EP1053344A1

EP1053344A1 - Mortierella

Info

Publication number: EP1053344A1
Application number: EP99901772A
Authority: EP
Inventors: Matthew Guy Duchars
Original assignee: Avecia Ltd
Current assignee: Avecia Ltd
Priority date: 1998-02-07
Filing date: 1999-01-21
Publication date: 2000-11-22
Also published as: JP2002502612A; BR9907701A; CA2320407A1; NO20003981D0; ID26203A; CN1296527A; KR20010040732A; AU2177299A; ZA99738B; GB9802561D0; NO20003981L; WO1999040215A1

Abstract

Adhesion of Mortierella to surfaces during culture is reduced by the use of morphologically compact variants.

Description

MORT1ERELLA THIS INVENTION relates to Mortierella.

Mortierella, a genus of filamentous fungus, is of industrial interest because at least some members produce lipids which contain residues of poly-unsaturated fatty acids, especially arachidonic acid and of other poly-unsaturated fatty acids metabolisable by human beings thereto, for example gamma linolenic acid. Such lipids and/or such acids derived therefrom are considered to be beneficial to human health when used as nutritional supplements. Addition of such lipids and/or acids to infant formula (artificial milk for human babies) has been proposed in order to simulate human mothers' milk more closely. The acids, lipids or the organisms themselves may be eaten, their poly- unsaturated fatty acids then becoming available by normal processes of digestion.

We have found that during fermentation Mortierella tends to adhere to plant surfaces. Thus, stirrers may become so encased with the fungal biomass as to become relatively ineffective and conduits may become blocked.

We have now discovered a form of Mortierella ("the desired organisms") substantially less subject to this problem.

Microscopic examination indicates that Mortierella filaments generally tend to be highly tangled, such that it is in general not possible to distinguish one individual from another in order to quantify morphological characteristics. However, non-tangled ends protrude from tangled masses, and examination of these indicates that the desired organisms are more highly branched than corresponding known organisms.

It can be shown by the following procedure that the desired organisms have a more compact morphology, corresponding to the greater extent of branching, than known varieties. It is not known why or how the desired organisms adhere less readily to solid surfaces. In order to identify desirable strains of Mortierella a number of strains may be cultured and under comparable conditions in liquid culture and plate culture. The nutrient medium for the plates may differ from that used in liquid culture. The area covered after a period of such plate culture is substantially less in the case of the desired organisms than in the case of other organisms of similar liquid culture growth rate. Typically the areas covered by a desired organism is less than half, and preferably less than one quarter of that covered by a known strain of Mortierella of similar growth rate in liquid culture. The morphology may be quantified more generally as a morphology index in which M = Kr μmax in which Kr is the average radial expansion in μm per hour on a plate and μmax is log_e 2 divided by the doubling times in hours (i.e. the time taken for the dry cell weight to double under unconstrained growth conditions i.e. its maximum growth rate) in liquid culture and at the same temperature using a medium for the liquid culture of composition

Concentration (gl^"1)

(NH₄)₂ SO₄ 3.0 MgSO₄.7H₂O 1.0

K₂SO₄ 1.3

H₃PO₄ 0.75 ml

Yeast Autolysate (BioSpringer) 3.0

Glucose 45.0 Trace Elements Solution 1.0

(Composition

MnSO₄.4H₂O 40 gl^"1

ZnSO₄.7H₂O 50 gl^"1

CuSO₄.5H₂O 5 gl^"1 Biotin 50 mg l^"1

H₂SO₄ 5 ml I^"1) at an initial pH of 6.8 which is prevented from falling by the addition of HaOH as necessary and the average value of Kr is based on measurements over a period of four days taken at 24 hr intervals on equal numbers of plates of the following compositions: (a) 1.9 (g/l) K₂HPO₄

1.6 (g/l) NaH₂PO₄

1.8 (g/l) (NH₄)₂SO₄

0.2 (g/l) MgSO₄.7H₂O (added filter-sterilised after autoclaving)

0.9 (mg/l) FeCI₃ 1.0 (ml/l) Trace Elements containing 720mg/l Ca, 5mg/l Cu, 23mg/l Zn to pH 7.2 with 2M HCI with glucose added to a final concentration of 45 g/l, yeast extract added to a final concentration of 5 g/l and Agar added to a final concentration of 15 g/l. (b) Potato extraci t 4.0 g/litre

Glucose 20.0 g/litre

Agar 15.0 g/litre pH 5.6 ± 0.2

(c) Malt extract 30.0 g/litre

Mycological peptone 5.0 g/litre

Agar 15.0 g/litre pH 5.4 ± 0.2

(d) 1.9 g/l K₂HPO₄

1.6 g/l NaH₂PO₄

0.1 g/l (NH₄)₂SO₄

0.2 g/l MgSO₄7H₂O (added filter sterilised 0.9 mg/l FeCI₃

1.0 ml/I Trace elements containing 720 mg/l Ca, 5 mg/l Cu, 23 mg/l Zn to pH 7.2 with 2M HCI

0.1 g/l yeast extract

45 g/l glucose

15 g/l agar

Values of M < 4 x 10³ μm and preferably < 3.5 x 10³ μm are preferred. If the colony on the plate is nearly circular, two perpendicular measures of radial expansion may suffice but more measures may be needed if it departs from circularity.

The invention comprises novel strains of Mortierella having a morphology index M as above defined of at most 4 x 10³ μm and preferably at most 3.5 x 10³ μm.

The suitable strains of Mortierella may be for example: M.alpina, M.bainieri, M.elongata, M.exigua, M.hygrophila, M.globalpina, M.minutissima, M.verticillata,

M.polycepuala and M.zyzchae. These may produce eicosapentaenoic acid, gamma linolenic acid (GLA) or preferably arachidonic acid and/or their derivatives, especially lipids containing their residues, depending on process conditions.

The invention further provides methods of selecting the novel Mortierella strains. One such method comprises culturing strains on agar plates under similar conditions and selecting those which have a small colony area. Strains which show little growth observed as a low density in the colony area should be disregarded. Strains so selected are the most likely to have a desirable morphology index. Suitable strains may be produced by prolonged culturing of Mortierella strains under conditions of nutrient limitation (especially nitrogen limitation), followed by selection of individuals of appropriate value of colony area and/or morphology. Such individuals may then be cultured suitably under conditions of nutrient limitation, and selection may be repeated if required.

The invention comprises a process of culturing Mortierella in a fermenter which comprises selecting an organism of compact morphology from the culture preferably after continuing culture until substantial adherence of Mortierella to surfaces of the fermenter has occurred and carrying out a subsequent fermentation of the said more compact organism. The procedure may be repeated as often as desired until an organism which has low adhesion to surfaces is obtained.

By "low adhesion to surfaces" is meant that the rate of removal of the organisms from surfaces under normal conditions of turbulence encountered in fermentation is sufficient to prevent a build-up of deposits on such surfaces. The invention also provides a process for producing a lipid which comprises a step of culturing Mortierella as above described.

In batch culture a medium is initially inoculated with organisms which are grown to a desired content of the organisms and then preferably further cultivated after exhaustion of one or more nutrients, and the biomass then harvested. The supply of one or more nutrients may be replenished during culture ("fed batch"). In continuous culture fresh nutrient(s) are added and product removed without discontinuing culture. Such addition and removal may be continuous or intermittent. The new variety of Mortierella is advantageous for batch or fed batch operation, but is particularly suitable for continuous culture. In each mode the new Mortierella forms a more homogeneous mixture and thus affords better mixing, and thus in general faster and/or greater biomass formation.

Continuous culture may be carried out in known manner, for example in an air lift fermenter (which has no moving parts but in which conduits may become clogged with known varieties) or in stirred vessels.

Organisms according to the invention may typically be grown at a pH of 4 to 10 and preferably 6 to 8. For lipid production the pH is preferably controlled to rise from an initial value of 5 to 6 to a final value of 6.5 to 7.5. The temperature may be 5-40 and preferably 20-30,°C. The organisms may be cultured on a solid medium but preferably a liquid medium is used. The carbon source in the liquid may comprise solubles, for example glucose, fructose, sucrose, maltose or soluble starch, or low-solubility carbohydrates, fatty acids or lipids and/or (in the case of organisms which metabolise hydrocarbons for growth) hydrocarbons. The nitrogen source may be complex, for example proteins, peptides and/or amino acids, for example yeast extract, meat extract, corn steep liquor; or simple for example nitrate and/or ammonium ions, ammonium ions being supplied for example as sulphate or gaseous ammonia, or it may be a mixture of complex and simple sources. The source of phosphate is suitably inorganic. Trace elements and vitamins may be included. The concentration (w/w on total medium) of carbon source is preferably at least 0.1%, possibly 30% or more, but preferably in the range 1-15%, for example 2-10%. The concentration (w/w on total medium) of nitrogen source is for example 0.01 to 1 , especially 0.1 to 0.4%, calculated as equivalent N. As nutrients are depleted additional nutrients may be added. A plurality of carbon sources may be employed. To produce enhanced yields of lipid, a phase of cultivation after exhaustion of a nutrient other than the carbon materials, especially nitrogen or phosphate, is preferably operated.

Culturing is aerobic, suitably in the presence of dissolved oxygen supplied as air, possibly enriched. The culture is suitably agitated for example by stirring or by flow of gas. Suitably an airlift fermenter (in which circulation of liquid is maintained by gas, for example oxygen or air, injection) is employed. Cultured cells are suitably subjected to extraction for example with an organic solvent, for example, hexane, an ester or an alcohol possibly in combination or succession. If desired, extraction may take place with a halogenated hydrocarbon, for example chloroform, but it is preferred to use hydrocarbons or alcohols or esters as extractants in order to avoid environmental problems. EXAMPLES

Mortierella Alpina ATCC 32222 was grown in continuous culture at pH 6.8, 26°C under aerobic conditions where the dissolved oxygen tension (DOT) was maintained at a level above 20% of air saturation. The culture was grown on SD4 medium, (see table below) with excess glucose added as primary carbon source such that the glucose was maintained at a standing concentration of 5 to 20 g/l in the culture throughout the fermentation and ammonium sulphate was the limiting substrate. The medium was added at a rate equivalent to a dilution rate of 0.05 h^"1. Each day samples of culture were removed from the fermenter, grown on S2GYE agar plates (see Appendix 2) and the resulting colonies carefully examined for colonial variants.

After 620 hours from inoculation of the continuous culture fermenter, colonial variants were observed. It was noted that these colonial variant colonies covered smaller areas than the original parent organism. Moreover, the colonial variants were more highly branched.

On continued cultivation for a further 165 hours, it was noted that colonial variant strains now constituted 88% of the population present. A pure culture of a new colonial variant was grown on S2GYE plates at 28°C.

After 7 days growth a spore suspension was prepared by aseptically transferring spores on the hyphae into sterile saline solution. A 10μl drop of the solution of spores was then transferred to the centre of agar plates containing the desired medium.

The plates were then incubated at 28°C and the rate of colony expansion measured each day, by measuring the diameter of the colony on each plate at 2 perpendicular positions. This was carried out on 4 different types of medium

(A) = S2GYE Agar

(B) = Potato Dextrose Agar

(C) = Malt Extract (D) = S2 Low N

(see Appendix 2). The same process was repeated with the original strain.

The radial growth rate was calculated each day and an average rate determined over the growth period (see Appendix 3). From the data it can be seen that the radial expansion rate of the new strain is significantly lower than that of the original strain. The new organism was cultivated in liquid medium (see appendix 4) at 26°C under aerobic conditions. A batch fermentation was used, with a starting pH of 6.8, in which the culture pH was prevented from falling by the addition of sterile 2 molar NaOH. No control was exerted to prevent a rise in pH. The maximum growth rate of the organism was determined by measurement of the CO₂ evolution rate (CER). Similarly the original organism was cultivated under the same conditions and its maximum growth rate determined by the same means. The results demonstrated that the growth rates were similar (0.06 h^"1 for original strain, 0.07 h^"1 for new strain) and that the new strain was less adhesive. Samples of culture were analysed after 6 days growth. The oil content of the biomass was determined by solvent extraction using 2:1 v/v mixture of dichloromethane and methanol and the amount of arachidonic acid contained within the extracted oil was determined by forming the methyl ester and analysis by gas liquid chromatography. The results are shown in the table below. TABLE

Oil Content (% w/w) Ara Content (% w/w)

Original Strain 32.5 31.8

New Strain 34.5 21.9

Using the above data it is possible to calculate a value for the morphology index (M) for each strain.

Original Strain M = K = 336 = 5.6 x 10 μm μmax 0.06

New Strain M = Kr = 153 = 2.2 x 10³ μm μmax 0.07

APPENDIX I SD4 Medium

Component Concentration (gl^"1)

(NH₄)₂ SO₄ 3.2

MgSO₄.7H₂O 0.5

K₂SO₄ 1.0

H₃PO₄ 1.0

Yeast Autolysate (BioSpringer) 0.5

Trace Elements Solution 1.0^*

*Trace Element Solution

Component Concentration (gl^"1)

MnSO₄.4H₂O 40

ZnSO₄.7H₂O 50

CuSO₄.5H₂O 5

Biotin 50 mg

H₂SO₄ 5 ml

APPENDIX 2 Seed 2 recipe

Seed 2 Medium

1.9 (g/l) K₂HPO₄ 1.6 (g/l) NaH₂PO₄

1.8 (g/l) (NH₄)₂SO₄

0.2 (g/l) MgSO .7H₂O (added filter-sterilised after autociaving)

0.9 (mg/l) FeCI₃

1.0 (ml/l) Fisons Trace Elements containing 720mg/l Ca, 5mg/l Cu, 23mg/l Zn to pH 7.2 with 2M HCI

Medium (A)

S2GYE was seed 2 medium with glucose added to a final concentration of 45 g/l. Yeast extract (Oxoid L21) added to a final concentration of 5 g/l and Agar (Oxoid

Bacteriological L11) added to a final concentration of 15 g/l. Medium (B)

Potato Dextrose Agar was obtained from Oxoid (CM 139). It is made up as follows:

Formula gm/litre

Potato extract 4.0

Glucose 20.0 Agar 15.0 pH 5.6 ± 0.2

Medium (C)

Malt extract Agar was obtained from Oxoid (CM59). It is made up as follows:

Formula gm/litre Malt extract 30.0

Mycological peptone 5.0

Agar 15.0 pH 5.4 ± 0.2

Medium D S2Low N was seed 2 with the level of ammonium sulphate changed to 0.1 gl^"1. Yeast extract (Oxoid L21) added to a final concentration of 0.1 g/l and glucose added to a final concentration of 45 g/l and again to a final concentration of 15 g/l. 10

APPENDIX 3

Comparison of radial colony expansion rate (Kr) for new strain and original strain of Mortierella on various media A-D.

Media Strain Time (h) Average

24 48 72 96

Medium A Original 500 335 270 235 335

(S2GYE) New 200 130 105 95 133

Medium B Original 390 345 265 350 337

(Potato Dextrose) New 275 175 140 130 180

Medium C Original 305 175 195 215 222

(Malt Extract) New 220 130 110 95 139

Medium D Original 700 450 315 340 451

(S2 Low N) New 250 155 125 110 160

The new strain was depos _ .i_t.e..d__ o - .n_ .8-111¹ January 1998 under deposit number IMI 378077 with Commonwealth Agricultural Bureau, International Mycological Institute, Ferry Lane, Kew, Surrey TW9 3AF, United Kingdom, (now of UK Centre (Egham), Bakeham Lane, Egham, Surrey, TW20 9TY)

11

APPENDIX 4

Concentration (gl^"1) (NH₄)₂ SO₄ 3.0 MgSO₄.7H₂O 1.0 K₂SO₄ 1.3 H₃PO₄ 0.75 ml

Yeast Autolysate (BioSpringer) 3.0 Glucose 45.0

Trace Elements Solution 1.0 (Composition MnSO₄.4H₂O 40 gl^"1 ZnSO₄.7H₂O 50 gl^"1 CuSO₄.5H₂O 5gl^"1 Biotin 50mgl r¹ H₂SO₄ 5mir ¹)

12

CABI BlOSCIENCE

A Division of

CAB INTERNAΠONAL

UK Centre (Egham), Bakeham Lane, Egham, Surrey, TWfaygrjpr Telephone: ++44 (0)1784470111 Fax: ++44 (0)178447WW

Dear Madam

NOTIFICATION OF ACCEPTANCE OF A DEPOSIT FOR THE PURPOSES OF PATENT PROCEDURE

Microorganism: Mortierella alpina IMI CC Number: 378077

Strain number: Strain 26C

The above designated microorganism, received on 12 January 1998 was accepted for deposit for patent purposes on 12 January 1998 in accordance with the terms and conditions set out in the Application form signed by you on 8 January 1998, a copy of which should have been retained by you.

Yours

DXrectOT

International Mycological

\_: c-so ces Collection DATΞ t- - Vf

GRRC PF3 ISSUE 3 I JAN 93 I Page 1 of 1

CABI BifttCTEKCETHI^rar j-

International !H \lWOT>rt g)Bg C--l Control (IIBC), Entomology (IIE), Parasitology (HP) and The International Mycological Institute (IMI) CABI BlOSCIE CE

A Dwiswn of CAB INTERNATIONAL

UK Centre (Egham), Bakeham Lane, Egham, Surrey, TW209TY Telephone: ++44 (0)1784470111 Fax: ++44 (0)1784470909

Dear Madam

VIABILITY STATEMENT FOR THE PURPOSE OF PATENT PROCEDURE

Microorganism: Mortierella alpina IMI CC Number: 378077

Strain number: 26C

The viability of the microorganism identified above was tested on 27 January 1998. On that date the said microorganism was viable.

Conditions under which the viability test has been performed (completed only if the test was negative).

Statement issued in accordance with microorganism deposits for the purposes of patent procedures under the Budapest Treaty, IMI Genetic Resource Reference Collection being an International Depositary authority recognized under the Treaty.

Yours faithfully /

T. ' " ^ I " . Director π

GRRC PF5 I ISSUE 3 1 JAN 93 Page 1 of 1

CABf BlIAU-ΛLt \nuψιm\i ' ' — =

International tf__8g_WSP8^__WCg<§ιc l Control (IIBC), Entomology (HE¹. Parasitology ιHP> and The International Mycological Institute (IMIi

Claims

14

1 A process of culturing Mortierella in a fermenter which comprises selecting an organism of compact morphology from the culture preferably after continuing culture until substantial adherence of Mortierella to surfaces of the fermenter has occurred and carrying out a subsequent fermentation of the said more compact organism.

2 A process as claimed in Claim 1 which comprises repeating the procedure until an organism which has a low adhesion to surfaces is obtained.

3 A variety of Mortierella characterised by a morphology index (M) of at most 4 x 10^"3 μm. 4 A method of selecting a variety of Mortierella having a low adhesion to solid surfaces which comprises growing a number of strains of Mortierella in both liquid and plate culture and selecting those which cover low areas of plate compared with strains of Mortierella of similar growth rate in liquid culture. 5 A process of producing a variety of Mortierella which comprises culturing a strain of Mortierella in a liquid medium under conditions of nutrient limitation, culturing individuals obtained thereby by plate culture, selecting at least one organism which has a small colony area relative to other plate cultures carried out under comparable conditions re-culturing that organism, and optionally repeating the process until an organism of low adherence to surfaces is obtained. 6 A process of producing lipids or foodstuffs comprising lipids which comprises a step of liquid culture of Mortierella in which the Mortierella is as claimed in Claim 3, is selected in accordance with Claim 4 or is produced in accordance with Claim 5. 7 A process as claimed in Claim 1 , 2 or 6 in which the culture of Mortierella is continuous. 8 A process as claimed in Claim 1 , 2, 5 or 6 or Mortierella as claimed in Claim 3, selected in accordance with Claim 4 or produced in accordance with Claim 5 in which the Mortierella is Mortierella Alpina.

9 A process as claimed in Claim 1 , 2, 6, 7 or 8 in which the Mortierella is cultured at a pH of 6 to 8 and at a temperature of 5 to 40°C in a liquid medium which comprises a soluble carbohydrate in the presence of dissolved oxygen. 15

10 A process in which lipids are recovered from Mortierella as claimed in Claim 3, selected in accordance with Claim 5 or produced in a process as claimed in any one of Claims 1 , 2, 6, 7, 8 or 9 by solvent extraction, preferably with an organic solvent.

11 Mortierella possessing the properties of the strain deposited with the International Mycological Institute as deposit IMI 378077 and variants, mutants and derivatives thereof.