CN110218691A

CN110218691A - One plant of genetic engineering bacterium for synthesizing altheine and its construction method and application

Info

Publication number: CN110218691A
Application number: CN201910425792.XA
Authority: CN
Inventors: 马江锋; 储乐乐; 方艳; 信丰学; 董维亮; 章文明; 陈可泉; 姜岷; 欧阳平凯
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2019-05-21
Filing date: 2019-05-21
Publication date: 2019-09-10

Abstract

The invention discloses the genetic engineering bacterium that one plant synthesizes altheine, classification naming is Escherichia coli △ fumABC △ arcA △ ptsG △ gltA △ argG/pAA, and deposit number is CCTCC NO:M2019170.Its building process includes knocking out starting strain fumarase encoding gene, DNA transcription in conjunction with regulatory factor encoding gene, glucose transport related gene, citrate synthase encoding gene and argininosuccinate synthetase encoding gene.Aspartic acid enzyme coding gene and asparagine synthetase A encoding gene are cloned on expression plasmid simultaneously, and by the recombinant plasmid transformed into the recombination bacillus coli for having knocked out gene before, obtain purpose bacterial strain.Altheine is synthesized using the engineering bacteria fermentation, realizes the route for using glucose to prepare altheine for raw material biosynthesis completely, efficient, green, environmental protection are economical.

Description

One plant of genetic engineering bacterium for synthesizing altheine and its construction method and application

Technical field

The invention belongs to genetic engineering and fermentation engineering fields, and in particular to one plant utilizes glucose biological synthesis system The genetic engineering bacterium and its construction method of standby altheine and application.

Background technique

Altheine is the hydroxyamide compounds of L-Aspartic acid, be widely used in food, medicine, chemical industry synthesis and The fields such as microculture.In terms of medicine, mainly as one of 20 kinds in amino acid transfusion, and there is treatment skin disease The effect of；In terms of food, it can be used as food additives for cold drink；Chemical industry mainly do molysite stabilizer and The film of ceramic surface；In the important source material that environmental protection industry (epi) is membrane for water treatment；Meanwhile it is also microculture and zooblast Cultivate important additive.With good market prospects.

The production of altheine at present is mainly raw material through being esterified ammonolysis using L-Aspartic acid, is refined, but its mistake Journey product yield is relatively low, and is related to a large amount of organic solvent, and pollution is big, and waste water is difficult.Bioanalysis, which has, reacts mild, conversion The advantages that rate is high, the three wastes are easy to handle, is the optimal production method of altheine.The monosaccharide such as glucose, xylose derive from can be again Raw biomass resource, abundance, screening or building, which obtain one plant, to prepare L- asparagus fern by fermentation method biosynthesis The production bacterial strain of amide has great importance.The present invention is based on this to construct the bacterium with high-performance bio synthesis altheine Strain can make full use of carbon source, reduce and be lost, while improving fermentating culturing process, increase the conversion ratio of target product, reach To efficient, green, environmentally friendly purpose, there is significant meaning.

Summary of the invention

The present invention claims the genetic engineerings that plant height effect biosynthesis altheine is to provide the technical issues of solution Bacterium, classification naming are Escherichia coli △ fumABC △ arcA △ ptsG △ gltA △ argG/pAA.

The present invention also technical problems to be solved are to provide the construction method of said gene engineering bacteria, knock out starting strain Fumarase encoding gene fumABC, DNA transcription combine regulatory factor encoding gene arcA, glucose transport related gene PtsG, citrate synthase encoding gene gltA and argininosuccinate synthetase encoding gene argG, while by Aspartase Encoding gene aspA and asparagine synthetase A encoding gene asnA is cloned into expression and constitutes recombinant plasmid, and is converted To the Escherichia coli starting strain for having knocked out fumABC gene, the genetic engineering bacterium, the deposit number of the starting strain are obtained For CCTCC NO:M 2018521, the fumA gene order is as shown in SEQ ID NO:1, the fumB gene order such as SEQ Shown in ID NO:2, the fumC gene order is as shown in SEQ ID NO:3, the aspA gene order such as SEQ ID NO:4 institute Show, for the asnA gene order as shown in SEQ ID NO:5, the arcA gene order is described as shown in SEQ ID NO:6 PtsG gene order is as shown in SEQ ID NO:7, and the gltA gene order is as shown in SEQ ID NO:8, the argG gene For sequence as shown in SEQ ID NO:9, the knockout of gene uses general CRISPR/Cas9 technology.Specific building and selection It is as follows:

(1) it is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:11 as primer, plasmid pTarget F is Template, PCR amplification obtain linear fragment 1；

(2) it is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:12 as primer, repeats step (1) and obtain Linear fragment 2；It is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:13 as primer, repeats step (1) and obtain Linear fragment 3；It is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:14 as primer, repeats step (1) and obtain Linear fragment 4；It is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:15 as primer, repeats step (1) and obtain Linear fragment 5；It is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:16 as primer, repeats step (1) and obtain Linear fragment 6；

(3) it will be attached after the SpeI digestion of linear fragment 1, and product after connection be transformed into competence, be coated with The LB flat screen of spectinomycin selects positive recombinant；

(4) numerous rear progress plasmid extraction is expanded into the LB liquid training that positive recombinant is inoculated in addition spectinomycin, obtains PTarget T1-1 plasmid；

(5) transformation and selection will be attached after the SpeI digestion of linear fragment 2 and goes out positive recombinant and carry out plasmid extraction, obtain Obtain pTarget T1-2 plasmid；Transformation and selection being attached after the SpeI digestion of linear fragment 3 and going out positive recombinant carry out matter Grain extracting, obtains pTarget T1-3 plasmid；Go out positive restructuring for transformation and selection is attached after the SpeI digestion of linear fragment 4 Son carries out plasmid extraction, obtains pTarget T1-4 plasmid；Go out transformation and selection is attached after the SpeI digestion of linear fragment 5 Positive recombinant carries out plasmid extraction, obtains pTarget T1-5 plasmid；It will be attached and turn after the SpeI digestion of linear fragment 6 Change filters out positive recombinant and carries out plasmid extraction, obtains pTarget T1-6 plasmid；

(6) it is arranged with nucleotides sequence shown in SEQ ID NO:17 and SEQ ID NO:18 as primer, genome of E.coli For template, PCR amplification obtains linear fragment 7；It is to draw with the column of nucleotides sequence shown in SEQ ID NO:19 and SEQ ID NO:20 Object, genome of E.coli are template, and PCR amplification obtains linear fragment 8；

(7) it is arranged with nucleotides sequence shown in SEQ ID NO:17 and SEQ ID NO:20 as primer, segment 7 and segment 8 Mixture is template, and over-lap PCR expands to obtain linear fragment 9；

(8) dephosphorylation carries out lucky cloth with segment 9 as carrier after using EcoRI to linearize pTarget T1-1 plasmid Gloomy one-step cloning selects positive recombinant with the LB flat screen of spectinomycin；

(9) numerous rear progress plasmid pumping is expanded into the LB liquid training that the positive recombinant in step (8) is inoculated in addition spectinomycin It mentions, obtains pTarget T2-1 plasmid；

It (10) is that primer repeats step (6) to step with the column of nucleotides sequence shown in SEQ ID NO:21-SEQ ID NO:24 Suddenly (9) obtain pTarget T2-2 plasmid；With the column of nucleotides sequence shown in SEQ ID NO:25-SEQ ID NO:28 for primer weight Multiple step (6) to step (9) obtain pTarget T2-3 plasmid；With nucleosides shown in SEQ ID NO:29-SEQ ID NO:32 Acid sequence is that primer repeats step (6) to step (9) acquisition pTarget T2-4 plasmid；With SEQ ID NO:33-SEQ ID Nucleotides sequence shown in NO:36 is classified as primer and repeats step (6) to step (9) acquisition pTarget T2-5 plasmid；With SEQ ID Nucleotides sequence shown in NO:37-SEQ ID NO:40 is classified as primer and repeats step (6) to step (9) acquisition pTarget T2-6 Plasmid；

(11) pCas plasmid is imported in the starting strain that deposit number is CCTCC NO:M 2018521, with kanamycins LB flat screen selects positive recombinant；

(12) positive recombinant in step (11) is induced with arabinose, is prepared as competence；

(13) by the competence of pTarget T2-1 plasmid steps for importing (12), with addition spectinomycin and kanamycins LB flat screen select positive recombinant；

(14) positive recombinant in step (13) is identified with the method for PCR, filters out aimed strain, and use PTarget T2-1 plasmid is eliminated in IPTG induction, obtains bacterial strain 1.

(15) bacterial strain 1 in step (14) is induced with arabinose, is prepared as competence；

(16) by the competence of pTarget T2-2 plasmid steps for importing (15), with addition spectinomycin and kanamycins LB flat screen select positive recombinant；

(17) positive recombinant in step (16) is identified with the method for PCR, filters out aimed strain, and use PTarget T2-2 plasmid is eliminated in IPTG induction, obtains bacterial strain 2.

(18) pTarget T2-3 plasmid, pTarget are imported in batches with the method for similar step (15) to step (17) T2-4 plasmid, pTarget T2-5 plasmid, pTarget T2-6 plasmid carry out gene knockout, and are eliminated and imported using IPTG induction Plasmid obtains gene knock-out bacterial strain Escherichia coli △ fumABC △ arcA △ ptsG △ gltA △ argG；

(19) it is arranged with nucleotides sequence shown in SEQ ID NO:41 and SEQ ID NO:42 as primer, genome of E.coli For template, PCR amplification obtains linear fragment 10；It is to draw with the column of nucleotides sequence shown in SEQ ID NO:43 and SEQ ID NO:44 Object, genome of E.coli are template, and PCR amplification obtains linear fragment 11；

(20) it is arranged with nucleotides sequence shown in SEQ ID NO:41 and SEQ ID NO:44 as primer, segment 10 and segment 11 Mixture be template, over-lap PCR expands to obtain linear fragment 12；

(21) dephosphorylation is as carrier after using EcoRI and HindIII to linearize pTrc99a plasmid, with segment 11 into Row Ji Busen one-step cloning selects positive recombinant with the LB flat screen of the resistance of benzyl containing ammonia；

(22) numerous rear progress plasmid is expanded into the LB liquid training that the positive recombinant in step (21) is inoculated in addition spectinomycin Extracting obtains pTrc99A-asnA-aspA recombinant plasmid；

(23) gene knock-out bacterial strain in step (18) is prepared as competence；By the pTrc99A- in step (22) AsnA-aspA plasmid is converted into competence, is selected positive recombinant with the LB flat screen of the resistance of benzyl containing ammonia, is obtained target gene Engineered strain.

The last technical problems to be solved of the present invention are to provide said gene engineering bacteria in biosynthesis altheine Application.

The engineering strain of above-mentioned high-performance bio synthesis altheine prepares the specific fermented and cultured of altheine Process is as follows:

(S1) coli strain that genetic engineering is transformed is transferred in LB culture medium, 10~12h of aerobic culture is obtained Primary seed solution；

(S2) primary seed solution is transferred in fermentor LB culture medium and is cultivated, obtain secondary seed solution；

(S3) when secondary seed solution OD600 to 8.5, fermentation medium is inoculated, inoculates fermentation medium, institute The formula for stating fermentation medium is as follows:

M9 culture medium: (NH₄)₂SO₄6g/L, Na₂HPO₄·12H₂O 15.2g/L, KH₂PO₄3g/L, NaCl0.5g/L, NH₄Cl 1g/L, MgSO₄2mM, CaCl₂0.1mM, 100g/L glucose, 0.3mM IPTG.

(S4) glucose 30g/L transformation stage fed-batch fermentation: is added after induction period.

In step (S1) and (S2), cultivation temperature is 35~37 DEG C, uses aerobic fermentation mode in step (S3) and (S4), Dissolved oxygen be 5~40% and fermentation process in temperature be 28~30 DEG C, incubation pH is adjusted to 7.0 with ammonium hydroxide.

The beneficial effects of the present invention are: innovative being constructed by way of gene knockout of the present invention is using glucose Raw material biology prepares the strain of altheine, and enhancing bacterial strain TCA recycles flux, inhibits fumaric acid synthesizing apple acid, reduces L- The consumption of aspartic acid achievees the purpose that increase L-Aspartic acid accumulation, then by aspartic acid enzyme coding gene aspA and asparagus fern Amide synthetase A encoding gene asnA is cloned on expression plasmid, while improving fermentating culturing process, is solved in biosynthesis The problem of ATP is supplied, finally improves the yield of altheine, the generation of by-product is effectively reduced, and has significant economical Property and social benefit, process route green, environmental protection.

Biomaterial of the present invention, classification naming Escherichia coli △ fumABC △ arcA △ ptsG △ gltA △ argG/pAA, has been preserved in China typical culture collection center (abbreviation CCTCC), deposit number are as follows: CCTCC NO:M 2019170, preservation date are as follows: on March 18th, 2019, preservation address are as follows: the Chinese Wuhan Wuhan University.

Specific embodiment

According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real It applies content described in example and is merely to illustrate the present invention, without sheet described in detail in claims should will not be limited Invention.

Embodiment 1

This example demonstrates that utilizing target matter of the overlapping pcr building comprising gene targeting sequence and homologous recombination segment Grain pTarget T2-1, pTarget T2-2, pTarget T2-3, pTarget T2-4, pTarget T2-5, pTarget The method of T2-6.

1, it is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:11 as primer, plasmid pTarget F is Template, PCR amplification obtain linear fragment 1；

2, it is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:12 as primer, repeats step 1 and obtain line Property segment 2；It is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:13 as primer, repeats step 1 and obtain linearly Segment 3；It is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:14 as primer, repeats step 1 and obtain linear piece Section 4；It is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:15 as primer, repeats step 1 and obtain linear fragment 5；It is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:16 as primer, repeats step 1 and obtain linear fragment 6；

3, it will be attached after the SpeI digestion of linear fragment 1, and product after connection be transformed into competence, coating is big The LB flat screen of miromycin selects positive recombinant；

4, numerous rear progress plasmid extraction is expanded into the LB liquid training that positive recombinant is inoculated in addition spectinomycin, obtains PTarget T1-1 plasmid；

5, transformation and selection will be attached after the SpeI digestion of linear fragment 2 and goes out positive recombinant and carry out plasmid extraction, obtain Obtain pTarget T1-2 plasmid；Transformation and selection being attached after the SpeI digestion of linear fragment 3 and going out positive recombinant carry out matter Grain extracting, obtains pTarget T1-3 plasmid；Go out positive restructuring for transformation and selection is attached after the SpeI digestion of linear fragment 4 Son carries out plasmid extraction, obtains pTarget T1-4 plasmid；Go out transformation and selection is attached after the SpeI digestion of linear fragment 5 Positive recombinant carries out plasmid extraction, obtains pTarget T1-5 plasmid；It will be attached and turn after the SpeI digestion of linear fragment 6 Change filters out positive recombinant and carries out plasmid extraction, obtains pTarget T1-6 plasmid；

6, it is arranged with nucleotides sequence shown in SEQ ID NO:17 and SEQ ID NO:18 as primer, genome of E.coli is Template, PCR amplification obtain linear fragment 7；It is arranged with nucleotides sequence shown in SEQ ID NO:19 and SEQ ID NO:20 as primer, Genome of E.coli is template, and PCR amplification obtains linear fragment 8；

7, arranged with nucleotides sequence shown in SEQ ID NO:17 and SEQ ID NO:20 as primer, segment 7 and segment 8 it is mixed Conjunction object is template, and over-lap PCR expands to obtain linear fragment 9；

8, dephosphorylation carries out Ji Busen with segment 9 as carrier after using EcoRI to linearize pTarget T1-1 plasmid One-step cloning selects positive recombinant with the LB flat screen of spectinomycin；

9, numerous rear progress plasmid extraction is expanded into the LB liquid training that the positive recombinant in step 8 is inoculated in addition spectinomycin, Obtain pTarget T2-1 plasmid；

It 10, is that primer repeats step 6 to step 9 with the column of nucleotides sequence shown in SEQ ID NO:21-SEQ ID NO:24 Obtain pTarget T2-2 plasmid；It is that primer repeats to walk with the column of nucleotides sequence shown in SEQ ID NO:25-SEQ ID NO:28 Rapid 6 to step 9 obtain pTarget T2-3 plasmid；It is with the column of nucleotides sequence shown in SEQ ID NO:29-SEQ ID NO:32 Primer repeats step 6 to step 9 and obtains pTarget T2-4 plasmid；With core shown in SEQ ID NO:33-SEQ ID NO:36 Nucleotide sequence is that primer repeats step 6 to step 9 acquisition pTarget T2-5 plasmid；With SEQ ID NO:37-SEQ ID NO: Nucleotides sequence shown in 40 is classified as primer and repeats step 6 to step 9 acquisition pTarget T2-6 plasmid.

Embodiment 2

This example demonstrates that the method for inducing the preparation competence of albumen containing cas9 using arabinose, specific steps include:

1, pCas plasmid is imported into the Escherichia coli recombinant strain to gene editing, the biomaterial is in Chinese patent (application Numbers 201811346790.3, applying date 2018.11.13) patent document in disclose, deposit number is CCTCC NO:M 2018521, positive recombinant is selected with the LB flat screen of kanamycins；

2, the arabinose of 30mM of the positive recombinant in above-mentioned steps is induced, bacterium culture 3- is shaken in 30 DEG C of environment 4 hours, OD value about 0.4~0.6 was prepared as competence.

Embodiment 3

This example demonstrates that knocking out parental E. coli fumarase encoding gene using CRISPR/Cas9 technology FumABC, DNA transcription combine regulatory factor encoding gene arcA, glucose transport related gene ptsG, citrate synthase coding The process of gene gltA and argininosuccinate synthetase encoding gene argG.

1, pTarget T2-1 plasmid is imported in the competence of albumen containing cas9, with adding spectinomycin and kanamycins LB flat screen selects positive recombinant.

2, positive recombinant among the above is identified with the method for PCR, filters out aimed strain.

3, the bacterial strain 1 filtered out in step 2 is transferred in the LB culture medium containing kanamycins for being added to IPTG induction and is trained After supporting, LB culture medium flat plate of the turning point extremely containing kanamycins and the LB culture medium containing kanamycins and spectinomycin simultaneously are flat respectively Plate filters out the recombinant bacterial strain 1 for having degraded pTarget T2-1 plasmid.

4, the recombinant bacterial strain for filtering out step 3 is induced with arabinose, is prepared as competence.

5, by the competence of pTarget T2-2 plasmid steps for importing 4, with the LB of addition spectinomycin and kanamycins Flat screen selects positive recombinant.

6, positive recombinant among the above is identified with the method for PCR, filters out aimed strain.

7, the bacterial strain 2 filtered out in step 6 is transferred in the LB culture medium containing kanamycins for being added to IPTG induction and is trained After supporting, LB culture medium flat plate of the turning point extremely containing kanamycins and the LB culture medium containing kanamycins and spectinomycin simultaneously are flat respectively Plate filters out the recombinant bacterial strain 2 for having degraded pTarget T2-2 plasmid.

8, with the method for similar step 5 to step 7 import in batches pTarget T2-3 plasmid, pTarget T2-4 plasmid, PTarget T2-5 plasmid, pTarget T2-6 plasmid carry out gene knockout, and are eliminated using IPTG induction and import plasmid, obtain Gene knock-out bacterial strain Escherichia coli △ fumABC △ arcA △ ptsG △ gltA △ argG.

Embodiment 4

This example demonstrates that building and the Escherichia coli △ fumABC/ of pTrc99A-asnA-aspA plasmid PTrc99A-asnA-aspA genetic engineering bacterium screening process.

1, it is arranged with nucleotides sequence shown in SEQ ID NO:41 and SEQ ID NO:42 as primer, genome of E.coli is Template, PCR amplification obtain linear fragment 10；It is to draw with the column of nucleotides sequence shown in SEQ ID NO:43 and SEQ ID NO:44 Object, genome of E.coli are template, and PCR amplification obtains linear fragment 11；

2, it is arranged with nucleotides sequence shown in SEQ ID NO:41 and SEQ ID NO:44 as primer, segment 10 and segment 11 Mixture is template, and over-lap PCR expands to obtain linear fragment 12；

3, dephosphorylation is carried out as carrier with segment 11 after using EcoRI and HindIII to linearize pTrc99a plasmid Ji Busen one-step cloning selects positive recombinant with the LB flat screen of the resistance of benzyl containing ammonia；

4, numerous rear progress plasmid extraction is expanded into the LB liquid training that the positive recombinant in step 3 is inoculated in addition spectinomycin, Obtain pTrc99A-asnA-aspA recombinant plasmid；

5, bacterial strain Escherichia coli △ fumABC △ arcA △ ptsG △ gltA △ argG is prepared as experiencing State；PTrc99A-asnA-aspA plasmid in step 4 is converted into competence, is selected with the LB flat screen of the resistance of benzyl containing ammonia Positive recombinant obtains target gene engineered strain.

Embodiment 5

This example demonstrates that improving altheine yield and related enzyme activity data comparison using aerobic fermentation.Specific step It is rapid as follows:

1, using LB culture medium by 1~2% (v/v) inoculum concentration from cryopreservation tube access triangular flask in, it is aerobic culture 10~ 12h is further seeded to seed fermentation tank (culture medium is also LB) by 1~2% (v/v) inoculum concentration, to thallus after 4~6h of culture Between OD600 to 2.5~4, by 5~10% inoculation induced mediums (M9 culture medium, IPTG induction), mended after induction period Add glucose 30g/L；

2, the control of seed culture process temperature is not required to adjust pH at 35~37 DEG C, in culture.Fermentation process uses aerobic hair Ferment mode, dissolved oxygen are 5~40%, and at 28~30 DEG C, incubation pH is controlled with ammonium hydroxide 7.0 the control of fermentation process temperature. Fermentation the results are shown in Table 1 for 36 hours, and starting strain and recombinant bacterial strain enzyme activity comparison result are shown in Table 2.

1 two kinds of strain fermentations of table produce altheine situation

From upper table as it can be seen that in the identical situation of fermentation condition, recombinant bacterial strain is mentioned than the altheine yield of starting strain It is high by 29.4%.

2 two kinds of bacterial strain enzyme activity of table compare

Bacterial strain	Sample time (h)	Aspartase (U/mL)	Asparagine synthetase A (U/mL)
				Starting strain	5	70.6	64.1
Recombinant bacterial strain	5	988.2	907.1

Sequence table

<110>Nanjing University of Technology

<120>one plants of genetic engineering bacteriums for synthesizing altheine and its construction method and application

<130> xb19052103

<160> 44

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1647

<212> DNA

<213>the fumA gene order () knocked out

<400> 1

atgtcaaaca aaccctttca ttatcaggct ccttttccac tcaaaaaaga tgatactgag 60

tattacctgc taaccagcga acacgttagc gtatctgaat ttgaagggca ggagattttg 120

aaagtcgcac ccgaagcgtt aactctgttg gcgcgccagg cgtttcatga tgcgtcgttc 180

atgctgcgtc cggcgcacca acaacaggtg gccgacattc tgcgtgaccc ggaggccagc 240

gaaaatgata aatatgtggc gctgcaattc ctgcgtaact ccgacatcgc ggcgaaaggc 300

gttctgccaa cctgtcagga taccggcacc gcgattattg ttggtaaaaa agggcagcgt 360

gtatggaccg gtggtggtga tgaagcggcg ctggcgcgcg gtgtctataa cacttatatc 420

gaagataatc tgcgctactc gcaaaacgcg ccgctggata tgtataaaga agtgaatacc 480

ggcaccaatc tgccagcgca gatcgatctt tatgccgttg atggcgacga atacaaattc 540

ctctgtatcg ccaaaggtgg tggttcggca aacaagacgt atctctatca ggaaaccaaa 600

gcgttactga cgccggggaa actgaaaaat tacctggttg agaagatgcg cacgctgggt 660

acggcggcct gtcctccgta tcatattgcg ttcgttattg gtggaacttc tgcagaaacg 720

aaccttaaaa cggtgaaact ggcttccgcg aaatactatg atgaactgcc aacggaaggg 780

aatgagcacg gtcaggcgtt ccgcgatgtg gaactggaaa aagaattgct gatcgaagcg 840

caaaatcttg gtctgggtgc gcagtttggt ggtaaatact tcgctcacga catccgcgtg 900

attcgcctgc cacgtcacgg cgcatcctgc ccggtcggta tgggcgtctc ctgctctgct 960

gaccgtaata tcaaagcgaa gatcaaccgt caggggatct ggatcgaaaa actggaacat 1020

aatccaggca aatatatccc ggaagagctg cgcaaagcgg gagaaggcga agcggtgcgc 1080

gttgacctta accgtccgat gaaagagatc ctcgcacagt tgtcgcagta tcccgtttct 1140

acacgcttat cgcttaacgg cacgattatc gtcggtcgtg atattgctca cgccaaactg 1200

aaagagcgga tggataacgg tgaagggctg ccgcagtaca tcaaagatca tccgatttac 1260

tacgcgggtc cggccaaaac gccggaaggt tatgcctccg gttctcttgg cccaacgacc 1320

gccggacgga tggattctta tgtcgatcaa ctgcaagcgc agggcggaag tatgatcatg 1380

ctggcgaaag gcaaccgcag ccagcaggtg acggatgcct gtaaaaaaca cggcggcttc 1440

taccttggca gtatcggtgg tccggccgct gtattggcgc agggaagtat taagagcctg 1500

gaatgtgttg aatatccgga actgggaatg gaagccatct ggaaaattga agtggaagat 1560

ttcccggcgt ttatccttgt ggatgataaa ggaaatgact tcttccagca gatacaactc 1620

acacaatgca cccgctgtgt gaaataa 1647

<210> 2

<211> 1647

<212> DNA

<213>the fumB gene order () knocked out

<400> 2

atgtcaaaca aaccctttat ctaccaggca cctttcccga tggggaaaga caataccgaa 60

tactatctac tcacttccga ttacgttagc gttgccgact tcgacggcga aaccatcctg 120

aaagtggaac cagaagccct gaccctgctg gcgcagcaag cctttcacga cgcttctttt 180

atgctccgcc cggcacacca gaaacaggtt gcggctattc ttcacgatcc agaagccagc 240

gaaaacgaca agtacgtggc gctgcaattc ttaagaaact ccgaaatcgc cgccaaaggc 300

gtgctgccga cctgccagga taccggcacc gcgatcatcg tcggtaaaaa aggccagcgc 360

gtgtggaccg gcggcggtga tgaagaaacg ctgtcgaaag gcgtctataa cacctatatc 420

gaagataacc tgcgctattc acagaatgcg gcgctggaca tgtacaaaga ggtcaacacc 480

ggcactaacc tgcctgcgca aatcgacctg tacgcggtag atggcgatga gtacaaattc 540

ctttgcgttg cgaaaggcgg cggctctgcc aacaaaacgt atctctacca ggaaaccaaa 600

gccctgctga ctcccggcaa actgaaaaac ttcctcgtcg agaaaatgcg taccctcggt 660

actgcagcct gcccgccgta ccatatcgcg tttgtgattg gcggtacgtc tgcggaaacc 720

aacctgaaaa ccgtcaagtt agcaagcgct cactattacg atgaactgcc gacggaaggg 780

aacgaacatg gtcaggcgtt ccgcgatgtc cagctggaac aggaactgct ggaagaggcc 840

cagaaactcg gtcttggcgc gcagtttggc ggtaaatact tcgcgcacga cattcgcgtt 900

atccgtctgc cacgtcacgg cgcatcctgc ccggtcggca tgggcgtctc ctgctccgct 960

gaccgtaaca ttaaagcgaa aatcaaccgc gaaggtatct ggatcgaaaa actggaacac 1020

aacccaggcc agtacattcc acaagaactg cgccaggccg gtgaaggcga agcggtgaaa 1080

gttgacctta accgcccgat gaaagagatc ctcgcccagc tttcgcaata cccggtatcc 1140

actcgtttgt cgctcaccgg caccattatc gtgggccgag atattgcaca cgccaagctg 1200

aaagagctga ttgacgccgg taaagaactt ccgcagtaca tcaaagatca cccgatctac 1260

tacgcgggtc cggcgaaaac ccctgccggt tatccatcag gttcacttgg cccaaccacc 1320

gcaggccgta tggactccta cgtggatctg ctgcaatccc acggcggcag catgatcatg 1380

ctggcgaaag gtaaccgcag tcagcaggtt accgacgcgt gtcataaaca cggcggcttc 1440

tacctcggta gcatcggcgg tccggcggcg gtactggcgc agcagagcat caagcatctg 1500

gagtgcgtcg cttatccgga gctgggtatg gaagctatct ggaaaatcga agtagaagat 1560

ttcccggcgt ttatcctggt cgatgacaaa ggtaacgact tcttccagca aatcgtcaac 1620

aaacagtgcg cgaactgcac taagtaa 1647

<210> 3

<211> 1404

<212> DNA

<213>the fumC gene order () knocked out

<400> 3

atgaatacag tacgcagcga aaaagattcg atgggggcga ttgatgtccc ggcagataag 60

ctgtggggcg cacaaactca acgctcgctg gagcatttcc gcatttcgac ggagaaaatg 120

cccacctcac tgattcatgc gctggcgcta accaagcgtg cagcggcaaa agttaatgaa 180

gatttaggct tgttgtctga agagaaagcg agcgccattc gtcaggcggc ggatgaagta 240

ctggcaggac agcatgacga cgaattcccg ctggctatct ggcagaccgg ctccggcacg 300

caaagtaaca tgaacatgaa cgaagtgctg gctaaccggg ccagtgaatt actcggcggt 360

gtgcgcggga tggaacgtaa agttcaccct aacgacgacg tgaacaaaag ccaaagttcc 420

aacgatgtct ttccgacggc gatgcacgtt gcggcgctgc tggcgctgcg caagcaactc 480

attcctcagc ttaaaaccct gacacagaca ctgaatgaga aatcccgtgc ttttgccgat 540

atcgtcaaaa ttggtcgtac tcacttgcag gatgccacgc cgttaacgct ggggcaggag 600

atttccggct gggtagcgat gctcgagcat aatctcaaac atatcgaata cagcctgcct 660

cacgtagcgg aactggctct tggcggtaca gcggtgggta ctggactaaa tacccatccg 720

gagtatgcgc gtcgcgtagc agatgaactg gcagtcatta cctgtgcacc gtttgttacc 780

gcgccgaaca aatttgaagc gctggcgacc tgtgatgccc tggttcaggc gcacggcgcg 840

ttgaaagggt tggctgcgtc actgatgaaa atcgccaatg atgtccgctg gctggcctct 900

ggcccgcgct gcggaattgg tgaaatctca atcccggaaa atgagccggg cagctcaatc 960

atgccgggga aagtgaaccc aacacagtgt gaggcattaa ccatgctctg ctgtcaggtg 1020

atggggaacg acgtggcgat caacatgggg ggcgcttccg gtaactttga actgaacgtc 1080

ttccgtccaa tggtgatcca caatttcctg caatcggtgc gcttgctggc agatggcatg 1140

gaaagtttta acaaacactg cgcagtgggt attgaaccga atcgtgagcg aatcaatcaa 1200

ttactcaatg aatcgctgat gctggtgact gcgcttaaca cccacattgg ttatgacaaa 1260

gccgccgaga tcgccaaaaa agcgcataaa gaagggctga ccttaaaagc tgcggccctt 1320

gcgctggggt atcttagcga agccgagttt gacagctggg tacggccaga acagatggtc 1380

ggcagtatga aagccgggcg ttaa 1404

<210> 4

<211> 1437

<212> DNA

<213>aspA gene order ()

<400> 4

atgtcaaaca acattcgtat cgaagaagat ctgttgggta ccagggaagt tccagctgat 60

gcctactatg gtgttcacac tctgagagcg attgaaaact tctatatcag caacaacaaa 120

atcagtgata ttcctgaatt tgttcgcggt atggtaatgg ttaaaaaagc cgcagctatg 180

gcaaacaaag agctgcaaac cattcctaaa agtgtagcga atgccatcat tgccgcatgt 240

gatgaagtcc tgaacaacgg aaaatgcatg gatcagttcc cggtagacgt ctaccagggc 300

ggcgcaggta cttccgtaaa catgaacacc aacgaagtgc tggccaatat cggtctggaa 360

ctgatgggtc accaaaaagg tgaatatcag tacctgaacc cgaacgacca tgttaacaaa 420

tgtcagtcca ctaacgacgc ctacccgacc ggtttccgta tcgcagttta ctcttccctg 480

attaagctgg tagatgcgat taaccaactg cgtgaaggct ttgaacgtaa agctgtcgaa 540

ttccaggaca tcctgaaaat gggtcgtacc cagctgcagg acgcagtacc gatgaccctc 600

ggtcaggaat tccgcgcttt cagcatcctg ctgaaagaag aagtgaaaaa catccaacgt 660

accgctgaac tgctgctgga agttaacctt ggtgcaacag caatcggtac tggtctgaac 720

acgccgaaag agtactctcc gctggcagtg aaaaaactgg ctgaagttac tggcttccca 780

tgcgtaccgg ctgaagacct gatcgaagcg acctctgact gcggcgctta tgttatggtt 840

cacggcgcgc tgaaacgcct ggctgtgaag atgtccaaaa tctgtaacga cctgcgcttg 900

ctctcttcag gcccacgtgc cggcctgaac gagatcaacc tgccggaact gcaggcgggc 960

tcttccatca tgccagctaa agtaaacccg gttgttccgg aagtggttaa ccaggtatgc 1020

ttcaaagtca tcggtaacga caccactgtt accatggcag cagaagcagg tcagctgcag 1080

ttgaacgtta tggagccggt cattggccag gccatgttcg aatccgttca cattctgacc 1140

aacgcttgct acaacctgct ggaaaaatgc attaacggca tcactgctaa caaagaagtg 1200

tgcgaaggtt acgtttacaa ctctatcggt atcgttactt acctgaaccc gttcatcggt 1260

caccacaacg gtgacatcgt gggtaaaatc tgtgccgaaa ccggtaagag tgtacgtgaa 1320

gtcgttctgg aacgcggtct gttgactgaa gcggaacttg acgatatttt ctccgtacag 1380

aatctgatgc acccggctta caaagcaaaa cgctatactg atgaaagcga acagtaa 1437

<210> 5

<211> 993

<212> DNA

<213>asnA gene order ()

<400> 5

atgaaaaccg cttacattgc caaacaacgt caaattagct tcgtgaaatc tcacttttct 60

cgtcaactgg aagaacgtct ggggctgatc gaagtccagg cgccgattct tagccgtgtg 120

ggggatggca cgcaggataa cttgtcgggc tgtgaaaaag cggtgcaggt aaaagtgaaa 180

gctctgcctg atgcccagtt cgaagtggtt cattcactgg cgaagtggaa acgtcagacc 240

ttagggcaac acgacttcag cgcgggcgaa gggctgtaca cgcacatgaa agcccttcgc 300

cccgatgaag accgtctttc tccgttgcac tcggtctatg ttgaccagtg ggactgggaa 360

cgcgtaatgg gcgacggtga gcgtcaattc tcgactctga aaagcacggt agaggcgatc 420

tgggcgggaa ttaaagcaac cgaagctgcg gttagcgaag agtttggcct ggcaccgttc 480

ctgccggatc agatccactt cgtacacagc caggagttac tgtctcgtta tccggatctt 540

gatgccaaag ggcgtgagcg ggcgatagcg aaagatcttg gcgcggtatt ccttgtcggg 600

attggcggca agctgagcga tggtcatcgc cacgacgtgc gcgcaccgga ttatgatgac 660

tggagcaccc cgtcagagct gggccatgcg ggtctgaacg gcgatattct ggtgtggaac 720

ccggtactgg aagatgcgtt tgagctttcc tccatgggga tccgtgtaga tgccgacacg 780

ctgaagcatc aactggcgct gaccggtgac gaagatcgcc tggagctgga gtggcatcag 840

gcgctgctgc gcggtgaaat gccgcagacc atcggcggcg gtatcggcca gtctcgtttg 900

actatgctgc tgctgcaact gccgcatatc ggccaggttc agtgtggagt atggccagct 960

gctgttcgcg agagcgtccc ttctctgctg taa 993

<210> 6

<211> 717

<212> DNA

<213>the arcA gene order () knocked out

<400> 6

atgcagaccc cgcacattct tatcgttgaa gacgagttgg taacacgcaa cacgttgaaa 60

agtattttcg aagcggaagg ctatgatgtt ttcgaagcga cagatggcgc ggaaatgcat 120

cagatcctct ctgaatatga catcaacctg gtgatcatgg atatcaatct gccgggtaag 180

aacggtcttc tgttagcgcg tgaactgcgc gagcaggcga atgttgcgtt gatgttcctg 240

actggccgtg acaacgaagt cgataaaatt ctcggcctcg aaatcggtgc agatgactac 300

atcaccaaac cgttcaaccc gcgtgaactg acgattcgtg cacgcaacct actgtcccgt 360

accatgaatc tgggtactgt cagcgaagaa cgtcgtagcg ttgaaagcta caagttcaat 420

ggttgggaac tggacatcaa cagccgttcg ttgatcggcc ctgatggcga gcagtacaag 480

ctgccgcgca gcgagttccg cgccatgctt cacttctgtg aaaacccagg caaaattcag 540

tcccgtgctg aactgctgaa gaaaatgacc ggccgtgagc tgaaaccgca cgaccgtact 600

gtagacgtga cgatccgccg tattcgtaaa catttcgaat ctacgccgga tacgccggaa 660

atcatcgcca ccattcacgg tgaaggttat cgcttctgcg gtgatctgga agattaa 717

<210> 7

<211> 1434

<212> DNA

<213>the ptsG gene order () knocked out

<400> 7

atgtttaaga atgcatttgc taacctgcaa aaggtcggta aatcgctgat gctgccggta 60

tccgtactgc ctatcgcagg tattctgctg ggcgtcggtt ccgcgaattt cagctggctg 120

cccgccgttg tatcgcatgt tatggcagaa gcaggcggtt ccgtctttgc aaacatgcca 180

ctgatttttg cgatcggtgt cgccctcggc tttaccaata acgatggcgt atccgcgctg 240

gccgcagttg ttgcctatgg catcatggtt aaaaccatgg ccgtggttgc gccactggta 300

ctgcatttac ctgctgaaga aatcgcctct aaacacctgg cggatactgg cgtactcgga 360

gggattatct ccggtgcgat cgcagcgtac atgtttaacc gtttctaccg tattaagctg 420

cctgagtatc ttggcttctt tgccggtaaa cgctttgtgc cgatcatttc tggcctggct 480

gccatcttta ctggcgttgt gctgtccttc atttggccgc cgattggttc tgcaatccag 540

accttctctc agtgggctgc ttaccagaac ccggtagttg cgtttggcat ttacggtttc 600

atcgaacgtt gcctggtacc gtttggtctg caccacatct ggaacgtacc tttccagatg 660

cagattggtg aatacaccaa cgcagcaggt caggttttcc acggcgacat tccgcgttat 720

atggcgggtg acccgactgc gggtaaactg tctggtggct tcctgttcaa aatgtacggt 780

ctgccagctg ccgcaattgc tatctggcac tctgctaaac cagaaaaccg cgcgaaagtg 840

ggcggtatta tgatctccgc ggcgctgacc tcgttcctga ccggtatcac cgagccgatc 900

gagttctcct tcatgttcgt tgcgccgatc ctgtacatca tccacgcgat tctggcaggc 960

ctggcattcc caatctgtat tcttctgggg atgcgtgacg gtacgtcgtt ctcgcacggt 1020

ctgatcgact tcatcgttct gtctggtaac agcagcaaac tgtggctgtt cccgatcgtc 1080

ggtatcggtt atgcgattgt ttactacacc atcttccgcg tgctgattaa agcactggat 1140

ctgaaaacgc cgggtcgtga agacgcgact gaagatgcaa aagcgacagg taccagcgaa 1200

atggcaccgg ctctggttgc tgcatttggt ggtaaagaaa acattactaa cctcgacgca 1260

tgtattaccc gtctgcgcgt cagcgttgct gatgtgtcta aagtggatca ggccggcctg 1320

aagaaactgg gcgcagcggg cgtagtggtt gctggttctg gtgttcaggc gattttcggt 1380

actaaatccg ataacctgaa aaccgagatg gatgagtaca tccgtaacca ctaa 1434

<210> 8

<211> 1284

<212> DNA

<213>the gltA gene order () knocked out

<400> 8

atggctgata caaaagcaaa actcaccctc aacggggata cagctgttga actggatgtg 60

ctgaaaggca cgctgggtca agatgttatt gatatccgta ctctcggttc aaaaggtgtg 120

ttcacctttg acccaggctt cacttcaacc gcatcctgcg aatctaaaat tacttttatt 180

gatggtgatg aaggtatttt gctgcaccgc ggtttcccga tcgatcagct ggcgaccgat 240

tctaactacc tggaagtttg ttacatcctg ctgaatggtg aaaaaccgac tcaggaacag 300

tatgacgaat ttaaaactac ggtgacccgt cataccatga tccacgagca gattacccgt 360

ctgttccatg ctttccgtcg cgactcgcat ccaatggcag tcatgtgtgg tattaccggc 420

gcgctggcgg cgttctatca cgactcgctg gatgttaaca atcctcgtca ccgtgaaatt 480

gccgcgttcc gcctgctgtc gaaaatgccg accatggccg cgatgtgtta caagtattcc 540

attggtcagc catttgttta cccgcgcaac gatctctcct acgccggtaa cttcctgaat 600

atgatgttct ccacgccgtg cgaaccgtat gaagttaatc cgattctgga acgtgctatg 660

gaccgtattc tgatcctgca cgctgaccat gaacagaacg cctctacctc caccgtgcgt 720

accgctggct cttcgggtgc gaacccgttt gcctgtatcg cagcaggtat tgcttcactg 780

tggggacctg cgcacggcgg tgctaacgaa gcggcgctga aaatgctgga agaaatcagc 840

tccgttaaac acattccgga atttgttcgt cgtgcgaaag acaaaaatga ttctttccgc 900

ctgatgggct tcggtcaccg cgtgtacaaa aattacgacc cgcgcgccac cgtaatgcgt 960

gaaacctgcc atgaagtgct gaaagagctg ggcacgaagg atgacctgct ggaagtggct 1020

atggagctgg aaaacatcgc gctgaacgac ccgtacttta tcgagaagaa actgtacccg 1080

aacgtcgatt tctactctgg tatcatcctg aaagcgatgg gtattccgtc ttccatgttc 1140

accgtcattt tcgcaatggc acgtaccgtt ggctggatcg cccactggag cgaaatgcac 1200

agtgacggta tgaagattgc ccgtccgcgt cagctgtata caggatatga aaaacgcgac 1260

tttaaaagcg atatcaagcg ttaa 1284

<210> 9

<211> 1344

<212> DNA

<213>the argG gene order () knocked out

<400> 9

atgacgacga ttctcaagca tctcccggta ggtcaacgta ttggtatcgc tttttctggc 60

ggtctggaca ccagtgccgc actgctgtgg atgcgacaaa agggagcggt tccttatgca 120

tatactgcaa acctgggcca gccagacgaa gaggattatg atgcgatccc tcgtcgtgcc 180

atggaatacg gcgcggagaa cgcacgtctg atcgactgcc gcaaacaact ggtggccgaa 240

ggtattgccg ctattcagtg tggcgcattt cataacacca ccggcggcct gacctatttc 300

aacacgacgc cgctgggccg cgccgtgact ggtaccatgc tggttgctgc gatgaaagaa 360

gatggcgtga atatctgggg tgacggtagc acctacaaag gaaacgatat cgaacgtttc 420

tatcgttatg gtctgctgac caatgctgaa ctgcagattt acaaaccgtg gcttgatact 480

gactttattg atgaactggg cggccgtcat gagatgtctg aatttatgat tgcctgcggt 540

ttcgactaca aaatgtctgt cgaaaaagcc tactccacag actccaacat gcttggtgca 600

acgcatgaag cgaaggatct ggaatacctc aactccagcg tcaaaatcgt caacccgatt 660

atgggcgtga aattctggga tgagagcgtg aagatcccgg cagaagaagt cacagtacgc 720

tttgaacaag gtcatccggt ggcgctgaac ggtaaaacct ttagcgacga cgtagaaatg 780

atgctggaag ctaaccgcat cggcggtcgt cacggcctgg gcatgagcga ccagattgaa 840

aaccgtatca tcgaagcgaa aagccgtggt atttacgaag ctccggggat ggcactgctg 900

cacattgcgt atgaacgcct gttgaccggt attcacaacg aagacaccat tgagcagtat 960

cacgcgcatg gtcgtcagtt gggccgtctg ctgtaccagg ggcgttggtt tgactcccag 1020

gcgctgatgc tgcgtgactc tctgcaacgc tgggttgcca gccagatcac tggtgaagtt 1080

accctggagc tgcgccgtgg gaacgattat tcaatcctga ataccgtctc agagaacctg 1140

acctacaagc cagagcgtct gacgatggaa aaaggcgact cggtgttctc gccagatgat 1200

cgtattggtc aattgaccat gcgtaacctg gatatcactg atacccgcga gaaacttttc 1260

ggttatgcca aaactggcct gctttcctcc tctgccgctt caggcgtgcc gcaggtggag 1320

aatctggaaa acaaaggcca gtaa 1344

<210> 10

<211> 28

<212> DNA

<213>amplimer sequence F ()

<400> 10

gatgactagt attataccta ggactgag 28

<210> 11

<211> 53

<212> DNA

<213>amplimer sequence R ()

<400> 11

ctagactagt atactatgat gaactgccaa gttttagagc tagaaatagc aag 53

<210> 12

<211> 53

<212> DNA

<213>amplimer sequence R ()

<400> 12

ctagactagt gtgagcgctt gctaacttga gttttagagc tagaaatagc aag 53

<210> 13

<211> 53

<212> DNA

<213>amplimer sequence R ()

<400> 13

ctagactagt actgtcccgt accatgaatc gttttagagc tagaaatagc aag 53

<210> 14

<211> 53

<212> DNA

<213>amplimer sequence R ()

<400> 14

ctagactagt gcgctgacct cgttcctgac gttttagagc tagaaatagc aag 53

<210> 15

<211> 53

<212> DNA

<213>amplimer sequence R ()

<400> 15

ctagactagt gcaggtattg cttcactgtg gttttagagc tagaaatagc aag 53

<210> 16

<211> 53

<212> DNA

<213>amplimer sequence R ()

<400> 16

ctagactagt gtggtattta cgaagctccg gttttagagc tagaaatagc aag 53

<210> 17

<211> 35

<212> DNA

<213>amplimer sequence F ()

<400> 17

gctttttttg aattctggat gaacctgaat ggaga 35

<210> 18

<211> 41

<212> DNA

<213>amplimer sequence R ()

<400> 18

ctgcacctgt atgttgcaga tgttctctca cttactgcct g 41

<210> 19

<211> 41

<212> DNA

<213>amplimer sequence F ()

<400> 19

caggcagtaa gtgagagaac atctgcaaca tacaggtgca g 41

<210> 20

<211> 31

<212> DNA

<213>amplimer sequence R ()

<400> 20

gatctaagct tatgggtgca tcgtcatttg c 31

<210> 21

<211> 35

<212> DNA

<213>amplimer sequence F ()

<400> 21

gctttttttg aattcatcgc catcgtggcg gtgta 35

<210> 22

<211> 42

<212> DNA

<213>amplimer sequence R ()

<400> 22

ccaggcgctg ggccgaagag gagcttccag cctgtaactc tg 42

<210> 23

<211> 42

<212> DNA

<213>amplimer sequence F ()

<400> 23

cagagttaca ggctggaagc tcctcttcgg cccagcgcct gg 42

<210> 24

<211> 31

<212> DNA

<213>amplimer sequence R ()

<400> 24

gatctaagct tttacgtccg cgcacgtaca t 31

<210> 25

<211> 35

<212> DNA

<213>amplimer sequence F ()

<400> 25

gctttttttg aattcgactg tactaacggt tgagt 35

<210> 26

<211> 40

<212> DNA

<213>amplimer sequence R ()

<400> 26

tttgacggtg gtaaagccga gtttgctacc taaattgcca 40

<210> 27

<211> 40

<212> DNA

<213>amplimer sequence F ()

<400> 27

tggcaattta ggtagcaaac tcggctttac caccgtcaaa 40

<210> 28

<211> 31

<212> DNA

<213>amplimer sequence R ()

<400> 28

gatctaagct tattctgctg attgcactga c 31

<210> 29

<211> 35

<212> DNA

<213>amplimer sequence F ()

<400> 29

gctttttttg aattcgatcg gttactggtg gaaac 35

<210> 30

<211> 40

<212> DNA

<213>amplimer sequence R ()

<400> 30

tctccccaac gtcttacgga aattgagagt gctcctgagt 40

<210> 31

<211> 40

<212> DNA

<213>amplimer sequence F ()

<400> 31

actcaggagc actctcaatt tccgtaagac gttggggaga 40

<210> 32

<211> 31

<212> DNA

<213>amplimer sequence R ()

<400> 32

gatctaagct tcagtaaagg ggtggaattt g 31

<210> 33

<211> 35

<212> DNA

<213>amplimer sequence F ()

<400> 33

gctttttttg aattcgttcc ggagacctgg cggca 35

<210> 34

<211> 42

<212> DNA

<213>amplimer sequence R ()

<400> 34

tttacaactt agcaatcaac cattaaggtc tccttagcgc ct 42

<210> 35

<211> 42

<212> DNA

<213>amplimer sequence F ()

<400> 35

aggcgctaag gagaccttaa tggttgattg ctaagttgta aa 42

<210> 36

<211> 31

<212> DNA

<213>amplimer sequence R ()

<400> 36

gatctaagct ttgaatttca caaggtgctt c 31

<210> 37

<211> 35

<212> DNA

<213>amplimer sequence F ()

<400> 37

gctttttttg aattcaatcc cactacgaag gccga 35

<210> 38

<211> 42

<212> DNA

<213>amplimer sequence R ()

<400> 38

agggcagggt tgatgtcgaa aaaataacac cctgcttaat ta 42

<210> 39

<211> 42

<212> DNA

<213>amplimer sequence F ()

<400> 39

taattaagca gggtgttatt ttttcgacat caaccctgcc ct 42

<210> 40

<211> 31

<212> DNA

<213>amplimer sequence R ()

<400> 40

gatctaagct ttccgcaatc cagcgccgtg t 31

<210> 41

<211> 41

<212> DNA

<213>amplimer sequence F ()

<400> 41

aggaaacaga ccatggaatt catgaaaacc gcttacattg c 41

<210> 42

<211> 52

<212> DNA

<213>amplimer sequence R ()

<400> 42

tttctacctc cttataaata gtttcctttt tacagcagag aagggacgct ct 52

<210> 43

<211> 54

<212> DNA

<213>amplimer sequence F ()

<400> 43

aaaggaaact atttataagg aggtagaaaa tgtcaaacaa cattcgtatc gaag 54

<210> 44

<211> 41

<212> DNA

<213>amplimer sequence R ()

<400> 44

tccgccaaaa cagccaagct tttactgttc gctttcatca g 41

Claims

1. the genetic engineering bacterium of one plant of synthesis altheine, classification naming is Escherichia coli △ fumABC △ ArcA △ ptsG △ gltA △ argG/pAA, deposit number are CCTCC NO:M 2019170.

2. genetic engineering bacterium according to claim 1, which is characterized in that knockout deposit number is CCTCC NO:M 2018521 Starting strain fumarase encoding gene fumABC, DNA transcription combine regulatory factor encoding gene arcA, glucose transport related Gene ptsG, citrate synthase encoding gene gltA and argininosuccinate synthetase encoding gene argG, while by asparagus fern ammonia Sour enzyme coding gene aspA and asparagine synthetase A encoding gene asnA are cloned on expression plasmid and constitute recombinant plasmid, and It is converted to the Escherichia coli starting strain for having knocked out fumABC gene, obtains the genetic engineering bacterium, the fumA gene Sequence is as shown in SEQ ID NO:1, and the fumB gene order is as shown in SEQ ID NO:2, the fumC gene order such as SEQ Shown in ID NO:3, the aspA gene order is as shown in SEQ ID NO:4, the asnA gene order such as SEQ ID NO:5 institute Show, for the arcA gene order as shown in SEQ ID NO:6, the ptsG gene order is described as shown in SEQ ID NO:7 GltA gene order is as shown in SEQ ID NO:8, and the argG gene order is as shown in SEQ ID NO:9.

3. the construction method of genetic engineering bacterium described in claim 1, which comprises the steps of:

(1) it is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:11 as primer, plasmid pTarget F is mould Plate, PCR amplification obtain linear fragment 1；

(2) it is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:12 as primer, repeats step (1) and obtain linearly Segment 2；It is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:13 as primer, repeats step (1) and obtain linearly Segment 3；It is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:14 as primer, repeats step (1) and obtain linearly Segment 4；It is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:15 as primer, repeats step (1) and obtain linearly Segment 5；It is arranged with nucleotides sequence shown in SEQ ID NO:10 and SEQ ID NO:16 as primer, repeats step (1) and obtain linearly Segment 6；

(3) it will be attached after the SpeI digestion of linear fragment 1, and product after connection be transformed into competence, be coated with grand sight The LB flat screen of mycin selects positive recombinant；

(5) it will be attached transformation and selection after the SpeI digestion of linear fragment 2 go out positive recombinant and carry out plasmid extraction, obtain PTarget T1-2 plasmid；Transformation and selection being attached after the SpeI digestion of linear fragment 3 and going out positive recombinant carry out plasmid Extracting obtains pTarget T1-3 plasmid；Go out positive recombinant for transformation and selection is attached after the SpeI digestion of linear fragment 4 Plasmid extraction is carried out, pTarget T1-4 plasmid is obtained；It will be attached transformation and selection after the SpeI digestion of linear fragment 5 and go out sun Property recon carry out plasmid extraction, obtain pTarget T1-5 plasmid；Conversion will be attached after the SpeI digestion of linear fragment 6 It filters out positive recombinant and carries out plasmid extraction, obtain pTarget T1-6 plasmid；

(6) it is arranged with nucleotides sequence shown in SEQ ID NO:17 and SEQ ID NO:18 as primer, genome of E.coli is mould Plate, PCR amplification obtain linear fragment 7；It is arranged for primer, greatly with nucleotides sequence shown in SEQ ID NO:19 and SEQ ID NO:20 Enterobacteria genome is template, and PCR amplification obtains linear fragment 8；

(7) mixing of segment 7 and segment 8 for primer is arranged with nucleotides sequence shown in SEQ ID NO:17 and SEQ ID NO:20 Object is template, and over-lap PCR expands to obtain linear fragment 9；

(8) dephosphorylation carries out lucky cloth gloomy one with segment 9 as carrier after using EcoRI to linearize pTarget T1-1 plasmid Step clone, selects positive recombinant with the LB flat screen of spectinomycin；

(9) numerous rear progress plasmid extraction is expanded into the LB liquid training that the positive recombinant in step (8) is inoculated in addition spectinomycin, obtains Obtain pTarget T2-1 plasmid；

It (10) is that primer repeats step (6) to step (9) with the column of nucleotides sequence shown in SEQ ID NO:21-SEQ ID NO:24 Obtain pTarget T2-2 plasmid；It is that primer repeats to walk with the column of nucleotides sequence shown in SEQ ID NO:25-SEQ ID NO:28 Suddenly (6) to step (9) obtain pTarget T2-3 plasmid；With nucleotides sequence shown in SEQ ID NO:29-SEQ ID NO:32 It is classified as primer and repeats step (6) to step (9) acquisition pTarget T2-4 plasmid；With SEQ ID NO:33-SEQ ID NO:36 Shown in nucleotides sequence be classified as primer repeat step (6) to step (9) obtain pTarget T2-5 plasmid；With SEQ ID NO: Nucleotides sequence shown in 37-SEQ ID NO:40 is classified as primer and repeats step (6) to step (9) acquisition pTarget T2-6 matter Grain；

(11) pCas plasmid is imported in the starting strain that deposit number is CCTCC NO:M 2018521, it is flat with the LB of kanamycins Screen selects positive recombinant；

(13) by the competence of pTarget T2-1 plasmid steps for importing (12), with the LB of addition spectinomycin and kanamycins Flat screen selects positive recombinant；

(14) positive recombinant in step (13) is identified with the method for PCR, filters out aimed strain, and use IPTG PTarget T2-1 plasmid is eliminated in induction, obtains bacterial strain 1；

(16) by the competence of pTarget T2-2 plasmid steps for importing (15), with the LB of addition spectinomycin and kanamycins Flat screen selects positive recombinant；

(17) positive recombinant in step (16) is identified with the method for PCR, filters out aimed strain, and use IPTG PTarget T2-2 plasmid is eliminated in induction, obtains bacterial strain 2；

(18) pTarget T2-3 plasmid, pTarget T2-4 matter are imported in batches with the method for similar step (15) to step (17) Grain, pTarget T2-5 plasmid, pTarget T2-6 plasmid carry out gene knockout, and are eliminated using IPTG induction and import plasmid, Obtain gene knock-out bacterial strain Escherichia coli △ fumABC △ arcA △ ptsG △ gltA △ argG；

(19) it is arranged with nucleotides sequence shown in SEQ ID NO:41 and SEQ ID NO:42 as primer, genome of E.coli is mould Plate, PCR amplification obtain linear fragment 10；It is arranged with nucleotides sequence shown in SEQ ID NO:43 and SEQ ID NO:44 as primer, Genome of E.coli is template, and PCR amplification obtains linear fragment 11；

(20) arranged with nucleotides sequence shown in SEQ ID NO:41 and SEQ ID NO:44 as primer, segment 10 and segment 11 it is mixed Conjunction object is template, and over-lap PCR expands to obtain linear fragment 12；

(21) dephosphorylation carries out Ji with segment 11 as carrier after using EcoRI and HindIII to linearize pTrc99a plasmid The gloomy one-step cloning of cloth selects positive recombinant with the LB flat screen of the resistance of benzyl containing ammonia；

(22) numerous rear progress plasmid extraction is expanded into the LB liquid training that the positive recombinant in step (21) is inoculated in addition spectinomycin, Obtain pTrc99A-asnA-aspA plasmid；

(23) gene knock-out bacterial strain in step (18) is prepared as competence；By the pTrc99A-asnA- in step (22) AspA plasmid is converted into competence, is selected positive recombinant with the LB flat screen of the resistance of benzyl containing ammonia, is obtained target gene engineering Bacterial strain.

4. the genetic engineering bacterium for synthesizing altheine described in claim 1 prepares answering in altheine in biosynthesis With.

5. application according to claim 4, which is characterized in that the process for preparing altheine is as follows:

(S1) genetic engineering bacterium is transferred in LB culture medium, 10~12h of aerobic culture obtains primary seed solution；

(S3) to secondary seed solution OD₆₀₀When to 8.5, induced medium is inoculated, the formula of the induced medium is as follows:

M9 culture medium: (NH₄)₂SO₄6g/L, Na₂HPO₄·12H₂O 15.2g/L, KH₂PO₄3g/L, NaCl 0.5g/L, NH₄Cl 1g/L, MgSO₄2mM, CaCl₂0.1mM, 100g/L glucose, 0.3mM IPTG；

6. application according to claim 5, which is characterized in that in step (S1) and (S2), cultivation temperature is 35~37 DEG C.

7. application according to claim 5, which is characterized in that in step (S3) and (S4), dissolved oxygen control 5~ 40%.

8. application according to claim 5, which is characterized in that temperature is 28~30 in step (S3) and (S4) fermentation process DEG C, incubation pH is adjusted to 7.0 with ammonium hydroxide.