Produced Thereof

Technical Field

The application relates to microbial genetic breeding technology and biological fermentation technology, particularly, a variant or mutant strain of microalgae for producing DHA (Docosahexaenoic acid).

Background

DHA (Docosahexaenoic acid, C22:6, (omega-3)) is an essential fatty acid, i.e. a long-chain fatty acids which cannot be synthesized in human body and has important physiological functions. DHA is a main component in human brain and retina. 20% of the total DHA is present in cerebral cortex and up to 50% in retina. Therefore, it plays an important role in development of nervous system and visual system, and maintenance of normal intelligence and vision function. DHA also has physiological functions in prevention of cardiovascular diseases, cancers, inflammation, etc. In addition, it is also an essential fatty acid required by a variety of marine fishes for growth and development, and it can improve the survival rate of fish fries and reduce the incidence of albinism. Traditionally, DHA is obtained from fish oil. However, the extraction of polyunsaturated fatty acids (PUFAs) from fish oil has some problems, such as unstable output, low yield, high costs and contamination of other ω-6 PUFAs. Due to the increasingly limited fisheriy resources, traditional resources of DHA cannot meet the growing need of DHA in market. Therefore, development of new resources of DHA has become a new hotspot.

Schizochytrium sp. is a marine microalgae (or pseudo-fungi) that has been developed as a commercial source for producing DHA and other polyunsaturated fatty acids (PUFAs). The biosafety of Schizochytrium sp. has been verified. Hammond et al. performed a series of tests with it on rats and rabbits, and found no side effects.

In the fermentation process, however, quality degradation of Schizochytrium sp. strain is unavoidable, which results in a decreased yield of DHA. Therefore, the continuous improvement of the strain quality (mainly growth rate, especially DHA content), is important for promoting sustainable development in Schizochytrium sp. industry.

Acetyl-CoA carboxylase (ACC, (EC 6.4.1.2)) is a key enzyme in fatty acid synthesis, and herbicide Quizalofop {2-[4-(6-chloro-2-Quinoxaline-oxy)-phenoxy] propionate} is an inhibitor of this enzyme. In the presence of Quizalofop, cells grow slowly or even die due to the disturbed fatty acid biosynthesis.

In recent years, some studies indicated that Quizalofop could be used to screen mutagenized microalgae for strains with increased EPA content.

Chaturvedi et al. (2004) mutagenized Nannochloropsis oculata with MNNG and screened for Quizalofop-resistant strains. Results from the screened strains showed a significantly increased EPA content.

In addition, Chaturvedi et al (2006) mutagenized Nannochloropsis oculata with EMS and screened for strains resistant to cerulenin and erythromycin. The resultant strains showed an increased EPA yield by 29% and 12%.

Cao Xiaohong et al (2007) treated diatom Nitzschia laevis with DMSO and Quizalofop, which results in that the EPA content of the algae increased from 3.00% to 3.58%.

So far, Schizochytrium sp. mutant strains which are resistant to Quizalofop have not been reported. Even if such Schizochytrium sp. mutants are present, it is still unknown whether the content of EPA or DHA is increased for a mutagenized strain undergoing Quizalofop selection. Furthermore, it is not clear whether or not a Schizochytrium sp. mutant induced by UV irradiation could survive Quizalofop selection.

Summary

The inventors of the present application surprisingly discovered that a Schizochytrium sp. variant or mutant with increased DHA content can be obtained by applying a mutagenesis of UV radiation to a Schizochytrium sp. strain and then performing a directional selection by using an inhibitor to the key enzyme in fatty acid synthesis (such as acetyl coenzyme A carboxylase), including but not limited to Quizalofop. Further, the obtained variants or mutants have high DHA contents, as well as improved growth rates compared with a naive Schizochytrium sp. strain.

Accordingly, in a first aspect, there is provided a method for producing a variant of Schizochytrium sp. strain having increased DHA content compared with a naive Schizochytrium sp. strain, comprising inducing mutagenesis in a Schizochytrium sp. strain with UV radiation to produce a mutant strain; contacting the mutant strain with an acetyl coenzyme A carboxylase inhibitor; and selecting a variant of Schizochytrium sp. strain having increased DHA content compared with a naive Schizochytrium sp. strain.

In an embodiment disclosed herein, the inhibitor to acetyl coenzyme A carboxylase is Quizalofop. In a particular embodiment, the selected variant having increased DHA content also has improved growth rate compared with that of a naive Schizochytrium sp. strain so that the efficiency of DHA production is increased compared with that of a naive Schizochytrium sp. strain.

In another embodiment disclosed herein, the variant of Schizochytrium sp. strain has higher DHA content as compared with that of its starting strain or a naive Schizochytrium sp. strain. Typically, the variant of Schizochytrium sp. strain has higher DHA content as compared with that of the starting or naive strain after UV radiation-induced mutagenesis with or without the selection by using an inhibitor to acetyl coenzyme A carboxylase.

In a second aspect, there is provided a variant of Schizochytrium sp. strain, such as strain 2010-0321 with a deposit reference number of CCTCC M 2011024 as deposited at the Chinese Center for Type Culture Collection (CCTCC) on January 21, 2011.

In an embodiment, the variant strain provided herein has increased DHA content compared with that in a naive Schizochytrium sp. strain. In another embodiment, the variant provided herein is obtained by the method as disclosed herein comprising inducing mutagenesis in a Schizochytrium sp. strain with UV radiation to produce a mutant strain, and contacting the mutant strain with an acetyl coenzyme A carboxylase inhibitor; and selecting a variant of the Schizochytrium sp. strain having increased DHA content and/or improved growth rate compared with a naive Schizochytrium sp. strain.

in a third aspect, there is provided a method for producing DHA, comprising culturing a variant of Schizochytrium sp. strain as disclosed herein in a culture medium, and optionally comprising collecting DHA from a biomass of the cultured variant of Schizochytrium sp. strain, or a culture medium thereof. According to an embodiment, it is also related to biomass produced by this method.

In another aspect, there is provided a food product. Particularly, the food product contains the biomass (i.e. having increased concentration of DHA compared with a naive Schizochytrium sp. strain) or DHA produced according to the method disclosed herein.

Brief Description of Figures

Figure 1 shows lethal effects of ultraviolet radiation on Schizochytrium sp.. Figure 2 shows the relationship between the concentration of Quizalofop and corresponding lethality rate of Schizochytrium sp..

Figure 3 shows the growth rates and DHA contents of a control strain (or a naive strain) and some variant strains.

Figure 4 shows the growth rates and DHA contents of a control strain and three variants in a 50-liter fermentor experiment.

Figure 5 shows alignment comparison of 18S rRNA sequences between a control strain and variant strain 2010-0321.

Detailed Descriptions

As used herein, the term "strain" refers to any culture, generally pure culture, of a microorganism such as algae or microalgae species including a Schhochytrium sp. strain obtained from a single cell or an isolated colony.

As used herein, the term 'Variant" or "mutant" of a reference strain X refers to any strain obtained from the reference strain X. In the context of the present application, the term "variant" more particularly refers to a strain obtained mainly by mutation and selection performed on a reference strain X, and the term "mutant" more particularly refers to a strain obtained by random or directed mutagenesis (for example UV radiation) applied to a reference strain X.

Once a mutant or variant possesses the features according to various aspects disclosed herein, particularly a higher or increased DHA content compared with a naive algae or microalgae species, particularly a Schizochytrium sp. strain, it falls within the protection scope claimed in the application.

As used herein, the term "food product" refers to any product intended for supplying human or animals with nutrition. In particular, food products include products intended for feeding infants, children, adolescents and adults. All or part of the food products disclosed herein may contain at least one biomass or DHA obtained by the method disclosed herein. The food products disclosed herein may also contain other ingredients usually used in the agriculture or food industry, such as additives, preservatives, fruits or fruit extracts, flavouring agents, colorants, thickeners, cereals, chocolate, etc.

In a particular embodiment, the food product is a dairy product.

As used herein, the term "dairy product" refers to, in addition to milk, any product derived from milk, such as, milk powder, cream, ice cream, butter, cheese, yogurt, fermented milk, or by-products derived from milk, such as lactoserum and casein as well as various prepared food products containing milk or milk fractions as main ingredient. The milk is generally from cows, but can also be obtained from other mammals, such as a goat, a ewe, a mare, a camel or a buffalo. The dairy products incorporate the biomass or DHA produced according to the method disclosed herein.

Microorganisms suitable for serving as a starting strain or a naive/control strain for the mutation mutagenesis and/or selection disclosed herein include heterotrophic microalgaes, including members of the genus Schizochytrium. A particular member of the genus Schizochytrium is Schizochytrium limacinum. Suitable organisms can be obtained from a number of public-available sources, including by collection from the natural environment. For example, Schizochytrium sp. which can be used in the present application includes Schizochytrium limacinum SR21, Schizochytrium sp. (S8) (ATCC 20889), Schizochytrium sp. (LC- M) (ATCC 18915) and Schizochytrium limacinum IFO 32693 (Honda et Yokochi, Institute for Fermentation (IFO), Osaka, Japan). Schizochytrium limacinum SR21 or Schizochytrium limacinum IFO 32693 is preferred.

As used herein, any microorganism or any specific type of organism includes wild-type strains, mutant strains or recombinant strains.

As used herein, the term "biomass" refers to a culture or cells in culture medium of algae or microalgae species, such as a Schizochytrium sp. strain, a variant of a Schizochytrium sp. strain, etc. as disclosed herein. Alternatively, the term "biomass" refers to at least partially dewatered algal or microalgal culture, or dewatered culture.

As used herein, the term "about" or "approximately" when used in conjunction with a numerical value refers to any value within 1, 5 or 10% variability of the referenced number.

As used herein, the verb "comprise" and its conjugations are used in their non-limiting sense, and mean that the followed items are included, but items not specifically mentioned are not excluded.

In addition, reference to an element by the mdefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless clearly otherwise indicated/implicated in the context. The indefinite article "a" or "an" thus usually means "at least one".

In an embodiment of mutation of Schizochytrium sp., a Schizochytrium sp. strain is exposed to ultraviolet radiation for about 10 to 140 sec (s), preferably about 20s to 120s, more preferably about 30s to 100s, and most preferably about 70s to 90s. In a particular embodiment, a Schizochytrium sp. strain is exposed to ultraviolet radiation for a period of time selected from the group consisting of about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 and 90 seconds. The survived colonies are collected and used in the directional selection by an acetyl coenzyme A carboxylase inhibitor, such as Quizalofop.

In an embodiment, Quizalofop is added to a culture medium at a certain concentration in order to select a Quizalofop-resistant strain. The concentration of Quizalofop used in the selection is in the range of about 5 μπιοΙ/L to about 100 μπιοΙ/L, or about 10 umol/L to about 90 umoVL, or about 50 μηιοΙ/L to 80 μπιοΙ/L in a culture medium. In a particular embodiment, the concentration of Quizalofop is selected from the group consisting of about 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 and 80 μπιοΙ/L in a culture medium. In a particular embodiment, Quizalofop is Quizalofop-p

{(R)-2-[4-(6-chloro-2-quinoxaliny)oxy]phenoxy]propanoate} , such as Quizalofop ethyl.

In a particular embodiment, the selection for Quizalofop-resistant Schizochytrium sp. colonies is conducted in a solid culture medium containing Quizalofop. Then, survived colonies are picked out from the solid culture medium and subsequently cultured in a liquid or semi-solid culture medium containing Quizalofop to verify the Quizalofop resistance.

The selected Quizalofop-resistant colonies are further screened for increased growth rate and DHA content compared with a naive strain such as a Schizochytrium sp. strain.

Accordingly, the Schizochytrium sp.variants obtained by the method disclosed herein comprising UV exposure and Quizalofop selection can produce a higher or increased amount of DHA as compared with a starting or naive Schizochytrium sp.strain. In particular embodiments, the Schizochytrium sp. variant can produce an amount of DHA higher than that produced by the starting or naive strain by at least about 10, 20, 25, 30, 35, 40%, or by at least about 20, 25, 30, 35, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70% in a 3 to 7-day, or a 5-day culture or biomass. In an embodiment, the amount of DHA produced by the Schizochytrium sp. variant is at least 1.0% (w/w), 2.0% (w/w), 3.0% (w/w), 3.5% (w/w), 4.0% (w/w), 4.5% (w/w), 5.0% (w/w), 5.5% (w/w), 6.0% (w/w) or 6.5% (w/w), or 3.0-6.5% (w w), based on the dry weight of the biomass of the Schizochytrium sp. variant.

In another embodiment, the growth rate of the selected Schizochytrium sp. variants are higher than those of the starting or naive strains by at least about 3, 4, 5, 6, 7, 8, 9 or 10%, preferably over a 3 to 7-day culture, more preferably over a 5-day culture.

In other embodiments of the invention, the selected Schizochytrium sp. variant strains have stable growth rate and ability to produce DHA at a high amount under different culture conditions, such as different concentrations of glucose, different nitrogen sources and different pH values as compared with those of the starting or naive strains.

In an optional embodiment, the 18S RNA of the Schizochytrium sp. variants can be analyzed to identify genetic variations as compared to the starting or naive strains.

In an embodiment, examples of the Schizochytrium sp. variants comprise but not limited to strains 321-1, 2010-0321, and 303-11, particularly strain 2010-0321.

In an embodiment, in order to produce biomass having higher amount of DHA, a Schizochytrium sp. variant disclosed herein, particularly a strain selected from the group consisting of strains 321-1, 2010-0321, 303-11, and any combination thereof, or strain 2010-0321, is cultured under a cultivation condition suitable for culturing Schizochytrium sp. strains.

In embodiments, the cultivation conditions may be established according to culturing methods well-known in the art, including the methods disclosed in U. S. Patent No. 5,130, 242 and U. S. Patent No. 7,022,512, which are incorporated herein by reference in their entirety, and optimal cultivation conditions may be readily determined by those skilled in the art. In other embodiments, cultivation may be carried out in any suitable fermentor, such as a stirred tank fermentor or an air-lift fermentor which provides oxygen source. The microorganisms can be agitated at a certain level, such that the concentration of dissolved oxygen is sufficient to support the growth of the culture and production of DHA, and at the same time the agitation does not shear or otherwise damage the microorganisms. Suitable level of dissolved oxygen is at least 10% of air saturation level. More particularly, level of dissolved oxygen is maintained from about 10% to about 50% of air saturation levels. An exemplary fermentor used in the method disclosed herein may provide an aeration rate of 1 WM with a rotating speed of about 70-100 rpm. In an embodiment, the capacity of the fermentor is at least 10-60 liters, such as 10, 20, 30, 40, 50 or 60 liters. A fermentor with the capacity of up to 100 or even 150 liters can be used.

Cultivation may be carried out at any temperature suitable for maintaining the survival of microorganisms. Particularly, microorganisms may be cultured at a temperature from about 15°C to about 34°C. Preferably, the cultivation temperature is maintained at about 20°C to about 28°C, more preferably about 22°C to about 27°C.

In an embodiment, the pH of the culture medium during fermentation can be from 4 to 10, such as from 5 to 8, preferably from 6 to 7.

Typically, the fermentation will last for 10 days or less, or 9 days or less, or 8 days or less. In some embodiments, the fermentation duration may be or be at least 3, 4, 5, 6 or 7 days.

Optionally, the fermentation duration may be 150 to 200 hours, such as 160 to 190 hours, or from 170 to 180 hours.

In embodiments, medium used in the method for culturing the Schizochytrium sp. variants disclosed herein is a liquid medium, which may comprise the components that can promote growth and production of DHA at commercially practicable scales, including those components disclosed in U. S. Patent No. 5,130, 242 and U. S. Patent No. 7,022, 512, all of which are incorporated herein by reference in their entirety.

Particularly, the medium used for culturing the Schizochytrium sp. variants disclosed herein to produce DHA comprise a carbon source, and an organic or inorganic nitrogen source.

In an embodiment, the carbon source comprises glucose, various starches, molasses, ground corn or a combination thereof. In another embodiment, the nitrogen source may include but not limited to nitrate, urea, ammonium salts, amino acids, yeast extracts and the like. An assimilable phosphorous (e.g. phosphate) and/or sulphur (e.g. sulphate) source may also be provided in the medium.

The medium may additionally contain other substances to facilitate the fermentation, for example a chelating agent (e.g. citric acid), an anti-foaming agent (e.g. soy bean oil), a vitamin (e.g. thiamine and or riboflavin), essential catalytic metals (for example, alkali earth metals such as magnesium or calcium, or zinc or iron and/or other metals such as cobalt and copper). In another embodiment, the medium also may contain a source of microbial growth factors, which are unspecified or specified compounds that can enhance the heterotrophic growth of unicellular microorganisms.

Exemplary medium for culturing Schizochytrium sp. may be found in Jiang and Chen, Process Biochemistry 35 (2000) 1205-1209; Jiang and Chen, Journal of Industrial Microbiology & Biotechnology, (1999) Vol. 23,508-513 ; Vazhappilly and Chen, Journal of the American Oil Chemists Society, (1998) Vol. 75, No. 3 p 393-397.

As an example, the medium used in the method disclosed herein contains glucose 40-60, yeast extract 10-15, glutamic acid 4-8, sodium chloride 2.4-4.0, bitter salt 3.0-6.0, and ammonia sulfate 3.0-6.0 (g/L medium). In an embodiment, the medium used in the methods disclosed herein consists of glucose 40-60, yeast extract 10-15, sodium glutamate 4-8, sodium chloride 2.4-4.0, bitter salt 3.0-6.0, and ammonia sulfate 3.0-6.0 (g L medium). An example of the medium used herein consists of glucose 60.0, yeast extract 15.0, sodium glutamate 4.0, sodium chloride 3.0, bitter salt 5.0, and ammonia sulfate 5.0 (g L medium).

The organisms or biomass may be harvested by means, such as centrifugation, flocculation, or filtration, and can be processed immediately or dried for future processing. Optionally, lipids can be extracted. As used herein, the term "lipid" includes phospholipids; free fatty acids; esters of fatty acids; triacylglycerols; diacylglycerides; monoacylglycerides; lysophospholipids; soaps; phosphatides; sterols and sterol esters; carotenoids; xanthophylls (e.g., oxycarotenoids); hydrocarbons; and other lipids known to one of ordinary skill in the art. As well understood by a skilled artisan, DHA disclosed herein can be in the form of these various lipids, and is not limited to a free fatty acid, which may be a form in the food product disclosed herein. Different forms or fractions of the lipids can be extracted, depending on the extraction technique that is used.

Lipids can be extracted with an effective amount of solvent. Among the suitable solvents used in the extraction, polar lipids (e.g., phospholipids) are generally extracted with polar solvents (e.g., chloroform/methanol), and neutral lipids (e.g., triacylglycerols) are generally extracted with non-polar solvents (e.g., hexane). A particular solvent is pure hexane. A suitable ratio of hexane to dry biomass is about 4 liters of hexane per kilogram of dry biomass. In a particular embodiment, hexane is mixed with the biomass in a stirred reactor at a temperature of about 50°C for about 2 hours. After mixing, the biomass is filtered and separated from the oil-containing hexane. Hexane is removed from the oil by distillation techniques. Conventional oilseed processing equipment is suitable to perform the filtering, separation and distillation. Additional processing steps can be performed if required or desirable for a particular application. Alternative methods for lipid recovery are described in the following references which are incorporated herein by reference in their entirety: PCT Publication WO 01/76715, entitled "Method for the Fractionation of Oil and Polar Lipid-Containing Native Raw Materials"; PCT Publication WO 01/76385, entitled "Method For The Fractionation Of Oil And Polar Lipid-Containing Native Raw Materials Using Alcohol And Centrifugation"; PCT Publication WO 00/153512, entitled "Solventless Extraction Process".

Although the present application is demonstrated by using specific organisms and methods, it is intended to include all the methods and organisms obtainable in light of the teachings disclosed herein. Any substitution, modification and optimization would be apparent to those of ordinary skill in the art without departing from the scope and spirit of the inventions. In addition, any element involved herein, such as steps, conditions or strains, could be combined in any manner as desired so as to achieve the object of the inventions.

The following examples are provided merely for the purposes of illustration and are not intended to limit the scope of the subject matters as claimed.

EXAMPLES

Example 1 Mutation effects of ultraviolet radiation on Schizochytrium sp.

Schizochytrium limacimim SR21, which was also used as a control or naive strain in the following Examples, was plated onto dishes, and exposed to UV radiation for 0 sec (control), 30 sec, 40 sec, 50 sec, 60 sec, 70 sec, 80 sec, 90 sec and 100 sec (experimental groups), respectively. After radiation, the dishes were kept in dark for 24h, and the numbers of colonies on each dish was counted. The number of colonies in the control group was defined as 100%, and a lethality rate in each experimental group was calculated accordingly (Figure 1). As shown in Figure 1, with prolonged radiation duration, higher lethality rate was observed in Schizochytrium sp., indicating a dose-dependent effect. Particularly, a radiation duration of 70-90 sec, which resulted in a lethality rate of 60%-80% in Schizochytrium sp., was used to induce mutagenesis in Schizochytrium sp..

The culture conditions are: A. culture medium consisting of

Glucose 55 g/L,

Yeast extract 10 g L,

Sodium glutamate 5 g L,

Sodium chloride 2.4 g/L,

Bitter salt 4.0 g/L,

Ammonia sulfate 4.0 g/L;

Water balance

B. culture temperature: 22-27°C; and

C. initial pH: 5.0-7.0.

Example 2 Quizalofop Selections on Schizochytrium sp.

Quizalofop ethyl was added into the medium for culturing Schizochytrium sp. at a concentration of 0 umol/L, 10μ mol/L, 30 μπιοΙ L, 50 μπιοΙ/L, 70 μιηοΙ L, 80 μπιοΙ/L and 90 umol/L. The number of colonies in the control group (0 μπιοΙ/L) was defined as 100%. Colonies in each group were counted, and the lethality rates were calculated based on the number of colonies in the control group (Figure 2). As shown in Figure 2, within the experimental range, a positive correlation between the lethality rates of Schizochytrium sp. and the concentrations of Quizalofop was observed. The concentration of Quizalofop used for selecting resistant strains was set to 50 μιηοΙ L to 80 μιηοΙ L.

For ease of operation, Quizalofop was added into the medium and the UV radiation-survived strains were plated for further directional selection.

The selected strains may be cultured in a liquid or semi-solid Quizalofop-containing medium to validate their Quizalofop resistance.

The culture conditions are:

A. culture medium consisting of

Glucose 60 g/L,

Yeast extract 15 g/L,

Sodium glutamate 4 g L,

Sodium chloride 3 g/L,

Bitter salt 5.0 g/L,

Ammonia sulfate 5.0 g/L;

Water balance B. culture temperature: 22-27°C; and

C. initial pH: 5.0-7.0.

Example 3 Differences between naive strains and variant strains in growth rate and DHA content

After selection and validation according to Example 2, more than 200 Quizalofop-resistant Schizochytrium sp. strains were obtained. Then these Quizalofop-resistant Schizochytrium sp. strains are screened for higher growth rate and DHA content for two rounds. 20-50 strains were screened out after the first round, and three after the second. Each of the three strains had growth rate and DHA content higher than that of the control (naive) strain by more than 10%.

The culture conditions are:

A. culture medium consisting of

Glucose 60 g L,

Yeast extract 15 g L,

Sodium glutamate 4 g L,

Sodium chloride 3 g L,

Bitter salt 5.0 g/L,

Ammonia sulfate 5.0 g/L;

Water balance

B. culture temperature: 22-27°C; and

C. initial pH: 5.0-7.0.

Figure 3 shows the biomass and DHA content of the control strain (left most) and some of the variants which underwent UV mutagenesis followed by Quizalofop selection. As shown in Figure 3, some of the mutant strains had growth rate and the DHA content lower than that of the control strain, e.g. 321-6, 321-7 and 321-8, and some had growth rate and the DHA content not significantly different from that of the control strain, e.g. 321-4 321-5 and 321-19. Among the mutants, many of them had increased DHA content, e.g. 321-1, 2010-0321, 321-12, 321-13, 321-14, 321-15, 321-16, 321-17 and 321-18. The outstanding strains are 321-1 and 2010-0321, which had growth rate increased by 10.5% and 31.5%, and DHA content increased by 64.5% and 66.4%, respectively.

In addition to these two mutant strains, mutant strain 303-11 also showed good growth rate and DHA content.

Three variant strains, i.e. strains 2010-0321, 321-1 and 303-11, were selected for the tests as described in Examples 4-7 to verify that the variants obtained by the method of the application had the properties of high DHA content and growth rate, and the above properties could be stably maintained under different culture conditions.

The general culture conditions used in the experiments in Examples 4 to 7 are

A. culture medium consisting of

Glucose 60 g/L,

Yeast extract 15 g L,

Sodium glutamate 4 g L,

Sodium chloride 3 g/L,

Bitter salt 5.0 g L,

Ammonia sulfate 5.0 g/L;

Water balance

B. culture temperature: 22-27°C;

C. initial pH: 5.0-7.0;

D. 50-L fermentor with aeration rate of 1VVM and rotating speed of 70-100 rpm; and

E. culture period: 4-7 days.

Example 4 The growth rates and DHA contents of the control strain and strains 321-1, 2010-0321 and 303-11 cultured under different concentrations of glucose.

Except for the concentration of glucose, the culture conditions are the same as those in the general conditions as described above.

In order to compare the response of the control (naive) strain and mutant strains 321-1, 2010-0321 and 303-11 to different culture conditions, and further confirm the advantage of the selected strains over the control strain, a series of tests and comparisons were conducted. In this Example, the growth rate and DHA content of the control strains and mutant strains cultured under different concentrations of carbon source were compared (Table 1), wherein the carbon source in the initial medium was replaced by the carbon source with different test concentrations. As shown in Table 1, when cultured under different concentrations of glucose (50 g L, 60 g L and 70 g L), the outcomes observed in all three mutants were superior to that in the control strain, in which 2010-0321 was the best, followed by 321-1 and 303-11.

Table 1. Growth rate and DHA content of the control strain and three mutant strains cultured under different concentrations of lucose

g L.d: weight of biomass per liter of medium per day.

w/w%: percentage of weight of DHA based on weight of dry cells.

Example 5: Growth rate and DHA content of the control strain and strains 321-1, 20104)321 and 303-11 cultured in different nitrogen sources

Except for the nitrogen source, the culture conditions are the same as the general conditions as described above.

The Example was conducted to compare the growth rate and DHA content of the control (naive) strain and mutant strains 321-1, 2010-0321 and 303-11 cultured in different nitrogen sources. The nitrogen source in the initial medium was replaced by following nitrogen sources: (a) Yeast extract (20 g L); (b) Yeast extract (20 g L)+Sodium glutamate (10 g LXc) Yeast extract(40 g L)+Sodium glutamate (10 g/L), and (d) Corn syrup(40 g/L)+Sodium glutamate (10 g L). As shown in Table 2, when cultured in different nitrogen sources, the growth rate and DHA content of mutant strains 321-1 and 2010-0321 were superior to those of the control strain, in which strain 2010-0321 was better than strain 321-1 in DHA content, while strain 303-11 showed a higher DHA content as compared to the control strain.

Table 2. Growth rate and DHA content of the control strain and three mutants cultured in different nitrogen sources

(a) 0») (c) (d)

Nitrogen

Biomass DHA Biomass DHA Biomass DHA Biomass DHA sources

(g/L-d) (w/w%) (g L-d) (w/w%) (g L-d) (w/w%) (g L-d) (w/w%) Control

4.78 4.00 5.04 4.98 6.54 2.45 5.96 1.84 strain

321-1 5.52 4.69 5.62 5.05 7.62 2.70 6.34 2.56

2010-0321 5.48 5.14 5.36 5.17 7.50 3.82 6.86 2.59

303-11 4.28 4.53 4.90 6.33 6.16 4.66 5.70 2.66 g L.d: weight of biomass per liter of medium per day.

w/w%: percentage of weight of DHA based on weight of dry cells.

Example 6 Growth rate and DHA content of the control strain and strains 321-1, 2010-0321 and 303-11 cultured under different pH values

Except for the pH value, the culture conditions are the same as those in the general conditions as described above.

This Example was conducted to assess the growth rate and DHA content of the control (naive) strain and three mutants cultured under different pH (Table 3). As can be seen from Table 3, strams 2010-0321 and 321-1 showed growth rate and DHA accumulation higher than those of the control strain, while mutant strain 303-11 exhibited slight advantage in DHA accumulation and no advantage in growth rate as compared to the control strain .

Table 3 Growth rate and DHA content of the strain control and three mutant strains cultured under different pH values

g L.d: weight of biomass per liter of medium per day.

w/w%: percentage of weight of DHA based on weight of dry cells.

Example 7 Growth rate and DHA content of the control strain and strains 321-1, 2010-0321 and 303-11 cultured in 50-L fermentors

Example 7 was conducted to assess the growth rate and DHA accumulation of the control (naive) strain and strains 321-1, 2010-0321 and 303-1 Icnltured in 50-L fermentors (Figure 4). The strains were cultured under the general conditions as described above. As compared to the control strain, strain 321-1 showed biomass increased by 9.5% and DHA content increased by 16.7%, respectively. As for strain 2010-0321, the biomass and DHA content increased by 6.7% and 48.1%, respectively. For strain 303-11, the biomass was substantially the same as the control strain, and the DHA content increased by only 7.9%. With a comprehensive comparison among the three mutant strains, 2010-0321 was most prominent, particularly in term of DHA content.

Example 8 Differences between the control strain and strain 2010-0321 in biochemical composition

Table 4 and Table 5 showed the differences between the control (naive) strain and strain 2010-0321 in biochemical composition, including the composition of proteins, amino acids and fatty acids. The content of proteins was determined by Kjeldahl method. The content of amino acids was assayed by an amino acid analyzer. The content of fat was analyzed by a lipid analyzer.

As shown in Table 4, strain 2010-0321 had protein content (23.8%) higher than mat of the control strain (22.3%). Consistently, strain 2010-0321 showed a higher level than the control strain in term of the content of most of the amino acids.

As shown in Table 5, strain 2010-0321 had remarkable differences from the control strain in the composition of fatty acids. Firstly, strain 2010-0321 had C16:0 content (8.2%) higher than that of the control strain (7.7%). Secondly, CI 6:1 was absent in 2010-0321, while the CI 6:1 content of the control strain was 1.0%. Thirdly, strain 2010-0321 had DHA (C22:6) content significantly higher than that of the control strain.

Table 4, Differences between the control strain and strain 2010-0321 in protein content and amino acid composition

Strains

Content (w/w%) Control 2010-0321

Protein content 22.3 23.8

Aspartic acid 1.42 1.49

Threonine 0.70 0.75

Serine 0.66 0.69 Glutamic acid 3.46 3.73

Glycine 0.71 0.80

Alanine 0.77 0.87

Amino acid Valine 0.78 0.70

composition Isoleucine 0.58 0.64

Leucine 0.94 1.03

Tyrosine 0.47 0.50

Phenylalanine 0.55 0.59

Lysine 0.55 0.52

Histidine 0.30 0.32

Arginine 0.57 0.48

Proline 0.58 0.67

Cystine 0.19 0.16

Methionine 0.21 0.23

Tryptophan 0.20 0.21

Total amino acids 13.56 14.46

Table 5 Difference between the control strain and strain 2010-0321 in fatty acid

Example 9 Comparison between the control strain and strain 2010-0321 in the sequence of lSS rR Agene

Total R As were extracted from test strains by lysis method. After reverse-transcribed to cDNAs, 18S RNA gene was amplified with forward primer 5 '-CCAACCTGGTTGATCCTGCCAGTA-3 ^* (SEQ ID NO: 3) and reverse primer 5'-CCTTGTTACGACTTCACCTTCCTCT-3' (SEQ ID NO: 4). The amplicons were recovered and used to transform E. coli DH 5a competent cells. Positive colonies were selected for sequencing. The sequences of 18 S RNA gene of the control (naive) strain and strain 2010-0321 were aligned and compared with Blast software. The results were shown in Figure 5. Figure 5 showed the alignment of 18S rRNA genes of the control strain and strain 2010-0321 for homology comparison. As shown in the figure, the 18S rRNA gene of the control strain (SEQ ID NO: 1) is 1757 bp in length, while the 18S rRNA gene of strain 2010-0321 (SEQ ID NO: 2) is 1751 bp in length, in which 9 base pairs varied. The percent homology between the 18S rRNA gene of the control strain and that of strain 2010-0321 was 99%.