CN110305823B - Method and strain for producing L-alanine by adopting lactic acid - Google Patents
Method and strain for producing L-alanine by adopting lactic acid Download PDFInfo
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Abstract
The invention discloses an engineering bacterium and application thereof in producing L-alanine, belonging to the technical field of biological engineering. The recombinant escherichia coli expresses exogenous L-lactate dehydrogenase and L-alanine dehydrogenase simultaneously, and strengthens and expresses a lactate transport gene and an NAD synthetic gene on the basis of host escherichia coli. On the basis of transforming an escherichia coli transfer and coenzyme synthesis system, the invention constructs the double-enzyme co-expression engineering bacteria and realizes the efficient production of L-alanine by adopting cheap raw materials.
Description
Technical Field
The invention relates to a method for producing L-alanine by adopting lactic acid and a strain, belonging to the technical field of biological engineering.
Background
L-alanine is an important amino acid and has wide application in the fields of chemical industry, food, medicine and the like.
At present, the production of L-alanine mainly comprises a chemical method, an enzyme conversion method and a fermentation method. The enzymatic conversion method is mainly produced by decarboxylation of aspartic acid, but aspartic acid itself has a higher valence. There is also a method for co-immobilizing malic acid enzyme and L-alanine dehydrogenase to produce malic acid as a substrate, but malic acid itself is expensive and industrial production is difficult to realize. The L-alanine produced by the fermentation of the genetically engineered bacteria by taking glucose as a raw material has the problems of low sugar acid conversion rate and difficult extraction caused by complex composition of fermentation liquor.
Disclosure of Invention
Based on the defects of various methods at present, the invention provides a production method for producing L-alanine by converting lactic acid, constructs double-enzyme coexpression engineering bacteria on the basis of modifying an escherichia coli transfer and coenzyme synthesis system, and realizes the high-efficiency production of L-alanine. The technical problem to be solved by the invention is to provide a recombinant bacterium capable of producing L-alanine by using cheap raw materials, and simultaneously, the invention also solves the technical problems of construction and application of the strain.
The first object of the present invention is to provide recombinant Escherichia coli capable of producing pure L-alanine at low cost; the recombinant Escherichia coli expresses exogenous L/D-lactate dehydrogenase and L-alanine dehydrogenase simultaneously.
In one embodiment, the exogenous L/D lactate dehydrogenase is a lactic acid bacteria-derived L/D-lactate dehydrogenase. The exogenous L-alanine dehydrogenase is L-alanine dehydrogenase derived from Bacillus, marine bacteria or actinomycetes.
In one embodiment, the L-lactate dehydrogenase is from Lactobacillus lactis ATCC 19257, Lactobacillus plantarum ATCC 14917.
In one embodiment, the amino acid sequence of the L-lactate dehydrogenase is the sequence of accession NO WP-003131075.1, KRL33571.1 at NCBI.
In one embodiment, the nucleotide sequence of the L-lactate dehydrogenase is that of an accession NO at NCBI: NZ _ JXJZ01000017REGION:18532..19509, AZEJ01000016REGION:16296.. 17249.
In one embodiment, the D-lactate dehydrogenase is from Lactobacillus lactis ATCC 19257, Lactobacillus plantarum ATCC 14917.
In one embodiment, the amino acid sequence of the D-lactate dehydrogenase is the sequence of accession NO WP-014573232.1, KRL35110.1 at NCBI.
In one embodiment, the nucleotide sequence of the D-lactate dehydrogenase is that of an accession NO at NCBI: NZ _ JXJZ01000008REGION: completion (31625..32602), AZEJ01000004REGION:38463.. 39455.
In one embodiment, the L-alanine dehydrogenase is from Bacillus megaterium DSM 319, Phaeobacterium hibens DSM 17395, Streptomyces corchorusii DSM 40340.
In one embodiment, the amino acid sequence of the L-alanine dehydrogenase is the sequence WP _013085206.1, WP _014881174.1, WP _014671828.1 for access NO at NCBI.
In one embodiment, the nucleotide sequence of the L-alanine dehydrogenase is that of an accession NO at NCBI: NC-014103 REGION: completion (4614172..4615293), NC-018290 REGION:3177716..3178831, NZ _ KQ948369REGION:24418.. 25542.
In one embodiment, the L-lactate dehydrogenase and L-alanine dehydrogenase are co-expressed by pETDuet-1.
In one embodiment, the recombinant E.coli is further enhanced to express one or more of a lactate transporter gene (a gene that transports lactate into the cell), an NAD synthesis gene (a key enzyme of the E.coli NAD synthesis pathway).
In one embodiment, the expression-enhanced gene is any one or more of lldP (lactate transporter gene), icsA (NAD synthesis gene), nadA (NAD synthesis gene).
In one embodiment, the host bacterium is Escherichia coli BL21(DE 3).
In one embodiment, the enhanced expression is achieved by adding a constitutive promoter in front of the gene to be enhanced on the genome of Escherichia coli BL21(DE 3).
In one embodiment, the lldP is access NO at NCBI: NC _012892REGION 3646638.. 3648293; icsA is NC _012892REGION: completion (2526116.. 2527330); nadA is NC _012892REGION:740487.. 741530.
The second object of the present invention is to provide a method for producing optically pure L-alanine by using the recombinant bacterium of the present invention.
In one embodiment, the L-alanine is produced by whole cell transformation.
In one embodiment, the whole cell transformation production system comprises 1-200g/L wet weight of cells, 1-300 g/L-lactic acid, 1-350g/L ammonium chloride, 4.0-9.0 pH, 15-40 ℃ and 250 rpm of shaking table rotation speed; the conversion time is 1-24 hours.
The third purpose of the invention is to provide the application of the recombinant bacterium in the fields of chemical industry, food, medicine and the like.
The fourth purpose of the invention is to provide the application of the method in the fields of chemical industry, food, medicine and the like.
The invention has the beneficial effects that:
the invention constructs a novel double-enzyme co-expression genetic engineering bacterium which can be applied to the production of L-alanine. The substrate selected by the invention is cheap, and the intracellular NAD content is high. The production process is simple, the raw materials are easy to obtain, and the method has a good industrial application prospect.
Detailed description of the preferred embodiments
The functional core of the escherichia coli engineering bacteria is that two enzymes can be co-expressed, namely Lactate dehydrogenase (Lactate dehydrogenase) and Alanine dehydrogenase (Alanine dehydrogenase). The principle is as follows: in the whole cell of the engineering bacteria, L-lactate dehydrogenase takes NAD in the bacteria as coenzyme to dehydrogenate L-lactate to generate L-alanine and NADH; the L-alanine dehydrogenase synthesizes pyruvic acid and ammonia into L-alanine, NADH is oxidized into NAD, and the regeneration of coenzyme NAD is realized. Simultaneously, the related genes on the genome of the Escherichia coli are knocked out or enhanced to promote the transfer of lactic acid and prevent the decomposition of L-alanine.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
1. the invention relates to a strain and a plasmid
Lactobacillus plantarum ATCC 14917, Lactobacillus lactis ATCC 19257, purchased from American type culture Collection ATCC, were purchased from Novagen corporation as pETDuet-1, pACYCDue-1, pCOLADuet-1, pRSFDuet-1 plasmid, and Escherichia coli BL21(DE 3). Bacillus megaterium DSM 319, Phaeobacterium hibernans DSM 17395, Streptomyces corchorusii DSM 40340 are available from the German Collection of microorganisms and strains DSMZ. pCasRed and pCRISPR-gDNA were purchased from Zhenjiang Aibiemeng Biotech Ltd.
2. Constitutive enhanced expression of related genes in E.coli
(1) Constitutive enhanced expression of lactic acid transporter gene in escherichia coli
In the whole cell transformation process, the L-alanine can be produced by dehydrogenation only by transferring lactic acid into cells, and the enhanced lactic acid transporter is beneficial to maintaining the high concentration of the intracellular lactic acid quickly and for a long time and is beneficial to dehydrogenation. The gene chosen was lldP, and access NO at NCBI was: NC _012892REGION:3646638.. 3648293.
(2) Overexpression of genes involved in NAD synthesis in Escherichia coli
In the process of lactate dehydrogenation, NAD is required to be used as coenzyme, the key enzyme of the synthesis pathway of the NAD of escherichia coli is enhanced and expressed, the level of the NAD in a bacterium can be improved, and the generation of L-alanine is facilitated. The selected genes are icsA and nadA. Access NO on NCBI is: NC-012892 REGION (2526116..2527330), NC-012892 REGION (740487.. 741530),
3. Selection of enzymes involved in the conversion of lactic acid to L-alanine
(1) Selection of lactate dehydrogenase
D/L-lactate dehydrogenases are widely present in many microorganisms, and lactate dehydrogenases, which usually use NAD (NADP) as a coenzyme, tend to synthesize lactate using pyruvate as a substrate, but some lactate dehydrogenases remove hydrogen from lactate to produce pyruvate when the lactate is in excess or the carbon source is lactate alone. Lactic acid includes both D and L, and L-lactic acid is more inexpensive to produce than D-lactic acid, and it is preferable in this patent to transfer hydrogen produced on L-lactic acid to coenzyme NAD or NADP using L-lactic acid as a substrate to produce NADH or NADPH. D-lactate can likewise be used as a substrate by a D-lactate dehydrogenase.
The L-lactate dehydrogenase gene llldh (with an amino acid sequence of WP _003131075.1) and lpldhh (with an amino acid sequence of KRL33571.1) are obtained from Lactobacillus lactis ATCC 19257 and Lactobacillus plantarum ATCC 14917 respectively, and expression products are used for dehydrogenation of L-lactic acid.
The D-lactate dehydrogenase genes llldh2 (the amino acid sequence is WP _014573232.1) and lpldhh 2 (the amino acid sequence is KRL35110.1) are respectively obtained from Lactobacillus lactis ATCC 19257 and Lactobacillus plantarum ATCC 14917, and the expression products are used for dehydrogenation of D-lactic acid.
(2) Selection of L-alanine dehydrogenase
Lactate dehydrogenase dehydrogenates lactate to generate pyruvate and NADH, L-alanine dehydrogenase reduces pyruvate to L-alanine by using NADH and ammonia, and NAD is regenerated, thereby realizing continuous reaction. The present invention obtains L-alanine dehydrogenase genes bmlad (amino acid sequence is WP _013085206.1), pilad (amino acid sequence is WP _014881174.1) and sclad (amino acid sequence is WP _014671828.1) from Bacillus megaterium DSM 319, Phaeobacterium hibernans DSM 17395 and Streptomyces corchorusii DSM 40340, respectively, and the expression products are used for synthesizing L-alanine.
4. Construction of co-expression system of L-lactate dehydrogenase and L-alanine dehydrogenase and culture of cells
Optionally one of each of the above selected L-lactate dehydrogenase and L-alanine dehydrogenase is co-expressed in a two-enzyme combination.
At present, multiple methods (an escherichia coli multigene co-expression strategy, journal of biological engineering in China, 2012, 32(4):117-122) are adopted for the escherichia coli multigene co-expression), the method is constructed by adopting an Liu-oriented epitaxy method (2016, Shanghai medical industry research institute, doctor paper, 2016, producing shikimic acid and resveratrol by transforming escherichia coli by using a synthetic biology technology), each gene comprises a T7 promoter and an RBS binding site in front, and a T7 terminator is arranged behind the gene. Theoretically, since each gene is preceded by T7 and RBS, the expression intensity of the gene is not greatly affected by the ranking. Each plasmid contains two genes, the constructed plasmids are thermally transduced into escherichia coli competent cells, and are coated on an antibiotic solid plate, and positive transformants are obtained through screening, so that the recombinant escherichia coli is obtained. And (3) culturing the cells: according to the classical recombinant Escherichia coli culture and induction expression scheme, transferring the recombinant Escherichia coli into LB fermentation medium (peptone 10g/L, yeast powder 5g/L, NaCl10g/L) according to the volume ratio of 2%, when the cell OD600After reaching 0.6-0.8, IPTG was added to a final concentration of 0.4mM, and expression-induced culture was carried out at 20 ℃ for 8 hours. After the induction expression was completed, the cells were collected by centrifugation at 8000rpm for 20 minutes at 20 ℃.
5. Whole cell transformation for producing L-alanine
The whole cell transformation system is as follows: the wet weight of the cells is 1-200g/L, the L/D-lactic acid is 1-300g/L, the pH is 4.0-9.0, the temperature is 15-40 ℃, and the rotating speed of a shaking table is 250 r/min; the conversion time is 1-24 hours.
6. Detection analysis of samples
Quantitative analysis of lactic acid was carried out according to the literature (determination of lactic acid and organic impurities [ J ] in fermentation lactic acid products by gas chromatography, proceedings of the Beijing institute of petrochemical engineering, 2003(03):46-50.)
Quantitative analysis of L-alanine was carried out according to the literature (determination of alanine content in Bromus by pre-column derivatization [ J ]. according to the guidance of traditional Chinese medicine, 2018,24(09):42-44.)
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more apparent, the present invention is described in detail below with reference to the embodiments. It should be noted that the specific embodiments described herein are only for explaining the present invention and are not used to limit the present invention.
Example 1
A medium expression strength constitutive Promoter (PG) in front of a corresponding gene on Escherichia coli BL21(DE3) genome is increased by adopting a method described in the document Large scale identification of an effective CRISPR/Cas-based multi-gene encoding protocol in Escherichia coli, Microbiological Cell industries, 2017,16(1):68, and the sequence is shown as SEQ ID NO: 22.
When the expression of the gene lldP is enhanced, Escherichia coli BL21(DE3) genome is used as a template, primers lldP-FF/lldP-FR, lldP-gpdA-F/lldP-gpdA-R, lldP-RF/lldP-RR are used to amplify upstream, promoter and downstream sequences, and lldP-FF and lldP-RR are used as primers to fuse into an expression frame containing a gpdA promoter. Then after being transformed into Escherichia coli BL21(DE3) together with plasmids pCasRed and pCRISPR-gDNA (containing lldP sgRNA), the Cas9/sgRNA induces the host to generate double-strand break at the lldP gene site, and recombinase Red integrates the gpdA promoter in front of the lldP gene, and the sequence is verified.
The following table is the corresponding index of the primer name and sequence number in the sequence listing.
TABLE 1 comparison of primer names with sequence Listing numbers
| Name (R) | Number in sequence listing |
| 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 gene transformation is completed, the co-expression plasmid is introduced. The induction of expression was carried out according to the following method: the induction expression method comprises the following steps: transferring the recombinant Escherichia coli into LB fermentation medium (peptone 10g/L, yeast powder 5g/L, NaCl10g/L) according to the volume ratio of 2%, when the cell OD600After reaching 0.6-0.8, IPTG was added to a final concentration of 0.4mM, and expression-induced culture was carried out at 20 ℃ for 8 hours. After the induction expression was completed, the mixture was centrifuged at 8000rpm for 20 minutes at 20 ℃The cells were collected. After the expression is finished, various cells are collected for transformation analysis, and the results are shown in Table 2. The whole cell transformation system is as follows: the wet weight of the cells is 10g/L, the L-lactic acid is 50g/L, the ammonium chloride is 350g/L, the pH value is 8.0, the temperature is 40 ℃, the rotating speed of a shaking table is 250 r/min; the conversion time was 12 hours.
TABLE 2 comparison of transformation results
| Bacterial strains | 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 was found that the modification of the sequence preceding the lldP gene to a constitutive promoter was also most effective. Escherichia coli BL21(DE3) (PG-lldP) was named Escherichia coli BLP.
Example 2
Constructing recombinant escherichia coli: first, genes encoding lactate dehydrogenase and L-alanine dehydrogenase were ligated to a two-gene expression plasmid pETDuet-1, respectively. Obtaining various double-gene co-expression recombinant plasmids, transforming the plasmids into escherichia coli BL21(DE3), and screening by using an ampicillin plate to obtain positive transformants, thus obtaining the recombinant escherichia coli.
After the induction of expression as in example 1, the cells were collected by centrifugation at 8000rpm for 20 minutes at 20 ℃.
The collected cells were analyzed for transformation, and the results are shown in Table 1. The whole cell transformation system is as follows: the wet weight of the cells is 100g/L, the L-lactic acid is 50g/L, the ammonium chloride is 100g/L, the pH value is 8.0, the temperature is 35 ℃, and the rotating speed of a shaking table is 250 r/min; the conversion time was 5 hours.
TABLE 3 comparison of L-alanine production efficiency of L-alanine from E.coli co-expressed with L-lactate dehydrogenase and L-alanine dehydrogenase
| Bacterial strains | 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 |
It can be seen from the above table that the effect is the best when the two genes pilad and llldh are combined.
Example 2
According to the strain construction method (each plasmid adopts different resistant plates to screen positive transformants according to the instruction) and the induction expression method described in example 1, each cell is collected for transformation analysis, and the results are shown in table 4. The whole cell transformation system is as follows: the wet weight of the cells is 50g/L, the L-lactic acid is 10g/L, the ammonium chloride is 20g/L, the pH value is 7.0, the temperature is 30 ℃, and the rotating speed of a shaking table is 250 r/min; the conversion time was 1 hour.
TABLE 4 comparison of various expression plasmids for the production of L-alanine by transformation
| Bacterial strains | 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 can be seen from the above table that co-expression using pETDuet-1 works best.
Example 5
The medium expression strength constitutive Promoter (PG) in E.coli before the gene for icsA and/or nadA was increased in Escherichia coli BLP according to the method of example 1, and the sequence is shown in SEQ ID NO: 22. The plasmid is then introduced.
When enhancing gene icsA expression, Escherichia coli BY genome is used as a template, primers icsA-FF/icsA-FR, icsA-gpdA-F/icsA-gpdA-R, icsA-RF/icsA-RR are used to amplify upstream, promoter and downstream sequences, and icsA-FF and icsA-RR are used as primers to fuse into an expression cassette containing a gpdA promoter. Then after being transferred into Escherichia coli BY together with plasmids pCasRed and pCRISPR-gDNA (containing icsAsgRNA), Cas9/sgRNA induces double strand break of the host at the icsA gene site, recombinase Red integrates the gpdA promoter in front of the icsA gene, and sequencing and verification are carried out.
When gene nadA expression is enhanced, an Escherichia coli BY genome is used as a template, primers nadA-FF/nadA-FR and nadA-gpdA-F/nadA-gpdA-R, nadA-RF/nadA-RR are used to amplify upstream, promoter and downstream sequences, and nadA-FF and nadA-RR are used as primers to fuse into an expression cassette containing a gpdA promoter. After transformation into Escherichia coli BY together with plasmids pCasRed, pCRISPR-gDNA (containing nadAsgRNA), Cas9/sgRNA induces double strand break at nadA gene site in the host, recombinase Red integrates gpdA promoter in front of nadA gene, and sequencing is performed for verification.
The following table is the corresponding index of the primer name and sequence number in the sequence listing.
TABLE 5 comparison of primer names with sequence Listing numbers
After the gene transformation is completed, the co-expression plasmid is introduced. Expression was induced according to the method described in example 1, and various types of cells were collected and subjected to transformation analysis, and the results are shown in Table 6. The whole cell transformation system is as follows: the wet weight of the cells is 20g/L, the L-lactic acid is 200g/L, the ammonium chloride is 350g/L, the pH is 9.0, the temperature is 30 ℃, and the rotating speed of a shaking table is 250 r/min; the conversion time was 24 hours.
TABLE 6 comparison of transformation results
| Bacterial strains | 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 |
As described above, it was found that the constitutive promoter was most effective when the sequences preceding both nadA and icsA genes were modified. Escherichia coli BLP (PG-icsA, PG-nadA) was named as Escherichia coli BLPN.
Example 6
According to the inducible expression method of the embodiment 1, thalli are collected after the induction expression of Escherichia coli BLPN/pETDuet-1-pilad-llldh is finished, and in a 100ml reaction system, the wet weight of the cells is 1g/L, the wet weight of the; the conversion time was 1 hour. As a result of the measurement, the L-alanine concentration was 23 mg/L.
Example 7
According to the inducible expression method of the embodiment 1, thalli are collected after the induction expression of Escherichia coli BLPN/pETDuet-1-pilad-llldh is finished, and in a 100ml reaction system, the wet weight of cells is 200g/L, the wet weight of L-lactic acid is 300g/L, ammonium amide is 350g/L, the pH value is 9.0, the temperature is 25 ℃, and the rotating speed of a shaking table is 250 r/min; the conversion time was 24 hours. As a result, the L-alanine concentration was 291 g/L.
Example 8
According to the induction expression method of the embodiment 1, thalli are collected after the induction expression of Escherichia coli BLPN/pETDuet-1-pilad-llldh2 is finished, and in a 100ml reaction system, the wet weight of cells is 200g/L, the wet weight of D-lactic acid is 300g/L, ammonium amide is 350g/L, the pH is 9.0, the temperature is 30 ℃, and the rotating speed of a shaking table is 250 r/min; the conversion time was 24 hours. As a result, the L-alanine concentration was 282 g/L. The L-alanine concentration of Escherichia coli BLPN/pETDuet-1-pilad-lpldh2 was 244g/L under the same conditions.
The modification and construction of the enzyme and its co-expressed genetically engineered bacteria, the culture medium composition and culture method of the bacteria, and the whole cell biotransformation described above are only preferred embodiments of the present invention, and are not intended to limit the present invention, and theoretically, other bacteria, filamentous fungi, actinomycetes, and animal cells can be used for genome modification and whole cell catalysis of multigene co-expression. Any modification, equivalent replacement, made within the principle and spirit of the present invention.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> method for producing L-alanine using lactic acid and strain
<160> 22
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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 (8)
1. The recombinant Escherichia coli is characterized in that the recombinant Escherichia coli expresses exogenous L-lactate dehydrogenase and L-alanine dehydrogenase simultaneously, and the amino acid sequence of the L-lactate dehydrogenase is the sequence of accession NO WP-003131075.1 and KRL33571.1 on NCBI; the amino acid sequence of the L-alanine dehydrogenase is the sequence of accession NO WP _013085206.1, WP _014881174.1 and WP _014671828.1 on NCBI, and the recombinant Escherichia coli also strengthens expression of a lactate transporter lldP.
2. The recombinant escherichia coli of claim 1, wherein the recombinant escherichia coli further enhances expression of any one or more of an NAD synthesis gene icsA and an nadA synthesis gene.
3. The recombinant Escherichia coli of claim 1, wherein the enhanced expression is achieved by adding a constitutive promoter in front of a gene to be enhanced on the genome of the host Escherichia coli.
4. The recombinant Escherichia coli of claim 1, wherein the recombinant Escherichia coli expresses a L-lactate dehydrogenase gene llldh and a L-alanine dehydrogenase gene pilad simultaneously, and enhances expression of lactate transporter genes lldP and NAD synthesis genes nadA.
5. A method for producing optically pure L-alanine by using the recombinant bacterium according to any one of claims 1 to 4.
6. The method of claim 5, wherein the method is carried out for whole cell transformation production; in the whole cell transformation production system, the wet weight of cells is 1-200g/L, the L/D lactic acid is 1-300g/L, the pH is 4.0-9.0, the temperature is 15-40 ℃, and the rotating speed of a shaking table is 250 revolutions per minute; the conversion time is 1-24 hours.
7. The recombinant bacterium of any one of claims 1-4 for use in chemical and food fields.
8. The method of claim 5 or 6, wherein the method is applied to the fields of chemical industry and food.
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Citations (4)
| 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 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3155112A1 (en) * | 2014-06-16 | 2017-04-19 | Invista Technologies S.à.r.l. | Process for producing glutarate and glutaric acid methyl ester |
-
2018
- 2018-11-16 CN CN201811366210.7A patent/CN110305823B/en active Active
Patent Citations (4)
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| Transport of L-Lactate, D-Lactate, and Glycolate by the LldP and GlcA Membrane Carriers of Escherichia coli;Maria Felisa. Nunez等;《Biochemical and Biophysical Research Communications》;20020228;第290卷(第2期);824-829 * |
| 大肠杆菌NAD+合成关键酶的克隆表达及发酵优化;施慧等;《微生物学报》;20161231;第57卷(第7期);1112-1125 * |
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