CN1065915C - Ethanol production by recombinant hosts - Google Patents

Abstract

Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

Description

Produce ethanol by recombinant host

The present invention is by Florida agricultural experiment centre, subsidize by following governmental fund: USDA alcohol fuel project fund 86CRCR12134,88-37233-3987 and 90-37233-5372 number, and basic energy resource office of Ministry of Energy fund FG05-86ER13574 number.Federal government keeps suitable right to the present invention.

The present invention is the part continuation application of following application: application number 07/670,821 (application on March 18th, 1991) and 07/624,277 (application on December 7 nineteen ninety).These two is the part continuation application of another application: application number 07/352,062 (application on May 15th, 1989, existing U.S. Patent number 5,000,000), this application itself is again the part continuation application of another application: application number 07/239,099 (on August 31st, 1988, application was now abandoned).These patent documents content is separately listed this paper in the form of document.

In the glycolytic cycle, cell converts simple carbohydrate such as glucose to pyruvic acid, and produces ATP and NADH only.When shortage has the electron transfer system of function, in the time of can not carrying out oxidative phosphorylation, at least 95% pyruvic acid can consume in short approach, regeneration of NAD ⁺, this is that continuation glycolysis-and generation ATP are necessary.These NAD ⁺The refuse that regeneration system rapidly generated is commonly referred to tunning.

The diversified tunning of generic microorganisms does not comprise organic acid such as lactic acid, acetate, succsinic acid and butyric acid; Also comprise neutral products such as ethanol, butanols, acetone and butynediol.In fact, the diversity of fermentation using bacteria product has been used as one of leading indicator of division bacteria.Referring to " uncle Jie Shi bacteriology classification manual " (Williams and Wilkins company, Baltimore, 1984.Hereinafter to be referred as " uncle Jie Shi handbook ") etc. handbook.

The fermentation end product has some essential features in common.Under the condition that originally product generates, their relative nontoxicitys; Yet along with the accumulation of product, its toxicity is increasing.Compare with pyruvic acid, they are in more reductive state because its direct precursor in glycolysis-as terminal electron acceptor.By the microorganisms tunning, constituted the longest in the biotechnology, the basis of the most successful application economically.Product comprises milk-product, meat product, beverage and fuel.In recent years, because new technology, area of biotechnology has obtained a lot of progress.Make the research worker can change the genetic composition of certain micro-organisms selectively.

Bacteria Escherichia coli (Escherichia coli) is a kind of important carrier in the biotechnology, is used for clone and modifying factor; It also is to be used to one of most important host who produces recombinant products.In recent years, the host range that is used for recombinant DNA research has been expanded to cover different bacteriums, yeast, fungi and eukaryotic cell.The present invention relates to use recombinant DNA technology, produce specific useful products by certain modified host.

Be used for modifying host's of the present invention DNA separation autokinesis fermentation single cell bacterium (Zymomonas mobilis), this bacterium usually appears in water and the honey.Zymomonas mobilis (Z.mobilis) is used for brew toddy and fermentation Folium Agaves variegatae juice manufacturing pulque (a kind of alcoholic Mexico beverage) for a long time always as inoculum.This microorganism also is used to produce alcohol fuel, and it is reported, its ethanol yield is apparently higher than yeast.

Although zymomonas mobilis (Z.mobilis) nutritional requirement is simple, and the energy synthesizing amino acid, Nucleotide and VITAMIN, the scope of the sugar of its energy metabolism is very limited, includes only glucose, fructose and sucrose usually.The substrate level phosphorylation that ferments after these sugar is its biosynthesizing and homeostatic unique energy.If do not have fermentable sugared zymomonas mobilis (Z.mobilis) even can not in rich medium such as nutrient broth, grow.

Zymomonas mobilis (Z.mobilis) is a kind of bacterium of obligate fermentation, and it lacks the oxidative phosphorylation system that function is arranged.As cereuisiae fermentum (Saccharomyces cerevisiae), zymomonas mobilis (Z.mobilis) produces ethanol and the main tunning of carbonic acid gas conduct.It produces ethanol by a short approach, only needs two kinds of enzymic activitys: pyruvic carboxylase and ethanol dehydrogenase.Pyruvic carboxylase is the key enzyme in this approach, and it makes pyruvic acid turn to the producing and ethanol approach.The non-oxidizable decarboxylation of pyruvic carboxylase catalysis pyruvic acid produces acetaldehyde and carbonic acid gas.Have the isozyme of two kinds of ethanol dehydrogenases to be present in this organism, they during the fermentation the catalysis acetaldehyde reduction become alcoholic acid reaction, make NADH be oxidized to NAD simultaneously ⁺Although the bacterium ethanol dehydrogenase is common in many organisms, the bacterium with pyruvic carboxylase is less.Improve in its research as the commercial exploitation rate of alcohol production bacterium at the movement transformation fermentation single cell bacterium, the achievement that obtains is very limited.

At present, most of alcohol fuels are to utilize cereuisiae fermentum (S.cerevisiae) or zymomonas mobilis (Z.mobilis) hexose from W-Gum or cane juice to produce.But this carbohydrate is the biomass sugar source of comparison costliness, also can be used as food.Starch and sugar are only represented the part of total reducing sugar in the plant.In the suchlike structures such as stem, leaf, husk, shell and corn cob of plant, the principal mode of carbohydrate is structural cell walls polymer, Mierocrystalline cellulose and hemicellulose.Discharge neutral carbohydrate admixture after these polymer hydrolysis, comprise glucose, wood sugar, seminose, semi-lactosi and pectinose.Occurring in nature still finds no all these sugar of independent organism metabolism fast and effeciently and generates ethanol or any other valuable single product.

The gene of coding alcoholdehydrogenase and pyruvic carboxylase is cloned respectively and is identified in the zymomonas mobilis (Z.mobilis), and obtains to express in E.coli.See Br_u and Sahm (1986a) Arch.Microbiol 144:296-301, (1986b) Arch.Microbiol.146:105-10; Conway etc. (1987a) J.Bacteriol 169:949-54; Conway etc. (1987b) J.Bacteriol.169:2591-97; Ingram and Conway (1988) Appl.Environ.Microbiol.54:397-404; Ingram etc. (1987) Appl.Environ.Microbiol.53:2420-25.

Br_u and Sahm (1986a) (on seeing) confirm that at first in the recombination bacillus coli (E.coli) of overexpression zymomonas mobilis (Z.mobilis) pyruvic carboxylase, though the alcohol concn that produces is very low, ethanol production can increase to some extent.Further this work has been expanded in research.Utilize other two kinds of intestinal bacteria, promptly chrysanthemum Erwinia (Erwiniachrysanthemi) and plant living Klebsiella (Klebsiella planticola) has obtained higher ethanol production from hexose, pentose and carbohydrate admixture.See Tolan and Finn (1987) Appl.Environ.Microbiol.53:2033-38,2039-44.

Therefore, when being raw material with simple carbohydrate, mentioned microorganism just has using value.But we find, and are most of cheap and biomass resource without cease is not with the monose form on the earth, but exist with the wood fibre prime form, and the latter mainly is the mixture of Mierocrystalline cellulose, hemicellulose and xylogen.Mierocrystalline cellulose is the same aggressiveness of glucose, and hemicellulose is comparatively complicated heteromers, not only comprises wood sugar (main ingredient), and comprises a considerable amount of pectinoses, seminose, glucose and semi-lactosi.According to estimates, utilize microorganism that the waste paper that is full of the U.S. and the saccharide residue in the domestic refuse are transformed after, the ethanol more than 10,000,000,000 gallons can be provided.The microorganism that changes the above-mentioned discussion of pipe can ferment effectively Mierocrystalline cellulose and the polymeric structure monose of hemicellulose in the lignocellulose, the method for saccharifying of improved lignocellulose is still a main research topic.

The method of saccharification lignocellulose comprises acid hydrolysis and enzymatic hydrolysis at present.Acid hydrolysis needs heat usually, and some shortcomings are arranged.Comprise consumes energy, produce sour refuse, and the toxigenicity compound hinders microbial fermentation subsequently.Thereby enzymatic hydrolysis is exactly a kind of ideal method.For example, enzyme can directly be added in the medium that comprises lignocellulosic material.

By the genetic engineering approach, make the microorganism of producing and ethanol or lactic acid add the saccharification proterties, the work of this respect is conceived to make the high-caliber enzyme amount of microorganism secretion in substratum always.In other words, modification has had the microorganism that the enzyme that will produce in the cell is transported to desired protein in the fermention medium, and these enzymes just can act on the polysaccharide substrate like this, produces monose and oligosaccharides.So adopt this method, be successfully to transform to not transporting these proteinic thalline because be difficult to.

Therefore, one of purpose of the present invention provides related microorganism exactly, and they can both change pyruvic acid over to the producing and ethanol approach effectively under aerobic and anaerobic growth condition.

Another object of the present invention promotes the recombinant host growth exactly, reduces the accumulation of unwanted meta-bolites in the substratum.

Another object of the present invention just provides recombinant host, and they can produce enough polysaccharidases in cell, and decomposition glucose or wood sugar oligomer make to generate ethanol by same host these degradation productions that can ferment.

In order to realize above-mentioned these and some other purpose, according to the present invention's first primary territory, provide a kind of non-intestinal bacteria (Escherichia coli) recombinant host, wherein comprising the allogeneic dna sequence DNA that coding produces ethanol dehydrogenase and pyruvic carboxylase.This host can express wherein, and allogeneic dna sequence DNA makes ethanol become this host's primary fermentation product to enough functional levels.

A favourable measure is the gene that has used encode in the zymomonas mobilis (Z.mobilis) ethanol dehydrogenase and pyruvic carboxylase in recombinant host.Other aspects, first field comprise that some have the plasmid of coding ethanol dehydrogenase and Pyruvate Decarboxylase Gene, and these plasmids help the acquisition of recombinant host among the present invention.

In second primary territory of the present invention, recombinant host also comprises coding protein involved DNA except the DNA that comprises coding ethanol dehydrogenase and pyruvic carboxylase, and these albumen mass-energy are transported and certain oligosaccharides of metabolism the host.After this DNA of host expresses reached certain level, the ethanol that host's metabolism oligosaccharides is produced became the primary fermentation product.Selected host comprises intestinal bacteria in this invention field, as Erwinia (Erwinia)) and Klebsiella (Klebsiella).Disaccharide and or three sugar that other selected host's energy metabolism is made up of five-carbon sugar and/or hexose monomer (as glucose, wood sugar) and maltose.

In the 3rd primary territory of the present invention, above-mentioned recombinant host also comprises the required DNA of one or more polysaccharidases of generation.The host produces polysaccharidase, subsequently it is discharged in the substratum.Can reduce the commercial enzyme consumption like this.These endonuclease capables are degraded into fermentable monose and oligosaccharides with raw material.

The DNA of coding polysaccharide can be host self, but is external under more susceptible shape, and is promptly allogenic.Useful polysaccharidase comprises cellulolytic enzyme, xylanase clastic enzyme and starch degrading enzyme.The host wants partly to secrete polysaccharidase at least, and perhaps polysaccharidase can accumulate in cell in a large number, discharges subsequently.Advantageously, the enzyme of accumulation is heat-staple in the cell, when needs, can discharge by the thermal induction cracking.The enzyme that the allogeneic dna sequence DNA codified is different, some enzyme secretions come out, and some enzymes accumulate.

Can carry out other modifies and promotes aforementioned host's alcoholic acid and produce.For example, the host also can comprise other allogeneic dna sequence DNA fragment, and its expressed protein product participates in transportation monose and/or oligosaccharides enters recombinant host.Equally, the host also can comprise in the glycolytic pathway other gene.Thus, can obtain high ethanol yield.

The present invention also provides and has utilized all aforementioned hosts to produce the alcoholic acid method.The method of relevant processing oligosaccharides raw material also is provided, oligosaccharides can have been changed into ethanol and simpler oligosaccharides and/or monose.A kind of method also is provided on the other hand, has utilized conversion host of the present invention in substratum, to produce ethanol, reduced the accumulation of acidic metabolite in the substratum.Provide a kind of method in addition, with the generation of functional protein in the recombinant host of zymomonas mobilis (Z.mobilis) the adhB gene that is increased in overexpression.Another aspect of the present invention comprises relevant operation design, will contain the fermentation of oligosaccharides biomass and generate ethanol.

Following detailed will make other purposes of the present invention, feature and advantage become apparent.But must understand, the detailed description of invention and embodiment are when showing the preferential use range of the present invention, only be that mode with illustrations provides, because in the spirit and scope of the invention, various changes and modification all are conspicuous for this area expert.

Fig. 1 shows the structure of the plasmid pLOI295 that comprises (Z.mobilis) in zymomonas mobilis coding pyruvic carboxylase and alcoholdehydrogenase gene.Abbreviation explanation: RI, EcoRI; H, the Hind III; B, Bam HI; T, terminator; Adh, the zymomonas mobilis alcoholdehydrogenase; Pdc, the zymomonas mobilis pyruvic carboxylase; Cat, the paraxin acyltransferase; Ap, β-Nei Xiananmei; Zm Pro frags has the zymomonas mobilis dna fragmentation of promoter activity; Col E1 is derived from the replication orgin of pBR322; Ori V is derived from the replication orgin of RSF1010; Plac, the lactose operon promotor; Pzm, the promotor of zymomonas mobilis; P?, concealed promotor on the carrier; Kb, kilobase.

Fig. 2 shows the several plasmids that comprise coding zymomonas mobilis pyruvic carboxylase and alcoholdehydrogenase gene.

Fig. 3 shows bacterial strain TC4 and comprises the growth and the product acid of the recon of coding producing and ethanol enzyme plasmid.

Fig. 4 shows the relation of recombinant host pyruvic carboxylase activity and extent of growth.Cell concentration after the growth in 24 hours is represented with 550 nanometers optical density value (O.D.550).

Fig. 5 shows the structure of pLOI510.

Fig. 6 shows the alcohol concn that Erwinia and Klebsiella producing and ethanol recon fermentation cellobiose produce.

Fig. 7 A, 7B and 7C show the construction of recombinant plasmid that comprises xyn Z and xyl B gene.

Fig. 8 A and 8B show the thin layer chromatography analysis result who utilizes intestinal bacteria (E.coli) KO11 (pLOI2003) hydrolyzed xylan.Bacterium is incubated in the Luria broth culture that comprises 4% birch xylan (pH6.0) at 45 ℃ (Fig. 8 A) or 60 ℃ (Fig. 8 B).

Fig. 9 A and 9B show recombination bacillus coli KO11 (Fig. 9 A) and urge childbirth Klebsiella (K.oxytoca) M5A1 (Fig. 9 B) to utilize the growing state of oligomeric xylose.

Figure 10 A and 10B show that reorganization urges childbirth Klebsiella (K.oxytoca) M5A1 to transform wood sugar and the xylan hydrolysis thing becomes ethanol.Figure 10 A shows growth, and Figure 10 B shows that ethanol produces.

Figure 11 A and 11B show by reorganization and urge childbirth Klebsiella (K.oxytoca) M5A1 bacterial strain to produce ethanol.Figure 11 A shows bacterial strain M5aI (pLOI555), has the pet gene bacterial strain P2 of integration, has the pet gene bacterial strain B1 of integration.Utilize glucose (100 grams per liter) to generate ethanol, Figure 11 B shows by bacterial strain P2 from cellobiose (100 grams per liter) fermentation generation ethanol.

Figure 12 shows after 120 hours, adds the plain enzyme of commercial fibre to the influence from the amount of SOLKA FLOCSW40 (50 grams per liter) producing and ethanol.

Figure 13 A, B and C show the thin layer chromatography analysis result who utilizes behind bacterial strain P2 (pCT 603T) hydrolysis and the fermented cellulose.Figure 13 A, the acid Mierocrystalline cellulose that rises; Figure 13 B, the alkali Mierocrystalline cellulose that rises; Figure 13 C, crystalline cellulose.

Figure 14 shows with cel D gene product pre-treatment and utilizes the promoter action of 1% cellulase to the ethanol generation that the result is with identical with the 5%CYTOLASE processing.

Figure 15 shows the structure of plasmid pLOI140 and pLOI141.They comprise the Starch debranching enzyme gene from alpha-amylase gene of bacstearothermophilus (Bacillus stearothermophilus) and hot anaerobic bacterium (Thermoanaerobinm brockii).

Figure 16 A and B show that respectively the ethanol of KO11 glucose fermentation and maltose produces and cell concentration.

Figure 17 shows the result of recombination bacillus coli (E:coli) KO11 (pLOI140) and KO11 (pLOI141) amylofermentation.(basic identical because of both results, only shown KO11 (pLOI141) data.)

Figure 18 shows (72 hours) in the fermenting process, the thin layer chromatography analysis result of KO11 (pLOI141) W-Gum fermentation.

The fermenting process synoptic diagram of Figure 19 for carrying out according to the present invention.

I. conventional carbohydrate metabolism

In most of animal and plants and bacterium, yeast and fungi, glucose earlier will be by the degraded of anaerobism approach before carrying out oxidation or fermentating metabolism.Prevailing this approach is called glycolysis-, comprises a series of enzymatic steps, by forming many intermediate products with six carbon glucose molecules, resolves into bimolecular C3compounds pyruvic acid.In this process, bimolecular NAD ⁺Be reduced into NADH.The clean reaction that glucose converts the pyruvic acid process to is:

Glucose+2Pi+2ADP+2NAD ⁺→ 2 pyruvic acid+2ATP+2NADH+2H ⁺

For glycolysis-is proceeded, the NAD that this process consumes ⁺Must be by the NADH oxidation regeneration.In the oxidative metabolism process, NADH passs oxygen hydrogen and forms water through series of steps usually.But most of organisms comprise other anaerobism approach, and glycolytic cycle is not continued when oxygen etc. does not exist.This anaerobic process are called fermentation, and homolactic fermentation may be one of modal anaerobically fermenting approach in many bacteriums and the animal.In the homolactic fermentation process, glucose finally is converted into bimolecular thricarbonate lactic acid.NADH is oxidized, and its hydrogen passes to pyruvic acid, generates lactic acid.The lactic fermentation regeneration of NAD ⁺The clean reaction of process is:

2 pyruvic acid+2NADH → 2 lactic acid+2NAD ⁺

As previously mentioned, producing and ethanol is biological, and they have another (ethanol) fermentation approach as zymomonas mobilis and cereuisiae fermentum, and glucose metabolism thus generates two molecules of ethanol and two molecule carbonic acid gas.Ethanol fermentation and lactic fermentation difference are regeneration of NAD ⁺Step.Ethanol fermentation needs two different steps.Pyruvic carboxylase makes pyruvic acid decomposition or acetaldehyde and carbonic acid gas.Ethanol dehydrogenase forwards hydrogen to acetaldehyde from NADH, and regeneration of NAD ⁺, and produce ethanol.The ethanol fermentation regeneration of NAD ⁺Process reaction is:

2 pyruvic acid → 2 acetaldehyde+2CO ₂

2 acetaldehyde+2NADH → 2 ethanol+2NAD ⁺

Clean reaction:

2 pyruvic acid+2NADH → 2 ethanol+2CO ₂+ 2NAD ⁺

The DNA of every kind of enzyme of encoding clones respectively, and the zymomonas mobilis pyruvic carboxylase is expressed by recombination method.As referring to Br_u and Sahm (1986a) (seing before).But, can not determine in not producing the biology (" non-producing and ethanol biology ") of ethanol as primary metabolite, whether glycolytic pathway can turn to significantly in the pyruvic acid stage, promptly allow to modify this biology to a certain extent, make it express two kinds of required enzymes of ethanol fermentation.

On the contrary, the someone thinks that perhaps this turning to can make recombinant host generation pyruvic acid and normal product hunger thereof.See Br_u and Sohm (1986a, b) (seing before).Also have a kind of possibility, do not producing originally in the alcoholic acid biology exactly, forward the turning to of carbon of producing and ethanol process from glycolysis-to, may upset the vital NAD/NADH ratio of be mutually related production capacity approach and biosynthetic pathway.

II. according to fermentation of the present invention

But, have been found that and can carry out genetically engineered the biology that carries out glycolysis-or similar procedure.According to the present invention, by transforming, in the pyruvic acid stage, the carbon of glycolytic cycle 95% can turn to a substantial route of synthesis.This approach comprises two kinds of enzymes by allos pyruvic carboxylase (pdc) and ethanol dehydrogenase (adh) genes encoding.The result produces a kind of genetically engineered bacteria, and it produces the primary fermentation product of ethanol as it.In other words, the ethanol of generation surpasses 50% of total tunning.

Find in addition, can select the host to carry out this heterogenous expression, rely on the ability of this body transporting of host and metabolism oligosaccharides, their more complicated raw materials that can ferment.(herein, " oligosaccharides " refers to a kind of molecule, and it comprises two or more sugar monomers.Include but not limited to disaccharide such as cellobiose, maltose and xylo-bioses, three sugar is as fiber three sugar and wooden three sugar, and polysaccharide such as Mierocrystalline cellulose, hemicellulose, starch, glycogen, pectin and the inulin of long-chain.) select like this and host transformed, its ability can further expand, can be by expressing the gene of one or more coding polysaccharidases in same host, oligosaccharides decomposition or the smaller oligosaccharides and/or the monose of polysaccharidase energy catalysis complexity.

(A) produce artificial producing and ethanol approach by genetic engineering: an importance of the present invention is one can make cell produce the alcoholic acid operon.A construct of the present invention is an example of this operon, is called " pet operon ".It comprises zymomonas mobilis encode respectively alcoholdehydrogenase and the active gene of pyruvic carboxylase, and regulates sequence accordingly, as promotor, operator gene, ribosome binding site and transcription terminator.Like this, constitute a pet operon by associated reaction fermentation single cell bacterium coding alcoholdehydrogenase and Pyruvate Decarboxylase Gene, the endogenous ethanol dehydrogenase of having abandoned in the former recombination system of dependence produces the alcoholic acid mode.And under aerobic and anaerobic condition, the recon that comprises the pet operon can both produce a large amount of ethanol.

In order to give the ability of microorganisms polysaccharidase (as zytase and cellulase), the gene that can add the required enzyme of coding is to modify producing and ethanol operon of the present invention.Perhaps, one or more polysaccharidase encoding gene groups can be gone into plasmid, transform the host who has had the operon (as mentioned above) that instructs the ethanol generation.(this respect content of the present invention sees following second joint (E) for details.) by another kind of approach, can selection itself have the host of the ability of expressing polysaccharidase as the host who transforms the producing and ethanol operon.Also having a kind of method is that the polysaccharase gene that will add is incorporated in the karyomit(e).

In single construct, the assortment of genes of the enzyme of coding one metabolism approach together, this method generally is applicable to different situations, can install to the gene from different seats together, constitutes manual maneuvering.Like this, the structure of pet operon, only an example of the present invention's application.For example, from the gene of different biological coding ethanol dehydrogenases, can with the assortment of genes of other pyruvic carboxylase of encoding together, to make up the operon of the required approach of coding.The operon of other approach of encoding also can make up.It is equally clear that for producing and ethanol approach discussed above, the gene of coding ethanol dehydrogenase and pyruvic carboxylase needn't be subjected to common control; They can be subjected to branch, and other is controlled, even is positioned at different plasmids or is positioned at different positions on the karyomit(e).Equally, the gene of coding polysaccharidase can be subjected to common or divide other to control, or is positioned at different plasmids, or on the karyomit(e) different positions.

The present invention relates on the other hand and utilizes the producing and ethanol recombinant host to produce recombinant polypeptide or protein effectively, in other words, the recon cell can be further with the gene transformation (these other genes can be positioned on the plasmid, or are incorporated in the karyomit(e)) of coding useful products (non-polysaccharidase).

Especially, the gene of coding producing and ethanol enzyme is common expresses high-levelly, and when anaerobic growth, domination is from the carbon stream of pyruvic acid and NADH oxidation.Under such condition, flowing of pyruvic acid carbon skeleton can turn to producing and ethanol from producing organic acid, as main tunning.

Like this, per unit cell protein acidifying degree produces rather than the organic acid generation by alcoholic acid, drops to minimum level.In the process of growing in the intensive culture of microorganism, the transfer of oxygen usually is a main limiting factor.Just because of this restriction, cause cultivating the generation and the pH value of supporting acid in the base and change.In contrast, among the present invention, express in the recombinant chou of producing and ethanol operon, allogenic producing and ethanol enzyme can turn to product acetaldehyde from glycolysis-with part acetone acid, reoxidizes NADH and produces ethanol, a kind ofly damages less meta-bolites.Like this, comprise the bacterial strain that oxidation phosphorylation function respiratory chain and ethanol produce enzyme, even can grow and reach higher cell density, because at regeneration of NAD ⁺All work with these two systems in the process that reduces the acid(ic)waste generation.The result that this intrinsic changes is that the convection current process control requires volume production less and the increase recombinant product.

The organic acid accumulation that produces from alternate manner is considered to be in the result that the anaerobic growth process is fermented.Even but under aerobic conditions, also can produce the acetate of appreciable amount.Like this, acetate may just produce gradually from the growth initial period, was not limited to the later stage, and the later stage cell density is very high, and anaerobic condition accounts for leading.Acid from glucose produces even under aerobic conditions, also can be limited in the growth in liquid and the solid medium, and this can be shown by the increase of final cell density in the substratum of phosphoric acid damping fluid.

Therefore, producing and ethanol transformant of the present invention also is the good host who produces recombinant products, even it under anaerobic, also only supervenes MIN acid.Many recombinant proteins and polypeptide comprise halfcystine or disulfide linkage, and its suitable folding or reaction is essential by being formed with active enzyme.Because oxygen promotes the formation of disulfide linkage, therefore this proteinoid of synthetic under anaerobic, separate and folding under controlled conditions before, it is undesired folding to have few opportunities, the recyclable thus more bioactive product that has.

Adh B is used for these constructs may has special superiority.The AdhB a kind of stress protein matter (An etc. (1991) FEBS LETTERS 282:205-208) of encoding.Shown already that stress protein matter helped the suitably folding to keep its biological activity of heterologous protein.(Lee etc. (1992) J.Biol.Chem.267:2849-2852).Therefore use adhB may promote in the reorganization biology the proteinic generation of function is arranged.

Under the aerobic conditions, the pyruvic acid that glycolysis-produces in the intestinal bacteria is mainly by pyruvate dehydrogenase complex and lactic dehydrogenase enzymes metabolism, and unnecessary acetyl-CoA is converted to acetate.See Gottschalk work BACTERIAL METABOLISM PP.210-80 (Springer-Verlag1986).The apparent milosevic constant km value of these two kinds of enzymes is respectively 0.4 and 7.2mM.The apparent Km value of zymomonas mobilis pyruvic carboxylase and equating of intestinal bacteria pyruvic oxidase, and be lower than the apparent Km value of serum lactic dehydrogenase, help acetaldehyde to produce like this.NAD under the aerobic conditions ⁺Regeneration mainly come from biosynthesizing and nadh oxidase (with the electron transfer system coupling, its performance Km value is 50 μ M).Zymomonas mobilis alcoholdehydrogenase performance Km value is than intestinal bacteria NAD ⁺The apparent Km value of oxydase will be hanged down four to five times, makes this enzyme of zymomonas mobilis can compete endogenous NADH storehouse effectively, becomes ethanol with reduction acetaldehyde.Thus, the character of zymomonas mobilis producing and ethanol enzyme and high-caliber relatively expression thereof are very suitable for making under aerobic conditions the carbon circulation to generating ethanol.

Under the anaerobic condition, the pyruvic acid that glycolysis-produces in the colibacillus bar is mainly by serum lactic dehydrogenase and the metabolism of pyruvic acid formic acid lyase.The apparent Km value of these two kinds of enzymes is higher 18 times and 5 times respectively than the apparent Km value of zymomonas mobilis pyruvic carboxylase.Similarly, participate in NAD in the intestinal bacteria ⁺Its Km value of the main enzyme of regenerated also all is significantly higher than the apparent Km value of zymomonas mobilis alcoholdehydrogenase.Like this, compare with oxidative phosphorylation, carbon (pyruvic acid) and reducing power (NADH) are had suitable competitive power, make the glycolysis-product flow to the producing and ethanol approach effectively from the producing and ethanol enzyme of zymomonas mobilis with the intestinal bacteria enzyme that ferments normally.

Have been found that, lactose and the main carbohydrate (being glucose, wood sugar, pectinose, semi-lactosi and seminose) that is present in Mierocrystalline cellulose and the hemicellulose, according to the present invention, the recombinant host that all can be able to be expressed producing and ethanol enzyme (as forming the enzyme of zymomonas mobilis ethanol approach discussed above) changes into ethanol.

When select being fit to carry out the host strain of alcohol production, must consider all factors according to the present invention.These factors comprise the substrate scope and to tolerance such as sugar tolerance, salt tolerance, anti-ethanol, anti-low pH and the thermotolerance of environment.For example, as described below, coli strain ATCC9637 (Waksman W strain) shows superior environmental resistance, although the amount of alcohol that produces from glucose is lower than other bacterial strains.Select strains A TCC9637 to have the unique ability of utilizing sucrose because of it.Advantageously, this characteristic of ATCC9637 makes this bacterial strain be used to ferment beet sugar, sucrose and other contain sucrose material.ATCC11303 (Luria B strain) and ATCC15244 (Kepes ML300 strain) when comprising pLOI297, produce the ethanol of highest level, and show the environmental resistance of acceptable level.In these two constructs, the plasmid quite stable, they are chosen as the optimal candidate bacterial strain with further improvement ethanol production.Two constructs all high level expression effectively produce the required zymomonas mobilis pyruvic carboxylase of ethanol.See (1987) such as Ingram.

Comprise recombination bacillus coli, all main sugars in the phytomass can be changed into ethanol from zymomonas mobilis ethanol approach.Its glucose and wood sugar change into alcoholic acid efficient and are higher than the cereuisiae fermentum of report in the past, see (1988) CRC Crit.Rev.Biotechnol 7:107-186 and pentose fermentation Yeast systems such as Lovitt.See Back (1989) Biotechnol.bioeng.Symp.17:617-627; Jeffries etc. (1988) Biotech-nol.Bioeng.31:502-506; Skoog etc. (1 988) Enzyme MicrobiolTechnol 10:66-79; Slininger etc. (1985) Biotechnol.Lett7:431-436).Recombination bacillus coli transforms wood sugar and becomes alcoholic acid efficient to be higher than the efficient of cereuisiae fermentum transforming glucose, as reports such as Lovitt (seing before).Obtain uncommon high ethanol yield (surpass theoretical value 100%) with wood-sugar fermentation, this katabolism that may ascribe compound nutritional matrix to has produced ethanol.But many amino acid become glycolysis-intermediate or pyruvic acid with complex medium component katabolism.The pyruvic acid of Sheng Chenging can be converted into ethanol then like this.

According to the present invention, pdc can transform with the adh gene and enter all different hosts, expresses with different promoters.Being subjected to the trainer to be easy to carry out these in this area transforms.For example, can easily pdc be inserted the various plasmids with different host ranges with the adh gene.The plasmid spy that can duplicate in intestinal bacteria and another kind of target organism who makes up is called " shuttle vectors ", because of it can duplicate in two or more hosts.These plasmids are convenient to obtain, and are easy to use.Most of common shuttling back and forth are combined as intestinal bacteria and other bacteriums, intestinal bacteria and yeast and intestinal bacteria and zooblast.These carriers are found in products catalogue, and know for this area expert.

Zymomonas mobilis pdc and adh gene have been inserted into Xanthomonas campestris (Xan-thomonas), Klebsiella (Klebsiella) and Erwinia (Erwinia) and have expressed therein.In other biology, also can obtain similar results easily.Directly modify as transform promotor to produce the suitableeest construct though may need to carry out some, according to the knowledge of the proteinic biochemical property of soluble cell matter and current relevant its expression, achieving success is in the contemplation.Express the change pyruvic acid for penetrating the foreign gene of required enzymic activity,, even can be incorporated in the karyomit(e), do not need plasmid just can express according to the present invention.See that second saves (C) (as follows).The pet gene construct that for example lacks promotor can be incorporated in the escherichia coli chromosome, just is positioned at after pyruvic acid formic acid lyase genes (pfl) promotor.Can be incorporated into similarly in most of biologies in pfl or other gene, the fragment of one section these target gene of these needs decides the site of integrating by homologous recombination.Remove the pfl gene, other target gene also can be used for integrating.Because can easily differentiate the construct of expressing pet on the indication flat board, this method is widely applicable for and fast and effeciently makes up the other biological body, is used for alcohol production.

There are many textbooks to describe the step of expression alien gene.Products catalogue is also listed relevant carrier, can be used for different organisms.Be used for the gram negative bacterium cloning vector, the gram positive bacterium cloning vector, the carrier of the extensive host's ability of tool, the fungi cloning vector, the insect cloning vector, zooblast cloning vector and vegetable cell cloning vector, these are all known by this area expert.Relevant products catalogue is easy to obtain, and therefrom can be scheduled to these cloning vectors, and this is known by expert in this area.For example, referring to Marino (1989) BioPharm 2:18-33; VECTORS:A SURVEY OF MOLECULARCLONING VECTORS ANO THEIR USES (" carrier: molecular cloning vector and application general view thereof ") (Butterworths 1988).

Utilize zymomonas mobilis gene above-mentioned to be probe, or better, active by observing on the indication flat board, as evaluation, skilled operators also can obtain pdc and adh gene or other similar with it gene in other source.For reaching the present invention's purpose, the gene source of coding alcohol dehydrogenase activity is also not serious, no matter is to separate from horse, yeast, people, insect, or other bacterium.Because the expression of alcohol dehydrogenase activity can directly be observed from acetaldehyde indication flat board, does not therefore need relevant order data, separates proteinic other gene of this enzymic activity of coding tool.Really, many alcohol dehydrogenase genes have been ripe dawn of this area expert, think evidence, to in March, 1991,252 kinds of adh genes (IntelliGenetics Inc., 700E.El Camino Drive have been enumerated in the Genbank database, Mountain View, CA 94040).

Zymomonas mobilis comprises the gene that two kinds of codings have the ethanol dehydrogenase of function, wherein a kind of adh evolution up and down row gene-correlation: clostridium acetobutylicum (Clostridiumacetobutylicum) butanols desaturase, intestinal bacteria methyl glycol oxidoreductase and yeast alcohol dehydrogenase ADH IV.Sequence has all been cloned and measured to all these genes.The another kind of alcohol dehydrogenase gene adh of zymomonas mobilis A is a kind of zinc ethanol dehydrogenase, has also been cloned and measured sequence.Have relevant according to dominant adh gene in the typical alcohol dehydrogenase gene of last and animal and plant and the yeast.AdhA gene and other alcohol dehydrogenase gene can replace the original adhB gene in this explanation.

By the same token, to achieve the object of the present invention, the active gene source of coding pyruvic carboxylase is also unimportant, and no matter it is from zymomonas mobilis, still from corn, and yeast or some other biological body.Life form is to evolve from the common ancestors, and this widely accepts, and is at large illustrated by the molecular genetics method.Not only organism can be arranged in systematic evolution tree according to its macrofeature, and during evolution, the ancestral gene of the tool specific function of the generation of evolving remains with the required conservative feature of these functions.This high conservative makes this area expert can utilize one or more members' of enzyme family raw information, from other organism, isolate function identical, involved enzyme in the heredity.The glycolysis-enzyme just belongs to the best example of this respect, because these enzymes are widely studied.

Really, utilize such method exactly,, successfully from corn, isolate Pyruvate Decarboxylase Gene according to zymomonas mobilis pdc and cereuisiae fermentum pdc pertinent data design dna probe.Referring to Kelly (1989) Plant Molecular Biology 13:213-22.The also available whole gene of other method is made probe.Because the proteinic synthetic of tool pyruvic carboxylase activity (pyruvic acid changes into acetaldehyde and carbonic acid gas) can directly observe from acetaldehyde indication flat board, do not need sequence data to find out other gene, although utilized sequence data to separate corn gene.Many Pyruvate Decarboxylase Gene with said function can be separated from other organism.These genes may be suitable for replacing the pdc gene of zymomonas mobilis, just just as there being several alcohol dehydrogenase genes to prove this similar function.To in March, 1991, the GenBank database has been enumerated at least 5 kinds of pdc genes.

As further improvement, the gene of the key ingredient of coding glucose transport in a kind of recombinant host available motion fermentation single cell bacterium among the present invention, promptly encode zymomonas mobilis glucokinase (glk) and glucose are assisted the gene transformation of diffusion albumen (glf).Particularly, glk and glf can be combined in the vertical son of a manual operation, as above-mentioned producing and ethanol operon pet, and are transformed in the recombinant chou that has the pet operon.The expression of glk/glf operon will cause film glucose flow to increase, and cause alcohol yied to increase thus.Equally, the gene of the key ingredient of coding oligosaccharides transport function among other host, or the gene of the key ingredient of coding glycolytic pathway according to the present invention, also can be used to further transform the host.Can estimate, with encoding stream by the trot rapid gene transformation host of degree after, will increase alcohol yied, improve the level of the glycolysis-intermediate of originating as the biosynthesizing skeleton.

(B) fermentation parameter: according to the present invention, allogenic producing and ethanol expression of gene level helps the recombinant host fermentation and produces ethanol.As mentioned above, with allogenic producing and ethanol gene insert suitable can not produce the alcoholic acid host after, host transformed can utilize oligosaccharides such as cellobiose, procellose, xylo-bioses and xylotriose to produce a considerable amount of ethanol.As described below, the alcohol concn that recombinant host produced among the present invention is 1M, high reached at 1.5M.

(C) exogenous origin gene integrator enters karyomit(e): carry with plasmid and compare, exogenous origin gene integrator has the benefit of several respects on karyomit(e).The plasmid construction body has many restrictions aspect commercial.Compare with chromogene, plasmid construction body itself is unstable, and is a kind of environmental hazard, a kind of source, storehouse of shifting the allos proterties.The unstable of plasmid construction body is often owing to the adding of multiple plasmid aggravates.However, because transformed host cell easily, the plasmid construction body is very useful in the initial experiment of allogeneic gene expression, the gene of the cellulase of for example encoding.But (for example, a kind of cellulase) screening usually needs to substitute the producing and ethanol gene that plasmid carries with the gene of chromosomal integration desired protein, resembles shown in the present invention for convenience.The producing and ethanol gene has been incorporated on the karyomit(e) of intestinal bacteria B strain (E.coliB), sees (1991) Appl.Environ.Microbiol.57:893-900 such as Ohta.Generally speaking, want earlier dna fragmentation of purifying, it comprises the goal gene that is positioned at the chloromycetin gene upstream and derives from one section homologous dna fragment of recipient cell.This dna fragmentation is connected to form and is used for transformation receptor behind the dna circle that does not have replicon.Like this, in intestinal bacteria (E.coli), the pfl gene can be used as acceptor gene, and the short-and-medium Sau 3A endonuclease bamhi at random of Klebsiella (Klebsiella) can be used for promoting homologous recombination after connection.

Paraxin (Cm) flat board with 20 mg/litre allows the recon of single copy integration grow the primary dcreening operation recon.The frequency that recon obtains may be very low.Ethanol produces the incipient statement of gene may be very low, is not enough to fermenting alcohol effectively.High-caliber expression can by on the flat board of 600-1000 mg/litre paraxin once the property screening obtain.The bacterial strain of Huo Deing is highly stable like this.Can predict, this method more suitably can successfully used in Klebsiella (Kebsiella) and Erwinia (Erwinia) bacterial strain equally.Tentative experiment to some wild type strains shows, electroporation can increase the transformation efficiency of plasmid but may reduce the frequency of chromosomal integration.(stating embodiment 16 as follows).

(D) host selects: as mentioned above, be suitable for being used for the biology of expressing heterologous pdc and adh gene and comprise the yeast of eukaryotic cell, non-natural producing and ethanol and bacterium that can not producing and ethanol, eukaryotic cell for example has zooblast, insect cell and fungal cell.For example, except intestinal bacteria (E.coli), the intestinal bacteria of other genus, as the chrysanthemum Erwinia (E.chrysathemi) of erwinia (Ewinia) and planting living Klebsiella (K.planticola) and urging childbirth Klebsiella (K.oxytoca) of Klebsiella (Klebsiella), all be attractive especially host, because they can utilize many different carbohydrates, comprise pentose.The host who has superiority can also select in wider scope, the bacterial classification of Flavobacterium (Xanthomonas) for example in the Gram-negative bacteria, the bacterial classification in bacillus (Bacil-lus), Clostridium (Clostridium) and the Cellulomonas (Lellulomonas) for example in the gram-positive microorganism.The all available suitable method for transformation of above-mentioned different types of host transforms.According to the present invention, some kind in the aforementioned micro organism has also possessed oligosaccharides fermentation has been alcoholic acid host's condition.More particularly, if (1) can produce oligosaccharides is shifted the enzyme that is positioned at cell (tenuigenin) that enters the necessary protein of cell and (2) energy metabolism oligosaccharides, so this microorganism can be selected as the host.The aforesaid microorganism that meets these conditions comprises urging childbirth Klebsiella (K.oxytoca) and planting living Klebsiella (K.planticola) of chrysanthemum Erwinia (E.chrysanthemi) in the intestinal bacteria and other Erwinia, Klebsiella (klebsiella).Urge childbirth Klebsiella self to produce xylobiase.Some intestinal bacteria (E.coli) are also satisfied these standards because they can transport with metabolism cellobiose, maltose and (or) trisaccharide maltose.Example is seen (1987) J.Bacteriol.169:2713-17 such as Hall.

By above-mentioned standard (1) and (2), can in the scope of broad, select the host, for example bacterial strain of Flavobacterium (Xanthomonas) and the bacillus pumilus that belongs to bacillus (Bacillus) (B.pumilus) in the gram-positive microorganism in the Gram-negative bacteria, subtilis (B.subtilis) and Bacillus coagulans (B.coagnlans), the acetone-butanol clostridium (Cl.acetobutyricum) that belongs to shuttle bacillus (Clostridium), gasproof clostridium (Cl.aerotolerans), heat fine clostridium (Cl.thermocellum), hot sulfhydrate clostridium (Cl.thermohydro-sulfurium) and pyrolysis sugar clostridium (Cl.thermosaccharolytium) belongs to the moist cellulomonas cartae (C.uda) of Cellulomonas (Lellulomonas), butyric acid arc cellulomonas cartae (C.butyrivibrio) and molten fiberoptic fiber Zymomonas mobilis (C.fibrisol-vens).Can do to have in host's the yeast Cryptococcus (Cryptococcus) white Cryptococcus (C.albidus), belong to kind, trunk pichia (Pichia stipitis) and the pullulan bud mould (Pullularia pullulans) of Candida (Monilia).The bacterium that can make other energy metabolism oligosaccharides of host also includes but not limited to Bacteroides succinogenes (Bacteroides succinogenes), hot anaerobic bacillus(cillus anaerobicus) belongs to the hot anaerobic bacillus(cillus anaerobicus) of ethanol (T.ethanolicus) of (Thermoanae-robacter), hot anaerobic bacterium belongs to the hot anaerobic bacterium (T.brochii) of (Thermoanaer-obium), the hot bacterioide of acetone ethene (T.acetoethylicus) of hot Bacteroides (Thermobac-teroides), the ruminococcus flavefaciens (R.flavefaciens) of Ruminococcus (Ruminococcus), the thermomonospora fusca (T.fusca) of Thermomonospora (Thermonospora) and the molten fiber acetify vibrios (A.cellulolyticus) of acetify Vibrio (Ace-tivibrio).The all existing suitable method for transformation of all these different hosts.

The document example relevant with the microorganism that meets host's standard seen (1989) Enzyme Microb.Tech-nol.11:626-44 and Beguin (1990) Ann.Rev.Microbiol.44:219-48 such as Biely (1985) Trendsin Biotech.3:286-90, Robsen.These documents content is separately put in order into this paper in this form with reference.This area expert will find that many other hosts are applicable to the present invention.Therefore, whether can transport with the metabolism oligosaccharides and just can select appropriate host by the test microorganism.The available several different methods of such screening realizes.Can for example, grow on suitable oligosaccharides substrate by test, those be not only monose can be rotated the microorganism enter cell can be screened to come out.Example is seen (1987) such as above-mentioned Hall.In addition, can screen the host by the activity of measuring desmo enzyme (as xylobiase).

Be applicable to that a Sample Filter of the present invention is to isolate Erwinia (Erwinia) bacterial strain that causes soft corruption with the minimum medium that comprises pectin from septic vegetable material.See Star, " The Genus Erwinia " is stated from PROKARYOTES second volume, 1260-71 page or leaf (Springer-Verlag1981).The isolated strains that can form pit in the pectin of calcium stable can further be identified.See " the uncle Jie Shi handbook first roll, 469-76 page or leaf.

Suitable Klebsiella (Klebsiella) bacterial strain for example can obtain from the useless small stream of ground such as the Pe-ny of Florida State and Palatka processing plant.Klebsiella (Klebsiella) is very abundant (Huntley etc. (1976) J.Water Pollution ControlFederation48:1766-71 in paper waste; Knittel etc. (1977) Appl.Environ.Micro-biol.34:557-63).The existence of a large amount of Klebsiellas (Klebsiella) will promote screening to have toxin immunity chemical and the useful especially bacterial strain that can utilize these advantages of oligosaccharides in these refuses.These bacterial strains can carry out primary dcreening operation by the growth on special sieve shape substratum, see Seidler " Klebsiella (non-medical aspect) " " prokaryotic organism " PROKARYOTES second volume 1166-72.Further can produce by indoles, Voges-Proskauer reaction and citric acid wait and identify.See " the uncle Jie Shi handbook first roll, 461-65 page or leaf.

Isolating Klebsiella (Klebsiella) and Erwinia (Erwinia) can come the ability of their degradation model substrates of comparison on the flat board of pH6.0 (30 ℃).The pattern substrate comprises carboxymethyl cellulose xylan orchid, methyl-umbrella shape glycosyl-β-D-glucopyranoside, methyl-umbrella shape glycosyl cellobiose, methyl-umbelliferose sill glucosides and methyl-umbrella shape glycosyl Arabinoside, and the required corresponding enzyme of degrading is endoglucanase, zytase, beta-glucosidase, cellobiohydrolase, xylosidase and arabinofuranosidase/xylosidase.Bacterial strain also can pass through the ability of its metabolism monose (sugar and pentose) and cellobiose, procellose, xylo-bioses and xylotriose and be screened.The method of available thin-layer chromatography is measured the level that disaccharides and trisaccharide are utilized in the tube culture that spends the night.Handle the xylan (sigma company) of birch with the zytase of thermal fiber clostridium (C.thermorellum) after, can produce xylosidase.

Use with the basic similar methods of screening intestinal bacteria B strains (E.coli B), can further filter out the promising bacterial strain of anti-ethanol, anti-halogen and heatproof.See (1989) Appl.Environ.Microbiol.55:1943-8 such as Altenhum; Beall etc. (1991) Biotechnol.﹠amp; Bioeng.38:296-303.Though most of isolated strains stored frozen in liquid nitrogen, most promising three bacterial strains are selected in each group comes out to do further operation.The bacterial strain of preserving can be used as the source of new polysaccharase gene, and is very useful in the research afterwards.

Best bacterial strain should keep the ability of effectively utilizing all lignocellulose components (disaccharides, trisaccharide and monose), can in alcohol concn, grow greater than 440 grams per liters, can tolerate the salt level of 0.3M, can tolerate the acetate of 0.2M, the enzyme and the minimum protease activity of tool that can tolerate 40 ℃ of high temperature and can produce high-caliber decomposition of cellulose, hemicellulose and pectin.Some bacterium also may include endogenous zytase and cellulase.After the producing and ethanol gene of external source was put in order, the suitableeest construct should utilize all proof sugar to produce ethanol, reaches more than 90% of theoretical yield, and keeps above-mentioned useful proterties.

Further research can be estimated the optimum pH and the temperature of excretory cellulase and measure thermostability.These data will help to design the optimum condition that hydrolysis and fermenting process are integrated.

(E) expression of allos polysaccharidase: as mentioned above, a preferential measure of the present invention is a recon of considering to produce energy expressing heterologous polysaccharidase, so that reduce the requirement of commercial enzyme in the oligosaccharides fermented product.Polysaccharidase for example comprises Mierocrystalline cellulose lyase, hemicellulose lyase, xylan lyase and starch degrading enzyme.

Wherein a favourable measure is that polysaccharidase is heat-staple.Consider that from this some the thermophilic protein gene (cel A, B, C and D) in the thermal fiber clostridium (Clostridium thermorellum) is high level expression in tenuigenin respectively.Collecting cell after the fermentation ends is heated to temperature (as 60 ℃) that they almost have maximum vigor and good stability so that heat-staple enzyme is discharged with these cells from cell, so just can collect the enzyme that reorganization produces.Then, these enzymes can be used within the predetermined time (as 12 hours) the complex polysaccharide substrate are worked.Remove the signal peptide part of goal gene, increase their expression vigor in intestinal bacteria (E.coli) greatly, and may with Erwinia (Erwinia) similar effect be arranged at Klebsiella (Klebsiella).

The present invention specially uses cel D gene.Transform the recon obtain with cel D among the present invention and can be used for the SOLKA-FLOC that ferments, the Solka-floc product that obtains after a kind of wood pulp mechanical disintegration purifying.Following embodiment 18 and shown in Figure 14, with there not being the recon of cel D gene to compare, the enzymic hydrolysis substrate with cel D coding can reduce about 1/5th with the plain enzyme dosage of commercial fibres in advance.According to the present invention, be used in the enzyme of the heterogenous expression of cell inner accumulation, quick start composite substrate saccharifying, this side removes can be used for any producing and ethanol recon that transforms with polysaccharase gene, and the expression product of its recovery can be used to cracking composite oligosaccharide (Mierocrystalline cellulose, hemicellulose, starch, glycogen, pectin, inulin etc.).Embodiment 17-19 as follows.

Like this, the cell of back fermentation can be used as the source of the required enzyme of back one-step fermentation process.In addition, do not need the enzyme secreting outside yet, and utilize enzyme to reclaim in intracellular accumulation.Just begun to occur the enzyme of high level expression, rather than slowly accumulation in growth and fermenting process.And the danger of microbial contamination is very little, because the microorganism that can utilize oligosaccharides seldom, so seldom there is the microflora of pollution to compete these sugar.

Another favourable measure is, the polysaccharidase that aforesaid producing and ethanol recon is expressed at least can merocrine secretion to the hydrolysis of catalysis extracellular, extracellular composite oligosaccharide.The product of such gene, the product exoglucanase of the product endoglucanase of cen A gene and cex gene for example can be increased in the vigor of conventional one or more enzymes that add in saccharification and the ethanol generative process.The gene that is applicable to the coding polysaccharidase of this purposes is the cellulose enzyme gene in the muck cellulomonas cartae (lellulomonas fimi), when conversion enters in Klebsiella (Klebslella) and the Erwinia (Erwinia), can be to extracellular part secretion (about 50% to 60%).In an Erwinia with the cex gene transformation (Erwinia) laboratory strains, the heterogenous expression product above 50% has been secreted in the substratum.

Excretory efficient can be melted mutually by the signal sequence of the pectate lyase in the Starch debranching enzyme in the same Klebsiella (Klebsiella) respectively and the Erwinia and be improved.See (1990) Gene 90:9-14 such as (1991) Proc.Nat ' lAcad.Sci.USA88:1079-83 such as (1990) Ann.Rev.Genetics 24:67-90, He such as Murooka (1990) Ferment.Technol.Ind.Appl.40-50, Pugsley and Saarilahti.

But special interest is to express the producing and ethanol transformant that heat-staple gene product can be expressed the excretory gene product again among the present invention.The existing cracking of this transformant, the vigor of enzyme can become oligosaccharides and monose with Mierocrystalline cellulose or other composite substrate cracking when pre-treatment, the vigor of Secretases is arranged again, constantly makes the substrate depolymerization during the fermentation.

Under special case, have heterologous gene and can cause that the sudden change of self proterties overexpression or the host of genetic modification are very useful.For example, can in Erwinia, screen the bacterial strain that can improve endogenous endoglucanase expression level by sudden change.In some situation, endoglucanase is suppressed by the glucose of high density.Can screen the mutant that disinthibites on glucose-carboxymethyl cellulose flat board, these mutant are not controlled by glucose concn, and wherein some bacterial strain also may the overexpression endoglucanase.

This area the expert know, this can utilize many different polysaccharide on the one hand according to the present invention.Equally, this one side according to the present invention, many conversion carriers all can use.Carry out necessary normal experiment and can select the host and the carrier of best fit.For example, can make up plasmid RSF1010 deutero-carrier, see (1987C) Appl.Environ.Microbiol.53:235-41 such as Conway, the wide character of its host range can allow heterologous gene is introduced in many Gram-negative bacterias, comprise all Erwinias and Klebsiella, its selective marker is a tetracyclin resistance.In contrast, puc deutero-plasmid is unstable in some Erwinia, and therefore, puc deutero-plasmid uses to such an extent that lack than RSF1010 deutero-plasmid.

Other is applicable to that polysaccharase gene of the present invention comprises the coding any gene with enzyme of Mierocrystalline cellulose lytic activity of example down:

Substrate
						The enzyme class	Crystalline cellulose	Carboxymethyl cellulose (CMC)	Non-crystalline cellulose	Cellotetrose	Cellobiose
Endoglucanase	-	+	+	+	-
Cellobiohydrolase	+	+／-	+	+	-
Beta-glucosidase	-	-	-	+	+

Endoglucanase random hydrolysis β-1,4, glycosidic link.It can not act on cellobiose, but can the hydrolysis fiber dextrin, phosphoric acid Mierocrystalline cellulose and Mierocrystalline cellulose substitute such as the carboxymethyl cellulose and the Natvosol of swelling.Some endoglucanase also can act on crystalline cellulose.

On the contrary, cellobiohydrolase acts on Mierocrystalline cellulose, specifically is the non-reducing end cutting-out cellobiose unit from chain.Cellobiohydrolase hydrolysis fiber dextrin, but hydrolysis fiber disaccharides not.Beta-glucosidase hydrolysis fiber disaccharides and cell-oligosaccharide generate glucose, but can not act on Mierocrystalline cellulose and high poly-cellodextrin.

Except the gene of coding cellulase in the above-mentioned thermal fiber clostridium (C.thermocellum), the gene of other coding Mierocrystalline cellulose lytic activity also has description.Example is seen Beguin (1990) Ann.Rev.Microbiol.44:219-48, and its content spy lists this paper in the mode of reference.These genes even can more easily obtain by the database of GenBanK.Till in March, 1991, comprise 13 endo glucanase genes, 3 cellobiose hydrolase genes and 3 beta-glucosidase genes in the database of GenBank.

Equally, according to the present invention, the gene of coding xylan lyase can be used as described above.The inscribe-1 of particularly encoding, the gene of xylan hydrolysis enzymes such as 4-beta-xylanase, xylobiase, α-glucuronidase, α-L-A Labaifunantangganmei, acetylase and acetyl xylan esterase.The coded product separately of the xylB gene of xynZ gene of example such as thermal fiber clostridium (C.thermocellum) and Butyrivibrio fibrisolvens (Butyvivibrio fibrisolvens), these two genes are all expressed in the transformant of producing and ethanol, (seeing embodiment 17) as described below.Equally, the gene of coding zytase is easy to obtain in the Gen-Bank clauses and subclauses, in March, 1991,7 inscribes-1 is arranged, 4-β xylanase gene, 5 xylobiase genes and 1 d-L-arabinofuranosidase gene.

Similarly, the present invention is equally applicable to use the gene of those coding amylolysis enzymes.This fermentoid and biogenetic derivation thereof and other character see Table.

Enzyme	Kind	The source	Quantity	Active
					α-Dian Fenmei	Liquefaction	Bacillus subtilus a	0.06% of starch weight	Reduce viscosity (cutting 1-4, pH5.5,70 ℃)
		Bacillus licheniformis	Starch heavy 0.06%	Reduce viscosity (92 ℃)
							Barley germ	The 0.5-1.0% that cereal is heavy	Reduce viscosity (60 ℃)
Beta-amylase	Saccharification	Barley germ b	Cereal heavy 20%	Produce maltose (cutting 1-6, pH5.5,60 ℃)
					Glucoamylase	Saccharification	Aspergillus niger b	0.18% (1.7 liters/ton) that starch is heavy	Produce glucose and (cut 1-6, pH5.0,60 ℃
Starch debranching enzyme	Saccharification	Acid branch sporeformer	0.2% 2.0 kg/ton that starch is heavy	Cut 1-6, pH5.0,60 ℃

The circumscribed amylase of a endo-amylase b

A-amylase of the example bacstearothermophilus described as follows of the enzyme of degraded starch and the Starch debranching enzyme of hot anaerobic bacterium.The gene of these enzymes is easy to obtain in GenBank, in March, 1991,190 a amylase genes, 3 beta-amylase genes is arranged among the GenBank, 8 glucoamylase genes and 3 Starch debranching enzyme genes.The gene of other polysaccharidase such as beta-glucanase, arabinofuranosidase/xylosidase, mannase, pectin hydrolase and pectate lyase is at GenBank and clone in the document of these genes and be easy to obtain.

According to the present invention, the polysaccharase gene of genus bacillus (Bacillus) the expression in escherichia coli of being everlasting is fine, so it also should be suitable for expressing in general intestinal bacteria.In fact, the polysaccharidase of many bacterial strain secretion numerous species of Bacillaceae comprises amylase, beta-glucanase and hemicellulase.The plain enzyme of many bacterial strain eccrine fibers, these bacterial strains comprise subtilis (B.subtilis), bacillus polymyxa (B.polymyxa), Bacillus licheniformis (B.lieheniformis) and bacillus cereus (B.cereus).See (1989) Enzyme Microb, Technol.11:626-44 such as Beguin (1990) (on seeing), Robson, these contents are put in order into this paper with the form of reference.If the syzygy of signal peptide sequence and heterologous gene does not have function in selected host such as Klebsiella and Erwinia, so usually use from host's signal peptide sequence and foreign gene merges and allogenic enzyme is secreted.

Benefit can be used and have to the heat-staple lytic enzyme of coding and other activity as the gene of endoglucanase (carboxymethylcelluloenzyme enzyme and nitrophenyl cellobiosidase), in the present invention in the bacstearothermophilus.This genoid has been identified and can have been obtained in Gen Bank and relevant document.See Beguin (1990) (on seeing).

(F) host's screening and conversion: in order to select the host of energy expressing heterologous polysaccharase gene, the ability that can test the recombinant bacterial strain fermented cellulose substrate SOLKA-FLOC that is integrated with the producing and ethanol gene among the present invention is used the plain enzymic hydrolysis Mierocrystalline cellulose of commercial fibres simultaneously.Make the corresponding dose curve of ethanol production and Mierocrystalline cellulose turnover ratio (commercial) with due to the effect of zymin.The plain zymin of industrial fiber (as SPEZYME, MULTIFECT and CYTO-LASE (Genencor product)) can be used for the in check fermenting experiment of these pH (35 ℃, pH6.0).

The interpolation of allos polysaccharidase can be finished by the described mode of embodiment 17-19.For example, after the polysaccharase gene that utilizes homologous recombination will have special benefits was integrated, initial recon screened with the tsiklomitsin of 5 mg/litre, and the tsiklomitsin of higher concentration is used to screen the mutant of high level expression goal gene.5-butyl-pyridine-2-formic acid can be used to the inactivation tetracycline resistance gene, so the gene of plurality of enzymes can add the host successively with identical method.Can estimate the effect of these constructs in a step and multistep cellulose fermentation process then.

Make up the gram-positive microorganism of producing and ethanol, as bacillus subtilis Pseudomonas or Cellulomonas bacterial strain, the normal route that adopts be after the chemomorphosis with the antibody screening of the pyruvic carboxylase of zymomonas mobilis (Z.mobilis), detect the clone that expression level has improved.There is the clone of high level expression further to study the machine profit that it forms this phenotype.The host that these mutant come in handy and should be suitable for expressing as other heterologous protein it is generally acknowledged that these hosts are safe (GRAS).

Transforming the producing and ethanol host with the allos cellulose enzyme gene when obtaining the intracellular reactive enzyme of high density, may need to use other conventional genetic manipulation and screening means, these means are looked different host with selected cellulase and different.For example, host's excretory proteolytic enzyme may become an obstacle of heterogenous expression among the present invention.Available SOME METHODS obtains to overcome substantially the mutant of this problem, for example uses the host of the indication flat board and the selection high level expression isodynamic enzyme of protease activity.

But the generation of any heterologous protein (comprising polysaccharidase) usually will influence host's growth velocity and efficient.This " gene burden " can reduce by the multiple construct of testing useful especially gene.The site of integrating also may have influence on " gene burden ", therefore, according to the present invention, should test the construct of different integration sites.

The construct that so obtains can be used for the cellulose fermentation test then, shown in embodiment 18, assesses the functioning efficiency (with commercial enzyme together) of Secretases and the efficient of fermentation.In the one-step fermentation process, commodity just add when the conversion host with the plain enzyme of producing and ethanol energy eccrine fiber inoculates together with enzyme, therefore, can test the ability that transforms host's excretory cellulase substitute goods cellulase.The producing and ethanol cell that contains thermophilic cellulase in the fermentation in advance added the effect that to test thermophilic cellulase in 60 ℃ the cellulosic suspensions.Sustainable for some time of insulation under the optimum condition of this specific enzymes was as 12 hours.In most of the cases, can reduce temperature and pH value to provide optimum condition to commercial enzyme.For example SPEZYME, MULTIFECT and CYTOLASE, and continue insulation 12 hours.Temperature is dropped to 35 ℃, after the pH value rises to 6.0, the producing and ethanol bacterium of recombinating is inoculated in the substratum.Can measure the alcoholic acid yield then.But this method change is to adapt to the special requirement that each enzyme reaches maximum activity.

The molecular genetic method of required usefulness is very ripe.Example is seen " molecular genetics laboratory manual " (1987) such as " laboratory manual falls in the molecule gram " (cold spring harbor laboratory, 1989), Ausubel such as Maniatis.Fermentation can be carried out on the pH level shown in above-mentioned and the embodiment.Ethanol can be used the solution-air measurement in chromatography, sees (1985) Appl.Environ.Microbiol.5:197-200 such as (1984) Biotechnol.Lett.3:177-82 such as Goel and Dombek.Monomer available colorimetry of sugar or high pressure liquid chromatography (HPLC) are measured, and see (1988) Methods Enzymol such as Ashwell (1966) Meth-ods Enzymol.8:93-95, Wood, 160.Oligosaccharides can be used high pressure liquid chromatography (HPLC) and thin layer chromatography analysis.Liquid culture can be carried out in complex medium (as the Luria substratum) or minimum medium.See " prokaryotic organism " second volume 1166-72 such as Starr, (1986) Biotechnol Bioeng.28:204-9 such as 1260-71 (Springer-Verlag 1981), Mizutani.Microorganism can grow contain 1.5% agar because of the body substratum on, be kept in-70 ℃ the glycerine.

The mensuration of cellulase is substantially according to (1988) J.Bacteriol.170:4576-81 such as (1988) Appl.Environ.Mi-crobiol.54:476-84 such as Curry and Gripenet.The active available p-nitrophenyl cellobiose operation mode substrate of exoglucanase is measured.Carboxymethylcelluloenzyme enzyme (endoglucanase) can discharge by reducing sugar to be measured.The also available traditional method of the activity of cellulase is promptly measured the reducing sugar that discharges on filter paper amount is measured.Methyl-umbelliferose derivative, carboxymethyl cellulose and pectic acid all can be used to do the indication flat board of qualitative comparison, to identify the bacterial strain that can produce the enzyme of high-caliber these substrates of hydrolysis.

(G) alcohol resistance and the ethanol production of raising transformant

The alcoholic acid peak concentration that the producing and ethanol recon produces among the present invention can surpass 1M.The ethanol that intestinal bacteria KO11 (E.coli KO11) transformant ferment lactose is produced can surpass 1.5M, and this bacterial strain preserving number is ATCC55124.Therefore, the alcohol resistance of producing and ethanol recon is particularly useful among increase the present invention.To film fat and (or) perhaps the change of stress protein matter can reach this purpose.The adh B gene of zymomonas mobilis (Z.mobilis) is a component of producing and ethanol operon used among the present invention, and it obviously is the stress protein matter that in the zymomonas mobilis temperature and ethanol is reacted that its coded product has been accredited as.See (1991) J.Bacteriol.173:5975-82 such as Haejung.The gene of the coding stress protein matter in other zymomonas mobilis should be useful equally.At last, expression in escherichia coli is mainly selected proteic gene, see (1986) Mol.Gen.Genet.202:435-45 such as (1988) Nucleic Acids Res.16:7545-61 such as (1989) J.Bcteriol.171:2748-55, Lipinska such as (1989) J.Bacteriol.171:1590-6, Ang such as Johnson and Fayet, can be used as corresponding gene in the probe separates zymomonas mobilis.

The lipid synthetic gene also might be also useful to alcohol resistance.Gene library is directly transformed the producing and ethanol bacterial strain, the a series of enrichments of going down to posterity of process in containing the fermention medium of excessive glucocorticoid, can screen and obtain the gene useful alcohol resistance, excessive sugar is in order to guarantee that those bacterium with ethanol resistance can further growth after alcohol concn surpasses the patience scope of original inoculum.Use chemostat also may select particularly advantageous to these.

The gene library of Lactobacillus homohiochii of high ethanol resistance (Lactobacillus homohiochii) and special-shaped rotten wine Bacterium lacticum (Lactobacillus heterohiochii), see In-gram (1990) Critical.Rev.Biotechnol, 9:305-19 also can test in enrichment process.Because these microbiological effect rice wine become sour, so they can tolerate 15% (V/V) ethanol (surpassing 2.5M).By this method, conventionally producing at least, every liter 80 gram alcoholic acid recombinant bacterial strain can obtain.

In addition, the glycolysis-enzyme of zymomonas mobilis can be used to improve the producing and ethanol recon among the present invention.That is to say that the one or more expression in 13 genes of coding glycolysis-enzyme should increase the glycolysis-flow and increase alcoholic acid output thus.

(H) fermentation procedure design: can design many fermentation procedures according to this paper and implement the present invention.Generally speaking, this operation is divided two steps at least: " saccharification " step, wherein, biomass homopolysaccharide enzyme contact back oligosaccharides be cracked into better simply oligosaccharides or (with) monose, next be " fermentation " step, wherein, simple oligosaccharides and/or monose are fermented into and are ethanol.Preferably these two steps are carried out respectively, because the optimum condition in each step differs widely.The optimum condition of saccharification is the about 50-60 of temperature ℃, the about 4.5-5.5 of pH value.The optimum condition of fermentation is the about 30-35 of temperature ℃, pH value about 6.0.

In order to improve the efficient of whole operation, may need to carry out to the saccharification stage recirculation of raw material from fermentation stage.Because the temperature of fermentation is about 30-35 ℃, so the high temperature fluid in saccharification stage earlier will be by entering fermentation stage again after the cooling of recirculation raw material in heat exchanger.Instantiation procedure is as described below and see Figure 19.

In Figure 19, the biomass that adds polysaccharidase and anticipated in the enzyme reactor 10, therein, the initial oligosaccharides in the biomass is cracked into simple oligosaccharides and monose.Enzyme reactor 10 temperature maintenance are at about 50-60 ℃, the about 4.5-5.0 of pH.Solid and mixtures of liquids enter solid/liquid separator 14 by passage 12 from enzyme reactor 10, solid and liquid separated (for example by centrifugal) in 14.

Isolated solid is got back to enzyme reactor 10 by passage 16, and the fluid in the separator enters heat exchanger 20 by passage 18, and fluid is cooled to about 30-35 ℃ in 20.Fluid is isolated high molecular weight polysaccharide through passage 22 to membrane separation apparatus 24 then, and the latter gets back to enzyme reactor 10 through passage 26.Membrane separation apparatus for example can be a ultra-filtration membrane, as (the Du Pont of E.I.Du Pont Company ^_) the CARRE filter membrane made, its separating power is approximately 10 ^-3To 10 ^-4Micron.Perhaps, can remove membrane separation apparatus 24, fluid directly enters fermentor tank 30.The sugar soln that all the other are made up of oligosaccharides and monose forwards fermentor tank 30 to through passage 28.But before entering fermentor tank, the pH value of sugar soln will be heightened by for example adding alkali.

Carbonic acid gas is got rid of via passage 32 in the fermentor tank.Fluid enters the distillation column (not shown) so that distill out ethanol by line 34.Mashed prod warp 36 in the fermentor tank enters solid-liquid separator 40 (a for example bench centrifuge).The microorganism that branches away in the separator 40 gets back to fermentor tank through passage 42.Fluid in the separator 40 enters heat exchanger 20 to cool off from the fluid in the solid-liquid separator 14.After leaving heat exchanger 20, fluidic pH is low to moderate the about 4.5-5.0 of pH value by adding the acid adjusting, and fluid enters enzyme reactor 10 then.Enzyme reactor 10 and fermentor tank 30 can have mixing tank 48 and 50, and the two can be any traditional kind.

Perhaps, on the successive basis, containing the about 0.5-5% of ethanol, often is that the liquid (beer) of 1-2% also can enter gasifier 54 from fermentor tank 30 through passage 52, in 54 the second alcohol and water gasified and warp 56 enter another the distillation stage (not shown).Alcohol concn in the line 56 reducible from 20% to 25%.Leave gasifier 54 and warp 58 is got back to the beer alcohol concn of fermentor tank 30 and often is less than 0.5%, also just be approximately 0.3% usually.By this way, alcohol concn maintains lower level in the fermentor tank, and alcoholic acid output maintains higher level.

Equally, it is lower thereby to the less inhibition of the reaction in the reactor to get back to the fluid alcohol concn of reactor 10 through passage 36,44 and 46.This successive processes drives by constantly add biomass in reactor 10.But to regularly remove the content in reactor 10 and the fermentor tank 30 so that remove the agglomerative solid and reduce contamination of heavy.

Like this, above-mentioned fermentation procedure can provide one to be the alcoholic acid effective ways with the biomass fermentation.This area expert know can add in the fermentation procedure many steps and (or) stage.For example, can comprise the biomass pretreatment stage, biomass is ground, hydrolysis (with or without steam and/or acid) with polysaccharidase and/or other enzyme processing etc.In addition, an initial step can be arranged, can be heated to cracking at the recombinant host cell of cell inner expression and accumulated polysaccharide enzyme to discharge polysaccharidase.Also have, can adopt the method for multistep fermentation, and additionally have solid to separate and recirculation unit with fluidic.

III. embodiment

The present invention is further described by following reference example, but can not be as restriction.All percentage ratios are weight ratio, and all solution proportion are volume ratio except that particularly pointing out.The additive method that relates among the embodiment is as described below:

Microorganism and growth conditions: this paper uses intestinal bacteria (Escherichia coli) TC ₄(seeing Conway et al, (1987a)) and contain the bacterium that derives of plasmid.The plasmid (PLOI284) that is loaded with the plasmid (pLOI276) of pyruvate decarboxylase gene from Zymomonas mobilis and is loaded with this bacterium alcoholdehydrogenase gene sees that Conway et al. (1987b) is described.

Bacterial strain and growth conditions: plasmid pUC18 and pUC19 see Yanisch-Perron etal, and ((1985) Gene 33:103-19) is described, and plasmid pLOI204 and pLOI295 see Conway et al. (1987b) and Ingram et al. (1987).Plasmid pLOI292, pLOI291, pLOI297, pLOI308, pLOI308-2, structure and the characteristic of pLOI308-5 and pLOI308-10 are seen below.

Culture (is seen Luria﹠amp at every liter of Luria substratum that contains 50 gram glucose under 37 ℃; M.Delbruck (1943) Genetics 28:491-511) cultivates in.The cell that is used for enzyme analysis and ferment-seeded is cultivated at the test tube of adorning 3 milliliters of LB (13 * 100 millimeters) under 37 ℃, and test tube places on the test tube turner.The culture of overnight incubation inserts fresh culture with 100 times of dilutions.Aerobic culture (adorning 50 milliliters of Luria nutrient solutions in 250 ml flasks) in reciprocating type water-bath, vibrate (per minute 120 change) cultivate.The anaerobism culture is cultivated in sealing serum bottle (adorning 100 milliliters of Luria nutrient solutions in 130 ml bottles), with 37 ℃ of cultivations of Clothoid type vibrator (per minute 150 changes).When anaerobism is cultivated, insert the gaseous product that No. 25 syringe needle is derived fermentation on the bottle stopper.

Thalli growth situation Spectronic70 spectrophotometer (Bausch ﹠amp; Lomb, Inc.Rochester NY) detects at the 550nm place, and cuvette is disposable culture test tube (10 * 75 millimeters).Under the experiment condition herein, an absorbance units is about as much as every milliliter and contains 0.25 milligram of cell protein.

The escherichia coli host that has each recombinant plasmid of the present invention is kept at American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rochville, its number of registration of Maryland 20852 USA. is as follows: the culture number of registration is collected date E.coli pLOI308-10 ATCC TC4 on May 15th, 67,983 1989 (pLOI292) ATCC 68237 nineteen ninety TC4 on February 23 (pLOI308-11) ATCC, 68238 nineteen ninety TC4 on February 23 (pLOI297) ATCC, 68239 nineteen ninety TC4 on February 23 (pLOI295) ATCC E.coli pLOI1510 on the 23rd ATCC 68484K.oxytoca in 68240 February nineteen ninety M5A1 ATCC68564 March 14 (pLOI555) in 1991

Collecting this culture must guarantee to have only during present patent application through United States Patent (USP) and trade-mark administration official and just can obtain this culture according to the personnel that 37CFR1.14 and 35USC122 authorize.In the present patent application or its registered country of achievement copy, can ask for this preservation culture by this state's patent law requirement.But must be clear that and obtain this culture and do not mean that to invade the patent right that government authorizes and use the present invention.

In addition, culture of the present invention will be preserved and disclose by the clause of Budapest microorganism preservation pact.Be that culture must be preserved very carefully and makes it to keep survival not contaminated at least five years after being asked for a sample providing at every turn, and keep survival not contaminated at least three ten years or in the term of a patent of the culture of regulation before the open date from preserving certainly.If owing to the problem of preservation condition causes providing when being asked for a sample, this preserves thing to preserve the obligated renewal of unit.From patent open from, all relevant disclosed restrictions of this culture cease to be in force automatically.

Genetic method: conversion, plasmid construction, DNA enzyme are cut and are analyzed all and undertaken by preceding method.Recon is selected on the solid plate (1.5% agar) that contains glucose (every liter of substratum 2 grams) and suitable antibiotic.Wherein the especially big bacterium colony of form promptly is considered to the recon with zymomonas mobilis producing and ethanol gene.If observe this recon on the acetaldehyde indicator medium, just can prove conclusively its reorganization proterties in the expression that does not contain poor growth on the Luria agar plate of glucose and alcohol dehydrogenase gene is arranged.

Enzyme is analyzed: the mensuration of the fragmentation of cell, heat inactivation and pyruvic carboxylase activity (to thermally-stabilised) is seen Conway et al. (1987b).The active mensuration of the alcoholdehydrogenase of the preparation of cell and catalysis ethanol oxidizing reaction is pressed preceding method, but cell washs in the potassium phosphate salt damping fluid that now adds solid ferrous ammonium sulphate solid (final concentration 0.5mM) and vitamin C sodium salt (final concentration 1mM) and fragmentation, sees Neale et al. (1986) Eur.J.Biochem.154:119-24.The prepared product that modified method obtains is measured the clean property of ethanol dehydrogenase immediately without depositing, and it is more much higher than what report than living.For calculating alcohol yied, (Lowry et al., J.Biol.Chem.193:265-75), the result represents with protein content in the culture cell concentration with good fortune quinoline phenol method mensuration.

Tunning is analyzed: with the clarification nutrient solution, having Millpore/Waters high-performance liquid chromatograph (the Millipore CorporationBed ford that specific refractory power detector and electronics and integration are analysed, MA) go up the mensuration tunning, output is separated in (long 300 millimeters on Aminex HPX-87H post, 7.8 millimeters of diameters) carry out on (purchasing Laboratories, Richmond CA) in Bio.Rad.Elution speed 0.25 ml/min (sample size 100 microlitres), 65 ℃ of temperature are determined the peak with safe criterion.Two peaks that occur prior to glucose reach the component that another the slow slightly unknown peak that occurred is non-inoculation medium between 45.4 to 45.8 minutes.The structure of embodiment 1. bacterial strains

The structure gene size of coding pyruvic carboxylase and alcoholdehydrogenase is respectively 1.7Kb and 1.1Kb.Encoded protein matter molecular weight is respectively 60KD and 40.1KD.These two genes are positioned at pUC18 derives on the plasmid, is subjected to the regulation and control (Fig. 1) of Lac promotor.The 1.4Kb that pLOI284 (being loaded with alcohol dehydrogenase gene) is gone up EcoR I and Sal I double digestion does not have the BamH1 site that promoter fragment insertion pLOI276 goes up the Pyruvate Decarboxylase Gene downstream, forms the recombinant chou of two genes.Selection has the bacterium colony of resistance to penbritin, checks the expression of ethanol dehydrogenase simultaneously on pararosaniline chloride-ethanol indicator medium that neoteric detection acetaldehyde generates.Have the bacterial strain that pLOI295 makes up plasmid, do not containing poor growth on the Luria agar plate of glucose (under the aerobic condition), but do not containing its stand density on the flat board of 2% glucose than all not much higher with the bacterial strain of the bacterium of plasmid and band pLOI276 or pLOI284 plasmid.

The recon of band pet operon grows up to bigger, less opaque bacterium colony on the Luria agar plate of glucose (under the aerobic condition) and is easy to detect containing because of it.The difference of this bacterium colony size and transparency aspect is to identify to have the significant notation of expressing ethanol dehydrogenase and pyruvic carboxylase plasmid recon simultaneously.

Whole base sequences of known pLOI295.The open reading frame of coding Pyruvate Decarboxylase Gene starts from the 163rd base in Lac promotor downstream, and sentences two terminator codons in the 85th base of the open reading frame upstream of coding alcoholdehydrogenase gene and finish.The upstream of open reading frame all has the sequence that is similar to ribosome bind site to these two genes being close to separately.The gene of coding alcoholdehydrogenase has a terminator codon, also has the palindromic sequence structure of 13 base pairs thereafter as transcription terminator.Embodiment 2. expression of zymomonas mobilis gene in intestinal bacteria

No matter Pyruvate Decarboxylase Gene and alcohol dehydrogenase gene are (respectively on pLOI276 and the pLOI284) or jointly in the intestinal bacteria of Lac promoter regulation, all high expression level individually.Wild-type e. coli does not have pyruvic carboxylase, and the ethanol dehydrogenase of the induction type of lower concentration is only arranged.When recombination bacillus coli was grown under the glucose condition is arranged, it is about 50% that the ratio of the zymomonas mobilis enzyme of its expression live to descend, and this is consistent to checking of Lac promotor with glucose.The ratio that the pyruvic carboxylase of pet operon near-end genes encoding is expressed in pLOI295 is lived than the Senior Three in pLOI276 doubly.Pet operon far-end gene, i.e. alcohol dehydrogenase gene, the ratio work that the enzyme of coding is expressed in pLOI295 is the twice in pLOI284.The glucose fermentation of embodiment 3. recombinant bacterial strains

It is ethanol that the expression of pet operon makes recombination bacillus coli main tunning when anaerobic growth.The main tunning of parent plant then is succsinic acid (1.5mM), lactic acid (18mM) and acetate (7mM) (Fig. 3 A).In the cell of band pLOI234 (being loaded with the alcoholdehydrogenase gene), observe same fermentation diagram (Fig. 3 c).In the pLOI276 host strain that is loaded with the coding Pyruvate Decarboxylase Gene, the ethanol peak is (18mM) clearly, accounts for 1/3 of fermentation accumulation product.The high-caliber generation of this ethanol is due to intestinal bacteria China and foreign countries source movement fermentation single cell bacterium pyruvic carboxylase and the endogenous ethanol dehydrogenase acting in conjunction.The pLOI295 host e. coli of band pet operon (containing zymomonas mobilis pyruvic carboxylase and alcohol dehydrogenase gene) produces a large amount of ethanol (750mM; 4.3% volume ratio), account for tunning more than 95%.

The intestinal bacteria high level expression ethanol dehydrogenase and the pyruvic carboxylase of band pet operon are being controlled the oxidation of NADH.Thereby the fermenting process of this bacterium can be transformed into the cereuisiae fermentum fermentative action identical with zymomonas mobilis.When normal fermentation is grown, pyruvic acid becomes acetyl-CoA by the pyruvate dehydrogenase complex role transformation, or become oxaloacetic acid (becoming succsinic acid then) by the phosphoric acid enol pyruvic acid carboxylase role transformation, or be formic acid and acetylcoenzyme, or be lactic acid by the serum lactic dehydrogenase role transformation by the pyruvate formate-lyase role transformation.Last a kind of transition pathway is the wild-type e. coli regeneration of NAD ⁺Main mode, but bacterium serum lactic dehydrogenase KmS value is quite high, is about 10mM to 1,000mM (Garvie, E.I. (1980) " Bacterial Lactate dehydrogenases, " Micro-biol.Rev.44:106-139; Tarmy, E.M_and N.O.Kaplan (1968) " Kinetics of Ecoli BD-lactate dehydrogenase and evidencefor pyruvate controlled change in conformation, " J.Biol.Chem.243:2587-2596).The Km value of zymomonas mobilis pyruvic carboxylase is 0.4mM (Bringer-Meyer, S., K.-L.Schimz, and H.Sahm (1986) " Pyruvate decarboxylase from Zymononas mobilis:Isolation andpartial characterization " Arch.Microbiol.146:105-110), this a large amount of enzymes adds that the pyruvic acid that lower Km value can constantly produce glycolysis-effectively changes ethanol into.

Under the culture condition with the promotion gaseous interchange, can obtain the culture of high-cell density in appropriate jolting or shake culture vessel.Under this condition, the nutrient solution final pH is 6.3 or higher, depends on the degree of gaseous interchange.Embodiment 4. plasmid constructions and the expression of zymomonas mobilis producing and ethanol enzyme in intestinal bacteria

Plasmid pLOI295 has the pyruvate decarboxylase gene from Zymomonas mobilis and the alcoholdehydrogenase gene of lac promoter regulation.This construct is called the pet operon, in the plasmids that make up other band different promoters as the source of producing and ethanol gene.EcoR I-Sal I fragment from pLOI295 band producing and ethanol gene, after filling and leading up end with the Klenow fragment of archaeal dna polymerase, insert the Sma I site of pUC19 (its lac promotor downstream is right after the pdc group), obtain being numbered the plasmid of pLOI293, this plasmid has the pet gene that a side is BamH I site.BamH I fragment inserting expressioning carrier pLOI204 with band coding producing and ethanol enzyme gene constitutes plastid pLOI291 and pLOI292 (direction of insertion is opposite).The complete sequence of ribosome bind site, two goal gene and the transcription terminator of an adhB far-end are arranged on this BamH I fragment.In pLOI292, these two expression of gene are subjected to the regulation and control of zymomonas mobilis promotor on the former expression vector.

Make up plasmid pLOI308, remove the lac promotor of control pet gene, but keep BamH I site, upstream, in order to inserting other promotors.With BamH I part enzymolysis pLOI293, handle through Klenow, remove the BamH I site of adhB gene far-end.Downcut no promotor BamH I (next-door neighbour pdc)-EcoR I (away from the adhB) fragment of band producing and ethanol gene from this plasmid, the directed pUC18 BamH I-EcoR I site of inserting produces pLOI308.This plasmid is low expression level adhB on acetaldehyde indication flat board, but does not show the macrocolony phenotype, this phenotype and other pet function plasmid pLOI295, and pLOI291 is relevant with pLOI292.

The zymomonas mobilis chromosomal DNA makes most of dna fragmentations less than 4Kb with SauBA part enzymolysis.Originate as promoter fragment with this DNA, be connected to pLOI308 dephosphorylation BamH I site without fractional separation.Containing on the Luria culture medium flat plate of glucose, having the amicillin resistance recon of expressing good pet operon and occur with the macrocolony form.Choose wherein three recon plasmid pLOI308-2, pLOI308-5 and pLOI308-10 study.Zymomonas mobilis dna fragmentation of tool promoter activity long respectively 6,2 and 2Kb in these plasmids.

The activity of pyruvic carboxylase and ethanol dehydrogenase in table 1 summary recombinant chou intestinal bacteria (E.coli) overnight culture.The pyruvic carboxylase vigor can be from the 0.37U/ milligram cell protein of TC4 (pLOI292) to TC4 (pLOI295) 8.23U.According to the pyruvic carboxylase vigor from big to small, the TC4 recombinant chou is arranged in order as follows: pLOI295＞pLOI308＞pLOI308-10＞pLOI308-2＞pLOI308-5＞pLOI292＞pLOI291.

Table 1

The expression plasmid pyruvic carboxylase ethanol dehydrogenase of the producing and ethanol enzyme of zymomonas mobilis in intestinal bacteria

Than living ^aThe % cell protein ^bThan living ^aThe % cell protein ^cPLOI291 0.37 0.4 0.21 0.02pLOI292 0.48 0.5 0.30 0.03pLOI308-2 2.26 2.3 1.54 0.21pLOI308-5 1.11 1.1 0.76 0.10pLOI308-10 6.5 6.5 2.51 0.34pLOI295 8.2 8.2 9.65 1.4 do not have 00 0.08

A. the mmole numerical table with every milligram of total cell protein per minute conversion of substrate shows

Suppose when b. calculating that pure enzyme is 100 than work

Pure enzyme when c. calculating behind the endogenous alcohol dehydrogenase activity of supposition deduction is 710 than work

In each recombinant bacterial strain with different plasmids, the alcohol dehydrogenase activity variation tendency is identical with pyruvic carboxylase.The alcohol dehydrogenase activity that records is intestinal bacteria endogenous enzyme and the coefficient vigor of zymomonas mobilis enzyme.Compare with the bacterial strain that has the zymomonas mobilis gene plasmid, not lower with observed enzyme activity level in the TCA bacterial strain of plasmid.Zymomonas mobilis enzymic activity (the endogenous alcohol dehydrogenase activity of deduction intestinal bacteria) scope is that 0.13IU/ milligram cell protein (the TC4 bacterial strain of band pLOI291) is to 9.6IU/ milligram cell protein (the TC4 bacterial strain of band pLOI295).Embodiment 5. contains the growth of the recombinant bacterial strain of zymomonas mobilis producing and ethanol gene

Change the alcoholic acid anabolism into by the grape catabolism of carbohydrate and also can influence the increment of thalline and the acidity variation of substratum.Though tunning is nontoxic relatively, might be accumulate to certain level during the fermentation and produces toxic effect.Bacterium is anaerobic growth in containing the LB nutrient solution of 10% glucose, the bacterial strain that does not contain plasmid can both reach every milliliter of nutrient solution with the bacterial strain that carries plasmid pLOI284 (gene that has the alcoholdehydrogenase of encoding) and contain 0.25 milliliter of proteic final concentration of mycetocyte after cultivating 48 hours, the pH value of liquid drops to 4.4 simultaneously.For the bacterial strain that carries plasmid pLOI276 (gene that has the pyruvic carboxylase of encoding), cell concn can increase twice, and the pH value then reduces to 4.5.The final cell concentration of carrying plasmid pLOI295 (promptly with the pet operon) bacterial strain is 2.5 mg/ml, and comparison will exceed 10 times according to bacterium, and final pH then drops to 4.7.As if when bacterial concentration reaches every milliliter when 2.5 milligrams of cell proteins are arranged, magnesium ion becomes limiting factor; If add the MgSO of 0.5mM this moment ₄Solution, then cell concn also can improve 1.5 times.

The growing state of recombinant bacterial strain under aerobic and two kinds of conditions of anaerobism all made the observation (see figure 3).(see Fig. 3 A and table 2) under aerobic culture condition, bacterial strain TC4 bred with per approximately 30 minutes monobasic speed in the quickest period of growth.Carry the derive bacterial strain TC4 of plasmid of plasmid pLOI308 and show equal maximum growth rate, the generation time is 26 to 30 minutes.Bacterial strain TC4 (pLOI295) growing state under same condition not good (71 minutes generation times), and with the part bacteriolyze.Bacterial strain TC4 (pLOI291) and TC4 (pLOI292) growth velocity are medium, and the generation time is 46 minutes.

Table 2

Maximum under aerobic and the anaerobism culture condition for the time,

The final pH value growth plasmid cell concentration of final cell concentration and nutrient solution ^aFor the time final condition (milligram albumen/milliliter) (branch) pH value aerobic do not have 0.7 29 4.4

pLOI291 0．7 46 5．3

pLOI292 1．3 46 5．1

pLOI295 1．1 71 5．7

pLOI308-2 1．7 27 5．5

pLOI308-5 0．8 30 5．0

PLOI308-10 2.5 26 5.0 anaerobism do not have 0.3 32 4.4

pLOI291 0．4 40 4．5

pLOI292 1．0 48 5．0

pLOI295 2．1 39 4．7

pLOI308-2 0．8 42 5．7

pLOI308-5 0．4 38 4．9

pLOI308 2．2 41 5．2

A. be determined to cultivate after 24 hours and carry out.

(see Fig. 3 B and table 2) under the anaerobism culture condition, the generation time that does not contain the bacterial strain TC4 of plasmid is 32 minutes, wants much shorter than the recombinant bacterial strain that contains the producing and ethanol enzyme.All recombinant bacterial strains are the maximum growth rate of exhibit comparable all, and the generation time all between 38 to 41 minutes, has only the growth velocity of bacterial strain TC4 (pLOI292) slow slightly, and the generation time is 48 minutes.

Except that bacterial strain TC4 (pLOI295), no matter all recombinant bacterial strains are that aerobic cultivation or anaerobism are cultivated, and after growth 12 hours, cell concentration all reaches level or even higher (the seeing Fig. 3 A and B) that equates with the bacterial strain TC4 that does not contain plasmid.Table 2 has been summed up the data of bacterial strain TC4 and heavy bacterial strain final cell concentration after growing 24 hours.Under aerobic condition, the TC4 bacterial strain that contains plasmid pLOI308-10 has maximum bacterial concentration, secondly is respectively the TC4 bacterial strain that contains plasmid pLOI308-2, contains the pLOI292 bacterial strain, contains the pLOI295 bacterial strain (thalline is the part cracking) and contain the TC4 bacterium of pLOI308-5.

Under anaerobic, the TC4 bacterium that contains pLOI308-10 is roughly the same with the final cell concentration of the TC4 bacterium that contains pLOI295, is respectively the TC4 bacterial strain that contains pLOI292, pLOI308-2, pLOI308-5 and pLOI291 secondly.

Fig. 4 has shown the pyruvic carboxylase activity level of somatic cells and the bacterial growth relation between the final cell concentration after 24 hours.Because the synthetic coupling mutually of the synthetic and alcoholdehydrogenase of pyruvic carboxylase in recombinant bacterial strain, thereby this curve is also represented zymomonas mobilis ZmobilisNAD ⁺Regeneration system rapidly is to the influence of final cell concentration.These data clearly illustrate that, no matter are aerobic condition or anaerobic condition, and the expression of zymomonas mobilis ethanol route of synthesis has all improved final cell concentration.In bacterial strain TC4 (pLOI308-10), the expression level of pyruvic carboxylase and alcoholdehydrogenase (being respectively 6.5IU and 2.5IU) under aerobic and anaerobic condition all near optimum level.Producing and ethanol expression of gene level even excessive among the bacterial strain TC4 (pLOI295) causes cell growth retardation under aerobic condition, and the cracking of part is arranged; Under anaerobic the speed of growth then slightly descends.

Bacterial strain TC4 (pLOI295) quickening of under anaerobic growing, and slight apparent cracking is arranged; And cracking takes place in poor growth simultaneously in the culturing bottle of quick oscillation, and this explanation is for this artificial bacterial strain that makes up, and highly Tong Qi growing environment is harmful to bacterium.Be reduced to originally 1/3rd as hunting speed, can reduce the cracking degree of recombinant bacterial strain significantly and improve the ultimate density of bacterium.Embodiment 6. producing and ethanol enzymes tie up to the acidification effect of the nutrient solution in the process of growth

Fig. 3 C has shown nutrient solution acidacidity change curve under the anaerobism culture condition.For the TC4 bacterial strain that does not contain plasmid, in initial 6 hours that cultivate, the pH value of solution descends rapidly; And for the derivative strain that contains the producing and ethanol gene, this downtrending greatly slows down.Initial 12 hours acidification is slowed down in containing the TC4 bacterium of pLOI295 at most, and next coming in order are respectively the TC4 that contains pLOI308-10, pLOI308-2 and the pLOI308-5 plasmid bacterium that derives.The survey of TC4 (pLOI291) and TC4 (pLOI292) bacterium shows that the result is not shown in the diagram, but lays respectively at below and the top of TC4 (pLOI308-5).Although the reorganization bacterium has higher ultimate density, they are low (seeing Table 2) than the nutrient solution of the TC4 bacterium that does not contain the producing and ethanol gene all in the acidity of growth nutrient solution after 24 hours under the anaerobic and aerobic condition.

Recombinant bacterial strain is when acidifying speed and degree descend, and its cell growth increases, though this explanation under the condition of highly ventilation, the decline of pH value also is the main factor of limiting growth.Following result of experiment is supported this view.The TC4 bacterium that does not contain plasmid grows in the nutrient solution of the 1M sodium phosphate buffer (pH7.0) that is added with 1/10 volume ratio, and its final cell concentration can increase by 85%.The addition that reduces damping fluid then the level of growth of bacterium between between the two above-mentioned.Embodiment 7. producing and ethanol enzymes system is to the influence of tunning

Table 3 has been summed up the analytical results of TC4 bacterium and reorganization bacterium 24 hours secondary fermentation products of cultivation under aerobic and anaerobic condition.Under aerobic condition, acetate is main tunning and in process of growth the constantly accumulation of TC4 bacterium in enriching nutrient solution that does not contain plasmid, alcoholic acid generate then detect less than.To containing the bacterial strain of zymomonas mobilis producing and ethanol gene, the growing amount of acetate greatly reduces, and ethanol then becomes main tunning.It is the highest to contain the amount of alcohol that the TC4 bacterium of pLOI308-10 produced, and next coming in order are respectively the TC4 bacterium that contains pLOI295, pLOI308-2, pLOI292, pLOI308-5 and pLOI291.Under aerobic condition, all bacterial strains also generate a spot of lactic acid (0.6 to 1.2mM).The TC4 bacterium that only contains plasmid pLOI308-10 accumulates a certain amount of succsinic acid, but only accounts for 1% of whole tunnings, and 94% tunning is an ethanol.

Table 3

The comparison of aerobic and anaerobic growth conditions bottom fermentation product

Tunning (mM (standard deviation)) growth conditions plasmid succsinic acid lactic acid acetate ethanol aerobic does not have 0.2 (0.1) 0.6 (0.2) 55 (2) Tr

pLOI308-2 Tr 1．2(0．3) 22(2) 98(3)

pLOI308-5 Tr 0．9(0．2) 43(3) 15(2)

pLOI308-10 4．9(0．5) 1．0(0．2) 17(2) 337(21)

pLOI295 Tr 1．1(0．4)?13(1) 114(10)

pLOI291 Tr 0．6(0．2)?34(3) 7(1)

PLOI292 Tr 1.3 (0.2) 30 (1.5) 24 (1) anaerobics do not have 0.9 (0.1) 22 (1) 7 (0.3) 0.4 (0.2)

pLOI308-2 0．8(0．1) 7(0．5) 4(0．3) 71(5)

pLOI308-5 0．3(0．1) 18(2) 6(1) 16(2)

pLOI308-10?5．0(0．4) 10(1) 1．2(0．2)?482(23)

pLOI295 2．2(0．20)?6(1) 3(0．3) 90(2)

pLOI291 1．1(0．1) 15(0．5) 7(0．2) 4(0．5)

pLOI292 2．3(0．2) 9(0．7) 7．2(0．3)?21(1)

Under anaerobic, to be the TC4 bacterium that do not contain plasmid adding the main tunning in the nutrient solution of enriching of glucose to lactic acid, and constantly accumulation in 24 hours of growth, and acetate, succsinic acid and alcoholic acid generate to some extent and reduce.In the reorganization bacterium that contains producing and ethanol enzyme system, the output of lactic acid significantly reduces, and has quite a large amount of ethanol to generate simultaneously.The amount of alcohol maximum that TC4 (pLOI308-10) generates accounts for 97% of fermentation soluble product total amount.It is consistent with result under the aerobic growth condition to test trend that each bacterial strain finds that ethanol generates.In fact, except that TC4 (pLOI308-10), growing amount when all bacterial strains are under anaerobic cultivated the amount of alcohol that generated in 24 hours than aerobic the cultivation is low, this may be because the total cell concentration of thalline that the anaerobism cultivation forms is less, cause the volume ratio of ethanol production to reduce, thereby reduced the alcoholic acid accumulating level.

Alcoholic acid growing amount under the anaerobic and aerobic condition (seeing Table 3) is directly connected to zymomonas mobilis producing and ethanol expression of gene level (seeing Table 1).As if in bacterial strain TC4 (pLOI308-10), the ethanol synthetic is expressed and is reached optimum level, the activity of pyruvic carboxylase is 6IU, and the activity of alcoholdehydrogenase is 2.5IU.

The bacterium incubation growth is to the somatic cells concentration level higher than the parent bacterium that does not contain plasmid but the intestinal bacteria TC4 that contains expressive movement fermentation single cell bacterium producing and ethanol gene derives.In the raising of final cell concentration, the nutrient solution in alcoholic acid semi-invariant and the process of growth acidification of nutrient solution to slow down rate all relevant with zymomonas mobilis producing and ethanol expression of gene level.Produce for reducing any problem relevant, except that plasmid pLOI295 (lac), all utilized allogeneic promoter to control expression of gene in the structure bacterial strain with transcriptional regulatory.It is that this expression level is significantly higher than zymomonas mobilis CP4 in plasmid pLOI308-10 (pyruvic carboxylase activity and alcoholdehydrogenase activity be respectively every milligram of cell protein 6.5IU and the 2.5IU) intestinal bacteria that expression level generates what all reach optimum regime aspect two at growth and ethanol, and the latter only comprises ethanol approach regeneration of NAD ⁺

The expression level of producing and ethanol gene in bacterial strain TC4 (pLOI295) as if too high (accounting for soluble cell proteic 17%).Under aerobic condition, the reduction of part cracking, poor growth and the ethanol production of the simultaneous thalline of this high-level genetic expression.This effect is eased after anaerobism is cultivated in the vibration and transferring to of slowing down.The apparent infringement that takes place under the condition of highly ventilation may cause exhausting of NADH that relation is arranged with the acting in conjunction of the active zymomonas mobilis second of apparent altitude n alcoholdehydrogenase II and endogenous nadh oxidase (with electron transfer system coupling mutually) with the part cracking.

As if as main tunning, alcoholic acid generates the growth velocity that can't oppositely influence intestinal bacteria TC4.Carrying the bacterial strain of pLOI308 (the containing the ColEI replicon) plasmid of deriving can express the pet operon and generate ethanol, it has the growth velocity identical with the parent bacterium under the growth conditions of aerobic interpolation glucose, and can reach the final cell concentration more taller than parent bacterium.Under aerobic condition, carry the strain growth of the plasmid pLOI291 that contains the RSF1010 replicon or pLOI292 will be slowly many.Because the producing and ethanol gene that these two structure bacterial strains are expressed and the amount of alcohol of generation are all low than plasmid pLOI308-10, this poor growth is attributable to the characteristic of carrier but not the expression level of pet-operon.The preparation of embodiment 8. additional strains

Test other coli strain, to select the good producing and ethanol bacterial strain of proterties.Coli strain through test comprises: ATCC8677, ATCC8739, ATCC9637, ATCC11303, ATCC11775, ATCC14948, ATCC15244 and ATCC23227.All bacterial strains are all cultivated in 30 ℃ shaking bath, nutrient solution is for containing the LB nutrient solution (Luria, S.E. and M.Delbruek (1943) Genetics28:491-511) of extractum carnis (10 grams per liter), yeast extract (5 grams per liter), Nacl (5 grams per liter) and fermentable sugars.Under situation about not being specifically noted, glucose and lactose add nutrient solution with the concentration of 100 grams per liters, and the working concentration of wood sugar then is 80 grams per liters.Various sugar are formulated in sterilization (121 ℃ following 15 minutes) separately in the distilled water to double working concentration.Wood sugar (SigmaChemical Co_St.Louis, MO) solution is acid, thereby will transfer to neutrality with NaOH solution before sterilization, otherwise solution can become brown sugared composition decomposition after sterilization.Use various sugars all to obtain identical fermentation result through autoclave sterilization or filtration sterilization.The survival of recombinant bacterial strain of carrying the gene of coding ethanol approach relevant enzyme in nutrient solution or on the solid plate requires to contain in the substratum fermentable sugars component.As shown, the final working concentration of tsiklomitsin is 10 mg/litre.Embodiment 9. additional strains are to the tolerance of environment

To control before ethanol synthetic plasmid imports eight above-mentioned different e. coli strains, compare their tolerances earlier severe environment.Checked that these bacterial strains are to Na-Cl, sugar, low pH and alcoholic acid resistivity.Table 4 has been listed the different concns of used NaCl and sugar, wherein also comprises in the original substratum both concentration.The pH value of original substratum is 6.8, is adjusted to required acidity with HCl during experiment.The acidic culture filtration sterilization.Ethanol is adding after autoclaved substratum cooling.Sugar component is sterilization separately.Thalline is incubated overnight in not containing the various sugar culture-mediums of fractions tested earlier, dilutes 60 times then and cultivates in 13 * 75mm test tube of 3mi test media is arranged.Measure nutrient solution after 48 hours in the O.D. at 550nm place value.O.D. equal 1.0 and promptly be equivalent to the tropina of 0.25 mg/ml and 0.33 milligram dry cell weight.In the environmental resistance test, final O.D. value is lower than 0.2 and has promptly reflected the bacterium multiplication less than secondary, and its growth can be ignored.

Table 4 has been summed up and carried out above-mentioned result of experiment in being contained the nutrient solution of glucose.The experimental result of carrying out in the nutrient solution that contains lactose or wood sugar is identical.Strains A TCC8677, ATCC8739 and ATCC11775 are responsive especially to the alcoholic acid restraining effect of lower concentration.Except that bacterial strain ATCC23227, other bacterial strain all still can double 2 to 4 times under pH4.0 acidity at least, and is wherein the strongest to the tolerance of low pH value with ATCC9637 and ATCC11775 again.All bacterial strains all have only limited growth in higher temperature after 45 ℃ cultivation grows up to down.All bacterial strains level that goes down to posterity in the temperature environment more than 45 ℃ is cultivated and all to be failed.All bacterial strains can both be grown in the nutrient solution that contains 20% Portugal's green onion sugar or 20% lactose or 12% wood sugar.

Intestinal bacteria are containing 100 grams per liters under table 4. chemistry and the physical coercion

The growing state of growing state stress conditions intestinal bacteria bacterium (ATCC numbering) in the LB training base fluid of glucose

8677 8739 9637 11303 11775 14948 15224 23227NaCl （ ／ ） 50 ＋ ＋＋ ＋＋ ＋＋ ＋＋ ＋＋ ＋＋ ＋＋60 0 ＋ ＋＋ ＋＋ ＋ ＋＋ ＋ ＋＋70 0 0 ＋ ＋ 0 ＋ ＋ ＋ （ ％ ） 3．8 ＋＋ ＋＋ ＋＋ ＋＋ ＋＋ ＋＋ ＋＋ ＋＋5．0 ＋＋ ＋＋ ＋ ＋ ＋ ＋ ＋ ＋6．3 0 ＋＋ ＋ ＋ ＋ ＋ ＋ 07．5 0 ＋ ＋ 0 0 ＋ 0 08．8 0 0 0 0 0 0 0 0pH4．50 ＋＋ ＋＋ ＋＋ ＋＋ ＋＋ ＋＋ ＋＋ ＋＋pH4．25 ＋＋ ＋＋ ＋＋ ＋ ＋＋ ＋＋ ＋＋ ＋pH4．00 ＋ ＋ ＋＋ ＋ ＋＋ ＋ ＋ 0pH3．75 0 0 ＋ 0 ＋ 0 0 0℃45 ＋＋ ＋＋ ＋＋ ＋＋ ＋ ＋＋ ＋＋ ＋＋47 ＋ ＋ ＋ ＋ ＋ ＋ ＋ ＋49 0 0 ＋ ＋ 0 0 ＋ ＋0＝O．D．550nm＋＝＋＋＝10．。

The utilization of sugar detects by two kinds of methods.For the bacterial strain that grows up to red single bacterium colony on the MacConkey agar that adds 2% sugared composition, its sugar utilizes ability can directly measure (Silhavy, T.J. and J.R.Beckwith (1985) Microbi-ol.Rev.49:398-418).Also can be according to the BiologEC plates method of the introduction on the product description (Biolog, Inc_Hayward, CA) detection somatic cells.The Biologplates method is quick and reliable, with the output of measuring NADH (promptly by certain tetrazolium salts to insoluble first _ conversion, as the index of substrate utilization.To 13 kinds of sugar of test, the result of two kinds of methods is in full accord.

The bacterial strain of all tests can both utilize glucose, semi-lactosi, seminose, pectinose, lactose, glucuronic acid and galacturonic acid.Bacterial strain 11775 can not utilize wood sugar.Maltose and trisaccharide maltose then can not be utilized by strains A TCC11303 and ATCC23227.All bacterial strains all have faint positive reaction ability to cellobiose.Sucrose is only utilized by strains A TCC9637.Sugar is utilized as result of study and gathers as table 5.Table 5 is taken the end value of intestinal bacteria growing state saccharic grain E.coli bacterial strain (ATCC numbering) O.D.550nm of plasmid pLOI297 and pLOI308-11

8,677 8,739 9,637 11,303 11,775 14,948 15,224 23227 glucose without 4.0 3.7 6.1 6.0 4.7 5.6 7.0 6.6 glucose pLOI297,10.0 10.5 10.5 10.0 9.5-9.5,10.2 glucose pLOI308-11,9.8 9.5 11.4 11.2-9.3,10.8 11.4 lactose without 4.3 3.8 7.5 6.0 4.5 6.1 7.0 6.4 lactose pLOI1297,13.0 6.8 11.6 10.8 7.6-10.5,7.0 lactose pLOI308-11,10.0 10.0 11.5 11.0-7.3,10.0 10.0 wood sugars without the genetic modification of 4.1 3.7 7.7 7.3 4.9 5.9 7.2 7.0 wood sugar pLOI297,8.1 10.6 10.8 10.6 4.7-11.0,11.0 wood sugar pLOI308-11,10.0 6.8 11.4 8.5-11.4,10.6 12.0 horizontal line registrations according to vacant embodiment 11 additional strains

Two novel plasmid (Maniatis have been made up by ordinary method, T., E.F.Fritsch, and J.Sambrook (1982) Molecular Cloning:a Laboratory Manual.Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), they all contain the resistant gene of antagonism penbritin and tsiklomitsin as selection markers.From plasmid pLOI308-10 (Ingram and Conway (1988), Supra) go up a 5.2Kb contained ethanol operon for synthesizing (pet-operon, comprise an endogenous zymomonas mobilis promotor, pyruvic carboxylase, ethanol dehydrogenase and transcription terminator) EcoR I endonuclease bamhi separate and insert on the EcoRI restriction enzyme site of pBR322 and be built into pLOI308-11.Plasmid pLOI297 then is by will be from plasmid pCOS2EMBL (Poustka, A_H.R.Rackwitz A.-M.Firschauf, and H.Lehrach (1984) Proc.Natl.acad.Sci.USA81:4129-4133) the 2.6Kb EcoR I endonuclease bamhi that contains tetracycline resistance gene inserts pLOI295, and (Ingram et al. (1987) is on Sal I restriction enzyme site supra) and make up.Before connection with sample with the Klenow fragment of E.coli archaeal dna polymerase handle can fill and lead up sticky end (Maniatis et al., supra).

According to Mandel and the described calcium chloride transformation (Mandel of Higa, M. and A.Higa (1970) J.Mol.Biol.53:159-162), change plasmid over to each E.coli bacterial strain, on the solid medium that contains 2% glucose and tsiklomitsin, select.The stability of plasmid is represented with subculture under the condition of no antibiotic selection still keeps the drug resistance marker after 25 generations thalline percentage ratio.

The bacterial strain of reorganization has obtained the plasmid of taking alcohol synthetic gene, contain grew 24 to 48 hours on the solid medium that adds sugar after, form king-sized, be the xanchromatic bacterium colony.In liquid medium, the contrast bacterium that the final cell concentration ratio of reorganization bacterium does not contain plasmid will exceed two to three times.Plasmid pLOI297 is to the transformant of strains A TCC14948, and plasmid pLOI308-11 then can't obtain all the time to the transformant of strains A TCC11775.The bacterial strain 11775 that can not utilize wood sugar to do.Its recon growth concentration when adding the culture condition of sugar with wood sugar as richness is not higher than the contrast bacterium, but its speed of growth is accelerated when adding lactose and glucose.

The detection of plasmid stability is carried out (seeing Table 6) after containing 25 generations of going down to posterity on the substratum of glucose.Two plasmids have identical replicon, can both very stably remain in all other bacterial strains except that ATCC8677 and ATCC8739.Table 6 plasmid pLOI297 and pLOI308-11 are not having antibiotic to select,

Contain the cell count (%) that stable ATCC bacterial strain after 25 generations of growth under the condition of glucose keeps plasmid

PLOI297 pLOI308-11 8,677 75 85 8,739 44 47 9,637 100 90 11,303 98 98 11,775 100--14948---97 15,224 99 100 23,227 91 100 dotted lines represent that data lack.The expression of embodiment 12 pyruvic carboxylases in the bacterial strain of hereditary change

The active mensuration of pyruvic carboxylase is according to aforementioned (Conway et al. (1987) sees before, and Neale et al. (1987) sees before), but when O.D.4.0 collecting cell, this moment, increment was about half of maximum.

The detection of zymomonas mobilis pyruvic carboxylase activity expression is carried out (seeing Table 7) after the growth containing under the culture condition of tsiklomitsin.To plasmid pLOI297, the zymomonas mobilis expression of gene is subjected to the control of intestinal bacteria lactose synthetic promoter; Plasmid pLOI308-11 then utilizes endogenous zymomonas mobilis promotor to regulate the expression of pet-operon.Strains A TCC11303 (pLOI297), ATCC11775 (pLOI297) and ATCC15224 (pLOI297) show activity are the highest.Table 7 is containing zymomonas mobilis under the growth conditions of glucose

Pyruvate Decarboxylase Gene take plasmid pLOI297 and

Express ATCC bacterial strain pyruvic carboxylase activity in the coli strain of pLOI308-11

PLOI297 pLOI308-118677 5.7 6.08739 0.8 1.49637 1.1 1.411303 16.7 2.111775 17.1--b14948---b 2.515224 16.3 1.823227 2.3 1.7 ^aEnzymic activity is with the international unit value representation of every milligram of tropina ^bDotted line is represented the ethanol production of the vacant embodiment 13 genetic modification bacterial strains of data

In the Luria nutrient solution in addition the potassiumphosphate of power pH7.0 slow in liquid to the 0.2M final concentration to be applicable to fermentation test.Phosphoric acid buffer, complex culture medium composition and sugar sterilize respectively and cool off after give mixing again.The working concentration of tsiklomitsin is 10 mg/litre.Inoculum is the bacterium colony of fresh separated, grows after 24 hours, with fermention medium washing to be measured, adds in the fermented liquid, and making its concentration is O.D.550nm about 1.0.Leavening temperature is 30 ℃ or 37 ℃.In 100 milliliters of Erlenmeyer flasks, adorn 80 milliliters of fermented solns,, insert No. 25 syringe needle on the stopper and derive the gas that fermentation produces with soft rubber ball plug mouth.The fermentation flask will immerse in the temperature controlled water bath device, stirs with 100rpm speed with magnetic stir bar.

Alcohol concn is measured with vapor-phase chromatography according to aforementioned.(Dombek, K, M. and L.O.Ingram (1985) Appl, Environ.Microbiol.51:197-200), measured value is represented with volume percent.Transformation efficiency according to adding sugar calculate, set per 20 gram glucose or wood sugar and generate 12.75 milliliters of (10.2 gram) ethanol or per 20 gram lactose to generate 13.5 milliliters of (10.8 gram) ethanol conversion be 100%.

All coli strains through genetic modification all produce a large amount of ethanol (seeing Table 8) when adding grape.The preliminary experiment that carries out with strains A TCC15244 (pLOI297) shows, contains 0.2M, and the nutrient solution of pH7.0 potassium phosphate buffer can make bacterial strain generate more ethanol.Estimate as using automatic acidity regulation system can obtain similar or better result to replace this potassium phosphate buffer.When glucose concn is 15%, to grow after 48 hours, the ethanol production of the bacterium of 30 ℃ of growths is more than 37 ℃ of bacterium that grow down.The fermentation test of lactose and wood sugar is only carried out under lower temperature is 30 ℃.As a whole, the thalline ethanol production of band plasmid pLOI297 will exceed the bacterium of band plasmid pLOI308-11.Growth is after 48 hours in 15% glucose, and it is maximum that strains A TCC11303 (pLOI297), ATCC11775 (pLOI297) and ATCC15224 (pLOI297) produce the alcoholic acid amount, accounts for the 5.8-6.9% of cumulative volume.In initial experiment, most of bacterial strains are relatively poor to the tolerance of wood sugar, thereby more promptly the carrying out in 8% wood sugar of fermenting experiment.Growth is after 72 hours in 8% wood sugar, and the amount of alcohol that strains A TCC9637 (pLOI297), ATCC11303 (pLOI297) and ATCC15224 (pLOI297) produce is maximum.All bacterial strains can both be grown in 15% lactose well.In lactose growth 48 little after, the amount of alcohol of strains A TCC11303 (pLOI297) and ATCC15224 (pLOI297) generation is maximum, is respectively 6.1% and 5.6% volume ratio.The coli strain that table 8 is taken plasmid pLOI297 and pLOI308-11 is containing

Glucose (48 hours), wood sugar (72 hours) and lactose (48 hours)

Growth conditions under through the ethanol productions of the ethanol production saccharic grain intestinal bacteria (ATCC numbering) of liquid fermenting (%, v/v)

8,677 8,739 9,637 11,303 11,775 14,948 15,224 2322715% glucose pLOI297 ^a2.4 4.7 4.2 4.3 4.8-4.8 0.915% glucose pLOI308-11 ^a3.6 1.4 2.1 1.3-3.4 2.8 1.315% glucose pLOI297 ^b3.2 4.7 4.1 5.8 6.9-6.1 3.115% glucose pLOI308-11 ^b5.8 5.0 3.5 1.5-3.8 3.0 3.215% lactose pLOI297 ^b2.3 4.4 5.3 6.1 4.5-5.6 3.715% lactose pLOI308-11 ^b2.3 2.1 3.4 0.9-2.9 2.7 3.08% wood sugar pLOI297 ^b0.9 4.1 4.8 5.2--4.8 1.28% wood sugar pLOI308-11 ^b2.0 2.8 2.8 1.2-2.0 3.5 1.0 dotted lines represent that the vacant a. insulation of data is incubated at 30 ℃ at 37 ℃ of b.

Relatively these results of study can find that strains A TCC11303 (pLOI297) and ATCC15224 (pLOI297) make up bacterial strain for the best of alcohol production.Set up the growth time one ethanol production graph of a relation (see figure 1) of these two bacterial strains in 12% glucose, 12% lactose and 8% wood sugar through measuring.Biomass has increased nearly ten times, and final concentration reaches every liter 3.6 gram dry weight.The rate of increase of biomass in wood sugar be half in glucose or lactose only.In these three kinds of sugar, the pass of ethanol production and thalli growth ties up to alcohol concn and reaches the 5% former linear dependence that roughly is.

With three groups of bacterial growths after 120 hours, the ethanol final concentration is in addition average, generates ethanol conversion (seeing Table 6) to calculate by sugar.The final alcohol concn of the culture of growing in 12% glucose is 7.2% (volumeization), is 94% of the theoretical yield of glucose fermentation.Lactose for 12%, alcoholic acid final concentration are 6.5%, are 80% of theoretical yield.For 8% wood sugar, its output can reach theoretical value 100% in addition higher.This in wood sugar poor growth and have the phenomenon of high yield may reflect in the thalline still to derive from the catabolic pyruvic acid of compound nutritional composition and transform to alcoholic acid except that generating the ethanol by the conversion of glucose to pyruvic acid.

By shown in Figure 1, calculate the ethanol production rate and gather and be table 9.Each is variant for alcoholic acid volume ratio production rate, per hour 1.4 grams per liters from glucose, per hour 0.64 grams per liter in the wood sugar.In three kinds of sugar, the alcoholic acid production rate all shows as maximum in the initial period of cultivating, and wherein maximum value is found in glucose, for per hour 2.1 restraining ethanol/gram dry cell weights.Utilize wood sugar can obtain the maximum ethanol yield of every relatively gram sugar, only surpassed the theoretical maximum yield of sugar relatively.Table 9 strains A TCC11303 (pLOI297) and ATCC15224 (pLOI297)

Produce alcoholic acid kinetic parameter mean value sugar volume ratio output ^aCompare production rate ^bYield ^cFormation efficiency ^dAmount of alcohol ^e12%glucoee 1.4 2.1 0.48 95 5812%lactoee 1.3 2.0 0.43 80 528%xyloee 0.6 1.3 0.52 102 42a gram ethanol/liter/hour b gram ethanol/gram dry cell weight/hour c gram ethanol/gram sugar The final alcohol concn of e (unit is a grams per liter)

ATCC11303 (pLOI297) has been carried out relevant experiment, utilized pectinose, semi-lactosi and seminose to generate the alcoholic acid situation with inspection.In 30 ℃, cultivate after 48 hours alcohol concn between 3% to 4%, but the end can be tested further.The pathways metabolism of these three kinds of carbohydrates is similar to the pathways metabolism of glucose and wood sugar, estimation can obtain close ethanol yield (Lin, E.CC. (1987) " Dissimilatory pathways for sugars; poly-ols; and carboxylates ", In F.C.Neidhardt, J.L.Ingraham, K.B.Low, B.Magasanik, and M.Schaechter (eds), Escherichia coliand Salmonella typhimurium, Vol 1, PP244-284 page or leaf American So-ciety for Microbiology, Washington, D.C.) alcohol production of embodiment 14 non-escherichia coli hosts

Because the high similarity of pathways metabolism between different strain can be accepted conversion in respect of a lot of parasitic fungis in advance, obtains foreign gene, send out product pure as it thereby produce ethanol.As described herein, when the host will use the conversion of adh and pdc gene, can estimate fully, can utilize suitable carrier to reach expression.In case express successfully, just can be and alcoholic acid output is made quite approaching estimation based on the consistence of pathways metabolism between different hosts.Alcoholic acid produces in the Klebsiella that embodiment 15 transforms

The original called after klebsiella pneumoniae M5A1 of the applied gram of the present invention Salmonella bacterial strain bacterial strain (Klebsiella pneumoniae M5A1, MahL, M.C_P.W.Wilson, M.A.Fife, W.H.Ewing (1965) J.Bacteriol.89:1482-1487).This bacterial strain is a kind of diazotroph, once is accredited as gas bacillus at first, renames according to its antigenic characteristic later on." after uncle's Jie Shi handbook offered some clarification on the new systematics standard of klebsiella pneumoniae, the species of this bacterial strain formed and have obtained further research from the 8th edition.Bacterial strain M5A1 can grow containing on the minimum medium of glucose in the time of 10 ℃, but can not produce gas from lactose in the time of 44.5 ℃.This bacterial strain indole reaction positive, and can utilize m-Salicylic acid salt or gentisate as unique carbon source is grown in the temperature of 30 ℃ or 37 ℃.Based on these test-results, M5A1 is named to urging childbirth Klebsiella (K.oxytoca).Except that the recombinant bacteria that has the plasmid that contains zymomonas mobilis (Z.mobilis) gene, bacterial strain is not adding the cultivation of going down to posterity on the Luria agar plate of sugar.The recombinant bacteria that contains adhB and pdc gene requires to contain in the substratum certain fermentable sugars and divides and could survive, and its bacterial classification goes down to posterity on the flat board that contains 2% glucose or wood sugar.Used antibiotic concentration is as follows: penbritin 50 mcg/ml, paraxin 40 mcg/ml, tsiklomitsin 12.5 mcg/ml.The expression of zymomonas mobilis adh II gene in the reorganization thalline screened by acetaldehyde indication flat board.Intestinal bacteria (Escherichia coli) bacterial strain TC4 is as the host of all plasmid construction bodies.

Because M5A1 has quite high resistance for penicillin and derivative thereof, we have made up and have had cat (Cm ^r) or tet (Tc ^r) shuttle vehicle of gene.By on the Sal of plasmid pLOI276 I site, inserting one section from pcos2EMBL (Poustka, A_H.R.Rackwitz, A.-M.Frischauf, B.Hohn, H.Lehrach (1984) Proc.Natl.Acad.Sci.USA 81:4129-4133) the EcoR I endonuclease bamhi of 2.6Kb can insert a tet gene again to the pLOI276 that contains zymomonas mobilis pdc gene.Sticky end can be handled and remove through the Klenow of e. coli dna polymerase fragment before ligation.The gained construct is proved conclusively through restricted enzyme cutting analysis, and is numbered pLOI560.

The intestinal bacteria B (ATCC11303) that studies show that in the past has endogenous low copy number plasmid.Zymomonas mobilis pdc and adh B gene and cat genosome are incorporated in these plasmids, can obtain useful novel vector.The structure of this plasmid requires to separate earlier one section Pst I enzyme fragment that does not contain promotor and carry the 4.6Kb of pdc, adh B and cat gene from pLOI510.This fragment does not possess copy function.Through after connect cyclisation, these fragments can be transformed among the intestinal bacteria B, screen Cm containing on the Luria agar plate of 2% glucose ^rResistance marker.Transformant is identified on acetaldehyde indication flat board, is formed the transformant that promptly is chosen as high-level adhB activity of gene expression of garnet bacterium colony.Prepare plasmid from these bacterial strains and move ability by transforming the corotation that large intestine TC4 tests its resistance marker and zymomonas mobilis gene again.All transformants show and are not with the pUC18 fragment that contains bla and Col E1 replicon all to the penbritin sensitivity.One of them plasmid, pLOI555 (8.4kb), the bacterium colony redness that forms on the acetaldehyde indication flat board is the darkest containing, and gives intestinal bacteria fabulous producing and ethanol ability, it seems and prepare the gained productive rate according to a small amount of plasmid, be to be present in the bacterium with low copy number.This plasmid and pLOI297 and pLOI560 can be used for conversion and urge childbirth Klebsiella M5A1, and with chlorampenicol resistant (Cm ^r) screened.

Three plasmid construction bodies that contain the gene of coding zymomonas mobilis PDC are transformed among the M5A1 and (see Table 10).The PDC specific activity pLOI555 transformant of two puc constructs (pLOI297 and pLOI560) transformant will exceed 8 times.Suppose the high specific 100U of being alive of pure PDC, then the shared cytoplasm protein ratio of this enzyme is 25% in M5A1 (pLOI297) and M5A1 (pLOI560), is 3.6% in M5A1 (pLOI555).Plasmid stability and pdc express in the table 10 M5A1 recombinant bacterial strain

The active proterties per-cent that keeps of PDC after 24 hours ^{A, b}Two sub-sampling b acetaldehyde proterties and resistance disappeared simultaneously when plasmid U/ milligram albumen (passage number) pLOI555 3.6 100 (38.5) 98 (68.5) pLOI297 28 52 (38.6) 10 (68.60) pLOI560 27 97 (32.9) 0 (62.9) a cultivated

The expression of zymomonas mobilis (Z.mobilis) gene in M5A1 can further be confirmed by the SDS-polyacrylamide gel electrophoresis.By comparing, can easily pick out the sample band of representing PDC and ADH II with original strain.Compare much widely with M5A1 (pLOI555) based on the PDC band of the recon of pUC, this result is consistent with the enzyme assay result.Though the ADH II is lacked than PDC content, the expression ratio of this zymomonas mobilis gene in M5A1 (pLOI297) also wants high in M5A1 (pLOI555).In the M5A1 that only has zymomonas mobilis pdc gene (pLOI560), detect less than band corresponding to the ADH II.

According to the yield in a small amount of plasmid preparation experiment, the copy number of inferring pLOI555 is less than 1/10th of other two recombinant chou copy numbers.Though this is supposition roughly, any is arranged is clearly, and promptly in the recombinant chou as carrier, the high level expression partly cause of PDC is that its copy number is higher.

Band pLOI555, pLOI297, or the thalline of pLOI560 contains 10% glucose and does not contain more than 60 generations of Luria substratum continuous passage of antibiotic at 30 ℃.Culture is through suitably coating on the Luria flat board that contains 2% glucose but do not contain antibiotic after the dilution.Use that the acetaldehyde indication is dull and stereotyped to detect whether bacterium colony keeps the producing and ethanol gene of zymomonas mobilis and to the resistance of suitable antibiotic.

Existing report is cloned in the zymomonas mobilis gene very unstable (Tolan in planting living Klebsiella (Klebsiella planticola) on the pBR322 deutero-carrier, J.S_R.K.Finn (1987) Appl.Eviron.Microbiol.53:2039-2044), can not be used for industrial production.The recon of band pLOI555 is then highly stable, and 98% has kept resistant gene and zymomonas mobilis gene in the flora.Though only detected adh II expression of gene by the acetaldehyde indicator medium, these recons have still kept the big phenotype of bacterium colony, show that pdc and adh II gene all express.

In the Luria substratum that contains 10% (w/v) glucose or wood sugar, 30 ℃, pH6.0 ferments under the 100rpm shaking culture condition.With single colony inoculation 30 ℃ of overnight incubation in static flask, obtain inoculum.Is OD550 1.0 (be equivalent to 330 milligrams of dry cell weights/liter) with inoculum inoculation fermentation solution to starting point concentration.With Bausch and Lomb Spectronic70 spectrophotometer monitoring thalli growth situation.

Alcohol concn is measured with gas liquid chromatography.When calculating transformation efficiency, suppose all sugar, and proofread and correct the volume change that the adding owing to alkali causes all by metabolism.The maximum theoretical that is produced amount of alcohol by wood sugar and glucose it is calculated that to every gram sugar 0.51 gram ethanol, is not counted in the part that is converted into CO2.Estimate according to the highest stage of activity with the productive rate that volume ratio is represented, represent the maximum activity value.All fermentation data are two batches of mean values with top fermentation in the chart.

Table 11 has shown that PDC of zymomonas mobilis and ADH II produce the alcoholic acid influence from glucose and wood sugar respectively to the M5A1 bacterial strain.Obviously, M5A (pLOL555) is best structure bacterial strain, and it utilizes glucose and wood sugar generation alcoholic acid maximum yield all to reach per hour 2.1 grams per liters.This recon as substrate, after 30 hours, can produce about 37 grams per liter ethanol with in these two kinds of sugar any, and secondary fermentation in 49 hours finishes substantially, productive rate be 45 gram ethanol/liter.

Table 11

Generate ethanol a plasmid/gene by the M5A1 recombinant bacterial strain from glucose and wood sugar ^bAlkali ^eTime ^dEthanol gets rate theory VP ^e30 hour cell amounts

(mmol (hour) (grams per liter) (gram/gram sugar) yield (grams per liter ethanol (gram/gram

/ gram sugar) (%)/hour) (grams per liter) sugar) glucose plasmid-free 1.0 48 15 0.16 31 1.1 15 0.044pLOI560 0.8 72 44 0.46 90 1.1 15 0.018/pdcpLOI297 0.7 72 50 0.52 102 1.3 24 0.020/pdc adhBpLOI555 1.0 48 48 0.50 98 2.1 43 0.040/pdc adhB wood sugar plasmid-frees 1.9 96 14 0.16 31 0.5 7 0.044pLOI560 0.7 96 37 0.38 75 0.5 5 0.025/pdcpLOI297 0.9 96 37 0.39 76 1.0 12 0.024/pdc adhB pLOI560 1.1 48 46 0.48 94 2.0 37 0.054/pdc adhB^aAccording to initial institute sugaring total ^bThe gene of marking embrace bacterium (Z.mobilis) from the motion fermentation list ^cKeep pH6.0 institute alkali consumption during fermentation ^dThe time of maximum concentration of ethanol ^eVP, maximum volume output when fermenting in batches

With regard to ethanol produced, the growth of M5A1 (pLOI555) had clear superiority.The growth of this recon and its parent strain are much at one.Different with parent plant is that cell density reaches maximum when cultivating 15 hours, continues then to descend.Owing to can not see the cracking of thalline, this decline may reflect that the specific refractory power that causes owing to alcohol accumulation descends.Do not need to add alkali with control pH value, the recon of band pdc and adh II, owing to reduced the generation of acid product (neutral tunning ratio is higher), its cell density can reach more than the twice of parent plant, as the result who observes in the intestinal bacteria.

Under maintenance same high efficient and ethanol final concentration situation, its maximum alcohol yied (per hour 2.1 grams per liters) almost is the twice of intestinal bacteria recon.Different with intestinal bacteria is that M5A1 (pLOI555) is with same speed xylose-fermenting and glucose.Under non-resistant selection situation, plasmid pLOI555 can be in M5A1 stable existence.With regard to ethanol produced, because the substrate scope of M5A1 is with colibacillary identical, the M5A1 recon had obvious and unexpected advantage.

Embodiment 16

Utilize Erwinia and Klebsiella producing and ethanol recon fermentation cellobiose to produce ethanol

This example has been described and has been utilized erwinia and the Klebsiella producing and ethanol recon a kind of oligosaccharides (cellobiose) that ferments.The used Erwinia of this example is a typical carrot soft rot Erwinia bacterial strain (Erwinia carotevara).Transform Erwinia with the pLOI555 described in the embodiment 15.This transforms the method for pressing (1988) Ag.and Biol Chem.52 (1) 293-4 such as Ho, uses Biorad electroporation device to change pLOI555 over to Erwinia (voltage 2.0kv, electric capacity 25 μ F, resistance 100 Ω).Obtain a large amount of transformants, transformation efficiency is greater than 100,000 transformants/μ g plasmid DNA.

The used Klebsiella of this example is given birth to Klebsiella M5A1 bacterial strain P2 for urging, and this bacterial strain is obtained by method described in the embodiment 18.

Press method among the embodiment 5,, ferment under the 100rpm oscillating condition containing the Luira substratum of 10% cellobiose (pH6,30 ℃).The alcohol concn that Erwinia and Klebsiella produce is respectively 1.113molar and 1.065 molar.This experimental result is seen Fig. 6, and alcohol concn is represented with g/L.Symbol ■ represents Erwinia, and represents Klebsiella.

Embodiment 17

Utilize single genetically engineered microbial fermentation polymer raw material to generate ethanol

This example has been described with two-step approach the polymer fermenting substrate has been become ethanol, and used single microorganism can produce the interior zytase of born of the same parents and produce ethanol as the primary fermentation product through after genetically engineered.Detailed process is that thermophilic bacterium thermal fiber clostridium xylanase gene excision upstream portion and lacZ gene N-end are merged to remove secretion signal.This recombination is aforesaid intestinal bacteria and urge in childbirth Klebsiella (pLOI555) can high level expression, a large amount of zytases of intracellular accumulation during ethanol fermentation.Collect the thalline contain zytase during fermentation ends, be added in the xylan solution of comparatively high temps to discharge the zytase of its tool saccharification.After the cooling, utilize microorganism of the same race that the hydrolysate fermentation is generated ethanol, produce the required zytase of subsequent saccharification effect simultaneously.

Microorganism and growth thereof:

This routine used bacterial isolates and plasmid are listed in the table 12.

Seedling strain of table 12 bacterium and plasmid bacterium/plasmid proterties source or reference bacillus coli DH 5 alpha lacZM15, the intestinal bacteria KO11 frd of recA Bethesda research laboratory, Cm ^r, Ipet ^aOhta etc. urge childbirth Klebsiella M5A1 Cm ^r, pet ^bEmbodiment 15, the same (pLOI1555) pCT1202 Ap ^r, xynZ ⁺PLOI1001 Ap such as Grepinet ^r, xyIB ⁺PLOI2000 Ap such as Utt ^r, xyIB ⁺Present embodiment pLOI2001 Ap ^r, lacZ, xynZ ⁺Present embodiment pLOI2002 Ap ^r, lacZ::xynZ ⁺Present embodiment pLOI2003 Ap ^r, lacZ::xynZ ⁺, the Xy-present embodiment

IB ^{+ a}Ipet represents that Z.mobilis pdc and adhB gene integration are on karyomit(e). ^bPet represents that Z.mobilis pdc and adhB gene are present on the plasmid pLOI555. ^cOhta etc. (1991) Appl.Environ.Microbiol.57:893-900. ^dGerpinet etc. (1988) J.Bacteriol.170:4582-4588. ^eUtt etc. (1991) Appl.Environ.Microbiol.57:1227-1234.

Bacterial strain added 50 gram wood sugars/liter the Luria substratum in cultivate.The intestinal bacteria transformant contain 50 milligrams of penbritins/liter or 40 milligrams of paraxin/liter the Luria agar plate on screen.Urge childbirth Klebsiella M5A1 transformant contain 1000 milligrams of penbritins/liter or 40 milligrams of paraxin/liter the Luria agar plate on select.

The recon that xylanase activity is arranged with the microtitre flat board that contains 4-methyl umbrella shape glycosyl-β-D furans cellobioside (100 mg/litre) (Millet (1985) FEMS Microbiol.Lett.29:145-149) screening.Equally, express the recon of the XylB gene of xylosidase and arabinofuranosidase/xylosidase with solid medium (Utt et al (1991) Appl.EnvironMierobiol57:1227-1234) screening that contains 4-methyl umbrella shape glycosyl-α-L-furans Arabinoside (20 mg/litre).Produce the fluorescence-causing substance (Umbelliferone) that is easy to detect under a kind of 340nm UV-light behind these substrates of positive colony hydrolysis.

Genetic manipulation and recombinant technology:

Standard method is all used in plasmid preparation, digestion with restriction enzyme, connection, conversion and gel electrophoresis.In all plasmid construction processes, all use e.colistraindh5 as the host.(Coy tests Products Co., Ltd to polymerase chain reaction at TempCycler Modle50, Ann Abor, the Michigan State., Ann Arbor MI) and contain Ge-neAmp Kit (test kit) (the Perkin Elmer Cetus of Taq polymerase, Norwalk carries out in CT).Amplification reaction solution is 100 μ l, and wherein dNTP respectively contains 2mM, and every kind of primer is 100pmol, template 20mg and 2.5UTaq polymerase.Product amplification cycles 30 times (94 ℃ 1 minute, 47 ℃ of 2 minutes and 72 ℃ 1 minute; Last 72 ℃ 3 minutes) back separates.

The mensuration of enzymic activity:

Xylan, xylopyranoside enzyme and furans Arabinoside enzymic activity are measured in the recon of 30 ℃ of incubated overnight.Centrifugal (7000g, 10 minutes) collect thalline, washed twice.Measuring the used washing lotion of xylanase activity is phosphoric acid-citric acid buffer agent (50mM potassiumphosphate and 12.5mM citric acid, pH6.3), mensuration xylosidase and Arabinoside enzymic activity are then selected phosphoric acid buffer for use, and (the 5mM sodium phosphate buffer contains the 100mM 2 mercapto ethanol, pH6.8).(20,000psi) broken twice, the lysate that obtains centrifugal (13,000g, 30 minutes, 4 ℃) is to remove bacterial chip in the French pressurized vessel for thalline.

Xylosidase and furans Arabinoside enzymic activity are pressed preceding method and are measured (Utt sees before).Xylanase activity is used with quadrat method and is measured, but substrate uses nitro-β-D-cellobioside.Xylanase activity is also by measuring hydrolysis birch xylan (Sigma ChemicalCompany St, Louis, Mo) (Bergmeyer compiles (1983) Methods of emzymatic analysis Vol. II to the reducing sugar amount of Chan Shenging, 3rd edition, P151-2, Verlay Chemie Weinheim.Germany) estimates.All activity are all shown with the mmole numerical table of per minute kind generation product.(Bradford (1976) Anal.Bioehem, 72:248-254) measure by method with Bradford for protein concn.

Thin layer chromatography separates the oligosaccharides wood sugar:

With trifluoroacetic acid partial hydrolysis 0.5% birch xylan (Domer (1988) MethEnzymol 160:176-180) preparation oligomeric xylose mixed solution.10 times of vacuum concentration at room temperature then, and get 1 μ l as standard.Use the solvent of forming by acetone, ethyl acetate and acetate (2: 1: 1) in the room temperature chromatographic separation on the oligomeric xylose Whatman150A silica-gel plate (Whatman Inc., Clifton, New Jersey).After the drying, as described in Bounias (Bounoas (1980) Anal Biochem106:291-295), make the oligosaccharide colour developing with naphthodiamide reagent.

Fermentation test:

The fermented liquid inoculum prepares 30 ℃ of static overnight incubation after by fresh single colony inoculation.To make O.D.550 be 0.1 or 0.3 to the initial inoculation amount in the fermented liquid, then the constant device of pH (350ml volume, pH6.0) (Beall et al (1991) the Biotechnol Bio-engin38:296-303 of stirring; Ohta et al (1991) Appl.Environ Micnosiol 57:893-900; With Ohta et al (1991) Appl.Gwiron Mierobiol57:2810-2815) 30 ℃ of insulations, contain paraxin in the fermented liquid or contain paraxin and penbritin simultaneously.

Xylan ferments with two-step approach, and elder generation is degradation of xylan at high temperature, then 30 ℃ of fermentations.Be degradation of xylan, thalline is centrifugal (7,000g, 10 minutes) collecting cell from cultivate 48 hours 350ml fermented liquid, is suspended in equal-volume and contains in the fresh Luria nutrient solution of 4% xylan (pH6.0), 60 ℃ of insulations 65 hours.After fermented liquid was cooled to 30 ℃, making postvaccinal O.D.550 with the recombinant microorganism inoculation was 0.3.

Take a sample to detect oligomeric xylose glycosides (thin layer chromatography), thalli growth (O.D.550) and ethanol at different time.Ethanol is measured with gas liquid chromatography (Dombek et al. (1985) Appl.Envion Microbiol51:197-200).

Be used for the hydrolyzed xylan construction of recombinant plasmid:

The natural xylan of hydrolysis needs the activity of two kinds of enzymes, i.e. endo-xylanase and xylosidase at least.Make up three plasmids and be applied to the xylan fermentation, in these plasmids, come from xynZ (zytase) gene of thermophile bacteria thermal fiber clostridium (Grepinet et al (1988) J.Bacteriol.170:4582-4588) and come from Butyrivibrio fibrisolvens (Utt, see before) xylB (xylosidase and arabinofuranosidase/xylosidase) gene, or single expression or form in the operon and to express (Fig. 7 A, 7B and 7C).

Fig. 7 A, 7B and 7C have set forth the construction of recombinant plasmid that is loaded with xynZ and xylB.The coding region is gone out by frame.Thermal fiber clostridium and Butyrivibrio fibrisolvens DNA represent that with fine rule thick line is represented carrier DNA.The tack connection site is represented with " X "." F " indicates the coding region of the lacZ ∷ xynZ fusion gene that does not change reading frame.

With xyIB gene subclone in pUC18.Specific practice is, downcut band ribose binding site and xyIB aminopeptidase gene terminal 0.3kbp Xbal-Pstl fragment among the pLOI1001 and with the 2.4kbp Pstl fragment of all the other xylB gene coding regions and translation termination, be inserted in the pUC18 multiple clone site Xbal between the Pst point of contact.Lac promotor control xylB expression of gene among the plasmid pLOI2000 that obtains.

Research in the past (Grepinet et al. sees before) has shown and has lacked coding secretion signal and a big segmental xylB gene of zytase N-terminal and a lacZ gene fusion that the expression of zytase can be enhanced.Handle tack by the 2.4kbp Sty I fragment that will have the pCT1202 that cuts aminoterminal xynZ gene through Klenow and be connected to pUC18 on the Pstl site that Klenow handles, made up similar lacZ ∷ xynZ genetic fusant.Among the plasmid pLOI2001 that obtains, the reading frame of two genes matches each other.

Before the plasmid that makes up zytase and xylosidase encoding gene while coexpression, be necessary to remove the transcription terminator at 30pb place, downstream, lacZ ∷ zynZ coding region and add a new Sst I site at fusion gene 3 ' end.These modifications utilize polymerase chain reaction to carry out.The pLOI2001 plasmid is a template, 5 '-GAATTCGAGCTCGGTAC-3 ' is 5 ' end primer, 5 '-GGGAGCTCCGGCATCATTATATCTG-3 ' is 3 ' end primer (comprising a new Sst I site).After Sst I enzyme was cut, this fragment was inserted the Sst I site in the multiple clone site of pUC18 and pLOI2000 respectively, constitutes plasmid pLOI2001 and pLOI2003 respectively.

The expression of the enzyme of relevant xylan degrading:

Measure the activity (table 13) of zytase, xylosidase and the arabinofuranosidase/xylosidase of in stable growth phase bacterial strain, expressing earlier as plasmid construction host's DH5 α bacterial strain.Compare with the bacterial strain of band pLOI2001 (only carrying the xynZ gene), xylanase activity descends 60% in the bacterial strain of band pLOI2003 (also carrying the xyIB gene in the xynZ downstream).Yet in the bacterial strain that has pLO2000 (only carrying the xyIB gene) and band pLOI2003 (the XynZ gene is also carried in the xyIB upstream) respectively, xylosidase and Arabinoside enzymic activity are close.

Table 13

The specific activity bacterial strain of the recombinase of degradation of xylan and plasmid are than live (mU/mg)

My Glycosylase xylan xylanase b of xylosidase

Enzyme ^a Bacillus coli DH 5 alpha 000 0pL,OII,200 1.2 2.2 0 0pL,OI2,001 00 1.4 12,4pL,OI2,003 1.5 2.5 0.5 48 Escherichia coli KO11 000 0pL,OI2,001 00 0.4 3,8pL,OI2,003 1.3 2.4 0.3 25pLOI2003^c1.1 1.9 0.3 47pLOI2003 ^C.dNd nd 0.8 93 urges childbirth Klebsiella M5A153 000 (pLOI555) and pLOI2001 49 0 0.4 3,9pL,OI2,003 56 2.6 0.2 24pLOI2001 ^c30 0 0.7 0pLOI2001 ^{C, d}Nd nd 1.8 144pLOI2003 ^c38 2.9 0.4 58pLOI2003 ^{C, d}Nd nd 0.8 144

Except that specializing, cell is grown in the pendulum bottle of the Luria substratum that contains 5% wood sugar, collects (stationary phase) after 20 hours; The activity of xylosidase and arabinofuranosidase/xylosidase is 30 ℃ of mensuration, and xylanase activity is 45 ℃ of mensuration.

^aXylanase activity is made substrate with p-nitrophenyl-β-D-cellobioside and is measured

^bXylanase activity is made substrate with 0.5% birch glycan and is measured

^cAfter the fermentation ends from the constant device of pH (the Luria medium pH 6.0 of 8% wood sugar) collecting cell.

^dXylanase activity is 60 ℃ of mensuration

Plasmid pLOI2001 and pLOI2003 are transformed among the producing and ethanol bacterial strain E.coli KO11 of producing and ethanol gene integration to the karyomit(e).Compare the expression activity (table 13) in the thalline in the constant device of pH in shaking culture stable growth phase thalline and after fermentation ends.Xylosidase, Arabinoside and xylanase activity and observed close in DH5 α, and in the bacterial strain of band pLOI2003 (the xyIB gene is carried in the downstream), xylanase activity descends.

Measure the expression activity in the K.oxytoca M5A1 bacterial strain of being with pLOI555 (carrying producing and ethanol gene among the Z.mobilis) simultaneously.Though do not know the replicon type of pLOI555, as if under the situation that another plasmid derived from pUC18 exists, pLO555 is very stable.Its arabinofuranosidase/xylosidase and xylosidase activity and observed roughly the same in E.coli.In M5A1, find abundant endogenous xylosidase.Compare with thalline in the constant fermented liquid of pH, xylosidase activity is higher in the thalline of shaking culture, and xylanase activity changes antithesis.

Under used test condition, Arabic glycosides activity ratio xylosidase activity is high 1.5 times to 1.7 times, with coming to the same thing of institute road (Utt et al seing before).The optimum temps of measuring endogenous zytase is 60 ℃ (Gerpinet et al. sees before), and the activity that records under 47 ℃ only is half of 60 ℃.From available data, the ratio that the hybrid xylanase specific activity beguine of calculating from the reducing sugar test records according to hydrolysis p-nitrophenyl-β-D-cellobioside is lived high about 100 times, and this shows that this enzyme preferentially utilizes endogenous substrate.

Recombinant bacterial strain is at high temperature to the hydrolysis of xylan:

KO11 (pLOI2003) bacterial strain is cultivated in the constant nutrient solution of the pH that contains 8% wood sugar, measures its xylan hydrolysis enzymic activity then.Thalline is suspended in equal-volume and contains in the fresh Luria nutrient solution of 4% xylan, cultivate down at 45 ℃ or 65 ℃, but the former is the top temperature of Butyrivibrio fibrisolvens zytase stable existence that the latter is the optimum temps of thermal fiber clostridium zytase.Though zytase and xylosidase are intracellular product, initial experiment shows that they are easy to be discharged in the substratum when being incubated down for 45 ℃ and 60 ℃.At different time sampling thin layer chromatography analysis hydrolysate (Fig. 8 A and 8B).

Fig. 8 A and 8B show the thin layer chromatography analysis result that the product that obtains behind E.coli KO11 (pLOI2003) the bacterial strain hydrolyzed xylan of cultivating in the Luria substratum (pH6.0) that contains 4% birch xylan at 45 ℃ (Fig. 8 A) or 60 ℃ (Fig. 8 B) is carried out.Every point sample hole application of sample 1 μ l, each below, hole is the time (with a hour expression) of cultivating.The acidic hydrolysis product of xylan is added in first hole (S) as standard.

Can go out wood sugar, xylo-bioses, xylotriose and Xylotetrose in the tunning, wherein xylo-bioses is main tunning with identifying.The monomer wood sugar accumulates lentamente.To comparison shows that of 24 hours and 48 hours posthydrolysis degree, the hydrolysis degree in the time of 45 ℃ is not as 60 ℃, although xylosidase instability 60 ℃ the time.

Although 60 ℃ of xylan hydrolysis after 65 hours are incomplete, this moment, zytase still kept higher vigor, was easy to detect as substrate with 4-methyl umbrella shape glycosylation beta-D-cellobioside.The rapid inactivation of xylosidase under this temperature.Add the bacterial lysate that contains these two kinds of enzymes and continue insulation 24 hours at 60 ℃, thin-layer chromatography is the result show, the xylan hydrolysis situation is constant.Therefore this oligosaccharides wood sugar product is the ultimate hydrolysate of hydrolysis Sigma birch glycan, may be that oxidized form product or substitute ingredient hinder its thorough hydrolysis.

Intestinal bacteria and the utilization of urging childbirth Klebsiella recombinant bacterial strain to the xylan hydrolysis product:

Carried out bench-scale testing, with the degree of assessment E.coli KO11 (pLOI2003) bacterial strain and K.oxytoca M5A1 bacterial strain metabolism oligosaccharides wood sugar.With bacterial strain insert 1 milliliter of xylan digest (60 ℃, 65 hours; The centrifugal thalline of removing is left and taken supernatant) 30 ℃ of static cultivations 48 hours.With thin layer chromatography analysis sample (Fig. 9 A and 9B).

Fig. 9 A and 9B have shown that intestinal bacteria KO11 (Fig. 9 A) and K.oxytoca M5A1 (Fig. 9 B) recombinant bacterial strain utilize the long situation of oligosaccharides wood consor respectively.Xylan (4%) is incubated at LB (pH6.0) altogether with intestinal bacteria KO11 bacterial strain (carrying pLOI2003), and after 65 hours, hydrolyzed solution inserts bacterium then through centrifugal after-filtration degerming, 30 ℃ of cultivations.Carry out thin layer chromatography analysis (being marked in below, corresponding point sample hole each sample time) every sampling in 24 hours.In first hole (S), add a kind of oligosaccharides xylan mixture as standard control.

Although there is active Butyrivibrio fibrisolvens xylosidase to have a recombination bacillus coli metabolism wood sugar.And wood sugar, xylo-bioses and xylotriose all can be urged the childbirth Klebsiella to utilize fully by producing and ethanol.Therefore select for use and urge the childbirth Klebsiella bacterium that derives to carry out changing alcoholic acid into and further study by xylan.

Urge of the fermentation of childbirth Klebsiella M5A1 derivative strain to wood sugar and xylan:

Two-step approach is adopted in the fermentation of xylan, is suspended in the Luria substratum that contains the poly-enzyme of 4% wood after Fa Jiao thalline is collected in advance and carries out saccharification (60 ℃, 65 hours).The xylan liquid inoculation back of saccharification is in 30 ℃ of fermentations (pH6.0).Use the parallel test that compares simultaneously, the centrifugal bacterial chip of removing before the inoculation, fermentation is carried out (being equivalent to the xylose concentration in 4% xylan in 4.47% wood sugar.The data of these fermentations see Table 14.

Figure 10 A and 10B have shown and urge childbirth Klebsiella M5A1 that wood sugar and xylan are converted to the alcoholic acid situation.The thalline that obtains of fermentation is as the enzyme source in advance, 60 ℃ of following hydrolyzed xylans 65 hours.Figure 10 A shows the growth of thalline, and Figure 10 B shows alcoholic acid output.Symbol: ▲, M5A1 (pLOI555) is to the fermentation of 4.47% wood sugar; Zero, M5A1 (pLOI555 and pLI2001) is to the fermentation of 4.4% wood sugar; ●, M5A1 (pLOI555 and PlOI2001) is to the fermentation of 4% xylan digest of not removing bacterial chip after the saccharification; is centrifugal to remove behind the bacterial chip M5A1 (pLOI555 and pLOI2001) to the fermentation of 4% xylan digest.

Table 14

Recombinant bacterial strain K.oxytoca M5A1 (pLOI555)

The ethanol yield of xylose-fermenting and wood oligose ^aThe substrate and the second plasmid alkali number time ethanol get rate theory yield VP cell yield

MM/g sugar h g/l g/g substrate (%) ^d(g/l/h) ^e(g/g sugar) wood sugar (4.47%) 1.2 36 22.6 0.51 99 1.29 0.07 independent pLOI555 band pLOI2001 1.5 48 21.7 0.49 95 1.02 0.08 band pLOI2003 1.3 36 20.6 0.47 91 0.83 0.07 xylans (40%) band pLOI2001 ^f0.7 24 7.7 0.19 34 0.37 0.04 band pLOI2003 ^f1.3 36 7.7 0.19 34 0.31 0.04 band pLOI2001 ^g1.3 60 7.8 0.20 34 0.28 nd are with pLOI2003 ^g1.1 60 7.9 0.20 35 0.30 nd

^aCalculate according to the totalling substrate ^bKeep the alkali number that pH6.0 consumes in the fermentation ^cReach the maximum concentration of ethanol required time ^dTheoretical yield (calculating by weight) is 0.51 to wood sugar, is 0.56 to xylan ^eMaximum volume in the fermentation compares output in batches ^fThe centrifugal hydrolyzed solution that removes the deglycation fragment ^gThe hydrolyzed solution that comprises the saccharification fragment.

Can see that M5A1 (pLOI555) generates ethanol expeditiously.Wood-sugar fermentation in 24 hours is complete substantially, can obtain the output into theoretical maximum 93%.When pLOI2001 or pLOI2003 existed simultaneously, growth and the ethanol production of this bacterial strain in wood sugar all can descend.This bacterial strain is after carrying pLOI2001, and required fermentation time extends to about 36 hours, and ethanol production is 91% of a theoretical value.This decline by monose generation alcohol yied has reflected that the expression exogenous enzyme is an added burden to recombinant bacterial strain.

With carrying plasmid pLOI2001 (the poly-enzyme (60 ℃, 65 hours) of M5A1 (pLOI555) hydrolysis wood that contains this glycoside enzyme gene and recombined xylanase gene.The thin-layer chromatography result of its hydrolysate shows that hydrolysis solution composition is identical with KO11 (pLOI2003) (seeing Fig. 9 A and 9B).These two M5A1 deutero-bacterium are identical to the degree of xylan hydrolysis.

Behind separately the microorganism, measure the growth and the alcoholic acid output (Figure 10 A and 10B) of thalline when inserting saccharification in the hydrolysate.In the clarification hydrolyzed solution, increment only is half in the peer-level wood sugar, and its fermenting speed slightly hurry up, and secondary fermentation in 24 hours is complete substantially.Producing the alcoholic acid yield by xylan and be about 1/3 of theoretical maximum, is 7.7 to 7.9g/l.

Oligomeric xylose in the fermented liquid is monitored with thin-layer chromatography, the situation of each composition identical with shown in Fig. 9 B (48 hours) during fermentation ends, and wherein wood sugar, xylo-bioses and the xylotriose metabolism that is done, Xylotetrose and longer oligosaccharides body still exist.

Discuss

This shows that the K.oxytoca M5A1 bacterial strain through highly transforming not only can be used as that the enzyme source is used for polymeric hydrolysis but also the xylan that can be used for fermenting generates ethanol.Existence and the transportation and the metabolism oligosaccharides wood sugar ability of xylosidase in the endogenous born of the same parents of this thalline were never reported in the past.It is reported that wild-type e. coli has phosphoric acid cellobiase (Hall et al. (1987) J.Bacteriol 169:2713-2717 in cellobiose phosphotransferase system and a kind of born of the same parents; Kricker et al. (1987) Genetics 115 419-429).May there be similar system in K.oxytoca M5A1 in the process of metabolism xylo-bioses and xylotriose.Movement system that exists among the K.oxytocaM5A1 and metabolism monomer, dimer and tripolymer xylosidase make it to have great advantage than intestinal bacteria or cereuisiae fermentum system aspect the required biomass further utilizing ethanol to produce.

The problem that is occurred when making it to degrade polymer and producing ethanol with the single microorganism of genetic engineering means transformation has in the past obtained solution in the two-step approach fermenting process of heat stable protein in using cell.High level synthesis secretion albumen usually can influence the normal cell physiological process, and excision N end secretion signal makes enzyme at cell inner expression, like this, can alleviate this problem.Because hydrolysis time is a limiting factor in conversion, high-caliber lytic enzyme will be beneficial in switching process and provide sugar with regard to maximum ground for quick fermentation at first most.In needing the process of enzyme secretion, the enzyme water gaging is flat very low when initial, and during near fermentation ends, accumulation reaches the highest.If but with the thalline that ferments in advance as the enzyme source, it is flat to reach the maximum enzyme water gaging at the very start.Use the thermophilic enzyme hydrolysis and reduce pollution and improve the hydrolysis rate except that helping, largest benefit is and can discharges intracellular enzyme easily from collect thalline.

Alcohol patience is individual often problem in the natural microbial of degraded cellulose and xylan.The ethanol of these microorganisms generally is less than 20 grams per liters (seeing Taillez et al (1989) Appl Environ.Microbiol.55:203-06).Although it is also not high to generate the alcoholic acid level by xylan, M5A1 (pLOI555) can produce 48 grams per liter ethanol (be about theoretical maximum yield 95%) from 100 grams per liter wood sugars at least.

It is lower, mainly incomplete owing to xylan hydrolysis than expection to produce the alcoholic acid total amount from birch xylan.After the saccharification 65 hours, undegradable oligomeric xylose is arranged seemingly.Though the zytase level that our recombinant chou is expressed is than Grepinet et al., it is low that (seing before) described best zytase merges the recombinant chou expression levels, and it still has enough expression levels.With 105 μ moles/ minutes hydrolysis rate, (40 grams per liters of xylan after 24 hours; The dehydration wood sugar of about 0.3moles) should be degraded to the poly-disaccharides of wood fully.The about 4.5g cell protein of fermentation gained thalline/liter, the xylan specific activity is 144 a μ moles/ minutes/gram cell protein, gross activity is 648umoles/ minute.Although zytase still has activity after 65 hours, still have undegradable oligomeric xylose to exist, adding new enzyme continuation insulation after 24 hours, the oligomeric xylose level is constant.Hydrolysis not exclusively may be owing to the competitive inhibition of oligomeric xylose to hydrolysis.In 45 ℃ of hydrolysis reaction, detect this competitive inhibition situation, under this temperature among zytase and the M5A1 enzyme of endogenous metabolism wood sugar, the poly-disaccharides of wood and the poly-trisaccharide of wood all keep active.Oligosaccharides composition after oligosaccharides composition under this condition and the M5A1 fermentation is identical all to also have the poly-tetrose of undegradable wood and the longer oligosaccharides of same level to have (data are unlisted).These oligosaccharides be it seems limiting hydrolysis, but the essence of the gene of limiting hydrolysis still belongs to the unknown.According to the index of manufacturers, its birch glycan contains 99% wood sugar.Investigator of the present invention infers that this product contains the oxidisability residue that generation still exists in storage and replacement process, (Chesson etc. (1983) J.Sci.Food.Agric34:1330-1340) after alkaline extraction and purification process.

Be about annual 1000000000 gallons (3,800,000,000 liters) Science251:1318-1323 such as () Lynd in U.S.'s ethanol fermentation output.Many bacteriums as giving birth to Klebsiella, intestinal bacteria, cereuisiae fermentum or zymomonas mobilis etc. through urging of genetic modification, all can be used for producing a series of zymoproteins, as the common property thing of ethanol fermentation.Calculate with the proteic minimum cell yield of every liter of beer 2 grams,, just can obtain at least 3,800,000 kilogram zymoprotein common property thing as long as 5% cell protein acquisition conversion is arranged.These enzymes are not limited in the enzyme of catalytic substrate depolymerization for fermentation usefulness, also can comprise other enzymes that some are in great demand, as be applied to stain remover industry, foodstuffs industry, pulping wood industry or produce the various enzymes of the industries such as infant industry of pharmaceutical chemicals with biological catalyst.If these markets can be developed, then the using value of these enzymes just can be worth considerably beyond being used to produce alcoholic acid at present.Embodiment 18 urges childbirth Klebsiella (Klebsiella oxytoca) by reorganization, it carries zymomonas mobilis (Z.mobilis) producing and ethanol gene that is incorporated on the karyomit(e) and the plasmid of expressing thermal fiber clostridium (Clostridium thermoeellum) heat-stable cellulase gene, and fermentation cellobiose, amorphous cellulose and crystalline cellulose are produced ethanol

In this experiment, zymomonas mobilis (Z.mobilis) producing and ethanol gene is integrated into and urges on childbirth Klebsiella (Klebsiella pneumonia) the M5A1 strain chromosome.Bacterial strain P2 is wherein best construct, and it is except also producing ethanol effectively from cellobiose from monomer sugar.This bacterial strain has been removed the needs to the external source beta-glucosidase to the utilization of cellobiose and procellose, has reduced the consumption of the plain enzyme of the required commercial fibre of fermentation SOLKA FLOC SW40 (being mainly crystalline cellulose).The plasmid that adds the gene that has coding thermal fiber clostridium (Clostridium thermocellum) endoglucanase, causing during the fermentation, the interior thermally-stabilised enzyme of cell accumulates as ethanol common property thing.Best construct P2 (pCT603T) comprises the celD gene, and it is used to amorphous cellulose is hydrolyzed into cellobiose, produces ethanol in the two stage fermentation process.Also P2 (pCT603T) is tested with the plain enzyme combined utilization of commercial fibre.With endoglucanase D SOLKAFLOC SW40 is carried out pre-treatment at 60 ℃, can reduce the required plain enzyme dosage of commercial fibre of fermentation SOLKA FLOC SW40, amplitude can reach 80%.The promoter action that the endoglucanase pre-treatment produces may be because due to the hydrolysis of amorphous domain, exposes more site thus and is subjected to the effect of fungal cellulase.As the component functionating of a complex body, therefore this enzyme and fungal enzyme form complex body to endoglucanase D probably in thermal fiber clostridium, perhaps combine with Mierocrystalline cellulose and produce more open architecture, are hydrolyzed.

Embodiment 17 illustrated urging childbirth Klebsiella M5A1, as the alcoholic acid common product, and the superiority of heat accumulation fiber clostridium zytase.In this example, the present inventor advances to urge among the childbirth Klebsiella M5A1 with the producing and ethanol gene integration, and the bacterium that explanation produces can change into ethanol with cellobiose effectively.This bacterium be it seems and can transport and metabolism cellobiose and procellose, removes the needs to the allos beta-glucosidase from.In this intasome, add plasmid, make to produce thermally-stabilised endoglucanase, thereby in the cellulose fermentation process, reduce needs the plain enzyme of commercial fibre with ethanol.

Bacterial classification and growth conditions: table 15 has been listed novel bacterial and plasmid that this institute uses.The bacterial classification length that has the producing and ethanol gene is containing on the Luria agar plate of 2% glucose (3); Other bacterial strain length are on the Luria of not sugaring agar plate.Except that specially illustrating, use paraxin (50 μ g/ml), tsiklomitsin (6 μ g/ml), and penbritin (50 μ g/ml) during selection.Routinely, intestinal bacteria (E.coli) urge the childbirth Klebsiella 30 ℃ of cultivations 37 ℃ of cultivations.The speed that the expression of zymomonas mobilis producing and ethanol operon indicates dull and stereotyped color to occur according to acetaldehyde detects (Conway etc. [1987] J.Bacteriol.169:2591-2597).The bacterium colony of screening tool cellulase activity is containing on the Luria agar plate of carboxymethyl cellulose earlier, cultivates 1 to 2 hour, and uses congo red staining (Wood etc. [1988] Meth.Enzymol.160:59-74) then for 55 ℃.

This research of table 15 bacterial strain uses therefor and plasmid bacterial isolates/plasmid characteristic source or reference bacterial strain are urged childbirth Klebsiella M5A1 Cm ^r, pet ^bEmbodiment 15 (pLOI555) S1 Cm ^r, Ipet ^aPresent embodiment S2 Cm ^r, Ipet ^aPresent embodiment S3 Cm ^r, Ipet ^aPresent embodiment P1 Cm ^r, Ipet ^aPresent embodiment P1 Cm ^r, Ipet ^aPresent embodiment P2 Cm ^r, Ipet ^aPresent embodiment B1 Cm ^r, Ipet ^aPresent embodiment plasmid pCOS2EMBL Tc ^rPoustka et al. ^cPLOI510 Cm ^rPet ^bOhta et al. ^dPCT105 Ap ^rCelA ⁺Cornet et al. ^ePCT207 Ap ^rCelB ⁺Jeffries ^fPCT301 Ap ^rTc ^rCelC ⁺Petre et al. ^gPCT603 Ap ^rTc ^rCelD ⁺Joliff et al. ^hPCT105T Ap ^rTc ^rCelA ⁺Present embodiment pCT207T Ap ^rTc ^rCelB ⁺Present embodiment pCT603T Ap ^rTc ^rCelD ⁺Present embodiment ^aIpet represents that Z.mobilis pdc and adhB gene integration are on karyomit(e). ^bPet represents that Z.mobilis pdc and adhB gene are on plasmid pLOI555. ^cPoustka etc. (1984) Proc.Natl.Acad.Sci.USA81:4129-4133. ^dOhta etc. (1991) APPl.Environ.Microbiol.57:2810-2815. ^eCornet etc. (1983) Bio/Technology1:589-594. ^fJeffries; Be stated from volumes such as J.F.Kennedy " timber and Mierocrystalline cellulose research: industrial utilization, biotechnology, structure and character " John Wiley ﹠amp; Sons, New York (1988). ^gPetre etc. (1986) Biochimie68:687-695. ^hJoliff etc. (1986) Bio/Technology4:896-900.

Genetics technology and plasmid construction: operate according to standard method.With the cyclized DNA transformed bacteria somatocyte that lacks all copy functions, thereby the pet gene integration is entered bacterial strain M5A1, wherein used cyclized DNA comprises three basal components: the 1) DNA of colibacillary pfl gene or M5A1; 2) the pet gene of Z.mobilis; 3) be used to the cat gene selected.Recon is selected with the substratum of 20 μ g/ml paraxin earlier, and expresses low-level Z.mobilis zymoprotein.(Ohta etc. [1991] Ap-pl.Environ.Microbiol.57:893-900) directly selects the resistance variety with the paraxin of 600 μ g/ml in colibacillary transformant, and its expression of exogenous gene greatly strengthens.Keep single bacterium colony that each independently integrates a high-level resistance of performance.

Tetracyclin resistance is added to respectively in the plasmid that contains celA gene (pCT105), celB gene (pCT207) and celD gene (pCT603).After plasmid pCT105 and pCT207 cut with BamH I enzyme, handle to obtain blunt end with the Klenow fragment of e. coli dna polymerase.Plasmid pBR322 forms blunt end to use with quadrat method after the EcoRI enzyme is cut.These three kinds of plasmids are cut with Sal I enzyme again, and the pBR322 that obtains is contained tet gene 5 ^r-terminal small segment is connected on the big fragment that is obtained by pCT105 and pCT207, is formed with the tet gene (earlier because of inserting thermal fiber clostridium DNA inactivation) of function, and the plasmid of structure is called pCT105T and pCT207T.With the 2.5kb EcoR I endonuclease bamhi that contains complete tet gene of plasmid pCOS2EMBL (Poustka etc. see before), be inserted into and form plasmid pCT603T on the EcoR I site unique among the plasmid pCT603.Plasmid pCT301 has cel C gene and function tet gene simultaneously, thereby need not to transform.The plasmid of all cellulase genes changes over to urges childbirth Klebsiella recon, all selects with tetracyclin resistance.

Cellulase activity: containing the inscribe dextran activity of bacteria culture fluid and aseptic culture fluid, is substrate with p-nitrophenyl-β-D-cellobioside, in 60 ℃ of mensuration (Petre etc. see before).The activity of endoglucanase D is also calculated according to the amount that is produced reducing sugar by amorphous cellulose.Amorphous cellulose (acid rise and alkali rise) prepares from SOLKA FLOC SW40 according to former method (Wood[1988] Meth.Enzymol.160:19-25).Reducing sugar is measured (Bergmeyer etc. [1983] pp.151-152 according to the Nelson-Somogyi method, be stated from Bergmeyer H.U. (volume) Methods of enzymatic analysis (enzymatic analysis method) Vol II .3rd edition (the second volume third edition), Verlag Chemie, Weinheim, the initial concentration of Germany. amorphous cellulose is measured (Wood[1988] .Meth Enzymol.160:87-116) as total reducing sugar according to phenol-sulfuric acid method.

Containing the ethanol enzyme is that the following method of relatively complying with of endoglucanase activity is carried out between gene and the recon of expressing the cel gene activity: in 18mm * 150mm culture test tube, put into about 15 milliliters usefulness 10% low viscosity carboxymethyl cellulose (Sigma Chemi-cal Company earlier, St.Louis, Mo.) solidified LB substratum repaves nutrient solution stationary phase with 2ml.After 48 hours, determine the liquefaction degree 55 ℃ of cultivations by the reversing test tube.

The cellulose hydrolysis product is according to the method for aforementioned mensuration xyloside and xylan hydrolysis thing, with thin layer chromatography analysis (Domer etc. [1988] Meth.Enzymol.160:355-362).Glucose, cellobiose and procellose can separate with oligosaccharides.Make standard with cellobiose and glucose.

Fermenting experiment: fermentation is carried out in the 500ml Fleaker of the constant device of band pH, and fermentation volume 350ml is substantially according to former described carrying out (Beall etc. see before).Test under 30 ℃, pH6.0,100rpm speed conditions with the Luria substratum that contains 10% glucose or 10% cellobiose.The ferment-seeded culture is grown in the 250ml flask that contains 50 ml Luria substratum (containing 4% glucose), 30 ℃ of static overnight incubation.After the mixing, survey cell concentration under 550nm, volume required to calculate, making starter bacteria concentration is 0.32mg bacterium dry weight/ml (O.D.550nm=1.0).The thalline of centrifugal this part volume of collection is with a small amount of nutrient solution suspension beginning fermentation in the constant incubator of each pH.

Sugar soln is sterilized by filtering separately.Cellulose fermentation comprises 50 grams per liter SOL-KA FLOC SW40, and (available from James River Corporation, Saddle Brook NJ), carries out at 35 ℃.Mierocrystalline cellulose is with the dry powder form autoclaving.For investigating the effectiveness of commodity enzyme in cellulose fermentation, CYTOLASE or MULTI-PECT when inoculation, have been added in addition.

Thalline number (O.D.550nm) and amount of alcohol (gas phase liquid chromatography (LC) wherein measured in the sampling back; According to Beall etc., see before).Alcohol concn is represented with grams per liter.Because adding volume so the ethanol yield that alkali increased fermentation in the fermenting process will do to proofread and correct and calculate with reference to total reducing sugar or cellulosic initial concentration.Saccharide residue need not to proofread and correct.Ethanol theoretical maximum yield through glycolysis-and fermentation is 0.51 gram/gram glucose, 0.536 gram/gram cellobiose, 0.56 gram/gram Mierocrystalline cellulose.Except that particularly pointing out, the result is twice or the mean value of more times fermentation.

The plain enzyme of commercial fibre: detected the plain enzymes of two kinds of commercial fibres, promptly CYTOLASEM104 and MULTIFECT CL (Genencor International, RollingMeadows, IL).Two kinds of enzymes all provide with solution state, are estimated as the Trichoderma longibranchiatum mutation nutrient solution of hanging oneself and suitably revising.By specification, two kinds of enzymes are all the mixture that comprises polygalacturonase, cellulase, hemicellulase.These zymins are used for food-processing through transformation at present.

Zymomonas mobilis (Z.mobilis) pet gene integration is to M5A1 karyomit(e): have three kinds of approach can with the pet gene integration in the karyomit(e) of M5A1 (Orsdov[1984] P461-465, be stated from N.R.Kreig and J.G.Holt and compile " the uncle Jie Shi handbook first roll.Owing to urge the intestinal bacteria and the affinity of childbirth Klebsiella very near, can be with colibacillary pfl gene as the potential homologous dna, with promote the similar regrouping process that is used for colibacillary method (Ohta etc., Appl.Environ.Microbiol.57:893-900).From plasmid pLOI510, isolate and carry the pet gene that is arranged in intestinal bacteria (E.coli) pfl gene and the 8.6kb Sal I endonuclease bamhi of cat gene.This fragment (do not contain and duplicate relevant gene) after connecting cyclisation, the recon that changes M5Al over to and directly select to obtain to integrate.The PstI endonuclease bamhi that also can separate a 5kb who only comprises the shorter sequence in intestinal bacteria pfl gene both sides is used with quadrat method and is integrated.The third method is will urge the homologous dna (the long Sau3A of 2kb is endonuclease bamhi at random) of giving birth to Klebsiella M5A1 to be connected to the long BamH I endonuclease bamhi that only contains pet gene and cat gene (non-e. coli dna) of a 4.6kb to be used for the bacterium conversion.In three kinds of methods, all use the LB solid plate that contains 2% glucose and 20 μ g/ml paraxin to select the bacterial strain of having integrated.The fragment of cutting through Sal I enzyme obtains 3 and integrates bacterial strains, and the Pst fragment is handled obtains 2 and integrate bacterial strains, and the segmental integration bacterial strain of BamH I is only chosen one.

After the incubated overnight, bacterium stationary phase of getting 0.1ml is coated with on the solid plate that contains 600 μ g/ml paraxin and 2% glucose, to select the bacterial strain of high expression level in liquid medium.Reservation is independently integrated the bigger single bacterium colony of form from each, and used restriction endonuclease is numbered when making up, i.e. S1, S2, S3, P1, P2 and B1.The conversion test of these single bacterium colonies being carried out preparing in a small amount again DNA to be determining existing of plasmid, and carries out the pet genetic expression experiment (table 16) on the acetaldehyde indicator flat board.Extract reorganization bacterium colony DNA,, then discard the pseudoconformity bacterial strain that this band carries the plasmid of cat gene if after enzyme is cut, confirmed to exist pLOI510 (estimating has a small amount of pollution in the gel electrophoresis purifying fragment).Starting strain and M5Al (pLOI555) (latter is an ideal producing and ethanol bacterium) in experiment respectively as negative, positive control strain.The producing and ethanol gene activity that bacterial strain B1 and P2 the express M5A1 (pLOI 555) that is on close level.

Table 16

The M5A1 bacterial strain of pet gene that contains integration is 10%

The fermentation reaction bacterial strain of (48 hours) in the glucose environment ^aThe alkali biomass ^bThe dull and stereotyped reaction of amount of alcohol acetaldehyde ^cPlasmid ^d

(mM／L)?(g／L) (g／L)M5A1 108 3．2 15 - -M5A1 91 5．1 45 ++++ +(pLO1555)S1 120 2．9 37 + -S2 171 3．2 44 ++ -S3 97 2．4 37 ++ -P1 120 2．0 38 ++ -P2 91 3．7 44 +++ -B1 63 4．0 47 ++++ -

^aThe data that provide mostly are the result that once experiment obtains.Wherein some data process in follow-on test repeats and average treatment.

^bBiomass is according to maximum value calculation (the about 0.32 grams per liter/O.D. unit of dry weight at O.D.550nm place.

^cThe relative determination value that the speed of relative movement that forms with color on the acetaldehyde indicator flat board in 30 ℃ is expressed as the pet operon.

^dOn sepharose, measure a small amount of preparation of DNA, and detect it is transferred to antimicrobial resistance bacillus coli DH 5 alpha in conversion process ability

Integrate the comparison of bacterial strain and M5A1 (pLOI555) bacterial strain glucose fermentation: cultivate after 48 hours, the bacterial strain that has four strains to integrate the pet gene produces and the same high-caliber ethanol of M5A1 (pLOI555) (seeing Table 16).Wherein two strain bacterium long the acid common property thing of generation is minimum to maximum density, the bacterium of this two strain called after P2 and B1 is selected to come out to do further investigation.

The glucose fermentation of bacterial strain P2 and B1 and cellobiose fermentation: checked the ferment ability (seeing Table 17) of 10% glucose and 10% cellobiose of bacterial strain P2 and B1.(see Table 17A) in the glucose fermentation process, this two strain is integrated bacterial strain and can both be produced than the high 3 times amount of alcohol of bacterium M5A1 of setting out, but aspect alcoholic acid yield and the volume ratio output all a little less than bacterial strain M5A1 (PLOI555) (containing the pet gene that plasmid carries).Unexpectedly, the integration of pet gene and high level expression among the bacterial strain B1 but are accompanied by the forfeiture of the ability of fermentation cellobiose.And bacterial strain P2 well-grown and generate ethanol (seeing Figure 11 B) when cellobiose exists, its yield reaches 96% of theoretical maximum.

Figure 11 A and Figure 11 B have illustrated the recombinant bacterial strain generation alcoholic acid situation that each urges childbirth Klebsiella M5A1.Figure 11 A has shown the amount of alcohol that is obtained by glucose (100 grams per liter).Marginal data: ●, bacterial strain M5A1 (pLOI555); ▲, integrated the P2 bacterial strain of pet gene; ■ has integrated the B1 bacterial strain of pet gene; Zero, contrast bacterium M5A1.Figure 11 B has shown that the P2 bacterial strain produces through the ethanol of cellobiose fermentation.Marginal data: ▲, amount of alcohol; △, cell concentration.

Table 17 urge childbirth Klebsiella M5A1 recombinant bacterial strain and

The ethanol production of intestinal bacteria KO11

Bacterial strain a	Substrate	Additive b	Volume ratio output c(grams per liter/hour)	The amount of alcohol maximum value e(grams per liter)	Yield d(gram/gram S)	The theoretical yield percentage ratio of % c
							P2	?5％SOLKA ?FLOC?SW40	5.0％ ?CYTOLA SE	0.52	16.9	0.36	64
P2	?5％sOLKA ?FLOC?SW40	10％ ?CYTOLA SE	0.50	16.3	0.35	62
							P2	?5％SOLKA ?FLoC?SW40	0.1％ ?MULTIF ECT	0.04	3.3	0.07	12
P2	?5％SOLKA ?FLOC?SW40	0.5％ ?Mutifect	0.18	5.9	0.13	23
							P2	?5％sOLKA ?FLOC?SW40	1.0％ ?MULTIF ECT	0.36	10.4	0.23	41
P2	?5％SOLKA ?FLOC?SW40	5.0％ ?MULTIF ECT	0.76	23.8	0.51	92
							P2	?5％SOLKA ?FLOC?SW40	10％ ?MULTIF ECT	0.86	32.5	0.69	123
KO11	?5％SOLKA ?FLOC?SW40	0.1％ ?CYTOLA SE	0.05	1.8	0.04	7
							KO11	?5％SOLKA ?FLOC?SW40	0.5％ ?CYTOLA SE	0.08	5.1	0.11	19
KO11	?5％SOLKA ?FLOC?SW40	1.0％ ?CYTOLA SE	0.13	6.2	0.13	23
							KO11	?5％SOLKA ?FLOC?SW40	5.0％ ?CYTOLA SE	0.51	14.7	0.31	56
KO11	?5％soLKA ?FLOC?SW40	10％ ?CYTOLA SE	0.32	8.0	0.17	30
							KO11	?5％sOLKA ?FLOC?SW40	0.1％ ?MULTIF ECT	0.06	2.2	0.05	8

Bacterial strain a	Substrate	Additive b	Volume ratio output c(grams per liter/hour)	The amount of alcohol maximum value e(grams per liter)	Yield d(gram/gram S)	The theoretical yield percentage ratio of % '
							KO11	?5％SOLKA ?FLOC?SW40	0.5％ ?MULTIF ECT	0.08	2.7	0.06	10
KO11	?5％SOLKA ?FLOC?SW40	1.0％ ?MULTIF ECT	0.18	5.6	0.12	21
							KO11	?5％SOLKA ?FLoCSW40	5.0％ ?MULTIF ECT	0.43	20.6	0.43	76
KO11	?5％SOLKA ?FLOC?SW40	10％ ?MULTIF ECT	0.84	30.7	0.63	112

^aThe fermentation of glucose and cellobiose is carried out at 30 ℃; Other fermenting process carries out at 35 ℃;

^bEnzyme adds in inoculation.

^cBetween 6 to 24 hours, calculate.

^dThe alkali lye that is used to dilute in the fermentation is proofreaied and correct.S (substrate) represents glucose, cellobiose or SOLKA FLOC SW40.

^eDuring calculating be: 51 gram ethanol/100 gram glucose, 53.5 gram ethanol/100 gram cellobioses, 28 gram ethanol/50 gram Mierocrystalline celluloses with the theoretical maximum yield.The substrate residue not being carried out data proofreaies and correct.

^fThe cell that SOLKA FLOC SW40 collects via pre-fermentation was 60 ℃ of pre-treatment 12 hours.Pre-fermentation can obtain thermal fiber clostridium celD product.Be cooled to after 35 ℃ the plain enzyme of commercial fibres that inserts bacterium liquid and add 1% volume ratio at nutrient solution and begin fermentation.

In the presence of the plain enzyme of the commercial fibre of different concns, urge the comparison to crystal form Mierocrystalline cellulose (SOLKA FLOC SW 40) fermentation of childbirth Klebsiella M5A1 recombinant bacterial strain and intestinal bacteria: the intestinal bacteria that contain the producing and ethanol gene can only utilize glucose.Urge the recon P2 of childbirth Klebsiella different therewith, its cellobiose that can also ferment does not need the external source beta-glucosidase.Utilize cellobiose to from improving degree with commercial enzyme by the output of cellulose raw producing and ethanol for mensuration, fermentation is a substrate with 50 gram SOLKA FLOC SW40, carries out (seeing Figure 12 and table 17) in CYTOLASE that contains 0-10% or MULTIFECT solution.These two kinds of zymins all contain the fungal enzyme mixture that a cover comprises endoglucanase, exoglucanase and beta-glucosidase.

Figure 12 shows that fermentation added the influence of the ethanol yield that the commercial fibre element transforms by 50 gram SOLKA FLOC SW40 in every liter of fermented liquid enzyme after 120 hours.Dotted line is only represented the ethanol maximum production by the cellulose conversion of adding.It mainly is owing to added MULTIFECT CL preparation after the suitable improvement as extra substrate that the ethanol of higher level generates.Marginal data: ▲, the fermentation of urging childbirth Klebsiella and MULTIFECT; ■, the fermentation of intestinal bacteria KO11 bacterial strain MULTIPECT; △, the fermentation of urging childbirth Klebsiella and CYTO-LASE; , the fermentation of intestinal bacteria KO11 bacterial strain and CYTOLASE.

When lower concentration, the effect of CYTOLASE is like more effective, but the preparation of high density has certain toxic action to bacterium.In all experiments, the character of urging the childbirth Klebsiella to integrate strains expressed is good than intestinal bacteria KO11 bacterial strain, although there is the plain zymin of the commercial fibre of beta-glucosidase to exist.Comprehensive The above results illustrates beta-glucosidase in the commercial cellulase preparation for other cellulase activity, and its activity does not reach to state of saturation.

When the MULTIFECT of peak concentration exists, observe the ethanol yield that obtains by Mierocrystalline cellulose (50g/L) and surpassed theoretical maximum.For studying this part extra alcoholic acid metabolism source, fermentative action the LB of not sugaring or enzyme nutrient solution and add 10%CYTO-LASE or the LB nutrient solution of 10%MULTIFECT but not sugaring in carry out respectively.All producing 1 grams per liter ethanol under the LB culture condition separately, and the amount of alcohol that produces is about 5 grams per liters under the culture condition that adds 10% cellulase.Thereby in containing the fermentative action of 10%MULTIFECT, there is the amount of about 4 grams per liters to derive from utilization in the ethanol of generation to commercial additive or enzyme stabilizers.Also may produce a certain proportion of ethanol by low concentration cellulose enzyme.The ethanol that deduction may be changed into by LB component and 10%MULTIFECT, the ethanol yield of cellulose fermentation is about 94% of theoretical maximum.

The expression of the clostridial cellulose gene of thermal fiber in the P2 bacterial strain: cellobiose can be utilized by bacterial strain P2 section, also is the primary product of the catalytic cellulase hydrolysis reaction of endoglucanase and exoglucanase.Though used bacterial strain all can not synthesize this two kinds of enzymes among the present invention.But after the pet gene integration is to the karyomit(e), help introducing the plasmid of the heterologous gene that comprises encoding such enzymes, thus at fermenting process with synthetic these the two kinds of enzymes of common property thing form.After having carried out the tetracyclin resistance selection, the expression to the clostridial four kinds of endoglucanase of thermophile bacteria thermal fiber detects again.The expression level of celD gene in intestinal bacteria is very high, and be also stronger in the expression of P2 bacterial strain.Somatic cells has kept most of endoglucanase vigor that is produced by these genes, and not getting rid of although known Ke Shi bacterial isolates has the protein secreting system has the part enzyme secretion outside born of the same parents.

The qualitative comparison of CMC Joule-Thomson effect is carried out in the tube culture in 55 ℃ of growths 48 hours, its result is with active to detect fixed result consistent, promptly effective with the recombinant bacterial strain of celD gene (pCT603T), secondly be the recombinant bacterial strain of band cel C gene (PCT301).The P2 strain growth situation of band cel B gene (pCT207T) is very poor, does not do further test.Cel A (pCT105T) recon has only slight Joule-Thomson effect.

The synthetic of heterologous gene products also done research to the influence of fermentative action.After having obtained to express the plasmid of any cel gene, thalline is final cell density loss 10-30% in 10% glucose fermentation, and ethanol production is decline (seeing Table 18) to some extent also.The infringement of celD expression of gene pair cell is minimum, and this moment, productive rate was every gram glucose 0.32 gram ethanol, was 62% of theoretical maximum.Being determined in 60 ℃ with right-oil of mirbane-β-D-cellobioside is that substrate carries out, and unit is every milliliter of micromole's number that contains bacteria culture fluid or generate hydrolyzate in the nutrient solution of bactofugation in the per minute.

Table 18

Thermal fiber clostridium endo glucanase gene

At P ₂Expressing gene plasmid activity (units per ml) in the bacterial strain

Thalline+no 0.2celA pCT105T 2.3 0.4 83celB pCT207T 17 0.8 95ce1C pCT301 41 6.2 85celD the pCT603T 49 13 73 of nutrient solution nutrient solution relevant cell per-cent contrast

The P2 bacterial strain of expressing the celD gene utilizes the hydrolysis and the fermentation of amorphous cellulose: the product of celD gene can not hydrolysis crystallization shape Mierocrystalline cellulose, but known this enzyme can hydrolysis amorphous cellulose (Beguin etc., Joliff etc., aforementioned).Test the bacterial strain P2 (pCT603T) that expresses this gene and transformed amorphous cellulose generation alcoholic acid ability.Ferment in batches by two-step approach, detected through phosphoric acid or alkali lye (sodium hydroxide) the SOLKA FLOCSW40 that bubble handles that rises and generated the alcoholic acid situation, glucose fermentation in advance in this experiment obtains P2 (pCT603T) thalline, so that endoglucanase D to be provided, produce ethanol with this thalline fermentation again.

The first step of reaction is bacterium to be contained rise Mierocrystalline cellulose or 32mg/ml alkali of 76mg/ml acid at 3ml rise in the cellulosic LB nutrient solution, cultivates 72 hours at 60 ℃ (pH6.0), and used bacterium is collected behind isopyknic glucose fermentation earlier and obtains.Be heated to 60 ℃, make cell inactivation, discharge endoglucanase D, also provide approximate optimum temperuture simultaneously for the enzyme effect.Detect the burst size of reducing sugar in the hydrolytic process.Though concentration of substrate is different the same, the acid cellulosic hydrolysis of rising is the alkali cellulosic twice that rises, and produces the normal cellobiose of the normal 60 μ molor of 240 μ molar respectively after cultivating 24 hours.Mierocrystalline cellulose is the ten minutes thickness after alkali rises, and makes difficulty of sampling ratio.Alkali was risen by cellulase hydrolysis after 72 hours, can produce the normal cellobiose of 110 μ molar.The above results shows that all its hydrolysis is all compared fully through the Mierocrystalline cellulose that acid is risen or alkali rises and handles.

Figure 13 A, B, C are the thin layer chromatography analysis result of Mierocrystalline cellulose after bacterial strain P2 (pCT603T) hydrolysis and fermentation utilization.To acid rise Mierocrystalline cellulose (Figure 13 A), alkali rise Mierocrystalline cellulose (Figure 13 B) and crystallization shape Mierocrystalline cellulose (Figure 13 C; SOLKA FLOC SW40) all checks.Article one road among every figure is the standard model of cellobiose and glucose.Among Figure 13 A and the B, be respectively the sample that different time is got in the hydrolysis amorphous cellulose process (being respectively 0,6,12,24,48 and 72 hour) by second to the 6th road; Article seven, the road is that the 6th the good sample cultivation liquid in road and bacterial strain P2 (pCT603T) are at the sample that obtain of 30 ℃ of fermentations after 24 hours.

Figure 13 C is the thin-layer chromatography result of crystallization shape Mierocrystalline cellulose (SOLKA FLOC SW40) after PZ (pCT603T) the bacterium co-fermentation of 1%MULTI-FECT and expression thermal fiber clostridium celD gene activity.Every the road sample is respectively: 2, and the SOLKA FLOC suspension of newly joining before the enzymolysis; 3, through 24 hours nutrient solution of 60 ℃ of enzymolysis of endoglucanase D; Enzyme is by providing through pre-fermented P2 (pCT603T); Article 4, the three, the road sample is again through P2 (pCT603T) bacterium fermentation 24 hours; 5, the sample after 35 ℃ 35 ℃ of P2 (pCT603T) bacterium fermentation 24 hours (total fermentation time 48 hours) again.

In a word, second to the 6th road shown respectively through acid and risen and the alkali Mierocrystalline cellulose the handled enzymolysis product situation at different time that rises among Figure 13 A and the 13B.Cellobiose all is main product in both cases, also produces a spot of glucose simultaneously.Acid risen also have behind the cellulase hydrolysis a spot of procellose to form.After these nutrient solutions are cooled to 35 ℃, insert P2 (pCT603T) bacterium of 50 microlitres, the condition bottom fermentation of not controlling at static and pH 24 hours.Glucose, cellobiose and procellose (seeing the 7th pore area among Figure 13 A and the B) during this period obtain the ethanol product of about 5 grams per liters.In alkali rose by Mierocrystalline cellulose, ethanol production was 0.16 gram/gram Mierocrystalline cellulose.The decline of pH may suppress alcoholic acid and generate, but the ethanol yield has reached 27% of theoretical maximum.

With the endogenous endoglucanase part of thermal fiber clostridium substitute goods cellulase: P2 (pCT603T) bacterial strain does not need to add in addition beta-glucosidase, itself can form endoglucanase D and come the hydrolysis amorphous cellulose, but when hydrolysis crystal form Mierocrystalline cellulose, then require other enzymic activity.In the two-step approach fermentation, the ability of having tested this bacterial strain part substitute goods cellulase.After collecting, be suspended in the LB nutrient solution that equal-volume contains 50 grams per liter SOLKA FLOC SW40 in advance, cultivate 24 hours postcooling to 35 ℃, insert same bacterial strain again, and add the plain enzyme of 1% commercial fibre at 60 ℃ through the somatic cells of glucose fermentation.CYTOLASE and MULTIFECT are obtained close result, and the ethanol yield is 17.4 grams per liters, is 65% (seeing Table 17) of theoretical maximum.

As shown in figure 14, use under the CYTOLASE situation, carry out pre-treatment with the celD gene product and can improve greatly and utilize 1% cellulase producing and ethanol amount, gained ethanol yield is equivalent to the ethanol yield of 5%CYTOLASE.Among Figure 14, Mierocrystalline cellulose (the SOL-KA FLOC SW40 of 50 grams per liters) is after CYTOLASE and the incomplete saccharification of endoglucanase D, by P2 bacterial strain and P2 (pCT603T) strain fermentation.Marginal data: △, per 100 milliliters of nutrient solutions add 5 milliliters of CYTOLASE when inoculation, and the P2 bacterium is fermented; , per 100 milliliters of nutrient solutions add 1 milliliter of CYTOLASE when inoculation, and the P2 bacterium is fermented; Zero, per 100 milliliters of nutrient solutions add 0.1 milliliter of CYTOLASE when inoculation, and the P2 bacterium is fermented; , SOLKA FLOC SW40 ferments after 12 hours through 60 ℃ of pre-treatment of P2 (pCT603T) bacterial strain, and the pre-fermentative action of bacterial strain warp is to provide thermal fiber clostridium endoglucanase D.After being cooled to 35 ℃, add the plain enzyme of commercial fibre that P2 (pCT603T) bacterial strain and every 100ml fermented liquid contain 1ml, begin fermentation.

Figure 13 C has shown the thin-layer chromatography result who takes a sample with MULTIFECT in the different steps of two-step approach fermentation.Glucose, cellobiose and procellose are equally by complete metabolism.Thereby via in the first step fermentation with ethanol common property deposits yields after the recombinant bacterial strain pre-treatment of endoglucanase D, the required plain enzyme dosage of commodity of saccharification can reduce 80% at most.

Discuss: knownly can limit fungus culture medium usually to cellulosic hydrolysis by the enzymatic hydrolysis reaction from the cellobiose to monose of beta-glucoside.In the required key enzyme of effective hydrocellulose, activity of beta-glucosidase is minimum usually, and is simultaneously also least stable.The accumulation of cellulase hydrolysis product cellobiose is competitive inhibitor (Eriksson etc. [1990] Microbiol and enzymatic degradation of woodand wood components.Springer-Verlag, the New York of the further hydrolysis of oligosaccharide; Jeffries sees before).Urging childbirth Klebsiella P2 bacterial strain is that the cellobiose of reporting at first that can fast and effeciently transform generates high-level alcoholic acid bacterium.

As if this bacterium can active transport cellobiose and procellose, thereby has reduced the accumulation of cellobiose, do not need to add the further depolymerization of exogenous enzyme again.Do not do research (Al[1989] J.Biotechnol.12:79-86) to urging the cellobiose movement system of childbirth in the Klebsiella, but carry the gene of coding phosphotransferase component and glucose 1-phosphate1-glycosides enzyme with the very near intestinal bacteria of its sibship, these two kinds of enzymes play a role in the metabolism of cellobiose, great majority experiments is recessive with this metabolism in the bacterial strain, but (the J.Bacteriol169:2713-2717 such as Hall that in natural microorganism, plays a role; Kricker etc.[1987] may similar gene (Al is with aforementioned) in action in the Klebsiella Geneties115:419-429), urging childbirth.

Ethanol produces relevant to after being incorporated into karyomit(e), and is synthetic by plasmid-encoded recombinant protein when helping ethanol fermentation.The application of thermal fiber clostridium cellulose enzyme gene is an example, and other more valuable recombinant protein all can be by same method production as lipase, proteolytic enzyme, glycosylhydrolase, zoohormone and biomedical product etc.When generating thermal fiber clostridium cellulase as the common property thing with the P2 bacterial strain, descending unexpectedly appears in fermentation efficiency.The reason of this phenomenon it be unclear that, although the present invention also observes same phenomenon urging the childbirth Klebsiella to transform in the bacterial strain with the producing and ethanol intestinal bacteria of hot anaerobic bacterium (Thermoanaerobium brockii) Starch debranching enzyme gene and many plasmids producing and ethanol of expression thermal fiber clostridium xylanase gene.

Thus, give birth to Klebsiella, improve by the Mierocrystalline cellulose conversion and generate the alcoholic acid approach based on urging in the previous embodiment.Known fiber disaccharides and procellose are the inhibitions of endoglucanase and exoglucanase, and these oligosaccharides of beta-glucoside enzyme catalysis are to the transformation of glucose monomer.But the producing and ethanol recombinant bacterial strain as the P2 bacterial strain, does not need the collecting process of separating of sugar, thereby does not need beta-glucosidase during metabolism, reduced cellobiose and the procellose end products restraining effect to cellulase simultaneously yet.Another advantage of using this bacterial strain is to have reduced contamination of heavy, because can utilize the bacterium specific energy of cellobiose or procellose to utilize the bacterial classification of monomer glucose to want much less comparatively speaking.Compared with the intestinal bacteria KO11 that can not utilize cellobiose, the function of hastening parturition transhipment that Klebsiella showed and decomposing cellobiose and procellose has reduced in the cellulose fermentation process demand to the commodity enzyme.

By utilizing additional fermentation common property thing endoglucanase, the consumption of the plain enzyme of commercial fibre can further reduce.From this angle, thermophilic enzyme is obviously particularly useful, because exist in a large number in the thalline of this enzyme after fermentation, need only just can discharge from thalline by activity form by elevated temperature, and active maximum under this temperature.With the pre-treatment of SOLKA FLOC SW40, can improve the efficient of Genencor cellulase greatly through pre-fermentation synthetic endoglucanase.Because endoglucanase D only has catalytic activity (Beguin etc. are with aforementioned) to cellulosic amorphous domain, therefore above-mentioned pre-treatment helps forming the plain enzyme effect of commercial fibre novel site.In thermal fiber clostridium, endoglucanase D is the component (Beguin etc. see before) of cellulase complex body.This enzyme also might be combined with each other with the fungoid enzyme or combine Mierocrystalline cellulose, thereby helps opening cellulosic structure, is hydrolyzed.

P2 (pCT603T) bacterial strain is constructed to generate the alcoholic acid optimum strain by cellulose fermentation, but its efficient is not as starting strain thermal fiber clostridium (Tailliez etc. [1989] Appl.Environ.Microbiol.55:203-211).Urge P2 (pCT603T) bacterial strain of childbirth Klebsiella not resemble thermal fiber clostridium, it does not have complete cellulase system, but with 1% commodity CYTOLASE or MULTIFECT co-fermentation after, ethanol yield that P2 (pCT603) is produced 35 ℃ of fermentations and ultimate density are all than the height of the thermal fiber clostridium mutant strain of anti-ethanol 65 ℃ of fermentations.This bacterial strain also can be done further improvement in heredity and process aspect.

Embodiment 19 utilizes the intestinal bacteria recon that carries the gene that can express thermally-stabilised glycated protein on the producing and ethanol gene that is integrated with zymomonas mobilis (Z.mobilis) on the karyomit(e) and the plasmid to produce ethanol from amylofermentation

But the coli strain of producing and ethanol can be at the thermophilic enzyme of cell inner expression starch saccharification after transforming.After the fermentation ends, these enzyme cells that contain of collecting are heated to the temperature (60-70 ℃) that enzyme is maximum activity, to discharge saccharifying enzyme.These microorganisms can be used as the source of enzyme in the yeast fermentation, and only produce small amount of ethanol common property product.

In this example, lack this advantage of protein secreting system and develop that can be at the producing and ethanol bacterial strain of heat-staple α-Dian Fenmei of cell inner expression and chitinase thereby utilize in the intestinal bacteria.Collect these cells after the fermentation ends and be heated to the temperature that enzyme is maximum activity, come starch saccharification to discharge heat-staple enzyme.

Bacterial strain and growth conditions: all ethanol fermentations all use intestinal bacteria KO11 (Ohta etc., 1991, see before) to carry out in sacchariferous Luria substratum.The plasmid of being with all marks in the bacterial strain.

Plasmid construction: made up two plasmid pLOI140 and pLOI141 (Figure 15 A and B) with standard method, they have the alpha-amylase gene [Mie-lenz etc. (1985) of bacstearothermophilus, U.S. Patent number 4,493,893] and the Starch debranching enzyme gene of hot anaerobic bacterium (Ther-moanaerobium brockii) [Coleman etc. (1986) U.S. Patent number 4,612,287; Coleman etc. (1987) J.Bacteriol 169:4302-7].The Hind III that has a segment length 5.4kb of amylase gene on the plasmid pWL625Amy (ATCC31,791) ^rEndonuclease bamhi is inserted into the pCPC90Z[Cole after partially digested, on an etc. (1986) see] Hind III site.Learning the plasmid that after insert in a site, obtains two genes of high level expression simultaneously only, this plasmid called after pLOI568 on the Luria flat board of starch-containing and amylopectin after relatively.Behind the partially digested pLOI568 of EcoR I enzyme, with (1985) Proc.Natl.Acad.Sci.USA81:4129-33 such as plasmid pCOS2EMBL[Poustka] the EcoRI endonuclease bamhi of the long 2.5kb of the preceding paragraph band tetracycline resistance gene inserts and forms plasmid pLOI140 and pLOI141.Last plasmid has the multiple sharp son of pBR322, and is highly stable in bacterial strain KO11, still has 96% cell to have this plasmid after 48 generations in growth under the condition that does not add antibiotic.

The used abbreviation of Figure 15 A and B is: amy, the gene of coding bacillus stearothermophilus (Bacillus stearothermophilus) α-Dian Fenmei; Pul, the gene of the hot anaerobic bacterium of encoding (Thermoanaerobium brockii) Starch debranching enzyme; Tet, tetracycline resistance gene; Amp, ampicillin resistance gene; Ori, the colE1 replicon.

Fermentation and analysis: fermentation is shaken in the bottle at 500 ml and is carried out according to former described method (Beall etc. are on seeing) substantially, shakes bottle as the constant device of pH (working volume 350ml).Substratum comprises the Luria substratum, add respectively 10% glucose, 5% glucose, 10% maltose, 5% maltose or 4.5% starch (30 ℃ of fermentations, pH6.0,100rpm).The cell concn that fermentation is inoculated is 0.03 milligram of dry weight/milliliter.Sugar is sterilized with filtration method in addition.Starch is unsterilised.

Cell concentration measure its O.D.550 value (1O.D. be equivalent to 0.32 the gram dry cell weight/liter).Total saccharification activity is used in 60 ℃ of fermentation mensuration reductive sugar amounts and decides (Bergmeyor etc. see before).Ethanol is measured (Dombek etc. see before) with gas-liquid chromatograph (GLC).Alcohol concn is represented with grams per liter.The ethanol yield is proofreaied and correct dilution by adding alkali during the fermentation, and calculates by total reducing sugar amount or total amount of starch, and disregards the sugar amount that does not have use.Fa Jiao maximum yield is 0.51 gram ethanol/gram glucose, 0.53 gram ethanol/gram maltose and 0.56 gram ethanol/gram starch approximately in theory.The result by twice or repeatedly the fermentation mean value and obtain.

Amylofermentation:, collect KO11 (pLOI140) and KO11 (pLOI141) cell and the stored frozen of in 10% glucose, having fermented in advance 72 hours for the starch that ferments.In 25 milliliters of pH values were 6.0 Luria substratum, 15.75 gram starch suspensions were in 325ml pH value is 6.0 Luria substratum these cell suspensions.Add half cell suspending liquid to starch suspension, in boiling water, stir insulation to 70 ℃ and 70 ℃ of insulations 5 minutes.Mixture is cooled to 60 ℃, adds remaining cell and continues insulation 24 hours at 60 ℃.Heating can make the recombination bacillus coli inactivation and discharge heat-staple enzymic hydrolysis starch.Starch cool to 30 after the saccharification ℃ and with the sterile distilled water adjusted volume to 350ml.

Inoculum is centrifugal collection after the overnight growth in 5% glucose, and is used for starting fermentation (0.03 gram viable cell dry weight/liter).Sampling is to check the alcoholic acid fermentation and to use thin layer chromatography analysis.

Use non-activated Whatman150A silica-gel plate (Whatman company, Clifton, New Jersey) separating glucose, maltose and trisaccharide maltose.Be separated in room temperature and carry out,, wherein contain sugared 5-50 microgram with 1 microlitre sample.Be separated in the mixture (2: 1: 1) of acetone, ethyl acetate and acetate and carry out, add 2% water before this mixture uses.Bounias (1980) Anal.Biochem.106:291-5 is seen in dry plate dyeing.Starch and sugar from Sigma chemical company (St.Louis, Mo).

With recombinant escherichia coli strain glucose fermentation and maltose: observe the situation of bacterial strain KO11 glucose fermentation and maltose, the results are shown in Figure 16A and B, it reflects ethanol production and cell concentration respectively.Used symbol is among the figure: ●, the bacterial strain KO11 in 10% glucose; The bacterial strain KO11 (pLOI140) of △ in 10% glucose; , the bacterial strain KO11 (pLOI141) in 10% glucose; Zero, the bacterial strain KO11 in 5% maltose (5% and 10% maltose comes to the same thing).

When though aerobic is cultivated intestinal bacteria to utilize the case study of maltose to get very clear, growth of bacterium and ethanol production are all slow in glucose in maltose.Yield in 10% maltose is less than 25% of theoretical maximum yield (53.5 gram ethanol/liter), and yield is less than 50% of theoretical maximum yield (26.8 gram ethanol/liter) in 5% maltose.Because the result who obtains in two kinds of maltose concentrations is similar, so can not be because the direct toxicity of maltose causes growing more slowly and fermenting.It is slower that maltose and glucose are compared fermentation, may be because other restriction as sugar transportation and glucose phosphorylation etc.

As Figure 16 A and B, behind the plasmid pLO1140 and pLOI141 of adding coding α-Dian Fenmei and Starch debranching enzyme, growth velocity and the ethanol production of bacterial strain KO11 in glucose all has reduction.Cell yield after 72 hours and ethanol yield all reduce about 1/3.

The expression of saccharifying enzyme: the potato starch suspension that is used in the Luria substratum (pH6.0) is made substrate (70 ℃), and the burst size of (72 hours) reduction polysaccharide can be used for measuring total saccharification activity of bacterial strain KO11 (pLOI140) and KO11 (pLOI141) after the fermentation ends.Two bacterium active similar, be 30 micromoles reducing sugar/milliliter nutrient solution/minute.Be intracellular reactive more than 90% wherein, but after being heated to 70 ℃, these activity are easy to be released.

Amylofermentation: test strain KO11 (pLOI140) and KO11 (pLOI141) in two-step approach, microorganism wherein can produce saccharifying enzyme can express the producing and ethanol enzyme again.Be collected in 10% glucose pre-fermentation cell, suspend the back as the enzyme source come hydrolysis equal-volume 4.5% starch cold suspension (the Luria substratum, pH6.0).The cell that adds half collection, mixture be heated to very soon 70 ℃ and in water-bath 70 ℃ the insulation about 5 minutes.Mixture moves on to 60 ℃, adds second half cell and is beneficial to hydrolysis, has tested the starch of corn, potato, paddy rice and wheat respectively, and its result is similar.Starch suspension keeps liquid in heating and hydrolytic process.Relative therewith, use not with starch base because of the KO11 bacterial strain, heat starch suspension completely solidified in back three minutes.

60 ℃ of insulations after 24 hours, will contain amylatic Luria substratum be cooled to 30 ℃ and inoculation with starting fermentation (pH6.0,100rpm, inoculum size be 0.03 gram dry cell weight/liter), (Beall etc., 1991, the same) as previously mentioned.In initial 24 hours, ethanol production is the 0.26-0.28 grams per liter, and all types of starch results are similar, and last ethanol yield is a 0.35-0.37 gram/gram starch, and maximum in theory yield is 0.56 gram/gram starch.The amylaceous fermented Figure 17 that the results are shown in.

Because identical in essence with the data that KO11 (pLOI141) obtains, so among Figure 17, the data of KO11 (pLOI141) have only been shown with bacterial strain KO11 (pLOI140).Used symbol is as follows among Figure 17: △, W-Gum (S-4126); , potato starch (2004); Zero, rice fecula (S-7260); ▲, wheat starch (S-5127); ■, W-Gum add 2mM CaCl ₂● KO11, glucose 5% compares.

In the two-step approach fermented maize starch, take out sample with thin layer chromatography analysis (Figure 18) in different steps.After 24 hours, hydrolysate has glucose, maltose, trisaccharide maltose, maltotetrose and long oligosaccharides.Glucose, maltose and trisaccharide maltose by KO11 (pLOI141) be used for the fermentation (72 hours).

Figure 18 shows the thin layer chromatography analysis result of W-Gum fermentation.Maltose and glucose are as standard substance.Glucose is denoted as G1 to G4 to maltotetrose.Article one, the road is that glucose and maltose are made standard substance; The second road is for using at the nutrient solution of 60 ℃ of saccharification after 24 hours; Article three, the road is 60 ℃ of nutrient solutions that saccharification was fermented 72 hours after 24 hours again.

Calcium ion usually can the stabilizing starch degrading enzyme.With containing 2mM CaCl ₂The Luria nutrient solution in the fermenting experiment that carries out of W-Gum show that fermentation rate slightly accelerates, yield is also higher, per hour be respectively 0.29 gram ethanol/liter and 0.40 restrain ethanol/gram starch.With traditional comparing with enzymic fermentation with yeast, the recombination bacillus coli fermentation is slower, and last alcohol concn is lower.85% of the current business method yield of using a kind of microorganism to be about to use yeast and additional enzymes (about 0.47 gram ethanol/gram starch) by the total ethanol yield of two-step approach fermentation starch.

This example shows that using recombination bacillus coli to produce the alcoholic acid fermentation can be used to produce the needed enzyme of part mashing, though also need further to improve.Because do not need aeration condition, so the generation of enzyme is very simple.The pH value identical (about pH6.0) that the optimum pH of heat-staple bacterial enzyme effect is required with Escherichia coli fermentation.Utilize these biologies, the residual starch in the low value raw material (as corn husk) can be used as substrate, and corn stem juice can be used as the source of composite nutrient.Of this sort operation can reduce the price of saccharifying enzyme and increase ethanol yield in the corn fermentation.

Claims (50)

1. the heterologous gene conversion with coding ethanol dehydrogenase and pyruvic carboxylase is different from colibacillary suitable Gram-negative host and produces the alcoholic acid method, it is characterized in that described host in enough functional level expressing said gene, is beneficial to described host and produces main tunning ethanol.

2. according to the process of claim 1 wherein that described host selects from erwinia, Klebsiella and xanthomonas.

3. according to the process of claim 1 wherein that described host selects from intestinal bacteria.

4. according to the process of claim 1 wherein that described host selects from erwinia and Klebsiella.

5. according to the method for claim 1, the recombinant host that wherein is transformed has first allogeneic dna sequence DNA of can encode described ethanol dehydrogenase and described pyruvic carboxylase, and described recombinant host can be expressed described allogeneic dna sequence DNA at enough functional levels and be produced main tunning ethanol to help described host.

6. according to the method for claim 5, wherein said recombinant host has urges childbirth Klebsiella M5A1 pLOI 555 to produce the alcoholic acid characteristics.

7. according to the method for claim 1, the plasmid that wherein said host is had coding ethanol dehydrogenase and Pyruvate Decarboxylase Gene transforms, wherein said plasmid can make described Gram-negative host produce described ethanol dehydrogenase and described pyruvic carboxylase at enough functional levels, is beneficial to described host and produces main tunning ethanol.

8. according to the method for claim 7, wherein said plasmid has the gene of encode in the zymomonas mobilis described ethanol dehydrogenase and described pyruvic carboxylase.

9. according to the method for claim 7, wherein said plasmid further has the lac promotor of the genetic expression of described coding ethanol dehydrogenase of control and pyruvic carboxylase.

10. according to the method for claim 7, wherein said plasmid has been named as pLOI555.

11. method according to claim 5, wherein said recombinant host comprises the plasmid that has coding ethanol dehydrogenase and Pyruvate Decarboxylase Gene, described plasmid can make described recombinant host produce described ethanol dehydrogenase and described pyruvic carboxylase at enough functional levels, is beneficial to described host and produces main tunning ethanol.

12. according to the method for claim 5, wherein said recombinant host

(A) also have the proteinic encoding gene that can make described host transhipment and metabolism oligosaccharides and

(B), make described host from the metabolism of described oligosaccharides, obtain main tunning ethanol at certain level expressing said gene and described allogeneic dna sequence DNA.

13. according to the method for claim 12, wherein said recombinant host is selected from intestinal bacteria.

14. according to the method for claim 13, wherein said recombinant host is selected from erwinia and Klebsiella.

15. according to the method for claim 14, wherein said recombinant host has urges the childbirth Klebsiella to produce the alcoholic acid characteristics.

16. according to the method for claim 12, wherein said oligosaccharides comprises the sugar monomer of selection from C5 and C6 sugar monomer.

17. according to the method for claim 16, wherein said oligosaccharides is made up of from the sugar monomer of glucose and wood sugar selection.

18. according to the method for claim 17, wherein said oligosaccharides is selected from dimer and tripolymer.

19. according to the method for claim 12, wherein said recombinant host also produces polysaccharidase.

20. according to the method for claim 19, wherein said recombinant host also has second allogeneic dna sequence DNA segment, its expression product is described polysaccharidase.

21. according to the method for claim 20, wherein said polysaccharidase is selected from a kind of Mierocrystalline cellulose lyase, a kind of xylan lyase and a kind of starch degrading enzyme.

22. method according to claim 21, wherein said polysaccharidase is selected from a kind of endo-dextranase, cellobiohydrolase, beta-glucosidase enzyme, interior-1,4-xylosidase, xylobiase, α-glucuronidase, α-L-A Labaifunantangganmei, acetylase, acetyl wood sugar esterase, α-Dian Fenmei, beta-amylase, glucoamylase, Starch debranching enzyme, beta-glucanase, hemicellulase, arabinofuranosidase/xylosidase, mannase pectin hydrolase and pectate lyase.

23. according to the method for claim 22, wherein said polysaccharidase is an expression of gene product in the following gene, these genes comprise celD, xynZ, xylB, a kind of alpha-amylase gene and a kind of Starch debranching enzyme gene.

24. method according to claim 23, wherein said celD and xynZ gene come the fine clostridium of self-heating, described xylB gene comes the fine genus bacillus of self-dissolving, and described alpha-amylase gene is from bacillus stearothermophilus, and described Starch debranching enzyme gene comes the self-heating anaerobic bacterium.

25. according to the method for claim 21, wherein said polysaccharidase is at least can the described polysaccharidase of merocrine secretion from the expression product of a kind of cellulose enzyme gene of muck cellulomonas cartae and described recombinant host.

26. according to any one method of claim 20-24, wherein said recombinant host also has other allogeneic dna sequence DNA fragment, it is relevant that its expression product and transhipment monose and/or oligosaccharides enter recombinant host.

27. according to any one method of claim 20-24, wherein said polysaccharidase to small part is secreted by described recombinant host.

28. according to any one method of claim 20-24, wherein said polysaccharidase has the accumulation of q.s in described recombinant host.

29. according to the method for claim 28, wherein said polysaccharidase is a kind of heat stable enzyme.

30. according to the method for claim 28, wherein said recombinant host also has other allogeneic dna sequence DNA fragment, its expression product is that described host at least can merocrine other polysaccharidase.

31. according to the method for claim 30, wherein said different polysaccharidase comprises the expression product of a kind of cellulose enzyme gene of muck cellulomonas cartae.

32. according to the method for claim 5, wherein said recombinant host also produces a kind of polysaccharidase.

33. according to the method for claim 32, wherein said recombinant host also is with another allogeneic dna sequence DNA fragment, its expression product is described polysaccharidase.

34. according to the method for claim 33, wherein said polysaccharidase is selected a group from Mierocrystalline cellulose lyase, wood sugar lyase, hemicellulose lyase and starch degrading enzyme composition.

35. method according to claim 34, wherein said polysaccharidase is selected from endo-dextranase, cellobiose lyase, beta-glucosidase enzyme, interior-1,4-β xylosidase, xylobiase, α-glucuronidase, α-L-A Labaifunantangganmei, acetylase, acetyl wood sugar esterase, α-Dian Fenmei, beta-amylase, glucoamylase, Starch debranching enzyme, beta-glucanase, arabinofuranosidase/xylosidase, mannase, pectin hydrolase and pectate lyase.

36. according to the method for claim 35, wherein said polysaccharidase is following expression of gene product, these genes are celD, xynZ, xylB, alpha-amylase gene and Starch debranching enzyme gene.

37. according to the method for claim 36, wherein said celD, xynZ and xylB gene derive from Clostridium thermocellum; Described alpha-amylase gene derives from bacstearothermophilus; And described Starch debranching enzyme gene gets the self-heating anaerobic bacterium.

38. according to the method for claim 35, wherein said polysaccharidase to small part is secreted by described recombinant host.

39. according to the method for claim 35, wherein said polysaccharidase is accumulation in large quantities in described recombinant host.

40. one kind from containing oligosaccharides biomass production alcoholic acid method, it is characterized in that comprising following steps:

A. in primary reactor, described biomass is contacted with polysaccharidase, becomes simpler oligosaccharides and/or monose with the oligosaccharides in the described biomass of degrading,

Wherein said contact is approximately 50 ℃ to 60 ℃ in temperature, and the pH value is approximately 4.5 to 5.0 times and carries out;

B. obtain a kind of sugar soln from described primary reactor, it comprises described simpler oligosaccharides and/or monose;

C. with described sugar soln introducing-fermentor tank, it comprises to make and describedly generates the alcoholic acid recombinant host from the fermentation of simple oligosaccharides and/or monose, described recombinant host has first allogeneic dna sequence DNA of can encode described ethanol dehydrogenase and described pyruvic carboxylase, described recombinant host can be expressed described allogeneic dna sequence DNA at enough functional levels and be produced main tunning ethanol to help described host, and

D. being approximately 30 ℃ to 35 ℃ and pH value in temperature is approximately and makes described simpler oligosaccharides and/or monose fermentation generation ethanol under 6.0 conditions.

41. according to the method for claim 40, its feature also is, takes out elementary liquid from described fermentor tank, and is used for cooling off the secondary liquid that comprises described simpler oligosaccharides and/or monose, and the latter is taken out from described primary reactor.

42. according to the method for claim 41, its feature is that also described elementary liquid is introduced into described primary reactor after the described secondary liquid of cooling.

43. according to the method for claim 40, wherein said recombinant host is selected from bacterium and yeast.

44. according to the method for claim 43, wherein said recombinant host is selected from gram negative bacterium.

45. according to the method for claim 44, wherein said recombinant host is selected from intestinal bacteria.

46. according to the method for claim 45, wherein said recombinant host is selected from erwinia and Klebsiella.

47. according to the method for claim 46, wherein said recombinant host has urges childbirth Klebsiella M5Al pLOI 555 to produce the alcoholic acid characteristics.

48. according to the method for claim 40, wherein said oligosaccharides comprises the sugar monomer that is selected from C5 and C6 sugar monomer.

49. according to the method for claim 48, wherein said oligosaccharides comprises the sugar monomer that is selected from glucose and wood sugar.

50. according to the method for claim 40, wherein said oligosaccharides is selected from dimer and tripolymer.

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