WO2018233531A1 - 微生物及其用途 - Google Patents

微生物及其用途 Download PDF

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WO2018233531A1
WO2018233531A1 PCT/CN2018/091113 CN2018091113W WO2018233531A1 WO 2018233531 A1 WO2018233531 A1 WO 2018233531A1 CN 2018091113 W CN2018091113 W CN 2018091113W WO 2018233531 A1 WO2018233531 A1 WO 2018233531A1
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lycopene
fermentation
medium
microorganism
gene
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French (fr)
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叶紫玲
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武汉臻智生物科技有限公司
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
    • C12P5/026—Unsaturated compounds, i.e. alkenes, alkynes or allenes
    • C—CHEMISTRY; METALLURGY
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • the present invention relates to the field of bioengineering.
  • the present invention relates to microorganisms and uses thereof. More specifically, the present invention relates to microorganisms, methods for obtaining lycopene, and uses of microorganisms for preparing lycopene.
  • lycopene mainly includes natural product extraction, chemical synthesis, and microbial fermentation.
  • the extraction of natural products is mainly through the extraction and purification of mature fruits to obtain lycopene.
  • this production method is affected by many uncontrollable factors such as climate, variety, geographical location, maturity, etc., with obvious seasonality and content instability, and The cost of large-scale planting and breeding is relatively high, and the content is usually low.
  • high-purity lycopene is very difficult in extraction and purification technology, which together make the finished product of lycopene very expensive.
  • the chemical synthesis method has the advantages that the raw materials are easy to obtain inexpensive, the reaction conditions are mild, the reaction rate is fast, and the product is easily separated from the reaction system, but the quality and safety of the product are difficult to control due to the double-selection stereoselectivity and the chemical residues of different degrees. Sex and scope of use are limited.
  • the microbial fermentation method mainly uses the biological metabolism of microorganisms to convert cheap raw materials such as glucose, starch and soybean cake powder into lycopene. This method is not affected by factors such as season, region and climate, and the raw materials are easy to obtain and the production cycle is short.
  • the process is simple, the cost is low, the product quality is controllable, the product is easy to purify, the safety is high, and the environmental pollution is less, which not only solves the problem of occupying a large amount of cultivated land due to planting plants, but also solves the disadvantages of chemical synthesis not being environmentally friendly. .
  • the lycopene produced by fermentation is a natural product whose activity is consistent with that of natural plant extracts and is considered to be the most promising method for lycopene production.
  • the present invention aims to solve at least one of the technical problems in the related art to some extent.
  • B. trispora is prone to degeneration during the passage, which leads to a decrease in lycopene production, accompanied by the synthesis of toxic components such as aflatoxin.
  • the growth cycle of these two microorganisms is comparable to that of yeast. It is longer with E. coli, which greatly reduces its production efficiency, which makes it more difficult to produce lycopene.
  • an object of the present invention is to provide a microorganism which produces high yield of lycopene, has a short production cycle, and has high production efficiency.
  • the invention proposes a microorganism.
  • the microorganism overexpresses the PaCrtE, PagCrtB and BtCrtI genes.
  • the production of lycopene is remarkably improved, the production cycle is short, and the production efficiency is high.
  • the microorganism may further include at least one of the following additional technical features:
  • the microorganism further overexpresses comprises selected from the group consisting of tHMG1, INO2, yap1, spt15-5, taf25-3, GapN, PYC2, SMAE1, MDH2, POS5, pntAB, ADH2, ACS6, ALD6, EUTE, At least one of ERG12, IDI1, ERG10, MVD1, ERG13, ERG8 genes.
  • the inventors have found that the microorganism further overexpresses at least one of the above genes, and the yield of lycopene is further increased.
  • the microorganism further comprises at least a gene selected from the group consisting of GAL1, GAL7, GAL10, GAL80, ROX1, VBA5, DOS2, Ypl062W, Yjl064W, Yer130C, Yer134C, Ynr063W, Exg1, Yor292C, Sfk1, Mef1 genes.
  • a gene selected from the group consisting of GAL1, GAL7, GAL10, GAL80, ROX1, VBA5, DOS2, Ypl062W, Yjl064W, Yer130C, Yer134C, Ynr063W, Exg1, Yor292C, Sfk1, Mef1 genes.
  • the microorganism is a yeast.
  • the growth cycle of the yeast is short, and the efficiency of producing lycopene by the microorganism of the embodiment of the present invention is further improved.
  • the microorganism further comprises an operably regulated ERG9 gene.
  • the operably regulated ERG9 gene includes, but is not limited to, a promoter that replaces the ERG9 gene, so that the expression of the ERG9 gene can be regulated as needed, so that the efficiency of the microorganism to produce lycopene is further improved.
  • the invention proposes a method of obtaining lycopene.
  • the method comprises: subjecting the microorganism described above to a fermentation treatment; and subjecting the fermentation treatment product to an extraction treatment to obtain the lycopene.
  • lycopene can be obtained with high yield and high efficiency, and the purity of lycopene is high.
  • the above method for obtaining lycopene may further include at least one of the following additional technical features:
  • the lycopene obtained by the method for obtaining lycopene according to an embodiment of the present invention has higher yield, higher efficiency, and higher purity.
  • the fermentation treatment is carried out by subjecting the microorganism to a basic fermentation treatment and a two-stage fed-batch fermentation treatment, which is carried out in a basic fermentation medium.
  • the two-stage fed-batch fermentation treatment is carried out by sequentially adding a first feed medium and a second feed medium on the basis of the basic fermentation medium, wherein the basic fermentation medium is Containing YPD medium containing 24 g/L peptone, 12 g/L yeast extract and 12 g/L glucose; the first feed medium is YPD medium containing 500 g/L glucose and 15 g/L yeast extract;
  • the second feed medium is 75% ethanol or 50% glycerol.
  • the extracting treatment comprises: performing the ultrasonication treatment and the organic extraction treatment on the fermentation treatment product.
  • the inventors found that the fermentation product was subjected to ultrasonication treatment, and the degradation of lycopene was greatly reduced compared with the method of cooking with hydrochloric acid, and the yield of lycopene obtained after the extraction treatment was further increased. improve.
  • the invention provides the use of the aforementioned microorganisms for the preparation of lycopene.
  • the production of lycopene is high, the production cycle is short, and the production efficiency is high.
  • FIG. 1 is a schematic structural view of a knockout box segment 1 according to Embodiment 1 of the present invention.
  • FIG. 2 is a schematic structural view of a knockout box segment 2 according to Embodiment 1 of the present invention.
  • FIG. 3 is a schematic structural diagram of a knockout box segment 3 according to Embodiment 1 of the present invention.
  • Figure 4 is a shake flask fermentation result of the second generation engineering strain
  • FIG. 5 is a block diagram showing the structure of a knockout box segment 4 according to Embodiment 4 of the present invention.
  • Figure 6 is a graph showing the results of screening of a fermentation medium according to an embodiment of the present invention.
  • the invention proposes a microorganism.
  • the microorganism overexpresses the PaCrtE, PagCrtB and BtCrtI genes.
  • the production of lycopene is high, the production cycle is short, and the production efficiency is high.
  • the microorganism may further be overexpressed and comprises selected from the group consisting of tHMG1, INO2, yap1, spt15-5, taf25-3, GapN, PYC2, SMAE1, MDH2, POS5, pntAB, ADH2, ACS6, ALD6 At least one of the EUTE, ERG12, IDI1, ERG10, MVD1, ERG13, ERG8 genes.
  • the microorganism may further be silenced to comprise a substrate selected from the group consisting of GAL1, GAL7, GAL10, GAL80, ROX1, VBA5, DOS2, Ypl062W, Yjl064W, Yer130C, Yer134C, Ynr063W, Exg1, Yor292C, Sfk1, At least one of the Mef1 genes.
  • the inventors have found that the microorganism further silences at least one of the above genes, and the yield of lycopene is further increased.
  • the inventors have found that the microorganism further overexpresses or silences at least one of the above genes, and the yield of lycopene is further significantly increased.
  • microorganisms described herein are used to produce lycopene, which has a significant increase in yield compared to the prior art and is capable of at least 2.5 g/L.
  • the microorganism may further comprise an operably regulated ERG9 gene.
  • the operably regulated ERG9 gene described herein refers to the replacement of the original promoter of ERG, thereby realizing the regulation of ERG9 expression by glucose, thereby increasing the production of lycopene.
  • Galactose induces or regulates the production of lycopene by glucose concentration; overexpression of INO2, yap1, spt15-5, taf25-3 gene is a stress-resistant gene, which can increase the yield of lycopene; overexpression of GapN, PYC2, SMAE1, One or more genes in MDH2, POS5, and pntAB can balance the reducing power in yeast and increase the yield of lycopene; overexpression of one or more genes in ADH2, ALD6, EUTE can increase the synthesis of lycopene The supply of body material increases the yield of lycopene; overexpression of one or more genes in ERG12, IDI1, ERG10, MVD1, ERG13, ERG8 can balance the MVA pathway in lycopene synthesis and increase lycopene Yield; silencing at least one of ROX1, VBA5, DOS2, Ypl062W, Yjl064W, Yer130C, Y
  • the invention proposes a method of obtaining lycopene.
  • the method comprises: subjecting the microorganism described above to a fermentation treatment; and subjecting the fermentation treatment product to an extraction treatment to obtain the lycopene.
  • lycopene can be obtained with high yield and high efficiency, and the purity of lycopene is high.
  • the fermentation treatment is carried out by a basic fermentation treatment and a two-stage fed-batch fermentation process, as follows:
  • the fermenter was placed in a small amount of pure water and then emptied at 115 degrees for 30 minutes.
  • the batch cultured fermentation broth and the feed liquid were prepared, the pH electrode of the fermenter was calibrated, and the respective tubes of the fermenter were clamped and sterilized at 115 degrees for 30 minutes.
  • Take out the fermenter connect the air line, perform a small amount of ventilation (about 0.1vvm), and turn on the cooling water device for cooling. When the temperature drops to about 50 degrees, the speed is turned on to 100 rpm. Calibrate the dissolved oxygen electrode.
  • the state of the unconnected line is set to 0% of the dissolved oxygen value, and then the rotational speed is raised to 600 rpm, and the ventilation is adjusted to 2 vvm, and the dissolved oxygen electrode line is connected, and the value is set to 100% after the dissolved oxygen is stabilized.
  • the aeration and speed were then reduced to the desired value for fermentation (ventilation 1.5 vvm, speed 300 rpm - 600 rpm).
  • the inoculation was started, the initial inoculation OD was adjusted to 0.5, and the required seed solution volume was calculated according to the formula.
  • the volume of the fermentation broth is 2500 mL and the OD value of the seed solution is n, the volume of the inoculum seed solution is 2500 x 0.5 / n mL).
  • the medium was controlled with a pH of 5.5 with 2 M NaOH, the initial aeration was 1.5 vvm, the initial agitation rate was set to 300 rpm, and the dissolved oxygen was maintained at 30% or more (associated with agitation rate, 300-600 rpm).
  • the feed glucose is started, and the initial feed rate is 10mL/L fermentation broth/h to maintain the residual concentration of glucose in the fermentation broth at 2-3g/L.
  • the OD600 value is measured once every two hours and the glucose content is measured once, and the feed rate is increased when the glucose concentration is below 1 g/L.
  • the feed glucose is stopped. At this time, the ethanol residue is monitored.
  • the ethanol concentration drops to 5-10 g/L, the ethanol glycerol is fed, and the initial feed rate is 2 mL/L. Fermentation liquid / h.
  • the ethanol and glycerol contents were then sampled every 4 hours, and the feed rate was adjusted to maintain ethanol and glycerol in the range of 5-10 g/L. After the color has changed, the product is detected to change the product, and the fermentation is terminated when the lycopene concentration is no longer increased.
  • the extracting treatment comprises: subjecting the fermented product to ultrasonication treatment and organic extraction treatment.
  • the inventors optimized the extraction process after the fermentation treatment. The inventors found that the degradation rate of lycopene was greatly reduced and the yield of lycopene was stabilized by the method of ultrasonic treatment compared with the method of cooking with hydrochloric acid.
  • organic extraction treatment refers to extracting the ultrasonication treatment product by using an organic solvent
  • the manner of organic extraction is not particularly limited.
  • the acetone solvent extracts lycopene from the sonicated product.
  • the invention provides the use of the microorganisms described above for the preparation of lycopene.
  • the inventors have confirmed through experiments that the microorganisms described in the present application have significant advantages in the production of lycopene, high yield of lycopene, low degradation rate of lycopene, and short production cycle.
  • the inventors detailed the construction process of the J1011-C-3 strain.
  • ⁇ LEU2:pGAL1-PaCrtE(Pantoea ananatis) knockout cassette ie, knock out the cassette fragment 1.
  • Recombination method recombines all fragments together and obtains ⁇ LEU2::pGAL1-PaCrtE(Pantoea ananatis) knockout cassette by restriction enzyme linearization, such as knockout cassette fragment 1 as shown in Figure 1.
  • Knockout cassette fragment 1 has SEQ. ID NO: The nucleotide sequence shown in 1.
  • the fragment was integrated into the yeast 30000B genome by the yeast homologous recombination mechanism, and the integration site was LEU2. Since LEU2 was not active in the original yeast, the integrated homologous left arm contained LEU2.
  • the complete gene was screened by SD-Leu solid plate (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, leucine-free mixed amino acid powder 1.3g/L, 2% agar powder). The transformants were purified by PCR after extracting the yeast genome, and the successfully verified strain was named J1011-C-1.
  • ⁇ URA3 pGAL1-PagCrtB (Pantoea agglomerans); pGAL10-BtCrtI (Blakeslea trispora) knock-out cassette, knock-out cassette fragment 2, which has the nucleotide sequence shown as SEQ ID NO: 2, was constructed.
  • the upstream and downstream primers were designed to amplify the fragments by 60-80 bp overlapping fragments, and then all the fragments were recombined by homologous recombination.
  • the ⁇ URA3::pGAL1-PagCrtB was obtained by enzyme linearization.
  • pGAL10-BtCrtI (Blakeslea trispora) knockout cassette, knockout cassette fragment 2 is shown in Figure 2.
  • the fragment was integrated into the yeast J1011-C-1 genome by the yeast homologous recombination mechanism, and the integration site was URA3.
  • the SD-HIS solid plate was used (synthetic yeast nitrogen source YNB6. 7g/L, glucose 20g/L, mixed amino acid powder lacking histidine 1.3g/L, 2% agar powder) were screened, and the obtained transformants were purified by PCR, and the yeast genome was extracted for PCR verification. Named J1011-C-2.
  • the ⁇ gal1, ⁇ gal7, ⁇ gal10:pGAL10-tHMG1 knockout cassette, ie knockout cassette fragment 3 was constructed, and the knockout cassette fragment 3 has the nucleotide sequence shown as SEQ ID NO: 3.
  • the upstream and downstream primers were designed to amplify the fragments by 60-80 bp overlapping fragments, and all the fragments were recombined by homologous recombination.
  • the ⁇ gal1, ⁇ gal7, ⁇ gal10 were obtained by linearization of the enzyme: : pGAL10-tHMG1 knockout fragment, the structure of knockout cassette fragment 3 is shown in Fig. 3.
  • the fragment was integrated into the yeast J1011-C-2 genome by the yeast homologous recombination mechanism, and the integration sites were GAL1, GAL7 and GAL10.
  • SD-Trp solid plate synthetic yeast
  • Nitrogen source YNB 6.7g / L, glucose 20g / L, mixed amino acid powder lacking tryptophan 1.3g / L, 2% agar powder) were screened, and the obtained transformants were purified by PCR, and the yeast genome was extracted for PCR verification.
  • the successfully verified strain was named J1011-C-3.
  • GAL80 is knocked out on the basis of knockout genes GAL1,7,10, in order to control the expression of genes by controlling the amount of glucose.
  • the resulting promoter information was used to construct expression that controls the carotenoid synthesis-related genes.
  • both the inducible promoter and the constitutive promoter were substantially identical in strength during the stationary phase. It can be seen that knocking out GAL80 can induce gene expression without adding galactose, and the induction intensity is comparable to that induced by galactose.
  • Yeast is a eukaryote, over-expression of the gene in the body needed to select appropriate promoter and terminator, after repeated experiments, the inventors selected the P GAL1, P GAL7, P GAL10 , P GAL1-10, P GAL10-1 And P HXT1 as a promoter for the expression gene.
  • P GAL1 having as SEQ ID NO: nucleotide sequence shown in 4 P GAL7 having as SEQ ID NO: nucleotide sequence shown in 5
  • P GAL10 having as SEQ ID NO: 6 shown nucleoside
  • the acid sequence P GAL1-10 has the nucleotide sequence shown as SEQ ID NO: 7
  • P GAL10-1 has the nucleotide sequence shown as SEQ ID NO: 8
  • P HXT1 has SEQ ID NO: 9 The nucleotide sequence shown.
  • any terminator such as T ADH2, T GAL10, T CYC1 , T GPM1 T PGK1, and the like
  • a combination of these elements can be realized with the overexpression of multiple genes.
  • the inventors adjusted on the J1011-C-3 strain based on the J1011-C-3 strain.
  • the specific characteristics of the second generation engineering strain are shown in Table 1.
  • the appropriate promoter and terminator were selected according to the design, and the fragments were amplified by PCR with the upstream and downstream primers, and the fragments were 60-80 bp overlapping each other, and then all the fragments were recombined by homologous recombination.
  • the corresponding fragments were obtained by restriction enzyme linearization, and the fragments were integrated into the yeast J1011-C-3 genome by yeast transformation using yeast homologous recombination mechanism. After transformation, screening was performed by screening resistant solid plates. The transformants were subjected to PCR verification by extracting the yeast genome after pure culture, and the successfully verified strains are shown in Table 1.
  • BtCrtI has a nucleotide sequence as shown in SEQ ID NO: 10
  • PagCrtB has a nucleotide sequence as shown in SEQ ID NO: 11
  • PaCrtE has a nucleotide sequence as shown in SEQ ID NO: 12.
  • the inventors detailed the experimental procedure for knocking out other genes on the basis of the above-mentioned engineered bacteria to obtain a silent genetically engineered strain.
  • the construction method is to construct a corresponding knockout cassette fragment by using a hygromycin resistance gene as a marker in a gene in need of inactivation, and the knockout cassette fragment is shown in FIG.
  • the fragment was integrated into the above-mentioned engineering bacteria by yeast transformation of lithium acetate, and finally the strain with suspected resistance was screened by using a plate containing hygromycin resistance, and the fourth-generation engineering strain was verified by PCR. See Table 2.
  • the inactivated gene sequence in the knockout cassette fragment can be downloaded from NCBI, so that the knockout box of Fig. 6 can be designed.
  • J1011-C-9, 10, 11, 13, 15, 19 have higher lycopene-producing properties than J1011-C-3.
  • the inventors detailed the experimental procedures associated with overexpression on the above engineered strains.
  • Example 4 Select the appropriate promoter and terminator to construct the expression cassette of the relevant gene in Table 3.
  • the yeast was transformed into the genome of the above-mentioned engineering strain by yeast transformation by the homologous recombination mechanism of yeast, and then transformed into a screening plate.
  • the obtained transformants were purified by PCR, and the yeast genome was extracted for PCR verification.
  • the successfully verified strains are shown in Table 3.
  • J1011-C-4 and J1011-C-20-32 have high lycopene-producing properties.
  • the INO2 gene has the nucleotide sequence shown in SEQ ID NO: 13
  • the gapN gene has the nucleotide sequence shown in SEQ ID NO: 14
  • the PYC2 gene has the nucleoside as shown in SEQ ID NO: 15.
  • the SMAE1 gene has the nucleotide sequence shown in SEQ ID NO: 16
  • the MDH2 gene has the nucleotide sequence shown in SEQ ID NO: 17
  • the POS5 gene has the nucleus as shown in SEQ ID NO:
  • the nucleotide sequence, the pntA gene has the nucleotide sequence shown in SEQ ID NO: 19
  • the pntB gene has the nucleotide sequence shown in SEQ ID NO: 20
  • the ADH2 gene has the sequence shown in SEQ ID NO: a nucleotide sequence
  • the ACS6 gene has the nucleotide sequence set forth in SEQ ID NO: 22
  • the ALD6 gene has the nucleotide sequence set forth in SEQ ID NO: 23
  • the EUTE gene has the sequence shown in SEQ ID NO: 24.
  • the ERG12 gene has the nucleotide sequence shown in SEQ ID NO: 25
  • the IDI1 gene has the nucleotide sequence shown in SEQ ID NO: 26
  • the ERG10 gene has the SEQ ID NO: 27
  • the nucleotide sequence shown, the MVD1 gene has the nucleotide sequence as shown in SEQ ID NO:
  • the ERG13 gene has the nucleotide sequence shown in SEQ ID NO: 29
  • the tHMG1 gene has the nucleotide sequence shown in SEQ ID NO: 30
  • the ERG8 gene has the nucleotide set as shown in SEQ ID NO:
  • the sequence, the yap1 gene has the nucleotide sequence shown as SEQ ID NO:32.
  • the spt15-5 gene has the nucleotide sequence shown as SEQ ID NO:51.
  • the taf25-3 gene has the nucleotide sequence shown as SEQ ID NO:52.
  • the inventors have confirmed through research that the new-generation engineering strain J1011-C-37-84 has high lycopene-producing properties.
  • the inventors have confirmed through research that the new-generation engineering strain J1011-C-85-138 has high lycopene-producing properties.
  • the inventors detailed the fermentation culture process of the engineered strains obtained in Examples 1 to 9.
  • the shake flask fermentation was carried out by two-stage seed culture, and the recombinant strain on the plate was picked into a PA bottle containing 5 mL of YPD medium, and the first-stage seed liquid was shaken by a 30-degree shaker, and after overnight culture (generally 14-18 h), the cells were cultured.
  • the seedlings were grown to a logarithmic growth phase (OD of about 5-8), and the strain was transferred to a 250 mL shake flask containing 50 mL of YPD medium at a 1% inoculum, and shaken to obtain a secondary seed solution.
  • the calculated volume was centrifuged, the supernatant was removed, and the cells were suspended in the corresponding fermentation medium and added to the corresponding 500 mL shake flask, and placed in a 30-degree shaker to start shake flask fermentation.
  • the cell concentration was measured at intervals of about 4 hours (about 4 hours), and the newspaper on the shake flask was removed after 8 hours. After about 48 hours, the sample was stored in a -80 degree refrigerator to determine the production of lycopene.
  • the inventors screened the manner in which the product obtained after the fermentation treatment was extracted, and the screening process was as follows.
  • Method 1 Remove the sample from the refrigerator and thaw it. Take 500 ⁇ L of the fermentation broth in a 15 mL centrifuge tube (pre-cooled on ice), centrifuge at 5000 g for 4 min for 2 min to collect the cells, and then resuspend the washed cells with 1 mL of pure water. Then, 1 mL of 3N HCl was added and the bath was boiled for 3 min to break the cells, and the HCl was removed by centrifugation and washed once with water.
  • Method 2 Take the sample from the refrigerator and thaw it. Take 500 ⁇ L of the fermentation broth in a 15 mL centrifuge tube (pre-cooled on ice), centrifuge at 5000 g for 4 min to collect the cells, remove the supernatant, and resuspend and wash the bacteria with 1 mL of pure water. body. Then add 4 mL of acetone (HPLC grade), 0.2 g of glass beads, 1% of antioxidant, shake for 5 min, then sonicate for 5-10 min in ice bath, then centrifuge at 5000 g for 2 min for 2 min, transfer the supernatant to a 50 mL centrifuge tube.
  • acetone HPLC grade
  • lycopene was detected by quaternary HPLC.
  • the detector was a UV detector.
  • the absorption wavelength of lycopene was 474 nm.
  • Column Agilent Zorbax C18 150 mm * 4.6 mm * 5 ⁇ m
  • mobile phase A acetonitrile
  • water 9:1
  • Method 1 The results showed that after the product of Method 1 was boiled with hydrochloric acid, the lycopene would degrade and the yield detection was unstable.
  • the samples obtained by this method were detected at intervals, and even degraded from 100 mg/L to 10 mg/L.
  • the samples are stable and are tested at intervals of only 100 mg/L to 90 mg/L.
  • the extraction scheme of Method 2 is more stable and better than Method 1.
  • the inventors optimized the conditions of the fermentation medium, and the experimental procedure is as follows:
  • the strain in the present embodiment adopts J1011-C-3
  • the seed culture medium adopts YPD medium
  • the fermentation medium adopts four kinds of culture medium optimized by shake flask to further explore the optimal medium of the fermenter level, various mediums.
  • the recipe is as follows:
  • 2.5L batch medium 2% peptone, 1% yeast extract, 0.8% KH 2 PO 4 and 2% glucose;
  • 1 L glucose feed medium 500 g/L glucose, 5 g/L MgSO 4 , 3.5 g/L K 2 SO 4 , 0.28 g/L Na 2 SO 4 , 10 g/L yeast extract.
  • 2.5L batch medium 60g peptone, 30g yeast extract and 30g glucose (more abundant than YPD);
  • 1L feed medium 1 500g / L glucose, 15g / L yeast extract
  • feed medium 2 300 mL of absolute ethanol, 100 ml of glycerol, 100 mL of pure water.
  • the upper tank culture adopts fed-batch method.
  • the first is batch culture. After the carbon source glucose is basically consumed, the feeding is started.
  • the feeding adopts two-stage feeding method, which is the glucose feeding stage (for the synthesis of biomass). And the ethanol glycerol feed stage (for product synthesis).
  • the specific method is as follows.
  • the fermenter was placed in a small amount of pure water and then emptied at 115 degrees for 30 minutes.
  • the batch cultured fermentation broth and the feed liquid were prepared, the pH electrode of the fermenter was calibrated, and the respective tubes of the fermenter were clamped and sterilized at 115 degrees for 30 minutes.
  • Take out the fermenter connect the air line, perform a small amount of ventilation (about 0.1vvm), and turn on the cooling water device for cooling. When the temperature drops to about 50 degrees, the speed is turned on to 100 rpm. Calibrate the dissolved oxygen electrode.
  • the state of the unconnected line is set to 0% of the dissolved oxygen value, and then the rotational speed is raised to 600 rpm, and the ventilation is adjusted to 2 vvm, and the dissolved oxygen electrode line is connected, and the value is set to 100% after the dissolved oxygen is stabilized.
  • the aeration and speed were then reduced to the desired value for fermentation (ventilation 1.5 vvm, speed 300 rpm - 600 rpm).
  • the inoculation was started, the initial inoculation OD was adjusted to 0.5, and the required seed solution volume was calculated according to the formula.
  • the volume of the fermentation broth is 2500 mL and the OD value of the seed solution is n, the volume of the inoculum seed solution is 2500 x 0.5 / n mL).
  • the medium was controlled with a pH of 5.5 with 2 M NaOH, the initial aeration was 1.5 vvm, the initial agitation rate was set to 300 rpm, and the dissolved oxygen was maintained at 30% or more (associated with agitation rate, 300-600 rpm).
  • Fermentation procedure When the glucose concentration drops to about 2g/L in batch culture, the feed glucose is started, and the initial feed rate is 10mL/L fermentation broth/h to maintain the residual concentration of glucose in the fermentation broth at 2-3g/L. Left and right (do not drop to 0g/L), measure the OD600 value every two hours and measure the glucose content once, and increase the feed rate when the glucose concentration is below 1g/L. When the OD value increases slowly (beginning to enter the stable phase), the feed glucose is stopped. At this time, the ethanol residue is monitored. When the ethanol concentration drops to 5-10 g/L, the ethanol glycerol is fed, and the initial feed rate is 2 mL/L. Fermentation liquid / h.
  • the ethanol and glycerol contents were then sampled every 4 hours, and the feed rate was adjusted to maintain ethanol and glycerol in the range of 5-10 g/L. After the color has changed, the product is detected to change the product, and the fermentation is terminated when the lycopene concentration is no longer increased.
  • the product detection method was the same as that of the second method in Example 9.
  • the medium No. 5 yield was better than the medium No. 2, so the medium was the optimal medium, and the fermentation strategy was an optimized two-step feed fermentation, which first supplemented the glucose long cells and supplemented the ethanol in the stable period of growth. To increase production.
  • the inventors fermented the constructed part of the engineered strain according to the optimal fermentation culture method obtained in Example 12, and the results are shown in Table 6.
  • J1011-C-77 2.69 J1011-C-79 2.89 J1011-C-81 2.85 J1011-C-82 2.95 J1011-C-87 2.69 J1011-C-91 2.66 J1011-C-95 3.01 J1011-C-99 2.78 J1011-C-120 2.85 J1011-C-123 2.96 J1011-C-126 2.79 J1011-C-132 2.56 J1011-C-138 2.98
  • the engineered strain constructed according to the embodiment of the present invention has high yield of lycopene.

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Abstract

本发明提出了一种微生物及其用途和获得番茄红素的方法，该微生物过表达PaCrtE，PagCrtB和BtCrtI基因。

Description

微生物及其用途
优先权信息
本申请请求2017年06月19日向中国国家知识产权局提交的、专利申请号为201710464916.6的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明涉及生物工程领域，具体的，本发明涉及微生物及其用途，更具体的，本发明涉及微生物、获得番茄红素的方法和微生物在制备番茄红素中的用途。
背景技术
番茄红素的开发生产主要有天然产物提取、化学合成、微生物发酵等方法。天然产物提取主要是通过成熟果实的萃取纯化获得番茄红素，然而这种生产方式受气候、品种、地理位置、成熟度等诸多不可控因素影响，具有明显的季节性，含量不稳定性，并且大面积种植、养殖的成本较高，含量通常也比较低。另外，高纯度的番茄红素在萃取纯化技术上非常困难，这些共同造成了番茄红素的成品十分昂贵。化学合成法具有原料易得价廉、反应条件温和、反应速率快、产物容易与反应体系分离的优点，但由于双键立体选择性难以控制及不同程度的化学试剂残留，其产品的质量、安全性及使用范围都受到了限制。微生物发酵法主要是利用微生物的生物代谢，将葡萄糖、淀粉、黄豆饼粉等廉价原料转化为番茄红素，这种方法不受季节、地域、气候等因素的影响，原料易获取、生产周期短、工艺操作简单、成本低廉、产物质量可控、产物易纯化、安全性高，并且环境污染较少，不仅解决了因种植植物等而占用大量耕地的问题，也解决了化学合成不环保的弊端。最重要的是，发酵法生产的番茄红素属于天然型产品，其活性与天然植物提取的活性成分一致，被认为是番茄红素生产最有前景的方法。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。
目前研究较多的番茄红素产生菌的主要是三孢布拉氏霉菌。但是三孢布拉氏霉菌在传代过程中容易发生退化现象，使得番茄红素产量减少，另伴有黄曲霉毒素等有毒成分的合成，另一方面，这两种微生物的生长周期相较于酵母和大肠杆菌较长，大大降低了其生产效率，从而造成了生产番茄红素的难度加大。
为此，本发明的一个目的在于提出了一种微生物，该微生物生产番茄红素的产量 高，生产周期短，生产效率高。
在本发明的第一方面，本发明提出了一种微生物。根据本发明的实施例，所述微生物过表达PaCrtE，PagCrtB和BtCrtI基因。利用根据本发明实施例的微生物，生产番茄红素的产量显著提高，生产周期短，生产效率高。
根据本发明的实施例，上述微生物还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述微生物进一步过表达包括选自tHMG1，INO2，yap1，spt15-5，taf25-3，GapN，PYC2，SMAE1，MDH2，POS5，pntAB，ADH2，ACS6，ALD6，EUTE，ERG12，IDI1，ERG10，MVD1，ERG13，ERG8基因的至少之一。发明人发现，所述微生物进一步过表达上述基因的至少之一，其番茄红素的产量进一步提高。
根据本发明的实施例，所述微生物进一步沉默包括选自GAL1，GAL7，GAL10，GAL80，ROX1，VBA5，DOS2，Ypl062W，Yjl064W，Yer130C，Yer134C，Ynr063W，Exg1，Yor292C，Sfk1，Mef1基因的至少之一。发明人发现，所述微生物进一步沉默上述基因的至少之一，其番茄红素的产量进一步提高。
根据本发明的实施例，所述微生物是酵母菌。酵母菌的生长周期短，利用本发明实施例的微生物生产番茄红素的效率进一步提高。
根据本发明的实施例，所述微生物进一步包括可操作调控ERG9基因。根据本发明的实施例，所述可操作调控ERG9基因包括但不限于替换ERG9基因的启动子，进而使得ERG9基因的表达可以根据需要进行调控，使微生物生产番茄红素的效率进一步提高。
在本发明的第二方面，本发明提出了一种获得番茄红素的方法。根据本发明的实施例，所述方法包括：将前面所述的微生物进行发酵处理；以及将发酵处理产物进行萃取处理，以便获得所述番茄红素。利用根据本发明实施例的获得番茄红素的方法，能够高产量、高效率获得番茄红素，且番茄红素的纯度高。
根据本发明的实施例，上述获得番茄红素的方法还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，利用根据本发明实施例的获得番茄红素的方法，获得的番茄红素产量更高、效率更高，纯度也更高。
根据本发明的实施例，所述发酵处理是通过如下方式实现的：将所述微生物进行基础发酵处理和两级分批补料发酵处理，所述基础发酵处理是在基本发酵培养基中进 行的，所述两级分批补料发酵处理是通过在所述基本发酵培养基基础上依次补加第一补料培养基和第二补料培养基实现的，其中，所述基本发酵培养基为含有含有24g/L蛋白胨，12g/L酵母提取物和12g/L葡萄糖的YPD培养基；所述第一补料培养基为含有500g/L葡萄糖和15g/L酵母提取物的YPD培养基；所述第二补料培养基为75％乙醇或50％甘油。发明人通过实验发现，采用上述发酵处理方式，微生物的扩增速率高、番茄红素的产率也进一步提高。
根据本发明的实施例，所述萃取处理包括：将所述发酵处理产物进行超声破碎处理及有机萃取处理。发明人对发酵产物进行萃取处理过程中发现，将发酵处理后产物进行超声破碎处理，相比于采用盐酸蒸煮的方法，番茄红素的降解大幅降低，萃取处理后获得的番茄红素的产量进一步提高。
在本发明的第三方面，本发明提出了前面所述微生物在制备番茄红素中的用途。如前所述，利用本发明实施例的微生物，生产番茄红素的产量高，生产周期短，生产效率高。
附图说明
图1是根据本发明实施例1的敲除盒片段1的结构示意图；
图2是根据本发明实施例1的敲除盒片段2的结构示意图；
图3是根据本发明实施例1的敲除盒片段3的结构示意图；
图4是第二代工程菌株摇瓶发酵结果；
图5是根据本发明实施例4的敲除盒片段4的结构示意图；以及
图6是根据本发明实施例的发酵培养基的筛选结果图。
发明详细描述
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
微生物
在本发明的第一方面，本发明提出了一种微生物。根据本发明的实施例，该微生物过表达PaCrtE，PagCrtB和BtCrtI基因。利用根据本发明实施例的微生物，生产番茄红素的产量高，生产周期短，生产效率高。
根据本发明的具体实施例，所述微生物还可以进一步过表达包括选自tHMG1，INO2， yap1，spt15-5，taf25-3，GapN，PYC2，SMAE1，MDH2，POS5，pntAB，ADH2，ACS6，ALD6，EUTE，ERG12，IDI1，ERG10，MVD1，ERG13，ERG8基因的至少之一。
根据本发明的再一具体实施例，所述微生物还可以进一步沉默包括选自GAL1，GAL7，GAL10，GAL80，ROX1，VBA5，DOS2，Ypl062W，Yjl064W，Yer130C，Yer134C，Ynr063W，Exg1，Yor292C，Sfk1，Mef1基因的至少之一。发明人发现，所述微生物进一步沉默上述基因的至少之一，其番茄红素的产量进一步提高。
发明人发现，所述微生物进一步过表达或沉默上述基因的至少一，其番茄红素的产量进一步显著提高。
本申请所述的微生物用于生产番茄红素，相比于现有技术，其产量得到了显著提高，能够至少达到2.5g/L。
根据本发明的具体实施例，所述微生物还可以进一步包括可操作调控ERG9基因。本申请所述的可操作调控ERG9基因是指替换ERG原始的启动子，从而实现通过葡萄糖调控ERG9的表达，从而增加番茄红素的产量。
需要说明的是，发明人通过实验，发现：过表达PaCrtE，PagCrtB，BtCrtI基因能够达到在酵母体内高效合成番茄红素的目的；沉默GAL1，7，10或GAL1，7，10，80能够达到利用半乳糖诱导或利用葡萄糖浓度调节生产番茄红素的目的；过表达INO2，yap1，spt15-5，taf25-3基因是抗逆基因，可以增加番茄红素的产量；过表达GapN，PYC2，SMAE1，MDH2，POS5，pntAB中的一个或多个基因能够平衡酵母体内的还原力，可以增加番茄红素的产量；过表达ADH2，ALD6，EUTE中的一个或多个基因能够增加合成番茄红素的前体物质的供应，增加番茄红素的产量；过表达ERG12，IDI1，ERG10，MVD1，ERG13，ERG8中的一个或多个基因，能够平衡有关番茄红素合成中的MVA途径，增加番茄红素的产量；沉默ROX1，VBA5，DOS2，Ypl062W，Yjl064W，Yer130C，Yer134C，Ynr063W，Exg1，Yor292C，Sfk1，Mef1基因的至少之一，能够从整体上调节酵母系统，从而增加番茄红素的产量。利用根据本发明实施例的微生物，番茄红素的产量得到显著提高。
获得番茄红素的方法
在本发明的第二方面，本发明提出了一种获得番茄红素的方法。根据本发明的实施例，该方法包括：将前面所述的微生物进行发酵处理；以及将发酵处理产物进行萃取处理，以便获得所述番茄红素。利用根据本发明实施例的获得番茄红素的方法，能够高产量、高效率获得番茄红素，且番茄红素的纯度高。
根据本发明的具体实施例，所述发酵处理是通过基础发酵处理和两级分批补料发酵处 理进行的，具体如下所述：
将发酵罐装入少量纯水后115度进行空灭30分钟。配制分批培养的发酵液以及补料液，校准发酵罐的pH电极，夹住发酵罐的各个管路，115度灭菌30分钟。取出发酵罐，连接空气管路，进行少量通气(0.1vvm左右即可)，同时打开冷却水装置进行冷却。待温度降至50度左右时，打开转速至100rpm。校准溶氧电极。未连接线路的状态设定溶氧值为0％，随后将转速升至600rpm，同时将通气量调至2vvm，连接溶氧电极线路，待溶氧稳定后设定该值为100％。随后将通气量和转速降低至发酵所需值(通气量1.5vvm，转速300rpm-600rpm)。先自然pH，将温度调节至30度。开始接种，初始接种OD调至0.5，所需种子液体积根据公式进行计算。(如发酵液体积为2500mL，种子液OD值为n，则接种种子液体积为2500x0.5/n mL)。开始发酵后，上罐时培养基用2M NaOH来控制pH值为5.5，初始通气量为1.5vvm，初始搅拌速率设置为300rpm，溶氧维持在30％以上(关联搅拌速率，300-600rpm)。
当分批培养时葡萄糖浓度降至2g/L左右时开始补料葡萄糖，初始补料速率为10mL/L发酵液/h，以维持发酵液中葡萄糖的残留浓度在2-3g/L左右(不要降至0g/L)，每两个小时取样测量一次OD600值和检测一次葡萄糖含量，当葡萄糖浓度低于1g/L时增加补料速率。当OD值增加缓慢时(开始进入稳定期)停止补料葡萄糖，此时开始监测乙醇残留量，当乙醇浓度降至5-10g/L时开始补料乙醇甘油，初始补料速率为2mL/L发酵液/h。随后每4h取样检测一次乙醇和甘油含量，当调整补料速率使乙醇和甘油维持在5-10g/L范围内。颜色有变化后开始提取产物检测产物变化，当番茄红素浓度不再增加时结束发酵。
其中，发明人发现，在基本发酵培养基中培养菌体，相比于现有技术的培养基，菌体的生长速度和生长状态均显著优于现有技术，补加的第一补料培养基，有效保证了菌体的快速和健康的生长，补加的第二补料培养基，有效提高了番茄红素的产量。根据本发明的再一具体实施例，所述萃取处理包括：将所述发酵处理后产物进行超声破碎处理以及有机萃取处理。发明人对发酵处理后的萃取过程进行了优化筛选，发明人发现，采用超声处理的方式相比于采用盐酸蒸煮的方式，番茄红素的降解速率大幅降低，番茄红素的产量稳定。另外，需要说明的是，本申请所述的“有机萃取处理”是指采取有机溶剂对超声破碎处理产物进行萃取，有机萃取的方式不受特别限制，如根据本发明的具体实施例，可以采用丙酮溶剂从超声破碎处理产物中萃取番茄红素。
用途
在本发明的第三方面，本发明提出了前面所述的微生物在制备番茄红素中的用途。发 明人经过实验证实，本申请所述的微生物在生产番茄红素方面具有显著的优势，番茄红素的产量高、番茄红素的降解速率低，生产周期短。
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1 第一代工程菌株J1011-C-3的构建
在本实施例中，发明人详细介绍了J1011-C-3菌株的构建过程。
首先构建ΔLEU2：pGAL1-PaCrtE(Pantoea ananatis)敲除盒，即敲除盒片段1，设计上、下游引物PCR扩增各片段，使其相互之间有60-80bp的重叠片段，再通过同源重组的方式将所有片段重组在一起，通过酶切线性化得到ΔLEU2：：pGAL1-PaCrtE(Pantoea ananatis)敲除盒，如敲除盒片段1如图1所示，敲除盒片段1具有如SEQ ID NO：1所示的核苷酸序列。利用酵母自身的同源重组机制将该片段通过醋酸锂法酵母转化分别整合到酵母30000B基因组上，整合位点为LEU2，由于原酵母中的LEU2没有活性，而整合后的同源左臂含有LEU2完整基因，固转化后采用SD-Leu固体板(合成酵母氮源YNB 6.7g/L，葡萄糖20g/L，缺亮氨酸的混合氨基酸粉末1.3g/L，2％琼脂粉)进行筛选，得到的转化子通过分纯培养后提取酵母基因组进行PCR验证，成功验证的菌株命名为J1011-C-1。
Figure PCTCN2018091113-appb-000001
Figure PCTCN2018091113-appb-000002
Figure PCTCN2018091113-appb-000003
Figure PCTCN2018091113-appb-000004
再构建ΔURA3：pGAL1-PagCrtB(Pantoea agglomerans)；pGAL10-BtCrtI(Blakeslea trispora)敲除盒，即敲除盒片段2，敲除盒片段2具有如SEQ ID NO：2所示的核苷酸序列。设计上、下游引物PCR扩增各片段，使其相互之间有60-80bp的重叠片段，再通过同源重组的方式将所有片段重组在一起，通过酶切线性化得到ΔURA3：：pGAL1-PagCrtB(Pantoea agglomerans)；pGAL10-BtCrtI(Blakeslea trispora)敲除盒，敲除盒片段2如图2所示。利用酵母自身的同源重组机制将该片段通过醋酸锂法酵母转化分别整合到酵母J1011-C-1基因组上，整合位点为URA3，转化后采用SD-HIS固体板(合成酵母氮源YNB6.7g/L，葡萄糖20g/L，缺组氨酸的混合氨基酸粉末1.3g/L，2％琼脂粉)进行筛选，得到的转化子通过分纯培养后提取酵母基因组进行PCR验证，成功验证的菌株命名为J1011-C-2。
Figure PCTCN2018091113-appb-000005
Figure PCTCN2018091113-appb-000006
Figure PCTCN2018091113-appb-000007
Figure PCTCN2018091113-appb-000008
Figure PCTCN2018091113-appb-000009
Figure PCTCN2018091113-appb-000010
再构建Δgal1，Δgal7，Δgal10:pGAL10-tHMG1敲除盒，即敲除盒片段3，敲除盒片段3具有如SEQ ID NO：3所示的核苷酸序列。设计上、下游引物PCR扩增各片段，使其相互之间有60-80bp的重叠片段，再通过同源重组的方式将所有片段重组在一起，通过酶切线性化得到Δgal1，Δgal7，Δgal10::pGAL10-tHMG1敲除片段，敲除盒片段3的结构如图3 所示。利用酵母自身的同源重组机制将该片段通过醋酸锂法酵母转化分别整合到酵母J1011-C-2基因组上，整合位点为GAL1，GAL7和GAL10，转化后采用SD-Trp固体板(合成酵母氮源YNB 6.7g/L，葡萄糖20g/L，缺色氨酸的混合氨基酸粉末1.3g/L，2％琼脂粉)进行筛选，得到的转化子通过分纯培养后提取酵母基因组进行PCR验证，成功验证的菌株命名为J1011-C-3.
Figure PCTCN2018091113-appb-000011
Figure PCTCN2018091113-appb-000012
Figure PCTCN2018091113-appb-000013
Figure PCTCN2018091113-appb-000014
Figure PCTCN2018091113-appb-000015
在以下实验中，发明人进一步对J1011-C-3菌株进行改造，具体如下所述：
实施例2 启动子和终止子的选择
为了能用半乳糖进行诱导，需要敲除代谢半乳糖的基因GAL1,GAL7,GAL10。因此特征启动子的背景菌株为敲除基因GAL1,7,10。半乳糖价格较高，并不适用于工业化生产，因此在敲除基因GAL1,7,10的基础上敲除GAL80，以期通过对葡萄糖量的控制达到对基因表达的调控。所得到的启动子信息用于构建控制类胡萝卜素合成相关基因的表达。
结果所示，在ΔGAL1/7/10/80菌株和ΔGAL1/7/10菌株中不论是诱导型启动子或是组成型启动子在稳定期强度基本一致。可见，敲除GAL80可以起到不加半乳糖即可诱导基因表达，诱导强度跟与半乳糖诱导强度相当。
酵母属于真核生物，在其体内过表达基因需要选择合适的启动子和终止子，经过反复多次实验，发明人选择了P GAL1，P GAL7，P GAL10，P GAL1-10，P GAL10-1和P HXT1作为表达基因的启动子。其中，P GAL1具有如SEQ ID NO：4所示的核苷酸序列，P GAL7具有如SEQ ID NO：5所示的核苷酸序列，P GAL10具有如SEQ ID NO：6所示的核苷酸序列，P GAL1-10具有如SEQ ID NO：7所示的核苷酸序列，P GAL10-1具有如SEQ ID NO：8所示的核苷酸序列，P HXT1具有如SEQ ID NO：9所示的核苷酸序列。
Figure PCTCN2018091113-appb-000016
Figure PCTCN2018091113-appb-000017
Figure PCTCN2018091113-appb-000018
Figure PCTCN2018091113-appb-000019
选择任意的终止子(比如T ADH2，T GAL10，T CYC1，T GPM1和T PGK1等)作为表达基因的终止子，这些元件的组合搭配能够实现多个基因的过表达。
实施例3 第二代工程菌株的构建
在本实施例中，发明人以J1011-C-3菌株为基础，在J1011-C-3菌株上调整
PaCrtE(Pantoea ananatis)，PagCrtB(Pantoea Agglomerans)和BtCrtI(Blakeslea trispora)这三个基因的拷贝数，获得第二代菌株。
其中第二代工程菌株的具体特性如表1所示。按照设计选择合适的启动子和终止子，用上、下游引物PCR扩增各片段，使其相互之间有60-80bp的重叠片段，再通过同源重组的方式将所有片段重组在一起，通过酶切线性化得到各相应片段，利用酵母自身的同源重组机制将该片段通过醋酸锂法酵母转化分别整合到酵母J1011-C-3基因组上，转化后采用筛选抗性固体板进行筛选，得到的转化子通过分纯培养后提取酵母基因组进行PCR验证，成功验证的菌株详见表1。
表1：第二代工程菌株
菌株 特性及敲除盒
J1011-C-5 J1011-C-3衍生菌株；在原基础上过表达BtCrtI.
J1011-C-6 J1011-C-3衍生菌株；在原基础上过表达BtCrtI和PagCrtB
J1011-C-7 J1011-C-3衍生菌株；在原基础上过表达BtCrtI和PagCrtB和PaCrtE
J1011-C-33 J1011-C-3衍生菌株；在原基础上过表达PagCrtB和PaCrtE
J1011-C-34 J1011-C-3衍生菌株；在原基础上过表达PaCrtE
J1011-C-35 J1011-C-3衍生菌株；在原基础上过表达BtCrtI和PaCrtE
J1011-C-36 J1011-C-3衍生菌株；在原基础上过表达PagCrtB.
其中，BtCrtI具有如SEQ ID NO：10所示的核苷酸序列，PagCrtB具有如SEQ ID NO：11所示的核苷酸序列，PaCrtE具有如SEQ ID NO：12所示的核苷酸序列。
Figure PCTCN2018091113-appb-000020
Figure PCTCN2018091113-appb-000021
Figure PCTCN2018091113-appb-000022
Figure PCTCN2018091113-appb-000023
更进一步，发明人通过实验验证，结果如图5所示，在摇瓶条件下，J1011-C-5，6，7和J1011-C-33～36具有比J1011-C-3更高产番茄红素的性能。
实施例4 沉默基因的工程菌株的构建
在本实施例中，发明人详细介绍了在上述工程菌的基础上敲除另外一些基因获得沉默基因工程菌株的实验过程。
在酿酒酵母中，有很多潜在的基因在敲除后对细胞积累番茄红素化合物有明显的影响，因此构建一系列背景菌株用于增加番茄红素产量。构建方法为在需要灭活的基因中以潮霉素抗性基因为标记构建相应的敲除盒片段，敲除盒片段见图4。将该片段通过醋酸锂法酵母转化分别整合到上述工程菌中，最后用含有潮霉素抗性的平板进行筛选得到疑似带有抗性的菌株，用PCR进行验证，得到的第四代工程菌株见表2。其中，敲除盒片段中的灭活基因序列可从NCBI上下载，从而可以设计得到图6的敲除框。
表2：沉默工程菌株特性
Figure PCTCN2018091113-appb-000024
更进一步，发明人通过实验验证，J1011-C-9,10,11,13,15,19都具有比J1011-C-3更高产番茄红素的性能。
实施例5 过表达基因的工程菌株的构建
在本实施例中，发明人详细介绍了在上述工程菌株上过表达相关的实验过程。
选择合适的启动子和终止子构建表3中相关基因的表达框，在实施例4的基础上选择合适的基因作为插入位点，通过设计上、下游引物PCR扩增各片段，使其相互之间有60-80bp的重叠片段，再通过同源重组的方式将所有片段重组在一起，通过酶切线性化得到即可实现沉默基因又能过表达基因的基因片段。利用酵母自身的同源重组机制将该片段通过醋酸锂法酵母转化分别整合到上述工程菌株基因组上，转化后采用筛选平板进行筛选，得到的转化子通过分纯培养后提取酵母基因组进行PCR验证，成功验证的菌株详见表3。
更进一步，发明人通过实验验证，J1011-C-4和J1011-C-20～32都具有高产番茄红素的性能。
表3：过表达基因的工程菌株
Figure PCTCN2018091113-appb-000025
其中，INO2基因具有如SEQ ID NO：13所示的核苷酸序列，gapN基因具有如SEQ ID NO：14所示的核苷酸序列，PYC2基因具有如SEQ ID NO：15所示的核苷酸序列，SMAE1基因具有如SEQ ID NO：16所示的核苷酸序列，MDH2基因具有如SEQ ID NO：17所示 的核苷酸序列，POS5基因具有如SEQ ID NO：18所示的核苷酸序列，pntA基因具有如SEQ ID NO：19所示的核苷酸序列，pntB基因具有如SEQ ID NO：20所示的核苷酸序列，ADH2基因具有如SEQ ID NO：21所示的核苷酸序列，ACS6基因具有如SEQ ID NO：22所示的核苷酸序列，ALD6基因具有如SEQ ID NO：23所示的核苷酸序列，EUTE基因具有如SEQ ID NO：24所示的核苷酸序列，ERG12基因具有如SEQ ID NO：25所示的核苷酸序列，IDI1基因具有如SEQ ID NO：26所示的核苷酸序列，ERG10基因具有如SEQ ID NO：27所示的核苷酸序列，MVD1基因具有如SEQ ID NO：28所示的核苷酸序列，ERG13基因具有如SEQ ID NO：29所示的核苷酸序列，tHMG1基因具有如SEQ ID NO：30所示的核苷酸序列，ERG8基因具有如SEQ ID NO：31所示的核苷酸序列，yap1基因具有如SEQ ID NO：32所示的核苷酸序列。spt15-5基因具有如SEQ ID NO：51所示的核苷酸序列。taf25-3基因具有如SEQ ID NO：52所示的核苷酸序列。
Figure PCTCN2018091113-appb-000026
Figure PCTCN2018091113-appb-000027
Figure PCTCN2018091113-appb-000028
Figure PCTCN2018091113-appb-000029
Figure PCTCN2018091113-appb-000030
Figure PCTCN2018091113-appb-000031
Figure PCTCN2018091113-appb-000032
Figure PCTCN2018091113-appb-000033
Figure PCTCN2018091113-appb-000034
Figure PCTCN2018091113-appb-000035
Figure PCTCN2018091113-appb-000036
Figure PCTCN2018091113-appb-000037
Figure PCTCN2018091113-appb-000038
Figure PCTCN2018091113-appb-000039
Figure PCTCN2018091113-appb-000040
Figure PCTCN2018091113-appb-000041
Figure PCTCN2018091113-appb-000042
Figure PCTCN2018091113-appb-000043
实施例8 新一代工程菌株的构建
根据实施例1～7，将有效果的改造进行叠加组合，如将有效果的敲除基因Ypl062W,Yer130C,Yer134c,Exg1等两两组合进行敲除，结果显示双基因敲除会得到产量更高的菌株。
在此基础上，我们将有效果的改造进行叠加组合，形成新一代的工程菌株，菌株特性如下表4.
表4.新一代工程菌株的特征
Figure PCTCN2018091113-appb-000044
Figure PCTCN2018091113-appb-000045
Figure PCTCN2018091113-appb-000046
Figure PCTCN2018091113-appb-000047
注：“√”代表该基因被敲除，"+"代表该基因过表达一个拷贝，"*"代表该基因过表达至少2个拷贝，“-”代表该基因不被操作(即不被敲除或不过表达)
更进一步，发明人通过研究证明，新一代工程菌株J1011-C-37～84具有高产番茄红素的性能。
实施例9 新二代工程菌株的构建
在上一代工程菌株的基础上，我们挑选了18个优势菌株，在此基础上构建了新二代的 工程菌株探究gapN和EUTE基因对番茄红素产量的影响，菌株特性如下表5.
表5.新二代工程菌株的特征
菌株名 原始菌株 过表达基因   产量(mg/L)
    GapN ADH2,EUTE  
J1011-C-85 J1011-C-35 √ - 296
J1011-C-86 J1011-C-35 - √ 276
J1011-C-87 J1011-C-35 √ √ 320
J1011-C-88 J1011-C-40 √ - 330
J1011-C-89 J1011-C-40 - √ 295
J1011-C-90 J1011-C-40 √ √ 297
J1011-C-91 J1011-C-42 √ - 295
J1011-C-92 J1011-C-42 - √ 270
J1011-C-93 J1011-C-42 √ √ 311
J1011-C-94 J1011-C-44 √ - 295
J1011-C-95 J1011-C-44 - √ 318
J1011-C-96 J1011-C-44 √ √ 325
J1011-C-97 J1011-C-47 √ - 296
J1011-C-98 J1011-C-47 - √ 301
J1011-C-99 J1011-C-47 √ √ 307
J1011-C-100 J1011-C-50 √ - 297
J1011-C-101 J1011-C-50 - √ 259
J1011-C-102 J1011-C-50 √ √ 315
J1011-C-103 J1011-C-56 √ - 280
J1011-C-104 J1011-C-56 - √ 268
J1011-C-105 J1011-C-56 √ √ 289
J1011-C-106 J1011-C-57 √ - 278
J1011-C-107 J1011-C-57 - √ 288
J1011-C-108 J1011-C-57 √ √ 296
J1011-C-109 J1011-C-63 √ - 307
J1011-C-110 J1011-C-63 - √ 298
J1011-C-111 J1011-C-63 √ √ 298
J1011-C-112 J1011-C-64 √ - 286
J1011-C-113 J1011-C-64 - √ 289
J1011-C-114 J1011-C-64 √ √ 301
J1011-C-115 J1011-C-73 √ - 318
J1011-C-116 J1011-C-73 - √ 296
J1011-C-117 J1011-C-73 √ √ 233
J1011-C-118 J1011-C-74 √ - 298
J1011-C-119 J1011-C-74 - √ 257
J1011-C-120 J1011-C-74 √ √ 325
J1011-C-121 J1011-C-75 √ - 296
J1011-C-122 J1011-C-75 - √ 297
J1011-C-123 J1011-C-75 √ √ 296
J1011-C-124 J1011-C-77 √ - 310
J1011-C-125 J1011-C-77 - √ 315
J1011-C-126 J1011-C-77 √ √ 311
J1011-C-127 J1011-C-79 √ - 313
J1011-C-128 J1011-C-79 - √ 299
J1011-C-129 J1011-C-79 √ √ 287
J1011-C-130 J1011-C-81 √ - 302
J1011-C-131 J1011-C-81 - √ 296
J1011-C-132 J1011-C-81 √ √ 297
J1011-C-133 J1011-C-82 √ - 298
J1011-C-134 J1011-C-82 - √ 286
J1011-C-135 J1011-C-82 √ √ 295
J1011-C-136 J1011-C-84 √ - 312
J1011-C-137 J1011-C-84 - √ 313
J1011-C-138 J1011-C-84 √ √ 318
注：“√”代表该基因被过表达，“-”代表该基因不被过表达
更进一步，发明人通过研究证明，新一代工程菌株J1011-C-85～138具有高产番茄红素 的性能。
实施例10 工程菌摇瓶培养发酵过程
在本实施例中，发明人详细介绍了实施例1～9所获得的工程菌株的发酵培养过程。
摇瓶发酵采用两级种子培养，将平板上的重组菌株挑到含有5mL YPD培养基的PA瓶中，30度摇床摇起一级种子液，过夜培养(一般14-18h)后，菌体长到对数生长期(OD在5-8左右)，再以1％的接种量将菌株转移到含有50mL YPD培养基的250mL摇瓶中，摇瓶培养得到二级种子液。约14-18h后，测二级种子的OD 600值，然后计算接种到含有200mL发酵培养基YPDG的500mL摇瓶中，使菌体终浓度OD600＝0.5，需要多少的二级种子液，然后取出算出的体积并离心，去掉上清，再用对应的发酵培养基将菌体悬浮并加入相应的500mL摇瓶中，置于30度摇床开始摇瓶发酵。每隔一段时间(约4h)取样测定细胞浓度，并在8h后将摇瓶上的报纸去掉，约48h后开始取样存于-80度冰箱留待测定番茄红素的产生情况。
实施例11 产物萃取方式比较
在本实施例中，发明人对发酵处理后所获得的产物进行萃取的方式进行了筛选，筛选过程如下所述。
方法一：从冰箱中取出样品解冻，取500μL发酵液于15mL离心管(冰上预冷)中，5000g 4度离心2min来收集菌体，去上清后再用1mL纯水重悬洗涤菌体，再加入1mL 3N的HCl并沸水浴3min来破碎细胞，离心去掉HCl后用水洗涤一次。再向其中加入1mL的丙酮(HPLC级别)，0.2g玻璃珠，1％的抗氧化剂，震荡5min，然后5000g 4度离心2min，转移上清到50mL的离心管中；再次重复加入上述提取液，并震荡，然后收集萃取液，直至菌体无明显黄色；混匀收集到的丙酮萃取液，取出2mL，12000rpm离心10分钟，再取上清1.2mL至棕色的HPLC进样瓶中，进行HPLC分析。
方法二：从冰箱中取出样品解冻，取500μL发酵液于15mL离心管(冰上预冷)中，5000g 4度离心2min来收集菌体，去上清后再用1mL纯水重悬并洗涤菌体。再向其中加入4mL的丙酮(HPLC级别)，0.2g玻璃珠，1％的抗氧化剂，震荡5min，然后冰浴超声5-10min，然后5000g 4度离心2min，转移上清到50mL的离心管中；再次重复加入上述提取液，并震荡，然后收集萃取液，直至菌体无明显黄色；混匀收集到的丙酮萃取液，取出2mL，12000rpm离心10分钟，再取上清1.2mL至棕色的HPLC进样瓶中，进行HPLC 分析。
检测方法：番茄红素的检测使用四元HPLC进行的，检测器是紫外检测器，番茄红素的吸收波长是474nm，色谱柱Agilent Zorbax C18(150mm*4.6mm*5μm)，流动相A(乙腈：水＝9：1)和流动相B(甲醇：异丙醇＝3：2)按如下条件分析：0-90％B(0-15min)，90％B(15-30min)，90％-0B(30-35min)，流速1mL/min。
结果显示：方法一中产物经盐酸煮过后，番茄红素会降解，产量检测不稳定，且该方法得到的样品间隔一段时间再检测，甚至能从100mg/L降解到10mg/L，方法二得到的样品比较稳定，间隔一段时间再检测，只从100mg/L到90mg/L。说明方法二的提取方案比方法一更稳定更好。
实施例12 工程菌发酵罐培养发酵方法比较
在本实施例中，发明人对发酵培养基的条件进行了优化筛选，实验过程如下所述：
本实施例中的菌种采用J1011-C-3，种子培养基采用YPD培养基，发酵培养基采用摇瓶优化过的4种培养基进一步探索发酵罐水平的最优培养基，各种培养基配方如下：
①培养基2号:YPD含盐培养基
2.5L分批培养基：2％蛋白胨，1％酵母提取物，0.8％KH 2PO 4和2％葡萄糖；
1L葡萄糖补料培养基：500g/L葡萄糖，5g/L MgSO 4，3.5g/L K 2SO 4，0.28g/L Na 2SO 4，10g/L酵母提取物。
1L乙醇补料培养基:100％乙醇。
②培养基5号：YPD丰富培养基
2.5L分批培养基：60g蛋白胨，30g酵母提取物和30g葡萄糖(比YPD更加丰富)；
1L补料培养基1：500g/L葡萄糖，15g/L酵母抽提物
500mL补料培养基2:300mL无水乙醇,100ml甘油，100mL纯水。
上罐培养采用fed-batch方式，首先是分批培养，待碳源葡糖糖基本消耗完后开始补料，补料采用两级补料方式，分别为葡萄糖补料阶段(用于合成生物量)和乙醇甘油补料阶段(用于产物合成)。具体方法如下。
将发酵罐装入少量纯水后115度进行空灭30分钟。配制分批培养的发酵液以及补料液，校准发酵罐的pH电极，夹住发酵罐的各个管路，115度灭菌30分钟。取出发酵罐，连接空气管路，进行少量通气(0.1vvm左右即可)，同时打开冷却水装置进行冷却。待温度降至50度左右时，打开转速至100rpm。校准溶氧电极。未连接线路的状态设定溶氧值为0％， 随后将转速升至600rpm，同时将通气量调至2vvm，连接溶氧电极线路，待溶氧稳定后设定该值为100％。随后将通气量和转速降低至发酵所需值(通气量1.5vvm，转速300rpm-600rpm)。先自然pH，将温度调节至30度。开始接种，初始接种OD调至0.5，所需种子液体积根据公式进行计算。(如发酵液体积为2500mL，种子液OD值为n，则接种种子液体积为2500x0.5/n mL)。开始发酵后，上罐时培养基用2M NaOH来控制pH值为5.5，初始通气量为1.5vvm，初始搅拌速率设置为300rpm，溶氧维持在30％以上(关联搅拌速率，300-600rpm)。
发酵程序：当分批培养时葡萄糖浓度降至2g/L左右时开始补料葡萄糖，初始补料速率为10mL/L发酵液/h，以维持发酵液中葡萄糖的残留浓度在2-3g/L左右(不要降至0g/L)，每两个小时取样测量一次OD600值和检测一次葡萄糖含量，当葡萄糖浓度低于1g/L时增加补料速率。当OD值增加缓慢时(开始进入稳定期)停止补料葡萄糖，此时开始监测乙醇残留量，当乙醇浓度降至5-10g/L时开始补料乙醇甘油，初始补料速率为2mL/L发酵液/h。随后每4h取样检测一次乙醇和甘油含量，当调整补料速率使乙醇和甘油维持在5-10g/L范围内。颜色有变化后开始提取产物检测产物变化，当番茄红素浓度不再增加时结束发酵。
产物检测方式与实施例9中方法二相同。
结果显示:培养基5号产量优于培养基2号，所以该培养基为最优培养基，发酵策略是优化的两步补料发酵，先补葡萄糖长菌体，在生长的稳定期补乙醇来提高产量。
实施例13 菌株的上罐发酵培养
在本实施例中，发明人按照实施例12中得到的最佳发酵培养方式对构建的部分工程菌株进行了发酵，结果如表6所示。
表6：
菌株名 发酵产量(g/L)
J1011-C-35 2.96
J1011-C-44 2.86
J1011-C-50 2.75
J1011-C-57 3.01
J1011-C-73 2.85
J1011-C-74 2.75
J1011-C-75 2.73
J1011-C-77 2.69
J1011-C-79 2.89
J1011-C-81 2.85
J1011-C-82 2.95
J1011-C-87 2.69
J1011-C-91 2.66
J1011-C-95 3.01
J1011-C-99 2.78
J1011-C-120 2.85
J1011-C-123 2.96
J1011-C-126 2.79
J1011-C-132 2.56
J1011-C-138 2.98
可以看出，根据本发明实施例构建的工程菌株具有高产番茄红素的性能
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (9)

  1. 一种微生物，其特征在于，过表达PaCrtE，PagCrtB和BtCrtI基因。
  2. 根据权利要求1所述的微生物，其特征在于，进一步过表达包括选自tHMG1，INO2，yap1，spt15-5，taf25-3，GapN，PYC2，SMAE1，MDH2，POS5，pntAB，ADH2，ACS6，ALD6，EUTE，ERG12，IDI1，ERG10，MVD1，ERG13，ERG8基因的至少之一。
  3. 根据权利要求1所述的微生物，其特征在于，进一步沉默包括选自GAL1，GAL7，GAL10，GAL80，ROX1，VBA5，DOS2，Ypl062W，Yjl064W，Yer130C，Yer134C，Ynr063W，Exg1，Yor292C，Sfk1，Mef1基因的至少之一。
  4. 根据权利要求1所述的微生物，其特征在于，所述微生物是酵母菌。
  5. 根据权利要求1所述的微生物，其特征在于，进一步包括可操作调控ERG9基因。
  6. 一种获得番茄红素的方法，其特征在于，包括：
    将权利要求1～5任一项所述的微生物进行发酵处理；以及
    将发酵处理产物进行萃取处理，以便获得所述番茄红素。
  7. 根据权利要求6所述的方法，其特征在于，所述发酵处理是通过如下方式实现的：
    将所述微生物进行基础发酵处理和两级分批补料发酵处理，所述基础发酵处理是在基本发酵培养基中进行的，所述两级分批补料发酵处理是通过在所述基本发酵培养基基础上依次补加第一补料培养基和第二补料培养基实现的，
    其中，所述基本发酵培养基为含有24g/L蛋白胨，12g/L酵母提取物和12g/L葡萄糖的YPD培养基；
    所述第一补料培养基为含有500g/L葡萄糖和15g/L酵母提取物的YPD培养基；
    所述第二补料培养基为75％乙醇和/或50％甘油。
  8. 根据权利要求6所述的方法，其特征在于，所述萃取处理包括：
    将所述发酵处理产物进行匀浆破碎或酶解破碎处理以及有机萃取处理。
  9. 权利要求1～5任一项所述微生物在制备番茄红素中的用途。
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