KR20210087145A - Composition for culturing chlamydomonas mutants and culture method thereof - Google Patents

Abstract

The present invention provides a culture composition optimal for culturing a mutant strain which is to increase the production of zeaxanthin from a mutant Chlamydomonas reinhardtii having a zeaxanthin-producing ability without producing lutein, a culture method thereof, and a method for producing zeaxanthin from the same. By using the culture composition and culture method of the present invention, compared to the case of using a conventional culture composition of Chlamydomonas reinhardtii, it is possible to produce 1.65 times or more improved zeaxanthin, so that it is possible to efficiently produce pigments at an industrial level, and zeaxanthin can be applied as a raw material for food, health functional food, and pharmaceuticals.

Description

Composition for culturing Chlamydomonas mutant and method thereof {COMPOSITION FOR CULTURING CHLAMYDOMONAS MUTANTS AND CULTURE METHOD THEREOF}

The present invention relates to a composition for culturing Chlamydomonas mutants capable of improving the zeaxanthin production of Chlamydomonas mutants having excellent zeaxanthin-producing ability, and a method for culturing Chlamydomonas mutants.

Macular degeneration is a disease that causes visual impairment due to degeneration of the macula, a nervous tissue located in the center of the inner retina of the eye. Macular degeneration is largely divided into non-exudative (dry) and exudative (wet), with about 80-90% of It corresponds to exudative macular degeneration. Non-exudative macular degeneration progresses slowly and the symptoms rarely worsen rapidly, but there is no special treatment, so health food can be of great help to alleviate symptoms and prevent further deterioration.

In particular, steady intake of macular pigment is known to be of great help in preventing or slowing the progression of macular degeneration. As a representative example, xanthophyll (xanthophyll, xanthophyll) is a carotenoid oxycarotenoid pigment produced by oxygenation of carotenoids. ), and the pigment belonging to xanthophylls includes lutein or zeaxanthin.

Animals cannot produce xanthophyll pigments and can only be consumed through food. Conventionally, xanthophyll pigments have been extracted from higher plants such as marigold, but the efficiency is very low in terms of time and cultivation area, and the price is high. there was To replace this, a method of producing macular pigment using a bacterial system has been devised, but the problem that the pigment obtained from bacteria is ultimately unsuitable for use as a food additive, and that the bacterial culture solution or bioreactor is also very expensive to maintain exist.

As a solution to this problem, a method using microalgae that produces its own pigment has been devised, but in the case of wild-type microalgae, the total amount of pigment produced is small, and it has the characteristic of producing lutein more abundantly than zeaxanthin, and each It is difficult to distinguish and separate the pigments.

Chlamydomonas mutant strain genetically engineered to overcome the limitations of these wild-type microalgae and to produce zeaxanthin alone among macular pigments was disclosed in Korean Registration Publication No. 10-1981935, but industrially pharmaceuticals or health functional foods Since the efficiency is insufficient to sufficiently produce the raw material of , the development of a technology for increasing the production of zeaxanthin through the mutant strain is additionally required.

No. 10-1981935

An object of the present invention for solving this problem is to provide a culture composition for culturing Chlamydomonas mutants having a high zeaxanthin content production ability, the present inventors have improved the production efficiency of zeaxanthin pigments from Chlamydomonas mutants. A new culture composition and culture method were established in order to increase the culture composition and culture method of the present invention, compared with the conventional culture composition and method, the culture time is reduced, and the production of zeaxanthin can be further improved by 65% or more. The present invention was completed by confirming experimentally.

In order to achieve the above object, the present invention provides a Chlamydomonas reinhardtii culture composition comprising nitrogen (N) 10.0mM to 17.0mM and phosphorus (P) 1.0mM to 2.5mM.

The culture composition comprises nitrogen (N) 13.5mM to 16.5mM and phosphorus (P) 1.0mM to 1.4mM; nitrogen (N) from 10.0 mM to 13.49 mM and phosphorus (P) from 1.9 mM to 2.5 mM; nitrogen (N) from 10.0 mM to 11.99 mM and phosphorus (P) from 1.9 mM to 2.5 mM; Or nitrogen (N) 12.0mM to 13.49mM and phosphorus (P) may be one comprising 1.9mM to 2.5mM.

The nitrogen _{may be included as NH 4} Cl, and the phosphorus may be included as K ₂ HPO ₄ and/or KH ₂ PO ₄ .

The phosphorus may include K ₂ HPO ₄ and KH ₂ PO ₄ together. In this case, the culture composition of the present invention may include NH ₄ Cl 10.0mM to 17.0mM _{as nitrogen and K 2} HPO ₄ 0.6mM to 1.45mM as phosphorus and KH ₂ PO ₄ 0.4mM to 1.05mM. In addition, the composition of the present invention comprises 13.5 _{mM to 16.5 mM of NH 4} Cl as nitrogen and 0.6 mM to 0.85 mM of K ₂ HPO ₄ and 0.4 mM to 0.55 mM of _{KH 2} PO _{4 as phosphorus; NH 4} Cl 10.0 mM to 13.49 mM as nitrogen and _{K 2} HPO ₄ 1.16 mM to 1.45 mM as phosphorus and KH ₂ PO ₄ 0.74 mM to 1.05 mM; _{10.0 mM to 11.99 mM of NH 4} Cl as nitrogen and 1.16 mM to 1.45 mM of K ₂ HPO _{4 as} phosphorus and 0.74 mM to 1.05 mM of _{KH 2} PO _{4 as phosphorus; or 12.0 mM to 13.49 mM of NH 4} Cl as nitrogen and 1.16 mM to 1.45 mM of K ₂ HPO _{4 as} phosphorus and 0.74 mM to 1.05 mM of _{KH 2} PO _{4 as phosphorus.}

The culture composition may further include calcium (Ca) and magnesium (Mg).

The calcium may be included in an amount of 0.47mM to 0.70mM, and the magnesium may be included in an amount of 0.55mM to 0.85mM. The calcium _{may be included as CaCl 2} hydrate, and the magnesium may be included as MgSO ₄ hydrate or MgCl ₂ hydrate.

The culture composition is NH ₄ Cl to 10.0mM 17.0mM, K ₂ HPO ₄ 0.6mM to about 1.45mM, KH ₂ PO ₄ 0.4mM to about 1.05mM, 0.47mM CaCl ₂ hydrate to 0.70mM, MgSO _4, and 0.55mM to 0.85 hydrate may contain mM.

The culture composition is NH ₄ Cl to 13.5mM 16.5mM and in a K ₂ HPO ₄ 0.6mM to about 0.85mM and KH ₂ PO ₄ 0.4mM to about 0.55mM, CaCl ₂ hydrate 0.601mM to 0.70mM, 0.75mM, and MgSO ₄ hydrate to 0.85 mM.

The culture composition is NH ₄ Cl to 10.0mM and the 13.49mM as K ₂ HPO ₄ 1.16mM to 1.45mM and KH ₂ PO ₄ 0.74mM to 1.05mM, 0.47mM CaCl ₂ hydrate to 0.6mM, MgSO ₄ and hydrates 0.550mM to 0.749 mM.

The culture composition is NH ₄ Cl 10.0mM to 11.99mM and phosphorus K ₂ HPO ₄ 1.16mM to 1.45mM and KH ₂ PO ₄ 0.74mM to 1.05mM, CaCl ₂ hydrate 0.47mM to 0.549mM, and MgSO ₄ hydrate 0.55mM to 0.649 mM.

The culture composition is NH ₄ Cl 12.0 mM to 13.49 mM and phosphorus K ₂ HPO ₄ 1.16 mM to 1.45 mM and KH ₂ PO ₄ 0.74 mM to 1.05 mM, CaCl ₂ hydrate 0.55 mM to 0.60 mM, and MgSO ₄ hydrate 0.650 mM to 0.749 mM.

The culture composition may further include glacial acetic acid as a carbon source. The glacial acetic acid may be included in an amount of 1.5 ml/L to 2.5 ml/L.

The culture composition is one or more trace elements selected from the group consisting of EDTA, iron (Fe), zinc (Zn), boron (B), manganese (Mn), copper (Cu), molybdenum (Mo), and cobalt (Co). may further include. Preferably, it may further include EDTA, iron (Fe), zinc (Zn), boron (B), manganese (Mn), copper (Cu), molybdenum (Mo), and cobalt (Co).

When the trace element is included in the culture composition, EDTA 133 μM to 137 μM, iron (Fe) 16 μM to 20 μM, zinc (Zn) 73 μM to 77 μM, boron (B) 183 μM to 187 μM, manganese (Mn) 24 μM to 28 μM, copper ( Cu) 5.0 μM to 8.0 μM, molybdenum (Mo) 4.0 μM to 7.0 μM, and/or cobalt (Co) 5.0 μM to 8.0 μM.

The trace element may be included in the form of a hydrate. Specifically, the EDTA may be included as EDTA hydrate; The iron may be included as _{FeSO 4} hydrate or FeCl _{3 hydrate;} The manganese may be included in MnCl ₂ hydrate; The zinc _{may be included as ZnSO 4} hydrate, and the copper may be included as CuCl ₂ hydrate or CuSO ₄ hydrate; The molybdenum is Na ₂ MoO ₄ can be included as a hydrate or hydrates and NaMoO _4; Further, the cobalt may be included in CoCl ₂ hydrate.

The culture composition may further include tris (hydroxymethyl) aminomethane (tris) as a buffer. The tris may be included in 2.0 g/L to 3.2 g/L.

The culture composition may have a pH of 7.0 to 7.4.

The Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) may be a mutant that does not produce lutein while having a zeaxanthin producing ability. The mutant may be a ZEP gene and a LCYE gene knocked out at the same time as a rain hard Tii (Chlamydomonas reinhardtii) mutant.

In this aspect, the culture composition of the present invention is a composition for culturing Chlamydomonas rain hard Tia mutants in which the ZEP gene and the LCYE gene are simultaneously knocked out; Alternatively, it may be a composition for culturing Chlamydomonas rain hard Tia mutant strains that do not produce lutein while having zeaxanthin-producing ability.

More specifically, the mutant is adenine (A) between the 816th base and the 817th base in the ZEP gene sequence of Chlamydomonas reinhardtii represented by SEQ ID NO: 1; the nucleotide sequence represented by SEQ ID NO: 3; or ZEP gene mutation into which the nucleotide sequence represented by SEQ ID NO: 5 is inserted; And the nucleotide sequence shown in SEQ ID NO: 9 between the 599th base and the 600th base in the LCYE gene sequence of Chlamydomonas reinhardtii represented by SEQ ID NO: 2; Or it may be one containing the LCYE gene mutation into which the nucleotide sequence represented by SEQ ID NO: 11 is inserted.

The zeaxanthin (zeaxanthin) producing ability, Chlamydomonas rain hard T T mutant that does not produce lutein, accession number KCTC 13626BP Chlamydomonas rain hard T T double ZEP / LCYE mutant 1 (dZL1) mutant number have.

The present invention also includes culturing a mutant Chlamydomonas reinhardtii having a zeaxanthin-producing ability without producing lutein in the culture composition of the present invention. Chlamydomonas reinhardtii mutant It provides a method of culturing.

In that the method is for obtaining a high content of zeaxanthin from a Chlamydomonas reinhardtii mutant, the present invention does not produce lutein in the culture composition of the present invention, but Chlamydomonas having a zeaxanthin-producing ability Rain hard Tia (Chlamydomonas reinhardtii) provides a zeaxanthin production method comprising culturing a mutant.

The method does not produce lutein in a culture composition containing nitrogen (N) 10.0mM to 17.0mM and phosphorus (P) 1.0mM to 2.5mM Chlamydomonas reinhardtii mutant strain having zeaxanthin-producing ability and culturing for 36 hours to 130 hours at a light intensity of 60 μE to 450 μE.

The mutant may be cultured for 60 hours to 130 hours at a light intensity of 60 μE to 160 μE, or 60 μE to 160 μE.

The mutant may be incubated for 36 hours to 60 hours at a light intensity of more than 160 μE to 450 μE. Alternatively, the mutant may be cultured for 36 hours to 60 hours at a light intensity of more than 160 μE to less than 250 μE.

The culture composition may further include one or more selected from the group consisting of calcium (Ca), magnesium (Mg), and glacial acetic acid as a carbon source.

The culture composition is NH ₄ Cl to 10.0mM 17.0mM, K ₂ HPO ₄ 0.6mM to about 1.45mM, KH ₂ PO ₄ 0.4mM to about 1.05mM, 0.47mM CaCl ₂ hydrate to 0.70mM, MgSO _4, and 0.55mM to 0.85 hydrate may contain mM.

The culture composition is NH ₄ Cl to 13.5mM 16.5mM and in a K ₂ HPO ₄ 0.6mM to about 0.85mM and KH ₂ PO ₄ 0.4mM to about 0.55mM, CaCl ₂ hydrate 0.601mM to 0.70mM, 0.75mM, and MgSO ₄ hydrate to 0.85 mM.

The culture composition is NH ₄ Cl to 10.0mM and the 13.49mM as K ₂ HPO ₄ 1.16mM to 1.45mM and KH ₂ PO ₄ 0.74mM to 1.05mM, 0.47mM CaCl ₂ hydrate to 0.6mM, MgSO ₄ and hydrates 0.550mM to 0.749 mM.

The culture composition is NH ₄ Cl 10.0mM to 11.99mM and phosphorus K ₂ HPO ₄ 1.16mM to 1.45mM and KH ₂ PO ₄ 0.74mM to 1.05mM, CaCl ₂ hydrate 0.47mM to 0.549mM, and MgSO ₄ hydrate 0.55mM to 0.649 mM.

The culture composition is NH ₄ Cl 12.0 mM to 13.49 mM and phosphorus K ₂ HPO ₄ 1.16 mM to 1.45 mM and KH ₂ PO ₄ 0.74 mM to 1.05 mM, CaCl ₂ hydrate 0.55 mM to 0.60 mM, and MgSO ₄ hydrate 0.650 mM to 0.749 mM.

The glacial acetic acid may be included in an amount of 1.5 ml/L to 2.5 ml/L.

The culture composition is one or more trace elements selected from the group consisting of EDTA, iron (Fe), zinc (Zn), boron (B), manganese (Mn), copper (Cu), molybdenum (Mo), and cobalt (Co). may further include. Preferably, it may further include EDTA, iron (Fe), zinc (Zn), boron (B), manganese (Mn), copper (Cu), molybdenum (Mo), and cobalt (Co).

The culture composition may have a pH of 7.0 to 7.4.

The present invention does not produce lutein and provides a culture composition for improving the zeaxanthin production of a mutant Chlamydomonas reinhardtii having a zeaxanthin-producing ability and a culture method using the same, thereby increasing the synthesis amount of zeaxanthin By improving by about 65% or more compared to the prior art, zeaxanthin can be efficiently provided as a raw material for medicines or health functional foods, and thus can be effectively utilized in industries such as food, pharmaceuticals and cosmetics.

Figures 1a to 1c confirm the growth ability according to the conditions for each light intensity of Chlamydomonas wild-type, ZEP and dZL mutants in TAP medium, Figure 1a is low light (60 μmol photons m ^-2 s ^-1 ), Figure 1b is medium light Figure (200 μmol photons m ^-2 s ^-1 ), Figure 1c shows the growth ability of Chlamydomonas wild-type, ZEP and dZL mutants at high light intensity (400 μmol photons m ^-2 s ^{-1 ).}
Figure 2 shows the biomass production per 1L according to the conditions for each light intensity of Chlamydomonas wild-type, ZEP and dZL mutants in TAP medium.
Figure 3a shows the production of zeaxanthin per 1L according to the conditions for each light intensity of Chlamydomonas wild-type, ZEP and dZL mutants in TAP medium.
Figure 3b shows the zeaxanthin content per biomass according to the conditions for each light intensity of Chlamydomonas wild-type, ZEP and dZL mutants in TAP medium.
4 shows the cell growth ability according to the control of the medium concentration of the dZL mutant when cultured at low light in each medium condition.
5 shows the production of zeaxanthin per 1 L according to the control of the medium concentration of the dZL mutant when cultured at low light in each medium condition.
This is the result of confirming the cell growth ability of each of the 16 medium compositions of the dZL mutant under low light conditions.
Figures 6a and 6b are low light (60 μmol photons m ^-2 s ^-1 ) conditions, nitrogen content in the medium increased by 25% (N25), 50% (N50), 75% (N75), or 100% (N100) The result of confirming each cell growth ability according to the phosphorus concentration ( FIG. 6a ) or the biomass production per 1L of the dZL mutant ( FIG. 6b ) is shown.
7a to 7d are low light (60 μmol photons m ⁻² s ⁻¹ ) conditions, nitrogen content of 25% (N25), 50% (N50), 75% (N75), or 100% (N100) increased medium shows the production of zeaxanthin per 1 L of the dZL mutant according to the phosphorus concentration.
8a to 8b show medium light (200 μmol photons m ⁻² s ⁻¹ ) conditions, nitrogen content of 25% (N25), 50% (N50), 75% (N75), or 100% (N100) increased medium shows the result of confirming each cell growth ability according to the phosphorus concentration in ( FIG. 8a ) or the biomass production per 1L of the dZL mutant ( FIG. 8b ).
9a to 9d show medium light intensity (200 μmol photons m ⁻² s ⁻¹ ) conditions, nitrogen content of 25% (N25), 50% (N50), 75% (N75), or 100% (N100) increased medium shows the production of zeaxanthin per 1 L of the dZL mutant according to the phosphorus concentration.
10A to 10B show a high luminance (400 μmol photons m ⁻² s ⁻¹ ) condition, a nitrogen content of 25% (N25), 50% (N50), 75% (N75), or 100% (N100) in an increased medium. The result of confirming each cell growth ability according to the phosphorus concentration (FIG. 10a) or the biomass production per 1L of the dZL mutant (FIG. 10b) is shown.
11A to 11D show a high light intensity (400 μmol photons m ⁻² s ⁻¹ ) condition, a nitrogen content of 25% (N25), 50% (N50), 75% (N75), or 100% (N100) in an increased medium. The zeaxanthin production per 1 L of the dZL mutant according to the phosphorus concentration is shown.

Since the present invention may have various changes and may have various forms, the specific examples and descriptions described below are only intended to aid the understanding of the present invention, and are not intended to limit the present invention to a specific disclosed form . It should be understood that the scope of the present invention includes all modifications, equivalents and substitutes included in the spirit and scope of the present invention.

Hereinafter, the present invention will be described in detail by way of Examples.

The present invention provides a Chlamydomonas reinhardtii culture composition comprising nitrogen (N) 10.0mM to 17.0mM and phosphorus (P) 1.0mM to 2.5mM.

In the present invention, the 'culture composition' means that a material for a specific purpose is additionally added and mixed to include a nutrient material required by a microorganism to be a culture target, that is, a culture body in order to culture a specific microorganism, medium, incubator Alternatively, it is used interchangeably with a culture medium, and is a concept that includes both a natural medium, a synthetic medium, or a selective medium.

The culture composition may include an element in the form of a hydrate. In the present invention, the term hydrate refers to a compound in which water is bound to another compound, and is also referred to as a hydrate. It may be monohydrate or dihydrate depending on the number of water molecules, and the degree of hydration may vary depending on conditions such as temperature or vapor pressure of water even in the same compound, and even for the same compound, all hydrates having different numbers of water molecules are included.

TAP medium or HS medium was used for culturing the conventional Chlamydomonas rain hard T T, but there is a limit to improving zeaxanthin production when using this. Culture composition for improving the zeaxanthin production ability from Chlamydomonas rain hard T T mutants. was newly implemented, and through this, the present invention was completed by experimentally confirming that zeaxanthin production of about 1.65 times or more is possible while lowering the incubation time than when using the conventional TAP medium.

Lutein is not produced in the culture composition of the present invention, in which the content of nitrogen is 1.5 times to 2 times or more, the amount of phosphorus is 1.25 times to 2 times or more, and the carbon source is increased by 2 times or more compared to the conventional TAP medium. It is possible to maximize the zeaxanthin-producing ability in Chlamydomonas rain hard Tia mutant having xanthine-producing ability.

As a specific example, the culture composition includes nitrogen (N) 13.5mM to 16.5mM and phosphorus (P) 1.1mM to 1.4mM; nitrogen (N) from 10.0 mM to 13.49 mM and phosphorus (P) from 1.9 mM to 2.5 mM; nitrogen (N) from 10.0 mM to 11.99 mM and phosphorus (P) from 1.9 mM to 2.5 mM; Or nitrogen (N) 12.0mM to 13.49mM and phosphorus (P) may be one comprising 1.9mM to 2.5mM.

The nitrogen _{may be included as NH 4} Cl, and the phosphorus may be included as K ₂ HPO ₄ and/or KH ₂ PO ₄ .

The phosphorus may include K ₂ HPO ₄ and KH ₂ PO ₄ together. In this case, the culture composition of the present invention may include NH ₄ Cl 10.0mM to 17.0mM _{as nitrogen and K 2} HPO ₄ 0.6mM to 1.45mM as phosphorus and KH ₂ PO ₄ 0.4mM to 1.05mM. In addition, the composition of the present invention comprises 13.5 _{mM to 16.5 mM of NH 4} Cl as nitrogen and 0.6 mM to 0.85 mM of K ₂ HPO ₄ and 0.4 mM to 0.55 mM of _{KH 2} PO _{4 as phosphorus; NH 4} Cl 10.0 mM to 13.49 mM as nitrogen and _{K 2} HPO ₄ 1.16 mM to 1.45 mM as phosphorus and KH ₂ PO ₄ 0.74 mM to 1.05 mM; _{10.0 mM to 11.99 mM of NH 4} Cl as nitrogen and 1.16 mM to 1.45 mM of K ₂ HPO _{4 as} phosphorus and 0.74 mM to 1.05 mM of _{KH 2} PO _{4 as phosphorus; or 12.0 mM to 13.49 mM of NH 4} Cl as nitrogen and 1.16 mM to 1.45 mM of K ₂ HPO _{4 as} phosphorus and 0.74 mM to 1.05 mM of _{KH 2} PO _{4 as phosphorus.}

The culture composition may further include calcium (Ca) and magnesium (Mg).

The calcium may be included in an amount of 0.47mM to 0.70mM, and the magnesium may be included in an amount of 0.55mM to 0.85mM. The calcium _{may be included as CaCl 2} hydrate, and the magnesium may be included as MgSO ₄ hydrate or MgCl ₂ hydrate.

The culture composition is NH ₄ Cl to 10.0mM 17.0mM, K ₂ HPO ₄ 0.6mM to about 1.45mM, KH ₂ PO ₄ 0.4mM to about 1.05mM, 0.47mM CaCl ₂ hydrate to 0.70mM, MgSO _4, and 0.55mM to 0.85 hydrate may contain mM.

The culture composition is NH ₄ Cl to 13.5mM 16.5mM and in a K ₂ HPO ₄ 0.6mM to about 0.85mM and KH ₂ PO ₄ 0.4mM to about 0.55mM, CaCl ₂ hydrate 0.601mM to 0.70mM, 0.75mM, and MgSO ₄ hydrate to 0.85 mM.

The culture composition is NH ₄ Cl to 10.0mM and the 13.49mM as K ₂ HPO ₄ 1.16mM to 1.45mM and KH ₂ PO ₄ 0.74mM to 1.05mM, 0.47mM CaCl ₂ hydrate to 0.6mM, MgSO ₄ and hydrates 0.550mM to 0.749 mM.

The culture composition is NH ₄ Cl 10.0mM to 11.99mM and phosphorus K ₂ HPO ₄ 1.16mM to 1.45mM and KH ₂ PO ₄ 0.74mM to 1.05mM, CaCl ₂ hydrate 0.47mM to 0.549mM, and MgSO ₄ hydrate 0.55mM to 0.649 mM.

The culture composition is NH ₄ Cl 12.0 mM to 13.49 mM and phosphorus K ₂ HPO ₄ 1.16 mM to 1.45 mM and KH ₂ PO ₄ 0.74 mM to 1.05 mM, CaCl ₂ hydrate 0.55 mM to 0.60 mM, and MgSO ₄ hydrate 0.650 mM to 0.749 mM.

The culture composition may further include glacial acetic acid as a carbon source. The glacial acetic acid may be included in an amount of 1.5 ml/L to 2.5 ml/L.

The culture composition is one or more trace elements selected from the group consisting of EDTA, iron (Fe), zinc (Zn), boron (B), manganese (Mn), copper (Cu), molybdenum (Mo), and cobalt (Co). may further include. Preferably, it may further include EDTA, iron (Fe), zinc (Zn), boron (B), manganese (Mn), copper (Cu), molybdenum (Mo), and cobalt (Co).

When the trace element is included in the culture composition, EDTA 133 μM to 137 μM, iron (Fe) 16 μM to 20 μM, zinc (Zn) 73 μM to 77 μM, boron (B) 183 μM to 187 μM, manganese (Mn) 24 μM to 28 μM, copper ( Cu) 5.0 μM to 8.0 μM, molybdenum (Mo) 4.0 μM to 7.0 μM, and/or cobalt (Co) 5.0 μM to 8.0 μM.

The trace element may be included in the form of a hydrate. Specifically, the EDTA may be included as EDTA hydrate; The iron may be included as _{FeSO 4} hydrate or FeCl _{3 hydrate;} The manganese may be included in MnCl ₂ hydrate; The zinc _{may be included as ZnSO 4} hydrate, and the copper may be included as CuCl ₂ hydrate or CuSO ₄ hydrate; The molybdenum is Na ₂ MoO ₄ can be included as a hydrate or hydrates and NaMoO _4; Further, the cobalt may be included in CoCl ₂ hydrate.

The culture composition may further include tris (hydroxymethyl) aminomethane (tris) as a buffer. The tris may be included in 2.0 g/L to 3.2 g/L.

The pH of the culture composition is not particularly limited as long as the Chlamydomonas rain hard Tia can survive and grow, but it is viable at pH 6 or higher, specifically pH 6 to pH 9, and preferably the pH of the culture composition is 6.5 to 7.5 or the pH may be from 7.0 to 7.4.

The culture method using the culture composition may be appropriately included in the present invention and may be used without limitation to a culture medium or a culture form, a method commonly used in the art. As an example, both a method of separately supplying carbon dioxide in aeration and a method of not separately supplying carbon dioxide while in contact with air may be used.

The Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) may be a mutant that does not produce lutein while having a zeaxanthin producing ability. The mutant may be a ZEP gene and a LCYE gene knocked out at the same time as a Rain hard T (Chlamydomonas reinhardtii) mutant.

In this aspect, the culture composition of the present invention is a composition for culturing Chlamydomonas rain hard Tia mutants in which the ZEP gene and the LCYE gene are simultaneously knocked out; Alternatively, it may be a composition for culturing Chlamydomonas rain hard Tia mutant strains that do not produce lutein while having zeaxanthin-producing ability.

The mutant of the present invention can be produced by knocking out the Zeaxanthin epoxidase ( ZEP ) gene and the Lycopene epsilon cyclase (LCYE) gene using RGNE RNPs and CRISPR gene editing technology rather than general mutagenesis. In this aspect, the mutant strain of the present invention may include mutations at specific positions of the ZEP ( Zeaxanthin epoxidase ) gene and the LCYE ( Lycopene epsilon cyclase) gene. In the case of using the CRISPR gene editing technology, it is possible to specifically knockout a gene at a desired gene location, so the mutant of the present invention is defined by mutation of the ZEP ( Zeaxanthin epoxidase ) gene and the LCYE ( Lycopene epsilon cyclase ) gene through the location. It can be specified, and the production of mutants from wild-type Chlamydomonas rain hard Tia microalgae is possible.

In the present invention, "mutation" refers to a change in a nucleotide sequence due to insertion, deletion, or substitution of a nucleotide into the original nucleotide sequence. Specifically, "insertion mutation" means that some nucleotides are inserted between the original nucleotide sequence. It means a mutation, and the number of the inserted bases may be different depending on the mutation, and thus is not limited thereto.

The mutant of the present invention may include an insertion mutation in which some bases are inserted between the 816th base and the 817th base in the ZEP gene sequence of Chlamydomonas reinhardtii represented by SEQ ID NO: 1, through which The ZEP gene can be knocked out.

More specifically, the mutant is adenine (A) between the 816th base and the 817th base in the ZEP gene sequence of Chlamydomonas reinhardtii represented by SEQ ID NO: 1; the nucleotide sequence represented by SEQ ID NO: 3; Alternatively, it may include a ZEP gene mutation into which the nucleotide sequence represented by SEQ ID NO: 5 is inserted.

In addition, the mutant is a nucleotide sequence represented by SEQ ID NO: 9 between the 599th base and the 600th base in the LCYE gene sequence of Chlamydomonas reinhardtii represented by SEQ ID NO: 2; Alternatively, the LCYE gene mutation into which the nucleotide sequence represented by SEQ ID NO: 11 is inserted may be included.

In addition, while having the zeaxanthin (zeaxanthin) producing ability, the Chlamydomonas rain hard T A mutant that does not produce lutein is a Chlamydomonas rain hard T T double ZEP / LCYE mutant 1 (dZL1) mutant of accession number KCTC 13626BP can be

The present invention also includes culturing a mutant Chlamydomonas reinhardtii having a zeaxanthin-producing ability without producing lutein in the culture composition of the present invention. Chlamydomonas reinhardtii mutant It provides a method of culturing.

In that the method is for obtaining a high content of zeaxanthin from a Chlamydomonas reinhardtii mutant, the present invention does not produce lutein in the culture composition of the present invention, but Chlamydomonas having a zeaxanthin-producing ability Rain hard Tia (Chlamydomonas reinhardtii) provides a zeaxanthin production method comprising culturing a mutant.

The zeaxanthin production method may further include; after the culturing step, isolating the Chlamydomonas rain hard Tia mutant of the present invention from the culture. The separated mutant may be further subjected to a processing step including drying.

In addition, the zeaxanthin production method may further include; extracting the pigment from the Chlamydomonas rain hard Tia mutant of the present invention, the culture of the mutant, the concentrate of the culture, or the dried product of the culture.

In addition, the zeaxanthin production method may further include a purification step after the extraction step, which may be performed by a conventional purification method in the art.

Zeaxanthin prepared through the concentration or drying step may be used as a raw material for food, health functional food, cosmetics, or pharmaceuticals.

The zeaxanthin production method may be carried out by employing other methods within a range that does not impair the effects of the present invention.

The method does not produce lutein in a culture composition containing nitrogen (N) 10.0mM to 17.0mM and phosphorus (P) 1.0mM to 2.5mM Chlamydomonas reinhardtii mutant strain having zeaxanthin-producing ability and culturing for 36 hours to 130 hours at a light intensity of 60 μE to 450 μE.

The mutant strain has a luminosity of greater than 160 μE to 450 μE; 60 μE to 160 μE; greater than 160 μE to 300 μE; or at a light intensity of 150 μE to 250 μE.

As the light source in the present invention, a conventional light source in the art may be used, and the type is not limited thereto.

More specifically, the mutant strain may be cultured for 60 hours to 130 hours at a light intensity of 60 μE to 160 μE. According to an embodiment of the present invention, it was confirmed that the intracellular zeaxanthin content was increased when cultured for 3 days or more under the light intensity condition.

The mutant may be incubated for 36 hours to 60 hours at a light intensity of more than 160 μE to 450 μE. Alternatively, the mutant strain is greater than 160 μE to 300 μE; or at a light intensity of greater than 300 μE to 450 μE for 36 to 60 hours. Through the examples of the present invention, it was confirmed that it had an excellent zeaxanthin production ability in the vicinity of 48 hours of incubation under medium or high light conditions.

The culture composition may further include one or more selected from the group consisting of calcium (Ca), magnesium (Mg), and glacial acetic acid as a carbon source.

The culture composition is NH ₄ Cl to 10.0mM 17.0mM, K ₂ HPO ₄ 0.6mM to about 1.45mM, KH ₂ PO ₄ 0.4mM to about 1.05mM, 0.47mM CaCl ₂ hydrate to 0.70mM, MgSO _4, and 0.55mM to 0.85 hydrate may contain mM.

The culture composition is NH ₄ Cl to 13.5mM 16.5mM and in a K ₂ HPO ₄ 0.6mM to about 0.85mM and KH ₂ PO ₄ 0.4mM to about 0.55mM, CaCl ₂ hydrate 0.601mM to 0.70mM, 0.75mM, and MgSO ₄ hydrate to 0.85 mM.

The culture composition is NH ₄ Cl to 10.0mM and the 13.49mM as K ₂ HPO ₄ 1.16mM to 1.45mM and KH ₂ PO ₄ 0.74mM to 1.05mM, 0.47mM CaCl ₂ hydrate to 0.6mM, MgSO ₄ and hydrates 0.550mM to 0.749 mM.

The culture composition is NH ₄ Cl 10.0mM to 11.99mM and phosphorus K ₂ HPO ₄ 1.16mM to 1.45mM and KH ₂ PO ₄ 0.74mM to 1.05mM, CaCl ₂ hydrate 0.47mM to 0.549mM, and MgSO ₄ hydrate 0.55mM to 0.649 mM.

The culture composition is NH ₄ Cl 12.0 mM to 13.49 mM and phosphorus K ₂ HPO ₄ 1.16 mM to 1.45 mM and KH ₂ PO ₄ 0.74 mM to 1.05 mM, CaCl ₂ hydrate 0.55 mM to 0.60 mM, and MgSO ₄ hydrate 0.650 mM to 0.749 mM.

The glacial acetic acid may be included in an amount of 1.5 ml/L to 2.5 ml/L.

The culture composition is one or more trace elements selected from the group consisting of EDTA, iron (Fe), zinc (Zn), boron (B), manganese (Mn), copper (Cu), molybdenum (Mo), and cobalt (Co). may further include. Preferably, it may further include EDTA, iron (Fe), zinc (Zn), boron (B), manganese (Mn), copper (Cu), molybdenum (Mo), and cobalt (Co).

The pH of the culture composition is not particularly limited as long as the Chlamydomonas rain hard Tia can survive and grow, but it is viable at pH 6 or higher, specifically pH 6 to pH 9, and preferably the pH of the culture composition is 7.0 to 7.4.

The culture method using the culture composition, except for the conditions described above, may be appropriately included in the present invention and used in a method commonly used in the art, and may be used without being limited to an incubator or culture type. As an example, both a method of separately supplying carbon dioxide in aeration and a method of not separately supplying carbon dioxide while in contact with air may be used.

The method for producing vegetable oil of the present invention may be carried out by employing other methods within the range that does not impair the effects of the present invention. The contents of the mutant strain and composition may be applied mutatis mutandis to the production method of the present invention.

Hereinafter, the present invention will be described in more detail according to Examples, which are merely illustrative of the present invention, and the content of the present invention is not to be construed as being limited by the Examples below.

Experimental Example 1. Preparation of strain

For the culture experiment for improving zeaxanthin productivity of the present invention, while having Chlamydomonas wild-type strain and zeaxanthin producing ability, Chlamydomonas reinhardtii mutant that does not produce lutein As an experiment, the ZEP gene and the LCYE gene were simultaneously knocked out of a Rain Hard Tii (Chlamydomonas reinhardtii) mutant (hereinafter referred to as 'ZEP/LCYE knockout mutant'), and a ZEP mutant was prepared.

Specifically, the ZEP/LCYE knockout mutant is Chlamydomonas rain hard Tia double ZEP/LCYE mutant 1 of the accession number KCTC 13626BP among the mutants in which the ZEP gene and the LCYE gene are simultaneously knocked out according to the method disclosed in Korean Registration Publication No. 10-1981935. (dZL1) mutant (hereinafter referred to as 'dZL strain' or 'ZEP/LCYE knockout mutant') was used.

Also, ZEP mutant was used as a Chlamydomonas lane hard tiahyi ZEP variation of the accession number of KCTC 13230BP 1 (△ Z1) ( Chlamydomonas reinhardtii ZEP mutant 1 (△ Z1)).

Experimental Example 2. Culture and biomass analysis of strains

Mixotrophic culture was used for the culture method by supplying photosynthesis and carbon source together, and in Test Example 1 to check the appropriate light intensity before confirming the culture solution composition, acetic acid was added to the TAP medium of Table 1 was added and cultured. Afterwards, in the experiment of Test Example 2 to confirm the medium composition, a strain culture experiment was performed according to each medium composition.

After making a medium having the composition shown in Table 1 below, autoclaving was performed under high pressure/sterilization, and cells in an active growth stage were used to make a concentration of 10 ⁶ cell/mL in the culture medium, and growth was started. The luminous intensity was given by using a fluorescent lamp according to each experimental condition. The solvent and dilution factor in which each component was dissolved are shown in Table 1.

The cell growth of the Chlamydomonas wild-type strain and the ZEP/LCYE knockout mutant and/or the ZEP mutant in the TAP medium of Table 1 or in the medium of the composition described in Test Example was compared by varying the brightness or medium composition. Specifically, cells were inoculated into 50 mL medium in a 250 mL flask ^{at 10 6} cells mL ^{-1 .} Thereafter, the cells were mixed nutrient culture on an orbital shaker at 25° C. under continuous white fluorescent light at 120 rpm.

However, the zeaxanthin production ability analysis test according to the nitrogen and phosphorus content of Test Example 3 was performed in the same manner as above except for inoculation and use in a 20 mL medium in a 100 mL flask.

Cell growth was determined by measuring the absorbance at 750 nm every 24 hours in a cell medium cultured at 25° C. in a specific light intensity and/or culture composition.

Experimental Example 3. Analysis of dye content

In order to confirm the zeaxanthin-producing ability of the strain according to the culture conditions of the present invention, the intracellular zeaxanthin content was analyzed in the culture conditions using HPLC.

For analysis of the dye, cells were harvested every 12 hours, and the dye was extracted by pipetting with 90% (v/v) acetone until the cells became colorless. The supernatant was analyzed using a Shimadzu Prominence HPLC model LC-20AD (Waters, USA) equipped with a Spherisorb 5.0 μm ODS1 4.6 x 250 mm cartridge column. Specifically, in order to separate the pigment, the total flow rate of the solvent was 1.2 mL per minute, and from 0 to 15 minutes, Tris at pH 8.0 was 14%, and acetonitrile was uniformly reduced from 84% to 0%, respectively, Methanol and ethyl acetate were started at 2% and increased to 68% and 32%, respectively, by the 15th minute. After that, hold this solvent ratio for 3 minutes (15th to 18th minutes), then for 1 minute (18th to 19th minutes) return the ratios of each solvent to the starting ratio, and then hold for the remaining 6 minutes. and post-run. Data were analyzed using a photodiode array detector (SPD-M20A, Shimadzu), and the concentration of zeaxanthin was calculated from absorbance at 445 nm and 670 nm.

Test Example 1. Comparative analysis of growth and zeaxanthin raw material content of zeaxanthin-producing Chlamydomonas mutants according to light intensity

According to the method described above, the cell growth of the Chlamydomonas wild-type strain, ZEP/LCYE knockout mutant, and parental ZEP mutant was compared by varying the brightness. Specifically, cells were inoculated into 50 mL medium in a 250 mL flask ^{at 10 6} cells mL ^{-1 .} Thereafter, the cells were exposed to continuous white fluorescent light (low intensity: 60 μmol photons m ^-2 s ^{-1 , medium intensity: 200 μmol photons m -2} s ^-1 , or high intensity: 400 μmol photons m ^-2 s ^-1 ) at 25° C. Mixed nutrition culture was performed on an orbital shaker at 120 rpm as conditions, and when 72 hours of culture had elapsed, cells were harvested to analyze cell density, zeaxanthin production and biomass.

(1) Effect of light intensity on cell density

As shown in FIGS. 1A to 1C , the change in cell density between the low light (FIG. 1A) and medium light (FIG. 1B) conditions was not significant, but the cell density was significantly increased at the high light (FIG. 1C). In high light compared to low light, wild-type Chlamydomonas strain showed increased cell density by 27%, ZEP strain by 33%, and dZL strain by 21%.

(2) Effect of light intensity on biomass

As shown in FIG. 2 , the difference in biomass between the strains in each luminous condition was not significant when culturing 72 hours elapsed, but the biomass increased as the luminosity of all three strains increased. Compared with low light intensity, 15 to 17% of biomass was improved at medium light intensity and 20 to 29% at high light intensity, and it was confirmed that cell density and biomass increased as the light intensity increased.

(3) Effect of light intensity on zeaxanthin production

However, as shown in FIGS. 3A to 3B , unlike an increase in biomass, an increase in intracellular zeaxanthin concentration was not observed as luminosity increased. Rather, it was analyzed that the content of zeaxanthin per dry weight was the highest in the cells of the low light condition, which had the lowest biomass, because the intracellular zeaxanthin concentration was the same in all light conditions.

Therefore, it is judged that the decrease in the content of zeaxanthin even though the biomass increased is because there is a factor that inhibits the growth of cells between the light conditions and the nutrients. 2 was performed.

Test Example 2. Analysis of zeaxanthin production ability according to the concentration of the medium composition

Under the low light conditions selected in Test Example 1 (60 μmol photons m ^-2 s ^-1 ), with the concentrations of nitrogen, phosphorus, and carbon in the TAP medium composition as variables, the ZEP/LCYE knockout mutant was cultured, and the zeaxanthin-producing ability was analyzed. did. Cells were cultured in the same manner as in Test Example 1, except that each of the compositions shown in Table 2 was used, and cell growth and zeaxanthin concentration were confirmed. Specifically, the absorbance of each medium cultured at 25 ° C., low light condition was measured at 750 nm every 12 hours to confirm the cell suit and shown in Figure 4, and the zeaxanthin concentration in the cells harvested every 24 hours of culture was confirmed in Figure 5 shown in

As a result of checking the cell growth every 12 hours after incubation, as shown in FIG. 4 , Opt1 had no difference in cell growth from the TAP medium, but Opt2 and Opt3 increased cell growth about 2 times compared to the TAP medium.

In addition, as shown in FIG. 5 , when cultured for 72 hours, Opt1 showed no difference in zeaxanthin productivity compared to TAP medium, similar to the cell growth results, and Opt2 was improved by 39% and Opt3 by 11%. In mixed nutrient culture at low light and Opt2, the ZEP/LCYE knockout mutant produced zeaxanthin at a concentration of 6.84 mg/L and biomass of 1.27 g/L, which was 30% and 79% improved compared to TAP culture, respectively.

However, in the case of OPT2 medium, cell normality increased and total zeaxanthin production increased, but the zeaxanthin content per cell unit was rather lower than that of TAP medium, which rather inhibited the production of zeaxanthin among medium components. Based on the OPT2 medium composition in Table 2 above, cell growth and zeaxanthin productivity were reconfirmed at low, medium, and high brightness with nitrogen (N) and phosphorus (P) content conditions as variables. experiment was performed.

Test Example 3. Confirmation of cell growth and zeaxanthin production according to nitrogen and phosphorus content

As shown in Tables 3 and 4 below, in a composition containing nitrogen as N25, N50, N75, and N100, a medium containing phosphorus at concentrations of P25, P50, P75, and P100, respectively, was prepared, thereby creating a total of 16 experimental media. For each experimental medium, in the same manner as in Experimental Example 1, ^{cells were inoculated into 20 mL medium in a 100 mL flask at 10 6} cells mL ^-1 , and continuously at 25° C. and each light condition on an orbital shaker at 120 rpm. white fluorescent light (low-light also: 60μmol photons m ^-2 s ^-1, heavy mineral Fig: 200μmol photons m ^-2 s ^-1, or high-intensity: 400μmol photons m ^-2 s ^-1) the mixture was incubated with nutritional research.

Media component 1L standard TAP Opt2 N25 N50 P25 P50 P75 P100 P25 P50 P75 P100 80x FBS mM NH ₄ Cl 7.5 9.375 11.25 CaCl ₂ 2H ₂ O 0.3375 0.421875 0.50625 MgSO ₄ 7H ₂ O 0.4 0.5 0.6 400X KPO ₄ mix mM K ₂ HPO ₄ 0.62 0.775 0.93 1.085 1.24 0.775 0.93 1.085 1.24 KH ₂ PO ₄ 0.41 0.5125 0.615 0.7175 0.82 0.5125 0.615 0.7175 0.82 200X Trace mineral μM EDTA2H ₂ O 135 FeSO ₄ 7H ₂ O 18 ZnSO ₄ 7H ₂ O 75 H ₃ BO ₃ 185 MnCl ₂ 4H ₂ O 26 CuCl ₂ 2H ₂ O 6.5 Na ₂ MoO ₄ 2H ₂ O 5.5 CoCl ₂ 6H ₂ O 6.5 Glacial acetic acid 1ml 2ml 2ml Tris 2.42g pH in the culture medium 7.2

Media component 1L standard TAP Opt2 N75 N100 P25 P50 P75 P100 P25 P50 P75 P100 80x FBS mM NH ₄ Cl 7.5 13.125 15 CaCl ₂ 2H ₂ O 0.3375 0.590625 0.675 MgSO ₄ 7H ₂ O 0.4 0.7 0.8 400X KPO ₄ mix mM K ₂ HPO ₄ 0.62 0.775 0.93 1.085 1.24 0.775 0.93 1.085 1.24 KH ₂ PO ₄ 0.41 0.5125 0.615 0.7175 0.82 0.5125 0.615 0.7175 0.82 200X Trace mineral μM EDTA2H ₂ O 135 FeSO ₄ 7H ₂ O 18 ZnSO ₄ 7H ₂ O 75 H ₃ BO ₃ 185 MnCl ₂ 4H ₂ O 26 CuCl ₂ 2H ₂ O 6.5 Na ₂ MoO ₄ 2H ₂ O 5.5 CoCl ₂ 6H ₂ O 6.5 Glacial acetic acid 1ml 2ml 2ml Tris 2.42g pH in the culture medium 7.2

The difference in cell growth according to time was confirmed by measuring the OD value of each medium at 750 nm at 24 hours, 48 hours and 72 hours after incubation, and at 48 hours (2 days) and 72 hours (3 days), each experimental group Cell biomass and intracellular zeaxanthin content were confirmed.

The results of culturing in a low light condition are shown in FIGS. 6A and 6B and FIGS. 7A to 7D . As shown in Figures 6a and 6b, it was confirmed that the cells grew up to the 3rd day through the increase in OD value and biomass up to 72 hours in all medium conditions during low-light culture. In addition, it was confirmed that the content of zeaxanthin also increased until the 3rd day as shown in FIGS. 7a to 7d. The treatment group showing the highest zeaxanthin production was the best as the 3rd day after culturing in N25/P100 showed a zeaxanthin content of 8.46±0.75 mg/L.

The results of culturing in medium light conditions are shown in FIGS. 8A and 8B and FIGS. 9A to 9D . As shown in Figures 8a and 8b, it was confirmed that OD and biomass were decreased on the 3rd day in the N25 and N50 medium composition during medium-light culture, and OD and biomass were maintained until the 3rd day in the N75 and N100 medium composition. . However, as shown in FIGS. 9A to 9D , it was confirmed that the zeaxanthin content was excellent at 8.00 mg or more per liter (L) in all media conditions of N50, N75, and N100. Therefore, it was confirmed that culturing in medium conditions of N50, N75, and N100 for less than 72 hours under medium light conditions was effective in increasing zeaxanthin production, and the treatment group showing the highest zeaxanthin production was 8.64±0.9 mg at N100/P25. /L, this was the highest zeaxanthin production among all culture conditions, with a 65% rise in TAP medium (low light, 3 days culture, 5.24 mg/L), OPT2 medium (low light, 3 days culture, 6.84 mg/L, 4 and 5) was also a result of increasing by 26%.

The results of culturing in high light conditions are shown in FIGS. 10A and 10B and FIGS. 11A to 11D . As shown in FIGS. 10a and 10b , only N75 increased OD and biomass up to 3 days, and N25, N50, and N100 showed OD and biomass maintained at 3 days. The production capacity of zeaxanthin also showed a tendency to slightly increase on the 3rd day only under the N75 condition, which was expected as a result of the increase in biomass. When cultured for 2 days in N100/P75 medium conditions, zeaxanthin production was 7.99±0.49 mg/L, indicating the highest viability.

SEQUENCE LISTING <110> Industry-University Cooperation Foundation Hanyang University Arca Eir Inc. <120> COMPOSITION FOR CULTURING CHLAMYDOMONAS MUTANTS AND CULTURE METHOD THEREOF <130> P19U10C1134 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 6693 <212> DNA <213> Chlamydomonas reinhardtii <400> 1 gcgacgcacg gctgggccaa attcgccaac ggcaggagac caaatcgatc gaggcgatct 60 tgcgaagttc tcggacaaat cgatcgcacc catagtgatt taagcattac atttgcccaa 120 ggcgtgagaa gtgcgaggcc cgaacggcta tgacgccaat gcgcagctta cgacatttaa 180 agcaaattat tcatacatca tacagcacgc ttatgtgaag aaagccagga ttttaggctc 240 tcgccccgat caagacgatc tccccattgc gaagttctcg gtttctttcc ggttcgcctg 300 ctccgtatga tttacctttg cgctacaaca gcgacttaaa cgacctacgt cgccttactg 360 tgtgcgcgta cgtgtttgta gctgtgagat agttttgtcc gcagcgtacc cgcaaataga 420 atgctcgcga gcacttacac gccctgtggc gttcgccagg tggcaggccg cacggttgca 480 gtgcccagca gcttggtcgc gccagtggca gtcgctcggt cgctggggtt ggcgccctac 540 gtccctgtat gtgagccttc tgcggcgctt ccggcctgcc agcagcctag cgggcgtcgt 600 catgttcaga ctgctgccac tctccgtgcc gacaacccca gctcggtcgc gcagctggtg 660 catcagaatg gaaaggggat gaaggttatt atcgccggcg cgggcatcgg cggcctggtg 720 ctagccgttg cacttctgaa gcagggcttc caggttcagg tctttgagcg cgacctgacg 780 gccatccgcg gcgagggcaa gtaccgtgga cccatccagg tgcgttcgcc ggaacaccaa 840 cgcgcttgtt tttgctgtgc cgcgaccat aactaggcct tatcttgagg tgttagcatg 900 tttagccagc gttggatctg tgtggcgagg ttggggtgag aacccttcct gtgtacctgc 960 tcgggcgtac cttgtgcccc accgctgact ggcttactta atgacaaaac gcaggttcaa 1020 agcaatgcgc tcgctgcgct ggaggctatc gatcccgagg tggccgcgga ggtgctgcgc 1080 gagggctgca tcactggcga ccgtatcaac gggctctgcg acggcctgac tggcgagtgg 1140 tgagtaggca atccagctgt gcatccagtc gcgcggttgc ggaggtcgtc tcgggaaacg 1200 cgacgtggcg tccactcgcc caaggagtgg tctcccgcag cgtggtctcc cgcagctcgg 1260 gtgcaacacc ctgccccctg ccgcgagcgc gctgcgcttg cttatgttgc gcagcggtgt 1320 gagttacaac agcttctgtt gaagagctgt catacgaagc acggcgcgct gtggcgctgc 1380 agccgtgctg tggaaactcc aacacctcca ccgccagcct gcgcacgcac acgcaataca 1440 ctcgcctcgt gtgccccctc ctcacacaac ggcatgtgac actcagtttt aactcttatt 1500 ttgacagctg agagctacac gcttgggtga atggggaggt ccttgatgtt tcgttgcact 1560 ccgtggctcc ggagtccgtg cggaccgtca cccacaaatg ggagcgcacg gctttcttgt 1620 gctgtctgcc ccgttagcca ctaactgcga atgaccttga cagtttactt tgctattttt 1680 ccttccaggt acgtcaagtt cgacacgttc cacccggcgg tcagcaaggg cctgccggtg 1740 acccgcgtca tcagccgcct cacgctgcag cagatcctgg ccaaagccgt ggagcggtga 1800 gccgtgcgcg cggtgtgatg gctttagcgt cagtgctagc atgggggttg gtgggtggta 1860 atcgcggcgc ccatggccgg gtagcagcgg ccgaaagctg gcgcagagcg cgcgttggac 1920 aagcggtcct gttgccggta tgggcacgag cagggcgctg gtgcgggcaa agggcagagt 1980 ggagttgcag agcagcgctg gcgtcggctg tgcgctctcc aaatggcctc gtggcattct 2040 gacgggacac atcctggaaa atagtagcgc acccaactgc tggtggctcc tcgtacaatc 2100 cccccaattt acaatcgctc gttctggctc gcagctacgg cggccccggc accatccaga 2160 acggctgcaa cgtgaccgag ttcacggagc gccgcaacga caccaccggc aacaacgagg 2220 tgagagcgtg ctaagaagag catgcacgtg gagcgtgtaa aattgtgtgg cctgaagcgg 2280 cagtgcctgc ggcatggact aggtggttgc agcatgctgc gcgcgtgggt tgccggtcag 2340 gaaaccgccg gaccgagccg cgcagattca gtcaggagcg gattaggaag tttgaaaaac 2400 agggttcgga gtgtgcaagc gggctcagga gctgtggtgc ctttctacac cggtcgccct 2460 accaggcacc cactgaaact gtaaaaccgt tgctgcgccg gcgatgccct ctacttcact 2520 aggtgactgt gcagctggag gacgggcgca cgtttgcggc cgacgtgctg gtgggcgccg 2580 acggcatctg gtccaagatc cgtaagcagc tcattggcga gaccaaggcc aactacagcg 2640 ggtacacctg ctacaccggt gagattattg accttcaagt tggaaggagg gagcgggggg 2700 agcggaatgg aaggaagcag cgtggacggg gcgcacggag gggaggggac tgcgggtcat 2760 agcgccgcct tgcggggcgt gaggagtgtt gggcggatat tcagttttct ttgcccaaga 2820 tcttcccaca atccgcgtgt gtctgacgcg ggatgtggcc cctgctgcca tggcttcgca 2880 ggcatctcgg actttacgcc ggcggacatt gcattgtgg gctaccgcgt gttcctgggc 2940 aacggccagt actttgtcag cagcgacgtg ggcaacggca agatgcagtg gtgagcggcg 3000 gcgggcgggc gagcgagggc tgcggggtct ggagggtgtg taccgggcgg aagggagggg 3060 aagggagggg aagggaaggc aggatgcagg cgagggcagg atgtgatggt gggaagaggg 3120 cgtggcgagc agcaactgga aaggtggtgg gtaaaaaaat ggtccatgaa tatggctcgg 3180 tacagttcaa agcatggaaa tggaacccgc cgtctgctgc accatgggcg tgagcgggga 3240 gtacgcgact cctggacagc cgtaacaatg cggatggcct caacaagcca ggagcggcac 3300 gaacccagct cacgagcgca cagcgtgcca ggacggcggc cggcaaggat gaaatgtttt 3360 tcctaatata aatgcggact cctgacgcat tatatccatt ttgccactga gccaaagaca 3420 catatataca cgtgcgccgc cgtcctgcgc cacagccgcc tagcgctccg gccgcgcccg 3480 gttccctcgg cgtcatgcgc tggagccccc tcgcaccctg caccgcaaag cccatcaaca 3540 ccacactcgt ccccacaccg cgagtcaccg ccactgcact cgctgtccct caacccgtca 3600 caatctcgcc gacacgcgat aacgaaccca cgcaggtacg gcttccacaa ggagccgtct 3660 ggcggcaccg accccgaggg cagccgcaag gcgcgcctgc tgcagatctt tggccactgg 3720 aacgacaacg tggtggacct gatcaaggcc acgcccgagg aggacgtgct gcgccgcgac 3780 atctttgaca ggtacggaaa aagggagagc ggggtggctg gagggcggga aagggcgaag 3840 gggcggagaa agaaatgact aggggatggt gttcatttgt gggattgaga ggggtccgcg 3900 gatcccggca gagggcgcca gtggcaaggc gtgggagtcg cggggcggac aatgctgggc 3960 caggggcgcc tagtcacccc gggacactgt ctcagtatgc cgccgtcccg gccgcgccgc 4020 acaggccgcc catcttcacc tggagcaagg gccgcgtggc cctgctgggc gacagcgcgc 4080 acgccatgca gcccaacctg ggccagggcg gctgcatggc cattgaggac gcctacgagc 4140 tggccatcga cctcagccgc gccgtgtccg acaaggccgg aaacgcggcg gcggtggacg 4200 tggagggcgt gctgcgcagc taccaggaca gccgcatttt gcgcgtcagc gccattcacg 4260 gcatggcggg tgagagctgc aaccagcgta gtcgggctgg gctgctgtgg gcagggtcgg 4320 gttgggttgg gcgcacgtgg gcggcgagtg tatgtgcagt gtgacgtgca cactatcata 4380 atactttatg ctcaccgcac cgcgccgcgc cgcaccacgc gccacaggca tggctgcctt 4440 catggccagc acctacaagt gctacctggg cgagggctgg agcaagtggg ttgaggggct 4500 gcgcatcccg caccccggcc gcgtggtggg gcggctggtg atgctgctca ccatgcccag 4560 cgtgctggag tgggtgctgg gcggcaacac cgaccacgtg gcgccgcacc gcaccagcta 4620 ctgctcgctg ggcgacaagc ccaaggtgag cggctgccgg gctgggggg ggtggaggga 4680 gaggaggagg attgcgggga gacgagggag ggcaaggcag gcgctgcctt cgtggatgca 4740 ccgccccgtc gttagcagga cctcaggaac tcgtccccaa aaccacaaca gaacccccaa 4800 tatcgcctct tccttcactg cttgtcacgc ctggtccgcc gaccgcaggc tttccccgag 4860 agccgcttcc ccgagttcat gaacaacgac gcctccatca tccgctcctc ccacgccgac 4920 tggctgctgg tggcggagcg cgacgccgcc acggccgccg ccgccaacgt gaacgccgcc 4980 accggcagca gcgccgccgc ggccgccgcc gccgacgtga acagcagctg ccagtgcaag 5040 ggcatctaca tggcggactc ggcggccctg gtgggccgct gcggcgccac ctcgcgcccc 5100 gcgctggccg tggacgacgt gcacgtcgcc gagagtcacg cgcaggtctg gcgcggcctc 5160 gccggcctcc ccccctcctc gtcgtccgcc tccaccgccg ccgcctctgc gtccgccgcc 5220 tcctctgccg ccagcggcac cgccagcacc ctgggcagct cggagggcta ctggctccgc 5280 gacctgggca gcggccgcgg cacctgggtc aacggcaagc gcctgcccga cggcgccacg 5340 gtgcagctgt ggcccggcga cgcggtggag ttcggccggc accccagcca cgaggtgttc 5400 aaggtgaaga tgcagcacgt gacgctgcgc agcgacgagc tcagcggcca ggcctacacc 5460 acgctcatgg tgggcaagat ccggaacaac gactacgtca tgcccgagtc gcggccggac 5520 ggcggcagcc agcagccggg ccgcctggtg acggcttaag cggcgccgtg cgtaagggcc 5580 ggcttacggg ggcggcagtg tcgctgtgga gggatggtct ggggtgggag gaatgggagg 5640 agagcggcgg gagcccgagg agcggagcgc tggaggcttg cggagcggca gcttgggaag 5700 agctgcggag agaggaagga gcgcagggcg cttggagcac gcgccagatt acgatcacgg 5760 cagcgcgagg cgcgcgtctg acttcgaagt ggtaaggaag atttcatgta tgattgcgtc 5820 gagggacacc gcaagtttta cgcgcggcgg agggagcctt ggggcataca acagtacgag 5880 cgggcgttgg tgagaaggtg gtcactccgt atgagaagat ggttactccg taccttcgtg 5940 agaagctgct gcgcacaagt tacgaaccta tctgtgtgga gagcccggta gtatatcagg 6000 ggcgagggtc atgaacgcga gtggcgagtc tgtgagcgcc aatttgttat gcggcataat 6060 ttcgcatcgg ggtattacgt ctacaaaatg ttgagctggc ttagcgcagg aggcaacacc 6120 tcaggcagaa tgtacgaatg tgtgcagaag ggcagagtca aggcagaggc ggagaagttg 6180 tcagggctgt gtgtggtttg gtcagggcgt ggctagatgg atatgagacc cgccgccgtc 6240 tccagattgt ggcggaggtg gaactctcgg cccccgcgcc agtccccgcg gccagcgcat 6300 cccgccatgc gggttgttgg ctggtgcatc gcgcggggtg tgctatgagt gtggaaacac 6360 tatgtcgcgt gtcgtgctga ggtctgttga gaggtttcgt cgtttgtgca tgtcctgtcc 6420 cggttggagt ttgagcgagg tggttcaaag tttttggatc gcgtgggaga gactgaaacg 6480 gtttggtgag aatggttgag acagaggttg ggcttggaaa ctggaggaga ggagcagcgt 6540 aactcgagga cgatgcagta gatgcaccac aacagttgtg gtgggcgcct ggagtaacac 6600 gcgtgccacc aacacgcaat tacagagatc cgtcatacag gagggatcat atgcgattta 6660 attttggttt tgcatttgta agacgttttc aca 6693 <210> 2 <211> 6652 <212> DNA <213> Chlamydomonas reinhardtii <400> 2 agagttgcac ataatccgct ggtgaaatct agcgtcggct ggggacgaag cgcccggtac 60 caaccacccc tgacatcgga gccagggttc tacgcgcat cagccccttt agctcgcttg 120 taggcgcact tacgcaacaa actcaatcct aagcaagcat tcgtagagcg ttgttggcta 180 ttcctggcca ctcgctgcac tcccactgca agcagggttg gttgcgagca taggtccgcg 240 catctgacaa tttttagtat agtcgggtat tcacttgtac cactgagcat aattagcagc 300 ataacagggc tgttgtgagc caggcgaggt accttctgtt tggtaccgtc tcgtcttccc 360 tgtagacgct tccagtcagg cgccaaacca aacaaaaatg cttcaccaga ttagcgcaaa 420 gcgctgcgga gcgagcggcg tcgtgcgtgg cgctgcgggc ctgcaagcct ttagcacaaa 480 gctcgtcgct cccttgccag cgcggcgagt ggcttgccag gttgtatcta cggagactga 540 ggcacctacc atcgtgcttg accggcccgg gaggaccgtt cagccggagc tctccattcc 600 agcggccctg aatcaagaaa tccgcgaggg gcactatgag gccgcgctgg tgaaggagca 660 ggtaactact cgcttgcgtt caggcccaat tgaacgcgcc ccaaatcgaa ttcggttcgt 720 tgtatgtgct caggcgaaca aggcggacgc ctctcaagcg gcaattgcgt ccgtattgaa 780 gcccgctgac accacggtca cagccgatgt gacaattgtt ggtgcgggcc cggctggcct 840 cttcctggct gcggagttgg gcaagcgggg catgagcgtg aacgtcctcg gtgagcggcc 900 gtcgtgtcaa acggacagcc cccccccccc cgcatgccct tcttgccacg caactgccgc 960 acggtcgctg ccaggcgcga cgcctgcatc taatggctct gtctcttgtc tcgcttgtca 1020 agtatcctga agctctgctg taatgccggg gcgtgtcttg ttgacagcgg ttcaatccaa 1080 atgcgtgcaa tcgctagtgt ggcccgcgtc catccactcc acacgcaacc agccagcgtg 1140 tctccgaggg actggcagac gcctgctgca tgccattgct cttgcagcgc tctgctgttg 1200 cacacatcag ccctcgcccc tgcgtacccc cgcggtggga ggcacggcca taatcaaacc 1260 caccccttcc gtgtcccatg aagccacacc agcgactgtg cagagacaca cacacacaca 1320 cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca 1380 cacacacgcg acaatgcctt atgctggact tacacacgca taatcccgca tataggcctg 1440 gacgtgccca ttgtgaacaa ctacggcgtg tggacggacg agttcgaggc cctgggcctg 1500 acccatacac tggagtgctc ctggcctgat gccgtgtgct acttcggcga gggcaaccag 1560 gtgcggggtg gggcggggcg tggtggagcg gggaggcaga gggcagacag ggttgcgtgc 1620 atcttgctgt ggtacgtagg gacctcttgt acgctcttgc atgtagcgaa acggtgtggc 1680 aggggggggct gcatgagtag cggcgcgagt gcacaagagt ccaacgagca gcagccctgg 1740 cgcccgtgcc agcgtcctgg cttcttacac gcgagtcttt tcttgttgtt tgtccaggtg 1800 agcgtgggcc gcggctacgg ccgtgtgagc cgccggctgc tgcgtgagca cctgctcaag 1860 atctgcgagg cggcgggcgt gcgcttcagc agcgccgagg tggcggacat caaggtgcgc 1920 gggcggggcg gggtggcggc gcagggtgtg gtggcgggtt gtggtctggt gtgcagttga 1980 ccgcaaaggg tctggctgac tggctggctg gtgcgagtac ggtgcttgag ctggatgagt 2040 cggccgactg gcaggagctt cactcactta ctgccatact gccaccccgc ccctgtccgc 2100 accatgcata ggttgtggag gagggcaagc tgactcagct caccaccaag gagggcgccg 2160 tgtacagcag caggtggggg aggggggggc ggcgggcagc agaagcggcc atgctaggca 2220 ctggctgtgg tgggggctgc gctgtagcaa cagcaatagc ggcatcagca gcagctgtgg 2280 gatacaaaaa gcgcacgtgc ctggctacgt ggcgctcgcg catgtcgtgc tctgacacct 2340 tgcgccctct tcacgcaacc ctcaggctga ccactctggc tgcgggtgcc gctgccggca 2400 agttcctgcg gtgagtgggt cgtccgcagc cctgggacag tgtctggtca cgtcacaaag 2460 atgtgaacgc cgtcttgctc tgccctgtgc cacctcccgg ctggagcctc ctccccactc 2520 ctcgcttctc acctcttctc cctctgccca cgcactccag ctatgaggag gacgcgccca 2580 ttgtcgccgc acaaaccgcg tacggcatcg aggcggaggt ggagggctat gacgccgcct 2640 acccgggcga cctcatgacc ttcatggatt tcaggtgcgg gcatgtgggc ttgtgtgtgg 2700 gtgcggatgg ttccaacacc aactaaacca agaccccagg ggggttgtag aggttagcag 2760 aatgcttgga gcatcaggcg agcgagcggc acgccaggga gcgaaagagc gagcaccaac 2820 agcgacgtga tgcacggtgc cggcgtactg gcggggaggg ggcgcgggca cgggggccga 2880 aacagccatc catgaatgcc ggggggcaaa tggtggcgtc gctgtggcct gacctggtcc 2940 cgctgtgttc ggccggctcc ccctcgcggc aggcgccacc acaccggcct gcacgacggc 3000 acggcgctca aattcgtgcc ggggcgccat cccaactcgg gcgacggcac gtggggcagc 3060 agcgaggagt gcccctcctt cctgtacgcc atgccgctgg gcggcaagcg cgtgttcctg 3120 gaggagacct gcctggtggc caagccggcg ctgccgttcg cggtgctcaa gcgccgcctc 3180 acacgccgcc tcaaggccat gggcatctcc gtgaccaagg tgtgtgcgga gaggggagga 3240 gaggggaggg agggagggag ggaggggagg gatagcgtct gaaagttcgt agaaacaaca 3300 ggccgatgtg accgaagcat gtgccctgac tccgcgccgc tctcacacgc atgcctggat 3360 cgccctcaga tccacgagga ggagtggagc tacatccctg tgggcggccc cctgccgctg 3420 cccgaccagg tgcgtgccgg ccagcgtaca gcactgtgtg gctttcggcg ggggctgtgc 3480 agagcgcgca catgagcatg cctggtgttt cctggtggta atgtgcgtaa tgtgcgctca 3540 gctgtcacgt caccctcgct cttccctcac ccgctgtcgc ctccacagtc ggtgacggcg 3600 ttcggcgccg ctgccaacct ggtgcacccc gccaccggct tcagcgtcag ccgctccttc 3660 cgtgaggcgc cgcaggtgcg tgcgggcacc gtgggtgggc acagggcggg agggcactgg 3720 gcacgcgggc actgggcagg cgggagaggg catggttgca ggcgcctgag gaatgagggt 3780 ttgggaggag atgcggggtc gggggccgtt gtggtggctg gggcttgcag ggcgaaagat 3840 gaggcggggg caggtcgttc gggggcctca aggatgtcag aggccgagcg gtccggtggg 3900 tgctccttat tggacgcgtt ctcctctctt cgcccctcct ctcctcattc ctcctttcct 3960 cacccctcct ccatcccccc ctgctgcaac tgtacctgcc tacgacttca cttcttaatt 4020 aatcatgaat gtaacccccc ctctcctcac ccccctcctc gccccgtcac taggtggcgg 4080 acgagctgca ggccgcgctg cgcgacgggc tggacgtgtc cgccgccagc cgccgcgtgt 4140 gggagcgcct gtggccgcag gagaagcgca cgcaggcctc cttccacgtg ttcggcatgg 4200 agctgctcgc cacgctggac ctcaacgcca ccaacgactt cttcaacacc ttcttccgcc 4260 tgccgccctt ctactggcgc ggcttcctgg cctctacgct cagcagcggg cagctgattg 4320 cctttgcgct ggtggtgttc acgctggcgc cctggaacat caagtacaag ctcattgagc 4380 atctgatcac gggtgggtgg aggaagggct gtgggtgcgg gtgcgggtgc agggggttgg 4440 cgggtgcggg tgcgggtgca ggtgcgggtg cggatgcgga tgcgggtccg ggtgcgggtg 4500 cgggtgcggg tgcaggggct ggcgggcgca ggggctggcg tctgtgtgtg tgggggggga 4560 ctgtagttta tctaaaccca actaaaccaa ggcctggcca gcccctaggg gggcagcggg 4620 tggcgggaag agggcaggag gcagtgggct gctgtcccaa gtgagccgaa gttcagcatg 4680 gggggcctgg aagggcggga tctggggtgg aagggcggcg gggcgggcag gtggggatgt 4740 ggcggcgttg agtctgcaag catgacctgt gtaggtgaag ctcccacgtc tgacgcttcc 4800 ctccccgaca ccaacgtcat gtctggcccc gcacgcagac cccgccggcg gctacctcat 4860 ccgtgcctac caggcgcagt gggacagctc tcaacaggcg ccgtccgccg cgactgccgc 4920 cgccgtgctg ctgctcagca acgagctgat gacgcgcgcg gtgctcaagg cgctggagag 4980 cggcagcgcg taagcactgc gttattggtc cccgtccccc ctgaatgaag gatgcacgag 5040 ggaggatggg gcaggagcat cagcagcagg agcaccagca gcggcagggg caagagcggc 5100 aggggcagga gcagacagta gacagaggcc gggctggcgg aaggattgga ggcgcgcgcg 5160 gttgcggcct tgtgatgtgc gcgtttgagc cttcgacacc gcatgcttga gacaatttga 5220 ttgcagcgga gaggaacgaa gaactttatc gttggaaagg gccgcccacc ggcggccgca 5280 tgacgcagca gcagcagcag tccaggagct gcggcggctg cagaagcagc aggagagcag 5340 cgctagctat gtcgtggtgg cggctgaaag gggggagcgg gtgcttaagt agctaccttt 5400 tgcccaggta acgtggctgt gcattgacgc cgctgcgagt ccgttgggtc cagcaacgcg 5460 ctgtcgtcgt tggtgcagtt gagattctgc ttcataggga tagtcacagc gagaggcatc 5520 gcagcgcgca ggctcttgat gcgcaggtgt gtggtgggct agaattcgtg ggggacaaca 5580 ctgttggttt gcatgtgcta tgtgtactga tggaatatgg tttgggcagc ttacacccgt 5640 acatgccaat ggtgtattca ctacgtcagc gtaagcggac cagcgatagg aatgcagcac 5700 ggtatggagg actaagcagg ggagtggtgg tgccattggc tggcgcagtg tgattgtcaa 5760 ccaaaagaga gcggccgtga gcgagatttt cgccccgatg acatcccggg gacggtacat 5820 gctttgtttc tgtgtagctt gtggcgtatg cgttgtggcc acgtacatgt gtgttgtttg 5880 tttcgtgtgt gtgcgtgtca tgtgattgac ctgttgcagt gcagatgcag ttgtttgctt 5940 ccagagctgt gtcaactagc ctggctcgcg cgccggggcg tggccaccag ggggcgtgat 6000 tgtgtgaagg ggctggaagg ggagccaggc agctctattg gagactccgc agcgcgggta 6060 ggtgcggcag gaagggcaca acgctgaaca ttggcggcaa agcgagcctg gaagtgcgag 6120 caatgttgac gaggattagg agagcgagcg agttgaccaa gaccagagaa ggccagaagg 6180 gagaagttgg ttgcagcatg gcgtgttgat gggatgggtc gatggggcgg ccccaggtag 6240 ctcgtgctgc tgcttggggc aatacgaccc aagtgctgaa gtgcagcgaa caggaagcgg 6300 cagcttgtca gcgcgcacgc tgctttgcgc tcgttgcggg aggacgccgt gtgcccgcgc 6360 aagctgcggg cccttctgca cttggtgccc gtaacattca tgttcaaccg ccgctctcct 6420 ggctgtcacc accagggatg tcccatggca ccaggactca ccagggctgt tgcacgagcg 6480 ccgggtgcgc gggcaagcag ggcacctcag gttggaggga atgaaatatg gtatgcgaca 6540 gggaccacaa gggtgtgggc cattcctggc cggtccatcc gcagggtggg ccgcgtgcag 6600 agccacctgt gattggttgt gctgccagct ccatgcgata gtaagatcgg gc 6652 <210> 3 <211> 42 <212> DNA <213> <220> <223> mutated ZEP gene sequence of Chlamydomonas reinhardtii <400> 3 gaaattaata agactcatta tattccggcg aacgcacctg ga 42 <210> 4 <211> 6735 <212> DNA <213> <220> <223> mutated ZEP gene sequence of Chlamydomonas reinhardtii <400> 4 gcgacgcacg gctgggccaa attcgccaac ggcaggagac caaatcgatc gaggcgatct 60 tgcgaagttc tcggacaaat cgatcgcacc catagtgatt taagcattac atttgcccaa 120 ggcgtgagaa gtgcgaggcc cgaacggcta tgacgccaat gcgcagctta cgacatttaa 180 agcaaattat tcatacatca tacagcacgc ttatgtgaag aaagccagga ttttaggctc 240 tcgccccgat caagacgatc tccccattgc gaagttctcg gtttctttcc ggttcgcctg 300 ctccgtatga tttacctttg cgctacaaca gcgacttaaa cgacctacgt cgccttactg 360 tgtgcgcgta cgtgtttgta gctgtgagat agttttgtcc gcagcgtacc cgcaaataga 420 atgctcgcga gcacttacac gccctgtggc gttcgccagg tggcaggccg cacggttgca 480 gtgcccagca gcttggtcgc gccagtggca gtcgctcggt cgctggggtt ggcgccctac 540 gtccctgtat gtgagccttc tgcggcgctt ccggcctgcc agcagcctag cgggcgtcgt 600 catgttcaga ctgctgccac tctccgtgcc gacaacccca gctcggtcgc gcagctggtg 660 catcagaatg gaaaggggat gaaggttatt atcgccggcg cgggcatcgg cggcctggtg 720 ctagccgttg cacttctgaa gcagggcttc caggttcagg tctttgagcg cgacctgacg 780 gccatccgcg gcgagggcaa gtaccgtgga cccatcgaaa ttaataagac tcattatatt 840 ccggcgaacg cacctggaca ggtgcgttcg ccggaacacc aacgcgcttg tttttgctgt 900 gccgcgacca tgaactaggc cttatcttga ggtgttagca tgtttagcca gcgttggatc 960 tgtgtggcga ggttggggtg agaacccttc ctgtgtacct gctcgggcgt accttgtgcc 1020 ccaccgctga ctggcttact taatgacaaa acgcaggttc aaagcaatgc gctcgctgcg 1080 ctggaggcta tcgatcccga ggtggccgcg gaggtgctgc gcgagggctg catcactggc 1140 gaccgtatca acgggctctg cgacggcctg actggcgagt ggtgagtagg caatccagct 1200 gtgcatccag tcgcgcggtt gcggaggtcg tctcgggaaa cgcgacgtgg cgtccactcg 1260 cccaaggagt ggtctcccgc agcgtggtct cccgcagctc gggtgcaaca ccctgccccc 1320 tgccgcgagc gcgctgcgct tgcttatgtt gcgcagcggt gtgagttaca acagcttctg 1380 ttgaagagct gtcatacgaa gcacggcgcg ctgtggcgct gcagccgtgc tgtggaaact 1440 ccaacacctc caccgccagc ctgcgcacgc acacgcaata cactcgcctc gtgtgccccc 1500 tcctcacaca acggcatgtg acactcagtt ttaactctta ttttgacagc tgagagctac 1560 acgcttgggt gaatggggag gtccttgatg tttcgttgca ctccgtggct ccggagtccg 1620 tgcggaccgt cacccacaaa tgggagcgca cggctttctt gtgctgtctg ccccgttagc 1680 cactaactgc gaatgacctt gacagtttac tttgctattt ttccttccag gtacgtcaag 1740 ttcgacacgt tccacccggc ggtcagcaag ggcctgccgg tgacccgcgt catcagccgc 1800 ctcacgctgc agcagatcct ggccaaagcc gtggagcggt gagccgtgcg cgcggtgtga 1860 tggctttagc gtcagtgcta gcatgggggt tggtgggtgg taatcgcggc gcccatggcc 1920 gggtagcagc ggccgaaagc tggcgcagag cgcgcgttgg acaagcggtc ctgttgccgg 1980 tatgggcacg agcagggcgc tggtgcgggc aaagggcaga gtggagttgc agagcagcgc 2040 tggcgtcggc tgtgcgctct ccaaatggcc tcgtggcatt ctgacgggac acatcctgga 2100 aaatagtagc gcacccaact gctggtggct cctcgtacaa tccccccaat ttacaatcgc 2160 tcgttctggc tcgcagctac ggcggccccg gcaccatcca gaacggctgc aacgtgaccg 2220 agttcacgga gcgccgcaac gacaccaccg gcaacaacga ggtgagagcg tgctaagaag 2280 agcatgcacg tggagcgtgt aaaattgtgt ggcctgaagc ggcagtgcct gcggcatgga 2340 ctaggtggtt gcagcatgct gcgcgcgtgg gttgccggtc aggaaaccgc cggaccgagc 2400 cgcgcagatt cagtcaggag cggattagga agtttgaaaa acagggttcg gagtgtgcaa 2460 gcgggctcag gagctgtggt gcctttctac accggtcgcc ctaccaggca cccactgaaa 2520 ctgtaaaacc gttgctgcgc cggcgatgcc ctctacttca ctaggtgact gtgcagctgg 2580 aggacgggcg cacgtttgcg gccgacgtgc tggtgggcgc cgacggcatc tggtccaaga 2640 tccgtaagca gctcattggc gagaccaagg ccaactacag cgggtacacc tgctacaccg 2700 gtgagattat tgaccttcaa gttggaagga gggagcgggg ggagcggaat ggaaggaagc 2760 agcgtggacg gggcgcacgg aggggagggg actgcgggtc atagcgccgc cttgcggggc 2820 gtgaggagtg ttgggcggat attcagtttt ctttgcccaa gatcttccca caatccgcgt 2880 gtgtctgacg cgggatgtgg cccctgctgc catggcttcg caggcatctc ggactttacg 2940 ccggcggaca ttgacattgt gggctaccgc gtgttcctgg gcaacggcca gtactttgtc 3000 agcagcgacg tgggcaacgg caagatgcag tggtgagcgg cggcgggcgg gcgagcgagg 3060 gctgcggggt ctggagggtg tgtaccgggc ggaagggagg ggaagggagg ggaagggaag 3120 gcaggatgca ggcgagggca ggatgtgatg gtgggaagag ggcgtggcga gcagcaactg 3180 gaaaggtggt gggtaaaaaa atggtccatg aatatggctc ggtacagttc aaagcatgga 3240 aatggaaccc gccgtctgct gcaccatggg cgtgagcggg gagtacgcga ctcctggaca 3300 gccgtaacaa tgcggatggc ctcaacaagc caggagcggc acgaacccag ctcacgagcg 3360 cacagcgtgc caggacggcg gccggcaagg atgaaatgtt tttcctaata taaatgcgga 3420 ctcctgacgc attatatcca ttttgccact gagccaaaga cacatatata cacgtgcgcc 3480 gccgtcctgc gccacagccg cctagcgctc cggccgcgcc cggttccctc ggcgtcatgc 3540 gctggagccc cctcgcaccc tgcaccgcaa agcccatcaa caccacactc gtccccacac 3600 cgcgagtcac cgccactgca ctcgctgtcc ctcaacccgt cacaatctcg ccgacacgcg 3660 ataacgaacc cacgcaggta cggcttccac aaggagccgt ctggcggcac cgaccccgag 3720 ggcagccgca aggcgcgcct gctgcagatc tttggccact ggaacgacaa cgtggtggac 3780 ctgatcaagg ccacgcccga ggaggacgtg ctgcgccgcg acatctttga caggtacgga 3840 aaaagggaga gcggggtggc tggagggcgg gaaagggcga aggggcggag aaagaaatga 3900 ctaggggatg gtgttcattt gtgggattga gaggggtccg cggatcccgg cagagggcgc 3960 cagtggcaag gcgtgggagt cgcggggcgg acaatgctgg gccaggggcg cctagtcacc 4020 ccgggacact gtctcagtat gccgccgtcc cggccgcgcc gcacaggccg cccatcttca 4080 cctggagcaa gggccgcgtg gccctgctgg gcgacagcgc gcacgccatg cagcccaacc 4140 tgggccaggg cggctgcatg gccattgagg acgcctacga gctggccatc gacctcagcc 4200 gcgccgtgtc cgacaaggcc ggaaacgcgg cggcggtgga cgtggagggc gtgctgcgca 4260 gctaccagga cagccgcatt ttgcgcgtca gcgccattca cggcatggcg ggtgagagct 4320 gcaaccagcg tagtcgggct gggctgctgt gggcagggtc gggttgggtt gggcgcacgt 4380 gggcggcgag tgtatgtgca gtgtgacgtg cacactatca taatacttta tgctcaccgc 4440 accgcgccgc gccgcaccac gcgccacagg catggctgcc ttcatggcca gcacctacaa 4500 gtgctacctg ggcgagggct ggagcaagtg ggttgagggg ctgcgcatcc cgcaccccgg 4560 ccgcgtggtg gggcggctgg tgatgctgct caccatgccc agcgtgctgg agtgggtgct 4620 gggcggcaac accgaccacg tggcgccgca ccgcaccagc tactgctcgc tgggcgacaa 4680 gcccaaggtg agcggctgcc gggctggggg ggggtggagg gagaggagga ggattgcggg 4740 gagacgaggg agggcaaggc aggcgctgcc ttcgtggatg caccgccccg tcgttagcag 4800 gacctcagga actcgtcccc aaaaccacaa cagaaccccc aatatcgcct cttccttcac 4860 tgcttgtcac gcctggtccg ccgaccgcag gctttccccg agagccgctt ccccgagttc 4920 atgaacaacg acgcctccat catccgctcc tcccacgccg actggctgct ggtggcggag 4980 cgcgacgccg ccacggccgc cgccgccaac gtgaacgccg ccaccggcag cagcgccgcc 5040 gcggccgccg ccgccgacgt gaacagcagc tgccagtgca agggcatcta catggcggac 5100 tcggcggccc tggtgggccg ctgcggcgcc acctcgcgcc ccgcgctggc cgtggacgac 5160 gtgcacgtcg ccgagagtca cgcgcaggtc tggcgcggcc tcgccggcct ccccccctcc 5220 tcgtcgtccg cctccaccgc cgccgcctct gcgtccgccg cctcctctgc cgccagcggc 5280 accgccagca ccctgggcag ctcggagggc tactggctcc gcgacctggg cagcggccgc 5340 ggcacctggg tcaacggcaa gcgcctgccc gacggcgcca cggtgcagct gtggcccggc 5400 gacgcggtgg agttcggccg gcaccccagc cacgaggtgt tcaaggtgaa gatgcagcac 5460 gtgacgctgc gcagcgacga gctcagcggc caggcctaca ccacgctcat ggtgggcaag 5520 atccggaaca acgactacgt catgcccgag tcgcggccgg acggcggcag ccagcagccg 5580 ggccgcctgg tgacggctta agcggcgccg tgcgtaaggg ccggcttacg ggggcggcag 5640 tgtcgctgtg gagggatggt ctggggtggg aggaatggga ggagagcggc gggagcccga 5700 ggagcggagc gctggaggct tgcggagcgg cagcttggga agagctgcgg agagaggaag 5760 gagcgcaggg cgcttggagc acgcgccaga ttacgatcac ggcagcgcga ggcgcgcgtc 5820 tgacttcgaa gtggtaagga agatttcatg tatgattgcg tcgagggaca ccgcaagttt 5880 tacgcgcggc ggagggagcc ttggggcata caacagtacg agcgggcgtt ggtgagaagg 5940 tggtcactcc gtatgagaag atggttactc cgtaccttcg tgagaagctg ctgcgcacaa 6000 gttacgaacc tatctgtgtg gagagcccgg tagtatatca ggggcgaggg tcatgaacgc 6060 gagtggcgag tctgtgagcg ccaatttgtt atgcggcata atttcgcatc ggggtattac 6120 gtctacaaaa tgttgagctg gcttagcgca ggaggcaaca cctcaggcag aatgtacgaa 6180 tgtgtgcaga agggcagagt caaggcagag gcggagaagt tgtcagggct gtgtgtggtt 6240 tggtcagggc gtggctagat ggatatgaga cccgccgccg tctccagatt gtggcggagg 6300 tggaactctc ggcccccgcg ccagtccccg cggccagcgc atcccgccat gcgggttgtt 6360 ggctggtgca tcgcgcgggg tgtgctatga gtgtggaaac actatgtcgc gtgtcgtgct 6420 gaggtctgtt gagaggtttc gtcgtttgtg catgtcctgt cccggttgga gtttgagcga 6480 ggtggttcaa agtttttgga tcgcgtggga gagactgaaa cggtttggtg agaatggttg 6540 agacagaggt tgggcttgga aactggagga gaggagcagc gtaactcgag gacgatgcag 6600 tagatgcacc acaacagttg tggtgggcgc ctggagtaac acgcgtgcca ccaacacgca 6660 attacagaga tccgtcatac aggagggatc atatgcgatt taattttggt tttgcatttg 6720 taagacgttt tcaca 6735 <210> 5 <211> 44 <212> DNA <213> <220> <223> mutated ZEP gene sequence of Chlamydomonas reinhardtii <400> 5 tagctctaaa acatccaggt gcgttcgccg gactatagtg agta 44 <210> 6 <211> 6737 <212> DNA <213> <220> <223> mutated ZEP gene sequence of Chlamydomonas reinhardtii <400> 6 gcgacgcacg gctgggccaa attcgccaac ggcaggagac caaatcgatc gaggcgatct 60 tgcgaagttc tcggacaaat cgatcgcacc catagtgatt taagcattac atttgcccaa 120 ggcgtgagaa gtgcgaggcc cgaacggcta tgacgccaat gcgcagctta cgacatttaa 180 agcaaattat tcatacatca tacagcacgc ttatgtgaag aaagccagga ttttaggctc 240 tcgccccgat caagacgatc tccccattgc gaagttctcg gtttctttcc ggttcgcctg 300 ctccgtatga tttacctttg cgctacaaca gcgacttaaa cgacctacgt cgccttactg 360 tgtgcgcgta cgtgtttgta gctgtgagat agttttgtcc gcagcgtacc cgcaaataga 420 atgctcgcga gcacttacac gccctgtggc gttcgccagg tggcaggccg cacggttgca 480 gtgcccagca gcttggtcgc gccagtggca gtcgctcggt cgctggggtt ggcgccctac 540 gtccctgtat gtgagccttc tgcggcgctt ccggcctgcc agcagcctag cgggcgtcgt 600 catgttcaga ctgctgccac tctccgtgcc gacaacccca gctcggtcgc gcagctggtg 660 catcagaatg gaaaggggat gaaggttatt atcgccggcg cgggcatcgg cggcctggtg 720 ctagccgttg cacttctgaa gcagggcttc caggttcagg tctttgagcg cgacctgacg 780 gccatccgcg gcgagggcaa gtaccgtgga cccatctagc tctaaaacat ccaggtgcgt 840 tcgccggact atagtgagta caggtgcgtt cgccggaaca ccaacgcgct tgtttttgct 900 gtgccgcgac catgaactag gccttatctt gaggtgttag catgtttagc cagcgttgga 960 tctgtgtggc gaggttgggg tgagaaccct tcctgtgtac ctgctcgggc gtaccttgtg 1020 ccccaccgct gactggctta cttaatgaca aaacgcaggt tcaaagcaat gcgctcgctg 1080 cgctggaggc tatcgatccc gaggtggccg cggaggtgct gcgcgagggc tgcatcactg 1140 gcgaccgtat caacgggctc tgcgacggcc tgactggcga gtggtgagta ggcaatccag 1200 ctgtgcatcc agtcgcgcgg ttgcggaggt cgtctcggga aacgcgacgt ggcgtccact 1260 cgcccaagga gtggtctccc gcagcgtggt ctcccgcagc tcgggtgcaa caccctgccc 1320 cctgccgcga gcgcgctgcg cttgcttatg ttgcgcagcg gtgtgagtta caacagcttc 1380 tgttgaagag ctgtcatacg aagcacggcg cgctgtggcg ctgcagccgt gctgtggaaa 1440 ctccaacacc tccaccgcca gcctgcgcac gcacacgcaa tacactcgcc tcgtgtgccc 1500 cctcctcaca caacggcatg tgacactcag ttttaactct tattttgaca gctgagagct 1560 acacgcttgg gtgaatgggg aggtccttga tgtttcgttg cactccgtgg ctccggagtc 1620 cgtgcggacc gtcacccaca aatgggagcg cacggctttc ttgtgctgtc tgccccgtta 1680 gccactaact gcgaatgacc ttgacagttt actttgctat ttttccttcc aggtacgtca 1740 agttcgacac gttccacccg gcggtcagca agggcctgcc ggtgacccgc gtcatcagcc 1800 gcctcacgct gcagcagatc ctggccaaag ccgtggagcg gtgagccgtg cgcgcggtgt 1860 gatggcttta gcgtcagtgc tagcatgggg gttggtgggt ggtaatcgcg gcgcccatgg 1920 ccgggtagca gcggccgaaa gctggcgcag agcgcgcgtt ggacaagcgg tcctgttgcc 1980 ggtatgggca cgagcagggc gctggtgcgg gcaaagggca gagtggagtt gcagagcagc 2040 gctggcgtcg gctgtgcgct ctccaaatgg cctcgtggca ttctgacggg acacatcctg 2100 gaaaatagta gcgcacccaa ctgctggtgg ctcctcgtac aatcccccca atttacaatc 2160 gctcgttctg gctcgcagct acggcggccc cggcaccatc cagaacggct gcaacgtgac 2220 cgagttcacg gagcgccgca acgacaccac cggcaacaac gaggtgagag cgtgctaaga 2280 agagcatgca cgtggagcgt gtaaaattgt gtggcctgaa gcggcagtgc ctgcggcatg 2340 gactaggtgg ttgcagcatg ctgcgcgcgt gggttgccgg tcaggaaacc gccggaccga 2400 gccgcgcaga ttcagtcagg agcggattag gaagtttgaa aaacagggtt cggagtgtgc 2460 aagcgggctc aggagctgtg gtgcctttct acaccggtcg ccctaccagg cacccactga 2520 aactgtaaaa ccgttgctgc gccggcgatg ccctctactt cactaggtga ctgtgcagct 2580 ggaggacggg cgcacgtttg cggccgacgt gctggtgggc gccgacggca tctggtccaa 2640 gatccgtaag cagctcattg gcgagaccaa ggccaactac agcgggtaca cctgctacac 2700 cggtgagatt attgaccttc aagttggaag gagggagcgg ggggagcgga atggaaggaa 2760 gcagcgtgga cggggcgcac ggaggggagg ggactgcggg tcatagcgcc gccttgcggg 2820 gcgtgaggag tgttgggcgg atattcagtt ttctttgccc aagatcttcc cacaatccgc 2880 gtgtgtctga cgcgggatgt ggcccctgct gccatggctt cgcaggcatc tcggacttta 2940 cgccggcgga cattgacatt gtgggctacc gcgtgttcct gggcaacggc cagtactttg 3000 tcagcagcga cgtgggcaac ggcaagatgc agtggtgagc ggcggcgggc gggcgagcga 3060 gggctgcggg gtctggaggg tgtgtaccgg gcggaaggga ggggaaggga ggggaaggga 3120 aggcaggatg caggcgaggg caggatgtga tggtgggaag agggcgtggc gagcagcaac 3180 tggaaaggtg gtgggtaaaa aaatggtcca tgaatatggc tcggtacagt tcaaagcatg 3240 gaaatggaac ccgccgtctg ctgcaccatg ggcgtgagcg gggagtacgc gactcctgga 3300 cagccgtaac aatgcggatg gcctcaacaa gccaggagcg gcacgaaccc agctcacgag 3360 cgcacagcgt gccaggacgg cggccggcaa ggatgaaatg tttttcctaa tataaatgcg 3420 gactcctgac gcattatatc cattttgcca ctgagccaaa gacacatata tacacgtgcg 3480 ccgccgtcct gcgccacagc cgcctagcgc tccggccgcg cccggttccc tcggcgtcat 3540 gcgctggagc cccctcgcac cctgcaccgc aaagcccatc aacaccacac tcgtccccac 3600 accgcgagtc accgccactg cactcgctgt ccctcaaccc gtcacaatct cgccgacacg 3660 cgataacgaa cccacgcagg tacggcttcc acaaggagcc gtctggcggc accgaccccg 3720 agggcagccg caaggcgcgc ctgctgcaga tctttggcca ctggaacgac aacgtggtgg 3780 acctgatcaa ggccacgccc gaggaggacg tgctgcgccg cgacatcttt gacaggtacg 3840 gaaaaaggga gagcggggtg gctggagggc gggaaagggc gaaggggcgg agaaagaaat 3900 gactagggga tggtgttcat ttgtgggatt gagaggggtc cgcggatccc ggcagagggc 3960 gccagtggca aggcgtggga gtcgcggggc ggacaatgct gggccagggg cgcctagtca 4020 ccccgggaca ctgtctcagt atgccgccgt cccggccgcg ccgcacaggc cgcccatctt 4080 cacctggagc aagggccgcg tggccctgct gggcgacagc gcgcacgcca tgcagcccaa 4140 cctgggccag ggcggctgca tggccattga ggacgcctac gagctggcca tcgacctcag 4200 ccgcgccgtg tccgacaagg ccggaaacgc ggcggcggtg gacgtggagg gcgtgctgcg 4260 cagctaccag gacagccgca ttttgcgcgt cagcgccatt cacggcatgg cgggtgagag 4320 ctgcaaccag cgtagtcggg ctgggctgct gtgggcaggg tcgggttggg ttgggcgcac 4380 gtgggcggcg agtgtatgtg cagtgtgacg tgcacactat cataatactt tatgctcacc 4440 gcaccgcgcc gcgccgcacc acgcgccaca ggcatggctg ccttcatggc cagcacctac 4500 aagtgctacc tgggcgaggg ctggagcaag tgggttgagg ggctgcgcat cccgcacccc 4560 ggccgcgtgg tggggcggct ggtgatgctg ctcaccatgc ccagcgtgct ggagtgggtg 4620 ctgggcggca acaccgacca cgtggcgccg caccgcacca gctactgctc gctgggcgac 4680 aagcccaagg tgagcggctg ccgggctggg ggggggtgga gggagaggag gaggattgcg 4740 gggagacgag ggagggcaag gcaggcgctg ccttcgtgga tgcaccgccc cgtcgttagc 4800 aggacctcag gaactcgtcc ccaaaaccac aacagaaccc ccaatatcgc ctcttccttc 4860 actgcttgtc acgcctggtc cgccgaccgc aggctttccc cgagagccgc ttccccgagt 4920 tcatgaacaa cgacgcctcc atcatccgct cctcccacgc cgactggctg ctggtggcgg 4980 agcgcgacgc cgccacggcc gccgccgcca acgtgaacgc cgccaccggc agcagcgccg 5040 ccgcggccgc cgccgccgac gtgaacagca gctgccagtg caagggcatc tacatggcgg 5100 actcggcggc cctggtgggc cgctgcggcg ccacctcgcg ccccgcgctg gccgtggacg 5160 acgtgcacgt cgccgagagt cacgcgcagg tctggcgcgg cctcgccggc ctccccccct 5220 cctcgtcgtc cgcctccacc gccgccgcct ctgcgtccgc cgcctcctct gccgccagcg 5280 gcaccgccag caccctgggc agctcggagg gctactggct ccgcgacctg ggcagcggcc 5340 gcggcacctg ggtcaacggc aagcgcctgc ccgacggcgc cacggtgcag ctgtggcccg 5400 gcgacgcggt ggagttcggc cggcacccca gccacgaggt gttcaaggtg aagatgcagc 5460 acgtgacgct gcgcagcgac gagctcagcg gccaggccta caccacgctc atggtgggca 5520 agatccggaa caacgactac gtcatgcccg agtcgcggcc ggacggcggc agccagcagc 5580 cgggccgcct ggtgacggct taagcggcgc cgtgcgtaag ggccggctta cgggggcggc 5640 agtgtcgctg tggagggatg gtctggggtg ggaggaatgg gaggagagcg gcgggagccc 5700 gaggagcgga gcgctggagg cttgcggagc ggcagcttgg gaagagctgc ggagagagga 5760 aggagcgcag ggcgcttgga gcacgcgcca gattacgatc acggcagcgc gaggcgcgcg 5820 tctgacttcg aagtggtaag gaagatttca tgtatgattg cgtcgaggga caccgcaagt 5880 tttacgcgcg gcggagggag ccttggggca tacaacagta cgagcgggcg ttggtgagaa 5940 ggtggtcact ccgtatgaga agatggttac tccgtacctt cgtgagaagc tgctgcgcac 6000 aagttacgaa cctatctgtg tggagagccc ggtagtatat caggggcgag ggtcatgaac 6060 gcgagtggcg agtctgtgag cgccaatttg ttatgcggca taatttcgca tcggggtatt 6120 acgtctacaa aatgttgagc tggcttagcg caggaggcaa cacctcaggc agaatgtacg 6180 aatgtgtgca gaagggcaga gtcaaggcag aggcggagaa gttgtcaggg ctgtgtgtgg 6240 tttggtcagg gcgtggctag atggatatga gacccgccgc cgtctccaga ttgtggcgga 6300 ggtggaactc tcggcccccg cgccagtccc cgcggccagc gcatcccgcc atgcgggttg 6360 ttggctggtg catcgcgcgg ggtgtgctat gagtgtggaa acactatgtc gcgtgtcgtg 6420 ctgaggtctg ttgagaggtt tcgtcgtttg tgcatgtcct gtcccggttg gagtttgagc 6480 gaggtggttc aaagtttttg gatcgcgtgg gagagactga aacggtttgg tgagaatggt 6540 tgagacagag gttgggcttg gaaactggag gagaggagca gcgtaactcg aggacgatgc 6600 agtagatgca ccacaacagt tgtggtgggc gcctggagta acacgcgtgc caccaacacg 6660 caattacaga gatccgtcat acaggaggga tcatatgcga tttaattttg gttttgcatt 6720 tgtaagacgt tttcaca 6737 <210> 7 <211> 1 <212> DNA <213> <220> <223> mutated ZEP gene sequence of Chlamydomonas reinhardtii <400> 7 a 1 <210> 8 <211> 6694 <212> DNA <213> <220> <223> mutated ZEP gene sequence of Chlamydomonas reinhardtii <400> 8 gcgacgcacg gctgggccaa attcgccaac ggcaggagac caaatcgatc gaggcgatct 60 tgcgaagttc tcggacaaat cgatcgcacc catagtgatt taagcattac atttgcccaa 120 ggcgtgagaa gtgcgaggcc cgaacggcta tgacgccaat gcgcagctta cgacatttaa 180 agcaaattat tcatacatca tacagcacgc ttatgtgaag aaagccagga ttttaggctc 240 tcgccccgat caagacgatc tccccattgc gaagttctcg gtttctttcc ggttcgcctg 300 ctccgtatga tttacctttg cgctacaaca gcgacttaaa cgacctacgt cgccttactg 360 tgtgcgcgta cgtgtttgta gctgtgagat agttttgtcc gcagcgtacc cgcaaataga 420 atgctcgcga gcacttacac gccctgtggc gttcgccagg tggcaggccg cacggttgca 480 gtgcccagca gcttggtcgc gccagtggca gtcgctcggt cgctggggtt ggcgccctac 540 gtccctgtat gtgagccttc tgcggcgctt ccggcctgcc agcagcctag cgggcgtcgt 600 catgttcaga ctgctgccac tctccgtgcc gacaacccca gctcggtcgc gcagctggtg 660 catcagaatg gaaaggggat gaaggttatt atcgccggcg cgggcatcgg cggcctggtg 720 ctagccgttg cacttctgaa gcagggcttc caggttcagg tctttgagcg cgacctgacg 780 gccatccgcg gcgagggcaa gtaccgtgga cccatcacag gtgcgttcgc cggaacacca 840 acgcgcttgt ttttgctgtg ccgcgaccat gaactaggcc ttatcttgag gtgttagcat 900 gtttagccag cgttggatct gtgtggcgag gttggggtga gaacccttcc tgtgtacctg 960 ctcgggcgta ccttgtgccc caccgctgac tggcttactt aatgacaaaa cgcaggttca 1020 aagcaatgcg ctcgctgcgc tggaggctat cgatcccgag gtggccgcgg aggtgctgcg 1080 cgagggctgc atcactggcg accgtatcaa cgggctctgc gacggcctga ctggcgagtg 1140 gtgagtaggc aatccagctg tgcatccagt cgcgcggttg cggaggtcgt ctcgggaaac 1200 gcgacgtggc gtccactcgc ccaaggagtg gtctcccgca gcgtggtctc ccgcagctcg 1260 ggtgcaacac cctgccccct gccgcgagcg cgctgcgctt gcttatgttg cgcagcggtg 1320 tgagttacaa cagcttctgt tgaagagctg tcatacgaag cacggcgcgc tgtggcgctg 1380 cagccgtgct gtggaaactc caacacctcc accgccagcc tgcgcacgca cacgcaatac 1440 actcgcctcg tgtgccccct cctcacacaa cggcatgtga cactcagttt taactcttat 1500 tttgacagct gagagctaca cgcttgggtg aatggggagg tccttgatgt ttcgttgcac 1560 tccgtggctc cggagtccgt gcggaccgtc acccacaaat gggagcgcac ggctttcttg 1620 tgctgtctgc cccgttagcc actaactgcg aatgaccttg acagtttact ttgctatttt 1680 tccttccagg tacgtcaagt tcgacacgtt ccacccggcg gtcagcaagg gcctgccggt 1740 gacccgcgtc atcagccgcc tcacgctgca gcagatcctg gccaaagccg tggagcggtg 1800 agccgtgcgc gcggtgtgat ggctttagcg tcagtgctag catgggggtt ggtgggtggt 1860 aatcgcggcg cccatggccg ggtagcagcg gccgaaagct ggcgcagagc gcgcgttgga 1920 caagcggtcc tgttgccggt atgggcacga gcagggcgct ggtgcgggca aagggcagag 1980 tggagttgca gagcagcgct ggcgtcggct gtgcgctctc caaatggcct cgtggcattc 2040 tgacgggaca catcctggaa aatagtagcg cacccaactg ctggtggctc ctcgtacaat 2100 ccccccaatt tacaatcgct cgttctggct cgcagctacg gcggccccgg caccatccag 2160 aacggctgca acgtgaccga gttcacggag cgccgcaacg acaccaccgg caacaacgag 2220 gtgagagcgt gctaagaaga gcatgcacgt ggagcgtgta aaattgtgtg gcctgaagcg 2280 gcagtgcctg cggcatggac taggtggttg cagcatgctg cgcgcgtggg ttgccggtca 2340 ggaaaccgcc ggaccgagcc gcgcagattc agtcaggagc ggattaggaa gtttgaaaaa 2400 cagggttcgg agtgtgcaag cgggctcagg agctgtggtg cctttctaca ccggtcgccc 2460 taccaggcac ccactgaaac tgtaaaaccg ttgctgcgcc ggcgatgccc tctacttcac 2520 taggtgactg tgcagctgga ggacgggcgc acgtttgcgg ccgacgtgct ggtgggcgcc 2580 gacggcatct ggtccaagat ccgtaagcag ctcattggcg agaccaaggc caactacagc 2640 gggtacacct gctacaccgg tgagattatt gaccttcaag ttggaaggag ggagcggggg 2700 gagcggaatg gaaggaagca gcgtggacgg ggcgcacgga ggggagggga ctgcgggtca 2760 tagcgccgcc ttgcggggcg tgaggagtgt tgggcggata ttcagttttc tttgcccaag 2820 atcttcccac aatccgcgtg tgtctgacgc gggatgtggc ccctgctgcc atggcttcgc 2880 aggcatctcg gactttacgc cggcggacat tgacattgtg ggctaccgcg tgttcctggg 2940 caacggccag tactttgtca gcagcgacgt gggcaacggc aagatgcagt ggtgagcggc 3000 ggcgggcggg cgagcgaggg ctgcggggtc tggagggtgt gtaccgggcg gaagggaggg 3060 gaagggaggg gaagggaagg caggatgcag gcgagggcag gatgtgatgg tgggaagagg 3120 gcgtggcgag cagcaactgg aaaggtggtg ggtaaaaaaa tggtccatga atatggctcg 3180 gtacagttca aagcatggaa atggaacccg ccgtctgctg caccatgggc gtgagcgggg 3240 agtacgcgac tcctggacag ccgtaacaat gcggatggcc tcaacaagcc aggagcggca 3300 cgaacccagc tcacgagcgc acagcgtgcc aggacggcgg ccggcaagga tgaaatgttt 3360 ttcctaatat aaatgcggac tcctgacgca ttatatccat tttgccactg agccaaagac 3420 acatatatac acgtgcgccg ccgtcctgcg ccacagccgc ctagcgctcc ggccgcgccc 3480 ggttccctcg gcgtcatgcg ctggagcccc ctcgcaccct gcaccgcaaa gcccatcaac 3540 accacactcg tccccacacc gcgagtcacc gccactgcac tcgctgtccc tcaacccgtc 3600 acaatctcgc cgacacgcga taacgaaccc acgcaggtac ggcttccaca aggagccgtc 3660 tggcggcacc gaccccgagg gcagccgcaa ggcgcgcctg ctgcagatct ttggccactg 3720 gaacgacaac gtggtggacc tgatcaaggc cacgcccgag gaggacgtgc tgcgccgcga 3780 catctttgac aggtacggaa aaagggagag cggggtggct ggagggcggg aaagggcgaa 3840 ggggcggaga aagaaatgac taggggatgg tgttcatttg tgggattgag aggggtccgc 3900 ggatcccggc agagggcgcc agtggcaagg cgtgggagtc gcggggcgga caatgctggg 3960 ccaggggcgc ctagtcaccc cgggacactg tctcagtatg ccgccgtccc ggccgcgccg 4020 cacaggccgc ccatcttcac ctggagcaag ggccgcgtgg ccctgctggg cgacagcgcg 4080 cacgccatgc agcccaacct gggccagggc ggctgcatgg ccattgagga cgcctacgag 4140 ctggccatcg acctcagccg cgccgtgtcc gacaaggccg gaaacgcggc ggcggtggac 4200 gtggagggcg tgctgcgcag ctaccaggac agccgcattt tgcgcgtcag cgccattcac 4260 ggcatggcgg gtgagagctg caaccagcgt agtcgggctg ggctgctgtg ggcagggtcg 4320 ggttgggttg ggcgcacgtg ggcggcgagt gtatgtgcag tgtgacgtgc acactatcat 4380 aatactttat gctcaccgca ccgcgccgcg ccgcaccacg cgccacaggc atggctgcct 4440 tcatggccag cacctacaag tgctacctgg gcgagggctg gagcaagtgg gttgaggggc 4500 tgcgcatccc gcaccccggc cgcgtggtgg ggcggctggt gatgctgctc accatgccca 4560 gcgtgctgga gtgggtgctg ggcggcaaca ccgaccacgt ggcgccgcac cgcaccagct 4620 actgctcgct gggcgacaag cccaaggtga gcggctgccg ggctgggggg gggtggaggg 4680 agaggaggag gattgcgggg agacgaggga gggcaaggca ggcgctgcct tcgtggatgc 4740 accgccccgt cgttagcagg acctcaggaa ctcgtcccca aaaccacaac agaaccccca 4800 atatcgcctc ttccttcact gcttgtcacg cctggtccgc cgaccgcagg ctttccccga 4860 gagccgcttc cccgagttca tgaacaacga cgcctccatc atccgctcct cccacgccga 4920 ctggctgctg gtggcggagc gcgacgccgc cacggccgcc gccgccaacg tgaacgccgc 4980 caccggcagc agcgccgccg cggccgccgc cgccgacgtg aacagcagct gccagtgcaa 5040 gggcatctac atggcggact cggcggccct ggtgggccgc tgcggcgcca cctcgcgccc 5100 cgcgctggcc gtggacgacg tgcacgtcgc cgagagtcac gcgcaggtct ggcgcggcct 5160 cgccggcctc cccccctcct cgtcgtccgc ctccaccgcc gccgcctctg cgtccgccgc 5220 ctcctctgcc gccagcggca ccgccagcac cctgggcagc tcggagggct actggctccg 5280 cgacctgggc agcggccgcg gcacctgggt caacggcaag cgcctgcccg acggcgccac 5340 ggtgcagctg tggcccggcg acgcggtgga gttcggccgg caccccagcc acgaggtgtt 5400 caaggtgaag atgcagcacg tgacgctgcg cagcgacgag ctcagcggcc aggcctacac 5460 cacgctcatg gtgggcaaga tccggaacaa cgactacgtc atgcccgagt cgcggccgga 5520 cggcggcagc cagcagccgg gccgcctggt gacggcttaa gcggcgccgt gcgtaagggc 5580 cggcttacgg gggcggcagt gtcgctgtgg agggatggtc tggggtggga ggaatgggag 5640 gagagcggcg ggagcccgag gagcggagcg ctggaggctt gcggagcggc agcttgggaa 5700 gagctgcgga gagaggaagg agcgcagggc gcttggagca cgcgccagat tacgatcacg 5760 gcagcgcgag gcgcgcgtct gacttcgaag tggtaaggaa gatttcatgt atgattgcgt 5820 cgagggacac cgcaagtttt acgcgcggcg gagggagcct tggggcatac aacagtacga 5880 gcgggcgttg gtgagaaggt ggtcactccg tatgagaaga tggttactcc gtaccttcgt 5940 gagaagctgc tgcgcacaag ttacgaacct atctgtgtgg agagcccggt agtatatcag 6000 gggcgagggt catgaacgcg agtggcgagt ctgtgagcgc caatttgtta tgcggcataa 6060 tttcgcatcg gggtattacg tctacaaaat gttgagctgg cttagcgcag gaggcaacac 6120 ctcaggcaga atgtacgaat gtgtgcagaa gggcagagtc aaggcagagg cggagaagtt 6180 gtcagggctg tgtgtggttt ggtcagggcg tggctagatg gatatgagac ccgccgccgt 6240 ctccagattg tggcggaggt ggaactctcg gcccccgcgc cagtccccgc ggccagcgca 6300 tcccgccatg cgggttgttg gctggtgcat cgcgcggggt gtgctatgag tgtggaaaca 6360 ctatgtcgcg tgtcgtgctg aggtctgttg agaggtttcg tcgtttgtgc atgtcctgtc 6420 ccggttggag tttgagcgag gtggttcaaa gtttttggat cgcgtgggag agactgaaac 6480 ggtttggtga gaatggttga gacagaggtt gggcttggaa actggaggag aggagcagcg 6540 taactcgagg acgatgcagt agatgcacca caacagttgt ggtgggcgcc tggagtaaca 6600 cgcgtgccac caacacgcaa ttacagagat ccgtcataca ggagggatca tatgcgattt 6660 aattttggtt ttgcatttgt aagacgtttt caca 6694 <210> 9 <211> 1870 <212> DNA <213> <220> <223> Mutated LCYE gene sequence of Chlamydomonas reinhardtii <400> 9 agtaccatca actgacgtta cattctgaca ggaaatgcca aggcgggtat caatctgttc 60 gggttgaccc gttggccaac acacgaatac atagtcacat ccccaaacac actagggagg 120 ggctcactgt tgcgttgcca agcggggcca gtcttgccgg atccttatca ggcgccgggg 180 gcggtgtccg gcggccccca gaggaactgc gccagttcct ccggatcggt gaagccggag 240 agatccagcg gggtctcctc gaacacctcg aagtcgtgca ggaaggtgaa ggcgagcagt 300 tcgcgggcga agtcctcggt ccgcttccac tgcgccccgt cgagcagcgc ggccaggatc 360 tcgcggtcgc cccggaaggc gttgagatgc agttgcacca ggctgtagcg ggagtctccc 420 gcatagacgt cggtgaagtc gacgatcccg gtgacctcgg tcgcggccag gtccacgaag 480 atgttggtcc cgtgcaggtc gccgtggacg aaccggggtt cgcggccggc cagcagcgtg 540 tccacgtccg gcagccagtc ctccaggcgg tccagcagcc ggggcgagag gtagccccac 600 ccgcggtggt cctcgacggt cgccgcgcgg cgttcccgca gcagttccgg gaagacctcg 660 gaatgggggg tgagcacggt gttcccggtc agcggcaccc tgtgcagccg gccgagcacc 720 cggccgagtt cgcgggccag ggcgagcagc gcgttccggt cggtcgtgcc gtccatcgcg 780 gaccgccagg tggtgccggt catccggctc atcaccaggt agggccacgg ccaggctccg 840 gtgccgggcc gcagctcgcc gcggccgagg aggcggggca ccggcaccgg ggcgtccgcc 900 aggaccgcgt acgcctccga ctccgacgcg aggctctccg gaccgcacca gtgctcgccg 960 aacagcttga tcaccgggcc gggctcgccg accagtacgg ggttggtgct ctcgccgggc 1020 acccgcagca ccggcggcac cggcagcccg agctcctcca gggctcggcg ggccagcggc 1080 tgcaaatgga aacggcgacg cagggttaga tgctgcttga gacagcgaca gaggagccaa 1140 aagccttcgt cgacacaatg cgggcgttgc aagtcaaatc tgcaagcacg ctgcctgatc 1200 cgccgggctt gctcgtcgac tcacctccca gaattcctgg tcgttccgca ggctcgcgta 1260 ggaatcatcc gaatcaatac ggtcgagaag taacagggat tcttgtgtca tgtttgcggg 1320 ttgtgactga aacgaagagg aagaggcgcc cccttagcgg agtgcgatca caagctcgag 1380 tggcctgtgt agaagtggta gtgatctagg tgtttgaata tggctttggt agctcgctat 1440 aatgtctttg caatcggggg cctggctatt taaacagcgc tcgccctgga gcggcatcgg 1500 agcgcccatg cagccccgaa ggagcttcgg ggggtcgaag catcatcggt gttgcatgca 1560 gcgccgggaa gccgtctcgc agcccgccct accttttgct ggaagtgtca tagcgcaaga 1620 aagacattcg cgcttcaaat acgcccagcc cgcccatgga gaaagaggcc aaaatcaacg 1680 gaggatcgtt acaaccaaca aaattgcaaa actcctccgc tttttacgtg ttgaaaaaga 1740 ctgatcagca cgaaacgggg agctaagcta ccgcttcagc acttgagagc agtatcttcc 1800 atccaccgcc gttcgtcagg gggcaaggct cagatcaacg agcgcctcca tttacacgga 1860 gcggaatcat 1870 <210> 10 <211> 8522 <212> DNA <213> <220> <223> Mutated LCYE gene sequence of Chlamydomonas reinhardtii <400> 10 agagttgcac ataatccgct ggtgaaatct agcgtcggct ggggacgaag cgcccggtac 60 caaccacccc tgacatcgga gccagggttc tacgcgcat cagccccttt agctcgcttg 120 taggcgcact tacgcaacaa actcaatcct aagcaagcat tcgtagagcg ttgttggcta 180 ttcctggcca ctcgctgcac tcccactgca agcagggttg gttgcgagca taggtccgcg 240 catctgacaa tttttagtat agtcgggtat tcacttgtac cactgagcat aattagcagc 300 ataacagggc tgttgtgagc caggcgaggt accttctgtt tggtaccgtc tcgtcttccc 360 tgtagacgct tccagtcagg cgccaaacca aacaaaaatg cttcaccaga ttagcgcaaa 420 gcgctgcgga gcgagcggcg tcgtgcgtgg cgctgcgggc ctgcaagcct ttagcacaaa 480 gctcgtcgct cccttgccag cgcggcgagt ggcttgccag gttgtatcta cggagactga 540 ggcacctacc atcgtgcttg accggcccgg gaggaccgtt cagccggagc tctccattca 600 gtaccatcaa ctgacgttac attctgacag gaaatgccaa ggcgggtatc aatctgttcg 660 ggttgacccg ttggccaaca cacgaataca tagtcacatc cccaaacaca ctagggaggg 720 gctcactgtt gcgttgccaa gcggggccag tcttgccgga tccttatcag gcgccggggg 780 cggtgtccgg cggcccccag aggaactgcg ccagttcctc cggatcggtg aagccggaga 840 gatccagcgg ggtctcctcg aacacctcga agtcgtgcag gaaggtgaag gcgagcagtt 900 cgcgggcgaa gtcctcggtc cgcttccact gcgccccgtc gagcagcgcg gccaggatct 960 cgcggtcgcc ccggaaggcg ttgagatgca gttgcaccag gctgtagcgg gagtctcccg 1020 catagacgtc ggtgaagtcg acgatcccgg tgacctcggt cgcggccagg tccacgaaga 1080 tgttggtccc gtgcaggtcg ccgtggacga accggggttc gcggccggcc agcagcgtgt 1140 ccacgtccgg cagccagtcc tccaggcggt ccagcagccg gggcgagagg tagccccacc 1200 cgcggtggtc ctcgacggtc gccgcgcggc gttccgcag cagttccggg aagacctcgg 1260 aatggggggt gagcacggtg ttcccggtca gcggcaccct gtgcagccgg ccgagcaccc 1320 ggccgagttc gcgggccagg gcgagcagcg cgttccggtc ggtcgtgccg tccatcgcgg 1380 accgccaggt ggtgccggtc atccggctca tcaccaggta gggccacggc caggctccgg 1440 tgccgggccg cagctcgccg cggccgagga ggcggggcac cggcaccggg gcgtccgcca 1500 ggaccgcgta cgcctccgac tccgacgcga ggctctccgg accgcaccag tgctcgccga 1560 acagcttgat caccgggccg ggctcgccga ccagtacggg gttggtgctc tcgccgggca 1620 cccgcagcac cggcggcacc ggcagcccga gctcctccag ggctcggcgg gccagcggct 1680 gcaaatggaa acggcgacgc agggttagat gctgcttgag acagcgacag aggagccaaa 1740 agccttcgtc gacacaatgc gggcgttgca agtcaaatct gcaagcacgc tgcctgatcc 1800 gccgggcttg ctcgtcgact cacctcccag aattcctggt cgttccgcag gctcgcgtag 1860 gaatcatccg aatcaatacg gtcgagaagt aacagggatt cttgtgtcat gtttgcgggt 1920 tgtgactgaa acgaagagga agaggcgccc ccttagcgga gtgcgatcac aagctcgagt 1980 ggcctgtgta gaagtggtag tgatctaggt gtttgaatat ggctttggta gctcgctata 2040 atgtctttgc aatcgggggc ctggctattt aaacagcgct cgccctggag cggcatcgga 2100 gcgcccatgc agccccgaag gagcttcggg gggtcgaagc atcatcggtg ttgcatgcag 2160 cgccgggaag ccgtctcgca gcccgcccta ccttttgctg gaagtgtcat agcgcaagaa 2220 agacattcgc gcttcaaata cgcccagccc gcccatggag aaagaggcca aaatcaacgg 2280 aggatcgtta caaccaacaa aattgcaaaa ctcctccgct ttttacgtgt tgaaaaagac 2340 tgatcagcac gaaacgggga gctaagctac cgcttcagca cttgagagca gtatcttcca 2400 tccaccgccg ttcgtcaggg ggcaaggctc agatcaacga gcgcctccat ttacacggag 2460 cggaatcatc agcggccctg aatcaagaaa tccgcgaggg gcactatgag gccgcgctgg 2520 tgaaggagca ggtaactact cgcttgcgtt caggcccaat tgaacgcgcc ccaaatcgaa 2580 ttcggttcgt tgtatgtgct caggcgaaca aggcggacgc ctctcaagcg gcaattgcgt 2640 ccgtattgaa gcccgctgac accacggtca cagccgatgt gacaattgtt ggtgcgggcc 2700 cggctggcct cttcctggct gcggagttgg gcaagcgggg catgagcgtg aacgtcctcg 2760 gtgagcggcc gtcgtgtcaa acggacagcc cccccccccc cgcatgccct tcttgccacg 2820 caactgccgc acggtcgctg ccaggcgcga cgcctgcatc taatggctct gtctcttgtc 2880 tcgcttgtca agtatcctga agctctgctg taatgccggg gcgtgtcttg ttgacagcgg 2940 ttcaatccaa atgcgtgcaa tcgctagtgt ggcccgcgtc catccactcc acacgcaacc 3000 agccagcgtg tctccgaggg actggcagac gcctgctgca tgccattgct cttgcagcgc 3060 tctgctgttg cacacatcag ccctcgcccc tgcgtacccc cgcggtggga ggcacggcca 3120 taatcaaacc caccccttcc gtgtcccatg aagccacacc agcgactgtg cagagacaca 3180 cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca cacacacaca 3240 cacacacaca cacacacgcg acaatgcctt atgctggact tacacacgca taatcccgca 3300 tataggcctg gacgtgccca ttgtgaacaa ctacggcgtg tggacggacg agttcgaggc 3360 cctgggcctg acccatacac tggagtgctc ctggcctgat gccgtgtgct acttcggcga 3420 gggcaaccag gtgcggggtg gggcggggcg tggtggagcg gggaggcaga gggcagacag 3480 ggttgcgtgc atcttgctgt ggtacgtagg gacctcttgt acgctcttgc atgtagcgaa 3540 acggtgtggc agggggggct gcatgagtag cggcgcgagt gcacaagagt ccaacgagca 3600 gcagccctgg cgcccgtgcc agcgtcctgg cttcttacac gcgagtcttt tcttgttgtt 3660 tgtccaggtg agcgtgggcc gcggctacgg ccgtgtgagc cgccggctgc tgcgtgagca 3720 cctgctcaag atctgcgagg cggcgggcgt gcgcttcagc agcgccgagg tggcggacat 3780 caaggtgcgc gggcggggcg gggtggcggc gcagggtgtg gtggcgggtt gtggtctggt 3840 gtgcagttga ccgcaaaggg tctggctgac tggctggctg gtgcgagtac ggtgcttgag 3900 ctggatgagt cggccgactg gcaggagctt cactcactta ctgccatact gccaccccgc 3960 ccctgtccgc accatgcata ggttgtggag gagggcaagc tgactcagct caccaccaag 4020 gagggcgccg tgtacagcag caggtggggg aggggggggc ggcgggcagc agaagcggcc 4080 atgctaggca ctggctgtgg tgggggctgc gctgtagcaa cagcaatagc ggcatcagca 4140 gcagctgtgg gatacaaaaa gcgcacgtgc ctggctacgt ggcgctcgcg catgtcgtgc 4200 tctgacacct tgcgccctct tcacgcaacc ctcaggctga ccactctggc tgcgggtgcc 4260 gctgccggca agttcctgcg gtgagtgggt cgtccgcagc cctgggacag tgtctggtca 4320 cgtcacaaag atgtgaacgc cgtcttgctc tgccctgtgc cacctcccgg ctggagcctc 4380 ctccccactc ctcgcttctc acctcttctc cctctgccca cgcactccag ctatgaggag 4440 gacgcgccca ttgtcgccgc acaaaccgcg tacggcatcg aggcggaggt ggagggctat 4500 gacgccgcct acccgggcga cctcatgacc ttcatggatt tcaggtgcgg gcatgtgggc 4560 ttgtgtgtgg gtgcggatgg ttccaacacc aactaaacca agaccccagg ggggttgtag 4620 aggttagcag aatgcttgga gcatcaggcg agcgagcggc acgccaggga gcgaaagagc 4680 gagcaccaac agcgacgtga tgcacggtgc cggcgtactg gcggggaggg ggcgcgggca 4740 cgggggccga aacagccatc catgaatgcc ggggggcaaa tggtggcgtc gctgtggcct 4800 gacctggtcc cgctgtgttc ggccggctcc ccctcgcggc aggcgccacc acaccggcct 4860 gcacgacggc acggcgctca aattcgtgcc ggggcgccat cccaactcgg gcgacggcac 4920 gtggggcagc agcgaggagt gcccctcctt cctgtacgcc atgccgctgg gcggcaagcg 4980 cgtgttcctg gaggagacct gcctggtggc caagccggcg ctgccgttcg cggtgctcaa 5040 gcgccgcctc acacgccgcc tcaaggccat gggcatctcc gtgaccaagg tgtgtgcgga 5100 gaggggagga gaggggaggg agggagggag ggaggggagg gatagcgtct gaaagttcgt 5160 agaaacaaca ggccgatgtg accgaagcat gtgccctgac tccgcgccgc tctcacacgc 5220 atgcctggat cgccctcaga tccacgagga ggagtggagc tacatccctg tgggcggccc 5280 cctgccgctg cccgaccagg tgcgtgccgg ccagcgtaca gcactgtgtg gctttcggcg 5340 ggggctgtgc agagcgcgca catgagcatg cctggtgttt cctggtggta atgtgcgtaa 5400 tgtgcgctca gctgtcacgt caccctcgct cttccctcac ccgctgtcgc ctccacagtc 5460 ggtgacggcg ttcggcgccg ctgccaacct ggtgcacccc gccaccggct tcagcgtcag 5520 ccgctccttc cgtgaggcgc cgcaggtgcg tgcgggcacc gtgggtgggc acagggcggg 5580 agggcactgg gcacgcgggc actgggcagg cgggagaggg catggttgca ggcgcctgag 5640 gaatgagggt ttgggaggag atgcggggtc gggggccgtt gtggtggctg gggcttgcag 5700 ggcgaaagat gaggcggggg caggtcgttc gggggcctca aggatgtcag aggccgagcg 5760 gtccggtggg tgctccttat tggacgcgtt ctcctctctt cgcccctcct ctcctcattc 5820 ctcctttcct cacccctcct ccatcccccc ctgctgcaac tgtacctgcc tacgacttca 5880 cttcttaatt aatcatgaat gtaacccccc ctctcctcac ccccctcctc gccccgtcac 5940 taggtggcgg acgagctgca ggccgcgctg cgcgacgggc tggacgtgtc cgccgccagc 6000 cgccgcgtgt gggagcgcct gtggccgcag gagaagcgca cgcaggcctc cttccacgtg 6060 ttcggcatgg agctgctcgc cacgctggac ctcaacgcca ccaacgactt cttcaacacc 6120 ttcttccgcc tgccgccctt ctactggcgc ggcttcctgg cctctacgct cagcagcggg 6180 cagctgattg cctttgcgct ggtggtgttc acgctggcgc cctggaacat caagtacaag 6240 ctcattgagc atctgatcac gggtgggtgg aggaagggct gtgggtgcgg gtgcgggtgc 6300 agggggttgg cgggtgcggg tgcgggtgca ggtgcgggtg cggatgcgga tgcgggtccg 6360 ggtgcgggtg cgggtgcggg tgcaggggct ggcgggcgca ggggctggcg tctgtgtgtg 6420 tgggggggga ctgtagttta tctaaaccca actaaaccaa ggcctggcca gcccctaggg 6480 gggcagcggg tggcgggaag agggcaggag gcagtgggct gctgtcccaa gtgagccgaa 6540 gttcagcatg gggggcctgg aagggcggga tctggggtgg aagggcggcg gggcgggcag 6600 gtggggatgt ggcggcgttg agtctgcaag catgacctgt gtaggtgaag ctccccacgtc 6660 tgacgcttcc ctccccgaca ccaacgtcat gtctggcccc gcacgcagac cccgccggcg 6720 gctacctcat ccgtgcctac caggcgcagt gggacagctc tcaacaggcg ccgtccgccg 6780 cgactgccgc cgccgtgctg ctgctcagca acgagctgat gacgcgcgcg gtgctcaagg 6840 cgctggagag cggcagcgcg taagcactgc gttattggtc cccgtccccc ctgaatgaag 6900 gatgcacgag ggaggatggg gcaggagcat cagcagcagg agcaccagca gcggcagggg 6960 caagagcggc aggggcagga gcagacagta gacagaggcc gggctggcgg aaggattgga 7020 ggcgcgcgcg gttgcggcct tgtgatgtgc gcgtttgagc cttcgacacc gcatgcttga 7080 gacaatttga ttgcagcgga gaggaacgaa gaactttatc gttggaaagg gccgcccacc 7140 ggcggccgca tgacgcagca gcagcagcag tccaggagct gcggcggctg cagaagcagc 7200 aggagagcag cgctagctat gtcgtggtgg cggctgaaag gggggagcgg gtgcttaagt 7260 agctaccttt tgcccaggta acgtggctgt gcattgacgc cgctgcgagt ccgttgggtc 7320 cagcaacgcg ctgtcgtcgt tggtgcagtt gagattctgc ttcataggga tagtcacagc 7380 gagaggcatc gcagcgcgca ggctcttgat gcgcaggtgt gtggtgggct agaattcgtg 7440 ggggacaaca ctgttggttt gcatgtgcta tgtgtactga tggaatatgg tttgggcagc 7500 ttacacccgt acatgccaat ggtgtattca ctacgtcagc gtaagcggac cagcgatagg 7560 aatgcagcac ggtatggagg actaagcagg ggagtggtgg tgccattggc tggcgcagtg 7620 tgattgtcaa ccaaaagaga gcggccgtga gcgagatttt cgccccgatg acatcccggg 7680 gacggtacat gctttgtttc tgtgtagctt gtggcgtatg cgttgtggcc acgtacatgt 7740 gtgttgtttg tttcgtgtgt gtgcgtgtca tgtgattgac ctgttgcagt gcagatgcag 7800 ttgtttgctt ccagagctgt gtcaactagc ctggctcgcg cgcccgggcg tggccaccag 7860 ggggcgtgat tgtgtgaagg ggctggaagg ggagccaggc agctctattg gagactccgc 7920 agcgcgggta ggtgcggcag gaagggcaca acgctgaaca ttggcggcaa agcgagcctg 7980 gaagtgcgag caatgttgac gaggattagg agagcgagcg agttgaccaa gaccagagaa 8040 ggccagaagg gagaagttgg ttgcagcatg gcgtgttgat gggatgggtc gatggggcgg 8100 ccccaggtag ctcgtgctgc tgcttggggc aatacgaccc aagtgctgaa gtgcagcgaa 8160 caggaagcgg cagcttgtca gcgcgcacgc tgctttgcgc tcgttgcggg aggacgccgt 8220 gtgcccgcgc aagctgcggg cccttctgca cttggtgccc gtaacattca tgttcaaccg 8280 ccgctctcct ggctgtcacc accagggatg tcccatggca ccaggactca ccagggctgt 8340 tgcacgagcg ccgggtgcgc gggcaagcag ggcacctcag gttggaggga atgaaatatg 8400 gtatgcgaca gggaccacaa gggtgtgggc cattcctggc cggtccatcc gcagggtggg 8460 ccgcgtgcag agccacctgt gattggttgt gctgccagct ccatgcgata gtaagatcgg 8520 gc 8522 <210> 11 <211> 94 <212> DNA <213> <220> <223> Mutated LCYE gene sequence of Chlamydomonas reinhardtii <400> 11 agtaccatca actgacgtta cattctgaca ggaaatgcca aggcgggtat caatctgttc 60 gggttgaccc gttggccaac acacgaatac atag 94 <210> 12 <211> 6746 <212> DNA <213> <220> <223> Mutated LCYE gene sequence of Chlamydomonas reinhardtii <400> 12 agagttgcac ataatccgct ggtgaaatct agcgtcggct ggggacgaag cgcccggtac 60 caaccacccc tgacatcgga gccagggttc tacgcgcat cagccccttt agctcgcttg 120 taggcgcact tacgcaacaa actcaatcct aagcaagcat tcgtagagcg ttgttggcta 180 ttcctggcca ctcgctgcac tcccactgca agcagggttg gttgcgagca taggtccgcg 240 catctgacaa tttttagtat agtcgggtat tcacttgtac cactgagcat aattagcagc 300 ataacagggc tgttgtgagc caggcgaggt accttctgtt tggtaccgtc tcgtcttccc 360 tgtagacgct tccagtcagg cgccaaacca aacaaaaatg cttcaccaga ttagcgcaaa 420 gcgctgcgga gcgagcggcg tcgtgcgtgg cgctgcgggc ctgcaagcct ttagcacaaa 480 gctcgtcgct cccttgccag cgcggcgagt ggcttgccag gttgtatcta cggagactga 540 ggcacctacc atcgtgcttg accggcccgg gaggaccgtt cagccggagc tctccattca 600 gtaccatcaa ctgacgttac attctgacag gaaatgccaa ggcgggtatc aatctgttcg 660 ggttgacccg ttggccaaca cacgaataca tagcagcggc cctgaatcaa gaaatccgcg 720 aggggcacta tgaggccgcg ctggtgaagg agcaggtaac tactcgcttg cgttcaggcc 780 caattgaacg cgccccaaat cgaattcggt tcgttgtatg tgctcaggcg aacaaggcgg 840 acgcctctca agcggcaatt gcgtccgtat tgaagcccgc tgacaccacg gtcacagccg 900 atgtgacaat tgttggtgcg ggcccggctg gcctcttcct ggctgcggag ttgggcaagc 960 ggggcatgag cgtgaacgtc ctcggtgagc ggccgtcgtg tcaaacggac agcccccccc 1020 cccccgcatg cccttcttgc cacgcaactg ccgcacggtc gctgccaggc gcgacgcctg 1080 catctaatgg ctctgtctct tgtctcgctt gtcaagtatc ctgaagctct gctgtaatgc 1140 cggggcgtgt cttgttgaca gcggttcaat ccaaatgcgt gcaatcgcta gtgtggcccg 1200 cgtccatcca ctccacacgc aaccagccag cgtgtctccg agggactggc agacgcctgc 1260 tgcatgccat tgctcttgca gcgctctgct gttgcacaca tcagccctcg cccctgcgta 1320 cccccgcggt gggaggcacg gccataatca aacccacccc ttccgtgtcc catgaagcca 1380 caccagcgac tgtgcagaga cacacacaca cacacacaca cacacacaca cacacacaca 1440 cacacacaca cacacacaca cacacacaca cacacacaca cgcgacaatg ccttatgctg 1500 gacttacaca cgcataatcc cgcatatagg cctggacgtg cccattgtga acaactacgg 1560 cgtgtggacg gacgagttcg aggccctggg cctgacccat acactggagt gctcctggcc 1620 tgatgccgtg tgctacttcg gcgagggcaa ccaggtgcgg ggtggggcgg ggcgtggtgg 1680 agcggggagg cagagggcag acagggttgc gtgcatcttg ctgtggtacg tagggacctc 1740 ttgtacgctc ttgcatgtag cgaaacggtg tggcaggggg ggctgcatga gtagcggcgc 1800 gagtgcacaa gagtccaacg agcagcagcc ctggcgcccg tgccagcgtc ctggcttctt 1860 acacgcgagt cttttcttgt tgtttgtcca ggtgagcgtg ggccgcggct acggccgtgt 1920 gagccgccgg ctgctgcgtg agcacctgct caagatctgc gaggcggcgg gcgtgcgctt 1980 cagcagcgcc gaggtggcgg acatcaaggt gcgcgggcgg ggcggggtgg cggcgcaggg 2040 tgtggtggcg ggttgtggtc tggtgtgcag ttgaccgcaa agggtctggc tgactggctg 2100 gctggtgcga gtacggtgct tgagctggat gagtcggccg actggcagga gcttcactca 2160 cttactgcca tactgccacc ccgcccctgt ccgcaccatg cataggttgt ggaggagggc 2220 aagctgactc agctcaccac caaggagggc gccgtgtaca gcagcaggtg ggggaggggg 2280 gggcggcggg cagcagaagc ggccatgcta ggcactggct gtggtggggg ctgcgctgta 2340 gcaacagcaa tagcggcatc agcagcagct gtgggataca aaaagcgcac gtgcctggct 2400 acgtggcgct cgcgcatgtc gtgctctgac accttgcgcc ctcttcacgc aaccctcagg 2460 ctgaccactc tggctgcggg tgccgctgcc ggcaagttcc tgcggtgagt gggtcgtccg 2520 cagccctggg acagtgtctg gtcacgtcac aaagatgtga acgccgtctt gctctgccct 2580 gtgccacctc ccggctggag cctcctcccc actcctcgct tctcacctct tctccctctg 2640 cccacgcact ccagctatga ggaggacgcg cccattgtcg ccgcacaaac cgcgtacggc 2700 atcgaggcgg aggtggaggg ctatgacgcc gcctacccgg gcgacctcat gaccttcatg 2760 gatttcaggt gcgggcatgt gggcttgtgt gtgggtgcgg atggttccaa caccaactaa 2820 accaagaccc caggggggtt gtagaggtta gcagaatgct tggagcatca ggcgagcgag 2880 cggcacgcca gggagcgaaa gagcgagcac caacagcgac gtgatgcacg gtgccggcgt 2940 actggcgggg agggggcgcg ggcacggggg ccgaaacagc catccatgaa tgccgggggg 3000 caaatggtgg cgtcgctgtg gcctgacctg gtcccgctgt gttcggccgg ctccccctcg 3060 cggcaggcgc caccacaccg gcctgcacga cggcacggcg ctcaaattcg tgccggggcg 3120 ccatcccaac tcgggcgacg gcacgtgggg cagcagcgag gagtgcccct ccttcctgta 3180 cgccatgccg ctgggcggca agcgcgtgtt cctggaggag acctgcctgg tggccaagcc 3240 ggcgctgccg ttcgcggtgc tcaagcgccg cctcacacgc cgcctcaagg ccatgggcat 3300 ctccgtgacc aaggtgtgtg cggagagggg aggagagggg agggagggag ggagggaggg 3360 gagggatagc gtctgaaagt tcgtagaaac aacaggccga tgtgaccgaa gcatgtgccc 3420 tgactccgcg ccgctctcac acgcatgcct ggatcgccct cagatccacg aggaggagtg 3480 gagctacatc cctgtgggcg gccccctgcc gctgcccgac caggtgcgtg ccggccagcg 3540 tacagcactg tgtggctttc ggcgggggct gtgcagagcg cgcacatgag catgcctggt 3600 gtttcctggt ggtaatgtgc gtaatgtgcg ctcagctgtc acgtcaccct cgctcttccc 3660 tcacccgctg tcgcctccac agtcggtgac ggcgttcggc gccgctgcca acctggtgca 3720 ccccgccacc ggcttcagcg tcagccgctc cttccgtgag gcgccgcagg tgcgtgcggg 3780 caccgtgggt gggcacaggg cgggagggca ctgggcacgc gggcactggg caggcgggag 3840 agggcatggt tgcaggcgcc tgaggaatga gggtttggga ggagatgcgg ggtcgggggc 3900 cgttgtggtg gctggggctt gcagggcgaa agatgaggcg ggggcaggtc gttcgggggc 3960 ctcaaggatg tcagaggccg agcggtccgg tgggtgctcc ttattggacg cgttctcctc 4020 tcttcgcccc tcctctcctc attcctcctt tcctcacccc tcctccatcc ccccctgctg 4080 caactgtacc tgcctacgac ttcacttctt aattaatcat gaatgtaacc ccccctctcc 4140 tcacccccct cctcgccccg tcactaggtg gcggacgagc tgcaggccgc gctgcgcgac 4200 gggctggacg tgtccgccgc cagccgccgc gtgtgggagc gcctgtggcc gcaggagaag 4260 cgcacgcagg cctccttcca cgtgttcggc atggagctgc tcgccacgct ggacctcaac 4320 gccaccaacg acttcttcaa caccttcttc cgcctgccgc ccttctactg gcgcggcttc 4380 ctggcctcta cgctcagcag cgggcagctg attgcctttg cgctggtggt gttcacgctg 4440 gcgccctgga acatcaagta caagctcatt gagcatctga tcacgggtgg gtggaggaag 4500 ggctgtgggt gcgggtgcgg gtgcaggggg ttggcgggtg cgggtgcggg tgcaggtgcg 4560 ggtgcggatg cggatgcggg tccgggtgcg ggtgcgggtg cgggtgcagg ggctggcggg 4620 cgcaggggct ggcgtctgtg tgtgtggggg gggactgtag tttatctaaa cccaactaaa 4680 ccaaggcctg gccagcccct aggggggcag cgggtggcgg gaagagggca ggaggcagtg 4740 ggctgctgtc ccaagtgagc cgaagttcag catggggggc ctggaagggc gggatctggg 4800 gtggaagggc ggcggggcgg gcaggtgggg atgtggcggc gttgagtctg caagcatgac 4860 ctgtgtaggt gaagctccca cgtctgacgc ttccctcccc gacaccaacg tcatgtctgg 4920 ccccgcacgc agaccccgcc ggcggctacc tcatccgtgc ctaccaggcg cagtgggaca 4980 gctctcaaca ggcgccgtcc gccgcgactg ccgccgccgt gctgctgctc agcaacgagc 5040 tgatgacgcg cgcggtgctc aaggcgctgg agagcggcag cgcgtaagca ctgcgttatt 5100 ggtccccgtc ccccctgaat gaaggatgca cgagggagga tggggcagga gcatcagcag 5160 caggagcacc agcagcggca ggggcaagag cggcaggggc aggagcagac agtagacaga 5220 ggccgggctg gcggaaggat tggaggcgcg cgcggttgcg gccttgtgat gtgcgcgttt 5280 gagccttcga caccgcatgc ttgagacaat ttgattgcag cggagaggaa cgaagaactt 5340 tatcgttgga aagggccgcc caccggcggc cgcatgacgc agcagcagca gcagtccagg 5400 agctgcggcg gctgcagaag cagcaggaga gcagcgctag ctatgtcgtg gtggcggctg 5460 aaagggggga gcgggtgctt aagtagctac cttttgccca ggtaacgtgg ctgtgcattg 5520 acgccgctgc gagtccgttg ggtccagcaa cgcgctgtcg tcgttggtgc agttgagatt 5580 ctgcttcata gggatagtca cagcgagagg catcgcagcg cgcaggctct tgatgcgcag 5640 gtgtgtggtg ggctagaatt cgtgggggac aacactgttg gtttgcatgt gctatgtgta 5700 ctgatggaat atggtttggg cagcttacac ccgtacatgc caatggtgta ttcactacgt 5760 cagcgtaagc ggaccagcga taggaatgca gcacggtatg gaggactaag caggggagtg 5820 gtggtgccat tggctggcgc agtgtgattg tcaaccaaaa gagagcggcc gtgagcgaga 5880 ttttcgcccc gatgacatcc cggggacggt acatgctttg tttctgtgta gcttgtggcg 5940 tatgcgttgt ggccacgtac atgtgtgttg tttgtttcgt gtgtgtgcgt gtcatgtgat 6000 tgacctgttg cagtgcagat gcagttgttt gcttccagag ctgtgtcaac tagcctggct 6060 cgcgcgcccg ggcgtggcca ccagggggcg tgattgtgtg aaggggctgg aaggggagcc 6120 aggcagctct attggagact ccgcagcgcg ggtaggtgcg gcaggaaggg cacaacgctg 6180 aacattggcg gcaaagcgag cctggaagtg cgagcaatgt tgacgaggat taggagagcg 6240 agcgagttga ccaagaccag agaaggccag aagggagaag ttggttgcag catggcgtgt 6300 tgatgggatg ggtcgatggg gcggccccag gtagctcgtg ctgctgcttg gggcaatacg 6360 acccaagtgc tgaagtgcag cgaacaggaa gcggcagctt gtcagcgcgc acgctgcttt 6420 gcgctcgttg cgggaggacg ccgtgtgccc gcgcaagctg cgggcccttc tgcacttggt 6480 gcccgtaaca ttcatgttca accgccgctc tcctggctgt caccaccagg gatgtcccat 6540 ggcaccagga ctcaccaggg ctgttgcacg agcgccgggt gcgcgggcaa gcagggcacc 6600 tcaggttgga gggaatgaaa tatggtatgc gacagggacc acaagggtgt gggccattcc 6660 tggccggtcc atccgcaggg tgggccgcgt gcagagccac ctgtgattgg ttgtgctgcc 6720 agctccatgc gatagtaaga tcgggc 6746 <210> 13 <211> 23 <212> DNA <213> Chlamydomonas reinhardtii <400> 13 cccatccagg tgcgttcgcc gga 23 <210> 14 <211> 23 <212> DNA <213> Chlamydomonas reinhardtii <400> 14 gccggagctc tccattccag cgg 23 <210> 15 <211> 1406 <212> PRT <213> <220> <223> cas9 protein <400> 15 Met Gly Ser Ser His His His His His His Val Tyr Pro Tyr Asp Val 1 5 10 15 Pro Asp Tyr Ala Glu Leu Pro Lys Lys Lys Arg Lys Val Gly Ile 20 25 30 Arg Ile Pro Gly Glu Lys Pro Asp Lys Lys Tyr Ser Ile Gly Leu Asp 35 40 45 Ile Gly Thr Asn Ser Val Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys 50 55 60 Val Pro Ser Lys Lys Phe Lys Val Leu Gly Asn Thr Asp Arg His Ser 65 70 75 80 Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr 85 90 95 Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg 100 105 110 Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met 115 120 125 Ala Lys Val Asp Asp Ser Phe Phe His Arg Leu Glu Glu Ser Phe Leu 130 135 140 Val Glu Glu Asp Lys Lys His Glu Arg His Pro Ile Phe Gly Asn Ile 145 150 155 160 Val Asp Glu Val Ala Tyr His Glu Lys Tyr Pro Thr Ile Tyr His Leu 165 170 175 Arg Lys Lys Leu Val Asp Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile 180 185 190 Tyr Leu Ala Leu Ala His Met Ile Lys Phe Arg Gly His Phe Leu Ile 195 200 205 Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp Val Asp Lys Leu Phe Ile 210 215 220 Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn 225 230 235 240 Ala Ser Gly Val Asp Ala Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys 245 250 255 Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys 260 265 270 Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro 275 280 285 Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu 290 295 300 Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile 305 310 315 320 Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp 325 330 335 Ala Ile Leu Leu Ser Asp Ile Leu Arg Val Asn Thr Glu Ile Thr Lys 340 345 350 Ala Pro Leu Ser Ala Ser Met Ile Lys Arg Tyr Asp Glu His His Gln 355 360 365 Asp Leu Thr Leu Leu Lys Ala Leu Val Arg Gln Gln Leu Pro Glu Lys 370 375 380 Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr 385 390 395 400 Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro 405 410 415 Ile Leu Glu Lys Met Asp Gly Thr Glu Glu Leu Leu Val Lys Leu Asn 420 425 430 Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile 435 440 445 Pro His Gln Ile His Leu Gly Glu Leu His Ala Ile Leu Arg Arg Gln 450 455 460 Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys 465 470 475 480 Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly 485 490 495 Asn Ser Arg Phe Ala Trp Met Thr Arg Lys Ser Glu Glu Thr Ile Thr 500 505 510 Pro Trp Asn Phe Glu Glu Val Val Asp Lys Gly Ala Ser Ala Gln Ser 515 520 525 Phe Ile Glu Arg Met Thr Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys 530 535 540 Val Leu Pro Lys His Ser Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn 545 550 555 560 Glu Leu Thr Lys Val Lys Tyr Val Thr Glu Gly Met Arg Lys Pro Ala 565 570 575 Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile Val Asp Leu Leu Phe Lys 580 585 590 Thr Asn Arg Lys Val Thr Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys 595 600 605 Lys Ile Glu Cys Phe Asp Ser Val Glu Ile Ser Gly Val Glu Asp Arg 610 615 620 Phe Asn Ala Ser Leu Gly Thr Tyr His Asp Leu Leu Lys Ile Ile Lys 625 630 635 640 Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp 645 650 655 Ile Val Leu Thr Leu Thr Leu Phe Glu Asp Arg Glu Met Ile Glu Glu 660 665 670 Arg Leu Lys Thr Tyr Ala His Leu Phe Asp Asp Lys Val Met Lys Gln 675 680 685 Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu 690 695 700 Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe 705 710 715 720 Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn Phe Met Gln Leu Ile His 725 730 735 Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile Gln Lys Ala Gln Val Ser 740 745 750 Gly Gln Gly Asp Ser Leu His Glu His Ile Ala Asn Leu Ala Gly Ser 755 760 765 Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr Val Lys Val Val Asp Glu 770 775 780 Leu Val Lys Val Met Gly Arg His Lys Pro Glu Asn Ile Val Ile Glu 785 790 795 800 Met Ala Arg Glu Asn Gln Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg 805 810 815 Glu Arg Met Lys Arg Ile Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln 820 825 830 Ile Leu Lys Glu His Pro Val Glu Asn Thr Gln Leu Gln Asn Glu Lys 835 840 845 Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg Asp Met Tyr Val Asp Gln 850 855 860 Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr Asp Val Asp His Ile Val 865 870 875 880 Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile Asp Asn Lys Val Leu Thr 885 890 895 Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp Asn Val Pro Ser Glu Glu 900 905 910 Val Val Lys Lys Met Lys Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys 915 920 925 Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly 930 935 940 Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile Lys Arg Gln Leu Val 945 950 955 960 Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln Ile Leu Asp Ser Arg 965 970 975 Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys Leu Ile Arg Glu Val Lys 980 985 990 Val Ile Thr Leu Lys Ser Lys Leu Val Ser Asp Phe Arg Lys Asp Phe 995 1000 1005 Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn Tyr His His Ala His 1010 1015 1020 Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala Leu Ile Lys Lys 1025 1030 1035 Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp Tyr Lys Val 1040 1045 1050 Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu Ile Gly 1055 1060 1065 Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn Phe 1070 1075 1080 Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg 1085 1090 1095 Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp 1100 1105 1110 Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro 1115 1120 1125 Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe 1130 1135 1140 Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile 1145 1150 1155 Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp 1160 1165 1170 Ser Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu 1175 1180 1185 Lys Gly Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly 1190 1195 1200 Ile Thr Ile Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp 1205 1210 1215 Phe Leu Glu Ala Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile 1220 1225 1230 Ile Lys Leu Pro Lys Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg 1235 1240 1245 Lys Arg Met Leu Ala Ser Ala Gly Glu Leu Gln Lys Gly Asn Glu 1250 1255 1260 Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu Tyr Leu Ala Ser 1265 1270 1275 His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn Glu Gln Lys 1280 1285 1290 Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu Ile Ile 1295 1300 1305 Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp Ala 1310 1315 1320 Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys 1325 1330 1335 Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu 1340 1345 1350 Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr 1355 1360 1365 Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala 1370 1375 1380 Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile 1385 1390 1395 Asp Leu Ser Gln Leu Gly Gly Asp 1400 1405 <210> 16 <211> 96 <212> RNA <213> <220> <223> sgRNA of ZEP <400> 16 uccggcgaac gcaccuggau guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugc 96 <210> 17 <211> 96 <212> RNA <213> <220> <223> sgRNA of LCYE <400> 17 ggagcucucc auuccagcgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu ggcaccgagu cggugc 96 <210> 18 <211> 76 <212> DNA <213> <220> <223> sgRNA <400> 18 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60 ggcaccgagt cggtgc 76

Claims (19)

Chlamydomonas reinhardtii culture composition comprising nitrogen (N) 10.0mM to 17.0mM and phosphorus (P) 1.0 mM to 2.5mM. According to claim 1,
The culture composition comprises nitrogen (N) 13.5 mM to 16.5 mM and phosphorus (P) 1.0 mM to 1.4 mM; nitrogen (N) from 10.0 mM to 13.49 mM and phosphorus (P) from 1.9 mM to 2.5 mM; nitrogen (N) from 10.0 mM to 11.99 mM and phosphorus (P) from 1.9 mM to 2.5 mM; Or nitrogen (N) 12.0mM to 13.49mM and phosphorus (P) 1.9mM to 2.5mM Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) culture composition comprising. According to claim 1,
The culture composition is Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) culture composition further comprising calcium (Ca) 0.47 mM to 0.70 mM and magnesium (Mg) 0.55 mM to 0.85 mM. According to claim 1,
The culture composition is Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) culture composition further comprising 1.5ml/L to 2.5ml/L of glacial acetic acid as a carbon source. According to claim 1,
The culture composition is one or more trace elements selected from the group consisting of EDTA, iron (Fe), zinc (Zn), boron (B), manganese (Mn), copper (Cu), molybdenum (Mo), and cobalt (Co). Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) culture composition further comprising a. 6. The method of claim 5,
The culture composition is EDTA 133 μM to 137 μM, iron (Fe) 16 μM to 20 μM, zinc (Zn) 73 μM to 77 μM, boron (B) 183 μM to 187 μM, manganese (Mn) 24 μM to 28 μM, copper (Cu) 5.0 μM to 8.0 μM , Molybdenum (Mo) 4.0 μM to 7.0 μM, and cobalt (Co) 5.0 μM to 8.0 μM Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) culture composition further comprising 8.0 μM. According to claim 1,
The Chlamydomonas rain hard Tii (Chlamydomonas reinhardtii) has a zeaxanthin (zeaxanthin) producing ability, does not produce lutein (lutein), the ZEP gene and the LCYE gene are knocked out at the same time rain hard Tii (Chlamydomonas reinhardtii) mutant cla Midomonas rain hard Tia (Chlamydomonas reinhardtii) culture composition. According to claim 1,
The mutant is adenine (A) between the 816th base and the 817th base in the ZEP gene sequence of Chlamydomonas reinhardtii represented by SEQ ID NO: 1; the nucleotide sequence represented by SEQ ID NO: 3; or ZEP gene mutation into which the nucleotide sequence represented by SEQ ID NO: 5 is inserted; And the nucleotide sequence shown in SEQ ID NO: 9 between the 599th base and the 600th base in the LCYE gene sequence of Chlamydomonas reinhardtii represented by SEQ ID NO: 2; Or a mutant strain containing the LCYE gene mutation into which the nucleotide sequence represented by SEQ ID NO: 11 is inserted, Chlamydomonas reinhardtii culture composition. In the culture composition according to any one of claims 1 to 9, Lutein is not produced and Chlamydomonas reinhardtii having a zeaxanthin-producing ability Chlamydomonas reinhardtii culturing a mutant; including Chlamydomonas rain hard A method of culturing a Tian (Chlamydomonas reinhardtii) mutant. 10. The method of claim 9,
A method of culturing the mutant strain at a light intensity of 60 μE to 450 μE for 36 hours to 130 hours, Chlamydomonas reinhardtii mutant strain. 10. The method of claim 9,
incubating the mutant at a light intensity of 60 μE to 160 μE for 60 hours to 130 hours; culturing the mutant for 36 to 60 hours at a light intensity of greater than 160 μE to 300 μE; Or Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) culturing a mutant comprising doing the mutant for 36 hours to 60 hours at a light intensity of more than 300 μE to 450 μE. 10. The method of claim 9,
The culture composition comprises nitrogen (N) 13.5 mM to 16.5 mM and phosphorus (P) 1.0 mM to 1.4 mM; nitrogen (N) from 10.0 mM to 13.49 mM and phosphorus (P) from 1.9 mM to 2.5 mM; nitrogen (N) from 10.0 mM to 11.99 mM and phosphorus (P) from 1.9 mM to 2.5 mM; Or nitrogen (N) 12.0mM to 13.49mM and phosphorus (P) 1.9 mM to 2.5mM, Chlamydomonas reinhardtii a method of culturing a mutant strain. 10. The method of claim 9,
The culture composition further comprises a calcium (Ca) 0.47 mM to 0.70mM and magnesium (Mg) 0.55 mM to 0.85mM, Chlamydomonas reinhardtii a method of culturing a mutant strain. 10. The method of claim 9,
The culture composition further comprises 1.5ml/L to 2.5ml/L of glacial acetic acid as a carbon source, Chlamydomonas reinhardtii) A method of culturing a mutant. 10. The method of claim 9,
The culture composition is one or more trace elements selected from the group consisting of EDTA, iron (Fe), zinc (Zn), boron (B), manganese (Mn), copper (Cu), molybdenum (Mo), and cobalt (Co). Which further comprises, Chlamydomonas rain hard Tia (Chlamydomonas reinhardtii) method of culturing a mutant. 16. The method of claim 15,
The culture composition is EDTA 133 μM to 137 μM, iron (Fe) 16 μM to 20 μM, zinc (Zn) 73 μM to 77 μM, boron (B) 183 μM to 187 μM, manganese (Mn) 24 μM to 28 μM, copper (Cu) 5.0 μM to 8.0 μM , Molybdenum (Mo) 4.0μM to 7.0μM, and cobalt (Co) 5.0μM to 8.0μM, Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) a method of culturing a mutant that further comprises 8.0μM. 10. The method of claim 9,
The mutant, while having a zeaxanthin (zeaxanthin) producing ability, the ZEP gene and the LCYE gene that do not produce lutein are simultaneously knocked out, Chlamydomonas reinhardtii) A method of culturing a mutant. 10. The method of claim 9,
The mutant is adenine (A) between the 816th base and the 817th base in the ZEP gene sequence of Chlamydomonas reinhardtii represented by SEQ ID NO: 1; the nucleotide sequence represented by SEQ ID NO: 3; or ZEP gene mutation into which the nucleotide sequence represented by SEQ ID NO: 5 is inserted; And the nucleotide sequence shown in SEQ ID NO: 9 between the 599th base and the 600th base in the LCYE gene sequence of Chlamydomonas reinhardtii represented by SEQ ID NO: 2; Or, the nucleotide sequence represented by SEQ ID NO: 11 will include the LCYE gene mutation is inserted, the mutant will, Chlamydomonas reinhardtii (Chlamydomonas reinhardtii) culturing a mutant strain. 10. In the culture composition according to any one of claims 1 to 9, without producing lutein, culturing Chlamydomonas reinhardtii mutants having zeaxanthin-producing ability; zeaxanthin production method comprising a.

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