阅读说明：本技术 基于杜氏藻代谢途径的链孢红素高产工程菌及其构建方法与应用 (Neurosporene high production bacteria and its construction method and application based on Du Shi algae metabolic pathway ) 是由 姜建国 陈浩宏 常旭 吴芳纯 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链孢红素高产工程菌及其构建方法与应用,涉及基因工程菌领域。本发明第一次实现全部利用来自杜氏藻(如杜氏巴氏藻或杜氏盐藻)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时,所述链孢红素高产工程菌的链孢红素的产率为2.4mg/g(细胞干重)；当杜氏藻为杜氏盐藻时,所述链孢红素高产工程菌的链孢红素的产率为3.1mg/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,这有利于链孢红素的大量积累,可用于实际应用生产。(The present invention discloses a kind of neurosporene high production bacteria based on Du Shi algae metabolic pathway and its construction method and application, is related to genetic engineering bacterium field.The present invention is realized all of the gene of the carotenoid approach from Du Shi algae (such as Du Shi Pasteur algae or Dunaliella salina) for the first time constructs engineering bacteria.When Du Shi algae is Du Shi Pasteur algae, the yield of the neurosporene of the neurosporene high production bacteria is 2.4mg/g (dry cell weight)；When Du Shi algae is Dunaliella salina, the yield of the neurosporene of the neurosporene high production bacteria is 3.1mg/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 neurosporene, can be used for practical application production.)

1. a kind of neurosporene 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 neurosporene 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 Cis--sigma carotene isomerase Ziso gene and the sigma carotene dehydrogenation for encoding amino acid sequence shown in SEQ ID NO:6 Enzyme Zds gene；

When based on Dunaliella salina (Dunaliella saline) metabolic pathway, the neurosporene 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:8: 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:10 shown in amino acid sequence 15- it is suitable Formula-sigma carotene isomerase Ziso gene and the sigma carotene dehydrogenase for encoding amino acid sequence shown in SEQ ID NO:12 Zds gene.

2. the neurosporene high production bacteria according to claim 1 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.

3. the neurosporene high production bacteria according to claim 1 or 2 based on Du Shi algae metabolic pathway, feature exist In:

When based on Dunaliella salina (Dunaliella saline) metabolic pathway, amino acid sequence shown in SEQ ID NO:8 is encoded The nucleotides sequence of yak base Mang ox base pyrophosphate synthetase Ggps gene is classified as the nucleotides sequence as shown in SEQ ID NO:7 Column；

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:10 is cis--nucleotide of sigma carotene isomerase Ziso gene Sequence is the nucleotide sequence as shown in SEQ ID NO:9；

Coding SEQ ID NO:12 shown in amino acid sequence sigma carotene dehydrogenase Zds gene nucleotides sequence be classified as Nucleotide sequence shown in SEQ ID NO:11.

4. the building side of the described in any item neurosporene high production bacterias based on Du Shi algae metabolic pathway of claims 1 to 3 Method, which comprises the steps of:

(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；

(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 building on pETduet-1 carrier, ammonia benzyl resistance obtains recombinant vector pETduet-ziso；

(3) it then by three recombinant vector cotransformations of step (2) building in e. coli bl21 (DE3), obtains based on Du The neurosporene 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)。

5. the construction method of the neurosporene high production bacteria according to claim 4 based on Du Shi algae metabolic pathway, It is characterized in that:

When construction of recombinant vector, it is respectively connected with ribosome bind site rbs sequence at 5 ' ends of 5 target gene, alternatively, connecting It is connected to T7_promoter sequence and ribosome bind site rbs sequence.

6. the described in any item neurosporene high production bacterias based on Du Shi algae metabolic pathway of claims 1 to 3 are in production chain Application in spore red pigment.

7. application according to claim 6, is characterized in that:

When Du Shi algae is Du Shi Pasteur algae (Dunaliella bardawil), the chain spore of the neurosporene high production bacteria The yield of red pigment is 2.4mg/g dry cell weight.

8. application according to claim 6, is characterized in that:

When Du Shi algae is Dunaliella salina (Dunaliella saline), the neurosporene of the neurosporene high production bacteria Yield be 3.1mg/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 neurosporene 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), the cDNA of sigma carotene dehydrogenase (Zds) gene and building The engineering bacteria of high yield neurosporene out.

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 providing a kind of based on Du Shi algae generation Thank to the neurosporene high production bacteria of approach.

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

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

A kind of neurosporene high production bacteria based on Du Shi algae metabolic pathway is based on Du Shi Pasteur algae (Dunaliella Bardawil) when metabolic pathway, the neurosporene high production bacteria contains amino shown in coding GenBank:APW83740.1 Yak base Mang ox base pyrophosphate synthetase (Ggps) gene of acid sequence encodes amino acid sequence shown in SEQ ID NO:2 Phytoene synthetase (Psy) gene, coding GenBank:ADD52599.1 shown in amino acid sequence octahydro tomato Phytoene dehydrogenase enzyme (Pds) gene, coding SEQ ID NO:4 shown in amino acid sequence 15- it is cis--sigma carotene isomerase (Ziso) sigma carotene dehydrogenase (Zds) gene of gene and amino acid sequence shown in coding SEQ ID NO:6；

When based on Dunaliella salina (Dunaliella saline) metabolic pathway, the neurosporene 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:8 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:10 The 15- of base acid sequence is cis--sigma carotene isomerase (Ziso) gene and coding SEQ ID NO:12 shown in amino acid sequence Sigma carotene dehydrogenase (Zds) 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；

Preferably, it when being based on Dunaliella salina (Dunaliella saline) metabolic pathway, encodes shown in SEQ ID NO:8 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:7 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:10 is cis--sigma carotene isomerase (Ziso) gene Nucleotides sequence is classified as the nucleotide sequence as shown in SEQ ID NO:9；

Encode the nucleotide sequence of sigma carotene dehydrogenase (Zds) gene of amino acid sequence shown in SEQ ID NO:12 For the nucleotide sequence as shown in SEQ ID NO:11；

The construction method of the neurosporene 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)；

(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 building on pETduet-1 carrier, ammonia benzyl resistance obtains recombinant vector pETduet-ziso；

(3) then by three recombinant vector cotransformations of step (2) building in e. coli bl21 (DE3), base is obtained In the neurosporene 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 5 target gene Site) sequence, alternatively, being respectively connected with T7 promoter (T7_promoter) sequence and rbs (ribosome bind site) sequence.

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

When Du Shi algae is Du Shi Pasteur algae (Dunaliella bardawil), the neurosporene high production bacteria The yield of neurosporene is 2.4mg/g (dry cell weight).

When Du Shi algae is Dunaliella salina (Dunaliella saline), the chain spore of the neurosporene high production bacteria The yield of red pigment is 3.1mg/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 neurosporene 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 neurosporene, and it is raw to can be used for practical application It produces.

Detailed description of the invention

Fig. 1 is pACYduet-Dbggps-Dbpsy of the present invention (A) and pCDFduet-Dbpds-Dbzds (B) structure figures.

Fig. 2 is pETduet-Dbziso structure figures of the present invention.

Fig. 3 is that the present invention is based on the neurosporene high production bacterias of Du Shi algae metabolic pathway and Bacillus coli communis BL21 (DE3) color compares；Wherein, A is the neurosporene high production bacteria based on Du Shi Pasteur's algae metabolic pathway；B is based on Du Shi The neurosporene high production bacteria of salt algae metabolic pathway.

Fig. 4 is neurosporene liquid phase figure；Wherein, A is embodiment 1；B is embodiment 2.

Fig. 5 is pACYduet1-Dsggps-Dspsy structure figures of the present invention.

Fig. 6 is pCDFduet1-Dspds-Dszds structure figures of the present invention.

Fig. 7 is pETduet1-Dsziso structure figures of the present invention.

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.