Cohen / Ratledge Single Cell Oils

Microbial and Algal Oils
2. Auflage 2015
ISBN: 978-1-63067-007-8
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, 516 Seiten

Featuring recognized academic and industrial experts in this cutting-edge field, this book reviews single cell oils (SCO) currently in the market. The text mainly focuses on the production of the long chain polyunsaturated fatty acids, Arachidonic acid, and Docosahexaenoinc acid. All chapters provide up to date references for navigating the vast amount of historic data available in the field. The authors provide real world examples of the commercial development and applications of various SCO in a variety of fields, from food ingredients and disease treatment to aquaculture and fish farming. It covers the essential information in this fast moving field giving details of the production of all the major SCOs, their extraction, purification, applications and safety evaluations. In addition, this new edition includes major coverage of the potential of SCOs for biofuels that may be of key significance in the coming years. - Includes sufficient detail on molecular breeding of yeasts and molds - Shows how microbial oils have gone from being academic curisoisties to being minor commodity oils - Presents details on the safey and nutrition of single cell oils for human and animal nutrition

Autoren/Hrsg.

Cohen, Zvi

Ratledge, Colin

Leseproben

2

Arachidonic Acid-Producing Creation of Mutants, Isolation of the Related Enzyme Genes, and Molecular Breeding

Eiji Sakuradania, Akinori Andob, Jun Ogawab and Sakayu Shimizua,     aDivision of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan; bResearch Division of Microbial Sciences, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan

Introduction

Polyunsaturated fatty acids (PUFAs) play important roles not only as structural components of membrane phospholipids but also as precursors of the eicosanoids of signaling molecules, including prostaglandins, thromboxanes, and leukotrienes, which are essential for all mammals. Fish oils, animal tissues, and algal cells are conventional, relatively rich sources of C20 PUFAs, which are not present in plants. For practical purposes, however, these conventional sources are not satisfactory with regard to either the lipid contents or the PUFA contents of the resultant lipids. To find more suitable sources of PUFAs in microbes, the first attempts at PUFA production with ?-linolenic acid (GLA, 18:3n-6) as the target were performed in the UK (Ratledge, 1992) and Japan (Suzuki et al., 1981), with fungi being used. Since then, various PUFAs have been studied with the aim of effective production. For example, arachidonic acid (AA, 20:4n-6), dihomo-?-linolenic acid (DGLA, 20:3n-6), and Mead acid (MA, 20:3n-9) are now commercially produced by using fungi (Certik et al., 1998;Certik & Shimizu, 1999; Sakuradani et al., 2005b; Shimizu & Yamada, 1990; Yamada et al., 1992), and docosahexaenoic acid (22:6n-3), docosapentaenoic acid (22:5n-6), and eicosapentaenoic acid (EPA, 20:5n-3) by using marine microorganisms, Labyrinthulae, and microalgae (Certik & Shimizu, 1999; Kyle et al., 1992; Nakahara et al., 1996; Raghukumar, 2008; Ratledge, 2004; Singh & Ward, 1997; Spolaore et al., 2006; Yazawa et al., 1992). Although success in this area over the last 25 years has generated much interest in the development of microbial fermentation processes, manipulation of the lipid compositions of microorganisms requires new biotechnological strategies to obtain high yields of the desired PUFAs.

The genus has been shown to be one of the promising single cell oil (SCO) sources rich in various types of C20 PUFAs (Amano et al., 1992; Shimizu & Jareonkitmongkol, 1995), after several strains were reported to be potential producers of AA in 1987 (Totani & Oba, 1987; Yamada et al., 1987). In particular, several strains have been extensively studied for the practical production of AA (Shinmen et al., 1989). Some of them are now used for the commercial production of SCO rich in AA. Among them, 1S-4 has a unique ability to synthesize a wide range of fatty acids and has several advantages not only as an industrial strain but also as a model for lipogenesis studies. The biosynthetic pathways for n-9, n-6, and n-3 PUFAs in 1S-4 are shown in Fig. 2.1a. The main product of the strain, AA, is synthesized through the n-6 pathway, which involves ?12 and ?6 desaturases, elongase (EL2), and ?5 desaturase. Depending on the conditions, the total amount of AA varies between 3 and 20 g/L (30–70% of the total cellular fatty acids), with 70–90% of the AA produced being present as triacylglycerols (Higashiyama et al., 1998, 2002; Shimizu et al., 2003b).

Fig. 2.1 Pathways for the biosynthesis of PUFAs in 1S-4 and its mutants.

Here, we describe recent progress in the breeding of commercially important arachidonic acid-producing strains, particularly approaches for creating desaturase and elongase mutants with unique pathways for PUFA biosynthesis involving conventional chemical mutagenesis and modern molecular genetics. Such mutants are useful not only for the regulation and overproduction of valuable PUFAs but also as excellent models for the elucidation of fungal lipogenesis.

Derivation of Mutants from 1S-4

A wide variety of mutants defective in desaturases (?9, ?12, ?6, ?5, and n-3) or elongase (EL1) or mutants with enhanced desaturase activities (?6 and ?5) have been derived from 1S-4 by treating the parental spores with -methyl--nitro--nitrosoguanidine (Jareonkitmongkol et al., 1992c). In addition, diacylglycerol-accumulating mutants and several lipid-excretive ones have been isolated by the same method. They are valuable both as producers of useful PUFAs (novel or already existing) and for providing valuable information on PUFA biosynthesis in this fungus (Certik et al., 1998). The main features of these mutants grown on glucose and the biosynthesis of various types of PUFAs by them are outlined below (see also Fig. 2.2).

Fig. 2.2 List of the mutants derived from 1S-4. Symbols in squares indicate apparent mutation sites. Fatty acids in square brackets are major fatty acids produced by the mutants. Abbreviations: arachidonic acid, AA; diacylglycerol, DG; dihomo-?-linolenic acid, DGLA; free fatty acid, FA; Mead acid, MA; and triacylglycerol, TG.

?9 Desaturase-Defective Mutants

Stearic acid (18:0) is the main fatty acid in the mycelial oil (up to 40%) produced by these mutants (Jareonkitmongkol et al., 2002). However, ?9 desaturase is not completely blocked. A total blockage would be lethal since low activity of the enzyme is necessary for introduction of the first double bond at the ninth carbon (from the carboxyl end) of the fatty acid chain to maintain cell viability (see next section).

?12 Desaturase-Defective Mutants

The attributes of ?12 desaturase-defective mutants include the absence of n-6 and n-3 PUFAs and high levels of n-9 PUFAs, such as oleic acid (18:1n-9), octadecadienoic acid (18:2n-9), eicosadienoic acid (20:2n-9), and MA, in their mycelia (Jareonkitmongkol et al., 1992a). Cultivation of these mutants under the optimal conditions yields a unique oil rich in large quantities of MA. However, the addition of either n-6 or n-3 fatty acids causes a rapid decrease in n-9 fatty acid formation by these mutants and an increase in the AA or EPA level, respectively, because of the substrate specificity of ?6 desaturase, which prefers linoleic acid, a-linolenic acid, and oleic acid, in that order (Jareonkitmongkol et al., 1993d). Therefore, the same mutants can be used for the production of an EPA-rich oil with a low AA level. a-Linolenic acid, when added exogenously (as linseed oil) to the medium, was efficiently converted to EPA, the final mycelial EPA/AA ratio being 2.5 (Jareonkitmongkol et al., 1993d).

Mutants with Enhanced Desaturase Activities

A mutant (209-7) with enhanced ?6 desaturase activity was isolated from a ?12 desaturase-defective mutant (Mut48) by selecting colonies with high MA contents after mutagenesis (Kawashima et al., 1997). ?6 desaturase activity is 1.4-fold elevated in this mutant, from which a mutant (JT-180) with elevated ?5 desaturase activity (3.3-fold) was obtained (see Fig. 2.2). Cultivation of JT-180 yields a large quantity of MA (2.6 g /L, 49% in oil) (Sakuradani et al., 2002). This mutant is used for the commercial production of MA.

?6 Desaturase-Defective Mutants

Mutants synthesizing a high level of linoleic acid and low concentrations of GLA, DGLA, and AA are considered to be defective in ?6 desaturase (Jareonkitmongkol et al., 1993c). These mutants are characterized by the accumulation of an eicosadienoic acid (20:2n-6) and a nonmethylene-interrupted n-6 eicosatrienoic acid (20:3?5). The latter PUFA is thought to be synthesized though elongation of linoleic acid and ?5 desaturation, as shown in Fig. 2.1b. In a similar manner, a nonmethylene-interrupted n-3 eicosatrienoic acid (20:4?5) can be produced from a-linolenic acid added to the medium (Jareonkitmongkol et al., 1993c).

?5 Desaturase-Defective Mutants

The fatty acid profiles of these mutants are characterized by a high DGLA level and a reduced concentration of AA (Jareonkitmongkol et al., 1993b). Production of DGLA by these mutants is advantageous because it does not require inhibitors and the yield is relatively high (4.1 g/L, 42% in oil; AA content, <1%)...