CN100560727C - Be used to produce the biological catalyst of D-lactic acid - Google Patents

Abstract

Problem of the present invention provides the high-efficiency method for producing of D-lactic acid, in addition, also provides optical purity height, highly selective D-production method of lectic acid that the organic acid by product is few.Cultivate the pyruvate formate-lyase inactivation or active reduce and derive from microorganism, FAD dependency D-lactic dehydrogenase enzyme deactivation or the active microorganism that reduces that the activity of the NADH dependency D-serum lactic dehydrogenase (ldhA) of escherichia coli is enhanced or except that the described feature of mentioned microorganism, have TCA circulation and malate dehydrogenase enzyme deactivation or actively reduce and aspartic acid ammonialyase inactivation or the active microorganism that reduces production D-lactic acid.The promotor that gene and the control of ldhA of will encoding on genome relates to the gene of the synthetic or amino acid bio synthetic protein expression of glycolysis-, biological nucleic acid is connected and strengthens the ldhA activity.

Description

Be used to produce the biological catalyst of D-lactic acid

Technical field

The present invention relates to the microorganism of highly selective High-efficient Production D-lactic acid and use the method for this microorganisms producing D-lactic acid.Specifically, relate to the method for the high lactic acid of High-efficient Production purity, particularly the few effective D-lactic acid manufacture method of the generation accumulation of pyruvic acid.

The invention still further relates to the production method of D-lactic acid, it is characterized in that, this method is used FAD dependency D-lactic dehydrogenase enzyme deactivation or the active microorganism that reduces.

In addition, the invention still further relates to the microorganism that produces D-lactic acid and use the D-production method of lectic acid of this microorganism, described microorganism does not produce impurity succsinic acid or fumaric acid when producing D-lactic acid.

Background technology

Biodegradable polymer poly lactic acid causes CO ₂In the time of problem, energy problem remarkableization, also get most of the attention as sustainability (sustainability), the corresponding type product of LCA (LCA (life cycle assessment) (life cycleassessment)), therefore just seeking effective and inexpensive method of manufacturing as the lactic acid of its raw material.

By the way, present industrial poly(lactic acid) is the L-lactic acid polymer, and lactic acid has L-lactic acid and D-lactic acid, and in recent years, D-lactic acid receives publicity as the intermediate of polymer raw material or agricultural chemicals, medicine.But in arbitrary purposes, all require to have high-optical-purity as the lactic acid of raw material.

The microorganism that occurring in nature exists milk-acid bacteria or thread fungus etc. effectively to produce lactic acid is stated bacterium in the use and makes in the method for lactic acid, and the method for practicability has been arranged.For example, as the known Lactbacillus of the having delbrueckii of microorganism that can effectively produce L-lactic acid etc., the microorganism that belongs to as the known Sporolactobacillus of having of the microorganism that can effectively produce D-lactic acid etc.The accumulation of lactic acid has all reached high level under any situation, but the by product beyond the lactic acid that contains in the purifying nutrient solution for example compounds such as acetic acid, ethanol, acetoin, pyruvic acid can't be removed in purge process fully, causes the quality of final product lactic acid to descend.In addition, because sneaking into of optical isomer causes optical purity decline also to become serious problem.

Descend for fear of aforesaid lactic acid purity, the amount of by-products that reduces when utilizing microorganisms producing is an effective means.If utilize the specific gene of the gene recombination technology destroy microorganisms of exploitation in recent years, just the possibility specificity hinders the production of by product.But at present, the gene disruption method can not easily be applicable to any microorganism, so can be not easy to be suitable in the microorganism of High-efficient Production lactic acid originally milk-acid bacteria or thread fungus etc.Its reason is that the mentioned microorganism genomic information is insufficient, and can not be extensive use of as the host of gene recombination.

Relative therewith, utilize genomic information abundant, as the sufficient escherichia coli of gene recombination host's real achievement, yeast, people's culturing cell etc., can carry out gene disruption with comparalive ease.Particularly from breeding rapid or cultivating the consideration of easy aspect, most preferably escherichia coli.Because the lactic acid of escherichia coli production has only the D-type, so when obtaining the high D-lactic acid of optical purity, be appropriate host.But the D-lactic acid-producing rate of the escherichia coli of wild-type is low, and produces the various organic acid by products beyond the D-lactic acid.In order to address this problem, once attempted utilizing gene recombination to change the pathways metabolism of escherichia coli, optionally High-efficient Production D-lactic acid.

People such as Chang (Chang, D.-E., et.al., Appl.Environ.Microbiol., Vol.65 (4), pp1384-1389 (1999)) use contains 5% glucose, reaches amino acid whose substratum, with the phosphotransacetylase (being designated hereinafter simply as pta) of escherichia coli, and 2 kinds of variants of Phosphoenolpyruvate carboxylase (being designated hereinafter simply as ppc) utilize aerated culture to make growing microorganism earlier, carrying out anaerobism then cultivates, simultaneously, append glucose, make the glucose in the substratum be no more than 5%, cultivate, produced the D-lactic acid of 62.2g/L through 60 hours.At this moment, becoming the transformation efficiency of D-lactic acid by conversion of glucose is 76%.

People such as Zhou (Zhou, S., et.al., Appl.Environ.Microbiol., Vol.69 (1), pp399-407 (2003)) preparation destroys the escherichia coli of pyruvate formate-lyase (being designated hereinafter simply as pfl), fumaric reductase (being designated hereinafter simply as frd), alcohol/aldehyde dehydrogenase (being designated hereinafter simply as adeE) and 4 kinds of enzymes of acetokinase (being designated hereinafter simply as ackA), its anaerobism in the minimal medium that contains 5% glucose was cultivated 168 hours, produce the D-lactic acid of 48.5g/L, and do not generate formic acid, succsinic acid, ethanol and acetic acid by product.But, though in this test successfully highly selective produce D-lactic acid, productivity is low, is 0.29g/L/hr, so can not satisfy the requirement of selection rate and productivity two aspects simultaneously.In addition, do not mention the by product pyruvic acid, it is also unclear that it reduces effect.Because pyruvic acid is the metabolic reaction matrix of D-lactic acid, therefore different with other by product organic acids, generate if excessively suppress it, then cause the generation of D-lactic acid itself to be suppressed.So, be difficult to the generation of pyruvic acid is suppressed at minimum.Usually, pyruvic acid is included in situation in the lactic acid monomer raw material with the form of impurity under, polymer polymerizing rate reduction etc. occurs and do not wish the problem that occurs, this is the fact well known to those skilled in the art, hence one can see that, and pyruvic acid is one of by product that must reduce.But the past can successfully be suppressed the report that the pyruvic acid by product generates again not about both keeping the High-efficient Production rate of D-lactic acid.

In a word, the maximum accumulation of the D-lactic acid of present known escherichia coli is 62.2g/L, and its production time is 60 hours.On the other hand, consider the milk-acid bacteria of using in the suitability for industrialized production of L-lactic acid or hyphomycetic L-lactic acid-producing rate as accumulation more than or equal to 100g/L, production time had to then think that the D-lactic acid accumulation of escherichia coli and production time still were in low-level in 24 hours.Past can realization and the report of the equal lactic acid-producing rate of milk-acid bacteria or thread fungus about escherichia coli, and also can hint use escherichia coli make the data of the accumulation of D-lactic acid above 100g/L.

When using colibacillus to carry out the D-lactic fermentation, the not existence of preferred enzyme usually.Reason is if there are electron acceptor(EA)s such as enzyme, then colibacillus nonfermented and breathing.Only under the non-existent situation of electron acceptor(EA)s such as enzyme, colibacillus could be only phosphorylation by the matrix level obtain energy (ATP), utilize the reducing power (NADH) that obtains in the glycolysis-, produce reductibility organic acids such as lactic acid.For the foregoing reasons, the D-lactic fermentation that utilizes colibacillus in the past nearly all is to cultivate by anaerobism to carry out.Though minority be by preceding half section for aerated culture, second half section are that two-section type that anaerobism is cultivated is cultivated and carried out, be in order to ensure enough biomasses by above-mentioned preceding half section aerated culture, final lactic fermentation remains to cultivate by anaerobism to be carried out.But during actual industrial production, in the amino acid source cereal vat liquor (being designated hereinafter simply as CSL) of the cheapness in adding substratum to etc., not only contain organic acid impurity, but also contain D-type, two kinds of lactic acid of L-type, but if anaerobism is cultivated, then L-lactic acid is by metabolism, and remains in the substratum.The katalaze enzyme L-serum lactic dehydrogenase (being designated hereinafter simply as lld) that is generated the reaction of pyruvic acid by the L-type is expressed under aeration condition, therefore, if have under aeration condition and also can carry out lactic acid fermented method efficiently, then can make bacterial metabolism be included in L-type in the substratum by using this method, generate the high D-type of optical purity, but, so far, also do not have to realize the technology of above-mentioned purpose.

About using pfl to destroy the lactic acid-producing of bacterial strain, in the report of Zhou etc., at first reported following content.That is, people such as Contag (Contag, P.R., et.al., Appl.Environ.Microiol., Vol.56 (12), pp3760-3765 (1990)) lactic acid that the unmanifest escherichia coli strain of pfl generates 35mM disclosed, and in the pfl variant, the productivity of lactic acid improves, and generates the lactic acid of 45mM.That is, in escherichia coli, because the pfl inactivation, the productivity of D-lactic acid improves, and this has become known content by disclosed data such as Contag.

The D-serum lactic dehydrogenase is divided into NADH dependency and FAD dependency according to the dependent difference to coenzyme.NADH dependency D-serum lactic dehydrogenase is the reaction of catalysis pyruvic acid generation D-lactic acid in vivo.The NADH dependency D-serum lactic dehydrogenase that derives from colibacillus is called ldhA especially.

Yang etc. (Yang, Y.T., et.al., Metab.Eng., Vol.1 (2), pp141-152 (1999)) have reported that will assemble the ldhA expression carrier imports escherichia coli, makes D-lactic acid accumulation improve about 8g/L, although very low thus.That is, by disclosed data such as Yang as can be known, in escherichia coli,, can improve the productivity of D-lactic acid by strengthening the ldhA activity.

In addition, according to [Bunch, PK. such as Bunch, Microbiology, Vol.143 (Pt 1), 187-195 (1997)] report, the escherichia coli that imports the ldhA expression carrier that derives from escherichia coli is owing to the importing of expression vector hinders its propagation.

In addition, as making D-serum lactic dehydrogenase (being designated hereinafter simply as ldh) the superfluous example of expressing in colibacillus that derives from colibacillus bacterium in addition, can enumerate [Kochhar such as Kochhar as the expression example of the ldh that derives from Lactobacillus helveticus, S., Eur.J.Biochem., (1992) 208,799-805] report or as [the Kochhar such as Kochhar of expression example of the ldh that derives from Lactobacillusbulgaricus, S., Biochem.Biophys.Res.Commun., (1992) 185,705-712] report, arbitrary example has all been studied the physicochemical property of the enzyme of expressing, but does not all mention the accumulation of D-type or pyruvic acid.

But, reduce about making pfl inactivation or activity, and the D-lactic acid-producing rate of the active microorganism of strengthening of ldhA still be not very clear.

Usually, in the gene and strengthening method of using expression vector, following inconvenience can take place: carrier comes off, the expression amount of goal gene descends, further cause the productivity of desired substance to reduce.Therefore, when being applied to D-lactic acid industrial production, have several problems that should solve in the enhancement method of the ldh gene of use expression vector, expectation replaces the gene and strengthening method of utilizing expression vector.But, also do not have and above-mentioned relevant report.

As the gene and strengthening method that replaces expression vector, can enumerate (Solem such as Solem, C., et.al., Appl.Environ.Microbiol., Vol.68 (5), pp2397-2403 (2002)) the promoter region with a certain gene on the genome of passing through of report is replaced as the method that promotor is arbitrarily strengthened this gene.But, if state in the use in the D-lactic acid manufacturing of ldhA gene and use present technique, then because the ldhA gene that utilizes this method to strengthen only is the single copy gene on the genome, compare with the enhancement method that utilizes the expression vector of expressing multi-copy gene, the active increase rate of ldhA is little, so, can be improved even those skilled in the art compared when also being difficult to infer D-lactic acid-producing rate and using expression vector.

In addition, the enzyme that utilizes the colibacillus purifying by parsing as can be known, the main catalysis of FAD dependency D-serum lactic dehydrogenase (being designated hereinafter simply as dld) and NADH dependency D-serum lactic dehydrogenase the reversed reaction of catalytic reaction, that is, and by the reaction of D-lactic acid generation pyruvic acid.Though people such as Shaw have obtained dld ruined large intestine bacterial strain JS150 and JS151, do not mention D-lactic acid-producing rate or pyruvic acid productivity (Shaw, L. in the above-mentioned bacterial strains, et.al., J.Bacteriol., Vol.121 (3), pp1047-1055 (1975)).In addition, people such as Barnes have reported dld relevant with the absorption of each seed amino acid or carbohydrate (Barnes, E.M., et.al., J.Biol.Chem., Vol.246 (17), pp5518-5522 (1971)), but do not mention the relation with the generation of D-lactic acid or pyruvic acid.

And, in database as the attached E.coliGenetic Stock Center of Yale university (CGSC) of one of large intestine bacterial strain preservation mechanism, if retrieval dld and two kinds of dissociants of pfl then can retrieve paper (Mat-Jan, the F. of Mat-Jan etc., et.al., J.Bacteriol., Vol.171 (1), pp342-348 (1989)), find but in fact read over the back, do not destroy the relevant explanation of bacterial strain for two kinds in this paper with dld and pfl.

About above-mentioned D-lactic acid, in utilizing the fermentative production of microorganism, do not realize meeting the productivity of industrial level and optionally report at present simultaneously, for example, do not have to keep the high productivity of D-lactic acid, can reduce again as the succsinic acid of main organic acid by product or the precedent of fumaric acid.

So, the applicant can suppress succsinic acid again with the productivity of not damaging D-lactic acid and be produced as purpose, further investigate, found that by under anaerobic, destruction can be suppressed the generation of succsinic acid fully as the gene of the enzyme malate dehydrogenase (malic acid dehydrogenase) (being designated hereinafter simply as mdh) of the reaction of catalysis oxaloacetic acid generation oxysuccinic acid under the prerequisite of not damaging D-lactic acid-producing rate.But,,, also can reduce the amount of fumaric acid by product so find to destroy the gene of aspartic acid ammonialyase (being designated hereinafter simply as aspA) owing to still generate the fumaric acid by product.

The paper of Courtright in 1970 etc. discloses the effect (Courtright, J.B.et.al., J.Baceriol., Vol.102 (3), pp722-728 (1970)) of mdh inactivation.Its disclosure is in the escherichia coli of mdh inactivation, though under anaerobic, the reactive behavior that oxaloacetic acid generates oxysuccinic acid is zero, and the reactive behavior that aspartic acid generates fumaric acid has improved on the contrary.Promptly, under anaerobic, the approach that generates succsinic acid comprises following 2 kinds of approach: generated the approach of fumaric acid, succsinic acid via oxysuccinic acid and generated the approach of fumaric acid, succsinic acid by oxaloacetic acid via aspartic acid by oxaloacetic acid, if mdh inactivation, a kind of approach is ended then, and a kind of approach in back is further activated.So the paper of Courtright etc. does not have open by making the mdh inactivation end the generation of succsinic acid.

Another one is about destroying the zymic technology (spy opens flat 11-056361 communique) of mdh gene about the prior art of the effect of mdh inactivation.This patent is by destroying zymic mdb gene, change the oxysuccinic acid amount that generates, but which kind of influence the growing amount of not mentioning succsinic acid is subjected to.

Even if those skilled in the art also are difficult to predict the generation that can suppress succsinic acid by the mdh of deactivation microorganism fully from existing knowledge.

In addition, about the effect of aspA inactivation, the past only discloses the situation (Dreyfus, L.A., et.al., J.Bacteriol., Vol.136 (2), pp757-764 (1978)) yersinia pestis.But the purport of this paper is because the aspA inactivation makes aspartic acid or glutamine be difficult to decompose in cell, the growing amount of fumaric acid is not studied.

Patent documentation 1: the spy opens flat 11-056361 communique

Non-patent literature 1:Chang, D.-E., et.al., Appl.Environ.Microbiol., Vol.65 (4), pp1384-1389 (1999)

Non-patent literature 2:Zhou, S., et.al., Appl.Environ.Microbiol., Vol.69 (1), pp 399-407 (2003)

Non-patent literature 3:Contag, P.R., et.al., Appl.Environ.Microbiol., Vol.56 (12), pp 3760-3765 (1990)

Non-patent literature 4:Yang, Y.T., et.al., Metab.Eng., Vol.1 (2), pp 141-152 (1999)

Non-patent literature 5:Bunch, P.K., et.al., Microbiology, Vol.143 (pt 1), pp187-195 (1997)

Non-patent literature 6:Kochhar, S., et.al., Eur.J.Biochem., Vol.208 (3), pp799-805 (1992)

Non-patent literature 7:Kochhar, S., et.al., Biochem.Biophys.Res.Commun., Vol.185 (2), pp705-712 (1992)

Non-patent literature 8:Solem, C., et.al., Appl.Environ.Microbiol., Vol.68 (5), pp2397-2403 (2002)

Non-patent literature 9:Shaw, L., et.al., J.Bacteriol., Vol.121 (3), pp1047-1055 (1975)

Non-patent literature 10:Barnes, E.M., et.al., J.Biol.Chem., Vol.246 (17), pp5518-5522 (1971)

Non-patent literature 11:Mat-Jan, F., et.al., Bacteriol., Vol.171 (1), pp342-348 (1989)

Non-patent literature 12:Courtright, J.B.et.al., Bacteriol., Vol.102 (3), pp722-728 (1970)

Non-patent literature 13:Dreyfus, L.A., et.al., J.Bacteriol., Vol.136 (2), pp757-764 (1978)

Summary of the invention

One of problem of the present invention provides the method for High-efficient Production D-lactic acid, and other problems of the present invention provide the D-production method of lectic acid of optical purity height, highly selective that the organic acid by product is few.

Other problems of the present invention provide the production method of D-lactic acid, and this method can reduce the accumulation that is not easy the pyruvic acid removed from the substratum that in the past utilized microorganism that lactic acid is accumulated as organic acid impurity.

Other problems of the present invention provide the production method of lectic acid that uses heterofermentative lactic bacteria to produce lactic acid effectively.Other problems of the present invention provide the production method of lectic acid that uses the high lactic acid of heterofermentative lactic bacteria production optical purity effectively.

Other problems of the present invention provide productivity that does not reduce desired substance and the D-production method of lectic acid that suppresses the generation of by product succsinic acid and/or fumaric acid.

Other problems of the present invention provide the stable D-lactate dehydrogenase gene enhancement method that replaces the enhancement method that uses expression vector, in addition, also provide the method for producing D-lactic acid more efficiently.

For solving above-mentioned problem, the inventor etc. further investigate, found that pyruvate formate-lyase (pfl) inactivation or active the reduction, and the bacterium that the activity that derives from the NADH dependency D-serum lactic dehydrogenase (ldhA) of escherichia coli is enhanced can produce D-lactic acid in than the short in the past time, and can realize the high accumulation that do not reach so far.Particularly about the method for ldhA increased activity, utilization on genome, will encode ldhA gene and control and glycolysis-, biological nucleic acid is synthetic or the promotor of the gene of the protein expression that amino acid bio is synthetic relevant is connected and make the microorganism of its expression, thus can be in than the time of the genetic expression enhancement method weak point that uses expression vector a large amount of D-lactic acid of generation.The cell inner expression amount of D-serum lactic dehydrogenase is more than method of the present invention in the method for using expression vector, but it is directly related with the High-efficient Production of D-lactic acid without any the expression of enzymes amount that the reason proof is high, and, it is shocking, in the present invention, even intracellular expression of enzymes amount is so not high, the productivity of D-lactic acid also significantly improves.

Next, the part of the pyruvic acid that exists in the discovery microbial culture mediums such as the inventor is actually the generation of dld catalysis D-lactic acid, and, by cultivating dld gene inactivation or the active microorganism that reduces in fact, compare with the host, the propagation of this microorganism is not suppressed, and obtains the nutrient solution that contains high-quality D-lactic acid of the pyruvic acid density loss in the substratum.By cultivating Pfl inactivation or active reduction and/or ldhA increased activity and dld gene inactivation or the active microorganism that reduces in fact, can obtain the high-quality D-lactic acid of the pyruvic acid density loss in the substratum.

And, the inventor has TCA circulation and malate dehydrogenase (malic acid dehydrogenase) (mdh) inactivation or active the reduction and aspartic acid ammonialyase (aspA) inactivation or the active mentioned microorganism that reduces by use, can not only keep high D-lactic acid-producing rate, the pair that can also suppress succsinic acid and fumaric acid generates, and has realized the present invention thus.

That is, the present invention includes following content.

[1] a kind of production method of lactic acid is characterized in that, having added the heterofermentative lactic bacteria of cultivating pyruvate formate-lyase (pfl) inactivation or active reduction more than 2 kinds or 2 kinds in the amino acid whose substratum, reclaims lactic acid from the culture that obtains.

[2] production method of [1] described lactic acid is characterized in that, heterofermentative lactic bacteria is an escherichia coli.

[3] production method of [2] described lactic acid is characterized in that, escherichia coli is MT-10934 (FERM BP-10057) strain.

[4] a kind of production method of D-lactic acid, it is characterized in that, the activity that cultivation derives from the NADH dependency D-serum lactic dehydrogenase (ldhA) of escherichia coli is enhanced and pyruvate formate-lyase (pfl) inactivation or the active bacterium that reduces, and reclaims D-lactic acid from the culture that obtains.

[5] production method of [4] described D-lactic acid, wherein, bacterium is an escherichia coli.

[6] production method of [4] or [5] described D-lactic acid is characterized in that, cultivates in the amino acid whose substratum having added more than 2 kinds or 2 kinds.

[7] a kind of microorganism, this microorganism is its FAD dependency D-serum lactic dehydrogenase (dld) inactivation that had originally or the active microorganism that reduces, it is characterized in that, pyruvate formate-lyase (pfl) inactivation or active reduce and/or derive from the NADH dependency D-serum lactic dehydrogenase (ldhA) of escherichia coli activity be enhanced.

[8] [7] described microorganism, this microorganism is a bacterium.

[9] [8] described microorganism, described bacterium is an escherichia coli.

[10] a kind of production method of D-lactic acid is characterized in that, cultivates each described microorganism of [7]～[9] in the liquid medium within, D-lactic acid is generated in nutrient solution and accumulates separation D-lactic acid from nutrient solution.

[11] production method of [10] described D-lactic acid is characterized in that, cultivates in the amino acid whose substratum having added more than 2 kinds or 2 kinds.

[12] a kind of production method of D-lactic acid is characterized in that, cultivates FAD dependency D-serum lactic dehydrogenase (dld) inactivation or the active microorganism that reduces in the liquid medium within, D-lactic acid is generated in nutrient solution and accumulates separation D-lactic acid from nutrient solution.

[13] [12] described method, wherein, microorganism is a bacterium.

[14] [13] described method, wherein, bacterium is an escherichia coli.

[15] a kind of microorganism, on the genome of this microorganism, coding derives from the gene of the NADH dependency D-serum lactic dehydrogenase (ldhA) of escherichia coli and expresses this NADH dependency D-serum lactic dehydrogenase (ldhA) by the promotor of the gene of the use control protein expression relevant with glycolysis, biological nucleic acid synthesis path or amino acid bio synthesis path.

[16] [15] described microorganism, this microorganism is an escherichia coli.

[17] [15] or [16] described microorganism is characterized in that, the pyruvate formate-lyase that this microorganism had originally (pfl) inactivation or active the reduction and/or FAD dependency D-serum lactic dehydrogenase (dld) inactivation or active the reduction.

[18] a kind of escherichia coli, in the genome of escherichia coli, the promotor of gene of using control to derive from the protein expression relevant with glycolysis, biological nucleic acid synthesis path or amino acid bio synthesis path of escherichia coli replaces the promotor of the gene of the NADH dependency D-serum lactic dehydrogenase (ldhA) that derives from escherichia coli of encoding, and expresses this NADH dependency D-serum lactic dehydrogenase (ldhA).

[19] [18] described escherichia coli, wherein, the promotor of the gene of the protein expression that control is relevant with glycolysis, biological nucleic acid synthesis path or the amino acid bio synthesis path of escherichia coli is the promotor that derives from the Glycerose 3-phosphate dehydrogenase gene of escherichia coli.

[20] [18] or [19] described escherichia coli is characterized in that, the pyruvate formate-lyase that this escherichia coli had originally (pfl) inactivation or active the reduction and/or FAD dependency D-serum lactic dehydrogenase (dld) inactivation or active the reduction.

[21] a kind of production method, this method are to utilize the method for each described microorganisms producing D-lactic acid in culture medium culturing [15]～[20].

[22] a kind of microorganism, this microorganism has TCA circulation and malate dehydrogenase (malic acid dehydrogenase) (mdh) inactivation or active the reduction, it is characterized in that the pyruvate formate-lyase of this microorganism (pfl) inactivation or active reduction the, and/or FAD dependency D-serum lactic dehydrogenase (dld) inactivation or active the reduction.

[23] [22] described microorganism, wherein, aspartic acid ammonialyase (aspA) inactivation that this microorganism had originally or active the reduction.

[24] [22] or [23] described microorganism, this microorganism is a bacterium.

[25] [24] described microorganism, wherein, bacterium is an escherichia coli.

[26] [25] described microorganism, wherein, the activity that derives from the NADH dependency D-serum lactic dehydrogenase (ldhA) of escherichia coli is enhanced.

[27] a kind of production method, this method are to utilize each described microorganism in culture medium culturing [22]～[26], produce the method for the organic acid compound in addition that produces in the TCA circulation thus.

[28] [27] described production method, wherein, the compound beyond the organic acid is a D-lactic acid.

[29] production method of each described lactic acid is characterized in that in [1]～[6], [10]～[14], [21], [28], and this method is to cultivate under aeration condition.

[30] production method of [29] described lactic acid is characterized in that, described aeration condition is, is the situation of object under at the water that is 30 ℃ with temperature, makes oxygen volume transmission quality coefficient K under the normal pressure by supplying with oxygen _LA is more than or equal to 1h ^-1, be less than or equal to 400h ^-1Condition in the scope.

[31] production method of each described lactic acid is characterized in that in [1]～[6], [10]～[14], [21], [28]～[30], and cultivating pH is 6～8.

Utilize the present invention to provide to have optionally microorganism of high D-lactic acid-producing rate and D-lactic acid.And, cultivate microorganism by the present invention's preparation, produce D-lactic acid, can produce highly purified D-lactic acid with existing method comparison more economically.

In addition, utilize the present invention that the bacterium that produces the few D-lactic acid of pyruvic acid growing amount can be provided.And, cultivate bacterial strain by the present invention's preparation, produce D-lactic acid, can produce chemical purity and the high D-lactic acid of optical purity with existing method comparison more economically.

In addition, the bacterial strain production lactic acid of use the present invention preparation can be made and method in the past compares the pyruvic acid content decline as impurity, the high-quality D-lactic acid fermentation liquid that purification process is few.

Utilize the present invention can not cause the productivity of the organic acid compound in addition of generation in the TCA circulation to reduce, the pair that can suppress succsinic acid and/or fumaric acid generates.Particularly the compound beyond the organic acid that produces in industrial production TCA circulation is under the situation of purpose, by reducing the kind and the amount of by product, can reduce the purifying cost of target product.

Description of drawings

Fig. 1 be in the nutrient solution of expression embodiment 20 D-lactic acid accumulation through the time graphic representation that changes.Among the figure, result with leg-of-mutton curve representation MG1655 Δ pfl Δ dld bacterial strain (embodiment 15), result with tetragonal curve representation MG1655 Δ pfl Δ dld/pGAPldhA bacterial strain (embodiment 18) has the result that circular curve representation MG1655 Δ pfl Δ dld/GAPpldh genome inserts strain (embodiment 19).

Fig. 2 be in the nutrient solution of expression embodiment 24 lactic acid accumulate concentration through the time graphic representation that changes.Among the figure, result with criss-cross curve representation Δ pfl Δ dld bacterial strain, result with circular curve representation Δ pfl Δ dld Δ mdh bacterial strain, result with leg-of-mutton curve representation Δ pfl Δ dld Δ ppc bacterial strain has the result of tetragonal curve representation Δ pfl Δ dld Δ frd bacterial strain.

Fig. 3 be in the nutrient solution of expression embodiment 24 succsinic acid accumulate concentration through the time graphic representation that changes.Among the figure, result with criss-cross curve representation Δ pfl Δ dld bacterial strain, result with circular curve representation Δ pfl Δ dld Δ mdh bacterial strain, result with leg-of-mutton curve representation Δ pfl Δ dld Δ ppc bacterial strain has the result of tetragonal curve representation Δ pfl Δ dld Δ frd bacterial strain.

Fig. 4 be in the nutrient solution of expression embodiment 25 lactic acid accumulate concentration through the time graphic representation that changes.Among the figure, result with circular curve representation Δ pfl Δ dld Δ mdh Δ asp bacterial strain, have the result that leg-of-mutton curve representation Δ pfl Δ dld Δ mdh Δ asp/GAPldhA genome inserts strain, have the result of tetragonal curve representation Δ pfl Δ dld Δ mdh bacterial strain.

Fig. 5 be in the nutrient solution of expression embodiment 25 fumaric acid accumulate concentration through the time graphic representation that changes.Among the figure, result with circular curve representation Δ pfl Δ dld Δ mdh Δ asp bacterial strain, have the result that leg-of-mutton curve representation Δ pfl Δ dld Δ mdh Δ asp/GAPldhA genome inserts strain, have the result of tetragonal curve representation Δ pfl Δ dld Δ mdh bacterial strain.

Embodiment

Describe the present invention below in detail.

Pyruvate formate-lyase among the present invention (pfl) is based on the enzyme that international biological chemistry associating (I.U.B.) enzyme committee report is belonged to enzyme number 2.3.1.54, is also referred to as the formic acid acetyltransferase.This enzyme is meant the general name of reversible catalysis by the enzyme of the reaction of pyruvic acid generation formic acid.

So-called inactivation is meant the state of activity below detectability of this enzyme that utilizes existing mensuration systems measurement among the present invention.

So-called reduction is meant sudden change and/or the gene recombination owing to the gene of this enzyme of coding among the present invention, and the state preceding with processing compares, the obvious state that descends of this enzymic activity.

So-called heterofermentative bacterium is meant that having fermentation decomposes carbohydrate, generates the lactic acid bacterium that is selected from the ability more than at least a kind or a kind in formic acid, acetate, succsinic acid, the ethanol in addition among the present invention.Particularly, as the heterofermentative bacterium among the present invention, preferred escherichia coli, as pyruvate formate-lyase (pfl) inactivation or the active heterofermentative bacterium that reduces, can enumerate the destruction strain or the escherichia coli MT-10934 of the pfl gene of any escherichia coli wild strain that can utilize preparations such as the method shown in the embodiment of the invention.

MT reconnaissance-the 10934th has been confirmed the active bacterial strain that reduces of pfl, can easily implement the present invention.Based on the budapest treaty that relates to the international authentications such as microbial preservation that are used for patented procedure, this bacterial strain is deposited in Independent Administrative Leged Industrial Technology Complex Inst patent biological preservation center that Ibaraki, Japan build ripple city east 1 fourth order a kind No. 1 central authorities 6 with the deposit number for FERM BP-10057 in putting down on November 8th, 14.

Independent variant about pfl, because MT-10934 has the character of HfrC, therefore as long as, dilute with the wild strain that has F-character arbitrarily, for example MG1655, W3110 etc. mix 2 hours in the LB substratum after, obtain single bacterium colony, the variant of therefrom selecting to wish gets final product.The pfl variant compares with wild strain in anaerobism is cultivated, and the growing amount of formic acid reduces, and therefore it can be selected as index and obtain

So-called cultivation is to use the microorganism among culture medium culturing the present invention among the present invention.As the substratum that uses,, be not particularly limited so long as the substratum that comprises carbon source, nitrogenous source, mineral ion and be used to produce the desired organic trace element of microorganism, nucleic acid, vitamins etc. of lactic acid gets final product.

So-calledly among the present invention be added with that amino acid whose substratum is meant the substratum more than at least 2 kinds or 2 kinds that contains in naturally occurring each seed amino acid more than 2 kinds or 2 kinds, also comprise the substratum of the hydrolyzate of crude substance such as containing yeast extract, casein hydrolysate, peptone, whey, waste molasses, cereal vat liquor or crude substance extract.In order to obtain more preferably result, preferably contain 0.5%～20%, more preferably contain 2%～15% at least a kind or the substratum of its mixture in yeast extract, peptone, whey, waste molasses, the cereal vat liquor of being selected from.Especially the interpolation of cereal vat liquor can obtain obvious effects, and the substratum that do not add salt such as ammonium sulfate this moment obtains better result on the contrary.Substratum is common liquid nutrient medium.

Culture condition changes according to the thalline of preparation, the difference of culture apparatus, and for example, when using the MT-10934 of the active reduction of pfl, culture temperature is preferably 20 ℃～40 ℃, more preferably cultivates under 25 ℃～35 ℃, and pH preferably utilizes NaOH, NH ₃Deng transferring to 6.0～7.2, more preferably 6.5～6.9, cultivate.Incubation time is not particularly limited, and is that thalline is fully bred and lactic acid generates the needed time.

In addition, when using the pfl gene disruption strain of escherichia coli wild strain MG1655 of pfl inactivation, can obtain maximum productivity under neutral or pH than neutral meta-alkalescence slightly, cultivating pH is 6.9～7.4, more preferably 7.1～7.3.Culture temperature can be higher than the temperature of MT-10934, can obtain maximum productivity 33 ℃～42 ℃ following cultivations.

Usually use the culture tank of energy controlled temperature, pH, aeration condition, stirring velocity during cultivation, but cultivation of the present invention is not limited to use culture tank.When using culture tank to cultivate, also can carry out in advance as required cultivating, be inoculated into then in the substratum in the culture tank of having modulated requirement in advance as pre-incubated kind.

MT-10934 generates formic acid in the pH zone of pH7～7.5, and the pfl gene disruption bacterial strain of MG1655 utilizes cultural method of the present invention not generate formic acid.So, use as the heterofermentation bacterium under the situation of escherichia coli, when in substratum, making the thalline generation lactic acid of employing near neutral pH, if as MT-10934, can observe the generation of formic acid, then when reality is made lactic acid, the pH of substratum is controlled in than neutrality slant acidity side slightly, if pfl gene disruption strain as MG1655, do not observe the generation of formic acid, then when reality is made lactic acid, the pH of substratum is controlled in neutrality or meta-alkalescence side slightly, can obtain peak performance thus.

So-called culture is meant thalline, nutrient solution and the handled thing thereof that utilizes aforesaid method to produce among the present invention.

From the cultures such as nutrient solution that obtain as mentioned above, reclaim the method for lactic acid, for example, if from nutrient solution, then can utilize common known method, for example, can adopt straight run distillation after the acidifying method, form carry out the distillatory method behind the rac-Lactide, add pure and mild catalyzer carry out carrying out after the esterification distillatory method, with the method for organic solvent extraction, utilize the isolating method of ion exchange column, utilize the method etc. of electrodialysis concentrating and separating or the method that aforesaid method is made up.In addition, utilize the thalline of method preparation of the present invention,, be considered to from culture, reclaim the part of the method for lactic acid so utilize this enzyme group further to produce, reclaim lactic acid owing to can produce the enzyme group that is fit to lactic acid-producing.

The so-called NADH dependency D-serum lactic dehydrogenase (ldhA) that derives from escherichia coli is to utilize pyruvic acid and NADH to generate the enzyme that derives from escherichia coli of D-lactic acid and NAD among the present invention, particularly, can enumerate by (Microbiology 143 (Pt 1) such as Bunch, 187-195 (1997)) gene that obtains, or the enzyme that to have genomic dna with escherichia coli be template, the gene that utilizes sequence number 3 and sequence number 4 to carry out the sequence that dna fragmentation was comprised of pcr amplification produces.

So-called ldhA increased activity among the present invention is meant the sudden change and/or the gene recombination of gene by coding ldhA, makes active obviously than the state enhanced state that carries out before the above-mentioned processing by the enzyme of the genes produce of coding ldhA.

Bacterium among the present invention is common prokaryotic cell prokaryocyte microorganism.

In the present invention, be enhanced and the example of pfl inactivation or the active bacterium that reduces, can enumerate the MT-10934/pGlyldhA described in the embodiment of the invention as the ldhA activity.This bacterial strain can be preferred for having the production method as the lactic acid of above-mentioned [1] described feature, promptly, being added with the heterofermentative lactic bacteria of cultivating pyruvate formate-lyase (pfl) inactivation or active reduction more than 2 kinds or 2 kinds in the amino acid whose substratum, from the culture that obtains, reclaim lactic acid.

As strengthening one of active method of ldhA among the present invention, following method is effective, promptly, the promotor of the gene of the protein expression that the gene of coding ldhA is relevant with glycolysis, biological nucleic acid synthesis path or amino acid bio synthesis path with control is connected, under this state, be assembled in the expression plasmid, again plasmid imported desirable bacterium.The promotor of the gene of the protein expression that control in such cases is relevant with glycolysis, biological nucleic acid synthesis path or amino acid bio synthesis path is meant the strong promoter that in bacterium, preferably plays a role usually in escherichia coli, even and in the presence of glucose, it is expressed and also is difficult for downtrod promotor, particularly, can enumerate the promotor or serine hydroxymethylase (glyA) promotor of glyceraldehyde-3-phosphate dehydrogenase.When the bacterium that obtains as mentioned above produces D-lactic acid under aeration condition, relatively, can not improve the accumulation of D-lactic acid when being enhanced, and reduce pyruvic acid concentration, simultaneously, improve the optical purity of D-lactic acid as impurity with the expression of ldhA.

FAD dependency D-serum lactic dehydrogenase (dld) among the present invention is meant in the presence of as the oxidized form flavin adenine dinucleotide of coenzyme, and catalysis is generated the general name of enzyme of the reaction of pyruvic acid by D-lactic acid.

Microorganism among the present invention is so long as have the big microorganism of ability that produces D-lactic acid and get final product, be not particularly limited, though also comprise it being not have the microorganism that produces D-lactic acid ability, can become microorganism with generation D-lactic acid ability by applying certain change.

As among the present invention with dld inactivation or active the reduction and/or pfl inactivation or active reduce and/or the ldhA activity is enhanced to the microorganism of feature, can enumerate escherichia coli MT-10994 (FERM BP-10058) bacterial strain.

The promotor of the gene of the protein expression that control is relevant with glycolysis, biological nucleic acid synthesis path or amino acid bio synthesis path among the present invention is meant the strong promoter that plays a role usually in microorganism, even and in the presence of glucose, it is expressed and also is difficult for downtrod promotor, particularly, can enumerate the promotor or the serine hydroxymethylase promotor of glyceraldehyde-3-phosphate dehydrogenase (below be sometimes referred to as GAPDH).

Promotor among the present invention be meant RNA polymerase with Sigma Factors in conjunction with and the position of transcriptional start.For example, the GAPDH promotor that derives from escherichia coli is designated as base 397-440 in the base sequence information of GenBankaccession number X02662.

The encode promotor of the gene of gene by on genome, using the control protein expression relevant of ldhA of the present invention with glycolysis, biological nucleic acid synthesis path or amino acid bio synthesis path, express this ldhA, make pfl inactivation or active the reduction, and/or dld inactivation or activity are reduced, with above-mentioned is the microorganism of feature, can enumerate escherichia coli MT-10994 (FERM BP-10058) bacterial strain.

Escherichia coli MT-10994 bacterial strain is connected with the GAPDH promoter function on genome by the ldhA gene expresses, or makes pflB, dld deactivation by gene disruption, so use this bacterial strain can easily implement the present invention.The budapest treaty that this bacterial strain is admitted in the world based on the microbial preservation that is used for patented procedure etc. is deposited in Independent Administrative Leged Industrial Technology Complex Inst patent biological preservation center that Ibaraki, Japan build ripple city east 1 fourth order a kind No. 1 central authorities 6 with the deposit number for FERM BP-10058 in putting down on March 19th, 16.

TCA circulation among the present invention is meant the metabolic pathway of large-scale carbon skeletons such as final complete oxidation sugar, lipid acid, multiple amino acids, is also referred to as tricarboxylic acid cycle, krebs circulation.

Malate dehydrogenase (malic acid dehydrogenase) among the present invention (mdh) is based on international biological chemistry associating (I.U.B.) enzyme committee report and is belonged to enzyme number 1.1.1.37, in the presence of as the oxidized form flavin adenine dinucleotide of coenzyme, reversibly catalysis is generated the general name of enzyme of the reaction of oxaloacetic acid by oxysuccinic acid.

As the mdh inactivation among the present invention or active reduction, pfl inactivation or reduction and/or dld inactivation or the active microorganism that reduces, can enumerate escherichia coli MT-10994 bacterial strain.This bacterial strain can be preferred for having the production method as the lactic acid of above-mentioned [1] described feature, promptly, being added with the heterofermentative lactic bacteria of cultivating pyruvate formate-lyase (pfl) inactivation or active reduction more than 2 kinds or 2 kinds in the amino acid whose substratum, from the culture that obtains, reclaim lactic acid.

Aspartic acid ammonialyase (aspA) among the present invention is united (I.U.B.) enzyme committee report based on international biological chemistry and is belonged to enzyme number 4.3.1.1, is also referred to as L-Aspartase.This enzyme is the general name of reversibly catalysis by the enzyme of the reaction of L-aspartic acid generation fumaric acid.

When cultivating the microorganism generation lactic acid that obtains among the present invention, though can not ventilate fully, the result preferably ventilates in order to obtain more preferably.Might not be in nutrient solution under the described aeration condition bubbling air, also comprise top ventilation, promptly, shape according to culture tank, one side is stir culture liquid moderately, Yi Bian change the gas cloud above the nutrient solution, makes the gas that comprises oxygen flow into the inside of culture tank.Under the situation of ventilating in liquid, the combination by interior pressure, agitating wing position, agitating wing shape, stirring velocity changes dissolved oxygen concentration, can obtain following optimum condition as index with the productivity of lactic acid and the organic acid content beyond the lactic acid etc. thus.For example, when escherichia coli MT-10934 bacterial strain is cultivated in the culture tank of smaller types such as the system culture apparatus BMJ-01 of ABLE society, when using the 500g nutrient solution, be 0.01vvm～1vvm, stirring velocity 50rpm～500rpm under the normal pressure at aeration condition, more preferably when 0.1vvm～0.5vvm, stirring velocity 100rpm under the normal pressure～400rpm, can obtain preferred result.Its condition is, under the situation of water as object with 30 ℃ of temperature, makes oxygen volume transmission quality coefficient K under the normal pressure by supplying with oxygen _LA is more than or equal to 1h ^-1, be less than or equal to 400h ^-1Scope in.

In addition, also comprise as other indexs of the suitableeest aeration condition, by regulating air flow, stirring velocity, thereby can make and utilize anaerobism to cultivate formic acid, acetate, succsinic acid, alcoholic acid content that the MT-10934 bacterial strain produces to be less than or equal to 5.0g/L, further preferably to be less than or equal to 1.0g/L, and can produce lactic acid.

In addition, also comprise as other indexs of the suitableeest aeration condition, by regulating air flow, stirring velocity, when in containing the substratum of 0.3% optical isomer L-lactic acid, cultivating MT-10934, be reduced to the interior L-of making concentration of lactic acid at 10～100 hours and be less than or equal to 0.02%.

Needn't remain above-mentioned aeration condition from cultivation initial stage to end, cultivate in part and implement in the operation to obtain preferred result.

In addition, by carrying out above-mentioned ventilation, can improve the productivity of lactic acid, and can reduce optical isomer.

Below, utilize embodiment to represent an example of the present invention, but the foregoing description there is no any qualification to the present invention.

[embodiment 1] utilizes the MT-10364 bacterial strain to produce lactic acid

Shown in the table 1 composed as follows of the substratum that is used to cultivate.

Table 1

In basal culture medium, contain the various organic acids of reducing sugar 0.34%, D-lactic acid 0.31%, L-lactic acid 0.31%, total free aminoacids 0.33% and trace after the acid hydrolysis that derives from the cereal extractive substance.

As pre-cultivation is that 25ml LB Broth is being housed, carry out the bacterium of planting of escherichia coli MT-10934 in the Erlenmeyer flask of Miller nutrient solution (Difco244620), speed stir culture with 120rpm is spent the night, in the 1L of the substratum that the above-mentioned composition of 475g is housed volumetrical culture tank (the system culture apparatus BMJ-01 of ABLE society), carry out full dose and plant bacterium then.Under atmospheric pressure, air flow is that 0.5vvm, stirring velocity are that 150rpm, culture temperature are that 31 ℃, pH are under the condition of 6.7 (utilizing NaOH to regulate), be cultured to the glucose completely consumed and fall.

After cultivating end,, utilize HPLC to measure organic acid content and optical purity in the nutrient solution that obtains according to ordinary method.The result is as shown in table 2.

Table 2

N.D：Not?detected

In The above results, the reason that total lactic acid production surpasses the glucose amount that adds when cultivating beginning is the carbon source of having utilized in the cereal extractive substance.But,, also can reach the turnover ratio more than 90% or 90% even all use reducing sugar, organic acid, amino acid in the cereal extractive substance.In addition, even use the nutrient solution that contains the optical isomer of organic acid impurity or lactic acid in the substratum, also can make the lactic acid that organic acid impurity is few, optical purity is high.

Need to prove that MG1655 can be used as ATCC47076 and buys from US mode bacterial classification collection center (ATCC).

[embodiment 2] ldhA expression vector and lactic acid produce the structure of bacterium

For obtaining (glyA) promotor of serine hydroxymethylase (serine hydroxymethyl transferase), with the colibacillus genomic dna as template, with sequence number 1, and sequence number 2 be probe, utilize the PCR method to increase, and with the fragment that restriction enzyme EcoRI cutting obtains, obtain the fragment of the coding glyA promotor of about 850bp thus.And, for obtaining the structure gene of ldhA, with the genomic dna of escherichia coli as template, with sequence number 3, and sequence number 4 be probe, utilize the PCR method to increase, and with the fragment that restriction enzyme EcoRI and HindIII cutting obtains, obtain the ldhA structure gene fragment of about 1.0kbp thus.Above-mentioned 2 fragments are mixed with the fragment of utilizing restriction enzyme EcoRI and HindIII cutting plasmid pUC18 to obtain, and utilize dna ligase to connect, be transformed into then in the colibacillus, obtain plasmid pGlyldhA.

The plasmid pGlyldhA that obtains is transformed in the escherichia coli MT-10934 bacterial strain, obtains lactic acid and produce bacterium MT-10934/pGlyldhA bacterial strain.

In addition, pUC18 can utilize ordinary method to extract from the ATCC37253 that collects the center available from the US mode bacterial classification to obtain.The MT-10934 bacterial strain is to be deposited in the biological preservation of the Independent Administrative Leged Industrial Technology Complex Inst's patent center that east, ripple city a kind of No. 1 central authorities 6 of 1 fourth order are built in the Ibaraki, Japan in putting down on November 8th, 14 with above-mentioned deposit number.

The lactic acid that [embodiment 3] utilize lactic acid to produce bacterium MT-10934/pGlyldhA bacterial strain generates

As pre-cultivation is that 25ml LB Broth is being housed, and carries out the bacterium of planting that lactic acid that embodiment 2 obtains produces bacterium MT-10934/pGlyldhA bacterial strain in the Erlenmeyer flask of Miller nutrient solution (Difco244620), utilizes the method identical with embodiment 1 to cultivate.After cultivating end,, utilize HPLC to measure the content and the optical purity of lactic acid according to ordinary method.The result is as shown in table 3.

Table 3

In The above results, the reason that total lactic acid production surpasses the glucose amount that adds when cultivating beginning is the carbon source of having utilized in the cereal extractive substance.But,, also can reach the turnover ratio more than 90% or 90% even all use reducing sugar, organic acid, amino acid in the cereal extractive substance.

The clone of the near zone of [embodiment 4] escherichia coli pfl gene

Whole base sequences of the genomic dna of escherichia coli are known (GenBankaccession number U00096), the base sequence of the gene of the pyruvate formate-lyase (below be also referred to as pfl) of coding escherichia coli also be in the news (GenBank accession numberAE000192).For the near zone of the base sequence of the gene (2283bp) of clones coding pfl, 4 kinds of oligonucleotide primers shown in the composition sequence numbers 5,6,7 and 8.The primer of sequence number 6,7 has Sph I recognition site in 5 ' end.

According to Current Protocols in Molecular Biology (JohnWiley﹠amp; Sons) described method prepares the genomic dna of escherichia coli MG1655 bacterial strain, the genomic dna 1 μ g that utilization obtains and primer with base sequence of sequence number 5, have the base sequence of sequence number 6 primer, have sequence number 7 base sequence primer and have the combination of primer of the base sequence of sequence number 8, use each 100pmol of above-mentioned primed DNA, under common condition, carry out PCR, about 1.8kbp that increases (below be also referred to as the pflB-L fragment), and the dna fragmentation of about 1.3kbp (below be also referred to as the pflB-R fragment).Utilize agarose electrophoresis to separate, reclaim above-mentioned dna fragmentation, utilize HindIII and Sph I cutting pflB-L fragment, utilize Sph I and Pst I cutting pflB-R.Utilize the T4DNA ligase enzyme to make above-mentioned 2 kinds of cutting fragments and thermal sensibility plasmid pTH18csl (GenBank accession nurmberAB019610) (Hashimoto-Gotoh, T., et.al., Gene, Vol.24 (1), pp185-191 (2000)) after HindIII and the reaction of Pst I cut substrate, be transformed in the escherichia coli DH5 α competent cell (competent cell), thereby obtain comprising near near segmental 2 the segmental plasmids fragment and 3 ' downstream of encoding the 5 ' upstream of gene of pflB, called after pTH Δ pfl.

The preparation of [embodiment 5] escherichia coli MG-1655 bacterial strain pfl gene disruption bacterial strain

The plasmid pTH Δ pfl that embodiment 4 is obtained is transformed in the escherichia coli MG1655 bacterial strain, can keep at cell under 30 ℃ of thermal sensibility plasmid, overnight incubation on the LB agar plate that contains 10 μ g/ml paraxin, transformant.With the transformant that obtains in the LB substratum in 30 ℃ cultivate 3 hours～spend the night, then, suitably dilute with LB liquid nutrient medium or physiological saline, be applied on the LB agar plate that contains 10 μ g/ml paraxin.Above-mentioned LB agar plate is cultivated down at can not holding temperature susceptibility plasmid 42 ℃, with the transformant that forms by genome outer-genome between identical reorganization, obtain the plasmid total length is assembled into the genomic bacterial strain of escherichia coli.

Obtain genomic dna by this bacterial strain, carry out pcr amplification with it as model, the anti-chloromycetin gene that discovery exists pTH18csl to have on genome, and with 5 ' the end near zone and the respectively identical zone of 3 ' end near zone of the gene of coding pflB, thereby confirm that this bacterial strain is that the plasmid total length is assembled into bacterial strain on the escherichia coli.

The plasmid total length is assembled into bacterial strain on the escherichia coli implants and the 100ml that 20ml do not contain the LB liquid nutrient medium of paraxin to be housed to be provided with in the flask of dash plate, it was cultivated 4 hours 30 ℃ of following joltings.Suitably dilute above-mentioned nutrient solution with the LB liquid nutrient medium that does not contain paraxin, then, be applied on the LB nutrient agar that does not contain paraxin.Select arbitrarily 96 with it at 42 ℃ of bacterium colonies of cultivating down breeding, it is bred not containing on the LB nutrient agar of paraxin and contain on the LB nutrient agar of paraxin respectively, select the bacterial strain of paraxin susceptibility.

Obtaining genomic dna from the bacterial strain of selecting, is that model carries out pcr amplification with it, selects the bacterial strain of the gene defect of coding pfl, with its called after MG1655 Δ pflB bacterial strain.

[embodiment 6] utilize the MG1655 Δ pfl bacterial strain that uses casein hydrolysate to produce lactic acid

As pre-cultivation is to prepare a plurality of 250g of being equipped with LB Broth, the Erlenmeyer flask of Miller nutrient solution (Difco244620), implant lactic-acid-producing strain MG1655, MG1655 Δ pfl bacterial strain therein respectively and embodiment 2 described plasmid pGlyldhA are assembled into 3 kinds of bacterial strains of MG1655 Δ pfl/pGlyldhA that MG1655 Δ pfl bacterial strain obtains according to ordinary method, under 30 ℃, after spending the night with the speed stir culture of 120rpm, in the 1L volumetrical culture tank of substratum shown in the 475g table 4 (the system culture apparatus BMJ-01 of ABLE society) is housed, carry out full dose respectively and plant bacterium.Under atmospheric pressure, air flow is that 0.5vvm, stirring velocity are that 200rpm, culture temperature are that 31 ℃, pH are under the condition of 6.7 (utilizing NaOH to regulate), cultivated 50 hours.After cultivating end,, utilize HPLC to measure the content and the optical purity of lactic acid in the gained nutrient solution according to ordinary method.The result is as shown in table 5.

Table 4

Table 5

[embodiment 7] use the generation of the lactic acid that utilizes MG1655 Δ pfl bacterial strain of cereal extractive substance

As pre-cultivation is respectively MG1655, MG1655 Δ pfl and MG1655 Δ pfl/pGlyldhA implantation to be equipped with in the Erlenmeyer flask of 25g nutrient solution, under 30 ℃, after spending the night with the speed stir culture of 120rpm, carry out full dose in the culture apparatus of the substratum shown in the 475g table 6 of in 1L volumetrical culture tank (the system culture apparatus BMJ-01 of ABLE society), packing into respectively and plant bacterium.Under atmospheric pressure, air flow is that 0.5vvm, stirring velocity are that 300rpm, culture temperature are that 35 ℃, pH are under the condition of 7.2 (utilizing NaOH to regulate), cultivated 24 hours.After cultivating end,, utilize lactic acid and pyruvic acid in the HPLC mensuration gained nutrient solution according to ordinary method.The result is as shown in table 7.

Table 6

Table 7

In The above results, the reason that total lactic acid production surpasses the glucose amount that adds when cultivating beginning is the carbon source of having utilized in the cereal extractive substance.But,, also can reach the turnover ratio more than 90% or 90% even all use reducing sugar, organic acid, amino acid in the cereal extractive substance.

Utilize the height of the lactic acid of MG1655 Δ pfl bacterial strain to accumulate production under [embodiment 8] high glucose concn

As pre-cultivation is MG1655 Δ pfl to be implanted be equipped with in the Erlenmeyer flask of 25g nutrient solution, after spending the night with the speed stir culture of 120rpm, be changed to by 10% and carry out full dose in the culture tank of the system culture apparatus BMJ-01 of ABLE society of 15% substratum and plant bacterium in that 475g glucose concn as shown in table 8 is housed.Under atmospheric pressure, air flow is that 0.5vvm, stirring velocity are that 300rpm, culture temperature are that 35 ℃, pH are under the condition of 7.2 (utilizing NaOH to regulate), be cultured to glucose and be consumed.After cultivating end,, utilize HPLC to measure lactic acid according to ordinary method.The result is as shown in table 9.

Table 8

Table 9

The reason that total lactic acid production surpasses the glucose amount that adds when cultivating beginning is the carbon source of having utilized in the cereal extractive substance.But,, also can reach the turnover ratio more than 90% or 90%, and reach this high accumulation that had not realized so far of 130g/L even all use reducing sugar, organic acid, amino acid in the cereal extractive substance.

[embodiment 9] utilize MG1655 Δ pfl bacterial strain research cereal extractive substance addition

As pre-cultivation is MG1655 Δ pfl to be implanted be equipped with in the Erlenmeyer flask of 25g nutrient solution, after spending the night with the speed stir culture of 120rpm, carry out full dose by the 1% system culture apparatus BMJ-01 of ABLE society that is changed to 10% substratum in for culture tank and plant bacterium in that 475g cereal extractive substance concentration as shown in table 10 is housed.Under atmospheric pressure, air flow is that 0.5vvm, stirring velocity are that 300rpm, culture temperature are that 35 ℃, pH are under the condition of 7.2 (utilizing NaOH to regulate), cultivated 24 hours.After cultivating end,, utilize HPLC to measure lactic acid according to ordinary method.The result is as shown in table 11.

Table 10

Table 11

Can be observed production rate in 1% cereal extractive substance Adding Area and descend, but in 24 hours, still reached this production rate that had not realized so far of 55g/L.In addition, with respect to the glucose that uses, the lactic acid turnover ratio maintains the level more than or equal to 90%.

[embodiment 10] utilize the influence of MG 1655 Δ pfl bacterial strains research aeration condition to glycolysis-speed

As pre-cultivation is MG1655 Δ pfl to be implanted be equipped with in the Erlenmeyer flask of 25g nutrient solution, after spending the night with the speed stir culture of 120rpm, carries out full dose and plant bacterium in the culture tank of the system culture apparatus BMJ-01 of ABLE society that substratum shown in the 475g table 12 is housed.Under atmospheric pressure and with condition shown in the table 13 is aeration condition, and culture temperature is that 35 ℃, pH are under the condition of 7.2 (utilizing NaOH to regulate), cultivates 24 hours.Utilize glucose CII-testwako (with the pure pharmaceutical worker's industry of light) to measure the remaining quantity of glucose.

Table 12

Table 13

Table 14

By this test as can be known, along with the raising of aeration condition, glycolysis-speed also improves, if excessively improve aeration condition, then sugar decomposition rate reduces.

[embodiment 11] utilize MG1655 Δ pfl/pGlyldhA bacterial strain research cereal extractive substance addition

As pre-cultivation is MG1655 Δ pflB/pGlyldhA to be implanted be equipped with in the Erlenmeyer flask of 25g nutrient solution, after spending the night with the speed stir culture of 120rpm, be changed to by 1% and carry out full dose in the culture tank of the system culture apparatus BMJ-01 of ABLE society of 10% substratum and plant bacterium in that 475g cereal extractive substance concentration as shown in Table 15 is housed.Under atmospheric pressure, air flow is that 0.5vvm, stirring velocity are that 300rpm, culture temperature are that 35 ℃, pH are under the condition of 7.2 (utilizing NaOH to regulate), cultivated 24 hours.After cultivating end,, utilize HPLC to measure D-lactic acid according to ordinary method.The result is shown in table 16.

Table 15

Table 16

Minimum in 1% cereal extractive substance Adding Area productivity, but in 24 hours, reach the production rate of the 58g/L that did not in the past have.In addition, with respect to the glucose that uses, the lactic acid turnover ratio maintains the level more than or equal to 90%.

[embodiment 12] utilize the influence of MG1655 Δ pfl/pGlyldhA bacterial strain research aeration condition to glycolysis-speed

As pre-cultivation is MG1655 Δ pflB/pGlyldhA to be implanted be equipped with in the Erlenmeyer flask of 25g nutrient solution, after spending the night with the speed stir culture of 120rpm, in the culture tank of the system culture apparatus BMJ-01 of ABLE society that substratum shown in the 475g table 17 is housed, carry out full dose and plant bacterium.Under atmospheric pressure and with condition shown in the table 18 is aeration condition, and culture temperature is that 35 ℃, pH are under the condition of 7.2 (utilizing NaOH to regulate), cultivates 24 hours.Utilize glucoseCII-testwako (with the pure pharmaceutical worker's industry of light) to measure the remaining quantity of glucose.

Table 17

Table 18

Table 19

By this test as can be known, along with the raising of aeration condition, glycolysis-speed also improves, if excessively improve aeration condition, then glycolysis-speed reduces.

The preparation of [embodiment 13] escherichia coli MG1655 strain dld genetically deficient bacterial strain

Utilization is carried out pcr amplification based on the CAACACCAAGCTTTCGCG (sequence number 9) of the gene information of the dld gene near zone of the genomic dna that derives from MG1655 bacterial strain preparation and TTCCACTCCTTGTGGTGGC (sequence number 10), AACTGCAGAAATTACGGATGGCAGAG (sequence number 11) and TGTTCTAGAAAGTTCTTTGAC (sequence number 12).The fragment that obtains is used restriction enzyme HindIII and Pst I, Pst I and Xba I cutting respectively, obtain the fragment of about 1140bp thus respectively.With this fragment and thermal sensibility plasmid pTH18csl (Hashimoto-Gotoh, T., et.al., Gene, Vol.241 (1), pp185-191 (2000)) through the fragment mixing that HindIII, Xba I cutting obtains, after the use ligase enzyme connects, be transformed in the DH5 α bacterial strain in 30 ℃, obtain the transformant of on the LB agar plate that contains 10 μ g/ml paraxin, growing.With the bacterium colony that obtains overnight incubation in 30 ℃ of LB liquid nutrient mediums that containing 10 μ g/ml paraxin, from the thalline that obtains, reclaim plasmid.This plasmid is transformed in the MG1655 bacterial strain in 30 ℃, obtains the transformant of on the LB agar plate that contains 10 μ g/ml paraxin, growing.The transformant that obtains is coated on the agar plate, in 30 ℃ of overnight incubation.Then, above-mentioned cultivation thalline is applied on the LB agar plate that contains 10 μ g/ml paraxin, obtains the bacterium colony of growing in 42 ℃.

Obtain again in the operation of 42 ℃ of single bacterium colonies of growing, select plasmid integral body by identical reorganization and be assembled into clone body in the karyomit(e).Do not have plasmid in the tenuigenin of this clone body.

Next, this clone body is coated on the LB agar plate, after 30 ℃ of following overnight incubation, is inoculated in the LB liquid nutrient medium (3ml/ test tube), cultivated 3～4 hours down in 42 ℃, jolting is cultivated.For obtaining single bacterium colony, this nutrient solution is suitably diluted (10 ^-2～10 ^-6About), be coated on the LB agar plate, in 42 ℃ of following overnight incubation, get bacterium colony.In the bacterium colony that occurs, select 100 bacterium colonies arbitrarily, make it, select the paraxin susceptibility clone body of only on the LB agar plate, breeding respectively breeding on the LB agar plate with on the LB agar plate that contains 10 μ g/ml paraxin.Utilize the fragment that contains about 2.0kbp of dld in the chromosomal DNA of pcr amplification above-mentioned purpose clone body again, select the bacterial strain of dld gene region disappearance, with the clone body that satisfies above-mentioned condition as the dld deletion mycopremna, and with the bacterial strain called after MG1655 Δ dld bacterial strain of gained.

[embodiment 14] utilize MG1655 Δ dld bacterial strain to produce D-lactic acid

As pre-cultivation is that 25ml LB Broth is being housed, and implants MG1655 bacterial strain or MG1655 Δ dld bacterial strain in the Erlenmeyer flask of Miller nutrient solution (Difco244620), spends the night with the speed stir culture of 120rpm.Various pre-nutrient solution global transfer in the culture tank of the system culture apparatus BMJ-01 of ABLE society of the substratum that 475g composition shown in table 20 is housed, are cultivated.Under atmospheric pressure, air flow is that 0.5vvm, stirring velocity are that 200rpm, culture temperature are that 31 ℃, pH are under the condition of 6.7 (utilizing NaOH to regulate), cultivated 96 hours.According to ordinary method, utilize HPLC to measure after 48 hours and the content of cultivating lactic acid, pyruvic acid, formic acid and acetate after finishing.MG1655 bacterial strain, MG1655 Δ dld bacterial strain are labeled as Wild, Δ dld respectively, and the result after 48 hours is shown in table 21, and the result who cultivates when finishing is shown in table 22.

Table 20

Table 21

Result after 48 hours

Table 22

Result after 96 hours

The preparation of [embodiment 15] escherichia coli MG1655pfl, dld genetically deficient bacterial strain

The plasmid pTH Δ pfl that obtains among the embodiment 4 is transformed in the MG1655 Δ dld bacterial strain, on the LB agar plate that contains 10 μ g/ml paraxin, grows, obtain transformant.The gained transformant is coated on the agar plate, in 30 ℃ of following overnight incubation.Then,, it is coated on the LB agar plate that contains 10 μ g/ml paraxin, obtains the bacterium colony of growing under 42 ℃ for obtaining above-mentioned cultivation thalline.By operating in the same manner with embodiment 5 and obtaining MG1655dld, pfl genetically deficient bacterial strain, with its called after MG1655 Δ pfl Δ dld bacterial strain from the gained bacterium colony.

[embodiment 16] ldhA expression vector is transformed in MG1655 Δ dld bacterial strain, the MG1655 Δ pfl Δ dld bacterial strain

Be transformed in MG1655 Δ dld bacterial strain, the MG1655 Δ pfl Δ dld bacterial strain by the plasmid pGlyldhA that will obtain among the embodiment 2, obtain MG1655 Δ dld/pGlyldhA bacterial strain, MG1655 Δ pfl Δ dld/pGlyldhA bacterial strain.

[embodiment 17] utilize MG1655 bacterial strain, MG 1655 Δ dld bacterial strains, MG1655 Δ pfl bacterial strain, MG1655 Δ pfl Δ dld bacterial strain, MG1655 Δ dld/pGlyldhA bacterial strain, MG1655 Δ pfl Δ dld/pGlyldhA bacterial strain to produce D-lactic acid

As pre-cultivation is that 25ml LB Broth is being housed, implant MG1655 bacterial strain, MG1655 Δ dld bacterial strain, MG1655 Δ pfl bacterial strain, MG1655 Δ pfl Δ dld bacterial strain, MG1655 Δ dld/pGlyldhA bacterial strain, MG1655 Δ pfl Δ dld/pGlyldhA bacterial strain in the Erlenmeyer flask of Miller nutrient solution, spend the night with the speed stir culture of 120rpm.Various pre-nutrient solution global transfer in the culture tank of the system culture apparatus BMJ-01 of ABLE society of the substratum that 475g composition shown in table 20 is housed, are cultivated.Under atmospheric pressure, air flow is that 0.5vvm, stirring velocity are that 200rpm, culture temperature are that 31 ℃, pH are under the condition of 6.7 (utilizing NaOH to regulate), cultivated 96 hours.After cultivating end,, utilize HPLC to measure the content of lactic acid, pyruvic acid, formic acid and acetate according to ordinary method.Result after 96 hours is shown in table 23.The bacterial strain name is expressed as A, B, C, D, E, F respectively.

Table 23

Various organic acid concentrations in the nutrient solution (the numerical value unit in the table all is g/L)

The structure of ldhA expression vector and ldhA expression vector transformant under the control of [embodiment 18] GAPDH promotor

The base sequence of the ldhA gene of escherichia coli is known (GenBank accessionnumber U36928).In order to obtain Glycerose 3-phosphate dehydrogenase (GAPDH) promotor, genomic dna with escherichia coli MG1655 bacterial strain is a template, utilize AACGAATTCTCGCAATGATTGACACGATTC (sequence number 13) and ACAGAATTCGCTATTTGTTAGTGAATAAAAGG (sequence number 14) to carry out pcr amplification, utilize restriction enzyme EcoRI to cut the dna fragmentation that obtains, obtain the fragment of the coding GAPDH promotor of about 100bp thus.Next, for obtaining D-serum lactic dehydrogenase structure gene (ldhA), genomic dna with escherichia coli MG1655 bacterial strain is a template, utilize GGAATTCCGGAGAAAGTCTTATGAAACT (sequence number 15) and CCCAAGCTTTTAAACCAGTTCGTTCGGGC (sequence number 16) to carry out pcr amplification, the dna fragmentation that utilizes restriction enzyme EcoRI and HindIII cutting to obtain, D-serum lactic dehydrogenase structure gene (ldh) fragment of the about 1.0kbp of acquisition thus.Above-mentioned 2 dna fragmentations are mixed with the fragment of utilizing restriction enzyme EcoRI and HindIII cutting plasmid pUC18 to obtain, and utilize ligase enzyme to connect, be transformed into then in the escherichia coli DH5 α competent cell (TAKARA BIO society system), on the LB agar plate that contains 50 μ g/ml penbritins, breed, obtain transformant.The bacterium colony that obtains on the LB liquid nutrient medium that contains 50 μ g/ml penbritins, in 30 ℃ of following overnight incubation, is reclaimed plasmid pGAPldhA from the thalline that obtains.This plasmid pGAPldhA is transformed in the MG1655 Δ pfl Δ dld bacterial strain,, obtains MG1655 Δ pfl Δ dld/pGA PldhA bacterial strain by overnight incubation on the LB agar plate that is containing 1 μ g/ml penbritin under 37 ℃.

The ldhA promotor is to the replacement of GAPDH promotor on the genome of [embodiment 19] escherichia coli MG1655 Δ pfl Δ dld bacterial strain

Whole base sequences of the genomic dna of escherichia coli are known (GenBankaccession number U00096), the base sequence of the ldhA gene of escherichia coli also be in the news (GenBank accession number U36928).Use is based on the AAGGTACCACCAGAGCGTTCTCAAGC (sequence number 17) and the GCTCTAGATTCTCCAGTGATGTTGAATCAC (sequence number 18) of the gene information preparation of 5 ' near zone of the ldhA gene that derives from escherichia coli MG1655 bacterial strain, with the escherichia coli genomic dna is that template is carried out PCR, the dna fragmentation of about 1000bp that increases thus.

In addition, utilization is based on the GGTCTAGAGCAATGATTCACACGATTCG (sequence number 19) of the sequence information of Glycerose 3-phosphate dehydrogenase (GAPDH) promotor of escherichia coli MG1655 bacterial strain preparation with based on the AACTGCAGGTTCGTTCTCATACACGTCC (sequence number 20) of the sequence information preparation of the ldhA gene of escherichia coli MG1655 bacterial strain, expression vector pGAPldhA with preparation among the embodiment 18 is a model, carry out pcr amplification, obtain the dna fragmentation of about 850bp of forming by the initiator codon near zone of GAPDH promotor and ldhA gene.

Utilize restriction enzyme KpnI and XbaI, XbaI and the above-mentioned fragment that obtains of PstI cutting respectively, this fragment is mixed with the fragment of utilizing KpnI and PstI cutting temperature susceptibility plasmid pTH18csl to obtain, and utilize ligase enzyme to connect, under 30 ℃, be transformed into then in the DH5 α competent cell (TAKARA BIO society system), on the LB agar plate that contains 10 μ g/ml paraxin, breed, obtain transformant.Bacterium colony overnight incubation on the LB liquid nutrient medium that is containing 10 μ g/ml paraxin under 30 ℃ with obtaining reclaims plasmid from the thalline that obtains, and called after pTH-GAPldhA.PTH-GAPldhA is transformed into MG1655 Δ pfl Δ dld bacterial strain under 30 ℃, and on 30 ℃ of LB agar plates that containing 10 μ g/ml paraxin overnight incubation, obtain transformant.The transformant that obtains is inoculated on the LB liquid nutrient medium that contains 10 μ g/ml paraxin, in 30 ℃ of overnight incubation.Next, for obtaining above-mentioned cultivation thalline, it is coated on the LB agar plate that contains 10 μ g/ml paraxin, 42 ℃ of breedings down obtain bacterium colony.With the gained bacterium colony in not containing the LB liquid nutrient medium of medicament in 30 ℃ of overnight incubation, and then be applied on the LB agar plate that does not contain medicament, 42 ℃ of breedings down obtain bacterium colony.

From the bacterium colony that occurs, select 100 bacterium colonies arbitrarily, it is bred not containing on the LB agar plate of medicament and contain on the LB agar plate of 10 μ g/ml paraxin respectively, select the clone body of paraxin susceptibility.Utilize the fragment of the about 800bp that comprises GAPDH promotor and ldhA gene in the chromosomal DNA of pcr amplification above-mentioned purpose clone body then, select the bacterial strain that the ldhA promoter region is replaced by the GAPDH promotor, the clone body called after MG1655 Δ pfl Δ dld/GAPldhA genome that satisfies above condition is inserted strain.

[embodiment 20] utilize MG1655 Δ pfl Δ dld bacterial strain, MG1655 Δ pfl Δ dld/pGAPldhA bacterial strain, MG1655 Δ pfl Δ dld/GAPldhA genome to insert strain and produce D-lactic acid

As pre-cultivation is that 25ml LB Broth is being housed, and implants MG1655 Δ pfl Δ dld bacterial strain, MG1655 Δ pfl Δ dld/pGAPldhA bacterial strain, MG1655 Δ pfl Δ dld﹠amp in the Erlenmeyer flask of Miller nutrient solution (Difco244620); The pflB/GAPldhA genome inserts strain, spends the night with the speed stir culture of 120rpm.Various pre-nutrient solution global transfer in the 1L volumetrical culture tank (the system culture apparatus BMJ-01 of ABLE society) that the substratum that 475g is made up of 120g/L glucose, 5% cereal extractive substance (japanese food chemical industry system) is housed, are cultivated.Under atmospheric pressure, air flow is that 0.5vvm, stirring velocity are that 200rpm, culture temperature are that 35 ℃, pH are under the condition of 7.2 (utilizing NaOH to regulate), be cultured to glucose and be consumed.After cultivating end,, utilize the D-lactic acid accumulation in the HPLC mensuration gained nutrient solution according to ordinary method.The result as shown in Figure 1.D-lactic acid is accumulated productivity and is respectively MG1655 Δ pfl Δ dld bacterial strain and at 30 hour was 113.5g/L at 48 hours for 115.6g/L, MG1655 Δ pfl Δ dld/GAPldhA genome insert strain for 109.0g/L, MG1655 Δ pfl Δ dld/pGAPldhA bacterial strain at 48 hours.

The preparation of [embodiment 21] escherichia coli MG1655 Δ pfl Δ dld Δ mdh bacterial strain

Whole base sequences of the genomic dna of escherichia coli are known (GenBankaccession number U00096), the base sequence of the mdh gene of escherichia coli also be in the news (GenBank accession number AE000403).For the base sequence near zone of the gene (939bp) of clones coding mdh, the oligonucleotide primer shown in synthetic 4 kinds of sequence numbers 21, sequence number 22, the sequence number 23 and 24.The primer that the primer that primer with base sequence of sequence number 21 has Kpn I recognition site in 5 ' end, have a base sequence of sequence number 22,23 has BamH I recognition site in 5 ' end, have a base sequence of sequence number 24 has Xba I recognition site in 5 ' end.

According to Current Protocols in Molecular Biology (JohnWiley﹠amp; Sons) described method prepares the genomic dna of escherichia coli MG1655 bacterial strain, the genomic dna 1 μ g that utilization obtains and the combination of sequence number 21 and sequence number 22, sequence number 23 and sequence number 24, and above-mentioned primed DNA respectively uses 100pmol, under common condition, carry out PCR, about 800bp that increases (below be also referred to as the mdh-L fragment), and the dna fragmentation of about 1000bp (below be also referred to as the mdh-R fragment).Utilize agarose electrophoresis to separate, reclaim above-mentioned dna fragmentation, utilize Kpn I and BamH I cutting mdh-L fragment, utilize BamH I and Xba I cutting mdh-R fragment.Utilize the T4DNA ligase enzyme to make above-mentioned 2 kinds of cutting fragments and thermal sensibility plasmid pTH18csl (GenBank accession number AB019610) (Hashimoto-Gotoh, T., et.al., Gene, Vol.241 (1), pp185-191 (2000)) after the product reaction of Kpn I and Xba I cutting, be transformed in the escherichia coli DH5 α competent cell (TAKARA BIO society system), thereby obtain having near the 5 ' upstream of gene of coding mdh near 2 segmental plasmids of the fragment fragment and 3 ' downstream, with its called after pTH Δ mdh.

Plasmid pTH Δ mdh is transformed in the escherichia coli MG1655 Δ pfl Δ dld bacterial strain, utilizes the method identical, the ruined MG1655 Δ of acquisition mdh gene pfl Δ dld bacterial strain with embodiment 5.With this bacterial strain called after MG1655 Δ pfl Δ dld Δ mdh bacterial strain.

The preparation of [comparative example 1] escherichia coli MG1655 Δ pflB Δ dld Δ ppc bacterial strain

Whole base sequences of the genomic dna of escherichia coli are known (GenBankaccession number U00096), the base sequence of the ppc gene of escherichia coli also be in the news (GenBank accession number AE000496).For the base sequence near zone of the gene (2652bp) of clones coding ppc, synthetic 4 kinds of oligonucleotide primers with the base sequence shown in sequence number 25,26,27 and 28.The primer of sequence number 26,27 has Xba I recognition site, sequence number 28 in 5 ' end primer has Sac I recognition site in 5 ' end.

Utilize the combination of genomic dna 1 μ g and sequence number 25 and sequence number 26, sequence number 27 and the sequence number 28 of escherichia coli MG1655 bacterial strain, and above-mentioned primed DNA respectively uses 100pmol, under common condition, carry out PCR, about 1450bp that increases (below be also referred to as the ppc-L fragment), and the dna fragmentation of about 750bp (below be also referred to as the ppc-R fragment).Utilize agarose electrophoresis to separate, reclaim above-mentioned dna fragmentation, utilize HindIII and Xba I cutting ppc-L fragment, utilize Xba I and SacI cutting ppc-R fragment.Utilize the T4DNA ligase enzyme to make above-mentioned 2 kinds of cutting fragments and thermal sensibility plasmid pTH18csl after the product reaction of HindIII and SacI cutting, be transformed in the escherichia coli DH5 α competent cell (TAKARA BIO society system), thereby obtain containing near near 2 the segmental plasmids of the fragment fragment and 3 ' downstream of encoding the 5 ' upstream of gene of ppc, with its called after pTH Δ ppc.

Plasmid pTH Δ ppc is transformed in the escherichia coli MG1655 Δ pfl Δ dld bacterial strain, obtains the ruined MG1655 Δ of ppc gene pfl Δ dld bacterial strain at last.With this bacterial strain called after MG1655 Δ pflB Δ dld Δ ppc bacterial strain.Need to prove that the detailed method that obtains this bacterial strain is based on the method described in the embodiments of the invention 5.

The preparation of [comparative example 2] escherichia coli MG1655 Δ pfl Δ dld Δ frd bacterial strain

Whole base sequences of the genomic dna of escherichia coli are known (GenBankaccession number U00096), the base sequence of the frd gene of escherichia coli also be in the news (GenBank accession number AE000487).The frd gene that lacks in the present embodiment is the gene of 4 kinds of genes of gene (360bp) of the gene (1809bp) that contains the frdA that encodes, the gene (735bp) of coding frdB, the gene (396bp) of coding frdC, the frdD that encodes.In order to clone the base sequence near zone of frd gene, synthetic 4 kinds of oligonucleotide primers with the base sequence shown in sequence number 29,30,31 and 32.The primer of sequence number 29 has EcoR I recognition site in 5 ' end, the primer of sequence number 30,31 has BamH I recognition site in 5 ' end, and the primer of sequence number 32 portion within it has the HindIII recognition site.

Utilize the combination of genomic dna 1 μ g and sequence number 29 and sequence number 30, sequence number 31 and the sequence number 32 of escherichia coli MG 1655 bacterial strains, and each 100pmol of above-mentioned primed DNA, under common condition, carry out PCR, about 600bp that increases (below be also referred to as the frd-L fragment), and the dna fragmentation of about 800bp (below be also referred to as the frd-R fragment).Utilize agarose electrophoresis to separate, reclaim above-mentioned dna fragmentation, utilize EcoR I and BamH I cutting frd-L fragment, utilize BamH I and HindIII cutting frd-R fragment.Utilize the T4DNA ligase enzyme to make above-mentioned 2 kinds of cutting fragments and thermal sensibility plasmid pTH18csl after the product reaction of EcoR I and HindIII cutting, be transformed in the escherichia coli DH5 α competent cell (TAKARABIO society system), thereby obtain having near the 5 ' upstream of gene of coding frd near 2 segmental plasmids of the fragment fragment and 3 ' downstream, with its called after pTH Δ frd.

Plasmid pTH Δ frd is transformed in the escherichia coli MG1655 Δ pfl Δ dld bacterial strain, obtains the ruined MG1655 Δ of frd genome pfl Δ dld bacterial strain at last.With this bacterial strain called after MG1655 Δ pfl Δ dld Δ frd bacterial strain.The detailed method that obtains this bacterial strain is based on the method described in the embodiment of the invention 5.

The preparation of [embodiment 22] escherichia coli MG1655 Δ pfl Δ dld Δ mdh Δ asp bacterial strain

Whole base sequences of the genomic dna of escherichia coli are known (GenBankaccession number U00096), the base sequence of the aspA gene of escherichia coli also be in the news (GenBank accession number AE000486).For the base sequence near zone of the gene (1482bp) of clones coding aspA, the oligonucleotide primer shown in synthetic 4 kinds of sequence numbers 33,34,35 and 36.

Utilize the combination of genomic dna 1 μ g and sequence number 33 and sequence number 34, sequence number 35 and the sequence number 36 of escherichia coli MG1655 bacterial strain, and above-mentioned primed DNA respectively uses 100pmol, under common condition, carry out PCR, about 910bp that increases (below be also referred to as the aspA-L fragment), and the dna fragmentation of about 1100bp (below be also referred to as the aspA-R fragment).Utilize agarose electrophoresis to separate, reclaim above-mentioned dna fragmentation, utilize DNA BluntingKit (TAKARA BIO society system) with aspA-L fragment and the segmental terminal smoothing of aspA-R after, utilize the T4 polynucleotide kinase, according to the ordinary method phosphorylase 15 ' end.On the other hand, thermal sensibility plasmid pTH18csl utilizes alkaline phosphatase to carry out dephosphorylation and handles after Sma I cutting.After utilizing the T4DNA ligase enzyme to make the plasmid reaction of 2 kinds of fragments of above-mentioned phosphorylation and dephosphorylation, be transformed in the escherichia coli DH5 α competent cell (TAKARABIO society system), thereby obtain containing near near 2 the segmental plasmids of the fragment fragment and 3 ' downstream of encoding the 5 ' upstream of gene of aspA, with its called after pTH Δ asp.

Plasmid pTH Δ asp is transformed in the escherichia coli MG1655 Δ pfl Δ dld Δ mdh bacterial strain, obtains the ruined MG1655 Δ of aspA genome pfl Δ dld Δ mdh bacterial strain at last.With this bacterial strain called after MG1655 Δ pfl Δ dld Δ mdh Δ asp bacterial strain.The detailed method that obtains this bacterial strain is based on the method described in the embodiment of the invention 5.

[embodiment 23] escherichia coli MG1655 Δ pfl Δ dld Δ mdh Δ asp bacterial strain/GAPldhA genome inserts the preparation of strain

The plasmid pTH-GAPldhA that obtains among the embodiment 19 is transformed under 30 ℃ in the MG1655 Δ pfl Δ dld Δ mdh Δ asp bacterial strain, and on the LB agar plate that contains 10 μ g/ml paraxin in 30 ℃ of following overnight incubation, transformant.The transformant that obtains is inoculated in the LB liquid nutrient medium that contains 10 μ g/ml paraxin, in 30 ℃ of overnight incubation.Next, in order to obtain above-mentioned cultivation thalline, it is coated on the LB agar plate that contains 10 μ g/ml paraxin, 42 ℃ of breedings down obtain bacterium colony.With the gained bacterium colony under 30 ℃ in not containing the LB liquid nutrient medium of medicament overnight incubation, be applied to then on the LB agar plate that does not contain medicament, 42 ℃ of down breedings obtain bacterium colony.

From the bacterium colony that occurs, select 100 bacterium colonies arbitrarily, breed not containing on the LB agar plate of medicament and contain on the LB agar plate of 10 μ g/ml paraxin respectively, select the clone body of paraxin susceptibility.Utilize the fragment of the about 800bp that comprises GAPDH promotor and ldhA gene in the chromosomal DNA of pcr amplification above-mentioned purpose clone body then, select the bacterial strain that the ldhA promoter region is replaced by the GAPDH promotor, the clone body called after MG1655 Δ pfl Δ dld Δ mdh Δ asp/GAPldhA genome that satisfies above condition is inserted strain.

[embodiment 24] utilize escherichia coli MG1655 Δ pfl Δ dld Δ mdh bacterial strain to produce D-lactic acid, reach succsinic acid

As pre-cultivation is at 4 25ml LB Broth to be housed, in the Erlenmeyer flask of Miller nutrient solution (Difco244620), implant the escherichia coli MG1655 Δ pfl Δ dld Δ frd bacterial strain that obtains in the escherichia coli MG1655 Δ pfl Δ dld Δ ppc bacterial strain that obtains in the escherichia coli MG1655 Δ pfl Δ dld Δ mdh bacterial strain that obtains among the escherichia coli MG1655 Δ pfl Δ dld bacterial strain that obtains among the embodiment 15, the embodiment 21, the comparative example 1, the comparative example 2 respectively, the speed stir culture with 120rpm under 30 ℃ is spent the night.Then, in 4 1L volumetrical culture tanks that substratum shown in the 475g table 24 is housed (the system culture apparatus BMJ-01 of ABLE society), implant the entire contents of above-mentioned flask respectively.Under atmospheric pressure, air flow is that 0.5vvm, stirring velocity are that 200rpm, culture temperature are that 35 ℃, pH are under the condition of 7.2 (utilizing NaOH to regulate), cultivated 32 hours.After cultivate finishing,, utilize HPLC to measure lactic acid in the nutrient solution that obtains, and the concentration of succsinic acid according to ordinary method.Lactic acid is accumulated the result as shown in Figure 2, succsinic acid accumulate the result as shown in Figure 3.

About lactic acid, 32 hours accumulation of Δ pfl Δ dld Δ mdh bacterial strain is 89g/L, has shown and the equal degree of accumulating of Δ pfl Δ dld bacterial strain, and in Δ pfl Δ dld Δ ppc bacterial strain, Δ pfl Δ dld Δ frd bacterial strain, has been respectively 56g/L, 71g/L.

About succsinic acid, 32 hours accumulation of Δ pfl Δ dld bacterial strain is 3.8g/L, and all the other 3 kinds of bacterial strains are not all seen and accumulated.

Table 24

(remainder: water)

[embodiment 25] utilize escherichia coli MG 1655 Δ pfl Δ dld Δ mdh Δ asp bacterial strains and MG1655 Δ pfl Δ dld Δ mdh Δ asp/GAPldhA genome to insert strain and produce D-lactic acid, reach fumaric acid

As pre-cultivation is at 3 25ml LB Broth to be housed, in the Erlenmeyer flask of Miller nutrient solution (Difco244620), implant respectively the escherichia coli MG1655 Δ pfl Δ dld Δ mdh Δ asp/GAPldhA genome that obtains among the escherichia coli MG1655 Δ pfl Δ dld Δ mdh Δ asp bacterial strain that obtains among the embodiment 22, the embodiment 23 insert strain, and embodiment 21 in the Δ pfl Δ dld Δ mdh that obtains, the speed stir culture with 120rpm under 30 ℃ is spent the night.Then, in 3 1L volumetrical culture tanks that the substratum shown in the 475g table 24 is housed (the system culture apparatus BMJ-01 of ABLE society), implant the entire contents of above-mentioned flask respectively.Under atmospheric pressure, air flow is that 0.5vvm, stirring velocity are that 200rpm, culture temperature are that 35 ℃, pH are under the condition of 7.2 (utilizing NaOH to regulate), cultivated 48 hours.After cultivate finishing,, utilize HPLC to measure lactic acid in the nutrient solution that obtains, and the concentration of fumaric acid according to ordinary method.Lactic acid is accumulated the result as shown in Figure 4, fumaric acid accumulate the result as shown in Figure 5.

About lactic acid, 48 hours accumulation of Δ pfl Δ dld Δ mdh Δ asp bacterial strain is 91g/L, 48 hours accumulation of Δ pfl Δ dld Δ mdh bacterial strain is 90g/L, the two has shown the equal degree of accumulating, and insert in the strain at Δ pfl Δ dld Δ mdh Δ asp/GAPldhA genome, 24 hours accumulation is 98g/L.

About fumaric acid, in Δ pfl Δ dld Δ mdh bacterial strain, 48 hours accumulation is 0.037g/L, and inserts in the strain at Δ pfl Δ dld Δ mdh Δ asp bacterial strain and Δ pfl Δ dld Δ mdh Δ asp/GAPldhA genome, and 48 hours accumulation is 0.01g/L.

Sequence table

＜110〉Mitsui Chemicals, Inc

＜120〉D-lactic acid-producing biological catalyst

<130>F000364

<160>36

<210>1

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>1

ggaattcgtc?gaccggctcc?agttcgaagc?tggt 34

<210>2

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>2

ggaattctga?ctcagctaac?aataaaattt?tt 32

<210>3

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>3

ggaattccgg?agaaagtctt?atgaaact 28

<210>4

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>4

cccaagcttt?taaaccagtt?cgttcgggc 29

<210>5

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>5

gcacgaaagc?tttgattacg 20

<210>6

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>6

ttattgcatg?cttagatttg?actgaaatcg 30

<210>7

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>7

ttattgcatg?cttatttact?gcgtacttcg 30

<210>8

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>8

aaggcctacg?aaaagctgca?g 21

<210>9

<211>18

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>9

caacaccaag?ctttcgcg 18

<210>10

<211>19

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>10

ttccactcct?tgtggtggc 19

<210>11

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>11

aactgcagaa?attacggatg?gcagag 26

<210>12

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>21

tgttctagaa?agttctttga?c 21

<210>13

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>13

aacgaattct?cgcaatgatt?gacacgattc?30

<210>14

<211>32

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>14

acagaattcg?ctatttgtta?gtgaataaaa?gg 32

<210>15

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>15

ggaattccgg?agaaagtctt?atgaaact 28

<210>16

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>16

cccaagcttt?taaaccagtt?cgttcggggc 30

<210>17

<211>26

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>17

aaggtaccac?cagagcgttc?tcaagc 26

<210>18

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>18

gctctagatt?ctccagtgat?gttgaatcac 30

<210>19

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>19

ggtctagagc?aatgattcac?acgattcg 28

<210>20

<211>28

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>20

aactgcaggt?tcgttctcat?acacgtcc 28

<210>21

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>21

aaaggtacca?gaataccttc?tgctttgccc?30

<210>22

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>22

aaaggatccc?ctaaactcct?tattatattg?30

<210>23

<211>30

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>23

aaaggatcca?aaccggagca?cagactccgg?30

<210>24

<211>

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>24

aaatctagaa?tcagatcatc?gtcgccttac?30

<210>25

<211>

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>25

acggagcatg?acggcaagc 19

<210>26

<211>

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>26

aatctagaca?ccccatctta?tcgtttg 27

<210>27

<211>

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>27

tttctagatc?ttcctcttct?gcaaaccc 28

<210>28

<211>

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>28

ctttgagctc?acgcgaggcc?aggttatc?28

<210>29

<211>

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>29

agtgaattct?cacagccagt?gcgccga 27

<210>30

<211>

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>30

agtggatccc?gcatcgccaa?tgtaaatcc 29

<210>31

<211>

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>31

agtggatccg?acattcctcc?agattgtttt?t?31

<210>32

<211>

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>32

ataacgcaag?aaagcttgtt?ga 22

<210>33

<211>

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>33

ttttgagctc?gatcaggatt?gcgttggtgg 30

<210>34

<211>

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>34

cgaacagtaa?tcgtacaggg 20

<210>35

<211>

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>35

tacgattact?gttcggcatc?gaccgaatac?ccgag?35

<210>36

<211>

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PCR primer

<400>36

tttttctaga?cctggcacgc?ctctcttctc 30

Claims (8)

1, a kind of production method of D-lactic acid, it is characterized in that, the activity that cultivation derives from the NADH dependency D-serum lactic dehydrogenase (ldhA) of escherichia coli is enhanced and pyruvate formate-lyase (pfl) inactivation or the active escherichia coli that reduces, and reclaims D-lactic acid from the culture that obtains.

2, the production method of D-lactic acid as claimed in claim 1 is characterized in that, cultivates in the amino acid whose substratum having added more than 2 kinds or 2 kinds.

3, a kind of escherichia coli, this escherichia coli is its FAD dependency D-serum lactic dehydrogenase (dld) inactivation that had originally or the active escherichia coli that reduces, it is characterized in that, pyruvate formate-lyase (pfl) inactivation or active reduce and/or derive from the NADH dependency D-serum lactic dehydrogenase (ldhA) of escherichia coli activity be enhanced.

4, a kind of production method of D-lactic acid is characterized in that, cultivates escherichia coli as claimed in claim 3 in the liquid medium within, D-lactic acid is generated in nutrient solution and accumulates separation D-lactic acid from nutrient solution.

5, the production method of D-lactic acid as claimed in claim 4 is characterized in that, cultivates in the amino acid whose substratum having added more than 2 kinds or 2 kinds.

6, as the production method of each described lactic acid in the claim 1,2,4,5, it is characterized in that, under aeration condition, cultivate.

7, the production method of lactic acid as claimed in claim 6 is characterized in that, described aeration condition is, is the situation of object under at the water that is 30 ℃ with temperature, makes oxygen volume transmission quality coefficient K under the normal pressure by supplying with oxygen _LA is more than or equal to 1h ^-1, be less than or equal to 400h ^-1Scope in condition.

As the production method of each described lactic acid in the claim 1,2,4,5, it is characterized in that 8, cultivating pH is 6～8.

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