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Desaturase-defective fungal mutants: useful tools for the regulation and overproduction of polyunsaturated fatty acids
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Desaturase-defective fungal mutants: useful tools for the regulation and overproduction of polyunsaturated fatty acids Milan Certik, Eiji Sakuradani and Sakayu Shimizu Increasing demand for biologically important polyunsaturated fatty acids has led to the search for alternative sources, especially fungi. Although successes in this area have induced interest in developing fungal fermentation processes, manipulation of their lipid composition requires new biotechnological strategies to achieve high yields of the desired polyunsaturated fatty acids. Fungal desaturase mutants with unique enzyme systems are useful not only for the regulation and overproduction of valuable polyunsaturated fatty acids but also because they are excellent models for the elucidation of fungal lipogenesis.
n recent years, considerable attention has been paid to polyunsaturated fatty acids (PUFAs), especially the essential fatty acids (EFAs). EFAs are distinguished by two main functions1,2. The first is their role in membrane structure, where EFAs confer fluidity and flexibility, and modulate the behaviour of certain membranebound proteins. However, more attention has been focused on the second role of EFAs, as precursors of a wide variety of metabolites (such as prostaglandins, leukotrienes and hydroxy-fatty acids) regulating critical biological functions. The roles played by EFAs make it apparent that they are necessary in every organ in the body. Because mammals lack the ability to insert double bonds between the ninth carbon (from the carboxyl end) and the terminal methyl group, they cannot synthesize linoleic (18:2 v-6) and a-linolenic (ALA; 18:3 v-3) acids (see Glossary). These two fatty acids are thus essential for mammals, which rely ultimately on plant, microbial and fish sources for their provision. The main oil sources that are relatively rich in C18 PUFAs are the seeds of some plants and moulds3, which predominantly synthesize g-linolenic acid (18:3 v-6) and ALA. By contrast, PUFAs above C18 cannot be synthesized by higher plants in any significant amounts owing to a lack of the requisite enzymes. On the other hand, fish oil, certain fungi, marine bacteria, algae and mosses may represent suitable potential sources of C20 PUFAs4–8.
I
Strategy for manipulating fungal PUFA formation Increasing demand for biologically active PUFAs has focused commercial attention on PUFA-based nutraceuticals and therapeutics, and on the provision of a suitable biosynthetic framework for their production. As an alternative to their conventional sources, concentration has focused on microorganisms. Oleaginous filamentous fungi, mainly zygomycetes, are thought to be one promising source of these essential compounds9–11, possessing several advantages: M. Certik, E. Sakuradani and S. Shimizu ([email protected]) are at the Division of Applied Life Sciences, Graduate School of Agricultural Sciences, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan.
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• an active PUFA-synthesizing apparatus; • PUFA production can be carried out throughout the year (no seasonal or climatic dependence); • high growth rates on a variety of substrates (including various waste materials); • simple metabolic regulation and control; • suitable model for studying and manipulating PUFA biosynthesis; • supply of more-concentrated PUFAs with controlled quality (pharmaceutical-grade PUFAs); • appropriate vehicles for cloning foreign genes for the production of specific PUFAs; • simultaneous upgrading of both PUFA and other products; • can be employed as macro- and micronutrient sources. Although comparative success in the fungal production of PUFAs (from screening to optimization of fermentation conditions) has led to a flourishing interest in developing fermentation processes and enabled several processes to attain commercial production levels2, two main problems still remain, relating to economic and marketing difficulties. Attempts to reduce the cost of microbial lipids (with emphasis on increasing the product value, using inexpensive substrates, screening for more-efficient strains and reducing the number of processing steps necessary for oil recovery from the cells) are continuing, and the manipulation of microbial PUFA composition is a rapidly growing field of biotechnology. However, the use of fungal lipids is still insufficient to meet industrial demand. Other strategies, therefore, should be combined with classical fermentations (Fig. 1). For example, mutation techniques resulting in the suppression or activation of specific fungal desaturases are beneficial not only for the production of tailor-made PUFAs but can also be used for studying PUFA biosynthesis in fungi12,13. Molecular engineering of parental strains and their mutants is a powerful tool for constructing novel microbial strains synthesizing economically valuable fatty acids14. Control of individual desaturation steps by the addition of desaturase inhibitors or activators to the growth media may also lead to directed PUFA modification15,16. Finally, manipulating PUFA composition by enzymatic treatment of pre-existing PUFA-containing oils is also
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attracting considerable attention (for example, the use of lipase-catalysed reactions in immobilized systems, twophase systems, membrane bioreactors and in combination with supercritical-carbon-dioxide extraction)17,18. Model for the regulation of PUFA biosynthesis The genus Mortierella is known to be one of the best producers of various types of PUFA10,13. In particular, Mortierella alpina 1S-4 has a unique capacity to synthesize the entire range of fatty acids and has several advantages as a model for these studies: • it is a highly oleaginous strain; • lipogenesis is simply regulated; • it is one of the most well-studied microorganisms producing PUFAs; • the strain is able to incorporate and transform exogenous fatty acids; • various desaturase mutants are available; • it is amenable to molecular-genetic study; • the strain can be used in an industrial scale. The biosynthetic pathway of v-9, v-6 and v-3 PUFAs in M. alpina 1S-4 is shown in Fig. 2. The main product of the strain, arachidonic acid (20:4 v-6; AA), is synthesized via the v-6 route, which involves D12 and D6 desaturases, elongase and D5 desaturase. Depending on the conditions, the total amount of AA varies between 3 and 13 g l21 (30–70% of the fatty acids), with 70–90% of the AA created being bound to triacylglycerols13. The highest yield of AA (13 g l21, corresponding to 220 mg g21 dry mycelia) was achieved in a 10 kl fermenter. The cultivation of the strain under certain conditions also leads to a variety of PUFAs being formed, for example: (1) the addition of D5 desaturase inhibitors to the media causes an increase in dihomo-g-linolenic acid (DGLA; 20:3 v-6) in the fungal oil16; (2) lowering the temperature (to 128C) with the simultaneous addition of ALA to the medium results in the production of eicosapentaenoic acid (EPA; 20:5 v-3)19; (3) the utilization of C15 and C17 n-alkanes by the strain yields PUFAs with odd number of carbons in their chain (total C17 and C19 fatty acids reached over 95% of mycelial fatty acids)20; and (4) when 1-hexadecene or 1-octadecene is the main carbon source, several v-1 PUFAs (20:5 v-1, 20:4 v-1, 16:1 v-1, 18:1 v-1 and 18:4 v-1) are produced21. Fatty-acid-desaturase-defective mutants of M. alpina Although the wild-type M. alpina 1S-4 is able to incorporate exogenous fatty acids and convert them into various PUFAs, the strain has a limited ability to produce new PUFAs or to increase existing PUFA formation. To extend these abilities, several desaturase mutants of M. alpina 1S-4 have been isolated following the treatment of the parental spores with N-methyl-N9nitro-N-nitrosoguanidine22. Other mutants [with defective desaturases (D5, D6, D9, D12 and v3), enhanced desaturase activities (D5, D6) or a combination] have been prepared13 and are worthwhile not only as producers of useful PUFAs (novel or already existing) but also for providing valuable information on PUFA biosynthesis in this fungus. They are also excellent tools for controlling and regulating exogenous-fatty-acid flow to targeted PUFAs13. TIBTECH DECEMBER 1998 (VOL 16)
Glossary v-x PUFA
The first double bond is located at the xth carbon from the methyl end of the PUFA Dm PUFA Non-methylene-interrupted double bond is located at the mth carbon from the carboxyl end of an v-x PUFA vx Desaturase Desaturase introducing double bonds at the xth carbon from the methyl end of the PUFA Dx Desaturase Desaturase introducing double bonds at the xth carbon from the carboxyl end of the PUFA
Fatty-acid profile of the mutant strains The biosynthesis of various types of PUFA by desaturase-defective mutants of M. alpina 1S-4 is presented in Table 1 and outlined on Fig. 2. The main features of these mutants grown on glucose can be summarized in the following manner. (1) The fatty-acid profile of the D5-desaturase-defective mutants is characterized by a high DGLA level and a reduced concentration of AA23. Production of DGLA by these mutants is advantageous because it does not require inhibitors and the yield is relatively high. (2) Attributes of D12-desaturase-defective mutants include the absence of v-6 and v-3 PUFAs and high levels of v-9 PUFAs, such as oleic (18:1 v-9), octadecadienoic (18:2 v-9), eicosadienoic (20:2 v-9) and mead (20:3 v-9) acids in their mycelia24. Of these, mead acid is known to be a precursor of LT3 leukotrienes and anti-inflammatory compounds25. (3) When both D5 and D12 desaturases are blocked, 20:2 v-9 is accumulated in large quantities26. (4) Mutants synthesizing a high level of linoleic acid and low concentrations of g-linolenic acid, DGLA and AA are considered to be defective in D6 desaturase27. Moreover, the accumulation of 20:2 v-6 and two nonmethylene-interrupted PUFAs [v-6 eicosatrienoic (20:3 D5) and v-3 eicosatetraenoic (20:4 D5)] acids Screening of microorganisms
Optimization of fermentation process (conditions, techniques, scale-up) Desaturase-defective mutants
Molecular-genetic approach Desired PUFAs
Desaturase inhibitors or activators
Enzymatic biotransformation
Figure 1 Strategy for the modification of microbial polyunsaturated fatty acids. Screening of potential microorganisms is an essential step and results in a limited number of strains for practical use. These strains can be used directly in process optimization or further modified by classical mutation techniques or molecular-engineering methods to produce the desired fatty acids. The fatty-acid profile may also be controlled by desaturase inhibitors or activators added to the medium and finally improved by enzymatic treatment of pre-existing oils.
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a
COOH 18:0
D9
D12
COOH
COOH
18:1w9
18:2w6
D6
D6
18:2w9
COOH
COOH
20:2w9
COOH
20:4w6
w-9 series
COOH
D6 18:2w9
EL
18:2w9
20:2w9
20:3w6
COOH
COOH
COOH
18:3w6
EL COOH
20:3w6
COOH
EL COOH
20:2w9
D5
COOH
20:3w6
D5 COOH
COOH
COOH
20:3w9
20:4w6
w-6 series
20:4w6
w-9 series
w-6 series
COOH
f
COOH 18:0
g
16:0
EL
D9
D12
COOH
w3
COOH
18:1w9
18:2w6
COOH
COOH
20:2w9
D5
18:3w6
COOH
EL
COOH
EL
18:1w9
D6
20:3w6
20:2w6
w3
D5 20:3D5
20:4w6
w-6 series
COOH
COOH
w3
D12
COOH 18:2w6
D6
18:2w9 COOH
18:3w6
COOH
20:3w3
EL
EL
D5
COOH
COOH
w-9 series
COOH
COOH
D5
20:3w9
18:0
D9
EL
EL
COOH
COOH 18:3w3
D6 18:2w9
D6
18:2w9
EL
COOH 18:2w6
D6 COOH
18:3w6
COOH
D12
COOH
D6 COOH
w-9 series
e
COOH
18:1w9
COOH
w-6 series
w-3 series
18:0
COOH
20:3w9
20:4w6
w-9 series
w-6 series
D9
D12
D5
20:3w9
COOH 20:5w3
20:4w6
18:2w6
20:2w9
D5
COOH
20:4w3
D5
d
EL COOH
EL
COOH
(low temp.)
COOH
EL COOH
COOH
COOH
w-9 series
D6
18:3w6
w3
20:3w6
20:3w9
18:1w9
COOH
18:4w3
D5
COOH
D6 COOH
COOH
18:0
18:2w6
COOH
COOH
w-3 series
COOH
18:1w9
D6
18:3w6
D5
D9
D12
COOH
20:2w9
20:5w3
c
COOH 18:0
18:3w3
EL
COOH
(low temp.)
w-6 series
D9
COOH
COOH
COOH 18:2w6
D6
18:2w9
D5 w3
COOH
D12
EL 20:4w3
D5 COOH
20:3w9
COOH
EL
20:3w6
D5
COOH 18:1w9
D6 18:4w3
EL COOH
COOH 18:3w3
D6
18:3w6
EL
D9
w3
b
COOH 18:0
COOH
20:2w9 20:4D5
w-3 series
COOH
20:3w6
COOH
D5
COOH
20:3w9
w-9 series
D5
COOH
20:4w6
w-6 series
Figure 2 Biosynthetic pathways of polyunsaturated fatty acids (PUFAs) in Mortierella alpina 1S-4 and its desaturase-defective mutants. Defective desaturases and characteristic fatty acids of the individual mutants are highlighted. Abbreviations: EL, elongation; D5, D6, D9, D12, v3, various fatty-acid desaturases. (a) The wild-type strain 1S-4 synthesizes arachidonic acid (AA) as the main fatty acid; lowering the temperature results in production of v-3 PUFAs, mainly eicosapentaenoic acid. The amount of v-9 PUFAs produced is almost negligible. (b) v3-Defective mutants do not form v-3 PUFAs. (c) D5-Desaturase-defective mutants cannot convert dihomo-g-linolenic acid (DGLA) to AA and DGLA is accumulated. (d) Mutants defective in D12 desaturase are unable to synthesize v-6 and v-3 PUFAs. Because the other desaturases (D6 and D5) and elongase are still active, v-9 PUFAs are accumulated where mead acid is the end product. (e) In mutants defective in both D5 and D12 desaturases, 20:2 v-9 is the final fatty acid in the PUFA-biosynthetic cascade. (f) Owing to a lack of D6 desaturase, linoleic acid is the main fatty acid in these mutants. However, linoleic acid is to some extent further metabolized to two non-methylene-interrupted fatty acids, 20:3 D5 v-6 and 20:4 D5 v-3. (g) D9-Desaturasedefective mutants synthesize high levels of stearic acid. Because this desaturase is not completely blocked, a small amount of v-6 PUFAs is also formed.
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Table 1. Production of some PUFAs by Mortierella alpina 1S-4 and its mutants PUFA series
v-3
PUFA name
20:3 20:4 (ETA) 20:4D5a 20:5 (EPA)
v-6
v-9
20:2 20:3 (DGLA) 20:3D5a 20:4 (AA) 18:2 20:2 20:3 (MA)
Strain
D6 mut D5 mut D1215 mut D6 mut 1S-4 D12 mut D6 mut 1S-4 D5 mut D6 mut 1S-4 D12 mut D1215 mut D1215 mut D12 mut
PUFA formation g21]b
[% in oil]
[mg
2.5 26 37 5 12 20 9 27 42 15 30–70 14 16 25 33
6.5 77 97 12 67 64 22 107 205 27 100–300 60 63 110 141
Remark [g
l21]
0.07 1.6 2.3 0.12 1.9 1.0 0.23 2.6 4.1 0.25 3–13 1.0 1.1 1.7 1.9
148C Linseed-oil addition Linseed-oil addition 148C 1128C, linseed-oil addition Linseed-oil addition 148C Inhibition of D5 by sesamin 1288C
aNon-methylene-interrupted
fatty acid. is, (mg fatty acid) (g dry biomass)21. Abbreviations: AA, arachidonic acid; DGLA, dihomo-g-linolenic acid; EPA, eicosapentaenoic acid; ETA, eicosatetraenoic acid; MA, mead acid; PUFA, polyunsaturated fatty acid. bThat
were found27. 20:3 D5 is thought to be synthesized by the elongation of linoleic acid and then D5 desaturation. The formation of 20:4 D5 might be initiated by conversion of linoleic acid to ALA (v-3 desaturation) followed by elongation and D5 desaturation. Although the biological activities of non-methylene-interrupted fatty acids are not fully understood, they are probably formed in response to a deficiency of v-3 and v-6 PUFAs. 20:3 D5 can be converted via cyclooxygenase to hydroxyfatty acids28 and may alter eicosanoid signalling29. (5) Mutants defective in v3 desaturation are unable to synthesize v-3 PUFAs when cultivated at low temperature30. (6) Finally, stearic acid (18:0) is the main fatty acid in the mycelial oil (up to 40%) of the D9-desaturasedefective mutants31. However, D9 desaturase is not completely blocked, because low activity of the enzyme is important for the introduction of the first double bond at the ninth carbon (from the carboxyl end) of the fatty-acid chain to maintain cell viability.
ALA. Even when grown at low temperature (which stimulates v-3-desaturase activity), ETA production is still low. On the other hand, the addition of linseed oil to the medium results in accumulation of ETA33. Nevertheless, DGLA and ETA formation is competitive and their ratio varies with the ratio of glucose and ALA added to the medium. Because an increase in glucose results in a greater DGLA concentration in the mycelial oil33, the conversion of LA (endogenously formed from glucose) to DGLA is much faster than the transformation of exogenous ALA to ETA. This suggests that D6 desaturase is more specific for LA than for ALA.
Biotransformation of exogenous fatty acids by the mutants The ability of all these mutants to utilize exogenous fatty acids may allow the preparation of various PUFAs in relatively large quantities. This could be very important, because there are various sources of easily available natural oils containing individual fatty-acid precursors. How the desaturase-defective mutants could be used to perform the transformation of exogenously added oils to a desired fatty acid is shown in the following examples (see also Table 1).
D12-Desaturase-defective mutants From a nutritional standpoint, an EPA-rich oil with a low AA level is preferred, because of the diverse biological activities of AA1. However, the AA concentration in the mycelial oil of M. alpina is usually higher than that of EPA, even when the parental strain is cultivated at low temperatures and in the medium supplemented with ALA. Therefore, as D12-desaturase-defective mutants cannot synthesize any v-3 or v-6 PUFAs when grown on glucose, these mutants could be used to solve this problem. Because the other desaturases and elongases are still active, ALA added exogenously (as linseed oil) to the medium is efficiently converted to EPA to give the mycelial EPA/AA ratio of 2.532. However, the addition of either v-3 or v-6 fatty acids causes a rapid decrease in v-9 fatty-acid formation by these mutants, because of the substrate specificity of D6 desaturase, which prefers LA and ALA over oleic acid32.
D5-Desaturase-defective mutants When these mutants are grown on glucose, DGLA is normally formed. However, eicosatetraenoic acid (ETA; 20:4 v-3) is the other potential end product of these mutants and can be synthesized either by v-3 desaturation of DGLA or through the v-3 route from
D5-D12-Desaturase-defective mutants Although growing D5-desaturase-defective mutants on linseed oil improves ETA production, the amount of DGLA in the mycelial oil is still high. Because ETA has a potential use as a precursor of prostaglandins34 and the separation of these two fatty acids is difficult, the
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formation of DGLA from glucose should be limited in order to increase the accumulation and improve the purification of ETA. Therefore, D5-D12desaturasedefective mutants can be cultivated with exogenously added ALA to give increased production of ETA.
D6-Desaturase-defective mutants The production of non-methylene-interrupted fatty acids by D6-desaturase-defective mutants depends on the cultivation temperature. An increase in the temperature (from 48C to 288C) is accompanied by a fall in 20:4 D5 (v-3 series), but a rise in non-methyleneinterrupted 20:3 D5 (v-6 series) levels. However, 20:4 D5 is effectively synthesized even at laboratory temperature when the mutants utilize exogenous ALA. The fatty-acid-biosynthetic apparatus of the mutants The prospects for efficient PUFA production depend on a knowledge of the mechanism by which PUFA biosynthesis is accomplished. This is, however, still poorly understood, although the main aspects of PUFA accumulation are known. There are several distinct pathways that could be involved in this process35: (1) de novo synthesis of fatty acids from glucose; (2) the incorporation of exogenous fatty acids directly into lipid structures; and (3) following desaturation and elongation of lipid sources. In addition, fatty-acid biohydrogenation and partial or total degradation (b oxidation) also contribute to this process35. It is well known that PUFA formation in fungi is catalysed by a multienzyme complex composed of three proteins: NADH–cytochrome-b5 reductase, cytochrome b5 and the terminal cyanide-sensitive desaturase. Simultaneously, enzyme system(s) providing the NADPH for desaturation and elongation reactions must be involved36. Membrane constituents such as phospholipids are accepted as the most likely form for fatty acids to be available for desaturation in fungi37. Once desaturation has occurred, acyltransferase reactions then facilitate the distribution of newly synthesized PUFAs to other cellular lipids, mainly triacylglycerols. Because these acylation processes are catalysed by separate acyltransferases, the acyltransferases and desaturases may be tightly bound and their cooperation could be responsible for the distribution of PUFA among individual lipid structures. The mutants of M. alpina 1S-4 are not only valuable as producers of some useful PUFAs; they also provide information on PUFA biosynthesis and regulation in this fungus. The PUFA-biosynthetic pathways in M. alpina 1S-4 and its desaturase-defective mutants are illustrated in Fig. 2. From observation of the D9-desaturase-defective mutant, it is evident that the first double bond is invariably inserted at the ninth carbon from the carboxyl end31. All three metabolic routes (v-3, v-6 and v-9) that are engaged in PUFA formation in this fungus share the same enzymes: D6 desaturase, elongase and D5 desaturase. However, the desaturases have a wide substrate specificity. For example, D5 desaturase can act on odd-numbered fatty acids, PUFAs with v-1 terminal double bonds or v-3, v-6 and v-9 fatty acids. On the other hand, varying specificities of individual desaturases (according to carbon number and location of double bonds in the fattyacid chain) modify the flux of C18 and C20 fatty acids to the end products. For instance, D6 desaturase, the rate-limiting step in PUFA formation, exhibits different
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specificities for oleic, linoleic and a-linolenic acids. Depending on the conditions, it may finally lead to varying amounts of v-3, v-6 and v-9 PUFAs being formed. There are two possible enzymes catalysing the conversion of C18 and C20 v-6 fatty acids to v-3 fatty acids: D15 and D17 desaturase, respectively. Nevertheless, the D15 and D17 desaturation in this fungus may be catalysed by one enzyme only, which recognizes the methyl end of both C18 and C20 v-6 fatty acids30. Thus, the enzyme is different from that found, for example, in Saprolegnia parasitica38, which is specific only for C20 fatty acids. This enzyme in M. alpina was thus described as an v3 desaturase (rather than a D desaturase) because it is specific for the methyl (not the carboxyl) terminus. Two routes for v-3 PUFA production operate in M. alpina. The first involves the conversion of an exogenous v-6 fatty acid into the corresponding v-3 fatty acid, and the second, the v-3-fatty-acid-biosynthetic pathway. The former route is activated at low temperature and has also been reported in other Mortierella species39,40; the latter route is independent of temperature and operates simultaneously with the former route at low temperatures. To summarize, the unique biotransforming enzyme systems of M. alpina 1S-4 and its desaturase-defective mutants can be used to construct useful v-3, v-6 or v-9 PUFAs from their precursors. They also indicate how it is possible to control the fatty-acid profile of fungal mutants and to regulate the flow of glucose or exogenous fatty acids to obtain a desired PUFA. Because of the simplicity of their metabolic system, these mutants are potentially ideal models for the elucidation of fungal lipogenesis. Presently, the studies performed with M. alpina and its mutants are focused on the molecular engineering of these desaturases and on the genes involved in the microsomal electron-transport system. Thus, these mutants may be used as vehicles for the cloning of desaturase genes, which could be introduced into either plants or animals to produce transgenic organisms with desired fatty-acid composition. Fungal desaturase-defective mutants other than Mortierella spp. Although the mutation strategy offers great advantages for manipulating fungal oils, most of the mutants (other than Mortierella spp.) that have been described have dominant defective D9-desaturase genes. The mutation of D9-desaturase genes led to a requirement for unsaturated fatty acids for fungal growth and to defective development and reduced respiration rates in some strains of Aspergillus niger and Neurospora crassa41,42. Moreover, some mutants, such as N. crassa42–44, Saccharomyces cerevisiae45 and Cryptococcus curvatus46, were unstable and quickly reverted to the wild-type growth habit. These mutants are used for studying membrane structures and functions47, as well as for elucidating the reaction mechanisms involved in fatty-acid biosynthesis48. Other approaches with D9-desaturase-defective mutants are focused on the development of strains producing highly saturated oils as a substitute for cocoa butter. This has resulted in the isolation of a strain of C. curvatus (which used to be called Apiotrichum curvatum) with an inactive D9 desaturase46. Although this mutant synthesizes oil with saturated fatty acids, it requires the addition of oleic acid to the medium, as this is an essential TIBTECH DECEMBER 1998 (VOL 16)
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component of the membrane system. However, a hybrid strain of C. curvatus with a partially active D9 desaturase is able to grow without oleic acid, and its fatty-acid composition is comparable to that of cocoa butter12. With the exception of D9 desaturase, the availability of desaturase-defective fungal mutants is rather limited. A recent analysis of five N. crassa mutants with altered PUFA synthesis indicated that multiple genes influence the PUFA composition in their membranes and storage lipids43. Also, a few new studies of the expression of plant D12 desaturase or animal D6 desaturase in S. cerevisiae reported the accumulation of linoleic and hexadecadienoic (16:2) acids49–51 or g-linolenic acid14, respectively. Future prospects Biotechnological strategies for the modification of microbial PUFAs represent a powerful tool not only for the study and regulation of complex metabolic pathways but also for the creation of novel microbial strains synthesizing economically valuable lipid metabolites. Comparative success in this area using mutation techniques and genetic engineering has resulted in the heterologous expression of desaturase genes from plants51,52 and animals14 in fungi and has allowed structure–function studies and the production of special fatty acids. Also, the introduction of microbial lipid-biosynthetic genes into specific subcellular locations of transgenic plants provides a useful means for performing unusual reactions and products in plants53. However, for both economic and biochemical reasons, the modification of fatty acids as a result of any form of manipulation must be correctly targeted into triacylglycerols and excluded from phospholipids. Therefore, further progress will depend on: (1) investigation of their regulatory mechanisms; (2) identification of the key steps limiting triacylglycerol biosynthesis; (3) understanding the mechanisms of enzyme action in PUFA biosynthesis; and (4) elucidation of the signalling systems of the lipidbiosynthetic machinery inside the cell. Currently, no aspect of these regulatory mechanisms is understood in any organism and thus this represents a challenging and potentially rewarding subject for the future research. References 1 Horrobin, D. F. (1995) Inform 6, 428–435 2 Gill, I. and Valivety, R. (1997) Trends Biotechnol. 15, 401–409 3 Ratledge, C. (1989) in Microbial Lipids (Vol. 2) (Ratledge, C. and Wilkinson, S. G., eds), pp. 567–668, Academic Press 4 Radwan, S. S. (1991) Appl. Microbiol. Biotechnol. 35, 421–430 5 Yamada, H., Shimizu, S., Shinmen, Y., Akimoto, K., Kawashima, H. and Jareonkitmongkol, S. (1992) in Industrial Applications of Single Cell Oils (Kyle, D. J. and Ratledge, C., eds), pp. 118–138, AOCS Press, Champaign, USA 6 Yazawa, K., Watanabe, K., Ishikawa, C., Kondo, K. and Kimura, S. (1992) in Industrial Applications of Single Cell Oils (Kyle, D. J. and Ratledge, C., eds), pp. 29–51, AOCS Press, Champaign, USA 7 Nakahara, T., Yokochi, T., Higashihara, T., Tanaka, S., Yaguchi, T. and Honda, D. (1996) J. Am. Oil Chem. Soc. 73, 11 8 Singh, A. and Ward, O. P. (1997) Adv. Appl. Microbiol. 45, 271–312 9 Certik, M., Sereke Berhan, S. and Sajbidor, J. (1993) Acta Biotechnol. 13, 193–196 10 Leman, J. (1997) Adv. Appl. Microbiol. 43, 195–243 11 Certik, M. and Shimizu, S. (1998) in Recent Research Developments in Oil Chemistry (Vol. 2) (Pandalai, S. G., ed.), pp. 89–103, Transworld Research Network, Trivandrum, India 12 Verwoert, I. I., Yjema, A., Valkenburg, J. A. C., Verbree, E. C., Nijkamp, H. J. J. and Smit, H. (1989) Appl. Microbiol. Biotechnol. 32, 327–333
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13 Shimizu, S. and Jareonkitmongkol, S. (1995) in Biotechnology in Agriculture and Forestry (Vol. 33) (Bajaj, Y. P. S., ed.), pp. 308–325, Springer-Verlag 14 Napier, J. A., Hey, S. J., Lacey, D. J. and Shewry, P. R. (1998) Biochem. J. 330, 611–614 15 Rolph, C. E., Moreton, R. S. and Harwood, J. L. (1990) Appl. Microbiol. Biotechnol. 34, 91–96 16 Shimizu, S., Akimoto, K., Sugano, M. and Yamada, H. (1993) in Essential Fatty Acids and Eicosanoids (Sinclair, A. and Gibson, R., eds), pp. 31–41, AOCS Press, Champaign, USA 17 Tanaka, Y., Hirano, J. and Funada, T. (1994) J. Am. Oil Chem. Soc. 71, 331–334 18 Shishikura, A., Fujimoto, K., Suzuki, T. and Arai, K. (1994) J. Am. Oil Chem. Soc. 71, 961–967 19 Shimizu, S., Kawashima, H., Akimoto, K., Shinmen, Y. and Yamada, H. (1989) Appl. Microbiol. Biotechnol. 32, 1–4 20 Shimizu, S., Kawashima, H., Akimoto, K., Shinmen, Y. and Yamada, Y. (1991) J. Am. Oil Chem. Soc. 68, 254–258 21 Shimizu, S., Jareonkitmongkol, S., Kawashima, H., Akimoto, K. and Yamada, Y. (1991) Arch. Microbiol. 156, 163–166 22 Jareonkitmongkol, S., Shimizu, S. and Yamada, H. (1992) J. Gen. Microbiol. 138, 997–1002 23 Jareonkitmongkol, S., Sakuradani, E. and Shimizu, S. (1993) Appl. Environ. Microbiol. 58, 4300–4304 24 Jareonkitmongkol, S., Kawashima, H., Shimizu, S. and Yamada, H. (1992) J. Am. Oil Chem. Soc. 69, 939–944 25 Cleland, L. G., Neumann, M. A., Gibson, R. A., Hamazaki, T., Akimoto, K. and James, M. J. (1995) Lipids 31, 829–837 26 Shimizu, S. (1995) J. Jpn Oil Chem. Soc. (Yukagaku) 44, 804–813 27 Jareonkitmongkol, S., Shimizu, S. and Yamada, H. (1993) Biochim. Biophys. Acta 1167, 137–141 28 Evans, R. W. and Sprecher, H. (1985) Prostaglandins 29, 431–441 29 Berger, A., Fenz, R. and German, J. B. (1993) J. Nutr. Biochem. 4, 409–420 30 Jareonkitmongkol, S., Sakuradani, E. and Shimizu, S. (1994) Arch. Microbiol. 161, 316–319 31 Jareonkitmongkol, S., Sakuradani, E., Kawashima, H. and Shimizu, S. (1993) Nippon Nogei Kagaku Kaishi 67, 531 32 Jareonkitmongkol, S., Shimizu, S. and Yamada, H. (1993) J. Am. Oil Chem. Soc. 70, 119–123 33 Kawashima, H., Kamada, N., Sakuradani, E., Jareonkitmongkol, S., Akimoto, K. and Shimizu, S. (1997) J. Am. Oil Chem. Soc. 74, 455–459 34 Oliw, E. H., Sprecher, H. and Hamberg, M. (1986) Acta Physiol. Scand. 127, 45–49 35 Certik, M., Balteszova, L. and Sajbidor, J. (1997) Lett. Appl. Microbiol. 25, 101–105 36 Kendrick, A. and Ratledge, C. (1992) Eur. J. Biochem. 209, 667–673 37 Kates, M., Pugh, E. L. and Ferrante, G. (1984) in Membrane Fluidity (Kates, M. and Manson, L. A., eds), pp. 379–395, Plenum 38 Gellerman, J. L. and Schlenk, H. (1979) Biochem. Biophys. Acta 573, 23–30 39 Bajpai, P. K., Bajpai, P. and Ward, O. P. (1992) J. Ind. Microbiol. 9, 11–18 40 Kotula, K. L. and Yi, M. (1994) J. Food Qual. 17, 101–114 41 Chattopadhyay, P., Banerjee, K. S. and Sen, K. (1985) Can. J. Microbiol. 31, 346–351 42 Goodrich-Tanrikulu, M., Stafford, A. E., Lin, J-T., Makapugay, M. I., Fuller, G. and McKeon, T. A. (1994) Microbiology 140, 2683–2690 43 Goodrich-Tanrikulu, M., Lin, J-T., Stafford, A. E., Makapugay, M. I., McKeon, T. A. and Fuller, G. (1995) Microbiology 141, 2307–2314 44 Scott, W. A. (1977) J. Bacteriol. 130, 1144–1148 45 Stutkey, J. E., McDonough, V. M. and Martin, C. E. (1989) J. Biol. Chem. 264, 16537–16544 46 Ykema, A., Verbree, E. C., Verwoert, I. I., van der Linden, K. H., Nijkamp, H. J. J. and Smit, H. (1990) Appl. Microbiol. Biotechnol. 33, 176–182 47 Gyorfy, Z. et al. (1997) Biochem. Biophys. Res. Commun. 237, 362–366 48 Anamnart, S., Tolstorukov, I., Kaneko, Y. and Harashima, S. (1998) J. Ferment. Bioeng. 85, 476–482 49 Covello, P. S. and Reed, D. W. (1996) Plant Physiol. 111, 223–226 50 Kajiwara, S., Shirai, A., Fuji, T., Toguri, T., Nakamura, K. and Ohtaguchi, K. (1996) Appl. Environ. Microbiol. 62, 4309–4313 51 Brown, A. P., Dann, R., Bowra, S. and Hills, M. J. (1998) J. Am. Oil Chem. Soc. 75, 77–82 52 Cahoon, E. B., Mills, L. A. and Shanklin, J. (1996) J. Bacteriol. 178, 936–939 53 Wang, C., Chin, C. K., Ho, C. T., Hwang, C. F., Polaschock, J. J. and Martin, C. E. (1996) J. Agric. Food Chem. 44, 3399–3402
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Desaturase-defective fungal mutants: useful tools for the regulation and overproduction of polyunsaturated fatty acids Milan Certik, Eiji Sakuradani and Sakayu Shimizu Increasing demand for biologically important polyunsaturated fatty acids has led to the search for alternative sources, especially fungi. Although successes in this area have induced interest in developing fungal fermentation processes, manipulation of their lipid composition requires new biotechnological strategies to achieve high yields of the desired polyunsaturated fatty acids. Fungal desaturase mutants with unique enzyme systems are useful not only for the regulation and overproduction of valuable polyunsaturated fatty acids but also because they are excellent models for the elucidation of fungal lipogenesis.
n recent years, considerable attention has been paid to polyunsaturated fatty acids (PUFAs), especially the essential fatty acids (EFAs). EFAs are distinguished by two main functions1,2. The first is their role in membrane structure, where EFAs confer fluidity and flexibility, and modulate the behaviour of certain membranebound proteins. However, more attention has been focused on the second role of EFAs, as precursors of a wide variety of metabolites (such as prostaglandins, leukotrienes and hydroxy-fatty acids) regulating critical biological functions. The roles played by EFAs make it apparent that they are necessary in every organ in the body. Because mammals lack the ability to insert double bonds between the ninth carbon (from the carboxyl end) and the terminal methyl group, they cannot synthesize linoleic (18:2 v-6) and a-linolenic (ALA; 18:3 v-3) acids (see Glossary). These two fatty acids are thus essential for mammals, which rely ultimately on plant, microbial and fish sources for their provision. The main oil sources that are relatively rich in C18 PUFAs are the seeds of some plants and moulds3, which predominantly synthesize g-linolenic acid (18:3 v-6) and ALA. By contrast, PUFAs above C18 cannot be synthesized by higher plants in any significant amounts owing to a lack of the requisite enzymes. On the other hand, fish oil, certain fungi, marine bacteria, algae and mosses may represent suitable potential sources of C20 PUFAs4–8.
I
Strategy for manipulating fungal PUFA formation Increasing demand for biologically active PUFAs has focused commercial attention on PUFA-based nutraceuticals and therapeutics, and on the provision of a suitable biosynthetic framework for their production. As an alternative to their conventional sources, concentration has focused on microorganisms. Oleaginous filamentous fungi, mainly zygomycetes, are thought to be one promising source of these essential compounds9–11, possessing several advantages: M. Certik, E. Sakuradani and S. Shimizu ([email protected]) are at the Division of Applied Life Sciences, Graduate School of Agricultural Sciences, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan.
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• an active PUFA-synthesizing apparatus; • PUFA production can be carried out throughout the year (no seasonal or climatic dependence); • high growth rates on a variety of substrates (including various waste materials); • simple metabolic regulation and control; • suitable model for studying and manipulating PUFA biosynthesis; • supply of more-concentrated PUFAs with controlled quality (pharmaceutical-grade PUFAs); • appropriate vehicles for cloning foreign genes for the production of specific PUFAs; • simultaneous upgrading of both PUFA and other products; • can be employed as macro- and micronutrient sources. Although comparative success in the fungal production of PUFAs (from screening to optimization of fermentation conditions) has led to a flourishing interest in developing fermentation processes and enabled several processes to attain commercial production levels2, two main problems still remain, relating to economic and marketing difficulties. Attempts to reduce the cost of microbial lipids (with emphasis on increasing the product value, using inexpensive substrates, screening for more-efficient strains and reducing the number of processing steps necessary for oil recovery from the cells) are continuing, and the manipulation of microbial PUFA composition is a rapidly growing field of biotechnology. However, the use of fungal lipids is still insufficient to meet industrial demand. Other strategies, therefore, should be combined with classical fermentations (Fig. 1). For example, mutation techniques resulting in the suppression or activation of specific fungal desaturases are beneficial not only for the production of tailor-made PUFAs but can also be used for studying PUFA biosynthesis in fungi12,13. Molecular engineering of parental strains and their mutants is a powerful tool for constructing novel microbial strains synthesizing economically valuable fatty acids14. Control of individual desaturation steps by the addition of desaturase inhibitors or activators to the growth media may also lead to directed PUFA modification15,16. Finally, manipulating PUFA composition by enzymatic treatment of pre-existing PUFA-containing oils is also
0167-7799/98/$ – see front matter © 1998 Elsevier Science. All rights reserved. PII: S0167-7799(98)01244-X
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attracting considerable attention (for example, the use of lipase-catalysed reactions in immobilized systems, twophase systems, membrane bioreactors and in combination with supercritical-carbon-dioxide extraction)17,18. Model for the regulation of PUFA biosynthesis The genus Mortierella is known to be one of the best producers of various types of PUFA10,13. In particular, Mortierella alpina 1S-4 has a unique capacity to synthesize the entire range of fatty acids and has several advantages as a model for these studies: • it is a highly oleaginous strain; • lipogenesis is simply regulated; • it is one of the most well-studied microorganisms producing PUFAs; • the strain is able to incorporate and transform exogenous fatty acids; • various desaturase mutants are available; • it is amenable to molecular-genetic study; • the strain can be used in an industrial scale. The biosynthetic pathway of v-9, v-6 and v-3 PUFAs in M. alpina 1S-4 is shown in Fig. 2. The main product of the strain, arachidonic acid (20:4 v-6; AA), is synthesized via the v-6 route, which involves D12 and D6 desaturases, elongase and D5 desaturase. Depending on the conditions, the total amount of AA varies between 3 and 13 g l21 (30–70% of the fatty acids), with 70–90% of the AA created being bound to triacylglycerols13. The highest yield of AA (13 g l21, corresponding to 220 mg g21 dry mycelia) was achieved in a 10 kl fermenter. The cultivation of the strain under certain conditions also leads to a variety of PUFAs being formed, for example: (1) the addition of D5 desaturase inhibitors to the media causes an increase in dihomo-g-linolenic acid (DGLA; 20:3 v-6) in the fungal oil16; (2) lowering the temperature (to 128C) with the simultaneous addition of ALA to the medium results in the production of eicosapentaenoic acid (EPA; 20:5 v-3)19; (3) the utilization of C15 and C17 n-alkanes by the strain yields PUFAs with odd number of carbons in their chain (total C17 and C19 fatty acids reached over 95% of mycelial fatty acids)20; and (4) when 1-hexadecene or 1-octadecene is the main carbon source, several v-1 PUFAs (20:5 v-1, 20:4 v-1, 16:1 v-1, 18:1 v-1 and 18:4 v-1) are produced21. Fatty-acid-desaturase-defective mutants of M. alpina Although the wild-type M. alpina 1S-4 is able to incorporate exogenous fatty acids and convert them into various PUFAs, the strain has a limited ability to produce new PUFAs or to increase existing PUFA formation. To extend these abilities, several desaturase mutants of M. alpina 1S-4 have been isolated following the treatment of the parental spores with N-methyl-N9nitro-N-nitrosoguanidine22. Other mutants [with defective desaturases (D5, D6, D9, D12 and v3), enhanced desaturase activities (D5, D6) or a combination] have been prepared13 and are worthwhile not only as producers of useful PUFAs (novel or already existing) but also for providing valuable information on PUFA biosynthesis in this fungus. They are also excellent tools for controlling and regulating exogenous-fatty-acid flow to targeted PUFAs13. TIBTECH DECEMBER 1998 (VOL 16)
Glossary v-x PUFA
The first double bond is located at the xth carbon from the methyl end of the PUFA Dm PUFA Non-methylene-interrupted double bond is located at the mth carbon from the carboxyl end of an v-x PUFA vx Desaturase Desaturase introducing double bonds at the xth carbon from the methyl end of the PUFA Dx Desaturase Desaturase introducing double bonds at the xth carbon from the carboxyl end of the PUFA
Fatty-acid profile of the mutant strains The biosynthesis of various types of PUFA by desaturase-defective mutants of M. alpina 1S-4 is presented in Table 1 and outlined on Fig. 2. The main features of these mutants grown on glucose can be summarized in the following manner. (1) The fatty-acid profile of the D5-desaturase-defective mutants is characterized by a high DGLA level and a reduced concentration of AA23. Production of DGLA by these mutants is advantageous because it does not require inhibitors and the yield is relatively high. (2) Attributes of D12-desaturase-defective mutants include the absence of v-6 and v-3 PUFAs and high levels of v-9 PUFAs, such as oleic (18:1 v-9), octadecadienoic (18:2 v-9), eicosadienoic (20:2 v-9) and mead (20:3 v-9) acids in their mycelia24. Of these, mead acid is known to be a precursor of LT3 leukotrienes and anti-inflammatory compounds25. (3) When both D5 and D12 desaturases are blocked, 20:2 v-9 is accumulated in large quantities26. (4) Mutants synthesizing a high level of linoleic acid and low concentrations of g-linolenic acid, DGLA and AA are considered to be defective in D6 desaturase27. Moreover, the accumulation of 20:2 v-6 and two nonmethylene-interrupted PUFAs [v-6 eicosatrienoic (20:3 D5) and v-3 eicosatetraenoic (20:4 D5)] acids Screening of microorganisms
Optimization of fermentation process (conditions, techniques, scale-up) Desaturase-defective mutants
Molecular-genetic approach Desired PUFAs
Desaturase inhibitors or activators
Enzymatic biotransformation
Figure 1 Strategy for the modification of microbial polyunsaturated fatty acids. Screening of potential microorganisms is an essential step and results in a limited number of strains for practical use. These strains can be used directly in process optimization or further modified by classical mutation techniques or molecular-engineering methods to produce the desired fatty acids. The fatty-acid profile may also be controlled by desaturase inhibitors or activators added to the medium and finally improved by enzymatic treatment of pre-existing oils.
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a
COOH 18:0
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Figure 2 Biosynthetic pathways of polyunsaturated fatty acids (PUFAs) in Mortierella alpina 1S-4 and its desaturase-defective mutants. Defective desaturases and characteristic fatty acids of the individual mutants are highlighted. Abbreviations: EL, elongation; D5, D6, D9, D12, v3, various fatty-acid desaturases. (a) The wild-type strain 1S-4 synthesizes arachidonic acid (AA) as the main fatty acid; lowering the temperature results in production of v-3 PUFAs, mainly eicosapentaenoic acid. The amount of v-9 PUFAs produced is almost negligible. (b) v3-Defective mutants do not form v-3 PUFAs. (c) D5-Desaturase-defective mutants cannot convert dihomo-g-linolenic acid (DGLA) to AA and DGLA is accumulated. (d) Mutants defective in D12 desaturase are unable to synthesize v-6 and v-3 PUFAs. Because the other desaturases (D6 and D5) and elongase are still active, v-9 PUFAs are accumulated where mead acid is the end product. (e) In mutants defective in both D5 and D12 desaturases, 20:2 v-9 is the final fatty acid in the PUFA-biosynthetic cascade. (f) Owing to a lack of D6 desaturase, linoleic acid is the main fatty acid in these mutants. However, linoleic acid is to some extent further metabolized to two non-methylene-interrupted fatty acids, 20:3 D5 v-6 and 20:4 D5 v-3. (g) D9-Desaturasedefective mutants synthesize high levels of stearic acid. Because this desaturase is not completely blocked, a small amount of v-6 PUFAs is also formed.
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Table 1. Production of some PUFAs by Mortierella alpina 1S-4 and its mutants PUFA series
v-3
PUFA name
20:3 20:4 (ETA) 20:4D5a 20:5 (EPA)
v-6
v-9
20:2 20:3 (DGLA) 20:3D5a 20:4 (AA) 18:2 20:2 20:3 (MA)
Strain
D6 mut D5 mut D1215 mut D6 mut 1S-4 D12 mut D6 mut 1S-4 D5 mut D6 mut 1S-4 D12 mut D1215 mut D1215 mut D12 mut
PUFA formation g21]b
[% in oil]
[mg
2.5 26 37 5 12 20 9 27 42 15 30–70 14 16 25 33
6.5 77 97 12 67 64 22 107 205 27 100–300 60 63 110 141
Remark [g
l21]
0.07 1.6 2.3 0.12 1.9 1.0 0.23 2.6 4.1 0.25 3–13 1.0 1.1 1.7 1.9
148C Linseed-oil addition Linseed-oil addition 148C 1128C, linseed-oil addition Linseed-oil addition 148C Inhibition of D5 by sesamin 1288C
aNon-methylene-interrupted
fatty acid. is, (mg fatty acid) (g dry biomass)21. Abbreviations: AA, arachidonic acid; DGLA, dihomo-g-linolenic acid; EPA, eicosapentaenoic acid; ETA, eicosatetraenoic acid; MA, mead acid; PUFA, polyunsaturated fatty acid. bThat
were found27. 20:3 D5 is thought to be synthesized by the elongation of linoleic acid and then D5 desaturation. The formation of 20:4 D5 might be initiated by conversion of linoleic acid to ALA (v-3 desaturation) followed by elongation and D5 desaturation. Although the biological activities of non-methylene-interrupted fatty acids are not fully understood, they are probably formed in response to a deficiency of v-3 and v-6 PUFAs. 20:3 D5 can be converted via cyclooxygenase to hydroxyfatty acids28 and may alter eicosanoid signalling29. (5) Mutants defective in v3 desaturation are unable to synthesize v-3 PUFAs when cultivated at low temperature30. (6) Finally, stearic acid (18:0) is the main fatty acid in the mycelial oil (up to 40%) of the D9-desaturasedefective mutants31. However, D9 desaturase is not completely blocked, because low activity of the enzyme is important for the introduction of the first double bond at the ninth carbon (from the carboxyl end) of the fatty-acid chain to maintain cell viability.
ALA. Even when grown at low temperature (which stimulates v-3-desaturase activity), ETA production is still low. On the other hand, the addition of linseed oil to the medium results in accumulation of ETA33. Nevertheless, DGLA and ETA formation is competitive and their ratio varies with the ratio of glucose and ALA added to the medium. Because an increase in glucose results in a greater DGLA concentration in the mycelial oil33, the conversion of LA (endogenously formed from glucose) to DGLA is much faster than the transformation of exogenous ALA to ETA. This suggests that D6 desaturase is more specific for LA than for ALA.
Biotransformation of exogenous fatty acids by the mutants The ability of all these mutants to utilize exogenous fatty acids may allow the preparation of various PUFAs in relatively large quantities. This could be very important, because there are various sources of easily available natural oils containing individual fatty-acid precursors. How the desaturase-defective mutants could be used to perform the transformation of exogenously added oils to a desired fatty acid is shown in the following examples (see also Table 1).
D12-Desaturase-defective mutants From a nutritional standpoint, an EPA-rich oil with a low AA level is preferred, because of the diverse biological activities of AA1. However, the AA concentration in the mycelial oil of M. alpina is usually higher than that of EPA, even when the parental strain is cultivated at low temperatures and in the medium supplemented with ALA. Therefore, as D12-desaturase-defective mutants cannot synthesize any v-3 or v-6 PUFAs when grown on glucose, these mutants could be used to solve this problem. Because the other desaturases and elongases are still active, ALA added exogenously (as linseed oil) to the medium is efficiently converted to EPA to give the mycelial EPA/AA ratio of 2.532. However, the addition of either v-3 or v-6 fatty acids causes a rapid decrease in v-9 fatty-acid formation by these mutants, because of the substrate specificity of D6 desaturase, which prefers LA and ALA over oleic acid32.
D5-Desaturase-defective mutants When these mutants are grown on glucose, DGLA is normally formed. However, eicosatetraenoic acid (ETA; 20:4 v-3) is the other potential end product of these mutants and can be synthesized either by v-3 desaturation of DGLA or through the v-3 route from
D5-D12-Desaturase-defective mutants Although growing D5-desaturase-defective mutants on linseed oil improves ETA production, the amount of DGLA in the mycelial oil is still high. Because ETA has a potential use as a precursor of prostaglandins34 and the separation of these two fatty acids is difficult, the
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formation of DGLA from glucose should be limited in order to increase the accumulation and improve the purification of ETA. Therefore, D5-D12desaturasedefective mutants can be cultivated with exogenously added ALA to give increased production of ETA.
D6-Desaturase-defective mutants The production of non-methylene-interrupted fatty acids by D6-desaturase-defective mutants depends on the cultivation temperature. An increase in the temperature (from 48C to 288C) is accompanied by a fall in 20:4 D5 (v-3 series), but a rise in non-methyleneinterrupted 20:3 D5 (v-6 series) levels. However, 20:4 D5 is effectively synthesized even at laboratory temperature when the mutants utilize exogenous ALA. The fatty-acid-biosynthetic apparatus of the mutants The prospects for efficient PUFA production depend on a knowledge of the mechanism by which PUFA biosynthesis is accomplished. This is, however, still poorly understood, although the main aspects of PUFA accumulation are known. There are several distinct pathways that could be involved in this process35: (1) de novo synthesis of fatty acids from glucose; (2) the incorporation of exogenous fatty acids directly into lipid structures; and (3) following desaturation and elongation of lipid sources. In addition, fatty-acid biohydrogenation and partial or total degradation (b oxidation) also contribute to this process35. It is well known that PUFA formation in fungi is catalysed by a multienzyme complex composed of three proteins: NADH–cytochrome-b5 reductase, cytochrome b5 and the terminal cyanide-sensitive desaturase. Simultaneously, enzyme system(s) providing the NADPH for desaturation and elongation reactions must be involved36. Membrane constituents such as phospholipids are accepted as the most likely form for fatty acids to be available for desaturation in fungi37. Once desaturation has occurred, acyltransferase reactions then facilitate the distribution of newly synthesized PUFAs to other cellular lipids, mainly triacylglycerols. Because these acylation processes are catalysed by separate acyltransferases, the acyltransferases and desaturases may be tightly bound and their cooperation could be responsible for the distribution of PUFA among individual lipid structures. The mutants of M. alpina 1S-4 are not only valuable as producers of some useful PUFAs; they also provide information on PUFA biosynthesis and regulation in this fungus. The PUFA-biosynthetic pathways in M. alpina 1S-4 and its desaturase-defective mutants are illustrated in Fig. 2. From observation of the D9-desaturase-defective mutant, it is evident that the first double bond is invariably inserted at the ninth carbon from the carboxyl end31. All three metabolic routes (v-3, v-6 and v-9) that are engaged in PUFA formation in this fungus share the same enzymes: D6 desaturase, elongase and D5 desaturase. However, the desaturases have a wide substrate specificity. For example, D5 desaturase can act on odd-numbered fatty acids, PUFAs with v-1 terminal double bonds or v-3, v-6 and v-9 fatty acids. On the other hand, varying specificities of individual desaturases (according to carbon number and location of double bonds in the fattyacid chain) modify the flux of C18 and C20 fatty acids to the end products. For instance, D6 desaturase, the rate-limiting step in PUFA formation, exhibits different
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specificities for oleic, linoleic and a-linolenic acids. Depending on the conditions, it may finally lead to varying amounts of v-3, v-6 and v-9 PUFAs being formed. There are two possible enzymes catalysing the conversion of C18 and C20 v-6 fatty acids to v-3 fatty acids: D15 and D17 desaturase, respectively. Nevertheless, the D15 and D17 desaturation in this fungus may be catalysed by one enzyme only, which recognizes the methyl end of both C18 and C20 v-6 fatty acids30. Thus, the enzyme is different from that found, for example, in Saprolegnia parasitica38, which is specific only for C20 fatty acids. This enzyme in M. alpina was thus described as an v3 desaturase (rather than a D desaturase) because it is specific for the methyl (not the carboxyl) terminus. Two routes for v-3 PUFA production operate in M. alpina. The first involves the conversion of an exogenous v-6 fatty acid into the corresponding v-3 fatty acid, and the second, the v-3-fatty-acid-biosynthetic pathway. The former route is activated at low temperature and has also been reported in other Mortierella species39,40; the latter route is independent of temperature and operates simultaneously with the former route at low temperatures. To summarize, the unique biotransforming enzyme systems of M. alpina 1S-4 and its desaturase-defective mutants can be used to construct useful v-3, v-6 or v-9 PUFAs from their precursors. They also indicate how it is possible to control the fatty-acid profile of fungal mutants and to regulate the flow of glucose or exogenous fatty acids to obtain a desired PUFA. Because of the simplicity of their metabolic system, these mutants are potentially ideal models for the elucidation of fungal lipogenesis. Presently, the studies performed with M. alpina and its mutants are focused on the molecular engineering of these desaturases and on the genes involved in the microsomal electron-transport system. Thus, these mutants may be used as vehicles for the cloning of desaturase genes, which could be introduced into either plants or animals to produce transgenic organisms with desired fatty-acid composition. Fungal desaturase-defective mutants other than Mortierella spp. Although the mutation strategy offers great advantages for manipulating fungal oils, most of the mutants (other than Mortierella spp.) that have been described have dominant defective D9-desaturase genes. The mutation of D9-desaturase genes led to a requirement for unsaturated fatty acids for fungal growth and to defective development and reduced respiration rates in some strains of Aspergillus niger and Neurospora crassa41,42. Moreover, some mutants, such as N. crassa42–44, Saccharomyces cerevisiae45 and Cryptococcus curvatus46, were unstable and quickly reverted to the wild-type growth habit. These mutants are used for studying membrane structures and functions47, as well as for elucidating the reaction mechanisms involved in fatty-acid biosynthesis48. Other approaches with D9-desaturase-defective mutants are focused on the development of strains producing highly saturated oils as a substitute for cocoa butter. This has resulted in the isolation of a strain of C. curvatus (which used to be called Apiotrichum curvatum) with an inactive D9 desaturase46. Although this mutant synthesizes oil with saturated fatty acids, it requires the addition of oleic acid to the medium, as this is an essential TIBTECH DECEMBER 1998 (VOL 16)
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component of the membrane system. However, a hybrid strain of C. curvatus with a partially active D9 desaturase is able to grow without oleic acid, and its fatty-acid composition is comparable to that of cocoa butter12. With the exception of D9 desaturase, the availability of desaturase-defective fungal mutants is rather limited. A recent analysis of five N. crassa mutants with altered PUFA synthesis indicated that multiple genes influence the PUFA composition in their membranes and storage lipids43. Also, a few new studies of the expression of plant D12 desaturase or animal D6 desaturase in S. cerevisiae reported the accumulation of linoleic and hexadecadienoic (16:2) acids49–51 or g-linolenic acid14, respectively. Future prospects Biotechnological strategies for the modification of microbial PUFAs represent a powerful tool not only for the study and regulation of complex metabolic pathways but also for the creation of novel microbial strains synthesizing economically valuable lipid metabolites. Comparative success in this area using mutation techniques and genetic engineering has resulted in the heterologous expression of desaturase genes from plants51,52 and animals14 in fungi and has allowed structure–function studies and the production of special fatty acids. Also, the introduction of microbial lipid-biosynthetic genes into specific subcellular locations of transgenic plants provides a useful means for performing unusual reactions and products in plants53. However, for both economic and biochemical reasons, the modification of fatty acids as a result of any form of manipulation must be correctly targeted into triacylglycerols and excluded from phospholipids. Therefore, further progress will depend on: (1) investigation of their regulatory mechanisms; (2) identification of the key steps limiting triacylglycerol biosynthesis; (3) understanding the mechanisms of enzyme action in PUFA biosynthesis; and (4) elucidation of the signalling systems of the lipidbiosynthetic machinery inside the cell. Currently, no aspect of these regulatory mechanisms is understood in any organism and thus this represents a challenging and potentially rewarding subject for the future research. References 1 Horrobin, D. F. (1995) Inform 6, 428–435 2 Gill, I. and Valivety, R. (1997) Trends Biotechnol. 15, 401–409 3 Ratledge, C. (1989) in Microbial Lipids (Vol. 2) (Ratledge, C. and Wilkinson, S. G., eds), pp. 567–668, Academic Press 4 Radwan, S. S. (1991) Appl. Microbiol. Biotechnol. 35, 421–430 5 Yamada, H., Shimizu, S., Shinmen, Y., Akimoto, K., Kawashima, H. and Jareonkitmongkol, S. (1992) in Industrial Applications of Single Cell Oils (Kyle, D. J. and Ratledge, C., eds), pp. 118–138, AOCS Press, Champaign, USA 6 Yazawa, K., Watanabe, K., Ishikawa, C., Kondo, K. and Kimura, S. (1992) in Industrial Applications of Single Cell Oils (Kyle, D. J. and Ratledge, C., eds), pp. 29–51, AOCS Press, Champaign, USA 7 Nakahara, T., Yokochi, T., Higashihara, T., Tanaka, S., Yaguchi, T. and Honda, D. (1996) J. Am. Oil Chem. Soc. 73, 11 8 Singh, A. and Ward, O. P. (1997) Adv. Appl. Microbiol. 45, 271–312 9 Certik, M., Sereke Berhan, S. and Sajbidor, J. (1993) Acta Biotechnol. 13, 193–196 10 Leman, J. (1997) Adv. Appl. Microbiol. 43, 195–243 11 Certik, M. and Shimizu, S. (1998) in Recent Research Developments in Oil Chemistry (Vol. 2) (Pandalai, S. G., ed.), pp. 89–103, Transworld Research Network, Trivandrum, India 12 Verwoert, I. I., Yjema, A., Valkenburg, J. A. C., Verbree, E. C., Nijkamp, H. J. J. and Smit, H. (1989) Appl. Microbiol. Biotechnol. 32, 327–333
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