CN113265344A - Genetic engineering bacterium for selectively producing retinol and construction method and application thereof - Google Patents

Abstract

The invention discloses a genetic engineering bacterium for selectively producing retinol, and a construction method and application thereof, belonging to the field of genetic engineering. The genetic engineering bacteria take an engineering strain for producing the beta-carotene as a starting bacteria, and introduce a beta-carotene 15, 15' -dioxygenase coding gene and an aldehyde reductase coding gene or/and a retinol dehydrogenase coding gene to realize the selective production of the retinol by a cell factory. In order to further improve the output of retinol, the genetic engineering bacteria also introduce a geranylgeranyl pyrophosphate synthase mutant coding gene and a coding gene of NADH kinase for cutting off mitochondrial positioning peptide at the N end, and simultaneously knock out a mevalonate pathway transcription inhibitor gene, thereby increasing the supply of precursor and coenzyme. The invention also provides a method for producing retinol by using the genetic engineering bacteria, and ferrous ions and antioxidants are added in the fermentation process, so that the yield of retinol is further improved.

Description

Genetic engineering bacterium for selectively producing retinol and construction method and application thereof

Technical Field

The invention relates to the fields of genetic engineering, metabolic engineering and microorganisms, in particular to a construction method and application of a genetic engineering bacterium capable of realizing selective production of retinol.

Background

Vitamin A (VA) generally refers to a substance with Retinol-like biological activity, generally consisting of 20 carbon structures with 4 consecutive isoprenoid unit side chains and one β -ionone ring, and is classified into Retinol (hydroxyl), retinal (aldehyde), retinoic acid (carboxyl) and retinyl ester (acyl) according to the difference of the 15-carbon-bonded groups. Due to the presence of a special conjugated double bond system, vitamin a is susceptible to degradation by oxidation and isomerization after exposure to light, oxygen and transition metal ions.

Vitamin A, as an essential vitamin of human body, plays an important role in maintaining visual function, participating in immune system, regulating cell proliferation and differentiation, stabilizing epithelial cell morphological function and other physiological activities.

Most of the commercial vitamin A on the market is chemically synthesized. At present, the traditional chemical synthesis method of vitamin A mainly comprises two processes of Roche and BASF. A common problem with both VA chemical synthesis routes is that the selectivity to the all-trans form with the highest VA titer is not high. The final product of the Roche route synthesis is all-trans isomer and 13-cis isomer with higher titer, and the cis isomer accounts for 80 percent. The BASF route has about 28% of 11-cis form in the backbone stage of the synthetic molecule, while the all-trans form is only 70%, and the cis-isomer from the C5 aldehyde ester in the subsequent witting reaction will produce 13-cis form, so that finally additional optical isomerisation is necessary to obtain the all-trans form.

The heterologous synthesis production of vitamin A by using microorganisms is an efficient and promising method. At present, the Vitamin A synthesized by Escherichia coli (Selective regeneration by modulating the composition of a Vitamin from microbial Engineered E.coli, Biotechnol Bioeng,2015) and Saccharomyces cerevisiae (Vitamin A Production by Engineered Saccharomyces cerevisiae via Two-Phase in Situ Extraction, ACS Synth Biol,2019) are used as hosts, the proportion of retinol accounts for 88% of the total yield at most, the yield is only 76.56mg/L, and the application value of the Vitamin A in the form of alcohol in the cosmetic field is much higher than that in the form of aldehyde.

Therefore, how to efficiently prepare vitamin A in an alcohol form by using microbial heterologous synthesis is a problem to be solved by those skilled in the art.

Disclosure of Invention

The invention aims to provide a genetic engineering bacterium capable of selectively producing retinol, and vitamin A in an alcohol form is efficiently prepared by a microbial heterologous synthesis mode.

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

the invention provides a genetic engineering bacterium for selectively producing Retinol, which takes an engineering strain for producing beta-carotene as a starting bacterium, and integrates a beta-carotene 15, 15 '-dioxygenase coding gene (beta-carotene 15, 15' -dioxygenase, BLH) and an Aldehyde reductase (Aldehyde reductase) coding gene or/and a Retinol dehydrogenase (Retinol dehydrogenase) coding gene on a chromosome;

or the genetic engineering bacteria take an engineering strain for producing the beta-carotene as a starting bacteria, and the host bacteria contain recombinant expression plasmids containing beta-carotene 15, 15' -dioxygenase coding genes and aldehyde reductase coding genes or/and retinol dehydrogenase coding genes.

The engineering strain for producing the beta-carotene by the spawn running is a strain with the function of producing the beta-carotene known in the field, wherein the condensation of geranylgeranyl pyrophosphate, the desaturation and isomerization of phytoene and the lycopene cyclization link are necessary ways for producing the retinol. On the basis, the invention utilizes a gene integration technology or a mode of introducing recombinant expression plasmids to ensure that beta-carotene 15, 15 ' -dioxygenase (BLH) and aldehyde reductase (YBBO), or beta-carotene 15, 15 ' -dioxygenase (BLH) and retinol dehydrogenase (ENV9), or beta-carotene 15, 15 ' -dioxygenase (BLH), aldehyde reductase (YBBO) and retinol dehydrogenase (ENV9) are successfully expressed in an engineering strain for producing beta-carotene and participate in the synthesis of retinol, thereby realizing the selective production of retinol by taking safe and efficient engineering bacteria as cell factories.

Preferably, the starting strain is an engineered strain Ycarot-02 producing beta-carotene, a publicly available material, and its construction method is described in the literature (Allation of metabolic bottenk by combinatorial engineering in Saccharomyces cerevisiae, Enzyme Microb Technol, 2017).

The invention obtains the coding gene segment of the enzyme which can be successfully expressed in the saccharomyces cerevisiae through gene cloning and codon optimization.

Furthermore, the nucleotide sequence of the beta-carotene 15, 15' -dioxygenase coding gene is shown as SEQ ID NO. 1. The present invention is codon optimized and synthesizes corresponding sequences according to the preference of the host cell. The nucleotide sequence of the aldehyde reductase coding gene is shown in SEQ ID NO.2, and the gene is derived from escherichia coli. The nucleotide sequence of the retinol dehydrogenase coding gene is shown in SEQ ID NO.3, and the gene is cloned from a saccharomyces cerevisiae genome for producing beta-carotene.

Furthermore, the genetically engineered bacterium also introduces a coding gene of geranylgeranyl pyrophosphate synthase mutant (CrtE03M) and a coding gene of NADH kinase (tPOS5) for cutting off N-terminal mitochondrial positioning peptide, and simultaneously, the mevalonate pathway transcription inhibitor (MOT3) gene is knocked out.

In order to further improve the yield of the retinol, the invention introduces geranylgeranyl pyrophosphate synthase mutant coding gene and truncated NADH kinase coding gene into saccharomyces cerevisiae, knocks out mevalonate pathway transcription inhibitor gene, and knocks out mevalonate pathway transcription inhibitor and over-expresses truncated NADH kinase to respectively increase the precursor and coenzyme (NADPH) required by retinol synthesis by over-expressing geranylgeranyl pyrophosphate synthase mutant, knocking out mevalonate pathway transcription inhibitor and over-expressing truncated NADH kinase, wherein the truncated NADH kinase is used for cutting off mitochondrion localization peptide at the N terminal thereof on the basis of NADH kinase so as to ensure that the truncated NADH kinase can play a role in cytoplasm.

Further, the nucleotide sequence of the geranylgeranyl pyrophosphate synthase mutant coding gene is shown as SEQ ID NO. 4; the nucleotide sequence of the encoding gene of the NADH kinase for cutting off the mitochondrial localization peptide at the N end is shown as SEQ ID NO. 5;

the CrtE03M gene and the POS5 gene are cloned from a saccharomyces cerevisiae genome for producing beta-carotene, primers are redesigned on the basis of the POS5 gene, and N-end mitochondrial localization peptide is deleted to obtain the coding gene of tPOS 5.

The nucleotide sequence of the mevalonate pathway transcription repressing factor gene is shown in SEQ ID NO. 8.

The invention also provides a method for constructing the genetic engineering bacteria for selectively producing the retinol, and the construction method comprises the following steps: taking an engineering strain for producing beta-carotene as a starting strain, and respectively integrating a beta-carotene 15, 15' -dioxygenase coding gene, an aldehyde reductase coding gene or/and a retinol dehydrogenase coding gene onto a chromosome of the engineering strain for producing the beta-carotene through integrating plasmids to obtain the genetic engineering strain for selectively producing the retinol;

or by taking an engineering strain for producing the beta-carotene as a host bacterium, and introducing a recombinant expression plasmid containing a beta-carotene 15, 15' -dioxygenase coding gene and an aldehyde reductase coding gene or/and a retinol dehydrogenase coding gene.

The invention introduces the coding genes of beta-carotene 15, 15' -dioxygenase (BLH) and endogenous retinol dehydrogenase (ENV9) or/and exogenous aldehyde reductase (YBBO) into the cells of an engineering strain for producing beta-carotene, so that the corresponding enzymes are successfully expressed in the engineering strain for producing beta-carotene, and the genetic engineering bacteria for selectively producing retinol are constructed.

Further, a geranylgeranyl pyrophosphate synthase mutant coding gene and a truncated NADH kinase coding gene are introduced into the engineering bacteria by using a gene integration or introduction recombinant expression plasmid mode; and knocking out mevalonate pathway transcription inhibitor genes by using a homologous recombination technology.

Specifically, the invention provides a construction method of a recombinant bacterium which integrates BLH, YBBO, ENV9, CrtE03M and tPOS5 and knocks out MOT3, wherein the construction method comprises the following steps:

(1) cloning a gene coding beta-carotene 15, 15' -dioxygenase with a nucleotide sequence shown as SEQ ID NO.1 to P of pUMRI-LPP1_GAL1The subsequent multiple cloning sites to obtain recombinant plasmid pUMRI-LPP 1-BLH;

(2) cloning a geranylgeranyl pyrophosphate synthase mutant coding gene with a nucleotide sequence shown as SEQ ID NO.4 and an aldehyde reductase coding gene with a nucleotide sequence shown as SEQ ID NO.2 to P of pUMRI-DPP1 respectively_GAL2And P_GAL7Obtaining a recombinant plasmid pUMRI-DPP1-CrtE03M-YBBO at the later multiple cloning site;

(3) cloning the upstream and downstream homologous arms of mevalonate pathway transcription inhibitor gene into pUMRI21 plasmid to construct pUMRI-MOT3, and cloning the coding gene of NADH kinase with nucleotide sequence shown as SEQ ID NO.5 and for cutting mitochondrial localization peptide at N end and the coding gene of retinol dehydrogenase with nucleotide sequence shown as SEQ ID NO.3 into P of pUMRI-MOT3_GAL10And P_GAL1Obtaining a recombinant plasmid pUMRI-MOT3-tPOS5-ENV9 from the later multiple cloning sites;

(4) the recombinant plasmids pUMRI-LPP1-BLH, pUMRI-DPP1-CrtE03M-YBBO and pUMRI-MOT3-tPOS5-ENV9 are sequentially transformed into an engineering strain Ycarot-02 for producing beta-carotene, and a recombinant bacterium which integrates a beta-carotene 15, 15' -dioxygenase coding gene, an aldehyde reductase coding gene, a retinol dehydrogenase coding gene, a geranylgeranyl pyrophosphate synthase mutant coding gene and a coding gene of NADH kinase for cutting off N-terminal mitochondrial positioning peptide in a chromosome and is knocked out a mevalonate pathway transcription inhibitor gene is the genetic engineering bacterium for selectively producing the retinol.

The plasmids pUMRI-LPP1, pUMRI-DPP1 and pUMRI21 are all publicly available materials. With P_GAL1、P_GAL10、P_GAL2、P_GAL7As a promoter, by pUMRIAnd (3) assembling a tool plasmid in series, and gradually integrating the gene segments into the chromosome of the host bacterium.

The invention also provides application of the genetic engineering bacteria for selectively producing the retinol in preparation of the retinol. Retinol is prepared from the fermentation culture of the genetically engineered strain constructed in the present invention.

The invention also provides a preparation method of the retinol, which comprises the following steps:

1) after the genetic engineering bacteria for selectively producing the retinol are subjected to expanded culture, the genetic engineering bacteria are inoculated into a fermentation medium containing ferrous ions, an extracting agent and an antioxidant are added, and shaking culture is performed to obtain fermentation liquor;

2) collecting the organic phase in the fermentation liquid, and separating to obtain the retinol.

In the step 1), the activated genetically engineered bacteria are subjected to amplification culture, the strains are inoculated in a fermentation culture medium, an extracting agent is added, two-phase fermentation culture is carried out, and the product retinol is dissolved in an organic phase extracting agent.

Ferrous ions are added into a fermentation medium to improve the activity of the beta-carotene 15, 15' -dioxygenase, which is beneficial to the growth of bacterial strains and the synthesis of retinol. Preferably, the concentration of ferrous ions in the YPD liquid medium is 1.26-1.80 mM. More preferably, the concentration of ferrous ions in the YPD liquid medium is 1.44 mM.

Preferably, the extractant is dodecane, and 2.5-10.0 mL of the extractant is added in every 50mL of culture solution; the antioxidant is dibutylhydroxytoluene (BHT), and 0.25-1.0 g of the antioxidant is added to 50mL of the culture solution.

The invention solves the problems of easy oxidation and easy isomerization of the retinol by adding BHT as an antioxidant in the culture process, and realizes the high yield of the retinol.

Preferably, the conditions for the two-phase fermentation are: culturing for 72-84 hours in a constant temperature shaking table at the speed of 200-250 rpm and the temperature of 28-30 ℃ in a dark place.

In step 2), high-yield and high-purity retinol is directly obtained from the upper organic phase through two-phase fermentation culture.

The invention has the following beneficial effects:

(1) the invention constructs a genetic engineering bacterium capable of realizing selective synthesis of retinol, takes an engineering strain for producing beta-carotene as a starting bacterium, introduces a beta-carotene 15, 15' -dioxygenase coding gene and an aldehyde reductase coding gene or a retinol dehydrogenase coding gene into the engineering bacterium for producing beta-carotene, and successfully expresses corresponding enzymes in the strain to participate in the synthesis of retinol.

In order to further improve the yield of the retinol, a coding gene of a geranylgeranyl pyrophosphate synthase mutant and a coding gene of NADH kinase for cutting off mitochondrial positioning peptide at the N end are introduced into an engineering strain for producing the beta-carotene, a mevalonate pathway transcription inhibitor gene is knocked out, and the yield of the retinol is improved by increasing the supply of precursors and coenzymes.

(2) The invention provides a method for realizing high retinol yield, which improves the activity of beta-carotene 15, 15' -dioxygenase by adding ferrous ions in the two-phase fermentation process taking dodecane as an extracting agent, and inhibits the oxidation of retinol by adding an antioxidant, so that the retinol yield is further improved. The yield of the synthesized retinol after the two-phase fermentation culture of the engineering strain constructed by the invention in the shake flask is superior to the report in the prior art, and the invention has good application prospect.

Drawings

FIG. 1 shows the backbone (A) and the construction result (B) required for the construction of pUMRI-MOT3 plasmid.

FIG. 2 shows integration sites involved in the construction of selective high-producing strains of retinol, (A) the construction of s.cerevisiae producing beta-carotene by the starting strain, and (B) the construction of s.cerevisiae Y03-43 producing retinol according to the present invention.

FIG. 3 shows the cofactor Fe required for the reaction of the BLH enzyme²⁺Is optimized.

FIG. 4 is a HPLC detection spectrum of a product of a selective retinol-producing strain Y03-43, wherein the upper, middle and lower liquid phase peak curves are respectively set in the experimental group, the retinal standard group and the retinol standard group.

FIG. 5 shows the ratio of retinol to retinal in the synthetic products of the retinol selective highly productive strain Y03-43 and other related strains. Y03 is a strain integrating a gene encoding beta-carotene 15, 15' -dioxygenase (BLH) in a beta-carotene-producing starting strain, Y03-33 is a strain integrating only a gene encoding aldehyde reductase (YBBO) in a Y03 strain, Y03-34 is a strain overexpressing only retinol dehydrogenase (ENV9) in a Y03 strain, Y03-251 is a strain overexpressing truncated NADH kinase (tPOS5) in a Y03 strain, Y03-252 is a strain overexpressing truncated NADH kinase (tPOS5) and a geranylgeranyl pyrophosphate synthase mutant (CrtE03M) in a Y03 strain, and Y03-42 is a strain overexpressing NADH kinase (tPOS5) and a geranylgeranyl pyrophosphate synthase mutant (CrtE03M) and flavonol dehydrogenase (ENV9) in a Y03 strain.

FIG. 6 shows the effect of different strains on retinol synthesis after addition of antioxidant BHT.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto. Unless otherwise specified, the experimental procedures used in the examples are those conventional in the art, and the starting materials and reagents are commercially available.

The engineering strain Ycarot-02 for producing beta-carotene is constructed in the early stage of the subject group, and the construction method is disclosed in the literature reference (allergy of metabolic flask by combinatorial engineering in Saccharomyces cerevisiae, Enzyme Microb Technol, 2017).

Example 1 cloning of genes required for retinol biosynthesis

1. Extraction of Escherichia coli and Saccharomyces cerevisiae genome DNA

1.1 extraction of Escherichia coli BL21 genome DNA is completed by a kit, and the specific steps are as follows:

(1) 1mL of Escherichia coli liquid cultured overnight was taken, added to a 1.5mL centrifuge tube, centrifuged at 8000rpm at room temperature for 1min, and the supernatant was discarded to collect the cells. Adding 180 mu L of lysozyme solution to resuspend the bacterial solution, and carrying out water bath at 37 ℃ for 30-60 min. Then 20. mu.L of protease K solution is added, and the mixture is shaken and mixed evenly. The cells were completely lysed by a water bath at 56 ℃ for 30 min.

(2) And in the water bath process, reversing and uniformly mixing the mixture every 10min until the mixture becomes clear and transparent, adding 20 mu L of RNase, standing the mixture at room temperature for 2-5 min, and removing RNA.

(3) Add 200. mu.L of Buffer BD and mix well by inversion.

(4) Add 200. mu.L of absolute ethanol and mix well by inversion.

(5) Putting the adsorption column into a collection tube, sucking all the solution and suspended matters into the adsorption column by using a pipette, standing for 2min, centrifuging at 12000rpm at room temperature for 1min, and pouring off the waste liquid in the collection tube.

(6) The adsorption column was returned to the collection tube, 500. mu.L of PW Solution was added, centrifuged at 10000rpm for 30s, and the filtrate was discarded.

(7) The adsorption column was returned to the collection tube, 500. mu.L of Wash Solution was added, centrifuged at 10000rpm for 30s, and the filtrate was discarded.

(8) And (4) idling and leaving residual Wash Solution.

(9) The adsorption column was placed in a new 1.5mL centrifuge tube, 75. mu.L of CE Buffer was added and left to stand for 3min, centrifuged at 12000rpm for 2min at room temperature, and the resulting DNA solution was collected and stored at-20 ℃.

1.2 the extraction of the genome DNA of saccharomyces cerevisiae (engineering strain Ycarot-02 for producing beta-carotene) is completed by a kit, and the method comprises the following steps:

(1) 1-3mL of yeast culture solution cultured for 20-24h was centrifuged at 12000rpm at room temperature for 5 min.

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

(3) Centrifuging at 12000rpm for 5min at room temperature, discarding the supernatant, adding 200 μ L Buffer YL and 50mg glass beads (0.4-0.6mm), vortex shaking for 3-5min, standing, and sucking the supernatant into a new 1.5mL centrifuge tube.

(4) Adding 25 μ L of protease K, mixing well, and incubating with shaking at 65 deg.C for 30 min.

(5) Add 5. mu.L RNase A and repeat the tube upside down and incubate 10min at room temperature.

(6) Add 220. mu.L Buffer YDL and 220. mu.L pure ethanol and vortex for 20 s.

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

(8) The adsorption column was returned to the collection tube, 500. mu.L of Buffer HB was added, centrifugation was carried out at 10000rpm for 30s, and the filtrate was discarded.

(9) The adsorption column was returned to the collection tube, 700. mu.L of DNA Wash Buffer was added, centrifuged at 10000rpm for 30s, and the filtrate was discarded.

(10) The above step was repeated, and the remaining DNA Wash Buffer was removed by idling.

(11) The adsorption column was placed in a new 1.5mL centrifuge tube, 75. mu.L of precipitation Buffer was added, and the mixture was allowed to stand at 65 ℃ for 3-5min, centrifuged at 12000rpm for 1min at room temperature, and the resulting DNA solution was collected and stored at-20 ℃.

2. The protein sequence of beta-carotene 15, 15' -dioxygenase (BLH) is found at NCBI, and a corresponding sequence is synthesized by using Saccharomyces cerevisiae as a host through codon optimization by a gene synthesis company, wherein the nucleotide sequence is shown as SEQ ID NO. 1.

3. The aldehyde reductase (YBBO) uses an escherichia coli genome as a template, retinol dehydrogenase (ENV9), NADH kinase (POS5) and geranylgeranyl pyrophosphate synthase mutant (CrtE03M) use a saccharomyces cerevisiae genome as a template, and high fidelity enzyme (Prime STARTM HS DNA polymerase) is adopted for PCR amplification.

Truncated NADH kinase (tPOS5) redesigns primer based on NADH kinase (POS5), and deletes N-terminal mitochondrion localization peptide.

The nucleotide sequence of the aldehyde reductase (YBBO) gene is shown as SEQ ID NO. 2; the nucleotide sequence of the retinol dehydrogenase (ENV9) gene is shown in SEQ ID NO. 3; the nucleotide sequence of the encoding gene of the geranylgeranyl pyrophosphate synthase mutant (CrtE03M) is shown as SEQ ID NO. 4; the nucleotide sequence of the truncated NADH kinase (tPOS5) gene is shown as SEQ ID NO.5, the coded amino acid sequence is shown as SEQ ID NO.6, and the nucleotide sequence of the NADH kinase (POS5) gene is shown as SEQ ID NO. 7.

Primer design is as follows:

TABLE 1 cloning of primers for genes required for retinol biosynthesis

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

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

Example 2 construction of pUMRI-MOT3 plasmid

The upstream and downstream homology arms of a transcription inhibitor (MOT3) used for constructing the plasmid pUMRI-MOT3 take a yeast genome as a template, a skeleton part used for the plasmid pUMRI-MOT3 takes a plasmid pUMRI21 as a template, high fidelity enzyme Prime STARTM HS DNA polymerase is adopted for PCR amplification, and the three fragments are connected by a Gibson Assembly kit. The construction is shown in figure 1. The nucleotide sequence of the mevalonate pathway transcription repressing factor gene is shown in SEQ ID NO. 8.

The specific primer design is as follows:

TABLE 2 primers used for construction of pUMRI-MOT3 plasmid

Example 3 construction of plasmids required for the retinol biosynthetic pathway

1. Enzyme digestion and gel recovery

The pUMRI series integration plasmids (pUMRI-LPP1 and pUMRI-DPP1 are constructed and preserved in the laboratory, and pUMRI-MOT3 is constructed in example 2) and the target fragment of the PCR product are subjected to double enzyme digestion by Takara restriction enzyme, the double enzyme digestion system is subjected to DNA gel recovery treatment after enzyme digestion according to the instruction of the Takara restriction enzyme, and the specific steps are carried out according to the instruction of an Axygen kit.

2. Enzyme linked to

The digested fragments and plasmids were ligated using T4 DNA ligase in the following ligation scheme (10. mu.l):

ligation was carried out at 22 ℃ for 30 min.

3. Transformation of

Adding 10 mu L of the ligation product into an escherichia coli competent solution, standing on ice for 15min, then performing heat shock at 42 ℃ for 90s, quickly placing in an ice bath for 3min, adding 1mL of LB liquid culture medium, recovering under a shaking table at 37 ℃ for 45min, then centrifuging, discarding part of supernatant, taking a proper amount of supernatant, coating the supernatant on a corresponding resistant LB flat plate, and standing in an incubator at 37 ℃ for 15 h.

Specifically, the enzyme digestion ligation is performed in the following manner:

cloning of a fragment of interest of beta-carotene 15, 15' -dioxygenase (BLH) into P of pUMRI-LPP1_GAL1The subsequent multiple cloning sites to obtain recombinant plasmid pUMRI-LPP 1-BLH;

p cloning of aldehyde reductase (YBBO) target fragment to pUMRI-DPP1_GAL7Obtaining a recombinant plasmid pUMRI-DPP1-YBBO at the later multiple cloning sites;

cloning of a fragment of retinol dehydrogenase (ENV9) of interest into P of pUMRI-DPP1_GAL7At the latter multiple cloning site, a recombinant plasmid pUMRI-DPP1-ENV9 was obtained.

P cloning of truncated NADH kinase (tPOS5) target fragment into pUMRI-MOT3_GAL10Obtaining a recombinant plasmid pUMRI-MOT3-tPOS5 from the later multiple cloning sites;

cloning of target fragment of geranylgeranyl pyrophosphate synthase mutant (CrtE03M) into P of pUMRI-DPP1_GAL2Obtaining a recombinant plasmid pUMRI-DPP1-CrtE03M at the later multiple cloning site;

cloning of target fragment of geranylgeranyl pyrophosphate synthase mutant (CrtE03M) into P of pUMRI-DPP1_GAL2The latter multiple cloning site, the P for cloning of the fragment of interest for aldehyde reductase (YBBO) into pUMRI-DPP1_GAL7The subsequent multiple cloning site to obtain recombinant plasmid pUMRI-DPP1-CrtE03M-YBBO；

P cloning of truncated NADH kinase (tPOS5) target fragment into pUMRI-MOT3_GAL10The latter multiple cloning site, the fragment of interest of retinol dehydrogenase (ENV9), was cloned P of pUMRI-MOT3_GAL1At the latter multiple cloning site, the recombinant plasmid pUMRI-MOT3-tPOS5-ENV9 was obtained.

Example 4 construction of Selective high yield Saccharomyces cerevisiae with retinol

The recombinant plasmid pUMRI-LPP1-BLH prepared in example 3 was transformed into the β -carotene-producing engineered strain Ycarot-02 so that BLH was integrated into the chromosome of the β -carotene-producing engineered strain to obtain Y03.

Transforming the recombinant plasmid pUMRI-DPP1-YBBO into Y03, so that the YBBO is integrated into Y03 chromosome to obtain Y03-33;

transforming the recombinant plasmid pUMRI-DPP1-ENV9 into Y03, so that ENV9 is integrated into Y03 chromosome, and obtaining Y03-34;

transforming the recombinant plasmid pUMRI-MOT3-tPOS5 into Y03, integrating tPOS5 into a Y03 chromosome, and knocking out MOT3 to obtain Y03-251;

the recombinant plasmid pUMRI-DPP1-CrtE03M was transformed into Y03-251 so that CrtE03M was integrated into Y03-251 chromosome, resulting in Y03-252.

The recombinant plasmids pUMRI-DPP1-CrtE03M and pUMRI-MOT3-tPOS5-ENV9 are sequentially transformed into Y03, so that CrtE03M, tPOS5 and ENV9 are integrated into a Y03 chromosome, and simultaneously, MOT3 is knocked out to obtain Y03-42.

The recombinant plasmids pUMRI-DPP1-CrtE03M-YBBO and pUMRI-MOT3-tPOS5-ENV9 prepared in example 3 were transformed into Y03 in sequence, CrtE03M, YBBO, tPOS5 and ENV9 were integrated into Y03, and at the same time, MOT3 was knocked out to obtain Y03-43, as shown in FIG. 2 (B).

The specific transformation method is as follows:

1. preparing saccharomyces cerevisiae competence: selecting single colony of beta-carotene producing yeast strain from YPD plate, inoculating to 5mL YPD test tube, culturing at 30 deg.C and 220rpm overnight, transferring to 50mL YPD shake flask according to 2% inoculum size, culturing at 30 deg.C and 220rpm for 4-5 hr, and culturing at OD₆₀₀Reaching about 2.0.

2. Converting saccharomyces cerevisiae:

(1) the ssDNA was heated in a metal bath at 100 ℃ for 5min and then rapidly cooled in ice for further use.

(2) 45mL of competent yeast liquid was put into a 50mL sterilized centrifuge tube, centrifuged at 4000rpm and 20 ℃ for 5min, and the supernatant was discarded.

(3) Washing with 20mL of sterile water, centrifuging to remove the supernatant, resuspending with 1mL of sterile water, packaging into 1.5mL sterile centrifuge tubes according to 100 μ L per tube, and centrifuging to remove the supernatant.

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

(5) mixing, and standing in metal bath at 42 deg.C for 40 min.

(6) Centrifuging at 12000rpm for 30s, discarding the supernatant, and adding 1mL YPD liquid culture medium to resuscitate for 2 h.

(7) Centrifuging at 12000rpm for 1min, discarding the supernatant, adding 1ml of sterile water for resuspension, and taking 100ul of the suspension to be spread on a corresponding resistant plate.

Example 5 optimization of ferrous ion concentration

A single colony of the engineered bacterium Y03-43 was picked from the streaked plate and inoculated into 5mL of YPD medium, and after the liquid in the test tube became turbid, the strain was cultured in 50mL of YPD liquid medium, and ferrous sulfate ions were added to the medium at final concentrations of 0.00mM, 0.18mM, 0.72mM, 1.08mM, 1.26mM, 1.44mM, and 1.80mM, and the mixture was cultured in a shaking table at 30 ℃ for 84 hours in the dark at 220 rpm.

As shown in FIG. 3, the growth of the engineered bacteria was optimal under 1.44mM condition, and total retinoid synthesis capacity was highest.

Example 6 two-phase fermentation culture of genetically engineered bacteria and extraction and analysis of products

1. A single colony of the engineered bacteria was picked from the streaked plate and inoculated into 5mL YPD medium, and after the tube became turbid, the strain was cultured in 50mL YPD liquid medium supplemented with 1.44mM ferrous ion. Two-phase fermentation was carried out with 2.5mL of dodecane supplemented with 1% (w/v) of dibutylhydroxytoluene (BHT) as an extractant, and the mixture was incubated in a constant temperature shaker at 220rpm and 30 ℃ for 84 hours in the absence of light.

2. Collecting yeast fermentation liquid into a centrifuge tube, centrifuging at 4000rpm for 5min, taking an organic phase layer, transferring into a brown 1.5mL centrifuge tube, diluting with acetone by a certain multiple, filtering with a 0.22 μm organic filter head, and detecting by HPLC. The product peaks correspond to the standard retinol and retinal, and the yield is determined after making a standard curve.

3. The conditions for detecting retinol in saccharomyces cerevisiae by HPLC were as follows:

the liquid phase analyzer is Shimadzu LC-20AT, the chromatographic column is YMC-Pack ODS-AQ C18-H column, the column temperature is 40 deg.C, the detection wavelength is 352nm, 95% methanol and 5% acetonitrile are used as mobile phase, and the flow rate is 0.6 mL/min.

As shown in FIGS. 4 and 5, the retinol content in the product of Y03-33 was 106.24mg/L, the retinal content was 3.69mg/L, the retinol content in the product of Y03-34 was 83.63mg/L, the retinal content was 46.51mg/L, the retinol content in the product of Y03-43 was 122.03mg/L, and the retinal content was 9.51 mg/L. As shown in FIG. 6, Y03-43 produced 122.03mg/L of retinol without BHT and 401.65mg/L of retinol with BHT, which is 99.86% of total vitamin A.

Sequence listing

<110> Zhejiang university

<120> genetic engineering bacterium for selectively producing retinol, and construction method and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 828

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgggtttaa tgttaattga ttggtgtgct ttagctttag ttgtttttat tggtttacct 60

catggtgctt tagatgctgc aatttcattt tctatgattt cttcagctaa aaggattgca 120

aggttagcag gtattttgtt gatttatttg ttgttagcaa ctgcattttt tttaatttgg 180

tatcaattac cagcattttc tttgttgatt tttttgttaa tttcaattat tcattttggt 240

atggcagatt ttaatgcatc accatctaaa ttgaaatggc ctcatattat tgctcatggt 300

ggtgttgtta cagtttggtt gccattaatt caaaaaaatg aagttactaa attgttttca 360

attttgacta atggtcctac tcctattttg tgggatattt tgttgatttt ttttttgtgt 420

tggtctattg gtgtttgttt acatacttat gaaactttga gatcaaaaca ttataatatt 480

gcatttgaat taattggttt gattttttta gcatggtatg cacctccatt agttacattt 540

gctacatatt tttgttttat tcattctagg aggcattttt catttgtttg gaaacaatta 600

caacacatgt catctaaaaa aatgatgatt ggttctgcta ttattttatc atgtacttct 660

tggttgattg gtggtggtat ttattttttt ctcaattcta aaatgattgc atcagaagct 720

gctttacaaa cagtttttat tggtttagca gctttgacag ttccacacat gattttaatt 780

gattttattt ttagaccaca ttcttcaagg attaaaatta aaaattaa 828

<210> 2

<211> 810

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 2

atgactcata aagcaacgga gatcctgaca ggtaaagtta tgcaaaaatc ggtcttaatt 60

accggatgtt ccagtggaat tggcctggaa agcgcgctcg aattaaaacg ccagggtttt 120

catgtgctgg caggttgccg gaaaccggat gatgttgagc gcatgaacag catgggattt 180

accggcgtgt tgatcgatct ggattcacca gaaagtgttg atcgcgcagc cgacgaggtg 240

atcgccctga ccgataattg tctgtatggg atctttaaca atgccggatt cggcatgtat 300

ggcccccttt ccaccatcag ccgtgcgcag atggaacagc agttttccgc caactttttc 360

ggcgcacacc agctcaccat gcgcctgtta cccgcgatgt taccgcacgg tgaagggcgt 420

attgtgatga catcatcggt gatgggatta atctccacgc cgggtcgtgg cgcttacgcg 480

gccagtaaat atgcgctgga ggcgtggtca gatgcactgc gcatggagct gcgccacagc 540

ggaattaaag tcagcctgat cgaacccggt cccattcgta ctcgcttcac cgacaacgtc 600

aaccagacgc aaagtgataa accagtcgaa aatcccggca tcgccgcccg ctttacgttg 660

ggaccggaag cggtggtgga caaagtacgc catgctttta ttagcgagaa gccgaagatg 720

cgctatccgg tgacgctggt gacctgggcg gtaatggtgc ttaagcgcct gctgccgggg 780

cgcgtgatgg acaaaatatt gcaggggtga 810

<210> 3

<211> 993

<212> DNA

<213> Saccharomyces cerevisiae (S. cerevisiae)

<400> 3

atgttagacc cacgaatatt gccatactac gacccggctg tggagaggaa gattgctgta 60

gtaacaggcg gaaatacggg tattgggtgg tatactgtct tgcatttgta tttgcatggg 120

tttgtcgttt atatttgtgg gagaaactct cacaagattt cgaaagcaat ccaggagata 180

ctggcagaag caaagaagag gtgccatgaa gatgacgacg gttcaagccc aggcgcgggc 240

ccaggtccaa gcattcagcg tctagggtca ctgcactata tccatttgga tttgacagac 300

ttaaaatgtg tggagagagc ggcacttaaa attctcaagc tggaagacca catagacgtg 360

cttgttaaca atgcagggat tatggcggtg cccttagaaa tgacgaagga cggatttgaa 420

gtgcaattgc agactaacta catttcgcac ttcatcttca cgatgagatt attgccttta 480

ctgcgccatt gtcgtggcag gatcatttcc ctgtcctcga taggccatca tctagagttc 540

atgtactgga aactgagcaa gacgtgggat tacaaaccta atatgctttt cacatggttt 600

aggtacgcga tgagtaaaac cgcgctaatc caatgcacga agatgttggc catcaaatac 660

cctgacgttc tttgtctctc cgttcatccg ggtctggtga tgaacacaaa cttattcagt 720

tattggacaa ggttacccat cgtcggtatt ttcttttggc tgttgttcca ggtcgtaggg 780

ttctttttcg gcgtatcgaa cgaacaaggt tcactagctt ctttgaagtg tgcattggac 840

ccgaatttat ctgtcgaaaa agataacggg aagtacttca ccacgggggg taaagaatct 900

aaatcgagct acgtttctaa caatgtcgac gaagcggcat cgacttggat ctggaccgtt 960

catcaactaa gagaccgtgg tttcgatata taa 993

<210> 4

<211> 1131

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atggattacg cgaacatcct cacagcaatt ccactcgagt ttactcctca ggatgatatc 60

gtgctccttg aaccgtatca ttacctagga aagaaccctg gaaaagaaat tcgatcacaa 120

ctcatcgagg ctttcaacta ttggttggat gtcaagaagg aggatctcga ggtcatccag 180

aacgttgttg gcatgctaca taccgctagc ttattaatgg acgatgtgga ggattcatcg 240

gtcctcaggc gtgggtcgcc tgtggcccat ctaatttacg ggattccgca gacaataaac 300

actgcaaact acgtctactt tctggcttat caagagatct tcaagcttcg cccaacaccg 360

atacccatgc ctgtaattcc tccttcatct gcttcgcttc aatcatccgt ctcctctgca 420

tcctcctcct cctcggcctc gtctgaaaac gggggcacgt caactcctaa ttcgcagatt 480

ccgttctcga aagatacgta tcttgataaa gtgatcacag acgagatgct ttccctccat 540

agagggcaag gcctggagct attctggaga gatagtctga cgtgtcctag cgaagaggaa 600

tatgtgaaaa tggttcttgg aaagacggga ggtttgttcc gtatagcggt cagattgatg 660

atggcaaagt cagaatgtga catagacttt gtccagcttg tcaacttgat ctcaatatac 720

ttccagatca gggatgacta tatgaacctt cagtcttctg agtatgccca taataagaat 780

tttgcagagg acctcacaga aggaaaattc agttttccca ctatccactc gattcatgcc 840

aacccctcat cgagactcgt catcaatacg ttgcagaaga aatcgacctc tcctgagatc 900

cttcaccgct gtgtaaacta catgcgcaca gaaacccact cattcgaata tactcaggaa 960

gtcctcaaca ccttgtcagg tgcactcgag agagaactag gaaggcttca aggagagttc 1020

gcagaagcta actcaaagat tgatcttgga gacgtagagt cggaaggaag aacggggaag 1080

aacgtcaaat tggaagcgat cctgaaaaag ctagccgata tccctctgtg a 1131

<210> 5

<211> 1197

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgagtacgt tggattcaca ttccctaaag ttacagagcg gctcgaagtt tgtaaaaata 60

aagccagtaa ataacttgag gagtagttca tcagcagatt tcgtgtcccc accaaattcc 120

aaattacaat ctttaatctg gcagaaccct ttacaaaatg tttatataac taaaaaacca 180

tggactccat ccacaagaga agcgatggtt gaattcataa ctcatttaca tgagtcatac 240

cccgaggtga acgtcattgt tcaacccgat gtggcagaag aaatttccca ggatttcaaa 300

tctcctttgg agaatgatcc caaccgacct catatacttt atactggtcc tgaacaagat 360

atcgtaaaca gaacagactt attggtgaca ttgggaggtg atgggactat tttacacggc 420

gtatcaatgt tcggaaatac gcaagttcct ccggttttag catttgctct gggcactctg 480

ggctttctat caccgtttga ttttaaggag cataaaaagg tctttcagga agtaatcagc 540

tctagagcca aatgtttgca tagaacacgg ctagaatgtc atttgaaaaa aaaggatagc 600

aactcatcta ttgtgaccca tgctatgaat gacatattct tacatagggg taattcccct 660

catctcacta acctggacat tttcattgat ggggaatttt tgacaagaac gacagcagat 720

ggtgttgcat tggccactcc aacgggttcc acagcatatt cattatcagc aggtggatct 780

attgtttccc cattagtccc tgctatttta atgacaccaa tttgtcctcg ctctttgtca 840

ttccgaccac tgattttgcc tcattcatcc cacattagga taaagatagg ttccaaattg 900

aaccaaaaac cagtcaacag tgtggtaaaa ctttctgttg atggtattcc tcaacaggat 960

ttagatgttg gtgatgaaat ttatgttata aatgaggtcg gcactatata catagatggt 1020

actcagcttc cgacgacaag aaaaactgaa aatgacttta ataattcaaa aaagcctaaa 1080

aggtcaggga tttattgtgt cgccaagacc gagaatgact ggattagagg aatcaatgaa 1140

cttttaggat tcaattctag ctttaggctg accaagagac agactgataa tgattaa 1197

<210> 6

<211> 398

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Ser Thr Leu Asp Ser His Ser Leu Lys Leu Gln Ser Gly Ser Lys

1 5 10 15

Phe Val Lys Ile Lys Pro Val Asn Asn Leu Arg Ser Ser Ser Ser Ala

20 25 30

Asp Phe Val Ser Pro Pro Asn Ser Lys Leu Gln Ser Leu Ile Trp Gln

35 40 45

Asn Pro Leu Gln Asn Val Tyr Ile Thr Lys Lys Pro Trp Thr Pro Ser

50 55 60

Thr Arg Glu Ala Met Val Glu Phe Ile Thr His Leu His Glu Ser Tyr

65 70 75 80

Pro Glu Val Asn Val Ile Val Gln Pro Asp Val Ala Glu Glu Ile Ser

85 90 95

Gln Asp Phe Lys Ser Pro Leu Glu Asn Asp Pro Asn Arg Pro His Ile

100 105 110

Leu Tyr Thr Gly Pro Glu Gln Asp Ile Val Asn Arg Thr Asp Leu Leu

115 120 125

Val Thr Leu Gly Gly Asp Gly Thr Ile Leu His Gly Val Ser Met Phe

130 135 140

Gly Asn Thr Gln Val Pro Pro Val Leu Ala Phe Ala Leu Gly Thr Leu

145 150 155 160

Gly Phe Leu Ser Pro Phe Asp Phe Lys Glu His Lys Lys Val Phe Gln

165 170 175

Glu Val Ile Ser Ser Arg Ala Lys Cys Leu His Arg Thr Arg Leu Glu

180 185 190

Cys His Leu Lys Lys Lys Asp Ser Asn Ser Ser Ile Val Thr His Ala

195 200 205

Met Asn Asp Ile Phe Leu His Arg Gly Asn Ser Pro His Leu Thr Asn

210 215 220

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

225 230 235 240

Gly Val Ala Leu Ala Thr Pro Thr Gly Ser Thr Ala Tyr Ser Leu Ser

245 250 255

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

260 265 270

Pro Ile Cys Pro Arg Ser Leu Ser Phe Arg Pro Leu Ile Leu Pro His

275 280 285

Ser Ser His Ile Arg Ile Lys Ile Gly Ser Lys Leu Asn Gln Lys Pro

290 295 300

Val Asn Ser Val Val Lys Leu Ser Val Asp Gly Ile Pro Gln Gln Asp

305 310 315 320

Leu Asp Val Gly Asp Glu Ile Tyr Val Ile Asn Glu Val Gly Thr Ile

325 330 335

Tyr Ile Asp Gly Thr Gln Leu Pro Thr Thr Arg Lys Thr Glu Asn Asp

340 345 350

Phe Asn Asn Ser Lys Lys Pro Lys Arg Ser Gly Ile Tyr Cys Val Ala

355 360 365

Lys Thr Glu Asn Asp Trp Ile Arg Gly Ile Asn Glu Leu Leu Gly Phe

370 375 380

Asn Ser Ser Phe Arg Leu Thr Lys Arg Gln Thr Asp Asn Asp

385 390 395

<210> 7

<211> 1245

<212> DNA

<213> Saccharomyces cerevisiae (S. cerevisiae)

<400> 7

atgtttgtca gggttaaatt gaataaacca gtaaaatggt ataggttcta tagtacgttg 60

gattcacatt ccctaaagtt acagagcggc tcgaagtttg taaaaataaa gccagtaaat 120

aacttgagga gtagttcatc agcagatttc gtgtccccac caaattccaa attacaatct 180

ttaatctggc agaacccttt acaaaatgtt tatataacta aaaaaccatg gactccatcc 240

acaagagaag cgatggttga attcataact catttacatg agtcataccc cgaggtgaac 300

gtcattgttc aacccgatgt ggcagaagaa atttcccagg atttcaaatc tcctttggag 360

aatgatccca accgacctca tatactttat actggtcctg aacaagatat cgtaaacaga 420

acagacttat tggtgacatt gggaggtgat gggactattt tacacggcgt atcaatgttc 480

ggaaatacgc aagttcctcc ggttttagca tttgctctgg gcactctggg ctttctatca 540

ccgtttgatt ttaaggagca taaaaaggtc tttcaggaag taatcagctc tagagccaaa 600

tgtttgcata gaacacggct agaatgtcat ttgaaaaaaa aggatagcaa ctcatctatt 660

gtgacccatg ctatgaatga catattctta cataggggta attcccctca tctcactaac 720

ctggacattt tcattgatgg ggaatttttg acaagaacga cagcagatgg tgttgcattg 780

gccactccaa cgggttccac agcatattca ttatcagcag gtggatctat tgtttcccca 840

ttagtccctg ctattttaat gacaccaatt tgtcctcgct ctttgtcatt ccgaccactg 900

attttgcctc attcatccca cattaggata aagataggtt ccaaattgaa ccaaaaacca 960

gtcaacagtg tggtaaaact ttctgttgat ggtattcctc aacaggattt agatgttggt 1020

gatgaaattt atgttataaa tgaggtcggc actatataca tagatggtac tcagcttccg 1080

acgacaagaa aaactgaaaa tgactttaat aattcaaaaa agcctaaaag gtcagggatt 1140

tattgtgtcg ccaagaccga gaatgactgg attagaggaa tcaatgaact tttaggattc 1200

aattctagct ttaggctgac caagagacag actgataatg attaa 1245

<210> 8

<211> 1486

<212> DNA

<213> Saccharomyces cerevisiae (S. cerevisiae)

<400> 8

atgaatgcgg accatcacct gcaacagcag cagcaacagc gacaacagca tcaacaacaa 60

cagcatcaac aacaacagca tcagcatcag catcaacagc agcagcacac gatattacaa 120

aatgtgtcga acactaacaa tatcggcagc gattcgctgg cgtcacagcc tttcaacacg 180

actactgttt cctctaacaa ggacgacgtt atggtgaact ctggggcaag agaacttcca 240

atgcccttac atcagcagca gtatatatac ccttactatc agtatacaag taataacagt 300

aacaacaata atgtgacggc tggtaacaat atgtctgcgt cgccgattgt ccataacaac 360

agcaacaaca gcaacaacag caatatttct gcttctgatt acactgtcgc aaacaacagt 420

actagcaata ataacaataa taataataat aacaacaata ataacaataa tattcaccca 480

aaccagttta ctgcggccgc aaatatgaac tcaaatgctg cagcggctgc ttattactcc 540

ttccccactg cgaatatgcc aataccgcaa caggatcaac aatatatgtt caatcctgct 600

tcatacataa gccattacta ttcagcagtt aacagcaata acaatggtaa taacgccgct 660

aacaatggca gcaacaactc ttctcactca gccccagccc cggcccccgg tccaccccat 720

caccggccat aaaggccatc accatcatag taatacacac aacaacctca acaatggtgg 780

tgctgtaaat acaaacaacg ctcctcagca ccatccaacg ataataacgg atcaatttca 840

attccaacta caacaaaacc cttctccaaa tttgaatctc aatattaacc cggcacaacc 900

tctgcatcta cctcctggtt ggaaaataaa cactatgccg caaccacgtc ctacgacagc 960

acctaaccat ccccctgcgc cggtgccttc ttcgaaccct gtggcctcga acttggttcc 1020

tgccccatca tcagaccata aatatatcca tcaatgccaa ttttgtgaga agtctttcaa 1080

aagaaaatca tggttgaaaa ggcacctatt gtcacactcg caacaaagac attttctatg 1140

cccttggtgc ttaagcaggc agaagagaaa agataatctt ttacagcata tgaaactcaa 1200

gcatacaaat tatttattag acgaactcaa gaaaaacaac atcatcttta actacaacaa 1260

ttcttcctcc tctaataata acaacgacaa taataataat aataacagca atagcgctag 1320

cggcagtggc ggtgccggtg ccgcggcggc agcagcaaca gctcccgaaa atgaagatgg 1380

aaacggttac gatacaaaca tcaagacttt aatcaatgat ggtgtactga ataaggacga 1440

cgttaaacgt gttttgaata accttattgt tagtcacaac aaatag 1486

Claims (10)

1. A genetic engineering bacterium for selectively producing retinol is characterized in that the genetic engineering bacterium takes an engineering strain for producing beta-carotene as a starting bacterium, and a beta-carotene 15, 15' -dioxygenase coding gene and an aldehyde reductase coding gene or/and a retinol dehydrogenase coding gene are integrated on a chromosome of the genetic engineering bacterium;

or the genetic engineering bacteria take an engineering strain for producing the beta-carotene as a starting bacteria, and the host bacteria contain recombinant expression plasmids containing beta-carotene 15, 15' -dioxygenase coding genes and aldehyde reductase coding genes or/and retinol dehydrogenase coding genes.

2. The genetically engineered bacterium that selectively produces retinol according to claim 1, wherein the nucleotide sequence of the gene encoding β -carotene 15, 15' -dioxygenase is represented by SEQ ID No. 1; the nucleotide sequence of the aldehyde reductase coding gene is shown as SEQ ID NO. 2; the nucleotide sequence of the retinol dehydrogenase encoding gene is shown in SEQ ID NO. 3.

3. The genetically engineered bacterium of claim 1 or 2, wherein a geranylgeranyl pyrophosphate synthase mutant coding gene, a gene coding for NADH kinase that cleaves the N-terminal mitochondrial localization peptide, and a mevalonate pathway transcription repressing factor gene are deleted are introduced.

4. The genetically engineered bacterium that selectively produces retinol according to claim 3, wherein the nucleotide sequence of the coding gene of the geranylgeranyl pyrophosphate synthase mutant is shown as SEQ ID No. 4; the nucleotide sequence of the encoding gene of the NADH kinase for cutting off the mitochondrial localization peptide at the N end is shown as SEQ ID NO. 5; the nucleotide sequence of the mevalonate pathway transcription repressing factor gene is shown in SEQ ID NO. 8.

5. The method for constructing genetically engineered bacteria for selective production of retinol according to claim 1, comprising: taking an engineering strain for producing beta-carotene as a starting strain, and respectively integrating a beta-carotene 15, 15' -dioxygenase coding gene, an aldehyde reductase coding gene or/and a retinol dehydrogenase coding gene onto a chromosome of the engineering strain for producing the beta-carotene through integrating plasmids to obtain the genetic engineering strain for selectively producing the retinol;

or by taking an engineering strain for producing the beta-carotene as a host bacterium, and introducing a recombinant expression plasmid containing a beta-carotene 15, 15' -dioxygenase coding gene and an aldehyde reductase coding gene or/and a retinol dehydrogenase coding gene.

6. The method for constructing genetically engineered bacteria for selective production of retinol as claimed in claim 5, comprising the steps of:

(1) cloning a gene coding beta-carotene 15, 15' -dioxygenase with a nucleotide sequence shown as SEQ ID NO.1 to P of pUMRI-LPP1_GAL1The subsequent multiple cloning sites to obtain recombinant plasmid pUMRI-LPP 1-BLH;

(2) geranylgeranyl char with nucleotide sequence shown as SEQ ID NO.4The coding gene of the mutant of the phosphate synthase and the coding gene of aldehyde reductase with the nucleotide sequence shown as SEQ ID NO.2 are respectively cloned to P of pUMRI-DPP1_GAL2And P_GAL7Obtaining a recombinant plasmid pUMRI-DPP1-CrtE03M-YBBO at the later multiple cloning site;

(3) cloning the upstream and downstream homologous arms of mevalonate pathway transcription inhibitor gene into pUMRI21 plasmid to construct pUMRI-MOT3, and cloning the coding gene of NADH kinase with nucleotide sequence shown as SEQ ID NO.5 and for cutting mitochondrial localization peptide at N end and the coding gene of retinol dehydrogenase with nucleotide sequence shown as SEQ ID NO.3 into P of pUMRI-MOT3_GAL10And P_GAL1Obtaining a recombinant plasmid pUMRI-MOT3-tPOS5-ENV9 from the later multiple cloning sites;

(4) sequentially mixing the recombinant plasmids pUMRI-LPP1-BLH,

Converting pUMRI-DPP1-CrtE03M-YBBO, pUMRI-MOT3-tPOS5-ENV9 into an engineering strain Ycarot-02 for producing beta-carotene, screening to obtain a recombinant bacterium which integrates a beta-carotene 15, 15' -dioxygenase coding gene, an aldehyde reductase coding gene, a retinol dehydrogenase coding gene, a geranylgeranyl pyrophosphate synthase mutant coding gene and an NADH kinase coding gene for cutting off mitochondrial localization peptides at the N end in a chromosome, and knocking out a mevalonate pathway transcription inhibitor gene, namely the genetic engineering bacterium for selectively producing the retinol.

7. Use of the genetically engineered bacterium of any one of claims 1 to 4 for the selective production of retinol in the preparation of retinol.

8. A preparation method of retinol is characterized by comprising the following steps:

1) carrying out amplification culture on the genetically engineered bacterium for selectively producing retinol according to any one of claims 1-4, inoculating the genetically engineered bacterium into a fermentation medium containing ferrous ions, adding an extracting agent and an antioxidant, and carrying out shake culture to obtain a fermentation broth;

2) collecting the organic phase in the fermentation liquid, and separating to obtain the retinol.

9. The process according to claim 8, wherein the concentration of ferrous ions in the fermentation medium is 1.26 to 1.80 mM; the extractant is dodecane, and 2.5-10.0 mL of the extractant is added into every 50mL of culture solution; the antioxidant is dibutyl hydroxy toluene, and 0.25-1.0 g of the antioxidant is added into each 50mL of culture solution.

10. The process for the preparation of retinol as in claim 8, wherein the two-phase fermentation conditions are: culturing for 72-84 hours in a constant temperature shaking table at the speed of 200-250 rpm and the temperature of 28-30 ℃ in a dark place.

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