CN109810958A

CN109810958A - The CCD2 mutant and its coded sequence in a kind of safflower source and application and the restructuring yeast strains for producing crocetin

Info

Publication number: CN109810958A
Application number: CN201910243448.9A
Authority: CN
Inventors: 元英进; 梁楠; 姚明东; 王颖; 肖文海
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2019-03-28
Filing date: 2019-03-28
Publication date: 2019-05-28
Anticipated expiration: 2039-03-28
Also published as: CN109810958B

Abstract

The present invention relates to gene engineering technology field, the CCD2 mutant and its coded sequence and application and the restructuring yeast strains for producing crocetin in a kind of safflower source are disclosed.CCD2 mutant of the present invention has one or more of V120F, Y190K/A, R192V/F, E211A, E212A, T290V, K320A and S323F/A/T site mutation.The present invention is transformed for the rate-limiting enzyme CCD2 in crocetin synthesis path, substrate by widening CCD2 is composed, it realizes from beta carotene and directly synthesizes crocetin, reconstruct is simplified to existing crocetin synthesis path, to solve the problems, such as that multistep reaction gross production rate is universal low；The efficiency that CCD2 is catalyzed former substrate zeaxanthin synthesis crocetin is improved simultaneously, and then obtains the yeast strain that a plant height produces crocetin.

Description

The CCD2 mutant and its coded sequence in a kind of safflower source and application and production The restructuring yeast strains of crocetin

Technical field

The present invention relates to gene engineering technology field, the CCD2 in more particularly to a kind of safflower source is mutated Body and its coded sequence and application and the restructuring yeast strains for producing crocetin.

Background technique

Crocetin (Crocetin) also known as crocetin or bayer acid, be saffron extract effective component it One, molecular formula C₂₀H₂₄O₄.The main source of crocetin is the column cap of safflower, but the resource of safflower in nature Very limited, column cap yield is even more extremely low, and each safflower all one's life can only three pistil stigmas of output.Average 25 Wan Duozang Safflower expends 40 the artificial of hour and extracts the saffron (saffron) that could obtain enough column cap production 1kg, content 97% crocetin market price is up to 13.6 ten thousand yuan/g, therefore safflower enjoys the good reputation of " plant gold ".

It is recorded in the classic medical book in China, safflower can be with activating microcirculation and removing stasis medicinal, tranquilizing the mind relieve stagnation, removing pattogenic heat from the blood and toxic material from the body, and the modern times cure Medicine field to the research of safflower it has also been found that safflower can be widely applied to prevent and treat cerebral thrombosis, vasculitis, myocardial infarction, It the diseases such as palpitates with fear demented.Crocetin is as one of the natural active compound in safflower, also because of its outstanding pharmacological effect It is known, is widely used in medicine, food processing and cosmetic industry.But the yield of crocetin is extremely low, causes Supply falls short of demand, the phenomenon of market shortage.

Currently, the main production process of crocetin has natural extraction, chemical synthesis and biosynthesis.Natural extract produces Measure it is extremely low, vulnerable to the place of production, weather, pick treatment process influence；Chemical synthesis mainly passes through hydrolysis crocetin dinethyl ester and obtains It arrives, by-product is more, and environmental pollution is serious, and synthesized crocetin can only cannot be used due to its lower purity as pigment Make food additives or drug.Therefore, constructing efficient Microbial cell factories synthesis crocetin is most promising production Method.

Nineteen eighty-two, Pfander etc. illustrate the natural biological synthesis path of crocetin；2003, ZCD existed for the first time It is found to identify in safflower, referred to as 7,8 (7', 8')-zeaxanthins crack dioxygenase；2014, Frusciante etc. Prove that ZCD cannot synthesize crocetin in Escherichia coli and corn embryosperm, and the CCD2 in newfound safflower source is demonstrate,proved Catalytic pyrolysis can be carried out at the 7 of zeaxanthin, 8 and 7', 8' in fact, synthesizes crocetin dialdehyde；2017, ChaiF etc. was making Realize the de novo formation of crocetin in brewer yeast for the first time using the CCD2 in safflower source, shake flask fermentation yield reaches 633ug/L(Chai F,et al. Heterologous biosynthesis and manipulation of crocetin In Saccharomyces cerevisiae [J] .Microbial Cell Factories, 2017), furthermore the research is also right Endogenous external source ALD is screened, and (crocetin yield is mentioned by 240 μ g/L to 633 μ g/L)；The expression for adjusting CCD2 and ALD is strong (crocetin yield is mentioned by 633 μ g/L to 1219 μ g/L) degree.

The CCD2 in safflower source is that presently found only one can be catalyzed zeaxanthin synthesis hiding in saccharomyces cerevisiae The CCD family member of safflower acid, but document report CCD2 can not be catalyzed beta carotene and lycopene in other sites, i.e., It does not include beta carotene and lycopene in the identifiable substrate spectrum of CCD2；Although making wine currently, realizing crocetin Synthesis in yeast, but pass through the long path day of lycopene-β-carrotene-zeaxanthin-safranal-crocetin The heterologous synthesis of right product faces the adaptation issues in path between gene and gene, between path gene and chassis.Meanwhile with The extension in path, gross production rate constantly reduce, yield remain unchanged it is very low.

Summary of the invention

In view of this, the purpose of the present invention is to provide a kind of CCD2 mutant in safflower source and its coded sequence, It, can be directly with β-when so that the CCD2 mutant and its coded sequence are for constructing production crocetin restructuring yeast strains Carrotene is substrate production crocetin and/or the efficiency for improving the production crocetin by substrate of zeaxanthin；

Another object of the present invention is that a kind of recombinant vector for expressing CCD2 mutant is provided, so that the recombination When carrier is for constructing production crocetin restructuring yeast strains, crocetin directly can be produced by substrate of beta carotene And/or improve the efficiency that crocetin is produced using zeaxanthin as substrate；

Another object of the present invention is that a kind of restructuring yeast strains for producing crocetin are provided, so that described heavy Group yeast strain can directly produce crocetin by substrate of beta carotene and/or improve by substrate of zeaxanthin and produce The efficiency of crocetin.

For achieving the above object, the invention provides the following technical scheme:

A kind of CCD2 mutant in safflower source has following one or more kinds of site mutation:

V120F、Y190K/A、R192V/F、E211A、E212A、T290V、K320A、S323F/A/T。

For the problem that current crocetin synthesis path is too long and not high to original substrate zeaxanthin catalytic efficiency, The present invention carries out mutation transformation to the rate-limiting enzyme CCD2 in crocetin route of synthesis, can directly utilize beta carotene Crocetin is produced for substrate, shortens synthesis path, and the efficiency that catalysis zeaxanthin generates crocetin can be improved.

Preferably, the CCD2 mutant in the safflower source is with S323F/A/T site mutation and/or has such as The site mutation of lower one or more:

V120F、Y190K/A、R192V/F、E211A、E212A、T290V、K320A。

In the specific embodiment of the invention, the CCD2 mutant in the safflower source has any one following situation Site mutation:

(1) S323F/A site mutation+R192V/F site mutation；

(2) S323F/A site mutation+T290V site mutation；

(3) S323F/A site mutation+R192V/F site mutation+T290V site mutation.

Meanwhile the present invention also provides the coded sequences of the CCD2 mutant in above-mentioned safflower source；Wild type safflower The coded sequence of the CCD2 in source is as shown in SEQ ID NO:1, wherein the base gtt of 358-360bp expresses CCD2 the 120th The valine (V) of position can sport ttc expression phenylalanine (F)；

The base tac of 568-570bp expresses CCD2 the 190th tyrosine (Y), can sport aag expression lysine (K) or sport gct expression alanine (A)；

The base aga of 574-576bp expresses CCD2 the 192nd arginine (R), can sport gtt expression valine (V) or sport ttc expression phenylalanine (F)；

The base gaa of 631-633bp expresses CCD2 the 211st glutamic acid (E), can sport gct expression alanine (A)；

The base gaa of 634-636bp expresses CCD2 the 212nd glutamic acid (E), can sport gct expression alanine (A)；

The base act of 868-870bp expresses CCD2 the 290th threonine (T), can sport gtt expression valine (V)；

The base aaa of 958-960bp expresses CCD2 the 320th lysine (K), can sport gct expression alanine (A)；

The base tcc of 967-969bp expresses CCD2 the 323rd serine (S), can sport ttc expression phenylpropyl alcohol ammonia Sour (F), it sports gct expression alanine (A) or sports act expression threonine (T).

By the way that the coded sequence of the CCD2 mutant is accessed carrier and is transformed into yeast strain, it can be achieved that the present invention Expected purpose, therefore the invention proposes the coded sequences of the CCD2 mutant to prepare the weight for expressing the CCD2 mutant Application in the restructuring yeast strains of group carrier and/or production crocetin.

According to the application of the coded sequence of the CCD2 mutant, the present invention provides a kind of recombinant vectors, on basis The coded sequence of the CCD2 mutant is accessed on carrier.

Preferably, the recombinant vector accessed on carrier is carrier promoter+CCD2 mutant coded sequence+ The expression cassette of terminator；In the specific embodiment of the invention, the preferred GAL promoter of promoter in the expression cassette, such as GAL10 Promoter, GAL7 promoter；The preferred TEF2 terminator of the terminator or PGI1 terminator.

In the yeast strain of production crocetin, ALD can be expressed by other carriers, can also be logical with CCD2 mutant Same recombinant vector expression is crossed, therefore also may include ALD coded sequence on the carrier is carrier；In the specific embodiment of the invention In, the ALD coded sequence is the Syn_ALD coded sequence (Synechocystis from synechocystis sp.PCC6803).Preferably, ALD coded sequence is connected to base with promoter+ALD coded sequence+terminator expression cassette On plinth carrier；In the specific embodiment of the invention, the preferred GAL promoter of promoter in the expression cassette, as GAL10 starts Son, GAL7 promoter；The preferred TEF2 terminator of the terminator or PGI1 terminator.

In the specific embodiment of the invention, the carrier is carrier selects pRS series plasmids, such as pRS416, pRS425K Deng.Based on same technical effect, the invention proposes the recombinant vectors to produce crocetin restructuring yeast strains in building In application.The yeast strain is preferably saccharomyces cerevisiae, more preferably CEN.PK series saccharomyces cerevisiae or BY series wine brewing ferment It is female.

In addition, the present invention also provides the construction method of the recombinant vector, in the coded sequence of the CCD2 mutant Both ends construct homology arm, and digestion carrier is carrier is linear carrier, are recombinated by yeast homologous the coded sequence being linked into base On plinth carrier.

More specific method is to construct the coded sequence+termination of upstream homology arm+promoter+CCD2 mutant Son+downstream homology arm expression cassette, the upstream homology arm described in carrier is carrier and downstream homology arm junction digestion become linear Carrier is recombinated by yeast homologous and the coded sequence of the CCD2 mutant is linked on carrier is carrier；Or

Construct upstream homology arm+promoter+CCD2 mutant coded sequence+terminator expression cassette or promoter Coded sequence+terminator+downstream homology arm expression cassette of+CCD2 the mutant, the upstream homology arm described in carrier is carrier With the digestion of terminator junction become linear carrier or the promoter described in carrier is carrier and downstream homology arm junction digestion at For linear carrier, is recombinated by yeast homologous and the coded sequence of the CCD2 mutant is linked on carrier is carrier；Or

Construct promoter+CCD2 mutant coded sequence+terminator expression cassette, on carrier is carrier described in Promoter and the digestion of terminator junction become linear carrier, are recombinated by yeast homologous by the code sequence of the CCD2 mutant Column are linked on carrier is carrier；

Meanwhile present invention provides the coded sequences and ALD coded sequence that access the CCD2 mutant simultaneously The construction method of recombinant vector, i.e., it is homologous in the coded sequence of the CCD2 mutant and the building of ALD coded sequence both ends respectively Arm, digestion carrier is carrier are linear carrier, are encoded the coded sequence of the CCD2 mutant and ALD by yeast homologous recombination Sequence is linked on carrier is carrier.In the specific embodiment of the invention, the construction method is building upstream homology arm+the first The+the first terminator of promoter+CCD2 mutant coded sequence, first terminator the+the second promoter+ALD coded sequence The expression cassette of+the second terminator, the upstream homology arm and the second terminator junction digestion on carrier is carrier become line Property carrier, recombinated by yeast homologous and the coded sequence of the CCD2 mutant and ALD coded sequence be linked into carrier is carrier On；The selection of first promoter, the second promoter, the first terminator and the second terminator is the same as aforementioned promoter and termination Son.

In above-mentioned construction method, according to each Genetic elements having on carrier is carrier, it can need to access accordingly Foreign gene or its expression cassette both ends add identical Genetic elements sequence, referred to as homology arm；For needing to import multiple external sources The situation of gene or its expression cassette guarantees that adjacent foreign gene or its expression cassette have the homology arm sequence of overlapping, finally again with The homology arm sequence that the linear carrier is carrier both ends of digestion have overlapping can connect together multiple foreign genes or its expression cassette Enter onto carrier is carrier.For easy consideration, the promoter and terminator that expression cassette also can be directly used as homology arm, Without additionally redesigning homology arm.

Illustrate by taking one of specific embodiment of the invention construction method as an example, (upstream is homologous for building HIS5 terminator Arm)+GAL10 promoter+CCD2 mutant coded sequence+TEF2 terminator, TEF2 terminator+GAL7 promoter+ The expression cassette of ALD coded sequence+PGI1 terminator, HIS5 terminator and the junction digestion of PGI1 terminator on carrier is carrier (selecting suitable enzyme according to actual restriction enzyme site) becomes linear carrier, is recombinated by yeast homologous by the CCD2 mutant Coded sequence and ALD coded sequence be linked on carrier is carrier, schematic diagram is shown in Fig. 1.

The present invention also provides a kind of restructuring yeast strains for producing crocetin, conversion has recombination of the present invention to carry Body；There is the recombinant vector of expression ALD as the yeast strain itself can express ALD or conversion, then need to only convert access The recombinant vector of the coded sequence of the CCD2 mutant；In specific conversion process, by by each expression cassette and The linear carrier is carrier of digestion is converted into yeast together, by homologous recombination machinery, recombination can be completed inside yeast and is carried The assembling of body and the expression for realizing albumen, therefore the restructuring yeast strains can be applicable in production crocetin.

In the method for production crocetin, directly adopt restructuring yeast strains of the present invention with zeaxanthin and/or Beta carotene is that substrate is fermented.Seed culture medium activation is generally first passed through, then is fermented by fermentation medium.

In the specific embodiment of the invention, the seed culture medium is synthetic yeast nitrogen source YNB6.7g/L, glucose 20g/L lacks the kilnitamin powder 2g/L of tryptophan, leucine, histidine and uracil；Wherein, kilnitamin powder Middle lacked amino acid screens label according to contained by carrier and determines；

The fermentation medium is 40g/L glucose, 20g/L peptone, 10g/L yeast extract, 10g/L D- galactolipin；

Specific production method is that the restructuring yeast strains are inoculated in 5mL seed culture medium, 30 DEG C, 250rpm is cultivated for 24 hours；With initial OD₆₀₀=0.2 is forwarded in 5mL fresh seeds culture medium, 30 DEG C, 250rpm culture 12h；With Initial OD₆₀₀=0.1 is forwarded in 50mL fermentation medium, 20 DEG C, fermented and cultured under the conditions of 250rpm；Fermentation time is 96- 144h, and extend yield at any time and increase.

From the above technical scheme, the present invention is transformed for the rate-limiting enzyme CCD2 in crocetin synthesis path, Substrate by widening CCD2 is composed, and is realized from beta carotene and is directly synthesized crocetin, synthesizes road to existing crocetin Diameter simplifies reconstruct, to solve the problems, such as that multistep reaction gross production rate is universal low；CCD2 is improved simultaneously is catalyzed former substrate zeaxanthin The efficiency of crocetin is synthesized, and then obtains the yeast strain that a plant height produces crocetin.

Detailed description of the invention

Fig. 1 show the building schematic diagram of recombinant vector of the present invention；

Fig. 2 show pRS416-C-01 plasmid map；

Fig. 3 show pRS416-A-01 plasmid map；

Fig. 4 show pRS425K-A-04 plasmid map；

Fig. 5 show the flow diagram for constructing restructuring yeast strains of the present invention；

Fig. 6 is shown to be sent out by the crocetin shaking flask of the recombinant Saccharomyces cerevisiae of substrate CCD2 simple point mutation of beta carotene Ferment result；

Fig. 7 is shown to be sent out by the crocetin shaking flask of the recombinant Saccharomyces cerevisiae of substrate CCD2 multipoint mutation of beta carotene Ferment result；

Fig. 8 is shown using zeaxanthin as the crocetin shake flask fermentation of the recombinant Saccharomyces cerevisiae of substrate CCD2 simple point mutation As a result；

Fig. 9 is shown to be shaken by the crocetin of the recombinant Saccharomyces cerevisiae of substrate CCD2 S323 site mutation of zeaxanthin Bottle fermentation results；

Figure 10 is shown to be sent out by the crocetin shaking flask of the recombinant Saccharomyces cerevisiae of substrate CCD2 multipoint mutation of zeaxanthin Ferment result.

Specific embodiment

The invention discloses a kind of CCD2 mutant in safflower source and its coded sequence and application and production safranine The restructuring yeast strains of acid are spent, those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.Especially need It is noted that all similar substitutions and modifications are apparent to those skilled in the art, they are considered as It is included in the present invention.Mutant, recombinant vector, bacterial strain and construction method of the present invention and application, which have passed through, preferably to be implemented Example is described, related personnel obviously can not depart from the content of present invention, in spirit and scope to mutant as described herein, Recombinant vector, bacterial strain and construction method and application are modified or appropriate changes and combinations, carry out implementation and application skill of the present invention Art.

Sequence appeared in the present invention preferably through saccharomyces cerevisiae codon optimization and suitably evades common restriction enzyme Enzyme site；

HIS5 terminator sequence is as shown in SEQ ID NO:2；

GAL10 promoter sequence is as shown in SEQ ID NO:3；

TEF2 terminator sequence is as shown in SEQ ID NO:4；

GAL7 promoter sequence is as shown in SEQ ID NO:5；

SynALD coded sequence is as shown in SEQ ID NO:6；

PGI1 terminator sequence is as shown in SEQ ID NO:7；

In addition, pRS416-C-01 plasmid complete genome sequence is as shown in SEQ ID NO:8, plasmid map is as shown in Figure 2；

For pRS416-A-01 plasmid complete genome sequence as shown in SEQ ID NO:9, plasmid map is as shown in Figure 3；

PRS425K-A-04 plasmid complete genome sequence is as shown in SEQ ID NO:10, and plasmid map is as shown in Fig. 4；

Above-mentioned each Genetic elements sequence can be linked in sequence according to expression cassette situation of the present invention, as corresponding expression The sequence of box.

Below with reference to embodiment, the present invention is further explained.

Embodiment 1: recombinant Saccharomyces cerevisiae bacterial strain of the present invention is constructed

1. chassis bacterial strain

The Wine brewing yeast strain or common Wine brewing yeast strain of being capable of high yield beta carotene and/or zeaxanthin may be selected (beta carotene and/or zeaxanthin need to be added when common strain fermentation as substrate)；

2. the acquisition of external source functional gene element and modulation conformity tool plasmid

CCD2 Genetic elements: (there is expression cassette HIS5t- thereon by template amplification acquisition of pRS416-C-01 plasmid GAL10p-CCD2-TEF2t)；

SynALD Genetic elements: (there is expression cassette TEF2t- thereon by template amplification acquisition of pRS425K-A-04 plasmid GAL7p-SynALD-PGI1t)；

Carrier is carrier is pRS416-A-01 plasmid, there is HIS5t-PGI1t Genetic elements thereon；

Above-mentioned each Genetic elements, plasmid or expression cassette also can be directly used fully synthetic mode and directly obtain.

3. the building of expression cassette and linearized vector

The building of expression cassette one: using pRS416-C-01 as template, required catastrophe point is introduced by CCD2 by PCR, is obtained CCD2_mut expression cassette HIS5t-GAL10p-CCD2_mut-TEF2t.It is specific as follows, using pRS416-C-01 as template, by setting Meter primer carries out two PCR respectively and reacts to obtain two DNA fragmentations, i.e. HIS5t-GAL10p- in the mutational site containing CCD2 CCD2_mut-frag1 and CCD2_mut-frag2-TEF2t, then carry out OEPCR as template using the two segments and react and can obtain To CCD2_mut expression cassette HIS5t-GAL10p-CCD2_mut-TEF2t.There is the expression cassette in multiple mutational sites referring to above-mentioned side Method building, difference be design primer number and PCR reaction times and obtained segments, finally pass through adjacent segment Between overlap with OEPCR reaction forming.

The building of expression cassette two: SynALD expression can be obtained with PstI/BamHI double digestion plasmid pRS425K-A-04 The addition of box TEF2t-GAL7p-SynALD-PGI1t, TEF2t are to be able to have homology arm with expression cassette one, make the two can Connection.

It linearizes the building of carrier is carrier: BamHI digested plasmid pRS416-A-01 is used, between HIS5t and PGI1t Disconnect, obtain linearisation carrier is carrier, expression cassette one and HIS5t on expression cassette two and PGI1t as homology arm can with it is linear Change carrier is carrier connection, forms recombinant vector.

4. producing the Wine brewing yeast strain of crocetin by yeast homologous recombination to construct

The mutant CCD2_mut gene of expression cassette one is carried together with expression cassette two and linearisation basis using Li-acetate method Body cotransformation makes mutant CCD2_mut gene and SynALD gene collectively reside in centromere plasmid into chassis bacterial strain PRS416, the yeast after conversion are coated with SC-TRP-LEU-HIS-URA solid panel (synthetic yeast nitrogen source YNB6.7g/L, glucose 20g/L lacks the kilnitamin powder 2g/L of tryptophan, leucine, histidine and uracil, 2% agar powder) it is sieved Choosing, obtained transformant scribing line, which divides after pure culture to choose single colonie and connect bacterium, extracts ferment in SC-TRP-LEU-HIS-URA fluid nutrient medium Matrix grain carries out PCR verifying, and PCR verifies correct yeast plasmid revolution Escherichia coli and proposes plasmid order-checking.

The present embodiment unitary construction flow diagram is shown in Fig. 5.

The crocetin shake flask fermentation of the recombinant Saccharomyces cerevisiae of embodiment 2:CCD2 simple point mutation tests (beta carotene Substrate)

1, test strain

Each group chassis bacterial strain is the bacterial strain SyBE_ of the high yield beta carotene in patent 201510435606.2 Sc14CY03。

Control strain 1: referring to the construction method building in embodiment 1, difference is, with wild type CCD1 gene replacement CCD2 gene, i.e. difference are that expression cassette one is HIS5t-GAL10p-CCD1-TEF2t, strain number SyBE_ Sc02070246；

Control strain 2: referring to the construction method building in embodiment 1, difference is that CCD2 gene is that wild type is unmutated Gene, i.e. difference are that expression cassette one is HIS5t-GAL10p-CCD2-TEF2t；

Recombinant bacterial strain 1 of the present invention: site mutation is selected as S323F, i.e. expression cassette one is HIS5t-GAL10p-CCD2_ Mut9 (S323F)-TEF2t, strain number SyBE_Sc02070192；

Recombinant bacterial strain 2 of the present invention: site mutation is selected as S323A, i.e. expression cassette one is HIS5t-GAL10p-CCD2_ Mut19 (S323A)-TEF2t, strain number SyBE_Sc02070248；

2, shake flask fermentation method and crocetin detection

Seed culture medium (SC-TRP-LEU-HIS-URA): synthetic yeast nitrogen source YNB6.7g/L, glucose 20g/L lack color Propylhomoserin, leucine, histidine and uracil kilnitamin powder 2g/L；

Fermentation medium: 40g/L glucose, 20g/L peptone, 10g/L yeast extract, 10g/L D- galactolipin.

Above-mentioned each group bacterial strain glycerol stock is inoculated in 5mL seed culture medium, is cultivated for 24 hours in 30 DEG C, 250rpm；With first Beginning OD600=0.2 is forwarded in 5mL fresh seeds culture medium, 30 DEG C, 250rpm culture 12h；Turned with initial OD 600=0.1 It is connected in 50mL fermentation medium, 20 DEG C, detect crocetin yield under the conditions of 250rpm when fermented and cultured 96h.

Crocetin extracting method: each fermentation flask takes 2mL fermentation liquid, and 12000rpm is centrifuged 2min and collects thallus, nothing It is extracted after bacterium washing twice to product.Method particularly includes: cell is resuspended with 3MHCl, is placed in boiling water bath and boils 2min, then Ice bath 3min immediately, quadruplication；Broken cell 12000rpm, 4 DEG C of centrifugation 5min are abandoned into supernatant, are added after sterile washing 2 times Enter 1mL acetone, and the 5min that is vortexed, it is repeated multiple times to become white to thallus；Centrifugal enrichment acetone phase, and with traditional vacuum concentrating instrument Acetone volatilization is clean, the DMF: methanol=1:7 of 400 μ L is added, and detected with HPLC with after 2 μm of organic membrane filtrations.

Crocetin HPLC detection method: BDS HYPERSIL ODS-2 C18 chromatographic column (150 mm × 4.6mm, 5 μm), Waters2489 UV/Vis detector；Waters e2695 pump；Mobile phase: methanol: ultrapure water (2% formic acid)=70:30；On Sample volume: 30 μ L；Flow velocity: 1mL/min；Column temperature: 40 DEG C；Detection wavelength: 430nm.

3, result

It is all realized according to two kinds of mutant S323F and S323A that Fig. 6 result can be seen that CCD2 from beta carotene Crocetin is synthesized, and the recombinant bacterial strain for importing wild type CCD2 and CCD1 does not have the generation of crocetin.

The crocetin shake flask fermentation of the recombinant Saccharomyces cerevisiae of embodiment 3:CCD2 multipoint mutation tests (beta carotene Substrate)

Recombinant bacterial strain is constructed according to the construction method of embodiment 1, chassis bacterial strain selects following CCD2 multiple spot with embodiment 2 Mutation, each group expression cassette one are as follows:

HIS5t-GAL10p-CCD2_mut24(R192F&S323F)-TEF2t；

HIS5t-GAL10p-CCD2_mut25(R192F&S323A)-TEF2t；

HIS5t-GAL10p-CCD2_mut26(T290V&S323F)-TEF2t；

HIS5t-GAL10p-CCD2_mut27(T290V&S323A)-TEF2t；

HIS5t-GAL10p-CCD2_mut28(R192F&T290V&S323F)-TEF2t；

HIS5t-GAL10p-CCD2_mut29(R192F&T290V&S323A)-TEF2t.

Strain number is followed successively by SyBE_Sc02070264-SyBE_Sc02070269；

As a result see Fig. 7, other mutation can make recombinant Saccharomyces cerevisiae crocetin output relatively single with S323A mutation combination Point mutation is improved significantly, and yield is up to 180ug/L or so.

The crocetin shake flask fermentation of the recombinant Saccharomyces cerevisiae of embodiment 4:CCD2 simple point mutation tests (zeaxanthin bottom Object)

Recombinant bacterial strain is constructed according to the construction method of embodiment 1, chassis bacterial strain selects document Fenghua Chai. Heterologous biosynthesis and manipulation of crocetin in Saccharomyces cere The bacterial strain SyBE_ of high-yield corn Huang matter in visiae.MicrobialCellFactories.2017.16 (1) Sc0123Cz14 selects following CCD2 simple point mutation, and each group expression cassette one is as follows:

HIS5t-GAL10p-CCD2_mut9(S323F)-TEF2t；

HIS5t-GAL10p-CCD2_mut10(V120F)-TEF2t；

HIS5t-GAL10p-CCD2_mut11(Y190K)-TEF2t；

HIS5t-GAL10p-CCD2_mut12(Y190A)-TEF2t；

HIS5t-GAL10p-CCD2_mut13(R192V)-TEF2t；

HIS5t-GAL10p-CCD2_mut14(R192F)-TEF2t；

HIS5t-GAL10p-CCD2_mut15(E211A)-TEF2t；

HIS5t-GAL10p-CCD2_mut16(E212A)-TEF2t；

HIS5t-GAL10p-CCD2_mut17(T290V)-TEF2t；

HIS5t-GAL10p-CCD2_mut18(K320A)-TEF2t；

HIS5t-GAL10p-CCD2_mut19(S323A)-TEF2t.

Strain number is followed successively by SyBE_Sc02070170 and SyBE_Sc02070177- SyBE_Sc02070186；

Control strain is selected from document Fenghua Chai.Heterologous biosynthesis and manipulation of crocetin in Saccharomyces cerevisiae.Microbial Cell Factories. the bacterial strain SyBE_Sc0123C050 (WT) of the production crocetin in 2017.16 (1)；

As a result see Fig. 8, the recombination wine brewing ferment of S323A, S323F, K320A, T290V, R192F and R192V simple point mutation Parent phase ratio SyBE_Sc0123C050, can be improved the efficiency of catalysis zeaxanthin production crocetin, and yield is mentioned Height, wherein being improved with two kinds of site mutations of S323A, S323F more significant.

The crocetin shake flask fermentation of the recombinant Saccharomyces cerevisiae of the S323 site mutation of embodiment 5:CCD2 tests (maize Matter substrate)

Recombinant bacterial strain is constructed according to the construction method of embodiment 1, chassis bacterial strain selects document Fenghua Chai. Heterologous biosynthesis and manipulation of crocetin in Saccharomyces The bacterial strain SyBE_ of high-yield corn Huang matter in cerevisiae.Microbial Cell Factories.2017.16 (1) Sc0123Cz14 selects the S323 site mutation of following CCD2, and each group expression cassette one is as follows:

HIS5t-GAL10p-CCD2_mut9(S323F)-TEF2t；

HIS5t-GAL10p-CCD2_mut19(S323A)-TEF2t

HIS5t-GAL10p-CCD2_mut21(S323H)-TEF2t；

HIS5t-GAL10p-CCD2_mut22(S323E)-TEF2t；

HIS5t-GAL10p-CCD2_mut23(S323T)-TEF2t.

Strain number is followed successively by SyBE_Sc02070110, SyBE_Sc02070111, SyBE_Sc02070249-SyBE_ Sc02070251；Simultaneously

As a result see that Fig. 9, the recombinant Saccharomyces cerevisiae of S323A, S323F, S323T simple point mutation compare SyBE_ Sc0123C050, can significantly improve the efficiency of catalysis zeaxanthin production crocetin, and yield significantly improves.

The crocetin shake flask fermentation of the recombinant Saccharomyces cerevisiae of embodiment 6:CCD2 multipoint mutation tests (zeaxanthin bottom Object)

Recombinant bacterial strain is constructed according to the construction method of embodiment 1, chassis bacterial strain selects document Fenghua Chai. Heterologous biosynthesis and manipulation of crocetin in Saccharomyces The bacterial strain SyBE_ of high-yield corn Huang matter in cerevisiae.Microbial Cell Factories.2017.16 (1) Sc0123Cz14 selects the multisite mutation of following CCD2, and each group expression cassette one is as follows:

HIS5t-GAL10p-CCD2_mut24(R192F&S323F)-TEF2t；

HIS5t-GAL10p-CCD2_mut25(R192F&S323A)-TEF2t；

HIS5t-GAL10p-CCD2_mut26(T290V&S323F)-TEF2t；

HIS5t-GAL10p-CCD2_mut27(T290V&S323A)-TEF2t；

HIS5t-GAL10p-CCD2_mut28(R192F&T290V&S323F)-TEF2t；

HIS5t-GAL10p-CCD2_mut29(R192F&T290V&S323A)-TEF2t.

Strain number is followed successively by SyBE_Sc02070252-SyBE_Sc02070257；

The recombinant Saccharomyces cerevisiae of the result is shown in Figure 10, above-mentioned each multipoint mutation combination compares SyBE_Sc0123C050, To significantly improve the efficiency of catalysis zeaxanthin production crocetin, yield is significantly improved.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Sequence table

<110>University Of Tianjin

<120>the CCD2 mutant and its coded sequence in a kind of safflower source and application and the recombination of production crocetin Yeast strain

<130> MP1833204

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1689

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

atggctaaca aggaagaagc cgaaaagaga aagaagaagc caaagccatt gaaagtcttg 60

atcaccaagg tcgatccaaa accaagaaag ggaatggctt cagttgcagt tgatttgttg 120

gaaaaggcct tcgtttactt gttatccggt aattctgcag ccgatagatc ttcctcttct 180

ggtagaagaa gaagaaagga gcactactac ttgtcaggaa attacgctcc agttggtcat 240

gaaactccac catcagatca tttgccaatc catggtagtc ttcctgagtg tttaaacgga 300

gtcttcttga gagttggccc aaatccaaaa ttcgctccag tagcaggtta taattgggtt 360

gatggagacg gaatgattca cggtttaaga atcaaggacg gtaaagctac ctacttgtct 420

agatacatca agacctccag gttcaagcaa gaagaatact tcggtagagc caagttcatg 480

aagattggcg atttgagagg actattgggt ttcttcacca tcttgatctt ggtcttgaga 540

accaccttga aggttatcga tatctcctac ggtagaggta caggtaatac agctttggtt 600

taccataacg gcttgttgtt ggctttgtca gaagaagaca agccatacgt tgttaaggtg 660

ttggaagacg gagatttgca aactttgggt atcctagact acgacaagaa gttgtcccat 720

ccttttactg ctcatccaaa aatcgatcca ttgacagacg agatgttcac tttcggttac 780

tctatctctc caccatactt gacctacaga gttatctcta aggacggcgt tatgcaagat 840

ccagttcaaa tttccatcac ctctccaact atcatgcacg atttcgctat caccgaaaac 900

tacgctatct tcatggactt gccactatac ttccaaccag aagaaatggt caagggcaaa 960

ttcgtttcct ctttccatcc aaccaagaga gctagaattg gagttttgcc aagatacgct 1020

aaagacgaac atccaattag gtggttcgac ttgccttctt gtttcatgac ccataacgct 1080

aacgcttggg aagaaaacga cgaagttgtt ctattcactt gcagattgga atccccagat 1140

ctagatatgt tgtcaggtcc agcagaagaa gaaatcggta atagcaagtc cgagttgtac 1200

gaaatgaggt tcaacttgaa gaccggtatt acctctcaaa agcaattgag cgttccatca 1260

gtcgatttcc caagaattaa ccagtcctac acaggtagaa agcaacaata cgtctactgc 1320

actttgggta acactaagat caagggcatc gttaagttcg acttgcaaat cgaaccagaa 1380

gcaggtaaaa ctatgttgga agttggaggt aacgttcaag gcattttcga attgggtcca 1440

agaagatacg gttcagaagc tatcttcgtc ccttgtcaac caggaattaa atcagacgaa 1500

gacgacggtt acttgatttt cttcgttcac gacgaaaaca acggtaaaag cgaagtcaac 1560

gtcattgacg ctaagactat gtcagcagaa ccagttgcag ttgttgaatt gccatctaga 1620

gttccatacg gttttcacgc attgttcttg aacgaagaag agttgcaaaa gcatcaagcc 1680

gaaacctaa 1689

<210> 2

<211> 200

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

atagattaat ttaaacagta tatgtacagt tttatatata tatatatata tatatacata 60

tataaagaaa cctgtgcgtt ttttgtattt tcaaatacat ttagttttgg cgccacttct 120

ataaaaggtc tcatgatatt gttacaatat caatttttct tcgtttctca tgccctgttt 180

ctatgataga ggatgaattt 200

<210> 3

<211> 668

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

tatagttttt tctccttgac gttaaagtat agaggtatat taacaatttt ttgttgatac 60

ttttatgaca tttgaataag aagtaataca aaccgaaaat gttgaaagta ttagttaaag 120

tggttatgca gcttttgcat ttatatatct gttaatagat caaaaatcat cgcttcgctg 180

attaattacc ccagaaataa ggctaaaaaa ctaatcgcat tattatccta tggttgttaa 240

tttgattcgt tgatttgaag gtttgtgggg ccaggttact gccaattttt cctcttcata 300

accataaaag ctagtattgt agaatcttta ttgttcggag cagtgcggcg cgaggcacat 360

ctgcgtttca ggaacgcgac cggtgaagac caggacgcac ggaggagagt cttccgtcgg 420

agggctgtcg cccgctcggc ggcttctaat ccgtacttca atatagcaat gagcagttaa 480

gcgtattact gaaagttcca aagagaaggt ttttttaggc taagataatg gggctcttta 540

catttccaca acatataagt aagattagat atggatatgt atatggtggt attgccatgt 600

aatatgatta ttaaacttct ttgcgtccat ccaaaaaaaa agtaagaatt tttgaaaatt 660

caatataa 668

<210> 4

<211> 500

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gagtaataat tattgcttcc atataatatt tttatatacc tcttattttt atgtattagt 60

taattaagta tttttatcta tctgcttatc attttctttt catatagggg gggttggtgt 120

tttcttgccc atcagattga tgtcctccaa ctcggcacta ttttacaaag ggtttttttg 180

taagagaagg agaagacaga tactaaacca tacgttactc gaaacaaaaa aaaaaaaaat 240

ggaaaaagct gctatcaaca aaagacggcc tcatcaaacc taaagaaacc atgtcagcgt 300

atgtatatac cttgtaattt acgtttcctt aaatcttctt tctactaacg ttttcattat 360

tctatactct atgaccaata aaaacagact gtactttcaa aatttaccca gtaggccagc 420

aaataaagaa aattatacca gattacttct gaaacacatt aatcccaaca acaagtatgc 480

cattaatccg tcgctacccc 500

<210> 5

<211> 725

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

tttgccagct tactatcctt cttgaaaata tgcactctat atcttttagt tcttaattgc 60

aacacataga tttgctgtat aacgaatttt atgctatttt ttaaatttgg agttcagtga 120

taaaagtgtc acagcgaatt tcctcacatg tagggaccga attgtttaca agttctctgt 180

accaccatgg agacatcaaa aattgaaaat ctatggaaag atatggacgg tagcaacaag 240

aatatagcac gagccgcgga gttcatttcg ttacttttga tatcactcac aactattgcg 300

aagcgcttca gtgaaaaaat cataaggaaa agttgtaaat attattggta gtattcgttt 360

ggtaaagtag agggggtaat ttttcccctt tattttgttc atacattctt aaattgcttt 420

gcctctcctt ttggaaagct atacttcgga gcactgttga gcgaaggctc attagatata 480

ttttctgtca ttttccttaa cccaaaaata agggaaaggg tccaaaaagc gctcggacaa 540

ctgttgaccg tgatccgaag gactggctat acagtgttca caaaatagcc aagctgaaaa 600

taatgtgtag ctatgttcag ttagtttggc tagcaaagat ataaaagcag gtcggaaata 660

tttatgggca ttattatgca gagcatcaac atgataaaaa aaaacagttg aatattccct 720

caaaa 725

<210> 6

<211> 1371

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

atgaataccg ccaaaactgt tgtcgctgaa caaagagatt tctttagaca aggtaaaact 60

aagtctgttc aagacagatt gaccgcttta gcaaaattga agactcaaat acaagctcaa 120

gaagaagaaa ttattaaggc attgaagcaa gattttggta aaccaacttt cgaatcttac 180

gtaaacgaaa tcttgggtgt tatcagagaa attaactact accaaaaaca tttgcaacaa 240

tggtccaagc cacaaagagt cggtacaaat ttgatggtat ttcctgccag tgctcaatta 300

agaccagaac ctttaggtgt agttttgatc atttctccat ggaactaccc tttctacttg 360

tgtttgatgc cattgatagg tgcaatcgct gctggtaact gcgtcgtagt taaaccatcc 420

gaatatactc ctgccatcag tggtgttatt acaagattga tccaaaacgt cttttcccca 480

gcatgggcca cagtcgtaga aggtgacgaa accatcagtc aacaattgtt acaagaaaag 540

ttcgaccata tctttttcac tggttcacct agagttggta gattgattat ggctgctgct 600

gctgaacaat taacaccagt taccttggaa ttaggtggta aatccccttg tgttgtcgat 660

agagaaatca atttgcaaga aacagctaag agaataatgt ggggtaaatt ggttaacgca 720

ggtcaaacct gtgtcgcccc agactatttg ttggtagaac aatcatgctt ggaacaattg 780

ttacctgctt tgcaacaagc aatccaaatg ttatttggtg aaaatccagc tcactctcct 840

gattacacaa gaatcgttaa ccaacaacaa tggtcaagat tggtatcttt gttgtcacat 900

ggtaaagtta taaccagagg tgaccataat gaaggtgaca gatatatcgc accaacttta 960

ataatcgatc cagacttaaa ctcacctttg atgcaagaag aaatcttcgg tccaatcttg 1020

cctatcttga cctaccaatc cttgagtgaa gccatagatt tcatcaacat caagccaaag 1080

cctttggctt tgtacttttt ctctaacaac agacaaaagc aagaagaaat attgcaatct 1140

acatcttcag gttcagtttg cttgaacgat atcttgttgc atttgactgt cacagacttg 1200

ccatttggtg gtgttggtga atctggtatg ggtagatatc acggtaaagc aactttcgat 1260

acattgtcca actacaagag tatcttgaga agaccattct ggggtgaaac aaatttgaga 1320

tactctcctt acggtaaaaa gatgaactta attaaaaagt tgttttcata a 1371

<210> 7

<211> 450

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

acaaatcgct cttaaatata tacctaaaga acattaaagc tatattataa gcaaagatac 60

gtaaattttg cttatattat tatacacata tcatatttct atatttttaa gatttggtta 120

tataatgtac gtaatgcaaa ggaaataaat tttatacatt attgaacagc gtccaagtaa 180

ctacattatg tgcactaata gtttagcgtc gtgaagactt tattgtgtcg cgaaaagtaa 240

aaattttaaa aattagagca ccttgaactt gcgaaaaagg ttctcatcaa ctgtttaaaa 300

ggaggatatc aggtcctatt tctgacaaac aatatacaaa tttagtttca aagatgaatc 360

agtgcgcgaa ggacataact catgaagcct ccagtatacc catcgatttg caagaaagat 420

actcgcactg gaagaaaaac actaaactac 450

<210> 8

<211> 3057

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

atagattaat ttaaacagta tatgtacagt tttatatata tatatatata tatatacata 60

tataaagaaa cctgtgcgtt ttttgtattt tcaaatacat ttagttttgg cgccacttct 120

ataaaaggtc tcatgatatt gttacaatat caatttttct tcgtttctca tgccctgttt 180

ctatgataga ggatgaattt tatagttttt tctccttgac gttaaagtat agaggtatat 240

taacaatttt ttgttgatac ttttatgaca tttgaataag aagtaataca aaccgaaaat 300

gttgaaagta ttagttaaag tggttatgca gcttttgcat ttatatatct gttaatagat 360

caaaaatcat cgcttcgctg attaattacc ccagaaataa ggctaaaaaa ctaatcgcat 420

tattatccta tggttgttaa tttgattcgt tgatttgaag gtttgtgggg ccaggttact 480

gccaattttt cctcttcata accataaaag ctagtattgt agaatcttta ttgttcggag 540

cagtgcggcg cgaggcacat ctgcgtttca ggaacgcgac cggtgaagac caggacgcac 600

ggaggagagt cttccgtcgg agggctgtcg cccgctcggc ggcttctaat ccgtacttca 660

atatagcaat gagcagttaa gcgtattact gaaagttcca aagagaaggt ttttttaggc 720

taagataatg gggctcttta catttccaca acatataagt aagattagat atggatatgt 780

atatggtggt attgccatgt aatatgatta ttaaacttct ttgcgtccat ccaaaaaaaa 840

agtaagaatt tttgaaaatt caatataaat ggctaacaag gaagaagccg aaaagagaaa 900

gaagaagcca aagccattga aagtcttgat caccaaggtc gatccaaaac caagaaaggg 960

aatggcttca gttgcagttg atttgttgga aaaggccttc gtttacttgt tatccggtaa 1020

ttctgcagcc gatagatctt cctcttctgg tagaagaaga agaaaggagc actactactt 1080

gtcaggaaat tacgctccag ttggtcatga aactccacca tcagatcatt tgccaatcca 1140

tggtagtctt cctgagtgtt taaacggagt cttcttgaga gttggcccaa atccaaaatt 1200

cgctccagta gcaggttata attgggttga tggagacgga atgattcacg gtttaagaat 1260

caaggacggt aaagctacct acttgtctag atacatcaag acctccaggt tcaagcaaga 1320

agaatacttc ggtagagcca agttcatgaa gattggcgat ttgagaggac tattgggttt 1380

cttcaccatc ttgatcttgg tcttgagaac caccttgaag gttatcgata tctcctacgg 1440

tagaggtaca ggtaatacag ctttggttta ccataacggc ttgttgttgg ctttgtcaga 1500

agaagacaag ccatacgttg ttaaggtgtt ggaagacgga gatttgcaaa ctttgggtat 1560

cctagactac gacaagaagt tgtcccatcc ttttactgct catccaaaaa tcgatccatt 1620

gacagacgag atgttcactt tcggttactc tatctctcca ccatacttga cctacagagt 1680

tatctctaag gacggcgtta tgcaagatcc agttcaaatt tccatcacct ctccaactat 1740

catgcacgat ttcgctatca ccgaaaacta cgctatcttc atggacttgc cactatactt 1800

ccaaccagaa gaaatggtca agggcaaatt cgtttcctct ttccatccaa ccaagagagc 1860

tagaattgga gttttgccaa gatacgctaa agacgaacat ccaattaggt ggttcgactt 1920

gccttcttgt ttcatgaccc ataacgctaa cgcttgggaa gaaaacgacg aagttgttct 1980

attcacttgc agattggaat ccccagatct agatatgttg tcaggtccag cagaagaaga 2040

aatcggtaat agcaagtccg agttgtacga aatgaggttc aacttgaaga ccggtattac 2100

ctctcaaaag caattgagcg ttccatcagt cgatttccca agaattaacc agtcctacac 2160

aggtagaaag caacaatacg tctactgcac tttgggtaac actaagatca agggcatcgt 2220

taagttcgac ttgcaaatcg aaccagaagc aggtaaaact atgttggaag ttggaggtaa 2280

cgttcaaggc attttcgaat tgggtccaag aagatacggt tcagaagcta tcttcgtccc 2340

ttgtcaacca ggaattaaat cagacgaaga cgacggttac ttgattttct tcgttcacga 2400

cgaaaacaac ggtaaaagcg aagtcaacgt cattgacgct aagactatgt cagcagaacc 2460

agttgcagtt gttgaattgc catctagagt tccatacggt tttcacgcat tgttcttgaa 2520

cgaagaagag ttgcaaaagc atcaagccga aacctaagag taataattat tgcttccata 2580

taatattttt atatacctct tatttttatg tattagttaa ttaagtattt ttatctatct 2640

gcttatcatt ttcttttcat ataggggggg ttggtgtttt cttgcccatc agattgatgt 2700

cctccaactc ggcactattt tacaaagggt ttttttgtaa gagaaggaga agacagatac 2760

taaaccatac gttactcgaa acaaaaaaaa aaaaaatgga aaaagctgct atcaacaaaa 2820

gacggcctca tcaaacctaa agaaaccatg tcagcgtatg tatatacctt gtaatttacg 2880

tttccttaaa tcttctttct actaacgttt tcattattct atactctatg accaataaaa 2940

acagactgta ctttcaaaat ttacccagta ggccagcaaa taaagaaaat tataccagat 3000

tacttctgaa acacattaat cccaacaaca agtatgccat taatccgtcg ctacccc 3057

<210> 9

<211> 5473

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accataccac agcttttcaa ttcaattcat catttttttt ttattctttt ttttgatttc 240

ggtttctttg aaattttttt gattcggtaa tctccgaaca gaaggaagaa cgaaggaagg 300

agcacagact tagattggta tatatacgca tatgtagtgt tgaagaaaca tgaaattgcc 360

cagtattctt aacccaactg cacagaacaa aaacctgcag gaaacgaaga taaatcatgt 420

cgaaagctac atataaggaa cgtgctgcta ctcatcctag tcctgttgct gccaagctat 480

ttaatatcat gcacgaaaag caaacaaact tgtgtgcttc attggatgtt cgtaccacca 540

aggaattact ggagttagtt gaagcattag gtcccaaaat ttgtttacta aaaacacatg 600

tggatatctt gactgatttt tccatggagg gcacagttaa gccgctaaag gcattatccg 660

ccaagtacaa ttttttactc ttcgaagaca gaaaatttgc tgacattggt aatacagtca 720

aattgcagta ctctgcgggt gtatacagaa tagcagaatg ggcagacatt acgaatgcac 780

acggtgtggt gggcccaggt attgttagcg gtttgaagca ggcggcagaa gaagtaacaa 840

aggaacctag aggccttttg atgttagcag aattgtcatg caagggctcc ctatctactg 900

gagaatatac taagggtact gttgacattg cgaagagcga caaagatttt gttatcggct 960

ttattgctca aagagacatg ggtggaagag atgaaggtta cgattggttg attatgacac 1020

ccggtgtggg tttagatgac aagggagacg cattgggtca acagtataga accgtggatg 1080

atgtggtctc tacaggatct gacattatta ttgttggaag aggactattt gcaaagggaa 1140

gggatgctaa ggtagagggt gaacgttaca gaaaagcagg ctgggaagca tatttgagaa 1200

gatgcggcca gcaaaactaa aaaactgtat tataagtaaa tgcatgtata ctaaactcac 1260

aaattagagc ttcaatttaa ttatatcagt tattacccta tgcggtgtga aataccgcac 1320

agatgcgtaa ggagaaaata ccgcatcagg aaattgtaaa cgttaatatt ttgttaaaat 1380

tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa 1440

tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca 1500

agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg 1560

gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta 1620

aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg 1680

cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa 1740

gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg 1800

gcgcgtcgcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 1860

cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg 1920

taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat 1980

acgactcact atagggcgaa ttgggtaccg ggccccccct cgagatagat taatttaaac 2040

agtatatgta cagttttata tatatatata tatatatata catatataaa gaaacctgtg 2100

cgttttttgt attttcaaat acatttagtt ttggcgccac ttctataaaa ggtctcatga 2160

tattgttaca atatcaattt ttcttcgttt ctcatgccct gtttctatga tagaggatga 2220

atttggatcc acaaatcgct cttaaatata tacctaaaga acattaaagc tatattataa 2280

gcaaagatac gtaaattttg cttatattat tatacacata tcatatttct atatttttaa 2340

gatttggtta tataatgtac gtaatgcaaa ggaaataaat tttatacatt attgaacagc 2400

gtccaagtaa ctacattatg tgcactaata gtttagcgtc gtgaagactt tattgtgtcg 2460

cgaaaagtaa aaattttaaa aattagagca ccttgaactt gcgaaaaagg ttctcatcaa 2520

ctgtttaaaa ggaggatatc aggtcctatt tctgacaaac aatatacaaa tttagtttca 2580

aagatgaatc agtgcgcgaa ggacataact catgaagcct ccagtatacc catcgatttg 2640

caagaaagat actcgcactg gaagaaaaac actaaactac gagctccagc ttttgttccc 2700

tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa 2760

attgttatcc gctcacaatt ccacacaaca taggagccgg aagcataaag tgtaaagcct 2820

ggggtgccta atgagtgagg taactcacat taattgcgtt gcgctcactg cccgctttcc 2880

agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 2940

gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 3000

ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 3060

gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 3120

aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 3180

gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 3240

ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 3300

cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 3360

cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 3420

gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 3480

cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 3540

agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg 3600

ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 3660

ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 3720

gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 3780

cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 3840

attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 3900

accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 3960

ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 4020

gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 4080

agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 4140

ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 4200

ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 4260

gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 4320

ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 4380

tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 4440

tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 4500

cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 4560

tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 4620

gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 4680

tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 4740

ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 4800

attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 4860

cgcgcacatt tccccgaaaa gtgccacctg ggtccttttc atcacgtgct ataaaaataa 4920

ttataattta aattttttaa tataaatata taaattaaaa atagaaagta aaaaaagaaa 4980

ttaaagaaaa aatagttttt gttttccgaa gatgtaaaag actctagggg gatcgccaac 5040

aaatactacc ttttatcttg ctcttcctgc tctcaggtat taatgccgaa ttgtttcatc 5100

ttgtctgtgt agaagaccac acacgaaaat cctgtgattt tacattttac ttatcgttaa 5160

tcgaatgtat atctatttaa tctgcttttc ttgtctaata aatatatatg taaagtacgc 5220

tttttgttga aattttttaa acctttgttt attttttttt cttcattccg taactcttct 5280

accttcttta tttactttct aaaatccaaa tacaaaacat aaaaataaat aaacacagag 5340

taaattccca aattattcca tcattaaaag atacgaggcg cgtgtaagtt acaggcaagc 5400

gatccgtcct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg 5460

aggccctttc gtc 5473

<210> 10

<211> 3048

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

gtagtttagt gtttttcttc cagtgcgagt atctttcttg caaatcgatg ggtatactgg 60

aggcttcatg agttatgtcc ttcgcgcact gattcatctt tgaaactaaa tttgtatatt 120

gtttgtcaga aataggacct gatatcctcc ttttaaacag ttgatgagaa cctttttcgc 180

aagttcaagg tgctctaatt tttaaaattt ttacttttcg cgacacaata aagtcttcac 240

gacgctaaac tattagtgca cataatgtag ttacttggac gctgttcaat aatgtataaa 300

atttatttcc tttgcattac gtacattata taaccaaatc ttaaaaatat agaaatatga 360

tatgtgtata ataatataag caaaatttac gtatctttgc ttataatata gctttaatgt 420

tctttaggta tatatttaag agcgatttgt tttatgaaaa caacttttta attaagttca 480

tctttttacc gtaaggagag tatctcaaat ttgtttcacc ccagaatggt cttctcaaga 540

tactcttgta gttggacaat gtatcgaaag ttgctttacc gtgatatcta cccataccag 600

attcaccaac accaccaaat ggcaagtctg tgacagtcaa atgcaacaag atatcgttca 660

agcaaactga acctgaagat gtagattgca atatttcttc ttgcttttgt ctgttgttag 720

agaaaaagta caaagccaaa ggctttggct tgatgttgat gaaatctatg gcttcactca 780

aggattggta ggtcaagata ggcaagattg gaccgaagat ttcttcttgc atcaaaggtg 840

agtttaagtc tggatcgatt attaaagttg gtgcgatata tctgtcacct tcattatggt 900

cacctctggt tataacttta ccatgtgaca acaaagatac caatcttgac cattgttgtt 960

ggttaacgat tcttgtgtaa tcaggagagt gagctggatt ttcaccaaat aacatttgga 1020

ttgcttgttg caaagcaggt aacaattgtt ccaagcatga ttgttctacc aacaaatagt 1080

ctggggcgac acaggtttga cctgcgttaa ccaatttacc ccacattatt ctcttagctg 1140

tttcttgcaa attgatttct ctatcgacaa cacaagggga tttaccacct aattccaagg 1200

taactggtgt taattgttca gcagcagcag ccataatcaa tctaccaact ctaggtgaac 1260

cagtgaaaaa gatatggtcg aacttttctt gtaacaattg ttgactgatg gtttcgtcac 1320

cttctacgac tgtggcccat gctggggaaa agacgttttg gatcaatctt gtaataacac 1380

cactgatggc aggagtatat tcggatggtt taactacgac gcagttacca gcagcgattg 1440

cacctatcaa tggcatcaaa cacaagtaga aagggtagtt ccatggagaa atgatcaaaa 1500

ctacacctaa aggttctggt cttaattgag cactggcagg aaataccatc aaatttgtac 1560

cgactctttg tggcttggac cattgttgca aatgtttttg gtagtagtta atttctctga 1620

taacacccaa gatttcgttt acgtaagatt cgaaagttgg tttaccaaaa tcttgcttca 1680

atgccttaat aatttcttct tcttgagctt gtatttgagt cttcaatttt gctaaagcgg 1740

tcaatctgtc ttgaacagac ttagttttac cttgtctaaa gaaatctctt tgttcagcga 1800

caacagtttt ggcggtattc attttttgag ggaatattca actgtttttt tttatcatgt 1860

tgatgctctg cataataatg cccataaata tttccgacct gcttttatat ctttgctagc 1920

caaactaact gaacatagct acacattatt ttcagcttgg ctattttgtg aacactgtat 1980

agccagtcct tcggatcacg gtcaacagtt gtccgagcgc tttttggacc ctttccctta 2040

tttttgggtt aaggaaaatg acagaaaata tatctaatga gccttcgctc aacagtgctc 2100

cgaagtatag ctttccaaaa ggagaggcaa agcaatttaa gaatgtatga acaaaataaa 2160

ggggaaaaat taccccctct actttaccaa acgaatacta ccaataatat ttacaacttt 2220

tccttatgat tttttcactg aagcgcttcg caatagttgt gagtgatatc aaaagtaacg 2280

aaatgaactc cgcggctcgt gctatattct tgttgctacc gtccatatct ttccatagat 2340

tttcaatttt tgatgtctcc atggtggtac agagaacttg taaacaattc ggtccctaca 2400

tgtgaggaaa ttcgctgtga cacttttatc actgaactcc aaatttaaaa aatagcataa 2460

aattcgttat acagcaaatc tatgtgttgc aattaagaac taaaagatat agagtgcata 2520

ttttcaagaa ggatagtaag ctggcaaagg ggtagcgacg gattaatggc atacttgttg 2580

ttgggattaa tgtgtttcag aagtaatctg gtataatttt ctttatttgc tggcctactg 2640

ggtaaatttt gaaagtacag tctgttttta ttggtcatag agtatagaat aatgaaaacg 2700

ttagtagaaa gaagatttaa ggaaacgtaa attacaaggt atatacatac gctgacatgg 2760

tttctttagg tttgatgagg ccgtcttttg ttgatagcag ctttttccat tttttttttt 2820

tttgtttcga gtaacgtatg gtttagtatc tgtcttctcc ttctcttaca aaaaaaccct 2880

ttgtaaaata gtgccgagtt ggaggacatc aatctgatgg gcaagaaaac accaaccccc 2940

cctatatgaa aagaaaatga taagcagata gataaaaata cttaattaac taatacataa 3000

aaataagagg tatataaaaa tattatatgg aagcaataat tattactc 3048

Claims

1. a kind of CCD2 mutant in safflower source, which is characterized in that have following one or more kinds of site mutation:

V120F、Y190K/A、R192V/F、E211A、E212A、T290V、K320A、S323F/A/T。

2. CCD2 mutant according to claim 1, which is characterized in that with S323F/A/T site mutation and/or have such as The site mutation of lower one or more:

V120F、Y190K/A、R192V/F、E211A、E212A、T290V、K320A。

3. expressing the coded sequence of CCD2 mutant as claimed in claim 1 or 2.

4. recombinant vector and/or production safflower that coded sequence described in claim 3 expresses the CCD2 mutant in preparation Application in the restructuring yeast strains of acid.

5. a kind of recombinant vector, which is characterized in that access coded sequence described in claim 3 on carrier is carrier.

6. recombinant vector according to claim 5, which is characterized in that also include ALD coded sequence on carrier is carrier.

7. application of the recombinant vector of claim 5 or 6 in building production crocetin restructuring yeast strains.

8. a kind of restructuring yeast strains for producing crocetin, which is characterized in that conversion has the right that 5 or 6 recombinations is required to carry Body.

9. application of the restructuring yeast strains described in claim 8 in production crocetin.

10. a kind of method for producing crocetin, which is characterized in that using the fermentation life of restructuring yeast strains described in claim 8 It produces.