTC

ATCTTACGCGATGGCTTAATGTGCGCGACCCACGGTTACCACATGGGTATTACGGCCGAAAACGT

GGCGAAAGAATACGGCATTACGCGCGAGATGCAGGATGAATTAGCACTGCACTCTCAGCGCAAA

GCAGCAGCCGCGATCGAGTCTGGTGCGTTTACGGCGGAAATCGTGCCAGTTAACGTGGTCACGC

GCAAGAAGACGTTCGTTTTCAGCCAGGACGAGTTCCCGAAGGCAAACAGCACCGCGGAGGCCTT

AGGTGCCTTACGCCCAGCCTTTGACAAAGCGGGCACGGTCACCGCCGGTAATGCGAGCGGCATC

AATGATGGTGCAGCGGCACTGGTCATCATGGAAGAGAGCGCCGCATTAGCAGCGGGTCTGACCC

CATTAGCGCGCATTAAATCTTATGCCAGCGGCGGCGTCCCACCAGCCCTGATGGGCATGGGTCCG

GTCCCAGCCACGCAAAAAGCCCTGCAATTAGCGGGCCTGCAACTGGCCGACATTGATCTGATCG

AGGCGAACGAGGCGTTTGCAGCGCAGTTCCTGGCGGTGGGTAAGAATCTGGGCTTCGACAGCGA

GAAAGTCAATGTGAACGGTGGCGCGATTGCGTTAGGCCATCCGATTGGTGCAAGCGGCGCACGC

ATCTTAGTGACGTTACTGCACGCCATGCAGGCACGCGACAAGACCTTAGGCCTGGCGACCTTATG

TATTGGTGGCGGTCAAGGTATCGCCATGGTGATCGAACGCCTGAACTGAAGATCTAGGAGGAAA

GCAAAATGAAACTGAGCACCAAGCTGTGCTGGTGTGGCATCAAGGGTCGCCTGCGCCCACAAAA

GCAGCAACAGCTGCACAACACGAACCTGCAAATGACCGAGCTGAAAAAGCAGAAGACGGCCGA

GCAAAAGACCCGCCCGCAGAACGTTGGCATCAAGGGCATCCAGATTTATATCCCGACGCAGTGT

GTCAACCAATCTGAGCTGGAGAAATTCGATGGCGTCAGCCAGGGTAAGTACACCATCGGCCTGG

GCCAGACCAACATGAGCTTCGTGAACGACCGTGAGGACATCTATTCTATGAGCCTGACGGTGCT

GTCTAAGCTGATCAAGAGCTACAACATCGACACGAATAAGATCGGTCGTCTGGAGGTGGGTACG

GAGACGCTGATTGACAAGAGCAAAAGCGTGAAGTCTGTCTTAATGCAGCTGTTCGGCGAGAACA

CGGATGTCGAGGGTATCGACACCCTGAACGCGTGTTACGGCGGCACCAACGCACTGTTCAATAG

CCTGAACTGGATTGAGAGCAACGCCTGGGATGGCCGCGATGCGATCGTCGTGTGCGGCGATATC

GCCATCTATGACAAGGGTGCGGCACGTCCGACCGGCGGTGCAGGCACCGTTGCGATGTGGATTG

GCCCGGACGCACCAATTGTCTTCGATTCTGTCCGCGCGTCTTACATGGAGCACGCCTACGACTTT

TACAAGCCGGACTTCACGAGCGAATACCCGTACGTGGACGGCCACTTCTCTCTGACCTGCTATGT

GAAGGCGCTGGACCAGGTTTATAAGTCTTATAGCAAAAAGGCGATTTCTAAGGGCCTGGTCAGC

GACCCGGCAGGCAGCGACGCCCTGAACGTGCTGAAGTATTTCGACTACAACGTGTTCCATGTCCC

GACCTGCAAATTAGTGACCAAATCTTATGGCCGCCTGTTATATAATGATTTCCGTGCCAACCCGC

AGCTGTTCCCGGAGGTTGACGCCGAGCTGGCGACGCGTGATTACGACGAGAGCCTGACCGACAA

GAACATCGAGAAGACCTTCGTCAACGTCGCGAAGCCGTTCCACAAAGAGCGTGTGGCCCAAAGC

CTGATCGTCCCGACCAACACGGGCAACATGTATACCGCGTCTGTCTACGCGGCATTCGCGAGCCT

GCTGAATTACGTCGGTTCTGACGACCTGCAGGGCAAGCGCGTTGGCCTGTTCAGCTACGGTAGCG

GCTTAGCGGCCAGCCTGTATAGCTGCAAAATTGTCGGCGACGTCCAGCACATCATCAAGGAGCT

GGACATCACCAACAAGCTGGCGAAGCGCATCACCGAGACGCCGAAAGATTACGAGGCAGCGAT

CGAGTTACGCGAGAATGCGCATCTGAAGAAGAACTTCAAGCCGCAAGGTAGCATCGAGCACCTG

CAGAGCGGCGTCTACTACCTGACGAACATTGACGACAAGTTCCGCCGTTCTTATGACGTCAAAAA

GTAACTAGTAGGAGGAAAACATCATGGTGCTGACGAACAAAACCGTCATTAGCGGCAGCAAGGT

GAAGTCTCTGAGCAGCGCCCAAAGCTCTAGCAGCGGCCCGTCTAGCAGCAGCGAGGAGGACGAC

AGCCGTGACATTGAGTCTCTGGACAAGAAGATCCGCCCGCTGGAGGAGTTAGAGGCCCTGCTGA

GCAGCGGCAACACCAAGCAGCTGAAGAACAAGGAAGTTGCAGCGCTGGTGATCCACGGTAAGC

TGCCACTGTATGCGCTGGAAAAGAAACTGGGCGATACGACGCGTGCGGTCGCGGTGCGTCGCAA

AGCCTTAAGCATCTTAGCGGAGGCCCCGGTGTTAGCCAGCGACCGCCTGCCGTACAAGAACTAC

GACTACGACCGCGTGTTTGGCGCGTGCTGCGAGAATGTCATTGGCTACATGCCGTTACCGGTTGG

TGTGATCGGCCCGCTGGTCATTGATGGCACGAGCTATCACATTCCAATGGCGACCACGGAAGGTT

GCTTAGTCGCCAGCGCCATGCGTGGCTGTAAGGCGATTAACGCCGGCGGTGGCGCGACGACCGT

GTTAACCAAGGATGGTATGACGCGCGGTCCGGTCGTCCGCTTCCCAACGCTGAAGCGCAGCGGC

GCGTGTAAGATTTGGCTGGATTCTGAGGAGGGCCAAAACGCGATCAAGAAAGCCTTCAACTCTA

CGAGCCGTTTCGCGCGTTTACAGCATATCCAGACCTGCCTGGCCGGCGACCTGCTGTTCATGCGC

TTCCGCACCACCACGGGCGATGCGATGGGCATGAACATGATCAGCAAGGGCGTCGAATATAGCC

TGAAACAAATGGTGGAAGAATATGGCTGGGAGGACATGGAGGTTGTCTCTGTGAGCGGCAACTA

TTGCACCGACAAGAAGCCGGCAGCCATTAACTGGATTGAGGGTCGCGGCAAAAGCGTCGTGGCA

GAAGCGACCATCCCAGGCGACGTGGTCCGTAAGGTTCTGAAGAGCGACGTCAGCGCCCTGGTTG

AGTTAAATATCGCGAAAAACCTGGTCGGCAGCGCGATGGCGGGCAGCGTGGGTGGCTTTAACGC

ACATGCAGCGAATCTGGTTACGGCGGTTTTCTTAGCCTTAGGTCAGGACCCAGCCCAAAATGTCG

AGAGCAGCAACTGCATTACCTTAATGAAAGAGGTTGACGGTGACCTGCGCATCAGCGTTTCTATG

CCGTCTATCGAGGTCGGCACGATCGGCGGCGGCACCGTTTTAGAACCGCAAGGTGCGATGCTGG

ATCTGCTGGGCGTGCGCGGCCCACATGCAACGGCCCCAGGCACCAATGCCCGCCAACTGGCCCG

TATCGTGGCCTGCGCGGTTCTGGCGGGTGAGCTGAGCCTGTGCGCCGCATTAGCCGCGGGCCATT

TAGTTCAATCTCACATGACCCACAACCGCAAGCCGGCAGAACCAACCAAGCCAAATAACCTGGA

CGCAACCGACATTAACCGTCTGAAGGATGGCAGCGTCACGTGCATTAAAAGCTGAGCATGCTAC

TAAGCTT

SEQ ID NO：2

The atoB (opt) of pAM52: mvaS:mvaA operon (5 ' to 3 ')

ATGAAGAACTGTGTGATTGTTTCTGCGGTCCGCACGGCGATCGGCAGCTTTAACGGCTCTTTAGC

GAGCACCTCTGCAATCGATCTGGGTGCGACGGTCATTAAGGCCGCCATTGAACGCGCCAAAATC

GACAGCCAGCACGTTGATGAGGTGATCATGGGCAATGTGTTACAAGCCGGCCTGGGTCAAAACC

CAGCGCGTCAAGCACTGTTAAAATCTGGTCTGGCCGAGACCGTGTGTGGCTTCACCGTCAATAAG

GTTTGCGGCTCTGGCCTGAAGAGCGTGGCCCTGGCAGCACAAGCGATTCAAGCCGGTCAGGCAC

AAAGCATCGTTGCGGGTGGCATGGAGAACATGTCTCTGGCGCCGTACTTATTAGATGCCAAAGC

CCGCAGCGGTTATCGCCTGGGCGATGGTCAGGTGTACGACGTCATCTTACGCGATGGCTTAATGT

GCGCGACCCACGGTTACCACATGGGTATTACGGCCGAAAACGTGGCGAAAGAATACGGCATTAC

GCGCGAGATGCAGGATGAATTAGCACTGCACTCTCAGCGCAAAGCAGCAGCCGCGATCGAGTCT

GGTGCGTTTACGGCGGAAATCGTGCCAGTTAACGTGGTCACGCGCAAGAAGACGTTCGTTTTCAG

CCAGGACGAGTTCCCGAAGGCAAACAGCACCGCGGAGGCCTTAGGTGCCTTACGCCCAGCCTTT

GACAAAGCGGGCACGGTCACCGCCGGTAATGCGAGCGGCATCAATGATGGTGCAGCGGCACTGG

TCATCATGGAAGAGAGCGCCGCATTAGCAGCGGGTCTGACCCCATTAGCGCGCATTAAATCTTAT

GCCAGCGGCGGCGTCCCACCAGCCCTGATGGGCATGGGTCCGGTCCCAGCCACGCAAAAAGCCC

TGCAATTAGCGGGCCTGCAACTGGCCGACATTGATCTGATCGAGGCGAACGAGGCGTTTGCAGC

GCAGTTCCTGGCGGTGGGTAAGAATCTGGGCTTCGACAGCGAGAAAGTCAATGTGAACGGTGGC

GCGATTGCGTTAGGCCATCCGATTGGTGCAAGCGGCGCACGCATCTTAGTGACGTTACTGCACGC

CATGCAGGCACGCGACAAGACCTTAGGCCTGGCGACCTTATGTATTGGTGGCGGTCAAGGTATC

GCCATGGTGATCGAACGCCTGAACTGAAGATCTAGGAGGAAAGCAAAATGACAATAGGTATCGA

CAAAATAAACTTTTACGTTCCAAAGTACTATGTAGACATGGCTAAATTAGCAGAAGCACGCCAA

GTAGACCCAAACAAATTTTTAATTGGAATTGGTCAAACTGAAATGGCTGTTAGTCCTGTAAACCA

AGACATCGTTTCAATGGGCGCTAACGCTGCTAAGGACATTATAACAGACGAAGATAAAAAGAAA

ATTGGTATGGTAATTGTGGCAACTGAATCAGCAGTTGATGCTGCTAAAGCAGCCGCTGTTCAAAT

TCACAACTTATTAGGTATTCAACCTTTTGCACGTTGCTTTGAAATGAAAGAAGCTTGTTATGCTGC

AACACCAGCAATTCAATTAGCTAAAGATTATTTAGCAACTAGACCGAATGAAAAAGTATTAGTT

ATTGCTACAGATACAGCACGTTATGGATTGAATTCAGGCGGCGAGCCAACACAAGGTGCTGGCG

CAGTTGCGATGGTTATTGCACATAATCCAAGCATTTTGGCATTAAATGAAGATGCTGTTGCTTAC

ACTGAAGACGTTTATGATTTCTGGCGTCCAACTGGACATAAATATCCATTAGTTGATGGTGCATT

ATCTAAAGATGCTTATATCCGCTCATTCCAACAAAGCTGGAATGAATACGCAAAACGTCAAGGT

AAGTCGCTAGCTGACTTCGCATCTCTATGCTTCCATGTTCCATTTACAAAAATGGGTAAAAAGGC

ATTAGAGTCAATCATTGATAACGCTGATGAAACAACTCAAGAGCGTTTACGTTCAGGATATGAA

GATGCTGTAGATTATAACCGTTATGTCGGTAATATTTATACTGGATCATTATATTTAAGCCTAATA

TCATTACTTGAAAATCGTGATTTACAAGCTGGTGAAACAATCGGTTTATTCAGTTATGGCTCAGG

TTCAGTTGGTGAATTTTATAGTGCGACATTAGTTGAAGGCTACAAAGATCATTTAGATCAAGCTG

CACATAAAGCATTATTAAATAACCGTACTGAAGTATCTGTTGATGCATATGAAACATTCTTCAAA

CGTTTTGATGACGTTGAATTTGACGAAGAACAAGATGCTGTTCATGAAGATCGTCATATTTTCTA

CTTATCAAATATTGAAAATAACGTTCGCGAATATCACAGACCAGAGTAACTAGTAGGAGGAAAA

CATCATGCAAAGTTTAGATAAGAATTTCCGACATTTATCTCGTCAACAAAAGTTACAACAATTGG

TAGATAAGCAATGGTTATCAGAAGATCAATTCGACATTTTATTGAATCATCCATTAATTGATGAG

GAAGTAGCAAATAGTTTAATTGAAAATGTCATCGCGCAAGGTGCATTACCCGTTGGATTATTACC

GAATATCATTGTGGACGATAAGGCATATGTTGTACCTATGATGGTGGAAGAGCCTTCAGTTGTCG

CTGCAGCTAGTTATGGTGCAAAGCTAGTGAATCAGACTGGCGGATTTAAAACGGTATCTTCTGAA

CGTATTATGATAGGTCAAATCGTCTTTGATGGCGTTGACGATACTGAAAAATTATCAGCAGACAT

TAAAGCTTTAGAAAAGCAAATTCATAAAATTGCGGATGAGGCATATCCTTCTATTAAAGCGCGTG

GTGGTGGTTACCAACGTATAGCTATTGATACATTTCCTGAGCAACAGTTACTATCTTTAAAAGTA

TTTGTTGATACGAAAGATGCTATGGGCGCTAATATGCTTAATACGATTTTAGAGGCCATAACTGC

ATTTTTAAAAAATGAATCTCCACAAAGCGACATTTTAATGAGTATTTTATCCAATCATGCAACAG

CGTCCGTTGTTAAAGTTCAAGGCGAAATTGACGTTAAAGATTTAGCAAGGGGCGAGAGAACTGG

AGAAGAGGTTGCCAAACGAATGGAACGTGCTTCTGTATTGGCACAAGTTGATATTCATCGTGCTG

CAACACATAATAAAGGTGTTATGAATGGCATACATGCCGTTGTTTTAGCAACAGGAAATGATAC

GCGTGGTGCAGAAGCAAGTGCGCATGCATACGCGAGTCGTGACGGACAGTATCGTGGTATTGCA

ACATGGAGATACGATCAAAAACGTCAACGTTTAATTGGTACAATAGAAGTGCCTATGACATTGG

CAATCGTTGGCGGTGGTACAAAAGTATTACCAATTGCTAAAGCTTCTTTAGAATTGCTAAATGTA

GATTCAGCACAAGAATTAGGTCATGTAGTTGCTGCCGTTGGTTTAGCACAGAACTTTGCAGCATG

TCGCGCGCTCGTTTCCGAAGGTATCCAGCAAGGCCATATGAGCTTGCAATATAAATCTTTAGCTA

TTGTTGTAGGTGCAAAAGGTGATGAAATTGCGCAAGTAGCTGAAGCATTGAAGCAAGAACCCCG

TGCGAATACACAAGTAGCTGAACGCATTTTACAAGAAATTAGACAACAATAG

SEQ ID NO：3

The ERG20-P of pAM498 _gAL-tHMGR inset (5 ' to 3 ')

GTTTAAACTACTATTAGCTGAATTGCCACTGCTATCGTTGTTAGTGGCGTTAGTGCTTGCATTCAA

AGACATGGAGGGCGTTATTACGCCGGAGCTCCTCGACAGCAGATCTGATGACTGGTCAATATATT

TTTGCATTGAGGCTCTGTTTGGAATTATATTTTGAGATGACCCATCTAATGTACTGGTATCACCAG

ATTTCATGTCGTTTTTTAAAGCGGCTGCTTGAGTCTTAGCAATAGCGTCACCATCTGGTGAATCCT

TTGAAGGAACCACTGACGAAGGTTTGGACAGTGACGAAGAGGATCTTTCCTGCTTTGAATTAGTC

GCGCTGGGAGCAGATGACGAGTTGGTGGAGCTGGGGGCAGGATTGCTGGCCGTCGTGGGTCCTG

AATGGGTCCTTGGCTGGTCCATCTCTATTCTGAAAACGGAAGAGGAGTAGGGAATATTACTGGCT

GAAAATAAGTCTTGAATGAACGTATACGCGTATATTTCTACCAATCTCTCAACACTGAGTAATGG

TAGTTATAAGAAAGAGACCGAGTTAGGGACAGTTAGAGGCGGTGGAGATATTCCTTATGGCATG

TCTGGCGATGATAAAACTTTTCAAACGGCAGCCCCGATCTAAAAGAGCTGACACCCGGGAGTTA

TGACAATTACAACAACAGAATTCTTTCTATATATGCACGAACTTTGTAATATGGAAGAAATTATGA

CGTACAAACTATAAAGTAAATATTTTACGTAACACATGGTGCTGTTGTGCTTCTTTTTCAAGAGA

ATACCAATGACGTATGACTAAGTTTAGGATTTAATGCAGGTGACGGACCCATCTTTCAAACGATT

TATATCAGTGGCGTCCAAATTGTTAGGTTTTGTTGGTTCAGCAGGTTTCCTGTTGTGGGTCATATG

ACTTTGAACCAAATGGCCGGCTGCTAGGGCAGCACATAAGGATAATTCACCTGCCAAGACGGCA

CAGGCAACTATTCTTGCTAATTGACGTGCGTTGGTACCAGGAGCGGTAGCATGTGGGCCTCTTAC

ACCTAATAAGTCCAACATGGCACCTTGTGGTTCTAGAACAGTACCACCACCGATGGTACCTACTT

CGATGGATGGCATGGATACGGAAATTCTCAAATCACCGTCCACTTCTTTCATCAATGTTATACAG

TTGGAACTTTCGACATTTTGTGCAGGATCTTGTCCTAATGCCAAGAAAACAGCTGTCACTAAATT

AGCTGCATGTGCGTTAAATCCACCAACAGACCCAGCCATTGCAGATCCAACCAAATTCTTAGCAA

TGTTCAACTCAACCAATGCGGAAACATCACTTTTTAACACTTTTCTGACAACATCACCAGGAATA

GTAGCTTCTGCGACGACACTCTTACCACGACCTTCGATCCAGTTGATGGCAGCTGGTTTTTTGTCG

GTACAGTAGTTACCAGAAACGGAGACAACCTCCATATCTTCCCAGCCATACTCTTCTACCATTTG

CTTTAATGAGTATTCGACACCCTTAGAAATCATATTCATACCCATTGCGTCACCAGTAGTTGTTCT

AAATCTCATGAAGAGTAAATCTCCTGCTAGACAAGTTTGAATATGTTGCAGACGTGCAAATCTTG

ATGTAGAGTTAAAAGCTTTTTTAATTGCGTTTTGTCCCTCTTCTGAGTCTAACCATATCTTACAGG

CACCAGATCTTTTCAAAGTTGGGAAACGGACTACTGGGCCTCTTGTCATACCATCCTTAGTTAAA

ACAGTTGTTGCACCACCGCCAGCATTGATTGCCTTACAGCCACGCATGGCAGAAGCTACCAAAC

AACCCTCTGTAGTTGCCATTGGTATATGATAAGATGTACCATCGATAACCAAGGGGCCTATAACA

CCAACGGGCAAAGGCATGTAACCTATAACATTTTCACAACAAGCGCCAAATACGCGGTCGTAGT

CATAATTTTTATATGGTAAACGATCAGATGCTAATACAGGAGCTTCTGCCAAAATTGAAAGAGCC

TTCCTACGTACCGCAACCGCTCTCGTAGTATCACCTAATTTTTTCTCCAAAGCGTACAAAGGTAA

CTTACCGTGAATAACCAAGGCAGCGACCTCTTTGTTCTTCAATTGTTTTGTATTTCCACTACTTAA

TAATGCTTCTAATTCTTCTAAAGGACGTATTTTCTTATCCAAGCTTTCAATATCGCGGGAATCATC

TTCCTCACTAGATGATGAAGGTCCTGATGAGCTCGATTGCGCAGATGATAAACTTTTGACTTTCG

ATCCAGAAATGACTGTTTTATTGGTTAAAACTGGTGTAGAAGCCTTTTGTACAGGAGCAGTAAAA

GACTTCTTGGTGACTTCAGTCTTCACCAATTGGTCTGCAGCCATTATAGTTTTTTCTCCTTGACGTT

AAAGTATAGAGGTATATTAACAATTTTTTGTTGATACTTTTATGACATTTGAATAAGAAGTAATA

CAAACCGAAAATGTTGAAAGTATTAGTTAAAGTGGTTATGCAGCTTTTGCATTTATATATCTGTT

AATAGATCAAAAATCATCGCTTCGCTGATTAATTACCCCAGAAATAAGGCTAAAAAACTAATCG

CATTATTATCCTATGGTTGTTAATTTGATTCGTTGATTTGAAGGTTTGTGGGGCCAGGTTACTGCC

AATTTTTCCTCTTCATAACCATAAAAGCTAGTATTGTAGAATCTTTATTGTTCGGAGCAGTGCGGC

GCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTGAAGACCAGGACGCACGGAGGAGAGTCTT

CCGTCGGAGGGCTGTCGCCCGCTCGGCGGCTTCTAATCCGTACTTCAATATAGCAATGAGCAGTT

AAGCGTATTACTGAAAGTTCCAAAGAGAAGGTTTTTTTAGGCTAAGATAATGGGGCTCTTTACAT

TTCCACAACATATAAGTAAGATTAGATATGGATATGTATATGGTGGTATTGCCATGTAATATGAT

TATTAAACTTCTTTGCGTCCATCCAAAAAAAAAGTAAGAATTTTTGAAAATTCAATATAAATGGC

TTCAGAAAAAGAAATTAGGAGAGAGAGATTCTTGAACGTTTTCCCTAAATTAGTAGAGGAATTG

AACGCATCGCTTTTGGCTTACGGTATGCCTAAGGAAGCATGTGACTGGTATGCCCACTCATTGAA

CTACAACACTCCAGGCGGTAAGCTAAATAGAGGTTTGTCCGTTGTGGACACGTATGCTATTCTCT

CCAACAAGACCGTTGAACAATTGGGGCAAGAAGAATACGAAAAGGTTGCCATTCTAGGTTGGTG

CATTGAGTTGTTGCAGGCTTACTTCTTGGTCGCCGATGATATGATGGACAAGTCCATTACCAGAA

GAGGCCAACCATGTTGGTACAAGGTTCCTGAAGTTGGGGAAATTGCCATCAATGACGCATTCAT

GTTAGAGGCTGCTATCTACAAGCTTTTGAAATCTCACTTCAGAAACGAAAAATACTACATAGATA

TCACCGAATTGTTCCATGAGGTCACCTTCCAAACCGAATTGGGCCAATTGATGGACTTAATCACT

GCACCTGAAGACAAAGTCGACTTGAGTAAGTTCTCCCTAAAGAAGCACTCCTTCATAGTTACTTT

CAAGACTGCTTACTATTCTTTCTACTTGCCTGTCGCATTGGCCATGTACGTTGCCGGTATCACGGA

TGAAAAGGATTTGAAACAAGCCAGAGATGTCTTGATTCCATTGGGTGAATACTTCCAAATTCAAG

ATGACTACTTAGACTGCTTCGGTACCCCAGAACAGATCGGTAAGATCGGTACAGATATCCAAGA

TAACAAATGTTCTTGGGTAATCAACAAGGCATTGGAACTTGCTTCCGCAGAACAAAGAAAGACT

TTAGACGAAAATTACGGTAAGAAGGACTCAGTCGCAGAAGCCAAATGCAAAAAGATTTTCAATG

ACTTGAAAATTGAACAGCTATACCACGAATATGAAGAGTCTATTGCCAAGGATTTGAAGGCCAA

AATTTCTCAGGTCGATGAGTCTCGTGGCTTCAAAGCTGATGTCTTAACTGCGTTCTTGAACAAAG

TTTACAAGAGAAGCAAATAGAACTAACGCTAATCGATAAAACATTAGATTTCAAACTAGATAAG

GACCATGTATAAGAACTATATACTTCCAATATAATATAGTATAAGCTTTAAGATAGTATCTCTCG

ATCTACCGTTCCACGTGACTAGTCCAAGGATTTTTTTTAACCCGGGATATATGTGTACTTTGCAGT

TATGACGCCAGATGGCAGTAGTGGAAGATATTCTTTATTGAAAAATAGCTTGTCACCTTACGTAC

AATCTTGATCCGGAGCTTTTCTTTTTTTTGCCGATTAAGAATTCGGTCGAAAAAAGAAAAGGAGAG

GGCCAAGAGGGAGGGCATTGGTGACTATTGAGCACGTGAGTATACGTGATTAAGCACACAAAGG

CAGCTTGGAGTATGTCTGTTATTAATTTCACAGGTAGTTCTGGTCCATTGGTGAAAGTTTGCGGCT

TGCAGAGCACAGAGGCCGCAGAATGTGCTCTAGATTCCGATGCTGACTTGCTGGGTATTATATGT

GTGCCCAATAGAAAGAGAACAATTGACCCGGTTATTGCAAGGAAAATTTCAAGTCTTGTAAAAG

CATATAAAAATAGTTCAGGCACTCCGAAATACTTGGTTGGCGTGTTTCGTAATCAACCTAAGGAG

GATGTTTTGGCTCTGGTCAATGATTACGGCATTGATATCGTCCAACTGCATGGAGATGAGTCGTG

GCAAGAATACCAAGAGTTCCTCGGTTTGCCAGTTATTAAAAGACTCGTATTTCCAAAAGACTGCA

ACATACTACTCAGTGCAGCTTCACAGAAACCTCATTCGTTTATTCCCTTGTTTGATTCAGAAGCAG

GTGGGACAGGTGAACTTTTGGATTGGAACTCGATTTCTGACTGGGTTGGAAGGCAAGAGAGCCC

CGAAAGCTTACATTTTATGTTAGCTGGTGGACTGACGCCGTTTAAAC

SEQ ID NO：4

The ERG13-P of pAM491 _gAL-tHMGR inset (5 ' to 3 ')

GTTTAAACTTGCTAAATTCGAGTGAAACACAGGAAGACCAGAAAATCCTCATTTCATCCATATTA

ACAATAATTTCAAATGTTTATTTGCATTATTTGAAACTAGGGAAGACAAGCAACGAAACGTTTTT

GAAAATTTTGAGTATTTTCAATAAATTTGTAGAGGACTCAGATATTGAAAAAAAGCTACAGCAAT

TAATACTTGATAAGAAGAGTATTGAGAAGGGCAACGGTTCATCATCTCATGGATCTGCACATGA

ACAAACACCAGAGTCAAACGACGTTGAAATTGAGGCTACTGCGCCAATTGATGACAATACAGAC

GATGATAACAAACCGAAGTTATCTGATGTAGAAAAGGATTAAAGATGCTAAGAGATAGTGATGA

TATTTCATAAATAATGTAATTCTATATATGTTAATTACCTTTTTTGCGAGGCATATTTATGGTGAA

GGATAAGTTTTGACCATCAAAGAAGGTTAATGTGGCTGTGGTTTCAGGGTCCATACCCGGGAGTT

ATGACAATTACAACAACAGAATTCTTTCTATATATGCACGAACTTGTAATATGGAAGAAATTATG

ACGTACAAACTATAAAGTAAATATTTTACGTAACACATGGTGCTGTTGTGCTTCTTTTTCAAGAG

AATACCAATGACGTATGACTAAGTTTAGGATTTAATGCAGGTGACGGACCCATCTTTCAAACGAT

TTATATCAGTGGCGTCCAAATTGTTAGGTTTTGTTGGTTCAGCAGGTTTCCTGTTGTGGGTCATAT

GACTTTGAACCAAATGGCCGGCTGCTAGGGCAGCACATAAGGATAATTCACCTGCCAAGACGGC

ACAGGCAACTATTCTTGCTAATTGACGTGCGTTGGTACCAGGAGCGGTAGCATGTGGGCCTCTTA

CACCTAATAAGTCCAACATGGCACCTTGTGGTTCTAGAACAGTACCACCACCGATGGTACCTACT

TCGATGGATGGCATGGATACGGAAATTCTCAAATCACCGTCCACTTCTTTCATCAATGTTATACA

GTTGGAACTTTCGACATTTTGTGCAGGATCTTGTCCTAATGCCAAGAAAACAGCTGTCACTAAAT

TAGCTGCATGTGCGTTAAATCCACCAACAGACCCAGCCATTGCAGATCCAACCAAATTCTTAGCA

ATGTTCAACTCAACCAATGCGGAAACATCACTTTTTTAACACTTTTCTGACAACATCACCAGGAAT

AGTAGCTTCTGCGACGACACTCTTACCACGACCTTCGATCCAGTTGATGGCAGCTGGTTTTTTGTC

GGTACAGTAGTTACCAGAAACGGAGACAACCTCCATATCTTCCCAGCCATACTCTTCTACCATTT

GCTTTAATGAGTATTCGACACCCTTAGAAATCATATTCATACCCATTGCGTCACCAGTAGTTGTTC

TAAATCTCATGAAGAGTAAATCTCCTGCTAGACAAGTTTGAATATGTTGCAGACGTGCAAATCTT

GATGTAGAGTTAAAAGCTTTTTTAATTGCGTTTTGTCCCTCTTCTGAGTCTAACCATATCTTACAG

GCACCAGATCTTTTCAAAGTTGGGAAACGGACTACTGGGCCTCTTGTCATACCATCCTTAGTTAA

AACAGTTGTTGCACCACCGCCAGCATTGATTGCCTTACAGCCACGCATGGCAGAAGCTACCAAA

CAACCCTCTGTAGTTGCCATTGGTATATGATAAGATGTACCATCGATAACCAAGGGGCCTATAAC

ACCAACGGGCAAAGGCATGTAACCTATAACATTTTCACAACAAGCGCCAAATACGCGGTCGTAG

TCATAATTTTTATATGGTAAACGATCAGATGCTAATACAGGAGCTTCTGCCAAAATTGAAAGAGC

CTTCCTACGTACCGCAACCGCTCTCGTAGTATCACCTAATTTTTTCTCCAAAGCGTACAAAGGTA

ACTTACCGTGAATAACCAAGGCAGCGACCTCTTTGTTCTTCAATTGTTTTGTATTTCCACTACTTA

ATAATGCTTCTAATTCTTCTAAAGGACGTATTTTCTTATCCAAGCTTTCAATATCGCGGGAATCAT

CTTCCTCACTAGATGATGAAGGTCCTGATGAGCTCGATTGCGCAGATGATAAACTTTTGACTTTC

GATCCAGAAATGACTGTTTTATTGGTTAAAACTGGTGTAGAAGCCTTTTGTACAGGAGCAGTAAA

AGACTTCTTGGTGACTTCAGTCTTCACCAATTGGTCTGCAGCCATTATAGTTTTTTCTCCTTGACG

TTAAAGTATAGAGGTATATTAACAATTTTTTGTTGATACTTTTATGACATTTGAATAAGAAGTAAT

ACAAACCGAAAATGTTGAAAGTATTAGTTAAAGTGGTTATGCAGCTTTTGCATTTATATATCTGT

TAATAGATCAAAAATCATCGCTTCGCTGATTAATTACCCCAGAAATAAGGCTAAAAAACTAATC

GCATTATTATCCTATGGTTGTTAATTTGATTCGTTGATTTGAAGGTTTGTGGGGCCAGGTTACTGC

CAATTTTTCCTCTTCATAACCATAAAAGCTAGTATTGTAGAATCTTTATTGTTCGGAGCAGTGCGG

CGCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTGAAGACCAGGACGCACGGAGGAGAGTCT

TCCGTCGGAGGGCTGTCGCCCGCTCGGCGGCTTCTAATCCGTACTTCAATATAGCAATGAGCAGT

TAAGCGTATTACTGAAAGTTCCAAAGAGAAGGTTTTTTTAGGCTAAGATAATGGGGCTCTTTACA

TTTCCACAACATATAAGTAAGATTAGATATGGATATGTATATGGTGGTATTGCCATGTAATATGA

TTATTAAACTTCTTTGCGTCCATCCAAAAAAAAAGTAAGAATTTTTGAAAATTCAATATAAATGA

AACTCTCAACTAAACTTTGTTGGTGTGGTATTAAAGGAAGACTTAGGCCGCAAAAGCAACAACA

ATTACACAATACAAACTTGCAAATGACTGAACTAAAAAAACAAAAGACCGCTGAACAAAAAAC

CAGACCTCAAAATGTCGGTATTAAAGGTATCCAAATTTACATCCCAACTCAATGTGTCAACCAAT

CTGAGCTAGAGAAATTTGATGGCGTTTCTCAAGGTAAATACACAATTGGTCTGGGCCAAACCAA

CATGTCTTTTGTCAATGACAGAGAAGATATCTACTCGATGTCCCTAACTGTTTTGTCTAAGTTGAT

CAAGAGTTACAACATCGACACCAACAAAATTGGTAGATTAGAAGTCGGTACTGAAACTCTGATT

GACAAGTCCAAGTCTGTCAAGTCTGTCTTGATGCAATTGTTTGGTGAAAACACTGACGTCGAAGG

TATTGACACGCTTAATGCCTGTTACGGTGGTACCAACGCGTTGTTCAACTCTTTGAACTGGATTG

AATCTAACGCATGGGATGGTAGAGACGCCATTGTAGTTTGCGGTGATATTGCCATCTACGATAAG

GGTGCCGCAAGACCAACCGGTGGTGCCGGTACTGTTGCTATGTGGATCGGTCCTGATGCTCCAAT

TGTATTTGACTCTGTAAGAGCTTCTTACATGGAACACGCCTACGATTTTTACAAGCCAGATTTCAC

CAGCGAATATCCTTACGTCGATGGTCATTTTTCATTAACTTGTTACGTCAAGGCTCTTGATCAAGT

TTACAAGAGTTATTCCAAGAAGGCTATTTCTAAAGGGTTGGTTAGCGATCCCGCTGGTTCGGATG

CTTTGAACGTTTTGAAATATTTCGACTACAACGTTTTCCATGTTCCAACCTGTAAATTGGTCACAA

AATCATACGGTAGATTACTATATAACGATTTCAGAGCCAATCCTCAATTGTTCCCAGAAGTTGAC

GCCGAATTAGCTACTCGCGATTATGACGAATCTTTAACCGATAAGAACATTGAAAAAACTTTTGT

TAATGTTGCTAAGCCATTCCACAAAGAGAGAGTTGCCCAATCTTTGATTGTTCCAACAAACACAG

GTAACATGTACACCGCATCTGTTTATGCCGCCTTTGCATCTCTATTAAACTATGTTGGATCTGACG

ACTTACAAGGCAAGCGTGTTGGTTTATTTTCTTACGGTTCCGGTTTAGCTGCATCTCTATATTCTT

GCAAAATTGTTGGTGACGTCCAACATATTATCAAGGAATTAGATATTACTAACAAATTAGCCAAG

AGAATCACCGAAACTCCAAAGGATTACGAAGCTGCCATCGAATTGAGAGAAAATGCCCATTTGA

AGAAGAACTTCAAACCTCAAGGTTCCATTGAGCATTTGCAAAGTGGTGTTTACTACTTGACCAAC

ATCGATGACAAATTTAGAAGATCTTACGATGTTAAAAAATAATCTTCCCCCATCGATTGCATCTT

GCTGAACCCCCTTCATAAATGCTTTATTTTTTGGCAGCCTGCTTTTTTAGCTCTCATTTAATAGA

GTAGTTTTTTAATCTATATACTAGGAAAACTCTTTATTTAATAACAATGATATATATATACCCGGG

AAGCTTTTCAATTCATCTTTTTTTTTTTTGTTCTTTTTTTTGATTCCGGTTTCTTTGAAATTTTTTTG

ATTCGGTAATCTCCGAGCAGAAGGAAGAACGAAGGAAGGAGCACAGACTTAGATTGGTATATAT

ACGCATATGTGGTGTTGAAGAAACATGAAATTGCCCAGTATTCTTAACCCAACTGCACAGAACA

AAAACCTGCAGGAAACGAAGATAAATCATGTCGAAAGCTACATATAAGGAACGTGCTGCTACTC

ATCCTAGTCCTGTTGCTGCCAAGCTATTTAATATCATGCACGAAAAGCAAACAAACTTGTGTGCT

TCATTGGATGTTCGTACCACCAAGGAATTACTGGAGTTAGTTGAAGCATTAGGTCCCAAAATTTG

TTTACTAAAAACACATGTGGATATCTTGACTGATTTTTCCATGGAGGGCACAGTTAAGCCGCTAA

AGGCATTATCCGCCAAGTACAATTTTTTACTCTTCGAAGACAGAAAATTTGCTGACATTGGTAAT

ACAGTCAAATTGCAGTACTCTGCGGGTGTATACAGAATAGCAGAATGGGCAGACATTACGAATG

CACACGGTGTGGTGGGCCCAGGTATTGTTAGCGGTTTGAAGCAGGCGGCGGAAGAAGTAACAAA

GGAACCTAGAGGCCTTTTGATGTTAGCAGAATTGTCATGCAAGGGCTCCCTAGCTACTGGAGAAT

ATACTAAGGGTACTGTTGACATTGCGAAGAGCGACAAAGATTTTGTTATCGGCTTTATTGCTCAA

AGAGACATGGGTGGAAGAGATGAAGGTTACGATTGGTTGATTATGACACCCGGTGTGGGTTTAG

ATGACAAGGGAGACGCATTGGGTCAACAGTATAGAACCGTGGATGATGTGGTCTCTACAGGATC

TGACATTATTATTGTTGGGTTTAAAC

SEQ ID NO：5

The TDTI-P of pAM493 _gAL-tHMGR inset (5 ' to 3 ')

GTTTAAACTACTCAGTATATTAAGTTTCGAATTGAAGGGCGAACTCTTATTCGAAGTCGGAGTCA

CCACAACACTTCCGCCCATACTCTCCGAATCCTCGTTTCCTAAAGTAAGTTTACTTCCACTTGTAG

GCCTATTATTAATGATATCTGAATAATCCTCTATTAGGGTTGGATCATTCAGTAGCGCGTGCGATT

GAAAGGAGTCCATGCCCGACGTCGACGTGATTAGCGAAGGCGCGTAACCATTGTCATGTCTAGC

AGCTATAGAACTAACCTCCTTGACACCACTTGCGGAAGTCTCATCAACATGCTCTTCCTTATTACT

CATTCTCTTACCAAGCAGAGAATGTTATCTAAAAACTACGTGTATTTCACCTCTTTCTCGACTTGA

ACACGTCCAACTCCTTAAGTACTACCACAGCCAGGAAAGAATGGATCCAGTTCTACACGATAGC

AAAGCAGAAAACACAACCAGCGTACCCCTGTAGAAGCTTCTTTGTTTTACAGCACTTGATCCATGT

AGCCATACTCGAAATTTCAACTCATCTGAAACTTTTCCTGAAGGTTGAAAAAGAATGCCATAAGG

GTCACCCGAAGCTTATTCACGCCCGGGAGTTATGACAATTACAACAACAGAATTCTTTCTATATA

TGCACGAACTTGTAATATGGAAGAAATTATGACGTACAAACTATAAAGTAAATATTTTACGTAAC

ACATGGTGCTGTTGTGCTTCTTTTTCAAGAGAATACCAATGACGTATGACTAAGTTTAGGATTTA

ATGCAGGTGACGGACCCATCTTTCAAACGATTTATATCAGTGGCGTCCAAATTGTTAGGTTTTGT

TGGTTCAGCAGGTTTCCTGTTGTGGGTCATATGACTTTGAACCAAATGGCCGGCTGCTAGGGCAG

CACATAAGGATAATTCACCTGCCAAGACGGCACAGGCAACTATTCTTGCTAATTGACGTGCGTTG

GTACCAGGAGCGGTAGCATGTGGGCCTCTTACACCTAATAAGTCCAACATGGCACCTTGTGGTTC

TAGAACAGTACCACCACCGATGGTACCTACTTCGATGGATGGCATGGATACGGAAATTCTCAAA

TCACCGTCCACTTCTTTCATCAATGTTATACAGTTGGAACTTTCGACATTTTGTGCAGGATCTTGT

CCTAATGCCAAGAAAACAGCTGTCACTAAATTAGCTGCATGTGCGTTAAATCCACCAACAGACC

CAGCCATTGCAGATCCAACCAAATTCTTAGCAATGTTCAACTCAACCAATGCGGAAACATCACTT

TTTAACACTTTTCTGACAACATCACCAGGAATAGTAGCTTCTGCGACGACACTCTTACCACGACC

TTCGATCCAGTTGATGGCAGCTGGTTTTTTGTCGGTACAGTAGTTACCAGAAACGGAGACAACCT

CCATATCTTCCCAGCCATACTCTTCTACCATTTGCTTTAATGAGTATTCGACACCCTTAGAAATCA

TATTCATACCCATTGCGTCACCAGTAGTTGTTCTAAATCTCATGAAGAGTAAATCTCCTGCTAGA

CAAGTTTGAATATGTTGCAGACGTGCAAATCTTGATGTAGAGTTAAAAGCTTTTTTAATTGCGTTT

TGTCCCTCTTCTGAGTCTAACCATATCTTACAGGCACCAGATCTTTTCAAAGTTGGGAAACGGAC

TACTGGGCCTCTTGTCATACCATCCTTAGTTAAAACAGTTGTTGCACCACCGCCAGCATTGATTGC

CTTACAGCCACGCATGGCAGAAGCTACCAAACAACCCTCTGTAGTTTGCCATTGGTATATGATAAG

ATGTACCATCGATAACCAAGGGGCCTATAACACCAACGGGCAAAGGCATGTAACCTATAACATT

TTCACAACAAGCGCCAAATACGCGGTCGTAGTCATAATTTTTATATGGTAAACGATCAGATGCTA

ATACAGGAGCTTCTGCCAAAATTGAAAGAGCCTTCCTACGTACCGCAACCGCTCTCGTAGTATCA

CCTAATTTTTTCTCCAAAGCGTACAAAGGTAACTTACCGTGAATAACCAAGGCAGCGACCTCTTT

GTTCTTCAATTGTTTTGTATTTCCACTACTTAATAATGCTTCTAATTCTTCTAAAGGACGTATTTTC

TTATCCAAGCTTTCAATATCGCGGGAATCATCTTCCTCACTAGATGATGAAGGTCCTGATGAGCT

CGATTGCGCAGATGATAAACTTTTGACTTTCGATCCAGAAATGACTGTTTTATTGGTTAAAACTG

GTGTAGAAGCCTTTTGTACAGGAGCAGTAAAAGACTTCTTGGTGACTTCAGTTTTCACCAATTGG

TCTGCAGCCATTATAGTTTTTTCTCCTTGACGTTAAAGTATAGAGGTATATTAACAATTTTTTGTT

GATACTTTTATGACATTTGAATAAGAAGTAATACAAACCGAAAATGTTGAAAGTATTTAGTTAAA

GTGGTTATGCAGCTTTTGCATTTATATATCTGTTAATAGATCAAAAATCATCGCTTCGCTGATTAA

TTACCCCAGAAATAAGGCTAAAAAACTAATCGCATTATTATCCTATGGTTGTTAATTTGATTCGTT

GATTTGAAGGTTTGTGGGGCCAGGTTACTGCCAATTTTTCCTCTTCATAACCATAAAAGCTAGTA

TTGTAGAATCTTTATTGTTCGGAGCAGTGCGGCGCGAGGCACATCTGCGTTTCAGGAACGCGACC

GGTGAAGACCAGGACGCACGGAGGAGAGTCTTCCGTCGGAGGGCTGTCGCCCGCTCGGCGGCTT

CTAATCCGTACTTCAATATAGCAATGAGCAGTTAAGCGTATTACTGAAAGTTCCAAAGAGAAGG

TTTTTTTAGGCTAAGATAATGGGGCTCTTTACATTTCCACAACATATAAGTAAGATTAGATATGG

ATATGTATATGGTGGTATTGCCATGTAATATGATTATTAAACTTCTTTGCGTCCATCCAAAAAAA

AAGTAAGAATTTTTGAAAATTCAATATAAATGACTGCCGACAACAATAGTATGCCCCATGGTGC

AGTATCTAGTTACGCCAAATTAGTGCAAAACCAAACACCTGAAGACATTTTGGAAGAGTTTCCTG

AAATTATTCCATTACAACAAAGACCTAATACCCGATCTAGTGAGACGTCAAATGACGAAAGCGG

AGAAACATGTTTTTCTGGTCATGATGAGGAGCAAATTAAGTTAATGAATGAAAATTGTATTGTTT

TGGATTGGGACGATAATGCTATTGGTGCCGGTACCAAGAAAGTTTGTCATTTAATGGAAAATATT

GAAAAGGGTTTACTACATCGTGCATTCTCCGTCTTTATTTTCAATGAACAAGGTGAATTACTTTTA

CAACAAAGAGCCACTGAAAAAATAACTTTCCCTGATCTTTGGACTAACACATGCTGCTCTCATCC

ACTATGTATTGATGACGAATTAGGTTTGAAGGGTAAGCTAGACGATAAGATTAAGGGCGCTATT

ACTGCGGCGGTGAGAAAACTAGATCATGAATTAGGTATTCCAGAAGATGAAACTAAGACAAGGG

GTAAGTTTCACTTTTTAAACAGAATCCATTACATGGCACCAAGCAATGAACCATGGGGTGAACAT

GAAATTGATTACATCCTATTTTATAAGATCAACGCTAAAGAAAACTTGACTGTCAACCCAAACGT

CAATGAAGTTAGAGACTTCAAATGGGTTTCACCAAATGATTTGAAAACTATGTTTGCTGACCCAA

GTTACAAGTTTACGCCTTGGTTTAAGATTATTTGCGAGAATTACTTATTCAACTGGTGGGAGCAA

TTAGATGACCTTTCTGAAGTGGAAAATGACAGGCAAATTCATAGAATGCTATAACAACGCGTCA

ATAATATAGGCTACATAAAAATCATAATAACTTTGTTATCATAGCAAAATGTGATATAAAACGTT

TCATTTCACCTGAAAAATAGTAAAAATAGGCGACAAAAATCCTTAGTAATATGTAAACTTTATTT

TCTTTATTTACCCGGGAGTCAGTCTGACTCTTGCGAGAGATGAGGATGTAATAATACTAATCTCG

AAGATGCCATCTAATACATATAGACATACATATATATATATATACATTCTATATATTCTTACCCA

GATTCTTTGAGGTAAGACGGTTGGGTTTTATCTTTTGCAGTTGGTACTATTAAGAACAATCGAAT

CATAAGCATTGCTTACAAAGAATACACATACGAAATATTAACGATAATGTCAATTACGAAGACT

GAACTGGACGGTATATTGCCATTGGTGGCCAGAGGTAAAGTTAGAGACATATATGAGGTAGACG

CTGGTACGTTGCTGTTTGTTGCTACGGATCGTATCTCTGCATATGACGTTATTATGGAAAACAGC

ATTCCTGAAAAGGGGATCCTATTGACCAAACTGTCAGAGTTCTGGTTCAAGTTCCTGTCCAACGA

TGTTCGTAATCATTTGGTCGACATCGCCCCAGGTAAGACTATTTTCGATTATCTACCTGCAAAATT

GAGCGAACCAAAGTACAAAACGCAACTAGAAGACCGCTCTCTATTGGTTCACAAACATAAACTA

ATTCCATTGGAAGTAATTGTCAGAGGCTACATCACCGGATCTGCTTGGAAAGAGTACGTAAAAA

CAGGTACTGTGCATGGTTTGAAACAACCTCAAGGACTTAAAGAATCTCAAGAGTTCCCAGAACC

AATCTTCACCCCATCGACCAAGGCTGAACAAGGTGAACATGACGAAAACATCTCTCCTGCCCAG

GCCGCTGAGCTGGTGGGTGAAGATTTGTCACGTAGAGTGGCAGAACTGGCTTGTAAAACTGTACT

CCAAGTGCAAAGATTATGCTAAGGAGAAGGGCATCATCATCGCAGACACTAAATTGTTTAAAC

SEQ ID NO：6

The ERG10-P of pAM495 _gAL-EGR12 inset (5 ' to 3 ')

GTTTAAACTATTGTGAGGGTCAGTTATTTCATCCAGATATAACCCGAGAGGAAACTTCTTAGCGT

CTGTTTTCGTACCATAAGGCAGTTCATGAGGTATATTTTCGTTATTGAAGCCCAGCTCGTGAATGC

TTAATGCTGCTGAACTGGTGTCCATGTCGCCTAGGTACGCAATCTCCACAGGCTGCAAAGGTTTT

GTCTCAAGAGCAATGTTATTGTGCACCCCGTAATTGGTCAACAAGTTTAATCTGTGCTTGTCCAC

CAGCTCTGTCGTAACCTTCAGTTCATCGACTATCTGAAGAAATTTACTAGGAATAGTGCCATGGT

ACAGCAACCGAGAATGGCAATTTCTACTCGGGTTCAGCAACGCTGCATAAACGCTGTTGGTGCC

GTAGACATATTCGAAGATAGGATTATCATTCATAAGTTTCAGAGCAATGTCCTTATTCTGGAACT

TGGATTTATGGCTCTTTTGGTTTAATTTCGCCTGATTCTTGATCTCCTTTAGCTTCTCGACGTGGGC

CTTTTTCTTGCCATATGGATCCGCTGCACGGTCCTGTTCCCTAGCATGTACGTGAGCGTATTTCCT

TTTAAACCACGACGCTTTGTCTTCATTCAACGTTTCCCATTGTTTTTTTCTACTATTGCTTTGCTGT

GGGAAAAACTTATCGAAAGATGACGACTTTTTCTTAATTCTCGTTTTAAGAGCTTGGTGAGCGCT

AGGAGTCACTGCCAGGTATCGTTTGAACACGGCATTAGTCAGGGAAGTCATAACACAGTCCTTTC

CCGCAATTTTCTTTTTCTATTACTCTTGGCCTCCTCTAGTACACTCTATATTTTTTTATGCCTCGGT

AATGATTTTCATTTTTTTTTTTTCCACCTAGCGGATGACTCTTTTTTTTTCTTAGCGATTGGCATTA

TCACATAATGAATTATACATTATATAAAGTAATGTGATTTCTTCGAAGAATATACTAAAGTTTAG

CTTGCCTCGTCCCCGCCGGGTCACCCGGCCAGCGACATGGAGGCCCAGAATACCCTCCTTGACAG

TCTTGACGTGCGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTTAGCCCATACATCCCC

ATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGGCGCGAAGCAAAAATTACGGCTCC

TCGCTGCAGACCTGCGAGCAGGGAAACGCTCCCCTCACAGACGCGTTGAATTGTCCCCACGCCG

CGCCCCTGTAGAGAAATATAAAAGGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTT

TTAAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAACCATGGCAGAACCA

GCCCAAAAAAAGCAAAAACAAACTGTTCAGGAGCGCAAGGCGTTTATCTCCCGTATCACTAATG

AAACTAAAATTCAAATCGCTATTTCGCTGAATGGTGGTTATATTCAAATAAAAGATTCGATTCTT

CCTGCAAAGAAGGATGACGATGTAGCTTCCCAAGCTACTCAGTCACAGGTCATCGATATTCACAC

AGGTGTTGGCTTTTTGGATCATATGATCCATGCGTTGGCAAAACACTCTGGTTGGTCTCTTATTGT

TGAATGTATTGGTGACCTGCACATTGACGATCACCATACTACCGAAGATTGCGGTATCGCATTAG

GGCAAGCGTTCAAAGAAGCAATGGGTGCTGTCCGTGGTGTAAAAAGATTCGGTACTGGGTTCGC

ACCATTGGATGAGGCGCTATCACGTGCCGTAGTCGATTTATCTAGTAGACCATTTGCTGTAATCG

ACCTTGGATTGAAGAGAGAGATGATTGGTGATTTATCCACTGAAATGATTCCACACTTTTTGGAA

AGTTTCGCGGAGGCGGCCAGAATTACTTTGCATGTTGATTGTCTGAGAGGTTTCAACGATCACCA

CAGAAGTGAGAGTGCGTTCAAGGCTTTGGCTGTTGCCATAAGAGAAGCTATTTCTAGCAATGGC

ACCAATGACGTTCCCTCAACCAAAGGTGTTTTGATGTGAAGTACTGACAATAAAAAGATTCTTGT

TTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGC

GTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGT

AATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATACTGCTGTCGATTCGATACTAACGCC

GCCATCCACCCGGGATGGTCTGCTTAAATTTCATTCTGTCTTCGAAAGCTGAATTGATACTACGA

AAAATTTTTTTTTGTTTCTCTTTCTATCTTTATTACATAAAACTTCATACACAGTTAAGATTAAAA

ACAACTAATAAATAATGCCTATCGCAAATTAGCTTATGAAGTCCATGGTAAATTCGTGTTTCCTG

GCAATAATAGATCGTCAATTTGTTGCTTTGTGGTAGTTTTATTTTCAAATAATTGGAATACTAGGG

ATTTGATTTTAAGATCTTTATTCAAATTTTTTGCGCTTAACAAACAGCAGCCAGTCCCACCCAAGT

CTGTTTCAAATGTCTCGTAACTAAAATCATCTTGCAATTTCTTTTTGAAACTGTCAATTTGCTCTT

GAGTAATGTCTCTTCGTAACAAAGTCAAAGAGCAACCGCCGCCACCAGCACCGGTAAGTTTTGT

GGAGCCAATTCTCAAATCATCGCTCAGATTTTTAATAAGTTCTAATCCAGGATGAGAAACACCGA

TTGAGACAAGCAGTCCATGATTTATTCTTATCAATTCCAATAGTTGTTCATACAGTTCATTATTAG

TTTCTACAGCCTCGTCATCGGTGCCTTTACATTTACTTAACTTAGTCATGATCTCTAAGCCTTGTA

GGGCACATTCACCCATGGCATCTAGAATTGGCTTCATAACTTCAGGAAATTTCTCGGTGACCAAC

ACACGAACGCGAGCAACAAGATCTTTTGTAGACCTTGGAATTCTAGTATAGGTTAGGATCATTGG

AATGGCTGGGAAATCATCTAAGAACTTAAAATTGTTTGTGTTTATTGTTCCATTATGTGAGTCTTT

TTCAAATAGCAGGGCATTACCATAAGTGGCCACAGCGTTATCTATTCCTGAAGGGGTACCGTGAA

TACACTTTTCACCTATGAAGGCCCATTGATTCACTATATGCTTATCGTTTTCTGACAGCTTTTCCA

AGTCATTAGATCCTATTAACCCCCCCAAGTAGGCCATAGCTAAGGCCAGTGATACAGAAATAGA

GGCGCTTGAGCCCAACCCAGCACCGATGGGTAAAGTAGACTTTAAAGAAAACTTAATATTCTTG

GCATGGGGGCATAGGCAAACAAACATATACAGGAAACAAAACGCTGCATGGTAGTGGAAGGAT

TCGGATAGTTGAGCTAACAACGGATCCAAAAGACTAACGAGTTCCTGAGACAAGCCATCGGTGG

CTTGTTGAGCCTTGGCCAATTTTTGGGAGTTTACTTGATCCTCGGTGATGGCATTGAAATCATTGA

TGGACCACTTATGATTAAAGCTAATGTCCGGGAAGTCCAATTCAATAGTATCTGGTGCAGATGAC

TCGCTTATTAGCAGGTAGGTTCTCAACGCAGACACACTAGCAGCGACGGCAGGCTTGTTGTACAC

AGCAGAGTGTTCACCAAAAATAATAACCTTTCCCGGTGCAGAAGTTAAGAACGGTAATGACATT

ATAGTTTTTTCTCCTTGACGTTAAAGTATAGAGGTATATTAACAATTTTTTGTTGATACTTTTATG

ACATTTGAATAAGAAGTAATACAAACCGAAAATGTTGAAAGTATTAGTTAAAGTGGTTATGCAG

CTTTTGCATTTATATATCTGTTAATAGATCAAAAATCATCGCTTCGCTGATTAATTACCCCAGAAA

TAAGGCTAAAAAACTAATCGCATTATTATCCTATGGTTGTTAATTTGATTCGTTGATTTGAAGGTT

TGTGGGGCCAGGTTACTGCCAATTTTTCCTCTTCATAACCATAAAAGCTAGTATTGTAGAATCTTTT

ATTGTTCGGAGCAGTGCGGCGCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTGAAGACCAG

GACGCACGGAGGAGAGTCTTCCGTCGGAGGGCTGTCGCCCGCTCGGCGGCTTCTAATCCGTACTT

CAATATAGCAATGAGCAGTTAAGCGTATTACTGAAAGTTCCAAAGAGAAGGTTTTTTTAGGCTAA

GATAATGGGGCTCTTTACATTTCCACAACATATAAGTAAGATTAGATATGGATATGTATATGGTG

GTATTGCCATGTAATATGATTATTAAACTTCTTTGCGTCCATCCAAAAAAAAAGTAAGAATTTTT

GAAAATTCAATATAAATGTCTCAGAACGTTTACATTGTATCGACTGCCAGAACCCCAATTGGTTC

ATTCCAGGGTTCTCTATCCTCCAAGACAGCAGTGGAATTGGGTGCTGTTGCTTTAAAAGGCGCCT

TGGCTAAGGTTCCAGAATTGGATGCATCCAAGGATTTTGACGAAATTATTTTTGGTAACGTTCTTT

CTGCCAATTTGGGCCAAGCTCCGGCCAGACAAGTTGCTTTGGCTGCCGGTTTGAGTAATCATATC

GTTGCAAGCACAGTTAACAAGGTCTGTGCATCCGCTATGAAGGCAATCATTTTGGGTGCTCAATC

CATCAAATGTGGTAATGCTGATGTTGTCGTAGCTGGTGGTTGTGAATCTATGACTAACGCACCAT

ACTACATGCCAGCAGCCCGTGCGGGTGCCAAATTTGGCCAAACTGTTCTTGTTGATGGTGTCGAA

AGAGATGGGTTGAACGATGCGTACGATGGTCTAGCCATGGGTGTACACGCAGAAAAGTGTGCCC

GTGATTGGGATATTACTAGAGAACAACAAGACAATTTTGCCATCGAATCCTACCAAAAATCTCA

AAAATCTCAAAAGGAAGGTAAATTCGACAATGAAATTGTACCTGTTACCATTAAGGGATTTAGA

GGTAAGCCTGATACTCAAGTCACGAAGGACGAGGAACCTGCTAGATTACACGTTGAAAAATTGA

GATCTGCAAGGACTGTTTTCCAAAAAGAAAACGGTACTGTTACTGCCGCTAACGCTTCTCCAATC

AACGATGGTGCTGCAGCCGTCATCTTGGTTTCCGAAAAAGTTTTGAAGGAAAAGAATTTGAAGC

CTTTGGCTATTATCAAAGGTTGGGGTGAGGCCGCTCATCAACCAGCTGATTTTACATGGGCTCCA

TCTCTTGCAGTTCCAAAGGCTTTGAAACATGCTGGCATCGAAGACATCAATTCTGTTGATTACTTT

GAATTCAATGAAGCCTTTTCGGTTGTCGGTTTGGTGAACACTAAGATTTTGAAGCTAGACCCATC

TAAGGTTAATGTATATGGTGGTGCTGTTGCTCTAGGTCACCCATTGGGTTGTTCTGGTGCTAGAGT

GGTTGTTACACTGCTATCCATCTTACAGCAAGAAGGAGGTAAGATCGGTGTTGCCGCCATTTGTA

ATGGTGGTGGTGGTGCTTCCTCTATTGTCATTGAAAAGATATGATTACGTTCTGCGATTTTCTCAT

GATCTTTTTCATAAAATACATAAATATATAAATGGCTTTATGTATAACAGGCATAATTTAAAGTT

TTATTTGCGATTCATCGTTTTTCAGGTACTCAAACGCTGAGGTGTGCCTTTTGACTTACTTTTCCC

GGGAGAGGCTAGCAGAATTACCCTCCACGTTGATTGTCTGCGAGGCAAGAATGATCATCACCGT

AGTGAGAGTGCGTTCAAGGCTCTTGCGGTTGCCATAAGAGAAGCCACCTCGCCCAATGGTACCA

ACGATGTTCCCTCCACCAAAGGTGTTCTTATGTAGTGACACCGATTATTTAAAGCTGCAGCATAC

GATATATATACATGTGTATATATGTATACCTATGAATGTCAGTAAGTATGTATACGAACAGTATG

ATACTGAAGATGACAAGGTAATGCATCATTCTATACGTGTCATTCTGAACGAGGCGCGCTTTCCT

TTTTTCTTTTTGCTTTTTCTTTTTTTTTCTCTTGAACTCGAGAAAAAAAATATAAAAGAGATGGAG

GAACGGGAAAAAGTTAGTTGTGGTGATAGGTGGCAAGTGGTATTCCGTAAGAACAACAAGAAA

AGCATTTCATATTATGGCTGAACTGAGCGA ACAAGTGCAAAATTTAAGCATCAACGACAACAAC

GAGAATGGTTATGTTCCTCCTCACTTAAGAGGAAAACCAAGAAGTGCCAGAAATAACAGTAGCA

ACTACAATAACAACAACGGCGGCGTTTAAAC

SEQ ID NO：7

The ERG8-P of pAM497 _gAL-EGR19 inset (5 ' to 3 ')

GTTTAAACTTTTCCAATAGGTGGTTAGCAATCGTCTTACTTTCTAACTTTTCTTACCTTTTACATTT

CAGCAATATATATATATATATTTCAAGGATATACCATTCTAATGTCTGCCCCTAAGAAGATCGTC

GTTTTGCCAGGTGACCACGTTGGTCAAGAAATCACAGCCGAAGCCATTAAGGTTCTTAAAGCTAT

TTCTGATGTTCGTTCCAATGTCAAGTTCGATTTCGAAAATCATTTAATTGGTGGTGCTGCTATCGA

TGCTACAGGTGTTCCACTTCCAGATGAGGCGCTGGAAGCCTCCAAGAAGGCTGATGCCGTTTTGT

TAGGTGCTGTGGGTGGTCCTAAATGGGGTACCGGTAGTGTTAGACCTGAACAAGGTTTACTAAA

AATCCGTAAAGAACTTCAATTGTACGCCAACTTAAGACCATGTAACTTTGCATCCGACTCTCTTTT

AGACTTATCTCCAATCAAGCCACAATTTGCTAAAGGTACTGACTTCGTTGTTGTCAGAGAATTAG

TGGGAGGTATTTACTTTGGTAAGAGAAAGGAAGACGTTTAGCTTGCCTCGTCCCCGCCGGGTCAC

CCGGCCAGCGACATGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTGCGCAGCTCAGGGG

CATGATGTGACTGTCGCCCGTACATTTAGCCCATACATCCCCATGTATAATCATTTGCATCCATAC

ATTTTGATGGCCGCACGGCGCGAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAGCAGGG

AAACGCTCCCCTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAATATAAAA

GGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGCTAGGATACAGT

TCTCACATCACATCCGAACATAAACAACCATGGCAGAACCAGCCCAAAAAAAGCAAAAACAAA

CTGTTCAGGAGCGCAAGGCGTTTATCTCCCGTATCACTAATGAAACTAAAATTCAAATCGCTATT

TCGCTGAATGGTGGTTATATTCAAATAAAAGATTCGATTCTTCCTGCAAAGAAGGATGACGATGT

AGCTTCCCAAGCTACTCAGTCACAGGTCATCGATATTCACACAGGTGTTGGCTTTTTGGATCATA

TGATCCATGCGTTGGCAAAACACTCTGGTTGGTCTCTTATTGTTGAATGTATTGGTGACCTGCACA

TTGACGATCACCATACTACCGAAGATTGCGGTATCGCATTAGGGCAAGCGTTCAAAGAAGCAAT

GGGTGCTGTCCGTGGTGTAAAAAGATTCGGTACTGGGTTCGCACCATTGGATGAGGCGCTATCAC

GTGCCGTAGTCGATTTATCTAGTAGACCATTTGCTGTAATCGACCTTGGATTGAAGAGAGAGATG

ATTGGTGATTTATCCACTGAAATGATTCCACACTTTTTGGAAAGTTTCGCGGAGGCGGCCAGAAT

TACTTTGCATGTTGATTGTCTGAGAGGTTTCAACGATCACCACAGAAGTGAGAGTGCGTTCAAGG

CTTTGGCTGTTGCCATAAGAGAAGCTATTTCTAGCAATGGCACCAATGACGTTCCCTCAACCAAA

GGTGTTTTGATGTGAAGTACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTGTATA

GTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGATTTATATTTTTTTTCGCCTC

GACATCATCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGT

GAATGCTGGTCGCTATACTGCTGTCGATTCGATACTAACGCCGCCATCCACCCGGGTTTCTCATTC

AAGTGGTAACTGCTGTTAAAATTAAGATATTTATAAATTGAAGCTTGGTCGTTCCGACCAATACC

GTAGGGAAACGTAAATTAGCTATTGTAAAAAAAGGAAAAGAAAAGAAAAGAAAAATGTTACAT

ATCGAATTGATCTTATTCCTTTGGTAGACCAGTCTTTGCGTCAATCAAAGATTCGTTTGTTTCTTG

TGGGCCTGAACCGACTTGAGTTAAAATCACTCTGGCAACATCCTTTTGCAACTCAAGATCCAATT

CACGTGCAGTAAAGTTAGATGATTCAAATTGATGGTTGAAAGCCTCAAGCTGCTCAGTAGTAAAT

TTCTTGTCCCATCCAGGAACAGAGCCAAACAATTTATAGATAAATGCAAAGAGTTTCGACTCATT

TTCAGCTAAGTAGTACAACACAGCATTTGGACCTGCATCAAACGTGTATGCAACGATTGTTTCTC

CGTAAAACTGATTAATGGTGTGGCACCAACTGATGATACGCTTGGAAGTGTCATTCATGTAGAAT

ATTGGAGGGAAAGAGTCCAAACATGTGGCATGGAAAGAGTTGGAATCCATCATTGTTTCCTTTGC

AAAGGTGGCGAAATCTTTTTCAACAATGGCTTTACGCATGACTTCAAATCTCTTTGGTACGACAT

GTTCAATTCTTTCTTTAAATAGTTCGGAGGTTGCCACGGTCAATTGCATACCCTGAGTGGAACTC

ACATCCTTTTTAATATCGCTGACAACTAGGACACAAGCTTTCATCTGAGGCCAGTCAGAGCTGTC

TGCGATTTGTACTGCCATGGAATCATGACCATCTTCAGCTTTTCCCATTTCCCAGGCCACGTATCC

GCCAAACAACGATCTACAAGCTGAACCAGACCCCTTTCTTGCTATTCTAGATATTTCTGAAGTTG

ACTGTGGTAATTGGTATAACTTAGCAATTGCAGAGACCAATGCAGCAAAGCCAGCAGCGGAGGA

AGCTAAACCAGCTGCTGTAGGAAAGTTATTTTCGGAGACAATGTGGAGTTTCCATTGAGATAATG

TGGGCAATGAGGCGTCCTTCGATTCCATTTCCTTTCTTAATTGGCGTAGGTCGCGCAGACAATTTT

GAGTTCTTTCATTGTCGATGCTGTGTGGTTCTCCATTTAACCACAAAGTGTCGCGTTCAAACTCAG

GTGCAGTAGCCGCAGAGGTCAACGTTCTGAGGTCATCTTGCGATAAAGTCACTGATATGGACGA

ATTGGTGGGCAGATTCAACTTCGTGTCCCTTTTCCCCCAATACTTAAGGGTTGCGATGTTGACGG

GTGCGGTAACGGATGCTGTGTAAACGGTCATTATAGTTTTTTCTCCTTGACGTTAAAGTATAGAG

GTATATTAACAATTTTTTGTTGATACTTTTATGACATTTGAATAAGAAGTAATACAAACCGAAAA

TGTTGAAAGTATTAGTTAAAGTGGTTATGCAGCTTTTGCATTTATATATCTGTTAATAGATCAAAA

ATCATCGCTTCGCTGATTAATTACCCCAGAAATAAGGCTAAAAAACTAATCGCATTATTATCCTA

TGGTTGTTAATTTGATTCGTTGATTTGAAGGTTTGTGGGGCCAGGTTACTGCCAATTTTTCCTCTT

CATAACCATAAAAGCTAGTATTGTAGAATCTTTATTGTTCGGAGCAGTGCGGCGCGAGGCACATC

TGCGTTTCAGGAACGCGACCGGTGAAGACCAGGACGCACGGAGGAGAGTCTTCCGTCGGAGGGC

TGTCGCCCGCTCGGCGGCTTCTAATCCGTACTTCAATATAGCAATGAGCAGTTAAGCGTATTACT

GAAAGTTCCAAAGAGAAGGTTTTTTTTAGGCTAAGATAATGGGGCTCTTTACATTTCCACAACATA

TAAGTAAGATTAGATATGGATATGTATATGGTGGTATTGCCATGTAATATGATTATTAAACTTCT

TTGCGTCCATCCAAAAAAAAAGTAAGAATTTTTGAAAATTCAATATAAATGTCAGAGTTGAGAG

CCTTCAGTGCCCCAGGGAAAGCGTTACTAGCTGGTGGATATTTAGTTTTAGATCCGAAATATGAA

GCATTTGTAGTCGGATTATCGGCAAGAATGCATGCTGTAGCCCATCCTTACGGTTCATTGCAAGA

GTCTGATAAGTTTGAAGTGCGTGTGAAAAGTAAACAATTTAAAGATGGGGAGTGGCTGTACCAT

ATAAGTCCTAAAACTGGCTTCATTCCTGTTTCGATAGGCGGATCTAAGAACCCTTTCATTGAAAA

AGTTATCGCTAACGTATTTAGCTACTTTAAGCCTAACATGGACGACTACTGCAATAGAAACTTGT

TCGTTATTGATATTTTCTCTGATGATGCCTACCATTCTCAGGAGGACAGCGTTACCGAACATCGTG

GCAACAGAAGATTGAGTTTTCATTCGCACAGAATTGAAGAAGTTCCCAAAACAGGGCTGGGCTC

CTCGGCAGGTTTAGTCACAGTTTTAACTACAGCTTTGGCCTCCTTTTTTGTATCGGACCTGGAAAA

TAATGTAGACAAATATAGAGAAGTTATTCATAATTTATCACAAGTTGCTCATTGTCAAGCTCAGG

GTAAAATTGGAAGCGGGTTTGATGTAGCGGCGGCAGCATATGGATCTATCAGATATAGAAGATT

CCCACCCGCATTAATCTCTAATTTGCCAGATATTGGAAGTGCTACTTACGGCAGTAAACTGGCGC

ATTTGGTTAATGAAGAAGACTGGAATATAACGATTAAAAGTAACCATTTACCTTCGGGATTAACT

TTATGGATGGGCGATATTAAGAATGGTTCAGAAACAGTAAAACTGGTCCAGAAGGTAAAAAATT

GGTATGATTCGCATATGCCGGAAAGCTTGAAAATATATACAGAACTCGATCATGCAAATTCTAG

ATTTATGGATGGACTATCTAAACTAGATCGCTTACACGAGACTCATGACGATTACAGCGATCAGA

TATTTGAGTCTCTTGAGAGGAATGACTGTACCTGTCAAAAGTATCCTGAGATCACAGAAGTTAGA

GATGCAGTTGCCACAATTAGACGTTCCTTTAGAAAAATAACTAAAGAATCTGGTGCCGATATCGA

ACCTCCCGTACAAACTAGCTTATTGGATGATTGCCAGACCTTAAAAGGAGTTCTTACTTGCTTAA

TACCTGGTGCTGGTGGTTATGACGCCATTGCAGTGATTGCTAAGCAAGATGTTGATCTTAGGGCT

CAAACCGCTGATGACAAAAGATTTTCTAAGGTTCAATGGCTGGATGTAACTCAGGCTGACTGGG

GTGTTAGGAAAGAAAAAGATCCGGAAACTTATCTTGATAAATAACTTAAGGTAGATAATAGTGG

TCCATGTGACATCTTTATAAATGTGAAGTTTGAAGTGACCGCGCTTAACATCTAACCATTCATCTT

CCGATAGTACTTGAAATTGTTCCTTTCGGCGGCATGATAAAATTCTTTTAATGGGTACAAGCTAC

CCGGGCCCGGGAAAGATTCTCTTTTTTTATGATATTTGTACATAAACTTTATAAATGAAATTCATA

ATAGAAACGACACGAAATTACAAAATGGAATATGTTCATAGGGTAGACGAAACTATATACGCAA

TCTACATACATTTATCAAGAAGGAGAAAAAGGAGGATGTAAAGGAATACAGGTAAGCAAATTG

ATACTAATGGCTCAACGTGATAAGGAAAAAGAATTGCACTTTAACATTAATATTGACAAGGAGG

AGGGCACCACACAAAAAGTTAGGTGTAACAGAAAATCATGAAACTATGATTCCTAATTTATATA

TTGGAGGATTTTCTCTAAAAAAAAAAAAATACAACAAATAAAAAACACTCAATGACCTGACCAT

TTGATGGAGTTTAAGTCAATACCTTCTTGAACCATTTCCCATAATGGTGAAAGTTCCCTCAAGAA

TTTTACTCTGTCAGAAACGGCCTTAACGACGTAGTCGACCTCCTCTTCAGTACTAAATCTACCAAT

ACCAAATCTGATGGAAGAATGGGCTAATGCATCATCCTTACCCAGCGCATGTAAAACATAAGAA

GGTTCTAGGGAAGCAGATGTACAGGCTGAACCCGAGGATAATGCGATATCCCTTAGTGCCATCA

ATAAAGATTCTCCTTCCACGTAGGCGAAAGAAACGTTAACACGTTTAAAC

SEQ ID NO：8

For carrying out codon optimized Artemisinin β-farnesene synthetic enzyme (5 ' to 3 ') at expression in escherichia coli

ATGGACACTCTGCCGATCTCTTCCGTAAGCTTTTCTTCCTCTACTTCCCCGCTGGTAGTCGATGAC

AAGGTTTCTACCAAACCTGATGTAATTCGTCACACTATGAACTTCAACGCATCTATCTGGGGCGA

TCAGTTCCTGACTTATGATGAACCGGAAGATCTGGTAATGAAAAAGCAACTGGTAGAAGAACTG

AAAGAAGAAGTTAAAAAGGAACTGATCACCATTAAGGGTAGCAACGAACCGATGCAGCACGTG

AAACTGATTGAACTGATCGATGCGGTTCAGCGTCTGGGTATTGCTTATCATTTTGAAGAGGAAAT

CGAGGAAGCTCTGCAACACATCCACGTAACCTACGGCGAACAATGGGTGGATAAAGAGAATCTG

CAGTCTATCAGCCTGTGGTTCCGCCTGCTGCGTCAGCAAGGTTTTCAATGTCTCTTCTGGCGTTTTC

AAAGACTTCATGGATGAAAAGGGCAAATTCAAGGAATCCCTGTGTAACGATGCGCAAGGTATCC

TGGCACTGTACGAAGCGGCCTTCATGCGTGTGGAAGACGAAACCATTCTGGACAACGCGCTGGA

ATTCACTAAAGTGCATCTGGACATCATCGCGAAAGATCCGTCCTGCGACTCCTCTCTGCGTACTC

AGATCCATCAAGCGCTGAAACAGCCGCTGCGTCGTCGCCTGGCACGTATTGAGGCTCTGCACTAT

ATGCCGATTTACCAGCAGGAAACCTCTCACGACGAAGTCCTGCTGAAACTGGCTAAACTGGACTT

CAGCGTTCTGCAATCTATGCACAAGAAAGAACTGTCCCACATCTGCAAATGGTGGAAAGATCTG

GATCTGCAAAACAAACTGCCGTACGTTCGTGACCGTGTTGTTGAGGGCTATTTTTGGATTCTGTC

CATCTACTATGAACCACAGCACGCGCGTACTCGCATGTTTCTGATGAAAACCTGCATGTGGCTGG

TTGTCCTGGACGACACCTTTGACAACTATGGTACGTACGAAGAACTGGAAATCTTCACCCAGGCC

GTGGAACGTTGGTCTATTTCCTGCCTGGATATGCTGCCGGAATACATGAAACTGATCTATCAAGA

ACTGGTTAACCTGCACGTGGAAATGGAAGAGTCTCTGGAGAAAGAAGGTAAAACTTACCAGATC

CACTACGTCAAGGAGATGGCGAAAGAACTGGTCCGTAACTATCTGGTCGAGGCGCGTTGGCTGA

AAGAGGGCTATATGCCGACTCTGGAAGAATACATGAGCGTATCCATGGTTACCGGCACCTACGG

CCTGATGATTGCGCGTTCCTACGTCGGCCGTGGTGATATTGTTACCGAAGATACCTTTAAGTGGG

TTTCTTCCTACCCGCCGATCATCAAAGCGTCTTGTGTCATCGTTCGCCTGATGGACGACATCGTTT

CTCACAAAGAGGAGCAAGAACGTGGTCACGTAGCATCTAGCATCGAATGCTACTCCAAAGAATC

CGGCGCGTCCGAAGAAGAAGCTTGCGAATACATCAGCCGTAAAGTTGAAGATGCCTGGAAAGTT

ATCAACCGCGAAAGCCTGCGTCCGACGGCGGTCCCGTTTCCGCTGCTGATGCCGGCAATCAACCT

GGCACGCATGTGTGAGGTTCTGTACAGCGTGAACGATGGTTTTACTCACGCGGAAGGTGACATG

AAGAGCTATATGAAGAGCTTCTTCGTACACCCTATGGTCGTATGA

SEQ ID NO：9

For carrying out codon optimized Picea excelsa α-farnesene synthetic enzyme (5 ' to 3 ') at expression in escherichia coli

ATGGACCTGGCAGTAGAAATTGCAATGGATCTGGCTGTTGATGATGTTGAACGTCGTGTGGGTGA

TTACCACTCTAACCTGTGGGACGACGACTTTATTCAGAGCCTGTCCACCCCGTACGGCGCCTCTT

CTTACCGCGAACGCGCTGAACGTCTGGTTGGTGAAGTTAAAGAAATGTTCACCTCTATTAGCATC

GAAGACGGCGAACTGACCTCTGACCTGCTGCAGCGTCTGTGGATGGTGGATAATGTAGAACGCC

TGGGTATCTCTCGTCACTTCGAAAACGAAATCAAAGCCGCGATCGATTACGTTTATTCTTACTGG

TCTGATAAAGGCATTGTTCGTGGTCGTGATAGCGCGGTTCCGGACCTGAATAGCATCGCGCTGGG

TTTCCGTACCCTGCGCCTGCATGGTTACACTGTTTCTAGCGATGTTTTTAAAGTTTTTCAGGACCG

TAAAGGCGAATTTGCTTGTTCTGCGATCCCGACCGAGGGTGATATTAAGGGCGTTCTGAACCTGC

TGCGTGCATCCTATATTGCATTCCCGGGTGAAAAAGTGATGGAGAAAGCACAGACCTTCGCTGC

AACCTACCTGAAAGAAGCACTGCAGAAAATCCAGGTTTCCAGCCTGAGCCGTGAGATCGAATAT

GTGCTGGAATATGGTTGGCTGACTAACTTCCCGCGTCTGGAAGCTCGTAACTATATCGATGTTTT

CGGTGAAGAAATCTGTCCGTACTTCAAAAAGCCGTGCATTATGGTTGATAAACTGCTGGAACTGG

CAAAACTGGAATTCAACCTGTTCCACTCTCTGCAGCAGACCGAACTGAAACATGTAAGCCGCTG

GTGGAAAGATTCTGGTTTTTCCCAGCTGACTTTCACCCGTCACCGTCACGTGGAATTCTACACCCT

GGCATCTTGTATCGCTATTGAGCCGAAACATTCCGCATTCCGTCTGGGCTTCGCGAAAGTGTGCT

ATCTGGGTATTGTGCTGGATGATATCTACGATACTTTTGGCAAGATGAAAGAACTGGAACTGTTT

ACCGCTGCTATTAAACGCTGGGACCCGTCTACTACTGAGTGCCTGCCGGAATATATGAAAGGCGT

TTATATGGCCTTCTATAACTGTGTAAATGAACTGGCCCTGCAGGCGGAGAAGACCCAGGGTCGTG

ACATGCTGAACTACGCGCGCAAAGCCTGGGAAGCACTGTTCGACGCGTTCCTGGAAGAAGCAAA

ATGGATTAGCTCCGGTTACCTGCCGACCTTCGAAGAATACCTGGAAAACGGCAAGGTATCTTTCG

GTTACCGTGCGGCCACTCTGCAGCCAATTCTGACTCTGGATATTCCGCTGCCGCTGCACATCCTG

CAGCAAATCGACTTCCCGTCTCGTTTTAACGATCTGGCTAGCAGCATCCTGCGTCTGCGTGGTGA

CATCTGCGGCTACCAGGCTGAACGTTCTCGTGGTGAAGAAGCCTCTTCCATCTCTTGCTATATGA

AAGATAATCCGGGCTCTACCGAAGAGGACGCGCTGTCTCACATTAACGCCATGATCTCTGATAAC

ATTAACGAGCTGAATTGGGAACTGCTGAAACCGAACTCTAATGTACCGATCTCCTCTAAGAAAC

ACGCGTTCGATATCCTGCGTGCGTTCTACCACCTGTACAAGTATCGTGACGGTTTCTCTATCGCTA

AGATCGAAACTAAGAACCTGGTGATGCGTACCGTTCTGGAGCCGGTACCGATGTAA

SEQ ID NO：10

Codon optimized Artemisinin β-farnesene synthetic enzyme (5 ' to 3 ') is carried out for expressing in yeast saccharomyces cerevisiae

GGATCCATGTCAACTTTGCCTATTTCTTCTGTGTCATTTTCCTCTTCTACATCACCATTAGTCGTGG

ACGACAAAGTCTCAACCAAGCCCGACGTTATCAGACATACAATGAATTTCAATGCTTCTATTTGG

GGAGATCAATTCTTGACCTATGATGAGCCTGAAGATTTAGTTATGAAGAAACAATTAGTGGAGG

AATTAAAAGAGGAAGTTAAGAAGGAATTGATAACTATCAAAGGTTCAAATGAGCCCATGCAGCA

TGTGAAATTGATTGAATTAATTGATGCTGTTCAACGTTTAGGTATAGCTTACCATTTTGAAGAAG

AGATCGAGGAAGCTTTGCAACATATACATGTTACCTATGGTGAACAGTGGGTGGATAAGGAAAA

TTTACAGAGTATTTCATTGTGGTTCAGGTTGTTGCGTCAACAGGGCTTTAACGTCTCCTCTGGCGT

TTTCAAAGACTTTATGGACGAAAAAGGTAAATTCAAAGAGTCTTTATGCAATGATGCACAAGGA

ATATTAGCCTTATATGAAGCTGCATTTATGAGGGTTGAAGATGAAACCATCTTAGACAATGCTTT

GGAATTCACAAAAGTTCATTTAGATATCATAGCAAAAGACCCATCTTGCGATTCTTCATTGCGTA

CACAAATCCATCAAGCCTTAAAACAACCTTTAAGAAGGAGATTAGCAAGGATTGAAGCATTACA

TTACATGCCAATCTACCAACAGGAAACATCTCATGATGAAGTATTGTTGAAATTAGCCAAGTTGG

ATTTCAGTGTTTTGCAGTCTATGCATAAAAAGGAATTGTCACATATCTGTAAGTGGTGGAAAGAT

TTAGATTTACAAAATAAGTTACCTTATGTACGTGATCGTGTTGTCGAAGGCTACTTCTGGATATTG

TCCATATACTATGAGCCACAACACGCTAGAACAAGAATGTTTTTGATGAAAACATGCATGTGGTT

AGTAGTTTTGGACGATACTTTTGATAATTATGGAACATACGAAGAATTGGAGATTTTTACTCAAG

CCGTCGAGAGATGGTCTATCTCATGCTTAGATATGTTGCCCGAATATATGAAATTAATCTACCAA

GAATTAGTCAATTTGCATGTGGAAATGGAAGAATCTTTGGAAAAGGAGGGAAAGACCTATCAGA

TTCATTACGTTAAGGAGATGGCTAAAGAATTAGTTCGTAATTTACTTAGTAGAAGCAAGATGGTTG

AAGGAAGGTTATATGCCTACTTTAGAAGAATACATGTCTGTTTCTATGGTTACTGGTACTTATGG

TTTGATGATTGCAAGGTCCTATGTTGGCAGAGGAGACATTGTTACTGAAGACACATTCAAATGGG

TTTCTAGTTACCCACCTATTATTAAAGCTTCCTGTGTAATAGTAAGATTAATGGACGATATTGTAT

CTCACAAGGAAGAACAAGAAAGAGGACATGTGGCTTCATCTATAGAATGTTACTCTAAAGAATC

AGGTGCTTCTGAAGAGGAAGCATGTGAATATATTAGTAGGAAAGTTGAGGATGCCTGGAAAGTA

ATCAATAGAGAATCTTTGCGTCCAACAGCCGTTCCCTTCCCTTTGTTAATGCCAGCAATAAACTT

AGCTAGAATGTGTGAGGTCTTGTACTCTGTTAATGATGGTTTTACTCATGCTGAGGGTGACATGA

AATCTTATATGAAGTCCTTCTTCGTTCATCCTATGGTCGTTTGACTCGAG

SEQ ID NO：11

Codon optimized Picea excelsa α-farnesene synthetic enzyme (5 ' to 3 ') is carried out for expressing in yeast saccharomyces cerevisiae

GGATCCATGGATTTGGCTGTTGAGATTGCAATGGACTTGGCTGTGGATGATGTCGAAAGAAGGG

TGGGCGATTACCATTCTAACTTATGGGACGATGATTTTATACAATCATTAAGTACTCCTTACGGT

GCTAGTTCATACAGAGAAAGAGCAGAGAGATTAGTGGGTGAGGTCAAAGAGATGTTTACATCCA

TCTCTATAGAGGATGGAGAATTAACAAGTGATTTATTACAGCGTTTGTGGATGGTTGATAATGTA

GAGAGATTGGGAATTTCCCGTCATTTTGAGAATGAGATCAAGGCAGCTATTGACTATGTCTATTC

CTACTGGAGTGATAAGGGCATCGTTAGAGGTAGGGATTCTGCAGTGCCTGATTTAAACTCTATTG

CCTTAGGTTTTAGAACATTGAGATTACATGGTTATACCGTCTCTTCCGACGTATTCAAAGTTTTTC

AAGATAGAAAGGGTGAATTCGCATGTAGTGCCATCCCAACTGAGGGCGACATTAAGGGTGTTTT

AAACTTGTTGAGAGCTTCATACATCGCCTTTCCAGGAGAAAAAGTTATGGAAAAAGCTCAAACA

TTTGCTGCTACTTATTTGAAAGAGGCATTGCAAAAGATTCAGGTTTCCTCTTTGTCACGTGAGATC

GAATACGTATTAGAATACGGCTGGTTGACAAACTTCCCTAGATTAGAAGCCAGAAACTATATCG

ATGTATTCGGTGAGGAGATTTGCCCATATTTCAAGAAGCCTTGTATAATGGTTGATAAGTTATTG

GAATTGGCTAAGTTAGAATTTAATTTATTTCACTCATTACAACAGACCGAATTGAAACACGTTTC

TAGATGGTGGAAAGACTCTGGCTTTTCTCAATTAACTTTTACACGTCATAGACATGTCGAATTCT

ATACATTGGCCTCCTGTATTGCAATTGAACCTAAACACTCTGCATTTAGGTTAGGTTTCGCCAAG

GTTTGCTACTTAGGCATCGTATTAGATGATATTTACGACACCTTCGGAAAGATGAAGGAATTAGA

ATTTATTCACTGCCGCAATAAAAAGATGGGACCCTTCAACAACAGAATGTTTACCAGAATACATG

AAAGGTGTGTACATGGCATTCTATAACTGCGTTAATGAATTGGCTTTACAAGCAGAGAAAACTCA

AGGAAGGGATATGTTAAACTACGCACGTAAGGCCTGGGAGGCTTTGTTCGATGCTTTTTTGGAAG

AAGCCAAATGGATATCTAGTGGTTACTTACCAACCTTCGAAGAATATTTAGAAAATGGTAAAGT

ATCTTTCGGATATAGAGCTGCAACTTTGCAGCCAATTTTAACTTTGGATATACCTTTGCCCTTGCA

TATTTTGCAGCAAATAGACTTCCCTTCTAGATTTAATGATTTAGCTAGTAGTATTTTGAGATTGCG

TGGCGATATATGCGGTTACCAAGCAGAGAGATCTAGAGGTGAGGAAGCATCATCTATAAGTTGC

TATATGAAGGATAATCCCGGTTCTACAGAAGAAGACGCCTTATCTCACATAAACGCTATGATATC

TGATAACATTAACGAGTTGAACTGGGAGTTATTAAAGCCAAATTCCAATGTTCCAATATCAAGTA

AAAAGCACGCTTTTGATATATTACGTGCTTTCTATCACTTGTATAAGTATCGTGATGGTTTTTCTA

TCGCAAAGATTGAAACTAAGAATTTGGTTATGAGAACTGTGTTGGAACCAGTACCAATGTGACTC

GAG

SEQ ID NO：12

From the KanMX-PMET3 region (5 ' to 3 ') of pAM328

GAATTCGCCCTTNTGGATGGCGGCGTTAGTATCGAATCGACAGCAGTATAGCGACCAGCATTCAC

ATACGATTGACGCATGATATTACTTTCTGCGCACTTAACTTCGCATCTGGGCAGATGATGTCGAG

GCGAAAAAAAATATAAATCACGCTAACATTTGATTAAAATAGAACAACTACAATATAAAAAAAC

TATACAAATGACAAGTTCTTGAAAACAAGAATCTTTTTATTGTCAGTACTGATTAGAAAAACTCA

TCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAG

CCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTAT

CGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAG

GTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGCTTATGC

ATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAAC

CAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGA

CAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTT

CACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTGCCGGGGATCGCAGTGGTGAGT

AACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCA

GCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAA

ACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTA

TCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAAAC

GTGAGTCTTTTCCTTACCCATGGTTGTTTATGTTCGGATGTGATGTGAGAACTGTATCCTAGCAAG

ATTTTAAAAGGAAGTATATGAAAGAAGAACCTCAGTGGCAAATCCTAACCTTTTATATTTCTCTA

CAGGGGCGCGGCGTGGGGACAATTCAACGCGTCTGTGAGGGGAGCGTTTCCCTGCTCGCAGGTC

TGCAGCGAGGAGCCGTAATTTTTGCTTCGCGCCGTGCGGCCATCAAAATGTATGGATGCAAATGA

TTATACATGGGGATGTATGGGCTAAATGTACGGGCGACAGTCACATCATGCCCCTGAGCTGCGC

ACGTCAAGACTGTCAAGGAGGGTATTCTGGGCCTCCATGTCGCTGGCCGGGTGACCCGGCGGGG

ACGAGGCAAGCTAAACAGATCTGATCTTGAAACTGAGTAAGATGCTCAGAATACCCGTCAAGAT

AAGAGTATAATGTAGAGTAATATACCAAGTATTCAGCATATTCTCCTCTTCTTTTGTATAAATCAC

GGAAGGGATGATTTATAAGAAAAATGAATACTATTACACTTCATTTACCACCCTCTGATCTAGAT

TTTCCAACGATATGTACGTAGTGGTATAAGGTGAGGGGGTCCACAGATATAACATCGTTTAATTT

AGTACTAACAGAGACTTTTGTCACAACTACATATAAGTGTACAAATATAGTACAGATATGACAC

ACTTGTAGCGCCAACGCGCATCCTACGGATTGCTGACAGAAAAAAAGGTCACGTGACCAGAAAA

GTCACGTGTAATTTTGTAACTCACCGCATTCTAGCGGTCCCTGTCGTGCACACTGCACTCAACAC

CATAAACCTTAGCAACCTCCAAAGGAAATCACCGTATAACAAAGCCACAGTTTTACAACTTAGTC

TCTTATGAAGTTACTTACCAATGAGAAATAGAGGCTCTTTCTCGAGAAATATGAATATGGATATA

TATATATATATATATATATATATATATATATGTAAACTTGGTTCTTTTTTAGCTTGTGATCTCTAGC

TTGGGTCTCTCTCTGTCGTAACAGTTGTGATATCGNAAGGGCGAATTC

SEQ ID NO：13

Primer 4-49mva Spel (5 ' to 3 ')

GCTACTAGTAGGAGGAAAACATCATGCAAAGTTTAGATAAGAATTTCCG

SEQ ID NO：14

Primer 4-49mva Xbal (5 ' to 3 ')

GCTTCTAGACTATTGTTGTCTAATTTCTTGTAAAATGCG

SEQ ID NO：15

Primer HMGS 3 ' Sa mvaS-AS (5 ' to 3 ')

TTGCATGATGTTTTCCTCCTACTAGTTACTCTGGTCTGTGATATTCGCGAAC

SEQ ID NO：16

Primer HMGS 5 ' Sa mvaS-S (5 ' to 3 ')

GAACTGAAGATCTAGGAGGAAAGCAAAATGACAATAGGTATCGACAAAATAAACT

SEO ID NO：17

Primer 19-25atoB Sfil-S (5 ' to 3 ')

GCTAGGCCATCCTGGCCATGAAGAACTGTGTGATTGTTTCTG

SEQ ID NO：18

Primer 19-25mvaA-AsiS1-AS (5 ' to 3 ')

GCTTGCGATCGCCGGCGGATTTGTCCTACTCAG

SEO ID NO：19

Primer 67-1A-C (5 ' to 3 ')

ACACTCGAGGAGGAATAAATGAGTTTTGATATTGCCAAATACCCG

SEQ ID NO：20

Primer 67-1B-C (5 ' to 3 ')

TGATGGTACCTTATGCCAGCCAGGCCTTGATTTTGGC

SEQ ID NO：21

Primer 67-1C-C (5 ' to 3 ')

ACTAGGTACCAGGAGGAATAAATGAAGCAACTCACCATTCTGGGC

SEQ ID NO：22

Primer 67-1D-C (5 ' to 3 ')

AATTGATGGGCCCTCAGCTTGCGAGACGCATCACCTC

SEQ ID NO：23

Primer 67-1E-C (5 ' to 3 ')

CATAAAGGGCCCAGGAGGAATAAATGGCAACCACTCATTTGGATG

SEQ ID NO：24

Primer 67-1F-C (5 ' to 3 ')

TATTGTTCATATGTTATGTATTCTCCTGATGGATGGTTCG

SEQ ID NO：25

Primer 67-1G-C (5 ' to 3 ')

AACTAACACATATGAGGAGGAATAAATGCGGACACAGTGGCCCTC

SEQ ID NO：26

Primer 67-1H-C (5 ' to 3 ')

TGTTAGTTACGCGTTTAAAGCATGGCTCTGTGCAATGG

SEQ IDNO：27

Primer 67-2A-C (5 ' to 3 ')

ACGGGATCCAGGAGGAATAAATGCGAATTGGACACGGTTTGACG

SEQ ID NO：28

Primer 67-2B-C (5 ' to 3 ')

TTTAGTTGGGCCCTCATTTTGTTGCCTTAATGAGTAGCGCC

SEQ ID NO：29

Primer 67-2C-C (5 ' to 3 ')

TACTAAGGGCCCAGGAGGAAATAATGCATAACCAGGCTCCAATTCAACG

SEQ ID NO：30

Primer 67-2D-C (5 ' to 3 ')

TCCGGGTACCTTATTTTTCAACCTGCTGAACGTCAATTCG

SEQ ID NO：31

Primer 67-2E-C (5 ' to 3 ')

AACAGGTACCAGGAGGAAATAATGCAGATCCTGTTGGCCAACC

SEQ ID NO：32

Primer 67-2F-C (5 ' to 3 ')

TGGATGAAGTCGACTTAATCGACTTCACGAATATCGACACGCAGC

SEQ ID NO：33

Primer 67-2G-C (5 ' to 3 ')

CATCAAGTCGACAGGAGGAAATAATGCAAACGGAACACGTCATTTTATTG

SEQ ID NO：34

Primer 67-2H-C (5 ' to 3 ')

TAATGCAAGCTTATTTAAGCTGGGTAAATGCAGATAATCG

SEQ ID NO：35

Primer 67-2I-C (5 ' to 3 ')

CAGTAAAGCTTAGGAGGAAATAATGGACTTTCCGCAGCAACTCG

SEQ ID NO：36

Primer 67-2J-C (5 ' to 3 ')

TAGTTCCATGGTTATTTATTACGCTGGATGATGTAGTCCGC

SEQ ID NO：37

Primer 9-156A (5 ' to 3 ')

ACATAGACGTCGGGAAAGCGAGGATCTAGGTAGGG

SEQ ID NO：38

Primer 9-156B (5 ' to 3 ')

TTCCCGCTCGAGGTGGCGGACCATATAGGCAGATCAG

SEQ ID NO：39

Primer 61-67-CPK001-G (5 ' to 3 ')

GTTTAAACTACTATTAGCTGAATTGCCACT

SEQ ID NO：40

Primer 61-67-CPK002-G (5 ' to 3 ')

ACTGCAAAGTACACATATATCCCGGGTGTCAGCTCTTTTAGATCGG

SEQ ID NO：41

Primer 61-67-CPK003-G (5 ' to 3 ')

CCGATCTAAAAGAGCTGACACCCGGGATATATGTGTACTTTGCAGT

SEQ ID NO：42

Primer 61-67-CPK004-G (5 ' to 3 ')

GTTTAAACGGCGTCAGTCCACCAGCTAACA

SEQ ID NO：43

Primer 61-67-CPK005-G (5 ' to 3 ')

GTTTAAACTTGCTAAATTCGAGTGAAACAC

SEQ ID NO：44

Primer 61-67-CPK006-G (5 ' to 3 ')

AAAGATGAATTGAAAAGCTTCCCGGGTATGGACCCTGAAACCACAG

SEQ ID NO：45

Primer 61-67-CPK007-G (5 ' to 3 ')

CTGTGGTTTCAGGGTCCATACCCGGGAAGCTTTTCAATTCATCTTT

SEQ ID NO：46

Primer 61-67-CPK008-G (5 ' to 3 ')

GTTTAAACCCAACAATAATAATGTCAGATC

SEQ ID NO：47

Primer 61-67-CPK009-G (5 ' to 3 ')

GTTTAAACTACTCAGTATATTAAGTTTCGA

SEQ ID NO：48

Primer 61-67-CPK010-G (5 ' to 3 ')

ATCTCTCGCAAGAGTCAGACTGACTCCCGGGCGTGAATAAGCTTCGGGTGACCCTTATGGCATTC

TTTTT

SEQ ID NO：49

Primer 61-67-CPK011-G (5 ' to 3 ')

AAAAAGAATGCCATAAGGGTCACCCGAAGCTTATTCACGCCCGGGAGTCAGTCTGACTCTTGCG

AGAGAT

SEQ ID NO：50

Primer 61-67-CPK012-G (5 ' to 3 ')

GTTTAAACAATTTAGTGTCTGCGATGATGA

SEQ ID NO：51

Primer 61-67-CPK013-G (5 ' to 3 ')

GTTTAAACTATTGTGAGGGTCAGTTATTTC

SEQ ID NO：52

Primer 61-67-CPK014alt-G (5 ' to 3 ')

GCGGGGACGAGGCAAGCTAAACTTTAGTATATTCTTCGAAGAAA

SEQ ID NO：53

Primer 61-67-CPK015alt-G (5 ' to 3 ')

TTTCTTCGAAGAATATACTAAAGTTTAGCTTGCCTCGTCCCCGC

SEQ ID NO：54

Primer 61-67-CPK017-G (5 ' to 3 ')

CGATACTAACGCCGCCATCCACCCGGGAGAGGCTAGCAGAATTACCCTCCACGTTGATTG

SEQ ID NO：55

Primer 61-67-CPK018-G (5 ' to 3 ')

GTTTAAACGCCGCCGTTGTTGTTATTGTAG

SEQ ID NO：56

Primer 61-67-CPK019-G (5 ' to 3 ')

GTTTAAACTTTTCCAATAGGTGGTTAGCAA

SEQ ID NO：57

Primer 61-67-CPK020-G (5 ' to 3 ')

GGGTGACCCGGCGGGGACGAGGCAAGCTAAACGTCTTCCTTTCTCTTACCAAAGT

SEQ ID NO：58

Primer 61-67-CPK021-G (5 ' to 3 ')

ACTTTGGTAAGAGAAAGGAAGACGTTTAGCTTGCCTCGTCCCCGCCGGGTCACCC

SEQ ID NO：59

Primer 61-67-CPK022-G (5 ' to 3 ')

AATATCATAAAAAAAGAGAATCTTTCCCGGGTGGATGGCGGCGTTAGTATCGAATCGACAGC

SEQ ID NO：60

Primer 61-67-CPK023-G (5 ' to 3 ')

GCTGTCGATTCGATACTAACGCCGCCATCCACCCGGGAAAGATTCTCTTTTTTTATGATATT

SEO ID NO：61

Primer 61-67-CPK024-G (5 ' to 3 ')

GTTTAAACGTGTTAACGTTTCTTTCGCCTACGTGGAAGGAGAATC

SEQ ID NO：62

Primer 61-67-CPK025-G (5 ' to 3 ')

TCCCCCCGGGTTAAAAAAAATCCTTGGACTAGTCA

SEQ ID NO：63

Primer 61-67-CPK031-G (5 ' to 3 ')

TCCCCCCGGGAGTTATGACAATTACAACAACAGAA

SEQ ID NO：64

Primer 61-67-CPK032-G (5 ' to 3 ')

TCCCCCCGGGTATATATATATCATTGTTAT

SEQ ID NO：65

Primer 61-67-CPK035-G (5 ' to 3 ')

TCCCCCCGGGAAAAGTAAGTCAAAAGGCAC

SEQ ID NO：66

Primer 61-67-CPK040-G (5 ' to 3 ')

TCCCCCCGGGATGGTCTGCTTAAATTTCAT

SEQ ID NO：67

Primer 61-67-CPK041-G (5 ' to 3 ')

TCCCCCCGGGTAGCTTGTACCCATTAAAAGAATTTTATCATGCCG

SEQ ID NO：68

Primer 61-67-CPK046-G (5 ' to 3 ')

TCCCCCCGGGTTTCTCATTCAAGTGGTAAC

SEQ ID NO：69

Primer 61-67-CPK047-G (5 ' to 3 ')

TCCCCCCGGGTAAATAAAGAAAATAAAGTT

SEQ ID NO：70

Primer 61-67-CPK050-G (5 ' to 3 ')

AATTTTTGAAAATTCAATATAAATGGCTTCAGAAAAAGAAATTAGGA

SEQ ID NO：71

Primer 61-67-CPK051-G (5 ' to 3 ')

TCCTAATTTCTTTTTCTGAAGCCATTTATATTGAATTTTCAAAAATT

SEQ ID NO：72

Primer 61-67-CPK052-G (5 ' to 3 ')

AGTTTTCACCAATTGGTCTGCAGCCATTATAGTTTTTTCTCCTTGACGTTA

SEQ ID NO：73

Primer 61-67-CPK053-G (5 ' to 3 ')

TAACGTCAAGGAGAAAAAACTATAATGGCTGCAGACCAATTGGTGAAAACT

SEQ ID NO：74

Primer 61-67-CPK054-G (5 ' to 3 ')

AATTTTTGAAAATTCAATATAAATGAAACTCTCAACTAAACTTTGTT

SEQ ID NO：75

Primer 61-67-CPK055-G (5 ' to 3 ')

AACAAAGTTTAGTTGAGAGTTTCATTTATATTGAATTTTCAAAAATT

SEQ ID NO：76

Primer 61-67-CPK056-G (5 ' to 3 ')

AATTTTTGAAAATTCAATATAAATGTCTCAGAACGTTTACATTGTAT

SEQ ID NO：77

Primer 61-67-CPK057-G (5 ' to 3 ')

ATACAATGTAAACGTTCTGAGACATTTATATTGAATTTTCAAAAATT

SEQ ID NO：78

Primer 61-67-CPK058-G (5 ' to 3 ')

TGCAGAAGTTAAGAACGGTAATGACATTATAGTTTTTTCTCCTTGACGTTA

SEQ ID NO：79

Primer 61-67-CPK059-G (5 ' to 3 ')

TAACGTCAAGGAGAAAAAACTATAATGTCATTACCGTTCTTAACTTCTGCA

SEQ ID NO：80

Primer 61-67-CPK060-G (5 ' to 3 ')

AATTTTTGAAAATTCAATATAAATGTCAGAGTTGAGAGCCTTCAGTG

SEQ ID NO：81

Primer 61-67-CPK061-G (5 ' to 3 ')

CACTGAAGGCTCTCAACTCTGACATTTATATTGAATTTTCAAAAATT

SEQ ID NO：82

Primer 61-67-CPK062-G (5 ' to 3 ')

GGTAACGGATGCTGTGTAAACGGTCATTATAGTTTTTTCTCCTTGACGTTA

SEQ ID NO：83

Primer 61-67-CPK063-G (5 ' to 3 ')

TAACGTCAAGGAGAAAAAACTATAATGACCGTTTACACAGCATCCGTTACC

SEQ ID NO：84

Primer 61-67-CPK064-G (5 ' to 3 ')

AATTTTTGAAAATTCAATATAAATGACTGCCGACAACAATAGTATGC

SEQ ID NO：85

Primer 61-67-CPK065-G (5 ' to 3 ')

GCATACTATTGTTGTCGGCAGTCATTTATATTGAATTTTCAAAAATT

SEQ ID NO：86

Primer A (5 ' to 3 ')

CCATGGACACTCTGCCGATCTCTTCCGTAAGC

SEQ ID NO：87

Primer B (5 ' to 3 ')

GAGCTCTCATACGACCATAGGGTGTACG

SEQ ID NO：88

Primer C (5 ' to 3 ')

CCATGGACCTGGCAGTAGAAATTGC

SEQ ID NO：89

Primer D (5 ' to 3 ')

GAGCTCTTACATCGGTACCGGCTCCAG

SEQ ID NO：90

Primer GW-52-84pAM326BamHI (5 ' to 3 ')

TAATAAGGATCCATGTCAACTTTGCCTATTTC

SEQ ID NO：91

Primer GW-52-84pAM326Nhel (5 ' to 3 ')

TTATAGCTAGCTCAAACGACCATAGGATGAAC

SEQ ID NO：92

Primer 61-67-CPK016-G (5 ' to 3 ')

CAATCAACGTGGAGGGTAATTCTGCTAGCCTCTCCCGGGTGGATGGCGGCGTTAGTATCG

SEQ ID NO：93

Primer 50-56-pw100-G (5 ' to 3 ')

GAGTGAACCTGCTGCCTGGCGTGCTCTGACTCAGTACATTTCATAGTGGATGGCGGCGTTAGTAT

C

SEQ ID NO：94

Primer 50-56-pw101-G (5 ' to 3 ')

CGTGTATACGTTTTCCGCTTCTGCTCTTCGTCTTTTCTCTTCTTCCGATATCACAACTGTTACGA

SEQ ID NO：95

Primer 61-67-CPK066-G (5 ' to 3 ')

GGTAAGACGGTTGGGTTTTATCTTTTGCAGTTGGTACTATTAAGAACAATCACAGGAAACAGCTA

TGACC

SEQ ID NO：96

Primer 61-67-CPK067-G (5 ' to 3 ')

TTGCGTTTTGTACTTTGGTTCGCTCAATTTTGCAGGTAGATAATCGAAAAGTTGTAAAACGACGG

CCAGT

Sequence table

<110> Amyris Biotechnologies Inc.

<120> comprises fuel composition and the preparation and application thereof of farnesane and farnesane derivative

<130>11836-012-228

<150>60/850,881

<151>2006-10-10

<150>60/860,854

<151>2006-11-21

<160>96

<170>FastSEQ for Windows Version 4.0

<210>1

<211>4247

<212>DNA

The artificial sequence of <213>

<220>

<223>MevT66 operon

<400>1

gaattcaaag gaggaaaata aaatgaagaa ctgtgtgatt gtttctgcgg tccgcacggc 60

gatcggcagc tttaacggct ctttagcgag cacctctgca atcgatctgg gtgcgacggt 120

cattaaggcc gccattgaac gcgccaaaat cgacagccag cacgttgatg aggtgatcat 180

gggcaatgtg ttacaagccg gcctgggtca aaacccagcg cgtcaagcac tgttaaaatc 240

tggtctggcc gagaccgtgt gtggcttcac cgtcaataag gtttgcggct ctggcctgaa 300

gagcgtggcc ctggcagcac aagcgattca agccggtcag gcacaaagca tcgttgcggg 360

tggcatggag aacatgtctc tggcgccgta cttattagat gccaaagccc gcagcggtta 420

tcgcctgggc gatggtcagg tgtacgacgt catcttacgc gatggcttaa tgtgcgcgac 480

ccacggttac cacatgggta ttacggccga aaacgtggcg aaagaatacg gcattacgcg 540

cgagatgcag gatgaattag cactgcactc tcagcgcaaa gcagcagccg cgatcgagtc 600

tggtgcgttt acggcggaaa tcgtgccagt taacgtggtc acgcgcaaga agacgttcgt 660

tttcagccag gacgagttcc cgaaggcaaa cagcaccgcg gaggccttag gtgccttacg 720

cccagccttt gacaaagcgg gcacggtcac cgccggtaat gcgagcggca tcaatgatgg 780

tgcagcggca ctggtcatca tggaagagag cgccgcatta gcagcgggtc tgaccccatt 840

agcgcgcatt aaatcttatg ccagcggcgg cgtcccacca gccctgatgg gcatgggtcc 900

ggtcccagcc acgcaaaaag ccctgcaatt agcgggcctg caactggccg acattgatct 960

gatcgaggcg aacgaggcgt ttgcagcgca gttcctggcg gtgggtaaga atctgggctt 1020

cgacagcgag aaagtcaatg tgaacggtgg cgcgattgcg ttaggccatc cgattggtgc 1080

aagcggcgca cgcatcttag tgacgttact gcacgccatg caggcacgcg acaagacctt 1140

aggcctggcg accttatgta ttggtggcgg tcaaggtatc gccatggtga tcgaacgcct 1200

gaactgaaga tctaggagga aagcaaaatg aaactgagca ccaagctgtg ctggtgtggc 1260

atcaagggtc gcctgcgccc acaaaagcag caacagctgc acaacacgaa cctgcaaatg 1320

accgagctga aaaagcagaa gacggccgag caaaagaccc gcccgcagaa cgttggcatc 1380

aagggcatcc agatttatat cccgacgcag tgtgtcaacc aatctgagct ggagaaattc 1440

gatggcgtca gccagggtaa gtacaccatc ggcctgggcc agaccaacat gagcttcgtg 1500

aacgaccgtg aggacatcta ttctatgagc ctgacggtgc tgtctaagct gatcaagagc 1560

tacaacatcg acacgaataa gatcggtcgt ctggaggtgg gtacggagac gctgattgac 1620

aagagcaaaa gcgtgaagtc tgtcttaatg cagctgttcg gcgagaacac ggatgtcgag 1680

ggtatcgaca ccctgaacgc gtgttacggc ggcaccaacg cactgttcaa tagcctgaac 1740

tggattgaga gcaacgcctg ggatggccgc gatgcgatcg tcgtgtgcgg cgatatcgcc 1800

atctatgaca agggtgcggc acgtccgacc ggcggtgcag gcaccgttgc gatgtggatt 1860

ggcccggacg caccaattgt cttcgattct gtccgcgcgt cttacatgga gcacgcctac 1920

gacttttaca agccggactt cacgagcgaa tacccgtacg tggacggcca cttctctctg 1980

acctgctatg tgaaggcgct ggaccaggtt tataagtctt atagcaaaaa ggcgatttct 2040

aagggcctgg tcagcgaccc ggcaggcagc gacgccctga acgtgctgaa gtatttcgac 2100

tacaacgtgt tccatgtccc gacctgcaaa ttagtgacca aatcttatgg ccgcctgtta 2160

tataatgatt tccgtgccaa cccgcagctg ttcccggagg ttgacgccga gctggcgacg 2220

cgtgattacg acgagagcct gaccgacaag aacatcgaga agaccttcgt caacgtcgcg 2280

aagccgttcc acaaagagcg tgtggcccaa agcctgatcg tcccgaccaa cacgggcaac 2340

atgtataccg cgtctgtcta cgcggcattc gcgagcctgc tgaattacgt cggttctgac 2400

gacctgcagg gcaagcgcgt tggcctgttc agctacggta gcggcttagc ggccagcctg 2460

tatagctgca aaattgtcgg cgacgtccag cacatcatca aggagctgga catcaccaac 2520

aagctggcga agcgcatcac cgagacgccg aaagattacg aggcagcgat cgagttacgc 2580

gagaatgcgc atctgaagaa gaacttcaag ccgcaaggta gcatcgagca cctgcagagc 2640

ggcgtctact acctgacgaa cattgacgac aagttccgcc gttcttatga cgtcaaaaag 2700

taactagtag gaggaaaaca tcatggtgct gacgaacaaa accgtcatta gcggcagcaa 2760

ggtgaagtct ctgagcagcg cccaaagctc tagcagcggc ccgtctagca gcagcgagga 2820

ggacgacagc cgtgacattg agtctctgga caagaagatc cgcccgctgg aggagttaga 2880

ggccctgctg agcagcggca acaccaagca gctgaagaac aaggaagttg cagcgctggt 2940

gatccacggt aagctgccac tgtatgcgct ggaaaagaaa ctgggcgata cgacgcgtgc 3000

ggtcgcggtg cgtcgcaaag ccttaagcat cttagcggag gccccggtgt tagccagcga 3060

ccgcctgccg tacaagaact acgactacga ccgcgtgttt ggcgcgtgct gcgagaatgt 3120

cattggctac atgccgttac cggttggtgt gatcggcccg ctggtcattg atggcacgag 3180

ctatcacatt ccaatggcga ccacggaagg ttgcttagtc gccagcgcca tgcgtggctg 3240

taaggcgatt aacgccggcg gtggcgcgac gaccgtgtta accaaggatg gtatgacgcg 3300

cggtccggtc gtccgcttcc caacgctgaa gcgcagcggc gcgtgtaaga tttggctgga 3360

ttctgaggag ggccaaaacg cgatcaagaa agccttcaac tctacgagcc gtttcgcgcg 3420

tttacagcat atccagacct gcctggccgg cgacctgctg ttcatgcgct tccgcaccac 3480

cacgggcgat gcgatgggca tgaacatgat cagcaagggc gtcgaatata gcctgaaaca 3540

aatggtggaa gaatatggct gggaggacat ggaggttgtc tctgtgagcg gcaactattg 3600

caccgacaag aagccggcag ccattaactg gattgagggt cgcggcaaaa gcgtcgtggc 3660

agaagcgacc atcccaggcg acgtggtccg taaggttctg aagagcgacg tcagcgccct 3720

ggttgagtta aatatcgcga aaaacctggt cggcagcgcg atggcgggca gcgtgggtgg 3780

ctttaacgca catgcagcga atctggttac ggcggttttc ttagccttag gtcaggaccc 3840

agcccaaaat gtcgagagca gcaactgcat taccttaatg aaagaggttg acggtgacct 3900

gcgcatcagc gtttctatgc cgtctatcga ggtcggcacg atcggcggcg gcaccgtttt 3960

agaaccgcaa ggtgcgatgc tggatctgct gggcgtgcgc ggcccacatg caacggcccc 4020

aggcaccaat gcccgccaac tggcccgtat cgtggcctgc gcggttctgg cgggtgagct 4080

gagcctgtgc gccgcattag ccgcgggcca tttagttcaa tctcacatga cccacaaccg 4140

caagccggca gaaccaacca agccaaataa cctggacgca accgacatta accgtctgaa 4200

ggatggcagc gtcacgtgca ttaaaagctg agcatgctac taagctt 4247

<210>2

<211>3669

<212>DNA

The artificial sequence of <213>

<220>

The atoB (opt) of <223>pAM52: mvaS:mvaA operon

<400>2

atgaagaact gtgtgattgt ttctgcggtc cgcacggcga tcggcagctt taacggctct 60

ttagcgagca cctctgcaat cgatctgggt gcgacggtca ttaaggccgc cattgaacgc 120

gccaaaatcg acagccagca cgttgatgag gtgatcatgg gcaatgtgtt acaagccggc 180

ctgggtcaaa acccagcgcg tcaagcactg ttaaaatctg gtctggccga gaccgtgtgt 240

ggcttcaccg tcaataaggt ttgcggctct ggcctgaaga gcgtggccct ggcagcacaa 300

gcgattcaag ccggtcaggc acaaagcatc gttgcgggtg gcatggagaa catgtctctg 360

gcgccgtact tattagatgc caaagcccgc agcggttatc gcctgggcga tggtcaggtg 420

tacgacgtca tcttacgcga tggcttaatg tgcgcgaccc acggttacca catgggtatt 480

acggccgaaa acgtggcgaa agaatacggc attacgcgcg agatgcagga tgaattagca 540

ctgcactctc agcgcaaagc agcagccgcg atcgagtctg gtgcgtttac ggcggaaatc 600

gtgccagtta acgtggtcac gcgcaagaag acgttcgttt tcagccagga cgagttcccg 660

aaggcaaaca gcaccgcgga ggccttaggt gccttacgcc cagcctttga caaagcgggc 720

acggtcaccg ccggtaatgc gagcggcatc aatgatggtg cagcggcact ggtcatcatg 780

gaagagagcg ccgcattagc agcgggtctg accccattag cgcgcattaa atcttatgcc 840

agcggcggcg tcccaccagc cctgatgggc atgggtccgg tcccagccac gcaaaaagcc 900

ctgcaattag cgggcctgca actggccgac attgatctga tcgaggcgaa cgaggcgttt 960

gcagcgcagt tcctggcggt gggtaagaat ctgggcttcg acagcgagaa agtcaatgtg 1020

aacggtggcg cgattgcgtt aggccatccg attggtgcaa gcggcgcacg catcttagtg 1080

acgttactgc acgccatgca ggcacgcgac aagaccttag gcctggcgac cttatgtatt 1140

ggtggcggtc aaggtatcgc catggtgatc gaacgcctga actgaagatc taggaggaaa 1200

gcaaaatgac aataggtatc gacaaaataa acttttacgt tccaaagtac tatgtagaca 1260

tggctaaatt agcagaagca cgccaagtag acccaaacaa atttttaatt ggaattggtc 1320

aaactgaaat ggctgttagt cctgtaaacc aagacatcgt ttcaatgggc gctaacgctg 1380

ctaaggacat tataacagac gaagataaaa agaaaattgg tatggtaatt gtggcaactg 1440

aatcagcagt tgatgctgct aaagcagccg ctgttcaaat tcacaactta ttaggtattc 1500

aaccttttgc acgttgcttt gaaatgaaag aagcttgtta tgctgcaaca ccagcaattc 1560

aattagctaa agattattta gcaactagac cgaatgaaaa agtattagtt attgctacag 1620

atacagcacg ttatggattg aattcaggcg gcgagccaac acaaggtgct ggcgcagttg 1680

cgatggttat tgcacataat ccaagcattt tggcattaaa tgaagatgct gttgcttaca 1740

ctgaagacgt ttatgatttc tggcgtccaa ctggacataa atatccatta gttgatggtg 1800

cattatctaa agatgcttat atccgctcat tccaacaaag ctggaatgaa tacgcaaaac 1860

gtcaaggtaa gtcgctagct gacttcgcat ctctatgctt ccatgttcca tttacaaaaa 1920

tgggtaaaaa ggcattagag tcaatcattg ataacgctga tgaaacaact caagagcgtt 1980

tacgttcagg atatgaagat gctgtagatt ataaccgtta tgtcggtaat atttatactg 2040

gatcattata tttaagccta atatcattac ttgaaaatcg tgatttacaa gctggtgaaa 2100

caatcggttt attcagttat ggctcaggtt cagttggtga attttatagt gcgacattag 2160

ttgaaggcta caaagatcat ttagatcaag ctgcacataa agcattatta aataaccgta 2220

ctgaagtatc tgttgatgca tatgaaacat tcttcaaacg ttttgatgac gttgaatttg 2280

acgaagaaca agatgctgtt catgaagatc gtcatatttt ctacttatca aatattgaaa 2340

ataacgttcg cgaatatcac agaccagagt aactagtagg aggaaaacat catgcaaagt 2400

ttagataaga atttccgaca tttatctcgt caacaaaagt tacaacaatt ggtagataag 2460

caatggttat cagaagatca attcgacatt ttattgaatc atccattaat tgatgaggaa 2520

gtagcaaata gtttaattga aaatgtcatc gcgcaaggtg cattacccgt tggattatta 2580

ccgaatatca ttgtggacga taaggcatat gttgtaccta tgatggtgga agagccttca 2640

gttgtcgctg cagctagtta tggtgcaaag ctagtgaatc agactggcgg atttaaaacg 2700

gtatcttctg aacgtattat gataggtcaa atcgtctttg atggcgttga cgatactgaa 2760

aaattatcag cagacattaa agctttagaa aagcaaattc ataaaattgc ggatgaggca 2820

tatccttcta ttaaagcgcg tggtggtggt taccaacgta tagctattga tacatttcct 2880

gagcaacagt tactatcttt aaaagtattt gttgatacga aagatgctat gggcgctaat 2940

atgcttaata cgattttaga ggccataact gcatttttaa aaaatgaatc tccacaaagc 3000

gacattttaa tgagtatttt atccaatcat gcaacagcgt ccgttgttaa agttcaaggc 3060

gaaattgacg ttaaagattt agcaaggggc gagagaactg gagaagaggt tgccaaacga 3120

atggaacgtg cttctgtatt ggcacaagtt gatattcatc gtgctgcaac acataataaa 3180

ggtgttatga atggcataca tgccgttgtt ttagcaacag gaaatgatac gcgtggtgca 3240

gaagcaagtg cgcatgcata cgcgagtcgt gacggacagt atcgtggtat tgcaacatgg 3300

agatacgatc aaaaacgtca acgtttaatt ggtacaatag aagtgcctat gacattggca 3360

atcgttggcg gtggtacaaa agtattacca attgctaaag cttctttaga attgctaaat 3420

gtagattcag cacaagaatt aggtcatgta gttgctgccg ttggtttagc acagaacttt 3480

gcagcatgtc gcgcgctcgt ttccgaaggt atccagcaag gccatatgag cttgcaatat 3540

aaatctttag ctattgttgt aggtgcaaaa ggtgatgaaa ttgcgcaagt agctgaagca 3600

ttgaagcaag aaccccgtgc gaatacacaa gtagctgaac gcattttaca agaaattaga 3660

caacaatag 3669

<210>3

<211>5050

<212>DNA

The artificial sequence of <213>

<220>

The ERG20-PGAL-tHMGR inset of <223>pAM498

<400>3

gtttaaacta ctattagctg aattgccact gctatcgttg ttagtggcgt tagtgcttgc 60

attcaaagac atggagggcg ttattacgcc ggagctcctc gacagcagat ctgatgactg 120

gtcaatatat ttttgcattg aggctctgtt tggaattata ttttgagatg acccatctaa 180

tgtactggta tcaccagatt tcatgtcgtt ttttaaagcg gctgcttgag tcttagcaat 240

agcgtcacca tctggtgaat cctttgaagg aaccactgac gaaggtttgg acagtgacga 300

agaggatctt tcctgctttg aattagtcgc gctgggagca gatgacgagt tggtggagct 360

gggggcagga ttgctggccg tcgtgggtcc tgaatgggtc cttggctggt ccatctctat 420

tctgaaaacg gaagaggagt agggaatatt actggctgaa aataagtctt gaatgaacgt 480

atacgcgtat atttctacca atctctcaac actgagtaat ggtagttata agaaagagac 540

cgagttaggg acagttagag gcggtggaga tattccttat ggcatgtctg gcgatgataa 600

aacttttcaa acggcagccc cgatctaaaa gagctgacac ccgggagtta tgacaattac 660

aacaacagaa ttctttctat atatgcacga acttgtaata tggaagaaat tatgacgtac 720

aaactataaa gtaaatattt tacgtaacac atggtgctgt tgtgcttctt tttcaagaga 780

ataccaatga cgtatgacta agtttaggat ttaatgcagg tgacggaccc atctttcaaa 840

cgatttatat cagtggcgtc caaattgtta ggttttgttg gttcagcagg tttcctgttg 900

tgggtcatat gactttgaac caaatggccg gctgctaggg cagcacataa ggataattca 960

cctgccaaga cggcacaggc aactattctt gctaattgac gtgcgttggt accaggagcg 1020

gtagcatgtg ggcctcttac acctaataag tccaacatgg caccttgtgg ttctagaaca 1080

gtaccaccac cgatggtacc tacttcgatg gatggcatgg atacggaaat tctcaaatca 1140

ccgtccactt ctttcatcaa tgttatacag ttggaacttt cgacattttg tgcaggatct 1200

tgtcctaatg ccaagaaaac agctgtcact aaattagctg catgtgcgtt aaatccacca 1260

acagacccag ccattgcaga tccaaccaaa ttcttagcaa tgttcaactc aaccaatgcg 1320

gaaacatcac tttttaacac ttttctgaca acatcaccag gaatagtagc ttctgcgacg 1380

acactcttac cacgaccttc gatccagttg atggcagctg gttttttgtc ggtacagtag 1440

ttaccagaaa cggagacaac ctccatatct tcccagccat actcttctac catttgcttt 1500

aatgagtatt cgacaccctt agaaatcata ttcataccca ttgcgtcacc agtagttgtt 1560

ctaaatctca tgaagagtaa atctcctgct agacaagttt gaatatgttg cagacgtgca 1620

aatcttgatg tagagttaaa agctttttta attgcgtttt gtccctcttc tgagtctaac 1680

catatcttac aggcaccaga tcttttcaaa gttgggaaac ggactactgg gcctcttgtc 1740

ataccatcct tagttaaaac agttgttgca ccaccgccag cattgattgc cttacagcca 1800

cgcatggcag aagctaccaa acaaccctct gtagttgcca ttggtatatg ataagatgta 1860

ccatcgataa ccaaggggcc tataacacca acgggcaaag gcatgtaacc tataacattt 1920

tcacaacaag cgccaaatac gcggtcgtag tcataatttt tatatggtaa acgatcagat 1980

gctaatacag gagcttctgc caaaattgaa agagccttcc tacgtaccgc aaccgctctc 2040

gtagtatcac ctaatttttt ctccaaagcg tacaaaggta acttaccgtg aataaccaag 2100

gcagcgacct ctttgttctt caattgtttt gtatttccac tacttaataa tgcttctaat 2160

tcttctaaag gacgtatttt cttatccaag ctttcaatat cgcgggaatc atcttcctca 2220

ctagatgatg aaggtcctga tgagctcgat tgcgcagatg ataaactttt gactttcgat 2280

ccagaaatga ctgttttatt ggttaaaact ggtgtagaag ccttttgtac aggagcagta 2340

aaagacttct tggtgacttc agtcttcacc aattggtctg cagccattat agttttttct 2400

ccttgacgtt aaagtataga ggtatattaa caattttttg ttgatacttt tatgacattt 2460

gaataagaag taatacaaac cgaaaatgtt gaaagtatta gttaaagtgg ttatgcagct 2520

tttgcattta tatatctgtt aatagatcaa aaatcatcgc ttcgctgatt aattacccca 2580

gaaataaggc taaaaaacta atcgcattat tatcctatgg ttgttaattt gattcgttga 2640

tttgaaggtt tgtggggcca ggttactgcc aatttttcct cttcataacc ataaaagcta 2700

gtattgtaga atctttattg ttcggagcag tgcggcgcga ggcacatctg cgtttcagga 2760

acgcgaccgg tgaagaccag gacgcacgga ggagagtctt ccgtcggagg gctgtcgccc 2820

gctcggcggc ttctaatccg tacttcaata tagcaatgag cagttaagcg tattactgaa 2880

agttccaaag agaaggtttt tttaggctaa gataatgggg ctctttacat ttccacaaca 2940

tataagtaag attagatatg gatatgtata tggtggtatt gccatgtaat atgattatta 3000

aacttctttg cgtccatcca aaaaaaaagt aagaattttt gaaaattcaa tataaatggc 3060

ttcagaaaaa gaaattagga gagagagatt cttgaacgtt ttccctaaat tagtagagga 3120

attgaacgca tcgcttttgg cttacggtat gcctaaggaa gcatgtgact ggtatgccca 3180

ctcattgaac tacaacactc caggcggtaa gctaaataga ggtttgtccg ttgtggacac 3240

gtatgctatt ctctccaaca agaccgttga acaattgggg caagaagaat acgaaaaggt 3300

tgccattcta ggttggtgca ttgagttgtt gcaggcttac ttcttggtcg ccgatgatat 3360

gatggacaag tccattacca gaagaggcca accatgttgg tacaaggttc ctgaagttgg 3420

ggaaattgcc atcaatgacg cattcatgtt agaggctgct atctacaagc ttttgaaatc 3480

tcacttcaga aacgaaaaat actacataga tatcaccgaa ttgttccatg aggtcacctt 3540

ccaaaccgaa ttgggccaat tgatggactt aatcactgca cctgaagaca aagtcgactt 3600

gagtaagttc tccctaaaga agcactcctt catagttact ttcaagactg cttactattc 3660

tttctacttg cctgtcgcat tggccatgta cgttgccggt atcacggatg aaaaggattt 3720

gaaacaagcc agagatgtct tgattccatt gggtgaatac ttccaaattc aagatgacta 3780

cttagactgc ttcggtaccc cagaacagat cggtaagatc ggtacagata tccaagataa 3840

caaatgttct tgggtaatca acaaggcatt ggaacttgct tccgcagaac aaagaaagac 3900

tttagacgaa aattacggta agaaggactc agtcgcagaa gccaaatgca aaaagatttt 3960

caatgacttg aaaattgaac agctatacca cgaatatgaa gagtctattg ccaaggattt 4020

gaaggccaaa atttctcagg tcgatgagtc tcgtggcttc aaagctgatg tcttaactgc 4080

gttcttgaac aaagtttaca agagaagcaa atagaactaa cgctaatcga taaaacatta 4140

gatttcaaac tagataagga ccatgtataa gaactatata cttccaatat aatatagtat 4200

aagctttaag atagtatctc tcgatctacc gttccacgtg actagtccaa ggattttttt 4260

taacccggga tatatgtgta ctttgcagtt atgacgccag atggcagtag tggaagatat 4320

tctttattga aaaatagctt gtcaccttac gtacaatctt gatccggagc ttttcttttt 4380

ttgccgatta agaattcggt cgaaaaaaga aaaggagagg gccaagaggg agggcattgg 4440

tgactattga gcacgtgagt atacgtgatt aagcacacaa aggcagcttg gagtatgtct 4500

gttattaatt tcacaggtag ttctggtcca ttggtgaaag tttgcggctt gcagagcaca 4560

gaggccgcag aatgtgctct agattccgat gctgacttgc tgggtattat atgtgtgccc 4620

aatagaaaga gaacaattga cccggttatt gcaaggaaaa tttcaagtct tgtaaaagca 4680

tataaaaata gttcaggcac tccgaaatac ttggttggcg tgtttcgtaa tcaacctaag 4740

gaggatgttt tggctctggt caatgattac ggcattgata tcgtccaact gcatggagat 4800

gagtcgtggc aagaatacca agagttcctc ggtttgccag ttattaaaag actcgtattt 4860

ccaaaagact gcaacatact actcagtgca gcttcacaga aacctcattc gtttattccc 4920

ttgtttgatt cagaagcagg tgggacaggt gaacttttgg attggaactc gatttctgac 4980

tgggttggaa ggcaagagag ccccgaaagc ttacatttta tgttagctgg tggactgacg 5040

ccgtttaaac 5050

<210>4

<211>5488

<212>DNA

The artificial sequence of <213>

<220>

The ERG13-PGAL-tHMGR inset of <223>pAM491

<400>4

gtttaaactt gctaaattcg agtgaaacac aggaagacca gaaaatcctc atttcatcca 60

tattaacaat aatttcaaat gtttatttgc attatttgaa actagggaag acaagcaacg 120

aaacgttttt gaaaattttg agtattttca ataaatttgt agaggactca gatattgaaa 180

aaaagctaca gcaattaata cttgataaga agagtattga gaagggcaac ggttcatcat 240

ctcatggatc tgcacatgaa caaacaccag agtcaaacga cgttgaaatt gaggctactg 300

cgccaattga tgacaataca gacgatgata acaaaccgaa gttatctgat gtagaaaagg 360

attaaagatg ctaagagata gtgatgatat ttcataaata atgtaattct atatatgtta 420

attacctttt ttgcgaggca tatttatggt gaaggataag ttttgaccat caaagaaggt 480

taatgtggct gtggtttcag ggtccatacc cgggagttat gacaattaca acaacagaat 540

tctttctata tatgcacgaa cttgtaatat ggaagaaatt atgacgtaca aactataaag 600

taaatatttt acgtaacaca tggtgctgtt gtgcttcttt ttcaagagaa taccaatgac 660

gtatgactaa gtttaggatt taatgcaggt gacggaccca tctttcaaac gatttatatc 720

agtggcgtcc aaattgttag gttttgttgg ttcagcaggt ttcctgttgt gggtcatatg 780

actttgaacc aaatggccgg ctgctagggc agcacataag gataattcac ctgccaagac 840

ggcacaggca actattcttg ctaattgacg tgcgttggta ccaggagcgg tagcatgtgg 900

gcctcttaca cctaataagt ccaacatggc accttgtggt tctagaacag taccaccacc 960

gatggtacct acttcgatgg atggcatgga tacggaaatt ctcaaatcac cgtccacttc 1020

tttcatcaat gttatacagt tggaactttc gacattttgt gcaggatctt gtcctaatgc 1080

caagaaaaca gctgtcacta aattagctgc atgtgcgtta aatccaccaa cagacccagc 1140

cattgcagat ccaaccaaat tcttagcaat gttcaactca accaatgcgg aaacatcact 1200

ttttaacact tttctgacaa catcaccagg aatagtagct tctgcgacga cactcttacc 1260

acgaccttcg atccagttga tggcagctgg ttttttgtcg gtacagtagt taccagaaac 1320

ggagacaacc tccatatctt cccagccata ctcttctacc atttgcttta atgagtattc 1380

gacaccctta gaaatcatat tcatacccat tgcgtcacca gtagttgttc taaatctcat 1440

gaagagtaaa tctcctgcta gacaagtttg aatatgttgc agacgtgcaa atcttgatgt 1500

agagttaaaa gcttttttaa ttgcgttttg tccctcttct gagtctaacc atatcttaca 1560

ggcaccagat cttttcaaag ttgggaaacg gactactggg cctcttgtca taccatcctt 1620

agttaaaaca gttgttgcac caccgccagc attgattgcc ttacagccac gcatggcaga 1680

agctaccaaa caaccctctg tagttgccat tggtatatga taagatgtac catcgataac 1740

caaggggcct ataacaccaa cgggcaaagg catgtaacct ataacatttt cacaacaagc 1800

gccaaatacg cggtcgtagt cataattttt atatggtaaa cgatcagatg ctaatacagg 1860

agcttctgcc aaaattgaaa gagccttcct acgtaccgca accgctctcg tagtatcacc 1920

taattttttc tccaaagcgt acaaaggtaa cttaccgtga ataaccaagg cagcgacctc 1980

tttgttcttc aattgttttg tatttccact acttaataat gcttctaatt cttctaaagg 2040

acgtattttc ttatccaagc tttcaatatc gcgggaatca tcttcctcac tagatgatga 2100

aggtcctgat gagctcgatt gcgcagatga taaacttttg actttcgatc cagaaatgac 2160

tgttttattg gttaaaactg gtgtagaagc cttttgtaca ggagcagtaa aagacttctt 2220

ggtgacttca gtcttcacca attggtctgc agccattata gttttttctc cttgacgtta 2280

aagtatagag gtatattaac aattttttgt tgatactttt atgacatttg aataagaagt 2340

aatacaaacc gaaaatgttg aaagtattag ttaaagtggt tatgcagctt ttgcatttat 2400

atatctgtta atagatcaaa aatcatcgct tcgctgatta attaccccag aaataaggct 2460

aaaaaactaa tcgcattatt atcctatggt tgttaatttg attcgttgat ttgaaggttt 2520

gtggggccag gttactgcca atttttcctc ttcataacca taaaagctag tattgtagaa 2580

tctttattgt tcggagcagt gcggcgcgag gcacatctgc gtttcaggaa cgcgaccggt 2640

gaagaccagg acgcacggag gagagtcttc cgtcggaggg ctgtcgcccg ctcggcggct 2700

tctaatccgt acttcaatat agcaatgagc agttaagcgt attactgaaa gttccaaaga 2760

gaaggttttt ttaggctaag ataatggggc tctttacatt tccacaacat ataagtaaga 2820

ttagatatgg atatgtatat ggtggtattg ccatgtaata tgattattaa acttctttgc 2880

gtccatccaa aaaaaaagta agaatttttg aaaattcaat ataaatgaaa ctctcaacta 2940

aactttgttg gtgtggtatt aaaggaagac ttaggccgca aaagcaacaa caattacaca 3000

atacaaactt gcaaatgact gaactaaaaa aacaaaagac cgctgaacaa aaaaccagac 3060

ctcaaaatgt cggtattaaa ggtatccaaa tttacatccc aactcaatgt gtcaaccaat 3120

ctgagctaga gaaatttgat ggcgtttctc aaggtaaata cacaattggt ctgggccaaa 3180

ccaacatgtc ttttgtcaat gacagagaag atatctactc gatgtcccta actgttttgt 3240

ctaagttgat caagagttac aacatcgaca ccaacaaaat tggtagatta gaagtcggta 3300

ctgaaactct gattgacaag tccaagtctg tcaagtctgt cttgatgcaa ttgtttggtg 3360

aaaacactga cgtcgaaggt attgacacgc ttaatgcctg ttacggtggt accaacgcgt 3420

tgttcaactc tttgaactgg attgaatcta acgcatggga tggtagagac gccattgtag 3480

tttgcggtga tattgccatc tacgataagg gtgccgcaag accaaccggt ggtgccggta 3540

ctgttgctat gtggatcggt cctgatgctc caattgtatt tgactctgta agagcttctt 3600

acatggaaca cgcctacgat ttttacaagc cagatttcac cagcgaatat ccttacgtcg 3660

atggtcattt ttcattaact tgttacgtca aggctcttga tcaagtttac aagagttatt 3720

ccaagaaggc tatttctaaa gggttggtta gcgatcccgc tggttcggat gctttgaacg 3780

ttttgaaata tttcgactac aacgttttcc atgttccaac ctgtaaattg gtcacaaaat 3840

catacggtag attactatat aacgatttca gagccaatcc tcaattgttc ccagaagttg 3900

acgccgaatt agctactcgc gattatgacg aatctttaac cgataagaac attgaaaaaa 3960

cttttgttaa tgttgctaag ccattccaca aagagagagt tgcccaatct ttgattgttc 4020

caacaaacac aggtaacatg tacaccgcat ctgtttatgc cgcctttgca tctctattaa 4080

actatgttgg atctgacgac ttacaaggca agcgtgttgg tttattttct tacggttccg 4140

gtttagctgc atctctatat tcttgcaaaa ttgttggtga cgtccaacat attatcaagg 4200

aattagatat tactaacaaa ttagccaaga gaatcaccga aactccaaag gattacgaag 4260

ctgccatcga attgagagaa aatgcccatt tgaagaagaa cttcaaacct caaggttcca 4320

ttgagcattt gcaaagtggt gtttactact tgaccaacat cgatgacaaa tttagaagat 4380

cttacgatgt taaaaaataa tcttccccca tcgattgcat cttgctgaac ccccttcata 4440

aatgctttat ttttttggca gcctgctttt tttagctctc atttaataga gtagtttttt 4500

aatctatata ctaggaaaac tctttattta ataacaatga tatatatata cccgggaagc 4560

ttttcaattc atcttttttt tttttgttct tttttttgat tccggtttct ttgaaatttt 4620

tttgattcgg taatctccga gcagaaggaa gaacgaagga aggagcacag acttagattg 4680

gtatatatac gcatatgtgg tgttgaagaa acatgaaatt gcccagtatt cttaacccaa 4740

ctgcacagaa caaaaacctg caggaaacga agataaatca tgtcgaaagc tacatataag 4800

gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc tatttaatat catgcacgaa 4860

aagcaaacaa acttgtgtgc ttcattggat gttcgtacca ccaaggaatt actggagtta 4920

gttgaagcat taggtcccaa aatttgttta ctaaaaacac atgtggatat cttgactgat 4980

ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta caatttttta 5040

ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca gtactctgcg 5100

ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt ggtgggccca 5160

ggtattgtta gcggtttgaa gcaggcggcg gaagaagtaa caaaggaacc tagaggcctt 5220

ttgatgttag cagaattgtc atgcaagggc tccctagcta ctggagaata tactaagggt 5280

actgttgaca ttgcgaagag cgacaaagat tttgttatcg gctttattgc tcaaagagac 5340

atgggtggaa gagatgaagg ttacgattgg ttgattatga cacccggtgt gggtttagat 5400

gacaagggag acgcattggg tcaacagtat agaaccgtgg atgatgtggt ctctacagga 5460

tctgacatta ttattgttgg gtttaaac 5488

<210>5

<211>4933

<212>DNA

The artificial sequence of <213>

<220>

The IDI1-PGAL-tHMGR inset of <223>pAM493

<400>5

gtttaaacta ctcagtatat taagtttcga attgaagggc gaactcttat tcgaagtcgg 60

agtcaccaca acacttccgc ccatactctc cgaatcctcg tttcctaaag taagtttact 120

tccacttgta ggcctattat taatgatatc tgaataatcc tctattaggg ttggatcatt 180

cagtagcgcg tgcgattgaa aggagtccat gcccgacgtc gacgtgatta gcgaaggcgc 240

gtaaccattg tcatgtctag cagctataga actaacctcc ttgacaccac ttgcggaagt 300

ctcatcaaca tgctcttcct tattactcat tctcttacca agcagagaat gttatctaaa 360

aactacgtgt atttcacctc tttctcgact tgaacacgtc caactcctta agtactacca 420

cagccaggaa agaatggatc cagttctaca cgatagcaaa gcagaaaaca caaccagcgt 480

acccctgtag aagcttcttt gtttacagca cttgatccat gtagccatac tcgaaatttc 540

aactcatctg aaacttttcc tgaaggttga aaaagaatgc cataagggtc acccgaagct 600

tattcacgcc cgggagttat gacaattaca acaacagaat tctttctata tatgcacgaa 660

cttgtaatat ggaagaaatt atgacgtaca aactataaag taaatatttt acgtaacaca 720

tggtgctgtt gtgcttcttt ttcaagagaa taccaatgac gtatgactaa gtttaggatt 780

taatgcaggt gacggaccca tctttcaaac gatttatatc agtggcgtcc aaattgttag 840

gttttgttgg ttcagcaggt ttcctgttgt gggtcatatg actttgaacc aaatggccgg 900

ctgctagggc agcacataag gataattcac ctgccaagac ggcacaggca actattcttg 960

ctaattgacg tgcgttggta ccaggagcgg tagcatgtgg gcctcttaca cctaataagt 1020

ccaacatggc accttgtggt tctagaacag taccaccacc gatggtacct acttcgatgg 1080

atggcatgga tacggaaatt ctcaaatcac cgtccacttc tttcatcaat gttatacagt 1140

tggaactttc gacattttgt gcaggatctt gtcctaatgc caagaaaaca gctgtcacta 1200

aattagctgc atgtgcgtta aatccaccaa cagacccagc cattgcagat ccaaccaaat 1260

tcttagcaat gttcaactca accaatgcgg aaacatcact ttttaacact tttctgacaa 1320

catcaccagg aatagtagct tctgcgacga cactcttacc acgaccttcg atccagttga 1380

tggcagctgg ttttttgtcg gtacagtagt taccagaaac ggagacaacc tccatatctt 1440

cccagccata ctcttctacc atttgcttta atgagtattc gacaccctta gaaatcatat 1500

tcatacccat tgcgtcacca gtagttgttc taaatctcat gaagagtaaa tctcctgcta 1560

gacaagtttg aatatgttgc agacgtgcaa atcttgatgt agagttaaaa gcttttttaa 1620

ttgcgttttg tccctcttct gagtctaacc atatcttaca ggcaccagat cttttcaaag 1680

ttgggaaacg gactactggg cctcttgtca taccatcctt agttaaaaca gttgttgcac 1740

caccgccagc attgattgcc ttacagccac gcatggcaga agctaccaaa caaccctctg 1800

tagttgccat tggtatatga taagatgtac catcgataac caaggggcct ataacaccaa 1860

cgggcaaagg catgtaacct ataacatttt cacaacaagc gccaaatacg cggtcgtagt 1920

cataattttt atatggtaaa cgatcagatg ctaatacagg agcttctgcc aaaattgaaa 1980

gagccttcct acgtaccgca accgctctcg tagtatcacc taattttttc tccaaagcgt 2040

acaaaggtaa cttaccgtga ataaccaagg cagcgacctc tttgttcttc aattgttttg 2100

tatttccact acttaataat gcttctaatt cttctaaagg acgtattttc ttatccaagc 2160

tttcaatatc gcgggaatca tcttcctcac tagatgatga aggtcctgat gagctcgatt 2220

gcgcagatga taaacttttg actttcgatc cagaaatgac tgttttattg gttaaaactg 2280

gtgtagaagc cttttgtaca ggagcagtaa aagacttctt ggtgacttca gttttcacca 2340

attggtctgc agccattata gttttttctc cttgacgtta aagtatagag gtatattaac 2400

aattttttgt tgatactttt atgacatttg aataagaagt aatacaaacc gaaaatgttg 2460

aaagtattag ttaaagtggt tatgcagctt ttgcatttat atatctgtta atagatcaaa 2520

aatcatcgct tcgctgatta attaccccag aaataaggct aaaaaactaa tcgcattatt 2580

atcctatggt tgttaatttg attcgttgat ttgaaggttt gtggggccag gttactgcca 2640

atttttcctc ttcataacca taaaagctag tattgtagaa tctttattgt tcggagcagt 2700

gcggcgcgag gcacatctgc gtttcaggaa cgcgaccggt gaagaccagg acgcacggag 2760

gagagtcttc cgtcggaggg ctgtcgcccg ctcggcggct tctaatccgt acttcaatat 2820

agcaatgagc agttaagcgt attactgaaa gttccaaaga gaaggttttt ttaggctaag 2880

ataatggggc tctttacatt tccacaacat ataagtaaga ttagatatgg atatgtatat 2940

ggtggtattg ccatgtaata tgattattaa acttctttgc gtccatccaa aaaaaaagta 3000

agaatttttg aaaattcaat ataaatgact gccgacaaca atagtatgcc ccatggtgca 3060

gtatctagtt acgccaaatt agtgcaaaac caaacacctg aagacatttt ggaagagttt 3120

cctgaaatta ttccattaca acaaagacct aatacccgat ctagtgagac gtcaaatgac 3180

gaaagcggag aaacatgttt ttctggtcat gatgaggagc aaattaagtt aatgaatgaa 3240

aattgtattg ttttggattg ggacgataat gctattggtg ccggtaccaa gaaagtttgt 3300

catttaatgg aaaatattga aaagggttta ctacatcgtg cattctccgt ctttattttc 3360

aatgaacaag gtgaattact tttacaacaa agagccactg aaaaaataac tttccctgat 3420

ctttggacta acacatgctg ctctcatcca ctatgtattg atgacgaatt aggtttgaag 3480

ggtaagctag acgataagat taagggcgct attactgcgg cggtgagaaa actagatcat 3540

gaattaggta ttccagaaga tgaaactaag acaaggggta agtttcactt tttaaacaga 3600

atccattaca tggcaccaag caatgaacca tggggtgaac atgaaattga ttacatccta 3660

ttttataaga tcaacgctaa agaaaacttg actgtcaacc caaacgtcaa tgaagttaga 3720

gacttcaaat gggtttcacc aaatgatttg aaaactatgt ttgctgaccc aagttacaag 3780

tttacgcctt ggtttaagat tatttgcgag aattacttat tcaactggtg ggagcaatta 3840

gatgaccttt ctgaagtgga aaatgacagg caaattcata gaatgctata acaacgcgtc 3900

aataatatag gctacataaa aatcataata actttgttat catagcaaaa tgtgatataa 3960

aacgtttcat ttcacctgaa aaatagtaaa aataggcgac aaaaatcctt agtaatatgt 4020

aaactttatt ttctttattt acccgggagt cagtctgact cttgcgagag atgaggatgt 4080

aataatacta atctcgaaga tgccatctaa tacatataga catacatata tatatatata 4140

cattctatat attcttaccc agattctttg aggtaagacg gttgggtttt atcttttgca 4200

gttggtacta ttaagaacaa tcgaatcata agcattgctt acaaagaata cacatacgaa 4260

atattaacga taatgtcaat tacgaagact gaactggacg gtatattgcc attggtggcc 4320

agaggtaaag ttagagacat atatgaggta gacgctggta cgttgctgtt tgttgctacg 4380

gatcgtatct ctgcatatga cgttattatg gaaaacagca ttcctgaaaa ggggatccta 4440

ttgaccaaac tgtcagagtt ctggttcaag ttcctgtcca acgatgttcg taatcatttg 4500

gtcgacatcg ccccaggtaa gactattttc gattatctac ctgcaaaatt gagcgaacca 4560

aagtacaaaa cgcaactaga agaccgctct ctattggttc acaaacataa actaattcca 4620

ttggaagtaa ttgtcagagg ctacatcacc ggatctgctt ggaaagagta cgtaaaaaca 4680

ggtactgtgc atggtttgaa acaacctcaa ggacttaaag aatctcaaga gttcccagaa 4740

ccaatcttca ccccatcgac caaggctgaa caaggtgaac atgacgaaaa catctctcct 4800

gcccaggccg ctgagctggt gggtgaagat ttgtcacgta gagtggcaga actggctgta 4860

aaactgtact ccaagtgcaa agattatgct aaggagaagg gcatcatcat cgcagacact 4920

aaattgttta gac 4933

<210>6

<211>6408

<212>DNA

The artificial sequence of <213>

<220>

The ERG10-PGAL-ERG12 inset of <223>pAM495

<400>6

gtttaaacta ttgtgagggt cagttatttc atccagatat aacccgagag gaaacttctt 60

agcgtctgtt ttcgtaccat aaggcagttc atgaggtata ttttcgttat tgaagcccag 120

ctcgtgaatg cttaatgctg ctgaactggt gtccatgtcg cctaggtacg caatctccac 180

aggctgcaaa ggttttgtct caagagcaat gttattgtgc accccgtaat tggtcaacaa 240

gtttaatctg tgcttgtcca ccagctctgt cgtaaccttc agttcatcga ctatctgaag 300

aaatttacta ggaatagtgc catggtacag caaccgagaa tggcaatttc tactcgggtt 360

cagcaacgct gcataaacgc tgttggtgcc gtagacatat tcgaagatag gattatcatt 420

cataagtttc agagcaatgt ccttattctg gaacttggat ttatggctct tttggtttaa 480

tttcgcctga ttcttgatct cctttagctt ctcgacgtgg gcctttttct tgccatatgg 540

atccgctgca cggtcctgtt ccctagcatg tacgtgagcg tatttccttt taaaccacga 600

cgctttgtct tcattcaacg tttcccattg tttttttcta ctattgcttt gctgtgggaa 660

aaacttatcg aaagatgacg actttttctt aattctcgtt ttaagagctt ggtgagcgct 720

aggagtcact gccaggtatc gtttgaacac ggcattagtc agggaagtca taacacagtc 780

ctttcccgca attttctttt tctattactc ttggcctcct ctagtacact ctatattttt 840

ttatgcctcg gtaatgattt tcattttttt tttttccacc tagcggatga ctcttttttt 900

ttcttagcga ttggcattat cacataatga attatacatt atataaagta atgtgatttc 960

ttcgaagaat atactaaagt ttagcttgcc tcgtccccgc cgggtcaccc ggccagcgac 1020

atggaggccc agaataccct ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt 1080

gactgtcgcc cgtacattta gcccatacat ccccatgtat aatcatttgc atccatacat 1140

tttgatggcc gcacggcgcg aagcaaaaat tacggctcct cgctgcagac ctgcgagcag 1200

ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg ccgcgcccct gtagagaaat 1260

ataaaaggtt aggatttgcc actgaggttc ttctttcata tacttccttt taaaatcttg 1320

ctaggataca gttctcacat cacatccgaa cataaacaac catggcagaa ccagcccaaa 1380

aaaagcaaaa acaaactgtt caggagcgca aggcgtttat ctcccgtatc actaatgaaa 1440

ctaaaattca aatcgctatt tcgctgaatg gtggttatat tcaaataaaa gattcgattc 1500

ttcctgcaaa gaaggatgac gatgtagctt cccaagctac tcagtcacag gtcatcgata 1560

ttcacacagg tgttggcttt ttggatcata tgatccatgc gttggcaaaa cactctggtt 1620

ggtctcttat tgttgaatgt attggtgacc tgcacattga cgatcaccat actaccgaag 1680

attgcggtat cgcattaggg caagcgttca aagaagcaat gggtgctgtc cgtggtgtaa 1740

aaagattcgg tactgggttc gcaccattgg atgaggcgct atcacgtgcc gtagtcgatt 1800

tatctagtag accatttgct gtaatcgacc ttggattgaa gagagagatg attggtgatt 1860

tatccactga aatgattcca cactttttgg aaagtttcgc ggaggcggcc agaattactt 1920

tgcatgttga ttgtctgaga ggtttcaacg atcaccacag aagtgagagt gcgttcaagg 1980

ctttggctgt tgccataaga gaagctattt ctagcaatgg caccaatgac gttccctcaa 2040

ccaaaggtgt tttgatgtga agtactgaca ataaaaagat tcttgttttc aagaacttgt 2100

catttgtata gtttttttat attgtagttg ttctatttta atcaaatgtt agcgtgattt 2160

atattttttt tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa 2220

tatcatgcgt caatcgtatg tgaatgctgg tcgctatact gctgtcgatt cgatactaac 2280

gccgccatcc acccgggatg gtctgcttaa atttcattct gtcttcgaaa gctgaattga 2340

tactacgaaa aatttttttt tgtttctctt tctatcttta ttacataaaa cttcatacac 2400

agttaagatt aaaaacaact aataaataat gcctatcgca aattagctta tgaagtccat 2460

ggtaaattcg tgtttcctgg caataataga tcgtcaattt gttgctttgt ggtagtttta 2520

ttttcaaata attggaatac tagggatttg attttaagat ctttattcaa attttttgcg 2580

cttaacaaac agcagccagt cccacccaag tctgtttcaa atgtctcgta actaaaatca 2640

tcttgcaatt tctttttgaa actgtcaatt tgctcttgag taatgtctct tcgtaacaaa 2700

gtcaaagagc aaccgccgcc accagcaccg gtaagttttg tggagccaat tctcaaatca 2760

tcgctcagat ttttaataag ttctaatcca ggatgagaaa caccgattga gacaagcagt 2820

ccatgattta ttcttatcaa ttccaatagt tgttcataca gttcattatt agtttctaca 2880

gcctcgtcat cggtgccttt acatttactt aacttagtca tgatctctaa gccttgtagg 2940

gcacattcac ccatggcatc tagaattggc ttcataactt caggaaattt ctcggtgacc 3000

aacacacgaa cgcgagcaac aagatctttt gtagaccttg gaattctagt ataggttagg 3060

atcattggaa tggctgggaa atcatctaag aacttaaaat tgtttgtgtt tattgttcca 3120

ttatgtgagt ctttttcaaa tagcagggca ttaccataag tggccacagc gttatctatt 3180

cctgaagggg taccgtgaat acacttttca cctatgaagg cccattgatt cactatatgc 3240

ttatcgtttt ctgacagctt ttccaagtca ttagatccta ttaacccccc caagtaggcc 3300

atagctaagg ccagtgatac agaaatagag gcgcttgagc ccaacccagc accgatgggt 3360

aaagtagact ttaaagaaaa cttaatattc ttggcatggg ggcataggca aacaaacata 3420

tacaggaaac aaaacgctgc atggtagtgg aaggattcgg atagttgagc taacaacgga 3480

tccaaaagac taacgagttc ctgagacaag ccatcggtgg cttgttgagc cttggccaat 3540

ttttgggagt ttacttgatc ctcggtgatg gcattgaaat cattgatgga ccacttatga 3600

ttaaagctaa tgtccgggaa gtccaattca atagtatctg gtgcagatga ctcgcttatt 3660

agcaggtagg ttctcaacgc agacacacta gcagcgacgg caggcttgtt gtacacagca 3720

gagtgttcac caaaaataat aacctttccc ggtgcagaag ttaagaacgg taatgacatt 3780

atagtttttt ctccttgacg ttaaagtata gaggtatatt aacaattttt tgttgatact 3840

tttatgacat ttgaataaga agtaatacaa accgaaaatg ttgaaagtat tagttaaagt 3900

ggttatgcag cttttgcatt tatatatctg ttaatagatc aaaaatcatc gcttcgctga 3960

ttaattaccc cagaaataag gctaaaaaac taatcgcatt attatcctat ggttgttaat 4020

ttgattcgtt gatttgaagg tttgtggggc caggttactg ccaatt tttc ctcttcataa4080

ccataaaagc tagtattgta gaatctttat tgttcggagc agtgcggcgc gaggcacatc 4140

tgcgtttcag gaacgcgacc ggtgaagacc aggacgcacg gaggagagtc ttccgtcgga 4200

gggctgtcgc ccgctcggcg gcttctaatc cgtacttcaa tatagcaatg agcagttaag 4260

cgtattactg aaagttccaa agagaaggtt tttttaggct aagataatgg ggctctttac 4320

atttccacaa catataagta agattagata tggatatgta tatggtggta ttgccatgta 4380

atatgattat taaacttctt tgcgtccatc caaaaaaaaa gtaagaattt ttgaaaattc 4440

aatataaatg tctcagaacg tttacattgt atcgactgcc agaaccccaa ttggttcatt 4500

ccagggttct ctatcctcca agacagcagt ggaattgggt gctgttgctt taaaaggcgc 4560

cttggctaag gttccagaat tggatgcatc caaggatttt gacgaaatta tttttggtaa 4620

cgttctttct gccaatttgg gccaagctcc ggccagacaa gttgctttgg ctgccggttt 4680

gagtaatcat atcgttgcaa gcacagttaa caaggtctgt gcatccgcta tgaaggcaat 4740

cattttgggt gctcaatcca tcaaatgtgg taatgctgat gttgtcgtag ctggtggttg 4800

tgaatctatg actaacgcac catactacat gccagcagcc cgtgcgggtg ccaaatttgg 4860

ccaaactgtt cttgttgatg gtgtcgaaag agatgggttg aacgatgcgt acgatggtct 4920

agccatgggt gtacacgcag aaaagtgtgc ccgtgattgg gatattacta gagaacaaca 4980

agacaatttt gccatcgaat cctaccaaaa atctcaaaaa tctcaaaagg aaggtaaatt 5040

cgacaatgaa attgtacctg ttaccattaa gggatttaga ggtaagcctg atactcaagt 5100

cacgaaggac gaggaacctg ctagattaca cgttgaaaaa ttgagatctg caaggactgt 5160

tttccaaaaa gaaaacggta ctgttactgc cgctaacgct tctccaatca acgatggtgc 5220

tgcagccgtc atcttggttt ccgaaaaagt tttgaaggaa aagaatttga agcctttggc 5280

tattatcaaa ggttggggtg aggccgctca tcaaccagct gattttacat gggctccatc 5340

tcttgcagtt ccaaaggctt tgaaacatgc tggcatcgaa gacatcaatt ctgttgatta 5400

ctttgaattc aatgaagcct tttcggttgt cggtttggtg aacactaaga ttttgaagct 5460

agacccatct aaggttaatg tatatggtgg tgctgttgct ctaggtcacc cattgggttg 5520

ttctggtgct agagtggttg ttacactgct atccatctta cagcaagaag gaggtaagat 5580

cggtgttgcc gccatttgta atggtggtgg tggtgcttcc tctattgtca ttgaaaagat 5640

atgattacgt tctgcgattt tctcatgatc tttttcataa aatacataaa tatataaatg 5700

gctttatgta taacaggcat aatttaaagt tttatttgcg attcatcgtt tttcaggtac 5760

tcaaacgctg aggtgtgcct tttgacttac ttttcccggg agaggctagc agaattaccc 5820

tccacgttga ttgtctgcga ggcaagaatg atcatcaccg tagtgagagt gcgttcaagg 5880

ctcttgcggt tgccataaga gaagccacct cgcccaatgg taccaacgat gttccctcca 5940

ccaaaggtgt tcttatgtag tgacaccgat tatttaaagc tgcagcatac gatatatata 6000

catgtgtata tatgtatacc tatgaatgtc agtaagtatg tatacgaaca gtatgatact 6060

gaagatgaca aggtaatgca tcattctata cgtgtcattc tgaacgaggc gcgctttcct 6120

tttttctttt tgctttttct ttttttttct cttgaactcg agaaaaaaaa tataaaagag 6180

atggaggaac gggaaaaagt tagttgtggt gataggtggc aagtggtatt ccgtaagaac 6240

aacaagaaaa gcatttcata ttatggctga actgagcgaa caagtgcaaa atttaagcat 6300

caacgacaac aacgagaatg gttatgttcc tcctcactta agaggaaaac caagaagtgc 6360

cagaaataac agtagcaact acaataacaa caacggcggc gtttaaac 6408

<210>7

<211>6087

<212>DNA

The artificial sequence of <213>

<220>

The ERG8-PGAL-ERG19 inset of <223>pAM497

<400>7

gtttaaactt ttccaatagg tggttagcaa tcgtcttact ttctaacttt tcttaccttt 60

tacatttcag caatatatat atatatattt caaggatata ccattctaat gtctgcccct 120

aagaagatcg tcgttttgcc aggtgaccac gttggtcaag aaatcacagc cgaagccatt 180

aaggttctta aagctatttc tgatgttcgt tccaatgtca agttcgattt cgaaaatcat 240

ttaattggtg gtgctgctat cgatgctaca ggtgttccac ttccagatga ggcgctggaa 300

gcctccaaga aggctgatgc cgttttgtta ggtgctgtgg gtggtcctaa atggggtacc 360

ggtagtgtta gacctgaaca aggtttacta aaaatccgta aagaacttca attgtacgcc 420

aacttaagac catgtaactt tgcatccgac tctcttttag acttatctcc aatcaagcca 480

caatttgcta aaggtactga cttcgttgtt gtcagagaat tagtgggagg tatttacttt 540

ggtaagagaa aggaagacgt ttagcttgcc tcgtccccgc cgggtcaccc ggccagcgac 600

atggaggccc agaataccct ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt 660

gactgtcgcc cgtacattta gcccatacat ccccatgtat aatcatttgc atccatacat 720

tttgatggcc gcacggcgcg aagcaaaaat tacggctcct cgctgcagac ctgcgagcag 780

ggaaacgctc ccctcacaga cgcgttgaat tgtccccacg ccgcgcccct gtagagaaat 840

ataaaaggtt aggatttgcc actgaggttc ttctttcata tacttccttt taaaatcttg 900

ctaggataca gttctcacat cacatccgaa cataaacaac catggcagaa ccagcccaaa 960

aaaagcaaaa acaaactgtt caggagcgca aggcgtttat ctcccgtatc actaatgaaa 1020

ctaaaattca aatcgctatt tcgctgaatg gtggttatat tcaaataaaa gattcgattc 1080

ttcctgcaaa gaaggatgac gatgtagctt cccaagctac tcagtcacag gtcatcgata 1140

ttcacacagg tgttggcttt ttggatcata tgatccatgc gttggcaaaa cactctggtt 1200

ggtctcttat tgttgaatgt attggtgacc tgcacattga cgatcaccat actaccgaag 1260

attgcggtat cgcattaggg caagcgttca aagaagcaat gggtgctgtc cgtggtgtaa 1320

aaagattcgg tactgggttc gcaccattgg atgaggcgct atcacgtgcc gtagtcgatt 1380

tatctagtag accatttgct gtaatcgacc ttggattgaa gagagagatg attggtgatt 1440

tatccactga aatgattcca cactttttgg aaagtttcgc ggaggcggcc agaattactt 1500

tgcatgttga ttgtctgaga ggtttcaacg atcaccacag aagtgagagt gcgttcaagg 1560

ctttggctgt tgccataaga gaagctattt ctagcaatgg caccaatgac gttccctcaa 1620

ccaaaggtgt tttgatgtga agtactgaca ataaaaagat tcttgttttc aagaacttgt 1680

catttgtata gtttttttat attgtagttg ttctatttta atcaaatgtt agcgtgattt 1740

atattttttt tcgcctcgac atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa 1800

tatcatgcgt caatcgtatg tgaatgctgg tcgctatact gctgtcgatt cgatactaac 1860

gccgccatcc acccgggttt ctcattcaag tggtaactgc tgttaaaatt aagatattta 1920

taaattgaag cttggtcgtt ccgaccaata ccgtagggaa acgtaaatta gctattgtaa 1980

aaaaaggaaa agaaaagaaa agaaaaatgt tacatatcga attgatctta ttcctttggt 2040

agaccagtct ttgcgtcaat caaagattcg tttgtttctt gtgggcctga accgacttga 2100

gttaaaatca ctctggcaac atccttttgc aactcaagat ccaattcacg tgcagtaaag 2160

ttagatgatt caaattgatg gttgaaagcc tcaagctgct cagtagtaaa tttcttgtcc 2220

catccaggaa cagagccaaa caatttatag ataaatgcaa agagtttcga ctcattttca 2280

gctaagtagt acaacacagc atttggacct gcatcaaacg tgtatgcaac gattgtttct 2340

ccgtaaaact gattaatggt gtggcaccaa ctgatgatac gcttggaagt gtcattcatg 2400

tagaatattg gagggaaaga gtccaaacat gtggcatgga aagagttgga atccatcatt 2460

gtttcctttg caaaggtggc gaaatctttt tcaacaatgg ctttacgcat gacttcaaat 2520

ctctttggta cgacatgttc aattctttct ttaaatagtt cggaggttgc cacggtcaat 2580

tgcataccct gagtggaact cacatccttt ttaatatcgc tgacaactag gacacaagct 2640

ttcatctgag gccagtcaga gctgtctgcg atttgtactg ccatggaatc atgaccatct 2700

tcagcttttc ccatttccca ggccacgtat ccgccaaaca acgatctaca agctgaacca 2760

gacccctttc ttgctattct agatatttct gaagttgact gtggtaattg gtataactta 2820

gcaattgcag agaccaatgc agcaaagcca gcagcggagg aagctaaacc agctgctgta 2880

ggaaagttat tttcggagac aatgtggagt ttccattgag ataatgtggg caatgaggcg 2940

tccttcgatt ccatttcctt tcttaattgg cgtaggtcgc gcagacaatt ttgagttctt 3000

tcattgtcga tgctgtgtgg ttctccattt aaccacaaag tgtcgcgttc aaactcaggt 3060

gcagtagccg cagaggtcaa cgttctgagg tcatcttgcg ataaagtcac tgatatggac 3120

gaattggtgg gcagattcaa cttcgtgtcc cttttccccc aatacttaag ggttgcgatg 3180

ttgacgggtg cggtaacgga tgctgtgtaa acggtcatta tagttttttc tccttgacgt 3240

taaagtatag aggtatatta acaatttttt gttgatactt ttatgacatt tgaataagaa 3300

gtaatacaaa ccgaaaatgt tgaaagtatt agttaaagtg gttatgcagc ttttgcattt 3360

atatatctgt taatagatca aaaatcatcg cttcgctgat taattacccc agaaataagg 3420

ctaaaaaact aatcgcatta ttatcctatg gttgttaatt tgattcgttg atttgaaggt 3480

ttgtggggcc aggttactgc caatttttcc tcttcataac cataaaagct agtattgtag 3540

aatctttatt gttcggagca gtgcggcgcg aggcacatct gcgtttcagg aacgcgaccg 3600

gtgaagacca ggacgcacgg aggagagtct tccgtcggag ggctgtcgcc cgctcggcgg 3660

cttctaatcc gtacttcaat atagcaatga gcagttaagc gtattactga aagttccaaa 3720

gagaaggttt ttttaggcta agataatggg gctctttaca tttccacaac atataagtaa 3780

gattagatat ggatatgtat atggtggtat tgccatgtaa tatgattatt aaacttcttt 3840

gcgtccatcc aaaaaaaaag taagaatttt tgaaaattca atataaatgt cagagttgag 3900

agccttcagt gccccaggga aagcgttact agctggtgga tatttagttt tagatccgaa 3960

atatgaagca tttgtagtcg gattatcggc aagaatgcat gctgtagccc atccttacgg 4020

ttcattgcaa gagtctgata agtttgaagt gcgtgtgaaa agtaaacaat ttaaagatgg 4080

ggagtggctg taccatataa gtcctaaaac tggcttcatt cctgtttcga taggcggatc 4140

taagaaccct ttcattgaaa aagttatcgc taacgtattt agctacttta agcctaacat 4200

ggacgactac tgcaatagaa acttgttcgt tattgatatt ttctctgatg atgcctacca 4260

ttctcaggag gacagcgtta ccgaacatcg tggcaacaga agattgagtt ttcattcgca 4320

cagaattgaa gaagttccca aaacagggct gggctcctcg gcaggtttag tcacagtttt 4380

aactacagct ttggcctcct tttttgtatc ggacctggaa aataatgtag acaaatatag 4440

agaagttatt cataatttat cacaagttgc tcattgtcaa gctcagggta aaattggaag 4500

cgggtttgat gtagcggcgg cagcatatgg atctatcaga tatagaagat tcccacccgc 4560

attaatctct aatttgccag atattggaag tgctacttac ggcagtaaac tggcgcattt 4620

ggttaatgaa gaagactgga atataacgat taaaagtaac catttacctt cgggattaac 4680

tttatggatg ggcgatatta agaatggttc agaaacagta aaactggtcc agaaggtaaa 4740

aaattggtat gattcgcata tgccggaaag cttgaaaata tatacagaac tcgatcatgc 4800

aaattctaga tttatggatg gactatctaa actagatcgc ttacacgaga ctcatgacga 4860

ttacagcgat cagatatttg agtctcttga gaggaatgac tgtacctgtc aaaagtatcc 4920

tgagatcaca gaagttagag atgcagttgc cacaattaga cgttccttta gaaaaataac 4980

taaagaatct ggtgccgata tcgaacctcc cgtacaaact agcttattgg atgattgcca 5040

gaccttaaaa ggagttctta cttgcttaat acctggtgct ggtggttatg acgccattgc 5100

agtgattgct aagcaagatg ttgatcttag ggctcaaacc gctgatgaca aaagattttc 5160

taaggttcaa tggctggatg taactcaggc tgactggggt gttaggaaag aaaaagatcc 5220

ggaaacttat cttgataaat aacttaaggt agataatagt ggtccatgtg acatctttat 5280

aaatgtgaag tttgaagtga ccgcgcttaa catctaacca ttcatcttcc gatagtactt 5340

gaaattgttc ctttcggcgg catgataaaa ttcttttaat gggtacaagc tacccgggcc 5400

cgggaaagat tctctttttt tatgatattt gtacataaac tttataaatg aaattcataa 5460

tagaaacgac acgaaattac aaaatggaat atgttcatag ggtagacgaa actatatacg 5520

caatctacat acatttatca agaaggagaa aaaggaggat gtaaaggaat acaggtaagc 5580

aaattgatac taatggctca acgtgataag gaaaaagaat tgcactttaa cattaatatt 5640

gacaaggagg agggcaccac acaaaaagtt aggtgtaaca gaaaatcatg aaactatgat 5700

tcctaattta tatattggag gattttctct aaaaaaaaaa aaatacaaca aataaaaaac 5760

actcaatgac ctgaccattt gatggagttt aagtcaatac cttcttgaac catttcccat 5820

aatggtgaaa gttccctcaa gaattttact ctgtcagaaa cggccttaac gacgtagtcg 5880

acctcctctt cagtactaaa tctaccaata ccaaatctga tggaagaatg ggctaatgca 5940

tcatccttac ccagcgcatg taaaacataa gaaggttcta gggaagcaga tgtacaggct 6000

gaacccgagg ataatgcgat atcccttagt gccatcaata aagattctcc ttccacgtag 6060

gcgaaagaaa cgttaacacg tttaaac 6087

<210>8

<211>1725

<212>DNA

The artificial sequence of <213>

<220>

<223> is for carrying out codon optimized Artemisinin 3-farnesene synthetic enzyme at expression in escherichia coli

<400>8

atggacactc tgccgatctc ttccgtaagc ttttcttcct ctacttcccc gctggtagtc 60

gatgacaagg tttctaccaa acctgatgta attcgtcaca ctatgaactt caacgcatct 120

atctggggcg atcagttcct gacttatgat gaaccggaag atctggtaat gaaaaagcaa 180

ctggtagaag aactgaaaga agaagttaaa aaggaactga tcaccattaa gggtagcaac 240

gaaccgatgc agcacgtgaa actgattgaa ctgatcgatg cggttcagcg tctgggtatt 300

gcttatcatt ttgaagagga aatcgaggaa gctctgcaac acatccacgt aacctacggc 360

gaacaatggg tggataaaga gaatctgcag tctatcagcc tgtggttccg cctgctgcgt 420

cagcaaggtt tcaatgtctc ttctggcgtt ttcaaagact tcatggatga aaagggcaaa 480

ttcaaggaat ccctgtgtaa cgatgcgcaa ggtatcctgg cactgtacga agcggccttc 540

atgcgtgtgg aagacgaaac cattctggac aacgcgctgg aattcactaa agtgcatctg 600

gacatcatcg cgaaagatcc gtcctgcgac tcctctctgc gtactcagat ccatcaagcg 660

ctgaaacagc cgctgcgtcg tcgcctggca cgtattgagg ctctgcacta tatgccgatt 720

taccagcagg aaacctctca cgacgaagtc ctgctgaaac tggctaaact ggacttcagc 780

gttctgcaat ctatgcacaa gaaagaactg tcccacatct gcaaatggtg gaaagatctg 840

gatctgcaaa acaaactgcc gtacgttcgt gaccgtgttg ttgagggcta tttttggatt 900

ctgtccatct actatgaacc acagcacgcg cgtactcgca tgtttctgat gaaaacctgc 960

atgtggctgg ttgtcctgga cgacaccttt gacaactatg gtacgtacga agaactggaa 1020

atcttcaccc aggccgtgga acgttggtct atttcctgcc tggatatgct gccggaatac 1080

atgaaactga tctatcaaga actggttaac ctgcacgtgg aaatggaaga gtctctggag 1140

aaagaaggta aaacttacca gatccactac gtcaaggaga tggcgaaaga actggtccgt 1200

aactatctgg tcgaggcgcg ttggctgaaa gagggctata tgccgactct ggaagaatac 1260

atgagcgtat ccatggttac cggcacctac ggcctgatga ttgcgcgttc ctacgtcggc 1320

cgtggtgata ttgttaccga agataccttt aagtgggttt cttcctaccc gccgatcatc 1380

aaagcgtctt gtgtcatcgt tcgcctgatg gacgacatcg tttctcacaa agaggagcaa 1440

gaacgtggtc acgtagcatc tagcatcgaa tgctactcca aagaatccgg cgcgtccgaa 1500

gaagaagctt gcgaatacat cagccgtaaa gttgaagatg cctggaaagt tatcaaccgc 1560

gaaagcctgc gtccgacggc ggtcccgttt ccgctgctga tgccggcaat caacctggca 1620

cgcatgtgtg aggttctgta cagcgtgaac gatggtttta ctcacgcgga aggtgacatg 1680

aagagctata tgaagagctt cttcgtacac cctatggtcg tatga 1725

<210>9

<211>1743

<212>DNA

The artificial sequence of <213>

<220>

<223> is for carrying out codon optimized Picea excelsa α-farnesene synthetic enzyme at expression in escherichia coli

<400>9

atggacctgg cagtagaaat tgcaatggat ctggctgttg atgatgttga acgtcgtgtg 60

ggtgattacc actctaacct gtgggacgac gactttattc agagcctgtc caccccgtac 120

ggcgcctctt cttaccgcga acgcgctgaa cgtctggttg gtgaagttaa agaaatgttc 180

acctctatta gcatcgaaga cggcgaactg acctctgacc tgctgcagcg tctgtggatg 240

gtggataatg tagaacgcct gggtatctct cgtcacttcg aaaacgaaat caaagccgcg 300

atcgattacg tttattctta ctggtctgat aaaggcattg ttcgtggtcg tgatagcgcg 360

gttccggacc tgaatagcat cgcgctgggt ttccgtaccc tgcgcctgca tggttacact 420

gtttctagcg atgtttttaa agtttttcag gaccgtaaag gcgaatttgc ttgttctgcg 480

atcccgaccg agggtgatat taagggcgtt ctgaacctgc tgcgtgcatc ctatattgca 540

ttcccgggtg aaaaagtgat ggagaaagca cagaccttcg ctgcaaccta cctgaaagaa 600

gcactgcaga aaatccaggt ttccagcctg agccgtgaga tcgaatatgt gctggaatat 660

ggttggctga ctaacttccc gcgtctggaa gctcgtaact atatcgatgt tttcggtgaa 720

gaaatctgtc cgtacttcaa aaagccgtgc attatggttg ataaactgct ggaactggca 780

aaactggaat tcaacctgtt ccactctctg cagcagaccg aactgaaaca tgtaagccgc 840

tggtggaaag attctggttt ttcccagctg actttcaccc gtcaccgtca cgtggaattc 900

tacaccctgg catcttgtat cgctattgag ccgaaacatt ccgcattccg tctgggcttc 960

gcgaaagtgt gctatctggg tattgtgctg gatgatatct acgatacttt tggcaagatg 1020

aaagaactgg aactgtttac cgctgctatt aaacgctggg acccgtctac tactgagtgc 1080

ctgccggaat atatgaaagg cgtttatatg gccttctata actgtgtaaa tgaactggcc 1140

ctgcaggcgg agaagaccca gggtcgtgac atgctgaact acgcgcgcaa agcctgggaa 1200

gcactgttcg acgcgttcct ggaagaagca aaatggatta gctccggtta cctgccgacc 1260

ttcgaagaat acctggaaaa cggcaaggta tctttcggtt accgtgcggc cactctgcag 1320

ccaattctga ctctggatat tccgctgccg ctgcacatcc tgcagcaaat cgacttcccg 1380

tctcgtttta acgatctggc tagcagcatc ctgcgtctgc gtggtgacat ctgcggctac 1440

caggctgaac gttctcgtgg tgaagaagcc tcttccatct cttgctatat gaaagataat 1500

ccgggctcta ccgaagagga cgcgctgtct cacattaacg ccatgatctc tgataacatt 1560

aacgagctga attgggaact gctgaaaccg aactctaatg taccgatctc ctctaagaaa 1620

cacgcgttcg atatcctgcg tgcgttctac cacctgtaca agtatcgtga cggtttctct 1680

atcgctaaga tcgaaactaa gaacctggtg atgcgtaccg ttctggagcc ggtaccgatg 1740

taa 1743

<210>10

<211>1737

<212>DNA

The artificial sequence of <213>

<220>

<223> carries out codon optimized Artemisinin β-farnesene synthetic enzyme for expressing in yeast saccharomyces cerevisiae

<400>10

ggatccatgt caactttgcc tatttcttct gtgtcatttt cctcttctac atcaccatta 60

gtcgtggacg acaaagtctc aaccaagccc gacgttatca gacatacaat gaatttcaat 120

gcttctattt ggggagatca attcttgacc tatgatgagc ctgaagattt agttatgaag 180

aaacaattag tggaggaatt aaaagaggaa gttaagaagg aattgataac tatcaaaggt 240

tcaaatgagc ccatgcagca tgtgaaattg attgaattaa ttgatgctgt tcaacgttta 300

ggtatagctt accattttga agaagagatc gaggaagctt tgcaacatat acatgttacc 360

tatggtgaac agtgggtgga taaggaaaat ttacagagta tttcattgtg gttcaggttg 420

ttgcgtcaac agggctttaa cgtctcctct ggcgttttca aagactttat ggacgaaaaa 480

ggtaaattca aagagtcttt atgcaatgat gcacaaggaa tattagcctt atatgaagct 540

gcatttatga gggttgaaga tgaaaccatc ttagacaatg ctttggaatt cacaaaagtt 600

catttagata tcatagcaaa agacccatct tgcgattctt cattgcgtac acaaatccat 660

caagccttaa aacaaccttt aagaaggaga ttagcaagga ttgaagcatt acattacatg 720

ccaatctacc aacaggaaac atctcatgat gaagtattgt tgaaattagc caagttggat 780

ttcagtgttt tgcagtctat gcataaaaag gaattgtcac atatctgtaa gtggtggaaa 840

gatttagatt tacaaaataa gttaccttat gtacgtgatc gtgttgtcga aggctacttc 900

tggatattgt ccatatacta tgagccacaa cacgctagaa caagaatgtt tttgatgaaa 960

acatgcatgt ggttagtagt tttggacgat acttttgata attatggaac atacgaagaa 1020

ttggagattt ttactcaagc cgtcgagaga tggtctatct catgcttaga tatgttgccc 1080

gaatatatga aattaatcta ccaagaatta gtcaatttgc atgtggaaat ggaagaatct 1140

ttggaaaagg agggaaagac ctatcagatt cattacgtta aggagatggc taaagaatta 1200

gttcgtaatt acttagtaga agcaagatgg ttgaaggaag gttatatgcc tactttagaa 1260

gaatacatgt ctgtttctat ggttactggt acttatggtt tgatgattgc aaggtcctat 1320

gttggcagag gagacattgt tactgaagac acattcaaat gggtttctag ttacccacct 1380

attattaaag cttcctgtgt aatagtaaga ttaatggacg atattgtatc tcacaaggaa 1440

gaacaagaaa gaggacatgt ggcttcatct atagaatgtt actctaaaga atcaggtgct 1500

tctgaagagg aagcatgtga atatattagt aggaaagttg aggatgcctg gaaagtaatc 1560

aatagagaat ctttgcgtcc aacagccgtt cccttccctt tgttaatgcc agcaataaac 1620

ttagctagaa tgtgtgaggt cttgtactct gttaatgatg gttttactca tgctgagggt 1680

gacatgaaat cttatatgaa gtccttcttc gttcatccta tggtcgtttg actcgag 1737

<210>11

<211>1755

<212>DNA

The artificial sequence of <213>

<220>

<223> carries out codon optimized Picea excelsa α-farnesene synthetic enzyme for expressing in yeast saccharomyces cerevisiae

<400>11

ggatccatgg atttggctgt tgagattgca atggacttgg ctgtggatga tgtcgaaaga 60

agggtgggcg attaccattc taacttatgg gacgatgatt ttatacaatc attaagtact 120

ccttacggtg ctagttcata cagagaaaga gcagagagat tagtgggtga ggtcaaagag 180

atgtttacat ccatctctat agaggatgga gaattaacaa gtgatttatt acagcgtttg 240

tggatggttg ataatgtaga gagattggga atttcccgtc attttgagaa tgagatcaag 300

gcagctattg actatgtcta ttcctactgg agtgataagg gcatcgttag aggtagggat 360

tctgcagtgc ctgatttaaa ctctattgcc ttaggtttta gaacattgag attacatggt 420

tataccgtct cttccgacgt attcaaagtt tttcaagata gaaagggtga attcgcatgt 480

agtgccatcc caactgaggg cgacattaag ggtgttttaa acttgttgag agcttcatac 540

atcgcctttc caggagaaaa agttatggaa aaagctcaaa catttgctgc tacttatttg 600

aaagaggcat tgcaaaagat tcaggtttcc tctttgtcac gtgagatcga atacgtatta 660

gaatacggct ggttgacaaa cttccctaga ttagaagcca gaaactatat cgatgtattc 720

ggtgaggaga tttgcccata tttcaagaag ccttgtataa tggttgataa gttattggaa 780

ttggctaagt tagaatttaa tttatttcac tcattacaac agaccgaatt gaaacacgtt 840

tctagatggt ggaaagactc tggcttttct caattaactt ttacacgtca tagacatgtc 900

gaattctata cattggcctc ctgtattgca attgaaccta aacactctgc atttaggtta 960

ggtttcgcca aggtttgcta cttaggcatc gtattagatg atatttacga caccttcgga 1020

aagatgaagg aattagaatt attcactgcc gcaataaaaa gatgggaccc ttcaacaaca 1080

gaatgtttac cagaatacat gaaaggtgtg tacatggcat tctataactg cgttaatgaa 1140

ttggctttac aagcagagaa aactcaagga agggatatgt taaactacgc acgtaaggcc 1200

tgggaggctt tgttcgatgc ttttttggaa gaagccaaat ggatatctag tggttactta 1260

ccaaccttcg aagaatattt agaaaatggt aaagtatctt tcggatatag agctgcaact 1320

ttgcagccaa ttttaacttt ggatatacct ttgcccttgc atattttgca gcaaatagac 1380

ttcccttcta gatttaatga tttagctagt agtattttga gattgcgtgg cgatatatgc 1440

ggttaccaag cagagagatc tagaggtgag gaagcatcat ctataagttg ctatatgaag 1500

gataatcccg gttctacaga agaagacgcc ttatctcaca taaacgctat gatatctgat 1560

aacattaacg agttgaactg ggagttatta aagccaaatt ccaatgttcc aatatcaagt 1620

aaaaagcacg cttttgatat attacgtgct ttctatcact tgtataagta tcgtgatggt 1680

ttttctatcg caaagattga aactaagaat ttggttatga gaactgtgtt ggaaccagta 1740

ccaatgtgac tcgag 1755

<210>12

<211>2121

<212>DNA

The artificial sequence of <213>

<220>

<223> is from the KanMX-PMET3 region of pAM328

<220>

<221>misc_feature

<222>13，2109

<223>n＝A，T，C or G

<400>12

gaattcgccc ttntggatgg cggcgttagt atcgaatcga cagcagtata gcgaccagca 60

ttcacatacg attgacgcat gatattactt tctgcgcact taacttcgca tctgggcaga 120

tgatgtcgag gcgaaaaaaa atataaatca cgctaacatt tgattaaaat agaacaacta 180

caatataaaa aaactataca aatgacaagt tcttgaaaac aagaatcttt ttattgtcag 240

tactgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt 300

atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca 360

gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat 420

acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt 480

gacgactgaa tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac 540

aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg 600

tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg 660

aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc 720

aggatattct tctaatacct ggaatgctgt tttgccgggg atcgcagtgg tgagtaacca 780

tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag 840

ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt 900

cagaaacaac tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg 960

cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa 1020

tcgcggcctc gaaacgtgag tcttttcctt acccatggtt gtttatgttc ggatgtgatg 1080

tgagaactgt atcctagcaa gattttaaaa ggaagtatat gaaagaagaa cctcagtggc 1140

aaatcctaac cttttatatt tctctacagg ggcgcggcgt ggggacaatt caacgcgtct 1200

gtgaggggag cgtttccctg ctcgcaggtc tgcagcgagg agccgtaatt tttgcttcgc 1260

gccgtgcggc catcaaaatg tatggatgca aatgattata catggggatg tatgggctaa 1320

atgtacgggc gacagtcaca tcatgcccct gagctgcgca cgtcaagact gtcaaggagg 1380

gtattctggg cctccatgtc gctggccggg tgacccggcg gggacgaggc aagctaaaca 1440

gatctgatct tgaaactgag taagatgctc agaatacccg tcaagataag agtataatgt 1500

agagtaatat accaagtatt cagcatattc tcctcttctt ttgtataaat cacggaaggg 1560

atgatttata agaaaaatga atactattac acttcattta ccaccctctg atctagattt 1620

tccaacgata tgtacgtagt ggtataaggt gagggggtcc acagatataa catcgtttaa 1680

tttagtacta acagagactt ttgtcacaac tacatataag tgtacaaata tagtacagat 1740

atgacacact tgtagcgcca acgcgcatcc tacggattgc tgacagaaaa aaaggtcacg 1800

tgaccagaaa agtcacgtgt aattttgtaa ctcaccgcat tctagcggtc cctgtcgtgc 1860

acactgcact caacaccata aaccttagca acctccaaag gaaatcaccg tataacaaag 1920

ccacagtttt acaacttagt ctcttatgaa gttacttacc aatgagaaat agaggctctt 1980

tctcgagaaa tatgaatatg gatatatata tatatatata tatatatata tatatatgta 2040

aacttggttc ttttttagct tgtgatctct agcttgggtc tctctctgtc gtaacagttg 2100

tgatatcgna agggcgaatt c 2121

<210>13

<211>49

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 4-49mvaA SpeI

<400>13

gctactagta ggaggaaaac atcatgcaaa gtttagataa gaatttccg 49

<210>14

<211>39

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 4-49mvaAR XbaI

<400>14

gcttctagac tattgttgtc taatttcttg taaaatgcg 39

<210>15

<211>52

<212>DNA

The artificial sequence of <213>

<220>

<223> primer HMGS 3 (initially) Sa mvaS-AS

<400>15

ttgcatgatg ttttcctcct actagttact ctggtctgtg atattcgcga ac 52

<210>16

<211>55

<212>DNA

The artificial sequence of <213>

<220>

<223> primer HMGS 5 (initially) Sa mvaS-S

<400>16

gaactgaaga tctaggagga aagcaaaatg acaataggta tcgacaaaat aaact 55

<210>17

<211>42

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 19-25atoB SfiI-S

<400>17

gctaggccat cctggccatg aagaactgtg tgattgtttc tg 42

<210>18

<211>33

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 19-25mvaA-AsiSI-AS

<400>18

gcttgcgatc gccggcggat ttgtcctact cag 33

<210>19

<211>45

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-1A-C

<400>19

acactcgagg aggaataaat gagttttgat attgccaaat acccg 45

<210>20

<211>37

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-1B-C

<400>20

tgatggtacc ttatgccagc caggccttga ttttggc 37

<210>21

<211>45

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-1C-C

<400>21

actaggtacc aggaggaata aatgaagcaa ctcaccattc tgggc 45

<210>22

<211>37

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-1D-C

<400>22

aattgatggg ccctcagctt gcgagacgca tcacctc 37

<210>23

<211>45

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-1E-C

<400>23

cataaagggc ccaggaggaa taaatggcaa ccactcattt ggatg 45

<210>24

<211>40

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-1F-C

<400>24

tattgttcat atgttatgta ttctcctgat ggatggttcg 40

<210>25

<211>45

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-1G-C

<400>25

aactaacaca tatgaggagg aataaatgcg gacacagtgg ccctc 45

<210>26

<211>38

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-1H-C

<400>26

tgttagttac gcgtttaaag catggctctg tgcaatgg 38

<210>27

<211>45

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-2A-C

<400>27

acgggatcca ggaggaataa atgcgaattg gacacggttt tgacg 45

<210>28

<211>41

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-2B-C

<400>28

tttagttggg ccctcatttt gttgccttaa tgagtagcgc c 41

<210>29

<211>49

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-2C-C

<400>29

tactaagggc ccaggaggaa ataatgcata accaggctcc aattcaacg 49

<210>30

<211>40

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-2D-C

<400>30

tccgggtacc ttatttttca acctgctgaa cgtcaattcg 40

<210>31

<211>43

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-2E-C

<400>31

aacaggtacc aggaggaaat aatgcagatc ctgttggcca acc 43

<210>32

<211>45

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-2F-C

<400>32

tggatgaagt cgacttaatc gacttcacga atatcgacac gcagc 45

<210>33

<211>50

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-2G-C

<400>33

catcaagtcg acaggaggaa ataatgcaaa cggaacacgt cattttattg 50

<210>34

<211>40

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-2H-C

<400>34

taatgcaagc ttatttaagc tgggtaaatg cagataatcg 40

<210>35

<211>44

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-2I-C

<400>35

cagtaaagct taggaggaaa taatggactt tccgcagcaa ctcg 44

<210>36

<211>41

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 67-2J-C

<400>36

tagttccatg gttatttatt acgctggatg atgtagtccg c 41

<210>37

<211>35

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 9-156A

<400>37

acatagacgt cgggaaagcg aggatctagg taggg 35

<210>38

<211>37

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 9-156B

<400>38

ttcccgctcg aggtggcgga ccatataggc agatcag 37

<210>39

<211>30

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK001-G

<400>39

gtttaaacta ctattagctg aattgccact 30

<210>40

<211>46

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK002-G

<400>40

actgcaaagt acacatatat cccgggtgtc agctctttta gatcgg 46

<210>41

<211>46

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK003-G

<400>41

ccgatctaaa agagctgaca cccgggatat atgtgtactt tgcagt 46

<210>42

<211>30

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK004-G

<400>42

gtttaaacgg cgtcagtcca ccagctaaca 30

<210>43

<211>30

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK005-G

<400>43

gtttaaactt gctaaattcg agtgaaacac 30

<210>44

<211>46

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK006-G

<400>44

aaagatgaat tgaaaagctt cccgggtatg gaccctgaaa ccacag 46

<210>45

<211>46

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK007-G

<400>45

ctgtggtttc agggtccata cccgggaagc ttttcaattc atcttt 46

<210>46

<211>30

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK008-G

<400>46

gtttaaaccc aacaataata atgtcagatc 30

<210>47

<211>30

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK009-G

<400>47

gtttaaacta ctcagtatat taagtttcga 30

<210>48

<211>70

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK010-G

<400>48

atctctcgca agagtcagac tgactcccgg gcgtgaataa gcttcgggtg acccttatgg 60

cattcttttt 70

<210>49

<211>70

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK011-G

<400>49

aaaaagaatg ccataagggt cacccgaagc ttattcacgc ccgggagtca gtctgactct 60

tgcgagagat 70

<210>50

<211>30

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK012-G

<400>50

gtttaaacaa tttagtgtct gcgatgatga 30

<210>51

<211>30

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK013-G

<400>51

gtttaaacta ttgtgagggt cagttatttc 30

<210>52

<211>44

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK014alt-G

<400>52

gcggggacga ggcaagctaa actttagtat attcttcgaa gaaa 44

<210>53

<211>44

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK015alt-G

<400>53

tttcttcgaa gaatatacta aagtttagct tgcctcgtcc ccgc 44

<210>54

<211>60

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK017-G

<400>54

cgatactaac gccgccatcc acccgggaga ggctagcaga attaccctcc acgttgattg 60

<210>55

<211>30

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK018-G

<400>55

gtttaaacgc cgccgttgtt gttattgtag 30

<210>56

<211>30

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK019-G

<400>56

gtttaaactt ttccaatagg tggttagcaa 30

<210>57

<211>55

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67CPK020-G

<400>57

gggtgacccg gcggggacga ggcaagctaa acgtcttcct ttctcttacc aaagt 55

<210>58

<211>55

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK021-G

<400>58

actttggtaa gagaaaggaa gacgtttagc ttgcctcgtc cccgccgggt caccc 55

<210>59

<211>62

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK022-G

<400>59

aatatcataa aaaaagagaa tctttcccgg gtggatggcg gcgttagtat cgaatcgaca 60

gc 62

<210>60

<211>62

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK023-G

<400>60

gctgtcgatt cgatactaac gccgccatcc acccgggaaa gattctcttt ttttatgata 60

tt 62

<210>61

<211>45

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK024-G

<400>61

gtttaaacgt gttaacgttt ctttcgccta cgtggaagga gaatc 45

<210>62

<211>35

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK025-G

<400>62

tccccccggg ttaaaaaaaa tccttggact agtca 35

<210>63

<211>35

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK031-G

<400>63

tccccccggg agttatgaca attacaacaa cagaa 35

<210>64

<211>30

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK032-G

<400>64

tccccccggg tatatatata tcattgttat 30

<210>65

<211>30

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK035-G

<400>65

tccccccggg aaaagtaagt caaaaggcac 30

<210>66

<211>30

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK040-G

<400>66

tccccccggg atggtctgct taaatttcat 30

<210>67

<211>45

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK041-G

<400>67

tccccccggg tagcttgtac ccattaaaag aattttatca tgccg 45

<210>68

<211>30

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK046-G

<400>68

tccccccggg tttctcattc aagtggtaac 30

<210>69

<211>30

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK047-G

<400>69

tccccccggg taaataaaga aaataaagtt 30

<210>70

<211>47

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK050-G

<400>70

aatttttgaa aattcaatat aaatggcttc agaaaaagaa attagga 47

<210>71

<211>47

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK051-G

<400>71

tcctaatttc tttttctgaa gccatttata ttgaattttc aaaaatt 47

<210>72

<211>51

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK052-G

<400>72

agttttcacc aattggtctg cagccattat agttttttct ccttgacgtt a 51

<210>73

<211>51

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK053-G

<400>73

taacgtcaag gagaaaaaac tataatggct gcagaccaat tggtgaaaac t 51

<210>74

<211>47

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK054-G

<400>74

aatttttgaa aattcaatat aaatgaaact ctcaactaaa ctttgtt 47

<210>75

<211>47

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK055-G

<400>75

aacaaagttt agttgagagt ttcatttata ttgaattttc aaaaatt 47

<210>76

<211>47

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK056-G

<400>76

aatttttgaa aattcaatat aaatgtctca gaacgtttac attgtat 47

<210>77

<211>47

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK057-G

<400>77

atacaatgta aacgttctga gacatttata ttgaattttc aaaaatt 47

<210>78

<211>51

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK058-G

<400>78

tgcagaagtt aagaacggta atgacattat agttttttct ccttgacgtt a 51

<210>79

<211>51

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK059-G

<400>79

taacgtcaag gagaaaaaac tataatgtca ttaccgttct taacttctgc a 51

<210>80

<211>47

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK060-G

<400>80

aatttttgaa aattcaatat aaatgtcaga gttgagagcc ttcagtg 47

<210>81

<211>47

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK061-G

<400>81

cactgaaggc tctcaactct gacatttata ttgaattttc aaaaatt 47

<210>82

<211>51

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK062-G

<400>82

ggtaacggat gctgtgtaaa cggtcattat agttttttct ccttgacgtt a 51

<210>83

<211>51

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK063-G

<400>83

taacgtcaag gagaaaaaac tataatgacc gtttacacag catccgttac c 51

<210>84

<211>47

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK064-G

<400>84

aatttttgaa aattcaatat aaatgactgc cgacaacaat agtatgc 47

<210>85

<211>47

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK065-G

<400>85

gcatactatt gttgtcggca gtcatttata ttgaattttc aaaaatt 47

<210>86

<211>32

<212>DNA

The artificial sequence of <213>

<220>

<223> primer A

<400>86

ccatggacac tctgccgatc tcttccgtaa gc 32

<210>87

<211>28

<212>DNA

The artificial sequence of <213>

<220>

<223> primer B

<400>87

gagctctcat acgaccatag ggtgtacg 28

<210>88

<211>25

<212>DNA

The artificial sequence of <213>

<220>

<223> primer C

<400>88

ccatggacct ggcagtagaa attgc 25

<210>89

<211>27

<212>DNA

The artificial sequence of <213>

<220>

<223> primer D

<400>89

gagctcttac atcggtaccg gctccag 27

<210>90

<211>32

<212>DNA

The artificial sequence of <213>

<220>

<223> primer GW-52-84pAM326 BamHI

<400>90

taataaggat ccatgtcaac tttgcctatt tc 32

<210>91

<211>32

<212>DNA

The artificial sequence of <213>

<220>

<223> primer GW-52-84pAM326 NheI

<400>91

ttatagctag ctcaaacgac cataggatga ac 32

<210>92

<211>60

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK016-G

<400>92

caatcaacgt ggagggtaat tctgctagcc tctcccgggt ggatggcggc gttagtatcg 60

<210>93

<211>66

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 50-56-pw100-G

<400>93

gagtgaacct gctgcctggc gtgctctgac tcagtacatt tcatagtgga tggcggcgtt 60

agtatc 66

<210>94

<211>65

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 50-56-pw101-G

<400>94

cgtgtatacg ttttccgctt ctgctcttcg tcttttctct tcttccgata tcacaactgt 60

tacga 65

<210>95

<211>70

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK066-G

<400>95

ggtaagacgg ttgggtttta tcttttgcag ttggtactat taagaacaat cacaggaaac 60

agctatgacc 70

<210>96

<211>70

<212>DNA

The artificial sequence of <213>

<220>

<223> primer 61-67-CPK067-G

<400>96

ttgcgttttg tactttggtt cgctcaattt tgcaggtaga taatcgaaaa gttgtaaaac 60

gacggccagt 70

Claims

1. fuel composition, it comprises or available from a kind of mixture, this mixture comprises:

A () has the isoprenoid compounds of formula (I) or (II):

Or its steric isomer, wherein Z is H, O-R or O-C (=O) R; R is H, alkyl, cycloalkyl, aryl, alkaryl or aralkyl;

(b) conventional oil component; With

(C) fuel dope;

Wherein fuel dope is selected from antioxidant, stablizer, low temperature flow promoter, ignition dope, defoamer, anti-hazing additive, inhibiter, deicing agent, smoke suppressant, drag reducing additive, metal passivator, dispersion agent, sanitising agent, demulsifying compound, marker, antistatic agent, sterilant and combination thereof;

Based on the cumulative volume of this fuel composition, the amount of this isoprenoid compounds is at least 1vol.%, and the amount of this conventional oil component is at least 5vol.%;

This fuel composition has the flash-point being equal to or greater than 38 DEG C, and has the initial boiling point between 100 DEG C to 200 DEG C.

2. fuel composition as claimed in claim 1, its T90 distillation temperature is 282 DEG C to 338 DEG C.

3. fuel composition as claimed in claim 1, wherein said isoprenoid compounds accounts for the 5vol.% to 90vol.% of the cumulative volume of this fuel composition.

4. fuel composition as claimed in claim 1, wherein said isoprenoid compounds accounts for the 5vol.% to 10vol.% of the cumulative volume of this fuel composition.

5. fuel composition as claimed in claim 1, wherein said isoprenoid compounds accounts for the 15vol.% to 25vol.% of the cumulative volume of this fuel composition.

6. fuel composition as claimed in claim 1, the cumulative volume that wherein said isoprenoid compounds accounts for this fuel composition is less than 75vol.%.

7. fuel composition as claimed in claim 1, wherein this fuel composition has the sulphur content being less than 20ppm based on its gross weight.

8. fuel composition as claimed in claim 1, wherein this fuel composition has and accounts for the aromatic content that its cumulative volume is less than 20%.

9. fuel composition as claimed in claim 1, wherein this fuel composition has the initial boiling point between 100 DEG C to 150 DEG C.

10. fuel composition as claimed in claim 1, wherein this fuel composition has the full boiling point being greater than 200 DEG C.

11. fuel compositions as claimed in claim 1, wherein said initial boiling point is between 100 DEG C and 150 DEG C, and this fuel composition has the full boiling point being greater than 300 DEG C.

12. fuel compositions as claimed in claim 1, wherein said conventional oil component comes from oil or coal.

13. fuel compositions as claimed in claim 12, wherein said conventional oil component comprises diesel oil fuel, petroleum diesel fuel, Jet A fuel, Jet A-1 fuel, kerosene or its combination.

14. fuel compositions as claimed in claim 1, wherein said conventional oil component comprises distillation diesel oil fuel, and wherein the amount of this distillation diesel oil fuel is at least 10% of this fuel composition gross weight.

15. fuel composition as claimed in claim 1, wherein said isoprenoid compounds is for having the compound of formula (IV):

Or its steric isomer, wherein R is H, alkyl, cycloalkyl, aryl, alkaryl or aralkyl.

16. fuel compositions as claimed in claim 15, wherein said R is C _1-c ₃alkyl.

17. fuel compositions as claimed in claim 16, wherein said R is methyl.

18. fuel composition as claimed in claim 1, wherein said isoprenoid compounds is for having the compound of formula (IV):

19. fuel composition, it comprises or available from a kind of mixture, this mixture comprises:

(a) by volume at least 50% conventional oil component;

(b) by volume at least 5% but be less than the formula (I) of 45% or the isoprenoid compounds of (II):

Or its steric isomer, wherein Z is H, O-R or O-C (=O) R; R is H, alkyl, cycloalkyl, aryl, alkaryl or aralkyl; With

(c) fuel dope;

Wherein fuel dope selects antioxidant, stablizer, low temperature flow promoter, ignition dope, defoamer, anti-hazing additive, inhibiter, deicing agent, smoke suppressant, drag reducing additive, metal passivator, dispersion agent, sanitising agent, demulsifying compound, marker, antistatic agent, sterilant and combination thereof;

20. fuel composition as claimed in claim 19, wherein said conventional oil component is JetA fuel, Jet A-1 fuel.

21. fuel compositions as claimed in claim 19, wherein said conventional oil component is diesel oil fuel.

22. fuel compositions as claimed in claim 21, wherein said diesel oil fuel is distillation diesel oil fuel.

23. fuel compositions as claimed in claim 19, wherein said isoprenoid compounds is

Or its steric isomer, wherein Z is OH, or

24. fuel composition as claimed in claim 23, wherein said isoprenoid compounds is formula (IV), and wherein R is C _1-c ₃alkyl.

25. fuel compositions as claimed in claim 24, wherein said R is methyl.

26. methods preparing fuel composition, it comprises the isoprenoid compounds by having formula (I) or (II)

Or its steric isomer, with conventional oil component, fuel dope or its combine and mix mutually, wherein Z is H, O-R or O-C (=O) R; R is H, alkyl, cycloalkyl, aryl, alkaryl or aralkyl; The isoprenoid compounds of wherein said formula (I) or (II) is by C ₁₅isoprenoid starting raw material obtains through chemical conversion;

27. method as claimed in claim 26, the T90 distillation temperature of wherein said fuel composition is 282 DEG C to 338 DEG C.

28. method as claimed in claim 26, the amount of wherein said isoprenoid compounds is the 5vol.% to 90vol.% of this fuel composition cumulative volume.

29. methods as claimed in claim 26, wherein said isoprenoid compounds is by C ₁₅isoprenoid starting raw material obtains through chemical conversion.

30. methods as claimed in claim 29, wherein said C ₁₅isoprenoid starting raw material is

Or its steric isomer.

31. method as claimed in claim 26, wherein said fuel composition has the sulphur content being less than 20ppm based on its gross weight.

32. methods as claimed in claim 26, wherein said C ₁₅isoprenoid starting raw material can obtain from biogenetic derivation.

33. methods as claimed in claim 32, wherein said C ₁₅isoprenoid starting raw material is

Or its steric isomer, it is hydrogenated or esterification generates

34. methods as claimed in claim 33, wherein R is C _1-c ₃alkyl.

35. methods as claimed in claim 34, wherein R is methyl.

36. methods as claimed in claim 32, wherein said C ₁₅isoprenoid starting raw material is

Or its steric isomer, it is hydrogenated or esterification generates

37. methods as claimed in claim 36, wherein R is C _1-c ₃alkyl.

38. methods as claimed in claim 37, wherein R is methyl.

39. methods as claimed in claim 26, wherein said conventional oil component is distillation diesel oil fuel.

40. fuel prepared by method according to claim 26.

41. fuel compositions as claimed in claim 1, it comprises at least two kinds of different compounds or its steric isomer, and described compound separately has formula (III), (IV) or (V):

Wherein R is C _1-c ₅alkyl, and the amount that described two kinds of compounds exist at least accounts for the 5vol.% of this fuel composition cumulative volume respectively.

42. comprise in oil engine, the fuel container be connected with this oil engine and fuel container as claim 1-25,41 and the vehicle of fuel composition described in 44-46 any one, wherein this fuel composition is used for providing power to this oil engine.

43. vehicles as claimed in claim 42, wherein said oil engine is diesel engine.

44. fuel compositions as claimed in claim 1, its T10 distillation temperature is 225 DEG C to 275 DEG C.

45. fuel composition as claimed in claim 1, the boiling point of wherein said fuel composition is 140 DEG C to 320 DEG C.

46. fuel compositions as claimed in claim 1, the boiling point of wherein said fuel composition is lower than 200 DEG C.

47. provide the method for power to engine, it comprises the step of the fuel composition as claimed in claim 1 that burns.

48. as claim 1-25, and 41 and fuel composition described in 44-46 any one, wherein isoprenoid compounds is Bioengineered C ₁₅isoprenoid compounds.

49. as claim 1-25, and 41 and fuel composition described in 44-46 any one, wherein isoprenoid compounds is prepared by farnesene, and wherein farnesene is by preparing with microorganism.

50. as claim 1-25, and 41 and fuel composition described in 44-46 any one, wherein isoprenoid compounds comes from monose.

51. fuel compositions as claimed in claim 50, wherein said monose is glucose, semi-lactosi, seminose, fructose, ribose or its combination.

52. prepare the method for fuel composition by monose, and it comprises the steps:

A () is being suitable for generating C ₁₅under the condition of isoprenoid starting raw material, this C can be generated ₁₅the cell of isoprenoid starting raw material contacts with described monose;

(b) hydrogenation and selective alkylation or this C of esterification ₁₅isoprenoid starting raw material, to form C ₁₅isoprenoid compounds; With

C () is by this C ₁₅isoprenoid compounds mixes to prepare this fuel composition with one or more fuel elements and one or more fuel dopes mutually, wherein this C ₁₅isoprenoid compounds is the compound with formula (I) or (II):

Or its steric isomer, wherein Z is H, O-R or O-C (=O) R; R is H, alkyl, cycloalkyl, aryl, alkaryl or aralkyl, wherein this C ₁₅isoprenoid compounds accounts for the amount at least 2vol.% of this fuel composition;

Wherein this C ₁₅isoprenoid starting raw material is the compound with one of following structural:

53. methods as claimed in claim 52, wherein said monose are glucose, semi-lactosi, seminose, fructose, ribose or its combination.