CN104388476B - A kind of method that high efficiency, high conversion produce 1,3 propane diols - Google Patents

Abstract

1,3 propane diols are generated the invention discloses the method that one kind produces 1,3 propane diols by Klebsiella pneumoniae CCTCC M2014574 glycerine convertings, including seed culture and ferment tank glycerine converting.The Klebsiella pneumoniae has high transformation efficiency.Further strengthen 3 hydracrylic acid metabolic pathways of Klebsiella pneumoniae by appropriateness, the excessive accumulation of the hydracrylic acid of accessory substance 3 is avoided while the accumulation for solving 3 hydroxy propanals, the Efficient Conversion of 1,3 propane diols is realized.Compared to existing method, the major advantage of method of the invention includes：Production concentration is high, and production intensity is big, high conversion rate.

Description

A kind of method that high efficiency, high conversion produce 1,3- propane diols

Technical field

The invention belongs to technical field of bioengineering, specifically, be on one kind by Klebsiella pneumoniae, and The 3- hydracrylic acid metabolic pathways high efficiency of appropriateness reinforcing Klebsiella pneumoniae, high conversion produce the side of 1,3- propane diols Method.

Background technology

1,3-PD (1,3-porpanediol, referred to as 1,3-PD) is a kind of important industrial chemicals, be can be used as molten Agent, antifreeze or protective agent, fine chemical material and new poly-vinegar --- poly terephthalic acid propane diols vinegar (PTT) and poly- ammonia The monomer of vinegar.PTT has proved to be a kind of new polyester material of excellent performance.Current 1,3-PD productions are main using biological conjunction Into.The main route of biological synthesis process is that 1,3-PD is converted glycerol into the presence of microorganism.At present, can be by nature The microorganism that glycerine is converted into 1,3-PD generally comprises：Klebsiella pneumoniae (Klebsiela pneumoniae), Freund Lemon bacterium (Citrobacter freundi), clostridium butyricum (Clostridium butyricum) etc..

Research shows that 1,3-PD of Klebsiella pneumoniae production ability is most strong, and also there are some related production bacterium the country Patent is planted, such as：1st, week victory etc., a kind of mangrove Klebsiella and its application (grant number in production 1,3-PD： CN102604863)；2nd, Liu Dehua etc. a, plant height produces the microorganism (publication number of 1,3-PD：CN103740609A).But Lack the major obstacle that the efficient production strain of high conversion is still restriction 1,3-PD industrialization.

In Klebsiella pneumoniae, 1,3-PD biosynthetic metabolism Study of way show：1. its glycerine first Oxidation has rigidity with reduction approach and distribution node, and characteristic of this rigidity regulation largely with strain itself has Close；2. the accumulation of the intermediate product 3-HPA in reduction approach is to influence 1,3-PD important factor.

Also there are some researchs for releasing the accumulation of intermediate product 3-HPA at present, such as：1. 1,3-PD oxidations are overexpressed also Protoenzyme；2. it is overexpressed 3-HPA dehydrogenase etc..But be overexpressed production of the 1,3- oxidoreducing enzyme to 1,3-PD and promote to make With very little, 3- hydracrylic acids (3-HP) accumulation can be caused again by being overexpressed 3- aldehyde radical propionic aldehyde dehydrogenases, so be difficult to obtain high 1, 3-PD yield.

The content of the invention

Present inventor obtains the Klebsiella pneumoniae that a plant height produces 1,3-PD under study for action by seed selection Klebsiela pneumoniae KG.Klebsiela pneumoniae KG were preserved in Chinese Typical Representative on November 18th, 2014 Culture collection (address：Wuhan City, Hubei Province Hongshan District Bayi Road, Wuhan University, China typical culture collection center Postcode：430072), preserving number CCTCC M2014574.The Klebsiella pneumoniae is used for glycerine converting and produces 1,3- There is higher transformation efficiency compared to other Klebsiellas during propane diols.Further appropriateness strengthens Klebsiella pneumoniae 3-HP metabolic pathways, appropriateness reinforcing 3-HP metabolic pathways had both solved the accumulation of 3-HPA and turn avoid excessive accessory substance 3-HP suppression, overcomes the shortcoming over-expressed in the past.

The present invention producing 1,3-propylene glycol by transforming glycerol method, including seed culture and ferment tank conversion it is sweet Oil generation 1,3-PD, this method is by Klebsiella pneumoniae CCTCC M2014574, and in citric acid pneumonia bar Appropriate overexpression 3-HPA dehydrogenase in bacterium, realizes high efficiency, high conversion production 1,3-PD.

According to the present invention, the appropriate overexpression is realized by the leakage expression of Tac promoters.

According to the present invention, described 3-HPA dehydrogenase gene can derive from Klebsiella pneumoniae, large intestine Bacillus.

Compared to the method that existing glycerine converting produces 1,3-PD, the major advantage of method of the invention includes：Product is dense Degree is high, and production intensity is big, high conversion rate.

Brief description of the drawings

Fig. 1:The 16srDNA of KG bacterial strains homology analysis

Embodiment

Below in conjunction with specific embodiment, the present invention is described in further detail.It should be understood that following examples are only used for Illustrate the present invention, not for restriction the scope of the present invention.

In example：

LB culture mediums are：

0.5% yeast extract, 1% tryptone, 1%NaCl, pH 7.0；Solid medium is added on this basis 1.2%-1.5% agar.

Slant medium is：

K₂HPO₄3H₂O 7g/L, (NH₄)₂SO₄1g/L, KH₂PO₄2g/L, MgCl₂ 7H₂O 0.1g/L, yeast extract 7g/L, it is micro- Each 0.3mL of secondary element, adjusts pH 7.0, agar 2g/L.

Seed culture medium is：

K₂HPO₄3H₂O 7g/L, (NH₄)₂SO₄1g/L, KH₂PO₄2g/L, MgCl₂ 7H₂O 0.1g/L, yeast extract 7g/L, it is micro- NaCl regulation osmotic pressure is added after each 0.3mL of secondary element, adjustment pH 7.0.

Fermentation tank culture medium is：

KCl 0.75g/L, NaH₂PO₄1.38g/L, (NH₄)₂SO₄5.35g/L, Na₂SO₄0.28g/L, MgSO₄6H₂O 0.26g/L, citric acid 0.42g/L, dusty yeast 2g/L, micro- each 0.3mL adjust pH 7.0.

The formula of trace element is as follows：

ZnCl₂34.2g/L, FeCl₃6H₂O 2.7g/L, MnCl₂4H₂O 10g/L, CuCl₂ 2H₂O 0.85g/L, CoCl₂2H₂O 23.8g/L, H₃BO₃0.31g/L, Na₂MoO₄0.25g/L

In embodiment, the method for determining dry cell weight in zymotic fluid is as follows：

Take 1.0mL zymotic fluids to dilute 7~10 times, using deionized water as control, read on 721 spectrophotometers in 620nm Take OD.The bacterium solution 10mL of different bacterium dense (i.e. different 620nm light absorption values) is taken, through thalline is collected by centrifugation, and is washed with deionized Two times washings, the thalline after being collected by centrifugation again is dried to constant weight in 80 DEG C of baking ovens.Weigh thalline make dry cell weight with OD₆₂₀Standard curve, and return out relational expression.The dry weight of later thalline is according to the OD of the bacterium solution of measure₆₂₀Value is by standard curve Regression relation is calculated.

The generating rate of 1,3- propane diols is defined as：The 1,3- propane diols grams units of zymotic fluid generation are per liter per hour g/L·h。

The conversion ratio of 1,3- propane diols is defined as：1,3- propane diols molal quantity of the consumption per the production of mole of glycerin raw material.

In example, the enzyme activity of beta galactose glycosidase is defined as：1U is 1 minute catalysis 1nmol ONPG under the conditions of 37 DEG C Enzyme amount.Enzyme amount (U) of specific enzyme activity unit (U/mg) definition contained by unit dry cell weight (mg).

The identification of embodiment 1, KG

With every physical and chemical indexs of the Gram-negative enteric microorganism physics and chemistry kit API20E (French Mei Liai) to KG Analyzed.Physics and chemistry authentication step is shown in Table 1 according to API20E operation manuals, qualification result.API20E handbooks are compareed, show that KG belongs to In Klebsiella pneumoniae.

Table 1：API20E physics and chemistry qualification results

With bacterium 16srDNA universal primers 27f (AGAGTTTGATCCTGGCTCA) and 1394r (TACGGCTACCTTGTTACGAGTT) using KG genomes as template PCR amplifications KG 16srNDA genes, glue reclaim purpose bar KG 16sNDA sequences (SEQ ID NO.1) are obtained after band, sequencing.16srNDA homologous comparison result shows that KG belongs to Cray Bai Shi pneumobacilluses (accompanying drawing 1).

Embodiment 2, KG can high conversion production 1,3-PD

KG is planted with compareing bacterium K.pneumoniae ATCC49790, access 250ml shaking flask (liquid amount 50ml) In son culture 20 hours, rear access 5L fermentation tanks (zymotic fluid liquid amount 2L), fermented according to process regulation as follows Process.

Initial glycerol concentration 60g/L, 35 DEG C of fermentation temperature；Throughput 1.0vvm；Speed of agitator 20rpm；In fermentation process In by adding NaOH solution control ph for 5.5~7.5.Fermentation each period by filling into various concentrations glycerite Control glycerol concentration in 10~60g/L, fermentation terminates for 30 hours.

Shown in fermentation results table 2：

Table 2：KG is compared with ATCC49790 fermentation

From Table 2, it can be seen that being compared with other 1,3-PD productions bacterium such as ATCC49790, KG bacterial strains 1,3-PD conversion Efficiency is substantially high, and conversion ratio is more than 0.6.

Embodiment 3, the expression plasmid for building the promoter of expression containing appropriateness Tac

Expression vector skeleton selects pET28a, the pGEX-4T-1 that Tac promoter sequences take.Contain BglII- with a pair The primer (GGAagatctACGTTATCGACTGCACGG, GGCgaattcCATGAATACTGTTTCCTGT) of EcoRI restriction enzyme sites, Tac starting region 260bp (SEQ ID NO.2) on pGEX-4T-1 is amplified to come, and is inserted into pET28a BglII- EcoRI restriction enzyme sites, replacing that the original T7 promoter regions of pET28a build can appropriate gene table in Klebsiella The expression vector pETac reached.

In order to prove pETac can in Klebsiella pneumoniae can appropriate expressing gene, it is right with reporter gene (lacZ) Promoter Tac is analyzed.LacZ (gene of half lactoside enzyme of coding) derives from Escherichia coli.With one containing HindIII-XhoI To primer (CACaagcttGCGTTTTACAACGTCGTGAC, CCGctcgagTTATTTTTGACACCAGACCA) from large intestine bar Pcr amplifies 3075bp lacZ gene and is inserted under pETac expression vector Tac promoters on bacterium BL21 genome Trip, construction recombination plasmid pETac::LacZ, and continue pETac::LacZ electricity is gone in KG, while pET28a electricity is gone into KG It is middle to be used as control.

Thalline is collected after the transformant of acquisition is cultivated 12 hours in LB culture mediums and determines beta galactose glucosides enzyme activity, it is real Test result such as table 3 so.

Table 3：Each transformant beta galactose glycosidase expression

Tac promoters are the inductivity strong promoters commonly used in escherichia expression system, and its startup needs luring for IPTG Lead.Although present invention discover that Tac promoters in klebostiella pneumoniae also can IPTG induction under high efficient expression, Tac Promoter has larger leakage expression in Klebsiella pneumoniae.Can also have good under conditions of no IPTG inductions Expression, can be used as " composing type " promoter moderately expressed in Klebsiella.

Embodiment 4, moderately strengthen the efficient production 1,3-PD of 3-HPA dehydrogenase in KG

Can be converted in Klebsiella pneumoniae 3-HPA to 3-HP 3- hydroxymalonate dehydrogenases can origin come from The aldA (SEQ ID NO.3) of klebsiella, puuC (SEQ ID NO.4), ydcW (SEQ ID NO.5)；And from big The aldH (SEQ ID NO.6) of enterobacteria, ydcW (SEQ ID NO.7) gene code.

Exemplified by moderately strengthening 3-HPA dehydrogenase using the puuC genes of pneumobacillus.Contained with a pair HindIII-XhoI restriction enzyme sites primer (CCCaagcttGCATGATGAATTTTCAGCACCT, CCGctcgagGTCAAGACTCCAGGGCAATCC) using KG genomes as template, pcr amplification puuC genes, and it is inserted into pETac Tac promoters downstream HindIII-XhoI sites, construction recombination plasmid pETac::PuuC, and will further build Recombinant plasmid electricity is gone in KG, and screens acquisition conversion bacterial strain KG/pETac::puuC.

With the fermentation condition of example 2, using recombinant bacterium KG/pETac::PuuC carries out fermenting experiment, experimental result such as table 4 It is shown.

Table 4：The fermentation results of appropriateness expression 3- hydroxymalonate dehydrogenases

From table 3 it is observed that (KG/pETac after appropriateness reinforcing 3- hydroxymalonate dehydrogenases::LacZ, is not induced), 1, 3-PD fermentations not only obtain high conversion ratio, and speed of production faster, it is more efficient.It is overexpressed 3- hydroxymalonate dehydrogenases 3-HP accumulation can be caused, is unfavorable for 1,3-PD fermentation on the contrary.

<110>East China University of Science

<120>A kind of method that high efficiency, high conversion produce 1,3- propane diols

<130> 2014

<160> 7

<170> PatentIn version 3.3

<210> 1

<211> 1551

<212> DNA

<213> K. pneumoniae KG

<400> 1

acttaaattg aagagtttga tcatggctca gattgaacgc tggcggcagg cctaacacat 60

gcaagtcgag cggtagcaca gagagcttgc tctcgggtga cgagcggcgg acgggtgagt 120

aatgtctggg aaactgcctg atggaggggg ataactactg gaaacggtag ctaataccgc 180

ataatgtcgc aagaccaaag tgggggacct tcgggcctca tgccatcaga tgtgcccaga 240

tgggattagc tagtaggtgg ggtaacggct cacctaggcg acgatcccta gctggtctga 300

gaggatgacc agccacactg gaactgagac acggtccaga ctcctacggg aggcagcagt 360

ggggaatatt gcacaatggg cgcaagcctg atgcagccat gccgcgtgtg tgaagaaggc 420

cttcgggttg taaagcactt tcagcgggga ggaaggcgat aaggttaata accttgtcga 480

ttgacgttac ccgcagaaga agcaccggct aactccgtgc cagcagccgc ggtaatacgg 540

agggtgcaag cgttaatcgg aattactggg cgtaaagcgc acgcaggcgg tctgtcaagt 600

cggatgtgaa atccccgggc tcaacctggg aactgcattc gaaactggca ggctagagtc 660

ttgtagaggg gggtagaatt ccaggtgtag cggtgaaatg cgtagagatc tggaggaata 720

ccggtggcga aggcggcccc ctggacaaag actgacgctc aggtgcgaaa gcgtggggag 780

caaacaggat tagataccct ggtagtccac gccgtaacga tgtcgatttg gaggttgtgc 840

ccttgaggcg tggcttccgg agctaacgcg ttaaatcgac gcctgggagt acggccgcaa 900

ggtttaaact caaatgaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt 960

cgatgcaacg cgaagaacct tacctggtct tgacatccac agaactttcc agagatggat 1020

tggtgccttc gggaactgtg agacaggtgc tgcatggctg tcgtcagctc gtgttgtgaa 1080

atgttgggtt aagtcccgca acgagcgcaa cccttatcct ttgttgccag cggtcaggcc 1140

gggaactcaa aggagactgc cagtgataaa ctggaggaag gtggggatga cgtcaagtca 1200

tcatggccct tacgaccagg gctacacacg tgctacaatg gcatatacaa agagaagcga 1260

cctcgcgaga gcaagcggac ctcataaagt atgtcgtagt ccggattgga gtctgcaact 1320

cgactccatg aagtcggaat cgctagtaat cgtagatcag aatgctacgg tgaatacgtt 1380

cccgggcctt gtacacaccg cccgtcacac catgggagtg ggttgcaaaa gaagtaggta 1440

gcttaacctt cgggagggcg cttaccactt tgtgattcat gactggggtg aagtcgtaac 1500

aaggtaaccg taggggaacc tgcggttgga tcacctcctt accttaaaga a 1551

<210> 2

<211> 260

<212> DNA

<213> E. coli

<400> 2

acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg 60

gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt 120

tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc 180

tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca 240

cacaggaaac agtattcatg 260

<210> 3

<211> 1440

<212> DNA

<213> K. pneumoniae

<400> 3

atgacagcac ccgttcaaca cccgatgtat attgatggcc agttcgtttc cggtcgcggc 60

gacggctgga tcgacgtgct taacccggcg accgaagcgc tgctgtcgcg gatcccggac 120

gggactgccg aagaggcgcg gctggcgatt gacgccgccg agcgcgccca gcccgcctgg 180

gaagcactgc cggcgattga gcgcgcgggc tggctgcgca agattgccgc gggtatccgc 240

cagcgtgctg aagagattgc cgggctgatc gtggctgaag gcggcaagat ccagcagctg 300

gcggcggtgg aagtcgcatt caccgctgac tatctcgact atatggccga atgggcgcgc 360

cgttacgaag gcgagatcgt gcagagcgat cgcccgggag agaatatcct cgtctttaaa 420

cgcgcgctgg gggtgaccac cgggatcctg ccgtggaact tcccgttctt tcttatcgcc 480

cgcaagctgg cgccggccct gatcaccggg aataccatcg tcattaagcc cagcgaattt 540

acgcccaata atgccatcgc ctttgccgag atcgtccatc aggttgggtt gccgaaaggg 600

gtctttaacc tggtgcttgg ccgcggagaa accgttggtc aggagctggc cggcaatccg 660

aaggtggcga tggttagcat gaccggtagc gtggcggcgg gagagaaaat tatggccgct 720

gcggcgaaaa atatcaccaa agtgtgcctc gagctcggcg gcaaagcgcc tgccattgtg 780

atggacgatg cggatctgga gctggcggtg aaagcggtgg tggactcgcg ggtgattaac 840

accgggcagg tgtgtaactg cgtcgagcgg gtctatgttc agcagggcat ttacgaccgc 900

ttcgtcaacc gcctcggcga ggcgatgaag gccgtgcagt ttggcgaccc ggcgacgcgg 960

gatgacatcg cgatggggcc gctgatcaac gcggcggcgc gggaccaggt ggcgggcaaa 1020

gtggcgaagg cggtggcgca gggggcgcgg gtggcgctgg gcggccagcc gctggagggc 1080

aaaggctatt tttatccgcc gaccctgctg ctggatgtgc gtcaggagat ggacattatc 1140

cacgaggaaa ccttcgggcc ggtgctgccg gtggtggcct tttcgaccct cgatgaggcg 1200

ctggcaatgg ccaatgacag cgattatggc ctgacctcct caatctatac ccgcgatctg 1260

aacgtggcga tgaaggcgat taagggactg aagttcggcg aaacctatat caaccgggaa 1320

aactttgagg cgatgcaggg tttccacgcc ggctggcgca aatcggggat cggcggcgct 1380

gatggccgcc acgggctgaa tgagtacctg cagacccagg tggtctatct gcaggcctga 1440

<210> 4

<211> 1488

<212> DNA

<213> K. pneumoniae

<400> 4

atgaattttc agcacctggc ttactggcag gaaaaagcga aaaacctggc cattgaaaca 60

cgcttattta ttaacggcga atattgcgcc gcggccgata ataccacctt tgagactatc 120

gaccctgccg cgcagcagac attagcccag gtcgcccgcg gtaaaaaagc cgacgtcgaa 180

cgggcggtga aagccgcgcg ccaggctttt gataacggcg actggtcgca ggcctccccc 240

gcacagcgta aagcgatcct cactcgcttt gctgatctga tggaggccca tcgtgaagag 300

ctggcgctgc tggaaacgct ggataccggc aaaccgattc gccacagcct gcgcgacgat 360

attcccggcg ccgcccgcgc cattcgctgg tatgccgaag cgctggataa agtctatggc 420

gaagtggccc ccaccggcag caacgagctg gcgatgatcg ttcgcgaacc aattggcgtg 480

atcgccgcgg tggtgccgtg gaacttcccg ctgctgctgg cctgctggaa actcggcccg 540

gcgctggcgg cgggcaatag cgtaatcctc aaaccctcgg aaaaatcgcc gctcaccgcc 600

ctgcgtctgg ccgggctggc gaaagaggcc ggcctgccgg acggcgtgtt gaacgtggtc 660

agcggctttg gccacgaggc cgggcaggcg ctggccctac atcctgatgt tgaagtcatc 720

accttcaccg gctccacccg caccggcaag cagctgctga aagacgccgg cgacagcaat 780

atgaagcgcg tgtggctgga agcgggcggc aagagcgcca acattgtctt cgccgattgc 840

ccggatctgc aacaagcggt tcgcgccacc gccggcggca tcttctacaa ccagggacag 900

gtgtgcatcg ccgggacccg tctgctgctc gaggagagca tcgctgacga gttcctggcg 960

cggctgaaag atgaggcgca acactggcag ccgggcaacc cgctcgatcc ggacaccacc 1020

atgggcatgc tgattgacaa tacccatgcc gacaacgtgc atagctttat tcgcggcggc 1080

gaaagccaaa gcaccctgtt cctcgacgga cggaaaaacc cgtggcctgc cgccgttggc 1140

ccgaccattt tcgttgacgt cgacccggca tcaaccctca gccgggaaga gatcttcggc 1200

ccggtgctgg tggtgacccg cttcaaaagc gaagaagagg cgctaaagct cgccaatgac 1260

agcgactacg gcttgggcgc cgcggtgtgg acccgcgatc tctcccgcgc ccaccgcatg 1320

agccgccgcc tgaaggccgg ctcggtcttc gtcaacaact ataacgatgg tgatatgacc 1380

gttccgttcg gcggctacaa gcagagcggc aacgggcgcg ataaatcgct gcacgcgctg 1440

gaaaaattca ccgaactgaa aaccatctgg attgccctgg agtcttga 1488

<210> 5

<211> 1428

<212> DNA

<213> K. pneumoniae

<400> 5

atgcaacaca acctattgat aaacggtaag ttagtggcag gtgaaggcga gaaggttccg 60

gtatataacc ctgcaaccgg cgaagtgatc ctggagattg ccgaagcgac agcggcccag 120

gtcgatgccg ccgttgaggc ggcggaccgg gcattcgatg cctggagtca gacgacgccg 180

aagacgcgtg cggaatgtct cctgaagctg gcggacgcca tctcggcgca ggctgaaacc 240

ctggcgcagc tggagtcgct gaactgcggc aagccgctgc actgcgtgat taatgatgaa 300

atgccggcca tcgccgacgt cttccgcttc ttcgccggcg ccgctcgctg cctgccgggg 360

atggcggccg gagagtatct cgaagggcat acctcaatga tccgccgcga tccggtgggc 420

gtggtggcct ccatcgcgcc gtggaactat ccgctgatga tggcggcctg gaagctggcc 480

ccggcgctgg cggcgggcaa ctgcgtagtg ataaaaccct cggagatcac cccgctgacg 540

gcgctgaagc tggcggagct ggcaaaagat atcttcccgg agggggttat caacgtgctg 600

tttggccgtg gtaaaacggt gggcgacccc ttgaccgccc acgttaaagt gcgaatggtc 660

tccctgaccg gatcgattgc caccggagcc catatcattg gccataccgc ctcgtcgatt 720

aaacgcaccc acatggagct ggggggtaag gcgccagtga tcgtttttga cgatgcggac 780

atcgatgcgg tggtggatgg ggtgcggacc ttcggttttt acaacgccgg ccaggactgc 840

acggccgcct gccggatcta cgcccagcag ggtatctatg accagctggt ggagaaactt 900

ggggcggcgg tggccagcct gaaaatgggg gcgccggagg acgctgccac cgaactgggt 960

ccgctgagct cgctggccca tcttgagcgc gttagcgcgg cggtggaagc cgcaagggcg 1020

ctgccgcaca ttaaggtggt caccggcggc agtcgggctg acggcgcggg ctattacttc 1080

cagccgacgc tgctcgccgg cgcccggcag gaagacgcta ttgtccagcg cgaagtgttt 1140

ggtccggtag tcagcgtgac gcctttcagc gatgaagcgc aggccctgag ctgggcgaat 1200

gactctcagt atggcctggc ctcctcggta tggacgaaag atgtcggtcg cgcccatcgt 1260

cttagcgcca ggctgcagta cggctgcacc tgggtcaata cccactttat gctggtcagc 1320

gaaatgccgc acggtggcca gaagctgtca ggctacggca aggacatgtc gatgtatggc 1380

cttgaggatt ataccgtggt ccgccatgtg atggttaagc atagctaa 1428

<210> 6

<211> 1488

<212> DNA

<213> E. coli

<400> 6

atgaattttc atcatctggc ttactggcag gataaagcgt taagtctcgc cattgaaaac 60

cgcttattta ttaacggtga atatactgct gcggcggaaa atgaaacctt tgaaaccgtt 120

gatccggtca cccaggcacc gctggcgaaa attgcccgcg gcaagagcgt cgatatcgac 180

cgtgcgatga gcgcagcacg cggcgtattt gaacgcggcg actggtcact ctcttctccg 240

gctaaacgta aagcggtact gaataaactc gccgatttaa tggaagccca cgccgaagag 300

ctggcactgc tggaaactct cgacaccggc aaaccgattc gtcacagtct gcgtgatgat 360

attcccggcg cggcgcgcgc cattcgctgg tacgccgaag cgatcgacaa agtgtatggc 420

gaagtggcga ccaccagtag ccatgagctg gcgatgatcg tgcgtgaacc ggtcggcgtg 480

attgccgcca tcgtgccgtg gaacttcccg ctgttgctga cttgctggaa actcggcccg 540

gcgctggcgg cgggaaacag cgtgattcta aaaccgtctg aaaaatcacc gctcagtgcg 600

attcgtctcg cggggctggc gaaagaagca ggcttgccgg atggtgtgtt gaacgtggtg 660

acgggttttg gtcatgaagc cgggcaggcg ctgtcgcgtc ataacgatat cgacgccatt 720

gcctttaccg gttcaacccg taccgggaaa cagctgctga aagatgcggg cgacagcaac 780

atgaaacgcg tctggctgga agcgggcggc aaaagcgcca acatcgtttt cgctgactgc 840

ccggatttgc aacaggcggc aagcgccacc gcagcaggca ttttctacaa ccagggacag 900

gtgtgcatcg ccggaacgcg cctgttgctg gaagagagca tcgccgatga attcttagcc 960

ctgttaaaac agcaggcgca aaactggcaa ccgggccatc cacttgatcc cgcaaccacc 1020

atgggcacct taatcgactg cgcccacgcc gactcggtcc atagctttat tcgggaaggc 1080

gaaagcaaag ggcaactgtt gttggatggc cgtaacgccg ggctggctgc cgccatcggc 1140

ccgaccatct ttgtggatgt ggacccgaat gcgtccttaa gtcgcgaaga gattttcggt 1200

ccggtgctgg tggtcacgcg tttcacatca gaagaacagg cgctacagct tgccaacgac 1260

agccagtacg gccttggcgc ggcggtatgg acgcgcgacc tctcccgcgc gcaccgcatg 1320

agccgacgcc tgaaagccgg ttccgtcttc gtcaataact acaacgacgg cgatatgacc 1380

gtgccgtttg gcggctataa gcagagcggc aacggtcgcg acaaatccct gcatgccctt 1440

gaaaaattca ctgaactgaa aaccatctgg ataagcctgg aggcctga 1488

<210> 7

<211> 1425

<212> DNA

<213> E. coli

<400> 7

atgcaacata agttactgat taacggagaa ctggttagcg gcgaagggga aaaacagcct 60

gtctataatc cggcaacggg ggacgtttta ctggaaattg ccgaggcatc cgcagagcag 120

gtcgatgctg ctgtgcgcgc ggcagatgca gcatttgccg aatgggggca aaccacgccg 180

aaagtgcgtg cggaatgtct gctgaaactg gctgatgtta tcgaagaaaa tggtcaggtt 240

tttgccgaac tggagtcccg taattgtggc aaaccgctgc atagtgcgtt caatgatgaa 300

atcccggcga ttgtcgatgt ttttcgcttt ttcgcgggtg cggcgcgctg tctgaatggt 360

ctggcggcag gtgaatatct tgaaggtcat acttcgatga tccgtcgcga tccgttgggg 420

gtcgtggctt ctatcgcacc gtggaattat ccgctgatga tggccgcgtg gaaacttgct 480

ccggcgctgg cggcagggaa ctgcgtagtg cttaaaccat cagaaattac cccgctgacc 540

gcgttgaagt tggcagagct ggcgaaagat atcttcccgg caggcgtgat taacatactg 600

tttggcagag gcaaaacggt gggtgatccg ctgaccggtc atcccaaagt gcggatggtg 660

tcgctgacgg gctctatcgc caccggcgag cacatcatca gccataccgc gtcgtccatt 720

aagcgtactc atatggaact tggtggcaaa gcgccagtga ttgtttttga tgatgcggat 780

attgaagcag tggtcgaagg tgtacgtaca tttggctatt acaatgctgg acaggattgt 840

actgcggctt gtcggatcta cgcgcaaaaa ggcatttacg atacgctggt ggaaaaactg 900

ggtgctgcgg tggcaacgtt aaaatctggt gcgccagatg acgagtctac ggagcttgga 960

cctttaagct cgctggcgca tctcgaacgc gtcagcaagg cagtagaaga ggcgaaagcg 1020

acagggcaca tcaaagtgat cactggcggt gaaaagcgca agggtaatgg ctattactat 1080

gcgccgacgc tgctggctgg cgcattacag gacgatgcca tcgtgcaaaa agaggtattt 1140

ggtccagtag tgagtgttac gcccttcgac aacgaagaac aggtggtgaa ctgggcgaat 1200

gacagccagt acggacttgc atcttcggta tggacgaaag atgtgggcag ggcgcatcgc 1260

gtcagcgcac ggctgcaata tggttgtacc tgggtcaata cccatttcat gctggtaagt 1320

gaaatgccgc acggtgggca gaaactttct ggttacggca aggatatgtc actttatggg 1380

ctggaggatt acaccgtcgt ccgccacgtc atggttaaac attaa 1425

Claims (2)

1. a kind of method of producing 1,3-propylene glycol by transforming glycerol, including seed culture and the life of ferment tank glycerine converting Into 1,3-PD, it is characterised in that this method is by Klebsiella pneumoniae CCTCCM2014574, and in citric acid Tac promoter leakage expression 3-HPA dehydrogenases are utilized in pneumobacillus, high efficiency, high conversion production 1 is realized, Ammediol.

2. according to the method described in claim 1, it is characterised in that the 3-HPA dehydrogenase gene derives from Cray Bai Shi pneumobacilluses or Escherichia coli.