KR20140013106A - Novel microalgae nostoc sp. knua003 and method for producing alkane, fatty alcohol, and fatty acid from the same - Google Patents

Abstract

The present invention relates to a novel microalgae Nostoc KNUA003 (Nostocsp. KNUA003) strain (KCTC 12063BP) and a method for producing heptadecane, hexadecenol, and fatty acids of C16-C18. The Nostoc KNUA003 strain of the present invention can autotrophically biosynthesize heptadecane (C17H36), which can directly be used as a fuel, while not requiring transesterification, as well as produce major components such as a fatty alcohol (hexadecenol (C20H40O)) and other general microalgae biodiesel components (palmitic acid (C16:0), palmitoleic acid (C16:1), linoleic acid (C18:2) and linolenic acid (C18:3)). Therefore, Nostocsp. KNUA003 can be effectively be used as raw material for producing biodiesel.

Description

Novel microalgae Nostoc KNUFA03 strain and method for producing alkanes, fatty alcohols and fatty acids therefrom {Novel Microalgae Nostoc sp. KNUA003 and method for producing alkane, fatty alcohol, and fatty acid from the same}

The present invention relates to a novel microalgal Nostoc sp. KNUA 003 strain and a method for producing _{heptadecane, hexadecenol and fatty acids of C 16} to C _{18 therefrom.}

Humanity is facing the double grief of global warming and depletion of energy sources due to reckless use of fossil fuels, and the worldwide demand for sustainable and renewable energy resources is increasing exponentially every year. In Korea, it has been actively developing new and renewable energy, but it is still in the beginning stage. Unlike fossil energy, bioenergy is eco-friendly energy that does not generate carbon dioxide, and organic carbon compounds in which energy is accumulated are generated through a photosynthesis process that captures carbon dioxide and solar energy, and energy is extracted from the stored biomass.

Bioenergy is renewable and non-toxic energy with almost no harmful components, and it is completely decomposed by microorganisms and produced by fixing carbon dioxide, thereby reducing the greenhouse effect. Currently, bioenergy research using terrestrial resources is being conducted, but policies and studies to use marine resources (microalgae and algae) are being conducted worldwide as limitations in the use of terrestrial resources such as competition with food resources and food cultivation area are revealed It is starting.

Bioenergy is typically bioethanol/butanol and biodiesel. Research on the production of bioethanol/butanol is being conducted centering on macroalgae, and biodiesel research is being conducted centering on microalgae. .

*In particular, microalgae, including cyanobacteria, are attractive candidates for biofuel production due to their higher photosynthetic efficiency and oil yield compared to land sources (Huntley and Redalje 2007; Li et al. 2008).

Biodiesel is produced in the form of methyl ester or ethyl ester by transesterification of fatty acid contained in biomass, and glycerol is produced as a by-product. Unlike petrodiesel, biodiesel is an eco-friendly energy with significantly low toxic substances emitted when burned.

The solar energy utilization efficiency of microalgae is 5%, about 25 times higher than 0.2% of land plants, and the carbon dioxide immobilization rate is 15 times that of pine trees, which is very efficient. Currently, microalgae are used not only as feed or biological fertilizer, but also as a variety of health foods. Microalgae have a photosynthetic mechanism similar to terrestrial plants in microscopic size, but are more effective in converting solar energy into biomass because they live in an aquatic environment where access to water, carbon dioxide and other nutrients is efficient.

Recently, research on the production of biodiesel using microalgae has been actively conducted overseas. In December 2007, Shell and HR Biopetroleum, a world refining company, jointly installed a microalgae culture facility in Hawaii for the purpose of producing biodiesel. In January 2008, Solazyme of the United States introduced a prototype called Soladiesel by Mercedes-Benz. It was announced that it would provide it to diesel vehicles to test its performance, and Chevron signed an agreement with Solazyme to develop joint technology. Recently, in France, a budget of 2.8 million euros has been allocated to the Shamash program for three years, and active research on the use of algae is being promoted. Along with the production of fatty acids from algae, research is underway to transform them into bioenergy.

In Korea, small and medium-sized companies are focusing on the development of biodiesel production process technology and the discovery of biodiesel energy, and research on biodiesel production using seaweed is currently underway. However, there are still many technical barriers that must be overcome.

One of the biggest challenges in microalgal biodiesel production is to reduce the cost of lipid extraction, which amounts to 90% of the total production energy consumption (Lardon et al. 2009). Algal biodiesel is generally obtained by transesterification of algal oil with alcohol in the presence of an acidic or basic catalyst (Meher et al. 2006; Demirbas 2007; Johnson & Wen 2009). Methanol, due to its low cost, is the most commonly used alcohol in this process (Demirbas 2005), and even cheap methanol has been reported to account for 47% of the total energy consumption in biodiesel production (de Souza et al. 2010). A recent study by Schirmer et al. (2010) has shown that some blue-green algae strains can naturally produce alkane-based hydrocarbons such as _{pentadecane (C 15} H ₃₂ ) and heptadecane (C ₁₇ H _{36 ).} Since petrodiesel consists of a mixture of alkanes having 8 to about 20 carbon atoms, alkanes synthesized from these blue-green algae can be used directly as biodiesel without converting triglycerides to liquid hydrocarbons, and thus can be used as a substitute for petroleum fuel. You will be able to play a role as

Thus, the inventors of the present invention constitute a filamentous nitrogen-fixed blue-green algae Nostoc sp. KNUA003 was aseptically isolated from summer bloom samples in Daecheong Lake, Korea, and this blue-green algae was shown to be capable of autotrophically producing not only heptadecane but also microalgal biodiesel components under optimal growth conditions. It is expected to contribute to bioenergy production as a biodiesel feedstock.

An object of the present invention is hepta-decane, hexadecane and senol having C ₁₆ to novel microalgae to produce fatty acids of C ₁₈ no stock sp. The purpose is to provide KNUA003. The KNUA003 can autotrophically produce alkane-based hydrocarbons such as _{heptadecane (C 17} H ₃₆ ) that can be directly used as fuel without requiring transesterification _{, and fatty alcohols (hexadecenol (C 20} H ₄₀ O)) and other common microalgal biodiesel components (palmitic acid (C16:0), palmitoleic acid (C16:1), lenoleic acid (C18:2) and linolenic acid (C18:3)) are also major It is produced by the KNUA003 strain as a fatty acid.

Another object of the present invention is to provide a method for producing _{heptadecane, hexadecenol, and fatty acids of C 16} to C _{18 from the novel Nostock KNUA003 strain.}

The invention hepta-decane, hexadecane and senol having C ₁₆ to microalgae of the furnace stock KNUA003 (Nostoc sp to produce fatty acids of C _18. KNUA003) A strain (KCTC 12063BP) is provided.

In addition, the present invention is the Nostoc KNUA003 (Nostoc sp. KNUA003) Strain Method of production of a hepta-decane, of hexa to senol and C ₁₆ to C ₁₈ fatty acid which comprises culturing (KCTC 12063BP) and to separate the fatty acids of heptanoic decane, hexadecane having senol and C ₁₆ to C ₁₈ from the culture medium Provides.

In addition, the present invention is the Nostoc KNUA003 (Nostoc sp. KNUA003) It provides a method for producing biodiesel, characterized in that fatty acid esters and glycerol are produced by transesterification _{of C 16} to C ₁₈ fatty acids produced in the strain (KCTC 12063BP).

Furthermore, an object of the present invention is to provide a biodiesel production method for producing fatty acid esters by transesterifying fatty acids of _{C 16} to C _{18 produced from the novel Nostock KNUA003 strain.}

Conventional microalgal biodiesel is generally obtained by transesterification of algal oil using alcohol in the presence of an acidic or basic catalyst, and this method has a disadvantage that the cost of lipid extraction is very high. there was. The present invention is a novel microalgae Nostock sp. Using the KNUA003 strain, not only can biodiesel be produced in the conventional way, but also alkane-based hydrocarbons such as heptadecane can be produced naturally and have the effect of directly producing biodiesel without conversion to liquid hydrocarbons. .

1 shows Nostoc KNUA003 (Nostoc sp. KNUA003) It is a diagram showing the microscopic observation results of the KCTC 12063BP strain.
Figure 2 shows Nostoc KNUA003 (Nostoc sp. KNUA003) is a diagram showing the difference in growth according to temperature conditions of the KCTC 12063BP strain.
Figure 3 shows Nostoc KNUA003 (Nostoc sp. KNUA003) It is a diagram showing the difference in growth according to the pH condition of the KCTC 12063BP strain.
Figure 4 is a diagram showing the difference in growth according to the salinity condition of the Nostoc KNUA003 (Nostoc sp. KNUA003) KCTC 12063BP strain.
5 is a diagram showing the GC/MS analysis results of Nostock KNUA003(a), Nostock PCC6720(b), and Nostock PCC7120(c).
6 is a diagram showing the electron impact (EI) mass spectra of heptadecane (a) and hexadecenol (b) produced by Nostock KNUA003.

The present invention is a microalgae producing heptadecane, hexadecenol and C ₁₆ to C ₁₈ fatty acids, Nostoc KNUA003 (Nostoc sp. KNUA003) strain (KCTC 12063BP) is provided.

The Nostoc KNUA003 (Nostoc sp. KNUA003) was collected from Daecheong Lake (36°22'N, 127°28'E) in September 2009, and then selected single colonies formed by streaking on agar and selected 16S rRNA, phycocyanin (PC) and rbcLX. It was identified through gene sequence analysis. The blue-green algae Nostok KNUA003 ( Nostoc sp. KNUA003) strain was deposited with the Korea Research Institute of Bioscience and Biotechnology (KCTC) on November 9, 2011 (accession number: KCTC 12063BP).

The Nostock KNUA003 strain produces an alkane hydrocarbon, preferably heptadecane (C ₁₇ H ₃₆ ). In addition, the Nostock KNUA003 strain is a major fatty acid having 16 carbon atoms and 0 or 1 double bonds (16:0, 16:1 fatty acids) and 18 carbon atoms and 2 or 3 double bonds (18:2 , 18:3 fatty acids). In addition, the Nostock KNUA003 strain produces a C ₂₀ fatty alcohol, preferably hexadecenol.

In addition, the present invention

1) culturing the Nostock KNUA003 strain;

2) extracting heptadecane, hexadecenol, and fatty acids of _{C 16} and C ₁₈ from the culture medium obtained in step 1); A method for producing _{heptadecane, hexadecenol and fatty acids of C 16} and C _{18 comprising} to provide.

As the growth conditions of the strain, the culture temperature may be 15 ℃ to 30 ℃, culture pH of 4.0 to 12, and can be cultured under NaCl of 0.2M or less. As the strain culture medium, a medium commonly used in the art may be used.

_{As a method for extracting heptadecane, hexadecenol, and fatty acids of C 16} to C ₁₈ from the culture medium of the strain, any method known in the art may be used. The extraction solvent may be selected from the group consisting of pentane, nucleic acid, cyclohexane, toluene, methylene chloride, ethyl acetate, acetone, chloroform, methanol, and a mixed solvent thereof.

In addition, the present invention can transesterify triglycerides produced from Nostock KNU003 strain to separate fatty acid esters and glycerol to prepare biodiesel. The fatty acid ester may preferably be a fatty acid methyl ester or a fatty acid ethyl ester, but is not limited thereto. The biodiesel may be produced in the form of methyl ester or ethyl ester by transesterification of fatty acids contained in biomass. The transesterification process refers to a method of transforming a large, branched molecular structure of fat contained in biomass into a small, straight-chain molecule required by a regular diesel engine. Triton X-100 or Tween 60 may be added as an emulsifier in the transesterification process, but is not limited thereto. The emulsifier promotes the interfacial stability of the bio-oil and mixes well, thereby increasing the reaction yield, thereby reducing the cost of recovering biodiesel. In addition, sodium hydroxide or potassium hydroxide may be used as a reaction catalyst for the transesterification process, but is not limited thereto.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

<Example>

1. Sample collection and separation of blue-green algae

Blue-green algae samples were collected from Daecheong Lake (36°22'N, 127°28'E) in September 2009. Thereafter, the collected sample was brought to the laboratory, and 1 ml of the sample was transferred to 100 ml BG-11 medium (Rippka et al. 1979) containing cycloheximide (cycloheximide, Sigma, St. Louis, MO, USA) at a concentration of 250 ug/ml. ) Was inoculated. The flasks were incubated with shaking at 160 rpm at 25 °C, and incubated until a pronounced biomass was observed.

A well-cultured culture solution (1.5 ml) was centrifuged at 3,000 g for 15 minutes (Centrifuge 5424, Eppendorf, Hamburg, Germany). Centrifuged pellets were inoculated on BG-11 agar containing imipenem (100ug/ml) (imipenem, Choongwae Pharma Corporation, Seoul, Korea) and cyclohexamide (20ug/ml), and cultured in the dark for 24 hours. Thus, the sterile culture was isolated. Thereafter, the plates were incubated at 25° C. in a light: dark (16 hours: 8 hours) cycle, and the growth of filamentous bodies was observed daily. When the Nostalgic filaments growing outside the plate grew to the extent that they could be seen with the naked eye, they were aseptically inoculated with new BG-11 plates to isolate them from contaminating bacteria (Hong et al. 2010). Thereafter, Nostock filamentous bodies were inoculated onto R2A and LB agar plates (Becton, Dickinson and Company, Sparks, MD, USA), and cultured in the dark for 14 days to confirm the sterility of the culture. The sterile culture was named KNUA003.

2. Morphology and Molecular Identification

For the morphological analysis of KNUA003, it was cultured without a nitrogen source in BG-11 medium for 20 days. Live cells were harvested and suspended in sterilized distilled water, and observed at 400 times magnification on a Zeiss Axioskop 2 optical microscope (Carl Zeiss, Standort Gottingen, Vertrieb, Germany) equipped with a differential interference contrast (DIC) optical lens.

The results are shown in FIG. 1.

As shown in Figure 1, strain KNUA003 is Nostoc . It has the general characteristics of the genus. The microorganism was composed of filamentous bodies having straight or slightly curved trichomes composed of vegetative cells in the form of beads, and both intercalary and terminal heterozygous cells were observed. The color was green to dark green, and densely aggregated filaments formed mats that can be observed with the naked eye. Overall, the morphological features of strain KNUA003 were found in Nostoc . Suggested that it belongs to the genus.

* In order to confirm the taxonomic location of the strain through a molecular biology approach, the identification of Nostoc sp. was performed by sequence analysis of 16S rRNA, rbcLX, and PC-IGS genes. For the amplification of 16S rRNA gene fragments, the primer sets CYA106F and CYA781R(a) and CYA781R(b) described by Nubel et al. (1997) were used. The phycocyanin encoding operon intergenic spacer (PC-IGS) region was amplified using the primer pairs PCβF and PCαR specific to blue-green algae (Neilan et al. 1995). The region of RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) rbc LX was amplified as described in the prior art using primers CW and CX (Rudi et al. 1998). Synthesis and DNA sequencing of the primers used in this study were performed by Macrogen (Macrogen, Seoul, Korea). Nostoc sp. PCC 6720 is from the Pasteur Culture Collection of Cyanobacteria, PCC, Paris, France, Anabaenopsis circularis NIES21 and Nostoc sp. NIES219 was purchased from the National Institute for Environmental Studies (NIES, Ibaraki, Japan), respectively. The DNA sequences obtained in this study were submitted to the NCBI database, and their accession numbers are shown in Tables 1 and 2.

Based on the 16S rRNA sequence data, strain KNUA003 was identified as Nostoc sp. PCC6720, Anabaenopsis circularis NIES21 and Nostoc sp. It showed 100% sequence homology with the strains HK-01 ( Nostoc sp. NIES2109 and the same strain). However, due to the characteristics of the 16S rRNA gene with a small variable region, other gene regions such as PC-IGS and RuBisCO rbc LX were analyzed. As a result, Nostoc sp. The blue-green algae most closely related to KNUA003, Nostoc sp. Only PCC6720 was selected for further analysis.

16S rRNA, rbcLX, PC-IGS BLAST Search results of KNUA003 Marker
gene Access number Closest match
(GenBank access number) Nesting
(%) sequence
Similarity
(%) 16S rRNA JF740671 Nostoc sp. PCC6720 (DQ185240)
Anabaenopsis circularis NIES21 (AF247595)
Nostoc sp. HK-01 (AB085687) ^a 100
100
100 100
100
100 PC-IGS JF740672 Nodularia spumigena KNUA005 (HM241946) 100 86 RuBisCO
rbc LX JN251745 Nostoc sp. PCC6720 (DQ185297) 72 100

Nostoc sp. HK-01 is Nostoc sp. The same strain as NIES-2109.

16S rRNA, PC-IGS, RuBisCO rbc LX sequence comparison result Blue-green algae Nostoc sp. Base sequence comparison with KNUA003 16S rRNA
PC-IGS
RuBisCO rbc LX
Nostoc sp. PCC6720 100% same
(DQ185240) 1 bp different
(JF740673) 100% same
(JN251746) Anabaenopsis circularis NIES21 100% same
(AF247595) 1 bp different
(JF740674) 2 bp different
(JN251747) Nostoc sp. NIES2109 100% same
(AB085687) 1 bp different
(JF740675) 1 bp different
(JN251748)

3. Physiological characteristics of strain KNUA 003

In order to confirm the physiological characteristics of strain KNUA 003, experiments were conducted while varying temperature, pH, salinity and carbon source. The seed culture (1 ml) of the 20-day-old strain KNUA003 was inoculated into BG-11 medium (100 ml) by repeating 3 times, and cultured for 20 days. In order to know the optimum temperature of the culture, the survival and growth of strain KNUA003 were observed at 5, 10, 15, 20, 25 and 30°C. Salinity and acid resistance tests were carried out at an optimum growth temperature of 25°C. Salinity and acidity were 0.1, 0.2, 0.3, and 0.4 M sodium chloride (NaCl) and a pH range of 3.0 to 13.0, respectively. The alternative carbon source availability test was also carried out as described in Miura and Yokota (2006) using glucose, sucrose, fructose, ribose, sodium acetate, glycerol and glycolic acid at 25°C. The optical density of the culture was measured at 750 nm in an Optimizer 2120UV spectrometer (Mecasys Co. Ltd., Daejeon, Korea) to measure the density of strain KNUA 003.

Results are shown in FIGS. 2 to 4 and Table 3.

Physiological characteristics of strain KNUA 003 Physiological factor growth Temperature
pH
NaCl resistance
Alternative carbon source availability:
Glucose
Sucrose
Ribose
Acetate
Fructose
Glycerol
Glycolic acid
Nitrogen-free-medium 15℃~30℃
pH 4 ~pH 12
Up to 0.2 M

×
×
×
×
○
×
×
×

2 to 4, and as shown in Table 3, the optimum growth temperature of the strain KNUA003 was 25 ℃, but it was possible to grow at 20 ℃ and 30 ℃. However, this microorganism was not able to survive below 10°C, and significantly delayed growth was observed in culture at 15°C. The growth pH ranged from pH 4.0 to 12.0, and the optimum pH was pH 7.4. Strain KNUA003 could not tolerate NaCl concentrations higher than 0.2 M. It was observed that strain KNUA003 can use fructose as an alternative carbon source.

4. Check GC/MS analysis results

For GC/MS analysis, Nostoc sp. KNUA003 was compared to two comparative strains, Nostoc sp. It was inoculated in BG-11 medium (pH 7.4) with PCC6720 and PCC7120 in three repetitions, and cultured at 25°C for 20 days. Cells were harvested by centrifugation at 3,220 g (Centrifuge 5810R, Eppendorf, Hamburg, Germany), and microalgal lipids were extracted using a partially modified Bligh-Dyer method (Lee et al. 2010). Specifically, a lipid component was extracted from the lyophilized blue-green algae biomass using a chloroform:methanol (2:1) solvent mixture. Biodiesel was obtained by transesterification between lipid and methanol, and potassium hydroxide (KOH) was used as a basic catalyst. The fatty acid composition of the culture was measured by GC/MS (Jeol JMS700 mass spectrometer equipped with Agilent 6890N GC) at the Daegu Center of the Korea Basic Science Institute (KBSI). Nostoc sp. PCC7120 is known to biosynthesize heptadecane, so this strain was included as a positive control strain for heptadecane production and analysis. In addition, strain Nostoc sp. PCC6720 was also selected as this strain was most closely associated with strain KNUA003 based on molecular evidence from 16S rRNA, PC-IGS and RuBisCO rbc LX (Table 2).

The results are shown in FIGS. 5, 6 and 4.

Nostoc sp. Alkanes, Fatty Acids, Fatty Alcohol Composition Components of KNUA003, PCC6720 and PCC7120 ingredient KNUA003(%) PCC6720(%) PCC7120(%) Heptadecane (C ₁₇ H ₃₆ ) 14.5 ±7.1 19.1 ±9.7 1.3 ±0.3 Myristic acid (C ₁₄ H ₂₈ O ₂ = C14:0) 0.3 ±0.1 0.3 ±0.2 0.2 ±0.0 Heneicosane (C ₂₁ H ₄₄ ) 2.7 ±1.7 1.5 ±1.2 2.0 ±1.2 Palmitoleic acid (C ₁₆ H ₃₀ O ₂ = C16:1) 17.6 ±3.5 14.0 ±4.5 25.4 ±1.7 Palmitic acid (C ₁₆ H ₃₂ O ₂ = C16:0) 25.1 ±5.7 22.0 ±1.9 25.3 ±4.8 Methyl palmitoleate (C ₁₇ H ₃₂ O ₂ ) 0.4 ±0.3 1.2 ±0.6 1.1 ±0.3 Heptadecanoic acid (C ₁₇ H ₃₄ O ₂ = C17:0) 0.1 ±0.1 0.8 ±0.1 0.0 ±0.0 Linoleic acid (C ₁₈ H ₃₂ O ₂ = C18:2) 8.5 ±2.4 7.2 ±3.2 13.8 ±1.9 Linolenic acid (C ₁₈ H ₃₀ O ₂ = C18:3) 17.6 ±5.8 15.5 ±5.4 24.5 ±2.8 Oleic acid (C ₁₈ H ₃₄ O ₂ = C18:1) 0.8 ±0.4 0.9 ±0.7 1.3 ±0.2 Hexadecenol (C ₂₀ H ₄₀ O) 11.1 ±6.5 13.9 ±4.7 3.9 ±0.7 Strearic acid (C ₁₈ H ₃₆ O ₂ = C18:0) 0.9 ±0.5 2.4 ±0.8 1.1 ±0.4

As shown in Fig. 5 and Table 4, the GC/MS results were obtained from all blue-green algae strains tested in this study, Nostoc sp. KNUA003(a), Nostoc sp. PCC7120(b), Nostoc sp. PCC6720 (c) is heptadecane (C ₁₇ H ₃₆ ), palmitic acid (C16:0), palmitoleic acid (C16: 1), linoleic acid (C18:2) _{with hexadecenol (C 20} H _{40 O).} And it was confirmed that fatty acids such as linolenic acid (C18:3) can be biosynthesized. In particular, strains KNUA003 and PCC6720 contained heptadecane and hexadecenol as their main lipid components.

As shown in Figure 6, it was possible to confirm the heptadecane (a) and hexadecenol (b) produced by Nostock KNUA003 using an electron impact (EI) mass spectra.

5. Detection and identification of genes related to alkane biosynthesis

Nostoc sp. In order to confirm that heptadecane was synthesized in KNUA003, genes related to alkanes biosynthesis were detected.

Aldehyde dicarbonylase and acyl-acyl carrier protein (ACP) reductase were used in Nostoc sp. In order to amplify from KNUA003, two primer sets were already prepared in Nostoc sp. It was designed based on PCC7120. Nostoc sp. In KNUA003, partial fragments of the two genes were successfully amplified by the above primer sets, and the primer sets used at this time were alr5283F-3 and alr5283R, and alr5284F-5 and alr5284R-5, respectively. Using Genome-walking PCR (Clontech, Mountain View, CA, USA), the sequence before and after each gene sequence obtained only partially through the PCR reaction was analyzed. Specifically, DNA was digested with restriction enzymes Dra I, Eco RV, Pvu II and Stu I restriction enzymes, and GenomeWalker adapters were attached to both ends of each DNA fragment. Each constructed GenomeWalker library was subjected to two nested-PCR reactions using adapter primers (AP1 and AP2) and two designed gene-specific primers (GSP1 and GSP2). Then, the secondary PCR products were cut out from the agarose gel and analyzed. Finally, Nostoc sp. Full length aldehyde dicarbonylase and acyl-ACP reductase including Open Reading Frames (ORFs) in KNUA003 and PCC6720 were applied to primer sets (KNUA003_5283F and KNUA003_5283R and KNUA003_5284F and KNUA003_5284R). Amplified by. All primers used in this study were designed using Primer3Plus software (Untergasser et al. 2007), and are shown in Table 5. The results are shown in Table 6.

Type of primer used in the experiment name Sequence (5'->3') Length (bp) gene alr5283F-3
(forward)
SEQ ID NO: 1 GGG GAA CAA GAA GCA TAC GA 20 partial aldehyde decarbonylase alr5283R-3
(reverse)
SEQ ID NO:2 TTA AGC TGC TGT AAG TCC GT 20 partial aldehyde decarbonylase alr5284F-5
(forward)
SEQ ID NO:3 CTT GCT TTT TAC CGG AGA TGC TAG C 25 partial acyl-ACP reductase alr5284R-5
(reverse)
SEQ ID NO:4 AGA GCA TAG ATT CCG CAA AAC AAG C 25 partial acyl-ACP reductase 5283_GSP1R
(upstream)
SEQ ID NO: 5 GTA TTC ATC TTT GAC TAC ACC CTC AGT 27 partial aldehyde decarbonylase GSP1 5283_GSP2R
(upstream)
SEQ ID NO: 6 GAA TCA GTA AGC AAG TGA CAA CGT TAC 27 partial aldehyde decarbonylase GSP2 5284_GSP1F
(downstream)
SEQ ID NO: 7 ATA CAC ACA CTG CCT ACA TTA TCT GTC 27 partial acyl-ACP reductase GSP1 5284_GSP2F
(downstream)
SEQ ID NO: 8 GAA AGC AAC AGT AGC TAT ATG TGG AG 26 Partial acyl-ACP reductase GSP2 5284_GSP1R
(upstream)
SEQ ID NO: 9 TAT AGC TAC TGT TGC TTT CGA CAG TTC 27 partial acyl-ACP reductase GSP1 5284_GSP2R
(upstream)
SEQ ID NO: 10 GAC AGA TAA TGT AGG CAG TGT GTG TAT 27 partial acyl-ACP reductase GSP2 KNUA003_5283F
(forward)
SEQ ID NO: 11 CGG TCT GTA CTG TTC TTA ATT CTG G 25 full aldehyde decarbonylase KNUA003_5283R
(reverse)
SEQ ID NO: 12 TTA AGC TGC TGT AAG TCC GTA GG 23 full aldehyde decarbonylase KNUA003_5284F
(forward)
SEQ ID NO: 13 ATC CCT GTT GCT GAT GAT TTT G 22 full acyl-ACP reductase KNUA003_5284R
(reverse)
SEQ ID NO: 14 GAC TTT GCT AAA GCA GCA GAG G 22 full acyl-ACP reductase

Nostoc sp. Aldehyde dicarbonylase gene and acyl-ACP reductase gene of KNUA003 and PCC6720 gene Translated amino acids Similarity
(Access number /% ID) Nostoc sp. KNUA003 (Access Number / Length) Nostoc sp. PCC6720
(Access Number / Length) Aldehyde
Dicarbonylase Hypothetical aldehyde dicarbonylase (JN391509 / 232 amino acids) ^b Hypothetical aldehyde dicarbonylase (JN581686 / 232 amino acids) ^c Hypothetical protein Ava_2533 [ Anabaena variabilis ATCC29413]
(YP 323043 / 86%) &
Hypothetical protein alr5283
[ Nostoc sp. PCC7120]
(NP 489323 / 86%) Acyl-ACP
Reductase Hypothetical acyl-ACP reductase
(JN391510 / 340 amino acids) ^d Hypothetical acyl-ACP reductase
(JN581687 / 340 amino acids) ^e The hypothetical protein Ava_2534 [ Anabaena variabilis ATCC29413]
(YP 323044 / 91%)

As shown in Table 6, Nostoc sp. The translated amino acid sequences of aldehyde dicarbonylase in KNUA003 (JN391509) and PCC6720 (JN581686) were identical to each other, and their closest matches were the hypothetical protein Ava_2533 (YP 323043) [ Anabaena variabilis ATCC29413] and the hypothetical protein alr5283 [ Nostoc sp. PCC7120] (NP 489323), the identity was 86%. For acyl-ACP reductase, Nostoc sp. The amino acid sequences translated from KNUA003 (JN391510) and PCC6720 (JN581687) showed 99.7% homology to each other (one amino acid difference), and their closest match was the hypothetical protein Ava_2534 [ Anabaena variabilis ATCC29413] (YP 323044 ), and the identity was 91%. Through this study, it was possible to prove the existence of aldehyde dicarbonylase and acyl-ACP reductase genes related to alkane biosynthesis in strain KNUA003. By using these genes to biosynthesize alkanes, it is more attractive in biofuel production. It is believed that a cost effective process can be achieved.

Korea Research Institute of Bioscience and Biotechnology KCTC12063BP 20111109

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Novel Microalgae Nostoc sp. KNUA003 and method for producing alkane, fatty alcohol, and fatty acid from the same <130> p106 <160> 14 <170> KopatentIn 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of partial aldehyde decarbonylase (alr5283F-3) <400> 1 ggggaacaag aagcatacga 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of partial aldehyde decarbonylase (alr5283R-3) <400> 2 ttaagctgct gtaagtccgt 20 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of partial acyl-ACP reductase (alr5284F-5) <400> 3 cttgcttttt accggagatg ctagc 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of partial acyl-ACP reductase (alr5284R-5) <400> 4 agagcataga ttccgcaaaa caagc 25 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Upstream primer of partial aldehyde decarbonylase GSP1(5283_GSP1R) <400> 5 gtattcatct ttgactacac cctcagt 27 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Upstream primer of partial partial aldehyde decarbonylase GSP2(5283_GSP2R) <400> 6 gaatcagtaa gcaagtgaca acgttac 27 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Downstream primer of partial acyl-ACP reductase GSP1(5284_GSP1F) <400> 7 atacacacac tgcctacatt atctgtc 27 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Downstream primer of Partial acyl-ACP reductase GSP2(5284_GSP2F) <400> 8 gaaagcaaca gtagctatat gtggag 26 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Upstream primer of partial acyl-ACP reductase GSP1(5284_GSP1R) <400> 9 tatagctact gttgctttcg acagttc 27 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Upstream primer of partial acyl-ACP reductase GSP2(5284_GSP2R) <400> 10 gacagataat gtaggcagtg tgtgtat 27 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of full aldehyde decarbonylase (KNUA003_5283F) <400> 11 cggtctgtac tgttcttaat tctgg 25 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of full aldehyde decarbonylase (KNUA003_5283R) <400> 12 ttaagctgct gtaagtccgt agg 23 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of full acyl-ACP reductase (KNUA003_5284F) <400> 13 atccctgttg ctgatgattt tg 22 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of full acyl-ACP reductase (KNUA003_5284R) <400> 14 gactttgcta aagcagcaga gg 22

Claims (2)

Hepta-decane, hexadecane and senol having C ₁₆ to microalgae of the furnace stock KNUA003 (Nostoc sp to produce fatty acids of C _18. KNUA003) strain (KCTC 12063BP). According to claim 1, wherein the C ₁₆ to C ₁₈ fatty acid is from the group consisting of palmitic acid (C16: 0), palmitoleic acid (C16: 1), linoleic acid (C18: 2) and linolenic acid (C18: 3) Nostok KNUA003 ( Nostoc sp. KNUA003) strain (KCTC 12063BP).

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