Polyunsaturated fatty acids (PUFAs) are considered to be useful for nutritional applications, pharmaceutical applications, industrial applications, and other purposes. However, the current supply of PUFAs from natural sources and from chemical synthesis is not sufficient for commercial needs. PUFAs derived from microorganism such as microalgae can be produced in large scale while avoiding the contamination issues associated with fish oils.
Polyketide synthase (PKS) systems are generally known in the art as enzyme complexes related to fatty acid synthase (FAS) systems, but which are often highly modified to produce specialized products that typically show little resemblance to fatty acids. It has now been shown, however, that polyketide synthase-like systems exist in marine bacteria and certain microalgae that are capable of synthesizing polyunsaturated fatty acids (PUFAs) from acetyl-CoA and malonyl-CoA. These systems are referred to herein as PUFA synthases, PUFA synthase systems, PUFA PKS systems, or PKS-like systems for the production of PUFAs, all of which are used interchangeably herein.
The PUFA PKS pathways for PUFA synthesis in Shewanella and another marine bacteria, Vibrio marinus, are described in detail in U.S. Pat. No. 6,140,486. The PUFA PKS pathways for PUFA synthesis in the eukaryotic Thraustochytrid, Schizochytrium sp. ATCC 20888 (hereinafter “Schizochytrium 20888”), is described in detail in U.S. Pat. No. 6,566,583. The PUFA PKS pathways for PUFA synthesis in eukaryotes such as members of Thraustochytriales, including the additional description of a PUFA PKS system in Schizochytrium 20888 and the identification of a PUFA PKS system in Thraustochytrium sp. ATCC 20892, including details regarding uses of these systems, are described in detail in U.S. Pat. No. 7,247,461, and in U.S. Pat. No. 7,642,074, respectively. The PUFA PKS pathways for PUFA synthesis in another eukaryotic Thraustochytrid, Schizochytrium sp. ATCC PTA-9695 (hereinafter “Schizochytrium 9695”), is described in detail in U.S. Patent Application Publication No. 2010-0266564, published Oct. 21, 2010 and in PCT Publication No. WO 2010/108114, published Mar. 19, 2010. U.S. Pat. No. 7,211,418, discloses the detailed structural description of a PUFA PKS system in Thraustochytrium sp. ATCC 20892, and further detail regarding the production of eicosapentaenoic acid (C20:5, n-3) (EPA) and other PUFAs using such systems. U.S. Pat. No. 7,217,856 discloses the structural and functional description of PUFA PKS systems in Shewanella olleyana and Shewanella japonica, and uses of such systems. These applications also disclose the genetic modification of organisms with the genes comprising the PUFA PKS pathway and the production of PUFAs by such organisms. Furthermore, U.S. Pat. No. 7,776,626 describes a PUFA PKS system in Ulkenia, and U.S. Pat. No. 7,208,590 describes PUFA PKS genes and proteins from Thraustochytrium aureum. Each of the above-identified applications is incorporated by reference herein in its entirety.
Accordingly, the basic domain structures and sequence characteristics of the PUFA synthase family of enzymes have been described, and it has been demonstrated that PUFA synthase enzymes are capable of de novo synthesis of various PUFAs (e.g., eicosapentaenoic acid (EPA; C20:5, n-3), docosahexaenoic acid (DHA; C22:6, n-3) and docosapentaenoic acid (DPAn-6; C22:5, n-6).
PUFA synthases produce long chain polyunsaturated tatty acids de novo from malonyl-CoA using NADPH (and perhaps NADH) as a reductant. These multi-subunit enzymes have been identified in both marine bacteria and in the eukaryotic Thraustochytrid group of marine algae (Metz et al., 2001, Science 293:290-293). All of the PUFA synthases identified to date contain multiple ACP domains upon which the fatty acids are assembled. ACP domains require attachment of a co-factor by a phosphopantetheinyl transferase (PPTase) in order to function. Individual PPTases can have ACP substrate preferences, and when expressing a PUFA synthase in a heterologous organism, it may be necessary to provide a PPTase that can recognize, and activate, its ACP domains.
Novel production of PUFAs in several heterologous host organisms has been achieved by expression of the genes encoding the PUFA synthase subunits along with an appropriate PPTase. Of particular interest here, is the PUFA synthase derived from Schizochytrium 20888 (Metz et at., 2001, Science 293:290-293). The primary products of this PUFA synthase are DHA and DPAn-6. Schizochytrium 20888 has been developed as a commercial source for oil enriched in DHA. The organism can accumulate high levels of oil (>60% of the biomass) and DHA can comprise >40% of the fatty acids present in that biomass (Barclay et al., Single Cell Oils, 2nd edition. 2010 AOCS Press, pgs 75-96). In the native organism the DHA to DPAn-6 ratio typically ranges between 2.3 to 2.7. Expression of the PUFA synthase subunits of Schizochytrium 20888, along with an appropriate PPTase (e.g., Hetl from Nostoc sp., see Hauvermale et a., 2008, Lipids 41: 739-747 and Metz et al., US Patent Application Publication No. 2013-0150599) in heterologous host cells has resulted in production of DHA and DPAn-6 in those cells. Although DHA and DPAn-6 are produced in cells of E. coli (Hauvermale et al., 2008, Lipids 41:739-747), and in yeast and higher plants (Metz et al., US Patent Application Publication No. 2013-0150599), the levels have not approached those observed in the native organism. Additionally, in both yeast and in plants, the DHA to DPAn-6 ratio is typically significantly lower than that observed in the native organism. It is possible that some factor (or factors) in addition to the activated subunits of the enzyme itself is present in the cells of Schizochytrium 20888 which facilitates activity of the enzyme.
Since additional factors involved in the PUFA synthesis mechanism can have implications for increasing the efficiency of and/or improving the production of PUFAs in an organism that has been genetically modified to produce such PUFAs, there is a need in the art for finding such factor/factors. Accordingly, there is also a need in the art for improved methods of production of PUFAs, including in microorganisms that have been genetically modified to produce such PUFAs, which take advantage of the activity of such mechanism.