CN114941006A - Streptomyces fuscosporivii recombinant expression plasmid, engineering bacterium and application - Google Patents
Streptomyces fuscosporivii recombinant expression plasmid, engineering bacterium and application Download PDFInfo
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- CN114941006A CN114941006A CN202111633710.4A CN202111633710A CN114941006A CN 114941006 A CN114941006 A CN 114941006A CN 202111633710 A CN202111633710 A CN 202111633710A CN 114941006 A CN114941006 A CN 114941006A
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12P19/44—Preparation of O-glycosides, e.g. glucosides
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly relates to a streptomyces limosus recombinant expression plasmid, an engineering bacterium and application. The invention successfully constructs an expression vector pSET152-groESp-accA by cloning a promoter groESp in a natamycin biosynthesis gene cluster of streptomyces fuscoporia and an acetyl coenzyme A carboxylase gene accA from an escherichia coli genome; the Streptomyces phaeoflavus engineering strain is obtained through a combined transfer experiment of Escherichia coli ET12567(pUZ8002) with an expression vector pSET152-groESp-accA and Streptomyces phaeoflavus, and the biosynthesis yield of natamycin is improved.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a streptomyces limonitidus recombinant expression plasmid, an engineering bacterium and application.
Background
Natamycin (Natamycin) is a natural, efficient and spectral antifungal agent, and can effectively inhibit the growth of yeast and mould at a lower dosage, thereby achieving an ideal preservative effect. Compared with artificial preservatives, natamycin has the characteristic of low solubility in water, so that natamycin can be applied to the preservation of the surface of food, and the flavor and the taste of the food are not influenced. In 1982, natamycin was officially approved by the FDA in the united states for use in food as a food preservative. In 1997, natamycin was officially approved as a preservative for food industry in our country. At present, natamycin is widely used in the production and preservation of food such as dairy products, meat products, fruit juice beverages and the like, and more than 50 countries which are approved for application are provided.
At present, the yield of natamycin is low, and researches show that the groESp promoter has low activity in primary metabolism and high activity in secondary metabolism, so that the primary metabolism of streptomyces is not influenced when the promoter is used, and the high-efficiency yield of related products is ensured in the secondary metabolism. The acetyl-CoA carboxylase gene accA can promote the conversion of acetyl-CoA to propionyl-CoA, thereby providing more short-chain fatty acids for the biosynthesis of natamycin. If the engineering strain is obtained through the expression of the gene, the yield of the natamycin can be greatly improved.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a streptomyces fuscoporia recombinant expression plasmid, engineering bacteria and application, wherein an expression vector pSET152-groESp-accA is successfully constructed by cloning a promoter groESp in a natamycin biosynthesis gene cluster of streptomyces fuscoporia and an acetyl coenzyme A carboxylase gene accA from an escherichia coli genome; through a combined transfer experiment of Enterobacter coli ET12567(pUZ8002) with an expression vector pSET152-groESp-accA and streptomyces fuscospora, the streptomyces fuscospora engineering strain is obtained, and the biosynthesis yield of natamycin is improved.
In one aspect, the invention provides a streptomyces fuscoporia recombinant expression plasmid, wherein a groESp promoter and an acetyl coenzyme A carboxylase gene accA are inserted into a pSET152 vector to construct a recombinant expression vector pSET 152-groESp-accA.
On the other hand, the invention provides an engineering bacterium containing the streptomyces fuscospora recombinant expression plasmid, which comprises the following preparation steps: mixing the escherichia coli containing the recombinant expression vector pSET152-groESp-accA with streptomyces fuseoflavus spores, uniformly coating the bacterial liquid on a culture medium containing MS, culturing for 13-20 h, then coating ddH2O, and continuously culturing for 3-5 days to screen out the engineering bacteria containing the recombinant expression vector pSET 152-groESp-accA.
The scheme is further improved, and the Escherichia coli and the Streptomyces phaeoflavus spores containing the recombinant expression vector pSET152-groESp-accA are mixed according to the ratio of 1: 1 mixing, and uniformly coating the bacterial solution on a mixture containing 10mM MgCL 2 The MS culture medium is cultured for 13-20 h at 29 ℃, then 1mL of ddH2O (containing 50 ug/mL-100 ug/mL nalidixic acid and 50ug/mL apramycin) is coated, and the engineering containing the recombinant expression vector pSET152-groESp-accA can be screened out after continuous culture for 3-5 days at 29 ℃.
On the other hand, the invention also provides application of the engineering bacteria in producing natamycin.
The preparation method for producing natamycin is as follows: preparing fresh spore suspension, inoculating to seed culture medium at 2-4% of inoculation amount, culturing at 29 deg.C and 200rpm for 2d, and inoculating to fermentation culture medium at 29 deg.C and 200rpm for fermentation culture with 5% of inoculation amount.
Wherein the seed culture medium comprises the following components in g/L: glucose 25, peptone 5, NaCl 10, pH 7.0.
The fermentation medium comprises the following components in g/L: glucose was 45, soytone was 10, pH 7.5.
The invention has the beneficial effects that:
(1) the expression vector pSET152-groESp-accA is successfully constructed by cloning a promoter groESp in a natamycin biosynthesis gene cluster of streptomyces fuscospora and an acetyl coenzyme A carboxylase gene accA from an escherichia coli genome; the engineering strain of the streptomyces flaviviridis is obtained by a combined transfer experiment of escherichia coli ET12567(pUZ8002) with an expression vector pSET152-groESp-accA and streptomyces flaviviridis; the groESp promoter in the biosynthesis gene cluster of the natamycin of the streptomyces fuscospora engineering strain is a more efficient promoter, the activity of the groESp promoter is weaker in primary metabolism, but the activity of the groESp promoter is higher than that of the promoter in the existing streptomyces in secondary metabolism, and the growth of the strain and the synthesis of the natamycin are facilitated. The acetyl-CoA carboxylase gene accA can promote the conversion of acetyl-CoA to propionyl-CoA, so that more short-chain fatty acids are provided for the biosynthesis of natamycin, and the superposition effect of a groESp promoter and the acetyl-CoA carboxylase gene accA further improves the biosynthesis of natamycin.
(2) The invention constructs a genetic engineering strain on the basis of a groESp promoter, realizes the synchronous growth of thalli and the synthesis of a target product natamycin in the process of secondary metabolism, ensures that more carbon flux is utilized in the secondary metabolism, greatly improves the yield of the natamycin, and improves the unit fermentation yield to 13.5g/L from the original 10 g/L.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A streptomyces fusciparum recombinant expression plasmid is characterized in that a groESp promoter and an acetyl coenzyme A carboxylase gene accA are inserted into a pSET152 vector to construct a recombinant expression vector pSET 152-groESp-accA.
Example 2
An engineering bacterium of the streptomyces fuscospora recombinant expression plasmid is prepared by the following steps:
will contain a recombinant expression vectorE.coli and S.fuscoporia spores of pSET152-groESp-accA were mixed in a 1: 1 mixing, and uniformly coating the bacterial solution on a mixture containing 8mM MgCL 2 The MS culture medium of (1) was cultured at 25 ℃ for 13 hours, then coated with 1mL of ddH2O (containing 50ug/mL to 100ug/mL nalidixic acid and 50ug/mL apramycin), and cultured at 25 ℃ for 3 days to screen out a cell containing the recombinant expression vector pSET 152-groESp-accA.
Example 3
The difference from the embodiment 2 lies in that the preparation steps of the engineering bacteria of the streptomyces limolividans recombinant expression plasmid are as follows:
coli containing the recombinant expression vector pSET152-groESp-accA was mixed with Streptomyces fuscoporia spores as follows: 1 mixing, and uniformly coating the bacterial solution on a mixture containing 15mM MgCL 2 The MS culture medium of (1) was cultured at 35 ℃ for 15 hours, then coated with 1mL of ddH2O (containing 50ug/mL to 100ug/mL nalidixic acid and 50ug/mL apramycin), and cultured at 35 ℃ for 4 days to screen out a cell containing the recombinant expression vector pSET 152-groESp-accA.
Example 4
The difference from the embodiment 2 lies in that the preparation steps of the engineering bacteria of the streptomyces limolividans recombinant expression plasmid are as follows:
e.coli containing the recombinant expression vector pSET152-groESp-accA was mixed with Streptomyces fuscoporia spores in a ratio of 1: 2 mixing, and uniformly coating the bacterial liquid on a mixture containing 10mM MgCL 2 The recombinant expression vector pSET152-groESp-accA can be screened by culturing the cells on the MS culture medium at 29 ℃ for 20h, then coating 1mLddH2O (containing 50ug/mL to 100ug/mL nalidixic acid and 50ug/mL apramycin), and continuously culturing the cells at 29 ℃ for 5 days.
Example 5
Whether the groESp promoter and the accA gene were successfully transferred into the genome of Streptomyces fuseoflavus was verified by PCR.
Example 6
The application of the engineering bacteria in the examples 2-4 to natamycin production.
The steps for producing natamycin are as follows: preparing fresh spore suspension, inoculating to seed culture medium at 3%, culturing at 29 deg.C and 200rpm for 2d, inoculating to fermentation culture medium at 5%, and fermenting at 29 deg.C and 200 rpm.
The seed culture medium comprises the following components in g/L: glucose 25, peptone 5, NaCl 10, pH 7.0.
The fermentation medium comprises the following components in g/L: glucose 45, soy peptone 10, pH 7.5.
Example 7
In contrast to example 6, the procedure for the production of natamycin is as follows: preparing fresh spore suspension, inoculating to seed culture medium at 2%, culturing at 25 deg.C and 200rpm for 1d, inoculating to fermentation culture medium at 3%, and fermenting at 25 deg.C and 200 rpm.
The seed culture medium comprises the following components in g/L: glucose 25, peptone 5, NaCl 10, pH 7.0.
The fermentation medium comprises the following components in g/L: glucose 45, soytone 10, pH 7.5.
Example 8
In contrast to example 6, the procedure for the production of natamycin is as follows: preparing fresh spore suspension, inoculating to seed culture medium at 5% inoculum size, culturing at 30 deg.C and 200rpm for 1d, inoculating to fermentation culture medium at 10% inoculum size, and fermenting at 30 deg.C and 200 rpm.
The seed culture medium comprises the following components in g/L: glucose 25, peptone 5, NaCl 10, pH 7.0.
The fermentation medium comprises the following components in g/L: glucose 45, soy peptone 10, pH 7.5.
Example 9
The fermentation yield in example 8 was measured, and the yield per unit fermentation was increased from 10g/L to 13.5g/L using Streptomyces fuscospora as a recombinant blank.
Although the present invention has been described in detail by way of the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Sequence listing
<110> Shandong Furuida Biotech Co., Ltd
<120> Streptomyces fuscosporuosus recombinant expression plasmid, engineering bacterium and application
<160> 3
<170> SIPOSequenceListing 1.0
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<211> 809
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cttccaccca tggccatgat ccgacctccc ccttcggaga tctcacgggg ttaactgtct 60
gaggtggcga ccaggtggat ccgtcgtcgc gggtgccgga cctgcccgtc gcgcgtagtt 120
ggcactcccc ggtggagagt gccaacgctg acactatgac gcggttagca ctcggtcaag 180
cggagtgcca atcggggtcc cggcctgtcg cccgggcagg gcgaataccg gccgcggggt 240
gcaggaattc ggacacacct ctcgcatcgg tggcaaccgt acgcaccgca cccacgtctt 300
ttcccgggac tgccgaaccg gatccggccc tgccgccagg gcccgcgact gcatgccgga 360
cgatgtagac cgacgacgta ccccccttcc ccgaggtctc caggaccagg gcagacccca 420
cagcggagga ctcgcgtgcg cggaccgacc agcgctttcc gggctgtccg gagaagggag 480
agcgacgtcg agagcaccat gaccttcgcc gctctcgccc aggctccgga ccgccgccgg 540
accgaggcct ggctgctcgc cctcgtcgtg ctgatcgccg tcttcggcta cgcctacgcc 600
ggcctgtcga tgaccggccg gctcccgtcc ggcctgcccc tcttcaccgt cgcgatgctc 660
tcggtcgcct ggctgccgca cctggtgctg cgccgcttcg cgccccgcgc cgacccgctg 720
ctcctgccgc tgacgacact gctgacggat ccgatatagg aatatcttcc acccatggcc 780
atgatccgat attcctatat cggatccgt 809
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gtgcgcaagg tgctcatcgc caatcgtggc gaaatcgctg tccgcgtggc ccgggcctgc 60
cgggacgccg ggatcgcgag cgtggccgtc tacgcggatc cggaccggga cgcgttgcac 120
gtccgtgccg ctgatgaggc gttcgccctg ggtggtgaca cccccgcgac cagctatctg 180
gacatcgcca aggtcctcaa agccgcgcgc gagtcgggcg cggacgccat ccaccccggc 240
tacgggttcc tctcggagaa cgccgagttc gcgcaggcgg tcctggacgc cggcctgatc 300
tggatcggcc cgcccccgca cgccatccgc gacctgggcg acaaggtcgc cgcccgccac 360
atcgcccagc gggccggcgc ccccctggtc gccggcaccc ccgaccccgt ctccggcgcg 420
gacgaggtcg tcgccttcgc caaggagcac ggcctgccca tcgccatcaa ggccgccttc 480
ggcggcggcg ggcgcggcct caaggtcgcc cgcaccctcg aagaggtgcc ggagctgtac 540
gactccgccg tccgcgaggc cgtggccgcc ttcggccgcg gggagtgctt cgtcgagcgc 600
tacctcgaca agccccgcca cgtggagacc cagtgcctgg ccgacaccca cggcaacgtg 660
gtcgtcgtct ccacccgcga ctgctccctc cagcgccgcc accaaaagct cgtcgaggag 720
gcccccgcgc ccttcctctc cgaggcccag acggagcagc tgtactcatc ctccaaggcc 780
atcctgaagg aggccggcta cgtcggcgcc ggcaccgtgg agttcctcgt cggcatggac 840
ggcacgatct ccttcctgga ggtcaacacc cgcctccagg tcgagcaccc ggtcaccgag 900
gaagtcgccg gcatcgacct ggtccgcgag atgttccgca tcgccgacgg cgaggaactc 960
ggctacgacg accccgccct gcgcggccac tccttcgagt tccgcatcaa cggcgaggac 1020
cccggccgcg gcttcctgcc cgcccccggc accgtcaccc tcttcgacgc gcccaccggc 1080
cccggcgtcc gcctggacgc cggcgtcgag tccggctccg tcatcggccc cgcctgggac 1140
tccctcctcg ccaaactgat cgtcaccggc cgcacccgcg ccgaggcact ccagcgcgcg 1200
gcccgcgccc tggacgagtt caccgtcgag ggcatggcca ccgccatccc cttccaccgc 1260
acggtcgtcc gcgacccggc cttcgccccc gaactcaccg gctccacgga ccccttcacc 1320
gtccacaccc ggtggatcga gacggagttc gtcaacgaga tcaagccctt caccacgccc 1380
gccgacaccg agacggacga ggagtcgggc cgggagacgg tcgtcgtcga ggtcggcggc 1440
aagcgcctgg aagtctccct cccctccagc ctgggcatgt ccctggcccg caccggcctg 1500
gccgccgggg cccgccccaa gcgccgcgcg gccaagaagt ccggccccgc cgcctcgggc 1560
gacaccctcg cctccccgat gcagggcacg atcgtcaaga tcgccgtcga ggagggccag 1620
gaagtccagg aaggcgacct catcgtcgta ctcgaggcga tgaagatgga acagcccctc 1680
aacgcccaca ggtccggcac catcaagggc ctcaccgccg aggtcggcgc ctccctcacc 1740
tccggcgccg ccatctgcga gatcaaggac tga 1773
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<211> 5715
<212> DNA
<213> pSET152
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atctacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca 60
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cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttg atcggcactt 180
tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattta tgcggtgtga 240
aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct 300
gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 360
agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 420
ttgtaaaacg acggccagtg ccaagcttgg gctgcaggtc gactctagag gatccgcggc 480
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gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 600
atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 660
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 720
tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 780
agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 840
aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 900
gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 960
tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 1020
cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 1080
ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 1140
cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 1200
atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 1260
agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 1320
gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 1380
gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 1440
tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 1500
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gattttggtc atgagattat caaaaaggat cttcacctag atccttttgg ttcatgtgca 1620
gctccatcag caaaagggga tgataagttt atcaccaccg actatttgca acagtgccgt 1680
tgatcgtgct atgatcgact gatgtcatca gcggtggagt gcaatgtcgt gcaatacgaa 1740
tggcgaaaag ccgagctcat cggtcagctt ctcaaccttg gggttacccc cggcggtgtg 1800
ctgctggtcc acagctcctt ccgtagcgtc cggcccctcg aagatgggcc acttggactg 1860
atcgaggccc tgcgtgctgc gctgggtccg ggagggacgc tcgtcatgcc ctcgtggtca 1920
ggtctggacg acgagccgtt cgatcctgcc acgtcgcccg ttacaccgga ccttggagtt 1980
gtctctgaca cattctggcg cctgccaaat gtaaagcgca gcgcccatcc atttgccttt 2040
gcggcagcgg ggccacaggc agagcagatc atctctgatc cattgcccct gccacctcac 2100
tcgcctgcaa gcccggtcgc ccgtgtccat gaactcgatg ggcaggtact tctcctcggc 2160
gtgggacacg atgccaacac gacgctgcat cttgccgagt tgatggcaaa ggttccctat 2220
ggggtgccga gacactgcac cattcttcag gatggcaagt tggtacgcgt cgattatctc 2280
gagaatgacc actgctgtga gcgctttgcc ttggcggaca ggtggctcaa ggagaagagc 2340
cttcagaagg aaggtccagt cggtcatgcc tttgctcggt tgatccgctc ccgcgacatt 2400
gtggcgacag ccctgggtca actgggccga gatccgttga tcttcctgca tccgccagag 2460
gcgggatgcg aagaatgcga tgccgctcgc cagtcgattg gctgagctca tgagcggaga 2520
acgagatgac gttggagggg caaggtcgcg ctgattgctg gggcaacacg tggagcggat 2580
cggggattgt ctttcttcag ctcgctgatg atatgctgac gctcaatgcc gtttggcctc 2640
cgactaacga aaatcccgca tttggacggc tgatccgatt ggcacggcgg acggcgaatg 2700
gcggagcaga cgctcgtccg ggggcaatga gatatgaaaa agcctgaact caccgcgacg 2760
tatcgggccc tggccagcta gctagagtcg acctgcaggt ccccggggat cggtcttgcc 2820
ttgctcgtcg gtgatgtact tcaccagctc cgcgaagtcg ctcttcttga tggagcgcat 2880
ggggacgtgc ttggcaatca cgcgcacccc ccggccgttt tagcggctaa aaaagtcatg 2940
gctctgccct cgggcggacc acgcccatca tgaccttgcc aagctcgtcc tgcttctctt 3000
cgatcttcgc cagcagggcg aggatcgtgg catcaccgaa ccgcgccgtg cgcgggtcgt 3060
cggtgagcca gagtttcagc aggccgccca ggcggcccag gtcgccattg atgcgggcca 3120
gctcgcggac gtgctcatag tccacgacgc ccgtgatttt gtagccctgg ccgacggcca 3180
gcaggtaggc cgacaggctc atgccggccg ccgccgcctt ttcctcaatc gctcttcgtt 3240
cgtctggaag gcagtacacc ttgataggtg ggctgccctt cctggttggc ttggtttcat 3300
cagccatccg cttgccctca tctgttacgc cggcggtagc cggccagcct cgcagagcag 3360
gattcccgtt gagcaccgcc aggtgcgaat aagggacagt gaagaaggaa cacccgctcg 3420
cgggtgggcc tacttcacct atcctgcccg gctgacgccg ttggatacac caaggaaagt 3480
ctacacgaac cctttggcaa aatcctgtat atcgtgcgaa aaaggatgga tataccgaaa 3540
aaatcgctat aatgaccccg aagcagggtt atgcagcgga aaagatccgt cgacctgcag 3600
gcatgcaagc tctagcgatt ccagacgtcc cgaaggcgtg gcgcggcttc cccgtgccgg 3660
agcaatcgcc ctgggtgggt tacacgacgc ccctctatgg cccgtactga cggacacacc 3720
gaagccccgg cggcaaccct cagcggatgc cccggggctt cacgttttcc caggtcagaa 3780
gcggttttcg ggagtagtgc cccaactggg gtaacctttg agttctctca gttgggggcg 3840
tagggtcgcc gacatgacac aaggggttgt gaccggggtg gacacgtacg cgggtgctta 3900
cgaccgtcag tcgcgcgagc gcgagaattc gagcgcagca agcccagcga cacagcgtag 3960
cgccaacgaa gacaaggcgg ccgaccttca gcgcgaagtc gagcgcgacg ggggccggtt 4020
caggttcgtc gggcatttca gcgaagcgcc gggcacgtcg gcgttcggga cggcggagcg 4080
cccggagttc gaacgcatcc tgaacgaatg ccgcgccggg cggctcaaca tgatcattgt 4140
ctatgacgtg tcgcgcttct cgcgcctgaa ggtcatggac gcgattccga ttgtctcgga 4200
attgctcgcc ctgggcgtga cgattgtttc cactcaggaa ggcgtcttcc ggcagggaaa 4260
cgtcatggac ctgattcacc tgattatgcg gctcgacgcg tcgcacaaag aatcttcgct 4320
gaagtcggcg aagattctcg acacgaagaa ccttcagcgc gaattgggcg ggtacgtcgg 4380
cgggaaggcg ccttacggct tcgagcttgt ttcggagacg aaggagatca cgcgcaacgg 4440
ccgaatggtc aatgtcgtca tcaacaagct tgcgcactcg accactcccc ttaccggacc 4500
cttcgagttc gagcccgacg taatccggtg gtggtggcgt gagatcaaga cgcacaaaca 4560
ccttcccttc aagccgggca gtcaagccgc cattcacccg ggcagcatca cggggctttg 4620
taagcgcatg gacgctgacg ccgtgccgac ccggggcgag acgattggga agaagaccgc 4680
ttcaagcgcc tgggacccgg caaccgttat gcgaatcctt cgggacccgc gtattgcggg 4740
cttcgccgct gaggtgatct acaagaagaa gccggacggc acgccgacca cgaagattga 4800
gggttaccgc attcagcgcg acccgatcac gctccggccg gtcgagcttg attgcggacc 4860
gatcatcgag cccgctgagt ggtatgagct tcaggcgtgg ttggacggca gggggcgcgg 4920
caaggggctt tcccgggggc aagccattct gtccgccatg gacaagctgt actgcgagtg 4980
tggcgccgtc atgacttcga agcgcgggga agaatcgatc aaggactctt accgctgccg 5040
tcgccggaag gtggtcgacc cgtccgcacc tgggcagcac gaaggcacgt gcaacgtcag 5100
catggcggca ctcgacaagt tcgttgcgga acgcatcttc aacaagatca ggcacgccga 5160
aggcgacgaa gagacgttgg cgcttctgtg ggaagccgcc cgacgcttcg gcaagctcac 5220
tgaggcgcct gagaagagcg gcgaacgggc gaaccttgtt gcggagcgcg ccgacgccct 5280
gaacgccctt gaagagctgt acgaagaccg cgcggcaggc gcgtacgacg gacccgttgg 5340
caggaagcac ttccggaagc aacaggcagc gctgacgctc cggcagcaag gggcggaaga 5400
gcggcttgcc gaacttgaag ccgccgaagc cccgaagctt ccccttgacc aatggttccc 5460
cgaagacgcc gacgctgacc cgaccggccc taagtcgtgg tgggggcgcg cgtcagtaga 5520
cgacaagcgc gtgttcgtcg ggctcttcgt agacaagatc gttgtcacga agtcgactac 5580
gggcaggggg cagggaacgc ccatcgagaa gcgcgcttcg atcacgtggg cgaagccgcc 5640
gaccgacgac gacgaagacg acgcccagga cggcacggaa gacgtagcgg cgtagcgaga 5700
cacccgggaa gcctg 5715
Claims (8)
1. A streptomyces fusciparum recombinant expression plasmid is characterized in that: the pSET152 vector was inserted with the groESp promoter and the acetyl-CoA carboxylase gene accA to construct a recombinant expression vector pSET 152-groESp-accA.
2. An engineered bacterium comprising the Streptomyces fuscospora recombinant expression plasmid of claim 1.
3. The engineering bacterium containing streptomyces fuscoporia recombinant expression plasmid according to claim 2, characterized in that: mixing the escherichia coli containing the recombinant expression vector pSET152-groESp-accA with streptomyces fuseoflavus spores, uniformly coating the bacterial liquid on a culture medium containing MS, culturing for 13-20 h, then coating ddH2O, and continuously culturing for 3-5 days to screen out the engineering bacteria containing the recombinant expression vector pSET 152-groESp-accA.
4. The engineering bacterium containing streptomyces fuscoporia recombinant expression plasmid according to claim 3, characterized in that: e.coli containing the recombinant expression vector pSET152-groESp-accA was mixed with Streptomyces fuscoporia spores in a ratio of 1: 1 mixing, and uniformly coating the bacterial solution on a mixture containing 10mM MgCL 2 The MS culture medium is cultured for 13-20 h at 29 ℃, then 1mLddH2O is coated, and the culture is continued for 3-5 days at 29 ℃, thus screening the engineering containing the recombinant expression vector pSET 152-groESp-accA.
5. Use of the engineered bacteria of any one of claims 1 to 4 for the production of natamycin.
6. The use of the engineered bacteria of claim 5 for the production of natamycin, characterized in that: preparing fresh spore suspension, inoculating to seed culture medium at 2-4%, culturing at 29 deg.C and 200rpm for 2 days, inoculating to fermentation culture medium at 5%, and fermenting at 29 deg.C and 200 rpm.
7. The use of the engineered bacteria of claim 6 for the production of natamycin, characterized in that: the seed culture medium comprises the following components in g/L: glucose 25, peptone 5, NaCl 10, pH 7.0.
8. The use of the engineered bacteria of claim 6 to produce natamycin, which is characterized by: the fermentation medium comprises the following components in g/L: glucose was 45, soy peptone was 10, pH 7.5.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117363552A (en) * | 2023-10-19 | 2024-01-09 | 山东福瑞达医药集团有限公司 | Streptomyces luteolin genetic engineering strain for high yield of natamycin and construction method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117363552A (en) * | 2023-10-19 | 2024-01-09 | 山东福瑞达医药集团有限公司 | Streptomyces luteolin genetic engineering strain for high yield of natamycin and construction method thereof |
| CN117363552B (en) * | 2023-10-19 | 2024-04-16 | 山东福瑞达医药集团有限公司 | Streptomyces luteolin gene engineering strain for producing natamycin and construction method thereof |
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