CN106434506A

CN106434506A - Building method for producing lycopene recombinant bacteria and application

Info

Publication number: CN106434506A
Application number: CN201610864886.3A
Authority: CN
Inventors: 张学礼; 李清艳; 唐金磊; 毕昌昊
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2016-09-29
Filing date: 2016-09-29
Publication date: 2017-02-22
Anticipated expiration: 2036-09-29
Also published as: CN106434506B

Abstract

The invention discloses a building method for producing lycopene recombinant bacteria and application. The lycopene recombinant bacteria are optional one of first lycopene recombinant bacteria, second lycopene recombinant bacteria and third lycopene recombinant bacteria, wherein first lycopene recombinant bacteria are obtained by increasing the expression and/or activity of (E)-4-hydroxyl-3-methyl-2-butenyl-geranylgeranyl pyrophosphate synthase genes ispG and the (E)-4-hydroxyl-3-methyl-2-butenyl-geranylgeranyl pyrophosphate reductase genes ispH in lycopene escherichia coli; the second lycopene recombinant bacteria are obtained by regulating the expression and/or activity of the crt operon of the first lycopene recombinant bacteria; the third lycopene recombinant bacteria are obtained by regulating the expression and/or activity of the glpD genes of the second lycopene recombinant bacteria. Experiments show that the terpene compound synthesizing ability of recombinant escherichia coli can be increased effectively by expressing the ispG and ispH genes in the recombinant escherichia coli with certain terpene compound synthesizing ability in a coordinated manner, and terpene compounds include beta-carotene, lycopene and other terpene compounds.

Description

A kind of construction method producing lycopene recombinant bacterium and application

Technical field

The invention belongs to biological technical field, more particularly, to a kind of construction method producing lycopene recombinant bacterium with should With.

Background technology

Terpenoid (terpenoids) is that a class present in nature is made up of for construction unit isoprene Compound.Many terpenoids have good pharmacologically active, are the principle active component of Chinese medicine and natural plant.Some Terpenoid has been developed over the active drug of wide clinical application.

Lycopene is a kind of typical terpenoid；It is the main component constituting tomato red pigments, be a kind of Outstanding antioxidant.Lycopene can prevent metabolite " free radical " to the injury caused by human tissue organ, can make For natural health care or medicine.Health food containing lycopene can prevent senile vision degeneration, anti-aging and pre- Anti- cardiovascular disease, lycopene is also respectively provided with certain suppression to digestive system cancer, cervical carcinoma, breast cancer, cutaneum carcinoma, carcinoma of urinary bladder etc. Effect.Lycopene is nontoxic, it can be added to as beta carotene ice cream, fruit syrup, hard candy, bread, biscuit, In the food such as cake, its nutritive value can be improved.Its health-care effect surpasses vitamin E and beta carotene.

Because terpenoid is with a wide range of applications, the market demand is huge, therefore efficiently produces terpenoid Method be study hotspot all the time.The production method of terpenoid mainly has three kinds at present：Chemical synthesis, plant carry Follow the example of and microbe fermentation method.Chemical synthesis technological process complexity, high energy consumption, pollution are big；On the other hand, terpenoid exists Content in plant is generally very low, and plant extraction method does great damage to wild plant resource；By contrast, fermentable Method is not limited by raw material, production process green cleans, and has very big advantage.Due to Escherichia coli (Escherichia Coli) genome sequence is announced completely, and genetic background and metabolic pathway are all perfectly clear, have culture medium requirement simply and give birth to The advantages of length is rapid, a lot of researchs are all chosen Escherichia coli and are carried out transformation production terpenoid as the bacterial classification that sets out.Fig. 1 is to draw After entering terpenes synthetic gene, Escherichia coli generate the route of synthesis of terpene compound.

Isopentenylpyrophosphate (IPP) and dimethyl propylene thiazolinyl pyrophosphoric acid (Dimethylallyl pyrophosphate, DMAPP) be all terpenoids precursor compound, be currently known two kinds of route of synthesis (Lee et al., 2002).One Kind be mevalonate pathway (Mevalonic Acid Pathway, MVA approach), be primarily present in fungi and plant cytosol or In endoplasmic reticulum.Another kind is MEP approach, is primarily present in bacterium, green alga and plant plastid.The starting material of this approach is 3- phosphorus Acid glycerol aldehyde and pyruvic acid, form about 5 through a series of enzymatics:1 IPP and DMAPP mixture.IPP is in isovaleryl Jiao's phosphorus Under the catalysis of acid isomer enzyme (Isopentenyl diphosphate isomerase, Idi), isomery becomes dimethyl propylene thiazolinyl Jiao's phosphorus Sour DMAPP.IPP and DMAPP is the basic C5 unit of terpenoid, can synthesize various terpenoids on this basis.

Escherichia coli have MEP approach, can synthesize precursor substance IPP and DMAPP of terpenoid, by terpenoid The synthetic gene of thing introduces Escherichia coli, and Escherichia coli can produce terpenoid.But due to Escherichia coli synthesis IPP and The poor ability of DMAPP, so the yield of terpenoid is generally very low.Improve the metabolic fluxes of MEP approach in Escherichia coli, carry The ability of high synthesis IPP and DMAPP, becomes and improves the key that Escherichia coli synthesize terpene compound yield.Multiple researchs are little Group find MEP approach in rate-limiting step, improve MEP approach in key gene expression intensity in, do a lot of work (Yoon et al.,2007；Ajikumar et al.,2010；Alper etal.,2005a；Alper et al.,2005b；Choi et al.,2010；Jin and Stephanopoulos,2007；Yuan et al.,2006).Research shows to improve in MEP approach 1- deoxidation-xylulose -5- phosphate synthase gene (dxs), 4- cytidine diphosphate (CDP) -2-C- methyl D antierythrite synthase gene And 4- cytidine diphosphate (CDP) -2-C- methyl D erythritol kinase gene (ispF), 2-C- methyl D-erythrite -2,4- (ispD) Ring pyrophosphate synthetase gene (ispE), the expression intensity of isovaleryl pyrophosphoric acid isomerase gene (idi) can improve restructuring large intestine Bacillus produces the ability of beta carotene.But improve 1- deoxidation-xylulose -5- phosphoric acid reduction isomerase gene in MEP approach (dxr), (E) -4- hydroxy-3-methyl -2- cyclobutenyl-pyrophosphate synthetase gene (ispG), (E) -4- hydroxy-3-methyl -2- The expression intensity of cyclobutenyl-pyrophosphoric acid reductase gene (ispH) reduces recombination bacillus coli on the contrary and produces beta carotene Ability (Yuan et al., 2006).

In order to identification of M EP by way of rate-limiting step, Yuan etc. utilize homologous recombination method, with T5 promoter in chromosome The promoter of the upper each gene that substituted for MEP approach respectively, after finding regulation and control dxs, idi, ispB, ispDF gene, β-carrot Plain yield has been respectively increased 100%, 40%, 20% and 40%.This 4 genes are combined after regulation and control with T5 promoter, β- Carotenoid production improves 6.3 times, reaches 6mg/g cellular dry weight (Yuan et al., 2006).Suh is adjusted with strong promoter T5 Control MEP by way of key gene dxs, idi and ispDF after, beta carotene yield increased improves 4.5 times of (Suh et al.,2012).In addition to the rate-limiting step in identified MEP approach, also have other albumen in possible MEP approach because high During expression, solubility is low, without finding speed limit (Zhou et al., 2012).Zhao 2013 research finds, in precursor substance When supply is not enough, downstream beta carotene synthetic gene is difficult to complete the experiment of high intensity promoter regulation, and works as precursor substance supply It is easy to realize high intensity promoter regulation downstream gene when sufficient, result, it is believed that bacterial strain condition changes, Escherichia coli product β- The rate-limiting step of carrotene can change (Zhao et al., 2013).

Content of the invention

One purpose of the present invention is to provide recombinant bacterium.

The present invention provide recombinant bacterium, for following 1) -3) in any one：

1) it is (E) -4- hydroxy-3-methyl -2- butylene in the Escherichia coli improved containing crt operator and produce lycopene The table of base-pyrophosphate synthetase gene ispG and (E) -4- hydroxy-3-methyl -2- cyclobutenyl-pyrophosphoric acid reductase gene ispH Reach and/or activity, obtain recombinant bacterium；

2) be regulation and control 1) shown in recombinant bacterium crt operator expression and/or activity, the recombinant bacterium obtaining；

3) be regulation and control 2) shown in the glpD gene expression of recombinant bacterium and/or activity, the recombinant bacterium obtaining.

In above-mentioned recombinant bacterium, described improve produce lycopene Escherichia coli in (E) -4- hydroxy-3-methyl -2- cyclobutenyl - The expression of pyrophosphate synthetase gene ispG and (E) -4- hydroxy-3-methyl -2- cyclobutenyl-pyrophosphoric acid reductase gene ispH And/or activity is to be realized by following (1) or (2)：

(1) insertion before the described ispG gene producing in lycopene Escherichia coli is started the sequence 23 of ispG gene expression Shown controlling element mRSL-4::IspG, and insertion before the described ispH gene producing in lycopene Escherichia coli is started The mRSL-14 shown in sequence 32 of ispH gene expression::ispH；

(2) insertion before the described ispG gene producing in lycopene Escherichia coli is started the sequence 21 of ispG gene expression Shown mRSL controlling element mRSL-1::IspG, and open inserting before the described ispH gene producing in lycopene Escherichia coli The mRSL-14 shown in sequence 32 of dynamic ispH gene expression::ispH；

Described regulation and control 1) shown in the crt operator expression of recombinant bacterium and/or activity be by 1) shown in crt behaviour in recombinant bacterium The promoter of vertical son replaces with inducible promoter；

Described regulation and control 2) shown in the glpD gene expression of recombinant bacterium and/or activity be by 2) shown in glpD base in recombinant bacterium The promoter of cause is substituted for artificial regulatory element M1-46.

Above-mentioned insertion position is insertion and is adjusted before gene, and is close to the initiation codon being adjusted gene.

In above-mentioned recombinant bacterium, described insertion is to carry out by the way of genome pinpoints editor or homologous recombination；

Or described genome fixed point editor is specially that ZFN edits, TALEN edits or CRISPR/Cas9 edits；

Or described homologous recombination is specially the homologous recombination of λ-red homologous recombination or the screening of sacB gene mediated or integrates matter The homologous recombination of grain mediation.

In above-mentioned recombinant bacterium, described inducible promoter is that the DNA containing inducible promoter Trc promoter and lacI divides Son；

Or the described nucleotides sequence containing inducible promoter Trc promoter and the DNA molecular of lacI is classified as sequence 33；

Or the nucleotides sequence of described artificial regulatory element M1-46 is classified as sequence 35；

Or described product lycopene Escherichia coli be Escherichia coli CAR001 bacterial strain beta carotene synthetic gene cluster in crtX With crtY gene knockout, build synthesis lycopene bacterial strain, then by alph-ketoglutaric acid dehydrase gene sucAB, succinic acid dehydrogenation The original controlling element of enzyme gene sdhABCD and transaldolase gene talB all replaces with what artificial regulatory element M1-46 obtained Bacterium, then uses RBS library regulation and control crt operator, dxs and idi gene respectively, and carries out combinatorial regulation acquisition；It is specially LYC010.

In above-mentioned recombinant bacterium, 3) preserving number of the recombinant bacterium shown in is CGMCC No.12883.

LYC029 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 19th in August in 2016 (abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101) preserving number is CGMCC No.12883, and Classification And Nomenclature is ETEC (Escherichiacoli).

Another object of the present invention is to provide a kind of method building above-mentioned recombinant bacterium, comprises the steps：

A-C：

A is following 1) or 2)：

1) before the ispG gene that will produce in lycopene Escherichia coli, insertion starts shown in the sequence 23 of ispG gene expression Controlling element mRSL-4::IspG, and insertion before the described ispH gene producing in lycopene Escherichia coli is started ispH base Because of the mRSL-14 shown in the sequence 32 of expression::IspH, obtains recombinant bacterium；

2) insertion before the described ispG gene producing in lycopene Escherichia coli is started the sequence 21 of ispG gene expression Shown mRSL controlling element mRSL-1::IspG, and open inserting before the described ispH gene producing in lycopene Escherichia coli The mRSL-14 shown in sequence 32 of dynamic ispH gene expression::IspH, obtains recombinant bacterium；

B, by A) promoter of crt operator in the recombinant bacterium that obtains replaces with inducible promoter, obtains recombinant bacterium；

C, by B) promoter of glpD gene in the recombinant bacterium that obtains is substituted for artificial regulatory element M1-46, obtains weight Group bacterium.

In said method, described insertion is to carry out by the way of genome pinpoints editor or homologous recombination；

In said method, described inducible promoter is that the DNA containing inducible promoter Trc promoter and lacI divides Son；

Or described product lycopene Escherichia coli be LYC010.

Application in producing lycopene for the above-mentioned recombinant bacterium is also the scope of protection of the invention.

The 3rd purpose of the present invention is to provide a kind of method producing lycopene.

The method that the present invention provides, comprises the steps：Ferment above-mentioned recombinant bacterium, obtains lycopene.

The experiment proves that, in the recombination bacillus coli with certain terpenoid synthesis capability, MEP approach Intermediate HMBPP intracellular accumulation, can cause cytotoxicity；Coordinated expression ispG and ispH gene, can effectively improve restructuring Colibacillary terpene compound synthesis capability, including beta carotene and lycopene, and other terpene compound.

Brief description

Fig. 1 is the route of synthesis of Escherichia coli generation terpene compound after introducing terpenes synthetic gene.

Fig. 2 is the transcriptional level built storehouse regulation and control and measure cell concentration, beta carotene fractional yield and corresponding gene；

A builds the cell concentration of selected bacterial strain and beta carotene fractional yield behind storehouse for dxr；B builds selected bacterial strain behind storehouse for dxr Dxr relative expression quantity；C builds the cell concentration of selected bacterial strain and beta carotene fractional yield behind storehouse for ispD；D builds behind storehouse for ispD Selected bacterial strain ispD relative expression quantity；E builds the cell concentration of selected bacterial strain and beta carotene fractional yield behind storehouse for ispE；F is IspE builds selected bacterial strain ispE relative expression quantity behind storehouse.The cell concentration that G builds selected bacterial strain behind storehouse for ispG is relative with beta carotene Yield；H builds selected bacterial strain ispG relative expression quantity behind storehouse for ispG；I builds the cell concentration of selected bacterial strain and β behind storehouse-recklessly for ispH Radish element fractional yield；J builds selected bacterial strain ispH relative expression quantity behind storehouse for ispH.

Fig. 3 is ispG and ispH assortment of genes regulation and control strain cell growth and beta carotene change of production.A is OD₆₀₀；B For beta carotene fractional yield.

Fig. 4 is ispG and ispH assortment of genes regulation and control bacterial strain yield of lycopene change in LYC010.

Fig. 5 is ispG and ispH assortment of genes regulation and control bacterial strain yield of lycopene change in LYC023 and LYC029.

Fig. 6 is zymotechnique figure.

Specific embodiment

Experimental technique used in following embodiments if no special instructions, is conventional method.

Material used, reagent etc. in following embodiments, if no special instructions, all commercially obtain.

Escherichia coli ATCC 8739 is in document " Gunsalus IC, Hand DB. (1941) .The use of bacteria in the chemical determination of total vitamin C.J Biol Chem.141: Mistake disclosed in 853-858. ", the public can obtain from Tianjin Institute of Industrial Biotechnology；

Recombinant bacterial strain M1-93 is in document " Lu, J., J.Tang, et al. (2012) .Combinatorial modulation of galP and glkgene expression for improved alternative glucose utilization.ApplMicrobiolBiotechnol93(6):Mistake disclosed in 2455-2462. ", the public can be from Tianjin industry Biotechnology research is obtained.

M1-93 artificial regulatory element sequences are shown in sequence 1.

Lycopene is purchased from Sigma, and catalog number is L9879.

2-C-Methyl-D-erythritol 4-phosphate (MEP) is purchased from Echelon Biosciences, product Catalog number (Cat.No.) is I-M051.

1-Hydroxy-2-methyl-2-buten-4-yl 4-diphosphate (HMBPP) is purchased from Echelon Biosciences, catalog number is I-M055.

1-Deoxy-D-xylulose 5-phosphate (DXP) is purchased from Echelon Biosciences, catalog number For I-M050.

4-Diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) is purchased from Echelon Biosciences, catalog number is I-M052.

2-C-Methyl-D-erythritol 2,4-cyclophosphate (MEcPP) is purchased from Echelon Biosciences, catalog number is I-M054.

Isopentenyl pyrophosphate triammonium salt solution (IPP) is purchased from Sigma, produces Product catalog number (Cat.No.) is I0503-1VL.

PXZ-CS plasmid is in document " Tan Z, Zhu X, Chen J, Li Q, Zhang X (2013) Activating phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in combination for improving succinate production.Appl Environ Microbiol79(16): Mistake disclosed in 4838-4844. ", the public can obtain from Tianjin Institute of Industrial Biotechnology.

PKD46 plasmid is in document " Datsenko, wanner.One-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products.Proc Natl Acad Sci USA.2000.97(12)：6640-6645；" disclosed in mistake, the public can obtain from Tianjin Institute of Industrial Biotechnology.

Recombination bacillus coli LYC010 is by crtX in recombination bacillus coli CAR001 bacterial strain beta carotene synthetic gene cluster With crtY gene knockout, build synthesis lycopene bacterial strain, then by alph-ketoglutaric acid dehydrase gene sucAB, succinic acid dehydrogenation The original controlling element of enzyme gene sdhABCD and transaldolase gene talB all replaces with what artificial regulatory element M1-46 obtained Bacterium, then uses RBS library regulation and control crt operator, dxs and idi gene respectively, and carries out combinatorial regulation acquisition；Concrete structure side Method is in Publication No. ZL 201410029600.0, invention entitled " one plant of production recombinant bacterium of lycopene and its application " Mistake disclosed in patent of invention, the public can obtain from Tianjin Institute of Industrial Biotechnology.In September in 2013 is preserved on the 23rd State's Microbiological Culture Collection administration committee common micro-organisms center (abbreviation CGMCC, address：BeiChen West Road, Chaoyang District, BeiJing City 1 Number institute 3, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.8238.

Recombinant bacterium CAR005 is to import beta carotene synthetic gene cluster and trc controlling element in Escherichia coli, then by β- The trc controlling element of carrotene synthetic gene cluster replaces with artificial regulatory element M1-93；Again by 1- deoxidation-xylulose -5- phosphorus The original controlling element of acid synthase gene dxs replaces with artificial regulatory element M1-37；Again by isovaleryl pyrophosphoric acid isomerase base Because the original controlling element of idi replaces with artificial regulatory element M1-46；Again by alph-ketoglutaric acid dehydrase gene sucAB, fourth two The original controlling element of dehydrogenase gene sdhABCD and transaldolase gene talB all replaces with artificial regulatory element M1-46 The bacterium obtaining, concrete construction method is in Publication No. CN 103087972A, the invention entitled " restructuring of production terpenoid Mistake disclosed in the patent of invention of microorganism and construction method ", the public can obtain from Tianjin Institute of Industrial Biotechnology.

Escherichia coli CAR005 with pKD46 is that plasmid pKD46 is imported the bacterium obtaining in Escherichia coli CAR005.

The preparation method of salt-free LB is as follows：

50% sucrose solution：Weigh 500g sucrose, on a small quantity after ultrapure water dissolves, be dissolved to 1L, 115 DEG C, sterilizing 20min.

10% salt-free sucrose LB culture medium：Weigh 5g yeast extract, 10g peptone, in 800ml water, 115 DEG C, sterilizes 20min.The 50% of 200ml sucrose solution is added after sterilizing.

6% salt-free sucrose LB culture medium：Weigh 5g yeast extract, 10g peptone, 15g agar powder, be dissolved in 880ml water, 115 DEG C, sterilize 20min.The 50% of 120ml sucrose solution is added after sterilizing.

In embodiment, chloramphenicol, ammonia benzyl mycin, the concentration of kanamycins are respectively 34 μ g/L, 50 μ g/L, 50 μ g/L.

The preparation method of fermentation medium is as follows：

1.10% glycerine mother liquor：Weigh 100g glycerine, plus deionized water is to 1L, 121 DEG C, sterilize 20min.

2. micro- mother liquor：.The composition of trace element solution：FeSO containing 10g in 1L solution₄·7H₂O、5.25g ZnSO₄·7H₂O、3.0g CuSO₄·5H₂O、0.5g MnSO₄·4H₂O、0.23g.

3. weigh dusty yeast：10g/l、K₂HPO₄：10.5g/l、(NH₄)₂HPO₄:6g/l, glycerine:15g/l, citric acid： In 700ml water, 121 DEG C, sterilize 1.84g/l, 10ml trace element mother liquor 20min.The MgSO of sterilizing is added after sterilizing₄· 7H₂The glycerine mother liquor of O solution and 100ml 10%, final plus sterilized water to 1L

MgSO4 7H2O solution:MgSO₄·7H₂O：5g is dissolved in 20ml deionized water, 121 DEG C, and sterilize 20min.

High density fermentation culture medium preparation method is as follows：

1. bed material culture medium：Dusty yeast：20g/l、KH₂PO₄：10.5g/l、(NH₄)₂HPO₄:6g/l, glycerine:30g/l, lemon Lemon acid：1.84g/l、MgSO₄·7H₂O:10g/L, 15ml/L trace element mother liquor.

2.100 micro- mother liquor again：FeSO containing 10g in 1L solution₄·7H₂O、5.25g ZnSO₄·7H₂O、3.0g CuSO₄·5H₂O、0.5g MnSO₄·4H₂O、0.23g.

3. supplemented medium：Glycerine：700g/l.

Primer used in table 1 present invention

Bacterial strain is built in table 2. present invention

The relation of embodiment 1, MEP approach ispG and ispH Gene expression intensities and beta carotene yield

In order to study CAR005 (Zhao J, Li Q, Sun T, et al.Engineering central metabolic modules of Escherichia coli for improving beta-carotene production.Metabolic engineering 2013；17:42-50.CAR001 and CAR005D comes from this article) in each gene expression of MEP approach strong Degree and the relation of beta carotene yield, set up mRS library regulation and control dxr, ispD, ispE, ispG and ispH gene, select 15 at random Individual bacterial strain, measures the yield of beta carotene, according to the yield of beta carotene, then selects several plants of representative bacterial strain real-time quantitatives PCR method, measures gene expression amount, the relation of the yield of research expression and beta carotene.

First, build dxr, ispD, ispE, ispG and ispH gene in mRS library regulation and control CAR005

The implementation case PCR amplifies mRS (mRNA stable region) library fragments, is inserted in and several treats the initial close of controlling gene Before numeral, retain original controlling element.It is referred to as mRNA stable region, this area's nucleotides kind between same -35th area of -10th area in prokaryotes Class sum purpose variation can affect the height of Gene Transcription in vitro, set up library in this region, can obtain different activities Promoter.This library sequence is shown in sequence 2.

Dxr-1, dxr-2, dxr-3 ... dxr-15 is respectively the dxr gene in CAR005 genome in Escherichia coli The mRSL-1 shown in table 2 of the front expression inserted respectively for starting dxr gene::dxr-mRSL-15::dxr.

IspD-1, ispD-2, ispD-3 ... ispD-15 is respectively the ispD in CAR005 genome in Escherichia coli The mRSL-1 shown in table 2 of the expression for starting ispD gene is inserted respectively before gene::ispD-mRSL-15::IspD,.

IspE-1, ispE-2, ispE-3 ... ispE-15 is respectively the ispE in CAR005 genome in Escherichia coli The mRSL-1 shown in table 2 of the expression for starting ispE gene is inserted respectively before gene::ispE-mRSL-15::ispE.

IspG-1, ispG-2, ispG-3 ... ispG-15 is respectively the ispG in CAR005 genome in Escherichia coli The mRSL-1 shown in table 2 of the expression for starting ispG gene is inserted respectively before gene::ispG-mRSL-15::ispG.

IspH-1, ispH-2, ispH-3 ... ispH-15 is respectively the ispG in CAR005 genome in Escherichia coli The mRSL-1 shown in table 2 of the expression for starting ispH gene is inserted respectively before gene::ispH-mRSL-15::ispH.

The all bacterium obtaining constitute library strains.

2nd, the yield of library strains beta carotene

Above-mentioned one each library strains obtaining are chosen single bacterium colony respectively in the test tube of the LB culture medium of 4ml, 30 DEG C, 250rpm incubated overnight；Then according to the inoculum concentration of 1% (volumn concentration), that is, 100 μ l bacterium solution, the bacterium solution in test tube is turned It is connected in the 100ml triangular flask containing 10ml nutrient solution, 30 DEG C, 250rpm cultivates；After culture 24h.Take 500 μ L bacterium solution in 13000rpm centrifugation 3min abandons supernatant, cleans thalline with aqua sterilisa, plus the resuspended thalline of 1ml acetone, under 55 DEG C of dark conditions Extraction 15min, 13000rpm centrifugation 10min collects supernatant.With CAR005 for comparison.

Using beta carotene absorption value in mensure supernatant under ultraviolet specrophotometer 453nm.

In the fractional yield=supernatant of beta carotene, beta carotene absorption value is multiplied by/supernatant cell turbidity (OD600nm)

The result of the fractional yield of dxr-1, dxr-2, dxr-3 ... dxr-15 beta carotene is shown in that Fig. 2A, Fig. 2A show, After the regulation and control of mRS library, the beta carotene yield of selected 15 plants of bacterium is 0.5 arriving of CAR005 to the dxr gene of CAR005 at random 0.99 times, cell concentration is 0.66 to 0.9 times of CAR005.Select the bacterial strain dxr-4 (controlling element that beta carotene yield is relatively low mRSL-4::Dxr sequence is shown in sequence 3) and dxr-6 (controlling element mRSL-6::Dxr sequence is shown in sequence 4), beta carotene yield Medium bacterial strain dxr-2 (controlling element mRSL-2::Dxr sequence is shown in sequence 5) and dxr-5 (controlling element mRSL-5::Dxr sequence Row are shown in sequence 6) and higher bacterial strain dxr-11 (the controlling element mRSL-11 of beta carotene yield::Dxr sequence is shown in sequence 7) and Dxr-15 (controlling element mRSL-15::Dxr sequence is shown in sequence 8) with real time quantitative PCR method measure dxr gene expression.

Shown in the result of the fractional yield of ispD-1 to ispD-15 beta carotene as Fig. 2 C, show, the ispD of CAR005 After the regulation and control of mRS library, the beta carotene yield of selected 15 plants of bacterium is 0.99 to 1.05 times of CAR005 to gene at random.Select β- Relatively low bacterial strain ispD-1 (the controlling element mRSL-1 of carotenoid production::IspD sequence is shown in sequence 9) and ispD-15 (regulation and control unit Part mRSL-15::IspD sequence is shown in sequence 10), medium bacterial strain ispD-6 (the controlling element mRSL-6 of beta carotene yield:: IspD sequence is shown in sequence 11) and ispD-13 (controlling element mRSL-13::IspD sequence is shown in sequence 12) and beta carotene yield Higher bacterial strain ispD-7 (controlling element mRSL-7::IspD sequence is shown in sequence 13) and ispD-9 (controlling element mRSL-9:: IspD sequence is shown in sequence 14) with real time quantitative PCR method measure ispD gene expression.

The result of the fractional yield of ispE-1 to ispE-15 beta carotene as shown in Figure 2 E, shows, the ispE of CAR005 After the regulation and control of mRS library, the beta carotene yield of selected 15 plants of bacterium is 0.97 to 1.09 times of CAR005 to gene at random.Select β- Relatively low bacterial strain ispE-5 (the controlling element mRSL-5 of carotenoid production::IspE sequence is shown in sequence 15) and ispE-15 (regulation and control unit Part mRSL-15::IspE sequence is shown in sequence 16), medium bacterial strain ispE-3 (the controlling element mRSL-3 of beta carotene yield:: IspE sequence is shown in sequence 17) and ispE-12 (controlling element mRSL-12::IspE sequence is shown in sequence 18) and beta carotene yield Higher bacterial strain ispE-6 (controlling element mRSL-6::IspE sequence is shown in sequence 19) and ispE-8 (controlling element mRSL-8:: IspE sequence is shown in sequence 20) with real time quantitative PCR method measure ispE gene expression.

The result of the fractional yield of ispG-1 to ispG-15 beta carotene as shown in Figure 2 G, shows, the ispG of CAR005 After the regulation and control of mRS library, the beta carotene yield of selected 15 plants of bacterium is 0.11 to 0.79 times of CAR005 to gene at random, cell Measure 0.47 to 0.91 times for CAR005.Select bacterial strain ispG-1 (the controlling element mRSL-1 that beta carotene yield is relatively low::ispG Sequence is shown in sequence 21) and ispG-13 (controlling element mRSL-13::IspG sequence is shown in sequence 22), beta carotene yield is relatively low Bacterial strain ispG-4 (controlling element mRSL-4::IspG sequence is shown in sequence 23) and ispG-5 (controlling element mRSL-5::IspG sequence See sequence 24), and bacterial strain ispG-11 (the controlling element mRSL-11 that beta carotene yield is relatively low::IspG sequence is shown in sequence 25) With ispG-14 (controlling element mRSL-14::IspG sequence is shown in sequence 26), measure ispG gene with real time quantitative PCR method Expression.

The result of the fractional yield of ispH-1 to ispH-15 beta carotene such as Fig. 2 I shows, the ispH gene warp of CAR005 After the regulation and control of mRS library, the beta carotene yield of selected 15 plants of bacterium is 0.98 to 1.06 times of CAR005 at random.Select ispH- at random 1 (controlling element mRSL-1::IspH sequence is shown in sequence 27), ispH-2 (controlling element mRSL-2::IspH sequence is shown in sequence 28), IspH-3 (controlling element mRSL-3::IspH sequence is shown in sequence 29), ispH-4 (controlling element mRSL-4::IspH sequence is shown in sequence Row 30), ispH-5 (controlling element mRSL-5::IspH sequence is shown in sequence 31) and ispH-14 (controlling element mRSL-14::ispH Sequence is shown in sequence 32) with real time quantitative PCR method measure ispH gene expression.

Above-mentioned each bacterium is the controlling element replacing with the promoter of respective gene inside bracket.

3rd, beta carotene yield and corresponding gene expression magnitude relation

Extract bacterial strain dxr-4, dxr-6, dxr-2, dxr-5, dxr-11 and dxr-15 total serum IgE, reverse transcription obtains cDNA's First chain；With the first chain cDNA as template, dxr-610-f/dxr-802-r is primer, with iQSYBR Green RT-PCR reagent Box (Bio-Rad Laboratories, Hercules, CA) is in real-time PCR (Bio-Rad CFX Connect Real Time System) enter performing PCR, meanwhile, the cDNA of the dxr with CAR005 measures as comparison, the expression of dxr in bacterial strain selected by mensure Amount.In order to ensure sample uniformity, with 16S RNA as reference gene, the primer for amplification is 16S-797-f/16S-963- R, the primer sequence is shown in Table 1.

Real-time quantitative PCR amplification program:

1)95℃10min,1cycle

2)95℃15s-60℃30s-72℃30s,40cycles

3) 60 DEG C -95 DEG C, 0.2 DEG C/s

ABI Prism 7000SDS software (Applied Biosystems) carries out data analysis.

The cDNA of CAR005 is diluted according to finite concentration, measures the relative expression quantity of dxr and 16S gene, draw standard Curve, calculates the relative expression quantity of dxr and 16S gene in each sample according to calibration curve, then calculates each sample and does three Individual repetition.

Dxr sample=dxr relative quantity/16S relative quantity

Dxr relative expression intensities=dxr sample/dxrCAR005

The relative expression quantity that bacterial strain corresponding gene is selected in library is measured respectively using same method, the primer is shown in Table 1.

Result is as shown in Figure 2.

Fig. 2 B shows, in 6 plants of bacterium selected by dxr gene mRS library of CAR005, dxr expression is dxr expression in CAR005 Amount 0.68 to 42.86 times.Dxr expression highest in the minimum bacterial strain dxr-4 of beta carotene yield, expression is 42.86 times of dxr expression in CAR005；And the expression of dxr gene is minimum in beta carotene yield highest dxr-15, For dxr expression in CAR005 0.68 times.Dxr expression is in inverse ratio with corresponding bacterial strain beta carotene yield, that is, in bacterial strain Dxr expression is higher, and its beta carotene yield is lower.

Fig. 2 D shows, in 6 plants of bacterium selected by ispD gene mRS library of CAR005, ispD expression is ispD table in CAR005 The amount of reaching 7 to 116 times.IspD gene mRS library institute roguing bacterium beta carotene yield and cell concentration and starting strain CAR005 Compare change less, and ispD expression has notable difference, it is therefore contemplated that ispD expression and beta carotene yield do not have Substantially correlation.

Fig. 2 F shows, in 6 plants of bacterium selected by ispE gene mRS library of CAR005, ispE expression is ispE table in CAR005 6 to 44.5 times of the amount of reaching.IspE gene mRS library institute roguing bacterium beta carotene yield and cell concentration and starting strain CAR005 Compare change less, and ispE expression has notable difference, it is therefore contemplated that ispE expression and beta carotene yield do not have Substantially correlation.

Fig. 2 H shows, in 9 plants of bacterium selected by ispG gene mRS library of CAR005, ispG expression is ispG table in CAR005 The amount of reaching 5.09 to 75.83 times.IspG expression highest in the minimum bacterial strain ispG-1 of beta carotene yield, expression is 75.83 times of ispG expression in CAR005；And in beta carotene yield highest ispG-11 the expression of ispG gene is Low, it is 5.09 times of ispG expression in CAR005.IspG expression and corresponding bacterial strain beta carotene yield are in inverse ratio, i.e. bacterium In strain, ispG expression is higher, and its beta carotene yield is lower.

Fig. 2 J shows, in 6 plants of bacterium selected by ispH gene mRS library of CAR005, ispH expression is ispH table in CAR005 The amount of reaching 4 to 36.4 times.IspH gene mRS library institute roguing bacterium beta carotene yield and cell concentration and starting strain CAR005 compares change less, and ispH expression has notable difference, it is therefore contemplated that ispH expression and beta carotene produce Amount does not have obvious correlation.

4th, the recombinant bacterium that ispG and ispH combinatorial regulation obtains

Respectively by the artificial regulatory element of the ispG of ispG-1, ispG-4 and ispG-11 in the regulation and control of ispG storehouse, with ispH storehouse The artificial regulatory element of the ispH of middle ispH-3, ispH-4 and ispH-14 combinatorial regulation in CAR005.

1st, the structure of recombination bacillus coli G1H3, G1H4, G1H14, G4H3, G4H4, G4H14, G11H3, G11H4 and G11H14 Build

Recombination bacillus coli G1H3 is insertion startup ispG gene before the ispG gene that will set out in bacterium CAR005 genome The mRSL controlling element mRSL-1 of expression::IspG (sequence 21), and insert before the ispH gene that will set out in bacterium CAR005 genome Enter to start the mRSL-3 of ispH gene expression::IspH (sequence 29)；

Recombination bacillus coli G1H4 is insertion startup ispG gene before the ispG gene that will set out in bacterium CAR005 genome The mRSL controlling element mRSL-1 of expression::IspG (sequence 21), and insert before the ispH gene that will set out in bacterium CAR005 genome Enter to start the mRSL-4 of ispH gene expression::IspH (sequence 30)；

Recombination bacillus coli G1H14 is insertion startup ispG gene before the ispG gene that will set out in bacterium CAR005 genome The mRSL controlling element mRSL-1 of expression::IspG (sequence 21), and insert before the ispH gene that will set out in bacterium CAR005 genome Enter to start the mRSL-14 of ispH gene expression::IspH (sequence 32).

Recombination bacillus coli G4H3 is insertion startup ispG gene before the ispG gene that will set out in bacterium CAR005 genome The mRSL controlling element mRSL-4 of expression::IspG (sequence 23), and insert before the ispH gene that will set out in bacterium CAR005 genome Enter to start the mRSL-3 of ispH gene expression::IspH (sequence 29).

Recombination bacillus coli G4H4 is insertion startup ispG gene before the ispG gene that will set out in bacterium CAR005 genome The mRSL controlling element mRSL-4 of expression::IspG (sequence 23), and insert before the ispH gene that will set out in bacterium CAR005 genome Enter to start the mRSL-4 of ispH gene expression::IspH (sequence 30).

Recombination bacillus coli G4H14 is insertion startup ispG gene before the ispG gene that will set out in bacterium CAR005 genome The mRSL controlling element mRSL-4 of expression::IspG (sequence 23), and insert before the ispH gene that will set out in bacterium CAR005 genome Enter to start the mRSL-14 of ispH gene expression::IspH (sequence 32).

Recombination bacillus coli G11H3 is insertion startup ispG gene before the ispG gene that will set out in bacterium CAR005 genome The mRSL controlling element mRSL-11 of expression::IspG (sequence 25), and before the ispH gene that will set out in bacterium CAR005 genome Insertion starts the mRSL-3 of ispH gene expression::IspH (sequence 29).

Recombination bacillus coli G11H4 is insertion startup ispG gene before the ispG gene that will set out in bacterium CAR005 genome The mRSL controlling element mRSL-11 of expression::IspG (sequence 25), and before the ispH gene that will set out in bacterium CAR005 genome Insertion starts the mRSL-4 of ispH gene expression::IspH (sequence 30).

Recombination bacillus coli G11H14 is insertion startup ispG base before the ispG gene that will set out in bacterium CAR005 genome MRSL controlling element mRSL-11 because of expression::IspG (sequence 25), and the ispH gene that will set out in bacterium CAR005 genome Front insertion starts the mRSL-14 of ispH gene expression::IspH (sequence 32).

Above-mentioned recombination bacillus coli, all with the method for two step homologous recombination, inserts artificial regulatory element, with G1H3, G1H4, As a example G1H14 construction method, specific as follows：

(1) with pXZ-CS plasmid as template, ispH-cat-up and ispH-cat down is primer, and PCR obtains 3000bp The DNA fragmentation I of left and right；After DpnI is processed, DNA fragmentation I electricity is gone in the Escherichia coli ispG-1 with pKD46.Take 200 μ l The bacterium solution of conversion is coated on the LB flat board containing chloramphenicol and ammonia benzyl, after 30 DEG C of incubated overnight, respectively containing chloramphenicol ammonia benzyl LB flat board and the LB flat board containing kanamycins on screen, obtain not long on kanamycins LB flat board, chloramphenicol ammonia benzyl LB Long clone on flat board, enters bacterium colony PCR checking using primer cat-up and ispH-395-down, and result recombinant bacterium is correct, and this is heavy Group bacterium can be used for second step homologous recombination for the Escherichia coli ispG-1 with cat-sacB before ispH gene, this bacterium.

(2) respectively with the genomic DNA of ispH-3, ispH-4 and ispH-14 as template, amplimer is ispH-440- Up/ispH-395-down, PCR amplification obtain 940bp about DNA fragmentation II；This fragment include corresponding controlling element and on Downstream 400bp about homology arm, this bar segment electricity is gone in the bacterial strain containing pKD46 that step () obtains.By transformed bacteria Liquid proceeds in 50ml salt-free LB+10%surcose culture medium, 37 DEG C, after 250rpm concussion and cultivate 24h, in salt-free LB+6% The flat lining out of surcose, 41 DEG C of incubated overnight, remove pKD46 plasmid.Respectively in the LB flat board containing chloramphenicol with without anti- Screen on the LB flat board of raw element, the clone not grown is obtained on the LB flat board containing chloramphenicol, enter performing PCR checking, using primer P- Up/ispH-395-down is verified.Screening verification is correct, sends to sequencing, the correct Strain Designation of sequencing is G1H3, G1H4, G1H14.In described case, the primer is shown in Table 1, builds bacterial strain and is shown in Table 2.

(3) above-mentioned (one), (two) methods described are adopted, with the artificial tune of the ispH of ispH-3, ispH-4 and ispH-14 Control element regulate and control ispH gene in ispG-4 and ispG-11 respectively, obtain recombination bacillus coli G4H3, G4H4, G4H14, G11H3, G11H4 and G11H14.The primer is shown in Table 1, and constructed bacterial strain is shown in Table 2.

2nd, the β of recombination bacillus coli G1H3, G1H4, G1H14, G4H3, G4H4, G4H14, G11H3, G11H4 and G11H14- Carrotene measures

By recombination bacillus coli G1H3, G1H4, G1H14, G4H3, G4H4, G4H14, G11H3, G11H4 and G11H14 according to The method of embodiment 1 carries out fermented and cultured, measures beta carotene yield, simultaneously with CAR005 for comparison, calculates beta carotene Fractional yield.Result is as shown in Figure 3.

Result shows, in G1H3, G1H4, G1H14, B- carotenoid production is respectively 0.16,0.47, the 0.55 of CAR005 Times, i.e. (in G1H3, G1H4, G1H14, ispG expression is 75.8 times of CAR005), beta carotene when ispG expression is high Yield with ispH gene expression amount improve and improve；In G4H3, G4H4, G4H14, B- carotenoid production is respectively CAR005 0.57,1.70,1.76 times (in G4H3, G4H4, G4H14, ispG expression is 10.84 times of CAR005), G11H3, G11H4 0.93,1.66,1.72 times that are respectively CAR005 with B- carotenoid production in G11H14 (in G11H3, G11H4 and G11H14 IspG expression is 5.09 times of CAR005)；When ispG expression is low, beta carotene yield increases with the expression of ispH Plus and increase, when ispH expression increase to a certain extent, then beta carotene yield is not further added by.And the cell of each bacterial strain Growth is identical with beta carotene output trend.

Beta carotene yield highest bacterial strain G4H14 is named as CAR015.

CAR015 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 19th in August in 2016 (abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101) preserving number is CGMCC No.12884, and Classification And Nomenclature number is ETEC (Escherichia coli).

Embodiment 2, ispG and ispH combinatorial regulation yield of lycopene

Find out in above-described embodiment 1 that ispG and ispH combinatorial regulation improves in the experiment of beta carotene yield, beta carotene Yield highest is combined as G4H14, i.e. mRSL-4::IspG and mRSL-14::Corresponding startup sub-portfolio in ispH, can be effective Improve ispG and ispH expression, whether that has same effect to all of terpenoid.

The present embodiment, uses mRSL-4::IspG and mRSL-14::IspH combinatorial regulation lycopene.

First, ispH gene in regulation and control LYC010

Recombination bacillus coli LYC013 is to insert before the ispH gene that will set out in bacterium LYC010 genome to be used for starting The mRSL-14 of ispH gene expression::IspH (sequence 32).

Above-mentioned replacement regulates and controls ispH gene by two-step method, builds LYC013 bacterial strain, comprises the following steps that.

(1) with pXZ-CS plasmid as template, ispH-cat-up and ispH-cat down is primer, and PCR obtains 3000bp The DNA fragmentation I of left and right；After DpnI is processed, DNA fragmentation I electricity is gone in the Escherichia coli LYC010 with pKD46.Take 200 μ l The bacterium solution of conversion is coated on the LB flat board containing chloramphenicol and ammonia benzyl, after 30 DEG C of incubated overnight, respectively containing chloramphenicol ammonia benzyl LB flat board and the LB flat board containing kanamycins on screen, obtain not long on kanamycins LB flat board, chloramphenicol ammonia benzyl LB Long clone on flat board, enters bacterium colony PCR checking using primer cat-up and ispH-395-down, and result recombinant bacterium is correct, and this is heavy Group bacterium can be used for second step homologous recombination for the Escherichia coli LYC010 with cat-sacB before ispH gene, this bacterium.

(2) with the genomic DNA of ispH-14 as template, amplimer is ispH-440-up/ispH-395-down, PCR amplification obtain 940bp about DNA fragmentation II；This fragment include corresponding controlling element and upstream and downstream 400bp about same Source arm, this bar segment electricity is gone in the bacterial strain containing pKD46 that step () obtains.Transformed bacteria solution is proceeded to the salt-free LB+ of 50ml In 10%surcose culture medium, 37 DEG C, after 250rpm concussion and cultivate 24h, in the flat lining out of salt-free LB+6%surcose, 41 DEG C incubated overnight, removes pKD46 plasmid.Screen on the LB flat board containing chloramphenicol and the LB flat board without antibiotic respectively, The clone not grown is obtained on the LB flat board containing chloramphenicol, enters performing PCR checking, carried out using primer P-up/ispH-395-down Checking.Screening verification is correct, sends to sequencing, and the correct Strain Designation of sequencing is LYC013.In described case, the primer is shown in Table 1, build bacterial strain and be shown in Table 2.

2nd, ispG gene in regulation and control LYC010 and LYC013

LYC011 is to insert before the ispG gene that will set out in bacterium LYC010 genome to be used for starting ispG gene expression MRSL controlling element mRSL-1::IspG (sequence 21).

LYC012 is to insert before the ispG gene that will set out in bacterium LYC010 genome to be used for starting ispG gene expression MRSL controlling element mRSL-4::IspG (sequence 23).

LYC014 is to insert before the ispG gene that will set out in bacterium LYC010 genome to be used for starting ispG gene expression MRSL controlling element mRSL-1::IspG (sequence 21), and insert before the ispH gene that will set out in bacterium LYC010 genome and be used for Start the mRSL-14 of ispH gene expression::IspH (sequence 32).

LYC015 is to insert before the ispG gene that will set out in bacterium LYC010 genome to be used for starting ispG gene expression MRSL controlling element mRSL-4::IspG (sequence 23), and insert before the ispH gene that will set out in bacterium LYC010 genome and be used for Start the mRSL-14 of ispH gene expression::IspH (sequence 32).

Above-mentioned each bacterial strain artificial regulatory element (respectively sequence 21 and sequence 23) of the ispG of ispG-1, ispG-4, Respectively from LYC010 and LYC013, two-step method regulates and controls ispG gene, builds LYC011, LYC012, LYC014, LYC015 bacterium Strain, comprises the following steps that.

(1) with pXZ-CS plasmid as template, ispG-cat-up and ispG-cat-down is primer, and PCR obtains 3000bp The DNA fragmentation I of left and right；After DpnI is processed, by DNA fragmentation I respectively electricity go to Escherichia coli LYC010 with pKD46, In LYC013.The bacterium solution taking 200 μ l conversions is coated on the LB flat board containing chloramphenicol and ammonia benzyl, after 30 DEG C of incubated overnight, respectively LB flat board containing chloramphenicol ammonia benzyl and the LB flat board containing kanamycins screen, obtains on kanamycins LB flat board not Long, long clone on chloramphenicol ammonia benzyl LB flat board, enters bacterium colony PCR checking, result using primer cat-up and ispG-305-down Recombinant bacterium is correct, and this recombinant bacterium is Escherichia coli LYC010, LYC013 with cat-sacB before ispG gene, for second step Homologous recombination.

(2) with the genomic DNA of ispG-1, ispG-4 as template, amplimer is ispG-471-up/ispG-305- Down, PCR amplification obtain 770bp about DNA fragmentation II；This fragment includes corresponding controlling element and upstream and downstream about 400bp The homology arm of left and right, this bar segment electricity is gone in the bacterial strain containing pKD46 that step () obtains.Transformed bacteria solution is proceeded to 50ml In salt-free LB+10%surcose culture medium, 37 DEG C, after 250rpm concussion and cultivate 24h, on salt-free LB+6%surcose flat board Line, 41 DEG C of incubated overnight, remove pKD46 plasmid.Respectively in the LB flat board containing chloramphenicol and the LB flat board without antibiotic Upper screening, obtains the clone not grown on the LB flat board containing chloramphenicol, enters performing PCR checking, using primer P-up/ispG-305- Down is verified.Screening verification is correct, sends to sequencing, the correct Strain Designation of sequencing is LYC011, LYC012, LYC014, LYC015.In described case, the primer is shown in Table 1, builds bacterial strain and is shown in Table 2.

IspG-1 is to replace with the promoter of the ispG gene in CAR005 genome in Escherichia coli shown in table 2 mRSL-1::IspG (controlling element mRSL-1::IspG sequence is shown in sequence 21).

IspG-4 replaces with the promoter of the ispG gene in CAR005 genome in Escherichia coli shown in table 2 mRSL-4::IspG (controlling element mRSL-4::IspG sequence is shown in sequence 23).

3rd, the lycopene of recombination bacillus coli LYC010, LYC011, LYC012, LYC014, LYC015 measures.

Recombination bacillus coli LYC011, LYC012, LYC014, LYC015 and LYC010 are chosen single bacterium colony train in the LB of 4ml In the test tube of foster base, 37 DEG C of 250rpm incubated overnight 24 hours；Then according to the inoculum concentration of volumn concentration 1%, will overnight Culture seed liquor be transferred in the little shaking flask of the culture medium of LB containing 10ml (100ml), 37 DEG C, 250rpm lucifuge culture 24h after, Yield of lycopene is measured by sampling.With LYC010 for comparison.

Assay method is as follows：

The bacterium solution taking 0.5ml culture is centrifuged 3min in 14000rpm, after sterile water wash, is suspended with 1ml acetone and precipitates, Extract 15 minutes under 55 DEG C of dark conditions, then sample is centrifuged 10 minutes under 14000rpm, supernatant takes on a red color, and bacterial strain warp After acetone extract, white is changed into from redness, by the supernatant containing lycopene after 0.45 μm of filtering with microporous membrane, uses HPLC Carry out assay.

Testing conditions：VWD detector, Symmetry C18 chromatographic column (250mm × 4.6mm, 5 μm), mobile phase is methyl alcohol： Acetonitrile：Dichloromethane (21:21:8), flow velocity 1.0mL/min, 30 DEG C of column temperature, Detection wavelength 480nm, minute is 20min. Each testing sample has 3 Duplicate Samples respectively, and experimental result takes from 3 parallel averages.Or with Symmetry C18 chromatogram Post (100mm × 4.6mm, 5 μm), mobile phase is methyl alcohol：Acetonitrile：Dichloromethane (21:21:8), flow velocity 1.2mL/min, measures 10min.

Pure Lycopene is purchased from sigma company of the U.S. (Cat.No.L9879).Through HPLC inspection, the appearance in sample Time identical with the appearance time of lycopene in standard items (appearance time is 11.3min).Each testing sample has 3 respectively Duplicate Samples, experimental result takes three parallel mean values.

Result is as shown in figure 4, LYC011 the and LYC012 bacterial strain of independent high expression ispG gene, its growth and lycopene Yield all receives suitable suppression, and cell concentration is the 76% and 91% of starting strain LYC010 respectively, and yield of lycopene is respectively The 12% of LYC010 and 40%；LYC014 and LYC015 is obtained, carefully after combinatorial regulation ispH on the basis of LYC011 and LYC012 Intracellular growth recovers, and yield of lycopene significantly improves, and yield of lycopene is respectively 7.97 times of starting strain (LYC010) With 4.43 times, the yield of lycopene of yield highest bacterial strain LYC015 is 44.38mg/L, and per cell dry weight yield is 27.97mg/g DCW, unit cell yield is 1.82 times of LYC010.

Embodiment 3, LYC023 strain construction and yield of lycopene measure

In LYC015 bacterial strain, the promoter of crt operator is composing type artificial regulatory element M1-93, by constitutive promoter It is replaced with inducible promoter trc promoter, this promoter is induced by IPTG.The implementation case research constitutive promoter and luring The impact to yield of lycopene for the conductivity type promoter.

First, crt operator promoter in LYC015 bacterial strain is replaced with inducible promoter

LYC023 is to insert before the ispG gene that will set out in bacterium LYC010 genome to be used for starting ispG gene expression MRSL controlling element mRSL-4::IspG (sequence 23), and insert before the ispH gene that will set out in bacterium LYC010 genome and be used for Start the mRSL-14 of ispH gene expression::IspH (sequence 32), and the promoter of crt operator in LYC010 genome is replaced It is changed to the DNA molecular containing inducible promoter Trc promoter and lacI (sequence 33), the recombinant bacterium obtaining.

From LYC015, two-step method regulates and controls crt operator, builds LYC023 bacterial strain, comprises the following steps that.

(1) with pXZ-CS plasmid as template, ldhA-cat-up and crtE-cat down is primer, and PCR obtains 3000bp The DNA fragmentation I of left and right；After DpnI is processed, DNA fragmentation I electricity is gone in the Escherichia coli LYC015 with pKD46.Take 500 μ l The bacterium solution of conversion is coated on the LB flat board containing chloramphenicol and ammonia benzyl, after 30 DEG C of incubated overnight, respectively containing chloramphenicol ammonia benzyl LB flat board and the LB flat board containing kanamycins on screen, obtain not long on kanamycins LB flat board, chloramphenicol ammonia benzyl LB Long clone on flat board, enters bacterium colony PCR checking using primer cat-up and crtE-340-down, and result recombinant bacterium is correct, and this is heavy Group bacterium can be used for second step homologous recombination for the Escherichia coli with cat-sacB before crt operator, this bacterium.

(2) with QL002 bacterial strain (Zhao J, Li Q, Sun T, et al.Engineering central metabolic modules of Escherichia coli for improving beta-carotene production.Metabolic engineering 2013；17:42-50.CAR001 and CAR005D comes from this article) STb gene be template, amplification draws Thing be ldhA-up/crtE-340-down, PCR amplification obtain 2340bp about DNA fragmentation II；This fragment includes Trc and opens Mover and lacI sequence and upstream and downstream 400bp about homology arm (Trc promoter and lacI sequence are shown in sequence 33), by this silver Section electricity goes in the bacterial strain containing pKD46 that step () obtains.Transformed bacteria solution is proceeded to 50ml salt-free LB+10%surcose training In foster base, 37 DEG C, after 250rpm concussion and cultivate 24h, in the flat lining out of salt-free LB+6%surcose, 41 DEG C of incubated overnight, go Except pKD46 plasmid.Screen on the LB flat board containing chloramphenicol and the LB flat board without antibiotic respectively, obtain containing chloramphenicol LB flat board on not grow clone, enter performing PCR checking, verified using primer ldhA-up/crtE-340-down.Screening is tested Card is correct, sends to sequencing, and the correct Strain Designation of sequencing is LYC023.In described case, the primer is shown in Table 1, builds bacterial strain and sees Table 2.

2nd, the lycopene of recombination bacillus coli LYC015 and LYC023 measures.

Recombination bacillus coli LYC015 and LYC023 is carried out fermented and cultured according to the method for embodiment 2, and will overnight train Foster seed liquor is transferred in the little shaking flask containing 10ml fermentation medium (100ml), 37 DEG C, 250rpm lucifuge culture 4h after, use The IPTG induction crt operator expression of 1mM, after cultivating 24 hours, takes a certain amount of bacterium to measure yield of lycopene.Result such as table Shown in 3.

Table 3 is abduction delivering crt operator and glpD regulation and control affect on yield of lycopene

As shown in table 3, LYC023 grows slightly good and LYC015, and OD600 is 1.06 times of LYC015, yield and specific yield Slightly reduce, be the 76% and 72% of LYC015 respectively, but thalline is more stable.

Case study on implementation 4, LYC029 strain construction and yield of lycopene measure

One .M1-46 artificial regulatory element regulates and controls the glpD gene of LYC023

Recombinant bacterium LYC029 is to insert before the ispG gene that will set out in bacterium LYC010 genome to be used for starting ispG gene The mRSL controlling element mRSL-4 of expression::IspG (sequence 23), and insert before the ispH gene that will set out in bacterium LYC010 genome Enter the mRSL-14 for starting ispH gene expression::IspH (sequence 32), and by the opening of crt operator in LYC010 genome Mover replaces with the DNA molecular containing inducible promoter Trc promoter and lacI (sequence 33), and by LYC010 genome The promoter of glpD gene is substituted for artificial regulatory element M1-46, the recombinant bacterium obtaining.

The promoter sequence of glpD gene is shown in sequence 34, and the artificial regulatory element M1-46 sequence changed is shown in sequence 35.

With the method for two step homologous recombination, insert artificial regulatory element, specific as follows：

(1) with pXZ-CS plasmid as template, glpD-cat-up and glpD-cat down is primer, and PCR obtains 3000bp The DNA fragmentation I of left and right；After DpnI is processed, DNA fragmentation I electricity is gone in the Escherichia coli LYC023 with pKD46.Take 200 μ l The bacterium solution of conversion is coated on the LB flat board containing chloramphenicol and ammonia benzyl, after 30 DEG C of incubated overnight, respectively containing chloramphenicol ammonia benzyl LB flat board and the LB flat board containing kanamycins on screen, obtain not long on kanamycins LB flat board, chloramphenicol ammonia benzyl LB Long clone on flat board, enters bacterium colony PCR checking using primer cat-up and glpD-373-I-r, and result recombinant bacterium is correct, and this is heavy Group bacterium can be used for second step homologous recombination for the Escherichia coli LYC023 with cat-sacB before glpD gene, this bacterium.

(2) with the genomic DNA of M1-46 as template, amplimer is glpD-p-up/glpD-RBS-down, and PCR expands Increase obtain 200bp about DNA fragmentation II；This fragment includes the initiation codon upstream and downstream of corresponding controlling element and glpD 50bp about homology arm, this bar segment electricity is gone in the bacterial strain containing pKD46 that step () obtains.Transformed bacteria solution is proceeded to In 50ml salt-free LB+10%surcose culture medium, 37 DEG C, after 250rpm concussion and cultivate 24h, put down in salt-free LB+6%surcose Lining out, 41 DEG C of incubated overnight, remove pKD46 plasmid.Respectively in the LB flat board containing chloramphenicol and the LB without antibiotic Screen on flat board, the clone not grown is obtained on the LB flat board containing chloramphenicol, enter performing PCR checking, using primer P-up/glpD- 373-I-r is verified.Screening verification is correct, sends to sequencing, and the correct Strain Designation of sequencing is LYC029.Institute in described case It is shown in Table 1 with primer, build bacterial strain and be shown in Table 2.

2nd, the lycopene of recombination bacillus coli LYC023 and LYC029 measures.

Recombination bacillus coli LYC023 and LYC029 is carried out fermented and cultured according to the method for embodiment 2, and will overnight train Foster seed liquor is transferred in the little shaking flask containing 10ml fermentation medium (100ml), 37 DEG C, 250rpm lucifuge culture 4h after, use The IPTG induction crt operator expression of 1mM, after cultivating 24 hours, takes a certain amount of bacterium to measure yield of lycopene.Result such as table Shown in 3.

As shown in table 3, after regulation and control glpD on the basis of LYC023, cell growth and yield of lycopene are all improved, growth 22% and 7% are adjusted with yield respectively.

LYC029 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 19th in August in 2016 (abbreviation CGMCC, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101) preserving number is CGMCC No.12883, and Classification And Nomenclature is ETEC (Escherichia coli).

Embodiment 5, the lycopene high density fermentation of recombination bacillus coli LYC023 and LYC029

By recombination bacillus coli LYC023 and LYC029 in 7L fermentation tank (Labfors 4；InforsBiotechnoligy Co.Ltd. carry out high density fermentation in).Method is as follows：

Technological process is as shown in fig. 6, specific as follows：

1. take lycopene bacterial classification from -80 DEG C of refrigerators, in the flat lining out of LB, be positioned over 15h in 37 DEG C of incubators.

2. picking single bacterium colony is inoculated in the triangular flask containing 120mlLB culture medium, is placed in 37 DEG C of shaking tables, and 250rpm cultivates Cause OD₆₀₀For 3.0-4.0, the seed liquor of the bacterium solution obtaining as high density fermentation..

3. the seed liquor of preparation is inoculated in 5L fermentation tank, the culture of 37 DEG C of cultivation temperature, pH be 7.0, dissolved oxygen constant 20%, dissolved oxygen and stirring and ventilation cascade adjust rotating speed by the intelligent control system of instrument and dissolved oxygen is maintained by ventilation 20%.In initial medium, carbon source exhausts rear dissolved oxygen and can raise suddenly, now opens feed supplement, adjusts feed supplement by DO-STAT method Dissolved oxygen is maintained suitable scope by speed.

4. 0.1mM IPTG induction is added when thalline OD grows to 90 about.48 hours fermentation ends of culture.

According to embodiment 2 method, the zymotic fluid of different time points is taken to measure cell yield of lycopene and OD₆₀₀.

Result as shown in figure 5, LYC023 cultivate 48 hours, thalline OD₆₀₀For 344, yield of lycopene is 3.14g/l, single Position yield 28.3mg/g (Fig. 5 A).LYC029 cultivates 48 hours, thalline OD₆₀₀For 420, yield of lycopene is 4.64g/l, relatively LYC023 improves 47.8%, specific yield 34.2mg/g, improves 20.8% (Fig. 5 B) compared with LYC023.

Claims

1. recombinant bacterium, for following 1) -3) in any one：

1) be improve containing crt operator and produce lycopene Escherichia coli in (E) -4- hydroxy-3-methyl -2- cyclobutenyl - The expression of pyrophosphate synthetase gene ispG and (E) -4- hydroxy-3-methyl -2- cyclobutenyl-pyrophosphoric acid reductase gene ispH And/or activity, obtain recombinant bacterium；

2. recombinant bacterium according to claim 1 it is characterised in that：Described raising produces (E) -4- in lycopene Escherichia coli Hydroxy-3-methyl -2- cyclobutenyl-pyrophosphate synthetase gene ispG and (E) -4- hydroxy-3-methyl -2- cyclobutenyl-pyrophosphoric acid The expression of reductase gene ispH and/or activity are to be realized by following (1) or (2)：

(1) insertion before the described ispG gene producing in lycopene Escherichia coli is started shown in the sequence 23 of ispG gene expression Controlling element mRSL-4::IspG, and insertion before the described ispH gene producing in lycopene Escherichia coli is started ispH base Because of the mRSL-14 shown in the sequence 32 of expression::ispH；

(2) insertion before the described ispG gene producing in lycopene Escherichia coli is started shown in the sequence 21 of ispG gene expression MRSL controlling element mRSL-1::IspG, and insertion before the described ispH gene producing in lycopene Escherichia coli is started The mRSL-14 shown in sequence 32 of ispH gene expression::ispH；

Described regulation and control 1) shown in the crt operator expression of recombinant bacterium and/or activity be by 1) shown in crt operator in recombinant bacterium Promoter replace with inducible promoter；

Described regulation and control 2) shown in the glpD gene expression of recombinant bacterium and/or activity be by 2) shown in glpD gene in recombinant bacterium Promoter is substituted for artificial regulatory element M1-46.

3. recombinant bacterium according to claim 1 and 2 it is characterised in that：

Described insertion is to carry out by the way of genome pinpoints editor or homologous recombination；

Or described homologous recombination is specially λ-red homologous recombination or the homologous recombination of sacB gene mediated screening or integrated plasmid is situated between The homologous recombination led.

4. according to described recombinant bacterium arbitrary in claim 1-3 it is characterised in that：

Described inducible promoter is the DNA molecular containing inducible promoter Trc promoter and lacI；

Or described product lycopene Escherichia coli be LYC010.

5. according to described recombinant bacterium arbitrary in claim 1-4 it is characterised in that：

3) preserving number of the recombinant bacterium shown in is CGMCC No.12883.

6. a kind of method building arbitrary described recombinant bacterium in claim 1-5, comprises the steps：

A-C：

A is following 1) or 2)：

1) before the ispG gene that will produce in lycopene Escherichia coli, insertion starts the tune shown in the sequence 23 of ispG gene expression Control element mRSL-4::IspG, and insertion before the described ispH gene producing in lycopene Escherichia coli is started ispH gene table The mRSL-14 shown in sequence 32 reaching::IspH, obtains recombinant bacterium；

2) insertion before the described ispG gene producing in lycopene Escherichia coli is started shown in the sequence 21 of ispG gene expression MRSL controlling element mRSL-1::IspG, and insertion before the described ispH gene producing in lycopene Escherichia coli is started The mRSL-14 shown in sequence 32 of ispH gene expression::IspH, obtains recombinant bacterium；

C, by B) promoter of glpD gene in the recombinant bacterium that obtains is substituted for artificial regulatory element M1-46, obtains recombinant bacterium.

7. method according to claim 6 it is characterised in that：

8. the method according to claim 6 or 7 it is characterised in that：Described inducible promoter is to start containing induction type Sub- Trc promoter and the DNA molecular of lacI；

Or described product lycopene Escherichia coli be LYC010.

9. application in producing lycopene for arbitrary described recombinant bacterium in claim 1-5.

10. a kind of method producing lycopene, comprises the steps：Arbitrary described recombinant bacterium in fermentation claim 1-5, Obtain lycopene.