CN108949656B - Engineering bacterium and application thereof in production of pyruvic acid - Google Patents
Engineering bacterium and application thereof in production of pyruvic acid Download PDFInfo
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Abstract
The invention discloses an engineering bacterium and application thereof in production of pyruvic acid, belonging to the technical field of biological engineering. The recombinant escherichia coli expresses exogenous L-lactate dehydrogenase and NADH oxidase at the same time, knocks out a pyruvate absorption gene on the basis of host escherichia coli, and further intensively expresses a lactate transport gene, a pyruvate transport gene and an NAD synthesis gene. On the basis of modifying an escherichia coli transfer and coenzyme synthesis system, the invention constructs the engineering bacteria co-expressed by double enzymes, realizes the high-efficiency production of pyruvic acid and reduces the generation of impurities.
Description
Technical Field
The invention relates to an engineering bacterium and application thereof in production of pyruvic acid, belonging to the technical field of biological engineering.
Background
Pyruvic acid (Pyruvate), also known as 2-oxopropionic acid or acetylformic acid, is a relatively weak organic acid. Pyruvic acid has wide application in chemical, food, medicine and other fields.
At present, chemical methods and biological methods are mainly used for producing pyruvic acid. The chemical method is used for producing pyruvic acid by oxidizing lactic acid under the action of a catalyst, but has higher cost and serious pollution (CN201510848841.2 and the like). The biological methods mainly include lactic acid conversion and direct fermentation. The fermentation of glucose as raw material to produce pyruvic acid (WO8901523, CN201310722490.1, CN201510205724.4, etc.) usually takes a long time, and the cost of extracting pyruvic acid from the impurity fermentation liquid system is high. Pyruvic acid can be produced by engineering bacteria or wild bacteria (WO9500656, CN200710156905.8, CN201110092393.X) containing lactate oxidase, etc., but hydrogen peroxide is produced in the process, and even if perhydrohydrogenase is co-expressed or is added externally, the pyruvic acid cannot be prevented from being oxidized to produce acetic acid, thereby influencing the yield and the purification effect.
The invention changes the transfer of substrates and products and the effective synthesis of coenzyme by transforming the Escherichia coli, and utilizes the L-type lactic acid which is a cheap substrate to convert and produce the pyruvic acid under the condition of not producing hydrogen peroxide.
Disclosure of Invention
Based on the defects of various methods at present, the invention provides a production method for converting lactic acid to produce pyruvic acid without producing hydrogen peroxide, 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 pyruvic acid. The invention aims to solve the technical problems of providing a recombinant bacterium capable of producing pyruvic acid and reducing impurity generation and constructing and applying the strain.
The first object of the present invention is to provide recombinant Escherichia coli capable of producing pure pyruvic acid at a low cost; the recombinant escherichia coli expresses exogenous L-lactate dehydrogenase and NADH oxidase at the same time, and a pyruvate absorption gene is knocked out on the basis of host escherichia coli.
In one embodiment, the exogenous L-lactate dehydrogenase is a lactic acid bacteria-derived L-lactate dehydrogenase. The exogenous NADH oxidase is NADH oxidase derived from lactobacillus.
In one embodiment, the lactate dehydrogenase is from Lactobacillus lactis ATCC 19257, Lactobacillus plantarum ATCC 14917.
In one embodiment, the amino acid sequence of the lactate dehydrogenase is the sequence of accession NO WP _003131075.1, KRL33571.1 at NCBI.
In one embodiment, the nucleotide sequence of the lactate dehydrogenase is that of an accession NO on NCBI: NZ _ JXJZ01000017REGION:18532..19509, AZEJ01000016REGION:16296.. 17249.
In one embodiment, the NADH oxidase is from Lactobacillus lactis ATCC 19257, Lactobacillus sanfranciscisciensis DSM20451, Lactobacillus brevicis ATCC 14869.
In one embodiment, the amino acid sequence of the NADH oxidase is the sequence WP _032950924.1, WP _056958268.1, ERK43827.1 on NCBI for access NO.
In one embodiment, the nucleotide sequence of said NADH oxidase is that of the accession NO on NCBI: NZ _ JXJZ01000002REGION: compensation (39571..40911), NZ _ AYYM01000013REGION: compensation (15875..17233), AWVK01000048REGION: compensation (50022.. 51416).
In one embodiment, the L-lactate dehydrogenase and NADH oxidase are co-expressed by pCOLADuet-1.
In one embodiment, the pyruvate-uptake gene (gene that transports pyruvate into a cell) is any one of btsT, ybdD, or a combination of both.
In one embodiment, the pyruvate uptake gene is an accession NO on NCBI: NC _012892REGION: completion (4496239..4498389) or NC _012892REGION:592652.. 592849.
In one embodiment, the recombinant E.coli further enhances expression of one or more of a lactate transporter gene (a gene that transports lactate into the cell), a pyruvate transporter gene (a gene that enhances transport of pyruvate to the outside of the cell), and 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), pykF (pyruvate transporter), 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; pykF is NC _012892REGION 1700961.. 1702373; icsA is NC _012892REGION: completion (2526116.. 2527330); nadA is NC _012892REGION:740487.. 741530.
The second purpose of the invention is to provide a method for producing optically pure pyruvic acid, which utilizes the recombinant bacterium of the invention.
In one embodiment, said producing pyruvate is produced by whole cell transformation.
In one embodiment, the whole cell transformation production system comprises 1-200g/L wet weight of cells, 1-100 g/L-lactic acid, 4.0-9.0 pH, 15-40 deg.C, and 250 rpm of shaking table; the conversion time is 1-24 hours.
The third purpose of the invention is to provide the application of the recombinant bacterium or the method of the invention 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 pyruvic acid. The selection scheme of the invention does not produce hydrogen peroxide, the cells are not easy to decompose the pyruvic acid, and the cells have higher NAD content. 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 NADH oxidase (NADH oxidase). 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 pyruvic acid and NADH; NADH oxidase oxidizes NADH to generate NAD, and the regeneration of coenzyme NAD is realized. Simultaneously, 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 pyruvic acid.
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, Lactobacillus brevis ATCC 14869, which are available 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). Lactobacillus sanfranciscensis DSM20451 was purchased from German Collection of microorganisms and cell cultures DSMZ. pCasRed and pCRISPR-gDNA were purchased from Zhenjiang Aibiemeng Biotech Ltd.
2. Knockout and constitutive enhanced expression of related genes in escherichia coli
(1) Knockout of pyruvate-absorptive genes of Escherichia coli
Pyruvate is a core compound in a main metabolic pathway and can be utilized or decomposed by a plurality of pathways, pyruvate genetic engineering bacteria usually knock out or weaken genes of related pathways to realize accumulation of pyruvate at present, but gene knock-out can influence normal growth of strains, and special culture media or culture methods are usually needed for pyruvate fermentation. The invention uses lactic acid as substrate to realize the whole cell enzyme conversion to produce pyruvic acid, in order to make the enzyme expression process normally proceed, the genes of decomposition and utilization path related to pyruvic acid in engineering bacteria are not modified, and only the gene transferring pyruvic acid into cell in colibacillus is knocked out, so that the strain can produce pyruvic acid and transfer it out of cell, but can not be absorbed into cell, thereby avoiding the decomposition of pyruvic acid. The genes selected were btsT and ybdD, with access NO at NCBI: NC _012892REGION: completion (4496239..4498389) and NC _012892REGION:592652.. 592849.
(2) Constitutive enhanced expression of escherichia coli lactic acid transport gene/pyruvic acid transport protein
In the whole cell transformation process, lactic acid needs to be transported into cells to perform dehydrogenation to produce pyruvic acid, 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 the dehydrogenation. The gene chosen was lldP, and access NO at NCBI was: NC _012892REGION:3646638.. 3648293. And simultaneously, the transport protein of pyruvic acid to the outside of cells is enhanced, the selected gene is pykF, and the accession NO on NCBI is as follows: NC _012892REGION:1700961.. 1702373.
(3) 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 the escherichia coli is enhanced and expressed, the level of the NAD in the bacteria can be improved, and the generation of pyruvic acid 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 associated with conversion of lactate to pyruvate
(1) Selection of L-lactate dehydrogenase
L-lactic acid is the cheapest organic acid, and pyruvic acid obtained by dehydrogenation has higher additional value. At present, L-lactate oxidase is mainly used for oxidizing L-lactate to produce pyruvic acid, hydrogen peroxide is produced in the process, and the hydrogen peroxide can oxidize the pyruvic acid to produce acetic acid. L-lactate dehydrogenase exists widely in various microorganisms, and lactate dehydrogenase, which generally uses NAD (NADP) as a coenzyme, tends to synthesize lactate using pyruvate as a substrate, but some lactate dehydrogenase removes hydrogen from lactate to produce pyruvate when lactate is excessive or the carbon source has only lactate, and transfers hydrogen produced on L-lactate to coenzyme NAD or NADP using L-lactate as a substrate to produce NADH or NADPH.
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 respectively obtained from Lactobacillus lactis ATCC 19257 and Lactobacillus plantarum ATCC 14917, and expression products are used for the dehydrogenation of lactic acid.
(2) Selection of NADH oxidase
Lactate dehydrogenase dehydrogenates lactate to pyruvate NADH. NADH needs to be oxidized by NADH oxidase to regenerate NAD, so that the reaction can be continued. The NADH oxidase has two types of water-producing type and hydrogen peroxide-producing type, and the water-producing type NADH oxidase does not produce hydrogen peroxide toxicity. The water-producing NADH oxidase genes lsnox (with an amino acid sequence of WP _056958268.1), llnox (with an amino acid sequence of WP _032950924.1) and lbnox (with an amino acid sequence of ERK43827) are obtained from Lactobacillus sanfranciscensis DSM20451, Lactobacillus lactis ATCC 19257 and Lactobacillus brevis ATCC 14869 respectively, and the expression products are used for NAD regeneration.
4. Construction of co-expression system of L-lactate dehydrogenase and NADH oxidase and culture of cells
Optionally one of each of the above selected L-lactate dehydrogenase and NADH oxidase 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, NaCl 10g/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. Production of pyruvic acid by whole cell transformation
The whole cell transformation system is as follows: the wet weight of the cells is 1-200g/L, the L-lactic acid is 1-100g/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 lactate and pyruvate was performed according to the literature (gas chromatography of serum lactate, pyruvate, succinate. proceedings of Lanzhou medical college, 1991(02):72-74)
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
Constructing recombinant escherichia coli: the genes encoding lactate dehydrogenase and NADH oxidase were first ligated separately to a two-gene expression plasmid pETDuet-1. 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, thereby obtaining the recombinant Escherichia coli.
The induction expression method comprises the following steps: transferring the recombinant Escherichia coli into LB fermentation medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L) at a volume ratio of 2%, when 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 ℃.
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 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 1 comparison of the efficiency of pyruvate production by E.coli with lactate dehydrogenase and NADH oxidase double-Gene coexpression
| Bacterial strains | 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 |
From the above table it can be seen that the effect is best when the two genes llnox 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 2. 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 pH 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 2 comparison of various expression plasmids for the production of pyruvate by transformation
| Bacterial strains | 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 |
As can be seen from the above table, the co-expression using pCOLADuet-1 performed the best.
Example 3
btsT and ybdD on Escherichia coli BL21(DE3) were subjected to single or double knockout according to the method described in the document Large scale validation of an effective CRISPR/Cas-based multi gene editing protocol in Escherichia coli, microbial Cell Factories,2017,16(1):68, wherein the plasmids used for gene knockout in the present invention are pCasRed and pCRISPR-gdna (btsT sgRNA) introduced together with the homology arm (btsdonor) onto Escherichia coli BL21(DE3), Cas9/sgRNA induced double strand breaks in the btsT gene site of the host, Red integrated btsT donor into the btsT gene, effecting gene knockout and sequencing of the recombinase. The bstT sgRNA, bstT donor, ybdD sgRNA and ybdD donor are respectively shown as SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31 and SEQ ID NO 32 of the sequence table.
After gene knockout, pCOLADuet-1-llnox-llldh plasmid was introduced into the corresponding strain for transformation comparison. Induced expression was performed according to the method described in example 1, and cells were collected for transformation analysis, with the results shown in Table 2. The whole cell transformation system in the transformation system is as follows: the wet weight of the cells is 200g/L, the L-lactic acid is 5g/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. After the conversion, it was determined that no L-lactic acid remained in the conversion solution, and all the L-lactic acid was converted.
TABLE 3 comparison of transformation results
| Bacterial strains | 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 |
It is clear that the transformation of the double knockout strain Escherichia coli BL21 (. DELTA.btst. DELTA.ybdD, DE3) is the best. This strain was named Escherichia coli BY.
Example 4
The medium expression strength constitutive Promoter (PG) in front of the corresponding gene on the Escherichia coli BY genome is increased before the glyceraldehyde-3-phosphate dehydrogenase gene (gpdA) of Escherichia coli, and the sequence is shown as SEQ ID NO:33, BY adopting the method described in the document Large scale identification of an effective CRISPR/Cas-based multi gene expression protocol in Escherichia coli, Microbiological Cell industries, 2017,16(1): 68.
When the expression of the gene lldP is enhanced, an Escherichia coli BY genome is used as a template, primers lldP-FF/lldP-FR and lldP-gpdA-F/lldP-gpdA-R, lldP-RF/lldP-RR are used to amplify an upstream sequence, a promoter and a downstream sequence, and the lldP-FF and the lldP-RR are used as primers to fuse into an expression frame containing a gpdA promoter. Then after being transformed into Escherichia coli BY together with plasmids pCasRed and pCRISPR-gDNA (containing lldP sgRNA), Cas9/sgRNA induces double strand break of host at lldP gene site, recombinase Red integrates gpdA promoter in front of lldP gene, and sequencing and verification are carried out.
When enhancing the expression of gene pykF, the Escherichia coli BY genome is used as a template, primers pykF-FF/pykF-FR, pykF-gpdA-F/pykF-gpdA-R, pykF-RF/pykF-RR are used for amplifying upstream, promoter and downstream sequences, and pykF-FF and pykF-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 pykF sgRNA), Cas9/sgRNA induces double strand break in the host at pykF gene site, recombinase Red integrates gpdA promoter in front of pykF 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 4 comparison of primer names with sequence Listing numbers
| Name (R) | Number in sequence listing |
| 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 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 4. 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 pH value is 8.0, the temperature is 40 ℃, and the rotating speed of a shaking table is 250 r/min; the conversion time was 12 hours.
TABLE 5 comparison of transformation results
| Bacterial strains | Concentration of pyruvic acid 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 is found that the modification of the sequences preceding both the lldP and pykF genes to constitutive promoters is most effective. Escherichia coli BY (PG-lldP, PG-pykF) was named as Escherichia coli BYLP
Example 5
The medium expression strength constitutive Promoter (PG) in E.coli before the gene for icsA and/or nadA was increased according to the method of example 4 before the gene for glyceraldehyde-3-phosphate dehydrogenase (gpdA) in Escherichia coli BYLP, and the sequence is shown in SEQ ID NO: 33. 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 6 comparison of primer names with sequence Listing numbers
| Name (R) | Number in sequence listing |
| 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 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 5. 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 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 7 comparison of transformation results
| Bacterial strains | 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 |
As described above, it was found that the constitutive promoter obtained by modifying only the sequence preceding nadA gene was most effective. Escherichia coli BYLP (PG-nadA) was named Escherichia coli BYLPN.
Example 6
According to the induced expression method in the embodiment 2, thalli are collected after the induced expression of Escherichia coli BYLPN/pCOLADuet-1-llnox-llldh is finished, and in a 100ml reaction system, the wet weight of cells is 1g/L, the L-lactic acid is 1g/L, the pH value is 4.0, the temperature is 15 ℃, and the rotating speed of a shaking table is 250 r/min; the conversion time was 1 hour. As a result of the measurement, the pyruvic acid concentration was 11 mg/L.
Example 7
According to the induced expression method in the embodiment 2, thalli are collected after the induced expression of Escherichia coli BYLPN/pCOLADuet-1-llnox-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 200g/L, the pH 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 of the measurement, the pyruvic acid concentration was 196 g/L. The acetone concentration of Escherichia coli BL21(DE3)/pCOLADuet-1-llnox-llldh under the same conditions was 162 g/L.
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
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aggagggaca gctgatagaa acagaagcca ctggagcacc tcaaaaacac catcatacac 960
taaatcagta agttggcagc atcaccccgt tttcagtacg ttacgtttca ctgtgagaat 1020
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Claims (5)
1. The recombinant escherichia coli is characterized by simultaneously expressing L-lactate dehydrogenase derived from lactic acid bacteria and NADH oxidase derived from lactic acid bacteria, and also simultaneously expressing an L-lactate dehydrogenase gene llnox and an NADH oxidase gene llldh, knocking out pyruvate absorption genes btsT and ybdD, and intensively expressing a lactate transport gene lldP, a pyruvate transport gene pykF and an NAD synthesis gene nadA.
2. 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.
3. A method for producing optically pure pyruvic acid, which comprises using the recombinant Escherichia coli of claim 1 or 2 to produce optically pure pyruvic acid.
4. The method of claim 3, 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-lactic acid is 1-100g/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.
5. The recombinant Escherichia coli of claim 1 or 2, or the method of claim 3 or 4, for preparing pyruvate-containing products in chemical, food, and pharmaceutical fields.
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| CN103710274A (en) * | 2013-12-24 | 2014-04-09 | 江南大学 | Genetically engineered bacterium for increasing yield of extracellular pyruvic acid and application thereof |
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| CN102199632A (en) * | 2011-04-13 | 2011-09-28 | 济南大学 | Method for preparing pyruvic acid by converting DL-lactic acid |
| WO2014029761A3 (en) * | 2012-08-20 | 2014-04-17 | Clariant Produkte (Deutschland) Gmbh | Cell-free and minimized metabolic reaction cascades for the production of chemicals |
| CN103710274A (en) * | 2013-12-24 | 2014-04-09 | 江南大学 | Genetically engineered bacterium for increasing yield of extracellular pyruvic acid and application thereof |
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