CN110305823A - Using the method and bacterial strain of production of lactic acid l-Alanine - Google Patents
Using the method and bacterial strain of production of lactic acid l-Alanine Download PDFInfo
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Abstract
The invention discloses a kind of engineering bacteria and its applications in production l-Alanine, belong to technical field of bioengineering.Recombination bacillus coli of the invention expresses external source l-lactate dehydrogenase and L-alanine dehydrogenase simultaneously, and overexpression Lactate Transport gene and NAD synthesize gene on the basis of host e. coli.The present invention constructs the engineering bacteria of double enzyme coexpressions, realizes and efficiently produce l-Alanine using cheap raw material on the basis of Escherichia coli transhipment and coenzyme synthetic system is transformed.
Description
Technical field
The present invention relates to the methods and bacterial strain using production of lactic acid l-Alanine, belong to technical field of bioengineering.
Background technique
L-Alanine is a kind of important amino acid, and chemical industry, food, medicine and other fields have a wide range of applications.
The production of l-Alanine mainly has chemical method, enzyme transforming process and fermentation method at present.Enzyme transforming process mainly passes through day
Aspartic acid decarboxylation production, but this personal value of aspartic acid is higher.Also have using malic acid as substrate, malate dehydrogenase and l-Alanine are taken off
The method that hydrogen enzyme fixes production altogether, but malic acid price itself is very high, is difficult to realize industrialized production.Genetic engineering bacterium is with grape
Sugar be fermenting raw materials produce l-Alanine then there is a problem of saccharic acid conversion ratio is low, fermentation liquid complicated composition cause to extract it is difficult.
Summary of the invention
Based on the defect of current various methods, the invention proposes the production methods that conversion lactic acid produces l-Alanine, are changing
On the basis of making Escherichia coli transhipment and coenzyme synthetic system, the engineering bacteria of double enzyme coexpressions is constructed, l-Alanine is realized
Efficient production.The recombination of cheap raw material production l-Alanine can be utilized technical problem to be solved by the invention is to provide a kind of
Bacterium, while the technical issues of the present invention also solves the building and application of the bacterial strain.
The first purpose of the invention is to provide can the inexpensive recombination bacillus coli for producing pure l-Alanine；The recombination
Escherichia coli express external source L/D- lactic dehydrogenase and L-alanine dehydrogenase simultaneously.
In one embodiment, the external source L/D lactic dehydrogenase is the L/D- lactic dehydrogenase of originating in lactic acid bacterium.Outside
The L-alanine dehydrogenase in source is the L-alanine dehydrogenase of bacillus, marine bacteria or actinomyces source.
In one embodiment, the l-lactate dehydrogenase from Lactococcus lactis ATCC 19257,
Lactobacillus plantarum ATCC 14917。
In one embodiment, the amino acid sequence of the l-lactate dehydrogenase is that accession NO is on NCBI
The sequence of WP_003131075.1, KRL33571.1.
In one embodiment, the nucleotide sequence of the l-lactate dehydrogenase is accession NO on NCBI are as follows:
The sequence of NZ_JXJZ01000017REGION:18532..19509, AZEJ01000016REGION:16296..17249.
In one embodiment, the D-lactic acid dehydrogenase from Lactococcus lactis ATCC 19257,
Lactobacillus plantarum ATCC 14917。
In one embodiment, the amino acid sequence of the D-lactic acid dehydrogenase is that accession NO is on NCBI
The sequence of WP_014573232.1, KRL35110.1.
In one embodiment, the nucleotide sequence of the D-lactic acid dehydrogenase is accession NO on NCBI are as follows:
NZ_JXJZ01000008REGION:complement(31625..32602)、AZEJ01000004REGION:
38463..39455 sequence.
In one embodiment, the L-alanine dehydrogenase from Bacillus megaterium DSM 319,
Phaeobacterinhibens DSM 17395、Streptomyces corchorusii DSM 40340。
In one embodiment, the amino acid sequence of the L-alanine dehydrogenase is that accession NO is on NCBI
WP_013085206.1, WP_014881174.1, WP_014671828.1 sequence.
In one embodiment, the nucleotide sequence of the L-alanine dehydrogenase is accession NO on NCBI
Are as follows: NC_014103REGION:complement (4614172..4615293), NC_018290REGION:
3177716..3178831、NZ_KQ948369REGION:24418..25542。
In one embodiment, the l-lactate dehydrogenase and L-alanine dehydrogenase are to be total to table by pETDuet-1
It reaches.
In one embodiment, the recombination bacillus coli also overexpression Lactate Transport gene is (Lactate Transport
To intracellular gene), NAD synthesis gene (key enzyme of Escherichia coli NAD route of synthesis) one or more.
In one embodiment, the gene of the overexpression is lldP (Lactate Transport gene), (NAD is synthesized icsA
Gene), any one or more in nadA (NAD synthesize 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；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 optical voidness l-Alanine, the method is to utilize this
The recombinant bacterium of invention.
In one embodiment, the production l-Alanine 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-
300g/L, ammonium chloride 1-350g/L, pH 4.0-9.0,15-40 DEG C of temperature, 250 revs/min of shaking speed；Transformation time 1-24
Hour.
Third object of the present invention is to provide recombinant bacterium of the present invention chemical industry, food, medicine and other fields application.
Fourth object of the present invention is to provide the method for the present invention in the application of chemical industry, food, medicine and other fields.
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 life of l-Alanine
It produces.The substrate that the present invention selects is cheap, and has higher NAD content into the cell.The production process is simple and raw material is easy to get, and has
There is 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), alanine dehydrogenase (Alanine dehydrogenase).Its principle are as follows: in engineering bacteria
Complete intracellular, Pfansteihl dehydrogenation is generated l-Alanine and NADH using endobacillary NAD as coenzyme by l-lactate dehydrogenase；The third ammonia of L-
Pyruvic acid, ammino are become l-Alanine by acidohydrogenase, and NADH, which is then aoxidized, generates NAD, realize the regeneration of coenzyme NAD.Simultaneously
Related gene in knockout or overexpression genome of E.coli promotes the transhipment of lactic acid and prevents the decomposition of l-Alanine.
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 is purchased from pETDuet-1, pACYCDue-1, pCOLADuet- of Novagen company
1, pRSFDuet-1 plasmid and Escherichia coli BL21 (DE3).Bacillus megaterium DSM 319,
Phaeobacterinhibens DSM 17395, Streptomyces corchorusii DSM 40340 are purchased from the micro- life of Germany
Object Culture Collection Center DSMZ.PCasRed, pCRISPR-gDNA are purchased from Zhenjiang Ai Bi dream Biotechnology Co., Ltd.
2, in Escherichia coli related gene composing type overexpression
(1) the composing type overexpression of Escherichia coli Lactate Transport gene
During resting cell, it need to enhance Lactate Transport to just can be carried out dehydrogenation production l-Alanine into the cell
Lactate Transport albumen facilitates the high concentration for maintaining lactic acid intracellular quickly and for a long time, is conducive to the progress of dehydrogenation.The base of selection
Because being lldP, the upper accession NO of NCBI are as follows: NC_012892REGION:3646638..3648293.
(2) 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 l-Alanine.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 l-Alanine relevant enzyme
(1) selection of lactic dehydrogenase
D/L- lactic dehydrogenase is widely present in multiple-microorganism, is usually the lactic dehydrogenase of coenzyme with NAD (NADP)
Enzyme is tended to using pyruvic acid as substrate synthesizing lactic acid, but some lactic dehydrogenase meetings when lactic acid excess or carbon source only have lactic acid
The hydrogen for taking off lactic acid generates pyruvic acid.Lactic acid has D and two kinds of L, and for D-ALPHA-Hydroxypropionic acid, Pfansteihl yield is bigger more honest and clean
Valence, the hydrogen generated on Pfansteihl is preferably passed to coenzyme NAD or NADP using Pfansteihl as substrate by this patent, to generate NADH
Or NADPH.Same D-ALPHA-Hydroxypropionic acid can also be utilized by substrate by D-lactic acid dehydrogenase.
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 Pfansteihl.
The present invention is from Lactococcus lactis ATCC 19257 and Lactobacillus plantarum ATCC
D-lactic acid dehydrogenase gene llldh2 (amino acid sequence is WP_014573232.1) and lpldh2 (ammonia are respectively obtained in 14917
Base acid sequence is KRL35110.1), expression product is used for the dehydrogenation of D-ALPHA-Hydroxypropionic acid.
(2) selection of L-alanine dehydrogenase
Lactic dehydrogenase dehydrogenation from lactic acid generates pyruvic acid and NADH, and L-alanine dehydrogenase utilizes NADH and ammonia, will
Pyruvic acid is reduced into l-Alanine, and NAD is then reproduced, to realize the lasting progress of reaction.The present invention is respectively from Bacillus
megaterium DSM 319、Phaeobacterinhibens DSM 17395、Streptomyces corchorusii DSM
L-alanine dehydrogenase gene bmlad (amino acid sequence is WP_013085206.1), pilad (amino acid sequence are obtained in 40340
Column are WP_014881174.1), sclad (amino acid sequence is WP_014671828.1), expression product is for synthesizing the third ammonia of L-
Acid.
4, the building of l-lactate dehydrogenase and L-alanine dehydrogenase coexpression system and the culture of cell
Double enzyme groups are carried out by class every in l-lactate dehydrogenase and L-alanine dehydrogenase selected above optional one to amount to
Expression.
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 l-Alanine
Resting cell system are as follows: wet cell weight 1-200g/L, L/D- lactic acid 1-300g/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
According to document progress, (gas chromatography measures lactic acid and organic impurities in fermentation method lactic product for Lactic acid quantification analysis
The Beijing Institute of Petrochemical Technology [J] journal, 2003 (03): 46-50.)
According to document progress, (pre-column derivatization method measures content [J] of alanine in sparrow brain for the quantitative analysis of l-Alanine
Traditional Chinese medicine Leader, 2018,24 (09): 42-44.)
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
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 increases Escherichia coli before corresponding to gene on Escherichia coli BL21 (DE3) genome
Medium expression intensity constitutive promoter (PG) before glyceraldehyde 3-phosphate dehydro-genase gene (gpdA), sequence such as SEQ ID NO:
Shown in 22.
When the lldP that enhances gene is expressed, using Escherichia coli BL21 (DE3) genome as template, with primer
LldP-FF/lldP-FR, lldP-gpdA-F/lldP-gpdA-R, lldP-RF/lldP-RR, amplify upstream, promoter, under
Sequence is swum, and is fused to the expression cassette containing gpdA promoter by primer of lldP-FF and lldP-RR.Then with plasmid
After pCasRed, pCRISPR-gDNA (sgRNA containing lldP) are transferred to Escherichia coli BL21 (DE3) together, Cas9/
SgRNA induce host lldP gene loci occur double-strand break, recombinase Red by gpdA promoter be integrated into lldP gene it
Before, and sequence verification.
Following table is the manipulative indexing of Primer and sequence table serial number.
1 Primer of table is compareed with sequence table serial number
| Title | It is numbered in sequence table |
| lldP sgRNA | SEQ ID NO:1 |
| lldP-FF | SEQ ID NO:4 |
| lldP-FR | SEQ ID NO:5 |
| lldP-gpdA-F | SEQ ID NO:6 |
| lldP-gpdA-R | SEQ ID NO:7 |
| lldP-RF | SEQ ID NO:8 |
| lldP-RR | SEQ ID NO:9 |
After the completion of genetic modification, co-expression plasmid is imported.Inducing expression: inducing expression side is carried out according to the following method
Method: be by volume by recombination bacillus coli 2% amount be transferred to LB fermentation medium (peptone 10g/L, yeast powder 5g/L,
NaCl10g/L in), as cell OD600After reaching 0.6-0.8, the IPTG of final concentration of 0.4mM is added, is trained in 20 DEG C of inducing expressions
Support 8h.After inducing expression, 20 DEG C, 8000rpm, cell is collected by centrifugation within 20 minutes.After expression collect various types of cells into
Row transformation assay, the results are shown in Table 2.Resting cell system are as follows: wet cell weight 10g/L, Pfansteihl 50g/L, ammonium chloride
350g/L, pH 8.0, temperature are 40 DEG C, shaking speed, 250 revs/min；Transformation time 12 hours.
2 conversion results of table compare
| Bacterial strain | L-Alanine concentration g/L |
| Escherichia coli BL21(DE3)(PG-lldP)/pETDuet-1-pilad-llldh | 7.3 |
| Escherichia coli BL21(DE3)/pETDuet-1-pilad-llldh | 0.1 |
From the above, it can be seen that simultaneously that the sequence alterations before lldP gene are best at the effect of constitutive promoter.It will
Escherichia coli BL21 (DE3) (PG-lldP) is named as Escherichia coli BLP.
Embodiment 2
Recombination bacillus coli building: it first by the gene of encoding lactate dehydrogenase and L-alanine dehydrogenase, is separately connected
Onto double gene expression plasmid pETDuet-1.Various double gene coexpression recombinant plasmids are obtained, plasmid is converted into Escherichia coli
Escherichiacoli BL21 (DE3) screens to obtain positive transformant to get recombination large intestine is arrived using ampicillin plate
Bacillus.
If embodiment 1 carries out inducing expression, 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, ammonium chloride 100g/L, pH 8.0, temperature are 35 DEG C, 250 revs/min of shaking speed；Transformation time 5 is small
When.
Table 3L- lactic dehydrogenase and L-alanine dehydrogenase double gene coexpression Escherichia coli generate the efficiency of l-Alanine
Compare
| Bacterial strain | L-Alanine g/L |
| Escherichia coli BLP/pETDuet-1-bmlad-llldh | 20.3 |
| Escherichia coli BLP/pETDuet-1-bmlad-lpldh | 30.3 |
| Escherichia coli BLPTDuet-1-pilad-llldh | 33.4 |
| Escherichia coli BLP/pETDuet-1-pilad-lpldh | 29.3 |
| Escherichia coli BLP/pETDuet-1-sclad-llldh | 23.0 |
| Escherichia coli BLP/pETDuet-1-scladl-pldh | 27.1 |
When two genes of pilad 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 4.Resting cell
System are as follows: wet cell weight 50g/L, Pfansteihl 10g/L, ammonium chloride 20g/L, pH 7.0, temperature are 30 DEG C, 250 turns of shaking speed/
Minute；Transformation time 1 hour.
Comparison of the various expression plasmids of table 4 for conversion production l-Alanine
| Bacterial strain | L-Alanine g/L |
| Escherichia coli BLP/pETDuet-1-pilad-llldh | 4.6 |
| Escherichia coli BLP/pACYCDuet-1-pilad-llldh | 2.6 |
| Escherichia coli BLP/pCOLADuet-1-pilad-llldh | 3.1 |
| Escherichia coli BLP/pRSFDuet-1-pilad-llldh | 2.2 |
| Escherichia coli BLP/pCDFDuet-1-pilad-llldh | 2.7 |
It is best using effect when pETDuet-1 coexpression as can be seen from the above table.
Embodiment 5
Large intestine bar will be increased before icsA and/or nadA gene in Escherichia coli BLP according to the method for example 1
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:22.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.
5 Primer of table is compareed with sequence table serial number
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 6.Resting cell system are as follows: wet cell weight 20g/L, Pfansteihl 200g/L,
Ammonification ammonium 350g/L, pH 9.0, temperature are 30 DEG C, 250 revs/min of shaking speed；Transformation time 24 hours.
6 conversion results of table compare
| Bacterial strain | L-Alanine g/L |
| Escherichia coli BLP(PG-icsA、PG-nadA)/pCOLADuet-1-pilad-llldh | 55.9 |
| Escherichia coli BLP(PG-icsA)/pCOLADuet-1-pilad-llldh | 35.1 |
| Escherichia coli BLP(PG-nadA)/pCOLADuet-1-pilad-llldh | 47.9 |
| Escherichia coli BLP/pCOLADuet-1-pilad-llldh | 29.4 |
From the foregoing, it will be observed that the effect that the sequence before nadA and icsA gene is transformed into constitutive promoter is best.It will
Escherichia coli BLP (PG-icsA, PG-nadA) is named as Escherichia coli BLPN.
Embodiment 6
According to derivational expression method described in embodiment 1, by Escherichia coli BLPN/pETDuet-1-pilad-
Thallus is collected after the completion of llldh inducing expression, in 100ml reaction system, wet cell weight 1g/L, Pfansteihl 1g/L, ammonification ammonium
1g/L, pH 4.0, temperature are 15 DEG C, 250 revs/min of shaking speed；Transformation time 1 hour.Measurement result, l-Alanine concentration
For 23mg/L.
Embodiment 7
According to derivational expression method described in embodiment 1, by Escherichia coli BLPN/pETDuet-1-pilad-
Thallus is collected after the completion of llldh inducing expression, in 100ml reaction system, wet cell weight 200g/L, Pfansteihl 300g/L, ammonia
Change ammonium 350g/L, pH9.0, temperature is 25 DEG C, 250 revs/min of shaking speed；Transformation time 24 hours.Measurement result, the third ammonia of L-
Acid concentration is 291g/L.
Embodiment 8
According to derivational expression method described in embodiment 1, by Escherichia coli BLPN/pETDuet-1-pilad-
Thallus is collected after the completion of llldh2 inducing expression, in 100ml reaction system, wet cell weight 200g/L, D-ALPHA-Hydroxypropionic acid 300g/L, ammonia
Change ammonium 350g/L, pH 9.0, temperature is 30 DEG C, 250 revs/min of shaking speed；Transformation time 24 hours.Measurement result, L- third
Propylhomoserin concentration is 282g/L.The L- third of Escherichia coli BLPN/pETDuet-1-pilad-lpldh2 under similarity condition
Propylhomoserin concentration is 244g/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 LISTING
<110>Southern Yangtze University
<120>method and bacterial strain of production of lactic acid l-Alanine are used
<160> 22
<170> PatentIn version 3.5
<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
cggctggcag gctgaagaag 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence
<400> 3
ttaacggcgt cggcttcggg 20
<210> 4
<211> 25
<212> DNA
<213>artificial sequence
<400> 4
aaatacaatc tctgtaggtt cttct 25
<210> 5
<211> 50
<212> DNA
<213>artificial sequence
<400> 5
tcggccactc atcaacatga ttcatgagtc tgttgctcat ctccttgtca 50
<210> 6
<211> 50
<212> DNA
<213>artificial sequence
<400> 6
tgacaaggag atgagcaaca gactcatgaa tcatgttgat gagtggccga 50
<210> 7
<211> 50
<212> DNA
<213>artificial sequence
<400> 7
cgtagttttg ttgccagaga ttcatggttt tctcctgtca ggaacgttcg 50
<210> 8
<211> 50
<212> DNA
<213>artificial sequence
<400> 8
cgaacgttcc tgacaggaga aaaccatgaa tctctggcaa caaaactacg 50
<210> 9
<211> 25
<212> DNA
<213>artificial sequence
<400> 9
taacacctga cccgcagtgt aaccg 25
<210> 10
<211> 25
<212> DNA
<213>artificial sequence
<400> 10
atgcgtctta tcaggcctac agtga 25
<210> 11
<211> 50
<212> DNA
<213>artificial sequence
<400> 11
tcggccactc atcaacatga ttcataatca ggctaccggc tggatgtacg 50
<210> 12
<211> 50
<212> DNA
<213>artificial sequence
<400> 12
cgtacatcca gccggtagcc tgattatgaa tcatgttgat gagtggccga 50
<210> 13
<211> 50
<212> DNA
<213>artificial sequence
<400> 13
agtcgagata aatcggtaat ttcatggttt tctcctgtca ggaacgttcg 50
<210> 14
<211> 50
<212> DNA
<213>artificial sequence
<400> 14
cgaacgttcc tgacaggaga aaaccatgaa attaccgatt tatctcgact 50
<210> 15
<211> 25
<212> DNA
<213>artificial sequence
<400> 15
aatgttcggc gcaccgtgtt ccagg 25
<210> 16
<211> 25
<212> DNA
<213>artificial sequence
<400> 16
tcgaatcctg cacgacccac cacta 25
<210> 17
<211> 50
<212> DNA
<213>artificial sequence
<400> 17
tcggccactc atcaacatga ttcatcgaca ttagcgtaat attcgctgtt 50
<210> 18
<211> 50
<212> DNA
<213>artificial sequence
<400> 18
aacagcgaat attacgctaa tgtcgatgaa tcatgttgat gagtggccga 50
<210> 19
<211> 50
<212> DNA
<213>artificial sequence
<400> 19
tgtctggatc aaacattacg ctcatggttt tctcctgtca ggaacgttcg 50
<210> 20
<211> 50
<212> DNA
<213>artificial sequence
<400> 20
cgaacgttcc tgacaggaga aaaccatgag cgtaatgttt gatccagaca 50
<210> 21
<211> 25
<212> DNA
<213>artificial sequence
<400> 21
catccacgga caatgcgcgc agctg 25
<210> 22
<211> 1100
<212> DNA
<213> Escherichia coli BL21(DE3)
<400> 22
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 L-alanine dehydrogenase.
2. recombination bacillus coli according to claim 1, which is characterized in that the outer exogenous lactate dehydrogenase comes for lactic acid bacteria
The lactic dehydrogenase in source, the L-alanine dehydrogenase of external source are the l-Alanine of bacillus, marine bacteria or actinomyces source
Dehydrogenase.
3. recombination bacillus coli according to claim 1 to 2, which is characterized in that the recombination bacillus coli is also strengthened
Express the one or more of Lactate Transport gene, NAD synthesis gene.
4. recombination bacillus coli according to claim 3, which is characterized in that the gene of the overexpression be lldP,
Any one or more in icsA, nadA.
5. recombination bacillus coli according to claim 3 or 4, 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.
6. recombination bacillus coli according to claim 2, which is characterized in that the recombination bacillus coli expresses L- simultaneously
Lactic acid dehydrogenase gene llldh and L-alanine dehydrogenase gene pilad, and overexpression Lactate Transport gene lldP,
NAD synthesizes gene nadA.
7. a kind of method for producing optical voidness l-Alanine, which is characterized in that the method is to utilize any institute of claim 1-6
The recombinant bacterium stated.
8. the method according to the description of claim 7 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, L/D lactic acid 1-300g/L, pH 4.0-9.0,15-40 DEG C of temperature,
250 revs/min of shaking speed；Transformation time 1-24 hours.
9. any recombinant bacterium of claim 1-6 is in the application of chemical industry, food, medicine and other fields.
10. method described in claim 7 or 8 is in the application of chemical industry, food, medicine and other fields.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60184393A (en) * | 1984-03-02 | 1985-09-19 | Ajinomoto Co Inc | Preparation of alanine |
| JPS6236196A (en) * | 1985-04-15 | 1987-02-17 | Ajinomoto Co Inc | Production of alanine |
| CN101324561A (en) * | 2007-06-13 | 2008-12-17 | 苏州艾杰生物科技有限公司 | D-lactic acid diagnosis/determination reagent kit and method for determining D-lactic acid concentration |
| CN105593361A (en) * | 2013-08-30 | 2016-05-18 | 巴斯夫欧洲公司 | Modified microorganism for improved production of alanine |
| CN106661597A (en) * | 2014-06-16 | 2017-05-10 | 英威达技术有限责任公司 | Process for producing glutarate and glutaric acid methyl ester |
-
2018
- 2018-11-16 CN CN201811366210.7A patent/CN110305823B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60184393A (en) * | 1984-03-02 | 1985-09-19 | Ajinomoto Co Inc | Preparation of alanine |
| JPS6236196A (en) * | 1985-04-15 | 1987-02-17 | Ajinomoto Co Inc | Production of alanine |
| CN101324561A (en) * | 2007-06-13 | 2008-12-17 | 苏州艾杰生物科技有限公司 | D-lactic acid diagnosis/determination reagent kit and method for determining D-lactic acid concentration |
| CN105593361A (en) * | 2013-08-30 | 2016-05-18 | 巴斯夫欧洲公司 | Modified microorganism for improved production of alanine |
| CN106661597A (en) * | 2014-06-16 | 2017-05-10 | 英威达技术有限责任公司 | Process for producing glutarate and glutaric acid methyl ester |
Non-Patent Citations (2)
| Title |
|---|
| MARIA FELISA. NUNEZ等: "Transport of L-Lactate, D-Lactate, and Glycolate by the LldP and GlcA Membrane Carriers of Escherichia coli", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
| 施慧等: "大肠杆菌NAD+合成关键酶的克隆表达及发酵优化", 《微生物学报》 * |
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