CN109486735A

CN109486735A - A kind of engineering lactic acid bacteria and its application that acid stress resistance improves

Info

Publication number: CN109486735A
Application number: CN201811364384.XA
Authority: CN
Inventors: 张娟; 杨佩珊; 刘为佳; 陈坚; 堵国成
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2018-11-16
Filing date: 2018-11-16
Publication date: 2019-03-19
Anticipated expiration: 2038-11-16
Also published as: CN109486735B

Abstract

The invention discloses engineering lactic acid bacterias and its application that a kind of acid stress resistance improves, belong to genetic engineering and microbial engineering field.The present invention is using the gene of code energy coupling factor transport protein ATP enzyme EcfA2 as purpose gene, using lactic acid bacteria as expressive host, successfully constructs a kind of engineering lactic acid bacteria that can be widely applied to prepare food, drug, feed and chemicals；The acid stress resistance of this engineering lactic acid bacteria is significantly improved, and improves 598.7 times compared with wild strain highest.

Description

A kind of engineering lactic acid bacteria and its application that acid stress resistance improves

Technical field

The present invention relates to engineering lactic acid bacterias and its application that a kind of acid stress resistance improves, belong to genetic engineering and micro- Technical field of bioengineering.

Background technique

Lactic acid bacteria is the common name of a kind of bacterium that a large amount of lactic acid can be generated using fermentable carbohydrate.This kind of bacterium exists Distributed in nature is extremely wide, has species diversity abundant.They are not only research classification, biochemical, heredity, molecular biosciences The ideal material with genetic engineering is learned, theoretically there is important learning value, and in food, medicine, feed, fining The essential industry field closely related with human lives such as product also has important application value.

But it produces in the industrial fermentation of lactic acid bacteria and plays a role in human gastrointestinal tract system as probiotics In the process, be inevitably faced with a variety of environment-stress from external environment, including acid stress, ethyl alcohol stress, Oxdative stress, Salt stress etc., these environment-stress seriously limit lactobacter growth performance.

And in numerous stress conditions that lactic acid bacteria faces, acid stress is the important stress conditions for influencing its physiological activity.

Acid stress is as caused by the acidic materials such as lactic acid bacteria metabolite lactic acid, acetic acid, with the metabolism growth of thallus Process, acidic materials are also generated and are accumulated therewith, and the acidic materials such as lactic acid, acetic acid of these accumulation are thin by passively diffusing into Cytoplasm is dissociated rapidly into lactic acid intracellular, acetic acid etc., is caused due to the usually more extracellular pH high 0.5~1.0 of pH intracellular The quick reduction of pH intracellular has seriously affected the physiological activity of cell, has greatly reduced so that cell faces serious acid stress The efficiency of lactobacillus food microorganism manufacture, wherein acid stress caused by the accumulation with lactic acid is one of most important stress.

For acid stress, to maintain the stability of lactobacillus-fermented production and improving production efficiency, the past, industrially usually PH is maintained to be in stable range by adding external source neutralizer during lactobacillus-fermented, for example, passing through addition alkali Property substance (ammonium hydroxide or NaOH) controls the pH value of yeasting.

However, the addition of alkaline matter frequently can lead to the accumulation of by-product, and the salt formed in by-product can be again Cause cell to be in hypertonic environment, to cause the generation of infiltration pressure stress, influences the growth and metabolism of thallus again.

Currently, the method for improving the acid stress resistances such as lactic acid, the acetic acid of lactic acid bacteria then mainly has: (1) mutation breeding, the party Method has the characteristics that easy, wide variety, but heavy workload, low efficiency are its major defects；(2) Biochemical Engineering strategy has Report that used external source adds aspartic acid to improve the acid stress tolerance of lactic acid bacteria, but the use of this method is caused and is produced into This increase；(3) metabolic engineering strategies, the method currently with metabolic engineering strategies raising lactic acid bacteria environment-stress mainly include Construct new metabolic pathway, expand existing metabolic pathway and weaken existing metabolic pathway, still, the method there are it is at high cost, at The low problem of power,

Therefore, it is badly in need of finding that a kind of new excellent effect, at low cost, success rate is high, easy to operate, workload is few, efficiency The high method that lactic acid bacteria acid stress resistance can be improved.

Summary of the invention

To solve the above problems, the present invention provides engineering lactic acid bacterias and its application that a kind of acid stress resistance improves. The present invention is with code energy coupling factor transport protein ATP enzyme EcfA2 (energy-coupling factor transport System ATP-binding protein) gene be that purpose gene using lactic acid bacteria as expressive host successfully constructs one kind It can be widely applied to prepare the engineering lactic acid bacteria of food, drug, feed and chemicals；The acid stress of this engineering lactic acid bacteria Resistance is significantly improved, and improves 598.7 times compared with wild strain highest.

Technical scheme is as follows:

The present invention provides a kind of acid stress resistance improve engineering lactic acid bacteria, the engineering bacteria include recombinant plasmid with Expressive host；The recombinant plasmid includes target gene and expression vector；The target gene is code energy coupling factor The gene of transport protein ATP enzyme EcfA2；The expressive host is lactic acid bacteria.

In one embodiment of the invention, the lactic acid bacteria is Lactococcus lactis.

In one embodiment of the invention, the lactic acid bacteria is Lactococcus lactis Lactococcus lactis NZ9000。

In one embodiment of the invention, the energy coupling factor delivery albumin A TP enzyme EcfA2 derives from lactic acid Galactococcus Lactococcus lactis NZ9000.

In one embodiment of the invention, the gene of the energy coupling factor delivery albumin A TP enzyme EcfA2 is encoded Nucleotide sequence as shown in SEQ ID NO.1.

In one embodiment of the invention, the amino acid sequence of the energy coupling factor delivery albumin A TP enzyme EcfA2 Column are as shown in SEQ ID NO.2.

In one embodiment of the invention, the expression vector is pNZ8148.

The present invention provides a kind of method for improving lactic acid bacteria acid stress resistance, the method is that scale is crossed in lactic acid bacteria Danone amount coupling factor transport protein ATP enzyme EcfA2.

In one embodiment of the invention, the gene of the code energy coupling factor transport protein ATP enzyme EcfA2 Nucleotide sequence as shown in SEQ ID NO.1.

In one embodiment of the invention, the overexpression is to first pass through code energy coupling factor transport protein The gene and expression vector establishment of ATP enzyme EcfA2 contain the gene weight of code energy coupling factor transport protein ATP enzyme EcfA2 Group plasmid, then recombinant plasmid is imported in lactic acid bacteria.

In one embodiment of the invention, the expression vector is pNZ8148.

The present invention provides anti-using a kind of above-mentioned acid stress that the method for improving lactic acid bacteria acid stress resistance is prepared Property improve lactic acid bacteria.

In one embodiment of the invention, the lactic acid bacteria that the acid stress resistance improves includes recombinant plasmid and expression Host；The recombinant plasmid includes target gene and expression vector；The target gene is the transhipment of code energy coupling factor The gene of albumin A TP enzyme EcfA2；The expressive host is lactic acid bacteria.

In one embodiment of the invention, the expression vector is pNZ8148.

The present invention provides it is above-mentioned it is a kind of improve lactic acid bacteria acid stress resistance method improve lactic acid bacteria acid stress resistance The application of aspect.

The present invention provides a kind of above-mentioned engineering lactic acid bacteria that acid stress resistance improves or a kind of above-mentioned raising lactic acid bacterias Application of the method for acid stress resistance in terms of preparing food, drug, feed and chemicals.

The utility model has the advantages that

(1) present invention firstly discovers that overexpression EcfA2 albumen is remarkably improved the acid stress of lactic acid bacteria in lactic acid bacteria Resistance；

(2) present invention has been obtained acid stress resistance and has been significantly improved by the overexpression EcfA2 albumen in Lactococcus lactis Recombinant Lactococcus lactis Lactococcus lactis NZ9000 (pNZ8148/ecfA2)；

(3) the Recombinant Lactococcus lactis Lactococcus lactis NZ9000 that benefit is obtained by the present invention (pNZ8148/ecfA2) it is significantly improved to the resistance of acid stress compared with wild type, the resistance of lactic acid is mentioned compared with wild type It is 598.7 times high.

Detailed description of the invention

Fig. 1: the structure chart of recombinant plasmid pNZ8148/ecfA2；

Fig. 2: the structure chart of recombinant plasmid pNZ8148/zitP；

Fig. 3: the structure chart of recombinant plasmid pNZ8148/zitQ；

Fig. 4: the structure chart of recombinant plasmid pNZ8148/bglF；

Fig. 5: the structure chart of recombinant plasmid pNZ8148/ganP；

The growth curve of Fig. 6: recombinant bacterial strain L lactis NZ9000 (pNZ8148/ecfA2) and control strain；

Fig. 7: recombinant bacterial strain L lactis NZ9000 (pNZ8148/zitP), L lactis NZ9000 (pNZ8148/ ) and the growth curve of control strain zitQ；

Fig. 8: recombinant bacterial strain L lactis NZ9000 (pNZ8148/bglF), L lactis NZ9000 (pNZ8148/ ) and the growth curve of control strain ganP；

Under the conditions of Fig. 9: pH 4.0 (lactic acid adjusting) recombinant bacterial strain L lactis NZ9000 (pNZ8148/ecfA2) with it is right According to the survival rate comparison of bacterial strain；

Under the conditions of Figure 10: pH 4.0 (lactic acid adjusting) recombinant bacterial strain L lactis NZ9000 (pNZ8148/zitP) with it is right According to the survival rate comparison of bacterial strain；

Under the conditions of Figure 11: pH 4.0 (lactic acid adjusting) recombinant bacterial strain L lactis NZ9000 (pNZ8148/zitQ) with it is right According to the survival rate comparison of bacterial strain；

Figure 12: recombinant bacterial strain L lactis NZ9000 (pNZ8148/ecfA2) and control strain acid stress before and after it is intracellular ATP content balance；

Figure 13: recombinant bacterial strain L lactis NZ9000 (pNZ8148/ecfA2) and control strain acid stress before and after it is intracellular Leucine content comparison；

Figure 14: recombinant bacterial strain L lactis NZ9000 (pNZ8148/ecfA2) and control strain acid stress before and after it is intracellular Aspartate content comparison.

Specific embodiment

The present invention will be further elaborated combined with specific embodiments below.

Lactococcus lactis Lactococcus lactis NZ9000 involved in following embodiments is ground from Holland NIZO Study carefully institute.

Culture medium involved in following embodiments is as follows:

Chloramphenicol plate: peptone (Oxoid company, Britain) 1% (m/v), yeast powder (Oxoid) 0.5% (m/v), chlorination Sodium 1% (m/v) and 2% (m/v) agar strip add 10 μ g/mL chloramphenicol of final concentration after sterilizing.

GM17 fluid nutrient medium: the glucose (Glucose) of 5 ‰ (m/v) of supplement in M17 culture medium (Oxoid).

GM17 chloramphenicol plate: the glucose (Glucose) and 2% of 5 ‰ (m/v) of supplement in M17 culture medium (Oxoid) (m/v) agar strip adds 10 μ g/mL chloramphenicol of final concentration after sterilizing.

Embodiment 1: the building of recombinant bacterial strain

Specific step is as follows:

(1) (the code energy coupling of the ecfA2 gene order as shown in SEQ ID NO.1 is obtained from ncbi database The gene of factor delivery albumin A TP enzyme EcfA2, for transporting the albumen of ATP on film), the zitP base as shown in SEQ ID NO.3 Because of sequence (encoding the gene of metal ion abc transport albumen permease ZitP, be used to transport the albumen of metal ion on film), such as ZitQ gene order shown in SEQ ID NO.4 (encodes the gene of metal ion abc transport ATP binding protein ZitQ, uses on film In transhipment metal ion albumen), the bglF gene order as shown in SEQ ID NO.5 (coding beta-glucosidase specificity PTS The gene of system IIABC component BglF, for transporting the albumen of beta-glucosidase on film), the ganP as shown in SEQ ID NO.6 Gene order (gene of encoding mannose abc transport albumen permease GanP, for transporting the albumen of maltose on film), according to Gene order design primer as shown in Table 1 respectively；

(2) it using the genome of L.lactis NZ9000 as template, is obtained respectively with the primer in table 1 by PCR amplification SEQID NO.1, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, genetic fragment shown in SEQ ID NO.6；

(3) PCR product and carrier pNZ8148 are subjected to double digestion, digestion products with the restriction enzyme in table 1 respectively After purified, it is attached；

(4) connection product is converted into Escherichia coli MC1061 (commercialization bacterial strain) competence, screens sun on chloramphenicol plate Property clone, verified through bacterium colony PCR and digestion verification, carry out sequencing identification after clip size is correct again, it is final to obtain containing correct The recombinant plasmid pNZ8148/ecfA2 (structure is as shown in Figure 1) of sequence, pNZ8148/zitP (structure is as shown in Figure 2), (structure is as schemed by pNZ8148/zitQ (structure is as shown in Figure 3), pNZ8148/bglF (structure is as shown in Figure 4), pNZ8148/ganP Shown in 5)；

(5) recombinant plasmid is extracted from recombination E.coli MC1061, electrotransformation competence L.lactis NZ9000 cell, Screening positive clone on chloramphenicol plate, is verified and digestion verification through bacterium colony PCR, after clip size is correct, is finally contained Bacterial strain L lactis NZ9000 (pNZ8148/ecfA2), the L lactis NZ9000 (pNZ8148/ of correct recombinant plasmid zitP)、L lactisNZ9000(pNZ8148/zitQ)、L lactis NZ9000(pNZ8148/bglF)、L lactis NZ9000(pNZ8148/ganP).Wherein, electrotransformation condition are as follows: mix, move into pre- with the competent cell of 40 μ in 1 μ L plasmid In cold electric revolving cup, 10min is placed on ice；Voltage 2000V is adjusted, capacitor 25 μ f, 200 Ω of resistance shock by electricity；Electric shock finishes Afterwards, it is added immediately into electric revolving cup and contains 20mM MgCl₂With 2mM CaCl₂GM17 culture medium (culture medium prescription: M17broth Culture medium+0.5%glucose)；30 DEG C of stationary culture 1.5h are subsequently placed in, are coated on the GM17 plate containing chloramphenicol, are trained Support 36h, the verifying of picking transformant.

1 primer of table and restriction enzyme site

Embodiment 2: the growth performance test of recombinant bacterial strain

Specific step is as follows:

(1) the bacterial strain L lactis for obtaining bacterial strain L lactis NZ9000 (pNZ8148) (control) and embodiment 1 NZ9000(pNZ8148/ecfA2)、L lactis NZ9000(pNZ8148/zitP)、L lactis NZ9000(pNZ8148/ ZitQ), Llactis NZ9000 (pNZ8148/bglF), L lactis NZ9000 (pNZ8148/ganP) are inoculated in respectively adds It is activated in the GM17 fluid nutrient mediums of 10 μ g/mL chloramphenicol, is placed on stationary culture in 30 DEG C of incubators and stays overnight；

(2) seed liquor obtained above is forwarded to by fresh chloramphenicol (10 μ g/mL) GM17 with 2% inoculum concentration respectively In fluid nutrient medium, 30 DEG C of stationary cultures；

(3) during the cultivation process, it samples at regular intervals, measures the OD value under 600nm wavelength；

(4) it cultivates to OD₆₀₀The nisin inducing expression transport protein of 10ng/mL is added when 0.4, using the time as abscissa, OD₆₀₀Value is ordinate, is drawn growth curve (growth curve that drafting obtains is as shown in figs 6-8).

As a result as shown in fig. 6, through growth performance analysis of experiments, recombinant bacterial strain L lactis NZ9000 (pNZ8148/ EcfA2 biomass and control strain) illustrates the overexpression EcfA2 albumen in L lactis NZ9000 without too big gap The growth performance of bacterial strain is not influenced.

As a result as shown in fig. 7, through growth performance analysis of experiments, recombinant bacterial strain L lactis NZ9000 (pNZ8148/ ZitP) and the biomass of Llactis NZ9000 (pNZ8148/zitQ) and control strain are without too big gap, illustrate in L Overexpression ZitP and ZitQ albumen do not influence the growth performance of bacterial strain in lactis NZ9000.

As a result as shown in figure 8, through growth performance analysis of experiments, recombinant bacterial strain L lactis NZ9000 (pNZ8148/ BglF) and the biomass of Llactis NZ9000 (pNZ8148/ganP) is significantly lower than control strain, illustrates in L lactis Overexpression BglF and GanP albumen influences the normal growth of bacterial strain in NZ9000.Therefore weight of the subsequent experimental to energy normal growth Group bacterial strain L lactisNZ9000 (pNZ8148/zitP) and L lactis NZ9000 (pNZ8148/zitQ) acid resistance into Row test.

Embodiment 3: tolerance test of the recombinant bacterial strain under lactic acid stress conditions

Specific step is as follows:

The bacterial strain L lactis that bacterial strain L lactis NZ9000 (pNZ8148) (control) and embodiment 1 are obtained NZ9000(pNZ8148/ecfA2)、L lactis NZ9000(pNZ8148/zitP)、lactis NZ9000(pNZ8148/ ZitQ) Fiber differentiation 6h respectively, is collected by centrifugation cell, is resuspended in after twice through 0.85% brine isometric new In the GM17 (containing 10 μ g/mL of chloramphenicol) of fresh pH 4.0 (lactic acid adjusting), different time is coerced；By the bacteria suspension after stress It is resuspended in after washing twice in isometric physiological saline, takes 10 μ L re-suspension liquids, dilute different gradient dibblings in GM17 chloramphenicol Viable count and survival rate are measured on plate (result is as shown in figs. 9-11)；

Survival rate=(N/N₀) × 100%；

Wherein, N₀It is work clump count of the bacteria suspension without acid stress processing on plate；N is grown on plate after coercing Work clump count.

As shown in figs. 9-11, it is analyzed through tolerance test, after coercing 4h in the GM17 of pH 4.0, recombinant bacterial strain L The survival rate of lactisNZ9000 (pNZ8148/ecfA2) is 598.7 times of control, illustrates recombinant bacterial strain L lactis NZ9000 (pNZ8148/ecfA2) significantly improves the tolerance of acid stress；After coercing 4h in the GM17 of pH 4.0, recombinant bacterium The survival rate of strain L lactis NZ9000 (pNZ8148/zitP) and lactis NZ9000 (pNZ8148/zitQ) is respectively pair According to 14.5 times and 9.4 times, illustrate recombinant bacterial strain L lactis NZ9000 (pNZ8148/zitP) and lactis NZ9000 (pNZ8148/zitQ) tolerance of acid stress is slightly improved.

Embodiment 4: the measurement of recombinant bacterial strain ATP content intracellular

Specific step is as follows:

The bacterial strain L lactis that bacterial strain L lactis NZ9000 (pNZ8148) (control) and embodiment 1 are obtained NZ9000 (pNZ8148/ecfA2) difference Fiber differentiation 6h, with isometric phosphate buffer (200mmolL^-1, pH 7.0) and it washes It washs 2 times to be resuspended in the GM17 (containing 10 μ g/mL of chloramphenicol) of isometric fresh pH 4.0 (lactic acid adjusting), stress is different It time, takes 4.0mL bacterium solution, centrifugation, washing, collects thallus and with liquid nitrogen pre-freeze liquid nitrogen pre-freeze, save backup.It is detected and is tried with ATP Agent box (green skies biotechnology research institute) measures the content of ATP intracellular.

As a result as shown in figure 12, recombinant bacterial strain ATP content intracellular is above control strain before and after acid stress, the results showed that weight Group bacterial strain can discharge more ATP, meet the needs of cell is to energy under stress conditions, and then enhance L.lactis The acid stress resistance of NZ9000.

Embodiment 5: the measurement of recombinant bacterial strain amino acid content intracellular

Specific step is as follows:

The bacterial strain L lactis that bacterial strain L lactis NZ9000 (pNZ8148) (control) and embodiment 1 are obtained NZ9000 (pNZ8148/ecfA2) difference Fiber differentiation 6h, with isometric phosphate buffer (200mmolL^-1, pH 7.0) and it washes It washs 2 times to be resuspended in the GM17 (containing 10 μ g/mL of chloramphenicol) of isometric fresh pH 4.0 (lactic acid adjusting), stress is different It time, takes 10.0mL bacterium solution, centrifugation, washing, collects thallus and with liquid nitrogen pre-freeze liquid nitrogen pre-freeze, save backup.Using efficient liquid Chromatography detects recombinant bacterial strain amino acid content intracellular.

The preparation of high performance liquid chromatograph test sample: take 1mL phosphate buffer that thallus is resuspended, bacteria suspension is transferred to broken Pipe vibrates smudge cells (4.0ms using FastPrep-24^-1) to bacteria suspension clarify；Centrifugation, takes 500 μ L of supernatant, adds and waits bodies The trichloroacetic acid (TCA) of 5% (m/v) of product, static placement 30min remove soluble protein；Centrifugation, takes supernatant, is filtered with water system Film (0.2 μm) is filled into spare in clean sample bottle.

High performance liquid chromatograph analysis method: OPA and boric acid carry out column front derivation；Column temperature is set as 40.0 DEG C；Flow velocity is set It is set to 1.0mLmin^-1；The wavelength of UV detector is 338nm；Chromatographic column selects ODS HYPERSIL (250.0mm × 4.6mm ×5.0μm)。

As a result as illustrated in figs. 13-14, recombinant bacterial strain leucine content intracellular is above control strain, bright ammonia before and after acid stress Competitive consumption pyruvic acid reduces formic acid Isoquant in sour synthesis process, can be relieved the stress pressure that cell is subject to；Before acid stress Recombinant bacterial strain aspartate content intracellular is above control strain afterwards, and aspartic acid disappears under the action of aspartate decarboxylase Consume H⁺, the carboxyl of the position β is taken off, alanine and CO are generated₂, effectively maintenance pH_iRelative equilibrium.Recombinant bacterial strain is by adjusting ammonia Base acid metabolic consumes H intracellular⁺, alkaline matter is generated, to maintain the stable state of pH intracellular, and then cell is helped to resist acid stress.

Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.

Sequence table

<110>Southern Yangtze University

<120>engineering lactic acid bacteria and its application that a kind of acid stress resistance improves

<160> 16

<170> PatentIn version 3.3

<210> 1

<211> 867

<212> DNA

<213>artificial sequence

<400> 1

atgattaaat ttgaaaaagt aaattatact tatcagccca acagcccttt tgccagtcgt 60

gccctttttg atattgattt agaagtcaaa aaaggaagtt acacggcttt aattgggcac 120

acaggttctg ggaaatcaac cttgttgcaa catctcaatg gtttgttgca accgactgag 180

ggtaaagtga ccgttggaga tattgttgta agttcaacaa gtaaacaaaa agagattaaa 240

cctgttcgca aaaaagtagg ggttgttttt caatttcctg aaagtcaact ttttgaggaa 300

acagtcttaa aagatgtggc ttttggccca caaaactttg gaattcctaa agaaaaagct 360

gaaaaaatcg ctgctgaaaa acttgaaatg gtcggtcttg cagatgaatt ttgggaaaaa 420

tctccttttg aactttcagg aggtcaaatg cgccgcgtcg ctattgctgg aattcttgct 480

atggaaccag aagttctagt gcttgatgaa ccaaccgcag gccttgaccc gaaagctaga 540

atcgaaatga tgcaactttt tgaaagtatt catcaatcag gacaaacagt tgtcttagtc 600

actcatttaa tggatgatgt ggctgattat gctgactatg tttatcttct tgaaaaaggg 660

catatcatta gctgcggtac tccgtcagac gtttttcaag aggttgattt tttgaaagca 720

catgaattgg gcgtgccaaa ggcgacgcat tttgctgacc aacttcaaaa aacaggagcg 780

gtcgcctttg aaaaattacc catcacacgg gctgagcttg ttactttact gacaagtctg 840

tcagtaaatt caggaggtga aaactga 867

<210> 2

<211> 288

<212> PRT

<213>artificial sequence

<400> 2

Met Ile Lys Phe Glu Lys Val Asn Tyr Thr Tyr Gln Pro Asn Ser Pro

1 5 10 15

Phe Ala Ser Arg Ala Leu Phe Asp Ile Asp Leu Glu Val Lys Lys Gly

20 25 30

Ser Tyr Thr Ala Leu Ile Gly His Thr Gly Ser Gly Lys Ser Thr Leu

35 40 45

Leu Gln His Leu Asn Gly Leu Leu Gln Pro Thr Glu Gly Lys Val Thr

50 55 60

Val Gly Asp Ile Val Val Ser Ser Thr Ser Lys Gln Lys Glu Ile Lys

65 70 75 80

Pro Val Arg Lys Lys Val Gly Val Val Phe Gln Phe Pro Glu Ser Gln

85 90 95

Leu Phe Glu Glu Thr Val Leu Lys Asp Val Ala Phe Gly Pro Gln Asn

100 105 110

Phe Gly Ile Pro Lys Glu Lys Ala Glu Lys Ile Ala Ala Glu Lys Leu

115 120 125

Glu Met Val Gly Leu Ala Asp Glu Phe Trp Glu Lys Ser Pro Phe Glu

130 135 140

Leu Ser Gly Gly Gln Met Arg Arg Val Ala Ile Ala Gly Ile Leu Ala

145 150 155 160

Met Glu Pro Glu Val Leu Val Leu Asp Glu Pro Thr Ala Gly Leu Asp

165 170 175

Pro Lys Ala Arg Ile Glu Met Met Gln Leu Phe Glu Ser Ile His Gln

180 185 190

Ser Gly Gln Thr Val Val Leu Val Thr His Leu Met Asp Asp Val Ala

195 200 205

Asp Tyr Ala Asp Tyr Val Tyr Leu Leu Glu Lys Gly His Ile Ile Ser

210 215 220

Cys Gly Thr Pro Ser Asp Val Phe Gln Glu Val Asp Phe Leu Lys Ala

225 230 235 240

His Glu Leu Gly Val Pro Lys Ala Thr His Phe Ala Asp Gln Leu Gln

245 250 255

Lys Thr Gly Ala Val Ala Phe Glu Lys Leu Pro Ile Thr Arg Ala Glu

260 265 270

Leu Val Thr Leu Leu Thr Ser Leu Ser Val Asn Ser Gly Gly Glu Asn

275 280 285

<210> 3

<211> 810

<212> DNA

<213>artificial sequence

<400> 3

atgtttgaat tattccagta tgattttatg aggaatgctc tcttagcagc gacagcaatt 60

tcaattttct cgccattact tggtgtattt cttgtcctgc gtagacaaag tctaatgtcg 120

gacacattat cgcatgtttc tttagcgggt gttgcctttg gcgtgctatt aagttggaat 180

ccaacgatta ctactttaat taccgtagtg attgctgcag tttttctaga atatttgaga 240

acgatttatc ataattacat ggaaattgcg acggcgattt tgatgtcggc gggtcttgct 300

attgctttat taattttaaa ttcttccaaa ggagcaacct ctgtaagttt agagcaatat 360

ttgtttggtt caattatcac aatttcactt tcgcaagtaa ttatgctctt tgttttggct 420

gcagttgttc tactcggatt tattctattt ttacgtccgc tttatgtgat gacatttgat 480

gaagatacgg catttgttga tggacttcca gttcgttgga tttcaattgc ctttaatatt 540

gtgacaggga ttgcgattgc actgatgatt ccggcagcgg gagctctatt ggtatcagcg 600

atcatggtgt taccagcttc gattgcgatg cgattaggaa aatctttcaa ggctgtgctt 660

tttatctcag ttattgttag ttttattggg ttaaatgctg gtttgattgc ttcttattat 720

atggatgcgc cagcctctgc cgcaataact ttgattttca ttgttctttt cttagttact 780

tcaagtttaa aacgattaat tagagggtaa 810

<210> 4

<211> 738

<212> DNA

<213>artificial sequence

<400> 4

atgagatata tcaatgttga aaatctgacc ttctactatg atagagagcc agtgttagaa 60

aatattagct accatgtgga ctctggtgaa tttgtcacct taactggaga aaatggagcg 120

gcaaaatcaa ctttaattaa aacaactttg ggtattttaa aaccaaagaa agggaaaata 180

acgatttctt ctaaaaataa tagaggagaa aagttacgga ttgcctacct tccgcagcaa 240

gttgcaagtt ttaacgcagg atttccgagt tctgttcatg aatttgtcat gagtggacgc 300

tatccaagac aggggtggtt taaaaaaatg ggcgctcatg atttagaaca tgtcaaggca 360

gctcttgatt cagttggtat gtgggactat cgcgataaac ggattggtga actttcggga 420

ggtcaaaagc aaaggattgc tattgcgaga atgtttgcta gtgaccctga tttatttatc 480

cttgatgaac cgacaactgg tatggatgat gtatcaagta gtgattttta tcagttgatg 540

catcatgcgg cccataagca tgggaaagca gtcctaatgg tcactcatga tcctgaggaa 600

gtgaaagatt tcgctgaccg taacattcat ctattaaaag acaaaaatgg aaagtttgct 660

tgttttgatt tgcatactga ccgtgataga gttctccaag aagagcaaga agaattagag 720

gagaaagcaa atgtttga 738

<210> 5

<211> 1566

<212> DNA

<213>artificial sequence

<400> 5

atggcaaatt attcacaact tgcgacagaa attatcgcaa atgtaggtgg cgctgagaat 60

gtcacaaaag ttattcactg tatcactcgt cttcgtttta ccttgaaaga caaagataaa 120

gcagatacgg cggcgattga agccttacct ggtgtcgctg gagctgttta taactcaaac 180

ttgaatcaat atcaagtagt tattggacaa gctgtagaag atgtttatga cgaggttgtt 240

gaacagcttg gagattcagt tgttgatgaa gatgcaacgg cgcaagcact tgctgcaaca 300

gcaccggcta gtggtaaaaa acaaaatcca attgttcatg ctttccaagt ggttattggg 360

acaattacag gttcgatgat tccaattatt ggtttacttg cggctggtgg gatgattaat 420

ggattattaa gtatctttgt taaaggaaat cgtttaattg aagtgattga ccctgcaagt 480

tcaacttacg tcattatctc aactctagca atgacaccat tttatttctt acctgtttta 540

gtaggatttt cagcagcaaa acaattagca cctaaagata ctgttttaca atttattggt 600

gctgctgttg gtggtttcat gattaatcca gggattacta acttggtaaa tgctcatgtt 660

ggaacaaatg cggccggtaa aaatgttgtt gttgaagcag cagctccagt agcaaatttc 720

cttggagtca cttttaatac aagttatttt ggaattccgg ttgctttgcc aagttatgct 780

tatacaattt tcccaatcat tgtggcggta gcaatcgcta aacctttgaa tgcttggttg 840

aaaaaggttt taccacttgc cttgcgtcca attttccaac cgatgattac tttcttcatc 900

actgcttcaa tcattttact cttggtcggt cctgttattt caacaatttc atctggtttg 960

tcattcgtta ttgaccatat cttgtcatta aacttaggga ttgcaagtat tatcgtcggt 1020

ggtttgtatc aatgtttggt tatatttggt ttgcactggt tggttgtacc acttatttca 1080

caagagttgg cagcaacagg agcaagctca cttaatatga ttgttagctt cacaatgctt 1140

gcgcaaggag ttggtgcctt gactgtcttc tttaaatcta aaaaagctga ccttaaagga 1200

ctttctgctc cagctgccat ttcggctttt tgtggagtaa ctgaacctgc catgtacgga 1260

attaacttga aatatgttcg cgtcttcatc atgtcttcaa ttggtgcagc aattggtgct 1320

gggattgccg gatttggtgg cttacaaatg tttggatttt cagggtcatt gattagtttt 1380

cctaacttta tctctaatcc attgacgcat catgcacctg cgggtaactt aatgctcttc 1440

tggattgcca ctgcggtatg tgctgttgcc actttcttat tagtttggtt ctttggttac 1500

aaggatactg atgtcatggg acaaggagtt gaacaaaaaa atgcatttaa ggatgctgta 1560

aaataa 1566

<210> 6

<211> 1383

<212> DNA

<213>artificial sequence

<400> 6

atgactaaaa agaaaaaaag aaaacaaacc gaaagtaatg tttctcctga agaaaaatct 60

attaaactac gtgaagtttt ccaaaaaggt aataccgtta caaaattaac tttcttcgtg 120

atgggcctga atcaaataaa aaacaaacag tgggtaaaag gatttacttt cttaattctt 180

gaaattgcat ttattggttg gcttcttttc tctggactta gtgctttttc tcttttgagt 240

agcttaggtc caaataaaac acttaaagaa acaacagacg ccaatggctt tccagttatt 300

attcaacccg atcactctgt tttgatttta ctttggggac tcattgcttg tcttgtcgtt 360

gttctcttta ttttacttta ctggttcaac tatcgttcaa acaaacatct ctactattta 420

gaacgggaag gcaaacatat ccctacaaat agagaagaac ttgcatccct acttgatgaa 480

aaactctatg cgacattaat ggctgttcct ttaattggag ttctagcttt cactgttttg 540

cctactgttt acatgatttc gatggctttc acaaactatg atcgtctaca tgctactgct 600

ttctcatgga ccggttttca agcctttggt aatgtcttaa ccggggattt agcgggaaca 660

ttcttccccg ttcttggttg gacattagta tgggcaattg tagcaacagc aacaacattt 720

ctcggtggtg ttttacttgc cttactcatt gagtcaactg gaattaaatt taaaggattc 780

tggagaacag tttttgttat cgtctttgcc gttccacaat ttgtaaccct attaatgatg 840

gcacaatttt tggaccaaca aggagctttt aatggaattt tgatgaatct tcatctaatt 900

tccaatccga tcaactttat tggtgcggct tctgacccaa tggttgcaag aatcactgtt 960

atatttgtta atatgtggat tggtatccct gtttcaatgc ttgtatctac agcaattatc 1020

caaaaccttc cccaagacca aatcgaagct gcacgtattg atggagcaaa tagtttaaat 1080

atcttccgtt ctatcacttt tcctcagatt ctctttgtta tgactcctgc attgattcaa 1140

caatttattg gtaacatcaa taacttcaat gttatttatc tactaacgca aggttggcca 1200

atgaatccaa actaccaagg agcaggttca accgaccttc ttgttacttg gctctacaac 1260

ctcgtctttg gtcaaactca acgttacaat gctgccgctg ttcttggtat cttgattttc 1320

attgttaatg catcaatttc attagtagca taccgtcgta ccaatgcatt taaggagggc 1380

taa 1383

<210> 7

<211> 47

<212> DNA

<213>artificial sequence

<400> 7

catgccatgg ggatgattaa atttgaaaaa gtaaattata cttatca 47

<210> 8

<211> 34

<212> DNA

<213>artificial sequence

<400> 8

ccaagctttc agttttcacc tcctgaattt actg 34

<210> 9

<211> 36

<212> DNA

<213>artificial sequence

<400> 9

catgccatgg ggatgtttga attattccag tatgat 36

<210> 10

<211> 35

<212> DNA

<213>artificial sequence

<400> 10

cccaagcttt taccctctaa ttaatcgttt taaac 35

<210> 11

<211> 37

<212> DNA

<213>artificial sequence

<400> 11

catgccatgg ggatgagata tatcaatgtt gaaaatc 37

<210> 12

<211> 29

<212> DNA

<213>artificial sequence

<400> 12

cccaagcttt caaacatttg ctttctcct 29

<210> 13

<211> 36

<212> DNA

<213>artificial sequence

<400> 13

catgccatgg ggatggcaaa ttattcacaa cttgcg 36

<210> 14

<211> 35

<212> DNA

<213>artificial sequence

<400> 14

cccaagcttt tattttacag catccttaaa tgcat 35

<210> 15

<211> 37

<212> DNA

<213>artificial sequence

<400> 15

catgccatgg ggatgactaa aaagaaaaaa agaaaac 37

<210> 16

<211> 31

<212> DNA

<213>artificial sequence

<400> 16

cccaagcttt tagccctcct taaatgcatt g 31

Claims

1. the engineering lactic acid bacteria that a kind of acid stress resistance improves, which is characterized in that the engineering bacteria includes recombinant plasmid and table Up to host；The recombinant plasmid includes target gene and expression vector；The target gene turns for code energy coupling factor Transport the gene of albumin A TP enzyme EcfA2；The expressive host is lactic acid bacteria.

2. the engineering lactic acid bacteria that a kind of acid stress resistance as described in claim 1 improves, which is characterized in that the energy is even Join factor delivery albumin A TP enzyme EcfA2 and derives from Lactococcus lactis Lactococcus lactis NZ9000.

3. the engineering lactic acid bacteria that a kind of acid stress resistance as claimed in claim 1 or 2 improves, which is characterized in that coding institute The nucleotide sequence of the gene of energy coupling factor delivery albumin A TP enzyme EcfA2 is stated as shown in SEQ ID NO.1.

4. the engineering lactic acid bacteria that a kind of acid stress resistance a method according to any one of claims 1-3 improves, which is characterized in that described Expression vector is pNZ8148.

5. a kind of method for improving lactic acid bacteria acid stress resistance, which is characterized in that the method is the overexpression in lactic acid bacteria Energy coupling factor delivery albumin A TP enzyme EcfA2.

6. a kind of method for improving lactic acid bacteria acid stress resistance as claimed in claim 5, which is characterized in that the energy coupling Factor delivery albumin A TP enzyme EcfA2 derives from Lactococcus lactis Lactococcus lactis NZ9000.

7. such as a kind of method for improving lactic acid bacteria acid stress resistance described in claim 5 or 6, which is characterized in that the excess It is expressed as first passing through the gene of code energy coupling factor transport protein ATP enzyme EcfA2 and expression vector establishment contains coding energy The gene recombination plasmid of coupling factor transport protein ATP enzyme EcfA2 is measured, then recombinant plasmid is imported in lactic acid bacteria.

8. the acid stress being prepared using a kind of method of any raising lactic acid bacteria acid stress resistance of claim 5-7 The lactic acid bacteria that resistance improves.

9. a kind of method of any raising lactic acid bacteria acid stress resistance of claim 5-7 is anti-in raising lactic acid bacteria acid stress Application in terms of property.

10. the engineering lactic acid bacteria or claim 5-7 that a kind of any acid stress resistance of claim 1-4 improves are any A kind of application of the method for raising lactic acid bacteria acid stress resistance in terms of preparing food, drug, feed and chemicals.