阅读说明：本技术 基于杜氏藻代谢途径的番茄红素高产工程菌及其构建方法与应用 (Lycopene high production bacteria and its construction method and application based on Du Shi algae metabolic pathway ) 是由 姜建国 陈浩宏 吴芳纯 谢红 于 2019-07-23 设计创作，主要内容包括：本发明公开一种基于杜氏藻代谢途径的番茄红素高产工程菌及其构建方法与应用。本发明公开一种类胡萝卜素异构酶Crtiso及应用,且第一次实现全部利用来自杜氏藻的类胡萝卜素途径的基因来构建工程菌。当杜氏藻为杜氏巴氏藻或杜氏盐藻时,所述番茄红素高产工程菌的番茄红素的产率为2.0mg/g细胞干重或3.8mg/g细胞干重。本发明采用的载体的启动子都是T7启动子,包含lacZ调控元件,其在IPTG作用的大肠杆菌BL21(DE3)中能够高表达,这有利于番茄红素的大量积累,可用于实际应用生产。同时本发明还鉴定了ziso和crtiso的功能,验证了植物番茄红素、β-胡萝卜素的生成必须有这两个酶的共同参与。(The present invention discloses a kind of lycopene high production bacteria based on Du Shi algae metabolic pathway and its construction method and application.The present invention discloses a kind of carotenoid isomerase Crtiso and application, and realizes construct engineering bacteria all of the gene of the carotenoid approach from Du Shi algae for the first time.When Du Shi algae is Du Shi Pasteur algae or Dunaliella salina, the yield of the lycopene of the lycopene high production bacteria is 2.0mg/g dry cell weight or 3.8mg/g dry cell weight.The promoter for the carrier that the present invention uses all is T7 promoter, includes lacZ controlling element, high can be expressed in the e. coli bl21 (DE3) of IPTG effect, this is conducive to a large amount of accumulation of lycopene, can be used for practical application production.The present invention also identifies the function of ziso and crtiso simultaneously, demonstrates plant lycopene, the generation of beta carotene must have the common participations of the two enzymes.)

1. a kind of carotenoid isomerase Crtiso, it is characterised in that: its amino acid sequence such as SEQ ID NO:8 or SEQ ID Shown in NO:16.

2. encoding the gene of carotenoid isomerase Crtiso described in claim 1, it is characterised in that: the core of the gene Nucleotide sequence is as shown in SEQ ID NO:7 or SEQ ID NO:15.

3. the application of carotenoid isomerase Crtiso described in claim 1, it is characterised in that:

The application includes one of following application:

Application of the carotenoid isomerase Crtiso in synthesis carotenoid；

Alternatively, 15- it is cis--sigma carotene isomerase Ziso and the carotenoid isomerase Crtiso synthesis class recklessly Application in radish element；

Alternatively, yak base Mang ox base pyrophosphate synthetase Ggps, phytoene synthetase Psy, phytoene Dehydrogenase Pds, 15- be cis--sigma carotene isomerase Ziso, sigma carotene dehydrogenase Zds and the carotenoid Application of the isomerase Crtiso in synthesis carotenoid.

4. application according to claim 3, it is characterised in that:

The amino acid sequence such as GenBank:APW83740.1 of the yak base Mang ox base pyrophosphate synthetase Ggps or Shown in SEQ ID NO:10；Preferably, the nucleotide sequence of gene such as GenBank:KX231795.1 or SEQ ID NO:9 institute Show；

The amino acid sequence of the phytoene synthetase Psy such as SEQ ID NO:2 or GenBank:AAB51287.1 It is shown；Preferably, the nucleotide sequence of gene is as shown in SEQ ID NO:1 or GenBank:U91900.1；

The amino acid sequence such as GenBank:ADD52599.1 or GenBank of the phytoene dehydrogenase Pds: Shown in CAA75094.1；Preferably, the nucleotide sequence of gene such as GenBank:GQ923693.1 or GenBank: Shown in Y14807.1；

The 15- is cis--the amino acid sequence such as SEQ ID NO:4 or SEQ ID NO:12 of sigma carotene isomerase Ziso It is shown；Preferably, the nucleotide sequence of gene is as shown in SEQ ID NO:3 or SEQ ID NO:11；

The amino acid sequence of the sigma carotene dehydrogenase Zds is as shown in SEQ ID NO:6 or SEQ ID NO:14；It is preferred that , the nucleotide sequence of gene is as shown in SEQ ID NO:5 or SEQ ID NO:13.

5. application according to claim 3 or 4, it is characterised in that:

The carotenoid is beta carotene or lycopene.

6. a kind of lycopene high production bacteria based on Du Shi algae metabolic pathway, it is characterised in that:

When based on Du Shi Pasteur algae (Dunaliella bardawil) metabolic pathway, the lycopene high production bacteria contains It encodes the yak base Mang ox base pyrophosphate synthetase Ggps gene of amino acid sequence shown in GenBank:APW83740.1, compile Phytoene synthetase Psy gene, the coding GenBank:ADD52599.1 of amino acid sequence shown in code SEQ ID NO:2 The phytoene dehydrogenase Pds gene of shown amino acid sequence, the 15- for encoding amino acid sequence shown in SEQ ID NO:4 The sigma carotene dehydrogenase of amino acid sequence shown in cis--sigma carotene isomerase Ziso gene, coding SEQ ID NO:6 Zds gene and the carotenoid isomerase Crtiso gene for encoding amino acid sequence shown in SEQ ID NO:8；

When based on Dunaliella salina (Dunaliella saline) metabolic pathway, the lycopene high production bacteria contains coding Yak base Mang ox base pyrophosphate synthetase Ggps gene, the coding GenBank of amino acid sequence shown in SEQ ID NO:10: The phytoene synthetase Psy gene of amino acid sequence shown in AAB51287.1, coding GenBank:CAA75094.1 institute Show amino acid sequence phytoene dehydrogenase Pds gene, coding SEQ ID NO:12 shown in amino acid sequence 15- it is suitable The sigma carotene dehydrogenase of amino acid sequence shown in formula-sigma carotene isomerase Ziso gene, coding SEQ ID NO:14 Zds gene and the carotenoid isomerase Crtiso gene for encoding amino acid sequence shown in SEQ ID NO:16.

7. the lycopene high production bacteria according to claim 6 based on Du Shi algae metabolic pathway, it is characterised in that:

When based on Du Shi Pasteur algae (Dunaliella bardawil) metabolic pathway, encode shown in GenBank:APW83740.1 The nucleotides sequence of the yak base Mang ox base pyrophosphate synthetase Ggps gene of amino acid sequence is classified as such as GenBank: Nucleotide sequence shown in KX231795.1；

Coding SEQ ID NO:2 shown in amino acid sequence phytoene synthetase Psy gene nucleotides sequence be classified as Nucleotide sequence shown in SEQ ID NO:1；

Encode the nucleotides sequence of the phytoene dehydrogenase Pds gene of amino acid sequence shown in GenBank:ADD52599.1 It is classified as the nucleotide sequence as shown in GenBank:GQ923693.1；

The 15- for encoding amino acid sequence shown in SEQ ID NO:4 is cis--nucleotides sequence of sigma carotene isomerase Ziso gene It is classified as the nucleotide sequence as shown in SEQ ID NO:3；

The nucleotides sequence of the sigma carotene dehydrogenase Zds gene of amino acid sequence shown in coding SEQ ID NO:6 is classified as such as SEQ Nucleotide sequence shown in ID NO:5；

Coding SEQ ID NO:8 shown in amino acid sequence carotenoid isomerase Crtiso gene nucleotides sequence be classified as Nucleotide sequence shown in SEQ ID NO:7；

When based on Dunaliella salina (Dunaliella saline) metabolic pathway, amino acid sequence shown in SEQ ID NO:10 is encoded The nucleotides sequence of yak base Mang ox base pyrophosphate synthetase Ggps gene be classified as the nucleotide as shown in SEQID NO:9 Sequence；

Encode the nucleotides sequence of the phytoene synthetase Psy gene of amino acid sequence shown in GenBank:AAB51287.1 It is classified as the nucleotide sequence as shown in GenBank:U91900.1；

Encode the nucleotides sequence of the phytoene dehydrogenase Pds gene of amino acid sequence shown in GenBank:CAA75094.1 It is classified as the nucleotide sequence as shown in GenBank:Y14807.1；

The 15- for encoding amino acid sequence shown in SEQ ID NO:12 is cis--nucleotide of sigma carotene isomerase Ziso gene Sequence is the nucleotide sequence as shown in SEQ ID NO:11；

Coding SEQ ID NO:14 shown in amino acid sequence sigma carotene dehydrogenase Zds gene nucleotides sequence be classified as Nucleotide sequence shown in SEQ ID NO:13；

The nucleotides sequence of the carotenoid isomerase Crtiso gene of amino acid sequence shown in coding SEQ ID NO:16 is classified as The nucleotide sequence as shown in SEQ ID NO:15.

8. the construction method of the lycopene high production bacteria based on Du Shi algae metabolic pathway described in claim 6 or 7, special Sign is, includes the following steps:

(1) be cloned into from Du Shi algae using related gene engineering means yak base Mang ox base pyrophosphate synthetase Ggps, Phytoene synthetase Psy, phytoene dehydrogenase Pds, 15- be cis--sigma carotene isomerase Ziso, ζ-recklessly Radish element dehydrogenase Zds, carotenoid isomerase Crtiso；

(2) by Ggps and Psy building on pACYduet-1 carrier, chlorampenicol resistant obtains recombinant vector pACYduet- ggps-psy；By Pds and Zds building on pCDFduet-1 carrier, streptomycin resistance obtains recombinant vector pCDFduet- pds-zds；By Ziso and Crtiso building on pETduet-1 carrier, ammonia benzyl resistance obtains recombinant vector pETduet- ziso-crtiso；

(3) it then by three recombinant vector cotransformations of step (2) building in e. coli bl21 (DE3), obtains based on Du The lycopene high production bacteria of family name's algae metabolic pathway；

The Du Shi algae is Du Shi Pasteur algae (Dunaliella bardawil) or Dunaliella salina (Dunaliella saline)。

9. the lycopene high production bacteria based on Du Shi algae metabolic pathway described in claim 6 or 7 is in production lycopene In application.

10. application according to claim 9, it is characterised in that:

When Du Shi algae is Du Shi Pasteur algae (Dunaliella bardawil), the tomato of the lycopene high production bacteria The yield of red pigment is 2.0mg/g dry cell weight；

When Du Shi algae is Dunaliella salina (Dunaliella saline), the lycopene of the lycopene high production bacteria Yield be 3.8mg/g dry cell weight.

Technical field

The present invention relates to genetic engineering bacterium fields, are related to the building clone system of Carotenoid in Plants metabolic pathway carrier System, in particular to a kind of lycopene high production bacteria and its construction method and application based on Du Shi algae metabolic pathway；Specifically It is related to a kind of Du Shi algae (such as Du Shi Pasteur algae (Dunaliella bardawil), Dunaliella salina (Dunaliella saline)) Middle yak base Mang ox base pyrophosphate synthetase (Ggps), phytoene synthetase (Psy), phytoene dehydrogenation Enzyme (Pds), 15- be cis--sigma carotene isomerase (Ziso), sigma carotene dehydrogenase (Zds), carotenoid isomerase (Crtiso) cDNA of gene and the engineering bacteria of high yield lycopene is constructed.

Background technique

Carotenoid molecule is the terpenoid containing 8 isoprene units, is widely present as organic pigment In the chloroplaset and chromoplast of plant and in some other photosynthetic tissues, such as algae, in part bacterium and fungi. Under given conditions, carotenoid content can reach the 14% of dry cell weight in Du Shi frustule.Its high-content may be with enzyme Transformation efficiency have very big relationship.

Summary of the invention

In order to overcome the disadvantages and deficiencies of the prior art, the primary purpose of the present invention is that one Carotenoids of offer are different Structure enzyme (Crtiso).

Another object of the present invention is to provide the applications of the carotenoid isomerase (Crtiso).

Another object of the present invention is to provide a kind of lycopene high production bacterias based on Du Shi algae metabolic pathway.

Another object of the present invention is to provide the above-mentioned lycopene high production bacterias based on Du Shi algae metabolic pathway Construction method.

A further object of the present invention is to provide the above-mentioned lycopene high production bacterias based on Du Shi algae metabolic pathway Using.

The purpose of the invention is achieved by the following technical solution:

The present invention provides a kind of carotenoid isomerase (Crtiso), amino acid sequence such as SEQ ID NO:8 or SEQ Shown in ID NO:16.

A kind of carotenoid isomerase (Crtiso) gene, nucleotide sequence such as SEQ ID NO:7 or SEQ ID Shown in NO:15.

Application of the carotenoid isomerase (Crtiso) in synthesis carotenoid.

Further, 15- it is cis--sigma carotene isomerase (Ziso) and carotenoid isomerase (Crtiso) closing At the application in carotenoid.

Further, yak base Mang ox base pyrophosphate synthetase (Ggps), phytoene synthetase (Psy), phytoene dehydrogenase (Pds), 15- it is cis--sigma carotene isomerase (Ziso), sigma carotene dehydrogenase (Zds) and carotenoid isomerase (Crtiso) is synthesizing the application in carotenoid.

Wherein, the amino acid sequence such as GenBank of yak base Mang ox base pyrophosphate synthetase (Ggps): Shown in APW83740.1 or SEQ ID NO:10；The nucleotide sequence of its gene such as GenBank:KX231795.1 or SEQ ID Shown in NO:9.

The amino acid sequence of phytoene synthetase (Psy) such as SEQ ID NO:2 or GenBank:AAB51287.1 It is shown；The nucleotide sequence of its gene is as shown in SEQ ID NO:1 or GenBank:U91900.1.

The amino acid sequence such as GenBank:ADD52599.1 or GenBank of phytoene dehydrogenase (Pds): Shown in CAA75094.1；The nucleotide sequence of its gene is as shown in GenBank:GQ923693.1 or GenBank:Y14807.1.

15- is cis--the amino acid sequence such as SEQ ID NO:4 or SEQ ID NO:12 of sigma carotene isomerase (Ziso) It is shown；The nucleotide sequence of its gene is as shown in SEQ ID NO:3 or SEQ ID NO:11.

The amino acid sequence of sigma carotene dehydrogenase (Zds) is as shown in SEQ ID NO:6 or SEQ ID NO:14；Its base The nucleotide sequence of cause is as shown in SEQ ID NO:5 or SEQ ID NO:13.

The carotenoid is beta carotene or lycopene.

The present invention provides a kind of lycopene high production bacteria based on Du Shi algae metabolic pathway, is based on Du Shi Pasteur algae When (Dunaliella bardawil) metabolic pathway, the lycopene high production bacteria contains coding GenBank: Yak base Mang ox base pyrophosphate synthetase (Ggps) gene, the coding SEQ ID of amino acid sequence shown in APW83740.1 Phytoene synthetase (Psy) gene of amino acid sequence shown in NO:2 encodes ammonia shown in GenBank:ADD52599.1 The 15- of amino acid sequence shown in phytoene dehydrogenase (Pds) gene of base acid sequence, coding SEQ ID NO:4 is cis-- The sigma carotene dehydrogenase of amino acid sequence shown in sigma carotene isomerase (Ziso) gene, coding SEQ ID NO:6 (Zds) carotenoid isomerase (Crtiso) gene of gene and amino acid sequence shown in coding SEQ ID NO:8；

When based on Dunaliella salina (Dunaliella saline) metabolic pathway, the lycopene high production bacteria contains Encode yak base Mang ox base pyrophosphate synthetase (Ggps) gene, the coding of amino acid sequence shown in SEQ ID NO:10 Phytoene synthetase (Psy) gene, the coding GenBank of amino acid sequence shown in GenBank:AAB51287.1: Phytoene dehydrogenase (Pds) gene of amino acid sequence shown in CAA75094.1 encodes ammonia shown in SEQ ID NO:12 The 15- of base acid sequence is cis--sigma carotene isomerase (Ziso) gene, amino acid sequence shown in coding SEQ ID NO:14 Sigma carotene dehydrogenase (Zds) gene and the carotenoid isomerase for encoding amino acid sequence shown in SEQ ID NO:16 (Crtiso) gene；

Preferably, when being based on Du Shi Pasteur algae (Dunaliella bardawil) metabolic pathway, GenBank is encoded: The nucleotide sequence of yak base Mang ox base pyrophosphate synthetase (Ggps) gene of amino acid sequence shown in APW83740.1 For the nucleotide sequence as shown in GenBank:KX231795.1；Wherein, GenBank:APW83740.1 and GenBank: Source Dunaliella salina record in KX231795.1 is wrong, and practical source should be Dunaliella bardawil；

Encode the nucleotides sequence of phytoene synthetase (Psy) gene of amino acid sequence shown in SEQ ID NO:2 It is classified as the nucleotide sequence as shown in SEQ ID NO:1；

Encode the core of phytoene dehydrogenase (Pds) gene of amino acid sequence shown in GenBank:ADD52599.1 Nucleotide sequence is the nucleotide sequence as shown in GenBank:GQ923693.1；Wherein, GenBank:ADD52599.1 with Source Dunaliella salina record in GenBank:GQ923693.1 is wrong, and practical source should be Dunaliella bardawil；

The 15- for encoding amino acid sequence shown in SEQ ID NO:4 is cis--core of sigma carotene isomerase (Ziso) gene Nucleotide sequence is the nucleotide sequence as shown in SEQ ID NO:3；

Encode the nucleotide sequence of sigma carotene dehydrogenase (Zds) gene of amino acid sequence shown in SEQ ID NO:6 For the nucleotide sequence as shown in SEQ ID NO:5；

Encode the nucleotides sequence of carotenoid isomerase (Crtiso) gene of amino acid sequence shown in SEQ ID NO:8 It is classified as the nucleotide sequence as shown in SEQ ID NO:7；

Preferably, it when being based on Dunaliella salina (Dunaliella saline) metabolic pathway, encodes shown in SEQ ID NO:10 The nucleotides sequence of yak base Mang ox base pyrophosphate synthetase (Ggps) gene of amino acid sequence is classified as such as SEQ ID NO:9 Shown in nucleotide sequence；

Encode the core of phytoene synthetase (Psy) gene of amino acid sequence shown in GenBank:AAB51287.1 Nucleotide sequence is the nucleotide sequence as shown in GenBank:U91900.1；Wherein, the source in GenBank:U91900.1 Dunaliella bardawil record is wrong, and practical source should be Dunaliella saline；

Encode the core of phytoene dehydrogenase (Pds) gene of amino acid sequence shown in GenBank:CAA75094.1 Nucleotide sequence is the nucleotide sequence as shown in GenBank:Y14807.1；Wherein, the source in GenBank:Y14807.1 Dunaliella bardawil record is wrong, and practical source should be Dunaliella saline；

The 15- for encoding amino acid sequence shown in SEQ ID NO:12 is cis--sigma carotene isomerase (Ziso) gene Nucleotides sequence is classified as the nucleotide sequence as shown in SEQ ID NO:11；

Encode the nucleotide sequence of sigma carotene dehydrogenase (Zds) gene of amino acid sequence shown in SEQ ID NO:14 For the nucleotide sequence as shown in SEQ ID NO:13；

Encode the nucleotide of carotenoid isomerase (Crtiso) gene of amino acid sequence shown in SEQ ID NO:16 Sequence is the nucleotide sequence as shown in SEQ ID NO:15；

The construction method of the lycopene high production bacteria based on Du Shi algae metabolic pathway, includes the following steps:

(1) yak base Mang ox base pyrophosphate synthetase is cloned into from Du Shi algae using related gene engineering means (Ggps), phytoene synthetase (Psy), phytoene dehydrogenase (Pds), 15- it is cis--sigma carotene isomery Enzyme (Ziso), sigma carotene dehydrogenase (Zds), carotenoid isomerase (Crtiso)；

(2) by Ggps and Psy building on pACYduet-1 carrier, chlorampenicol resistant obtains recombinant vector pACYduet- ggps-psy；By Pds and Zds building on pCDFduet-1 carrier, streptomycin resistance obtains recombinant vector pCDFduet- pds-zds；By Ziso and Crtiso building on pETduet-1 carrier, ammonia benzyl resistance obtains recombinant vector pETduet- ziso-crtiso；

(3) then by three recombinant vector cotransformations of step (2) building in e. coli bl21 (DE3), base is obtained In the lycopene high production bacteria of Du Shi algae metabolic pathway.

Preferably, the Du Shi algae is Du Shi Pasteur algae (Dunaliella bardawil) or Dunaliella salina (Dunaliella saline)。

Preferably, when construction of recombinant vector, rbs (ribosomes combination is respectively connected in the N-terminal (5 ' end) of 6 target gene Site) sequence, alternatively, being respectively connected with T7 promoter (T7_promoter) sequence and rbs (ribosome bind site) sequence.

Application of the lycopene high production bacteria based on Du Shi algae metabolic pathway in production lycopene.

When Du Shi algae is Du Shi Pasteur algae (Dunaliella bardawil), the lycopene high production bacteria The yield of lycopene is 2.0mg/g (dry cell weight).

When Du Shi algae is Dunaliella salina (Dunaliella saline), the tomato of the lycopene high production bacteria The yield of red pigment is 3.8mg/g (dry cell weight).

The present invention has the following advantages and effects with respect to the prior art:

The present invention is realized for the first time all of the carotenoids for coming from Du Shi algae (such as Du Shi Pasteur algae or Dunaliella salina) The gene of plain approach constructs engineering bacteria.And Du Shi algae (such as Du Shi Pasteur algae or Dunaliella salina) be known production lycopene most One of high plant.The promoter for the carrier that the present invention uses all is T7 promoter, includes lacZ controlling element, is made in IPTG It high can be expressed in e. coli bl21 (DE3), this is conducive to a large amount of accumulation of lycopene, and it is raw to can be used for practical application It produces.The present invention also identifies the function of ziso and crtiso simultaneously, demonstrate plant lycopene, the generation of beta carotene must There must be the common participation of the two enzymes.

Detailed description of the invention

Fig. 1 is pACYduet-Dbggps-Dbpsy of the present invention (A) and pETduet-Dbziso-Dbcrtiso (B) building Figure.

Fig. 2 is pCDFduet-Dbpds-Dbzds of the present invention (A) and pCDFduet-Dbpds-Dbzds-Dblycb (B) structure Build figure.

Fig. 3 is pETduet-Dbziso of the present invention (A) and pETduet-Dbcrtiso (B) structure figures.

Fig. 4 is that the present invention is based on the carotenoid high production bacterias and Bacillus coli communis of Du Shi Pasteur's algae metabolic pathway BL21 (DE3) color compares, wherein A is beta carotene high production bacteria；B be lycopene high production bacteria (1: BL21 (DE3) bacterial strain, 2: lycopene superior strain)；C is to combine the purpose bacterial strain 3., 4., 5., 6., 7., 8. obtained.

Fig. 5 is beta carotene liquid phase figure；Wherein, A is embodiment 1；B is embodiment 2.

Fig. 6 is lycopene liquid phase figure；Wherein, A is embodiment 1；B is embodiment 2.

Fig. 7 is pACYduet1-Dsggps-Dspsy structure figures of the present invention.

Fig. 8 is pCDFduet1-Dspds-Dszds structure figures of the present invention.

Fig. 9 is pCDFduet1-Dspds-Dszds-Dslycb structure figures of the present invention.

Figure 10 is pETduet1-Dsziso structure figures of the present invention.

Figure 11 is pETduet1-Dscrtiso structure figures of the present invention.

Figure 12 is pETduet1-Dsziso-Dscrtiso structure figures of the present invention.

Figure 13 is the carotenoid high production bacteria and Bacillus coli communis the present invention is based on dunaliella salina metabolic pathway BL21 (DE3) color compares, wherein A is beta carotene high production bacteria；B is lycopene high production bacteria；C is combination 3., the purpose bacterial strain that 4., 5., 6., 7., 8. obtains.

Specific embodiment

Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited In this.

The test method of specific experiment condition is not specified in the following example, usually according to conventional laboratory conditions or according to system Make experiment condition proposed by factory.Used material, reagent etc., unless otherwise specified, for the reagent obtained from commercial channels And material.