CN1345929A - Novel engineering colibacillus G830 - Google Patents

Abstract

The present invention discloses a microbe applicable to high-density fermentation. On the chromosome of the described microbe the exogenous hyaline vibratile bacterium haemogluobin gene is integrated, and the main pathway of acetic metabolism of said micribe is blocked. Said invented engineering bacterium G 830 not only can elimiante acetic detrimental accumulation, but also can raise its capability for utilizing oxygen greatly, so that it more suitable for expression of exogenous protein under the condition of high-density fermentation. Said invention also discloses a new method for producing integrated exogenous gene (for example hyaline vibratile bacterium haemoglobin gene)in genome.

Description

New engineering colibacillus G 830

The present invention relates to bioengineering field, relate more specifically to intestinal bacteria are transformed being applicable to the method for high density fermentation, the engineering bacteria of acetate metabolism pathway deficiency of and consequent new integrated Vitreoscilla hemoglobin-(Pta-Ack).

The high density fermentation technology of bacillus coli gene engineering bacteria is to obtain a large amount of genetically engineered reorganization purpose products, basis as recombinant vaccine, medicine, the animal and plant growth factor and toolenzyme etc., being that the gene engineering product scale operation that moves towards the industrialization is necessary, is one of focus technology of bioengineering field research.

The research of genetically engineered high density fermentation starts from the seventies, three kinds of modes of general employing: batch culture, cultured continuously and batch feeding are cultivated, influence factor (Yee L, Blanch H W.Recombinant protein expression in high cell density fed-batchcultures of Escherichia coli.Biotechnology (N Y) .1992 of proteolysis enzyme activity, protein excretion and protein yield by the control composition of cell culture medium and fermentation condition such as temperature, pH and some other; 10 (2): 1550-1556; Lee SY..High cell-density culture of Escherichia coli.Trends Biotechnol.1996; 14 (3): 98-105High cell density growth of micro-organisms; Bunch AW.High cell densitygrowth of micro-organisms.Biotechnol Genet Eng Rev.1994; 12:535-61; Park SJ, Georgiou G, Lee SY.Secretory production of recombinant protein by a high celldensity culture of a protease negative mutant Escherichia coli strain.BiotechnolProg 1999; 15 (2): 164-167; Riesenberg D, Schulz V, Knorre WA, Pohl HD, Korz D, Sanders EA, Ross A, Deckwer WD.High cell density cultivation of Escherichia coliat controlled specific growth rate.J Biotechnol.1991; 20 (1): 17-27).

The high density fermentation system of hitherto reported, along with improving constantly of yeast culture density, occur formation that dissolved oxygen deficiency, carbon dioxide content improve, stimulate acetate, reductions of fermentor tank mixing efficiency, heat production etc. in the fermentor tank and cause the reduction of engineering bacteria growth velocity, wherein dissolved oxygen and harmful secondary metabolite acetate are to engineering bacterium fermentation (the Lee S Y.High cell-density culture of Escherichia coli.TrendsBiotechnol.1996 that has the greatest impact; 14 (3): 98-105).This shows how to reduce the generation and the accumulation of acetate during the fermentation, improve engineering bacteria utilization to oxygen under conditions of high density, express significant for culture density that improves genetic engineering bacterium and product.Usually adopt the feed supplement control mode to regulate at present such as factor (Markl H such as dissolved oxygen deficiency, the variations of pH value, Zenneck C, Dubach A, Ogbonna J C.Cultivation of Escherichia coli to high cell densities in a dialysis reactor.ApplMicrobiol Biotechnol.1993; 39 (1): 48-52; Miroux B, Walker J E.Over-productionof proteins in Escherichia coli:mutant hosts that allow synthesis of some membraneproteins and globular proteins at high levels.J Mol Biol.1996; 260 (3): 289-298).But these class methods often are subjected to the restriction of external device condition, can not tackle the problem at its root.Utilize molecular biological method that the pathways metabolism of engineering bacteria is transformed, be expected the fundamentally fermentation level of betterment works bacterium.

When intestinal bacteria are substratum with different carbon sources, its pathways metabolism is different (Fraenkel D G.Glycolysis, pentose phosphate pathway, and Emtner Doudoroff pathway inEscherichia coli and Salmonella typhimurium:Cellcular and Molecular Biology.1987; Pp 142-150.American Society for Microbiology, Washington DC; Knappe J.Anaerobic dissimilation of pyruvate in Escherichia coli and Salmonella tvpimurium:Cellcular and Molecular Biology.1987; Pp 151-155.American Society forMicrobiology, Washington DC; Nimmo H G.The tricarboxylic acid cycle andanaplerotic reactions in Escherichia coli and Salmonella typimurium:Cellcular andMolecular Biology.1987; Pp 156-169.American Society for Microbiology, Washington DC; Nunn W D.Two-carbon compounds and fatty acids as carbonsources in Escherichia coli and Salmonella typimurium:Cellcular and MolecularBiology.1987; Pp 285-301.American Society for Microbiology, WashingtonDC).When being carbon source with glucose, great majority enter tricarboxylic acid cycle by glycolytic pathway, also have some metabolic intermediate product such as pyruvic acid, are the pta-ack approach by phosphate acetyltransferase (pta) and E.C. 2.7.2.1 (ack), product acetate.When being sole carbon source with the pyruvic acid, the first meta-bolites acetyl-CoA that produces produces a large amount of acetate (Guest I R. Anaerobio growth of Escherichiacoli K12 with fumarate as terminal electron acceptor:Genetic studies withmenaquinone and fluoroacetate-resistant mutants.J Gen Microbiol.1979 by the pta-ack approach; 115:259-271; Brown T D.et al.The enzymatic interconversion of acetate andacetylcoenzyme A in Escherichia coli.J Gen Microbiol.1979; 102:327-336).When being sole carbon source with acetate, acetate is by reverse pta-ack approach metabolism.

The following pathways metabolism of pta-ack approach catalysis:

Acetyl-CoA+Pi＜=〉acetyl-Pi+CoA

Acetyl-Pi+ADP＜=〉acetate+ATP

The pta-ack approach is except being a kind of important pathways metabolism, also determining the concentration of acetylphosphate base in born of the same parents, this is phosphate donor (Fox D K, the et al.Phosphate transferbetween acetate kinase and enzyme I of the bacterial phosphotransferase system.JBiol Chem.1977 of phosphotransferase system; 261:13498-13503).From colibacillary physiological metabolism path analysis, in the genetic engineering bacterium process of high-density fermentation, thalline mainly produces acetate by the Pta-Ack approach, its acetate accumulation of the engineering bacteria of Pta-Ack pathways metabolism defective reduces significantly, and nectar degree and expression of recombinant proteins amount all have raising by a relatively large margin.Because it is unfavorable to the growth of thalline by the acetate that the pta-ack approach produces, therefore, the bacterial strain of some pta or ack defective and two defectives has obtained and has studied the accumulation degree of its acid product, the expression of most of strain fermentation density and product all increase (Regan L, et al.Fluxanalysis of microbial metabolic pathways using a wisual programming environment.J Biotechnol.1995; 151-16l; Rhee J S, et al.Characterization and evaluation fa pta (phosphotransacetylase) negative mutant of Escherichia coli HB 101 as productionhost of foreign lipase.Appl Microbiol Biotechnol.1994; 42 (1): 100-107; San K Y, et al.Acetic acid reduction using phosphate-acetyltransferase-deficient and acetate-kinase-deficient E.coli, and metabolic engineering for high cell densityfermentation.Ann NY Acad Sci.1994; 721:257-267; Neway J O, et al.Improvedexpression of human interleulin-2 in high-cell-density fermentor cultures ofEscherichia coli K-12 by a phosphotransacetylase mutant.Appl Environ Microbiol.1990; 56 (5): 1296-1302; Rhie H G, et al.The function of ackA and pta genes isnecessary for phoy (3-hydroxybutyrate-co-3-hydroxyvalerate) synthesis inrecombinant pha+Escherichia coli.Can J Microbiol.1995; 200-206; Jiang Lan. Ph D dissertation .1997; Pp58-71), but this class engineering bacteria is higher when cell density in process of high-density fermentation, substratum is more and more during thickness, and the transmission of oxygen is difficulty further also, still faces the oxygen in the environment is utilized problem.

In view of the oxygen supply deficiency is a ubiquitous problem in the intestinal bacteria high density fermentation, by interpolation and the direct air that in fermentor tank, feeds pure oxygen or be rich in oxygen of regulating nutritive substance, make dissolved oxygen level maintain certain level, high density fermentation condition (Bauer S, the Shiloach J.Maximal exponential growth rate and yield of E.coli obtainable in a bench-scalefermentor.Biotechnol Bioeng.1974 of engineering bacteria have been improved; 169 (7): 939-941; Fass R, Clem T R, Shiloch J.Use of a novel air separation system in a fed-batch fermentative culture ofEscherichia coli.Appl Environ Microbiol.1989; 55 (5): 1305-1307; Qoronfleh MW.Dissolved oxygen concentration affects the accumulation of HIV-1 recombinantproteins in Escherichia coli.Appl Biochem Biotechnol.1999; 80 (2): 107-20.).But these measures under the long again situation of comparatively large-scale fermentor tank, incubation time, cause production cost relatively just higher in industrialized production.Therefore, utilize molecular biology method, more effective in actual applications by improving cell respiratory metabolism under the oxygen deprivation condition.

Vitreoscilla hemoglobin (abbreviating " VHb " as) is that a kind of oxygen of finding in the prokaryotic organism is conjugated protein, its mechanism of action may be under the hypoxia condition VHb oxygen among can combining environmental, play effect (the Wakabayashi S that rich long-pending oxygen utilizes for host cell, Matsubara H, Webster D A.Primary sequence of a dimeric bacterial haemogloin from Vitreoscilla.Nature.1986; 322:481-483).Under the high density fermentation condition, to analyze from the microenvironment of thalli growth, the insufficient phenomenon of oxygen supply is even more serious.Come from this angle, the utilization of Vitreoscilla hemoglobin is a kind of very significant have the raising fermentation density of widespread use potentiality and method that product is expressed.The expression of VHb in intestinal bacteria promoted host's growth and Recombinant Protein Expression, the Vitreoscilla hemoglobin gene is inserted in the plasmid of various resistances and copy type and expresses, under the oxygen deprivation condition, improve expression amount (Demodena S J, the et al.The production of Cephaloporin Cby acremonium chrysogenum in improved by the intraacellular expression of abacteria hemoglobin.Bio/Technology.1993 of fermentation density and purpose product albumen; 11:926-929:Dikshit K L, et al.Cloning, characterization and expression of bacterial globin gene from Vitreoscillain E.coli.Gene.1988; 70:377-386; Fieschko J, et al.Production of human alphaconsensus interferon in recombinant E.coli.Chem Eng Commun.1986; 45:229-231; Khosla C, et al.Expression of intracellular hemoglobin improves proteinsynthesis in oxygen-limited E.coli.Bio/Technology.1990; 8:849-853).Yet, host cell reaches because the proteic overexpression of VHb because carrying two kinds of plasmids that contain target protein gene and VHb gene, bring serious physiological load to cell, thereby weaken the function of Vitreoscilla hemoglobin gene (abbreviating " vgb gene " as), in born of the same parents, form inclusion body as the VHb albumen of overexpression.The vgb gene is inserted be built in the chromosomal DNA of host cell integrated engineering bacteria (Wu Yi. Ph D dissertation: the function of Vitreoscilla hemoglobin gene, use and mechanism research.1990; Pp21-34.), avoid this negative interaction.

Therefore, this area presses for that exploitation is new transforms being applicable to the method for high density fermentation intestinal bacteria, and the consequent new engineering bacteria that is applicable to high density fermentation.

One object of the present invention just provides a kind ofly to be transformed intestinal bacteria, thereby makes intestinal bacteria be more suitable for the method for high density fermentation.

Another object of the present invention provides the colibacillus engineering of new suitable high density fermentation.

In a first aspect of the present invention, a kind of microbial process that genome conformity has foreign gene that produces is provided, it comprises step:

(a) provide an integrated recombinant plasmid, this plasmid contains (i) responsive to temperature type replicon, and this replicon duplicates under the temperature and suppressing to duplicate under the temperature and suppressed duplicating; (ii) chromogene homologous fragment A of described microorganism and homologous fragment B; Foreign gene (iii) to be integrated; And (iv) 2 kinds or multiple (preferably being the 2-5 kind, more preferably is the 2-3 kind) resistance screening marker gene (as paraxin (cam) and kantlex (kan) resistant gene);

(b), suppressing to filter out common intasome under the temperature with described integrative plasmid transformed host cell; For example when the resistance screening marker gene was paraxin (cam) and kantlex (kan) resistant gene, the phenotype of intasome was altogether: suppressing cam under the temperature ^R, kan ^RDuplicating cam under the temperature ^R, kan ^R

(c) the common intasome that filters out in the culturing step (b) under duplicating temperature dissociates common intasome; Produce and split body,

(d) filter out the fractionation body; For example when the resistance screening marker gene was paraxin (cam) and kantlex (kan) resistant gene, the phenotype that splits the body body was: suppressing cam under the temperature ^S, kan ^RDuplicating cam under the temperature ^R, kan ^R

(e) suppressing cultured continuously fractionation body under the temperature, recombinant plasmid is lost, thereby produced the microorganism that chromosomal integration has foreign gene.For example when the resistance screening marker gene was paraxin (cam) and kantlex (kan) resistant gene, phenotype was: suppressing cam under the temperature ^S, kan ^RDuplicating cam under the temperature ^S, kan ^R

Preferably, after step (e), also comprise the step that microorganism is identified.

In the present invention, described microbial staining body dna homolog Segment A is selected from down group with homologous fragment B: two fragments of two fragments of same gene, different adjacent genes or operon structure gene.

In a second aspect of the present invention, a kind of microorganism is provided, at the genome conformity of this microorganism the Vitreoscilla hemoglobin gene of external source is arranged, and this microorganism is an acetate metabolism pathway deficiency type.

In a preference of the present invention, the genotype of described microorganism is F-dcm ompT hsdS (r _B ^-, m _B ^-) Δ (pta-ack) ∷ rrnBT ₁T ₂Δ (thrAB) ∷ vhb-kan ^R

In Figure of description,

Fig. 1 is the synoptic diagram that carries out the general policies of gene replacement among the present invention.

Fig. 2 A-2I has shown the structure of plasmid pMAK705, pthrA, pthrB, pthrA-thrB, pBR322-VHb, pACYC-VHb, pET-VHb, pVHb and pVHb-Kan respectively.

Fig. 3 has shown the 2% agarose gel electrophoresis figure that the direction of insertion of the kantlex that inserts in the pVHb-Kan plasmid (Kan) resistant gene is carried out the PCR evaluation.From left to right, each swimming lane is respectively molecular weight marker thing, primer to the amplified production of ThrB3-KMRM and the amplified production of ThrB3-KMRP.

Fig. 4 carries out the electrophorogram that PCR identifies to intestinal bacteria G830.

Fig. 5 has shown the result who intestinal bacteria G830 is carried out the Western engram analysis.Fig. 5 A is the 12%SDS-PAGE electrophorogram to the full cell extract of PA1 and G830, and wherein the M swimming lane is the molecular weight marker thing.Fig. 5 B is for to carry out Western engram analysis photo to PA1 and G830.

Fig. 6 is carbon monoxide (CO) the differential spectra analysis chart of G830 or PA1 cell extract.CO+ represents that carbon monoxide is arranged; CO-represents not have carbon monoxide.

Fig. 7 is the thr operon of intestinal bacteria G830 and the structure collection of illustrative plates of Pta-Ack operon.

The influence that Fig. 8 VHb integrates and the Pta-Ack defective is grown to engineering bacteria, wherein,, G830; O, PA1; Δ, BL21.Fig. 8 A is the high-solubility oxygen condition.In shaking bottle,, cultivate down for 300 rev/mins in 37 ℃; Fig. 8 B is the low dissolved axygen condition.In shaking bottle,, cultivate down for 100 rev/mins in 37 ℃.

Fig. 9 has shown that at e. coli bl21 the SDS-PAGE of prolyl endopeptidase among PA1 and the G830 (PEP) expression analyzes and Western engram analysis result.Fig. 9 A is the SDS-PAGE electrophorogram of the prolyl endopeptidase (PEP) of expression in e. coli bl21 (pPEP), and wherein swimming lane M is the molecular weight marker thing; Fig. 9 B is the WES engram analysis of the prolyl endopeptidase (PEP) of expression in e. coli bl21 (pPEP); Fig. 9 C is the SDS-PAGE electrophorogram of the prolyl endopeptidase (PEP) of expression in intestinal bacteria PA1 (pPEP); Fig. 9 D is the SDS-PAGE electrophorogram of the prolyl endopeptidase (PEP) of expression in intestinal bacteria G830 (pPEP); Fig. 9 E accounts for the per-cent of total protein for prolyl endopeptidase (PEP) in various engineering bacterias, △ wherein, BL21; Zero, PA1; ●, G830.

Figure 10 has shown under the high density fermentation condition, the influence that VHb integrates and the Pta-Ack defective is grown to engineering bacteria.Figure 10 A is BL21, the DO2 graphic representation of PA1 and G830.Figure 10 B is BL21, the growth curve chart of PA1 and G830.

Figure 11 under the high density fermentation condition, the expression of recombinant protein prolyl endopeptidase (PEP).Figure 11 A is the DO21 curve of PA1 (pPEP) and G830 (pPEP); Figure 11 B is G830 (pPEP) (●) and PA1 (pPEP) growth curve (O); Figure 11 C is the expression electrophorogram of PEP in G830 and PA1.

As used herein, " microorganism " comprises bacterium, yeast, eukaryotic cell, and they all can be used for the inventive method.Preferably, described microorganism is bacterium or yeast, more preferably is intestinal bacteria.

As used herein, " foreign gene " refers to the gene that described host cell does not contain originally.In the methods of the invention, to foreign gene without any special restriction.Preferable foreign gene is to improving the helpful gene of microbial fermentation ability, for example Vitreoscilla hemoglobin gene etc.

As used herein, " resistance screening marker gene " refers to be used to screen the purpose resistant gene, for example various resistance screening marker gene known in the art such as paraxin (cam), kantlex (kan) resistant gene, ampicillin resistance gene.

As used herein, " responsive to temperature type replicon " refers under a certain temperature reproducible, and duplicates downtrod replicon in another temperature.The temperature that can duplicate is " duplicating temperature "; Duplicate downtrod temperature and be " inhibition temperature ".Responsive to temperature type replicon known in the art or that find in the future all can be used for the present invention.In a preference of the present invention, described responsive to temperature type replicon comprises pSC101rep (ts) replicon, and duplicating temperature is 30 ℃, is 44 ℃ and suppress temperature.

As used herein, " intasome altogether " refers to plasmid and karyomit(e) generation homologous recombination, forms a quasi-microorganism of plasmid-karyomit(e) mixture.

As used herein, " fractionation body " produces through the karyomit(e) of variation and a quasi-microorganism of plasmid after referring to plasmid and the exchange of karyomit(e) generation homology.

The inventor discovers, damaged and import Vitreoscilla hemoglobin gene and can overcome separately defective by making up the pta-ack pathways metabolism, remove on the one hand the grow accumulation of disadvantageous acetate of engineering bacteria, improve engineering bacteria ability of utilizing to oxygen in microenvironment on the other hand.The present invention fully utilizes molecular biology method, by the homologous recombination between plasmid and the chromosomal homologous fragment, Vitreoscilla hemoglobin gene is imported on the threonine operon gene location of the damaged engineering bacteria PA1 of pta-ack pathways metabolism, made up and be applicable to high density fermentation, integrated Vitreoscilla hemoglobin vgb gene and block the engineering bacteria G830 of acetate metabolism approach Pta-Ack gene.

Method principle of the present invention

The integration method of e. coli chromosomal dna normally inserts the foreign gene dna segment in swivel base gene or the phage DNA earlier, carries this external source random integration in host chromosome DNA by transposon or phage again.This method screening operation amount is big, integrates the position instability, also may cause the disappearance of some gene and influences the cell normal physiological function.Here we made up a kind of simple, efficiently karyomit(e)-plasmid homologous recombination method with the vgb gene integration to host chromosome.

At first utilize and carry temperature sensitive pSC101 rep (ts) replicon (this replicon duplicates at 30 ℃, and is suppressed at 44 ℃), resistance marker is the plasmid (cam of paraxin (cam) _{44 ℃} ^S _{30 ℃} ^R) make up one and contain bacterial chromosome gene a, b (a, b can be two fragments of same gene, also can be the structure gene of two adjacent genes or operon) and target sequence or the integrated recombinant plasmid of goal gene (ε) integrated.

Recombinant plasmid transformed is to suitable host bacterium, in case homologous gene is recombinated on bacterial chromosome homologous fragment that plasmid is entrained and the host chromosome, because the entrained pSC101 rep of plasmid (ts) replicon is suppressed at 44 ℃, so be easy under 44 ℃ of culture condition, screen common intasome (the cointegrates) (cam that forms between plasmid and the karyomit(e) ^R), the chlorampenicol resistant that common intasome shows is started by chromosome duplication.

44 ℃ of common intasomies that screen are continued to cultivate down at 30 ℃, make common intasome dissociate (resolve); Filter out then under 30 ℃ of culture condition and on the paraxin flat board, grow, and growth is repressed under 44 ℃ of conditions, is the fractionation body (resolution products) that common intasome splits fully.Because after plasmid and karyomit(e) disintegrated, duplicating then of paraxin started by pSC101 rep (ts).Plasmid after the reorganization can be by being lost 44 ℃ of following cultured continuously.By between homologous fragment and target sequence, inserting other suitable resistant gene such as kan ^R,, only need filter out that phenotype is cam under 30 ℃ and 44 ℃ of conditions as the positive mark ^SKan ^RBacterial strain, further through identifying that methods analysts such as PCR, amino acid nutrient defective, pathways metabolism defective, Western trace, integrated target sequence activation analysis identify, can significantly reduce the workload of screening integron to it.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment

1 materials and methods

1.1 material

1.1.1 bacterial classification and plasmid bacterial classification and plasmid are referring to table 1

Table 1 bacterium and plasmid

Bacterium or plasmid	Feature
		??DH5α	supE44ΔlacU169(80?lacZΔM15)hsdR17?recA1?endA1?gyrA96?thi-1?relAl
??BL21	?F -dcm?ompT?hsdS(r B -，m B -)
		????PA1	?F -dcm?ompT?hsdS(r B -，m B -)Δ(pta-ack)∷rrnBT tT 2(Jiang Lan, the same)
????G830	F -dcm?ompT?hsdS(r B -，m B -)Δ(pta-ack)∷rrnBT 1T 2Δ(thrAB)∷vgb-kan R
		?pMAK705	Responsive to temperature type pSC101 replication initiation; 30 ℃ are duplicated, and 44 ℃ of inhibition are duplicated, 5510 bp, cam R(Carol?M?H，et.al.?New?method?for?generating?deletions?and?gene replacements?in?Esherchia?coli.??J?Bacteriol.1989；171(9)：4617-4622)
pACYC184	P15A replication initiation, 4245 bp, cam Rtet R
		??pET5α	PBR322 replication initiation, 4184 bp amp R
??pUC4K	PUC replication initiation, 3966 bp, kan Ramp R
		????pthrA	The pMAK705 plasmid of deriving contains the thrA partial sequence with pcr amplification, 6354 bp, cam 30℃ R 44℃ S
??pthrB	The pMAK705 plasmid of deriving contains the thrB partial sequence with pcr amplification, 6245 bp, cam 30℃ R 44℃ S
		pthrA-thrB	The pMAK705 that obtains from pUCthrA-thrB subclone thrA-thrB, 7091 bp, cam 30℃ R 44℃ S
pBR322- ????VHb	The pBR322 plasmid of deriving contains the VHb gene, 5134bp, amp R
		?pACYC- ????VHb	The pACYC184 plasmid of deriving is from pBR322-VHb subclone VHb, 5016 bp, cam R?tet S
pET-VHb	The pET5 α that obtains from the pACYC-VHb subclone VHb plasmid of deriving, 5287 bp, amp R
		??pVHb	The pthrA-thrB that obtains from the pET-VHb subclone VHb gene plasmid of deriving, 8649 bp, cam 30℃ R 44℃ S
pVHb-Kan	Subclone Kan resistant gene from pUC4K is plasmid and the pthrA-thrB that obtains derives, 9901 bp, cam 30℃ R 44℃ S
		??pPEP	The PGEM plasmid of deriving is expressed the prolyl endopeptidase gene, amp R

* each construction of recombinant plasmid sees below

1.1.2 the anti-VHb antibody of rabbit is given by E.Bailey (Switzerland), tires 1: 400; Mouse-anti prolyl endopeptidase antibody is given by Chen Changqing researcher (Shanghai Research Center of Biotechnology); The anti-mountain of enzyme di-sheep anti mouse IgG-AP and goat anti-rabbit igg-AP are all available from Huamei Bio-Engrg Co..

1.1.3 substratum

Transform at plasmid, in the processes such as recombinant screen and VHb integration, paraxin, ammonia benzyl mycin and the tsiklomitsin concentration in the LB substratum is respectively 20mg/L, 100mg/L, 34mg/L.

1.1.4 test kit

Shanghai is magnificent reclaims the recovery that Kit, plasmid extraction test kit, genome DNA extraction test kit and glue recovery test kit are respectively applied for the recovery of PCR product, plasmid extraction and purifying, reach DNA restriction enzyme digestion sample along the PCR of bio-engineering corporation product.

1.1.5 main agents

Restriction enzyme, Taq enzyme, T4 dna ligase etc. are available from Promega company.

1.2 method

1.2.1 VHb integrates the screening of bacterial strain

(A) transform: transform host bacterium PA1 with integrated recombinant plasmid pVHb-Kan; Converted product directly inserts the liquid LB substratum that 100ml contains 20mg/L paraxin and 50mg/L kantlex, and rotary shaking table 200r/min cultivates 48h for 30 ℃.

(B) screening of intasome (plasmid-chromosome cointegrates): because the insertion of kalamycin resistance gene, alleviated the workload of screening correct intasome greatly, phenotype is cam under 30 ℃ and 44 ℃ of two kinds of culture condition by directly filtering out ^SKan ^RRecombinant bacterial strain, the bacterial strain of this phenotype then is required integron, and the recombinant plasmid after the exchange is lost owing to the growth under 44 ℃ is suppressed.The 48h culture is diluted to 10 ^-4, 10 ^-6Diluents at different levels are inhaled 100 μ l respectively and are coated with LB flat board (50mg/L Kan), and each extent of dilution is coated with 20 flat boards, and wherein 5 place 30 ℃ of incubators to cultivate, and all the other 15 place 44 ℃ of cultivations.Behind the 36h, note down each and dull and stereotyped go up the single colony number that occurs, and calculate average single colony number under 30 ℃ and the 44 ℃ of culture temperature.Integration efficiency is calculated as follows altogether:

Be total to integration efficiency=44 ℃ of average single colony number/30 ℃ of average single colony number * 100%

(C) folding split (resolution products) evaluation: from 10 single bacterium colonies of picking on 44 ℃ of flat boards to 100ml LB liquid nutrient medium (20mg/L kan), 200r/min on rotary shaking table, 30 ℃ of overnight incubation; Get 0.1ml and spend the night bacterium, on 30 ℃ of rotary shaking tables, continue to cultivate 1h, get the coating of 0.1ml culture, place 30 ℃ of thermostat containers to cultivate to the fresh LB substratum of 100ml; Or 30 ℃ cultivate 48h after, get an amount of culture coated plate, place 30 ℃ of thermostat containers to cultivate.Single bacterium colony of 44 ℃ of appearance put respectively receive 30 ℃ and 44 ℃ and contain 20mg/L paraxin LB flat board, therefrom filter out 30 ℃ and 44 ℃ of kan ^RCam ^SBacterial strain, this phenotype bacterial strain is required intasome.Note down each and dull and stereotyped go up the single colony number that occurs, and calculate kan under 30 ℃ and the 44 ℃ of culture temperature ^RCam ^SThe average single colony number of phenotype bacterial strain is calculated as follows:

Integration efficiency=kan ^RCam ^SAverage single colony number/kan ^RAverage single colony number * 100%

1.2.2 the auxotrophic evaluation of Threonine

(A) perfect medium (every liter) 2g glucose, 10g peptone, 5g yeast powder, 5gNaCl;

(B) minimum medium (every liter) 2g glucose, 0.5g Trisodium Citrate, 7g K ₂HPO ₄, 2gKH ₂PO ₄, 10g (NH ₄) ₂SO ₄, 1g MgSO ₄.7H ₂O, each seed amino acid (Methionin, methionine(Met), aspartic acid, leucine, Isoleucine, proline(Pro), L-glutamic acid) is 20mg, 5mg VITMAIN B1,0.5mg vitamin H;

(C) minimum medium+Threonine (every liter) adds the 20mg Threonine in minimum medium.

Liquid culture is diluted to 10 ^-6, be coated on the perfect medium flat board, after 16 hours, connect 100 single bacterium colonies respectively to minimum medium flat board and minimum medium+Threonine flat board.Only containing on the minimum medium of Threonine and could grow, the bacterial strain of then not growing on the minimum medium that does not contain Threonine is the Threonine auxotrophic strain.

1.2.3 VHb activation analysis method

Basically press Sharon K.M, et al.Biotechcology.1991; The described method of 9:473-476 is carried out.Each engineering bacteria is incubated overnight under 37 ℃ of hypoxia conditions, centrifugal collection thalline, resuspended with an amount of PBS damping fluid, behind the ultrasonic disruption thalline, centrifugal collection supernatant, add an amount of vat powder, the supernatant extract is carried out the analysis of CO differential spectra, sample is divided into isopyknic two parts, a copy of it feeds CO blistering reaction 5min, measure the differential spectra of two duplicate samples at 380-450nm with scanning spectrophotometer behind the CO association reaction, determine VHb concentration according to 420nm place spectrum peak, calculation formula is:

C _VHb(mg/ml)＝A ₄₂₀/E ₄₂₀

Wherein, E ₄₂₀For 1cm cuvette 1mg/ml VHb differential spectra absorption peak, get E ₄₂₀=3.6.

1.2.4 Western engram analysis

Culture under the separation 1ml hypoxia condition of sample is resuspended in 100 microlitres, 1 * SDS sample-loading buffer ((200 μ L50% (W/V) glycerine, 100 μ L 10%SDS, 10 μ L beta-mercaptoethanols, 100 μ L, 0.1% tetrabromophenol sulfonphthalein, 250 μ L 0.5M Tris (pH 6.8), the aseptic H of 340 μ L ₂O), through the 15%SDS-PAGE electrophoretic separation.

Detecting Vitreoscilla hemoglobin detects with anti-VHb antibody of rabbit and the anti-goat anti-rabbit igg-AP of enzyme di-; Prolyl endopeptidase detects with mouse-anti prolyl endopeptidase antibody and the anti-mountain of enzyme di-sheep anti mouse IgG-AP.

1.2.5 acetic acid content is measured by Boechringer TC Acetate Kit explanation and is measured.

1.2.6 high density fermentation condition

1.2.6.1 media components

A LB substratum (LB) 10g/L peptone; The 5g/L yeast extract paste; 10g/L NaCl;

B minimum medium (BM) 5g/L peptone; 2.5g/L yeast extract paste; 10g/L NaCl;

C supplemented medium (AM) 300g/L peptone; The 150g/L yeast extract paste; 10g/L NaCl;

40% glucose

1.2.6.2 culture condition

The A shake flask fermentation

Seed is 37 ℃ of overnight incubation, and inoculation 1% bacterium that spends the night shakes bottle to 100ml LB/250ml, and 100 or the rotary shaking table of 250rpm, cell density is definite at the optical density(OD) A600 of 600nm by culture.

The B ferment tank

Seed is 37 ℃ of overnight incubation, and inoculation 1% is spent the night bacterium to 1 L BM/2.5L fermentor tank, and cell density is determined by the optical density(OD) A600 of culture at 600nm.

1.2.7 the assay of recombinant protein

The broken thalline of ultrasonication, sample separates through the 12%SDS-polyacrylamide gel electrophoresis, after Xylene Brilliant Cyanine G dyes decolouring, detects the content of each expression product with four stars glue imaging analysis systematic quantification.

Embodiment 1

The integrated construction of recombinant plasmid of VHb

(1) clone of thrA and thrB gene

Choose the thrA of threonine operon, B Gene Partial sequence is as homologous fragment, according to Katinka (Katinka M, Cossart P, Sibilli L, et al., Nucleotide sequence of the thrA gene ofEscherichia coli., Proc.Natl.Acad.Sci.U.S.A 1980; 77 (10): 5730-5733) and Cossart (Cossart P., Katinka M., Yaniv M., Nucleotide sequence of the thrB gene of E.coli, and its two adjacent regions; The thrAB and thrBC jurnctions.Nucleic AcidsRes.1981; 9 (2): the 339-47) sequence of Bao Dao thrA and thrB, design following PCR primer (table 2):

The primer of table 2 amplification thrA and thrB gene fragment

Primer	The selective enzyme restriction site	Be incorporated into the site of template	Sequence
				ThrA5	KpnI	187-215	5′-TCGGTACCCATGCGAGTGTTGAAGTTCG-3′
ThrA3	BamHI	1024-1053	5′-TACGGATCCTTAATCAGGCAAGGGATCTGG-3′
				ThrB5	PstI	2881-2907	5′-GACCTGCAGGTAAGCAAATTCCAGTGG-3′
ThrB3	HindIII	3614-3641	5′-TGCAAGCTTGCTGACCTGCTCGTTGTGA-3′

ThrA, the clone of thrB: as template, primer amplifies the thrA PCR product of 866bp to ThrA5-ThrA3 with PA1 karyomit(e), and primer amplifies the thrB PCR product of 760bp to ThrB5-ThrB3; After the PCR product reclaimed with KIT, thrA, thrB were cloned into the temperature sensitive carrier of pMAK705 (pSC101 temperature sensitive replicon, the cam that handle through the respective limits restriction endonuclease respectively respectively through KpnI+BamHI and PstI+HindIII double digestion _{30 ℃} ^R _{44 ℃} ^S) (Fig. 2 A), connect product and transform DH5 α, on the IPTG-X-gal flat board, filter out white colony, after restriction enzyme is identified, obtain recombinant plasmid pthrA (Fig. 2 B) and pthrB (Fig. 2 C) respectively, and two recons are checked order, sequencing result is consistent with report.The corresponding restriction enzyme site of thrB from the PstI-HindIII site subclone of plasmid pthrB to pthrA makes up pthrAB (Fig. 2 D).

(2) subclone of VHb gene

Identify integrated VHb gene for convenience later on, kept suitable restricted endoenzyme site in VHb gene both sides.

The structure of pACYC-VHb: with HindIII+SaII double digestion pBR322-VHb (Fig. 2 E), reclaim about 1.4 kbVHb fragments, and the pACYC184 that the same double digestion of itself and warp is handled is connected, transform DH5 α, picking list bacterium colony from the 50mg/L chlorampenicol resistant flat board, receive respectively on paraxin flat board and tsiklomitsin (34mg/L) flat board, filter out cam ^RTet ^SRecon is cut evaluation through enzyme again, this recombinant plasmid called after pACYC-VHb (Fig. 2 F).

The structure of pET-VHb: with the two pACYC-VHb that cut of XbaI+SalI, reclaim about 1.5kbVHb fragment, and the pET5 α that the same double digestion of itself and warp is handled is connected, transform DH5 α, picking list bacterium colony on the 100mg/L ammonia benzyl resistant panel, cut evaluation through enzyme again, this recombinant plasmid called after pET-VHb (Fig. 2 G).

2.1.3 the structure of recombinant plasmid pVHb

Cut pET-VHb with BamHI+SalI is two, reclaim about 1.57kbVHb fragment, and it is connected with the pthrA-thrB that handles through same double digestion, transform DH5 α, picking list bacterium colony on 30 ℃ of 20mg/L chlorampenicol resistant flat boards is cut evaluation through enzyme again, this recombinant plasmid called after pVHb (Fig. 2 H).

2.1.4 the structure of integrated recombinant plasmid pVHb-Kan

In order effectively to filter out required correct clone, 1.2kb kan is inserted in the SalI site between VHb and thrB ^RGene fragment is as the positive mark, and this resistant gene obtains by cut pUC4K with the SalI enzyme, and recombinant plasmid is pVHb-Kan (Fig. 2 I).

The evaluation of Kan gene fragment direction of insertion: design Kan forward primer KMRP (consistent) and reverse primer KMRM (with the Kan transcriptional orientation opposite) with the Kan transcriptional orientation, right with ThrB3 primer formation PCR primer respectively, with pVHb-Kan is template, PCR result shows, primer amplifies the PCR product of a treaty 900bp to KMRP-ThrB3, consistent with the expectation size, and KMRM-ThrB3 does not have amplified production (Fig. 3).Therefore, Kan is that forward inserts.

KMRP：5′-CGAGCAAGACGTTTCCCGTTGAATATG-3′

KMRM：5′-CTGGCAGAGCATTACGCTGACTTGA-3′

Embodiment 2

The screening of G830 bacterial strain and evaluation

(1) phenotypic screen of integrated recombinant bacterial strain (phenotype)

Integrated recombinant plasmid carries temperature sensitive low copy replicon and can duplicate at 30 ℃; and under 44 ℃ of culture condition reproducible not; therefore carry the derive host bacterium of plasmid of this plasmid or this replicon and under 30 ℃ of conditions, have chlorampenicol resistant; and under 44 ℃ of culture condition to the paraxin sensitivity, promptly cam ( _{30 ℃} ^R, _{44 ℃} ^S).Utilize this characteristic, filter out that phenotype is kan under 30 ℃ and 44 ℃ of culture condition ^RCam ^SBacterial strain, this phenotype be consistent through the recombinant bacterial strain of correctly integrating, dissociating and plasmid is eliminated.At extent of dilution is 10 ^-4Down, the frequency of integrating altogether is

The frequency that correct folding divides and plasmid is eliminated is 2.4%

(table 3) filters out the bacterial strain that three strains meet this phenotype altogether, called after G830 (sequence number No.1-3).The The selection result of the integrated VHb bacterial strain of table 4

Wherein the intestinal bacteria G830 of No.1 is preserved in Chinese typical culture collection center (China, Wuhan) on November 10th, 1999, and preserving number is CCTCC NO:M99012.

(2) identify

Choose the No.1 bacterial strain further through identifying PCR, Threonine auxotrophy, acetate metabolism approach (Pta-Ack) defective, Western engram analysis, CO differential spectra Analysis and Identification.

(A) identify PCR

Having designed the forward primer of VHb and Kan, is the ingenious design reverse primer of ThrC5 '-end ThrC3 on the karyomit(e) being right after 3 of ThrB '-end, has only correct integrated bacterial strain, just may amplify the PCR product; Reverse primer ThrB3 in contrast, primer sequence and estimate that binding site is as shown in table 4.

Identify that PCR result shows that G830 No.1 bacterial strain has amplified the pcr amplification product (Fig. 4, swimming lane 3-5) of 2223 bp (primer is to VHb5-ThrC3) and 1039 bp (primer is to KMRP-ThrC3), size is all consistent with expectation; Under similarity condition, PA1 (pVHb-Kan) does not amplify these two specific PCR products (Fig. 4, swimming lane 6).Above result proves that VHb-Kan has been incorporated into the ThrA-ThrB site.

Table 4 is used to identify the primer of PCR

Primer	The template binding site template binding site of estimating	Sequence
			VHb5	PA1 does not have pVHb-Kan 2526-2554 G830 2526-2554	5′-ACAGGCACGCCTGCAAACAATTCACGAA-3′
KMRP	PA1 does not have pVHb-Kan 3710-3736	5′-CGAGCAAGACGTTTCCCGTTGAATATG-3′

	G830????3710-3736
			ThrC3	PA1 4724-4749 pVHb-Kan does not have G830 4724-4749	5′-CAATAAACGCCGAGAGGATCTTCGCA-3′
ThrB3	PA1?????????????4583-4611 pVHb-Kan????????583-4611 G830????????????4583-4611	5′-TGCAAGCTTGCTGACCTGCTCGTTGTGA-3′

(B) the auxotrophic evaluation of Threonine

The nutrient solution of G830 and PA1 is diluted to 10 respectively ^-6, be coated on the perfect medium, behind the 16h, connect 100 single bacterium colonies respectively to minimum medium and minimum medium+Threonine.PA1 is equal well-grown in two kinds of substratum, and G830 only could grow on the minimum medium of Threonine containing, on the minimum medium that does not contain Threonine, then do not grow by (table 5), this explanation since the site-directed integration of VHb-Kan gene between the threonine operon ThrA-ThrB of starting strain PA1, thereby make it become Threonine auxotrophy bacterial strain.

The auxotrophic evaluation of table 5 Threonine

	Minimum medium	Minimum medium+Threonine
			??PA1	????+	????+
G830	????-	????+
			Annotate :+, growth;-, can not grow

(C) evaluation of acetate metabolism Pta-Ack approach disappearance

Method by Boehringer TC acetate test kit is measured BL21 respectively, the content of acetate in PA1 and the G830 fermented liquid.This method utilizes in the test kit final product NADH of the specific reaction of acetate in the component and fermented liquid to measure the content of acetate in the raising of the absorption value at 340nm wavelength place.The result shows (table 6), acetic acid content has only the 9-12% of the acetate that BL2 produces in PA1 and the G830 fermented liquid, illustrate because transcription termination signal rrnBT1T2 is inserted between the Pta-AcK of BL21, cause the defective of the main acetate metabolism approach Pta-AcK of original starting strain, obviously reduced the accumulation of poisonous secondary metabolite acetate.

Table 6 is measured the acetate production of BL21, PA1 and G830 with the UV method

Sample	?A 600	?A 0	?A 1	?A 2	?A 1-A 0	?A 2-A 0	?ΔA	?C(mg/L)	Per-cent (%)
										Blank		?0.002	?0.226	?0.238	?0.224	?0.236
??BL21	?2.12	?0.035	?0.26	?0.415	?0.225	?0.38	?0.22	???67.02	????100
										??PA1	?2.55	?0.032	?0.251	?0.277	?0.219	?0.245	?0.02	????6.20	????9

??G830

?2.74

??0.03

??0.247

??0.276

??0.217

??0.246

??0.03

??7.85

????12

(D) Western engram analysis

G830 and PA1 be at 37 ℃, incubated overnight under the 100r/min hypoxia condition, and sample is (Fig. 5 A) after the 12%SDS-PAGE electrophoretic separation, and electrotransfer is to hybridizing nylon membrane, by test kit explanation carrying out Western engram analysis.Through the anti-VHb antibody of rabbit, goat anti-rabbit igg-AP hybridization, the NBT-BCIP chemical colour reaction, Fig. 5 B result shows that the G830 sample has specific band at 15 kD places, size is consistent with VHb, proves that integrated VHb obtains to express on G830 karyomit(e).

(E) the CO differential spectra is analyzed

G830 and PA1 be incubated overnight under 37 ℃ of hypoxia conditions, and centrifugal collection thalline is resuspended with an amount of PBS damping fluid, and behind the ultrasonic disruption thalline, centrifugal collection supernatant adds an amount of vat powder, respectively G830 and PA1 supernatant extract is carried out the analysis of CO differential spectra.As shown in Figure 6, the differential spectra after the result shows expression product VHb albumen in the G830 extract and CO combines peak value occurs at the 420nm place, and starting strain PA1 does not have such differential spectra characteristic.Therefore, integrated VHb obtains to express in G830, and biologically active.

(F) G830 genotype

Result according to (A)-(E) identifies draws out the thr operon of G830 and the structure collection of illustrative plates (Fig. 7) and genotype: the F thereof of Pta-Ack operon ^-Dcm ompT hsdS (r _B ^-, m _B ^-) Δ (pta-ack) ∷ rrnBT ₁T ₂Δ (thrAB) ∷ vhb-kan ^R

Embodiment 3

VHb integration and Pta-Ack defective are to the growth of G830 and the influence of expression of recombinant proteins

(A) shake flask fermentation

(1) VHb integration and Pta-Ack defective are to the growth effect of G830

Shake flask fermentation is cultivated E.coli BL21, PA1, G830 respectively under hyperoxia and hypoxia condition, A is measured in appropriate time sampling at interval ₆₀₀The result is shown in Fig. 8 A and 8B.

The result shows, under the hyperoxia culture condition, PA1, the growth vigor of G830 is embodied in the logarithm later stage, and BL21 has more early entered stationary phase (Fig. 8 A), and this explanation is because the accumulation of the acetate that BL21 produced has suppressed the growth of engineering bacteria, and preceding two kinds of recombinant bacterial strains are because the defective of acetate metabolism approach, obviously reduced the accumulation of acetate, thereby removed the growth-inhibiting of acetate to a certain extent engineering bacteria.And under the hypoxemia culture condition, in logarithmic phase and logarithm later stage, G830 has all embodied obvious growth advantage (Fig. 8 B), and the VHb albumen that the integration of this explanation VHb and the defective of Pta-Ack acetate metabolism approach are expressed and the minimizing of acetate accumulation have promoted the growth of engineering bacteria.

(2) integrated VHb promotes expression of recombinant proteins

The gene expression plasmid that a kind of allos Periplasmic secretion albumen is Punctiform aerogenic monad prolyl endopeptidase (PEP) is distinguished Transformed E .coli BL21, PA1, G830, ferment in shaking bottle by the hypoxemia culture condition, measure 3,6,9,12 respectively, the expression of 24h prolyl endopeptidase in engineering bacteria.SDS-PAGE electrophoretic analysis and Western engram analysis show that prolyl endopeptidase obtains to express (Fig. 9 A-D) in three kinds of engineering bacterias; In the 3-24h induction time, engineering bacteria G830 and PA1 all can promote the expression of prolyl endopeptidase, and G830 can keep the stable high expression level of prolyl endopeptidase; The prolyl endopeptidase that G830, PA1 and BL21 express on average accounts for 54.1%, 52.3% and 16.6% (Fig. 9 E) of total protein respectively.

Embodiment 4

The research of G830 high density fermentation

(1) VHb integration and Pta-Ack defective are to the growth effect of G830

Under the high density fermentation condition, cultivate E.coli BL21, PA1, G830, appropriate time sampling at interval.In the later stage of high density fermentation, the respiratory intensity of G830 (the dissolved oxygen saturation ratio of nutrient solution) is different with BL21 and PA1, and the former DO2 is reduced to zero, and then both obviously weaken (Figure 10 A) to the metabolic capacity of oxygen; BL21 and PA1 have only a logarithmic phase, entered stationary phase subsequently, and second logarithmic phase (Figure 10 B) have appearred in G830 under the dissolved oxygen limiting condition.

It is higher to work as cell density in process of high-density fermentation, substratum is more and more during thickness, the transmission of oxygen is also difficult further in the thalline, the difference of kinetic property proves that once more the metabolism of VHb pair cell has tangible influence under the oxygen deprivation condition, do not express the cellular respiration of VHb expression slows down at low dissolved oxygen level, whole energy metabolism level reduces, and the expression of VHb makes cell still keep original respiratory intensity, this explanation VHb may participate in some step of bacterial respiratory chain under little oxygen condition, make cell be in the higher state of bio-oxidation efficient, thereby shown growth vigor.Nectar degree and the dry cell weight of G830 have improved 55.5% and 47.2% (table 7) than BL21 respectively.Therefore, the defect project bacterium G830 of the integration of VHb and Pta-Ack acetate metabolism approach has remarkable advantages in high density fermentation.Table 7 VHb integration and Pta-Ack defective are to each engineering bacteria

The influence of maximum bacterial density and dry cell weight

	Cell density		Dry cell weight
					Bacterial strain	????A 600	????％	????g/L	????％
??BL21	????84.5	????100	????42.4	????100
					????PA1	????98.7	????116.8	????52.7	????124.3
????G830	????131.4	????155.5	????62.4	????147.2

Embodiment 5

Integrated VHb promotes expression of recombinant proteins

(1) G830 carries the breath state of expression plasmid

PPEP is transformed into G830 and PA1 respectively with the expression of recombinant proteins plasmid, carries out high density fermentation by same fermentation parameter among the embodiment 4 in the 2.5L fermentor tank, the dissolved oxygen saturation ratio DO21 curve of record fermenting process, result and empty host bacterium basically identical.Carry the G830 (pPEP) of plasmid and the oxygen metabolism curve basically identical of G830, the situation of the oxygen in the nutrient solution all occurs making full use of, promptly oxygen saturation reaches to the stagnation point null value; And also basically identical of the oxygen metabolism curve of PA1 (pPEP) and PA1 reaches some stagnation point to the oxygen utilization in the nutrient solution and has promptly reached state of saturation, can not make full use of the free oxygen in the nutrient solution, and bacterial classification is to the utilization of oxygen descend (Figure 11 A).But G830 (pPEP) obviously faster than G830, may be because the energy metabolism of host bacterium at initial period quickened in the intervention of expression plasmid to the accretion rate of oxygen.Come from metabolism situation oxygen, restriction engineering bacteria high density fermentation parameter is except that dissolved oxygen level (oxygen delivery capacity that depends on equipment), a very important characteristic that parameter is exactly an engineering bacteria itself, be the power of different strains, whether import as the totally different VHb that just is of the fermentation result of G830 of the present invention and PA1 for oxygen or other factor metabolic capacity.

(2) G830 carries the upgrowth situation of expression plasmid

In process of high-density fermentation, by the turbidity (A of time sampling and measuring thalline ₆₀₀).The result shows that two logarithmic phases all appear in the growth curve feature basically identical of G830 (pPEP) and G830, and the highest nectar degree reaches 84.7 (A ₆₀₀), dry cell weight is 43.8 (g/L); And control group PA1 (pPEP) and PA1 also basically identical of growth curve feature, the highest nectar degree reaches 66.3 (A ₆₀₀), dry cell weight is 33.1 (g/L) (Figure 11 B, tables 8).

Maximum cell density and the dry cell weight of table 8 engineering bacteria G830 (pPEP) and PA1 (pPEP)

	Cell density (A 600)	Dry cell weight (g/L)
			??PA1(pPEP)	????66.3	????33.1
??G830(pPEP)	????84.7	????43.8

(3) recombinant plasmid stability in G830

Good genetic engineering bacterium should possess can make the expression of recombinant proteins plasmid keep certain stability in metabolic process.Culture when the highest nectar is spent is diluted to 10 respectively ^-9, 10 ^-12With 10 ^-15, respectively each extent of dilution sample is drawn 100 μ l and is applied to 3 kan ^R, 3 kan ^R+ amp ^RThe LB flat board, 37 ℃ of overnight incubation, single bacterium colony number that record occurs, PA1 (pPEP) culture then is applied to LB flat board and amp ^RThe LB flat board.

The result shows, in the later stage of high density fermentation, among the G830 sustainment rate of plasmid pPEP on average 67.3%, and among the PA1 sustainment rate of plasmid pPEP on average at 73.2% (table 9).The inventor inferred in the high-density later stage, because the nectar degree is dense, microbiotic is very little for the selection pressure influence of plasmid in the engineering bacteria, carried out duplicating of plasmid by the genetic mechanism of bacterial classification itself to a great extent and distributed.

Table 9 under the high density fermentation condition, the stability of recombinant plasmid pPEP in G830 and PA1

Extent of dilution 10 -15	The host selects		Plasmid is selected		Stabilization efficiency
							????/	??kan R	??amp R	?amp R+kan R	????％
G830(pPEP)	???N/A	???226.5	???N/A	????152.5	????67.3
						?PA1(pPEP)	????172	????N/A	????126	????N/A	????73.2

* annotate 10 ^-9With 10 ^-12The bacterium colony that extent of dilution occurs down is too much, can't count.

(4) Recombinant Protein Expression

One of key problem of high density fermentation is exactly a Recombinant Protein Expression.After G830 when the highest nectar is spent (pPEP) and PA1 (pPEP) sample preparation, analyze Recombinant Protein Expression (Figure 11 C) with the 12%SDS-PAGE gel electrophoresis, and analyze through four stars glue imaging analysis systematic quantification, PEP all obtains to express preferably in G830 and PA1, accounts for 43.3% and 39.5% of bacterial protein respectively.

Discuss

Experimental result shows, temperature sensitive integrated recombination system is as the efficient ways of homologous gene exchange between a kind of plasmid-karyomit(e), not only can be applied to the disappearance (Knockout) of chromogene, also can be incorporated into specific dna fragmentation on chromosomal definite position, the positive sifting property of this method has been simplified the screening operation amount greatly.Because the threonine operon gene extensively exists on bacterial chromosome, the plasmid pVHb-kan of Gou Jianing also can be used for the vgb gene integration to other bacterial chromosome here.

The difference of cell pneodynamics character under the oxygen deprivation condition proves that once more the metabolism of VHb pair cell has tangible influence.Do not express the cellular respiration of VHb expression slows down at low dissolved oxygen level, whole energy metabolism level reduces, the expression of VHb makes cell still keep original respiratory intensity under very low dissolved oxygen level, this explanation VHb may participate in some step of bacterial respiratory chain under little oxygen condition, make cell be in the higher state of bio-oxidation efficient, thereby shown growth vigor.The research of Kallio and Bailey (Kallio P T, etal.Intracellular expression of Vitreoscilla hemoglobin alters E.coli energymetabolism under oxygen-limited conditions.Eur J Biochem.1994; 219:201-208) also hold identical viewpoint, several energy parameters and VHb are closely related in the intestinal bacteria under oxygen limit condition, express the cell of VHb, its H ⁺/ O, stride film Δ pH and ATP concentration be former colibacillary 1.5-2 doubly, this result is quite consistent to the estimation of respiratory intensity with us.The concrete mode and the mechanism of action of further understanding VHb intervention cellular metabolism will be very useful to research VHb physiological function.

Fermenting process is one of important step of gene engineering product industrialization, also is that an influence factor is maximum, the link that process is the most complicated, and its basic goal will reduce the energy and raw material consumption exactly, improves product yield, the industrialization of implementation procedure.High density fermentation becomes one of research focus of genetically engineered fermentation over past ten years.The accumulation of harmful product and the restriction of dissolved oxygen are the major obstacles of high-density culture in the fermenting process.It is the common pattern of high density fermentation that stream adds pattern (Fed-batch fermentation), will be to control the growth of cell by the stream Calais that regulates supplemented medium, the output of harmful side product, prolong the exponential phase of growth of cell, improve cell density, (Batch fermentation) has advantage (Xu B through batch fermentation, JahicM, Blomsten G, Enfors SO.Glucose overflow metabolism and mixed-acidfermentation in aerobic large-scale fed-batch processes with Escherichia coli.ApplMicrobiol Biotechnol 1999; 51 (5): 564-571; 33 Panda AK, Khan RH, Rao KB, Totey SM.Kinetics of inclusion body production in batch and high cell density fed-batch culture of Escherichia coli expressing ovine growth hormone.J Biotechno1.1999; 75 (2-3): 161-72).The stream of supplemented medium is added with many patterns: by rule of thumb data or pre-establish feed supplement, by control such as process parameter pH, dissolved oxygen (DO), respiratory quotient (RQ) feed supplement, calculate (34 Riesenberg D.High-cell-density cultivation of Escherichia coli.Curr Opion Biotechnol.1991 such as decision, online detection limit matrix or harmful side product concentration control feed supplement by material balance; 2 (3): 380-384; Knorre WA, Deckwer WD, Korz D, Pohl HD, Riesenberg D, Ross A, Sanders E.Schulz V.High cell density fermentation of recombinant Escherichiacoli with computer-controlled optimal growth rate.Ann N Y Acad Sci.1991; 646:300-6; Macdonald H L, Neway J O.Effects of medium quality on the expression ofhuman interleukin-2 at high cell density in fermentor cultures of Escherichia coliK-12.Appl Environ Microbiol.1990; 56 (3): 640-645; DeLisa MP, Li J, Rao G, Weigand WA, Bentley WE.Monitoring GFP-operon fusion protein expressionduring high cell density cultivation of Escherichia coli using an on-line opticalsensor.Biotechnol Bioeng.1999; 65 (1): 54-64; Hewitt CJ, Nebe-Von Caron G, Nienow AW, McFarlane CM.Use of multi-staining flow cytometry to characterisethe physiological state of Escherichia coli W3110 in high cell density fed-batchcultures.Biotechnol Bioeng 1999; 63 (6): 705-711).

The pattern of process parameter feedback control is adopted in feed supplement among the present invention control, and the stream of controlling carbon source glucose and other raw material (being mainly nitrogenous source) according to pH and dissolved oxygen adds, and the stream rate of acceleration is by computer controlled automatic.Equation be rule of thumb and the pattern that combines with reference to other model design.Because the complicacy of high density fermentation control, the optimization of process needs the continuous summary and the research of practical experience.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Claims (10)

1. one kind produces the microbial process that genome conformity has foreign gene, it is characterized in that it comprises step:

(a) provide an integrated recombinant plasmid, this plasmid contains (i) responsive to temperature type replicon, and this replicon duplicates under the temperature and suppressing to duplicate under the temperature and suppressed duplicating; (ii) chromogene homologous fragment A of described microorganism and homologous fragment B; Foreign gene (iii) to be integrated; And (iv) 2 kinds or multiple resistance screening marker gene;

(b), suppressing to filter out the common intasome that to grow under the temperature with described integrative plasmid transformed host cell;

(c) the common intasome that filters out in the culturing step (b) under duplicating temperature dissociates common intasome;

(d) filter out and duplicating under the temperature growth and suppressing the repressed fractionation body of growth under the temperature;

(e) suppressing cultured continuously fractionation body under the temperature, recombinant plasmid is lost, thereby produced the microorganism that genome conformity has foreign gene.

2. the method for claim 1 is characterized in that, also comprises the step that microorganism is identified after step (e).

3. the method for claim 1 is characterized in that, described microorganism is intestinal bacteria.

4. the method for claim 1 is characterized in that, described responsive to temperature type replicon comprises pSC101 rep (ts) replicon, and duplicating temperature is 30 ℃, is 44 ℃ and suppress temperature.

5. the method for claim 1 is characterized in that, described foreign gene comprises Vitreoscilla hemoglobin gene.

6. the method for claim 1 is characterized in that, described microbial staining body dna homolog Segment A is selected from down group with homologous fragment B: two fragments of two fragments of same gene, different adjacent genes or operon structure gene.

7. a microorganism is characterized in that, is integrated with the Vitreoscilla hemoglobin gene of external source on the karyomit(e) of described microorganism, and this microorganism is an acetate metabolism Pta-Ack pathway deficiency type.

8. microorganism as claimed in claim 7 is characterized in that described microorganism comprises intestinal bacteria.

9. microorganism as claimed in claim 7 is characterized in that, the genotype of described microorganism is F ^-DcmompT hsdS (r _B ^-, m _B ^-) Δ (pta-ack) ∷ rrnBT ₁T ₂Δ (thrAB) ∷ vhb-kan ^R

10. microorganism as claimed in claim 7 is characterized in that, it is esherichia coli G830, and preserving number is CCTCC NO:M99012.

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