CN115927130A - Synechococcus JS50 capable of secreting and producing fructose at high yield and preparation method thereof - Google Patents

Abstract

The invention relates to synechococcus JS50 with fructose secretion capacity and high fructose yield, which takes PCC7942 as an original strain, knocks out fructokinase, and over-expresses sucrose hydrolase, sucrose phosphate synthase and sucrose phosphate phosphorylase. By knocking out fructokinase, PCC7942 can have the capacity of high-yield fructose and transporting fructose to the outside of cells, and by over-expressing sucrose hydrolase, sucrose phosphate synthase and sucrose phosphate phosphorylase, the fructose yield of the engineering algae strain can be greatly improved. The constructed algal strain JS50 can produce 3.9g/L of fructose in the culture system after 12 days of culture. The carbon dioxide and water are directly converted into fructose by taking solar energy as a drive on a single platform, the effects of carbon sequestration and emission reduction and fructose production are achieved, and the engineering application potential of the cyanobacteria optical drive carbon sequestration and sugar production technology is improved.

Description

Synechococcus JS50 capable of secreting and producing fructose at high yield and preparation method thereof

Technical Field

The invention relates to the field of synthetic biology, in particular to synechococcus JS50 with fructose secretion capacity and high fructose yield and a preparation method thereof.

Background

Fructose is widely applied in food, medicine, chemical industry and other industries, and especially can be used for preventing diabetes, obesity, decayed tooth and other diseases. At present, fructose products on the market have two forms of high fructose syrup and crystalline fructose. The production of the high fructose syrup takes starch as a main raw material, and comprises the steps of firstly utilizing alpha-amylase and glucoamylase to hydrolyze the starch into glucose through a chemical catalytic synthesis route, and then utilizing glucose isomerase to convert the glucose into the high fructose syrup containing 42 percent of fructose and 58 percent of glucose. Crystalline fructose (fructose with purity of more than 97%) is fine powdery crystal, and is produced by extracting a product from a glucose-fructose mixed system on the basis of high fructose syrup, and the product is prepared by a series of operations such as chromatographic separation, enrichment, concentration, crystallization, screening and the like. Therefore, the development of a novel efficient and green fructose synthesis technology has important significance for reducing the economic and environmental cost of the fructose industry and improving the product competitiveness and market acceptance.

Cyanobacteria is a prokaryotic microorganism for carrying out plant type oxygen release photosynthesis, and compared with microalgae and higher plants, cyanobacteria has the advantages of simple structure, rapid growth, convenient genetic modification and the like, and thus, cyanobacteria becomes a photosynthetic synthesis platform for biological energy and biological-based chemicals with great potential. However, no potential high fructose content cyanobacteria has been found in the current research, nor has the ability to secrete fructose outside been found. Niedeholtmeyer et al formed a fructose secretion pathway by transferring hexose transporter Glf gene into Synechococcus PCC7942, so that the bacteria could secrete fructose outwards, but even under optimal conditions, it produced extracellular fructose only at a maximum of 28.8mg/L, and the fructose level decreased with increasing cell density, which did not meet the requirements of industrial production (Niedeholteyer, H., wolfstadter, B.T., savage, D.F., silver, P.A., and Way, J.C., engineering to synthesis and export hydrophic products, l.environ.Microbiol.,2010.76 (11): p.3462-3466).

Therefore, there is a need for a new cyanobacterium that has a high fructose yield and secretes fructose outside the cell.

Disclosure of Invention

We have found during our studies on synechococcus PCC7942 that when fructokinase of this alga is knocked out, the mutant unexpectedly shows very high fructose production and essentially all of these fructose is secreted extracellularly, resulting in fructose levels in the culture system of up to several hundred milligrams per liter. On the basis, the alga is further subjected to metabolic engineering to obtain an engineering alga strain JS50 with ultrahigh fructose yield.

Based on the work, the invention provides a method for preparing synechococcus with fructose secretion capacity and high fructose yield, which takes synechococcus PCC7942 as a starting strain, and the fructokinase of the starting strain is knocked out. Preferably, sucrose hydrolase, sucrose phosphate synthase and sucrose phosphate phosphorylase are also overexpressed in the starting algal strain. By knocking out fructokinase, synechococcus PCC7942 can have the capacity of high-yield fructose and transporting fructose to the outside of cells, and by over-expressing sucrose hydrolase, sucrose phosphate synthase and sucrose phosphate phosphorylase, the fructose yield of the engineering algal strain can be greatly improved.

In a specific embodiment, the fructokinase has an amino acid sequence shown as SEQ ID NO.1 and a nucleic acid sequence shown as SEQ ID NO. 2.

In a specific embodiment, the sucrose hydrolase has the amino acid sequence shown as SEQ ID NO.4 and the nucleic acid sequence shown as SEQ ID NO. 5.

In a specific embodiment, the sucrose phosphate synthase has an amino acid sequence as shown in SEQ ID NO.6 and a nucleic acid sequence as shown in SEQ ID NO. 7.

In a specific embodiment, the sucrose phosphate phosphorylase has an amino acid sequence shown in SEQ ID NO.8 and a nucleic acid sequence shown in SEQ ID NO. 9.

Although specific amino acid sequences and nucleic acid sequences of sucrose hydrolase, sucrose phosphate synthase, and sucrose phosphate phosphorylase are listed in the above several embodiments, one skilled in the art, after reading the disclosure of the present application and appreciating the spirit of the present application, can select appropriate amino acid or nucleic acid sequences within existing and future gene pools for overexpression in algal strains to increase fructose yield and productivity of algal strains.

In a specific embodiment, the overexpression of the above-mentioned genes is driven by a strong promoter, forming a gene expression cassette. The strong promoter may be selected from a library of existing or future promoters, as long as the selected promoter is strongly expressed in the PCC7942 cells. For example, constitutive strong promoters such as the cpcB1 promoter, the cpcB560 promoter and the rbcL promoter may be selected as the promoter, and inducible strong promoters such as the lac promoter and the trc promoter may be selected as the promoter.

The gene expression cassette described above may be inserted into the genomic DNA of PCC7942, for example, into some functional genes or into a non-functional neutral platform (referring to sites that do not produce any phenotype following some insertion or deletion mutations identified from the genome). The gene expression cassette described above may also be present in an independently replicable form, e.g., on a replicable plasmid in PCC 7942. The gene expression cassettes may constitute separate operons or may form polycistrons under one operon.

In a toolIn one embodiment, the ATP synthase F of the starting strain ₀ F ₁ The 252 th amino acid of the alpha subunit is mutated from cysteine to phenylalanine. The yield of the algal strain itself can be improved by the mutation.

The invention also provides synechococcus with fructose secretion capacity and high fructose yield, which is prepared by the method.

The invention also provides a method for producing fructose by using the synechococcus.

In one embodiment, the culture conditions for synechococcus are as follows: the culture medium is BG11, the culture temperature is 25-35 deg.C, and the illumination is 100-300 μmol photons m ^-2 s ^-1 Culturing in the presence of CO gas ₂ The gas mixture with air, for example, may contain 3% CO ₂ And 97% air.

In a particular embodiment, one or more supplements of the nutrient components of the medium are performed during the culturing process. For example, medium nutrients may be doubled after 4 days of culture.

The culture conditions can not only improve the fructose yield of the engineering algae, but also ensure that the growth of the engineering algae is not influenced.

The invention constructs an engineering alga JS50 with high fructose yield by taking synechococcus PCC7942 as an original alga strain. The strain can produce fructose content of 3.9g/L in the culture system after culturing for 12 days, and can extract fructose from the culture system more easily because fructose is basically secreted to the outside of cells. Therefore, the invention realizes that carbon dioxide and water are directly converted into fructose by taking solar energy as drive on a single platform, achieves the effects of carbon sequestration and emission reduction and fructose production, and improves the engineering application potential of the cyanobacteria optical drive carbon sequestration and sugar production technology.

Drawings

FIG. 1 is a plasmid map of pJS 11.

FIG. 2 is a plasmid map of pJS 15.

FIG. 3 is a plasmid map of pJS 32.

FIG. 4 is a plasmid map of pSS18.

FIG. 5 is an electrophoretogram of PCR amplification products for the fructokinase knockout site of JS05.

FIG. 6 shows the result of detecting the mutation site of strain JS50, wherein A is the electrophoresis photograph of the PCR amplification product of the fructose kinase knockout site; b is an electrophoresis picture of a PCR amplification product of the NS1 site; c is an electrophoresis picture of a PCR amplification product of the NS2 site; d is ATP synthase F ₀ F ₁ And (3) sequencing results of the alpha subunit near the 252 th amino acid.

FIG. 7 is an HPLC peak profile of a saccharide standard.

FIG. 8 is a graph showing the culture growth curve (A), the extracellular fructose content (B) and the intracellular fructose content (C) of wild-type PCC7942 (WT) and fructokinase knockout strain JS05.

FIG. 9 is a graph showing the statistics of the culture growth curve (A) and the extracellular fructose content (B) of JS50 and JS50 after the addition of one-fold nutrients on day4 (JS 50-day 4-2X).

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

1. Plasmid construction

1.1 construction of fructokinase knockout plasmid pJS11: the fructokinase gene (the amino acid sequence is shown as SEQ ID NO.1, and the nucleic acid sequence is shown as SEQ ID NO. 2) and the upstream and downstream sequences thereof are found from the PCC7942 genome. Primers were designed based on these sequences and the upstream homology arm (FK-N) and downstream homology arm (FK-C) of fructokinase were amplified using the PCC7942 genome as a template. The recombinant plasmid pJS11 was obtained by inserting the chloramphenicol resistance fragment between the upstream and downstream homology arms of fructokinase and integrating it into the pUC19 backbone, and the plasmid map is shown in FIG. 1.

1.2 construction of Invertase over-expression plasmid pJS15: firstly, obtaining kanamycin resistant fragment and constitutive strong promoter P _cpcB1 (SEQ ID NO. 3), then obtaining a sucrose hydrolase (Invertase) gene fragment (the amino acid sequence is shown as SEQ ID NO.4, the nucleic acid sequence is shown as SEQ ID NO. 5) by using a PCC7942 genome as a template for amplification, and the upper part of a neutral site (a site which does not generate phenotype due to deletion mutation) NS1A downstream homology arm and a downstream homology arm. The recombinant plasmid pJS15 is obtained by inserting an overexpression frame of the Invertase gene between the upstream homology arm and the downstream homology arm of NS1, and integrating the resistant fragment as a screening marker into the pUC19 skeleton, and the plasmid map is shown in FIG. 2.

1.3 construction of Sps and Spp overexpression plasmid pJS32: firstly, a spectinomycin resistant fragment and a strong promoter P are obtained _cpcB1 And secondly, amplifying sucrose phosphate synthase gene segments Sps (the amino acid sequence is shown as SEQ ID NO.6, and the nucleic acid sequence is shown as SEQ ID NO. 7) and sucrose phosphate phosphorylase Spp (the amino acid sequence is shown as SEQ ID NO.8, and the nucleic acid sequence is shown as SEQ ID NO. 9) by taking the genome of the PCC6803 as a template. By ligation to obtain P _cpcB1 And (3) driving expression frames of the Sps gene and the Spp gene, wherein the expression frames are inserted between the upstream and downstream homologous arms of a neutral site NS2, a resistance fragment is used as a screening marker and is integrated onto a pUC19 framework to obtain a recombinant plasmid pJS32, and the plasmid map is shown in figure 3.

1.4 construction of AtpA Point mutation plasmid pSS18: amplification of ATP synthase F from PCC7942 ₀ F ₁ Alpha subunit (AtpA), and mutating the 252 th amino acid from cysteine (C) to phenylalanine (F), wherein the amino acid sequence is shown as SEQ ID NO.10, and the nucleic acid sequence is shown as SEQ ID NO.11, so as to construct a point mutation plasmid pSS18. The plasmid map is shown in FIG. 4.

2. Construction of transgenic algal strains

The plasmid pJS11 was transformed into wild type PCC7942 to obtain algal strain JS05. The method comprises the following steps:

1) 1mL of the mixture was taken in the logarithmic growth phase (OD) ₇₃₀ About 1), centrifuging 5000g for 5min to collect cells, washing the cells 2 times with fresh BG11 medium, discarding the supernatant, and resuspending the cell pellet in 250. Mu.L BG11 solution; BG11 Medium: from 1.5g L ^-1 NaNO ₃ ,40mg L ^-1 K ₂ HPO ₄ ·3H ₂ O,36mg L ^-1 CaCl ₂ ·2H ₂ O,6mg L ^-1 Citric acid, 6mg L ^-1 Ammonium ferric citrate, 1mg L ^-1 EDTA disodium salt, 20mg L ^-1 Na ₂ CO ₃ ,2.9mg L ^-1 H ₃ BO ₃ ,1.8mg L ^-1 MnCl ₂ ·4H ₂ O,0.22mg L ^-1 ZnSO ₄ ·7H ₂ O,0.39mg L ^-1 Na ₂ MoO ₄ ·2H ₂ O,0.079mg L ^-1 CuSO ₄ ·5H ₂ O and 0.01mg L ^{- 1} CoCl ₂ ·6H ₂ And (C) O.

2) To the resuspended algal solution was added 2. Mu.L of plasmid pJS11 (plasmid concentration 100. Mu.g/mL);

3) Wrapping the EP tube added with the plasmid by using tin foil paper, and incubating for 20 hours at 30 ℃ in a shaking table;

4) The incubated transformation products were plated on BG11 plates with corresponding resistance (chloramphenicol: 5. Mu.g/mL), 30 ℃ 100. Mu. Mol phosns m ^-2 s ^-1 After culturing under the condition, transformants grow up after 4-5 days, the transformants are selected and streaked on a fresh BG11 plate (chloramphenicol: 5. Mu.g/mL), and completely separated algae strains are obtained by screening.

The wild-type PCC7942 was transformed with the plasmids pJS11, pJS15 and pJS32, and on that basis, the transformation was continued with the plasmid pSS18, resulting in the algal strain JS50. The method comprises the following steps:

1) 1mL of the solution was taken in logarithmic growth phase (OD) ₇₃₀ About 1), centrifuging 5000g for 5min to collect cells, washing the cells 2 times with fresh BG11 medium, discarding the supernatant, and resuspending the cell pellet in 250. Mu.L BG11 solution;

2) To the resuspended algal solution was added 2. Mu.L each of the three plasmids: plasmids pJS11, pJS15, pJS32 (plasmid concentration 100. Mu.g/mL);

3) Wrapping the EP tube added with the plasmid with tin foil paper, and incubating for 20 hours at 30 ℃ in a shaking table;

4) The incubated transformation products were plated on BG11 plates with corresponding resistance (spectinomycin: 10. Mu.g/mL, chloramphenicol: 5. Mu.g/mL, kanamycin: 10. Mu.g/mL), 30 ℃, 100. Mu. Mol photons m ^-2 s ^-1 Culturing under the condition that transformants grow up after 4-5 days, selecting the transformants to streak on a fresh BG11 plate (spectinomycin: 10 mug/mL, chloramphenicol: 5 mug/mL, kanamycin: 10 mug/mL), and obtaining completely separated algae strains through screening;

5) Transforming pSS18 into the engineering strain obtained in 4), and the method is similar to the previous method; completely separated algae strains are obtained by screening.

As shown in fig. 5, in JS05, fructokinase was completely knocked out. As shown in FIG. 6, JS50 had fructokinase knocked out, and Invertase gene, sps and Spp genes were overexpressed, and ATP synthase F ₀ F ₁ The 252 th amino acid in the alpha subunit (AtpA) was mutated from cysteine to phenylalanine.

3. JS05 fructose secretion

A column type photoreactor is used, the photoreactor is a round bottom glass tube made of common glass and having the diameter of 3cm and the length of 20cm, the total liquid loading of the photoreactor can reach 100mL, and the liquid loading in the experimental process is 65mL. The seed solution was BG11 culture in logarithmic growth phase (supplemented with chloramphenicol: 5. Mu.g/mL) at initial inoculum concentration OD ₇₃₀ 2, BG11 medium (antibiotic addition), 150. Mu. Mol photons m at 30 ℃ ^-2 s ^-1 Introducing mixed gas (3% CO) ₂ +97% air), or by adding 150mM NaCl to the medium at the beginning of the culture.

And monitoring the growth of the strain in the column type illumination culture process of JS05, drawing a strain growth curve, and simultaneously carrying out quantitative analysis on the extracellular fructose content and the intracellular fructose content.

Analysis of fructose content using HPLC method: taking 1mL of algae solution of the engineering strain in the culture process, centrifuging at 13000rpm for 10min, transferring the centrifuged supernatant into another clean 1.5mL of EP tube, and determining the content of extracellular fructose; diluting the fructose extracted from the cells, and detecting by liquid chromatography (using Agilent high performance liquid chromatograph 1260, equipped with differential detector, and HPX-87H sugar analysis column with mobile phase of 5mM H ₂ SO ₄ Solution flow rate of 0.5 mL/min), it was shown by preliminary experiments that fructose peaks around 20min (fig. 7).

As shown in fig. 8, the growth curves of the mutant strain JS05 cultured in the normal BG11 medium and BG11 medium supplemented with 150mM NaCl were not very different. In the early stage of culture, the addition of a salt culture medium can lead the strain to accumulate higher intracellular fructose content (6-12 days), but by 14 days, the intracellular fructose content of each group has no significant difference, which indicates that the intracellular fructose content accumulation reaches a limit value along with the extension of the culture time.

Unexpectedly, no extracellular fructose secretion was observed in the wild type with or without salt, whereas in JS05 we observed higher concentrations of extracellular fructose in both conditions, especially in JS05 culture system up to 387mg/L after 14 days of culture in salt addition. This suggests that the fructokinase knock-out not only dramatically and unexpectedly increased the fructose production of synechococcus PCC7942, but also activated the pathway for fructose secretion out of the cell.

4. JS50 secretion fructose

A column type photoreactor is used, the column type photoreactor is a round bottom glass tube which is made of common glass and has the diameter of 3cm and the length of 20cm, the total liquid loading of the column type photoreactor can reach 100mL, and the liquid loading in the column type photoreactor in the experimental process is 65mL. The seed solution was BG11 culture (supplemented with the corresponding antibiotic: spectinomycin, 10. Mu.g/mL; kanamycin, 10. Mu.g/mL; chloramphenicol, 5. Mu.g/mL) in logarithmic growth phase at the initial inoculation concentration OD ₇₃₀ For 2, the medium is BG11 (added with antibiotics) at 25-35 deg.C and 100-300. Mu. Mol phosns m ^-2 s ^-1 Under the conditions, the mixed gas is introduced (3% CO) ₂ +97% air). Meanwhile, BG11 nutrient supplement treatment was performed up to day4 of the culture. Fructose assay was as above.

And (3) monitoring the growth of the strain in the column type illumination culture process of JS50, drawing a strain growth curve, and simultaneously carrying out quantitative analysis on the extracellular fructose content.

The results are shown in FIG. 9, after 12 days of culture, the content of fructose secreted by JS50 extracellular cells can reach 2.7g/L, and the sugar yield can reach 3.9g/L at the maximum after nutrient supplement. Use of a p-free ATP synthase F ₀ F ₁ Similar results were obtained for algal strains with alpha subunit (AtpA) mutated, only the growth rate of algal strains was slightly slower.

The result shows that on the basis of a knockout strain, a fructose production pathway in PCC7942 is strengthened through genetic operation (sucrose hydrolase Invertase, synechocystis PCC 6803-derived sucrose phosphate synthase genes Sps and sucrose phosphate phosphorylase Spp are driven by a strong promoter), so that the algal strain JS50 with ultrahigh fructose yield is obtained, after culture conditions are optimized (culture medium nutrients are supplemented in the culture process), the extracellular fructose content of the JS50 can be increased to thousands of times of the total fructose content of a wild type strain, and is increased by more than 10 times and up to 3.9g/L compared with the fructokinase knockout strain.

It should be noted that in the examples of the present invention, we describe the genetic manipulation of synechococcus PCC7942 by specific methods, including the use of specific methods, specific selection markers, specific definition of the sequence of the enzyme in the overexpressed fructose production pathway and the specific location of the insertion of the overexpressed fragment. However, these examples should not be used to limit the scope of the present invention. After reading the disclosure and comprehending the spirit of the present application, one skilled in the art can select existing or future genetic manipulation methods, gene sequences, resistance fragments and appropriate insertion sites, and can obtain such high fructose producing algal strains by knocking out fructokinase in PCC7942 and overexpressing appropriate sucrose hydrolase, sucrose phosphate synthase and sucrose phosphate phosphorylase.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.

Sequence listing

<110> institute for bioenergy and Process in Qingdao of Chinese academy of sciences

<120> synechococcus JS50 capable of secreting and producing fructose with high yield and preparation method thereof

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 324

<212> PRT

<213> Synechococcus PCC7942

<400> 1

Met Thr Ala Gln Ala Pro Thr Val Ile Cys Leu Gly Glu Met Leu Trp

1 5 10 15

Asp Cys Leu Ala Asn Asp Arg Asp Leu Pro Ala Glu Gln Val Gln Ser

20 25 30

Trp Thr Pro Trp Pro Gly Gly Ala Pro Ala Asn Val Ala Thr Ala Leu

35 40 45

Val Lys Leu Gly Val Gln Ser Gly Leu Ile Thr Cys Leu Gly Ser Asp

50 55 60

Pro Glu Gly Asp Arg Leu Phe Gln Val Leu Gln Asp Asn Gly Val Ala

65 70 75 80

Ile Ala Ala Val Gln Arg His Pro Arg Leu Pro Thr Arg Gln Val Gln

85 90 95

Val Leu Arg Thr Ala Gln Gly Asp Arg Gln Phe Gly Gly Phe Gly Ala

100 105 110

Ile Ala Cys Asp Gln Phe Ala Asp Thr Glu Leu Gln Ala Glu Gln Leu

115 120 125

Pro Gln Ala Trp Phe Arg Leu Ala Lys Val Leu Cys Leu Gly Thr Ile

130 135 140

Pro Leu Ala Tyr Pro Ser Ser Ala Ile Ala Ala Gln Gln Ala Leu Thr

145 150 155 160

Trp Ala Asn Gln Gln Gly Met Thr Val Leu Leu Asp Val Asn Trp Arg

165 170 175

Pro Ser Phe Trp Pro Asp Pro Ser Leu Ala Pro Asp Arg Ile Arg Ala

180 185 190

Ile Leu Pro Gln Ile Gln Gln Leu Lys Leu Ala Arg Glu Glu Ala Glu

195 200 205

Trp Leu Phe Asp Thr Val Asp Pro Ala Thr Ile Gln Ala Arg Gln Pro

210 215 220

Gln Leu Gln Ala Val Leu Ile Thr Asp Gly Asp Arg Gly Cys His Tyr

225 230 235 240

Trp Val Ala Gly Trp Gln Gly His Cys Pro Ser Phe Pro Val Lys Ala

245 250 255

Ile Asp Thr Thr Gly Ala Gly Asp Ala Phe Val Ala Ala Trp Leu Ala

260 265 270

Gln Asn Thr Gln Met Asp Phe Gln Trp Thr Ser Ala Glu Gln Val Gln

275 280 285

Thr Ala Ile Arg Trp Ala Ser Ala Ala Gly Ala Leu Thr Thr Leu Asn

290 295 300

Pro Gly Ala Ile Ala Ala Gln Pro Thr Ser Asp Ala Ile Gln Ser Leu

305 310 315 320

Leu Ser Lys Thr

<210> 2

<211> 975

<212> DNA

<213> Synechococcus PCC7942

<400> 2

atgacagccc aagcgccaac ggtcatctgt ctgggagaaa tgctctggga ttgtttggcc 60

aacgatcgcg atcttccggc ggaacaggtg caaagttgga caccttggcc ggggggtgct 120

cccgccaatg ttgcaacggc gctagtcaag cttggtgttc agtcgggact gattacctgc 180

ctcggttctg atcctgaagg cgatcgcctc tttcaagttc tccaagacaa tggcgtggcg 240

atcgcggcgg tgcagcggca tccccgcctg ccgacccgac aggtgcaagt gctgcgaacc 300

gctcagggcg atcgccagtt cggagggttt ggcgcgatcg cttgcgatca atttgcggat 360

acggaactgc aagcggagca gttgccgcag gcttggtttc gtttggccaa agtgctctgc 420

ttaggcacga ttccgctggc ttatcccagt agtgcgatcg cggcgcagca agcgctgact 480

tgggccaatc agcagggaat gaccgttctc ttggatgtca actggcgacc aagtttctgg 540

cctgatccca gtttggctcc cgatcgcatt cgtgccatcc tgcctcaaat tcagcagttg 600

aaattagcga gagaagaagc ggaatggctg ttcgacacgg ttgatccagc aacaattcaa 660

gcccgacaac cgcagctaca agcagtattg attacggatg gcgatcgcgg ctgccactac 720

tgggttgctg gttggcaggg tcactgtccc tcttttccgg taaaggcgat cgatacgacc 780

ggcgcgggag atgcatttgt cgctgcttgg ctagcgcaaa acacgcaaat ggacttccag 840

tggacgagcg cagagcaggt gcaaacagcg attcgctggg ctagtgctgc tggtgccttg 900

acgaccctga atcctggcgc gatcgcagct cagccgactt ctgatgctat tcaatcttta 960

ctatctaaaa cttaa 975

<210> 3

<211> 626

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ctacagcctg ggttcctcat gactctcagt ctcagctgca tgctggttgc ttgccctgct 60

gccggtccca tgagccagcg atcgctcagt tcaagaatgt aaatgaatat tacaaacctt 120

cagctcgtaa cgatttcaga tccaaaagat gtagctgttg ggtcagccat aagtttttct 180

tcaacttctc aagcgatcgt cgaccatcaa cttaaagcat ctttacaaag ctagagattg 240

accctagtcg tggacgactt acagattcag gattgatacg gccaatcccg atcgcgatcg 300

ctctaaatcc ccgtcagtca gagcttcaca atttttagcg aatcttgtgg ccgcgatcgt 360

tgtataagaa tgccaaggca actggataag gttcactaat cgttgctaag cgacagtgaa 420

ctgcgccaat tgcctgacag gcccctctcg tttaacaaac gatttaatgt aaatcattgt 480

taagagtctc tcacaatcga gagttttctt gaagaatgat ggggacggtt caggtgcagg 540

gtttccctgc tagagaatgc gaaaaaaccg cgttctcgtt ttaggaatcg agagtcaata 600

aaagtcgaga acaggagact ggttga 626

<210> 4

<211> 463

<212> PRT

<213> Synechococcus PCC7942

<400> 4

Met Pro Asp Ser Val Val Leu Pro Ala Thr Leu Gln Thr Ala Leu Gln

1 5 10 15

Thr Ala Glu Gln Leu Leu Trp Asp Arg Ala Leu Val Arg Tyr His Asp

20 25 30

Gln Trp Ala Gly Ala Ile Ala Ala Leu Pro Glu Asp Gln Glu Leu Ala

35 40 45

Ala Ala Asn Tyr Arg Glu Ile Phe Ile Arg Asp Asn Val Pro Val Met

50 55 60

Leu Tyr Leu Leu Leu Gln Gly Lys Thr Asp Val Val Arg Asp Phe Leu

65 70 75 80

Gln Leu Ser Leu Ser Leu Gln Ser Gln Ala Leu Gln Thr Tyr Gly Ile

85 90 95

Leu Pro Thr Ser Phe Val Cys Glu Glu Thr His Cys Val Ala Asp Tyr

100 105 110

Gly Gln Arg Ala Ile Gly Arg Val Val Ser Ala Asp Pro Ser Leu Trp

115 120 125

Trp Pro Val Leu Leu Gln Ala Tyr Arg Arg Ala Ser His Asp Asp Ala

130 135 140

Phe Val His Ser Pro Thr Val Gln Gln Gly Leu Gln Arg Leu Leu Ala

145 150 155 160

Phe Leu Leu Arg Pro Val Phe Asn Gln Asn Pro Leu Leu Glu Val Pro

165 170 175

Asp Gly Ala Phe Met Val Asp Arg Pro Leu Asp Val Ala Gly Ala Pro

180 185 190

Leu Glu Ile Gln Val Leu Leu Tyr Gly Ala Leu Arg Ala Cys Gly Gln

195 200 205

Leu Leu Gln Tyr Thr Glu Ala Ala Asn Ala Ala His Val Gln Ala Arg

210 215 220

Arg Leu Arg Gln Tyr Leu Cys Trp His Tyr Trp Val Thr Pro Asp Arg

225 230 235 240

Leu Arg Arg Trp Gln Gln Trp Pro Thr Glu Glu Phe Gly Asp Arg Ser

245 250 255

His Asn Pro Tyr Asn Ile Gln Pro Ile Ala Ile Pro Asp Trp Val Glu

260 265 270

Pro Trp Leu Gly Glu Ser Gly Gly Tyr Phe Leu Gly Asn Ile Arg Ala

275 280 285

Gly Arg Pro Asp Phe Arg Phe Phe Ser Leu Gly Asn Leu Leu Ala Ile

290 295 300

Val Phe Asp Val Leu Pro Leu Asn Gln Gln Gly Ala Ile Leu Arg Leu

305 310 315 320

Ile Leu Gln Asn Glu Ala Gln Ile Leu Gly Gln Val Pro Leu Arg Leu

325 330 335

Cys Tyr Pro Ala Leu Thr Gly Ser Ala Trp Lys Ile Leu Thr Gly Cys

340 345 350

Asp Pro Lys Asn Gln Pro Trp Ser Tyr His Asn Gly Gly Ser Trp Pro

355 360 365

Ser Leu Leu Trp Tyr Leu Ser Ala Ala Val Leu His Tyr Gln Gln Arg

370 375 380

Gly Gly Asp Arg Asn Leu Cys Gln Val Trp Leu Asn Lys Leu Gln His

385 390 395 400

Tyr His Thr Gln Gln Cys Glu Gln Leu Pro Gly Asp Glu Trp Pro Glu

405 410 415

Tyr Tyr Glu Gly Gln Asp Ser Val Gln Ile Ala Thr Arg Ala Cys Arg

420 425 430

Tyr Gln Thr Trp Thr Phe Thr Gly Leu Leu Leu Asn His Ala Leu Leu

435 440 445

Ser Gln Pro Gln Gly Ile Gln Leu Leu Ser Leu Arg Gly Leu Pro

450 455 460

<210> 5

<211> 1392

<212> DNA

<213> Synechococcus PCC7942

<400> 5

atgcccgatt ctgttgtgct gcccgctacg ctgcagaccg cgctgcaaac agcggagcag 60

ttactttggg atcgggcctt ggttcgctat cacgatcagt gggcgggggc gatcgcggca 120

ctgcctgaag atcaggagtt ggcggcagcg aactaccgcg aaatctttat tcgcgacaac 180

gtgccggtga tgctctacct gctgttgcag ggcaaaactg acgttgtccg cgacttcttg 240

caactgtcgc tttctctcca gagccaggca ctgcaaacct atggcattct gccgaccagt 300

ttcgtctgtg aggaaaccca ctgcgttgct gactatggtc agcgggcgat cgggcgggtg 360

gtttctgctg accctagcct ttggtggccg gtgctgctac aggcctatcg gcgggcctcc 420

catgatgatg cctttgtcca cagtccgact gttcagcagg ggttacagcg gttgctggct 480

ttcctgctgc gtccggtttt caaccaaaac ccactgctcg aggtgcccga tggggccttc 540

atggtcgatc gtcccttgga tgtggcgggc gcacctttag aaattcaagt cctgctctac 600

ggggcactgc gggcttgtgg gcagttgctg caatacaccg aagcggccaa tgctgcccat 660

gtgcaagccc gtcgcctgcg gcagtatctc tgctggcact actgggtgac gcccgatcgc 720

ctgcgacgct ggcagcagtg gcccaccgaa gaatttggcg atcgcagcca taacccctac 780

aacattcagc cgatcgccat ccctgactgg gttgaacctt ggctgggtga gtcgggtggc 840

tacttcctag ggaacatacg ggcaggacgt cctgacttcc gcttttttag ccttggcaat 900

ttgctggcga tcgttttcga tgtgcttccg ctcaatcagc agggtgcgat tctgcgcttg 960

attttgcaga acgaagccca gattttgggc caagtgccgt tgcggctctg ctatcccgct 1020

ttaaccggat cggcgtggaa aatcctgacg ggttgcgatc ctaaaaatca gccttggtcc 1080

tatcacaacg gtggtagttg gccatccctg ctttggtatc tcagtgcggc ggtcttgcac 1140

taccaacagc ggggaggcga tcgcaatctc tgtcaggtct ggctgaataa gcttcagcac 1200

taccacactc agcagtgcga gcaactccct ggcgatgagt ggccagagta ctacgagggt 1260

caggactcgg tccagattgc tactcgcgcc tgccgttatc agacttggac gtttacggga 1320

ttgctgctga atcacgcact gctctcgcag ccccagggca ttcaactgct gagtctgcgg 1380

ggcttaccct aa 1392

<210> 6

<211> 720

<212> PRT

<213> Synechocystis PCC6803

<400> 6

Met Ser Tyr Ser Ser Lys Tyr Ile Leu Leu Ile Ser Val His Gly Leu

1 5 10 15

Ile Arg Gly Glu Asn Leu Glu Leu Gly Arg Asp Ala Asp Thr Gly Gly

20 25 30

Gln Thr Lys Tyr Val Leu Glu Leu Ala Arg Ala Leu Val Lys Asn Pro

35 40 45

Gln Val Ala Arg Val Asp Leu Leu Thr Arg Leu Ile Lys Asp Pro Lys

50 55 60

Val Asp Ala Asp Tyr Ala Gln Pro Arg Glu Leu Ile Gly Asp Arg Ala

65 70 75 80

Gln Ile Val Arg Ile Glu Cys Gly Pro Glu Glu Tyr Ile Ala Lys Glu

85 90 95

Met Leu Trp Asp Tyr Leu Asp Asn Phe Ala Asp His Ala Leu Asp Tyr

100 105 110

Leu Lys Glu Gln Pro Glu Leu Pro Asp Val Ile His Ser His Tyr Ala

115 120 125

Asp Ala Gly Tyr Val Gly Thr Arg Leu Ser His Gln Leu Gly Ile Pro

130 135 140

Leu Val His Thr Gly His Ser Leu Gly Arg Ser Lys Arg Thr Arg Leu

145 150 155 160

Leu Leu Ser Gly Ile Lys Ala Asp Glu Ile Glu Ser Arg Tyr Asn Met

165 170 175

Ala Arg Arg Ile Asn Ala Glu Glu Glu Thr Leu Gly Ser Ala Ala Arg

180 185 190

Val Ile Thr Ser Thr His Gln Glu Ile Ala Glu Gln Tyr Ala Gln Tyr

195 200 205

Asp Tyr Tyr Gln Pro Asp Gln Met Leu Val Ile Pro Pro Gly Thr Asp

210 215 220

Leu Glu Lys Phe Tyr Pro Pro Lys Gly Asn Glu Trp Glu Thr Pro Ile

225 230 235 240

Val Gln Glu Leu Gln Arg Phe Leu Arg His Pro Arg Lys Pro Ile Ile

245 250 255

Leu Ala Leu Ser Arg Pro Asp Pro Arg Lys Asn Ile His Lys Leu Ile

260 265 270

Ala Ala Tyr Gly Gln Ser Pro Gln Leu Gln Ala Gln Ala Asn Leu Val

275 280 285

Ile Val Ala Gly Asn Arg Asp Asp Ile Thr Asp Leu Asp Gln Gly Pro

290 295 300

Arg Glu Val Leu Thr Asp Leu Leu Leu Thr Ile Asp Arg Tyr Asp Leu

305 310 315 320

Tyr Gly Lys Val Ala Tyr Pro Lys Gln Asn Gln Ala Glu Asp Val Tyr

325 330 335

Ala Leu Phe Arg Leu Thr Ala Leu Ser Gln Gly Val Phe Ile Asn Pro

340 345 350

Ala Leu Thr Glu Pro Phe Gly Leu Thr Leu Ile Glu Ala Ala Ala Cys

355 360 365

Gly Val Pro Ile Val Ala Thr Glu Asp Gly Gly Pro Val Asp Ile Ile

370 375 380

Lys Asn Cys Gln Asn Gly Tyr Leu Ile Asn Pro Leu Asp Glu Val Asp

385 390 395 400

Ile Ala Asp Lys Leu Leu Lys Val Leu Asn Asp Lys Gln Gln Trp Gln

405 410 415

Phe Leu Ser Glu Ser Gly Leu Glu Gly Val Lys Arg His Tyr Ser Trp

420 425 430

Pro Ser His Val Glu Ser Tyr Leu Glu Ala Ile Asn Ala Leu Thr Gln

435 440 445

Gln Thr Ser Val Leu Lys Arg Ser Asp Leu Lys Arg Arg Arg Thr Leu

450 455 460

Tyr Tyr Asn Gly Ala Leu Val Thr Ser Leu Asp Gln Asn Leu Leu Gly

465 470 475 480

Ala Leu Gln Gly Gly Leu Pro Gly Asp Arg Gln Thr Leu Asp Glu Leu

485 490 495

Leu Glu Val Leu Tyr Gln His Arg Lys Asn Val Gly Phe Cys Ile Ala

500 505 510

Thr Gly Arg Arg Leu Asp Ser Val Leu Lys Ile Leu Arg Glu Tyr Arg

515 520 525

Ile Pro Gln Pro Asp Met Leu Ile Thr Ser Met Gly Thr Glu Ile Tyr

530 535 540

Ser Ser Pro Asp Leu Ile Pro Asp Gln Ser Trp Arg Asn His Ile Asp

545 550 555 560

Tyr Leu Trp Asn Arg Asn Ala Ile Val Arg Ile Leu Gly Glu Leu Pro

565 570 575

Gly Leu Ala Leu Gln Pro Lys Glu Glu Leu Ser Ala Tyr Lys Ile Ser

580 585 590

Tyr Phe Tyr Asp Ala Ala Ile Ala Pro Asn Leu Glu Glu Ile Arg Gln

595 600 605

Leu Leu His Lys Gly Glu Gln Thr Val Asn Thr Ile Ile Ser Phe Gly

610 615 620

Gln Phe Leu Asp Ile Leu Pro Ile Arg Ala Ser Lys Gly Tyr Ala Val

625 630 635 640

Arg Trp Leu Ser Gln Gln Trp Asn Ile Pro Leu Glu His Val Phe Thr

645 650 655

Ala Gly Gly Ser Gly Ala Asp Glu Asp Met Met Arg Gly Asn Thr Leu

660 665 670

Ser Val Val Val Ala Asn Arg His His Glu Glu Leu Ser Asn Leu Gly

675 680 685

Glu Ile Glu Pro Ile Tyr Phe Ser Glu Lys Arg Tyr Ala Ala Gly Ile

690 695 700

Leu Asp Gly Leu Ala His Tyr Arg Phe Phe Glu Leu Leu Asp Pro Val

705 710 715 720

<210> 7

<211> 2163

<212> DNA

<213> Synechocystis PCC6803

<400> 7

atgagctatt catcaaaata cattttacta attagtgtcc atggtttaat tcggggagaa 60

aaccttgagt tgggcagaga tgccgacacc ggcgggcaaa ccaaatatgt gctggaactg 120

gcccgggcct tggtaaaaaa tccccaggtg gccagggtgg atttgctgac ccgtttaatt 180

aaagatccca aagtagatgc agattatgcc cagcctagag aactcattgg cgatcgggcc 240

cagattgttc gcattgagtg cggcccggag gaatatattg ccaaggaaat gctctgggac 300

tatttggata attttgctga ccatgccctg gactatctca aagaacagcc cgaactgccc 360

gatgtcatcc atagccatta cgccgatgcg ggttacgtgg gcaccagact ttctcaccaa 420

ttgggtattc ctttggtgca caccggacat tccctgggtc gtagtaagcg cacccgtctc 480

ctgctcagtg ggattaaagc cgacgaaatt gaaagccgtt acaatatggc ccgccggatt 540

aacgcggagg aagaaaccct aggatcagcg gcgagggtga ttaccagtac ccatcaggaa 600

atcgcagaac agtacgccca atacgactat taccagccag accagatgtt ggttattccc 660

cccggcactg atttagaaaa gttttatccc cccaaaggga acgagtggga aacgcccatt 720

gttcaagagt tgcaacgatt tctacggcat ccccgtaagc ctattatcct cgctttgtcc 780

cgaccggatc cccgcaaaaa tatccataaa ttaattgcag cctatggcca gtccccgcag 840

ttacaggccc aggccaattt ggtcattgtg gcgggcaatc gggatgacat cacggatcta 900

gaccaggggc cgagggaagt actgacggat ttactgttga ccattgaccg ttacgatctc 960

tacggcaaag tggcttaccc caaacagaat caggcggagg atgtgtatgc tttgtttcgc 1020

ctcactgctt tatcccaggg agtatttatc aatccggctt tgacggaacc ctttggttta 1080

actttgattg aagcggcggc ctgtggtgtg cccattgtgg ccacggagga tgggggcccg 1140

gtggatatta tcaaaaattg tcagaatggc tatctaatta atcccctcga tgaagtggat 1200

attgcggata aattgctcaa agtactaaac gacaaacaac aatggcaatt cctttctgaa 1260

agtggtctag agggagttaa gcgccattat tcttggcctt cccacgttga aagttattta 1320

gaagccatca acgctctgac ccaacagact tcagtgctga aacgtagtga tttaaagcgg 1380

cggcggactt tgtactataa cggtgccctg gttactagtt tggaccaaaa tttactgggg 1440

gcattacagg ggggattacc gggcgatcgc cagacgttgg acgaattact ggaagtgctg 1500

tatcaacatc gaaaaaatgt cggcttttgc attgccactg ggagaagatt ggattcggtg 1560

ctgaaaattt tgcgggagta tcgcattccc caaccggata tgttgatcac cagcatgggc 1620

acggaaattt attcttcccc ggatttgatc cccgaccaga gttggcgcaa tcacattgat 1680

tatttgtgga accgtaacgc cattgtgcgt attttggggg aattacccgg tttagccctc 1740

caacccaagg aagaactgag cgcctataaa attagctatt tctacgatgc ggcgatcgcc 1800

cctaacctag aagaaattcg gcaactgttg cataaagggg aacaaaccgt aaataccatc 1860

atttcctttg gtcaattttt ggatattctg cccatccgag cttccaaagg ctatgctgtg 1920

cgttggttga gccaacagtg gaatattccc ctggagcacg ttttcaccgc cggaggatcg 1980

ggagccgacg aagatatgat gcggggtaac accctttccg tcgtcgtggc taaccgtcac 2040

catgaggaac tttctaatct aggggagatc gaaccgattt atttttccga aaaacgttac 2100

gccgccggta ttctggacgg tctggcccat taccgcttct ttgagttgtt agaccccgtt 2160

taa 2163

<210> 8

<211> 244

<212> PRT

<213> Synechocystis PCC6803

<400> 8

Met Arg Gln Leu Leu Leu Ile Ser Asp Leu Asp Asn Thr Trp Val Gly

1 5 10 15

Asp Gln Gln Ala Leu Glu His Leu Gln Glu Tyr Leu Gly Asp Arg Arg

20 25 30

Gly Asn Phe Tyr Leu Ala Tyr Ala Thr Gly Arg Ser Tyr His Ser Ala

35 40 45

Arg Glu Leu Gln Lys Gln Val Gly Leu Met Glu Pro Asp Tyr Trp Leu

50 55 60

Thr Ala Val Gly Ser Glu Ile Tyr His Pro Glu Gly Leu Asp Gln His

65 70 75 80

Trp Ala Asp Tyr Leu Ser Glu His Trp Gln Arg Asp Ile Leu Gln Ala

85 90 95

Ile Ala Asp Gly Phe Glu Ala Leu Lys Pro Gln Ser Pro Leu Glu Gln

100 105 110

Asn Pro Trp Lys Ile Ser Tyr His Leu Asp Pro Gln Ala Cys Pro Thr

115 120 125

Val Ile Asp Gln Leu Thr Glu Met Leu Lys Glu Thr Gly Ile Pro Val

130 135 140

Gln Val Ile Phe Ser Ser Gly Lys Asp Val Asp Leu Leu Pro Gln Arg

145 150 155 160

Ser Asn Lys Gly Asn Ala Thr Gln Tyr Leu Gln Gln His Leu Ala Met

165 170 175

Glu Pro Ser Gln Thr Leu Val Cys Gly Asp Ser Gly Asn Asp Ile Gly

180 185 190

Leu Phe Glu Thr Ser Ala Arg Gly Val Ile Val Arg Asn Ala Gln Pro

195 200 205

Glu Leu Leu His Trp Tyr Asp Gln Trp Gly Asp Ser Arg His Tyr Arg

210 215 220

Ala Gln Ser Ser His Ala Gly Ala Ile Leu Glu Ala Ile Ala His Phe

225 230 235 240

Asp Phe Leu Ser

<210> 9

<211> 735

<212> DNA

<213> Synechocystis PCC6803

<400> 9

atgcgacagt tattgctaat ttctgacctg gacaatacct gggtcggaga tcaacaagcc 60

ctggaacatt tgcaagaata tctaggcgat cgccggggaa atttttattt ggcctatgcc 120

acggggcgtt cctaccattc cgcgagggag ttgcaaaaac aggtgggact catggaaccg 180

gactattggc tcaccgcggt ggggagtgaa atttaccatc cagaaggcct ggaccaacat 240

tgggctgatt acctctctga gcattggcaa cgggatatcc tccaggcgat cgccgatggt 300

tttgaggcct taaaacccca atctcccttg gaacaaaacc catggaaaat tagctatcat 360

ctcgatcccc aggcttgccc caccgtcatc gaccaattaa cggagatgtt gaaggaaacc 420

ggcatcccgg tgcaggtgat tttcagcagt ggcaaagatg tggatttatt gccccaacgg 480

agtaacaaag gtaacgccac ccaatatctg caacaacatt tagccatgga gccgtctcaa 540

accctggtgt gtggggactc cggcaatgat attggcttat ttgaaacttc cgctcggggt 600

gtcattgtcc gtaatgccca gccggaatta ttgcactggt atgaccaatg gggggattct 660

cgtcattatc gggcccaatc gagccatgct ggcgctatcc tagaggcgat cgcccatttc 720

gattttttga gctga 735

<210> 10

<211> 505

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Met Val Ser Ile Arg Pro Asp Glu Ile Ser Ser Ile Ile Arg Gln Gln

1 5 10 15

Ile Glu Gln Tyr Ser Gln Asp Val Lys Val Glu Asn Val Gly Thr Val

20 25 30

Leu Gln Val Gly Asp Gly Ile Ala Arg Ile Tyr Gly Leu Gln Gln Val

35 40 45

Met Ser Gly Glu Leu Val Glu Phe Glu Asp Gly Thr Thr Gly Ile Ala

50 55 60

Leu Asn Leu Glu Glu Asp Asn Val Gly Ala Val Leu Met Gly Glu Gly

65 70 75 80

Arg Asn Ile Gln Glu Gly Ser Thr Val Lys Ala Thr Gly Lys Ile Ala

85 90 95

Gln Ile Pro Val Gly Asp Ala Leu Val Gly Arg Val Val Ser Pro Leu

100 105 110

Gly Ala Pro Leu Asp Gly Lys Gly Glu Ile Ala Ala Thr Glu Asn Arg

115 120 125

Leu Ile Glu Ser Pro Ala Pro Gly Ile Ile Ala Arg Arg Ser Val His

130 135 140

Glu Pro Met Gln Thr Gly Ile Thr Ala Ile Asp Ala Met Ile Pro Ile

145 150 155 160

Gly Arg Gly Gln Arg Glu Leu Ile Ile Gly Asp Arg Gln Thr Gly Lys

165 170 175

Thr Ala Ile Ala Ile Asp Thr Ile Leu Asn Gln Lys Gly Glu Asp Val

180 185 190

Ile Cys Val Tyr Val Ala Ile Gly Gln Lys Ala Ser Ser Val Ala Asn

195 200 205

Ile Ile Glu Val Leu Arg Glu Arg Gly Ala Leu Asp Tyr Thr Val Val

210 215 220

Val Ala Ala Asn Ala Ser Glu Pro Ala Thr Leu Gln Tyr Leu Ala Pro

225 230 235 240

Tyr Ala Gly Ala Ala Ile Ala Glu Tyr Phe Met Phe Lys Gly Lys Ala

245 250 255

Thr Leu Val Ile Tyr Asp Asp Leu Thr Lys Gln Ala Gln Ala Tyr Arg

260 265 270

Gln Met Ser Leu Leu Leu Arg Arg Pro Pro Gly Arg Glu Ala Tyr Pro

275 280 285

Gly Asp Val Phe Tyr Leu His Ser Arg Leu Leu Glu Arg Ala Ala Lys

290 295 300

Leu Ser Asp Ala Leu Gly Gly Gly Ser Met Thr Ala Leu Pro Val Ile

305 310 315 320

Glu Thr Gln Ala Gly Asp Val Ser Ala Tyr Ile Pro Thr Asn Val Ile

325 330 335

Ser Ile Thr Asp Gly Gln Ile Phe Leu Ser Ser Asp Leu Phe Asn Ser

340 345 350

Gly Leu Arg Pro Ala Ile Asn Val Gly Ile Ser Val Ser Arg Val Gly

355 360 365

Ser Ala Ala Gln Thr Lys Ala Ile Lys Lys Ile Ala Gly Thr Leu Lys

370 375 380

Leu Glu Leu Ala Gln Phe Asp Glu Leu Ala Ala Phe Ala Gln Phe Ala

385 390 395 400

Ser Asp Leu Asp Lys Ala Thr Gln Asn Gln Leu Ala Arg Gly Gln Arg

405 410 415

Leu Arg Glu Leu Leu Lys Gln Pro Gln Phe Ser Pro Leu Ile Leu Ala

420 425 430

Glu Gln Val Ala Val Val Tyr Ala Gly Val Lys Gly Leu Ile Asp Glu

435 440 445

Ile Pro Val Asn Gln Val Thr Ala Phe Val Ser Glu Leu Arg Ser Tyr

450 455 460

Leu Lys Thr Ser Lys Pro Glu Phe Ile Glu Lys Val Gln Ser Ser Lys

465 470 475 480

Gln Leu Asp Asp Ala Ala Glu Ala Leu Leu Lys Glu Ala Ile Ala Glu

485 490 495

Val Lys Lys Asn Ile Leu Ala Ala Val

500 505

<210> 11

<211> 1518

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atggttagca tcagacccga cgaaattagc agcatcattc gtcagcagat tgagcagtac 60

agccaagacg tcaaagttga aaacgtcggt accgttctgc aggtgggtga cggcattgcc 120

cgcatctatg gtctgcagca ggtgatgtct ggcgagttgg tggagttcga agatggcacc 180

accggcatcg cgctcaactt ggaagaagac aacgtcggtg cggtgttgat gggcgaaggc 240

cgcaacatcc aagagggcag cacggttaaa gcgaccggca aaatcgctca gattccggtc 300

ggtgatgcac tggttggccg tgtggtcagc cccttgggtg cgccgttgga cggcaaaggt 360

gaaattgcgg caaccgaaaa tcgcctgatc gaatcgccgg ctcctggcat cattgcgcgt 420

cgctcggtgc atgagcccat gcagaccggt attaccgcga tcgacgcgat gattccgatc 480

ggccggggcc agcgcgagct gatcatcggt gaccgtcaga ccggcaaaac cgcaatcgcg 540

atcgacacga ttctgaacca gaaaggcgaa gacgtaattt gcgtctacgt cgcgatcggt 600

cagaaagcct cttcggttgc caacatcatt gaagtcctgc gggaacgcgg tgccctcgat 660

tacaccgtgg tcgttgcggc caacgcttca gaaccggcaa cgctgcaata cctggctccc 720

tacgccggtg ctgcgatcgc cgagtacttt atgttcaaag gcaaagccac cttggtcatc 780

tacgatgact tgaccaagca agcgcaggct taccgccaga tgtcgctgct gctgcgtcgt 840

ccgcccggtc gggaagctta ccccggcgat gttttctacc tccacagccg tctgctggaa 900

cgcgccgcta aactgtcgga cgctcttggt ggtggcagca tgactgccct gccggtgatc 960

gaaacccaag ccggtgacgt ctcggcctac attccgacca acgtgatctc gattacggac 1020

ggtcaaatct tcctgtcctc tgacctgttc aactcgggtc tgcgtccggc tattaacgtc 1080

ggtatctcgg ttagccgggt cggttccgct gctcaaacca aggcgatcaa gaaaattgct 1140

ggcacattga agctggaatt ggctcagttt gatgagctgg cggcctttgc tcagtttgca 1200

tctgacttgg acaaagccac tcaaaaccag ttggctcgcg gtcagcgtct gcgtgagctg 1260

ctgaaacagc ctcagttctc gccgttgatt ctggcggaac aagtagcagt ggtctacgcc 1320

ggtgttaaag gtctgatcga cgagattccg gtcaaccaag tcacggcctt cgtttctgag 1380

ctgcgctcct acctgaagac cagcaagcct gagttcatcg agaaagttca aagctcgaaa 1440

caactcgatg acgccgctga agcattgctg aaagaagcga tcgcggaagt gaagaaaaac 1500

atcttggctg ctgtctag 1518

Claims (10)

1. A method for preparing synechococcus with fructose secretion capacity and high fructose yield is characterized in that synechococcus PCC7942 is taken as an original strain, and fructokinase of the original strain is knocked out.

2. The method according to claim 1, wherein the fructokinase has an amino acid sequence as shown in SEQ ID No. 1.

3. The method according to claim 1, wherein a sucrose hydrolase is also overexpressed in the starting algal strain, the amino acid sequence of said sucrose hydrolase being represented by SEQ ID No. 4.

4. The method according to claim 1, wherein a sucrose phosphate synthase is also overexpressed in the starting algal strain, the sucrose phosphate synthase having an amino acid sequence as set forth in SEQ ID No. 6.

5. The method according to claim 1, wherein sucrose phosphate phosphorylase is also overexpressed in the starting algal strain, and the amino acid sequence of sucrose phosphate phosphorylase is represented by SEQ ID No. 8.

6. The method of claim 1, wherein the ATP synthase F of the starting strain is further introduced _O F ₁ The 252 th amino acid of the alpha subunit is mutated from cysteine to phenylalanine.

7. Synechococcus having fructose-secreting ability and high fructose production, which is produced by the method of any one of claims 1 to 6.

8. A method for producing fructose, comprising the steps of culturing the synechococcus of claim 7 and allowing said synechococcus to produce fructose.

9. The method of claim 8, wherein the nannochloropsis is cultured under the following conditions: the culture medium is BG11, the culture temperature is 25-35 deg.C, and the illumination is 100-300 μmol photons m ^-2 s ^-1 Culturing in the presence of CO gas ₂ Mixed gas with air.

10. The method of claim 9, wherein the one or more additional media nutrient supplements are performed during the culturing.

Images