Algae that produce free fatty acids, and method for producing fatty acids

Abstract

To provide a novel free fatty acid-producing algae with excellent FFA productivity, and a method for producing free fatty acids using the same free fatty acid-producing algae. [Solution] Free fatty acid-producing algae in which either or both of the enzymes that convert acyl ACP to acyl aldehyde (AAR) and the enzyme that converts lipid aldehyde to alkane (ADO) are functionally impaired or functionally deficient, and further, free fatty acid-producing algae into which either or both of the katG gene and sodB gene have been introduced, and a method for producing fatty acids using these free fatty acid-producing algae.

Description

[Technical Field]

[0001] The present invention relates to free fatty acid-producing algae and a method for producing fatty acids using free fatty acid-producing algae. [Background technology]

[0002] In an effort to reduce dependence on fossil fuels, the development of sustainable energy production technologies is progressing worldwide. Among these efforts, algae capable of accumulating lipids such as triacylglycerol (TAG) within their cells are attracting attention. The lipids accumulated in the cells of such algae can be used as raw materials for jet fuel and biodiesel fuel (BDF: registered trademark). However, while these algae accumulate lipids such as TAG within their cells, extracting the accumulated lipids requires the recovery and drying of cultured algal cells and extraction with organic solvents (intracellular production method). In particular, the recovery and drying of algal cells and the extraction with organic solvents require more than 50% of the energy administered for fuel production, which is an obstacle to practical application.

[0003] Therefore, extracellular production systems for free fatty acids (FFA) using genetically modified algae are attracting attention. Figure 6 shows a schematic diagram of the fatty acid metabolic pathway in genetically modified algae, which is a conventional extracellular production system. Algae synthesize membrane lipids by synthesizing acyl-ACP from CO2 assimilated through photosynthesis. FFA is cleaved from membrane lipids by lipase, but FFA is converted back to acyl-ACP by acyl-ACP synthase (Aas) and reused in membrane lipid biosynthesis, so FFA is not normally released outside the cell. However, if the acyl-ACP synthase gene (aas) is disrupted, FFA accumulates inside the cell, and excess FFA is released outside the cell. Furthermore, by introducing an exogenous thioesterase gene (tes), it becomes possible to directly cleave FFA from acyl-ACP, greatly increasing FFA production.

[0004] In Non-Patent Document 1, the present inventors described a type of cyanobacterium, Synechococcus elongatus PCC7942 strain. In Non-Patent Literature 2, the dAS1 strain, derived from the SPc strain of PCC 7942 (hereinafter also referred to as strain 7942), is genetically modified to lack endogenous acyl-ACP synthase (Aas). We propose the dAS1T strain, which has excellent FFA production capacity and incorporates esA. Non-patent document 3 describes using NA3, a strain of 7942 lacking a nitrate ion transporter, as the parent strain, and describes the deficiency of acyl ACP synthase (Aas) and E. coli-derived thioesterase (`tes). We propose a dAS2T / pRND1 strain with superior extracellular FFA release ability, which is created by introducing a plasmid (pRND1) for overexpressing a transporter (rndA1B1) for effluxing FFA into the extracellular space of a dAS2T strain that has been introduced with A).

[0005] As described above, extracellular production systems that release FFA outside the cell enable the production of biofuels with higher energy efficiency than intracellular production methods. In Patent Document 1, the present inventors have proposed the non-genetically modified dAS1_g21r strain (accession number FERM BP-22463), into which DNA encoding lipases (galp1, galp2) and transporters (rndA, rndB) that are naturally present in algae have been introduced. They have also proposed the genetically modified dAS1_g21r_KS strain (accession number FERM BP-22489), which exhibits superior photosynthetic ability under strong light conditions, by further introducing DNA encoding catalase (katG) and DNA encoding superoxide dimstase (sodB) into the dAS1_g21r strain (accession number FERM BP-22463). [Prior art documents] [Patent Documents]

[0006]

Patent Document 1

Non-Patent Document

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

[0008] An object of the present invention is to provide a novel free fatty acid-producing alga having excellent FFA productivity and a method for producing free fatty acids using the free fatty acid-producing alga. Means for Solving the Problems

[0009] The means for solving the problems of the present invention are as follows. 1. A free fatty acid-producing alga, characterized in that either or both of an enzyme (AAR) that converts acyl-ACP to acyl-aldehyde and an enzyme (ADO) that converts lipid aldehyde to alkane have reduced function or loss of function.[[ID=2))0]] 2. Either or both of an enzyme (AAR) that converts acyl-ACP to acyl-aldehyde and an enzyme (ADO) that converts lipid aldehyde to alkane have reduced function or loss of function, and a free fatty acid-producing alga, characterized in that either or both of the katG gene and the sodB gene are introduced. 3. The free fatty acid-producing alga according to 1. or 2., characterized in that the Synechococcus elongatus PCC7942 strain (Synechococcus elongatus PCC 7942) is used as a parent strain. 4. A free fatty acid-producing alga according to any one of 1 to 3, characterized in that one or more DNA selected from the group consisting of DNA encoding lipases naturally present in algae and DNA encoding transporters that naturally excrete free fatty acids outside the cell has been introduced. 5. The free fatty acid-producing algae described in 1, characterized in that it is the dAS1_G1R_dADR strain (receipt number FERM ABP-22506). 6. The free fatty acid-producing algae according to 2, characterized in that it is strain dAS1_G1R_dADR_KS (receipt number FERM ABP-22507). 7. A method for producing fatty acids, characterized by using free fatty acid-producing algae as described in any of sections 1 to 6. [Effects of the Invention]

[0010] Acyl ACP is used not only in FFA synthesis but also in alkane synthesis. However, in the free fatty acid-producing algae of the present invention, alkane synthesis is suppressed, and acyl ACP is mainly used for free fatty acid production, resulting in superior free fatty acid productivity. Furthermore, free fatty acid-producing algae into which either or both of the katG gene and sodB gene have been introduced have enhanced photosynthetic capacity, enabling photosynthesis even under strong light conditions and allowing for more efficient production of free fatty acids. [Brief explanation of the drawing]

[0011] [Figure 1] A schematic diagram of the metabolic pathway of free fatty acid-producing algae according to the present invention. [Figure 2] A graph showing the change in bacterial count over time in Experiment 1. [Figure 3] A graph showing the change in free fatty acid concentration over time in Experiment 1. [Figure 4] A graph showing the change in bacterial count over time in Experiment 2. [Figure 5] A graph showing the time course of free fatty acid concentration in Experiment 2. [Figure 6] A schematic diagram of the fatty acid metabolic pathway in genetically modified algae, which is a conventional extracellular production system. [Modes for carrying out the invention]

[0012] Figure 1 shows a schematic diagram of the fatty acid metabolic pathway in the free fatty acid-producing algae of the present invention. The first free fatty acid-producing algae of the present invention has reduced or missing function in either the enzyme that converts acyl ACP to acyl aldehyde (AAR), or the enzyme that converts lipid aldehyde to alkane (ADO). The second free fatty acid-producing algae of the present invention has reduced or complete dysfunction of either the enzyme that converts acyl ACP to acyl aldehyde (AAR) or the enzyme that converts lipid aldehyde to alkane (ADO), or both, and furthermore, either the katG gene or the sodB gene, or both, have been introduced. Hereinafter, the first and second free fatty acid-producing algae of the present invention will be collectively referred to as the free fatty acid-producing algae of the present invention.

[0013] The free fatty acid-producing algae of the present invention have reduced or missing function in either the enzyme that converts acyl ACP to acyl aldehyde (AAR: acyl ACP reductase, SEQ ID NO: 1), or the enzyme that converts lipid aldehyde to alkane (ADO: aldehyde deformylation oxygenase, SEQ ID NO: 3). In the free fatty acid-producing algae of the present invention, the method for reducing or eliminating the function of either AAR, ADO, or both is not particularly limited. For example, the gene encoding AAR, aar (SEQ ID NO: 2), and the gene encoding ADO, ado (SEQ ID NO: 4), can be reduced by a method such as incorporating DNA containing homologous sequences around the aar and ado genes, into which an antibiotic resistance cassette is inserted to delete the aar and ado genes, into the genomic DNA through natural transformation.

[0014] AAR and ADO are enzymes involved in alkane synthesis using acyl-ACP as a starting material. This alkane synthesis pathway, like the FFA synthesis pathway, uses acyl-ACP as a starting material and competes with the FFA synthesis pathway. The free fatty acid-producing algae of the present invention have the alkane synthesis pathway that competes with the FFA synthesis pathway suppressed, and acyl-ACP is mainly consumed in the FFA production pathway, resulting in superior FFA productivity. The free fatty acid-producing algae of the present invention, compared to similar strains except that both ADR and ADO function normally, exhibit the same characteristics at a light intensity of 200 μE / m². 2 The free fatty acid production after 28 days of culture in s is preferably 1.05 times or more, more preferably 1.1 times or more, and even more preferably 1.15 times or more.

[0015] The second free fatty acid-producing algae of the present invention, like the first free fatty acid-producing algae, exhibits excellent FFA productivity because the alkane synthesis pathway that competes with the FFA synthesis system is suppressed. Furthermore, the second type of free fatty acid-producing algae, in addition to suppressing the alkane synthesis pathway, has either the katG gene, the sodB gene, or both introduced. The katG gene (SEQ ID NO: 5) is DNA encoding catalase, and the SodB gene (SEQ ID NO: 6) is DNA encoding superoxide dimstase. In other words, the katG gene and the sodB gene encode catalase and superoxide dimstase, enzymes that remove reactive oxygen species generated by photosynthesis. By introducing either or both of the katG and sodB genes, the second type of free fatty acid-producing algae can efficiently remove reactive oxygen species generated by photosynthesis and perform photosynthesis efficiently even under strong light, thus exhibiting superior FFA productivity under strong light conditions.

[0016] The second free fatty acid-producing algae, compared to a similar strain except that it did not have the katG and sodB genes introduced, performed better at a light intensity of 700 μE / m². 2 The free fatty acid production after 28 days of culture in s is preferably 1.5 times or more, more preferably 1.7 times or more, and even more preferably 1.9 times or more.

[0017] In this invention, there are no particular restrictions on the algae used as parent stocks, and genera such as Synechococcus and Synechocystis can be used. Specifically, Synechococcus elongatus strain PCC7942 can be cited. In addition, wild algae inhabiting the site where free fatty acids are produced can be collected and used as parent stocks.

[0018] Algae typically possess multiple types of lipases and multiple types of transporters, including multiple DNAs that encode lipases and multiple DNAs that encode transporters that expel free fatty acids from the cell. The free fatty acid-producing algae of the present invention preferably have one or more DNA selected from the group consisting of DNA encoding lipase (hereinafter also referred to as lipase DNA) and DNA encoding a transporter that excretes free fatty acids into the cell (hereinafter also referred to as transporter DNA) introduced into the parent strain of the algae used. This achieves either or both of the following: enhanced FFA excision through high expression of lipase, enhanced excretion of FFA into the cell through high expression of the transporter. It is estimated that strain 7942 has 22 DNA molecules as lipase DNA, including at least galp1 (SEQ ID NO: 7) and galp2 (SEQ ID NO: 8).

[0019] In this invention, the DNA introduced into the parent algae is one or more selected from the group consisting of lipase DNA and transporter DNA that are naturally present in the parent algae. For example, one or more lipase DNAs naturally present in the parent algae can be introduced, one or more transporter DNAs naturally present in the parent algae can be introduced, or one or more lipase DNAs naturally present in the parent algae and one or more transporter DNAs naturally present in the parent algae can be introduced. One or more types that can increase free fatty acid production can be selected from the lipase DNA and transporter DNA present in the parent algae. The method of gene introduction is not particularly restricted and can be carried out by spontaneous transformation, homologous recombination, etc. In this case, by attaching a modified psbAII promoter, which encodes the D1 reaction center protein of photosystem II, to the upstream of the DNA to be incorporated, by removing the negative element sequence and making it constitutively expressible, the introduced lipase DNA and transporter DNA can be strongly expressed.

[0020] The dAS1_G1R_dADR strain can be used as the first free fatty acid-producing algae of the present invention. The dAS1_G1R_dADR strain was received on August 9, 2024, under receipt number FERM ABP-22506 by the Patent Microorganism Depositary Center (NPMD) of the National Institute of Technology and Evaluation (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan (Postal Code 292-0818)). The dAS1_G1R_dADR strain is a free fatty acid-producing algae with strain 7942 as its parent strain. The dAS1_G1R_dADR strain is a strain of the dAS1_galp1_rndAB strain described in Patent Document 1, in which both the enzyme that converts acyl ACP to acyl aldehyde (AAR) and the enzyme that converts lipid aldehyde to alkane (ADO) are functionally deficient. The dAS1_galp1_rndAB strain was created by introducing galp1 (sequence number 7), which is the DNA encoding Galp1 lipase, and rndA (sequence number 9) and rndB (sequence number 10), which are the DNA encoding transporters that expel free fatty acids from cells, into strain 7942 as rndAB (sequence number 11).

[0021] As the second free fatty acid-producing alga of the present invention, the dAS1_G1R_dADR_KS strain can be used. The dAS1_G1R_dADR_KS strain is a free fatty acid-producing alga in which the katG gene (SEQ ID NO: 5) and the sodB gene (SEQ ID NO: 6) are introduced into the dAS1_G1R_dADR strain. The dAS1_G1R_dADR_KS strain has been received as accession number FERM ABP-22507 at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (NPMD) (Room 120, 2-5-8 Kazusa Kamashima, Kisarazu City, Chiba Prefecture, Japan (Postal Code 292-0818)) on August 9, 2024.

[0022] · Method for producing free fatty acids By culturing the free fatty acid-producing alga of the present invention, fatty acids can be produced. The temperature during culturing is, for example, 15°C or higher and 40°C or lower, preferably 20°C or higher, more preferably 22°C or higher, even more preferably 24°C or higher, and preferably 38°C or lower, more preferably 36°C or lower, even more preferably 34°C or lower. The pH during culturing is, for example, pH 7.5 or higher and 12.0 or lower, preferably pH 7.8 or higher, more preferably pH 8.0 or higher, and preferably pH 11.0 or lower, more preferably pH 10.5 or lower, even more preferably pH 10.0 or lower.

[0023] The free fatty acid-producing alga of the present invention produces free fatty acids by photosynthesis. Therefore, it is necessary to irradiate light for photosynthesis during culturing. In the case of the first free fatty acid-producing alga, the light intensity is 30 μE / m 2 · s or more and 1500 μE / m 2 · s or less is preferable, 50 μE / m 2 · s or more is more preferable, 100 μE / m 2 · s or more is even more preferable, 150 μE / m 2 · s or more is even more preferably, and also 1200 μE / m 2 · s or less is more preferable, 1000 μE / m 2 · s or less is even more preferable, 800 μE / m 2• Less than s is even more preferable, 600 μE / m 2 • Less than s is even more preferable, 400 μE / m 2 • s or less is even more preferable.

[0024] In the case of the second free fatty acid-producing algae, the light intensity is 100 μE / m 2 ·s or more 1500μE / m 2 Preferably less than s, and 150 μE / m 2 • s or higher is more preferable, 200 μE / m 2 More preferably s or higher, and 250 μE / m 2 • More preferably s or higher, 300 μE / m 2 • More preferably s or higher, and also 1200 μE / m 2 ・s or less is more preferable, 1000 μE / m 2 • More preferably less than s, and 800 μE / m 2 • s or less is even more preferable.

[0025] The free fatty acid-producing algae of the present invention produce fatty acids from water and carbon dioxide through photosynthesis. Therefore, from the viewpoint of improving fatty acid productivity, a high carbon dioxide concentration is preferable, and it is preferable to blow in gas with a carbon dioxide concentration of 0.04% (v / v) or higher. The carbon dioxide concentration in the blown-in gas is more preferably 0.1% (v / v) or higher, even more preferably 0.5% (v / v) or higher, even more preferably 1.0% (v / v) or higher, and even more preferably 1.5% (v / v) or higher. On the other hand, if the carbon dioxide concentration becomes too high, the growth of the algae may be inhibited. Therefore, the carbon dioxide concentration in the blown-in gas is preferably 4.0% (v / v) or lower, more preferably 3.5% (v / v) or lower, and even more preferably 3.0% (v / v) or lower. [Examples]

[0026] Non-patent document 1 describes the creation of the dAS1_sacB strain, derived from the SPc strain of Synechococcus elongatus PCC 7942 (hereinafter also referred to as strain 7942), a type of cyanobacterium, by genetically modifying the coding region of the endogenous acyl-ACP synthase gene (aas) to introduce a kanamycin resistance gene (nptI) and a gene encoding levansculasase (sacB), which induces cell death in the presence of sucrose.

[0027] A DNA plasmid was constructed by introducing rndAB (SEQ ID NO: 11), which is connected to the psbAII promoter, into the deletion region of the aas gene as described above. The constructed DNA plasmid was introduced into the dAS1_sacB strain described in Non-Patent Literature 1, and the dAS1_rndAB strain was prepared by selection in sucrose-supplemented medium.

[0028] A DNA plasmid was constructed by tandemly linking galp1 (SEQ ID NO: 7) and rndAB (SEQ ID NO: 11), both linked to the psbAII promoter, within the deletion region of the aas gene. The constructed DNA plasmid was introduced into the dAS1_rndAB strain, and G1R (dAS1_galp1_rndAB) strain was generated by selection in sucrose-supplemented medium.

[0029] The enzyme aar (SEQ ID NO: 2), which encodes the enzyme that converts acyl ACP to acyl aldehyde (AAR), and the enzyme ado (SEQ ID NO: 4), which encodes the enzyme that converts lipid aldehyde to alkane (ADO), are both encoded by the ado / aar operon. The ado / aar operon of the G1R strain was disrupted by inserting the kanamycin resistance gene, creating the dAS1_G1R_dADR strain (dAS1_galp1_rndAB_dado / aar strain).

[0030] DNA plasmids were constructed by introducing the katG gene (SEQ ID NO: 5) and sodB gene (SEQ ID NO: 6), connected to the psbAII promoter, and a chloramphenicol resistance gene into the 0084 gene region, which is the neutral site of strain 7942. The constructed plasmids were introduced into the NS2 neutral site of the genome of the G1R_dADR strain, and the strains were selected using chloramphenicol-supplemented medium to create the dAS1_G1R_dADR_KS strain.

[0031] For cultivation, a basic medium partially modified from BG-11 medium was used (Suzuki I., Kikuchi H., Nakanishi S., Fujita Y., Sugiyama T., Omata T. (1995), A novel nitrite reductase gene from the cyanobacterium Plectonema boryanum. J. Bacteriol. 177: 6137-6143). The pH of the basic culture medium was adjusted to pH 8.2 by adding potassium hydroxide using TES, a good buffer. When inoculating the algae, potassium nitrate was added as a nitrogen source to achieve a final potassium ion concentration of 15 mM.

[0032] Experiment 1 • Example 1 After placing 50 mL of basic culture medium in a 90 mL glass culture tube and sterilizing it by autoclaving (121°C, 15 minutes), the dAS1_G1R_dADR strain was inoculated, resulting in a culture density of 70 μE / m². 2 Under continuous light irradiation of s, OD 730 The cells were cultured until the volume reached 0.5-1.0 (pre-culture). Next, transfer the pre-culture medium to the new base medium (OD). 730 The cells were inoculated so that the ratio was 0.05, and isopropyl myristate was added as a topcoat to recover free fatty acids from the culture medium. 2% (v / v) CO2 supply from a gas cylinder and 200 μE / m² of warm white LED light 2 The cells were incubated at 32°C for 48 days under continuous light irradiation of -s.

[0033] • Comparative Example 1 The procedure was the same as in Example 1, except that the dAS1 strain described in Non-Patent Document 1 was used. • Comparative Example 2 The procedure was the same as in Example 1, except that the dAS1_rndAB strain was used. • Comparative Example 3 The procedure was the same as in Example 1, except that the G1R strain was used.

[0034] FFA concentration was measured using the Free Fatty Acid Quantification Kit (Biovision). OD 730 (Bacterial turbidity) and FFA concentration were measured periodically. The dry cell weight (DCW, g / L) is calculated as 0.218 × OD, according to Non-Patent Document 4. 730 It was calculated using the formula +0.014. The results are shown in Table 1, Figures 2 and 3.

[0035] [Table 1]

[0036] ·result The first free fatty acid-producing algae strain of the present invention, strain dAS1_G1R_dADR, exhibited superior FFA production compared to the other strains used in Comparative Examples 1-3. In particular, Comparative Example 3 used the G1R strain, which is similar to the first strain except for functional deficiencies in ADO and ADR. However, while the growth of the dAS1_G1R_dADR strain (Example 1) was equivalent to that of the G1R strain (Comparative Example 3), the FFA production was more than 1.3 times (=589 / 432).

[0037] Experiment 2 The first free fatty acid-producing algae strain, dAS1_G1R_dADR, and the second free fatty acid-producing algae strain, dAS1_G1R_dADR_KS, were supplied with 2% (v / v) CO2 from a gas cylinder and 200 μE / m³ of warm-white LED light. 2 · s, 400 μE / m 2 · s, 700 μE / m2 The cultures were incubated at 32°C for 16–28 days under continuous light irradiation of -s.

[0038] The dry cell weight (DCW, g / L) is 0.218 × OD, according to Non-Patent Document 4. 730 It was calculated using the formula +0.014. The results are shown in Table 2, Figures 4 and 5. [Table 2]

[0039] ·result The first free fatty acid-producing algae, strain dAS1_G1R_dADR, showed a decrease in both cell dry weight and FFA production as light intensity increased, reaching 200 μE / m². 2 A light intensity of approximately s was considered suitable. The second free fatty acid-producing alga, strain dAS1_G1R_dADR_KS, showed a decrease in cell dry weight when light intensity increased, but still maintained a cell dry weight of 700 μE / m². 2 Even under strong light conditions, the cells grew sufficiently, with a dry weight of 0.92 g / L, and furthermore, FFA production reached 432 mg / L. The dAS1_G1R_dADR_KS strain showed a 700 μE / m² difference compared to the dAS1_G1R_dADR strain. 2 Under strong light conditions, both the dry cell weight and FFA production increased by approximately twofold, confirming that FFA production is possible under a wide range of light conditions.

Claims

1. Free fatty acid-producing algae characterized by having reduced or missing function of either or both of the enzymes that convert acyl-ACP to acyl aldehyde (AAR) and the enzyme that converts lipid aldehyde to alkane (ADO).

2. If either or both of the enzymes that convert acyl-ACP to acylaldehyde (AAR) or lipid aldehyde to alkane (ADO) are impaired or deficient in function, Free fatty acid-producing algae characterized by the introduction of either the katG gene, the sodB gene, or both.

3. The free fatty acid-producing alga according to claim 1 or 2, characterized in that the parent strain is Synechococcus elongatus PCC 7942.

4. The free fatty acid-producing algae according to claim 1 or 2, characterized in that one or more DNA selected from the group consisting of DNA encoding lipases naturally present in algae and DNA encoding transporters that naturally excrete free fatty acids outside the cell is introduced.

5. The free fatty acid-producing algae according to claim 1, characterized in that it is the dAS1_G1R_dADR strain (receipt number FERM ABP-22506).

6. The free fatty acid-producing algae according to claim 2, characterized in that it is strain dAS1_G1R_dADR_KS (receipt number FERM ABP-22507).

7. A method for producing fatty acids, characterized by using the free fatty acid-producing algae described in claim 1 or 2.

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