CN109370969A - A kind of recombination klebsiella is preparing the application in 1,3-PD - Google Patents
A kind of recombination klebsiella is preparing the application in 1,3-PD Download PDFInfo
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Abstract
The invention discloses a kind of recombination klebsiellas in preparation 1, application in 3-propanediol, more particularly to a kind of recombination klebsiella for being overexpressed lactobacillus fermenti glycerol channel protein gene glpF in the application of production 1,3-PD, belong to genetic engineering field.Over-express vector with glpF gene in lactobacillus fermenti is transferred in klebsiella by present invention application gene engineering method, to improve its turn-over capacity to glycerol, improves the yield of 1,3-PD in klebsiella.Recombinant bacterium is up to 76g/L through 5L fermentor fed-batch fermentation, 1,3-PD yield.The invention has successfulLY changed the turn-over capacity in klebsiella to glycerol, provides brand-new thinking for breeding 1,3-PD superior strain.
Description
Technical field
The present invention relates to a kind of recombination klebsiellas to prepare the application in 1,3-PD, and in particular in Cray
In Bai Shi bacillus heterogenous expression lactobacillus fermenti glycerol channel protein gene glpF with improve 1,3-PD yield method and
Using belonging to gene engineering technology field.
Background technique
1,3-PD (1,3-PDO) is one of six big petrochemical industry new products generally acknowledged in the world, in weaving, plastics and food
Packaging etc. has broad application prospects, and functions primarily as the monomer of synthesis polyester, polyethers and polyurethane, it
The polypropylene terephthalate (PTT) being polymerized with terephthalic acid (TPA) is the precursor of the high-quality polyester material of synthesizing new
Matter.
The production method of 1,3-PD has chemical synthesis and biotransformation method.Biotransformation method produces 1,3-PD
Relatively easy with synthesis technology, reaction condition is milder, does not generate toxic intermediary, the more environmentally friendly advantage of production process,
Have become current research hotspot.Bacterial strain for producing 1,3-PD is bacterium, mainly there is klebsiella, Freund
Lemon bacterium, enterobacter agglomerans, Lactobacillus brevis, lactobacillus buchneri, clostridium butyricum and clostridium pasteurianum etc., they can only
Using glycerol 1,3-PD cannot be directly generated by cheap carbon sources such as carbohydrates.Wherein klebsiella has relatively high
The substrate transformation rate and production intensity, thus obtained more concern.
However, being that production purpose product is usual with a significant challenge of micro-organisms bulk chemical such as bio-fuel
It is toxic to cell.Known many short chain alcohols are reduced cell viability by damaging cells film and interfere necessary physiology course.Cause
This, cell must be balanced against producing and survive, and reduce purpose product potential production.
1,3-PD synthesizes in klebsiella, is the metabolite of growth coupling, when klebsiella benefit
When glycerol being used to produce 1,3-PD as carbon source, the disadvantages of it is lower that there are yield, and conversion ratio is lower, and in klebsiella
As the accumulation of 1,3-PD can inhibit the transhipment of substrate when fermenting and producing.And currently, the transformation to klebsiella mentions
The PRODUCTION TRAITS of high 1,3-PD is concentrated mainly on the two aspects of the regeneration of metabolic pathway and reducing power, but existing
Reforming mode can not improve the transfer efficiency of substrate.Turn therefore it provides a kind of yield and conversion ratio are higher, can improve substrate
The method for transporting the production 1,3-PD of efficiency, has great importance for the production application of 1,3-PD.
Summary of the invention
The first purpose of the invention is to provide a kind of recombination klebsiellas, which is characterized in that being will be from hair
The glycerol channel protein gene glpF of kefir milk bacillus (Lactobacillus fermentum) is connected on over-express vector, will
Connection product is transferred to obtained in klebsiella (Klebsiella Peneumoniae).
In one embodiment of the invention, the amino acid sequence of the glycerol channel albumen such as SEQ ID NO.2 institute
Show, the nucleotide sequence of the glycerol channel protein gene glpF is as shown in SEQ ID NO.1.
In one embodiment of the invention, the over-express vector is that sequence is introduced in pET-28a carrier such as
Tac promoter shown in SEQ ID NO.3.
In one embodiment of the invention, the construction method of the over-express vector are as follows: bis- with EcoRI and BamHI
Digestion pET28a and sequence the tac promoter as shown in SEQ ID NO.3, after digestion, PCR connects to obtain over-express vector
pEtac-28a(+)。
A second object of the present invention is to provide a kind of construction method of above-mentioned recombination klebsiella, the methods
Include:
(1) glycerol channel protein gene glpF, nucleotide sequence such as sequence table are cloned from lactobacillus fermenti genome
Shown in SEQ ID NO.1；
(2) glpF gene is connected with above-mentioned over-express vector pEtac-28a, and connection product is transformed into Cray primary
In family name bacillus.
In one embodiment of the invention, the conversion is using electroporated method.
Third object of the present invention is to provide above-mentioned recombination klebsiellas to prepare answering in 1,3-PD
With.
In one embodiment of the invention, the application is to be to recombinate klebsiella using glycerol as substrate
Biocatalyst carries out resting cell by the way of batch feeding.
In one embodiment of the invention, the application includes that bacterium solution is inoculated into 5L fermentor with 4% inoculum concentration
In, anaerobism, fed-batch fermentation recombinates klebsiella, and glycerol adding is flowed, keeps it in 15-30g/L, 37 DEG C, 150rpm,
Ferment 48h.
Over-express vector with glpF gene in lactobacillus fermenti is transferred to gram by present invention application gene engineering method
In thunder Bai Shi bacillus, to improve its turn-over capacity to glycerol, the yield of 1,3-PD in klebsiella is improved.Weight
For group bacterium through 5L fermentor fed-batch fermentation, 1,3-PD yield is up to 76g/L.The invention has successfulLY changed citric acid bar
To the turn-over capacity of glycerol in bacterium, brand-new thinking is provided for breeding 1,3-PD superior strain.
Detailed description of the invention
Fig. 1: wild type klebsiella fed-batch fermentation Methanogenesis.
Fig. 2: it is overexpressed recombination klebsiella feed supplement in 5L fermentor of the glpF gene in lactobacillus fermenti source
Batch fermentation Methanogenesis.
Fig. 3: it is overexpressed recombination klebsiella feed supplement point in 5L fermentor of the glpF gene of Escherichia coli
Wholesale ferment Methanogenesis.
Fig. 4: it is overexpressed the recombination klebsiella of the endogenous glpF gene fed-batch fermentation generation in 5L fermentor
Thank to product analysis.
Specific embodiment
(1) culture medium
5L fermentor (gL-1): glycerol 20, yeast extract 8, glucose 6, MgSO42, (NH4)2SO42, KH2PO47.5
FeSO4·7H2O 0.005, VB120.015, trace element solution 0.1mLL-1, use 10molL-1KOH tune pH value is extremely
8.5。
Trace element solution (gL-1): Na2MoO4·2H2O 0.35, CoCl2·6H2O 2, NiCl2·6H2O 0.25,
H3BO30.6, MnCl2·4H2O 1, CuCl20.2, ZnCl2 0.7。
(2) tunning detection method
Biomass estimation: dry cell weight (DCW) is according to spectrophotometer measurement OD600Value and dry weight corresponding relationship formula
1OD600=0.36gL-1。
Product measurement: 1,3-PDO, 2,3-BDO, acetic acid, glycerol, succinic acid and cream in HPLC method detection tunning are utilized
The concentration of acid, ultraviolet to detect with differential refraction detector, exchange column type used is organic acid ion exchange column, and work column temperature:
60℃；Prepare flowing phase concentration: 5mmolL-1H2SO4；Setting detection flow velocity: 0.6mLmin-1。
The preparation of the recombination klebsiella of embodiment 1
According to lactobacillus fermenti genome sequence using primer P1-XhoI (sequence information is as shown in SEQ ID NO.4) and
P2-SalI (sequence information is as shown in SEQ ID NO.5) PCR amplification obtains lactobacillus fermenti glpF gene order segment.
PCR reaction system is following (50 μ L): 25 μ L Prime STAR Max Premix (2 ×), 15pmol primer,
150ng template, adding distilled water to 50 μ L, (primer is the synthesis of Shanghai Sheng Gong bioengineering Co., Ltd, remaining is public purchased from TaKaRa
Department), amplification reaction condition is as follows: 98 DEG C, 10s；55 DEG C, 15s, 72 DEG C, 1kb/min, 30 circulations.
Obtained pcr amplification product is subjected to electrophoresis verifying, using the recycling examination of UNIQ-10 pillar DNA glue after being proved to be successful
Agent box (Shanghai Sheng Gong bioengineering Co., Ltd) carries out gel extraction.
Reclaimer illustrates according to kit: cutting out the centrifuge tube that target fragment is put into 1.5mL with blade, adds in conjunction with buffering
Liquid is placed in 55 DEG C of heating water baths 10min, every 2min and mixes primary；Glue is transferred to the UNIQ- covered in collecting pipe after melting completely
In 10 columns, it is placed at room temperature for 2min；Under 8000rpm, it is centrifuged 1min；The waste liquid in collecting pipe is outwelled, 500 μ L flushing liquors are added,
Under 8000rpm, room temperature is centrifuged 1min, which is repeated once；The waste liquid in collecting pipe is outwelled, UNIQ-10 column is put into together
In one collecting pipe, 12000rpm is centrifuged 15s；UNIQ-10 column is put into the centrifuge tube of a new 1.5mL, in pillar film
The distilled water of 50 μ L is added dropwise in center, is placed at room temperature for 3min；12000rpm is centrifuged 1min, and the liquid in centrifuge tube is to recycle
DNA fragmentation.
Purchase commercialization pET-28a, artificial synthesized sequence information tac promoter as shown in SEQ ID NO.3.With
The tac promoter fragment of EcoRI and BamHI double digestion pET-28a plasmid and PCR is connected by digestion and carries out pEtac-28a
The building of (+) expression vector, by the over-express vector built XhoI and Sal I double digestion, reaction system is following (50 μ L):
5 μ L QucikCut Buffer (10 ×), 1 μ L XhoI, 1 μ L Sal I, 30 μ L pEtac-28a (+) plasmid fragments add double steamings
Water is to 50 μ L (enzyme be purchased from TaKaRa company).Reaction condition is as follows: 37 DEG C, 3h.Cycle-Pure Kit is used after digestion is complete
(200) column QIAquick Gel Extraction Kit (Shanghai Sheng Gong bioengineering Co., Ltd) is recycled.Reclaimer illustrates according to kit: enzyme
The centrifuge tube that product is put into 1.5mL is cut, adds 250mLCP buffer, is gently centrifuged 1min；The adsorption column being transferred in collecting pipe
In, it is placed at room temperature for 2min；10000rpm is centrifuged 2min；Product is collected into adsorption column again, and 10000rpm is centrifuged 2min；It outwells
Waste liquid in collecting pipe, is added 700 μ L DNA Wash Buffer, 10000rpm room temperatures and is centrifuged 1min, which is repeated one
It is secondary；The waste liquid in collecting pipe is outwelled, adsorption column column is put into the same collecting pipe, 10000rpm is centrifuged 15s；Adsorption column is put
In the centrifuge tube for entering new 1.5mL, lid is opened, 65 DEG C, stands 10min；The distilled water of 50 μ L, room is added dropwise in pillar film center
Temperature places 3min；8000rpm is centrifuged 1min, and the liquid in centrifuge tube is pEtac-28a (+) segment of digestion recycling.
The glpF sequence fragment of recycling carries out double digestion with XhoI and SalI, and reaction system is following (50 μ L): 5 μ L
QucikCut Buffer (10 ×), 1 μ L XhoI, 1 μ L SalI, 30 μ L glpF sequence fragments, adding distilled water to 50 μ L, (enzyme is purchased
From TaKaRa company).Reaction condition is as follows: 37 DEG C, 3h.Cycle-Pure Kit (200) column reclaim reagent is used after digestion is complete
Box (Shanghai Sheng Gong bioengineering Co., Ltd) is recycled.Reclaimer illustrates according to kit: digestion products are put into 1.5mL
Centrifuge tube, add 250mL CP buffer, 6000rpm is centrifuged 1min；It is transferred in the adsorption column in collecting pipe, is placed at room temperature for
2min；10000rpm is centrifuged 2min；Product is collected into adsorption column again, and 10000rpm is centrifuged 2min；It outwells in collecting pipe
Waste liquid is added 700 μ L DNA Wash Buffer, 10000rpm room temperatures and is centrifuged 1min, which is repeated once；Outwell collection
Adsorption column column is put into the same collecting pipe by the waste liquid in pipe, and 10000rpm is centrifuged 15s；Adsorption column is put into new 1.5mL
Centrifuge tube in, open lid, 65 DEG C, stand 10min；The distilled water of 50 μ L is added dropwise in pillar film center, is placed at room temperature for 3min；
8000rpm is centrifuged 1min, and the liquid in centrifuge tube is the DNA fragmentation of digestion recycling.
The glpF sequence fragment and pEtac-28a (+) segment of digestion using T4 ligase (be purchased from TaKaRa company) according to
Connection work is completed in specification instruction, and linked system is as follows: 1 μ L T4 ligase, 1 μ L T4 connection buffer, 1 μ L pEtac-
28a (+) segment, 7 μ L glpF sequence fragments, adds distilled water to 10 μ L.Reaction condition is as follows: 16 DEG C, 12h.And by connection product
E. coli jm109 is converted, coating blocks on that plate, cultivates 8h at 37 DEG C.Picking single colonie carries out PCR verifying, primer P1
And P2, select the correct single colonie of stripe size to connect LB Shake flask medium.37 DEG C, 8h, 150rpm, extract plasmid (plasmid extraction
Kit is purchased from Shanghai Sheng Gong bioengineering Co., Ltd), carry out the verifying of XhoI and SalI double digestion double digestion.
Klebsiella is converted after being proved to be successful, method for transformation uses electroporated method.Steps are as follows: picking single colonie
It is inoculated in LB liquid medium, 37 DEG C, 150rpm cultivates 10-16h activation, and the seed liquor after activation is turned by 2% inoculum concentration
It is connected in the LB culture medium of 50mL, culture to OD600Value is about 0.4-0.6 or so.Bacterium night is put into ice and places 15-20min,
Then 4 DEG C, thalline were collected by centrifugation by 8000rpm, discards supernatant liquid.With the 2-3 (centrifugation every time of 10% glycerol washing thalline of pre-cooling
After put static 5min on ice).Supernatant to the greatest extent is abandoned, thallus is resuspended in the 10% pre- cold glycerol that 2-3mL is added, and is transferred to sterilized
In the centrifuge tube of 1.5mL, every 100 μ L of pipe is used for electrotransformation.5 μ L of plasmid to be transformed is added, places 10-15min on ice.1mm
Electric revolving cup dry in superclean bench, and be put into ice cooling 20min.Electricity is added in the competent cell for being mixed with plasmid to turn
In cup, under 2500V voltage, shock by electricity 5ms or so, the whole bacterium solutions of suction after the LB culture medium of addition 1mL mixes after the completion of electric shock, and 37
1h is cultivated after DEG C, is then coated on corresponding kalamycin resistance plate, and the recombination citric acid of expression glpF gene is obtained
Bacillus, 37 DEG C, 150rpm cultivates 8h.
The fermentation of embodiment 2 is overexpressed the recombination klebsiella of the glpF gene from lactobacillus fermenti
Bacterium solution obtained in embodiment 1 is inoculated into 5L fermentor, anaerobism, fed-batch fermentation with 4% inoculum concentration, is flowed
Glycerol adding keeps it in 15-30g/L, and 37 DEG C, 150rpm, ferment 48h.Not timing sampling measures OD by spectrophotometric600And
Tunning is surveyed by HPLC.As depicted in figs. 1 and 2, compared with wild type, it is overexpressed the Cray of lactobacillus fermenti glpF gene
The 1,3-PD concentration of Bai Shi bacillus is increased to 76g/L by 55g/L, and glycerol conversion yield is increased to 60% by 56%.
The result shows that being overexpressed lactobacillus fermenti glycerol channel protein gene glpF, Ke Yiti in klebsiella
High klebsiella production 1,3-PD ability and glycerol transfer efficiency.
Comparative example 1
The glycerol channel protein gene glpF of Escherichia coli is overexpressed in klebsiella, by itself and overexpression
Carrier pEtac-28a (+) connection, is transferred in klebsiella, obtains recombination klebsiella.The recombination gram that fermentation obtains
Thunder Bai Shi bacillus produces 1,3-PD.
Using genome of E.coli DNA as template, use primer P3-XhoI (sequence information is as shown in SEQ ID NO.6)
Escherichia coli glpF gene order segment (NCBI- is obtained with P4-SalI (sequence information is as shown in SEQ ID NO.7) PCR amplification
GeneID:948422)。
It is consistent in remaining step and embodiment 1 and embodiment 2, the obtained recombination for being overexpressed Escherichia coli glpF gene
In 5L fermentation cylinder for fermentation, yield does not change significantly klebsiella compared to wild type, as shown in Figure 3.Knot
Fruit shows the glycerol channel protein gene glpF of heterogenous expression Escherichia coli, and obtained recombinant bacterium is applied to preparation
1,3-PD cannot obtain good effect.
Comparative example 2
Endogenous glycerol channel protein gene glpF is overexpressed in klebsiella, by itself and over-express vector
PEtac-28a (+) connection, is transferred in klebsiella, obtains recombination klebsiella.Ferment obtained recombination Cray primary
Family name bacillus produces 1,3-PD.
Using klebsiella genomic DNA as template, primer P5-XhoI (sequence information such as SEQ ID NO.8 institute is used
Show) and the endogenous glpF gene order segment (sequence of P6-SalI (sequence information is as shown in SEQ ID NO.9) progress PCR amplification acquisition
Column information is as shown in SEQ ID NO.10).
It is consistent in remaining step and embodiment 1 and embodiment 2, the obtained recombination Cray for being overexpressed endogenous glpF gene
For Bai Shi bacillus in 5L fermentation cylinder for fermentation, 1,3-PDO yield is 65g/L, and the fermentation than recombinant bacterium provided by the invention produces
Measure low 17%.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>a kind of recombination klebsiella is preparing the application in 1,3-PD
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 711
<212> DNA
<213> Lactobacillus fermentum
<400> 1
atgcatcaat tatttgcgga attaatgggg actgccctca tgatcgtctt tggggtcggg 60
gttcacgccg acaccgtctt aaatgacacg aagtaccacg gttcaggaca tttatttgcc 120
atcacaacat gggcatttgg catctcaatc gttttattca tttttccaac catctgcctg 180
aacccagcca tggcatttgc gcaattctta ctggggaaca tgagctttag ccggttcatt 240
gaagtctcga tttttgaact ggctggtggg gtcattggcg ccgtcattgt gtacatcatg 300
tacgccgacc aatttaagca ctcctacgac aatatcgacc cagtgacgat ccgaaacatt 360
ttctcaacgg gtcctggtgt ccggaacctg ccacggaact tcttcgtcga attctttgac 420
accttcattt tcctgacggc gatcatggtc atcgtgacca tcaagacacc cggcatcatg 480
ccaattggca tcggactgct ggtctgggcc attgggatgg gacttggggg cccaactggg 540
ttcgccatga accaagcgcg ggacctcggc ccacgaattg cttttgccat cctgccattg 600
aagaacaagg cggcagccga ttggcaatac gggttgcttg tgcctggtat cgcaccgttc 660
tttggtgccg cggcagccgt catttttgca aaattttatc ttggacttta a 711
<210> 2
<211> 236
<212> PRT
<213> Lactobacillus fermentum
<400> 2
Met His Gln Leu Phe Ala Glu Leu Met Gly Thr Ala Leu Met Ile Val
1 5 10 15
Phe Gly Val Gly Val His Ala Asp Thr Val Leu Asn Asp Thr Lys Tyr
20 25 30
His Gly Ser Gly His Leu Phe Ala Ile Thr Thr Trp Ala Phe Gly Ile
35 40 45
Ser Ile Val Leu Phe Ile Phe Pro Thr Ile Cys Leu Asn Pro Ala Met
50 55 60
Ala Phe Ala Gln Phe Leu Leu Gly Asn Met Ser Phe Ser Arg Phe Ile
65 70 75 80
Glu Val Ser Ile Phe Glu Leu Ala Gly Gly Val Ile Gly Ala Val Ile
85 90 95
Val Tyr Ile Met Tyr Ala Asp Gln Phe Lys His Ser Tyr Asp Asn Ile
100 105 110
Asp Pro Val Thr Ile Arg Asn Ile Phe Ser Thr Gly Pro Gly Val Arg
115 120 125
Asn Leu Pro Arg Asn Phe Phe Val Glu Phe Phe Asp Thr Phe Ile Phe
130 135 140
Leu Thr Ala Ile Met Val Ile Val Thr Ile Lys Thr Pro Gly Ile Met
145 150 155 160
Pro Ile Gly Ile Gly Leu Leu Val Trp Ala Ile Gly Met Gly Leu Gly
165 170 175
Gly Pro Thr Gly Phe Ala Met Asn Gln Ala Arg Asp Leu Gly Pro Arg
180 185 190
Ile Ala Phe Ala Ile Leu Pro Leu Lys Asn Lys Ala Ala Ala Asp Trp
195 200 205
Gln Tyr Gly Leu Leu Val Pro Gly Ile Ala Pro Phe Phe Gly Ala Ala
210 215 220
Ala Ala Val Ile Phe Ala Lys Phe Tyr Leu Gly Leu
225 230 235
<210> 3
<211> 29
<212> DNA
<213>artificial synthesized
<400> 3
ttgacaatta atcatcggct cgtataatg 29
<210> 4
<211> 32
<212> DNA
<213>artificial synthesized
<400> 4
ccgctcgaga tgcatcaatt atttgcggaa tt 32
<210> 5
<211> 38
<212> DNA
<213>artificial synthesized
<400> 5
acgcgtcgac ttaaagtcca agataaaatt ttgcaaaa 38
<210> 6
<211> 39
<212> DNA
<213>artificial synthesized
<400> 6
gcgtcgacgg gtttcatatg agtcaaacat caaccttga 39
<210> 7
<211> 29
<212> DNA
<213>artificial synthesized
<400> 7
ccctcgagtt acagcgaagc tttttgttc 29
<210> 8
<211> 35
<212> DNA
<213>artificial synthesized
<400> 8
acgcgtcgac atgagccaaa catcaacctt aaaag 35
<210> 9
<211> 40
<212> DNA
<213>artificial synthesized
<400> 9
ccctcgaggc gtcgacttac agcgaagctt tatgttgagt 40
<210> 10
<211> 852
<212> DNA
<213> Klebsiella Peneumoniae
<400> 10
atgagccaaa catcaacctt aaaaggccag tgcatcgcag agttcctcgg taccgggttg 60
ttgatctttt tcggcgttgg gtgcgtggct gcgctcaagg tcgcgggagc cagcttcggg 120
caatgggaaa tcagcatcat ctggggtctg ggcgtcgcca tggcgatcta cctgaccgct 180
ggggtctccg gtgcgcacct taaccctgcg gtaactatcg cactctggct gttcgcctgc 240
ttcgatggcc gcaaagtggt cccttttatc atttcgcaat tcgctggcgc cttttgcgct 300
gcggcattag tttacgggct ttactacaat cttttcctcg attatgaaac cacccaccat 360
atggtccgcg gcagcgtaga aagcctcgat ctggccggca tcttctccac ctatccgaac 420
ccgcatatca attttgtgca ggccttcgcg gtagagatgg tgattaccgc tatcctgatg 480
ggcgtcatcc tggcgctgac cgacgatggc aacggcgtgc cgcgcggccc gcttgctccg 540
ctgctgatcg gcctgctgat tgcggtgatt ggcgcctcca tgggaccgct gaccggcttc 600
gccatgaacc cggcgcgtga tatcggcccg aaagctttcg cctggctggc cggctggggc 660
gacgtcgcct tcaccggcgg caaagatatt ccttatttcc tggtgccgct gtgcgcaccg 720
gtggtcggcg cggcgctggg cgcattcagc tatcgtaagc tgattggccg tcacctgcct 780
tgcgacacct gcgtggagga agagcaacag agcccctcct cttccaccac tcaacataaa 840
gcttcgctgt aa 852
Claims (10)
1. a kind of recombination klebsiella, which is characterized in that be that will derive from lactobacillus fermenti (Lactobacillus
Fermentum glycerol channel protein gene glpF) is connected on over-express vector pEtac-28a, and connection product is transferred to gram
Obtained in thunder Bai Shi bacillus (Klebsiella Peneumoniae).
2. recombination klebsiella according to claim 1, which is characterized in that the glycerol channel protein gene glpF
Nucleotide sequence as shown in SEQ ID NO.1.
3. recombination klebsiella according to claim 1 or 2, which is characterized in that the over-express vector pEtac-
28a is that sequence tac promoter as shown in SEQ ID NO.3 is introduced in pET-28a carrier.
4. recombination klebsiella according to claim 3, which is characterized in that the over-express vector pEtac-28a
Construction method are as follows: use EcoRI and BamHI double digestion pET28a and sequence the tac promoter as shown in SEQ ID NO.3, enzyme
After cutting, PCR connects to obtain over-express vector pEtac-28a (+).
5. the construction method of any recombination klebsiella of claim 1-4, which is characterized in that the method packet
It includes:
(1) glycerol channel protein gene glpF, nucleotide sequence such as sequence table SEQ are cloned from lactobacillus fermenti genome
Shown in ID NO.1；
(2) glpF gene is connected with the over-express vector pEtac-28a in claim 4, and connection product is transformed into gram
In thunder Bai Shi bacillus.
6. construction method according to claim 5, which is characterized in that the conversion is using electroporated method.
7. application of any recombination klebsiella of claim 1-4 in production 1,3-PD.
8. application according to claim 7, which is characterized in that the application is using glycerol as substrate, to recombinate Cray primary
Family name bacillus is biocatalyst, and resting cell is carried out by the way of batch feeding.
9. application according to claim 8, which is characterized in that the application includes being inoculated into bacterium solution with 4% inoculum concentration
In 5L fermentor, anaerobism, fed-batch fermentation recombinates klebsiella, flows glycerol adding, keeps it in 15-30g/L, and 37
DEG C, 150rpm, ferment 48h.
10. any recombination klebsiella of claim 1-4 is in the application of weaving, plastics and food packaging applications.
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