CN108949840B - Engineering bacterium and application thereof in production of p-hydroxycinnamic acid - Google Patents
Engineering bacterium and application thereof in production of p-hydroxycinnamic acid Download PDFInfo
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Abstract
The invention discloses an engineering bacterium and application thereof in production of p-hydroxycinnamic acid, and belongs to the technical field of biological engineering. The invention provides a recombinant bacterium capable of producing p-hydroxycinnamic acid at low cost; the recombinant strain simultaneously expresses 4 enzymes which are respectively tyrosine phenol lyase, tyrosine ammonia lyase, L-lactate dehydrogenase and NADH oxidase; furthermore, the recombinant bacterium of the invention also removes the phenolic substance decomposition gene, and strengthens and expresses any one or more of lactic acid transport gene, phenol transport gene and coenzyme synthesis related gene. The invention realizes the high-efficiency production of the hydroxycinnamic acid, and the method has simple process, less impurities and important industrial application value.
Description
Technical Field
The invention relates to an engineering bacterium and application thereof in producing p-hydroxy cinnamic acid, belonging to the technical field of biological engineering.
Background
P-hydroxycinnamic acid (3- (4-hydroxyphenyl) -2-propenoic acid), a major component of lignocellulose, is produced. Research shows that the composition can reduce the formation of nitrosamine as carcinogen and has anticancer effect.
At present, p-hydroxycinnamic acid (CN201110337186.6, JP200423154) is synthesized mainly by a chemical synthesis method, and has larger pollution. Also obtained by hydrolysis of biomass feedstocks (WO/2017/170549).
Currently, the synthesis of p-hydroxycinnamic acid by biological methods is the hottest direction, such as the method of converting tyrosine into p-hydroxycinnamic acid by tyrosine ammonia lyase (US20170166936 and US20080213846), the method of converting cinnamic acid into p-hydroxycinnamic acid by microorganisms (US 20030170834), and the method of synthesizing p-hydroxycinnamic acid by using glucose as a raw material by Escherichia coli engineering bacteria (EP1589112 and WO/2002/090523). These processes are subject to improvement to reduce cost and increase product purity.
Disclosure of Invention
Based on the defects of various methods at present, the invention provides a novel production method of p-hydroxycinnamic acid, constructs a multienzyme coexpression engineering bacterium, and realizes the high-efficiency production of p-hydroxycinnamic acid. The invention aims to solve the technical problems of providing a recombinant strain capable of efficiently producing p-hydroxycinnamic acid by using a cheap substrate and constructing and applying the strain.
The first purpose of the invention is to provide a recombinant bacterium capable of producing p-hydroxycinnamic acid at low cost; the recombinant strain expresses 4 enzymes simultaneously, namely tyrosine phenol lyase, tyrosine ammonia lyase, L-lactate dehydrogenase and NADH oxidase.
In one embodiment, the L-lactate dehydrogenase is from Lactococcus lactis ATCC 19257.
In one embodiment, the amino acid sequence of the L-lactate dehydrogenase is the sequence WP-003131075.1 for access NO 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.
In one embodiment, the NADH oxidase is from Lactococcus lactis ATCC 19257.
In one embodiment, the amino acid sequence of the NADH oxidase is the sequence WP _032950924.1 for access NO on NCBI.
In one embodiment, the nucleotide sequence of said NADH oxidase is that of the accession NO on NCBI: NZ _ JXJZ01000002REGION: completion (39571.. 40911).
In one embodiment, the tyrosol lyase is from Erwinia herbicoloa ATCC 214344.
In one embodiment, the amino acid sequence of the tyrosol lyase is accession NO at NCBI as P31011.2.
In one embodiment, the tyrosine ammonia lyase is from Rhodobacter sphaeroides ATCC BAA-808.
In one embodiment, the amino acid sequence of tyrosine ammonia lyase is the sequence with access NO WP _011339422.1 on NCBI.
In one embodiment, the nucleotide sequence of tyrosine ammonia lyase is the nucleotide sequence of accession NO on NCBI: NC-007494 REGION: completion (668571.. 670142).
In one embodiment, the recombinant bacterium comprises genes encoding tyrosine phenol lyase, tyrosine ammonia lyase, NADH oxidase and L-lactic acid dehydrogenase, which are connected to 2 plasmids, and then the recombinant plasmids are transformed into host escherichia coli to obtain the recombinant engineering bacterium.
In one embodiment, the NADH oxidase gene and the L-lactate dehydrogenase gene are expressed after being ligated to plasmid pACYCDue-1, and the tyrosine ammonia lyase gene and the tyrosine phenol lyase gene are expressed after being ligated to plasmid pETDuet-1.
In one embodiment, the host bacterium is Escherichia coli BL21(DE 3).
In one embodiment, the recombinant bacterium further knocks out a phenolic substance-decomposing gene.
In one embodiment, the knockout phenolic substance resolving gene is any one of hpaD, mhpB, or a combination of both.
In one embodiment, the nucleotide sequence of the phenolics-decomposing gene is that on NCBI the accession NO: NC _012892REGION: completion (4505585..4506436) and NC _012892REGION:339806.. 340750.
In one embodiment, the recombinant bacterium further enhances expression of any one or more of a lactate transporter gene, a phenol transporter gene, and a coenzyme synthesis-related gene.
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 expression-enhanced gene is any one or more of lldP (lactate transporter gene), hpaX (phenol transporter gene), mhpT (phenol transporter gene), nadA (NAD synthesis gene), pdxJ (pyridoxal phosphate synthesis gene).
In one embodiment, the lldP is access NO at NCBI: NC _012892REGION 3646638.. 3648293; hpaX is; NC _012892REGION: compensation (4502025.. 4503401); mhpT NC-012892 REGION 344788.. 345999; nadA is NC _012892REGION 740487.. 741530; pdxJ is NC _012892REGION: completion (2567591.. 2568322).
In one embodiment, the recombinant bacterium is an escherichia coli host with hpaD and mhpB knocked out, and the recombinant bacterium is enhanced to express lldP, hpaX, mhpT, nadA and pdxJ, and simultaneously express tyrosine phenol lyase, tyrosine ammonia lyase, L-lactate dehydrogenase and NADH oxidase.
The second purpose of the invention is to provide a method for producing p-hydroxycinnamic acid, wherein the method utilizes the recombinant bacterium of the invention.
In one embodiment, the production of p-hydroxycinnamic acid is carried out by whole cell transformation.
In one embodiment, the whole cell transformation production system has a cell wet weight of 1-200g/L, a phenol concentration of 1-200g/L, an L-lactic acid concentration of 1-200g/L, a pH of 6.0-9.0, and an ammonium ion concentration of 1-30 g/L; reacting at 15-40 deg.c for 1-48 hr. And (4) determining the yield of the p-hydroxycinnamic acid by liquid chromatography after the conversion is finished.
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 four-enzyme co-expression genetic engineering bacterium which can be applied to the production of p-hydroxycinnamic acid. 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 engineering bacteria is that 4 enzymes can be expressed simultaneously, namely tyrosine phenol lyase, tyrosine ammonia lyase, NADH oxidase and L-lactate 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 pyruvic acid and NADH; the tyrosine phenol lyase catalyzes pyruvic acid, ammonia radical ions and phenol to generate L-tyrosine; the L-tyrosine is deaminated by tyrosine ammonia lyase to generate p-hydroxycinnamic acid; NADH oxidase dehydrogenates NADH to realize the regeneration of coenzyme NAD. Simultaneously, related genes on the genome of the escherichia coli are knocked out or enhanced to promote substrate transport and reduce phenolic substance decomposition.
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
Escherichia coli BL21(DE3), Rhodobacter sphaeroides ATCC BAA-808, Lactobacillus lactis ATCC19257, Erwinia herbicolo ATCC 214344, which were purchased from American type culture Collection ATCC. pETDuet-1, pACYCDue-1 plasmids and Escherichia coli BL21(DE3) were purchased from Novagen. 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 Escherichia coli phenolic substance decomposition gene
The phenolic substances in the invention are all very easily decomposed by enzymes in Escherichia coli, and according to the literature (Biodegradation of Aromatic Compounds by Escherichia coli, Microbiol Mol Biol Rev.2001,65(4): 523-. The genes selected were hpaD and mhpB, with access NO at NCBI: NC _012892REGION: completion (4505585..4506436) and NC _012892REGION:339806.. 340750.
(2) Constitutive enhanced expression of lactic acid and phenol transport genes of escherichia coli
In the whole cell transformation process, the substrate is required to be transported into the cell, and the enhanced lactic acid transporter is beneficial to quickly and long-term maintaining the high concentration of the intracellular substrate and facilitating the reaction. The lactate transport-associated gene was selected to be lldP, with access NO at NCBI as: NC _012892REGION:3646638.. 3648293. Genes related to phenol transport are hpaX and mhpT, and access NO on NCBI is: NC _012892REGION: completion (4502025..4503401) and NC _012892REGION:344788.. 345999.
(3) Constitutive enhanced expression of important genes related to Escherichia coli coenzyme synthesis
NADH is required to be used as coenzyme in the reduction process of NADH oxidase, the key enzyme of the NAD synthetic pathway of escherichia coli is enhanced and expressed, the NAD level in the bacteria can be improved, and the generation of p-hydroxycinnamic acid is facilitated. The gene of choice is nadA. Access NO on NCBI is: NC _012892REGION:740487.. 741530.
The pyridoxal phosphate (amine) is a coenzyme of tyrosine phenol lyase, and the core gene pdxJ in the coenzyme pathway is overexpressed, thereby being beneficial to the synthesis of L-tyrosine. Access NO on NCBI is: NC _012892REGION: completion (2567591.. 2568322).
3. Selection of enzymes in a four-enzyme coupled catalytic reaction
(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, and hydrogen peroxide is produced in the process to further oxidize the pyruvic acid and destroy enzymes in bacteria. In general, lactate dehydrogenase whose coenzyme is NAD (NADP) tends to synthesize lactate using pyruvate as a substrate, but if lactate is excessive, the lactate dehydrogenase removes hydrogen from lactate to produce pyruvate. The L-lactate dehydrogenase gene llldh (amino acid sequence is WP _003131075.1) was obtained from Lactococcus lactis ATCC 19257.
(2) Selection of tyrosine phenol lyase
Tyrosine phenol lyase (Tyrosine phenol lyase, TPL, E.C.4.1.99.2), also known as beta-tyrosinase, can catalyze L-Tyrosine to perform beta-elimination reaction to generate phenol, pyruvic acid and ammonia. The reaction is reversible, and phenol, pyruvic acid and ammonia can generate L-tyrosine under the catalysis of tyrosine phenol lyase. The invention clones tyrosine phenol lyase genes ehtpl from Erwinia herbicoloa ATCC 214344 respectively, and the amino acid sequence of the genes is P31011.2.
(3) Selection of tyrosine ammonia lyase
Tyrosine Ammonia Lyase (Tyrosine Ammonia Lyase) can produce corresponding p-hydroxycinnamic acid and p-hydroxycinnamic acid by non-oxidative deamination of Tyrosine, dopa, etc. The tyrosine ammonia lyase rstal (amino acid sequence is WP-011339422.1) derived from Rhodobacter sphaeroides ATCC BAA-808 was selected in the present invention.
(4) 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 invention obtains a water-producing NADH oxidase gene llox (with an amino acid sequence of WP _032950924.1) from Lactococcus lactis ATCC19257 respectively, and an expression product is used for NAD regeneration.
4. Construction of Co-expression System and culture of cells
The tyrosine ammonia lyase, the tyrosine phenol lyase, the L-lactate dehydrogenase and the NADH oxidase which are selected above are subjected to four-enzyme co-expression.
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 coli multigene co-expression), the method is constructed by adopting the method of Liu-oriented epitaxy (2016, Shanghai medical industry research institute, doctor paper) for 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 each gene. Theoretically, since each gene is preceded by T7 and RBS, the expression intensity of the gene is not greatly affected by the ranking. Adopting two plasmids, namely pACYCDue-1 plasmid and pETDue-1 plasmid, wherein each plasmid contains two genes, simultaneously carrying out heat transfer on the constructed plasmids, introducing the plasmids into escherichia coli competent cells, coating the escherichia coli competent cells on a solid plate with double antibodies (Kan and Cm), and screening to obtain the plasmidAnd obtaining the recombinant escherichia coli by the positive transformant. 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 ℃.
4. Production of p-hydroxy cinnamic acid by whole cell transformation
The system for cell transformation production is as follows: the wet weight of the cells is 1-200g/L, the concentration of phenol is 1-200g/L, the concentration of L-lactic acid is 1-200g/L, the pH value is 6.0-9.0, and the concentration of ammonium ions is 1-30 g/L; reacting at 15-40 deg.c for 1-48 hr. And (4) determining the yield of the p-hydroxycinnamic acid by liquid chromatography after the conversion is finished. The solubility of p-hydroxycinnamic acid is low, and a large amount of acid solution is needed to be used for determination after complete dissolution.
5. Detection analysis of samples
Quantitative analysis of hydroxycinnamic acid: the conversion solution is detected and analyzed by a Perkinelmer Series 200 high performance liquid chromatograph, and is matched with an ultraviolet detector. The chromatographic conditions are as follows: the mobile phase is methanol-0.1% formic acid water (40:60), and the detection wavelength is 280nm, with a flow rate of 1ml/min, a column temperature of 30 deg.C, a sample volume of 20 μ l, and a chromatography column (4.6 × 250mm, 5 μm) of Megres C18 Hanbang.
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
The hpaD and mhpB on Escherichia coli BL21(DE3) were single or double knocked out according to the methods described in the document Large scale identification of an effective CRISPR/Cas-based Multi gene editing protocol in Escherichia coli. Microbiological Cell industries, 2017,16(1): 68. The plasmid for gene knockout is pCasRed, pCRISPR-gDNA (hpaD sgRNA) and a homology arm (hpaD donor) are introduced into Escherichia coli BL21(DE3) together, Cas9/sgRNA induces a host to generate double-strand break at an hpaD gene site, recombinase Red integrates the hpaD donor to the hpaD gene, gene knockout is realized, and sequencing verification is carried out. The hpaD sgRNA, hpaD donor, mhpB sgRNA, mhpB donor are shown in sequence tables SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12 and SEQ ID NO 13 respectively. mhpB was knocked out in the same way.
A solution having a pH of 8, 2g/L of phenol or p-hydroxycinnamic acid and a wet cell mass of 100g/L was prepared, and the concentration was measured after leaving at 35 ℃ for 10 hours. The remaining amounts of phenol and p-hydroxycinnamic acid in the reaction system are shown in Table 1.
TABLE 1 residual concentrations of different strains after substrate and product decomposition
| Bacterial strains | Phenol g/L | P-hydroxy cinnamic acid g/L |
| Escherichia coli BL21(DE3) | 1.1 | 0.8 |
| Escherichia coli BL21(ΔhpaDΔmhpB,DE3) | 1.8 | 1.9 |
| Escherichia coli BL21(ΔhpaD,DE3) | 1.4 | 1.5 |
| Escherichia coli BL21(ΔmhpB,DE3) | 1.5 | 1.4 |
It is clear that Escherichia coli BL21(Δ hpaD Δ mhpB, DE3) works best and is named Escherichia coli HM.
Example 2
Constructing recombinant escherichia coli: genes encoding tyrosine phenol lyase, tyrosine ammonia lyase, NADH oxidase and L-lactate dehydrogenase were first ligated to plasmids pETDuet-1 or pACYCDuet-1, respectively. Obtaining two kinds of double-gene co-expression recombinant plasmids, transforming Escherichia coli HM by the two kinds of plasmids, and screening by using chloramphenicol and ampicillin plates to obtain positive transformants, thus 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 ℃.
After the induction expression of the recombinant escherichia coli is finished, collecting thalli, and in a reaction volume of 100ml, the wet weight of cells is 20g/L, the concentration of phenol is 10g/L, the concentration of L-lactic acid is 10g/L, the pH value is 8.0, and the concentration of ammonium ions is 30 g/L; the reaction was carried out at 35 ℃ for 12 hours. And (4) determining the yield of the p-hydroxycinnamic acid by liquid chromatography after the conversion is finished.
TABLE 2 comparison of various recombinant bacteria
| Recombinant bacterium | P-hydroxy cinnamic acid g/L |
| Escherichia coli HM/pETDuet-1-ehtpl-llldh+pACYCDuet-1-rstal-llnox | 6.7 |
| Escherichia coli HM/pETDuet-1-ehtpl-rstal+pACYCDuet-1-llldh-llnox | 6.9 |
| Escherichia coli HM/pETDuet-1-ehtpl-llnox+pACYCDuet-1-rstal-llldh | 8.1 |
| Escherichia coli HM/pETDuet-1-rstal-llldh+pACYCDuet-1-ehtpl-llnox | 5.0 |
| Escherichia coli HM/pETDuet-1-rstal-llnox+pACYCDuet-1-ehtpl-llldh | 6.6 |
| Escherichia coli HM/pETDuet-1-llnox-llldh+pACYCDuet-1-rstal-ehtpl | 6.1 |
Example 3
The medium expression strength constitutive Promoter (PG) in front of the corresponding gene of Escherichia coli 3-glyceraldehyde phosphate dehydrogenase gene (gpdA) is increased on Escherichia coli HM genome 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, and the sequence is shown as SEQ ID NO: 9.
When the expression of the gene lldP is enhanced, an Escherichia coli HM 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 HM 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 the expression of the enhanced gene hpaX is carried out, a method similar to the expression of the enhanced gene lldP is adopted, an upstream sequence, a promoter sequence and a downstream sequence are amplified firstly, and a primer is designed and fused into an expression frame containing a gpdA promoter. Then after being transformed into Escherichia coli HM together with plasmids pCasRed and pCRISPR-gDNA (containing hpaX sgRNA), Cas9/sgRNA induces double strand break of host at hpaX gene locus, recombinase Red integrates gpdA promoter in front of hpaX gene, and sequencing verifies
When enhancing the expression of the gene mhpT, a method similar to the method for enhancing the expression of the gene lldP is adopted, an upstream sequence, a promoter and a downstream sequence are amplified, and a primer is designed and fused into an expression frame containing a gpdA promoter. Then after being transformed into Escherichia coli HM together with plasmids pCasRed and pCRISPR-gDNA (containing mhpT sgRNA), Cas9/sgRNA induces double strand break of host at mhpT gene site, recombinase Red integrates gpdA promoter in front of mhpT, and sequencing verifies
The following table is the corresponding index of the primer name and sequence number in the sequence listing.
TABLE 3 comparison of primer names with sequence Listing numbers
| Name (R) | Number in sequence listing |
| lldP sgRNA | SEQ ID NO:1 |
| hpaX sgRNA | SEQ ID NO:14 |
| mhpT sgRNA | SEQ ID NO:15 |
| lldP-FF | SEQ ID NO:3 |
| lldP-FR | SEQ ID NO:4 |
| lldP-gpdA-F | SEQ ID NO:5 |
| lldP-gpdA-R | SEQ ID NO:6 |
| lldP-RF | SEQ ID NO:7 |
| lldP-RR | SEQ ID NO:8 |
Expression was induced according to the method described in example 2, and various types of cells were collected for transformation analysis, and the results are shown in Table 4. The whole cell transformation system in the transformation system is as follows: the wet weight of the cells is 10g/L, the L-lactic acid is 200g/L, the phenol is 10g/L, the pH 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 4 comparison of transformation results
The most effective Escherichia coli HM (PG-lldP, PG-hpaX, PG-mhpT) was named Escherichia coli PXT.
Example 4
According to the method of example 3, the medium expression strength constitutive Promoter (PG) in front of nadA and pdxJ genes in Escherichia coli PXT is increased, and the sequence is shown as SEQ ID NO: 9. The plasmid is then introduced.
When the expression of gene nadA is enhanced, the method similar to the method for enhancing the expression of gene lldP in example 3 is adopted, the upstream, promoter and downstream sequences are amplified firstly, and the primer fusion is designed to be an expression frame containing the gpdA promoter. Then after being transformed into Escherichia coli PXT together with plasmids pCasRed and pCRISPR-gDNA (containing nadA-gRNA), Cas9/sgRNA induces double strand break of host at nadA gene site, recombinase Red integrates gpdA promoter in front of nadA gene, and sequencing verifies
When the expression of the gene pdxJ is enhanced, the method similar to the method for enhancing the expression of the gene lldP in example 3 is adopted, the upstream, promoter and downstream sequences are amplified firstly, and a primer fusion is designed to be an expression frame containing a gpdA promoter. Then after being transformed into Escherichia coli PXT together with plasmids pCasRed and pCRISPR-gDNA (containing pdxJ-gRNA), Cas9/sgRNA induces double strand break of host at pdxJ gene site, recombinase Red integrates gpdA promoter in front of pdxJ gene, and sequencing verifies
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
| Name (R) | Number in sequence listing |
| nadA sgRNA | SEQ ID NO:2 |
| pdxJ sgRNA | 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 2, and various types of cells were collected for transformation analysis, and the results are shown in Table 6. The whole cell transformation system in the transformation system is as follows: the wet weight of the cells is 20g/L, the L-lactic acid is 200g/L, the phenol 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 6 comparison of transformation results
The best Escherichia coli PXT (PG-nadA, PG-pdxJ) was named Escherichia coli NJ.
Example 6
According to the inducible expression method of the embodiment 2, thalli are collected after the induction expression of Escherichia coli NJ/pETDuet-1-ehtpl-llnox + pACYCDuet-1-rstal-llldh is completed, and in a 100ml reaction system, the wet weight of the cells is 1g/L, the L-lactic acid is 1g/L, the phenol is 1g/L, the pH value is 6.0, 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 concentration of p-hydroxycinnamic acid was 81 mg/L.
Example 7
According to the inducible expression method described in example 2, after the strains in Table 7 are induced to express, collecting thalli, and putting the thalli in a 100ml reaction system, wherein the wet weight of cells is 200g/L, the L-lactic acid is 200g/L, the phenol is 200g/L, the pH is 8.5, the temperature is 40 ℃, and the rotating speed of a shaking table is 250 revolutions per minute; the conversion time was 48 hours. The result was determined after the precipitate was completely diluted and dissolved.
TABLE 7 comparison of transformation results
| Bacterial strains | P-hydroxy cinnamic acid g/L |
| Escherichia coli NJ/pETDuet-1-ehtpl-llldh+pACYCDuet-1-rstal-llnox | 344.9 |
| Escherichia coli NJ/pETDuet-1-ehtpl-rstal+pACYCDuet-1-llldh-llnox | 338.3 |
| Escherichia coli NJ/pETDuet-1-ehtpl-llnox+pACYCDuet-1-rstal-llldh | 376.9 |
| Escherichia coli NJ/pETDuet-1-rstal-llldh+pACYCDuet-1-ehtpl-llnox | 337.4 |
| Escherichia coli NJ/pETDuet-1-rstal-llnox+pACYCDuet-1-ehtpl-llldh | 351.4 |
| Escherichia coli NJ/pETDuet-1-llnox-llldh+pACYCDuet-1-rstal-ehtpl | 346.3 |
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
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Claims (7)
1. A method for producing p-hydroxy cinnamic acid is characterized in that recombinant Escherichia coli is used for producing the p-hydroxy cinnamic acid by taking phenol as a substrate; wherein the recombinant escherichia coli expresses exogenous tyrosine phenol lyase, tyrosine ammonia lyase, L-lactate dehydrogenase and NADH oxidase at the same time, and genes related to decomposition of phenolic compounds are knocked out on the basis of host escherichia coli; the tyrosine phenol lyase and the L-lactate dehydrogenase are co-expressed by a pETDuet-1 vector; the tyrosine ammonia lyase and the NADH oxidase are co-expressed by a pACYCDue-1 vector; the amino acid sequence of the L-lactate dehydrogenase is an access NO of WP-003131075.1 sequence on NCBI, the amino acid sequence of the NADH oxidase is an access NO of WP-032950924.1 sequence on NCBI, the amino acid sequence of the tyrosine phenol lyase is an access NO of P31011.2 on NCBI, and the amino acid sequence of the tyrosine ammonia lyase is a sequence of the access NO of WP-011339422.1 on NCBI; the genes related to phenolic compound decomposition are any one or the combination of hpaD and mhpB; the nucleotide sequences of genes hpaD and mhpB related to phenolic compound decomposition are respectively that access NO on NCBI is: NC _012892REGION: 4505585..4506436 and NC _012892REGION:339806.. 340750.
2. The method of claim 1, wherein the recombinant Escherichia coli further enhances expression of one or more of a lactate transporter gene, a phenol transporter gene, an NAD synthetic gene, and a pyridoxal phosphate synthetic gene; the gene with enhanced expression is any one or more of a lactate transport gene lldP, a phenol transport gene hpaX, a phenol transport gene mhpT, an NAD synthetic gene nadA and a pyridoxal phosphate synthetic gene pdxJ; the access NO of lldP at NCBI is: NC _012892REGION 3646638.. 3648293; hpaX is; NC _012892REGION 4502025.. 4503401; mhpT NC-012892 REGION 344788.. 345999; nadA is NC _012892REGION 740487.. 741530; pdxJ is NC _012892REGION: 2567591.. 2568322.
3. The method of claim 2, wherein the enhanced expression is achieved by pre-increasing a constitutive promoter in a gene to be enhanced on the genome of E.coli in the host.
4. The method according to claim 1, wherein the host E.coli is Escherichia coli BL21(DE 3).
5. The method of claim 1, wherein said producing is by whole cell transformation; in the whole cell conversion production system, the wet weight of cells is 1-200g/L, the concentration of phenol is 1-200g/L, the concentration of L-lactic acid is 1-200g/L, the pH value is 6.0-9.0, and the concentration of ammonium ions is 1-30 g/L; reacting at 15-40 deg.c for 1-48 hr.
6. Use of the process according to any one of claims 1 to 5 for the preparation of p-hydroxycinnamic acid.
7. A recombinant bacterium, comprising: on the basis of an escherichia coli host with hpaD and mhpB knocked out, a lactate transport gene lldP, a phenol transport gene hpaX, a phenol transport gene mhpT, an NAD synthetic gene nadA and a pyridoxal phosphate synthetic gene pdxJ are intensively expressed, and tyrosine phenol lyase, tyrosine ammonia lyase, L-lactate dehydrogenase and NADH oxidase are simultaneously expressed; the tyrosine phenol lyase and the L-lactate dehydrogenase are co-expressed by a pETDuet-1 vector; the tyrosine ammonia lyase and the NADH oxidase are co-expressed by a pACYCDue-1 vector; the amino acid sequence of the L-lactate dehydrogenase is an access NO of WP-003131075.1 sequence on NCBI, the amino acid sequence of the NADH oxidase is an access NO of WP-032950924.1 sequence on NCBI, the amino acid sequence of the tyrosine phenol lyase is an access NO of P31011.2 on NCBI, and the amino acid sequence of the tyrosine ammonia lyase is a sequence of the access NO of WP-011339422.1 on NCBI; the nucleotide sequences of the phenolic substance decomposition genes hpaD and mhpB are respectively that the accession NO on NCBI is: NC _012892REGION: 4505585..4506436 and NC _012892REGION:339806.. 340750; the access NO of lldP at NCBI is: NC _012892REGION 3646638.. 3648293; hpaX is; NC _012892REGION 4502025.. 4503401; mhpT NC-012892 REGION 344788.. 345999; nadA is NC _012892REGION 740487.. 741530; pdxJ is NC _012892REGION: 2567591.. 2568322.
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