CN103993048B - With the method for the bacterial fermentation production L-threonine for changing aconitase controlling element - Google Patents

Abstract

The present invention provides the methods of production of L-threonine by fermentation comprising on transformation bacterial chromosomeacnAThe controlling element of the wild type of gene makes the expression quantity of its aconitase A reduce but not disappear；With produce L-threonine with the bacterial fermentation of transformation.In addition, the present invention also provides the methods and applications as derived from this method, and the bacterium etc. that can be used in these methods and applications.

Description

With the method for the bacterial fermentation production L-threonine for changing aconitase controlling element

Technical field

The invention belongs to field of amino acid fermentation, specifically, the present invention relates to the method for fermenting and producing l- threonine and Its derivative methods and applications, and the bacterium etc. that can be used in these methods and applications.

Background technique

By bacterium (e.g., the bacillus of the Escherichia coli of the Escherichia and Corynebacterium) fermentation for producing l- threonine Industrial application has been obtained to produce l- threonine.These bacteriums can be the bacterium separated from nature, be also possible to Have both at the same time by the bacterium, or both that mutagenesis or genetic engineering transformation obtain.In current document report, pass through gene work The attention of journey transformation is concentrated mainly onAKIII、thrR(referring to Chinese patent 94102007 and 99121353 etc.) on equal genes, It has no for L-threonine production and pays close attention to aconitase (e.g., aconitase A) and its encoding gene.

Aconitase is an enzyme in tricarboxylic acid cycle, and two step of enzymatic chemical reaction, respectively citric acid convert Isocitric acid is converted into for aconitic acid and aconitic acid.It is currently known in Escherichia,acnA(its nucleotide sequence is such as gene Shown in SEQ ID No:1) coding aconitase A, but may be since its metabolism is too far apart from final l- threonine product, Intermediate supersession branch is too many and complicated, and does not attract people's attention always in l- Threonine Fermentation.

The present inventor has especially relied on some fortune, has chanced on by studying for a long period of time and practicingacnAThe regulation of gene The transformation of element can aid in the yield for improving l- threonine；However, the prior art otherwise by increase copy and/or fixed point Mutation imports the beneficial enzyme gene of expression quantity and/or enzymatic activity raising or by knocking out unfavorable gene to be allowed to enzyme activity Property and/or expression quantity disappear, but it is different, the inventors discovered that,acnAThe controlling element of gene cannot be simply It improves or knocks out, after especially knocking outacnAGene is not expressed so that bacterial growth is difficult, it is difficult to practical application, therefore develop New is directed toacnAThe method of the controlling element of gene, improves the yield of l- threonine with this, and this method with it is existing The chromosome transformation site of the bacterium of a large amount of high yield L-threonines of transformation does not conflict, and can be superimposed the effect of raising, thus It can be used for bacterial fermentation production l- threonine in practice.

Summary of the invention

The technical problem to be solved in the present invention is that provide new fermenting and producing l- threonine method and its relevant side Method, including the method relative to the fermenting and producing amount that bacterium raising l- threonine is not transformed, the bacterium of transformation is in fermenting and producing l- Application in threonine, the bacterium of transformation relative to application bacterium is not transformed improved the fermenting and producing amount of l- threonine, and/ Or, the method etc. of transformation bacterium.In addition, the present invention also provides the polynucleotides that can be used in the above method, carrier and/or Bacterium etc..

Specifically, in a first aspect, the present invention provides the methods of fermenting and producing l- threonine comprising:

(1) it is transformed on bacterial chromosomeacnAThe controlling element of the wild type of gene, the rhizome of Chinese monkshood for the bacterium for obtaining transformation The expression quantity of sour enzyme A is reduced but is not disappeared；With,

(2) bacterial fermentation obtained from step (1) transformation produces l- threonine.

Herein, term " transformation " refers to it being that the object being accordingly modified changes, to reach certain effect Fruit.The frequency of gene of the frequency of controlling element of the transformation on chromosome much smaller than transformation on chromosome, but two The technological means that person is transformed is almost the same, including but not limited to, mutagenesis, rite-directed mutagenesis, and/or homologous recombination, and preferably Both after being.These technological means are recorded in extensively in molecular biology and microbiology document, and there are many even commodity Change.In a specific embodiment of the invention, according to the principle of homologous recombination, the pKOV being commercialized using Addgene company PUC pUC is transformed, and will be transformed on bacterial chromosomeacnAThe controlling element of the wild type of gene, being transformed into can The new controlling element that the expression quantity of the aconitase A for the bacterium for obtaining transformation is reduced but do not disappeared.Therefore, in text herein In, preferably transformation is the transformation carried out by homologous recombination.

The present inventor passes through the discovery that studies for a long period of time, so thatacnAThe expression quantity of the aconitase A of coded by said gene disappears, will It causes bacterium itself to grow difficulty, or even can not grow/breed.Therefore, " transformation " of the invention is thin relative to what is be not transformed The expression quantity of bacterium, the aconitase A for the bacterium for obtaining transformation is reduced but is not disappeared, the rhizome of Chinese monkshood for the bacterium for preferably obtaining transformation The expression quantity of sour enzyme A reduces by 20% ~ 95%, more preferably reduces by 50% ~ 90%, such as reduces by 65%, 70% or 80%.

Correspondingly, the present invention also provides other application or methods.For example, the present invention provides raisings in second aspect The method of the amount of fermentation of l- threonine comprising:

(1) it is transformed on bacterial chromosomeacnAThe controlling element of the wild type of gene, the rhizome of Chinese monkshood for the bacterium for obtaining transformation The expression quantity of sour enzyme A is reduced but is not disappeared；With,

(2) bacterial fermentation obtained from step (1) transformation generates l- threonine.

Important metabolite of the l- threonine as bacterium, most of bacteriums can more or less ferment generate it is a certain amount of L- threonine.Although the bacterium of low yield L-threonine is not suitable for producing l- threonine with having an economic benefit, by this hair Bright method can still improve the amount of fermentation of l- threonine, can still use for the place insensitive to economic benefit.When So, herein, preferred bacterium is the bacterium of high yield L-threonine.By means of the invention it is also possible to further increase its production Amount.In addition, in method or application of the invention, in addition on transformation bacterial chromosomeacnAThe controlling element of the wild type of gene In addition, other transformations can be no longer carried out, such as the wild type that can not be even transformed on bacterial chromosomeacnAGene.Example Such as, it for particularly with the bacterium of high yield L-threonine, is only transformed on bacterial chromosomeacnAThe regulation of the wild type of gene Element.

For another example, in the third aspect, application of the bacterium obtained the present invention provides transformation in fermenting and producing l- threonine, Wherein, the transformation acquisition is on transformation bacterial chromosomeacnAThe controlling element of the wild type of gene and obtain, and make to change The expression quantity for making the aconitase A of the bacterium of acquisition is reduced but is not disappeared.

The bacterium that transformation obtains can be applied individually to any in fermenting and producing l- threonine, can also produce L-threonine with other Bacterium mixed fermentive produce l- threonine, or otherwise be applied to fermenting and producing l- threonine in.Herein, such as It is not particularly limited (as do not limited with " transformation obtain "), term " bacterium " is the bacterium before not being transformed or be transformed, chromosome 'sacnAControlling element before and after gene locus is the controlling element of wild type.It is preferred that herein, bacterium is the thin of production threonine Bacterium such as produces the bacterium that threonine is more than 0.5g/L.

Also such as, in fourth aspect, the present invention provides the bacteriums that transformation obtains to improve answering for the amount of fermentation of l- threonine With, wherein the transformation acquisition is on transformation bacterial chromosomeacnAThe controlling element of the wild type of gene and obtain, and make The expression quantity that the aconitase A of the bacterium obtained is transformed is reduced but is not disappeared.

Herein, bacterium is preferably Escherichia bacteria, more preferably Escherichia coli.Since the prior art does not almost have Have and paid close attention to bacterium in l- threonine production/fermentationacnAThe gene of the controlling element of gene, the chromosome of transformation is most It concentrates on other gene locis, or even is all not concerned with controlling element, therefore bacterium (especially Escherichia in the prior art Belong to bacterium, such as Escherichia coli)acnAThe controlling element not being reported before and after gene without wild type.In tool of the invention In body embodiment, no matter the bacterium of high yield or low yield L-threonine, as long as havingacnAThe regulation member of the wild type of gene Part is transformed by means of the present invention, and the amount of fermentation of L-threonine can be made to be improved.

More constitutionally, at the 5th aspect, the present invention provides the methods of transformation bacterium comprising the Bacterial stain is transformed On bodyacnAThe expression quantity of the controlling element of the wild type of gene, the aconitase A for the bacterium for obtaining transformation reduces but does not disappear It loses.

The bacterium that the method for fifth aspect present invention is transformed and is obtained can be used in fermenting and producing or generate L-threonine.Cause This, in the 6th aspect, the bacterium obtained the present invention provides the transformation of the method for fifth aspect present invention.

The controlling element of wild type can be promoter, enhancer or the promoter region sequence of upstream region of gene, can also be with It is downstream of gene sequence relevant to expression regulation.Preferably, herein, the controlling element of the wild type is wild type Promoter.In a specific embodiment of the invention, the nucleotide sequence of the promoter of the wild type such as SEQ ID No:1 institute Show, the amount of fermentation that L-threonine is improved after being replaced is proved.

It studies and confirms by the present inventor, it is highly preferred that herein, on the transformation bacterial chromosomeacnAGene Wild type controlling element be will be on chromosomeacnAThe controlling element of the wild type of gene replaces with weak transcriptional regulatory element, It is preferred that weak transcripting promoter is replaced with, such as nucleotide sequence weak transcripting promoter as shown in SEQ ID No:2.

It is preferred, therefore, that the production threonine bacterium of sixth aspect present invention, does not include wild as shown in SEQ ID No:1 The nucleotide sequence of the promoter of type.

The production threonine bacterium of more preferable sixth aspect present invention, it includes the polynucleotides of following 7th aspect.

In addition, the present invention also provides the substances such as the polynucleotides that can be used in the above method and/or carrier.For example, At the 7th aspect, the present invention provides polynucleotides, the nucleotide sequence of the polynucleotides is as shown in SEQ ID No:2.Again Such as, in eighth aspect, the present invention provides carriers, and it includes the polynucleotides of seventh aspect present invention.

The beneficial effects of the present invention are open up and facts have proved the side of the amount of fermentation of new raising L-threonine Formula is all suitable for the bacterium of high yield and low yield L-threonine, and the bacterium with a large amount of high yield L-threonines of existing transformation Chromosome transformation site do not conflict, observed can be superimposed the effect for improving yield, to can be used in practice thin Bacterium fermenting and producing l- threonine, it is easy to promote and utilize.

In order to make it easy to understand, the present invention will be described in detail by specific embodiment below.It needs to refer in particular to Out, these descriptions are only exemplary description, and are not meant to limit the scope of the invention.Opinion according to this specification It states, many variations of the invention, change will be apparent from for those skilled in the art.

In addition, the present invention refers to open source literature, these documents are their full text in order to more clearly describe the present invention Content is included in and is referred to herein, just looks like that repeated description herein has been excessively for their full text.

Specific embodiment

The contents of the present invention are further illustrated by the following examples.Such as not specified, technology used in embodiment The conventional means and commercially available common instrument, reagent that means are well known to those skilled in the art, reference can be made to " Molecular Cloning: A Laboratory Guide (the 3rd edition) " (Science Press), " Microbiology Experiment (the 4th edition) " (Higher Education Publishing House) and corresponding instrument and The reference such as manufacturers instruction of reagent.

Embodiment is constructed to be replaced with the weaker promoter of transcriptional activityacnAPromoter

By rightE. coli In K12 W3110acnAUpstream sequence analysis, we devise weak transcripting promoter (sequence As shown in SEQ ID No:2) and entrust Institute of Micro-biology, the Chinese Academy of Sciences to synthesize and be built into pMD-19T plasmid and (it is raw to be purchased from Dalian treasured Object company) in, to replaceacnAPromoter region (the sequence such as SEQ ID No:1 institute of the wild type of gene ORF upstream 196bp Show), to weaken wild typeacnAThe intensity of gene.

Specifically, with the wild-type e. coli of extractingE. coli K12 W3110 genome chromosome is template, with Primer P1 and P2, P3 and P4 carry out PCR amplification respectively, obtain two DNA fragmentations that length is respectively 486 bp and 619 bp (being respectively designated as Up2 and Down2 segment).To contain the pMD-19T plasmid of above-mentioned weak transcripting promoter, carried out with P5 and P6 PCR amplification obtains the weak transcripting starting sub-piece (being named as P segment) that length is 161 bp.Wherein, PCR as follows into Row: 94 DEG C are denaturalized 30 s(second), 52 DEG C are annealed 30 s(second) and 72 DEG C of extensions 30 s(seconds) (30 recycle).

By above-mentioned three DNA fragmentations after agarose gel electrophoresis isolates and purifies, then with the mixing of above-mentioned Up2 and P segment The segment (being named as Up-P segment) of 622bp is about by Overlap PCR amplification using P1 and P6 as primer for template.Its In, PCR is carried out as follows: 94 DEG C of denaturation 30s(seconds), 52 DEG C are annealed 30 s(second) and 72 DEG C of extensions 60 s(seconds) (30 A circulation).

Up-P and Down2 segment after agarose gel electrophoresis is isolated and purified is that template passes through using P1 and P4 as primer Overlap PCR amplification is about the segment (being named as Up-P-down segment) of 1240bp.Wherein, PCR is carried out as follows: 94 DEG C are denaturalized 30 s(second), 52 DEG C are annealed 30 s(second) and 72 DEG C of extensions 60 s(seconds) (30 recycle).

Above-mentioned primer sequence used is as follows:

P1: 5’-CGCGGATCCGTGATGGCGATTATATGAGG-3’

P2:5’-GGTTTCTTAGACGTCGGATTGAGAAAACGCGCCCATCCAGGA-3’

P3:5’-ATCAGCAGGACGCACTGACCCATTAAGGAGGAGCTATGTCG-3’

P4: 5’-ATTGCGGCCGCTCCATTCACCGTCCTGCAATT-3’

P5:5’-TCCTGGATGGGCGCGTTTTCTCAATCCGACGTCTAAGAAACC-3’

P6:5’-CGACATAGCTCCTCCTTAATGGGTCAGTGCGTCCTGCTGAT-3’

Up-P-down segment and pKOV plasmid after agarose gel electrophoresis is isolated and purified (are purchased from Addgene public affairs Department) it uses respectivelyBamHI/NotI double digestion connects after agarose gel electrophoresis isolates and purifies, and obtains the carrier for importing PKOV-Up-P-Down, and send sequencing company to carry out sequencing identification carrier pKOV-Up-P-Down, show that it contains correctly Weak transcriptional promoter sequence, saves backup.

By the pKOV-Up-P-Down plasmid built, electrotransformation enters common low yield L-threonine respectivelyE. coli MG442 bacterial strain (is purchased from Russia's National Industrial Organism Depositary (VKPM), deposit number CMIM B-1628；Its building side Method can be found in US4278765A) and high yield L-threonineE. coli BKIIM B-3996 bacterial strain (is purchased from Russia's antibiotic Research institute, registration number 1867；Its construction method can be found in US5175107A) (confirm on the two strain chromosomes through sequencing Remain with the acnA gene (i.e. 1333855 to 1336530 in the record such as Genbank U00096.2) and thereon of wild type Downstream components), after 30 DEG C, 100 rpm, 2 h that recover in LB culture medium, according to the quotient of the pKOV plasmid of Addgene company Product guide picks out the monoclonal of the homologous recombination positive, confirms the wild type on its chromosome through sequencingacnAThe upstream of gene Promoter is replaced by weak transcripting promoter, respectively obtainsacnA(production L-threonine low/high) Escherichia coli of promoter mutation. Through detecting, in different medium, the expression quantity of the aconitase of the two bacterial strains has 65 ~ 80% decline.

The experiment of effect example Threonine Fermentation

It willE. coli BKIIM B-3996 bacterial strain,E. coli MG442 bacterial strain and the bacterial strain of Examples 1 and 2 difference It is seeded in culture medium described in 25mL table 1, cultivates 12 h in 37 DEG C, 200rpm.Then the culture of 1 mL culture medium is taken to connect Kind is in the culture medium described in 25 mL tables 1, in 37 DEG C, 36 h of 200rpm culture culture.When cultivating completion, surveyed by HPLC Determine the generation of l- threonine.

1 culture medium prescription of table

Ingredient	g/L
		Glucose	40
Ammonium sulfate	12
		Potassium dihydrogen phosphate	0.8
Epsom salt	0.8
		Ferrous sulfate heptahydrate	0.01
Manganese sulfate monohydrate	0.01
		Yeast extract	1.5
Calcium carbonate	0.5
		L- methionine	0.5
KOH	pH 7.0

As a result,E. coli The l- Soviet Union of the bacterial strain of BKIIM B-3996 bacterial strain and the high yield l- threonine of building embodiment Propylhomoserin yield is respectively 16.2 g/L and 18.0g/L；E. coli MG442 bacterial strain and the low yield l- threonine for constructing embodiment The l- production amount of threonine of bacterial strain be respectively 1.9 g/L and 2.3 g/L, it is seen then that no matter for high yield or low yield L-threonine Original strain,acnAThe Escherichia coli of the promoter mutation of gene both contribute to the raising of l- production amount of threonine.Especially mesh The preceding engineered strain for producing L-threonine, does not report and was transformedacnAThe promoter of gene, thus to further increase L- Soviet Union ammonia Acid yield brings wide prospect.

<110>Ningbo Eppen Biotech Co., Ltd.

<120>with the method for the bacterial fermentation production L-threonine for changing aconitase controlling element

<130> CN

<160> 2

<170> PatentIn version 3.5

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<212> DNA

<213>Escherichia coli

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gttatcaaat cgttacgcga tgtttgtgtt atctttaata ttcaccctga agagaatcag 180

ggcttcgcaa ccctgt 196

<210> 2

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<213>Escherichia coli

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caatccgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc 60

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agtgatagag atactgagca catcagcagg acgcactgac c 161

Claims (8)

1. the method for production of L-threonine by fermentation comprising:

(1) transformation produces the controlling element of the wild type of acnA gene on the bacterial chromosome of L-threonine, obtains transformation thin The expression quantity of the aconitase A of bacterium is reduced but is not disappeared, wherein the transformation produces acnA base on the bacterial chromosome of L-threonine The controlling element of the wild type of cause is that the promoter of the wild type of acnA gene on chromosome is replaced with to nucleotide sequence such as SEQ Weak transcripting promoter shown in ID No:2；With,

(2) bacterial fermentation obtained from step (1) transformation produces L-threonine；

And wherein, the formula that culture medium used is produced described in step (2) is as follows:

Ingredient g/L Glucose 40 Ammonium sulfate 12 Potassium dihydrogen phosphate 0.8 Epsom salt 0.8 Ferrous sulfate heptahydrate 0.01 Manganese sulfate monohydrate 0.01 Yeast extract 1.5 Calcium carbonate 0.5 L-methionine 0.5, The pH value that KOH adjusting culture medium is added reaches pH 7.0

。

2. method described in claim 1, wherein the nucleotide sequence of the promoter of the wild type such as SEQ ID No:1 institute Show.

3. method described in claim 1, wherein the bacterium is Escherichia bacteria.

4. method as claimed in claim 3, wherein the bacterium is Escherichia coli.

5. the method for improving the amount of fermentation of L-threonine comprising:

(1) transformation produces the controlling element of the wild type of acnA gene on the bacterial chromosome of L-threonine, obtains transformation thin The expression quantity of the aconitase A of bacterium is reduced but is not disappeared, wherein the transformation produces acnA base on the bacterial chromosome of L-threonine The controlling element of the wild type of cause is that the promoter of the wild type of acnA gene on chromosome is replaced with to nucleotide sequence such as SEQ Weak transcripting promoter shown in ID No:2；With,

(2) bacterial fermentation obtained from step (1) transformation generates L-threonine；

And wherein, the formula of the culture medium used in fermenting described in step (2) is as follows:

Ingredient g/L Glucose 40 Ammonium sulfate 12 Potassium dihydrogen phosphate 0.8 Epsom salt 0.8 Ferrous sulfate heptahydrate 0.01 Manganese sulfate monohydrate 0.01 Yeast extract 1.5 Calcium carbonate 0.5 L-methionine 0.5, The pH value that KOH adjusting culture medium is added reaches pH 7.0

。

6. method described in claim 5, wherein the nucleotide sequence of the promoter of the wild type such as SEQ ID No:1 institute Show.

7. method described in claim 5, wherein the bacterium is Escherichia bacteria.

8. method of claim 7, wherein the bacterium is Escherichia coli.