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

Abstract

To provide a novel free fatty acid-producing algae with excellent productivity in producing unsaturated fatty acids, and a method for producing fatty acids using the same free fatty acid-producing algae. [Solution] Free fatty acid-producing algae into which the galp3 gene (SEQ ID NO: 1) has been introduced, and a method for producing fatty acids characterized by 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 8 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). 【0006】 Furthermore, in Patent Document 2, the present inventors have introduced DNA encoding a lipase (galp1) naturally present in algae and DNA encoding transporters (rndA, rndB) naturally present in algae, resulting in a dAS1_G1R_dADR strain (accession number FERM BP-22506) in which both the enzyme that converts acyl ACP to fatty aldehyde (AAR) and the enzyme that converts fatty aldehyde to alkane (ADO), which are involved in the alkane synthesis pathway that competes with FFA synthesis, are functionally deficient. They have also proposed the dAS1_G1R_dADR_KS strain (accession number FERM BP-22507, hereinafter also referred to as the G1R_dADR_KS strain) in which katG and sodB are introduced into the dAS1_G1R_dADR_KS strain. [Prior art documents] [Patent Documents] 【0007】 [Patent Document 1] Japanese Patent Publication No. 2024-127776 [Patent Document 2] Patent application No. 2024-153265 [Non-patent literature] 【0008】 [Non-Patent Document 1] Takatani N, Use K, Kato A, Ikeda K, Kojima K, Aichi M, et al.(2015) Essential role of acyl-ACP synthetase in acclimation of the cyanobacterium Synechococcus elongatus strain PCC 7942 to high-light conditions. Plant Cell Physiol. 56:1608-15. [Non-Patent Document 2] Kato A, Use K, Takatani N, Ikeda K, Matsuura M, Kojima K, Aichi M, et al. (2016) Modulation of the balance of fatty acid production and secretion is crucial for enhancement of growth and productivity of the engineered mutant of the cyanobacterium Synechococcus elongatus. Biotechonol for Biofu. Vol. 9, 91 [Non-Patent Document 3] Kato A, Takatani N, Use K, Uesaka K, Ikeda K, Chang Y, et al. (2015) Identification of a cyanobacterial RND-type efflux system involved in export of free fatty acids. Plant Cell Physiol. 56:2467-77. [Non-Patent Document 4] Kato A, Takatani N, Ikeda K, Maeda SI, Omata T (2017) Removal of the product from the culture medium strongly enhances free fatty acid production by genetically engineered Synechococcus elongatus. Biotechnol Biofuels 10:141. [Summary of the Invention] [Problems to be Solved by the Invention] 【0009】 The fatty acids produced by the free fatty acid-producing algae reported by the present inventors in Non-Patent Documents 1 to 3 and Patent Document 1 mainly contain palmitoleic acid (16:1). Unsaturated fatty acids are excellent as starting materials for the chemical industry because they can undergo chemical reactions at the double bond portion. An object of the present invention is to provide a novel free fatty acid-producing alga having excellent productivity of unsaturated fatty acids and a method for producing a fatty acid using the free fatty acid-producing alga. 【Means for Solving the Problems】 【0010】 The means for solving the problems of the present invention are as follows. 1. A free fatty acid-producing alga characterized in that the galp3 gene (SEQ ID NO: 1) is introduced. 2. The free fatty acid-producing alga according to 1., characterized in that the Synechococcus elongatus PCC7942 strain (Synechococcus elongatus PCC 7942) is used as a parent strain. 3. The free fatty acid-producing alga according to 1. or 2., characterized in that DNA encoding a transporter that excretes free fatty acids inherent in the alga extracellularly is introduced. 4. The free fatty acid-producing alga according to any one of 1. to 3., characterized in that it is the dAS1_galp3_rndAB strain (Accession No. FERM BP-22509). 5. The free fatty acid-producing alga according to any one of 1. to 4., characterized in that either or both of an enzyme (AAR) that converts acyl-ACP to fatty aldehyde and an enzyme (ADO) that converts fatty aldehyde to alkane have reduced function or loss of function. 6. The free fatty acid-producing alga according to any one of 1. to 5., characterized in that it is the dAS1_galp1_galp3_rndAB_katG_sodB_dado / aar strain (Accession No. FERM BP-22510). 7. A method for producing a fatty acid, characterized by using the free fatty acid-producing alga according to any one of 1. to 6. 【Effects of the Invention】 【0011】 The free fatty acid-producing algae of the present invention produce a large amount of unsaturated fatty acids and exhibit excellent unsaturated fatty acid productivity. [Brief explanation of the drawing] 【0012】 [Figure 1] A schematic diagram of the metabolic pathway of free fatty acid-producing algae into which the galp3 gene, the present invention, has been introduced. [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 composition of free fatty acids produced by free fatty acid-producing algae in Experiment 2. [Figure 5] A schematic diagram of the metabolic pathway of free fatty acid-producing algae in which the galp3 gene, the present invention, has been introduced, resulting in functional deficiencies in both the enzyme that converts acyl ACP to fatty aldehyde (AAR) and the enzyme that converts fatty aldehyde to alkane (ADO). [Figure 6] A graph showing the change in bacterial count over time in Experiment 3. [Figure 7] A graph showing the time course of free fatty acid concentration in Experiment 3. [Figure 8] 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] 【0013】 Figure 1 shows a schematic diagram of the fatty acid metabolic pathway in the free fatty acid-producing algae of the present invention. The free fatty acid-producing algae of this invention have the galp3 gene (SEQ ID NO: 1) introduced into them. The galp3 gene is a gene that encodes a lipase that cleaves FFA from membrane lipids, and although the mechanism is unknown, the free fatty acid-producing algae into which the galp3 gene has been introduced show improved productivity of unsaturated fatty acids. 【0014】 In this invention, there are no particular restrictions on the algae used as parent stock, and genera such as Synechococcus and Synechocystis can be used. Specifically, Synechococcus elongatus PCC7942 strain (hereinafter also referred to as strain 7942) can be used. In addition, wild algae inhabiting the site where free fatty acids are produced can be collected and used as parent stock. 【0015】 The free fatty acid-producing algae of the present invention only need to have the galp3 gene introduced, and other lipase DNA can also be introduced. However, in order to prevent the creation of algae that do not exist in nature due to the incorporation of foreign genes, it is preferable to introduce lipase DNA that is naturally present in the parent algae used. Algae usually have multiple types of lipases and therefore multiple types of lipase DNA. For example, strain 7942 is estimated to have 22 DNA molecules as lipase DNA, including at least galp3, galp1 (SEQ ID NO: 2), and galp2 (SEQ ID NO: 3). 【0016】 The free fatty acid-producing algae of the present invention can be modified to introduce one or more types of DNA encoding transporters that release free fatty acids into the extracellular space (hereinafter also referred to as transporter DNA). By introducing transporter DNA, free fatty acids can be efficiently released into the extracellular space, preventing the death of free fatty acid algae due to high intracellular free fatty acid concentrations. It is preferable to introduce transporter DNA that is naturally present in the parent algae used as the transporter DNA. 【0017】 The free fatty acid-producing algae of the present invention can be modified by introducing either the katG gene, the sodB gene, or both. The katG gene (SEQ ID NO: 4) is DNA encoding catalase, and the SodB gene (SEQ ID NO: 5) 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 gene and the sodB gene, reactive oxygen species generated by photosynthesis can be efficiently removed, enabling efficient photosynthesis even under strong light conditions and improving FFA productivity under strong light conditions. 【0018】 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 introduced, by removing the negative element sequence and making it constitutively expressible, the DNA to be introduced can be strongly expressed. 【0019】 As the free fatty acid-producing algae of the present invention, strain dAS1_galp3_rndAB (hereinafter also referred to as strain G3R) can be used. Strain G3R is a non-genetically modified free fatty acid-producing algae with Synechococcus elongatus strain PCC7942 as its parent strain. Strain G3R was created by introducing galp3 (sequence number 1), which is DNA encoding Galp3 lipase, and rndA (sequence number 6) and rndB (sequence number 7), which are DNA encoding transporters that expel free fatty acids from cells, into the parent strain as rndAB (sequence number 8). Strain G3R was internationally deposited with the National Institute of Technology and Evaluation (IPOD) (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan (postal code 292-0818)) on October 2, 2024, under accession number FERM BP-22509. 【0020】 The free fatty acid-producing algae of the present invention may have reduced or missing function in either or both of the enzymes that convert acyl ACP to fatty aldehyde (AAR: acyl ACP reductase, SEQ ID NO: 9) and the enzyme that converts fatty aldehyde to alkane (ADO: aldehyde deformylating oxygenase, SEQ ID NO: 10). It is preferable that both AAR and ADO are reduced or missing function, and it is more preferable that both AAR and ADO are missing function. Figure 5 shows a schematic diagram of the fatty acid metabolic pathway in the free fatty acid-producing algae of the present invention, in which both AAR and ADO are functionally deficient. 【0021】 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. In free fatty acid-producing algae in which either AAR or ADO, or both, are functionally impaired or lacking, the alkane synthesis pathway that competes with the FFA synthesis pathway is suppressed, and acyl-ACP is mainly consumed in the FFA production pathway, resulting in improved FFA productivity. 【0022】 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, a method for eliminating the function of both can be carried out by spontaneously transforming the genomic DNA into DNA containing homologous sequences around the aar and ado genes, into which an antibiotic resistance cassette has been inserted to delete the aar and ado genes, thereby reducing the function of the aar gene (sequence number 11) and the ado gene (sequence number 12). 【0023】 As the free fatty acid-producing algae of the present invention, the dAS1_galp1_galp3_rndAB_katG_sodB_dado / aar strain (hereinafter also referred to as the G13R_dADR_KS strain) can be used. The G13R_dADR_KS strain is a strain in which galp3 has been introduced into the dAS1_G1R_dADR_KS strain (accession number FERM BP-22507) described in Patent Document 2. The G13R_dADR_KS strain was internationally deposited with the National Institute of Technology and Evaluation (IPOD) (2-5-8 Kazusa-Kamatari, Kisarazu City, Chiba Prefecture, Japan (postal code 292-0818)) on October 2, 2024, under accession number FERM BP-22510. 【0024】 • Methods for producing fatty acids Fatty acids can be produced by cultivating the free fatty acid-producing algae of the present invention. The cultivation temperature can be adjusted according to the type of free fatty acid-producing algae, for example, between 15°C and 40°C. Lower cultivation temperatures reduce algal activity and decrease fatty acid production, but tend to increase the proportion of unsaturated fatty acids in the produced fatty acids. Cultivation temperatures of 17°C or higher are preferred, 19°C or higher are more preferred, 35°C or lower are preferred, 30°C or lower are more preferred, 28°C or lower are even more preferred, and 26°C or lower are even more preferred. The pH during cultivation is, for example, between 7.5 and 12.0, preferably 7.8 or higher, more preferably 8.0 or higher, preferably 11.0 or lower, more preferably 10.5 or lower, and even more preferably 10.0 or lower. 【0025】 The free fatty acid-producing algae of the present invention produce free fatty acids through photosynthesis. Therefore, it is necessary to irradiate them with light for photosynthesis during cultivation. In the case of free fatty acid-producing algae in which neither the katG gene nor the sodB gene has been introduced, the light intensity is 30 μE / m 2 ·s or more 1500μE / m 2 Preferably less than 50 μE / m 2 • s or higher is more preferable, 100 μE / m2 ·s or more is more preferable, and 150 μE / m 2 ·s or more is even more preferable, and also 1200 μE / m 2 ·s or less is more preferable, and 1000 μE / m 2 ·s or less is even more preferable, and 800 μE / m 2 ·s or less is even more preferable, and 600 μE / m 2 ·s or less is even more preferable, and 400 μE / m 2 ·s or less is even more preferable. 【0026】 In the case of free fatty acid-producing algae into which either or both of the katG gene and the sodB gene are introduced, the light intensity is 100 μE / m 2 ·s or more and 1500 μE / m 2 ·s or less is preferable, and 150 μE / m 2 ·s or more is more preferable, and 200 μE / m 2 ·s or more is even more preferable, and 250 μE / m 2 ·s or more is even more preferable, and 300 μE / m 2 ·s or more is even more preferable, and also 1200 μE / m 2 ·s or less is more preferable, and 1000 μE / m 2 ·s or less is even more preferable, and 800 μE / m 2 ·s or less is even more preferable. 【0027】 The free fatty acid-producing algae of the present invention produce fatty acids from water and carbon dioxide by photosynthesis. Therefore, from the viewpoint of improving the productivity of fatty acids, it is preferable that the carbon dioxide concentration is high, and it is preferable to blow in a gas having a carbon dioxide concentration of 0.04% (v / v) or more. The carbon dioxide concentration in the gas to be blown in is more preferably 0.1% or more, even more preferably 0.5% or more, even more preferably 1.0% or more, and even more preferably 1.5% or more. On the other hand, if the carbon dioxide concentration becomes too high, the growth of algae may be inhibited. Therefore, the carbon dioxide concentration in the gas to be blown in is preferably 4.0% or less, more preferably 3.5% or less, and even more preferably 3.0% or less. [Examples] 【0028】 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. 【0029】 A DNA plasmid was constructed by introducing rndAB (SEQ ID NO: 8), 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. 【0030】 A DNA plasmid was constructed by tandemly linking galp3 (SEQ ID NO: 1) and rndAB (SEQ ID NO: 8), 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 G3R (dAS1_galp3_rndAB) strain was generated by selection in sucrose-supplemented medium. 【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 G3R 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 (IM) 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 from a warm white LED light / m 2 The cells were incubated at 25°C for 34 days under continuous light irradiation of -s. 【0033】 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. FFA concentration was measured using the Free Fatty Acid Quantification Kit (Biovision). The results are shown in Table 1, Figures 2 and 3. 【0034】 [Table 1] 【0035】 On day 26 of culture, FFA production was 354 mg / L, and the FFA production rate was 11.3 mg-F The yield was FA / g-DCW / day. This was equivalent to the growth and free fatty acid production capacity of the dAS1_galp1_rndAB strain (hereinafter referred to as the G1R strain) and dAS1_galp2_rndAB strain created in Patent Document 1. 【0036】 Experiment 2: Composition of Free Fatty Acids • Comparative Example 1 The culture 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 culture procedure was the same as in Example 1, except that the dAS1_rndAB strain described in Patent Document 1 was used. • Comparative Example 3 The culture was carried out in the same manner as in Example 1, except that the G1R strain described in Patent Document 1 was used. 【0037】 The composition of FFA was identified by non-targeted lipidomics analysis using LC / MS (LC / Q-TOF MS). Table 2 and Figure 4 show the total amount and composition of free fatty acids produced by each strain of Example 1 and Comparative Examples 1-3 after 20 days of culture. [Table 2] 【0038】 The G3R strain, a free fatty acid-producing algae of the present invention, and the G1R strain used in Comparative Example 3, exhibited excellent free fatty acid productivity and produced large quantities of free fatty acids. The G3R strain, a free fatty acid-producing algae of the present invention, produced a larger total amount of unsaturated fatty acids compared to Comparative Examples 1-3, demonstrating superior unsaturated fatty acid productivity. 【0039】 Experiment 3 The G1R_dADR_KS strain described in Patent Document 2 was introduced with galp3 in the same manner as described above, and selected using sucrose-supplemented medium to produce the dAS1_galp1_galp3_rndAB_katG_sodB_dado / aar strain (G13R_dADR_KS strain). • Example 2 The G13R_dADR_KS strain was tested at a light intensity of 400 μE / m². 2·s -1 The cells were cultured at 25°C under two-phase culture conditions with isopropyl myristate (IM) as a top layer. • Comparative Example 4 The culture procedure was the same as in Example 2, except that the G1R_dADR_KS strain described in Patent Document 2 was used. In the same manner as in Experiment 1, OD 730 (Bacterial turbidity) and FFA concentration were measured periodically. The results are shown in Table 3, Figures 6 and 7. 【0040】 [Table 3] 【0041】 Example 2 (G13R_dADR_KS strain), which represents the present invention, showed slightly improved growth compared to Comparative Example 4 (G1R_dADR_KS strain). While FFA production did not increase after 20 days in Comparative Example 4, it continued to increase steadily after 20 days in Example 2, reaching 684 mg-FFA / L, an FFA production rate of 12.3 mg-FFA / g-DCW / day, and a biomass-to-fat ratio of 0.37 g / g on day 30 of cultivation. These values ​​were obtained at a light intensity of 400 μE / m². 2 ·s -1 Under these conditions, it was superior to the alkane synthesis inhibitory strain described in Patent Document 2. 【0042】 Experiment 4 For the bacterial strains of Example 2 and Comparative Example 4, an inorganic salt medium containing nitrate was used, and the light intensity was 200 μE / m². 2 ·s -1 The cells were cultured for 20 days under two-phase conditions with isopropyl myristate (IM) overlay at 25°C. The composition of FFA was identified in the same manner as in Experiment 2. The results are shown in Table 4. 【0043】 [Table 4] 【0044】 The G13R_dADR_KS strain (Example 2) and the G1R_dADR_KS strain (Comparative Example 4), which are free fatty acid-producing algae of the present invention, exhibit enhanced inhibition of the alkane synthesis pathway that competes with the FFA synthesis system and improved reactive oxygen species scavenging ability compared to the G3R strain (Example 1) and the G1R strain (Comparative Example 3), respectively, resulting in a significant increase in free fatty acid production. The free fatty acid-producing algae of the present invention (G13R_dADR_KS strain) produced less free fatty acids than the G1R_dADR_KS strain, but produced more unsaturated fatty acids, demonstrating superior unsaturated fatty acid productivity.

Claims

[Claim 1] Algae that produce free fatty acids, characterized by the introduction of the galp3 gene (SEQ ID NO: 1). [Claim 2] The free fatty acid-producing alga according to claim 1, characterized in that Synechococcus elongatus PCC 7942 strain is used as the parent strain. [Claim 3] The free fatty acid-producing algae according to claim 1, characterized in that DNA encoding a transporter that excretes free fatty acids, which are naturally present in algae, is introduced. [Claim 4] The free fatty acid-producing algae according to claim 3, characterized in that it is the dAS1_galp3_rndAB strain (accession number FERM BP-22509). [Claim 5] The free fatty acid-producing algae according to claim 1, characterized in that either or both of the enzymes that convert acyl ACP to fatty aldehyde (AAR) and the enzyme that converts fatty aldehyde to alkane (ADO) are functionally impaired or functionally deficient. [Claim 6] The free fatty acid-producing algae according to claim 5, characterized in that it is the dAS1_galp1_galp3_rndAB_katG_sodB_dado / aar strain (accession number FERM BP-22510). [Claim 7] A method for producing fatty acids, characterized by using free fatty acid-producing algae as described in any one of claims 1 to 6.

Images