CN105586351A - Cyanobacteria aliphatic hydrocarbon key synthesis gene and application thereof - Google Patents
Cyanobacteria aliphatic hydrocarbon key synthesis gene and application thereof Download PDFInfo
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Abstract
The invention discloses a cyanobacteria aliphatic hydrocarbon key synthesis gene and application thereof. The aliphatic hydrocarbon synthesis gene includes the coding genes of fatty acyl-acyl carrier protein reductase (AAP) and fatty aldehyde deformylating oxygenase (ADO). The invention also relates a contractor, wherein the contractor comprises a promoter having activity and the aliphatic hydrocarbon key synthesis gene controlled by the promoter. By converting the contractor into escherichia coli, the genetic engineering escherichia coli can have an aliphatic hydrocarbon synthesis capacity. By converting the at least one contractor into the cyanobacteria, the aliphatic hydrocarbon yield of the genetic engineering cyanobacteria can be significantly improved. The cyanobacteria hydrocarbon production key gene disclosed by the invention can provide a new candidate gene resource for directly synthesizing aliphatic hydrocarbon novel bio-fuels from microorganisms.
Description
Technical field
The present invention relates to energy microorganism germ plasma resource and genetic resources field, specifically a kind of cyanobacteria fatHydrocarbon synthesizes key gene.
Background technology
The energy is the pillar of modern industry, and the worsening shortages of the traditional energy taking fossil fuel as representative is restriction ChinaThe bottleneck of sustainable economic development, the application of reproducible bio-fuel becomes alleviating energy crisis and improves ecological ringOne of the optimal selection in border. The fossil fuel such as widely used gasoline, diesel oil and aviation kerosine in transportationMain component be the aliphatic hydrocarbon of certain chain lengths scope, aliphatic hydrocarbon has high-energy-density, agent of low hygroscopicity, low volatilizationProperty, with the available engine advantage such as compatible mutually, be the best substitute (Keaslingand of traditional petrifying liquid fluid fuelChou2008)。
It may be the material of biogenetic derivation, wherein aliphatic hydrocarbon in a large number that oil, ancient sediments and meteoritic abundance have comprisedAlso be one of member of greatest concern. It is thin that scientist has once proposed rotten algae for the origin of traditional fossil energyBorn of the same parents are theories (HanandCalvin1969) of its constituent. And from the latter stage sixties in last century start aboutIn cyanobacteria, the research of aliphatic hydrocarbon is mainly around the different cyanobacterias of analysis and identification (or other Eukaryotic Algaes)Aliphatic hydrocarbon composition and content launch. The minimum dry cell weight that accounts for of aliphatic hydrocarbon content in the wild cyanobacteria of having reported at present0.01% (Wintersetal.1969), reach as high as 0.26% (Coatesetal.2014). Its chain length is found in researchMainly concentrate between C15 to C20, and taking C17 as main (Hanetal.1968).
As energy microflora of new generation, cyanobacteria has following advantage: (1) and synthesising biological fuelThe differences such as heterotrophic microorganism reactor Escherichia coli, saccharomyces cerevisiae, cyanobacteria can utilize solar energy can dioxyChange carbon as carbon source for growth, possess abundant species, cultivate the advantages such as cost is low, vitality is strong, growth rate is fast.(2) consider from genetic engineering angle, utilize the micro-algae of eucaryon of solar energy and carbon dioxide, high planting with sameThing difference, cyanobacteria is as the host of synthesizing aliphatic hydrocarbons, and its genetic manipulation is more simple and easy, can lose on a large scalePass transformation. (3) cyanobacteria has genetic background relatively clearly. More than 130 strain cyanobacterias are at present announcedWhole genome sequence (Shihetal.2013).
Have benefited from the fast development of molecular biology, bioinformatics and metabolic engineering technology etc., at present in indigo plantIn bacterium, identify two aliphatic hydrocarbon route of synthesis. One is present in most cyanobacterias, is mainly carried by acyl acyl groupBody protein reductase (Fattyacyl-acylcarrierproteinreductase, AAR) and fatty aldehyde piptonychia acyl group addOxygenase (Fattyaldehydedeformylatingoxygenase, ADO) catalysis completes, and its product is mainly straight chainAlkane, side chain alkane and alkene (unsaturated bond is positioned at carbochain inside) are (Schirmeretal.2010). Another approachBe present in minority cyanobacteria, mainly by terminal olefin synzyme (terminalolefinsynthase, OLS) catalysisComplete, its product be terminal olefin (unsaturated bond is positioned at carbochain end) (Mendez-Perezetal.2011).
Based on the qualification of above-mentioned aliphatic hydrocarbon route of synthesis, carry by genetic engineering modified and metabolic engineering optimization at presentThe high aliphatic hydrocarbon output of restructuring cyanobacteria. Taking cytoalgae PCC6803 as example, result of study confirms, recombinant bacteriumThe output of strain can reach 9 times above (Wangetal.2013) of wild strain. But genetic engineering cyanobacteria is synthetic at presentThe level of aliphatic hydrocarbon also reaches far away the level of commercial Application, produces the excavation of hydrocarbon cyanobacteria germ plasm resource and genetic resourcesFurther to improve cyanobacteria aliphatic hydrocarbon combined coefficient and abundant one of the multifarious key factor of hydrocarbon of producing.
Summary of the invention
The object of the invention is to provide a kind of cyanobacteria aliphatic hydrocarbon to synthesize key gene.
For achieving the above object, the technical solution used in the present invention is:
A kind of cyanobacteria hydrocarbon-producing genes: hydrocarbon-producing genes comprises encoding gene (aar) and the fat of acyl ACP reductaseThe encoding gene (ado) of fat aldehyde piptonychia acyl group oxygenase.
The encoding gene of the encoding gene (aar) of described acyl ACP reductase and fatty aldehyde piptonychia acyl group oxygenase(ado) be cyanobacteria NostoccalcicolaFACHB389, Leptolyngbyasp.NIES-30, PhormidiumambiguumNIES-2119，ChroogloeocystissiderophilaNIES-1031，Calothrixsp.The aar of NIES-2101 or Scytonemasp.NIES-2130 and ado.
The encoding gene of the encoding gene (aar) of described acyl ACP reductase and fatty aldehyde piptonychia acyl group oxygenase(ado) there is SEQIDNO:1 and 2, SEQIDNO:3 and 4, SEQIDNO:5 and 6, SEQIDNO:7With 8, SEQIDNO:9 and 10 or SEQIDNO:11 and 12 shown in sequence.
A kind of construct, construct comprises promoter, and the coding of the acyl ACP reductase of this promoter controlThe encoding gene (ado) of gene (aar) and fatty aldehyde piptonychia acyl group oxygenase.
The promoter of described construct is derive from the PcpcB promoter of cyanobacteria or derive from colibacillary T7Promoter.
The encoding gene of the encoding gene (aar) of described acyl ACP reductase and fatty aldehyde piptonychia acyl group oxygenase(ado) be cyanobacteria NostoccalcicolaFACHB389, Leptolyngbyasp.NIES-30, PhormidiumambiguumNIES-2119，ChroogloeocystissiderophilaNIES-1031，Calothrixsp.The aar of NIES-2101 or Scytonemasp.NIES-2130 and ado.
A genetic engineering bacterium for energy synthesizing aliphatic hydrocarbons, engineering bacteria comprises above-mentioned construct.
Further say so, described construct is imported in Escherichia coli by conversion.
Further say so, described construct is imported to e. coli bl21 (DE3) fadE mutant strain by conversionIn.
In the application of a kind of described cyanobacteria hydrocarbon-producing genes as synthesizing aliphatic hydrocarbons.
The beneficial effect that the present invention has:
Using cyanobacteria as microbial expression system synthesis aliphatic hydrocarbon, be to utilize solar energy stabilizing carbon dioxide synthetic rawThe process of thing fuel. This process is the production process of the clean biometric energy, is not subject to the restriction of insufficient raw material, can not increaseAdd carbon emission. Technical scheme of the present invention can be above-mentioned bioenergy preparation process valuable candidate gene money is providedSource.
Meanwhile, cyanobacteria produces hydrocarbon key gene (acyl ACP reductase/fatty aldehyde piptonychia acyl group oxygenase coding baseBecause of) clone have following important function: one, for to further investigate synthetic generation of cyanobacteria aliphatic hydrocarbon from molecular levelIn the mechanism of thanking and cyanobacteria born of the same parents, the physiological function of aliphatic hydrocarbon is laid a good foundation. Two, can understand different cyanobacteria bacterial strainsProduce hydrocarbon characteristic, and resolve the regularity of distribution of different structure aliphatic hydrocarbon in cyanobacteria.
Therefore, the present invention collects cyanobacteria microorganism resource by extensive, and by genome sequencing and hydrocarbon-producing genesClone, is intended to for the cyanobacteria liquid fuel synthetic system with solar energy, carbon dioxide and the direct synthesizing aliphatic hydrocarbons of waterCandidate's germplasm and genetic resources are provided.
Brief description of the drawings
Fig. 1 is the aliphatic hydrocarbon constituent and properties of the algae strain NostoccalcicolaFACHB389 based on GC-MS mensuration.
Fig. 2 is the aliphatic hydrocarbon constituent and properties of the algae strain Leptolyngbyasp.NIES-30 based on GC-MS mensuration.
Fig. 3 be the algae strain PhormidiumambiguumNIES-2119 that measures based on GC-MS aliphatic hydrocarbon composition andCharacteristic.
Fig. 4 is the aliphatic hydrocarbon group of the algae strain ChroogloeocystissiderophilaNIES-1031 based on GC-MS mensurationBecome and characteristic.
Fig. 5 is the aliphatic hydrocarbon constituent and properties of the algae strain Calothrixsp.NIES-2101 based on GC-MS mensuration.
Fig. 6 is the aliphatic hydrocarbon constituent and properties of the algae strain Scytonemasp.NIES-2130 based on GC-MS mensuration.
Fig. 7 is the basic structure of recombinant vector pTZ60. Recombinant vector pTZ60 is as primer, with Nostoc taking 389-1/2CalcicolaFACHB389 genomic DNA is template, and pcr amplification goes out 2065bp fragment, is cloned intoPEASY-blunt carrier, Transformed E .coliTrans1-T1 phage resistance competent cell and obtaining.
Fig. 8 is the basic structure of recombinant vector pTZ61. Recombinant vector pTZ61 is taking 30-1/2 as primer, withLeptolyngbyasp.NIES-30 genomic DNA is template, and pcr amplification goes out 1949bp fragment, is cloned intoPEASY-blunt carrier, Transformed E .coliTrans1-T1 phage resistance competent cell and obtaining.
Fig. 9 is the basic structure of recombinant vector pTZ62. Recombinant vector pTZ62 is taking 2119-1/2 as primer, withPhormidiumambiguumNIES-2119 genomic DNA is template, and pcr amplification goes out 1914bp fragment,Be cloned into pEASY-blunt carrier, Transformed E .coliTrans1-T1 phage resistance competent cell and obtaining.
Figure 10 is the basic structure of recombinant vector pTZ63. Recombinant vector pTZ63 is taking 1031-1/2 as primer, withChroogloeocystissiderophilaNIES-1031 genomic DNA is template, and pcr amplification goes out 1911bp sheetSection, is cloned into pEASY-blunt carrier, Transformed E .coliTrans1-T1 phage resistance competent cell and obtaining.
Figure 11 is the basic structure of recombinant vector pTZ64. Recombinant vector pTZ64 is taking 2101-1/2 as primer, withCalothrixsp.NIES-2101 genomic DNA is template, and pcr amplification goes out 1831bp fragment, is cloned intoPEASY-blunt carrier, Transformed E .coliTrans1-T1 phage resistance competent cell and obtaining.
Figure 12 is the basic structure of recombinant vector pTZ65. Recombinant vector pTZ65 is taking 2130-1/2 as primer, withScytonemasp.NIES-2130 genomic DNA is template, and pcr amplification goes out 1887bp fragment, is cloned intoPEASY-blunt carrier, Transformed E .coliTrans1-T1 phage resistance competent cell and obtaining.
Figure 13 is the basic structure of recombinant vector pTZ68. Cut pTZ60 and reclaim Nostoc with BamHI and XhoI enzymeThe hydrocarbon-producing genes fragment ado-aar of calcicolaFACHB389, cuts pET21b also with BamHI and XhoI enzymeReclaim carrier part, both connect structure and obtain recombinant plasmid pTZ68.
Figure 14 is the basic structure of recombinant vector pTZ69. Cut pTZ61 and reclaim with BamHI and XhoI enzymeThe hydrocarbon-producing genes fragment ado-aar of Leptolyngbyasp.NIES-30, cuts pET21b with BamHI and XhoI enzymeAnd reclaiming carrier part, both connect structure and obtain recombinant plasmid pTZ69.
Figure 15 is the basic structure of recombinant vector pTZ70. Cut pTZ62 and reclaim with BamHI and XhoI enzymeThe hydrocarbon-producing genes fragment ado-aar of PhormidiumambiguumNIES-2119, cuts with BamHI and XhoI enzymePET21b also reclaims carrier part, and both connect structure and obtain recombinant plasmid pTZ70.
Figure 16 is the basic structure of recombinant vector pTZ71. Cut pTZ63 and reclaim with BamHI and XhoI enzymeThe hydrocarbon-producing genes fragment ado-aar of ChroogloeocystissiderophilaNIES-1031, with BamHI and XhoIEnzyme is cut pET21b and is reclaimed carrier part, and both connect structure and obtain recombinant plasmid pTZ71.
Figure 17 is the basic structure of recombinant vector pTZ72. Cut pTZ64 and reclaim with BamHI and NotI enzymeThe hydrocarbon-producing genes fragment ado-aar of Calothrixsp.NIES-2101, cuts pET21b with BamHI and NotI enzymeAnd reclaiming carrier part, both connect structure and obtain recombinant plasmid pTZ72.
Figure 18 is the basic structure of recombinant vector pTZ73. Cut pTZ65 and reclaim with BamHI and XhoI enzymeThe hydrocarbon-producing genes fragment ado-aar of Scytonemasp.NIES-2130, cuts pET21b with BamHI and XhoI enzymeAnd reclaiming carrier part, both connect structure and obtain recombinant plasmid pTZ73.
Figure 19 is the basic structure of recombinant vector pTZ66. Cut pTZ60 with NdeI enzyme and fill, then cut with XhoI enzymeAnd reclaim the hydrocarbon-producing genes fragment ado-aar of NostoccalcicolaFACHB389, be cloned into through same restrictionsIn the pXX47 of restriction endonuclease processing, build and obtain recombinant plasmid pTZ66.
Figure 20 is the aliphatic hydrocarbon constituent and properties of the hydrocarbon-producing genes engineer's blue bacterium ZT187 based on GC-MS mensuration.
Figure 21 is the aliphatic hydrocarbon volume analysis of genetic engineering cyanobacteria ZT187.
Sequence table information:
SEQIDNO:1 is the fatty aldehyde piptonychia acyl group oxygenase encoding gene ado of NostoccalcicolaFACHB389Sequence.
SEQIDNO:2 is the order of the acyl ACP reductase encoding gene aar of NostoccalcicolaFACHB389Row.
SEQIDNO:3 is the fatty aldehyde piptonychia acyl group oxygenase encoding gene ado of Leptolyngbyasp.NIES-30Sequence.
SEQIDNO:4 is the sequence of the acyl ACP reductase encoding gene aar of Leptolyngbyasp.NIES-30.
SEQIDNO:5 is the fatty aldehyde piptonychia acyl group oxygenase encoding gene of PhormidiumambiguumNIES-2119The sequence of ado.
SEQIDNO:6 is the acyl ACP reductase encoding gene aar of PhormidiumambiguumNIES-2119Sequence.
SEQIDNO:7 is that the fatty aldehyde piptonychia acyl group oxygenase of ChroogloeocystissiderophilaNIES-1031 is compiledThe sequence of code gene ado.
SEQIDNO:8 is the acyl ACP reductase encoding gene of ChroogloeocystissiderophilaNIES-1031The sequence of aar.
SEQIDNO:9 is the order of the fatty aldehyde piptonychia acyl group oxygenase encoding gene ado of Calothrixsp.NIES-2101Row.
SEQIDNO:10 is the sequence of the acyl ACP reductase encoding gene aar of Calothrixsp.NIES-2101.
SEQIDNO:11 is the fatty aldehyde piptonychia acyl group oxygenase encoding gene ado of Scytonemasp.NIES-2130Sequence.
SEQIDNO:12 is the sequence of the acyl ACP reductase encoding gene aar of Scytonemasp.NIES-2130.
SEQIDNO:13 is the nucleotide sequence of primer 389F.
SEQIDNO:14 is the nucleotide sequence of primer 389R.
SEQIDNO:15 is the nucleotide sequence of primer 30F.
SEQIDNO:16 is the nucleotide sequence of primer 30R.
SEQIDNO:17 is the nucleotide sequence of primer 2 119F.
SEQIDNO:18 is the nucleotide sequence of primer 2 119R.
SEQIDNO:19 is the nucleotide sequence of primer 1031F.
SEQIDNO:20 is the nucleotide sequence of primer 1031R.
SEQIDNO:21 is the nucleotide sequence of primer 2 101F.
SEQIDNO:22 is the nucleotide sequence of primer 2 101R.
SEQIDNO:23 is the nucleotide sequence of primer 2 130F.
SEQIDNO:24 is the nucleotide sequence of primer 2 130R.
SEQIDNO:25 is ado-aar and the intervening sequence thereof of NostoccalcicolaFACHB389.
SEQIDNO:26 is ado-aar and the intervening sequence thereof of Leptolyngbyasp.NIES-30.
SEQIDNO:27 is ado-aar and the intervening sequence thereof of PhormidiumambiguumNIES-2119.
SEQIDNO:28 is ChroogloeocystissiderophilaNIES-1031's and intervening sequence.
SEQIDNO:29 is ado-aar and the intervening sequence thereof of Calothrixsp.NIES-2101.
SEQIDNO:30 is ado-aar and the intervening sequence thereof of Scytonemasp.NIES-2130.
SEQIDNO:31 is the amino acid sequence of the acyl ACP reductase of FischerellamajorNIES-592.
SEQIDNO:31 is the amino acid order of the fatty aldehyde piptonychia acyl group oxygenase of NostoccalcicolaFACHB389Row.
SEQIDNO:32 is the amino acid sequence of the acyl ACP reductase of NostoccalcicolaFACHB389.
SEQIDNO:33 is the amino acid sequence of the fatty aldehyde piptonychia acyl group oxygenase of Leptolyngbyasp.NIES-30.
SEQIDNO:34 is the amino acid sequence of the acyl ACP reductase of Leptolyngbyasp.NIES-30.
SEQIDNO:35 is the amino of the fatty aldehyde piptonychia acyl group oxygenase of PhormidiumambiguumNIES-2119Acid sequence.
SEQIDNO:36 is the amino acid order of the acyl ACP reductase of PhormidiumambiguumNIES-2119Row.
SEQIDNO:37 is the fatty aldehyde piptonychia acyl group oxygenase of ChroogloeocystissiderophilaNIES-1031Amino acid sequence.
SEQIDNO:38 is the amino of the acyl ACP reductase of ChroogloeocystissiderophilaNIES-1031Acid sequence.
SEQIDNO:39 is the amino acid sequence of the fatty aldehyde piptonychia acyl group oxygenase of Calothrixsp.NIES-2101.
SEQIDNO:40 is the amino acid sequence of the acyl ACP reductase of Calothrixsp.NIES-2101.
SEQIDNO:41 is the amino acid sequence of the fatty aldehyde piptonychia acyl group oxygenase of Scytonemasp.NIES-2130.
SEQIDNO:42 is the amino acid sequence of the acyl ACP reductase of Scytonemasp.NIES-2130.
SEQIDNO:43 is the nucleotide sequence of primer ado-1.
SEQIDNO:44 is the nucleotide sequence of primer ado-2.
SEQIDNO:45 is the nucleotide sequence of primer aar-1.
SEQIDNO:46 is the nucleotide sequence of primer aar-2.
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail. Embodiment described herein saysBright property and non-restrictive form provides. It will be understood to those of skill in the art that can change or revise multiple non-keyParameter and obtain the result of similitude.
Relational language
In the present invention, except as otherwise noted, otherwise Science and Technology noun used herein has this area skillThe implication that art personnel understand conventionally. And, cyanobacteria cultivation used herein, molecular biology, analysisLearn experimental implementation step and be widely used conventional steps in corresponding field. Meanwhile, in order to understand better thisBright, definition and the explanation of relational language are provided below.
As used herein, " cyanobacteria (Cyanobacterium) " is the photoautotrophic prokaryotic micro-organisms of a class,It can utilize solar energy, stabilizing carbon dioxide. Cyanobacteria is also referred to as blue-green algae. In the present invention, " cyanobacteria "" blue-green algae " is used interchangeably.
As used herein, " germ plasm resource (Germplasmresources) " is that all have certain germplasm or baseThe general name of cause reproducible biotype. Germ plasm resource claims again genetic resources. Include but not limited to: ancient groundSide's kind, new commercial variety of cultivating, important genetic stocks, wild kindred plant, microorganism etc.
As used herein, " aliphatic hydrocarbon " is the hydrocarbon with aliphatic compound base attribute. In moleculeBetween carbon atom, link catenulate carbon skeleton, open and the hydrocarbon of Cheng Huan not at two ends, is called open-chain hydrocarbons, is called for short chain hydrocarbon. CauseFor fat has this structure, so be also called fat chain hydrocarbon. Some cyclic hydrocarbon is different from aromatic hydrocarbon in nature, and tenDivide similar fat chain hydrocarbon, this class cyclic hydrocarbon is alicyclic. Like this, aliphatic hydrocarbon just becomes all hydrocarbon except aromatic hydrocarbonGeneral name.
As used herein, " acyl carrier protein (acylcarrierprotein, ACP) " is generally abbreviated as ACP.From the beginning synthetic fatty acid, is to carry out under the catalysis of the aliphatic acid synthesis system of the complex that contains plurality of enzymes, andIn the stage of its acyl group condensation, acyl-CoA is not as direct substrate, but is transferred to the acyl in complexBase carrier albumen reacts. ACP is difficult for dissociating out from combined enzyme agent in animal or yeast, but at large intestine barBacterium can be separated, and molecular weight is 8847 dalton, is made up of 77 amino acid residues. 4 '-phosphoric acid pantothenic acid sulfydrylThe generation that the serine of ethamine and protein portion closes ACP with fat bond is as follows: 4 of coacetylase '-phosphopan tetheine mercaptoBase ethamine is transferred to the ACP of apoenzyme by the reaction of enzyme.
As used herein, " acyl ACP reductase (Fattyacyl-ACPreductase, AAR) " is blue thinA key enzyme in bacterium aliphatic hydrocarbon route of synthesis, catalysis acyl ACP is converted into fatty aldehyde. Coding cyanobacteria acylThe gene of ACP reductase is well known in the art, includes but not limited to: derive from cyanobacteria FischerellamajorNIES-592，NostoccalcicolaFACHB389，Leptolyngbyasp.NIES-30，PhormidiumambiguumNIES-2119，ChroogloeocystissiderophilaNIES-1031，Calothrixsp.The acyl ACP reductase of NIES-2101 and Scytonemasp.NIES-2130.
As used herein, " fatty aldehyde piptonychia acyl group oxygenase (Fattyaldehydedeformylatingoxygenase,ADO) be " key enzyme in cyanobacteria aliphatic hydrocarbon route of synthesis, catalysis fatty aldehyde is converted into aliphatic hydrocarbon, belongs toIn class ferritin superfamily. The gene of coding cyanobacteria fatty aldehyde piptonychia acyl group oxygenase is well known in the art, bagDraw together but be not limited to: deriving from cyanobacteria FischerellamajorNIES-592, NostoccalcicolaFACHB389，Leptolyngbyasp.NIES-30，PhormidiumambiguumNIES-2119，ChroogloeocystisSiderophilaNIES-1031, the fat of Calothrixsp.NIES-2101 and Scytonemasp.NIES-2130Aldehyde piptonychia acyl group oxygenase.
As used herein, " carrier (vector) " refers to and DNA fragmentation (for example, genes of interest) can be insertedWherein allow DNA (for example, genes of interest) to transfer to a kind of nucleic acid delivery vehicle in donee's cells thereby enter.Carrier can be by transforming, and transduction or transfection import host cell, and the DNA fragmentation that it is carried is thin hostIn born of the same parents, obtain and express. Carrier is well known to a person skilled in the art, includes but not limited to: plasmid; Bacteriophage; KeThis plasmid etc.
" homogeneity " or " homogeneity percentage " refers between two amino acid sequences or between two nucleotide sequencesSequence homogeneity. For determining the homogeneity percentage of two amino acid sequences or two nucleic acid, with relatively order of the bestSequence is compared. Homogeneity percentage between two sequences is the same position that had by these sequencesFunction (, the sum of the number/position of homogeneity percentage=same position (for example, lap position) of number× 100). For example, " homogeneity percentage " calculates by following manner: in comparison window, compare two through the bestThe sequence of comparison, is determined at the number that occurs the position of identical nucleotide base or same amino acid residue in two sequencesOrder is to produce the number of matched position, total number (, the window by the number of matched position divided by position in comparison windowSize), thereby and result be multiplied by 100 produce sequence homogeneity percentages. For sequence relativelyGood comparison can be undertaken by following: for example, and the algorithm (Smithand of local homology of Smith and WatermanWaterman1981); Sequence analysis algorithm (the Needlema.SbandWunsch of Needleman and Wunsch1970); The similarity searching method (PearsonandLipman1988) of Pearson and Lipman; These algorithmsComputerization implement (for example, WisconsinGeneticsSoftwarePackage, GeneticsComputerGroup, 575ScienceDr., Madison, GAP, BESTFIT in Wis., FASTA, BLASTP,BLASTN and TFASTA).
It is at least about 50% that the related homogeneity percentage of embodiment of the present invention comprises, or at least about 60%,At least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least largeApproximately 90%, or higher, for example about 95%, or about 96%, or about 97%, or about 98%, or approximately99%, for example at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％。
Embodiment 1: the cultivation of cyanobacteria and biomass estimation
The cultivation of cyanobacteria and physiological index determining:
Described table 1 cyanobacteria bacterial strain adopts ventilation training method to cultivate, and algae strain is seeded to is equipped with 300mLBG11 trainingSupport in the 500mL triangular flask of base, at 30 DEG C, 30 μ E.m-2.s-1Under continuous light, cultivate 8 to 10 days. For measuringIts increment, the cyanobacteria suspension that 10ml is fully mixed, the nitrocellulose filter of weighing in advance through 0.45 μ m(ddH2O washing three times, 110 DEG C of hyperthermia dryings) after suction filtration, put 110 DEG C, dry 24 hours, and weigh itDry weight. Finally using dry cell weight as weigh its biomass with reference to index. Bacterial strain and originate as shown in table 1.
Algae (bacterium) strain and source thereof that table 1 uses
aFACHB:FreshwaterAlgalCultureCollectionofTheInstituteOfHy drobiology (Chinese Academy of Sciences aquatileResearch institute's algae kind storehouse)
bNIES-MCC：TheNationalInstituteforEnvironmentalStudiesofJapan-MicrobialCultureCollection (institute of microbiology of Japanese Environment research institute)
Embodiment 2: aliphatic hydrocarbon composition and the assay of cyanobacteria
Respectively collect above-mentioned 200mL in plateau and the cyanobacteria fully mixing (in table 1), use 10mLddH2OResuspended above-mentioned cyanobacteria respectively, and be placed on ice-water bath ultrasonic disruption 10min (10son, 10soff). BrokenThe broken rear 30 μ g n-eicosane hydrocarbon that add respectively, as interior mark, add 10mL chloroform after mixing: methyl alcohol (V:V is 2:1), concuss mixed after 1-2 hour. Then 6, the centrifugal 10min of 000g, is transferred to new by lower floor's organic phaseIn test tube, put nitrogen at 55 DEG C and dry up. Add 1ml aliphatic hydrocarbon that n-hexane dissolution is carried, then through 0.22 μ m filterMembrane filtration, is transferred in 2ml gas phase bottle. Adopt Agilent7890A gas chromatograph-mass spectrometer (GC-MS) (GC-MS)Measure aliphatic hydrocarbon, GC-MS test program is: 40 DEG C of 1min; Rise to 200 DEG C with 5 DEG C of min-1; With 25 DEG CMin-1 to 240 DEG C; 240 DEG C maintain 15min. Chromatographic column is that (μ m × 0.25,30m × 250 μ m) for HP-INNOWax;Carrier gas is helium, and gas flow rate is 1mL/min, and sample size is 1 μ L, and injector temperature is 250 DEG C. Each algae strainAliphatic hydrocarbon GC-MS analyzes collection of illustrative plates as shown in Figures 1 to 6, and aliphatic hydrocarbon composition and content are summarized in table 2.
The each algae strain of table 2 aliphatic hydrocarbon composition and output (mg/gDCW)
a7-Methylheptadecane
b4-Ethyltetradecane
cMay be 5-Methylpetadecane, 6-Methylpetadecane, 7-Methylpetadecane, 4-Ethyltetradecane
dN.D: (Notdetectable) do not detected
Embodiment 3: cyanobacteria gene order-checking and the sequence retrieval of producing hydrocarbon key gene ado-aar
(1) the genomic extraction step of cyanobacteria is as follows:
Collect each bacterium liquid that 50ml left and right fully mixes, the centrifugal collection of 5,000g, with 1.8ml solution A (50mMTris-Cl+50mMNa2EDTA+1MNaCl) resuspended, then use the homogenate of 2ml Potter-Elvehjem Tissue Grinders appropriateness, by threadCell homogenates is dispersed to unicellular state.
Then proceed as follows: the cell suspension of above-mentioned each cyanobacteria and algae strain is divided and is filled to 6 EppendorfIn pipe (300 μ l/ pipe), every pipe adds the 10% sarcosyl aqueous solution (Sarkosyl solution) to wholeConcentration is 0.1%, then at 4 DEG C, preserves 1hr, with the centrifugal 15min of 10,000g, sedimentation cell; Use 1mlTwice of solution A washed cell;
After washing, again cyanobacteria and algae strain cell are suspended from respectively to the solution of 250 μ l (buffer A+25% sucrose)In, room temperature is placed 1hr; Add lysozyme to resuspended solution (final concentration 10-20mg/ml), and in 37 DEG C of insulations15min, with 750 μ l buffer B (10mMTris-Cl+50mMNa2EDTA) diluting cells suspensions, and again in37 DEG C of insulation 30min; Add 10%SDS to final concentration be 1%, carefully stir evenly, continue insulation 1hr, until outstandingLiquid becomes sticky, and adds Proteinase K (final concentration 100 μ g/ml) to suspension, then is incubated 2hr; With phenol/chloroform (1:1)Extracting once, then is used chloroform extracting once, centrifugal suct clearly after, by the absolute ethyl alcohol precipitate nucleic acids of 2 times of volumes,-20 DEG C are spent the night; With the DNA of 12,000rpm centrifugation, then precipitation is dissolved in TE buffer solution, use RNaseTreatment Solution, then uses phenol/chloroform (1:1) extracting once, then uses chloroform extracting once, adds 5MNaCl to wholeConcentration is 1M, then uses the ice cold ethanol precipitation DNA of 2 times of volumes of liquid in pipe, and-20 DEG C are spent the night, again centrifugal heavyShallow lake DNA, and be dissolved in 100 μ lTE buffer solutions.
(2) gene order-checking: each bacterial strain genome sequencing entrusts Shanghai Sheng Gong bioengineering limited company to complete.
(3) produce hydrocarbon key gene ado-aar sequence retrieval
Produce hydrocarbon key gene ado and the special degenerate primer (SEQIDNO:43 to SEQID of aar with cyanobacteriaNO:46) retrieval cyanobacteria NostoccalcicolaFACHB389, Leptolyngbyasp.NIES-30,PhormidiumambiguumNIES-2119，ChroogloeocystissiderophilaNIES-1031，CalothrixSp.NIES-2101 or Scytonemasp.NIES-2130 genome sketch, obtained above-mentioned each algae strain ado andAar. Gained gene has the sequence as shown in SEQIDNO:1 to SEQIDNO:12.
Embodiment 4: cyanobacteria produces the clone of hydrocarbon key gene ado-aar
(1) for the design of the primer of the ado-aar that increases
Because ado and aar are adjacent gene in all cyanobacterias of having identified, clone ado so adopt simultaneouslyStrategy with aar. For the definite ado-aar sequence of embodiment 3, design is as SEQIDNO:13 to SEQIDPrimer sequence shown in NO:24, for the product hydrocarbon key gene of the described algae strain of increasing.
(2) pcr amplification and carry the structure of ado-aar sequence recombinant plasmid
With the gene of primer 389-1/2 (SEQIDNO:13/14) amplification NostoccalcicolaFACHB389Group DNA, the fragment of acquisition 2065bp, extension amplification outcome enters pEASYBlunt carrier (ammonia benzyl/block that to resistProperty), build and obtain recombinant plasmid pTZ60 (as shown in Figure 7).
With the genome of primer 30-1/2 (SEQIDNO:15/16) amplification Leptolyngbyasp.NIES-30DNA, obtains the fragment of 1949bp, and extension amplification outcome enters pEASYBlunt carrier (ammonia benzyl/block that resistance),Build and obtain recombinant plasmid pTZ61 (as shown in Figure 8).
With primer 2 119-1/2 (SEQIDNO:17/18) amplification PhormidiumambiguumNIES-2119Genomic DNA, obtain the fragment of 1914bp, extension amplification outcome enter pEASYBlunt carrier (ammonia benzyl/Block that resistance), build and obtain recombinant plasmid pTZ62 (as shown in Figure 9).
With primer 1031-1/2 (SEQIDNO:19/20) amplification ChroogloeocystissiderophilaThe genomic DNA of NIES-1031, the fragment of acquisition 1911bp, extension amplification outcome enters pEASYBlunt and carriesBody (ammonia benzyl/block that resistance), builds and obtains recombinant plasmid pTZ63 (as shown in figure 10).
With the genome of primer 2 101-1/2 (SEQIDNO:21/22) amplification Calothrixsp.NIES-2101DNA, obtains the fragment of 1831bp, and extension amplification outcome enters pEASYBlunt carrier (ammonia benzyl/block that resistance),Build and obtain recombinant plasmid pTZ64 (as shown in figure 11).
With the genome of primer 2 130-1/2 (SEQIDNO:23/24) amplification Scytonemasp.NIES-2130DNA, obtains the fragment of 1887bp, and extension amplification outcome enters pEASYBlunt carrier (ammonia benzyl/block that resistance),Build and obtain recombinant plasmid pTZ65 (as shown in figure 12).
Above-mentioned PCR all uses FastPfuFlyDNA polymerase, and amplification condition is as follows:
95℃，5min；(95℃,30sec，55℃,30sec，72℃,3min)30×；72℃，10min
(3) conversion of the preparation of Escherichia coli competence and plasmid
1) competent escherichia coli cell
Competent escherichia coli cell (TransT1 competence) is purchased from Beijing Quanshijin Biotechnology Co., Ltd
2) conversion of recombinant plasmid
The competent cell that takes out-80 DEG C of preservations, is placed in ice and melts 5min.
To add respectively in the competent cell melting above-mentioned steps (2) obtain the different amplified productions of 5-10 μ l (asShown in SEQIDNO:25 to SEQIDNO:30) with the connecting fluid of pEASYBlunt carrier, mix gently,In ice-water bath, place 30min.
42 DEG C of heat shocks 90 seconds, then ice-water bath 2min immediately.
Add 900 μ lLB fluid nutrient mediums, 37 DEG C, 200rpm concussion recovery 1 hour.
The centrifugal 3min of 5000rpm, abandons supernatant, and precipitation is resuspended in 100 μ lLB culture mediums, coating ammonia benzyl(50 μ g/mL)/block that (50 μ g/mL) dual anti-solid plate.
Flat-plate inverted is placed in the incubator of 37 DEG C and cultivates after 12-16 hour and grow bacterium colony.
(4) transform Escherichia coli with recombinant plasmid pTZ60, pTZ61, pTZ62, pTZ63, pTZ64 and pTZ65TransT1 competence, obtain respectively bacterial strain ZT166, the ZT167, ZT168, the ZT169 that contain hydrocarbon-producing genes,ZT170 and ZT171.
Embodiment 5: cyanobacteria produces expression and the product hydrocarbon situation analysis of hydrocarbon key gene ado-aar in Escherichia coli
(1) structure of ado-aar expression vector
Cut pTZ60 and reclaim the hydrocarbon-producing genes of NostoccalcicolaFACHB389 with BamHI and XhoI enzymeFragment ado-aar, cuts pET21b and reclaims carrier part with BamHI and XhoI enzyme, and both connect conversion large intestineBacillus competent cell (TransT1 competence), builds and obtains recombinant plasmid pTZ68 (as shown in figure 13)
Cut pTZ61 and reclaim the hydrocarbon-producing genes sheet of Leptolyngbyasp.NIES-30 with BamHI and XhoI enzymeSection ado-aar, cuts pET21b and reclaims carrier part with BamHI and XhoI enzyme, and both connect conversion large intestine barBacterium competence cell (TransT1 competence), builds and obtains recombinant plasmid pTZ69 (as shown in figure 14)
Cut pTZ62 and reclaim the product hydrocarbon of PhormidiumambiguumNIES-2119 with BamHI and XhoI enzymeGenetic fragment ado-aar, cuts pET21b and reclaims carrier part with BamHI and XhoI enzyme, and both connect conversionCompetent escherichia coli cell (TransT1 competence), builds and obtains recombinant plasmid pTZ70 (as Figure 15 instituteShow)
Cut pTZ63 and reclaim ChroogloeocystissiderophilaNIES-1031 with BamHI and XhoI enzymeHydrocarbon-producing genes fragment ado-aar, cut pET21b and reclaim carrier part, Liang Zhelian with BamHI and XhoI enzymeSwitching through competent escherichia coli cell (TransT1 competence), builds and obtains recombinant plasmid pTZ71 (as figureShown in 16)
Cut pTZ64 and reclaim the hydrocarbon-producing genes fragment of Calothrixsp.NIES-2101 with BamHI and NotI enzymeAdo-aar, cuts pET21b and reclaims carrier part with BamHI and NotI enzyme, and both connect conversion Escherichia coliCompetent cell (TransT1 competence), builds and obtains recombinant plasmid pTZ72 (as shown in figure 17)
Cut pTZ65 and reclaim the hydrocarbon-producing genes sheet of Scytonemasp.NIES-2130 with BamHI and XhoI enzymeSection ado-aar, cuts pET21b and reclaims carrier part with BamHI and XhoI enzyme, and both connect conversion large intestine barBacterium competence cell (TransT1 competence), builds and obtains recombinant plasmid pTZ73 (as shown in figure 18)
(2) expression of ado-aar in Escherichia coli
Host Strains: Escherichia coli fadE is the encoding gene of acyl CoA dehydrogenase, knocking out of fadE can make cellAccumulate acyl CoA, be conducive to the generation (Duanetal. of derivative of fatty acid (for example fatty-acid ethyl ester, fatty alcohol)2011). Therefore, the related experiment of ado-aar functional verification of the present invention is selected with BL21 (DE3)FadE mutant strainAs host cell. Transform with recombinant plasmid pTZ68, pTZ69, pTZ70, pTZ71, pTZ72 and pTZ73BL21 (DE3) △ fadE mutant strain, obtain respectively genetic engineering produce hydrocarbon coli strain ZT181, ZT182,ZT183, ZT184, ZT185 and ZT186.
Culture medium: used medium is the improvement M9 culture medium that is applicable to recombinant bacterium product hydrocarbon, and it has following composition: 6g/LNa2HPO4,3g/LKH2PO4,0.5g/LNaCl,2g/LNH4Cl,0.25g/LMgSO4·7H2O,11mg/LCaCl2,27mg/LFeCl3·6H2O,2mg/LZnCl2·4H2O,2mg/LNa2MoO4·2H2O,1.9mg/LCuSO4·5H2O,0.5mg/LH3BO3, 1mg/Lthiamine, 200mMBis-Tris (pH7.25) and0.1％(v/v)Triton-X100。
Induction and cultivation: by constructed ZT181, ZT182, ZT183, ZT184, ZT185 and ZT186 bacteriumStrain is respectively being inoculated in (containing 50 μ g/mL ampicillins) in 5mL liquid improvement M9 culture medium, 30 DEG C, 250rpmCultivate 12h, be then forwarded in 20mL improvement M9 culture medium and (contain by 1:50 (v/v) inoculative proportion nutrient solution50 μ g/mL ampicillins), be cultured to logarithmic phase (OD600Be about 0.6～0.8) after, add 0.5mMIPTGDerivant, 30 DEG C, 250rpm cultivates 24h, and gained bacterium liquid extracts for aliphatic hydrocarbon.
(3) gene engineering colibacillus aliphatic hydrocarbon extracts and volume analysis
Get the above-mentioned each hydrocarbon-producing genes engineering colon bacillus of 10mL in plateau in 20mL small beaker, be placed inOn ice-water bath, ultrasonic disruption 10min (10son, 10soff). After fragmentation, add respectively 50 μ g n-eicosane hydrocarbonAs interior mark, after mixing, add 10mL chloroform: methyl alcohol (V:V is 2:1), concuss mixes 1-2 hourAfter. Then 6, the centrifugal 10min of 000g, is transferred to lower floor's organic phase in new test tube, puts nitrogen at 55 DEG C and dries up.Add 100 μ L aliphatic hydrocarbons that n-hexane dissolution is carried, then, through 0.22 μ m membrane filtration, be transferred to 2ml gas phase littleIn bottle (built-in 200 μ L bushing pipes). Adopt Agilent7890A gas chromatograph-mass spectrometer (GC-MS) (GC-MS) to measureAliphatic hydrocarbon, GC-MS test program is: 40 DEG C of 1min; Rise to 200 DEG C with 5 DEG C/min; With 25 DEG C/minTo 240 DEG C; 240 DEG C maintain 15min. Chromatographic column is that (μ m × 0.25,30m × 250 μ m) for HP-INNOWax; CarryGas is helium, and gas flow rate is 1mL/min, and sample size is 1 μ L, and injector temperature is 250 DEG C. Each bacterial strain fatFat hydrocarbon composition and aliphatic hydrocarbon total output are summarized in table 3.
Table 3. gene engineering colibacillus produces hydrocarbon situation analysis
The above results confirms the NostoccalcicolaFACHB389 that derives from disclosed in this invention,Leptolyngbyasp.NIES-30，PhormidiumambiguumNIES-2119，ChroogloeocystisSiderophilaNIES-1031, the aar of Calothrixsp.NIES-2101 or Scytonemasp.NIES-2130All can normal expression in Escherichia coli with ado gene order, make the Escherichia coli that can not produce hydrocarbon obtain aliphatic hydrocarbonSynthesis capability.
The synthetic key gene of embodiment 6:NostoccalcicolaFACHB389 aliphatic hydrocarbon is pattern cyanobacteriaThe expression of Synechocystissp.PCC6803 and the situation analysis of product hydrocarbon
(1) structure of ado-aar expression vector
Cut pTZ60 with NdeI enzyme and fill, then cut and reclaim NostoccalcicolaFACHB389 with XhoI enzymeHydrocarbon-producing genes fragment ado-aar, be cloned into pXX47 through the processing of same restrictions restriction endonuclease (for example referring inState's application for a patent for invention 201310595856.3) (provide cyanobacteria genome neutrality locus integration platform, resistance sieveSelect mark and strong promoter) in, build and obtain recombinant plasmid pTZ66.
(2) expression of ado-aar in cyanobacteria
Host Strains: Synechocystissp.PCC6803, transforms Synechocystissp. with recombinant plasmid pTZ66PCC6803 obtains genetic engineering and produces hydrocarbon cyanobacteria ZT187 (spectinomycin resistance).
Culture medium: it is BG11 culture medium that cyanobacteria is cultivated used medium, and it has following composition: 1.5gL-1NaNO3,40mgL-1K2HPO4·3H2O,36mgL-1CaCl2·2H2O,6mgL-1Citric acid, 6mgL-1LemonLemon acid iron ammonium, 1mgL-1EDETATE DISODIUM, 20mgL-1NaCO3,2.9mgL-1H3BO3,1.8mgL-1MnCl2·4H2O,0.22mgL-1ZnSO4·7H2O,0.39mgL-1NaMoO4·2H2O,0.079mgL-1CuSO4·5H2O and 0.01mgL-1CoCl2·6H2O. In solid medium, add 8mMTES buffer,0.3％Na2S203With 1.5% agar powder.
Method for transformation: get in exponential phase (OD730Be about 0.5～1.0) Synechocystissp.PCC6803 wild-type cell 5mL, centrifugal collecting cell; With fresh BG11 culture medium washed cell twice, then willCell is resuspended in 0.5mLBG11 culture medium. Get the resuspended liquid of 0.2mL cell and be placed in new PCR pipe, addIn 2～3, recombinant plasmid pTZ66, mixes, and is placed in 30 DEG C, 30 μ 0,-2s-1Under illumination condition, incubation 4 is littleTime. Then mixture is coated on the nitrocellulose filter being layered on BG11 flat board, and be placed in 30 DEG C, 30Nitric acid fibre-2s-1Under illumination condition, cultivate 24 hours. Then, nitrocellulose filter is transferred to the solid of grand resistanceOn flat board, after 30 DEG C, 30 property, will-2s-1Illumination condition under continue to cultivate 5～7 days, by transformant from flatOn plate, choose, cultivate 5～7 days in the upper line of fresh BG11 flat board (grand resistance); Finally access grand anti-Property liquid B G11 medium culture, extract genome, after PCR checking genotype is correct, get plateau bacterium liquid, useDetect in aliphatic hydrocarbon.
(3) genetic engineering cyanobacteria produces hydrocarbon situation and volume analysis
Genetic engineering cyanobacteria aliphatic hydrocarbon extracting method is with embodiment 2, and different is to add 100 μ g n-eicosane hydrocarbonAs interior mark.
Genetic engineering cyanobacteria ZT187 aliphatic hydrocarbon volume analysis: measure through GC-MS, will derive from NostocThe synthetic key gene ado-aar of aliphatic hydrocarbon of calcicolaFACHB389 is integrated into type strain SynechocystisSp.PCC6803 postgenome:
Aspect product hydrocarbon diversity: make Host Strains obtain the synthesis capability (Figure 20) of pentadecane (C15).
Aspect aliphatic hydrocarbon output: the total output of Host Strains aliphatic hydrocarbon is significantly improved, be about 2 of wild-type strainDoubly (Figure 21).
Above-mentioned data acknowledgement product hydrocarbon provided by the present invention key gene increases the product hydrocarbon diversity of cyanobacteria aliphatic hydrocarbon,Improve the aliphatic hydrocarbon output of cyanobacteria.
The present invention bases oneself upon the present Research of the genetic engineering cyanobacteria synthctic fat hydro carbons new bio energy, be inspired inThe qualification of aliphatic hydrocarbon route of synthesis in cyanobacteria, had obtained but the 6 strain cyanobacterias works of genome the unknown already with this teamFor research object, systematically analyze its aliphatic hydrocarbon composite character and output. And based on the analysis of sequence homogeneity, Quan JiBecause of group order-checking and round pcr, obtain the product hydrocarbon key gene sequence of described cyanobacteria bacterial strain. Meanwhile, at large intestineIn bacillus, gained hydrocarbon-producing genes has been carried out to functional verification, and based on gained sequence construct efficient synthesizing aliphatic hydrocarbonsGenetic engineering cyanobacteria. Do not wish to be bound by any theory, inventor thinks gained cyanobacteria bacterial strain and produce hydrocarbon keyGene order will be for utilizing the direct synthctic fat hydro carbons of microorganism synthesis system bio-fuel that valuable candidate is providedGenetic resources.
Meanwhile, on hydrocarbon-producing genes of the present invention basis, also can add the encoding gene of terminal olefin synzyme(olefinssynthase, ols), makes it pass through the synthetic terminal olefin (Mendez-Perez of class I type polyketide synthases approachEtal.2011), and then also can be acquisition fat hydrocarbon bio-fuel valuable candidate gene resource is provided.
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Claims (10)
1. a cyanobacteria hydrocarbon-producing genes, is characterized in that:
Hydrocarbon-producing genes comprises the encoding gene (aar) of acyl ACP reductase and fatty aldehyde piptonychia acyl group oxygenaseEncoding gene (ado).
2. by cyanobacteria hydrocarbon-producing genes claimed in claim 1, it is characterized in that:
The encoding gene of the encoding gene (aar) of described acyl ACP reductase and fatty aldehyde piptonychia acyl group oxygenase(ado) be cyanobacteria NostoccalcicolaFACHB389, Leptolyngbyasp.NIES-30, PhormidiumambiguumNIES-2119，ChroogloeocystissiderophilaNIES-1031，Calothrixsp.The aar of NIES-2101 or Scytonemasp.NIES-2130 and ado.
3. by the cyanobacteria hydrocarbon-producing genes described in claim 1 or 2, it is characterized in that:
The encoding gene of the encoding gene (aar) of described acyl ACP reductase and fatty aldehyde piptonychia acyl group oxygenase(ado) there is SEQIDNO:1 and 2, SEQIDNO:3 and 4, SEQIDNO:5 and 6, SEQIDNO:7With 8, SEQIDNO:9 and 10 or SEQIDNO:11 and 12 shown in sequence.
4. a construct, is characterized in that:
Construct comprises promoter, and the encoding gene (aar) of the acyl ACP reductase of this promoter controlEncoding gene (ado) with fatty aldehyde piptonychia acyl group oxygenase.
5. by construct claimed in claim 4, it is characterized in that:
The promoter of described construct is derive from the PcpcB promoter of cyanobacteria or derive from colibacillary T7Promoter.
6. by the construct described in claim 4 or 5, it is characterized in that: the volume of described acyl ACP reductaseThe encoding gene (ado) of code gene (aar) and fatty aldehyde piptonychia acyl group oxygenase is cyanobacteria NostoccalcicolaFACHB389，Leptolyngbyasp.NIES-30，PhormidiumambiguumNIES-2119，ChroogloeocystissiderophilaNIES-1031, Calothrixsp.NIES-2101 or Scytonemasp.The aar of NIES-2130 and ado.
7. a genetic engineering bacterium for synthesizing aliphatic hydrocarbons, is characterized in that: engineering bacteria comprises above-mentioned construct.
8. by the genetic engineering bacterium of energy synthesizing aliphatic hydrocarbons claimed in claim 7, it is characterized in that:
Described construct is imported in Escherichia coli by conversion.
9. by the genetic engineering bacterium of energy synthesizing aliphatic hydrocarbons claimed in claim 8, it is characterized in that: by described structureBody imports in e. coli bl21 (DE3) fadE mutant strain by conversion.
10. in a cyanobacteria hydrocarbon-producing genes claimed in claim 1 application as synthesizing aliphatic hydrocarbons.
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