CN113604374B

CN113604374B - Genetically engineered bacterium for efficiently producing carotenoid, construction method and application thereof

Info

Publication number: CN113604374B
Application number: CN202110958208.4A
Authority: CN
Inventors: 叶丽丹; 于洪巍; 陈明凯
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-08-20
Filing date: 2021-08-20
Publication date: 2023-09-15
Anticipated expiration: 2041-08-20
Also published as: CN113604374A

Abstract

The invention discloses a genetically engineered bacterium for efficiently producing carotenoid, and a construction method and application thereof, and belongs to the technical field of genetic engineering. The genetically engineered bacterium takes engineering strains producing carotenoid as starting bacteria, and a chromosome of the genetically engineered bacterium is integrated with a multidirectional drug resistance transcription factor coding gene PDR1 or PDR3; or the genetically engineered bacterium takes engineering strains producing carotenoid as starting bacteria, and host bacteria contain recombinant expression plasmids containing multidirectional drug resistance transcription factor coding genes PDR1 or PDR 3. According to the invention, the multidirectional drug-resistant transcription factor coding gene PDR1 or PDR3 is introduced into the engineering strain for producing carotenoid, so that the corresponding transcription regulatory factor is successfully expressed in the strain, the synthesis of carotenoid is promoted, the yield of carotenoid is obviously improved, and the method has a good application prospect.

Description

Genetically engineered bacterium for efficiently producing carotenoid, construction method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a genetic engineering bacterium capable of realizing efficient production of carotenoid, and a construction method and application thereof.

Background

With the development of transgenic technology, the production of carotenoids by using fungi produced by genetic engineering methods has become a hot spot of research. Carotenoids, as liposoluble products, tend to cause cell damage after accumulation within the cell membrane reaches a certain threshold. It was found that carotenoids synthesized heterologously in Saccharomyces cerevisiae intercalate into cell membranes (Investigation of cellular targeting of carotenoid pathway enzymes in Pichia pastoris, J Biotechnol, 2009). Thus, carotenoid synthesis and accumulation often stress yeast cells, thereby limiting their biological production efficiency.

The family of multidrug resistance (pleiotropic drug resistance, PDR) proteins, as part of the regulation of the naturally occurring stress tolerance system of saccharomyces cerevisiae, plays an important role in the resistance of yeast to a large number of cytotoxic compounds (Characterization of three pleiotropic drug resistance transporter genes and their participation in the azole resistance of Mucor circinelloides, front Cell Infect Microbiol, 2021).

Transcriptomics research analysis found that expression of PDR transporter was up-regulated in high β -carotene producing saccharomyces cerevisiae engineering strains, and trace amounts of β -carotene were detected extracellular, whereas knockout of PDR10 gene significantly reduced β -carotene production (Heterologous carotenoid production in Saccharomyces cerevisiae induces the pleiotropic drug resistance stress response, yeast, 2010). The expression of Saccharomyces cerevisiae PDR10 gene in rhodosporidium can promote the synthesis of carotenoid and secrete part of torulasis, beta-carotene and rhodosporin to outside cells (Engineering Rhodosporidium toruloides with a membrane transporter facilitates production and separation of carotenoids and lipids in a bi-phase culture, appl Microbiol Biotechnol, 2016). Similarly, overexpression of PDR transporter in s.cerevisiae engineering strains also promotes synthesis and secretion of β -carotene, which yields up to 32% (Engineering endogenous ABC transporter with improving ATP supply and membrane flexibility enhances the secretion of beta-carotene in Saccharomyces cerevisiae, biotechnol Biofuels, 2020).

However, the PDR transporter acts as a membrane protein, the excessive expression increases the metabolic burden of cells, and the content of the secreted carotenoid is very low, so that the conventional regulation strategy is difficult to form.

Therefore, how to regulate the biosynthesis of carotenoids by multidrug resistance proteins is a problem that needs to be addressed by those skilled in the art.

Disclosure of Invention

The invention aims to provide a genetically engineered bacterium for efficiently producing carotenoid, which utilizes a microorganism heterologous synthesis mode to efficiently prepare the carotenoid.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the invention provides a genetic engineering bacterium for efficiently producing carotenoid, which takes an engineering strain for producing carotenoid as a starting bacterium, and a chromosome of the genetic engineering bacterium is integrated with a multidirectional drug resistance transcription factor coding gene PDR1 or PDR3;

or the genetically engineered bacterium takes engineering strains producing carotenoid as starting bacteria, and host bacteria contain recombinant expression plasmids containing multidirectional drug resistance transcription factor coding genes PDR1 or PDR3;

the nucleotide sequence of the gene PDR1 for encoding the multi-drug resistance transcription factor is shown as SEQ ID NO.1, and the nucleotide sequence of the gene PDR3 for encoding the multi-drug resistance transcription factor is shown as SEQ ID NO. 2.

The engineering strain for producing carotenoid by the starting strain adopts a strain with carotenoid producing function which is known in the field. The carotenoids include, but are not limited to, beta-carotene, canthaxanthin, astaxanthin.

The invention utilizes the gene integration technology or the way of introducing recombinant expression plasmid to enable the multidirectional drug-resistant transcription factor PDR1 or PDR3 to be successfully expressed in engineering strains for producing carotenoid and participate in the synthesis of the carotenoid, thereby realizing the mass production of the carotenoid by taking safe and efficient saccharomyces cerevisiae as a cell factory.

Further, the engineering strain for producing carotenoid is an engineering strain for producing beta-carotene, an engineering strain for producing canthaxanthin or an engineering strain for producing astaxanthin.

Preferably, the beta-carotene producing engineering strain adopts beta-carotene producing engineering strain Ycarot-02, which is a publicly available material, and its construction method is referred to in references (Alleviation of metabolic bottleneck by combinatorial engineering enhanced astaxanthin synthesis in Saccharomyces cerevisiae, enzyme Microb Technol, 2017).

Preferably, the cantharidin yellow-producing engineering strain takes the beta-carotene-producing engineering strain as a starting strain, and a beta-carotene ketolase encoding gene is integrated on a chromosome of the engineering strain; alternatively, the host bacterium contains a recombinant expression plasmid containing a gene encoding a beta-carotene ketolase.

Preferably, the nucleotide sequence of the beta-carotene ketolase encoding gene is shown as SEQ ID NO. 3.

Preferably, the astaxanthin-producing engineering strain adopts an astaxanthin-producing engineering strain YPP-17, which is a publicly available material, and the construction method thereof is described in the literature (Directed coevolution of beta-Carotene ketolase and hydroxylase and its application in temperature-regulated biosynthesis of astaxanthin, J Agric Food Chem, 2019).

The invention also provides a method for constructing the genetically engineered bacterium for efficiently producing carotenoid, which comprises the following steps: taking engineering strains producing carotenoid as starting bacteria, integrating multidirectional drug resistance transcription factor coding genes PDR1 or PDR3 on the chromosome of the engineering strains producing carotenoid through an integration plasmid to obtain the genetic engineering bacteria producing carotenoid efficiently;

or taking engineering strains producing carotenoid as starting bacteria, and introducing recombinant expression plasmids containing multidirectional drug resistance transcription factor coding genes PDR1 or PDR3;

the nucleotide sequence of PDR1 is shown as SEQ ID NO.1, and the nucleotide sequence of PDR3 is shown as SEQ ID NO. 2.

The multidirectional drug-resistant transcription factor encoding genes PDR1 and PDR3 are cloned from the Saccharomyces cerevisiae genome producing beta-carotene.

According to the invention, the multidirectional drug-resistant transcription factor coding gene PDR1 or PDR3 is introduced into the cell of the engineering strain for producing carotenoid, so that the corresponding transcription control factor is expressed in the engineering strain for producing carotenoid, participates in carotenoid synthesis, and a genetic engineering strain capable of efficiently producing carotenoid is constructed.

Specifically, the construction method comprises the following steps:

(1) Coding multidirectional drug-resistant transcription factor with nucleotide sequence shown as SEQ ID NO.1Gene PDR1 or multidirectional drug resistance transcription factor coding gene PDR3 with nucleotide sequence shown as SEQ ID NO.2 is cloned to pUMRI-YPL062W P _GAL1 The later multiple cloning site to obtain recombinant plasmid pUMRI-YPL062W-PDR1 or pUMRI-YPL062W-PDR3;

(2) The recombinant plasmid pUMRI-YPL062W-PDR1 or pUMRI-YPL062W-PDR3 is transformed into engineering strain for producing carotenoid, and recombinant bacteria integrated with multidirectional drug resistance transcription factor coding genes in chromosomes are obtained through screening, namely the genetic engineering bacteria for efficiently producing carotenoid.

The plasmid pUMRI-YPL062W is a publicly available material. The gene segment of the gene PDR1 or PDR3 encoding the multidrug resistance transcription factor is integrated on the host bacterial chromosome by pUMRI series assembling tool plasmid.

Furthermore, the engineering strain for producing carotenoid is engineering strain for producing beta-carotene, cantharidin yellow or astaxanthin.

Further, the construction method of the cantharidin yellow-producing engineering strain comprises the following steps:

1) Cloning a beta-carotene ketolase encoding gene with a nucleotide sequence shown as SEQ ID NO.3 into P of PUMRI-DPP1 _GAL1 The later multiple cloning site to obtain recombinant plasmid PUMRI-DPP1-OBKTM29;

2) Converting the recombinant plasmid PUMRI-DPP1-OBKTM29 into an engineering strain for producing beta-carotene, and screening to obtain recombinant bacteria integrated with a beta-carotene ketolase coding gene in a chromosome, namely the engineering strain for producing cantharidin yellow.

The plasmid PUMRI-DPP1 is a publicly available material and is provided with a promoter P _GAL1 Terminator T _CYC1 . The beta-carotene ketolase encoding gene was integrated into the host bacterial chromosome by pUMRI series of assembly tool plasmids.

The invention also provides application of the genetically engineered bacterium for efficiently producing carotenoid in preparing carotenoid. The various carotenes are prepared from fermentation cultures of the genetically engineered strains constructed by the invention.

Specifically, the application includes: after the genetically engineered bacteria for efficiently producing carotenoid are subjected to amplification culture, inoculating the genetically engineered bacteria into YPD liquid culture medium, and performing shake culture to obtain fermentation liquor; and collecting thalli in the fermentation liquor, and extracting corresponding carotenoid after cell disruption.

The carotenoid in the cell disruption liquid is extracted by using an organic phase extractant, preferably acetone.

When cantharidin and astaxanthin are produced, iron ions can be added into the fermentation medium, which is beneficial to improving the activity of beta-carotene ketolase and improving the product yield.

Preferably, the conditions of fermentation are: culturing at 200-250 rpm and 28-30 deg.c in a constant temperature shaking table for 72-84 hr.

Compared with the prior art, the invention has the beneficial effects that:

the invention constructs a genetic engineering bacterium capable of realizing high-efficiency synthesis of carotenoid, wherein the carotenoid can be but is not limited to beta-carotene, canthaxanthin and astaxanthin. The invention takes engineering strain producing carotenoid as starting strain, introduces multidirectional drug-resistant transcription factor coding gene PDR1 or PDR3, and the corresponding transcription regulatory factor is successfully expressed in the strain, thereby promoting the synthesis of carotenoid, remarkably improving the yield of carotenoid and having good application prospect.

Drawings

FIG. 1 shows the construction of the pUMRI-YPL062W-PDR1 and pUMRI-YPL062W-PDR3 plasmids as a framework (A) and the results of construction (B) and (C).

FIG. 2 shows the backbone (A) and the construction result (B) required for constructing pUMRI-DPP1-OBKTM29 plasmid.

FIG. 3 shows the integration sites involved in the construction of the starting strain β -carotene-producing Saccharomyces cerevisiae (A) and astaxanthin-producing Saccharomyces cerevisiae (B).

FIG. 4 shows the integration sites involved in the construction of high-yield beta-carotene yeasts in example 1.

FIG. 5 is an integration site involved in the construction of high yielding yellow cantharis yeast in example 2.

FIG. 6 shows the integration sites involved in the construction of astaxanthin-producing yeasts in example 3.

FIG. 7 shows the HPLC detection spectrum (A) and the yield analysis result (B) of the beta-carotene-producing yeast constructed in example 1, wherein the upper and lower liquid phase peak curves in (A) are respectively an experimental group and a beta-carotene standard group.

FIG. 8 shows the HPLC detection pattern (A) and yield analysis result (B) of the cantharidin yellow high-yield strain constructed in example 2, wherein the upper and lower liquid phase peak pattern curves in (A) are respectively an experimental group and a cantharidin yellow standard group.

FIG. 9 is a product HPLC detection spectrum (A) and yield analysis result (B) of the astaxanthin-high-producing strain constructed in example 3, wherein upper and lower liquid phase peak pattern curves in (A) are an experimental group and an astaxanthin standard group, respectively.

Detailed Description

The invention will be further illustrated with reference to specific examples. The following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.

The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

The engineering strain Ycarot-02 for producing beta-carotene is constructed in advance for the subject group, and the construction method is described in documents (Alleviation of metabolic bottleneck by combinatorial engineering enhanced astaxanthin synthesis in Saccharomyces cerevisiae, enzyme Microb Technol, 2017). The integration sites involved in strain construction are shown in FIG. 3 (A).

Astaxanthin-producing engineering strains YPP-17 were previously constructed for this subject group, and methods for their construction are described in the literature (Directed coevolution of beta-Carotene ketolase and hydroxylase and its application in temperature-regulated biosynthesis of astaxanthin, J Agric Food Chem, 2019). The integration sites involved in strain construction are shown in FIG. 3 (B).

Plasmids pUMRI-DPP1 and pUMRI-YPL062W were previously constructed for this subject group, and the construction method thereof was described in the literature (Construction of a controllable beta-carotene biosynthetic pathway by decentralized assembly strategy in Saccharomyces cerevisiae. Biotechnol Bioeng, 2014).

EXAMPLE 1 construction of beta-carotene highly productive Saccharomyces cerevisiae

1. Cloning of multidirectional drug-resistant transcription factor PDR1 and PDR3 genes

1.1 extraction of Saccharomyces cerevisiae genomic DNA

The extraction of the genome DNA of the saccharomyces cerevisiae (engineering strain producing beta-carotene) is completed by a kit, and the specific steps are as follows:

(1) Taking 1-3mL of yeast culture solution for 20-24h, and centrifuging at 12000rpm for 5min at room temperature.

(2) The supernatant was discarded, 480. Mu.L Buffer SE, 10. Mu.L mercaptoethanol, 20. Mu.L muramidase were added, and the pellet was resuspended and incubated at 30℃for 30min.

(3) Centrifuge at 12000rpm for 5min at room temperature, discard supernatant, add 200. Mu.L Buffer YL and 50mg glass beads (0.4-0.6 mm), vortex for 3-5min, rest, and aspirate supernatant to a new 1.5mL centrifuge tube.

(4) 25. Mu.L of Proteinase K was added and mixed well and incubated with shaking at 65℃for 30min.

(5) 5. Mu.L of RNase A was added and the tube was inverted upside down and incubated at room temperature for 10min.

(6) 220. Mu.L Buffer YDL and 220. Mu.L pure ethanol were added and vortexed for 20s.

(7) Placing the adsorption column into a collecting tube, sucking all the supernatant into the adsorption column by using a pipette, centrifuging at 10000rpm for 1min, and discarding the supernatant.

(8) The adsorption column was put back into the collection tube, 500. Mu.L Buffer HB was added, centrifuged at 10000rpm for 30s, and the filtrate was discarded.

(9) The column was put back into the collection tube, 700. Mu. L DNA Wash Buffer was added, centrifuged at 10000rpm for 30s, and the filtrate was discarded.

(10) The previous step is repeated, the idle running is carried out, and the residual DNA Wash Buffer is left.

(11) The adsorption column was placed in a new 1.5mL centrifuge tube, 75. Mu.L of an absorption Buffer was added, the mixture was allowed to stand at 65℃for 2min, and the mixture was centrifuged at 12000rpm at room temperature for 1min, and the obtained DNA solution was collected and stored at-20 ℃.

1.2 find the gene sequences of multidirectional drug-resistant transcription factors PDR1 and PDR3 in a yeast genome database (Saccharomyces genome database, SGD), and adopt Saccharomyces cerevisiae genome as a template to carry out PCR amplification by adopting high-fidelity enzyme (Prime STARTM HS DNA polymerase), wherein the nucleotide sequence is shown as SEQ ID NO.1 and SEQ ID NO. 2.

Primer design is as follows:

TABLE 1 primers for cloning of the multidirectional drug resistance transcription factor PDR1 and PDR3 genes

The PCR reaction system (50. Mu.L) was as follows:

the PCR procedure was as follows: (1) pre-denaturation at 98℃for 2min; (2) denaturation at 98 ℃,10s; annealing at 55 ℃ for 20s; extension at 72℃at 1kb/min,30 cycles; (3) extending at 72 ℃ for 5min; (4) preserving at4 ℃ for 1min.

2. Construction of recombinant plasmids

2.1 enzyme digestion and glue recovery

The pUMRI series integrative plasmid PUMRI-YPL062W (plasmid backbone is shown in FIG. 1A) was constructed by laboratory and stored in E.coli Top 10, and E.coli Top 10 plasmid extraction was completed by the kit.

The pUMRI series integrated plasmid and the target fragment of the PCR product are subjected to double digestion by Takara restriction enzyme, a double digestion system is used according to the specification of the Takara restriction enzyme, and after digestion, the system is subjected to DNA gel recovery treatment, and specific steps are carried out according to the specification of the Axygen kit.

2.2 enzyme Linked

The digested fragments and plasmids were ligated using TakaraT4 DNA ligase, and the ligation system (10. Mu.l) was as follows:

and the connection is carried out for 50min at 22 ℃.

2.3 conversion

Adding 10 mu L of the ligation product into the competent solution of escherichia coli, standing on ice for 15min, performing heat shock at 42 ℃ for 90s, rapidly placing in an ice bath for 5min, adding 1mL of LB liquid medium, uniformly mixing, resuscitating for 50min at 37 ℃ by a shaking table, centrifuging, discarding part of supernatant, taking a proper amount of supernatant, coating on a corresponding resistant LB plate, and standing in a 37 ℃ incubator for 12h.

Specifically, the enzyme digestion and ligation are performed in the following manner:

the target fragment of the multidirectional drug resistance transcription factor gene PDR1 is cloned to the P of pUMRI-YPL062W _GAL1 The latter multiple cloning site, the recombinant plasmid pUMRI-YPL062W-PDR1 was obtained. pUMRI-YPL062W-PDR3 was constructed as described above. The recombinant plasmid structure is shown in FIG. 1 (B) and (C).

3. Construction of beta-carotene high-yield Saccharomyces cerevisiae

And (3) respectively converting the recombinant plasmids pUMRI-YPL062W-PDR1 and pUMRI-YPL062W-PDR3 constructed in the step (2) into the engineering strain Ycarot-02 for producing beta-carotene, so that the PDR1 or PDR3 is integrated into the chromosome of the engineering strain for producing beta-carotene to obtain Ycarot-02-1 and Ycarot-02-3. The integration sites involved in strain construction are shown in FIG. 4.

The specific transformation method is as follows:

1. and (3) preparing saccharomyces cerevisiae competence: single colonies of beta-carotene producing yeast strains were picked from YPD plates and inoculated into 5mL YPD tubes, cultured at 30℃at 220rpm overnight, transferred to 50mL YPD shake flasks at 2% inoculum size, cultured at 30℃at 220rpm for 4-5h, OD ₆₀₀ Reaching about 2.0.

2. The saccharomyces cerevisiae conversion step comprises the following steps:

(1) The ssDNA is placed in a metal bath at 100 ℃ and heated for 5min, and then is rapidly placed in ice for cooling for standby.

(2) 40mL of competent yeast solution was centrifuged at 3000rpm and 4℃for 5min in a 50mL sterile centrifuge tube, and the supernatant was discarded.

(3) Washing with 15mL of sterile water once, centrifuging to discard the supernatant, re-suspending with 1mL of sterile water, sub-packaging into 1.5mL sterile centrifuge tubes at 100 μL per tube, and centrifuging to discard the supernatant.

(4) The following conversion systems (360. Mu.L) were added to the centrifuge tube in sequence:

(5) Mixing thoroughly, and standing in a water bath at 42 deg.C for 40min.

(6) Centrifuging at 12000rpm for 1min, discarding supernatant, adding 1mL YPD liquid culture medium, mixing, and recovering for 2h in a 37 ℃.

(7) Centrifuge at 12000rpm for 1min, discard supernatant, add 1ml sterile water to resuspend, take 100ul of bacteria liquid and spread on the corresponding resistance plate.

Example 2 construction of Saccharomyces cerevisiae, which produces cantharis yellow

1. Cloning of the beta-carotene ketolase encoding Gene OBKTM29

The beta-carotene ketolase encoding gene (beta-carotene ketolase variant, OBKTM 29) is obtained by optimizing the earlier stage of the subject group, and the nucleotide sequence is shown as SEQ ID NO. 3.

The target gene fragment was obtained by PCR, the PCR reaction system was the same as in example 1, and plasmid P416XWP04-OBKTM used as a template was deposited in the laboratory, and the construction method thereof was as described in references (Directed coevolution of beta-Carotene ketolase and hydroxylase and its application in temperature-regulated biosynthesis of astaxanthin, J Agric Food Chem, 2019).

TABLE 2 primers for cloning of beta-carotene ketolase encoding gene OBKTM29

2. Construction of recombinant plasmid pUMRI-DPP1-OBKTM29

The pUMRI series of integrative plasmids pUMRI-DPP1 (plasmid backbone as shown in FIG. 2A) was constructed and stored by laboratory. Plasmid extraction, cleavage and ligation were performed as in example 1.

OBKTM29 geneCloning of the target fragment of (C) into the P of pUMRI-DPP1 _GAL1 The latter multiple cloning site, the recombinant plasmid pUMRI-DPP1-OBKTM29 was obtained. The recombinant plasmid structure is shown in FIG. 2 (B).

3. Construction of Saccharomyces cerevisiae producing cantharis yellow

The recombinant plasmids pUMRI-DPP1-OBKTM29 constructed in the step 2 are respectively transformed into Ycarot-02, ycarot-02-1 and Ycarot-02-3 constructed in the example 1, so that the OBKTM29 is respectively integrated into chromosomes of Ycarot-02, ycarot-02-1 and Ycarot-02-3 to obtain Y1-0, Y1-1 and Y1-3. The transformation procedure is as in example 1. The integration sites involved in strain construction are shown in FIG. 5.

EXAMPLE 3 construction of astaxanthin-high-yielding Saccharomyces cerevisiae

The construction method is the same as in example 1 except that Ycarot-02 in the engineering strain producing beta-carotene from the original strain is replaced with the engineering strain YPP-17 producing astaxanthin, so that PDR1 or PDR3 is integrated into the chromosome of the engineering strain producing astaxanthin, and YPP-17-1 and YPP-17-3 are obtained. The integration sites involved in strain construction are shown in FIG. 6.

Test example 1 fermentation culture of genetically engineered bacteria and extraction and analysis of products

1. Single colonies of engineering bacteria are selected from streaked plates and inoculated into 5mL of YPD medium, and after the liquid in a test tube is turbid, the strains are cultured in 50mL of YPD liquid medium. Fermentation was carried out at 220rpm in a shaking table at 30℃for 84 hours.

2. 0.5ml of yeast fermentation broth was collected into a 2ml centrifuge tube, centrifuged at 12000rpm for 2min, the supernatant was discarded, then washed twice with 1ml of distilled water, the discarded supernatant was centrifuged, about 0.5ml volume of the beads (0.1 mm and 0.5mm of each half of zirconia beads) was added to the centrifuge tube, 200. Mu.L of acetone was added to the centrifuge tube, and the cells were resuspended.

3. Placing the centrifuge tube filled with the beads and the thalli in a full-automatic rapid sample grinding instrument, grinding for 5min at 65 HZ.

4. After grinding, 800. Mu.L of acetone was added to the centrifuge tube, thoroughly mixed, and placed under ultrasound for 10min.

5. After sonication, the tube was placed in a centrifuge at 12000rpm for 2min at4℃and the supernatant was collected into a new 2mL centrifuge tube.

6. Adding 1mL of acetone into the original 2mL centrifuge tube, re-extracting for one time until the color of the thalli becomes white, fully and uniformly mixing, carrying out ultrasonic treatment for 10min, centrifuging at 12000rpm for 2min, and then sucking the supernatant into the new centrifuge tube in the step 5 by using a pipette.

7. The mixed extract was filtered through a 0.22 μm organic filter and subjected to HPLC. The following conditions were used to detect various carotenes and their content in the Saccharomyces cerevisiae engineering strain by HPLC. The liquid phase analyzer is Shimadzu LC-20AT, and the chromatographic column is Amethylst C18-H column (4.6X105 mm,5 μm) with 470nm detection wavelength. Gradient elution is adopted, wherein the mobile phase is A pump acetonitrile and pure water (9:1), the B pump is methanol and isopropanol (3:2), the flow rate is 1mL/min, and the gradient elution conditions are as follows: 0-15min, the mobile phase of the pump B rises from 0 to 90%;15-27min, the mobile phase of the pump B remains unchanged to 90%;27-28min, the mobile phase of the pump B is reduced from 90% to 0;27-35min, the b pump mobile phase remained unchanged at 0.

As shown in FIG. 7, the yield of beta-carotene in the Ycarot-02-3 was 97.15mg/L, which was 3.3 times higher than that of the starting strain Ycarot-02.

As shown in FIG. 8, the canthaxanthin yield in Y1-3 is 102.84mg/L, which is 75% higher than that of the control strain Y1-0.

As shown in FIG. 9, the yield of astaxanthin in YPP-17-1 was 14.96mg/L, which was 36% higher than that of the control strain YPP-17.

Sequence listing

<110> university of Zhejiang

<120> genetically engineered bacterium for efficiently producing carotenoid, construction method and application thereof

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ttcaacagcc aaagctttaa agactataca agagcttttt ctttcctgac aatcgtcact 2460

cgtatcatgc ttttggcata cggagagtcc tctagcacca acctcgatgt tatctccaaa 2520

tatattgatg agaatgcgcc agatttaaaa ggtataatag aacttgtctt ggatacaaac 2580

tcttgcgctt atcgattttt attagaacct gttcagaagt caggattcca tttaacggtc 2640

tcacaaatgt tgaaaaatag aaagtttcag gaaccactaa tgtcaaatga agataataag 2700

cagatgaagc ataattcggg aaaaaatttg aatccggatc tcccaagtct taagacgggc 2760

acttcatgct tactaaatgg cattgaatcg cctcaattgc cattcaatgg tcgctcagca 2820

ccttccccag tgagaaacaa ctcactaccg gagtttgcac aattgccttc atttaggtca 2880

ttatccgtgt ctgatatgat caatcccgat tacgcgcaac caacaaatgg gcaaaataat 2940

acgcaagtcc aatctaataa accaatcaat gctcagcagc aaatacccac ttcagtacaa 3000

gtaccattta tgaacacaaa tgaaatcaat aacaacaaca acaacaacaa caacaataaa 3060

aacaatatta acaatattaa caacaacaac agtaacaatt tttctgcaac tagttttaat 3120

ttggggacac tagatgaatt tgttaataac ggtgatcttg aggacctcta cagtatcctg 3180

tggagcgacg tttatccaga tagttaa 3207

<210> 2

<211> 2931

<212> DNA

<213> Saccharomyces cerevisiae (S. Cerevisiae)

<400> 2

atgaaagtga agaaatcaac tagatcaaaa gtttcgacag catgtgtcaa ttgcagaaaa 60

aggaaaatca aatgcacagg taaatatcca tgtaccaact gcatttctta cgattgtacg 120

tgtgtattcc taaaaaaaca tttaccgcag aaggaggata gttcccagtc tttgcctact 180

acagctgttg ctccaccctc ttcccacgcc aatgtagagg cttcagcaga tgtacagcat 240

ctggacactg cgattaagct agataatcaa tattacttca aactgatgaa cgacctgata 300

cagactccag tctctccgag tgcgacgcat gctcctgata cttccaataa tcctactaat 360

gataataata ttctctttaa agatgattcc aaatatcaaa atcaactggt tacgtatcaa 420

aatattctga caaatttgta cgctctgccg ccttgtgatg acactcagct cttgattgat 480

aaaacgaagt cgcagttgaa taacctgatt aacagttgga atcccgaaat aaactacccc 540

aagctttcca gtttctctcc tcgcccacaa agatcgatag aaacgtatct tttaaccaac 600

aagtatagaa ataaaataca catgacgagg ttctcctttt ggacagacca aatggttaaa 660

tcacaaagtc cagattcatt tctagccacc actccactag tagatgaagt atttggtctt 720

ttctctccaa tacaggcttt ttcactaaga ggtataggat atttaattaa aaaaaatatc 780

gaaaacacgg gttcatcgat gttaatagat acaaaggaaa ctatttatct aatattaaga 840

ttgtttgatt tgtgttatga acatttgatc caaggttgca tctctatttc taatccatta 900

gagaactatc ttcaaaaaat aaagcaaact cctactacga cggcatctgc tagtttgcct 960

acttccccag cacctttatc taacgattta gtcatttctg ttattcatca actacctcag 1020

ccatttatac aatcgattac cgggtttacg actactcaat tgatagaaaa tttacatgat 1080

tcattttcga tgtttcgaat agttactcaa atgtatgctc aacataggaa gcgctttgcg 1140

gaatttttaa accaagcttt ctccttgccc catcaagaaa agagtgtttt attctcgtca 1200

ttctgctcat cagaatatct tctatctact ctttgttacg catactacaa tgttacccta 1260

tatcacatgt tggacataaa cactttagat tacctagaga ttttagtgtc attgctagaa 1320

atccaaaatg aaattgatga gcgttttgga tttgaaaaaa tgctagaagt tgcggttaca 1380

tgctccacta agatgggatt gtctcgttgg gagtattatg ttggaataga cgaaaatact 1440

gccgaacgga gaagaaaaat atggtggaaa atatacagtc tggaaaagcg ttttttaact 1500

gatcttggtg atttatcctt aataaatgaa catcaaatga attgtctctt gccgaaggat 1560

ttcagggaca tgggattcat taaccataaa gaatttttaa cgaaaattgg tacgtcctct 1620

ttatcaccgt catcgcccaa gctaaaaaac ttgtcattgt ccaggcttat tgaatatggt 1680

gagttagcga tagcccaaat tgttggagat tttttttcag agactcttta taatgagaaa 1740

ttcacgtctt tagaagtatc cgttaaaccc acaattatca gacaaaagtt attggagaaa 1800

gtttttgagg acattgaatc ttttaggtta aaattggcca aaataaagct tcacacctca 1860

agagtttttc aagtagctca ctgcaaatat ccagaatatc caaaaaacga tctaattgaa 1920

gcagctaaat ttgtaagtta ccataaaaat acatggttct ccatcttggg tgctgttaac 1980

aatcttattg ctaggctatc tgaagatcca gaggtgataa ctgagcaaag catgaaatat 2040

gcgaatgaaa tgtttcaaga atggagggaa attaatcaat tcttaataca ggttgatact 2100

gattttattg tttgggcatg tttggacttt tatgaactga tatttttcgt gatggcttca 2160

aaattttatg tggaagaccc gcacatcact ttagaggatg ttatcaacac tttgaaagtt 2220

tttaagagaa taactaacat tatttctttt tttaataata atttggacga gaaggattat 2280

gattgtcaaa ctttcaggga gttttcgaga agttcgagtt tggttgccat atccataaga 2340

atcatatttt taaaatactg ctatgccgaa caaattgata gagccgaatt catcgaacgt 2400

ttgaaagaag ttgaaccggg tctaagtgac cttttgcgtg agttttttga tacccgctct 2460

tttatttaca ggtacatgtt gaaatccgtt gaaaaatcag gctttcattt aataattaga 2520

aaaatgttag aaagcgacta taaatttttg tatagagaca aattggccac tggtaatatt 2580

ccagaccaag gaaattcaag ccaaatttct cagttgtatg acagtactgc tccttcatac 2640

aacaatgctt ctgcctcagc agcaaactca ccgttgaagt tatcgtcttt gttgaactct 2700

ggagaggaat cgtacactca agacgcatca gaaaatgttc catgtaatct gcggcatcaa 2760

gatcgatcgt tacaacagac aaaaagacaa cattctgcgc ctagccaaat aagcgctaat 2820

gagaataata tatacaactt gggtacttta gaggagtttg tcagcagtgg tgacctgact 2880

gatttatatc atactctgtg gaatgacaat acttcatatc ccttcttatg a 2931

<210> 3

<211> 942

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

atggttgaac aaaagggttc tgaagctgct gcttcttctc cagacgtttt gagagcttgg 60

gctactcaat accacatgcc atctgaatct tctgacgctg ctcgtcccgc tctcaagcac 120

gcttacaagc caccagcttc tgacgctaag ggtatcacta tggctttgac tatcatcggt 180

acttggactg ctgttttctt gcacgctatc ttccaaatca gattgccaac ttctatggac 240

caattgcatt ggttaccagt aagcgaagct actgctcaat tgttgggtgg ttcttcttct 300

ttgttgcaca tcgctgctgt tttcatcgtt ttggaatttt tgtacactgg tttgttcatc 360

actactcacg acgctatgca cggcactatc gctctcagac acagacagtt gaacgacttg 420

ttgggtaaca tctgtatctc tttgtacgct tggttcgact actctatgtt gcgcagaaag 480

cactgggaac accacaacca cactggtgaa gttggtaagg acccagactt ccacaagggt 540

aacccaggtt tggttccatg gttcgcttct ttcatgagtt cgtacatgag cttgtggcaa 600

ttcgcgagat tggcttggtg ggctgttgtt atgcaaatgt tgggtgctcc aatggctaac 660

ttgttggttt tcatggctgc tgctccaatc ttgtctgctt tcagattgtt ctacttcggt 720

acttacttgc cacacaagcc agaaccaggt ccagctgctg gttctcaaga tatggcttgg 780

ttcagagcta agacttctga agctagtgac gttatgagct tcttgacttg ttaccacttc 840

gacttgcact gggaacacca cagatggcca tacgctccat ggtggcaatt gccacactgt 900

agaagattgt ctggtagagg tttggttcca gctttggctt aa 942

Claims

1. A genetically engineered bacterium for efficiently producing carotenoid, wherein the carotenoid is beta-carotene, canthaxanthin or astaxanthin, is characterized in that the genetically engineered bacterium takes an engineering strain producing beta-carotene, canthaxanthin or astaxanthin as a starting bacterium, and a multidirectional drug-resistant transcription factor coding gene is integrated and overexpressed on a chromosomePDR3；

Or the genetically engineered bacterium takes an engineering strain for producing beta-carotene, cantharidin yellow or astaxanthin as a starting bacterium, and the host bacterium contains a multidirectional drug-resistant transcription factor coding genePDR3Recombinant expression plasmids of (a);

wherein, the gene for encoding the multidirectional drug-resistant transcription factorPDR3The nucleotide sequence of (2) is shown as SEQ ID NO. 2.

2. The genetically engineered strain for efficiently producing carotenoids according to claim 1, wherein the cantharidin yellow-producing engineered strain is a strain which starts from a beta-carotene-producing engineered strain and has a beta-carotene ketolase encoding gene integrated on its chromosome; alternatively, the host bacterium contains a recombinant expression plasmid containing a gene encoding a beta-carotene ketolase.

3. The genetically engineered bacterium for efficiently producing carotenoids according to claim 2, wherein the nucleotide sequence of the gene encoding β -carotene ketolase is shown in SEQ ID No. 3.

4. The genetically engineered strain for efficient production of carotenoids according to claim 2, wherein the engineering strain for producing β -carotene is engineering strain Ycarot-02; the engineering strain for producing astaxanthin adopts engineering strain YPP-17.

5. The method for constructing genetically engineered bacterium capable of producing carotenoids efficiently according to any one of claims 1 to 4Characterized by comprising: the engineering strain producing beta-carotene, cantharidin yellow or astaxanthin is used as starting strain, and the gene encoding the multidirectional drug-resistant transcription factor is integrated with plasmidPDR3Integrating the strain into the chromosome of engineering strain to obtain the genetically engineered strain for efficiently producing beta-carotene, cantharidin yellow or astaxanthin;

or taking engineering strain producing beta-carotene, cantharidin yellow or astaxanthin as starting strain, introducing gene containing multidirectional drug-resistant transcription factor codingPDR3Recombinant expression plasmids of (a);

wherein ,PDR3the nucleotide sequence of (2) is shown as SEQ ID NO. 2.

6. The construction method according to claim 5, comprising the steps of:

(1) Multidirectional drug-resistant transcription factor coding gene with nucleotide sequence shown as SEQ ID NO.2PDR3Cloning into pUMRI-YPL062W _GAL1 The later multiple cloning site to obtain recombinant plasmid pUMRI-YPL062W-PDR3;

(2) The recombinant plasmid pUMRI-YPL062W-PDR3 is transformed into engineering strain producing beta-carotene, cantharidin yellow or astaxanthin, and the recombinant strain integrating the gene encoding the multi-directional drug-resistant transcription factor in chromosome is obtained by screening, thus the genetically engineered strain for efficiently producing carotenoid is obtained.

7. The construction method according to claim 5, wherein the construction method of cantharidin yellow producing engineering strain comprises:

8. The use of the genetically engineered bacterium for the efficient production of carotenoids according to claims 1-4 for the preparation of β -carotene, canthaxanthin or astaxanthin.

9. The use according to claim 8, comprising: after the genetically engineered bacteria for efficiently producing carotenoid are subjected to amplification culture, inoculating the genetically engineered bacteria into YPD liquid culture medium, and performing shake culture to obtain fermentation liquor; and collecting thalli in the fermentation liquor, and extracting corresponding beta-carotene, cantharidin yellow or astaxanthin after cell disruption.