CN103740633B - Recombinant bacterium and the application thereof of Lyeopene are produced in one strain - Google Patents
Recombinant bacterium and the application thereof of Lyeopene are produced in one strain Download PDFInfo
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Abstract
The invention discloses recombinant bacterium and application thereof that Lyeopene is produced in a strain.Recombinant bacterium 1 disclosed by the invention, builds according to the method comprised the steps: the enzyme improving yak base yak base diphosphate synthase in recombination bacillus coli is lived; The method that in described raising recombination bacillus coli, the enzyme of yak base yak base diphosphate synthase is lived is that the original controlling element of the yak base yak base diphosphate synthase gene crtE in described recombination bacillus coli is replaced with artificial regulatory element 1.Bacterial strain yield of lycopene disclosed by the invention is high, has very high production application and is worth.
Description
Technical field
The present invention relates to recombinant bacterium and application thereof that Lyeopene is produced in a strain.
Background technology
Lyeopene is the main component forming tomato red pigments, and belonging to " carotenoid " material, is a kind of outstanding antioxidant.The cancellation effect of Lyeopene to singlet oxygen is 2 times of β-carotene, 100 times of vitamin-E.Lyeopene also has function of anti-cancer and cancer prevention, enhancing body immunizing power and anti-ageing physiological function of waiting for a long time, and has important use at food, makeup and field of medicaments.The injury that Lyeopene can prevent meta-bolites " radical " from causing human tissue organ, because it has anti-radical effect, therefore can be used as natural health care or medicine, current European market there is the tens of kinds of protective foodss containing Lyeopene and medicine.Protective foods containing Lyeopene can prevent senile vision degeneration, anti-ageing and prevention cardiovascular diseases, and Lyeopene also all has certain restraining effect to digestive tract cancer, cervical cancer, mammary cancer, skin carcinoma, bladder cancer etc.Lyeopene is nontoxic, and it is the same with β-carotene adds in the food such as ice-creams, fruit syrup, hard candy, bread, biscuit, cake, can improve its nutritive value.The research and development of its related products in recent years have become the focus of new drug research in the world.
The production of Lyeopene mainly contains the methods such as natural extract, chemosynthesis, fermentable in the world at present.Content of lycopene in tomato is very low, only containing 20 grams of Lyeopenes in usual tomato per ton; And natural extract method productive rate is low, expensive, cannot satisfy the demands.Though chemical synthesis cost declines to some extent but there is certain insecurity.By contrast, microbe fermentation method is not subject to the restriction of raw material, production process green cleans, and has very large advantage.Along with the fast development of biotechnology, utilizing fermentable to produce Lyeopene becomes one of current study hotspot.
Microbe fermentation method divides again two kinds.One uses wild strain to ferment.The microorganism that current fermentative production Lyeopene is relatively commonly used is trispore Bruce mould (Blakesleatrispora), and output is at about 1g/L, but fermentation period is longer.Another kind builds recombinant microorganism to ferment.Along with the development that metabolic engineering and synthesising biological in recent years learn a skill, the kind of recombinant microorganism synthesis terpenoid and output are in continuous lifting.Because intestinal bacteria (Escherichiacoli) genome sequence is announced completely, genetic background and pathways metabolism are all perfectly clear, have substratum requirement simple, grow the advantages such as rapid, become the most frequently used recombinant microorganism.At present; a lot of research is all in intestinal bacteria, introduce yak base yak base tetra-sodium (Geranyl-geranyldiphosphate; GGPP) synthase gene (crtE), phytoene desaturase gene (phytoenedesaturase; crtI); phytoene synthase gene (Phytoenesynthase; crtB) gene, and then (Ajikumaretal., 2008 are transformed to it; Dasetal., 2007; Keasling, 2008; Leeetal., 2002).Fig. 1 is the route of synthesis that after introducing Lyeopene synthetic gene, intestinal bacteria generate Lyeopene.
The method utilizing metabolic engineering to improve yield of lycopene at present mainly improves the expression intensity of pathways metabolism key gene, improve recombination bacillus coli synthesis Lyeopene precursor substance isopentenylpyrophosphate (Isopentenyldiphosphate, and dimethyl propylene thiazolinyl tetra-sodium (Dimethylallylpyrophosphate IPP), DMAPP) ability, thus improve Lyeopene synthesis capability.
The scheme improving recombination bacillus coli synthesis IPP and DMAPP ability mainly contains two kinds: a kind of is the mevalonate pathway (MevalonicAcidPathway, MVA approach) introducing external source, as MVA approach (Martinetal., 2003 of yeast saccharomyces cerevisiae; Yoonetal., 2007; Yoonetal., 2009); Another kind is by improving the methylerythritolphosphate(MEP of intestinal bacteria self) in approach the expression intensity of key gene and the supply that improves MEP approach precursor compound glyceraldehyde 3-phosphate and pyruvic acid improve efficiency (Ajikumaretal., 2010 of intestinal bacteria self MEP approach; Alperetal., 2005a; Alperetal., 2005b; Choietal., 2010; JinandStephanopoulos, 2007; Yuanetal., 2006).The overexpression isopentenylpyrophosphate isomerase genes (idi) such as Wang and 1-deoxidation-xylulose-5-phosphate synthase gene (dxs), and the crtE of ancient green-ball bacterium (Archaeoglobus.fulgidus) of being glimmered by orthogenesis optimization, the output of Lyeopene is made to be improved largely (Wangetal., 2000).Kim etc. express idi gene by high copy number plasmid, low copy plasmid expression Lyeopene synthetic gene, and by the output increased of Lyeopene to 260mg/L, unit cell content reaches 5.7mg/gDCW(Kimetal., and 2009).Yoon etc. are with the downstream part gene of the MVA approach of plasmid form overexpression streptococcus pneumoniae (Streptococcuspneumoniae), and add mevalonic acid as the middle precursor substance producing Lyeopene, by the output increased of Lyeopene to 102mg/L, unit cell content reaches 22mg/gDCW(Yoonetal., and 2006).Kim etc. are with plasmid form overexpression idi and dxs gene, and 3-Hydroxymethylglutaryl list acyl CoA synthase gene (mvaS) introduced from enterococcus faecalis (Enterococcusfaecalis), 3-Hydroxymethylglutaryl list acyl CoA reductase gene (mvaA), from the Mevalonic kinase gene (mvaK1) of S.pneumoniae, Phosphomevalonic kinase gene (mvaK2) and bisphosphate mevalonic acid dehydrase gene (mvaD), with in the minimal medium that is carbon source containing 10g/L glucose and 7.5g/L pectinose, high density fermentation, yield of lycopene reaches 1350mg/L, unit cell content reaches 32mg/gDCW(Kimetal., 2011).Cho etc. are on the basis of introducing crtEIB, the plasmid of mvaA, mvaS, mvaK1, mvaK2 and mvaD5 the foreign gene carrying MVA approach is with the addition of in recombination bacillus coli, Lyeopene unit cell content is brought up to 38.1mg/gDCW (Choetal., 2010).
Up to the present, the intestinal bacteria of bibliographical information produce yield of lycopene and reach as high as 1350mg/L, and unit cell content is up to 38.1mg/gDCW.In order to reduce the cost of fermentative Production Lyeopene, also need the output and the throughput rate that improve Lyeopene further.
Summary of the invention
The object of this invention is to provide recombinant bacterium and application thereof that Lyeopene is produced in a strain.
Recombinant bacterium 1 provided by the invention, builds according to the method comprised the steps: the enzyme improving yak base yak base diphosphate synthase in recombination bacillus coli is lived;
The method that in described raising recombination bacillus coli, the enzyme of yak base yak base diphosphate synthase is lived is that the original controlling element of the yak base yak base diphosphate synthase gene crtE in described recombination bacillus coli is replaced with artificial regulatory element 1;
The original controlling element of described yak base yak base diphosphate synthase gene crtE is as shown in SEQIDNo.10;
The nucleotide sequence of described artificial regulatory element 1 is as shown in SEQIDNo.11;
The construction process of described recombination bacillus coli is as follows:
(1) the zeaxanthin glycosyltransferase gene crtX in β-carotene synthetic gene in intestinal bacteria CAR001 bunch and lycopene beta cyclase gene crtY is replaced to artificial RBS sequence;
The sequence of described zeaxanthin glycosyltransferase gene crtX is as shown in SEQIDNo.1;
The sequence of described lycopene beta cyclase gene crtY is as shown in SEQIDNo.2;
Described artificial RBS sequence is as shown in SEQIDNo.3;
Described β-carotene synthetic gene bunch is the gene cluster be made up of yak base yak base diphosphate synthase gene crtE, zeaxanthin glycosyltransferase gene crtX, lycopene beta cyclase gene crtY, phytoene desaturase gene crtI and phytoene synthase gene crtB;
(2) enzyme improving ketoglurate dehydrogenase in step (1) recombination bacillus coli that obtains is lived;
The original controlling element of the alph-ketoglutaric acid dehydrase gene sucAB that the method that in the recombination bacillus coli that described raising step (1) obtains, ketoglurate dehydrogenase enzyme is lived is specially in the recombination bacillus coli described step (1) obtained replaces with artificial regulatory element M1-46;
The original controlling element of described alph-ketoglutaric acid dehydrase gene sucAB is as shown in SEQIDNo.4;
(3) enzyme improving transaldolase in step (2) recombination bacillus coli that obtains is lived;
The original controlling element of the transaldolase gene talB that the method that in the recombination bacillus coli that described raising step (2) obtains, the enzyme of transaldolase is lived is specially in the recombination bacillus coli described step (2) obtained replaces with artificial regulatory element M1-46;
The original controlling element of described transaldolase gene talB is as shown in SEQIDNo.6;
(4) enzyme improving succinate dehydrogenase in step (3) recombination bacillus coli that obtains is lived, and obtains object recombination bacillus coli;
The original controlling element of the succinate dehydrogenase gene sdhABCD that the method that in the recombination bacillus coli that described raising step (3) obtains, the enzyme of succinate dehydrogenase is lived is specially in the recombination bacillus coli described step (3) obtained replaces with artificial regulatory element M1-46;
The original controlling element of described succinate dehydrogenase gene sdhABCD is as shown in SEQIDNo.7;
The nucleotide sequence of described artificial regulatory element M1-46 is as shown in SEQIDNo.5.
Recombinant bacterium 2 also belongs to protection scope of the present invention, builds according to the method comprised the steps: the enzyme of 1-deoxidation in the recombinant bacterium 1 described in raising-xylulose-5-phosphate synthase is lived;
The method of the enzyme work of 1-deoxidation in described raising recombinant bacterium 1-xylulose-5-phosphate synthase is specially and the original controlling element of the 1-deoxidation in described recombination bacillus coli-xylulose-5-phosphate synthase gene dxs is replaced with artificial regulatory element 2;
The original controlling element of described 1-deoxidation-xylulose-5-phosphate synthase gene dxs is as shown in SEQIDNo.8;
The nucleotide sequence of described artificial regulatory element 2 is as shown in SEQIDNo.12.
Recombinant bacterium 3 also belongs to protection scope of the present invention, builds: the original controlling element of the isovaleryl tetra-sodium isomerase gene idi in recombination bacillus coli is replaced with artificial regulatory element X, obtains object recombinant bacterium 3 according to the method comprised the steps;
The original controlling element of described isovaleryl tetra-sodium isomerase gene idi is as shown in SEQIDNo.5;
The nucleotide sequence of described artificial regulatory element X is as shown in SEQIDNo.9;
Described recombination bacillus coli is described recombinant bacterium 2.
Recombinant bacterium 4 also belongs to protection scope of the present invention, builds: the original controlling element of the isovaleryl tetra-sodium isomerase gene idi in recombination bacillus coli is replaced with artificial regulatory element 4 according to the method comprised the steps;
The original controlling element of described isovaleryl tetra-sodium isomerase gene idi is as shown in SEQIDNo.5;
The nucleotide sequence of described artificial regulatory element 4 is as shown in SEQIDNo.13;
Described recombination bacillus coli is described recombinant bacterium 2.
Recombinant bacterium 5 also belongs to protection scope of the present invention, the intestinal bacteria of this bacterium to be deposit number be CGMCCNo.8238.
The method of producing Lyeopene also belongs to protection scope of the present invention: comprise the steps:, by an above-mentioned arbitrary described recombinant bacterium fermentation culture, to obtain Lyeopene.
In aforesaid method, it is as follows that the fermention medium in described fermentation culture often rises composition:
10g glycerine, 1.7g citric acid, 10.5gKH
2pO
43H
2o, 6g (NH
4)
2hPO
4, 3.44gMgSO
47H
2o, 10ml trace element solution, surplus is water;
Often liter of composition of described trace element solution is as follows: 10gFeSO
47H
2o, 5.25gZnSO
47H
2o, 3.0gCuSO
45H
2o, 0.5gMnSO
44H
2o, 0.23gNa
2b
4o
710H
2o, 2.0gCaC1
2, 0.1g (NH
4)
6mo
7o
24, surplus is water.
The condition of described fermentation is specially temperature 30 DEG C, and air flow quantity is 4L/min, and dissolved oxygen is 30%, pH is 7.0;
Described pH carries out regulating particular by ammoniacal liquor;
In described fermentation, glycerol concentration remains on below 0.2g/L, and the output of acetic acid maintains below 0.1g/L.
In above-mentioned arbitrary described method, described method also comprises the step of adding supplemented medium, and it is as follows that described supplemented medium often rises composition: 500g glycerine, 15g peptone, 30g yeast extract, 30gMgSO
47H
2o, surplus is water.
The application of above-mentioned arbitrary described recombinant bacterium in production Lyeopene also belongs to protection scope of the present invention.
Bacterial strain yield of lycopene provided by the invention is high, has very high production application and is worth.
Accompanying drawing explanation
Fig. 1 is the approach that after introducing Lyeopene synthetic gene, intestinal bacteria generate Lyeopene.
Fig. 2 is Lyeopene bioassay standard curve.
Fig. 3 is the schematic diagram that two step homologous recombination techniques knock out crtXY gene.A is the first step homologous recombination, and B is second step homologous recombination.
Fig. 4 is after the dxs gene of use two step homologous recombination technique to LYC005 bacterial strain carries out the regulation and control of RBS library, the change of yield of lycopene.
Fig. 5 is after the idi gene of use two step homologous recombination technique to LYC005 bacterial strain carries out the regulation and control of RBS library, the change of yield of lycopene.
Fig. 6 is after the crtE gene of use two step homologous recombination technique to LYC005 bacterial strain carries out the regulation and control of RBS library, the change of yield of lycopene.
Fig. 7 is after the dxs gene of use two step homologous recombination technique to LYC008 bacterial strain carries out the regulation and control of RBS library, the change of yield of lycopene.
Fig. 8 is after the idi gene of use two step homologous recombination technique to LYC009 bacterial strain carries out the regulation and control of RBS library, the change of yield of lycopene.
Fig. 9 is that recombination bacillus coli LYC010 high density fermentation produces Lyeopene.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Recombinant bacterial strain M1-46 and M1-93 is disclosed in document " Lu; J.; J.Tang; etal. (2012) .CombinatorialmodulationofgalPandglkgeneexpressionforimp rovedalternativeglucoseutilization.ApplMicrobiolBiotechn ol93 (6): 2455-2462. ", and the public can obtain from Tianjin Institute of Industrial Biotechnology.
Lyeopene is purchased from Sigma, and catalog number is L9879.
PXZ-CS plasmid is disclosed in document " TanZ; ZhuX; ChenJ; LiQ; ZhangX (2013) Activatingphosphoenolpyruvatecarboxylaseandphosphoenolpy ruvatecarboxykinaseincombinationforimprovingsuccinatepro duction.ApplEnvironMicrobiol79 (16): 4838-4844. ", and the public can obtain from Tianjin Institute of Industrial Biotechnology.
PKD46 plasmid is at document " Datsenko, wanner.One-stepinactivationofchromosomalgenesinEscherich iacoliK-12usingPCRproducts.ProcNatlAcadSciUSA.2000.97 (12): 6640-6645; " in be disclosed, the public can obtain from Tianjin Institute of Industrial Biotechnology.
Recombination bacillus coli CAR001 publication number be CN103087972A, denomination of invention is be disclosed in the patent of invention of " producing the recombinant microorganism of terpenoid and construction process ", the public can obtain from Tianjin Institute of Industrial Biotechnology.The preparation method of recombination bacillus coli CAR001 is shown in that publication number is CN103087972A, denomination of invention is [0104] to [0244] section in the patent of invention of " recombinant microorganism and the construction process of producing terpenoid ".
The preparation method of salt-free LB is as follows:
The sucrose solution of 1.50%: take 500g sucrose, is dissolved in 1L ultrapure water, 115 DEG C, sterilizing 20min.
The salt-free sucrose LB substratum of 2.10%: take 5g yeast extract paste, 10g peptone in 800ml water, 115 DEG C, sterilizing 20min.The sucrose solution of 50% of 200ml is added after sterilizing.
The salt-free sucrose LB substratum of 3.6%: take 5g yeast extract paste, 10g peptone, 15g agar powder, be dissolved in 880ml water, 115 DEG C, sterilizing 20min.The sucrose solution of 50% of 120ml is added after sterilizing.
In embodiment, the concentration of paraxin, ammonia benzyl mycin, kantlex is 34 μ g/L, 50 μ g/L, 50 μ g/L respectively.
Embodiment 1, the fermentation of product Lyeopene recombination bacillus coli and the mensuration of Lyeopene
One, the drafting of Lyeopene typical curve
The standard substance 50mg accurately taking Lyeopene adds 1ml methylene dichloride and dissolves, and is transferred to by solution in the brown volumetric flask of 250ml, is settled to 250ml with sherwood oil, be mixed with the storing solution (being stored in-80 DEG C of refrigerators) of 200 μ g/ml.During use with acetone carry out doubling dilution (2 ×, 4 ×, 8 ×, 16 ×, 32 ×), the filtering with microporous membrane of 0.45 μm in HPLC sample injection bottle, carry out HPLC detection (adopt
c18 post (4.6 × 250mm, 5 μm); Column temperature: 30 DEG C; Moving phase: methyl alcohol: acetonitrile: methylene dichloride=21:21:8(volume ratio); Flow velocity: 1ml/min; Sample size: 20 μ l; Sample injection time: 20min; DAD lamp detects; Determined wavelength is 480nm, and with standard substance peak area for ordinate zou, lycopene concentration (mg/L) is X-coordinate, obtains the typical curve of Lyeopene, as shown in Figure 2.
Two, the mensuration of recombination bacillus coli Lyeopene
Choose single bacterium colony (according to fermentation strain situation, add corresponding microbiotic or not added with antibiotic) by producing the recombination bacillus coli of Lyeopene in the test tube of the LB substratum of 4ml, 37 DEG C of 250rpm incubated overnight 24 hours; Then according to the inoculum size of volumn concentration 1%, the seed liquor of incubated overnight is transferred in the little shaking flask containing 10mlLB substratum (100ml), 37 DEG C, 250rpm lucifuge cultivates after 24h, sampling and measuring yield of lycopene.Measuring method is as follows:
Get the bacterium liquid of 0.5ml cultivation in the centrifugal 3min of 14000rpm, after sterile water wash, suspend with 1ml acetone and precipitate, 15 minutes are extracted under 55 DEG C of dark conditions, then by sample under 14000rpm centrifugal 10 minutes, supernatant takes on a red color, and bacterial strain becomes white from redness after acetone extract, by the supernatant containing Lyeopene after the filtering with microporous membrane of 0.45 μm, carry out assay with HPLC; When HPLC checks, the appearance time in sample identical with the appearance time of Lyeopene in standard substance (appearance time is 11.3min).Each testing sample has 3 Duplicate Samples respectively, and experimental result gets three parallel mean values.
Embodiment 2, knock out crtX and the crtY genes produce Lyeopene of recombination bacillus coli CAR001
Recombination bacillus coli CAR001 is replaced zeaxanthin glycosyltransferase gene crtX in described β-carotene synthetic gene bunch and lycopene beta cyclase gene crtY to obtain by crtX and the crtY gene constructed recombinant bacterium LYC001 recombinant bacterium LYC001 one, knocking out recombination bacillus coli CAR001.Zeaxanthin glycosyltransferase gene crtX is as shown in SEQIDNo.1, and lycopene beta cyclase gene crtY is as shown in SEQIDNo.2.Adopt the method for two step homologous recombination, knock out crtX and crtY gene, between yak base yak base diphosphate synthase gene crtE and Phytoene dehydrogenase gene crtI, insert the artificial RBS sequence (shown in SEQIDNo.3) being applicable to genetic expression in intestinal bacteria simultaneously.By two to general primer, the method for PCR is utilized to amplify fragment for two step homologous recombination.
Before utilizing CrtE-taa-cat-f(to comprise to knock out gene locus, 5 ' end of 50bp base and cat-sacB gene plays 20bp homologous fragment) and CrtI-atg-cat-r(comprise knock out gene locus after 3 ' end homologous fragment of 50bp base and cat-sacB gene) amplify DNA fragmentation I, carry out the first step homologous recombination, crtX and crtY gene to be knocked out is replaced with cat-sacB fragment, as shown in Figure 3A.
CrtEI-RBS-f(is utilized to comprise 20bp homologous fragment after crtE gene 3 ' end 50bp base, artificial RBS sequence and crtI gene start codon ATG) and CrtI-484-r primer, amplify DNA fragmentation II, carry out second step homologous recombination, cat-sacB fragment is replaced with artificial RBS sequence (SEQIDNo.3), as shown in Figure 3 B.
Concrete steps are as follows:
(1) with pXZ-CS plasmid for template, to increase about 3500bpDNA fragment I with primer CrtE-taa-cat-f and CrtI-atg-cat-r, this fragment is the DNA fragmentation I containing chloromycetin gene and Polylevulosan sucrose transferase gene.
Primer sequence is:
CrtE-taa-cat-f：
5’-ACCATTTTGTTCAGGCCTGGTTTGAGAAAAAACTCGCTGCCGTCAGTTAATGTGACGGAAGATCACTTCGCA-3’；
CrtI-atg-cat-r：
5’-GCCAGAGCCAGACCACCAAAGCCTGCGCCAATTACTGTAGTTCTATTCATTTATTTGTTAACTGTTAATTGTCCT-3’。
Preparation with the intestinal bacteria CAR001 of pKD46: preparation method is shown in that publication number is CN103087972A, denomination of invention is in the patent of invention of " recombinant microorganism and the construction process of producing terpenoid " [0253].
By above-mentioned obtain the cleaning of pcr amplification product DNA fragmentation I PCR cleaning reagent box after, DpnI process, then by electric for the DNA fragmentation I intestinal bacteria CAR001 gone to pKD46.Getting the bacterium liquid that 200 μ l transform is coated on the LB flat board containing paraxin and ammonia benzyl, after 30 DEG C of incubated overnight, dull and stereotyped at the LB containing paraxin and ammonia benzyl and screen containing on the LB flat board of kantlex respectively, obtain on kantlex LB flat board not long, clone long on paraxin and ammonia benzyl LB flat board, primer cat-up and CrtI-484-r is used to carry out bacterium colony PCR checking, result recombinant bacterium is correct, the pcr amplified fragment imported and bacterium generation homologous recombination, by crtX and crtY gene knockout, make crtE and crtI gene in bacterium middle with cat-sacB gene, by this recombinant bacterium called after cat-sacB-CAR001, this bacterium can be used for second step homologous recombination.
Checking primer sequence is:
cat-up：5’-TGTGACGGAAGATCACTTCGCA-3’；
CrtI-484-r：5’-GCCTGAAGTTTTGCCAGCTG-3’。
(2) with the genomic dna of recombinant bacterial strain CAR001 for template, with CrtEI-RBS-f and CrtI-484-r for primer, carry out pcr amplification, obtain DNA fragmentation II.
Primer sequence is:
CrtEI-RBS-f：
5’-ACCATTTTGTTCAGGCCTGGTTTGAGAAAAAACTCGCTGCCGTCAGTTAACTAAGGAGATATACCATGAATAGAACTACAGTAAT-3’；
CrtI-484-r：5’-GCCTGAAGTTTTGCCAGCTG-3’。
DNA fragmentation II electricity goes to step the cat-sacB-CAR001 that () obtains.Proceeded to by transformed bacteria liquid in the salt-free LB+10% sucrose medium of 50ml, 37 DEG C, 250rpm concussion cultivates after 24h, salt-free LB+6% sucrose plate is rule, and 41 DEG C of incubated overnight, remove pKD46 plasmid.Screening with not containing on antibiotic LB flat board at the LB flat board containing paraxin respectively, obtaining, containing clone not long on the LB flat board of paraxin, carrying out PCR checking, use primer crtE-540-f and CrtI-484-r verifies.
crtE-540-f：5’-TGACGCCATTCTGATGACCA-3’；
CrtI-484-r：5’-GCCTGAAGTTTTGCCAGCTG-3’。
Be LYC001 by Strain Designation correct for sequence verification.
Two, recombination bacillus coli LYC001 fermentative production Lyeopene
Recombination bacillus coli LYC001 is chosen single bacterium colony in the test tube of the LB substratum of 4ml, 37 DEG C, 250rpm incubated overnight; Then according to the inoculum size of volumn concentration 1%, the seed liquor of incubated overnight be transferred in the 100ml triangular flask containing 10mlLB nutrient solution, 37 DEG C, 250rpm lucifuge cultivates after 24h, sampling and measuring yield of lycopene, each sample does three repetitions.The yield of lycopene of LYC001 is measured according to the method for embodiment 1.
The yield of lycopene that the results are shown in Table 1, LYC001 reaches 10.49mg/L, and Lyeopene accounts for dry cell weight and reaches 6.52mg/g.
The expression intensity of the alph-ketoglutaric acid dehydrase gene of embodiment 3, raising recombination bacillus coli LYC001
One, the expression intensity improving the alph-ketoglutaric acid dehydrase gene of recombination bacillus coli LYC001 builds recombination bacillus coli LYC002
Recombinant bacterium LYC002 changes the original controlling element of the sucAB gene of LYC001 into artificial regulatory element M1-46 to obtain, and the original controlling element of sucAB gene is as shown in SEQIDNo.4, and artificial regulatory element M1-46 is as shown in SEQIDNo.5.
Change the original controlling element of sucAB gene in LYC001 into artificial regulatory element M1-46 according to the two step homologous recombination methods similar to embodiment 2, concrete grammar is as follows:
(1) with pXZ-CS plasmid for template, SucAB-cat-up and SucAB-cat – down is primer, and PCR obtains the DNA fragmentation I of about 3400bp; After DpnI process, then DNA fragmentation I electricity is gone to the intestinal bacteria LYC001 with pKD46.Getting the bacterium liquid that 200 μ l transform is coated on the LB flat board containing paraxin and ammonia benzyl, after 30 DEG C of incubated overnight, dull and stereotyped at the LB containing paraxin ammonia benzyl and screen containing on the LB flat board of kantlex respectively, obtain on kantlex LB flat board not long, clone long on paraxin ammonia benzyl LB flat board, use primer cat-up(sequence identical with embodiment 2) and sucAB-r enter bacterium colony PCR and verify, result recombinant bacterium is correct, this recombinant bacterium is the intestinal bacteria LYC001 with cat-sacB before sucAB gene, by its called after cat-sacB-LYC001, this bacterium can be used for second step homologous recombination.
(2) with the genomic dna of M1-46 for template, amplimer is SucAB-up-P/SucAB-RBS-down, and pcr amplification obtains the DNA fragmentation II of about 200bp; DNA fragmentation II electricity go to step that (one) obtain containing in the cat-sacB-LYC001 of pKD46.Proceeded to by transformed bacteria liquid in the salt-free LB+10%surcose substratum of 50ml, 37 DEG C, 250rpm concussion cultivates after 24h, at the flat lining out of salt-free LB+6%surcose, 41 DEG C of incubated overnight, remove pKD46 plasmid.Screening with not containing on antibiotic LB flat board at the LB flat board containing paraxin respectively, obtaining, containing clone not long on the LB flat board of paraxin, carrying out PCR checking, use primer P-up and sucAB-r verifies.Obtain correct recombination bacillus coli LYC002.
The primer sequence of amplification is as follows:
SucAB-cat-up：5’-GGTAGTATCCACGGCGAAGTAAGCATAAAAAAGATGCTTAAGGGATCACGTGTGACGGAAGATCACTTCGCA-3’；
SucAB-cat–down：5’-CCAGAGAGGTAAGAAGAGTCCAACCAGGCTTTCAAAGCGCTGTTCTGCATTTATTTGTTAACTGTTAATTGTCCT-3’。
sucAB-up-P：5’-GGTAGTATCCACGGCGAAGTAAGCATAAAAAAGATGCTTAAGGGATCACGTTATCTCTGGCGGTGTTGAC-3’；
sucAB-RBS-down：
5’-CCAGAGAGGTAAGAAGAGTCCAACCAGGCTTTCAAAGCGCTGTTCTGCATAGCTGTTTCCTGGTT-3’。
The PCR primers designed sequence of above-mentioned restructuring is:
P-up：5’-TTATCTCTGGCGGTGTTGAC-3’；
sucAB-r：5’-GAAATATTCACGCGTTTGAG-3’。
PCR is verified correct bacterial strain send order-checking, after sequence verification is correct, the controlling element obtaining the sucAB gene of LYC001 changes the bacterial strain of artificial regulatory element M1-46 into, by its called after recombination bacillus coli LYC002.
Two, recombination bacillus coli LYC002 produces Lyeopene
Recombination bacillus coli LYC002 is carried out fermentation culture according to the method for embodiment 1, measures yield of lycopene.The results are shown in Table 1, recombination bacillus coli LYC002 produces yield of lycopene and reaches 15.36mg/L, unit cell content reaches 10.67mg/g, and after replacing the original regulating and controlling sequence of the sucAB of LYC001, the unit cell content of the Lyeopene of the LYC002 obtained improves 64% relative to LYC001.
The alph-ketoglutaric acid dehydrase gene of embodiment 4, combinatorial regulation recombination bacillus coli LYC002 and the expression intensity of transaldolase gene (talB)
One, the alph-ketoglutaric acid dehydrase gene of combinatorial regulation recombination bacillus coli LYC002 and the expression intensity of transaldolase gene build recombination bacillus coli LYC003
Recombination bacillus coli LYC003 changes the original controlling element of the talB gene of LYC002 into artificial regulatory element M1-46, and the original controlling element of talB gene is as shown in SEQIDNo.6; Artificial regulatory element M1-46 is as shown in SEQIDNo.5.
According to the two step homologous recombination methods similar to embodiment 2, concrete grammar is as follows:
The amplification template of the first step homologous recombination is pXZ-CS plasmid, and primer is talB-cat-up and talB-cat – down, and checking primer is cat-up and talB-300-down.
The amplimer sequence of homologous recombination is:
talB-cat-up：5’-AGTCTCGCCTGGCGATAACCGTCTTGTCGGCGGTTGCGCTGACGTTGCGTCGTGTGTGACGGAAGATCACTTCGCA-3’；
talB-cat–down：5’-TCATGATAGTATTTCTCTTTAAACAGCTTGTTAGGGGGATGTAACCGGTCTGCTTATTTGTTAACTGTTAATTGTCCT-3’。
After PCR primer DpnI process, electricity goes to the intestinal bacteria LYC002 with pKD46.The bacterium liquid getting 200 μ l conversions is coated on the LB flat board containing paraxin ammonia benzyl, after 30 DEG C of incubated overnight, dull and stereotyped at the LB containing paraxin ammonia benzyl and screen containing on the LB flat board of kantlex respectively, obtain on kantlex LB flat board not long, clone long on paraxin ammonia benzyl LB flat board, primer cat-up and talB-300-down is used to carry out bacterium colony PCR checking, result recombinant bacterium is correct, this recombinant bacterium is the intestinal bacteria LYC002 with cat-sacB before talB gene, and this bacterium can be used for second step homologous recombination.
The amplification template of second step homologous recombination is the genomic dna of M1-46, with talB-up-P and talB-RBS-down for primer, and amplification second step homology band.
The amplimer sequence of homologous recombination is:
talB-up-P：5’-AGTCTCGCCTGGCGATAACCGTCTTGTCGGCGGTTGCGCTGACGTTGCGTCGTGTTATCTCTGGCGGTGTTGAC-3’；
talB-RBS-down：5’-AGTGTCGGCCACTACGGTGGTGTACTGACGAAGGGAGGTCAATTTGTCCGTCATAGCTGTTTCCTGGTT-3’。
The primers designed of above-mentioned second step homologous recombination is P-up and talB-300-down.
talB-300-down:5’-GACGCTTCGGTGTCATAGGAAAG-3’；
PCR primer is the DNA fragmentation II of about 200bp; DNA fragmentation II electricity go to step that (one) obtain containing in the first step gained intermediate strains of pKD46.Proceeded to by transformed bacteria liquid in the salt-free LB+10% sucrose medium of 50ml, 37 DEG C, 250rpm concussion cultivates after 24h, salt-free LB+6% sucrose plate is rule, and 41 DEG C of incubated overnight, remove pKD46 plasmid.Screening with not containing on antibiotic LB flat board at the LB flat board containing paraxin respectively, obtaining, containing clone not long on the LB flat board of paraxin, carrying out PCR checking, use primer P-up and talB-300-down verifies.After the recombinant bacterium sequence verification that second step obtains is correct, the Strain Designation original controlling element of the talB gene of LYC002 being changed into artificial regulatory element M1-46 is recombination bacillus coli LYC003.
Two, recombination bacillus coli LYC003 produces Lyeopene
Recombination bacillus coli LYC003 is carried out fermentation culture according to the method for embodiment 1, measures yield of lycopene.
The results are shown in Table 1, recombination bacillus coli LYC003 unit cell content reaches 11.03mg/g, and after sucAB and the talB combinatorial regulation of LYC001, the unit cell content of the Lyeopene of the LYC003 obtained improves 70% relative to LYC001.
The expression intensity of the succinate dehydrogenase gene (sdhABCD) of embodiment 5, combinatorial regulation recombination bacillus coli LYC003
One, the structure of recombination bacillus coli LYC005
The original controlling element of the sdhABCD gene of recombination bacillus coli LYC003 is replaced with artificial regulatory element M1-46 by recombination bacillus coli LYC005, and the original controlling element of sdhABCD gene is as shown in SEQIDNo.7.Artificial regulatory element M1-46 is as shown in SEQIDNo.5.
According to the two step homologous recombination methods similar to embodiment 2, concrete grammar is as follows:
The amplification template of the first step homologous recombination is pXZ-CS plasmid, and primer is SdhABCD-cat-up and SdhABCD-cat – down, and checking primer is cat-up and sdhABCD-230-down.
Homologous recombination primer:
SdhABCD-cat-up：5’-ACTTTGTTGAATGATTGTCAAATTAGATGATTAAAAATTAAATAAATGTTTGTGACGGAAGATCACTTCGCA-3’；
SdhABCD-cat–down：5’-TTAACAGGTCTTTGTTTTTTCACATTTCTTATCATGAATAACGCCCACATTTATTTGTTAACTGTTAATTGTCCT-3’。
After PCR primer DpnI process, electricity goes to the intestinal bacteria LYC003 with pKD46.Getting the bacterium liquid that 200 μ l transform is coated on the LB flat board containing paraxin and ammonia benzyl, after 30 DEG C of incubated overnight, dull and stereotyped at the LB containing paraxin ammonia benzyl and screen containing on the LB flat board of kantlex respectively, obtain on kantlex LB flat board not long, clone long on paraxin ammonia benzyl LB flat board, use primer cat-up and sdhABCD-230-down to enter bacterium colony PCR to verify, result recombinant bacterium is correct, this recombinant bacterium is the intestinal bacteria LYC003 with cat-sacB before sdhABCD gene, and this bacterium can be used for second step homologous recombination.
The template of second step homologous recombination amplification is the genomic dna of M1-46, with SdhABCD-up-P and SdhABCD-RBS-down for primer, and amplification second step homology band.
Homologous recombination primer:
SdhABCD-up-P：5’-ACTTTGTTGAATGATTGTCAAATTAGATGATTAAAAATTAAATAAATGTTTTATCTCTGGCGGTGTTGAC-3’；
SdhABCD-RBS-down:5’-TTAACAGGTCTTTGTTTTTTCACATTTCTTATCATGAATAACGCCCACATAGCTGTTTCCTGGTT-3’。
In the primers designed of above-mentioned two steps, sdhABCD-230-down sequence is as follows:
sdhABCD-230-down：5’-AATTTGACGAAGAAGCTGC-3’；
Template bacterial strain is recombinant bacterial strain M1-46, amplimer is SdhABCD-up-P/SdhABCD-RBS-down, obtains the DNA fragmentation II of about 200bp; DNA fragmentation II electricity goes in the first step gained intermediate strains containing pKD46.Proceeded to by transformed bacteria liquid in the salt-free LB+10%surcose substratum of 50ml, 37 DEG C, 250rpm concussion cultivates after 24h, at the flat lining out of salt-free LB+6%surcose, 41 DEG C of incubated overnight, remove pKD46 plasmid.Screen with not containing on antibiotic LB flat board at the LB flat board containing paraxin respectively, obtain clone not long on the LB flat board containing paraxin, carry out PCR checking, use primer P-up and sdhABCD-230-down to verify, obtain the fragment of about 320bp for positive.After the recombinant bacterium sequence verification that second step obtains is correct, the Strain Designation controlling element of the sdhABCD gene of LYC003 being replaced with artificial regulatory element M1-46 is recombination bacillus coli LYC005.
Two, recombination bacillus coli LYC005 produces Lyeopene
Recombination bacillus coli LYC005 is carried out fermentation culture according to the method for embodiment 1, measures yield of lycopene.
The results are shown in Table 1, recombination bacillus coli LYC005 unit cell content reaches 11.53mg/gDCW, and after sucAB, talB and sdhABCD assortment of genes regulation and control of LYC003, the unit cell content of the Lyeopene of the LYC005 obtained improves 76% relative to LYC001.
The dxs Gene expression intensities of embodiment 6, two-step approach regulation and control recombination bacillus coli LYC005 improves Lyeopene and produces
One, the dxs Gene expression intensities of two-step approach regulation and control recombination bacillus coli LYC005
According to the two step methods of homologous recombination similar to embodiment 2, dxs gene original artificial regulatory element M1-37 of recombination bacillus coli LYC005 is replaced with RBS controlling element library.The sequence of M1-37 is as shown in SEQIDNo.8.The sequence in RBS controlling element library is as shown in SEQIDNo.9.
Concrete grammar is as follows:
The amplification template of the first step homologous recombination is pXZ-CS plasmid, and primer is dxs-cat-up and dxs-cat – down, and checking primer is cat-up and dxs-381-down.
Homologous recombination primer:
dxs-cat-up：5’-ACTACATCATCCAGCGTAATAAATAAACAATAAGTATTAATAGGCCCCTGTGTGACGGAAGATCACTTCGCA-3’；
dxs-cat-down：5’-GTGGAGTCGACCAGTGCCAGGGTCGGGTATTTGGCAATATCAAAACTCATTTATTTGTTAACTGTTAATTGTCCT-3’；
After PCR primer DpnI process, electricity goes to the intestinal bacteria LYC005 with pKD46.Getting the bacterium liquid that 200 μ l transform is coated on the LB flat board containing paraxin and ammonia benzyl, after 30 DEG C of incubated overnight, dull and stereotyped at the LB containing paraxin ammonia benzyl and screen containing on the LB flat board of kantlex respectively, obtain on kantlex LB flat board not long, clone long on paraxin ammonia benzyl LB flat board, primer cat-up and dxs-381-down is used to carry out bacterium colony PCR checking, result recombinant bacterium is correct, this recombinant bacterium is the intestinal bacteria LYC005 with cat-sacB before dxs gene, and this bacterium can be used for second step homologous recombination.
The template of second step homologous recombination amplification is the genomic dna of M1-93, homologous recombination primer is dxs-up-P and dxs-RBSL-down, upstream primer dxs-up-P comprise wait 50bp base outside dxs gene original promoter and M1-93(SEQIDNo.10) 5 ' end of artificial regulatory element play 20bp homologous fragment.3 ' the end that downstream primer dxs-RBSL-down comprises the 50bp base (+1 to+50 district of gene) after the initiator codon of regulation and control dxs gene, the degenerate primer fragment (5 '-CAGGAGRNNNNNN-3 ') in RBS district and the artificial regulatory element of M1-93 plays 20bp homologous fragment.This can amplify one group of RBS district to primer is the artificial regulatory element annexing sequence, carries out second step homologous recombination, can obtain dxs library by this set of pieces.
Second step homologous recombination primer sequence is as follows:
dxs-up-P：5’-ACTACATCATCCAGCGTAATAAATAAACAATAAGTATTAATAGGCCCCTGTTATCTCTGGCGGTGTTGAC-3’；
dxs-RBSL-down：
5’-GTGGAGTCGACCAGTGCCAGGGTCGGGTATTTGGCAATATCAAAACTCATNNNNNNYCTCCTGGTTTAAACGTACATG-3’；
In the primers designed of above-mentioned two steps, the sequence of dxs-381-down is:
dxs-381-down：5’-GATGGAGGTTGATGAATGC-3’；
With the genomic DNA template of M1-93 bacterial strain, pcr amplification goes out RBS and mixes fragment, proceeds in the competent cell of the first step obtained strains by electricity after this fragment purification, in 250mL triangular flask (containing the salt-free sucrose medium of 50mL), cultivate 12h.Then bacterium liquid is diluted, is applied on 2 salt-free sucrose plate, 37 DEG C of incubated overnight.Each flat board obtains about 300 recons, and about totally 600 recons, are dxs library.Random picking 100 is cloned on paraxin flat board and carries out primary dcreening operation, and more than 90% all for reassembling into merit bacterial strain (can not at paraxin grow on plates).Select 5 bacterial strain order-checkings at random, the sequence in RBS region is different, shows that the diversity in storehouse is abundanter.
Random choose 30 mono-clonals, PCR checking is carried out with primer p-up/dxs-381-down, identify after second time homologous recombination that the fragment obtaining about 470bp is for positive, selecting 15 correct, that color is redder clone designation of checking is dxs1-dxs15, for measuring yield of lycopene.
Two, the 15 strain dxs library strains that recombination bacillus coli LYC005 sets out produce Lyeopene
By the dxs1-dxs15 obtained totally 15 strain recombination bacillus colis carry out fermentation culture according to the method for embodiment 1, measure yield of lycopene, take simultaneously LYC005 as contrast, the Lyeopene fractional yield result of 15 strain bacterium is as shown in Figure 4.
Fig. 4 shows, LYC005 is after the controlling element of dxs gene replaces with artificial regulatory element, the yield of lycopene of the 15 strain bacterium obtained is 0.73 to 1.13 times of the yield of lycopene of LYC005, wherein there is the yield of lycopene of 6 strain bacterium higher than the yield of lycopene of contrast LYC005, the yield of lycopene of the bacterial strain dxs8 that output is the highest is 18.34mg/L, and unit cell content reaches 13.08mg/g.
The idi Gene expression intensities of embodiment 7, two-step approach regulation and control recombination bacillus coli LYC005 improves Lyeopene and produces
One, according to two step methods of homologous recombination similar to Example 6, idi gene original artificial regulatory element M1-46 (SEQIDNo.5) of recombination bacillus coli LYC005 is replaced with the RBS controlling element library (SEQIDNo.9) described in embodiment 6.
The amplification template of the first step homologous recombination is pXZ-CS plasmid, and primer is idi-cat-up and idi-cat-down, and checking primer is cat-up and idi-483-down.
Homologous recombination primer:
idi-cat-up：5’-TCACTTGGTTAATCATTTCACTCTTCAATTATCTATAATGATGAGTGATCTGTGACGGAAGATCACTTCGCA-3’；
idi-cat-down：5’-CCCGTGGGAACTCCCTGTGCATTCAATAAAATGACGTGTTCCGTTTGCATTTATTTGTTAACTGTTAATTGTCCTTG-3’；
After product D pnI process, electricity goes to the intestinal bacteria LYC005 with pKD46.Getting the bacterium liquid that 200 μ l transform is coated on the LB flat board containing paraxin and ammonia benzyl, after 30 DEG C of incubated overnight, dull and stereotyped at the LB containing paraxin ammonia benzyl and screen containing on the LB flat board of kantlex respectively, obtain on kantlex LB flat board not long, clone long on paraxin ammonia benzyl LB flat board, use primer cat-up and idi-483-down to enter bacterium colony PCR to verify, result recombinant bacterium is correct, this recombinant bacterium is the intestinal bacteria LYC005 with cat-sacB before idi gene, and this bacterium can be used for second step homologous recombination.
The template of second step homologous recombination amplification is the genomic dna of M1-93, and homologous recombination primer is idi-u
p-P and idi-RBSL-down, this can amplify one group of RBS district to primer is the promoter element annexing sequence, carries out second step homologous recombination, can obtain idi library by this set of pieces.
Second step homologous recombination primer sequence is as follows:
idi-up-P：
5’-TCACTTGGTTAATCATTTCACTCTTCAATTATCTATAATGATGAGTGATCTTATCTCTGGCGGTGTTGAC-3’；
idi-RBSL-down：
5’-CCCGTGGGAACTCCCTGTGCATTCAATAAAATGACGTGTTCCGTTTGCATNNNNNNYCTCCTGGTTTAAACGTACATG-3’；
In the checking primer of above-mentioned two steps, idi-483-down sequence is:
idi-483-down：5’-CGTGGCATCAATACCGTGTA-3’；
With the genomic DNA template of M1-93 bacterial strain, pcr amplification goes out RBS and mixes fragment, proceeds in the competent cell of the first step obtained strains by electricity after this fragment purification, in 250mL triangular flask (containing the salt-free sucrose medium of 50mL), cultivate 12h.Then bacterium liquid is diluted, is applied on 2 salt-free sucrose plate, 37 DEG C of incubated overnight.Each flat board obtains about 300 recons, and about totally 600 recons, are idi library.Random picking 100 is cloned on paraxin flat board and carries out primary dcreening operation, and more than 90% all for reassembling into merit bacterial strain (can not at paraxin grow on plates).Select 5 bacterial strain order-checkings at random, the sequence in RBS region is different, shows that the diversity in storehouse is abundanter.
Random choose 30 mono-clonals, PCR checking is carried out with primer p-up/idi-483-down, identify after second time homologous recombination that the fragment obtaining about 570bp is for positive, selecting 15 correct, that color is redder clone designation of checking is idi1-idi15, for measuring yield of lycopene.
Two, the 15 strain idi library strains that recombination bacillus coli LYC005 sets out produce Lyeopene
By the idi1-idi15 obtained totally 15 strain recombination bacillus colis carry out fermentation culture according to the method for embodiment 1, measure yield of lycopene, take simultaneously LYC005 as contrast, the Lyeopene fractional yield result of 15 strain bacterium is as shown in Figure 5.
Fig. 5 shows, in selected 15 bacterial strains, there is the yield of lycopene of 8 strain bacterium all higher than the yield of lycopene of contrast LYC005, between the yield of lycopene of 15 bacterial strains is 0.67 times to 1.13 times of LYC005, wherein the yield of lycopene of idi9 is the highest, and output is 18.28mg/L, unit cell content reaches 13.01mg/g.
The crtE Gene expression intensities of embodiment 8, two-step approach regulation and control recombination bacillus coli LYC005 improves Lyeopene and produces
One, according to two step methods of homologous recombination similar to Example 6, crtE gene original artificial regulatory element M1-93 (SEQIDNo.10) of recombination bacillus coli LYC005 is replaced with the RBS controlling element library (SEQIDNo.9) described in embodiment 6.
The amplification template of the first step homologous recombination is pXZ-CS plasmid, and primer is ldhA-up-cat and crtE-sacB-down, and checking primer is cat-up and crtE-340-down.
Homologous recombination primer
ldhA-up-cat：
5’-ATTAAATTTGAAATTTTGTAAAATATTTTTAGTAGCTTAAATGTGATTCATGTGACGGAAGATCACTTCGCA-3’；
crtE-sacB-down：5’-GCATCGCTGTGTATGAAATTGACGTGTTGTTCTGCACAGACCGTCATCATTTATTTGTTAACTGTTAATTGTCCTTG-3’；
After product D pnI process, electricity goes to the intestinal bacteria LYC005 with pKD46.Getting the bacterium liquid that 200 μ l transform is coated on the LB flat board containing paraxin and ammonia benzyl, after 30 DEG C of incubated overnight, dull and stereotyped at the LB containing paraxin ammonia benzyl and screen containing on the LB flat board of kantlex respectively, obtain on kantlex LB flat board not long, clone long on paraxin ammonia benzyl LB flat board, use primer cat-up and crtE-sacB-down to enter bacterium colony PCR to verify, result recombinant bacterium is correct, this recombinant bacterium is the intestinal bacteria LYC005 with cat-sacB before crtE gene, and this bacterium can be used for second step homologous recombination.
The template of second step homologous recombination amplification is the genomic dna of M1-93, and homologous recombination primer is ldhA-up-P and crtE-RBSL-down, and this can amplify one group of RBS district to primer is the promoter element annexing sequence.
Second step homologous recombination primer sequence is as follows:
ldhA-up-P：5’-TTCAATTAAATTTGAAATTTTGTAAAATATTTTTAGTAGCTTAAATGTGATTCATTATCTCTGGCGGTGTTGAC-3’；
crtE-RBSL-down：5’-GCATCGCTGTGTATGAAATTGACGTGTTGTTCTGCACAGACCGTCATCATNNNNNNYCTCCTGGTTTAAACGTACATG-3’；
In the primers designed of above-mentioned two steps, crtE-340-down sequence is:
crtE-340-down：5’-CGACATGTTCACCATACTG-3’；
With the genomic DNA template of M1-93 bacterial strain, pcr amplification goes out RBS and mixes fragment, proceeds in the competent cell of the first step obtained strains by electricity after this fragment purification, in 250mL triangular flask (containing the salt-free sucrose medium of 50mL), cultivate 12h.Then bacterium liquid is diluted, is applied on 2 salt-free sucrose plate, 37 DEG C of incubated overnight.Each flat board obtains about 300 recons, and about totally 600 recons, are crtE library.Random picking 100 is cloned on paraxin flat board and carries out primary dcreening operation, and more than 90% all for reassembling into merit bacterial strain (can not at paraxin grow on plates).Select 5 bacterial strain order-checkings at random, the sequence in RBS region is different, shows that the diversity in storehouse is abundanter.
Random choose 30 mono-clonals, PCR checking is carried out with primer p-up/crtE-340-down, identify after second time homologous recombination that the fragment obtaining about 420bp is for positive, selecting 15 correct, that color is redder clone designation of checking is crtE1-crtE15, for measuring yield of lycopene.
Two, the 15 strain crtE library strains that recombination bacillus coli LYC005 sets out produce Lyeopene
According to embodiment 1 method fermentation culture selected by crtE1-crtE15 totally 15 strain bacterial strains, take simultaneously LYC005 as contrast, and measure the fractional yield of Lyeopene, result is as shown in Figure 6.
Fig. 6 shows, the yield of lycopene of selected crtE1-crtE15 totally 15 strain bacterial strains is 0.88 times of the yield of lycopene of LYC005 to 1.14 times, there is the yield of lycopene of 3 strain bacterium higher than the yield of lycopene of LYC005, wherein crtE9 yield of lycopene is the highest, output is 20.9mg/L, unit cell content reaches 13.2mg/g, is LYC008 by this Strain Designation.In LYC008, the artificial RBS controlling element sequence of crtE is as shown in SEQIDNo.11.
The expression intensity of dxs and the idi gene of embodiment 9, artificial RBS controlling element storehouse regulation and control recombination bacillus coli LYC008 improves Lyeopene and produces
Embodiment 6 to embodiment 8 shows, from LYC005, after individually regulating and controlling dxs, idi and crtE gene, yield of lycopene all improves to some extent, and three genes is built storehouse regulation and control at same bacterial strain, must obtain higher output yield.
One, the present embodiment is from recombination bacillus coli LYC008, in strict accordance with the RBS controlling element library regulation and control dxs gene of method described in embodiment 6 of embodiment 6, obtain recombinating the bacterial strain that successfully also sequence verification is correct, 15 strains are selected to change the promotor of the dxs gene of LYC008 the bacterial strain of artificial RBS controlling element at random, respectively by its called after crtE9-dxs1, crtE9-dxs2, crtE9-dxs3, crtE9-dxs4, crtE9-dxs5, crtE9-dxs6, crtE9-dxs7, crtE9-dxs8, crtE9-dxs9, crtE9-dxs10, crtE9-dxs11, crtE9-dxs12, crtE9-dxs13, crtE9-dxs14, crtE9-dxs15.
Above-mentioned 15 strain bacterium are carried out fermentation culture according to the method for embodiment 1, and measure yield of lycopene, Simultaneously test LYC005 and LYC008(crtE9) yield of lycopene, result is as shown in Figure 7.After the dxs gene regulating of LYC008, the yield of lycopene of 15 strain bacterium is 0.77 to 1.15 times of the yield of lycopene of LYC005, the bacterial strain that output is the highest is crtE9-dxs12, its yield of lycopene reaches 24.34mg/L, unit cell content reaches 13.30mg/g, improves 15% relative to LYC005, improves 104% relative to LYC001, be LYC009 by this Strain Designation, wherein the controlling element sequence of dxs is as shown in SEQIDNo.12.
Two, from LYC009, in strict accordance with the method for embodiment 7, with artificial RBS controlling element library regulation and control idi gene, obtain recombinating the bacterial strain that successfully also sequence verification is correct, 15 strains are selected to be distinguished called after crtE9-dxs12-idi1 at random, crtE9-dxs12-idi2, crtE9-dxs12-idi3, crtE9-dxs12-idi4, crtE9-dxs12-idi5, crtE9-dxs12-idi6, crtE9-dxs12-idi7, crtE9-dxs12-idi8, crtE9-dxs12-idi9, crtE9-dxs12-idi10, crtE9-dxs12-idi11, crtE9-dxs12-idi12, crtE9-dxs12-idi13, crtE9-dxs12-idi14, crtE9-dxs12-idi15.
15 strain bacterium are carried out fermentation culture according to the method for embodiment 1, and measure yield of lycopene, Simultaneously test LYC005, LYC008(crtE9) and yield of lycopene LYC009(crtE9-dxs12), result is as shown in Figure 8.Fig. 8 shows, after the idi gene regulating of LYC009, the yield of lycopene of 15 strain bacterium is 1.05 times of the yield of lycopene of LYC005 to 1.32 times, the bacterial strain that output is the highest is crtE9-dxs12-idi7, and its output reaches 26.33mg/L, and unit cell content reaches 15.22mg/g, 32% is improve relative to the yield of lycopene of LYC005, improve 133% relative to LYC001, is LYC010 by this Strain Designation, shown in the controlling element sequence SEQIDNo.13 of wherein idi.
Three, LYC010 is through being accredited as colon bacillus (Escherichiacoli), this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 23rd, 2013 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCCNo.8238.
The high density fermentation of embodiment 10, recombination bacillus coli LYC010
By recombination bacillus coli LYC010 at 7L fermentor tank (Labfors4; InforsBiotechnoligyCo.Ltd.) high density fermentation is carried out in.Method is as follows:
One, single bacterium colony is chosen in the LB substratum of 4ml, 37 DEG C, 250rpm incubated overnight.
Two, be the inoculum size of 1% according to volumn concentration, be transferred in the 250ml triangular flask containing 50mlLB nutrient solution by the bacterium liquid in 500 μ l test tubes, 37 DEG C, 250rpm cultivates 12h, and the bacterium liquid obtained is the seed liquor of high density fermentation.
Three, 7L ferments canned 3L fermention medium, 300ml seed liquor, adopts synthetic medium to carry out high density fermentation to it.
Fermention medium: by glycerine, citric acid, KH
2pO
43H
2o, (NH
4)
2hPO
4, MgSO
47H
2o, trace element solution and water form; The content of each component is as follows: 10g/L glycerine, 1.7g/L citric acid, 10.5g/LKH
2pO
43H
2o, 6g/L (NH
4)
2hPO
4, 3.44g/LMgSO
47H
2o, 10ml/L trace element solution, surplus is water.
Trace element solution: by FeSO
47H
2o, ZnSO
47H
2o, CuSO
45H
2o, MnSO
44H
2o, Na
2b
4o
710H
2o, CaC1
2, (NH
4)
6mo
7o
24form with water; The content of each component is as follows: 10g/LFeSO
47H
2o, 5.25g/LZnSO
47H
2o, 3.0g/LCuSO
45H
2o, 0.5g/LMnSO
44H
2o, 0.23g/LNa
2b
4o
710H
2o, 2.0g/LCaC1
2, 0.1g/L (NH
4)
6mo
7o
24, surplus is water.
Leavening temperature 30 DEG C, air flow quantity 4L/min, dissolved oxygen controls 30%.In order to make dissolved oxygen control 30%, rotating speed need with dissolved oxygen coupling, rotating speed remains on 400-1200rpm, dissolved oxygen bounce-back (primary carbon source has utilized) after rotating speed raise gradually, reach and remain on 1200rpm always.5M ammoniacal liquor is used to control the pH of fermentation 7.0.
Supplemented medium: by glycerine, peptone, yeast extract, MgSO
47H
2o forms; The content of each component is as follows: 500g/L glycerine, 15g/L peptone, 30g/L yeast extract, 30g/LMgSO
47H
2o, surplus is water.Supplemented medium is added when the glycerine in fermention medium is soon finished time.
What fermentation adopted is simulation exponential fed-batch feed profile, and whole fermenting process makes glycerol concentration remain on below 0.2g/L, and make the output of acetic acid maintain below 0.1g/L, average feed rate is 25ml/h.Whole fermenting process is feed supplement 2.56L altogether, and 5M ammoniacal liquor consumes 500ml, and in fermented liquid, the final volume of substratum is 6.2L.
After fermentation starts 10h, 2ml fermented liquid is got every 6h, in the centrifugal 3min of 14000rpm, with aqua sterilisa cleaning, by supernatant evacuation, be stored in-20 DEG C of refrigerators, survey in content of lycopene forward direction sample and add 1ml acetone, under 55 DEG C of dark conditions, extract 15min, the centrifugal 10min of 14000rpm, get supernatant and use HPLC(AgilentTechnologies high performance liquid chromatograph 1260Infinity) survey content of lycopene, concrete grammar is as embodiment 1.
As shown in Figure 9, the high density fermentation cycle is 116h to fermentation results, and cell enters stationary phase at 90h, the highest OD
600be 192.5, dry cell weight is 73g/LDCW.Yield of lycopene increases always, finally reaches 3.517g/L, and unit cell content reaches 50.57mg/g.
Table 1 recombination bacillus coli produces Lyeopene
Claims (9)
1. recombinant bacterium builds according to the method comprised the steps: the enzyme improving yak base yak base diphosphate synthase in recombination bacillus coli is lived;
The method that in described raising recombination bacillus coli, the enzyme of yak base yak base diphosphate synthase is lived is that the original controlling element of the yak base yak base diphosphate synthase gene crtE in described recombination bacillus coli is replaced with artificial regulatory element 1;
The original controlling element of described yak base yak base diphosphate synthase gene crtE is as shown in SEQIDNo.10;
The nucleotide sequence of described artificial regulatory element 1 is as shown in SEQIDNo.11;
The construction process of described recombination bacillus coli is as follows:
(1) the zeaxanthin glycosyltransferase gene crtX in β-carotene synthetic gene in intestinal bacteria CAR001 bunch and lycopene beta cyclase gene crtY is replaced to artificial RBS sequence;
The sequence of described zeaxanthin glycosyltransferase gene crtX is as shown in SEQIDNo.1;
The sequence of described lycopene beta cyclase gene crtY is as shown in SEQIDNo.2;
Described artificial RBS sequence is as shown in SEQIDNo.3;
Described β-carotene synthetic gene bunch is the gene cluster be made up of yak base yak base diphosphate synthase gene crtE, zeaxanthin glycosyltransferase gene crtX, lycopene beta cyclase gene crtY, phytoene desaturase gene crtI and phytoene synthase gene crtB;
(2) enzyme improving ketoglurate dehydrogenase in step (1) recombination bacillus coli that obtains is lived;
The original controlling element of the alph-ketoglutaric acid dehydrase gene sucAB that the method that in the recombination bacillus coli that described raising step (1) obtains, ketoglurate dehydrogenase enzyme is lived is specially in the recombination bacillus coli described step (1) obtained replaces with artificial regulatory element M1-46;
The original controlling element of described alph-ketoglutaric acid dehydrase gene sucAB is as shown in SEQIDNo.4;
(3) enzyme improving transaldolase in step (2) recombination bacillus coli that obtains is lived;
The original controlling element of the transaldolase gene talB that the method that in the recombination bacillus coli that described raising step (2) obtains, the enzyme of transaldolase is lived is specially in the recombination bacillus coli described step (2) obtained replaces with artificial regulatory element M1-46;
The original controlling element of described transaldolase gene talB is as shown in SEQIDNo.6;
(4) enzyme improving succinate dehydrogenase in step (3) recombination bacillus coli that obtains is lived, and obtains object recombination bacillus coli;
The original controlling element of the succinate dehydrogenase gene sdhABCD that the method that in the recombination bacillus coli that described raising step (3) obtains, the enzyme of succinate dehydrogenase is lived is specially in the recombination bacillus coli described step (3) obtained replaces with artificial regulatory element M1-46;
The original controlling element of described succinate dehydrogenase gene sdhABCD is as shown in SEQIDNo.7;
The nucleotide sequence of described artificial regulatory element M1-46 is as shown in SEQIDNo.5.
2. recombinant bacterium builds according to the method comprised the steps: the enzyme improving 1-deoxidation in recombinant bacterium 1 according to claim 1-xylulose-5-phosphate synthase is lived;
The method of the enzyme work of 1-deoxidation in described raising recombinant bacterium according to claim 1 1-xylulose-5-phosphate synthase is specially and the original controlling element of the 1-deoxidation in described recombinant bacterium 1 according to claim 1-xylulose-5-phosphate synthase gene dxs is replaced with artificial regulatory element 2;
The original controlling element of described 1-deoxidation-xylulose-5-phosphate synthase gene dxs is as shown in SEQIDNo.8;
The nucleotide sequence of described artificial regulatory element 2 is as shown in SEQIDNo.12.
3. recombinant bacterium builds according to the method comprised the steps: the original controlling element of the isovaleryl tetra-sodium isomerase gene idi in recombination bacillus coli is replaced with artificial regulatory element X, obtains object recombinant bacterium 3;
The original controlling element of described isovaleryl tetra-sodium isomerase gene idi is as shown in SEQIDNo.5;
The nucleotide sequence of described artificial regulatory element X is as shown in SEQIDNo.9;
Described recombination bacillus coli is recombinant bacterium 2 according to claim 2.
4. recombinant bacterium builds according to the method comprised the steps: the original controlling element of the isovaleryl tetra-sodium isomerase gene idi in recombination bacillus coli is replaced with artificial regulatory element 4;
The original controlling element of described isovaleryl tetra-sodium isomerase gene idi is as shown in SEQIDNo.5;
The nucleotide sequence of described artificial regulatory element 4 is as shown in SEQIDNo.13;
Described recombination bacillus coli is recombinant bacterium 2 according to claim 2.
5. recombinant bacterium 4 according to claim 4, is characterized in that: the intestinal bacteria of this bacterium to be deposit number be CGMCCNo.8238.
6. produce the method for Lyeopene: comprise the steps:, by arbitrary for a claim 1-5 described recombinant bacterium fermentation culture, to obtain Lyeopene.
7. method according to claim 6, is characterized in that: it is as follows that the fermention medium in described fermentation culture often rises composition:
10g glycerine, 1.7g citric acid, 10.5gKH
2pO
43H
2o, 6g (NH
4)
2hPO
4, 3.44gMgSO
47H
2o, 10ml trace element solution, surplus is water;
Often liter of composition of described trace element solution is as follows: 10gFeSO
47H
2o, 5.25gZnSO
47H
2o, 3.0gCuSO
45H
2o, 0.5gMnSO
44H
2o, 0.23gNa
2b
4o
710H
2o, 2.0gCaC1
2, 0.1g (NH
4)
6mo
7o
24, surplus is water;
The condition of described fermentation is specially temperature 30 DEG C, and air flow quantity is 4L/min, and dissolved oxygen is 30%, pH is 7.0;
Described pH carries out regulating particular by ammoniacal liquor;
In described fermentation, glycerol concentration remains on below 0.2g/L, and the output of acetic acid maintains below 0.1g/L.
8. the method according to claim 6 or 7, is characterized in that: described method also comprises the step of adding supplemented medium, and it is as follows that described supplemented medium often rises composition: 500g glycerine, 15g peptone, 30g yeast extract, 30gMgSO
47H
2o, surplus is water.
9. the arbitrary described recombinant bacterium of claim 1-5 is producing the application in Lyeopene.
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| CN105087408B (en) * | 2015-09-10 | 2018-08-21 | 武汉生物技术研究院 | A kind of yeast strain producing beta carotene and its application |
| CN106544348B (en) * | 2015-09-23 | 2020-03-31 | 中国科学院微生物研究所 | Isopentenyl pyrophosphate isomerase gene and application thereof |
| CN105969709A (en) * | 2016-03-28 | 2016-09-28 | 中国科学院微生物研究所 | Recombinant escherichia coli strain and method for preparing lycopene by adopting same |
| CN107164254B (en) * | 2016-09-13 | 2020-05-15 | 湖北广济药业股份有限公司 | Microorganisms and uses thereof |
| CN106434506B (en) * | 2016-09-29 | 2019-12-31 | 中国科学院天津工业生物技术研究所 | Construction method and application of recombinant bacterium for producing lycopene |
| CN108588106B (en) * | 2018-05-08 | 2022-03-11 | 华东理工大学 | Lycopene high-yield strain, preparation method and application thereof |
| CN112852694B (en) * | 2020-10-26 | 2022-04-26 | 中国科学院天津工业生物技术研究所 | Construction and application of astaxanthin synthetic strain |
| CN112646764B (en) * | 2020-12-18 | 2022-09-20 | 山东大学 | Recombinant lactococcus lactis for producing lycopene and application thereof |
| CN112779295B (en) * | 2020-12-31 | 2022-12-13 | 广东博沃特生物科技有限公司 | High-density fermentation medium for producing lycopene saccharomyces cerevisiae |
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