CN104388476B - A kind of method that high efficiency, high conversion produce 1,3 propane diols - Google Patents
A kind of method that high efficiency, high conversion produce 1,3 propane diols Download PDFInfo
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- CN104388476B CN104388476B CN201410719724.1A CN201410719724A CN104388476B CN 104388476 B CN104388476 B CN 104388476B CN 201410719724 A CN201410719724 A CN 201410719724A CN 104388476 B CN104388476 B CN 104388476B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
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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
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.
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