CN105586351A - Cyanobacteria aliphatic hydrocarbon key synthesis gene and application thereof - Google Patents

Abstract

The invention discloses a key gene for aliphatic hydrocarbon synthesis derived from cyanobacteria and its application. The aliphatic hydrocarbon synthesis genes include fatty acyl-ACP reductase (Fatty?acyl?carrier?protein?reductase, AAR) and fatty aldehyde deformyloxygenase (Fatty?aldehyde?deformylating?oxygenase, ADO) coding genes . The present invention also relates to a construct comprising an active promoter and a key gene for aliphatic hydrocarbon synthesis controlled by the promoter. Transforming the construct into Escherichia coli can make the genetically engineered Escherichia coli obtain aliphatic hydrocarbon synthesis ability. The aliphatic hydrocarbon production of the genetically engineered cyanobacteria can be significantly improved by transforming the at least one construct into the cyanobacteria. The key genes for hydrocarbon production of cyanobacteria disclosed by the invention will provide new candidate gene resources for microorganisms to directly synthesize new biofuels of aliphatic hydrocarbons.

Description

A key gene for cyanobacterial aliphatic hydrocarbon synthesis and its application

technical field

The invention relates to the field of energy microbial germplasm resources and gene resources, in particular to a key gene for cyanobacteria aliphatic hydrocarbon synthesis.

Background technique

Energy is the pillar of modern industry. The increasing shortage of traditional energy represented by fossil fuels is the bottleneck restricting the sustainable development of my country's economy. The application of renewable biofuels has become one of the best choices to alleviate the energy crisis and improve the ecological environment. Fossil fuels such as gasoline, diesel and aviation kerosene widely used in the transportation industry are mainly composed of aliphatic hydrocarbons of a specific chain length range, which have high energy density, low hygroscopicity, low volatility, and are compatible with existing engines It is the best substitute for traditional petrochemical liquid fuels (Keasling and Chou2008).

Petroleum, ancient sediments, and meteorites contain a large number of substances that may be of biological origin, among which aliphatic hydrocarbons are also one of the most concerned members. Scientists have proposed the theory that decayed algae cells are its constituents for the origin of traditional fossil energy (Han and Calvin 1969). Since the late 1960s, the research on aliphatic hydrocarbons in cyanobacteria mainly revolves around the analysis and identification of the composition and content of aliphatic hydrocarbons in different cyanobacteria (or other eukaryotic algae). The reported content of aliphatic hydrocarbons in wild cyanobacteria is as low as 0.01% of dry cell weight (Winters et al. 1969) and as high as 0.26% (Coates et al. 2014). Studies have found that the chain length is mainly concentrated between C15 and C20, and mainly C17 (Hanetal.1968).

As a new generation of energy microbial system, cyanobacteria have the following advantages: (1) Unlike Escherichia coli and Saccharomyces cerevisiae, which are heterotrophic microbial reactors that synthesize biofuels, cyanobacteria can use solar energy and can grow with carbon dioxide as a carbon source, and have a variety of , low cultivation cost, strong vitality, fast growth rate and other advantages. (2) From the perspective of genetic engineering, unlike eukaryotic microalgae and higher plants that can also utilize solar energy and carbon dioxide, cyanobacteria are hosts for synthesizing aliphatic hydrocarbons, and their genetic manipulation is simpler and large-scale genetic modification can be carried out. (3) Cyanobacteria have a relatively clear genetic background. The whole genome sequences of more than 130 strains of cyanobacteria have been published (Shih et al. 2013).

Thanks to the rapid development of molecular biology, bioinformatics and metabolic engineering technologies, two aliphatic hydrocarbon synthesis pathways have been identified in cyanobacteria. One exists in most cyanobacteria, and is mainly catalyzed by fatty acyl-acylcarrier protein reductase (Fattyacyl-acylcarrierprotein reductase, AAR) and fatty aldehyde deformyloxygenase (Fattyaldehydedeformylatingoxygenase, ADO), and its products are mainly linear alkanes, Side-chain alkanes and alkenes (unsaturated bonds located inside the carbon chain) (Schirmer et al. 2010). Another pathway exists in a few cyanobacteria, mainly catalyzed by terminal olefin synthase (OLS), whose product is terminal olefins (unsaturated bonds at the end of the carbon chain) (Mendez-Perez et al. 2011).

Based on the above identification of aliphatic hydrocarbon synthesis pathways, the production of aliphatic hydrocarbons in recombinant cyanobacteria has been improved through genetic engineering and metabolic engineering optimization. Taking Synechocystis sp. PCC6803 as an example, the research results confirmed that the yield of the recombinant strain was more than 9 times that of the wild strain (Wang et al. 2013). However, the level of aliphatic hydrocarbon synthesis by genetically engineered cyanobacteria is far from the level of industrial application. The mining of germplasm resources and genetic resources of hydrocarbon-producing cyanobacteria is an important factor to further improve the efficiency of aliphatic hydrocarbon synthesis in cyanobacteria and enrich the diversity of hydrocarbon production. one.

Contents of the invention

The purpose of the present invention is to provide a key gene for aliphatic hydrocarbon synthesis of cyanobacteria.

To achieve the above object, the technical solution adopted in the present invention is:

A cyanobacteria hydrocarbon-producing gene: the hydrocarbon-producing gene includes the coding gene (aar) of fatty acyl ACP reductase and the coding gene (ado) of fatty aldehyde deformyl oxygenase.

The coding gene (aar) of the fatty acyl ACP reductase and the coding gene (ado) of fatty aldehyde deformyl oxygenase are cyanobacteria NostoccalcicolaFACHB389, Leptolyngbyasp.NIES-30, PhormidiumambiguumNIES-2119, ChroogloeocystissiderophilaNIES-1031, Calothrixsp.NIES - aar and ado of 2101 or Scytonemasp.NIES-2130.

The coding gene (aar) of the fatty acyl ACP reductase and the coding gene (ado) of fatty aldehyde deformyl oxygenase have SEQIDNO:1 and 2, SEQIDNO:3 and 4, SEQIDNO:5 and 6, SEQIDNO:7 and 8, the sequences shown in SEQ ID NO: 9 and 10 or SEQ ID NO: 11 and 12.

A construct, which comprises a promoter, and the coding gene (aar) of fatty acyl ACP reductase and the coding gene (ado) of fatty aldehyde deformyl oxygenase controlled by the promoter.

The promoter of the construct is the PcpcB promoter derived from cyanobacteria or the T7 promoter derived from Escherichia coli.

The coding gene (aar) of the fatty acyl ACP reductase and the coding gene (ado) of fatty aldehyde deformyl oxygenase are cyanobacteria NostoccalcicolaFACHB389, Leptolyngbyasp.NIES-30, PhormidiumambiguumNIES-2119, ChroogloeocystissiderophilaNIES-1031, Calothrixsp.NIES - aar and ado of 2101 or Scytonemasp.NIES-2130.

A genetically engineered bacterium capable of synthesizing aliphatic hydrocarbons, the engineered bacterium contains the above construct.

Furthermore, the construct was introduced into E. coli by transformation.

Furthermore, the construct was introduced into Escherichia coli BL21(DE3)fadE mutant strain by transformation.

In the application of the cyanobacteria hydrocarbon-producing gene as a synthetic aliphatic hydrocarbon.

The beneficial effects that the present invention has:

Using cyanobacteria as a microbial expression system to synthesize aliphatic hydrocarbons is the process of using solar energy to fix carbon dioxide to synthesize biofuels. This process is a production process of clean bio-energy, which is not restricted by the shortage of raw materials and will not increase carbon emissions. The technical solution of the present invention can provide valuable candidate gene resources for the above bioenergy production process.

At the same time, the cloning of key genes for hydrocarbon production in cyanobacteria (fatty acyl-ACP reductase/fatty aldehyde deformyloxygenase encoding genes) has the following important functions: 1. To study the metabolic mechanism of cyanobacteria aliphatic hydrocarbon synthesis at the molecular level and lay the foundation for the physiological function of aliphatic hydrocarbons in cyanobacteria. 2. Understand the hydrocarbon production characteristics of different cyanobacteria strains, and analyze the distribution of aliphatic hydrocarbons with different structures in cyanobacteria.

Therefore, the present invention aims to provide candidate germplasm and genes for the cyanobacteria liquid fuel synthesis system that uses solar energy, carbon dioxide and water to directly synthesize aliphatic hydrocarbons by extensively collecting cyanobacteria strain resources, and by means of whole genome sequencing and hydrocarbon-producing gene cloning resource.

Description of drawings

Figure 1 shows the aliphatic hydrocarbon composition and characteristics of the algal strain Nostoccalcicola FACHB389 determined based on GC-MS.

Fig. 2 is the aliphatic hydrocarbon composition and characteristics of algal strain Leptolyngbyasp. NIES-30 determined based on GC-MS.

Fig. 3 is the aliphatic hydrocarbon composition and characteristics of algal strain Phormidium ambiguum NIES-2119 determined based on GC-MS.

Fig. 4 is the aliphatic hydrocarbon composition and characteristics of algal strain Chroogloeocystissiderophila NIES-1031 determined based on GC-MS.

Fig. 5 is the aliphatic hydrocarbon composition and characteristics of algae strain Calothrixsp.NIES-2101 determined based on GC-MS.

Fig. 6 is the aliphatic hydrocarbon composition and characteristics of algae strain Scytonemasp.NIES-2130 determined based on GC-MS.

Figure 7 is the basic structure of the recombinant vector pTZ60. The recombinant vector pTZ60 is obtained by using 389-1/2 as a primer and NostoccalcicolaFACHB389 genomic DNA as a template to amplify a 2065bp fragment by PCR, clone it into the pEASY-blunt vector, and transform it into E.coliTrans1-T1 phage-resistant competent cells.

Figure 8 is the basic structure of the recombinant vector pTZ61. The recombinant vector pTZ61 uses 30-1/2 as a primer and Leptolyngbyasp.NIES-30 genomic DNA as a template to amplify a 1949bp fragment by PCR, clone it into pEASY-blunt vector, and transform E.coliTrans1-T1 phage-resistant competent cells And get.

Figure 9 shows the basic structure of the recombinant vector pTZ62. The recombinant vector pTZ62 is obtained by using 2119-1/2 as a primer and PhormidiumambiguumNIES-2119 genomic DNA as a template to amplify a 1914bp fragment by PCR, clone it into pEASY-blunt vector, and transform E.coliTrans1-T1 phage-resistant competent cells .

Figure 10 shows the basic structure of the recombinant vector pTZ63. The recombinant vector pTZ63 is obtained by using 1031-1/2 as a primer and ChroogloeocystissiderophilaNIES-1031 genomic DNA as a template to amplify a 1911bp fragment by PCR, clone it into pEASY-blunt vector, and transform E.coliTrans1-T1 phage-resistant competent cells .

Figure 11 shows the basic structure of the recombinant vector pTZ64. The recombinant vector pTZ64 uses 2101-1/2 as a primer and Calothrixsp.NIES-2101 genomic DNA as a template to amplify a 1831bp fragment by PCR, clone it into pEASY-blunt vector, and transform E.coliTrans1-T1 phage-resistant competent cells And get.

Figure 12 shows the basic structure of the recombinant vector pTZ65. The recombinant vector pTZ65 uses 2130-1/2 as a primer and Scytonemasp.NIES-2130 genomic DNA as a template to amplify a 1887bp fragment by PCR, clone it into pEASY-blunt vector, and transform E.coliTrans1-T1 phage-resistant competent cells And get.

Figure 13 is the basic structure of the recombinant vector pTZ68. pTZ60 was digested with BamHI and XhoI and the hydrocarbon-producing gene fragment ado-aar of NostoccalcicolaFACHB389 was recovered, pET21b was digested with BamHI and XhoI and the vector part was recovered, and the recombinant plasmid pTZ68 was constructed by ligation of the two.

Figure 14 is the basic structure of the recombinant vector pTZ69. pTZ61 was digested with BamHI and XhoI and the hydrocarbon-producing gene fragment ado-aar of Leptolyngbyasp.NIES-30 was recovered, pET21b was digested with BamHI and XhoI and the vector part was recovered, and the recombinant plasmid pTZ69 was obtained by ligation of the two.

Figure 15 is the basic structure of the recombinant vector pTZ70. pTZ62 was digested with BamHI and XhoI and the hydrocarbon-producing gene fragment ado-aar of PhormidiumambiguumNIES-2119 was recovered, pET21b was digested with BamHI and XhoI and the vector part was recovered, and the recombinant plasmid pTZ70 was constructed by ligation of the two.

Figure 16 shows the basic structure of the recombinant vector pTZ71. pTZ63 was digested with BamHI and XhoI and the hydrocarbon-producing gene fragment ado-aar of ChroogloeocystissiderophilaNIES-1031 was recovered, pET21b was digested with BamHI and XhoI and the vector part was recovered, and the recombinant plasmid pTZ71 was obtained by ligation of the two.

Figure 17 shows the basic structure of the recombinant vector pTZ72. Digest pTZ64 with BamHI and NotI and recover the hydrocarbon-producing gene fragment ado-aar of Calothrixsp.

Figure 18 is the basic structure of the recombinant vector pTZ73. pTZ65 was digested with BamHI and XhoI and the hydrocarbon-producing gene fragment ado-aar of Scytonemasp.NIES-2130 was recovered, pET21b was digested with BamHI and XhoI and the vector part was recovered, and the recombinant plasmid pTZ73 was obtained by ligation of the two.

Figure 19 shows the basic structure of the recombinant vector pTZ66. pTZ60 was digested with NdeI to make up the level, and then digested with XhoI to recover the hydrocarbon-producing gene fragment ado-aar of NostoccalcicolaFACHB389, cloned into pXX47 treated with the same restriction endonuclease, and constructed the recombinant plasmid pTZ66.

Fig. 20 is the aliphatic hydrocarbon composition and characteristics of the hydrocarbon-producing genetically engineered cyanobacteria ZT187 determined based on GC-MS.

Figure 21 is the analysis of the aliphatic hydrocarbon production of the genetically engineered cyanobacteria ZT187.

Sequence listing information:

SEQ ID NO: 1 is the sequence of the gene ado encoding fatty aldehyde deformyloxygenase of Nostoccalcicola FACHB389.

SEQ ID NO: 2 is the sequence of the acyl-ACP reductase coding gene aar of Nostoccalcicola FACHB389.

SEQ ID NO: 3 is the sequence of the gene ado encoding fatty aldehyde deformyloxygenase of Leptolyngbyasp.NIES-30.

SEQ ID NO: 4 is the sequence of the acyl-ACP reductase coding gene aar of Leptolyngbyasp.NIES-30.

SEQ ID NO: 5 is the sequence of the gene ado encoding fatty aldehyde deformyloxygenase of Phormidium ambiguum NIES-2119.

SEQ ID NO: 6 is the sequence of the acyl-ACP reductase coding gene aar of Phormidium ambiguum NIES-2119.

SEQ ID NO: 7 is the sequence of the gene ado encoding fatty aldehyde deformyloxygenase of ChroogloeocystissiderophilaNIES-1031.

SEQ ID NO: 8 is the sequence of the acyl-ACP reductase coding gene aar of ChroogloeocystissiderophilaNIES-1031.

SEQ ID NO: 9 is the sequence of ado, the fatty aldehyde deformyloxygenase encoding gene of Calothrixsp.NIES-2101.

SEQ ID NO: 10 is the sequence of the acyl-ACP reductase coding gene aar of Calothrixsp.NIES-2101.

SEQ ID NO: 11 is the sequence of the gene ado encoding fatty aldehyde deformyloxygenase of Scytonemasp.NIES-2130.

SEQ ID NO: 12 is the sequence of the fatty acyl-ACP reductase coding gene aar of Scytonemasp.NIES-2130.

SEQ ID NO: 13 is the nucleotide sequence of primer 389F.

SEQ ID NO: 14 is the nucleotide sequence of primer 389R.

SEQ ID NO: 15 is the nucleotide sequence of primer 30F.

SEQ ID NO: 16 is the nucleotide sequence of primer 30R.

SEQ ID NO: 17 is the nucleotide sequence of primer 2119F.

SEQ ID NO: 18 is the nucleotide sequence of primer 2119R.

SEQ ID NO: 19 is the nucleotide sequence of primer 1031F.

SEQ ID NO: 20 is the nucleotide sequence of primer 1031R.

SEQ ID NO: 21 is the nucleotide sequence of primer 2101F.

SEQ ID NO: 22 is the nucleotide sequence of primer 2101R.

SEQ ID NO: 23 is the nucleotide sequence of primer 2130F.

SEQ ID NO: 24 is the nucleotide sequence of primer 2130R.

SEQ ID NO: 25 is the ado-aar of Nostoccalcicola FACHB389 and its spacer sequence.

SEQ ID NO: 26 is the ado-aar of Leptolyngbyasp.NIES-30 and its spacer sequence.

SEQ ID NO: 27 is the ado-aar of Phormidium ambiguum NIES-2119 and its spacer sequence.

SEQ ID NO: 28 is Chroogloeocystissiderophila NIES-1031 and its spacer sequence.

SEQ ID NO: 29 is the ado-aar of Calothrixsp.NIES-2101 and its spacer sequence.

SEQ ID NO: 30 is the ado-aar of Scytonemasp.NIES-2130 and its spacer sequence.

SEQ ID NO: 31 is the amino acid sequence of the fatty acyl-ACP reductase of FischerellamajorNIES-592.

SEQ ID NO: 31 is the amino acid sequence of the fatty aldehyde deformyloxygenase of Nostoccalcicola FACHB389.

SEQ ID NO: 32 is the amino acid sequence of the fatty acyl-ACP reductase of Nostoccalcicola FACHB389.

SEQ ID NO: 33 is the amino acid sequence of the fatty aldehyde deformyloxygenase of Leptolyngbyasp.NIES-30.

SEQ ID NO: 34 is the amino acid sequence of the fatty acyl-ACP reductase of Leptolyngbyasp.NIES-30.

SEQ ID NO: 35 is the amino acid sequence of the fatty aldehyde deformyloxygenase of Phormidium ambiguum NIES-2119.

SEQ ID NO: 36 is the amino acid sequence of the fatty acyl-ACP reductase of Phormidium ambiguum NIES-2119.

SEQ ID NO: 37 is the amino acid sequence of the fatty aldehyde deformyloxygenase of Chroogloeocystissiderophila NIES-1031.

SEQ ID NO: 38 is the amino acid sequence of the fatty acyl-ACP reductase of Chroogloeocystissiderophila NIES-1031.

SEQ ID NO: 39 is the amino acid sequence of the fatty aldehyde deformyloxygenase of Calothrix sp. NIES-2101.

SEQ ID NO: 40 is the amino acid sequence of the fatty acyl-ACP reductase of Calothrixsp.NIES-2101.

SEQ ID NO: 41 is the amino acid sequence of the fatty aldehyde deformyloxygenase of Scytonemasp.NIES-2130.

SEQ ID NO: 42 is the amino acid sequence of the fatty acyl-ACP reductase of Scytonemasp.NIES-2130.

SEQ ID NO: 43 is the nucleotide sequence of primer ado-1.

SEQ ID NO: 44 is the nucleotide sequence of primer ado-2.

SEQ ID NO: 45 is the nucleotide sequence of primer aar-1.

SEQ ID NO: 46 is the nucleotide sequence of primer aar-2.

detailed description

Embodiments of the present invention will be described in detail below in conjunction with examples. The examples described herein are provided by way of illustration and not limitation. Those skilled in the art will appreciate that a number of non-critical parameters can be changed or modified to obtain similar results.

related terms

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the experimental operation steps of cyanobacteria culture, molecular biology, and analytical chemistry used in this paper are all routine steps widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, "Cyanobacterium" is a class of photoautotrophic prokaryotic microorganisms capable of harnessing solar energy to fix carbon dioxide. Cyanobacteria are also known as cyanobacteria. In the present invention, "cyanobacteria" and "cyanobacteria" are used interchangeably.

As used herein, "germplasm resources (Germplasm resources)" is a general term for all biological types that have certain germplasm or genes and are capable of reproduction. Germplasm resources are also called genetic resources. Including but not limited to: ancient local varieties, newly cultivated promotion varieties, important genetic materials, wild relatives, microorganisms, etc.

As used herein, an "aliphatic hydrocarbon" is a hydrocarbon having the basic attributes of an aliphatic compound. The carbon atoms in the molecule are connected to form a chain-like carbon frame, and the hydrocarbons whose two ends are opened without forming a ring are called open-chain hydrocarbons, or chain hydrocarbons for short. Because fats have this structure, they are also called aliphatic chain hydrocarbons. Some cyclic hydrocarbons are different from aromatic hydrocarbons in nature, but very similar to aliphatic chain hydrocarbons. Such cyclic hydrocarbons are called alicyclic hydrocarbons. In this way, aliphatic hydrocarbons become the general term for all hydrocarbons except aromatic hydrocarbons.

As used herein, "acylcarrier protein (ACP)" is generally abbreviated as ACP. The de novo synthesis of fatty acids is catalyzed by the fatty acid synthesis system of a complex containing multiple enzymes, and in the stage of its acyl condensation, the acyl CoA is not used as a direct substrate, but is transferred to the acyl group in the complex The carrier protein is reactive. ACP is not easily dissociated from the enzyme complex in animals or yeast, but can be separated in Escherichia coli, with a molecular weight of 8847 Daltons and composed of 77 amino acid residues. The formation of 4'-phosphopantethein and serine in the protein part is lipid-bonded to ACP as follows: 4'-phosphopantethein of coenzyme A is transferred to apo-ACP by the enzyme reaction.

As used herein, "Fattyacyl-ACPreductase (AAR)" is a key enzyme in the aliphatic hydrocarbon synthesis pathway of cyanobacteria, which catalyzes the conversion of fatty acyl-ACP into fatty aldehyde. Genes encoding cyanobacterial fatty acyl-ACP reductase are well known in the art, including but not limited to: derived from cyanobacteria FischerellamajorNIES-592, NostoccalcicolaFACHB389, Leptolyngbyasp.NIES-30, PhormidiumambiguumNIES-2119, ChroogloeocystissiderophilaNIES-1031, Calothrixsp.NIES-2101 and Scytonemasp. NIES-2130 acyl-ACP reductase.

As used herein, "Fattyaldehydedeformylatingoxygenase (ADO)" is a key enzyme in the aliphatic hydrocarbon synthesis pathway of cyanobacteria, which catalyzes the conversion of fatty aldehydes into aliphatic hydrocarbons, and belongs to the ferritin-like protein superfamily. Genes encoding cyanobacteria fatty aldehyde deformyloxygenase are well known in the art, including but not limited to: derived from cyanobacteria FischerellamajorNIES-592, NostoccalcicolaFACHB389, Leptolyngbyasp.NIES-30, PhormidiumambiguumNIES-2119, ChroogloeocystissiderophilaNIES-1031, Calothrixsp. Fatty aldehyde deformyloxygenases of NIES-2101 and Scytonemasp.NIES-2130.

As used herein, "vector" refers to a nucleic acid delivery vehicle into which a DNA segment (eg, a gene of interest) can be inserted, thereby allowing the DNA (eg, a gene of interest) to be transferred into a recipient cell. The vector can be introduced into host cells by transformation, transduction or transfection, so that the DNA fragments it carries can be expressed in the host cells. Vectors are well known to those skilled in the art, including but not limited to: plasmids; bacteriophages; cosmids and the like.

"Identity" or "percent identity" refers to the sequence identity between two amino acid sequences or between two nucleic acid sequences. To determine the percent identity of two amino acid sequences or two nucleic acids, the sequences are aligned for optimal comparison purposes. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (ie, percent identity = number of identical positions/total number of positions (eg, overlapping positions) x 100). For example, "percent identity" is calculated by comparing two optimally aligned sequences over a comparison window and determining the number of positions where the same nucleotide base or the same amino acid residue occurs in the two sequences To generate the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window (ie, the size of the window), and the result is multiplied by 100 to generate the percent sequence identity. Optimal alignment of sequences for comparison can be performed by, for example, the local homology algorithm of Smith and Waterman (Smithand Waterman 1981); the homology alignment algorithm of Needleman and Wunsch (Needlema. Sband Wunsch 1970); Pearson and Lipman's Similarity Search Method (Pearson and Lipman 1988); computerized implementations of these algorithms (eg, GAP, BESTFIT, FASTA, BLASTP, BLASTN, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.).

Percentages of identity contemplated by embodiments of the present invention include at least about 50%, or at least about 60%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or higher, such as about 95%, or about 96%, or about 97%, or about 98%, or about 99%, 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 and biomass determination of cyanobacteria

Cultivation and physiological index determination of cyanobacteria:

The cyanobacteria strains in Table 1 were cultured by aeration culture, and the algae strains were inoculated into 500 mL Erlenmeyer flasks filled with 300 mL of BG11 medium, and cultured at 30°C and 30 μE.m ^-2 .s ^-1 under continuous light for 8 to 10 days. In order to measure the growth amount, 10ml of the well-mixed cyanobacteria suspension was suction-filtered through a 0.45μm pre-weighed nitrocellulose membrane (washed three times with ddH ₂ O, dried at 110°C), and placed at 110°C. Dry it for 24 hours and measure its dry weight. Finally, the dry weight of the bacteria was used as a reference index to measure the biomass. The strains and their sources are shown in Table 1.

Algae (bacteria) strains used in Table 1 and their sources

^a FACHB: FreshwaterAlgalCultureCollectionofTheInstituteOfHydrobiology

^b NIES-MCC: The National Institute for Environmental Studies of Japan-MicrobialCultureCollection (Institute of Microbial Culture, Japan Academy of Environmental Sciences)

Embodiment 2: the aliphatic hydrocarbon composition and content determination of cyanobacteria

Collect 200 mL of the above-mentioned cyanobacteria in the plateau stage and well mixed (in Table 1), resuspend the above-mentioned cyanobacteria in 10 mL of ddH ₂ O, place them on an ice-water bath, and ultrasonically disrupt them for 10 min (10son, 10soff). After crushing, add 30 μg of n-eicosane as an internal standard, and after mixing, add 10 mL of chloroform:methanol (V:V is 2:1), shake and mix vigorously for 1-2 hours. Then centrifuge at 6,000 g for 10 min, transfer the lower organic phase to a new test tube, and dry it with nitrogen at 55°C. Add 1ml of n-hexane to dissolve the extracted aliphatic hydrocarbons, then filter through a 0.22μm membrane filter, and transfer to a 2ml gas phase vial. Use Agilent7890A gas chromatography-mass spectrometry (GC-MS) to measure aliphatic hydrocarbons. The GC-MS test program is: 40°C for 1min; 5°C min-1 to 200°C; 25°C min-1 to 240°C; Maintain at 240°C for 15 minutes. The chromatographic column is HP-INNOWax (30m×250μm×0.25μm); the carrier gas is helium, the gas flow rate is 1mL/min, the injection volume is 1μL, and the inlet temperature is 250°C. The GC-MS analysis patterns of aliphatic hydrocarbons of each algae strain are shown in Figures 1 to 6, and the composition and content of aliphatic hydrocarbons are summarized in Table 2.

Table 2 The composition and yield of aliphatic hydrocarbons in each algal strain (mg/gDCW)

^a 7-Methylheptadecane

^b 4-Ethyltetradecane

^c may be 5-Methylpetadecane, 6-Methylpetadecane, 7-Methylpetadecane, 4-Ethyltetradecane

^d ND: Not detected (Notdetectable)

Example 3: Sequencing of the cyanobacteria genome and sequence retrieval of the key gene ado-aar for hydrocarbon production

(1) The extraction steps of the cyanobacteria genome are as follows:

Collect about 50ml of well-mixed bacterial solution, centrifuge at 5,000g, resuspend with 1.8ml solution A (50mM Tris-Cl+50mMNa ₂ EDTA+1MNaCl), and then use a 2ml tissue homogenizer to properly homogenize the filamentous cells The homogenate was dispersed to a single cell state.

Then proceed as follows: the cell suspension of each of the above-mentioned cyanobacteria and algal strains is divided into 6 Eppendorf tubes (300 μl/tube), and 10% sodium lauryl sarcosine aqueous solution (Sarkosyl solution) is added to each tube. The final concentration is 0.1%, and then stored at 4°C for 1 hr, centrifuged at 10,000g for 15 min to pellet the cells; wash the cells twice with 1ml solution A;

After washing, resuspend the cyanobacteria and algae cells in 250 μl (buffer A+25% sucrose) solution, and place at room temperature for 1 hr; add lysozyme to the resuspension solution (final concentration 10-20 mg/ml), and Incubate at 37°C for 15min, dilute the cell suspension with 750μl buffer B (10mM Tris-Cl+50mMNa2EDTA), and incubate at 37°C for 30min; add 10% SDS to a final concentration of 1%, stir carefully, and continue to incubate for 1hr until The suspension becomes viscous, add proteinase K to the suspension (final concentration 100 μg/ml), and keep warm for 2 hours; extract once with phenol/chloroform (1:1), then extract once with chloroform, centrifuge and suck the supernatant, Precipitate nucleic acid with 2 times the volume of absolute ethanol, overnight at -20°C; centrifuge the precipitated DNA at 12,000 rpm, then dissolve the precipitate in TE buffer, treat the solution with RNase, and then pump it with phenol/chloroform (1:1) Extract once, then extract once with chloroform, add 5M NaCl to a final concentration of 1M, then precipitate DNA with 2 times the volume of liquid in the tube with ice-cold ethanol, overnight at -20°C, centrifuge again to precipitate DNA, and dissolve it in 100 μl TE buffer middle.

(2) Genome sequencing: Whole genome sequencing of each strain was entrusted to Shanghai Sangon Bioengineering Co., Ltd. to complete.

(3) Key gene ado-aar sequence search for hydrocarbon production

Retrieve the cyanobacteria NostoccalcicolaFACHB389, Leptolyngbyasp.NIES-30, PhormidiumambiguumNIES-2119, ChroogloeocystissiderophilaNIES-1031, Calothrixsp.NIES-2101 or Scytonemasp with the specific degenerate primers (SEQIDNO:43 to SEQIDNO:46) of the key genes of cyanobacteria hydrocarbon production ado and aar . The draft genome of NIES-2130 obtained the ado and aar of the above-mentioned algae strains. The resulting gene has the sequences shown in SEQ ID NO:1 to SEQ ID NO:12.

Embodiment 4: Cloning of key gene ado-aar of cyanobacteria hydrocarbon production

(1) Design of primers for amplifying ado-aar

Because ado and aar are adjacent genes in all identified cyanobacteria, the strategy of simultaneously cloning ado and aar was adopted. For the ado-aar sequence determined in Example 3, primer sequences as shown in SEQIDNO: 13 to SEQIDNO: 24 were designed to amplify the key genes for hydrocarbon production of the algal strain.

(2) PCR amplification and construction of a recombinant plasmid carrying the ado-aar sequence

The genomic DNA of NostoccalcicolaFACHB389 was amplified with primer 389-1/2 (SEQIDNO: 13/14) to obtain a 2065bp fragment, and the amplified product was cloned into the pEASYBlunt vector (ampicillin/kanna resistance), and the recombinant plasmid pTZ60 (as shown in the figure) was constructed and obtained 7).

The genomic DNA of Leptolyngbyasp.NIES-30 was amplified with primer 30-1/2 (SEQIDNO: 15/16) to obtain a 1949bp fragment, and the amplified product was cloned into the pEASYBlunt vector (ampicillin/Kana resistance) to construct a recombinant plasmid pTZ61 (shown in Figure 8).

The genomic DNA of PhormidiumambiguumNIES-2119 was amplified with primer 2119-1/2 (SEQIDNO:17/18) to obtain a 1914bp fragment, and the amplified product was cloned into the pEASYBlunt vector (ampicillin/kanna resistance) to construct the recombinant plasmid pTZ62 ( as shown in Figure 9).

The genomic DNA of ChroogloeocystissiderophilaNIES-1031 was amplified with primer 1031-1/2 (SEQIDNO:19/20) to obtain a 1911bp fragment, and the amplified product was cloned into the pEASYBlunt vector (ampelin/Kana resistance) to construct the recombinant plasmid pTZ63 ( as shown in Figure 10).

The genomic DNA of Calothrixsp.NIES-2101 was amplified with primer 2101-1/2 (SEQIDNO: 21/22) to obtain a 1831bp fragment, and the amplified product was cloned into the pEASYBlunt vector (ampelin/Kana resistance) to construct a recombinant plasmid pTZ64 (shown in Figure 11).

The genomic DNA of Scytonemasp.NIES-2130 was amplified with primer 2130-1/2 (SEQIDNO:23/24) to obtain a 1887bp fragment, and the amplified product was cloned into the pEASYBlunt vector (ampelin/Kana resistance) to construct a recombinant plasmid pTZ65 (shown in Figure 12).

The above PCRs all use FastPfuFly DNA polymerase, and the amplification conditions are as follows:

95°C, 5min; (95°C, 30sec, 55°C, 30sec, 72°C, 3min) 30×; 72°C, 10min

(3) Competent preparation of Escherichia coli and transformation of plasmid

1) Competent cells of Escherichia coli

Escherichia coli competent cells (TransT1 competent) were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

2) Transformation of recombinant plasmids

Take out a competent cell stored at -80°C and put it on ice to thaw for 5 minutes.

Add 5-10 μl of different amplification products (as shown in SEQ ID NO: 25 to SEQ ID NO: 30) and pEASYBlunt carrier connection solution obtained in the above step (2) to the melted competent cells, mix gently, and place in an ice-water bath 30min.

Heat shock at 42°C for 90 seconds, then immediately ice-water bath for 2 minutes.

Add 900 μl LB liquid medium, shake and recover for 1 hour at 37°C and 200 rpm.

Centrifuge at 5000 rpm for 3 minutes, discard the supernatant, resuspend the pellet in 100 μl LB medium, and coat the ampicillin (50 μg/mL)/Kana (50 μg/mL) double antibody solid plate.

Place the plate upside down in an incubator at 37°C and incubate for 12-16 hours before colonies grow.

(4) Transform Escherichia coli TransT1 competently with recombinant plasmids pTZ60, pTZ61, pTZ62, pTZ63, pTZ64 and pTZ65, that is, obtain strains ZT166, ZT167, ZT168, ZT169, ZT170 and ZT171 containing hydrocarbon-producing genes respectively.

Example 5: Expression of key gene ado-aar in Escherichia coli and analysis of hydrocarbon production in cyanobacteria

(1) Construction of ado-aar expression vector

Digest pTZ60 with BamHI and XhoI and recover the hydrocarbon-producing gene fragment ado-aar of NostoccalcicolaFACHB389, digest pET21b with BamHI and XhoI and recover the vector part, connect the two to transform Escherichia coli competent cells (TransT1 competent), and construct a recombinant plasmid pTZ68 (as shown in Figure 13)

Digest pTZ61 with BamHI and XhoI and recover the hydrocarbon-producing gene fragment ado-aar of Leptolyngbyasp.NIES-30, digest pET21b with BamHI and XhoI and recover the vector part, and connect the two to transform Escherichia coli competent cells (TransT1 competent), Construction obtains recombinant plasmid pTZ69 (as shown in Figure 14)

Digest pTZ62 with BamHI and XhoI and recover the hydrocarbon-producing gene fragment ado-aar of PhormidiumambiguumNIES-2119, digest pET21b with BamHI and XhoI and recover the vector part, connect the two to transform Escherichia coli competent cells (TransT1 competent), and construct Recombinant plasmid pTZ70 (as shown in Figure 15)

Digest pTZ63 with BamHI and XhoI and recover the hydrocarbon-producing gene fragment ado-aar of ChroogloeocystissiderophilaNIES-1031, digest pET21b with BamHI and XhoI and recover the vector part, connect the two to transform Escherichia coli competent cells (TransT1 competent), and construct Recombinant plasmid pTZ71 (as shown in Figure 16)

Digest pTZ64 with BamHI and NotI and recover the hydrocarbon-producing gene fragment ado-aar of Calothrixsp. Construct and obtain recombinant plasmid pTZ72 (as shown in Figure 17)

Digest pTZ65 with BamHI and XhoI and recover the hydrocarbon-producing gene fragment ado-aar of Scytonemasp.NIES-2130, digest pET21b with BamHI and XhoI and recover the vector part, and connect the two to transform Escherichia coli competent cells (TransT1 competent), Construct and obtain recombinant plasmid pTZ73 (as shown in Figure 18)

(2) Expression of ado-aar in Escherichia coli

Host bacteria: Escherichia coli fadE is the coding gene of fatty acyl-CoA dehydrogenase. The knockout of fadE can make cells accumulate fatty acyl-CoA, which is beneficial to the production of fatty acid derivatives (such as fatty acid ethyl esters, fatty alcohols) (Duan et al.2011) . Therefore, the relevant experiments of ado-aar functional verification of the present invention are selected with BL21 (DE3) The fadE mutant was used as host cells. The BL21(DE3)△fadE mutant strains were transformed with the recombinant plasmids pTZ68, pTZ69, pTZ70, pTZ71, pTZ72 and pTZ73, and the genetically engineered hydrocarbon-producing Escherichia coli strains ZT181, ZT182, ZT183, ZT184, ZT185 and ZT186 were respectively obtained.

Medium: The medium used is an improved M9 medium suitable for hydrocarbon production by recombinant bacteria, which has the following components: 6g/LNa ₂ HPO ₄ , 3g/LKH ₂ PO ₄ , 0.5g/LNaCl, 2g/LNH ₄ Cl, 0.25g /LMgSO ₄ 7H ₂ O,11mg/LCaCl ₂ ,27mg/LFeCl ₃ 6H ₂ O,2mg/LZnCl ₂ 4H ₂ O,2mg/LNa ₂ MoO ₄ 2H ₂ O,1.9mg/LCuSO ₄ 5H ₂ O, 0.5 mg/LH ₃ BO ₃ , 1 mg/Lthiamine, 200 mM Bis-Tris (pH 7.25) and 0.1% (v/v) Triton-X100.

Induction and culture: Inoculate the constructed ZT181, ZT182, ZT183, ZT184, ZT185 and ZT186 strains in 5 mL liquid modified M9 medium (containing 50 μg/mL ampicillin), culture at 30 °C, 250 rpm for 12 h, and then press 1 : 50 (v/v) inoculum ratio culture solution was transferred to 20 mL of modified M9 medium (containing 50 μg/mL ampicillin), cultured to the logarithmic phase (OD ₆₀₀ was about 0.6 to 0.8), and 0.5 mMIPTG inducer was added , 30°C, 250rpm cultured for 24h, the obtained bacterial liquid was used for the extraction of aliphatic hydrocarbons.

(3) Extraction and yield analysis of genetically engineered Escherichia coli aliphatic hydrocarbons

Take 10 mL of the above-mentioned hydrocarbon-producing genetically engineered Escherichia coli in the plateau stage into a 20 mL small beaker, place on an ice-water bath, and ultrasonically break for 10 min (10son, 10soff). After crushing, add 50 μg of n-eicosane as an internal standard, and after mixing, add 10 mL of chloroform:methanol (V:V is 2:1), shake and mix vigorously for 1-2 hours. Then centrifuge at 6,000 g for 10 min, transfer the lower organic phase to a new test tube, and dry it with nitrogen at 55°C. Add 100 μL of n-hexane to dissolve the extracted aliphatic hydrocarbons, then filter through a 0.22 μm filter membrane, and transfer to a 2 ml gas phase vial (built-in 200 μL liner). Use Agilent7890A gas chromatography-mass spectrometry (GC-MS) to measure aliphatic hydrocarbons. The GC-MS test program is: 40°C for 1min; 5°C/min to 200°C; 25°C/min to 240°C; 240°C Maintain for 15 minutes. The chromatographic column is HP-INNOWax (30m×250μm×0.25μm); the carrier gas is helium, the gas flow rate is 1mL/min, the injection volume is 1μL, and the inlet temperature is 250°C. The composition of aliphatic hydrocarbons and the total production of aliphatic hydrocarbons of each strain are summarized in Table 3.

Table 3. Analysis of hydrocarbon production by genetically engineered Escherichia coli

The above results confirm that the aar and ado gene sequences disclosed in the present invention derived from NostoccalcicolaFACHB389, Leptolyngbyasp.NIES-30, PhormidiumambiguumNIES-2119, ChroogloeocystissiderophilaNIES-1031, Calothrixsp.NIES-2101 or Scytonemasp.NIES-2130 can be normal in Escherichia coli Expression, so that Escherichia coli, which cannot produce hydrocarbons, acquires the ability to synthesize aliphatic hydrocarbons.

Example 6: Expression of NostoccalcicolaFACHB389 aliphatic hydrocarbon synthesis key gene in model cyanobacteria Synechocystissp.PCC6803 and analysis of hydrocarbon production

(1) Construction of ado-aar expression vector

Digest pTZ60 with NdeI to make up the level, and then digest with XhoI to recover the hydrocarbon-producing gene fragment ado-aar of NostoccalcicolaFACHB389, and clone it into pXX47 treated with the same restriction enzyme (for example, see Chinese invention patent application 201310595856.3) (provided by Cyanobacteria Genome neutral site integration platform, resistance selection marker and strong promoter), the recombinant plasmid pTZ66 was constructed.

(2) Expression of ado-aar in cyanobacteria

Host bacteria: Synechocystissp.PCC6803. Synechocystissp.PCC6803 was transformed with recombinant plasmid pTZ66 to obtain genetically engineered hydrocarbon-producing cyanobacteria ZT187 (spectinomycin resistance).

Medium: The medium used for cyanobacteria culture is BG11 medium, which has the following components: 1.5gL ^-1 NaNO ₃ , 40mgL ^-1 K ₂ HPO ₄ 3H ₂ O, 36mgL ^-1 CaCl ₂ 2H ₂ O, 6mgL ^{- 1} citric acid, 6mgL ^-1 ferric ammonium citrate, 1mgL ^-1 EDTA disodium salt, 20mgL ^-1 NaCO ₃ , 2.9mgL ^-1 H ₃ BO ₃ , 1.8mgL ^-1 MnCl ₂ 4H ₂ O, 0.22mgL ^-1 ZnSO ₄ ·7H ₂ O, 0.39 mgL ⁻¹ NaMoO ₄ ·2H ₂ O, 0.079 mgL ⁻¹ CuSO ₄ ·5H ₂ O and 0.01 mgL ⁻¹ CoCl ₂ ·6H ₂ O. Add 8mMTES buffer, 0.3% Na ₂ S ₂ 0 ₃ and 1.5% agar powder to the solid medium.

Transformation method: Take 5 mL of Synechocystissp.PCC6803 wild-type cells in the logarithmic growth phase (OD ₇₃₀ is about 0.5-1.0), and collect the cells by centrifugation; wash the cells twice with fresh BG11 medium, and then resuspend the cells in 0.5 mL of BG11 medium. Take 0.2 mL of the cell suspension and place it in a new PCR tube, add 2 to 3, the recombinant plasmid pTZ66, mix well, and incubate for 4 hours at 30°C, 30 μ0, ^-2 s ^-1 light. Then the mixture was spread on the nitrocellulose membrane spread on the BG11 plate, and placed at 30°C, 30 nitrocellulose ^-2 s ^-1 light conditions and cultured for 24 hours. Then, transfer the nitrocellulose membrane to a solid plate with spectacular resistance. After 30°C and 30°C, continue to cultivate for 5 to 7 days under the light condition of ^-2 s ^-1 , and pick the transformant from the plate. , cultured by streaking on a fresh BG11 plate (Spectacular resistance) for 5-7 days; finally inserted into the Spectacular-resistant liquid BG11 medium for culture, extracting the genome, and after PCR verification of the correct genotype, take the plateau bacterial liquid for use in Aliphatic hydrocarbon detection.

(3) Hydrocarbon production and yield analysis of genetically engineered cyanobacteria

The method for extracting aliphatic hydrocarbons from genetically engineered cyanobacteria is the same as in Example 2, except that 100 μg of n-eicosane is added as an internal standard.

Analysis of aliphatic hydrocarbon production in genetically engineered cyanobacteria ZT187: after GC-MS determination, the key gene ado-aar derived from NostoccalcicolaFACHB389 for aliphatic hydrocarbon synthesis was integrated into the genome of model strain Synechocystissp.PCC6803:

In terms of hydrocarbon production diversity: the host bacteria obtained the ability to synthesize pentadecane (C15) (Figure 20).

In terms of the production of aliphatic hydrocarbons: the total production of aliphatic hydrocarbons of the host bacteria is significantly increased, which is about 2 times that of the wild-type strain (Figure 21).

The above data prove that the key genes for hydrocarbon production provided by the present invention increase the diversity of hydrocarbon production of cyanobacteria aliphatic hydrocarbons and increase the production of aliphatic hydrocarbons of cyanobacteria.

The present invention is based on the current research status of genetically engineered cyanobacteria for the synthesis of aliphatic hydrocarbon new bioenergy sources, inspired by the identification of aliphatic hydrocarbon synthesis pathways in cyanobacteria, taking 6 strains of cyanobacteria that our team has obtained but whose genomes are unknown as research objects, and systematically analyzed The characteristics and yield of its aliphatic hydrocarbon synthesis were studied. And based on sequence identity analysis, whole genome sequencing and PCR technology, the key gene sequence for hydrocarbon production of the cyanobacteria strain was obtained. At the same time, the obtained hydrocarbon-producing gene was functionally verified in Escherichia coli, and a genetically engineered cyanobacteria for efficiently synthesizing aliphatic hydrocarbons was constructed based on the obtained sequence. Without wishing to be bound by any theory, the inventors believe that the obtained cyanobacteria strains and key gene sequences for hydrocarbon production will provide valuable candidate gene resources for the direct synthesis of aliphatic hydrocarbon biofuels using microbial synthesis systems.

At the same time, on the basis of the hydrocarbon-producing gene of the present invention, the coding gene (olefinssynthase, ols) of terminal olefin synthase can also be added to make it synthesize terminal olefins through the type I polyketide synthase pathway (Mendez-Perez et al. 2011), Furthermore, it can also provide valuable candidate gene resources for obtaining aliphatic hydrocarbon biofuels.

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Claims (10)

1. a cyanobacteria hydrocarbon-producing gene, characterized in that: Hydrocarbon-producing genes include acyl-ACP reductase coding gene (aar) and fatty aldehyde deformyl oxygenase coding gene (ado). 2. by the cyanobacteria hydrocarbon production gene described in claim 1, it is characterized in that: The coding gene (aar) of the fatty acyl ACP reductase and the coding gene (ado) of fatty aldehyde deformyl oxygenase are cyanobacteria NostoccalcicolaFACHB389, Leptolyngbyasp.NIES-30, PhormidiumambiguumNIES-2119, ChroogloeocystissiderophilaNIES-1031, Calothrixsp.NIES - aar and ado of 2101 or Scytonemasp.NIES-2130. 3. by the cyanobacteria hydrocarbon-producing gene described in claim 1 or 2, it is characterized in that: The coding gene (aar) of the fatty acyl ACP reductase and the coding gene (ado) of fatty aldehyde deformyl oxygenase have SEQIDNO:1 and 2, SEQIDNO:3 and 4, SEQIDNO:5 and 6, SEQIDNO:7 and 8, the sequences shown in SEQ ID NO: 9 and 10 or SEQ ID NO: 11 and 12. 4. A construct characterized in that: The construct comprises a promoter, and the gene encoding fatty acyl ACP reductase (aar) and the encoding gene (ado) of fatty aldehyde deformyl oxygenase controlled by the promoter. 5. The construct according to claim 4, characterized in that: The promoter of the construct is the _PcpcB promoter derived from cyanobacteria or the T7 promoter derived from Escherichia coli. 6. by the described construct of claim 4 or 5, it is characterized in that: the coding gene (aar) of described fatty acyl ACP reductase and the coding gene (ado) of fatty aldehyde deformyl oxygenase are cyanobacteria NostoccalcicolaFACHB389 aar and ado of , Leptolyngbyasp.NIES-30, PhormidiumambiguumNIES-2119, ChroogloeocystissiderophilaNIES-1031, Calothrixsp.NIES-2101 or Scytonemasp.NIES-2130. 7. A genetically engineered bacterium for synthesizing aliphatic hydrocarbons, characterized in that: the engineered bacterium contains the above construct. 8. the genetically engineered bacterium capable of synthesizing aliphatic hydrocarbons according to claim 7, is characterized in that: The construct was introduced into E. coli by transformation. 9. The genetic engineering bacterium capable of synthesizing aliphatic hydrocarbons according to claim 8, characterized in that: said construct is introduced into Escherichia coli BL21 (DE3) fadE mutant strain by transformation. 10. A cyanobacteria hydrocarbon-producing gene as claimed in claim 1 is used in the application of synthetic aliphatic hydrocarbons.