CN108949656A - A kind of engineering bacteria and its application in production pyruvic acid - Google Patents
A kind of engineering bacteria and its application in production pyruvic acid Download PDFInfo
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Abstract
The invention discloses a kind of engineering bacteria and its applications in production pyruvic acid, belong to technical field of bioengineering.Recombination bacillus coli of the invention expresses external source l-lactate dehydrogenase and nadh oxidase simultaneously, and knocked out pyruvic acid on the basis of host e. coli and absorbed gene, further go back overexpression Lactate Transport gene, pyruvic acid transporter gene, NAD synthesis gene.The present invention constructs the engineering bacteria of double enzyme coexpressions on the basis of Escherichia coli transhipment and coenzyme synthetic system is transformed, and realizes efficiently producing and reducing impurity generation for pyruvic acid.
Description
Technical field
The present invention relates to a kind of engineering bacteria and its applications in production pyruvic acid, belong to technical field of bioengineering.
Background technique
Pyruvic acid (Pyruvate) also known as Acetylformic acid or acetyl formic acid are a kind of organic acids that acidity is weaker.Acetone
Acid has a wide range of applications in chemical industry, food, medicine and other fields.
The production of pyruvic acid mainly has chemical method and bioanalysis at present.Chemical method passes through Oxidation of Lactic under the action of catalyst
Pyruvic acid is produced, but higher cost is seriously polluted (CN201510848841.2 etc.).Bioanalysis mainly has lactic acid conversion method and straight
Connect fermentation method.Using glucose as fermenting raw materials produce pyruvic acid (WO8901523, CN201310722490.1,
CN201510205724.4 etc.) usually fermentation time is longer, and pyruvic acid higher cost is extracted from impurity fermentation liquid system.
With the engineering bacteria containing lactate oxidase etc. or wild mushroom (WO9500656, CN200710156905.8,
CN201110092393.X), can produce pyruvic acid, but hydrogen peroxide can be generated during this, though coexpression over hydrogenation hydrogen enzyme or
Outside addition over hydrogenation hydrogen enzyme not can avoid pyruvic acid yet and be oxidized generation acetic acid, to influence yield and purification effect.
The present invention is synthesized with to the transformation of Escherichia coli, changing it excessively to the transhipment of substrate and product and the effect of coenzyme, and
Production pyruvic acid is converted in the case where not producing hydrogen peroxide using cheap substrate L-type lactic acid.
Summary of the invention
Based on the defect of current various methods, the invention proposes the lifes that the conversion lactic acid for not producing hydrogen peroxide produces pyruvic acid
Production method constructs the engineering bacteria of double enzyme coexpressions, realizes on the basis of Escherichia coli transhipment and coenzyme synthetic system is transformed
The efficient production of pyruvic acid.Pyruvic acid can be produced and to reduce impurity raw technical problem to be solved by the invention is to provide a kind of
At recombinant bacterium, while the technical issues of the invention solves the building of the bacterial strain and applications.
The first purpose of the invention is to provide can the inexpensive recombination bacillus coli for producing pure pyruvic acid；The recombination is big
Enterobacteria expresses external source l-lactate dehydrogenase and nadh oxidase simultaneously, and has knocked out third on the basis of host e. coli
Keto acid resorption gene.
In one embodiment, the external source l-lactate dehydrogenase is the l-lactate dehydrogenase of originating in lactic acid bacterium.External source
Nadh oxidase be originating in lactic acid bacterium nadh oxidase.
In one embodiment, the lactic dehydrogenase from Lactococcus lactis ATCC 19257,
Lactobacillus plantarum ATCC 14917。
In one embodiment, the amino acid sequence of the lactic dehydrogenase is that accession NO is WP_ on NCBI
003131075.1, the sequence of KRL33571.1.
In one embodiment, the nucleotide sequence of the lactic dehydrogenase is accession NO on NCBI are as follows: NZ_
The sequence of JXJZ01000017REGION:18532..19509, AZEJ01000016REGION:16296..17249.
In one embodiment, the nadh oxidase from Lactococcus lactis ATCC 19257,
Lactobacillus sanfranciscensis DSM20451、Lactobacillus brevis ATCC 14869。
In one embodiment, the amino acid sequence of the nadh oxidase is that accession NO is WP_ on NCBI
032950924.1, WP_056958268.1, ERK43827.1 sequence.
In one embodiment, the nucleotide sequence of the nadh oxidase is accession NO on NCBI are as follows: NZ_
JXJZ01000002REGION:complement(39571..40911)、NZ_AYYM01000013REGION:complement
The sequence of (15875..17233), AWVK01000048REGION:complement (50022..51416).
In one embodiment, the l-lactate dehydrogenase and nadh oxidase are co-expressed by pCOLADuet-1
's.
In one embodiment, pyruvic acid absorption gene (pyruvic acid is transported to intracellular gene) is
Any one in btsT, ybdD or two kinds of combinations.
In one embodiment, it is accession NO on NCBI that the pyruvic acid, which absorbs gene, are as follows: NC_
012892REGION:complement (4496239..4498389) or NC_012892REGION:592652..592849.
In one embodiment, the recombination bacillus coli also overexpression Lactate Transport gene is (Lactate Transport
To intracellular gene), pyruvic acid transporter gene (enhancing pyruvic acid toward extracellular transporter gene), NAD synthesize gene
The one or more of (key enzyme of Escherichia coli NAD route of synthesis).
In one embodiment, the gene of the overexpression is lldP (Lactate Transport gene), (pyruvic acid turns pykF
Transport gene), icsA (NAD synthesize gene), any one or more in nadA (NAD synthesizes gene).
In one embodiment, the host strain is Escherichia coli BL21 (DE3).
In one embodiment, the overexpression is by by Escherichia coli BL21 (DE3) genome
Increase constitutive promoter before the gene of upper need to strengthen expression.
In one embodiment, lldP accession NO on NCBI are as follows: NC_012892REGION:
3646638..3648293；PykF is NC_012892REGION:1700961..1702373；IcsA is NC_
012892REGION:complement(2526116..2527330)；NadA is NC_012892REGION:
740487..741530。
A second object of the present invention is to provide a kind of method for producing optics pure pyruvic acid, the method is to utilize this hair
Bright recombinant bacterium.
In one embodiment, the production pyruvic acid is to carry out resting cell production.
In one embodiment, in the system of the resting cell production, wet cell weight 1-200g/L, Pfansteihl 1-
100g/L, pH 4.0-9.0,15-40 DEG C of temperature, 250 revs/min of shaking speed；Transformation time 1-24 hours.
Third object of the present invention is to provide recombinant bacteriums of the present invention or the method for the present invention in chemical industry, food, medicine etc.
The application in field.
Beneficial effects of the present invention:
The present invention constructs a kind of novel double enzyme co-expression gene engineering bacterias, which can be applied to the production of pyruvic acid.
Selection scheme of the present invention does not produce hydrogen peroxide, and cell is not easily decomposed pyruvic acid, and has higher NAD content into the cell.The life
Production process is simple and raw material is easy to get, and has good industrial applications prospect.
Specific embodiment
The leitungskern of colibacillus engineering of the invention is can to co-express two kinds of enzymes, respectively lactic dehydrogenase
(Lactate dehydrogenase), nadh oxidase (NADH oxidase).Its principle are as follows:, L- entirely intracellular in engineering bacteria
Pfansteihl dehydrogenation is generated pyruvic acid and NADH using endobacillary NAD as coenzyme by lactic dehydrogenase；Nadh oxidase is by NADH oxygen
Metaplasia realizes the regeneration of coenzyme NAD at NAD.While the related gene on knockout or overexpression genome of E.coli promotees
Into lactic acid transhipment and prevent the decomposition of pyruvic acid.
In order to solve the above technical problems, The technical solution adopted by the invention is as follows:
1, bacterial strain and plasmid according to the present invention
Lactobacillus plantarum ATCC 14917 purchased from American Type Culture Collecti ATCC,
Lactococcus lactis ATCC 19257, Lactobacillus brevis ATCC 14869 are purchased from Novagen company
PETDuet-1, pACYCDue-1, pCOLADuet-1, pRSFDuet-1 plasmid and Escherichia coli BL21
(DE3).Lactobacillus sanfranciscensis DSM20451 is purchased from Germany Microbiological Culture Collection Center DSMZ.
PCasRed, pCRISPR-gDNA are purchased from Zhenjiang Ai Bi dream Biotechnology Co., Ltd.
2, the knockout of related gene and composing type overexpression in Escherichia coli
(1) Escherichia coli pyruvic acid absorbs the knockout of gene
Pyruvic acid is the core compound in main metabolic pathway, can be utilized or be decomposed by number of ways, at present pyruvic acid
Genetic engineering bacterium is usually the accumulation for knocking out or weakening the gene realization pyruvic acid of relational approach, but the knockout of gene will affect bacterium
The normal growth of strain, it usually needs special culture medium or cultural method carry out pyruvate fermentation.The present invention is using lactic acid the bottom of as
Object realizes full cell enzymatic conversion production pyruvic acid, in order to allow expression of enzymes process to be normally carried out, relevant point of pyruvic acid in engineering bacteria
Solution and the genes of utilization ways do not make an amendment, only pyruvic acid to be transported to intracellular gene knockout in Escherichia coli, from
And allow bacterial strain to be able to produce pyruvic acid and transport out cell, but cannot reabsorb into cell, so as to avoid the decomposition of pyruvic acid.
The gene of selection is btsT and ybdD, the upper accession NO of NCBI are as follows: NC_012892REGION:complement
(4496239..4498389) and NC_012892REGION:592652..592849.
(2) Escherichia coli Lactate Transport gene/pyruvic acid transport protein composing type overexpression
, need to be Lactate Transport to just can be carried out dehydrogenation production pyruvic acid into the cell during resting cell, enhancing is newborn
Acid transporter albumen facilitates the high concentration for maintaining lactic acid intracellular quickly and for a long time, is conducive to the progress of dehydrogenation.The gene of selection
It is lldP, the upper accession NO of NCBI are as follows: NC_012892REGION:3646638..3648293.It is past to enhance pyruvic acid simultaneously
Extracellular transport protein, the gene of selection are pykF, the upper accession NO of NCBI are as follows: NC_012892REGION:
1700961..1702373。
(3) overexpression of Escherichia coli NAD synthesis related gene
It is needed during lactic dehydrogenase using NAD as coenzyme, the key enzyme of overexpression Escherichia coli NAD route of synthesis,
It is horizontal that endobacillary NAD can be improved, to be conducive to the generation of pyruvic acid.The gene of selection has icsA, nadA.On NCBI
Accession NO are as follows: NC_012892REGION:complement (2526116..2527330), NC_012892REGION:
740487..741530、
3, lactic acid is converted into the selection of pyruvic acid relevant enzyme
(1) selection of l-lactate dehydrogenase
Pfansteihl is organic acid the most cheap, after dehydrogenation at pyruvic acid added value with higher.At present mainly with
Pfansteihl oxydasis Pfansteihl produces pyruvic acid, generates hydrogen peroxide in the process, and hydrogen peroxide can oxide acetylacetonate acid
Generate acetic acid.L-lactate dehydrogenase is widely present in multiple-microorganism, usually with NAD (NADP) be coenzyme lactic dehydrogenase more
It is intended to using pyruvic acid as substrate synthesizing lactic acid, but some lactic dehydrogenases can be taken off when lactic acid excess or carbon source only have lactic acid
The hydrogen of lactic acid generates pyruvic acid, and the hydrogen generated on Pfansteihl is passed to coenzyme NAD or NADP using Pfansteihl as substrate, thus raw
At NADH or NADPH.
The present invention is from Lactococcus lactis ATCC 19257 and Lactobacillus plantarum ATCC
L-lactate dehydrogenase gene llldh (amino acid sequence is WP_003131075.1) and lpldh (amino are respectively obtained in 14917
Acid sequence is KRL33571.1), expression product is used for the dehydrogenation of lactic acid.
(2) selection of nadh oxidase
Lactic dehydrogenase dehydrogenation from lactic acid generates pyruvic acid NADH.NADH needs to be regenerated by nadh oxidase oxidation
NAD, to realize the lasting progress of reaction.Nadh oxidase, which has, produces two kinds of peroxidating Hydrogen of water type and production, produces the NADH of water type
Oxidizing ferment will not generate hydrogen peroxide toxicity.The present invention is respectively from Lactobacillus sanfranciscensis
DSM20451, Lactococcus lactis ATCC 19257, it obtains in Lactobacillus brevis ATCC 14869
Production water type nadh oxidase gene lsnox (amino acid sequence is WP_056958268.1), (amino acid sequence is WP_ to llnox
032950924.1), lbnox (amino acid sequence is ERK43827), expression product are used for the regeneration of NAD.
4, the building of l-lactate dehydrogenase and nadh oxidase coexpression system and the culture of cell
Double enzyme combination coexpressions are carried out by class every in l-lactate dehydrogenase and nadh oxidase selected above optional one.
At present Escherichia coli polygenes coexpression there are many method, (Escherichia coli polygenes coexpression strategy, China are raw
Object engineering magazine, 2012,32 (4): 117-122), (synthetic biology technological transformation Escherichia coli are raw using Liu Xianglei by the present invention
Produce shikimic acid and resveratrol, 2016, Shanghai Institute of Pharmaceutical Industry, doctoral thesis) the method building, before each gene
Comprising T7 promoter and RBS binding site, T7 terminator is had after gene.Theoretically speaking because each gene before have T7 and
RBS, thus the expression intensity of gene influenced by arrangement order it is little.It include two genes on each plasmid, by what is built
Plasmid heat is transduceed in competent escherichia coli cell, and is coated on antibiotic solid plate, and screening obtains positive transformant,
Obtain recombination bacillus coli.The culture of cell: it according to classical recombination bacillus coli culture and inducing expression scheme, will recombinate
Escherichia coli are that 2% amount is transferred to LB fermentation medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/ by volume
L in), as cell OD600After reaching 0.6-0.8, the IPTG of final concentration of 0.4mM is added, in 20 DEG C of inducing expression culture 8h.It lures
After leading expression, 20 DEG C, 8000rpm, cell is collected by centrifugation within 20 minutes.
5, resting cell produces pyruvic acid
Resting cell system are as follows: wet cell weight 1-200g/L, Pfansteihl 1-100g/L, pH 4.0-9.0, temperature 15-40
DEG C, 250 revs/min of shaking speed；Transformation time 1-24 hours.
6, the detection and analysis of sample
The quantitative analysis of Lactic acid and Pyruvic acid carries out (the gas-chromatography survey of serum lactic, pyruvic acid, succinic acid according to document
Determine the Lanzhou medical college journal, 1991 (02): 72-74)
In order to which technical problems, technical solutions and advantages to be solved are more clearly understood, tie below
Embodiment is closed, the present invention will be described in detail.It should be noted that specific embodiment described herein is only to explain
The present invention is not intended to limit the present invention.
Embodiment 1
Recombination bacillus coli building: it first by the gene of encoding lactate dehydrogenase and nadh oxidase, is connected respectively to double
On gene expression plasmid pETDuet-1.Various double gene coexpression recombinant plasmids are obtained, plasmid is converted into Escherichia coli
Escherichia coli BL21 (DE3) screens to obtain positive transformant using ampicillin plate to get big to recombination
Enterobacteria.
Derivational expression method: being that 2% amount is transferred to LB fermentation medium (peptone by recombination bacillus coli by volume
10g/L, yeast powder 5g/L, NaCl 10g/L) in, as cell OD600After reaching 0.6-0.8, it is added final concentration of 0.4mM's
IPTG, in 20 DEG C of inducing expression culture 8h.After inducing expression, 20 DEG C, 8000rpm, cell is collected by centrifugation within 20 minutes.
The cell being collected into carries out transformation assay, and the results are shown in Table 1.Resting cell system are as follows: wet cell weight 100g/
L, Pfansteihl 50g/L, pH 8.0, temperature are 35 DEG C, 250 revs/min of shaking speed；Transformation time 5 hours.
Compared with 1 lactic dehydrogenase of table generates the efficiency of pyruvic acid with nadh oxidase double gene coexpression Escherichia coli
| Bacterial strain | Pyruvic acid g/L |
| Escherichia coli BL21(DE3)/pETDuet-1-lsnox-llldh | 19.5 |
| Escherichia coli BL21(DE3)/pETDuet-1-lsnox-lpldh | 26.1 |
| Escherichia coli BL21(DE3)/pETDuet-1-llnox-llldh | 30.4 |
| Escherichia coli BL21(DE3)/pETDuet-1-llnox-lpldh | 27.0 |
| Escherichia coli BL21(DE3)/pETDuet-1-lbnox-llldh | 18.9 |
| Escherichia coli BL21(DE3)/pETDuet-1-lbnoxl-pldh | 26.7 |
When two genes of llnox and llldh are combined, effect is best as can be seen from the above table.
Embodiment 2
According to strain construction method described in embodiment 1, (all kinds of plasmids are sieved according to specification using different resistant panels
Select positive transformant) and derivational expression method, it collects various types of cells and carries out transformation assay, the results are shown in Table 2.Resting cell
System are as follows: wet cell weight 50g/L, Pfansteihl 10g/L, pH 7.0, temperature are 30 DEG C, 250 revs/min of shaking speed；When conversion
Between 1 hour.
Comparison of the various expression plasmids of table 2 for conversion production pyruvic acid
| Bacterial strain | Pyruvic acid g/L |
| Escherichia coli BL21(DE3)/pETDuet-1-llnox-llldh | 2.2 |
| Escherichia coli BL21(DE3)/pACYCDuet-1-llnox-llldh | 1.9 |
| Escherichia coli BL21(DE3)/pCOLADuet-1-llnox-llldh | 2.6 |
| Escherichia coli BL21(DE3)/pRSFDuet-1-llnox-llldh | 2.1 |
| Escherichia coli BL21(DE3)/pCDFDuet-1-llnox-llldh | 1.6 |
It is best using effect when pCOLADuet-1 coexpression as can be seen from the above table.
Embodiment 3
According to document Large scale validation of an efficient CRISPR/Cas-based multi
gene editing protocol in Escherichia coli.Microbial Cell Factories,2017,16
(1): method described in 68 by Escherichia coli BL21 (DE3) btsT and ybdD carry out single or double knockout,
In, the plasmid of gene knockout used in the present invention is pCasRed and pCRISPR-gDNA (btsT sgRNA) and homology arm (btsT
Donor it) imports on Escherichia coli BL21 (DE3) together, Cas9/sgRNA induces host and sends out in btsT gene loci
BtsT donor is integrated on btsT gene by raw double-strand break, recombinase Red, realizes the knockout of gene, and sequence verification.
BstT sgRNA, bstT donor, ybdD sgRNA, ybdD donor are respectively such as sequence table SEQ ID NO:29, SEQ ID
NO:30, SEQ ID NO:31, shown in SEQ ID NO:32.
After gene knockout, pCOLADuet-1-llnox-llldh plasmid is imported in corresponding bacterial strain and carries out conversion comparison.
Inducing expression is carried out according to method described in example 1, cell is collected and carries out transformation assay, the results are shown in Table 2.In transformation system
Resting cell system are as follows: wet cell weight 200g/L, Pfansteihl 5g/L, pH 9.0, temperature are 30 DEG C, 250 revs/min of shaking speed
Clock；Transformation time 24 hours.It measures in conversion fluid after conversion and is remained without Pfansteihl, is all converted.
3 conversion results of table compare
| Bacterial strain | Pyruvic acid g/L |
| Escherichia coli BL21(ΔbtsTΔybdD,DE3)/pCOLADuet-1-llnox-llldh | 4.6 |
| Escherichia coli BL21(ΔbtsT,DE3)/pCOLADuet-1-llnox-llldh | 4.1 |
| Escherichia coli BL21(ΔybdD,DE3)/pCOLADuet-1-llnox-llldh | 4.4 |
| Escherichia coli BL21(DE3)/pCOLADuet-1-llnox-llldh | 3.8 |
Obviously the changing effect of double knock-out bacterial strain Escherichia coli BL21 (Δ btsT Δ ybdD, DE3) is most
It is good.It is Escherichia coli BY by this Strain Designation.
Embodiment 4
Using document Large scale validation of an efficient CRISPR/Cas-based multi
gene editing protocol in Escherichia coli.Microbial Cell Factories,2017,16
(1): method described in 68 will correspond to the 3- phosphoric acid for increasing Escherichia coli before gene on Escherichia coli BY genome
Medium expression intensity constitutive promoter (PG) before glyceraldehyde dehydrogenase gene (gpdA), sequence such as SEQ ID NO:33 institute
Show.
When the lldP that enhances gene is expressed, using Escherichia coli BY genome as template, with primer lldP-FF/
LldP-FR, lldP-gpdA-F/lldP-gpdA-R, lldP-RF/lldP-RR amplify upstream, promoter, downstream sequence, and
The expression cassette containing gpdA promoter is fused to by primer of lldP-FF and lldP-RR.Then with plasmid pCasRed,
After pCRISPR-gDNA (sgRNA containing lldP) is transferred to Escherichia coli BY together, Cas9/sgRNA induces host and exists
Double-strand break occurs for lldP gene loci, before gpdA promoter is integrated into lldP gene by recombinase Red, and sequence verification.
When the pykF that enhances gene is expressed, using Escherichia coli BY genome as template, with primer pykF-FF/
PykF-FR, pykF-gpdA-F/pykF-gpdA-R, pykF-RF/pykF-RR amplify upstream, promoter, downstream sequence, and
The expression cassette containing gpdA promoter is fused to by primer of pykF-FF and pykF-RR.Then with plasmid pCasRed,
After pCRISPR-gDNA (sgRNA containing pykF) is transferred to Escherichia coli BY together, Cas9/sgRNA induces host and exists
Double-strand break occurs for pykF gene loci, before gpdA promoter is integrated into pykF gene by recombinase Red, and sequence verification.
Following table is the manipulative indexing of Primer and sequence table serial number.
4 Primer of table is compareed with sequence table serial number
| Title | It is numbered in sequence table |
| lldP sgRNA | SEQ ID NO:1 |
| pykF sgRNA | SEQ ID NO:2 |
| lldP-FF | SEQ ID NO:5 |
| lldP-FR | SEQ ID NO:6 |
| lldP-gpdA-F | SEQ ID NO:7 |
| lldP-gpdA-R | SEQ ID NO:8 |
| lldP-RF | SEQ ID NO:9 |
| lldP-RR | SEQ ID NO:10 |
| pykF-FF | SEQ ID NO:11 |
| pykF-FR | SEQ ID NO:12 |
| pykF-gpdA-F | SEQ ID NO:13 |
| pykF-gpdA-R | SEQ ID NO:14 |
| pykF-RF | SEQ ID NO:15 |
| pykF-RR | SEQ ID NO:16 |
After the completion of genetic modification, co-expression plasmid is imported.According to method inducing expression described in embodiment 1, collect each
Class cell carries out transformation assay, and the results are shown in Table 4.Resting cell system are as follows: wet cell weight 10g/L, Pfansteihl 50g/L,
PH8.0, temperature are 40 DEG C, 250 revs/min of shaking speed；Transformation time 12 hours.
5 conversion results of table compare
| Bacterial strain | Concentrations of pyruvate g/L |
| Escherichia coli BY(PG-lldP)/pCOLADuet-1-llnox-llldh | 7.3 |
| Escherichia coli BY(PG-pykF)/pCOLADuet-1-llnox-llldh | 6.5 |
| Escherichia coli BY (PG-lldP, PG-pykF)/pCOLADuet-1-llnox-llldh | 8.4 |
| Escherichia coli BY/pCOLADuet-1-llnox-llldh | 6.2 |
From the above, it can be seen that simultaneously that the sequence alterations before lldP and pykF gene are best at the effect of constitutive promoter.It will
Escherichia coli BY (PG-lldP, PG-pykF) is named as Escherichia coli BYLP
Embodiment 5
Large intestine bar will be increased before icsA and/or nadA gene in Escherichia coli BYLP according to the method for example 4
Medium expression intensity constitutive promoter (PG) before the glyceraldehyde 3-phosphate dehydro-genase gene (gpdA) of bacterium, sequence such as SEQ ID
Shown in NO:33.Then plasmid is imported again.
When the icsA that enhances gene is expressed, using Escherichia coli BY genome as template, with primer icsA-FF/
IcsA-FR, icsA-gpdA-F/icsA-gpdA-R, icsA-RF/icsA-RR amplify upstream, promoter, downstream sequence, and
The expression cassette containing gpdA promoter is fused to by primer of icsA-FF and icsA-RR.Then with plasmid pCasRed,
After pCRISPR-gDNA (containing icsAsgRNA) is transferred to Escherichia coli BY together, Cas9/sgRNA induces host and exists
Double-strand break occurs for icsA gene loci, before gpdA promoter is integrated into icsA gene by recombinase Red, and sequence verification.
When the nadA that enhances gene is expressed, using Escherichia coli BY genome as template, with primer nadA-FF/
NadA-FR, nadA-gpdA-F/nadA-gpdA-R, nadA-RF/nadA-RR amplify upstream, promoter, downstream sequence, and
The expression cassette containing gpdA promoter is fused to by primer of nadA-FF and nadA-RR.Then with plasmid pCasRed,
After pCRISPR-gDNA (containing nadAsgRNA) is transferred to Escherichia coli BY together, Cas9/sgRNA induces host and exists
Double-strand break occurs for nadA gene loci, before gpdA promoter is integrated into nadA gene by recombinase Red, and sequence verification.
Following table is the manipulative indexing of Primer and sequence table serial number.
6 Primer of table is compareed with sequence table serial number
| Title | It is numbered in sequence table |
| icsA sgRNA | SEQ ID NO:3 |
| nadA sgRNA | SEQ ID NO:4 |
| icsA-FF | SEQ ID NO:17 |
| icsA-FR | SEQ ID NO:18 |
| icsA-gpdA-F | SEQ ID NO:19 |
| icsA-gpdA-R | SEQ ID NO:20 |
| icsA-RF | SEQ ID NO:21 |
| icsA-RR | SEQ ID NO:22 |
| nadA-FF | SEQ ID NO:23 |
| nadA-FR | SEQ ID NO:24 |
| nadA-gpdA-F | SEQ ID NO:25 |
| nadA-gpdA-R | SEQ ID NO:26 |
| nadA-RF | SEQ ID NO:27 |
| nadA-RR | SEQ ID NO:28 |
After the completion of genetic modification, co-expression plasmid is imported.According to method inducing expression described in embodiment 1, collect each
Class cell carries out transformation assay, and the results are shown in Table 5.Resting cell system are as follows: wet cell weight 20g/L, Pfansteihl 200g/L,
PH 9.0, temperature are 30 DEG C, 250 revs/min of shaking speed；Transformation time 24 hours.
7 conversion results of table compare
| Bacterial strain | Pyruvic acid g/L |
| Escherichia coli BYLP(PG-icsA、PG-nadA)/pCOLADuet-1-llnox-llldh | 39.2 |
| Escherichia coli BYLP(PG-icsA)/pCOLADuet-1-llnox-llldh | 27.5 |
| Escherichia coli BYLP(PG-nadA)/pCOLADuet-1-llnox-llldh | 44.3 |
| Escherichia coli BYLP/pCOLADuet-1-llnox-llldh | 26.1 |
From the foregoing, it will be observed that only that the sequence alterations before nadA gene are best at the effect of constitutive promoter.It will
Escherichia coli BYLP (PG-nadA) is named as Escherichia coli BYLPN.
Embodiment 6
According to derivational expression method described in embodiment 2, by Escherichia coli BYLPN/pCOLADuet-1-
Thallus is collected after the completion of llnox-llldh inducing expression, in 100ml reaction system, wet cell weight 1g/L, Pfansteihl 1g/L,
PH 4.0, temperature are 15 DEG C, 250 revs/min of shaking speed；Transformation time 1 hour.Measurement result, concentrations of pyruvate 11mg/
L。
Embodiment 7
According to derivational expression method described in embodiment 2, by Escherichia coli BYLPN/pCOLADuet-1-
Thallus is collected after the completion of llnox-llldh inducing expression, in 100ml reaction system, wet cell weight 200g/L, Pfansteihl
200g/L, pH 9.0, temperature are 25 DEG C, 250 revs/min of shaking speed；Transformation time 24 hours.Measurement result, pyruvic acid are dense
Degree is 196g/L.Escherichia coli BL21 (DE3)/pCOLADuet-1-llnox-llldh acetone under similarity condition
Concentration is 162g/L.
The transformation and building of above-described enzyme and its co-expression gene engineering bacteria, the culture medium composition of thallus and culture side
Method and Whole Cell Bioconversion are only presently preferred embodiments of the present invention, are not intended to restrict the invention, theoretically speaking its
Its bacterium, filamentous fungi, actinomyces, zooblast can carry out the transformation of genome, and for the complete of polygenes coexpression
Cell catalysis.All made any modifications, equivalent replacement within principle and spirit of the invention.
Sequence table
<110>Southern Yangtze University
<120>a kind of engineering bacteria and its application in production pyruvic acid
<130> 2018.3.15
<160> 33
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
gattgccacc gtccacgagg 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
ggtatcaagc aggatagcgg 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence
<400> 3
cggctggcag gctgaagaag 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence
<400> 4
ttaacggcgt cggcttcggg 20
<210> 5
<211> 25
<212> DNA
<213>artificial sequence
<400> 5
aaatacaatc tctgtaggtt cttct 25
<210> 6
<211> 50
<212> DNA
<213>artificial sequence
<400> 6
tcggccactc atcaacatga ttcatgagtc tgttgctcat ctccttgtca 50
<210> 7
<211> 50
<212> DNA
<213>artificial sequence
<400> 7
tgacaaggag atgagcaaca gactcatgaa tcatgttgat gagtggccga 50
<210> 8
<211> 50
<212> DNA
<213>artificial sequence
<400> 8
cgtagttttg ttgccagaga ttcatggttt tctcctgtca ggaacgttcg 50
<210> 9
<211> 50
<212> DNA
<213>artificial sequence
<400> 9
cgaacgttcc tgacaggaga aaaccatgaa tctctggcaa caaaactacg 50
<210> 10
<211> 25
<212> DNA
<213>artificial sequence
<400> 10
taacacctga cccgcagtgt aaccg 25
<210> 11
<211> 25
<212> DNA
<213>artificial sequence
<400> 11
gacaattatt tgtgactttc attgc 25
<210> 12
<211> 50
<212> DNA
<213>artificial sequence
<400> 12
tcggccactc atcaacatga ttcataaact tgctttctgg gcgcactgcc 50
<210> 13
<211> 50
<212> DNA
<213>artificial sequence
<400> 13
ggcagtgcgc ccagaaagca agtttatgaa tcatgttgat gagtggccga 50
<210> 14
<211> 50
<212> DNA
<213>artificial sequence
<400> 14
tgcaaacaat tttggtcttt ttcatggttt tctcctgtca ggaacgttcg 50
<210> 15
<211> 50
<212> DNA
<213>artificial sequence
<400> 15
cgaacgttcc tgacaggaga aaaccatgaa aaagaccaaa attgtttgca 50
<210> 16
<211> 25
<212> DNA
<213>artificial sequence
<400> 16
gtttgcaacg tcaccggctt ctgcg 25
<210> 17
<211> 25
<212> DNA
<213>artificial sequence
<400> 17
atgcgtctta tcaggcctac agtga 25
<210> 18
<211> 50
<212> DNA
<213>artificial sequence
<400> 18
tcggccactc atcaacatga ttcataatca ggctaccggc tggatgtacg 50
<210> 19
<211> 50
<212> DNA
<213>artificial sequence
<400> 19
cgtacatcca gccggtagcc tgattatgaa tcatgttgat gagtggccga 50
<210> 20
<211> 50
<212> DNA
<213>artificial sequence
<400> 20
agtcgagata aatcggtaat ttcatggttt tctcctgtca ggaacgttcg 50
<210> 21
<211> 50
<212> DNA
<213>artificial sequence
<400> 21
cgaacgttcc tgacaggaga aaaccatgaa attaccgatt tatctcgact 50
<210> 22
<211> 25
<212> DNA
<213>artificial sequence
<400> 22
aatgttcggc gcaccgtgtt ccagg 25
<210> 23
<211> 25
<212> DNA
<213>artificial sequence
<400> 23
tcgaatcctg cacgacccac cacta 25
<210> 24
<211> 50
<212> DNA
<213>artificial sequence
<400> 24
tcggccactc atcaacatga ttcatcgaca ttagcgtaat attcgctgtt 50
<210> 25
<211> 50
<212> DNA
<213>artificial sequence
<400> 25
aacagcgaat attacgctaa tgtcgatgaa tcatgttgat gagtggccga 50
<210> 26
<211> 50
<212> DNA
<213>artificial sequence
<400> 26
tgtctggatc aaacattacg ctcatggttt tctcctgtca ggaacgttcg 50
<210> 27
<211> 50
<212> DNA
<213>artificial sequence
<400> 27
cgaacgttcc tgacaggaga aaaccatgag cgtaatgttt gatccagaca 50
<210> 28
<211> 25
<212> DNA
<213>artificial sequence
<400> 28
catccacgga caatgcgcgc agctg 25
<210> 29
<211> 20
<212> DNA
<213>artificial sequence
<400> 29
gcggtagttg cattacgtcg 20
<210> 30
<211> 120
<212> DNA
<213>artificial sequence
<400> 30
atacgaaaaa actattcaag cacataccct gggtgattct cggaatcatc ggtgcattct 60
agcacgtcag cgccctgtgg atcgtggtcg cctctgtgtc ggtgtatctg gtggcgtatc 120
<210> 31
<211> 20
<212> DNA
<213>artificial sequence
<400> 31
cggaaaatat ttaggtcagg 20
<210> 32
<211> 120
<212> DNA
<213>artificial sequence
<400> 32
ccgcatccgg cactctttca gcaacatggt tagcggaggc caagatgttt gattcactgg 60
cgaagctgat gattggtatg cctgattacg acaactatgt cgaacatatg cgggttaatc 120
<210> 33
<211> 1100
<212> DNA
<213> Escherichia coli BL21(DE3)
<400> 33
atgaatcatg ttgatgagtg gccgatcgct acgtgggaag aaaccacgaa actccattgc 60
gcaatacgct gcgataacca gtaaaaagac cagccagtga atgctgattt gtaaccttga 120
atatttattt tccataacat ttcctgcttt aacataattt tccgttaaca taacgggctt 180
ttctcaaaat ttcattaaat attgttcacc cgttttcagg taatgactcc aacttattga 240
tagtgtttta tgttcagata atgcccgatg actttgtcat gcagctccac cgattttgag 300
aacgacagcg acttccgtcc cagccgtgcc aggtgctgcc tcagattcag gttatgccgc 360
tcaattcgct gcgtatatcg cttgctgatt acgtgcagct ttcccttcag gcgggattca 420
tacagcggcc agccatccgt catccatatc accacgtcaa agggtgacag caggctcata 480
agacgcccca gcgtcgccat agtgcgttca ccgaatacgt gcgcaacaac cgtcttccgg 540
agcctgtcat acgcgtaaaa cagccagcgc tggcgcgatt tagccccgac atagccccac 600
tgttcgtcca tttccgcgca gacgatgacg tcactgcccg gctgtatgcg cgaggttacc 660
gactgcggcc tgagtttttt aagtgacgta aaatcgtgtt gaggccaacg cccataatgc 720
gggcagttgc ccggcatcca acgccattca tggccatatc aatgattttc tggtgcgtac 780
cgggttgaga agcggtgtaa gtgaactgca gttgccatgt tttacggcag tgagagcaga 840
gatagcgctg atgtccggcg gtgcttttgc cgttacgcac caccccgtca gtagctgaac 900
aggagggaca gctgatagaa acagaagcca ctggagcacc tcaaaaacac catcatacac 960
taaatcagta agttggcagc atcaccccgt tttcagtacg ttacgtttca ctgtgagaat 1020
ggagattgcc catcccgcca tcctggtcta agcctggaaa ggatcaattt tcatccgaac 1080
gttcctgaca ggagaaaacc 1100
Claims (10)
1. a kind of recombination bacillus coli, which is characterized in that the recombination bacillus coli expresses external source l-lactate dehydrogenase simultaneously
And nadh oxidase, and knocked out pyruvic acid on the basis of host e. coli and absorbed gene.
2. recombination bacillus coli according to claim 1, which is characterized in that the external source l-lactate dehydrogenase is lactic acid bacteria
The l-lactate dehydrogenase in source, the nadh oxidase of external source are the nadh oxidase of originating in lactic acid bacterium.
3. recombination bacillus coli according to claim 1, which is characterized in that the pyruvic acid absorb gene be btsT,
Any one in ybdD or two kinds of combinations.
4. recombination bacillus coli according to claim 1 to 3, which is characterized in that the recombination bacillus coli is also strengthened
Express the one or more of Lactate Transport gene, pyruvic acid transporter gene, NAD synthesis gene.
5. recombination bacillus coli according to claim 4, which is characterized in that the gene of the overexpression be lldP,
Any one or more in pykF, icsA, nadA.
6. recombination bacillus coli according to claim 4 or 5, which is characterized in that the overexpression is by by host
Increase constitutive promoter before the gene of need to strengthen expression on genome of E.coli.
7. recombination bacillus coli according to claim 2, which is characterized in that the recombination bacillus coli expresses L- simultaneously
Lactic acid dehydrogenase gene llnox and nadh oxidase gene llldh has knocked out pyruvic acid and has absorbed gene btsT and ybdD, and
Overexpression Lactate Transport gene lldP, pyruvic acid transporter gene pykF, NAD synthesize gene nadA.
8. a kind of method for producing optics pure pyruvic acid, which is characterized in that the method is any described using claim 1-8
Recombinant bacterium.
9. according to the method described in claim 8, it is characterized in that, the method is to carry out resting cell production；It is described complete
In the system of cell transformation production, wet cell weight 1-200g/L, Pfansteihl 1-100g/L, pH 4.0-9.0,15-40 DEG C of temperature,
250 revs/min of shaking speed；Transformation time 1-24 hours.
10. recombinant bacterium as claimed in claim 1 to 7 or claim 8-9 the method are led in chemical industry, food, medicine etc.
The application in domain.
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