CN102154191B - 2S-methylmalonyl-coenzyme A producing engineered pseudomonas - Google Patents

Abstract

The invention provides 2S-methylmalonyl-coenzyme A producing engineered pseudomonas, which is constructed by integrating a propionyl-CoA carboxylase coding pccB gene derived from sterptomyces coelicolor, a carboxylase composite A subunit coding accA1 gene, and a screening marker gene into the genome of pseudomonas putida KT2440. A basis is provided for the synthesis of a secondary metabolite of which the heterologous expression uses 2S-methylmalonyl-coenzyme A as a precursor unit. And the 2S-methylmalonyl-coenzyme A producing engineered pseudomonas has a high application value.

Description

Produce the false unit cell engineering bacteria of 2S-methylmalonyl CoA

Technical field

The present invention relates to the genetically engineered field, specifically, relate to a kind of false unit cell engineering bacteria of the 2S-of producing methylmalonyl CoA.

Background technology

Derive from actinomycetic secondary metabolite and be the important source of medicine clinically, about 70% antibacterials belong to this type of.Because the complicacy of these secondary metabolite structures adopts high, the complex operation of synthetic these compound costs of chemical process, can not satisfy the needs that a large amount of commercializations is produced.And adopt original bacterium to carry out the production of secondary metabolites; Often thalli growth speed slow, yield poorly, the difficult cultivation, genetic background is unintelligible, meta-bolites is unintelligible; And be difficult to carry out genetic manipulation, these factors are restricting the Application and Development to valuable secondary metabolite.In recent years, the heterogenous expression of secondary metabolite biological synthesis gene cluster becomes the effective means that improves compound output or transform compound targetedly.

In heterogenous expression host's selection; Because the use of pseudomonas and actinomycetes codon is close; Posttranslational modification is arranged, safety non-toxic (like type strain pseudomonas putida Pseudomonas putida KT2440), growth is fast; Advantages such as the secondary metabolite background is clear become one of first-selected host bacterium.

P.putida KT2440 expressing heterologous product has successful example; As finding to have at least eight PKS types after the whole genome sequence analysis of slime bacteria Stigmatella aurantiaca DW4/3-1; Biosynthesis gene a small bundle of straw, etc. for silkworms to spin cocoons on of NRPS type or PKS/NRPS heterozygous; But Myxothiazol compound is had in the conventional fermentation and the separation and purification of product, and the analysis that certain " silencer a small bundle of straw, etc. for silkworms to spin cocoons on " is knocked out back gained variant finds that its HPLC collection of illustrative plates lacks a characteristic peak than former strain.After this " silencer a small bundle of straw, etc. for silkworms to spin cocoons on " is cloned by RulfMuller study group; Be converted among the P.putidaKT2440, induce down, obtained PKS/NRPS type compound Myxochromide at phenylformic acid; The 2 days output of fermenting has just reached 40mg/mL, and 7 days output of former strain fermentation is merely 8mg/mL.Therefore, can obtain compound new, that potential using value is arranged in large quantities, be the further pharmacodynamic study source that furnishes ample material through gene engineering method.

But pseudomonas putida can not provide the complicated required small molecules precursor of secondary metabolite biosynthesizing all sidedly.As a lot of secondary metabolites need (2S-Methylmalonyl-CoA 2S-mmCoA) as extender unit, and can not detect the existence of 2S-Methylpropanedioic acid coenzyme A in pseudomonas putida with 2S-Methylpropanedioic acid coenzyme A.Though existing report can carry out chemosynthesis 2S-methylmalonyl CoA; But the chemosynthesis cost is big; Technical difficulty is high, and is difficult to obtain single optical isomer product, and because its unstable is difficult to directly supply with mikrobe as synthetic substrate utilization; And the 2S-methylmalonyl CoA generates in cell and can directly be used for the synthetic of secondary metabolite, can reduce fermentation costs greatly.Therefore Synthetic 2 S-methylmalonyl CoA is optimal selection in heterogenous expression host bacterium; Structure can produce the engineering strain of 2S-Methylpropanedioic acid coenzyme A; Be heterogenous expression need with the basis as the secondary metabolite of precursor substance, value has a wide range of applications.

The biosynthetic pathway that two 2S-Methylpropanedioic acid coenzyme As are arranged in the known organism body.The one, the MCM approach; Be initial compounds promptly with the succinyl-coenzyme A in the tricarboxylic acid cycle; Effect through methylmalonyl-CoA isomerase (MCM) and methylmalonyl-CoA isomerase mixture protected protein (MeaB) generates the 2R-methylmalonyl CoA, and the effect through methylmalonyl CoA isomerase (Epi) generates 2S-Methylpropanedioic acid coenzyme A again.Other one is the PCC approach; Promptly at first generate (or with the degraded of odd bits lipid acid) and obtain propionyl coenzyme A from L-L-Ala, L-Isoleucine or L-methionine(Met); Then the effect through carboxylase generates 2S-Methylpropanedioic acid coenzyme A in the presence of ATP, and the gene of participating in this process comprises propionyl-CoA carboxylase gene and carboxylase mixture A subunit gene.

For heterogenous expression need be with 2S-Methylpropanedioic acid coenzyme A as the unitary myxothiazol of precursor; Germany Rolf Muller study group is integrated into mcm, epi and three genes of meaB of cluster in the full genome of slime bacteria Sorangium cellulosumSoce56 in the genome of pseudomonas P.putida KT2440 through homologous recombination; After the obtained strains fermentation, show the 2S-Methylpropanedioic acid coenzyme A that can produce 4.68nmol/mL through gc and mass spectrum logotype (GC/MS) detection.Yet the output of this bacterial strain 2S-Methylpropanedioic acid coenzyme A is lower, has directly influenced the output of final secondary metabolite.

Summary of the invention

The false unit cell engineering bacteria that the purpose of this invention is to provide a kind of 2S-of producing methylmalonyl CoA.

In order to realize the object of the invention; A kind of false unit cell engineering bacteria that produces the 2S-methylmalonyl CoA of the present invention, it is that the accA1 gene of the pccB gene of the coding propionyl-CoA carboxylase that derives from streptomyces coelicolor (Sterptomyces coelicolor) and coding carboxylase mixture A subunit is integrated in the genome of pseudomonas putida (Pseudomonasputida) KT2440 with the selection markers gene and obtains.Wherein, said selection markers gene is kalamycin resistance gene or qingfengmeisu qiong resistant gene.

Preferably, aforesaid engineering bacteria is pccB gene and accA1 gene are integrated into the zone between the rpe gene and trpE gene in the pseudomonas putida KT2440 genome with the selection markers gene and obtain.Wherein, rpe gene and trpE gene encode respectively ribulose monophosphate 3-isomerase and o-amino benzoyl acid synthase are the gph gene of coding phosphoric acid ethanol Phosphoric acid esterase between rpe gene and the trpE gene.The gph gene is nonessential gene, and promptly the disappearance of this gene does not influence the physiology and the biochemical function of bacterial strain.

The cultural method of the false unit cell engineering bacteria of aforementioned product 2S-methylmalonyl CoA is: said engineering bacteria is inoculated in the LB substratum, under 30 ℃, cultivates.

The object of the invention can also adopt following technical measures further to realize.

The present invention provides a kind of pseudomonas putida (Pseudomonas putida) Sino-PCC of the 2S-of producing methylmalonyl CoA, preserving number CGMCC NO.4517.It obtains through following method: will derive from streptomyces coelicolor the encoding sox propionyl-CoA carboxylase pccB gene and coding carboxylase mixture A subunit the accA1 gene with kalamycin resistance gene through recombination and integration to the genome of pseudomonas putida KT2440.Kalamycin resistance gene and pccB gene and accA1 gene link together, as the selection markers of gene integration bacterial strain.

Sino-PCC ferments to pseudomonas putida, and show through gc and mass spectrum logotype (GC/MS) detection: Sino-PCC can produce the 2S-Methylpropanedioic acid coenzyme A up to 6.24nmol/mL, and this output is higher than the output of bibliographical information.

The present invention is through engineered method; PccB gene and the accA1 gene of coding carboxylase mixture A subunit that will derive from the coding propionyl-CoA carboxylase of streptomyces coelicolor (Sterptomyces coelicolor) are integrated in the genome of pseudomonas putida (Pseudomonas putida) KT2440 with the selection markers gene; Structure obtains producing the false unit cell engineering bacteria of 2S-methylmalonyl CoA; For heterogenous expression need provide the foundation as the synthetic of the unitary secondary metabolite of precursor with 2S-Methylpropanedioic acid coenzyme A, value has a wide range of applications.

Description of drawings

Fig. 1 is that the genes involved of PCC approach biosynthesizing 2S-methylmalonyl CoA is integrated into the synoptic diagram in the P.putida KT2440 genome, and wherein: oriT combines to shift fragment, and sacB is a 6-fructosyl transferase gene; Bla is an ampicillin resistance gene; Rpe is a ribulose monophosphate 3-isomerase gene, and Km is a kalamycin resistance gene, and accA1 is the gene of coding propionyl group coenzyme A carboxylase α subunit; PccB is the gene of coding propionyl group coenzyme A carboxylase; TrpE is the anthranilic acid synthase gene, and gph is a phosphoric acid ethanol phosphatase gene, and pp_0414 and pp_0418 are the not clear and definite genes of function in the P.putida KT2440 genome; The PCR primer that AS1, L1, L2, K2, AS2, R1, R2 and AS3 are designed for checking P.putida Sino-PCC strain gene type.

Fig. 2 is the agarose gel electrophoresis figure of P.putida Sino-PCC strain gene type analysis, wherein: 1.DL500DNA Marker; 2.DL2000DNA Marker; 3.AS1 1.2kb with the L2 primer amplification; 4.L1 2.1kb with the K2 primer amplification; 5.AS2 2.6kb with the R2 primer amplification; 6.R1 1.5kb with the AS3 primer amplification.

Fig. 3 is the GC/MS analytical results figure that P.putida Sino-PCC produces 2S-mmCoA, and wherein: A is the GC/MS analytical results figure of P.putida KT2440 fermented liquid (blank); B is the GC/MS figure as a result of P.putida Sino-PCC fermented liquid, and MS101 is the molecular ion peak of propanedioic acid, and MS104 is the molecular ion peak of interior mark deuterium for propanedioic acid.

Fig. 4 is time-production pattern that P.putida Sino-PCC produces the 2S-methylmalonyl CoA.

Embodiment

Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.

The term that uses in following examples only if other explanation is arranged, generally has the implication of those of ordinary skills' common sense.

In following examples; Various processes and the method do not described in detail are ordinary methods as known in the art; The source of agents useful for same, trade(brand)name and being necessary listed its moity; Indicate when occurring first that all used thereafter identical reagent is like no specified otherwise, and is all identical with the content of indicating first.

Bacterial strain that uses in following examples and plasmid are disclosed bacterial strain and plasmid:

1.Escherichia coli DH10B: genotype F-mcrA (mrr-hsdRMS-mcrBC) 80dlacZ M15lacX74deoR recA1araD139 (ara, leu) 7697galU galK rpsL endA1nupG.Document: LifeTechnologies, Inc.Focus (1990) 12, and 19.Available from American I nvitrogen company.

2.E.coli HB101/pRK2073: document: Marx CJ; Lidstrom ME.evelopment of improved versatile broad-host-range vectors for use inmethylotrophs and other Gram-negative bacteria.icrobiology.2001,147 (Pt 8): 2065-75.Provide by Mary professor Lidstrom of Washington, DC university.

3.P.putida KT2440: document: Nelson KE; Weinel C; Paulsen IT etc.; Complete genome sequence and comparative analysis of themetabolically versatile Pseudomonas putida KT2440.Environ Microbiol2002,4:799-808.Doctor KarenNelson by U.S. The Institute for Genomic Research provides.

4.S.coelicolor A3 (2): document: Bentley SD; Chater KF; Cerdeno-Tarraga AM etc., Complete genome sequence of the modelactinomycete Streptomyces coelicolor A3 (2) .Nature.2002,417:141-147.Provide by Keith doctor Chater of Britain John Innes institute.

5.pBluescript II KS (-): document: Alting-Mees MA, Short JM.pBluescript II:gene mapping vectors.Nucleic Acids Res, 1989,17:9494.Available from U.S. Novagen company.

6.pACYC177: document: Chang AC; Cohen SN.Construction andcharacterization of amplifiable multicopy DNA cloning vehicles derivedfrom the P15A cryptic miniplasmid.1978; J Bacteriol; 134,1141-1156.Available from U.S. NEB company.

7.pEX100Tlink: document: Quenee L; Lamotte D; Polack B.CombinedsacB-based negative selection and cre-lox antibiotic marker recycling forefficient gene deletion in pseudomonas aeruginosa.Biotechniques.2005,38 (1): 63-7.Benoit professor Polack by French Centre Hospitalier Universitaire provides.

8. the Genbank of pseudomonas putida KT2440 genome sequence reception number (Accession No.) is NC_002947.

Embodiment 1 makes up and contains the plasmid with the genes involved of PCC approach biosynthesizing 2S-methylmalonyl CoA

1. the preparation of competence intestinal bacteria DH10B and DNA transform

37 ℃ of shaken overnight in inoculation fresh streak culture single bacterium colony to the 2ml LB liquid nutrient medium go among the 50ml LB with 1/50 volume on flat board, and shaking culture is about 0.6 to thalline OD600.Bacterium liquid is poured in the centrifuge tube of precooling, ice bath 10 minutes, 4 ℃, centrifugal 5 minutes of 5000rpm abandons supernatant.Glycerine washing precipitation twice with 10% is suspended in 10% glycerine of 200 μ l at last, every pipe packing 50 μ l.

DNA is connected in the electric transformed competence colibacillus cell that product adds to the DH10B that 50 μ l melt on ice, flick mixing.Mixed solution is transferred in the 1mm electricity revolving cup of precooling on ice, electric shock transforms.Electricity conversion condition: 1mm electricity revolving cup, 200 Ω, 1800V, the Gene PulserIIR of Bio-Rad company electricity conversion instrument.Add 1ml LB liquid nutrient medium to electric revolving cup, the piping and druming mixing is transferred to solution in the aseptic 1.5ml eppendorf pipe, and 37 ℃ or 30 ℃ of shaking culture are after 60 minutes, and conversion fluid is coated on the corresponding antibiotic resistant panel, 37 ℃ or 30 ℃ of cultivations.Picking list bacterium colony is cultivated in containing corresponding antibiotic liquid nutrient medium, extracts plasmid, the enzyme evaluation of cutting and check order.

The composition of LB substratum (g/ml, %): peptone 1, yeast extract 0.5, sodium-chlor 1 adds 1.5% agar during solid culture, 115 ℃ of sterilization 20min.

2.Sterptomyces the extraction of coelicolor A3 (2) genomic dna

To line on the ISPII solid plate at-70 ℃ of frozen S.coelicolor A3 (2), cultivate more than 2 days for 30 ℃; Cultivate from the one ferfas piece of picking on the solid plate to 20mL ISPII liquid nutrient medium, 30 ℃ of 220rpm/min shaking tables were cultivated about 30 hours; Get the 1.0ml thalline to the 1.5mL centrifuge tube, 13200rpm is centrifugal, 30 seconds, collects thalline; Thalline is resuspended in 467L TE damping fluid, and mixing adds 30mL 10%SDS (sodium laurylsulfonate), adds the Proteinase K solution of 3mL20mg/mL, mixing, and 37 ℃ are incubated 1 hour; Add equal-volume phenol: chloroform extracting, mixing, the centrifugal 10min of 13200rpm; Get supernatant, repeat above-mentioned steps; Get supernatant, add the extracting of equal-volume chloroform, mixing, the centrifugal 10min of 13200rpm; Get supernatant, add the 5M sodium-acetate of 1/10 times of volume, the Virahol of 0.6 times of volume, the flocks of mixing to adularescent produces; With the flocks of glass stick picking, use 70% washing with alcohol; Flocks is dissolved in an amount of TE damping fluid, 50 ℃ of 30min; Place 37 ℃ to treat that DNA dissolves fully.

The composition of ISPII substratum (g/ml, %): yeast extract 0.3, malt extract 1, glucose 1, pH7.3 adds 1.5% agar during solid culture, 115 ℃ of sterilization 20min.

The composition of TE damping fluid: the 10mM Tri(Hydroxymethyl) Amino Methane Hydrochloride, the 1mM edetate disodium, pH 8.0.

3. make up the plasmid of the genes involved that contains PCC approach biosynthesizing 2S-methylmalonyl CoA

The design primer:

L1：5′-GGG GAGCTCGGGAGCCCTGTTTGCCTTTGCC-3′

L2：5′-GGG GGATCCGCATGCCGATCTCGCGGATGTTGTTGAC-3′

Introduce the SacI site at 5 ' end respectively, introduce BamHI and SphI site (representing) with underscore at 3 ' end.With P.putida KT2440 genomic dna is template, and L1 and L2 are the rpe gene region that primer amplification goes out 1.0kb, after cutting with SacI and BamHI enzyme, is cloned into SacI and the BamHI site of pBluescript IIKS (-), obtains recombinant plasmid pSino-100.

The design primer:

K1：5′-GGG GAGCTCGCA-3′

K2：5′-GGG GGATCCTTAGAAAAACTCATCGAGCATC-3′

Introduce SacI and SphI site at 5 ' end respectively, introduce BamHI site (representing) with underscore at 3 ' end.With pACYC177 is template, and K1 and K2 are the Km gene that primer amplification goes out 1.0kb, after cutting with SacI and BamHI enzyme, is cloned into SacI and the BamHI site of pBluescript II KS (-), obtains recombinant plasmid pSino-101.

The design primer:

AC1：5′-GGG TCTAGAAGATCTGTGCGCAAGGTGCTCATCGCC-3′

AC2：5′-GGG CTGCAGTCAGTCCTTGATCTCGCAGATG-3′

Introduce XbaI and BglII site at 5 ' end respectively, introduce PstI site (representing) with underscore at 3 ' end.With the S.coelicolor genomic dna is template, and AC1 and AC2 are the accA1 gene that primer amplification goes out 1.8kb, after XbaI and PstI enzyme are cut, is cloned into XbaI and the PstI site of pBluescript II KS (-), obtains recombinant plasmid pSino-102.

The design primer:

PB1：5′-GGG CTGCAGATGTCCGAGCCGGAAGAGCAG-3′

PB2：5′-GGG CTCGAGTTACAGGGGGATGTTGCCGTG-3′

Introduce the PstI site at 5 ' end respectively; Introduce XhoI site (representing) at 3 ' end with underscore; With the S.coelicolor genomic dna is template, and PB1 and PB2 are the pccB gene that primer amplification goes out 1.8kb, after PstI and XhoI enzyme are cut; Be cloned into PstI and the XhoI site of pBluescript II KS (-), obtain recombinant plasmid pSino-103.

The design primer:

R1：5′-GGG CTCGAGCTGCGCCTGGCCGCTG-3′

R2：5′-GGG TCTAGAGTGCTCGGCAATTTCCTTGTCGTC-3′

Introduce the XhoI site at 5 ' end respectively, introduce XbaI site (representing) with underscore at 3 ' end.With P.putida KT2440 genomic dna is template; R1 and R2 are the trpE gene region that primer amplification goes out 1.0kb; After cutting with XhoI and XbaI enzyme cutting enzyme, be cloned into XhoI and the XbaI enzyme cutting site of pBluescript II KS (-), obtain recombinant plasmid pSino-104.

The cloning host bacterium of above plasmid is DH10B, and all cut the exactness with its calling sequence of cloning of sequence verification through enzyme.

SacI and SphI enzyme are cut the rpe gene fragment that pSino-100 obtains 1.0kb; SphI and BamH enzyme are cut the Km gene fragment that pSino-101 obtains 1.0kb; Two fragments connect SacI and the BamHI site of rear clone to pBluescript II KS (-), obtain recombinant plasmid pSino-105.

PstI and XhoI enzyme are cut the pccB gene fragment that pSino-103 obtains 1.6kb; The XhoI-XbaI enzyme is cut the trpE gene fragment that pSino-104 obtains 1.0kb; Two fragments connect PstI and the XbaI site of rear clone to pBluescript II KS (-), obtain recombinant plasmid pSino-106.

SacI and BamHI enzyme are cut the insertion fragment that pSino-105 obtains 2.0kb; BglII and PstI enzyme are cut the accA1 gene fragment that pSino-102 obtains 1.8kb; PstI and XbaI enzyme cutting pSino-106 obtain the insertion fragment of 2.6kb; Three fragments connect SacI and the XbaI site of rear clone to pBluescript II KS (-), obtain recombinant plasmid pSino-107.

The 6.4kb fragment cloning of SacI and XbaI enzyme cutting pSino-108 gained finally is used for the homologous recombination plasmid and is got pSino-108 to the SacI and XbaI site of pEX100Tlink.

More than 50 μ l PCR reaction systems:

dd?H ₂O 38.2μl

10 * PCR reaction buffer, 5 μ l

10mM?dNTP 1.3μl

Upstream primer (final concentration 0.75mM) 1.5 μ l

Downstream primer (final concentration 0.75mM) 1.5 μ l

Template (the about 100ng of plasmid, the about 200ng of genomic dna) 2 μ l

Pfu polysaccharase (5U/ μ l) 0.5 μ l

The PCR reaction conditions: 95 ℃ of sex change 5 minutes, (95 ℃ 45 seconds, 60 ℃ 1 minute, 72 ℃ of 1kb PMs), totally 34 circulations, 72 ℃ were extended 10 minutes.The PCR appearance is available from Bio-rad company, and model is PTC-200.

After reaction finishes, detect with 1% agarose gel electrophoresis of the ethidium bromide (EB) that adds 20 μ g/ml.

Embodiment 2

The method of above-mentioned gained plasmid through three close conjugal transfers is converted into P.putidaKT2440; Under the negative screening effect of 10% sucrose; PCC approach genes involved gets the genome that homologous recombination is integrated into P.putida KT2440 through rpe gene and trpE gene, obtains engineering strain Sino-PCC.

1. the triparental mating method transforms P.putida KT2440

The P.putida KT2440 that from frozen pipe, rules respectively is dull and stereotyped in LB, and 30 ℃, overnight cultures; Line E.coli HB101/pRK2073 is dull and stereotyped in the LB that contains 50 μ g/mL Streptomycin sulphates, and 37 ℃, overnight cultures; Line E.coli DH10B/pSino-108 is dull and stereotyped in the LB that contains 30 μ g/mL kantlex, and 37 ℃, overnight cultures.Choose the single colony inoculation of P.putida KT2440 in 2mL LB liquid nutrient medium, 30 ℃, 220rpm, shaking culture is spent the night; Choose the single colony inoculation of E.coliHB101/pRK2073 and contain in the LB liquid nutrient medium of 50 μ g/mL Streptomycin sulphates in 2mL, 37 ℃, 220rpm, shaking culture is spent the night; Choose single bacterium colony E.coli DH10B/pSino-108 in the LB liquid nutrient medium that contains 30 μ g/mL kantlex, 37 ℃, shaking culture is spent the night.

Get the fresh bacterium liquid of E.coli HB101/pRK2073, E.coli DH10B/pSino-108 and the 1.4mL P.putida KT2440 of 1mL respectively; Centrifugal respectively; Supernatant discarded is washed twice of thalline removing residual microbiotic with LB, and is used the LB suspension thalline of 1mL respectively.The suspension bacteria liquid of 500 μ l E.coli HB101/pRK2073 and 500 μ l E.coli DH10B/pSino-108 is mixed; 37 ℃ of incubation 30min; Mix with the P.putida KT2440 bacterium liquid that 1mL has suspended, centrifugal, 100 μ l LB suspension blended thalline; And small area coats on the LB flat board, places 30 ℃ of about 15h.

Scrape the lawn of getting on the LB flat board with the 1mL sterilized water; Getting the dilution of 20 μ l bacteria suspensions coats on the flat board of the PMM that contains 50 μ g/mL paraxin and 30 μ g/mL kantlex; Cultivate about 20h for 30 ℃; Picking list bacterium colony contains incubated overnight in the LB liquid nutrient medium of 50 μ g/mL paraxin and 30 μ g/mL kantlex at 1mL, gets 50 μ l dilution and coats on the PMM flat board that contains 50 μ g/mL paraxin and 30 μ g/mL kantlex and 10% sucrose.

It is as shown in Figure 1 that the genes involved of PCC approach biosynthesizing 2S-methylmalonyl CoA is integrated into the genomic synoptic diagram of P.putida KT2440.

The composition of PMM substratum (g/ml, %): potassium hydrogenphosphate 0.7, potassium primary phosphate 0.3, Trisodium Citrate 0.05, MAGNESIUM SULPHATE HEPTAHYDRATE 99.5 0.1, ammonium sulfate 0.1, glucose 0.4, agar 1.5, pH7.3,115 ℃ of sterilization 20min.

2. the acquisition of engineering strain Sino-PCC

SacB genes encoding 6-fructosyl transferase among the pSino-108, this enzyme catalysis sucrose is the Polylevulosan to the toxic effect of pseudomonas, the bacterial strain that therefore contains pSino-108 can not be grown containing on the substratum of sucrose.Under the screening of kalamycin resistance; The homologous fragment generation homologous recombination of rpe gene that pSino-108 is last and trpE gene and P.putida KT2440; The Km-accA1-pccB gene integration is to P.putida KT2440 genome, on the genome between rpe gene and the trpE gene gph gene be removed.

With gained list colony inoculation overnight cultures in the LB liquid nutrient medium that contains 30 μ g/mL kantlex, extract genome, carry out the PCR checking.Design of primers is following: AS1:5 '-ATCTCGCCCATGGTGGTGCCG-3 ', for being directed against pp 0414 gene order designed primer; L2 is the downstream primer of amplification rpe gene; L1 is the upstream primer of amplification rpe gene; K2 is the downstream primer of amplification km gene; AS2:5 '-GTGCTCGGCAATTTCCTTGTCGTC-3 ' is the downstream primer of amplification accA1 gene; R2 is the downstream primer of amplification trpE gene; R1 is the upstream primer of amplification trpE gene; AS3:5 '-CTGCGCCTGGCCGCTGCCGGC-3 ' is for being directed against pp_0418 gene intermediate sequence designed primer.

Obtain the correct transgenic engineered bacteria of four pnca gene types altogether; Appoint and get a strain called after P.putida Sino-PCC; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center at present; Datun Road, Chaoyang District, Beijing City, address institute of microbiology of the Chinese Academy of Sciences, deposit number CGMCC NO.4517, preservation date on December 28th, 2010.

The agarose gel electrophoresis figure of P.putida Sino-PCC strain gene type analysis is as shown in Figure 2.

Embodiment 3

Fermentation culture P.putida Sino-PCC is through the content of mmCoA in the GC/MS mensuration different fermentations time cell.

1. the fermentation culture of transformant

The transformant mono-clonal picking of double exchange is contained to 2mL in the LB liquid nutrient medium of 30 μ g/mL kantlex, 30 ℃, 180rpm, shaking culture is spent the night; Contain in the LB liquid nutrient medium of 30 μ g/mL kantlex to 100mL with 1: 1000 dilution proportion; 30 ℃, 180rpm, shaking culture; In 10h, 20h, 40h, 60h, 80h, 100h sampling, cultivate as contrast with the former strain of P.putidaKT2440 simultaneously respectively.OD is measured in the sampling back ₆₀₀, the centrifugal 5min of residue bacterium liquid 7000rpm collects thalline.

2. the extraction of 2S-methylmalonyl CoA in the born of the same parents

In thalline, add 3mL pH7.050mM phosphate buffered saline buffer, mixing; With the thalline ultrasonication, become clear to bacterium liquid; Add 50mL methyl alcohol, the 180rpm vibration shakes up 1 hour; Methanol solution is filtered with funnel; Liquid 45 ℃ of evaporates to dryness on Rotary Evaporators with filtration obtains add the 1ml dissolve with methanol behind the evaporate to dryness, obtain extract.

3.2S-the derivatization treatment of methylmalonyl CoA and internal standard substance

Get 300 μ l extracts, add internal standard substance 10nmol methyl deuterium for propanedioic acid, mixing, vacuum is drained; Add 400 μ l hexanaphthenes, the acid propyl carbinol of 100 μ l, mixing; In sample bottle 80 ℃, reaction 2h is cooled to room temperature; The Na that adds 500 μ l 6% ₂CO ₃Solution, mixing, centrifugal, two are separated; Get upper organic phase.

4.P.putida Sino-PCC produces the GC/MS of 2S-mmCoA and analyzes

Dimethyl malonate and 2S-methylmalonyl CoA react the dibutyl ester that the back generates the Methylpropanedioic acid that can be used for the GC/MS analysis with propyl carbinol under acidic conditions.Select for use deuterium for Methylpropanedioic acid as internal standard substance; On mass spectrum, the molecular ion peak of 2S-methylmalonyl CoA derivatize is MS101, and the internal standard substance deuterium is MS104 for the molecular ion peak of Methylpropanedioic acid derivatize; Noiseless between the two, and in this mass range, do not have other fragment and disturb.

The GC/MS analysis condition is following: the 7000A type makings analyser that adopts U.S. Agilent company.Chromatographic condition: chromatographic column: DB5MS (30 μ m * 250 μ m * 0.25 μ m); Input mode: pulse is not shunted, pulsating pressure: 14psi, burst length: 0.5min; Carrier gas: helium; Post flow: 1mL/min; Injector temperature: 300 ℃.Heating schedule: 70 ℃ of starting temperatures (5 ℃); 5 ℃/min rises to 170 ℃; 30 ℃/min rises to 300 ℃, keeps 5min; 30 ℃/min is cooled to 70 ℃.Mass spectrum condition: solvent delay 3min; EM voltage 1094V; Mass range 50-550; 230 ℃ of ion source temperatures; 150 ℃ of quadrupole temperature; Sample size 1.0 μ l.

According to the ratio of MS101 and MS104 integral area, can calculate the content of 2S-methylmalonyl CoA.Experiment finds that P.putida Sino-PCC fermented sample detects the MS101 quasi-molecular ions, and its fragment peak type is consistent with expection, and former strain does not detect the MS101 quasi-molecular ions, explains that the engineering strain fermentation has produced 2S-mmCoA.The GC/MS analytical results of P.putida Sino-PCC generation 2S-methylmalonyl coenzyme is as shown in Figure 3.This shows that P.putida Sino-PCC fermentation has produced the 2S-methylmalonyl CoA, and former strain P.putida KT2440 fails to detect the 2S-methylmalonyl CoA.

Time-the production pattern of P.putida Sino-PCC generation 2S-methylmalonyl CoA is as shown in Figure 4, and the content that Fig. 4 is illustrated in 40 hours interior 2S-methylmalonyl CoAs of born of the same parents is the highest, reaches 6.24nmol/mL.

Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.

Claims (2)

1. produce the false unit cell engineering bacteria of 2S-methylmalonyl CoA, it is pseudomonas putida (Pseudomonasputida) Sino-PCC, preserving number CGMCC NO.4517.

2. the cultural method of the said engineering bacteria of claim 1 is characterized in that, said engineering bacteria is inoculated in the LB substratum, under 30 ℃, cultivates.

Images