JP5894666B2 - Method for mutagenesis of Schizochytrium sp. And mutants produced therefrom - Google Patents

Description

  The present application relates to microbial genetic breeding techniques and microbial fermentation techniques, and in particular to microbial algae mutants or mutants for producing DHA (docosahexaenoic acid).

  DHA (docosahexaenoic acid, C22: 6, (omega-3)) is an essential fatty acid, that is, a long-chain fatty acid, which cannot be synthesized in the human body and has important physiological functions. DHA is a major component in the human brain and retina, with 20% of all DHA present in the cerebral cortex and up to 50% in the retina. Thus, DHA plays an important role in the development of the nervous and visual systems and in the maintenance of normal intelligence and visual functions. DHA also has physiological functions in the prevention of cardiovascular disease, cancer, granulation seed production and the like. In addition, DHA is an essential fatty acid required for the growth and development of various marine fish, and can improve the survival rate of juvenile fish and reduce the incidence of pigment deficiency. Traditionally, DHA is obtained from fish oil. However, extracting polyunsaturated fatty acids (PUFA) from fish oil has several problems, such as unstable extraction, low yield, high cost, and other ω-6 PUFA contamination There is. Due to the increasing constraints on fishery resources, conventional DHA resources cannot cope with increasing demand for DHA in the market. Therefore, there is an interest in developing new DHA resources.

  Schizochytrium sp. Is a marine microalgae (or mimic fungus) that has been developed as a commercial source for the production of DHA and other polyunsaturated fatty acids (PUFA). The biosafety of Schizochytrium sp. Has been demonstrated. Hammond et al. Conducted a series of tests using it on rats and rabbits and found no side effects.

  However, in the fermentation process, quality degradation of the Schizochytrium sp. Strain is unavoidable, leading to a reduction in DHA yield. Therefore, continuous improvement in strain quality (mainly growth rate, especially DHA content) is important to promote sustainable development in the Schizochytrium sp. Industry.

  Acetyl-CoA carboxylase (ACC, (EC 6.4.1.2) is an important enzyme in fatty acid synthesis and the herbicide Quizalofop {2- [4- (6-chloro-2-quinoxaline-oxy ) -Phenoxy] propionate} is an inhibitor of this enzyme, and in the presence of quizalofop, cells slow down and even die due to disturbances in fatty acid biosynthesis.

  Recently, some studies have shown that quizalofop can be used to select mutated microalgae for strains with increased EPA content.

  Chaturvedi et al. (2004) mutated Nannochloropsis oculata at MNNG and screened for quizalofop-resistant strains. Results from the selected strains showed a significantly increased EPA content.

  In addition, Chaturvedi et al. (2006) mutated Nannochloropsis oculata with EMS and conducted a selection test for strains resistant to cerulenin and erythromycin. The resulting strain showed an EPA yield increased by 29% and 12%.

  Cao Xiaohong et al. (2007) resulted in the treatment of the diatom plant Nitzschia laevis with DMSO and kizarohop, increasing the alga EPA content from 3.00% to 3.58%.

  So far, no Schizochytrium sp. Mutant resistant to quizalofop has been reported. Even if such a Schizochytrium sp. Mutant exists, it is still unclear whether the content of EPA or DHA is increased for mutants undergoing quizalofop selection. Furthermore, it is not clear whether Schizochytrium sp. Mutants induced by UV irradiation can withstand quizalofop selection.

  The inventor of the present application surprisingly applied Schizochytrium sp. Mutants or mutants with increased DHA content applied mutant induction of UV irradiation to Schizochytrium sp. Strains. And then found that it can be obtained by performing directional selection using inhibitors (including but not limited to quizalofop) to key enzymes in fatty acid synthesis (eg, acetyl coenzyme A carboxylase). Furthermore, the resulting mutant or mutant has a high DHA content and an improved growth rate compared to an untreated Schizochytrium sp. Strain.

  Accordingly, in a first aspect, there is provided a method for producing a mutant Schizochytrium sp. Strain having an increased DHA content compared to an untreated Schizochytrium sp. Strain, the method comprising: Inducing mutagenesis in a Schizochytrium sp. Strain using ultraviolet radiation to produce a mutant strain, contacting the mutant strain with an acetyl coenzyme A carboxylase inhibitor, and Selecting a mutant of the Schizochytrium sp. Strain having an increased DHA content compared to an untreated Schizochytrium sp. Strain.

  In an embodiment disclosed herein, the inhibitor for acetyl coenzyme A carboxylase is quizalofop. In certain embodiments, selected mutants with increased DHA content also have an increased growth rate compared to the growth rate of an untreated Schizochytrium sp. Strain, resulting in DHA production. Efficiency is increased compared to the DHA production efficiency of an untreated Schizochytrium sp. Strain.

  In another embodiment disclosed herein, the DHA content of a mutant of the Schizochytrium sp. Strain is compared to that of the initial or untreated Schizochytrium sp. Strain. Higher. Typically, the DHA content of a mutant of a Schizochytrium sp. Strain is the starting strain after mutagenesis induced by UV radiation with or without selection by use of an inhibitor to acetyl coenzyme A Or higher compared to that of the untreated strain.

In a second aspect, a mutant of a Schizochytrium sp. Strain is provided, for example deposited at the Chinese Center for Type Culture Collection (CCTCC) on January 21, 2011. And the strain 2010-0321 having the deposit reference number CCTCC M 2011024, on which the request for transfer to the deposit under the Budapest Treaty has been received .

  In one embodiment, the mutant strain provided herein had an increased DHA content compared to that of an untreated Schizochytrium sp. Strain. In another embodiment, the mutants provided herein are obtained by a method as disclosed herein, which method comprises using ultraviolet radiation to produce a mutant strain, Schizochytrium. sp.) and inducing mutation induction in the strain and contacting the mutant strain with an acetyl-coenzyme A carboxylase inhibitor, and increased DHA content and / or compared to an untreated Schizochytrium sp. Or selecting a variant of a Schizochytrium sp. Strain having an improved growth rate.

  In a third aspect, a method of producing DHA is provided, the method comprising culturing a mutant of a Schizochytrium sp. Strain as disclosed herein in a culture medium, and Optionally, harvesting DHA from the cultured mutant biomass of a Schizochytrium sp. Strain, or culture medium thereof. According to one embodiment, the present invention also relates to biomass produced by this method.

  In another aspect, a food product is provided. Specifically, the food product contains biomass (ie, having an increased concentration of DHA compared to an untreated Schizochytrium sp. Strain) or DHA produced by the methods disclosed herein. .

Shows the lethal effect of ultraviolet irradiation on Schizochytrium sp. Shows the relationship between the concentration of quizalofop and the lethality of the corresponding Schizochytrium sp. Shows the growth rate and DHA content of the control strain (or untreated strain) and several mutant strains. Shows the growth rate and DHA content of the control strain and the three variants in a 50 L fermentor experiment. Shows the alignment of the 18S rRNA sequence between the control strain and the mutant strain 2010-0321.

  As used herein, the term “strain” refers to any culture, generally algae or microbes containing a Schizochytrium sp. Strain obtained from a single cell or an isolated colony. Represents a pure culture of microorganisms such as algae.

  The term “variant” or “mutant” of reference strain X, as described herein, refers to any strain derived from reference strain X. In the context of this application, the term “variant” more particularly refers to a strain obtained mainly by mutation and selection carried out on the reference strain X, and the term “mutant” more particularly , Represents a strain obtained by random or direct mutagenesis (eg UV irradiation) applied to reference strain X.

  The mutant or variant is once compared to features according to the various aspects disclosed herein, specifically untreated algae or microalgal species, specifically a Schizochytrium sp. Strain. Possessing the characteristic of higher or increased DHA content is within the scope of protection claimed in this application.

  As used herein, the term “food product” refers to any product intended to provide nutrition to a human or animal. In particular, food products include products intended for feeding infants, children, young people and adults. All or a portion of the food product disclosed herein can contain at least one biomass or DHA obtained by the methods disclosed herein. The food products disclosed herein also include other ingredients commonly used in agriculture or the food industry, such as additives, preservatives, fruits or fruit extracts, flavoring agents, colorants, thickeners, Can contain barley, chocolate and the like.

  In certain embodiments, the food product is a dairy product.

  As used herein, the term “dairy product” refers to any product derived from milk in addition to milk, such as milk powder, cream, ice cream, butter, cheese, yogurt, fermented milk, etc., or milk. Secondary products such as whey and casein and various cooked food products containing milk or milk components as the main ingredients. Milk is generally derived from dairy cows, but can also be obtained from other animals such as goats, ewes, mares, camels, or wild cows. Dairy products utilize biomass or DHA produced by the methods disclosed herein.

  Microorganisms disclosed herein that serve as primary strains or untreated / control strains, or suitable for mutant / mutagenesis and / or selection, include members of the genus Schizochytrium. For example, heterotrophic microalgae. A specific member of the genus Schizochytrium is Schizochytrium limacinum. Suitable microorganisms can be obtained from a number of publicly available resources, including collection from the natural environment. For example, Schizochytrium sp. That can be used in the present application includes Schizochytrium limacinum SR21, Schizochytrium sp. (S8) (ATCC 208z, SCC LC-RM) (ATCC 18915) and Schizochytrium limacinum IFO 32693 (Honda et Yokochi, IFO: Institute for Fermentation, Osaka, Japan). Schizochytrium limacinum SR21 or Schizochytrium limacinum IFO 32693 is more preferred.

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

  As used herein, the term “biomass” refers to a species of algae or microalgae, eg, a Schizochytrium sp. Strain, a Schizochytrium sp. Variant. Represents a culture or cells in a culture medium such as Alternatively, the term “biomass” refers to an at least partially dehydrated algae or microalgae, or a dehydrated culture.

  As used herein, the term “about” or “approximately”, when used in conjunction with a numerical value, refers to any value that is within 1, 5 or 10% variation of the referenced numerical value. Represent.

  As used herein, the verb “including” and its conjugations are used in a non-limiting sense and mean that the following items are included, and items not specifically mentioned are not excluded.

  Furthermore, an element following the indefinite article “a” or “an” does not exclude the possibility of one or more elements being present unless clearly indicated / linked in context. Thus, the indefinite article “a” or “an” usually means “at least one”.

  In a mutant embodiment of Schizochytrium sp., A Schizochytrium sp. Strain is about 10-140 seconds, preferably about 20-120 seconds, more preferably about 30-100 seconds, and most Preferably, it is exposed to ultraviolet radiation for about 70 to 90 seconds. In certain embodiments, the Schizochytrium sp. Strain is about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86. , 87, 88, 89, and 90 seconds. Surviving colonies are collected and used for directional selection with acetyl coenzyme A carboxylase inhibitors such as quizalofop.

  In one embodiment, quizalofop is added to the culture medium at a specific concentration to select quizalofop-resistant strains. The concentration of quizalofop used for selection is about 5 μmol / L to about 100 μmol / L, or about 10 μmol / L to about 90 μmol / L, or about 50 μmol / L to 80 μmol / L in the culture medium. L range. In certain embodiments, the concentration of quizalofop is about 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 in the culture medium. 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 and 80 μmol / L. In a particular embodiment, the quizalofop is quizalofop-p {(R) -2- [4- (6-chloro-2-quinoxalinyl) oxy] phenoxy} propanoate}, for example quizalofop ethyl.

  In certain embodiments, the selection of quizalofop-resistant Schizochytrium sp. Colonies is performed in a solid culture medium containing quizalofop. The surviving colonies are then picked from the solid culture medium and subsequently cultured in a liquid or semi-solid culture medium containing quizalofop to confirm quizalofop resistance.

  The selected quizalofop-resistant colonies are further selected for increased growth rate and increased DHA content compared to untreated strains, such as Schizochytrium sp.

  Therefore, Schizochytrium sp. Mutants obtained by the methods disclosed herein, including ultraviolet irradiation and quizalofop selection, are more in comparison to the initial or untreated Schizochytrium sp. Strains. High or increased amounts of DHA can be produced. In certain embodiments, a Schizochytrium sp. Variant has a higher amount of DHA in a 3-7 day or 5 day culture or biomass than the amount produced by the starting or untreated strain, At least about 10, 20, 25, 30, 35, 40%, or 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%, higher amounts of DHA can be produced. In one embodiment, the amount of DHA produced by a Schizochytrium sp. Mutant is at least 1.0% (w / w) based on the dry weight of the Schizochytrium sp. ), 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).

  In another embodiment, the growth rate of the selected Schizochytrium sp. Is preferably increased in growth of the starting or untreated strain in cultures over 3-7 days, more preferably over 5 days. At least about 3, 4, 5, 6, 7, 8, 9 or 10% higher than the speed.

  In other embodiments, the selected Schizochytrium sp. Variant has different culture conditions, such as different glucose concentrations, different nitrogen sources and different pH values compared to the starting or untreated strain. Has the ability to produce DHA at a stable growth rate and in high amounts.

  In an optional embodiment, 18S RNA of a Schizochytrium sp. Variant can be analyzed to identify genetic variation compared to the starting or untreated strain.

  In one example, examples of Schizochytrium sp. Mutants include, but are not limited to 321-1, 2010-0321, and 303-11, especially 2010-0321.

  In one embodiment, to produce biomass with higher amounts of DHA, a Schizochytrium sp. Variant disclosed herein, among others, strains 321-1, 2010-0321, 303-11, A strain selected from the group consisting of, and any combination thereof, or strain 2010-0321 is cultured under culture conditions suitable for culturing Schizochytrium sp.

  In embodiments, the culture conditions include those disclosed in US Pat. Nos. 5,130,242 and 7,022,512, which are incorporated herein by reference in their entirety. It can be established according to culture methods well known in the art, and optimal culture conditions can be easily determined by those skilled in the art. In other embodiments, culturing can be performed in any suitable fermentor, such as an agitated tank fermentor or an airlift fermentor supplied with an oxygen source. Microorganisms can be agitated at a given concentration so that the dissolved oxygen concentration is sufficient to support culture growth and DHA production, and at the same time the agitation will not shear or damage the microorganisms There is no. A preferred concentration of dissolved oxygen is at least 10% of the saturation concentration of air. More specifically, the dissolved oxygen concentration is maintained at about 10% to about 50% of the air saturation concentration. A typical fermentor used in the methods disclosed herein is provided with an aeration rate of 1 VVM using a rotational speed of about 70-100 rpm.

  In one embodiment, the capacity of the fermentor is at least 10-60 L, such as 10, 20, 30, 40, 50 or 60 L. Fermenters with a capacity of up to 100 or even 150 L can be used.

  Culturing can be carried out at any suitable temperature that maintains the survival of the microorganism. Specifically, the microorganism can be cultured at a temperature of about 15 ° C to about 34 ° C. Preferably, the culture temperature is maintained at about 20 ° C to about 28 ° C, more preferably about 22 ° C to about 27 ° C.

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

  In general, fermentation is continued within 10 days, or within 9 days, or within 8 days. In some embodiments, the fermentation duration may be at least 3, 4, 5, 6, or 7 days.

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

  In an embodiment, the medium used in the method of culturing a Schizochytrium sp. Variant disclosed herein is a liquid medium, which itself promotes growth and production of DHA on a commercially viable scale. Components, which can be included and are incorporated by reference in their entirety, U.S. Pat. Nos. 5,130,242 and 7,022,512, all of which are hereby incorporated by reference in their entirety. Those components disclosed in the document are mentioned.

  In particular, the medium used to culture the Schizochytrium sp. Mutants disclosed herein to produce DHA includes a carbon source and an organic or inorganic nitrogen source.

  In one embodiment, the carbon source includes glucose, various starches, molasses, ground corn, or combinations thereof. In another embodiment, the nitrogen source includes, but is not limited to, nitrates, ureas, ammonium salts, amino acids, yeast extracts, and the like. Sources of assimilable phosphorus compounds (eg phosphates) and / or sulfur (eg sulfates) may also be provided in the medium.

  The medium can be other materials such as chelating agents (eg citric acid), antifoaming agents (eg soybean oil), vitamins (eg thiamine / riboflavin) important catalytic metals (eg magnesium) to promote fermentation. Or an alkaline earth such as calcium, or other metals such as zinc or iron and / or cobalt and copper).

  In other embodiments, the medium may also contain a source of microbial growth factor that is an unspecified or specified compound that can enhance heterotrophic growth of unicellular microorganisms. it can.

  Representative media for culturing Schizochytrium sp. Are Process Biochemistry by Jiang and Chen, 35 (2000) 1205-1209; Journal of Biooligol, 19 by Jiang and Chen, Biobiol. 23, 508-513; Journal of the American Oil Chemistry Society by Vazhappilly and Chen, (1998) Vol. 75, no. 3p393-397.

  As an example, the medium used in the methods disclosed herein is glucose 40-60, yeast extract 10-15, glutamic acid 4-8, salt 2.4-4.0, and salt 3 0.0 to 6.0 and ammonium sulfate 3.0 to 6.0 (g / L medium). In one embodiment, the medium used in the methods disclosed herein is glucose 40-60, yeast extract 10-15, sodium glutamate 4-8, sodium chloride 2.4-4.0, and salt 3 0.0 to 6.0, and ammonium sulfate 3.0 to 6.0 (g / L medium). Examples of media used herein include glucose 60.0, yeast extract 15.0, sodium glutamate 4.0, salt 3.0, niga salt 5.0, and ammonium sulfate 5.0 (g / L medium).

  The organism or biomass can be harvested by means such as, for example, centrifugation, coagulation sedimentation, 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; fatty acid esters; triacylglycerols; diacyl glycerides; monoacyl glycerides; lysophospholides; soaps; ); Hydrocarbons; and other lipids known to those skilled in the art. As will be appreciated by those skilled in the art, the DHA disclosed herein may exist in the form of these various lipids and may be in the form of free fatty acids that may be in the food product disclosed herein. It is not limited. Different forms or fractions of lipids can be extracted, depending on the extraction technique used.

  Lipids can be extracted using an effective amount of solvent. Among suitable solvents used for extraction, polar lipids (eg phospholipids) are generally extracted with polar solvents (eg chloroform / methanol) and neutral lipids (eg triacylglycerol) are generally Extract with a non-polar solvent (eg, hexane). A special solvent is pure hexane. A suitable ratio of hexane to dry biomass is about 4 liters (L) of hexane per kg of dry biomass. In a special embodiment, hexane is mixed with 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 for carrying out filtration, separation and distillation. Additional processing steps can be performed if necessary or desired for a particular application. Alternative methods for lipid recovery are described in the following references, which are hereby incorporated by reference in their entirety: The document is WO 01/76715, entitled “Method for the Fractionation of Oil and Polar Lipid-Containing Native Raw Materials”; WO 01/76715; No. 76385, title “Method For The Fractionation Of Oil And Polar Lipid-Containing Native Raw Materials US Public Alcohol Publication”. 00/153512 Pamphlet , Entitled "solventless extraction process (Solventless Extraction Process)", it is.

  While this application has been demonstrated using specific organisms and methods, it is intended to encompass all of the methods and organisms obtained in light of the teachings disclosed herein. Those skilled in the art will readily recognize any permutations, modifications and optimizations without departing from the scope and spirit of the present invention. In addition, any element involved in the specification, such as a process, condition, or strain, may be combined in any manner as necessary to achieve the objectives of the present invention.

  The following examples are provided for illustrative purposes only and are not intended to limit the scope of the subject matter as claimed.

EXAMPLE 1 Mutation effect of UV irradiation on Schizochytrium sp. Schizochytrium limacinum SR21 (which is also used as a control or untreated strain in the following examples) on a dish Plated and exposed to UV irradiation for 0 seconds (control), 30 seconds, 40 seconds, 50 seconds, 60 seconds, 70 seconds, 80 seconds, 90 seconds, and 100 seconds (experimental group), respectively. After irradiation, the dishes were kept in the dark for 24 hours and the number of colonies on each dish was counted. The number of colonies in the control group was defined as 100%, and the mortality in each experimental group was calculated based on that (FIG. 1). As can be seen in FIG. 1, higher lethality was observed in Schizochytrium sp. As the irradiation period was extended, indicating a dose-dependent effect. Specifically, continued irradiation for 70 to 90 seconds (resulting in a mortality rate of 60 to 80% in Schizochytrium sp.), Which leads to mutation induction in Schizochytrium sp. Used to trigger.

The culture conditions are
A. A culture medium,
Glucose 55g / L
Yeast extract 10g / L
Sodium glutamate 5g / L
Sodium chloride 2.4g / L
4.0 g / L of salt
Ammonium sulfate 4.0 g / L
A culture medium consisting of the remainder of the water,
B. Culturing temperature: 22-27 ° C. and C.I. Initial pH: 5.0-7.0
It is.

Example 2 Selection of quizalofop for Schizochytrium sp. Concentration 0 μmol / L, 10 μmol / L, 30 μmol / L, 50 μmol of quizalofopethyl in a medium for culturing Schizochytrium sp. / L, 70 μmol / L, 80 μmol / L, and 90 μmol / L. The number of colonies in the control group (0 μmol / L) was defined as 100%. The colonies in each group were counted and the mortality was calculated based on the number of colonies in the control group (FIG. 2). As seen in FIG. 2, a positive correlation was observed between Schizochytrium sp. Lethality and the concentration of quizalofop within the scope of the experiment. The concentration of quizalofop used to select resistant strains was set at 50 μmol / L to 80 μmol / L.

  For ease of manipulation, kuzarohop was added into the medium and strains that survived UV irradiation were plated for further directional selection.

  The selected strains can be cultured in a medium containing liquid or semi-solid quizalofop to demonstrate their quizalofop resistance.

The culture conditions are
A. A culture medium,
Glucose 60g / L
Yeast extract 15g / L
Sodium glutamate 4g / L
Sodium chloride 3g / L
Niga salt 5.0g / L
Ammonium sulfate 5.0 g / L
A culture medium consisting of the remainder of the water,
B. Culturing temperature: 22-27 ° C. and C.I. Initial pH: 5.0-7.0
It is.

Example 3 Difference in growth rate and DHA content between untreated and mutant strains After selection and demonstration in Example 2, more than 200 quizalofop-resistant Schizochytrium sp. Strains were obtained. . These quizalofop-resistant Schizochytrium sp. Strains were then selected for two rounds for higher growth rate and DHA content. After the first round, 20 to 50 strains were selected, and after the second round, three strains were selected. The growth rate and DHA content of each of the three strains was more than 10% higher than the control (untreated) strain.

The culture conditions are
A. A culture medium,
Glucose 60g / L
Yeast extract 15g / L
Sodium glutamate 4g / L
Sodium chloride 3g / L
Niga salt 5.0g / L
Ammonium sulfate 5.0 g / L
A culture medium consisting of the remainder of the water,
B. Culture temperature: 22-27 ° C
C. Initial pH: 5.0-7.0
It is.

  Figure 3 shows the biomass and DHA content of the control strain (leftmost) and some of the mutants that received quizalofop selection following UV irradiation mutagenesis. As shown in FIG. 3, the growth rate and DHA content of some of the mutant strains (eg 321-6, 321-7 and 321-8) are lower than that of the control strain and some (eg 321-4 The growth rate and DHA content of 321-5 and 321-19) are not significantly different from those of the control strain. Among the mutants, many of them had increased DHA content (eg, 321-1, 2010-0321, 321-12, 321-13, 321-14, 321-15, 321-16, 321 -17 and 321-18). Prominent strains are 321-1 and 2010-0321, which have growth rates increased by 10.5% and 31.5%, respectively, and DHA increased by 64.5% and 66.4%, respectively. Had a content.

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

  Three mutant strains, 2010-0321, 321-1 and 321-11, were selected for the tests described in Examples 4-7, and the mutants obtained by the method of the present application had a high DHA content. It has been demonstrated that it has characteristics with growth rate and that the above characteristics can be stably maintained under different culture conditions.

The general culture conditions used for the experiments in Examples 4-7 are:
A. A culture medium,
Glucose 60g / L
Yeast extract 15g / L
Sodium glutamate 4g / L
Sodium chloride 3g / L
Niga salt 5.0g / L
Ammonium sulfate 5.0 g / L
A culture medium consisting of the remainder of the water,
B. Culture temperature: 22-27 ° C
C. Initial pH: 5.0-7.0,
D. A 50 L fermentor with an aeration rate of 1 VVM and a rotational speed of 70-100 rpm,
E. Culture period: 4-7 days.

Example 4 Growth Rate and DHA Content for Control Strains and 321-1, 2010-0321 and 303-11, Cultured under Different Concentrations of Glucose Other than the concentration of glucose, the culture conditions were as described above. Same as condition.

  To compare the response of the control (untreated) strain to the mutant strains 321-1, 2010-0321 and 303-11 to different culture conditions and to further confirm the advantages of the selected strain over the control strain, A series of tests and comparisons were performed. This example compares the growth rate and DHA content of control and mutant strains cultured under different concentrations of carbon source (Table 1), where the carbon source in the initial medium is different. Replaced with carbon sources at 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 results observed in all three mutants are Outstanding than the results, among them, 2010-0321 was the best, followed by 321-1 and 303-11.

Example 5 Growth rate and DHA content of control strains and strains 321-1, 2010-0321 and 303-11 cultured in different nitrogen sources The culture conditions were the same as those described above except for the nitrogen source. Are the same.

  This example was performed to compare the growth rate and DHA content of control (untreated) strains and mutant strains 321-1, 2010-0321 and 303-11 cultured in different nitrogen sources. The nitrogen source in the initial medium was replaced with the following nitrogen sources: (a) yeast extract (20 g / L); (b) yeast extract (20 g / L) + sodium glutamate (10 g / L); c) 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 grown in different nitrogen sources, the growth rate and DHA content of mutant strains 321-1 and 2010-0321 exceeded those of the control strain, among which strain 2010- 0321 was superior to strain 321-1 in DHA content, while strain 303-11 showed a higher DHA content compared to the control strain.

Example 6 Growth rate and DHA content of control strains and strains 321-1, 2010-0321 and 303-11 cultured under different pH values Except for the pH value, the culture conditions were those of the above general conditions Are the same.

  This example was performed to evaluate the growth rate and DHA content of control (untreated) and three mutant strains cultured under different pH (Table 3). As shown in Table 3, strains 2010-0321 and 321-1 showed higher growth rates and accumulation of DHA than that of the control strain, while mutant strain 303-11 was compared to the control strain. , Showed a slight advantage in DHA accumulation and no advantage in growth rate.

Example 7 Growth rate and DHA content of control strains and strains 321-1, 2010-0321 and 303-11 cultured in a 50 L fermentor Control (untreated) strain and strain 321-1 cultured in a 50 L fermentor Example 7 was performed to evaluate the growth rate and DHA accumulation with 2010-0321 and 303-11 (FIG. 4). Strains were cultured under general conditions as described above. Compared to the control strain, strain 321-1 showed a 9.5% increased biomass and a 16.7% increased DHA content, respectively. For strain 2010-0321, the biomass and DHA content increased by 6.7% and 48.1%, respectively. For strain 303-11, the biomass was almost the same as the control strain, and the DHA content increased only 7.9%. Comprehensive comparison among the three mutant strains, 2010-0321 was the best, especially with respect to DHA content.

Example 8 Differences between control strain and strain 2010-0321 in biochemical composition Tables 4 and 5 show control (untreated) strains and strains in biochemical composition including protein, amino acid and fatty acid composition The difference with 2010-0321 was shown. The protein content was determined by the Kjeldahl method. The amino acid content was quantified by an amino acid analyzer. The fat content was analyzed by a lipid analyzer.

  As shown in Table 4, the protein content (23.8%) in strain 2010-0321 was higher than that of the control strain (22.3%). Continuing, strain 2010-0321 showed higher concentrations than the control strain for most amino acid content.

  As shown in Table 5, the strain 2010-0321 was significantly different in fatty acid composition from the control strain. First, the strain 2010-0321 had a higher C16: 0 content (8.2%) than that of the control strain (7.7%). Secondly, C16: 1 was not present in 2010-0321, while the C16: 1 content of the control strain was 1.0%. Third, the strain 2010-0321 had a significantly higher DHA (C22: 6) content than that of the control strain.

Example 9 Comparison of 18S rRNA gene sequence between control strain and strain 2010-0321 Total RNA was extracted from the test strain by the lysis method. After reverse transcription into cDNA, the 18S RNA gene was amplified with forward primer 5′-CCAACCTGGTTGATCCTGCCAGTA-3 ′ (SEQ ID NO: 3) and reverse primer 5′-CCTTGTTACGAACTTCACCTCTCCTCT-3 ′ (SEQ ID NO: 4). Amplicons (amplicons) were collected and used to transform E. coli DH5α competent cells. Positive colonies were selected for sequencing. The sequences of the 18S RNA gene of the control (untreated) and strain 2010-0321 were aligned and compared with Blast software. The results are shown in FIG.

  FIG. 5 shows the alignment of 18S rRNA genes for homology comparison between the control strain and strain 2010-0321. As shown in the figure, the 18S rRNA gene of the control strain (SEQ ID NO: 1) is 1757 bp long, while the 18S rRNA gene of the strain 2010-0321 (SEQ ID NO: 2) is 1751 bp long and contains 9 bases The pair was changing. The% homology between the control strain 18S rRNA gene and the strain 2010-0321 18S rRNA gene was 99%.

Claims (21)

  Schizochytrium sp. Strain identified as 2010-0321 and deposited at the Chinese Traditional Culture Storage Center with the deposit reference number of CCTCC M 2011024.   The Schizochytrium strain according to claim 1, wherein the Schizochytrium strain has an increased DHA content and / or improved growth rate characteristics compared to an untreated Schizochytrium sp. Strain.   3. The Schizochytrium strain has a different protein content, a different amino acid composition and / or a different fatty acid composition compared to an untreated Schizochytrium sp. Strain. Schizochytrium sp. A method for producing a mutant of a Schizochytrium sp. Strain, comprising:
Exposing the Schizochytrium sp. Strain to ultraviolet radiation to induce mutation induction in the Schizochytrium sp. Strain to produce a mutant strain;
Contacting the mutant strain with an acetyl coenzyme A carboxylase inhibitor;
Selecting a mutant of the Schizochytrium sp. Strain having an increased DHA content and / or an improved growth rate compared to an untreated Schizochytrium sp. Strain.   5. The method of claim 4, wherein the acetyl coenzyme A carboxylase inhibitor is quizalofop.   6. The method of claim 5, wherein the quizalofop is used at a concentration in the range of 10 [mu] mol / L to 90 [mu] mol / L.   7. A method according to any one of claims 4 to 6, wherein the variant has an increased DHA content and an improved growth rate.   The method according to any one of claims 4 to 7, wherein the variants have different protein contents, proteins with different amino acid compositions and / or different fatty acid compositions.   9. The method according to any one of claims 4 to 8, wherein the Schizochytrium sp. Strain is exposed to ultraviolet radiation for between 30 seconds and 100 seconds. A method for producing DHA comprising:
A Schizochytrium sp. Strain according to any one of claims 1 to 3 is cultured under conditions suitable for culturing a Schizochytrium sp. Strain for producing DHA. Including a method. A method for producing DHA comprising:
Exposing a Schizochytrium sp. Strain to ultraviolet radiation to induce mutagenesis in the Schizochytrium sp. Strain to produce a mutant strain;
Contacting the mutant strain with an acetyl coenzyme A carboxylase inhibitor;
Selecting a mutant of a Schizochytrium sp. Strain having an increased DHA content and / or an improved growth rate compared to an untreated Schizochytrium sp. Strain,
Culturing the mutant under conditions suitable for culturing a Schizochytrium sp. Strain for producing DHA,
Including a method. Collecting DHA from the biomass of the cultured mutant of a Schizochytrium sp. Strain or variant, or from the culture medium of a Schizochytrium sp. Strain,
The method according to claim 10 or 11, further comprising: The condition is
A culture medium comprising a carbon source of 50 to 70 g / L and a nitrogen source of 10 to 20 g / L, a culture temperature of 20 ° C. to 28 ° C.,
And / or pH: 3 to 10,
The method according to claim 10 or 11, comprising: The culture medium is 50 to 70 glucose in g / L,
10-30 yeast extracts
10-20 sodium glutamate
Sodium chloride from 2.4 to 4.0,
3.0 to 6.0 salt
3.0 to 6.0 ammonium sulfate,
14. The method of claim 13, comprising.   A method for producing biomass,
Culturing the Schizochytrium sp. Strain according to any one of claims 1 to 3 under conditions suitable for culturing a Schizochytrium sp. Strain for producing biomass.
Including a method.   Biomass, which is a culture or a cell in a culture medium of the Schizochytrium sp. Strain according to any one of claims 1 to 3. 17. A food product comprising the biomass of claim 16 , wherein the food product comprises a product intended to feed infants, children, adolescents and adults, or the food product is a dairy product. . What Schizochytrium Shokukabu der according to any one of claims 1 to 3, by the Schizochytrium Shokukabu or mutant, the amount of DHA which Ru produced in cultures over 3-7 days, untreated Schizochytrium genus ( Schizochytrium sp.) than the amount of DHA which Ru produced by strain, at least 10% higher, Schizochytrium Shokukabu. 15. The method according to any one of claims 4 to 14, wherein the amount of DHA produced by the Schizochytrium strain or mutant in culture over 3-7 days is reduced to an untreated Schizochytrium sp. ) A method that is at least 10% higher than the amount of DHA produced by the strain . The Schizochytrium strain according to any one of claims 1 to 3 and 18 , wherein the growth rate of the Schizochytrium strain or variant of the Schizochytrium sp. Strain is cultured in 3-7 days. A Schizochytrium strain that is at least 3% higher than the growth rate of the untreated Schizochytrium sp. Strain .   The method according to any one of claims 4 to 14 and 19, wherein the growth rate of the Schizochytrium sp. Or mutant of a Schizochytrium sp. Strain is 3 to 7 days in culture. A method that is at least 3% higher than said growth rate of an untreated Schizochytrium sp. Strain.

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