CN107586795A - A kind of method of fermentation by saccharomyces cerevisiae production phloretin - Google Patents

A kind of method of fermentation by saccharomyces cerevisiae production phloretin Download PDF

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CN107586795A
CN107586795A CN201710934514.8A CN201710934514A CN107586795A CN 107586795 A CN107586795 A CN 107586795A CN 201710934514 A CN201710934514 A CN 201710934514A CN 107586795 A CN107586795 A CN 107586795A
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CN107586795B (en
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陈贤情
刘晓楠
江会锋
王筱
王文
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Jiaxing Xin Baylet Biotechnology Co Ltd
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Jiaxing Xin Baylet Biotechnology Co Ltd
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Abstract

The present invention relates to a kind of method of fermentation by saccharomyces cerevisiae production phloretin, using para hydroxybenzene propionic acid as raw material, S. cervisiae is Host Strains, is completed by the catalysis of some enzymes in host bacterial.The invention difference from existing technology is：1) using cost degradation compound as raw material, the technique cost of raw material is low；2) to transform microorganism as technical foundation, in microbial body carrying out biology enzyme catalyzes and synthesizes, and avoids large-scale extracting and developing, purge process, from production cost and it is environmentally friendly in terms of be easily controlled；3) this technique is fewer than other method in the scale and quantity of production equipment, is easy to industry conversion；4) this technique only isolates and purifies process in the final step of production, and purifying products technique is simple, and product quality is more preferable than conventional method purity, content is higher；5) building-up process does not have discharging of waste liquid, environment-friendly, pollution-free, sustainable production.

Description

A kind of method of fermentation by saccharomyces cerevisiae production phloretin
Technical field
The present invention relates to a kind of biological synthesis method of phloretin, particularly a kind of fermentation by saccharomyces cerevisiae production root skin The method of element.
Background technology
Phloretin molecular formula：C15H14O5；English name：Phloretin；CAS：60-82-2；Molecular weight：274.28；Physics Property：Methanol, ethanol and acetone are soluble in, chloroform is slightly soluble in, is insoluble in water, petroleum ether and benzene, square prismatic or flaky crystal (second Alcohol or methanol).
Phloretin is a kind of external new type natural skin-whitening agents for researching and developing out recently, another natural after ursin Cosmetics, it is distributed mainly in the pericarp and root skin branches and leaves of the rich fruits such as apple, pears.It is mainly used as skin natural whiting Agent, it can suppress tyrosinase activity, melanin activity, have desalination effect to various skin splash.It is found according to current research Also anti-inflammatory, immunosupress, cardiovascular protection etc. act on.The whole world is no less than 20 tons per annual consumption, and demand is every year with 20% speed Increase, has the larger market demand.
At present, the technique of existing extraction phloretin, there are sour water solution, enzymolysis and directly extraction.It is more with shaddock ped in actual production Glycosides is raw material, and Raney Ni is catalyst, in sodium hydroxide solution, catalytic hydrogenation synthesis aurantiin dihydrochalcone, and Ran Hou In hydrochloric acid solution, aurantiin dihydrochalcone hydrolyzes to obtain phloretin.There is the discharge of chemical waste fluid in the method, chemical contamination is tight Weight.Also it is using apple skin as raw material, sour water solution extracts drying again after being extractant, concentrate by ethanol solution, obtains root Pi Su, the method uses sour water solution, seriously polluted.Present invention seek to address that this technical problem, there is provided a kind of conditioned response is gentle, The high phloretin biological synthesis method of combined coefficient.
The content of the invention
It is an object of the invention to solve the deficiencies in the prior art, there is provided a kind of side of fermentation by saccharomyces cerevisiae production phloretin Method, this method yield is high, pollution-free, with short production cycle.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of method of fermentation by saccharomyces cerevisiae production phloretin, the synthetic method are made using para hydroxybenzene propionic acid as raw material Brewer yeast bacterium is Host Strains, is completed by the catalysis of some enzymes in host bacterial；
The Host Strains build acquisition as follows：
1) according to saccharomyces cerevisiae codon preference optimization gene Ha4CL and EbCHS codon, then opened using two-way Mover, it is gene constructed in saccharomyces cerevisiae expression YCplac22 by two, carrier 1 is obtained, expression vector YCplac22 is carried The ammonia benzyl chloramphenicol resistance of Escherichia coli and the selection markers trp genes of saccharomyces cerevisiae；
2) malonic acid is converted into two genes RlmatB and RlmatC of malonyl coenzyme A according to saccharomyces cerevisiae codon Preference is optimized, and two genes then are implemented in into saccharomyces cerevisiae expression YCplac33 simultaneously, obtain carrier 2, table Up to ammonia benzyl mycin resistant genes of the carrier YCplac33 with Escherichia coli and the selection markers ura genes of saccharomyces cerevisiae；
3) above-mentioned carrier 1 and 2 is transferred in wild type Saccharomyces cerevisiae W303 simultaneously, according to the selection markers of saccharomyces cerevisiae Trp and ura genes, screening positive clone, then extract saccharomyces cerevisiae genome, further PCR checkings；
4) pathway key gene SeACS of the ethanol to malonyl coenzyme A will be strengthenedL641P, ScADH2, ScALD6, ScACC1S659A, s1157AThe YPRCdelta15 integration sites of the 16th article of chromosome of saccharomyces cerevisiae are incorporated into, while integrate leu screenings Marker gene, obtain Saccharomyces cerevisiae host bacterium.
The synthetic route of the fermentation by saccharomyces cerevisiae production phloretin of the present invention is as follows, as shown in Figure 1：
Preferably, in step 1) Ha4CL and EbCHS genes according to the amino acid sequence of gene in sunflower and fleabane flower come Synthesis.
Preferably, two genes RlmatB and RlmatC come from rhizobium leguminosarum in step 2).
Preferably, SeACS in step 4)L641PGene comes from salmonella, ScADH2, ScACC1S659A, S1157AAnd ScALD6 Gene comes from saccharomyces cerevisiae.
The method that fermentation by saccharomyces cerevisiae produces phloretin, the synthetic method step are as follows：
1) according to saccharomyces cerevisiae codon preference optimization gene Ha4CL and EbCHS codon, then opened using two-way Mover, it is gene constructed in saccharomyces cerevisiae expression YCplac22 by two, carrier 1 is obtained, expression vector YCplac22 is carried The ammonia benzyl chloramphenicol resistance of Escherichia coli and the selection markers trp genes of saccharomyces cerevisiae；
2) malonic acid is converted into two genes RlmatB and RlmatC of malonyl coenzyme A according to saccharomyces cerevisiae codon Preference is optimized, and two genes then are implemented in into saccharomyces cerevisiae expression YCplac33 simultaneously, obtain carrier 2, table Up to ammonia benzyl mycin resistant genes of the carrier YCplac33 with Escherichia coli and the selection markers ura genes of saccharomyces cerevisiae；
3) above-mentioned carrier 1 and 2 is transferred in wild type Saccharomyces cerevisiae W303 simultaneously, according to the selection markers of saccharomyces cerevisiae Trp and ura genes, screening positive clone, then extract saccharomyces cerevisiae genome, further PCR checkings；
4) pathway key gene SeACS of the ethanol to malonyl coenzyme A will be strengthenedL641P, ScADH2, ScALD6, ScACC1S659A, s1157AThe YPRCdelta15 integration sites of the 16th article of chromosome of saccharomyces cerevisiae are incorporated into, while integrate leu screenings Marker gene, obtain Saccharomyces cerevisiae host bacterium；
5) Saccharomyces cerevisiae host bacterium is cultivated, induced expression；
4) para hydroxybenzene propionic acid substrate is added in the bacterium solution after expression, continues to react 20-26h.
5) the phloretin extracting and developing in reaction solution is purified.
Preferably, phloretin is extracted from zymotic fluid using methanol, the volume ratio 1: 1 of methanol and zymotic fluid.
The gene that this patent is related to is At4CL, EbCHS, MdDBR, HaPAL and CcC4H (being shown in Table 1).
The species used in the patent of table 1 and gene brief introduction
The beneficial effects of the invention are as follows：The present invention prepares phloretin difference from prior art and is：
1) using cost degradation compound as raw material, the technique cost of raw material is low.
2) to transform microorganism as technical foundation, catalyzing and synthesizing for biology enzyme is carried out in microbial body, is avoided extensive Extracting and developing, purge process, from production cost and it is environmentally friendly in terms of be easily controlled.
3) this technique is fewer than other method in the scale and quantity of production equipment, simple to operate, is easy to industry and turns Change.
4) this technique only isolates and purifies process in the final step of production, and purifying products technique is simple, product quality ratio Conventional method purity is more preferable, content is higher.
5) building-up process does not have discharging of waste liquid, environment-friendly, pollution-free, sustainable production, and present invention process is from economy, ring Border and occupational health angle are that excellent industrialized production shows the way line.
Brief description of the drawings
Fig. 1 is the phloretin biosynthesis route map of the present invention；
Fig. 2 (a) is the YCplac22-Ha4CL-EbCHS plasmid maps in the present invention；
Fig. 2 (b) is the YCplac33-RlmatB-RlmatC plasmid maps in the present invention；
Fig. 3 is the liquid phase result figure of synthetic sample of the present invention；
Fig. 4 is the mass spectral results figure of synthetic sample of the present invention.
Embodiment
Below by specific embodiment, and with reference to accompanying drawing, technical scheme is described in further detail.With Under to the present invention principle and feature be described, the given examples are served only to explain the present invention, be not intended to limit the present invention Scope.
The plasmid construction of embodiment 1.
According to the nucleic acid of amino acid sequence and saccharomyces cerevisiae codon preference the design corresponding gene provided in the present invention Sequence, send commercial company to synthesize gene Ha4CL and EbCHS, the homology arm of design 20bp or so sizes, tried using one-step cloning Agent box, by two it is gene constructed obtain carrier 1 on carrier YCplac22, as shown in Fig. 2 (a), expression vector YCplac22 is carried The ammonia benzyl chloramphenicol resistance of Escherichia coli and the selection markers trp genes of saccharomyces cerevisiae；
The malonic acid for coming from rhizobium leguminosarum is converted into two genes RlmatB and RlmatC of malonyl coenzyme A Optimized according to saccharomyces cerevisiae codon preference, two genes are then implemented in saccharomyces cerevisiae expression simultaneously YCplac33, carrier 2 is obtained, as shown in Fig. 2 (b), expression vector YCplac33 carries the ammonia benzyl chloramphenicol resistance base of Escherichia coli The selection markers ura genes of cause and saccharomyces cerevisiae；
Above-mentioned carrier 1 and 2 is transferred in wild type Saccharomyces cerevisiae W303 simultaneously.
The conversion process of embodiment 2.
1) picking single bacterium colony shakes training 12h overnight in corresponding culture medium；
2) OD600 of the bacterium solution of culture value is measured with spectrophotometric；
3) it is transferred to by 0.2OD of first OD600 values in 50mL fresh YPAD culture mediums；
4) activation 4-5 hours allow saccharomyces cerevisiae to rise in value two generation bacterium solution OD600 values in 0.8-0.9；
3600rpm centrifugations 5min collects thalline, 25mL ddH2O washes (ddH used twice2O is preferably same day sterilizing)；
5) 1mL water is resuspended in sterile 1.5mL centrifuge tubes；(being used in super-clean bench)
6) 13000rpm centrifuges 30s and collects thalline；
7) often the μ L of pipe 100 are used to convert for 1mL water resuspension packing, and the bacterium solution of packing is centrifuged into 20s on palm centrifuge abandons On reset and add transformation system；
(operation must be completed in super-clean bench above, ensure gnotobasis)
8) transformation system matches：
PEG3350 (50% (W/V filtration sterilizations)) 240ul
LiAc 1.0M (filtration sterilization) 36ul
SSDNA(2.0mg/ml) 50ul
Cotransformation DNA fragmentation and water 34ul
Total 360ul；
9) fragment needed for cotransformation, add 400ng mixing by each fragment and mix；
10) thalline is resuspended with the transformation system mixed, 20min is incubated in 30 DEG C of incubators；
11) 42 DEG C of heat shock 40min apply corresponding screen plate, and inversion, which is put in 30 DEG C of incubators, cultivates 2-3 days, son to be transformed It is longer, monoclonal is selected, proposes genome checking；
The integration process of embodiment 3
1) saccharomyces cerevisiae genome is extracted, by the YPRCdelta15 integration site upstream and downstream of No. 16 chromosomes of saccharomyces cerevisiae Homology arm extracts fragment, fragment upstream 1, segments downstream 2 by PCR.
2) by fragment upstream and screening label leu genes, gene (including promoter and terminator) SeACSL641P, ScADH2, ScALD6, ScACClS659A, S1157AIt is transferred to together in saccharomyces cerevisiae W303 by homology arm, in saccharomyces cerevisiae body, Using homologous recombination, the genetic fragment of importing can be incorporated into the genome of saccharomyces cerevisiae by saccharomyces cerevisiae itself.
Embodiment 4 synthesizes phloretin
1) Saccharomyces cerevisiae host bacterium is cultivated, para hydroxybenzene propionic acid and malonic acid substrate is added in bacterium solution after incubation, React 120-160h；
2) the phloretin extracting and developing in reaction solution is purified, extracted from zymotic fluid phloretin using methanol, The volume ratio 1: 1 of methanol and zymotic fluid.
The liquid phase and mass spectral results for the sample that embodiment 4 synthesizes are as shown in Figure 3 and Figure 4.
Described is only presently preferred embodiments of the present invention, is not intended to limit the invention, every in the spiritual and former of the present invention Within then, any modification, equivalent substitution and improvements done etc. all should be in the row of protection scope of the present invention.
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Pro Ala Ile Ser Pro Gly Ala Val Gly Ala Ala Pro Gly Gly Pro Leu
1395 1400 1405
Gly Ala Pro Gly Leu Ala Leu Leu Ala Leu Ala Val Ser Ser Ala Gly
1410 1415 1420
Ile Ala Ile Ile Ile Leu Ala Pro Gly Thr Gly Ala Pro Val Pro Leu
1425 1430 1435 1440
Ala Ala Leu Ile Ala Ala Val Ser Gly Thr Val Ile Leu Thr Gly Met
1445 1450 1455
Thr Thr Gly Val Leu Ala Ala Leu Gly Gly Thr Val Pro Leu Ser Leu
1460 1465 1470
Gly Leu Pro Gly Ser Met His Leu Ala Pro Ile Ala Thr Pro Thr Pro
1475 1480 1485
Val Leu Gly Thr Leu Gly Pro Leu Ala Thr Leu Ala His Leu Met Gly
1490 1495 1500
Thr Thr Thr Val Thr Ala Pro Pro Gly Leu Pro Ala Gly Ala Ser Ser
1505 1510 1515 1520
Ser Gly Thr Leu Ala Pro Ser Ala Ala Val Leu Leu Thr Ala Ala Pro
1525 1530 1535
Pro Ile Ser Ala Gly Leu Ile Gly Ala Gly Ala Gly Gly Leu Thr Gly
1540 1545 1550
Val Gly Ala Gly Pro Gly Ala Ala Ala Ile Gly Met Val Ala Pro Leu
1555 1560 1565
Ile Thr Val Leu Thr Pro Gly Thr Pro Ala Gly Ala Gly Pro Val Val
1570 1575 1580
Val Ala Ala Ala Ile Thr Pro Leu Ile Gly Ser Pro Gly Pro Gly Gly
1585 1590 1595 1600
Ala Gly Pro Pro Ala Leu Val Thr Gly Thr Ala Ala Leu Ala Gly Ile
1605 1610 1615
Pro Ala Ile Thr Leu Ala Ala Ala Ser Gly Ala Ala Ile Gly Met Ala
1620 1625 1630
Gly Gly Ile Val Pro Leu Pro Gly Val Ala Thr Ala Ala Ala Ala Ala
1635 1640 1645
Pro Ala Leu Gly Pro Gly Thr Leu Thr Leu Thr Ser Gly Gly Met Gly
1650 1655 1660
Thr Leu Leu Leu Pro Ala Leu Gly Ala Ser Val Leu Thr Gly Ala Thr
1665 1670 1675 1680
Val Ile Ala Gly Gly Gly Ala Pro Val Ile Leu Thr Ile Ile Gly Ser
1685 1690 1695
Gly Ala Gly Leu Gly Val Gly Cys Leu Ala Gly Ser Gly Leu Ile Ala
1700 1705 1710
Gly Ala Thr Ser Ala Ala Thr His Ala Ile Pro Thr Ile Thr Leu Val
1715 1720 1725
Thr Cys Ala Ser Val Gly Ile Gly Ala Thr Leu Val Ala Leu Gly Gly
1730 1735 1740
Ala Ala Ile Gly Val Gly Gly Gly Pro Ile Ile Leu Thr Gly Ala Pro
1745 1750 1755 1760
Ala Ile Ala Leu Met Leu Gly Ala Gly Val Thr Thr Ser Ala Leu Gly
1765 1770 1775
Leu Gly Gly Thr Gly Ile Met Thr Ala Ala Gly Val Ser His Leu Thr
1780 1785 1790
Ala Val Ala Ala Leu Ala Gly Val Gly Leu Ile Val Gly Thr Met Ser
1795 1800 1805
Thr Val Pro Ala Leu Ala Ala Met Pro Val Pro Ile Leu Gly Thr Leu
1810 1815 1820
Ala Thr Thr Ala Ala Pro Val Ala Pro Thr Pro Thr Ala Ala Gly Thr
1825 1830 1835 1840
Thr Ala Val Ala Thr Met Ile Gly Gly Ala Gly Thr Gly Ser Gly Pro
1845 1850 1855
Gly Thr Gly Leu Pro Ala Leu Gly Ser Pro Pro Gly Thr Leu Ser Gly
1860 1865 1870
Thr Ala Leu Gly Val Val Val Gly Ala Ala Ala Leu Gly Gly Ile Pro
1875 1880 1885
Leu Gly Val Ile Gly Val Gly Thr Ala Thr Val Gly Ala Leu Ile Pro
1890 1895 1900
Ala Ala Pro Ala Ala Pro Ala Ser Ala Gly Thr Leu Ile Gly Gly Pro
1905 1910 1915 1920
Gly Gly Val Thr His Pro Ala Ser Ala Pro Leu Thr Ala Gly Ala Ile
1925 1930 1935
Ala Ala Pro Ala Ala Gly Gly Gly Leu Pro Met Met Ile Leu Ala Ala
1940 1945 1950
Thr Ala Gly Pro Ser Gly Gly Gly Ala Ala Met Pro Ala Gly Val Leu
1955 1960 1965
Leu Thr Gly Ser Pro Ile Val Ala Ala Leu Val Ala Thr Leu Gly Pro
1970 1975 1980
Ile Ile Ile Thr Ile Pro Pro Thr Gly Gly Leu Ala Gly Gly Ser Thr
1985 1990 1995 2000
Val Val Val Ala Pro Thr Ile Ala Ala Ala Gly Met Gly Met Thr Ala
2005 2010 2015
Ala Val Ala Ala Ala Ala Gly Val Leu Gly Pro Gly Gly Met Val Gly
2020 2025 2030
Ile Leu Pro Ala Ala Gly Leu Leu Leu Ala Thr Met Ala Ala Leu Ala
2035 2040 2045
Ala Leu Thr Ala Gly Leu Ala Ser Gly Leu Ser Ala Leu Ser Leu Ala
2050 2055 2060
Pro Gly Val His Gly Gly Ile Ser Leu Gly Leu Ala Ala Ala Gly Ala
2065 2070 2075 2080
Gly Leu Leu Pro Ile Thr Gly Gly Ile Ser Leu Gly Pro Ala Ala Leu
2085 2090 2095
His Ala Ala Ser Ser Ala Met Val Ala Leu Gly Val Ile Ser Leu Gly
2100 2105 2110
Leu Gly Thr Thr Gly Ala Ala Ala Pro Pro Pro Thr Ala Leu Ala Ala
2115 2120 2125
Ala Leu Ala Gly Gly Thr Leu Ile Leu Ala Leu Ser His Gly Val Gly
2130 2135 2140
Gly Ala Ser Ala Leu Gly Leu Ile Ala Ala Ile Ala Ser Thr Thr Pro
2145 2150 2155 2160
Ala Ser Val Ala His Gly Ala Ala Ala Gly Val Ala Thr Thr Ile Gly
2165 2170 2175
Gly Ala Thr Leu Thr Leu Ala Ala Leu Leu Leu Gly Leu Leu Leu Gly
2180 2185 2190
Ser Pro Ala Gly Ala Leu Ala Leu Leu Ile Ala Ser Ala His Ala Ala
2195 2200 2205
Ala Ile Ala Gly Leu Ser Gly Val Ile Leu Met Leu Ser Thr Ala Ala
2210 2215 2220
Leu Gly Leu Leu Leu Leu Thr Leu Leu
2225 2230
<210> 4
<211> 652
<212> PRT
<213>Saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 4
Met Ser Gly Thr His Leu His Ala Ile Pro Ala Ala Ile Ala Ala Ala
1 5 10 15
Cys Leu Ile Ala Pro Gly Gly Thr Gly Thr Leu Thr Leu Gly Ser Ile
20 25 30
Ala Ala Pro Ala Thr Pro Thr Gly Gly Gly Gly Leu Ile Leu Ala Thr
35 40 45
Ile Thr Pro Thr Gly Leu Val Leu Ala Thr Ser Pro Ala Pro Gly Ala
50 55 60
Val Ser Ile Leu Thr Thr Gly Ala Gly Thr Leu Ala Leu Ala Ala Ala
65 70 75 80
Cys Leu Ala Ala His Leu Gly Gly Ala Gly Ala Ala Thr Ala Ile Ile
85 90 95
Thr Gly Gly Ala Ala Ala Ser Gly Ser Leu His Ile Ser Thr Ala Gly
100 105 110
Leu His Ala Ala Val Cys Ala Pro Ala Ala Thr Leu Leu Ala Leu Gly
115 120 125
Ile Leu Leu Gly Ala Val Val Ala Ile Thr Met Pro Met Val Pro Gly
130 135 140
Ala Ala Val Ala Met Leu Ala Cys Ala Ala Ile Gly Ala Val His Ser
145 150 155 160
Val Ile Pro Gly Gly Pro Ser Pro Gly Ala Val Ala Gly Ala Ile Ile
165 170 175
Ala Ser Ser Ser Ala Leu Val Ile Thr Ala Ala Gly Gly Val Ala Ala
180 185 190
Gly Ala Ser Ile Pro Leu Leu Leu Ala Val Ala Ala Ala Leu Leu Ala
195 200 205
Pro Ala Val Thr Ser Val Gly His Val Ile Val Leu Leu Ala Thr Ala
210 215 220
Ser Ala Ile Ala Thr Gly Gly Gly Ala Ala Leu Thr Thr Ala Ala Leu
225 230 235 240
Ile Gly Leu Ala Ser Pro Gly His Gly Pro Gly Ala Met Ala Ala Gly
245 250 255
Ala Pro Leu Pro Ile Leu Thr Thr Ser Gly Ser Thr Gly Leu Pro Leu
260 265 270
Gly Val Leu His Thr Thr Gly Gly Thr Leu Val Thr Ala Ala Thr Thr
275 280 285
Pro Leu Thr Val Pro Ala Thr His Pro Gly Ala Ile Thr Thr Cys Thr
290 295 300
Ala Ala Val Gly Thr Val Thr Gly His Ser Thr Leu Leu Thr Gly Pro
305 310 315 320
Leu Ala Cys Gly Ala Thr Thr Leu Met Pro Gly Gly Val Pro Ala Thr
325 330 335
Pro Thr Pro Ala Ala Met Cys Gly Val Val Ala Leu His Gly Val Ala
340 345 350
Ile Leu Thr Thr Ala Pro Thr Ala Ile Ala Ala Leu Met Ala Gly Gly
355 360 365
Ala Leu Ala Ile Gly Gly Thr Ala Ala Ser Ser Leu Ala Ile Leu Gly
370 375 380
Ser Val Gly Gly Pro Ile Ala Pro Gly Ala Thr Gly Thr Thr Thr Leu
385 390 395 400
Leu Ile Gly Leu Gly Leu Cys Pro Val Val Ala Thr Thr Thr Gly Thr
405 410 415
Gly Thr Gly Gly Pro Met Ile Thr Pro Leu Pro Gly Ala Ile Gly Leu
420 425 430
Leu Ala Gly Ser Ala Thr Ala Pro Pro Pro Gly Val Gly Pro Ala Leu
435 440 445
Val Ala Ala Gly Gly His Pro Gly Gly Gly Ala Thr Gly Gly Ala Leu
450 455 460
Val Ile Thr Ala Ser Thr Pro Gly Gly Ala Ala Thr Leu Pro Gly Ala
465 470 475 480
His Gly Ala Pro Gly Gly Thr Thr Pro Ser Thr Pro Leu Ala Met Thr
485 490 495
Pro Ser Gly Ala Gly Ala Ala Ala Ala Gly Ala Gly Thr Thr Thr Ile
500 505 510
Thr Gly Ala Val Ala Ala Val Leu Ala Val Ser Gly His Ala Leu Gly
515 520 525
Thr Ala Gly Ile Gly Ser Ala Leu Val Ala His Pro Leu Ile Ala Gly
530 535 540
Ala Ala Val Val Gly Ile Pro His Ala Ile Leu Gly Gly Ala Ile Thr
545 550 555 560
Ala Thr Val Thr Leu Ala His Gly Gly Gly Pro Ser Pro Gly Leu Thr
565 570 575
Ala Gly Val Ala Ala Thr Val Ala Leu Gly Ile Gly Pro Leu Ala Thr
580 585 590
Pro Ala Val Leu His Thr Thr Ala Ser Leu Pro Leu Thr Ala Ser Gly
595 600 605
Leu Ile Met Ala Ala Ile Leu Ala Leu Ile Ala Ala Gly Ala Thr Ser
610 615 620
Ala Leu Gly Ala Thr Ser Thr Leu Ala Ala Pro Gly Val Val Gly Leu
625 630 635 640
Pro Leu Gly Gly Leu Gly Ala Ile Ala Met Pro Ser
645 650
<210> 5
<211> 348
<212> PRT
<213>Saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 5
Met Ser Ile Pro Gly Thr Gly Leu Ala Ile Ile Pro Thr Gly Ser Ala
1 5 10 15
Gly Leu Leu Gly His Leu Ala Ile Pro Val Pro Leu Pro Leu Pro Ala
20 25 30
Gly Leu Leu Ile Ala Val Leu Thr Ser Gly Val Cys His Thr Ala Leu
35 40 45
His Ala Thr His Gly Ala Thr Pro Leu Pro Thr Leu Leu Pro Leu Val
50 55 60
Gly Gly His Gly Gly Ala Gly Val Val Val Gly Met Gly Gly Ala Val
65 70 75 80
Leu Gly Thr Leu Ile Gly Ala Thr Ala Gly Ile Leu Thr Leu Ala Gly
85 90 95
Ser Cys Met Ala Cys Gly Thr Cys Gly Leu Gly Ala Gly Ser Ala Cys
100 105 110
Pro His Ala Ala Leu Ser Gly Thr Thr His Ala Gly Ser Pro Gly Gly
115 120 125
Thr Ala Thr Ala Ala Ala Val Gly Ala Ala His Ile Pro Gly Gly Thr
130 135 140
Ala Leu Ala Gly Val Ala Pro Ile Leu Cys Ala Gly Ile Thr Val Thr
145 150 155 160
Leu Ala Leu Leu Ser Ala Ala Leu Ala Ala Gly His Thr Ala Ala Ile
165 170 175
Ser Gly Ala Ala Gly Gly Leu Gly Ser Leu Ala Val Gly Thr Ala Leu
180 185 190
Ala Met Gly Thr Ala Val Leu Gly Ile Ala Gly Gly Pro Gly Leu Gly
195 200 205
Gly Leu Pro Thr Ser Leu Gly Gly Gly Val Pro Ile Ala Pro Thr Leu
210 215 220
Gly Leu Ala Ile Val Ser Ala Val Val Leu Ala Thr Ala Gly Gly Ala
225 230 235 240
His Gly Ile Ile Ala Val Ser Val Ser Gly Ala Ala Ile Gly Ala Ser
245 250 255
Thr Ala Thr Cys Ala Ala Ala Gly Thr Val Val Leu Val Gly Leu Pro
260 265 270
Ala Gly Ala Leu Cys Ser Ser Ala Val Pro Ala His Val Val Leu Ser
275 280 285
Ile Ser Ile Val Gly Ser Thr Val Gly Ala Ala Ala Ala Thr Ala Gly
290 295 300
Ala Leu Ala Pro Pro Ala Ala Gly Leu Val Leu Ser Pro Ile Leu Val
305 310 315 320
Val Gly Leu Ser Ser Leu Pro Gly Ile Thr Gly Leu Met Gly Leu Gly
325 330 335
Gly Ile Ala Gly Ala Thr Val Val Ala Thr Ser Leu
340 345
<210> 6
<211> 500
<212> PRT
<213>Saccharomyces cerevisiae (Saccharomyces cerevisiae)
<400> 6
Met Thr Leu Leu His Pro Ala Thr Ala Gly Pro Val Leu Ile Thr Leu
1 5 10 15
Pro Ala Gly Leu Thr Thr Gly Gly Pro Thr Gly Leu Pro Ile Ala Ala
20 25 30
Leu Pro Met Leu Ala Gly Ala Gly Leu Thr Thr Pro Val Gly Ala Pro
35 40 45
Ser Thr Gly Ala Thr Val Cys Gly Val Ser Ser Ala Thr Thr Gly Ala
50 55 60
Val Gly Thr Ala Ile Gly Cys Ala Ala Ala Ala Pro His Ala Thr Gly
65 70 75 80
Thr Ala Thr Gly Ala Pro Ala Gly Ala Gly Ala Leu Leu Ser Leu Leu
85 90 95
Ala Ala Gly Leu Gly Ser Gly Ile Ala Leu Val Ser Ser Ile Gly Ala
100 105 110
Leu Ala Ala Gly Leu Thr Leu Ala Leu Ala Ala Gly Ala Val Thr Ile
115 120 125
Ala Ile Ala Cys Leu Ala Ala Ala Ala Ala Thr Ala Ala Leu Val Ala
130 135 140
Gly Ala Thr Ile Ala Thr Gly Ala Gly Thr Met Ala Pro Thr Thr Leu
145 150 155 160
Gly Pro Ile Gly Val Cys Gly Gly Ile Ile Pro Thr Ala Pro Pro Ile
165 170 175
Met Met Leu Ala Thr Leu Ile Ala Pro Ala Leu Ala Met Gly Ala Val
180 185 190
Cys Ile Leu Leu Pro Ala Ala Val Thr Pro Leu Ala Ala Leu Thr Pro
195 200 205
Ala Ser Leu Cys Leu Leu Val Gly Ile Pro Ala Gly Val Val Ala Ile
210 215 220
Val Pro Gly Pro Gly Ala Thr Val Gly Ala Ala Leu Thr Ala Ala Pro
225 230 235 240
Ala Ile Ala Leu Leu Ala Pro Thr Gly Ser Thr Gly Val Gly Leu Ser
245 250 255
Val Ala Val Ala Ser Ser Gly Ser Ala Leu Leu Leu Ile Thr Leu Gly
260 265 270
Leu Gly Gly Leu Ser Ala His Leu Val Pro Ala Ala Ala Ala Ile Leu
275 280 285
Leu Thr Leu Pro Ala Leu Val Ala Gly Ile Pro Leu Ala Ala Gly Gly
290 295 300
Ile Cys Ser Ser Gly Ser Ala Ile Thr Val Gly Gly Gly Ile Thr Ala
305 310 315 320
Gly Leu Leu Ala Ala Pro Leu Ala Thr Leu Gly Thr Gly Ile Leu Val
325 330 335
Gly Ala Pro Pro Ala Leu Ala Ala Pro Gly Gly Ala Ile Thr Ala Ala
340 345 350
Gly Gly Pro Ala Thr Ile Met Ala Thr Ile Ala Ile Gly Leu Leu Gly
355 360 365
Gly Ala Leu Ile Leu Thr Gly Gly Gly Leu Val Gly Ala Leu Gly Thr
370 375 380
Pro Ile Ala Pro Thr Val Pro Thr Ala Val Ala Gly Ala Met Ala Ile
385 390 395 400
Val Leu Gly Gly Ile Pro Gly Pro Val Val Thr Val Ala Leu Pro Leu
405 410 415
Thr Leu Gly Gly Gly Val Gly Met Ala Ala Ser Ser Gly Pro Gly Leu
420 425 430
Gly Ser Gly Ile Gly Thr Gly Ser Leu Ser Thr Gly Leu Leu Val Ala
435 440 445
Leu Met Leu Leu Ala Gly Thr Val Thr Ile Ala Thr Thr Ala Ala Pro
450 455 460
Ala Ser Ala Val Pro Pro Gly Gly Val Leu Gly Ser Gly Thr Gly Ala
465 470 475 480
Gly Met Gly Gly Gly Val Thr His Ala Thr Thr Gly Val Leu Ala Val
485 490 495
Ala Ile Leu Leu
500

Claims (7)

  1. A kind of 1. method of fermentation by saccharomyces cerevisiae production phloretin, it is characterised in that the synthetic method is with para hydroxybenzene propionic acid It is raw material with malonic acid, S. cervisiae is Host Strains, is completed by the catalysis of some enzymes in host bacterial；
    The Host Strains build acquisition as follows：
    1) according to saccharomyces cerevisiae codon preference optimization gene Ha4CL and EbCHS codon, two-way startup is then utilized Son, it is gene constructed in saccharomyces cerevisiae expression YCplac22 by two, carrier 1 is obtained, expression vector YCplac22 is with big The ammonia benzyl chloramphenicol resistance of enterobacteria and the selection markers trp genes of saccharomyces cerevisiae；
    2) malonic acid is converted into two genes RlmatB and RlmatC of malonyl coenzyme A according to saccharomyces cerevisiae codon preference Property optimize, two genes are then implemented in saccharomyces cerevisiae expression YCplac33 simultaneously, obtain carrier 2, expression carries The selection markers ura genes of ammonia benzyl mycin resistant gene and saccharomyces cerevisiae of the body YCplac33 with Escherichia coli；
    3) above-mentioned carrier 1 and 2 is transferred in wild type Saccharomyces cerevisiae W303 simultaneously, according to the selection markers trp of saccharomyces cerevisiae and Ura genes, screening positive clone, then extract saccharomyces cerevisiae genome, further PCR checkings；
    4) pathway key gene SeACS of the ethanol to malonyl coenzyme A will be strengthenedL641P, ScADH2, ScALD6, ScACC1S659A , S1157AThe YPRCdelta15 integration sites of the 16th article of chromosome of saccharomyces cerevisiae are incorporated into, while integrate leu riddled basins, Obtain Saccharomyces cerevisiae host bacterium.
  2. 2. the method for fermentation by saccharomyces cerevisiae production phloretin according to claim 1, it is characterised in that the synthetic method Synthetic route it is as follows：
  3. 3. the method for fermentation by saccharomyces cerevisiae production phloretin according to claim 1, it is characterised in that in step 1) Ha4CL and EbCHS genes synthesize according to the amino acid sequence of gene in sunflower and fleabane flower.
  4. 4. the method for fermentation by saccharomyces cerevisiae production phloretin according to claim 1, it is characterised in that two in step 2) Gene RlmatB and RlmatC come from rhizobium leguminosarum.
  5. 5. the method for fermentation by saccharomyces cerevisiae production phloretin according to claim 1, it is characterised in that in step 4) SeACSL641PGene comes from salmonella, ScADH2, ScACC1S659A, S1157ASaccharomyces cerevisiae is come from ScALD6 genes.
  6. 6. the method for fermentation by saccharomyces cerevisiae production phloretin according to claim 1, it is characterised in that the synthetic method Step is as follows：
    1) according to saccharomyces cerevisiae codon preference optimization gene Ha4CL and EbCHS codon, two-way startup is then utilized Son, it is gene constructed in saccharomyces cerevisiae expression YCplac22 by two, carrier 1 is obtained, expression vector YCplac22 is with big The ammonia benzyl chloramphenicol resistance of enterobacteria and the selection markers trp genes of saccharomyces cerevisiae；
    2) malonic acid is converted into two genes RlmatB and RlmatC of malonyl coenzyme A according to saccharomyces cerevisiae codon preference Property optimize, two genes are then implemented in saccharomyces cerevisiae expression YCplac33 simultaneously, obtain carrier 2, expression carries The selection markers ura genes of ammonia benzyl mycin resistant gene and saccharomyces cerevisiae of the body YCplac33 with Escherichia coli；
    3) above-mentioned carrier 1 and 2 is transferred in wild type Saccharomyces cerevisiae W303 simultaneously, according to the selection markers trp of saccharomyces cerevisiae and Ura genes, screening positive clone, then extract saccharomyces cerevisiae genome, further PCR checkings；
    4) pathway key gene SeACS of the ethanol to malonyl coenzyme A will be strengthenedL641P, ScADH2, ScALD6, ScACC1S659A , S1157AThe YPRCdelta15 integration sites of the 16th article of chromosome of saccharomyces cerevisiae are incorporated into, while integrate leu riddled basins, Obtain Saccharomyces cerevisiae host bacterium；
    5) Saccharomyces cerevisiae host bacterium is cultivated, para hydroxybenzene propionic acid and malonic acid substrate, reaction is added in bacterium solution after incubation 120-160h。
    6) the phloretin extracting and developing in reaction solution is purified.
  7. 7. the method for fermentation by saccharomyces cerevisiae according to claim 6 production phloretin, it is characterised in that using methanol by root Pi Su extracts from zymotic fluid, the volume ratio 1: 1 of methanol and zymotic fluid.
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CN108004274A (en) * 2018-01-19 2018-05-08 嘉兴欣贝莱生物科技有限公司 A kind of fermentation by saccharomyces cerevisiae produces acrylic acid
CN108004274B (en) * 2018-01-19 2021-06-04 嘉兴欣贝莱生物科技有限公司 Method for producing acrylic acid by fermentation of saccharomyces cerevisiae
CN108384814A (en) * 2018-03-02 2018-08-10 重庆大学 A kind of preparation method of phloretin
CN108384814B (en) * 2018-03-02 2022-05-17 重庆大学 Preparation method of phloretin
CN109180457A (en) * 2018-08-08 2019-01-11 嘉兴欣贝莱生物科技有限公司 A kind of process for separating and purifying of biosynthesis phloretin
CN109180457B (en) * 2018-08-08 2021-10-26 嘉兴欣贝莱生物科技有限公司 Separation and purification process for biologically synthesizing phloretin
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CN110117550B (en) * 2019-01-09 2023-04-07 嘉兴欣贝莱生物科技有限公司 Process for producing phloretin based on saccharomyces cerevisiae fermentation and saccharomyces cerevisiae
CN110960452A (en) * 2019-12-26 2020-04-07 嘉兴欣贝莱生物科技有限公司 Yeast extract containing phloretin and preparation process and application thereof
CN112899314A (en) * 2021-02-04 2021-06-04 陕西师范大学 Method for promoting recombinant yarrowia lipolytica to synthesize phloretin
CN112899314B (en) * 2021-02-04 2022-06-17 陕西师范大学 Method for promoting recombinant yarrowia lipolytica to synthesize phloretin

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Inventor after: Chen Xianqing

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Inventor before: Liu Xiaonan

Inventor before: Jiang Huifeng

Inventor before: Wang Xiao

Inventor before: Wang Wen

PE01 Entry into force of the registration of the contract for pledge of patent right
PE01 Entry into force of the registration of the contract for pledge of patent right

Denomination of invention: A method for fermenting wine yeast to produce root bark extract

Granted publication date: 20211105

Pledgee: China Co. truction Bank Corp Jiaxing branch

Pledgor: JIAXING SYNBIOLAB BIOTECHNOLOGY CO.,LTD.

Registration number: Y2024330000750