CN112111442B - Coli having integrated amino acid oxidase and glucose dehydrogenase - Google Patents
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Abstract
The invention discloses escherichia coli integrated with amino acid oxidase and glucose dehydrogenase, belonging to the technical field of bioengineering. The invention integrates the L-amino acid oxidase and glucose dehydrogenase genes into the chromosome of the escherichia coli by adopting a synthetic biology technical means in a multi-site and multi-copy way, so that the escherichia coli has better growth characteristics, and the final quantity of the strain cultured in a high density can be increased by more than 30% compared with that of the original strain. The engineering bacteria are applied to the production of the salvianic acid A, the yield of the salvianic acid A can reach 87.46mM, and meanwhile, the engineering bacteria can also well produce the hydroxytyrosol, and the yield of the hydroxytyrosol can reach 54.43mM.
Description
Technical Field
The invention relates to escherichia coli integrated with amino acid oxidase and glucose dehydrogenase, belonging to the technical field of bioengineering.
Background
L-amino acid oxidase is widely found in bacteria, fungi, mammalian cells, snake venom, insect toxins, and algae (L-amino acid oxidase as biocatalyst: astream to farappl. Microbiol. Biotechnol.2013, 97:9323-41). Most L-amino acid oxidases oxidize amino acids to produce hydrogen peroxide which is toxic to themselves or cells in the environment. There is also a class of hydrogen peroxide-free amino acid oxidase in nature, which is derived from Proteus sp, providencia sp, morganella sp, etc. In these bacterial cells, it is reported by the literature that, after the amino acid is dehydrogenated to the corresponding keto acid, electrons are transferred to cytochrome oxidase through the membrane respiratory chain and finally combined with oxygen to produce water, and these enzymes are also called L-amino acid dehydrogenase or amino acid deaminase.
Glucose dehydrogenase is a member of the short-chain alcohol dehydrogenase family, found on the coenzyme NAD (P) + And the like, can catalyze the conversion of beta-D-glucose into D-gluconolactone, and the D-gluconolactone generated by the reaction can spontaneously further hydrolyze into gluconic acid. Currently, glucose dehydrogenases can be classified into 3 types according to the kind of the coenzyme: (1) NAD (P) + Microorganisms containing such enzymes are Gluconobacter suboxydans, haloferax mediterranei, bacillus cereus, bacillus megaterium, bacillus subtilis, etc., depending on the type (EC 1.1.1.47); (2) FAD (FAD) + The dependent type (EC 1.1.99.10), the microorganism containing the enzyme has Aspergillus niger, burkholderia cepacia and the like; (3) Examples of the PQQ-dependent type (EC 1.1.5.2) include Pseudomonas fluorescens, acinetobacter calcoaceticus, mythylobacterium extorquens and the like. FAD (FAD) + The dependent hydrogen peroxide-generating cell damage, PQQ dependent is less efficient, whereas NAD (P) + The high stability of the dependent glucose dehydrogenase activity generally provides coenzyme regeneration for whole cell catalytic reactions.
The engineering strain constructed by using L-amino acid oxidase or glucose dehydrogenase at present usually adopts a plasmid expression system, and the plasmid expression system needs antibiotics to maintain the stability of plasmids and inducers to induce the expression of genes, expensive inducers increase the production cost, and the addition of antibiotics and inducers also increase the complexity of production. Therefore, in order to span these defects, it is necessary to construct a chromosome-integrated engineering strain (Techniques for chromosomal integration and expression optimization in Escherichia coll. Biotechnol Bioeng 2018,115 (10), 2467-2478).
The invention relates to L-amino acid oxidase and NAD (P) -based enzyme + The dependent glucose dehydrogenase is integrated on the most commonly used protein expression host escherichia coli, so that the growth characteristics of the escherichia coli are changed, and the escherichia coli has stronger environment adaptability and better application characteristics. The strain has important industrial application prospect.
Disclosure of Invention
The invention integrates the L-amino acid oxidase and glucose dehydrogenase which do not produce hydrogen peroxide into the most commonly used protein expression host escherichia coli, thereby changing the growth characteristics of the escherichia coli and leading the escherichia coli to have stronger environment adaptability and better application characteristics. The strain has important industrial application prospect.
The invention provides an escherichia coli recombinant bacterium integrated with amino acid oxidase and glucose dehydrogenase. Meanwhile, the invention aims to solve the technical problems of construction and application of the strain.
One of the purposes of the invention is to provide a genetically engineered bacterium integrating amino acid oxidase and glucose dehydrogenase, which is escherichia coli utilizing chromosome integration technology to express L-amino acid oxidase and glucose dehydrogenase in a multi-site and multi-copy constitutive mode, so that the escherichia coli has better growth characteristics.
In one embodiment, the L-amino acid oxidase is an L-amino acid oxidase from Proteus mirabilis ATCC 29906 (abbreviated as pmaao), an L-amino acid oxidase from Cosenzaea myxofaciens ATCC 19692 (abbreviated as cmaao). The glucose dehydrogenase is glucose dehydrogenase from Gluconobacter suboxydans ATCC 621 (abbreviated as gsgdh), from Haloferax mediterranei ATCC 33500 (abbreviated as hmgdh), or from Bacillus subtilis ATCC 13952 (abbreviated as bsgdh).
In one embodiment, the amino acid sequence of the L-amino acid oxidase is as set forth in NCBI accession No. WP_004244224.1, OAT 30925.1. The amino acid sequence of glucose dehydrogenase is shown as the sequence shown in NCBI with access NO WP_041112217.1, AFK18021.2 and AKC 45923.1.
In one embodiment, the nucleotide sequence of the L-amino acid oxidase, e.g., an accession No. at NCBI, is: NZ_GG668576REGION 1350390, 1351805, LXEN01000066REGION 20563, 21963. The nucleotide sequence of glucose dehydrogenase, e.g., accession NO on NCBI is: NZ_ CP004373.1REGION:2428736..2429536, CP001868.2REGION:262147..262932, CP011115.1REGION:445341.. 446126.
In one embodiment, the E.coli host is E.coli BL21, E.coli JM109, E.coli DH 5. Alpha. Or E.coli Top10.
In one embodiment, the genetically engineered bacterium is integrated at the dkgB, eaeH, lysO, bluF, tam, glsB, rcs, mntH, nupG, pitB, rbsA or pgi locus; the dkgB is located at chromosome 229167 ～ 229970; the eaeH is located at chromosome 314357 ～ 315244; the lysO is located at chromosome 913958 ～ 914857; the blu f is located at chromosome 1214264 ～ 1215475; the tam is located at chromosome 1607346 ～ 1608104; the glsB is located at chromosome 1612325 ～ 1613251; the rcs is located at chromosome 2316177 ～ 2316827; the mntH is located at chromosome 2511468 ～ 2512706; the nupG is located at chromosome 3105714 ～ 3106970; the pitB is located at chromosome 3134872 ～ 3136371; the rbsA is located at chromosome 3933778 ～ 3935283; the pgi is located at chromosome 4233758 ～ 4235407.
In one embodiment, the genetically engineered bacterium integrates 9 copies of pmaao and gsgdh at glsB on the genome, 6 copies of pmaao and gsgdh at nupG and 9 copies of pmaao and gsgdh at pitB.
In one embodiment, the genetically engineered bacterium also expresses other genes or enzymes of interest.
In one embodiment, the genetically engineered bacterium further expresses lactate dehydrogenase, amino acid decarboxylase, and alcohol dehydrogenase.
In one embodiment, the genetically engineered bacterium expresses lactate dehydrogenase via pET20B, pColdII, pETDuet-1 or pACYCDuet-1. The amino acid decarboxylase and alcohol dehydrogenase are expressed by pcoldi.
The second object of the present invention is to provide a method for constructing the genetically engineered bacterium, wherein genes encoding L-amino acid oxidase and glucose dehydrogenase are integrated on the chromosome of E.coli BL21 (DE 3).
In one embodiment, the integration is at any one of the following: dkgB, eaeH, lysO, bluF, tam, glsB, rcs, mntH, nupG, pitB, rbsA, pgi.
In one embodiment, the method comprises the steps of:
(1) Constructing an amino acid oxidase and glucose dehydrogenase gene integration frame; the integration frame is as follows: an integration site left arm, an amino acid oxidase gene, a glucose dehydrogenase gene, an integration site right arm;
(2) Transforming the amino acid oxidase and glucose dehydrogenase gene integration frame constructed in the step (1) into an E.coli electrotransformation competent cell; the E.coli is E.coli BL21, E.coli JM109, E.coli DH5 alpha or E.coli Top10.
In one embodiment of the present invention, a ribosome binding site is also contained between the amino acid oxidase gene and the glucose dehydrogenase gene.
The third object of the present invention is to provide an application of any of the above genetically engineered bacteria in the field of fermentation.
In one embodiment, the use is as a cell catalyst to participate in whole cell catalytic reactions.
In one embodiment, the use is the production of tanshinol using L-dopa as a substrate.
In one embodiment, the use is the production of hydroxytyrosol using L-dopa as a substrate.
The beneficial effects are that: the escherichia coli engineering strain constructed by the invention integrates the target gene into a chromosome, and antibiotics are not required to be added to maintain the plasmid stability of the strain, so that the operation steps and the production cost are reduced. And the strain growth after integrating the amino acid oxidase and glucose dehydrogenase is higher than that of the original escherichia coli strain, and the final amount of the strain cultured in high density is 31.82% higher than that of the original strain. The engineering bacteria are applied to the production of the salvianic acid A, the yield of the salvianic acid A can reach 87.46mM, and meanwhile, the engineering bacteria can also well produce the hydroxytyrosol, and the yield of the hydroxytyrosol can reach 54.43mM.
Detailed Description
The strain and plasmid related to the invention:
proteus mirabilis ATCC 29906, cosenzaea myxofaciens ATCC 19692,Gluconobacter suboxydans ATCC 621, haloferax mediterranei ATCC 33500, bacillus subtilis ATCC 13952 available from the American type culture Collection ATCC. pET-20B, pKD from Novagen, the PCP20 plasmid and T-Vector pMD109 (Simple) from TaKaRa were used for construction of the chromosomal integration plasmid.
The invention will be further illustrated with reference to specific examples, but the invention is not limited to the examples.
Example 1: screening of chromosomal integration sites
1. The chromosome of E.coli BL21 (DE 3) having a total sequence length of 4.6Mbp was divided into six regions, and two sites were selected for integration of L-amino acid oxidase and glucose dehydrogenase in each region, and then activity measurement was performed on the strain having the L-amino acid oxidase and glucose dehydrogenase integrated at each site, thereby determining the optimal integration site. The regions and sites grouped are 0 to 0.76Mbp (dkgB, eaeH), 0.76 to 1.53Mbp (lysO, blu), 1.53 to 2.28Mbp (tam, glsB), 2.28 to 3.04Mbp (rcs, mntH), 3.04 to 3.8Mbp (nupG, pitB), 3.8 to 4.6Mbp (rbsA, pgi), respectively.
(1) Extraction of plasmids
Plasmid extraction was performed according to the instructions on the plasmid miniprep kit from Shanghai JieRui Corp.
(2) Construction of pIn-LA-cat-RA plasmid
The primers used in the construction are listed in Table 1. The chloramphenicol resistance gene was amplified from the pKD3 plasmid using the primers ApaI-EcoRV-cat (F) and cat-AflII-AvrII-XbaI (R). The amplified chloramphenicol resistance gene was ligated into a T-Vector pMD19 (Simple) Vector, and the plasmid was designated as pIn-cat.
Using the Escherichia coli BL (DE 3) genome as a template, primers ApaI-NarI-LA1 (F) and LA1-EcoRV (R), apaI-NarI-LA2 (F) and LA2-EcoRV (R), apaI-NarI-LA3 (F) and LA3-EcoRV (R), apaI-NarI-LA4 (F) and LA4-EcoRV (R), apaI-NarI-LA5 (F) and LA5-EcoRV (R), apaI-NarI-LA6 (F) and LA6-EcoRV (R), apaI-NarI-LA7 (F) and LA7-EcoRV (R) were used, respectively, apaI-NarI-LA8 (F) and LA8-EcoRV (R), apaI-NarI-LA9 (F) and LA9-EcoRV (R), apaI-NarI-LA10 (F) and LA10-EcoRV (R), apaI-NarI-LA11 (F) and LA11-EcoRV (R), apaI-NarI-LA12 (F) and LA12-EcoRV (R) were amplified to obtain gene fragments of 1000bp left of each integration site, and the obtained gene fragments were designated as LA1, LA2, LA3, LA4, LA5, LA6, LA7, LA8, LA9, LA10, LA11 and LA12, respectively. With Escherichia coli BL (DE 3) genome as template, primers AvrII-RA1 (F) and RA1-NarI-XbaI (R), avrII-RA2 (F) and RA2-NarI-XbaI (R), avrII-RA3 (F) and RA3-NarI-XbaI (R), avrII-RA4 (F) and RA4-NarI-XbaI (R), avrII-RA5 (F) and RA5-NarI-XbaI (R), avrII-RA6 (F) and RA6-NarI-XbaI (R), avrII-RA7 (F) and RA7-NarI-XbaI (R), avrII-RA8 (F) and RA8-NarI-XbaI (R), avrII-RA9 (F) and RA9-NarI (R), avrII-RA5 (F) and RA5-NarI-XbaI (R), and RA 5-NarI-RA 6 (F) were amplified as the respective RA1, RA2, RA11 and RA12 (R) and RA11 were amplified, respectively. The left homology arms LA 1-12 and the vector pIn-cat were digested with ApaI and EcoRV endonucleases, and then the digested LA1-12 were ligated to the digested vector pIn-cat, respectively, and the resulting plasmids were designated pIn-LA1-cat, pIn-LA2-cat, pIn-LA3-cat, pIn-LA4-cat, pIn-LA5-cat, pIn-LA6-cat, pIn-LA7-cat, pIn-LA8-cat, pIn-LA9-cat, pIn-LA10-cat, pIn-LA11-cat, pIn-LA12-cat, respectively. The following homology arms RA were ligated between the AvrII and XbaI sites of vectors pIn-LA1-cat, pIn-LA2-cat, pIn-LA3-cat, pIn-LA4-cat, pIn-LA5-cat, pIn-LA6-cat, pIn-LA7-cat, pIn-LA8-cat, pIn-LA9-cat, pIn-LA10-cat, pIn-LA11-cat, pIn-LA12-cat, respectively, and the obtained plasmids were named pIn-LA1-cat-RA1, pIn-LA2-cat-RA2, pIn-LA3-cat-RA3, pIn-LA4-cat-RA4, pIn-LA5-cat-RA5, pIn-LA6-cat-RA6, pIn-LA7-cat 7-LA 8-RA 8, 3575-RA 8-RA 35-RA 10-RA 35-75, 3512-RA 10-RA 11-cat, and 3575-RA 10-RA 12-RA 11-cat, respectively.
The enzyme digestion system in the experimental operation process is as follows: 10 Xcut buffer 3. Mu.L, DNA 4. Mu.L, endonucleases 0.5. Mu.L each, sterile water 2. Mu.L total 30. Mu.L. Cutting in a water bath at 37 ℃ for 1h. The DNA fragments were cloned onto plasmids and transformed into e.coli DH5 a competent cells. The connection system is as follows: 10X DNA ligase buffer 2.5.5. Mu.L of DNA fragment 8. Mu.L, 2. Mu.L of vector DNA, 1. Mu.L of T4 DNA library, and a total of 25. Mu.L of sterile water 11.5. Mu.L. And the connection is carried out for 12 to 16 hours in a water bath at the temperature of 16 ℃.
TABLE 1 primer names and sequences
2. Acquisition of integration fragments at different sites
(1) Extraction of plasmids
Plasmid extraction was performed according to the instructions on the plasmid miniprep kit from Shanghai JieRui Corp.
(2) Construction of the integrative plasmid
The primers used in the construction are listed in Table 2. The L-amino acid oxidase pmaao was amplified using primers EcoRV-pmaao (F) and pmaao-SacI (R), and then ligated at EcoRV and SacI sites of the plasmid pET-20B, and the resulting plasmid was designated pET-pmaao. The glucose dehydrogenase gsgdh with RBS was amplified using the primers SalI-RBS-gsgdh (F) and gsgdh-NotI (R), and then ligated at the SalI and NotI sites of the plasmid pET-pmao, the resulting plasmid was designated pET-pmao-gsgdh.
(3) Acquisition of integration fragments
The primers used in the construction are listed in Table 2. An integrated fragment containing the left arm, right arm, kana antibiotic gene, pmaao gene and gsgdh gene was obtained, pET-pmaao-gsgdh was amplified using primers AflII-T7-pmaao-gsgdh (F) and T7-pmaao-gsgdh-avrII (R) to obtain pmaao containing the T7 promoter, ribosome binding site and T7 terminator (sequence as NCBI Reference Sequence: WP_ 004244224.1) and gsgdh (sequences such as NCBI Reference Sequence:WP_ 041112217.1) fragments T7-rbs-pmaao-rbs-gsgdh-Tm, then ligating T7-rbs-pmaao-rbs-gsgdh-Tm into the AflII and AvrII sites of plasmids pIn-LA1-cat-RA1, pIn-LA2-cat-RA2, pIn-LA3-cat-RA3, pIn-LA4-cat-RA4, pIn-LA5-cat-RA5, pIn-LA6-cat-RA6, pIn-LA7-cat-RA7, pIn-LA8-cat-RA8, pIn-LA9-cat-RA9, pIn-LA10-cat-RA10, pIn-LA11-cat-RA11, pIn-LA12-cat-RA12, respectively. The resulting plasmids were designated as pIn-LA 1-cat-pmao-gsdh-RA 1, pIn-LA 2-cat-pmao-gsdh-RA 2, pIn-LA 3-cat-pmao-gsdh-RA 3, pIn-LA 4-cat-pmao-gsdh-RA 4, pIn-LA 5-cat-pmao-gsdh-RA 5, pIn-LA 6-cat-pmao-gsdh-RA 6, pIn-LA 7-cat-pmao-gsdh-RA 7, pIn-LA 8-pmao-gsdh-RA 8, pIn-LA 9-cat-pmao-gsgh-RA 9, pIn-LA 10-cat-pmao-gsdh-RA 10-RA 11-pallet-RA 12-cat-11-pallet-RA.
TABLE 2 primer names and sequences
3. Integration of L-amino acid oxidase and glucose dehydrogenase
The restriction enzyme NarI was used to treat plasmids pIn-LA1-cat-pmaao-gsgdh-RA1, pIn-LA2-cat-pmaao-gsgdh-RA2, pIn-LA3-cat-pmaao-gsgdh-RA3, pIn-LA 4-cat-pmaao-gsdh-RA 4, pIn-LA 5-cat-pmaao-gsdh-RA 5, pIn-LA 6-cat-pmaao-gsdh-RA 6, pIn-LA7-cat-pmaao-gsgdh-RA7, pIn-LA 8-cat-pmaao-gsdh-RA 8, pIn-LA 9-cat-pmao-gsgh-RA 9, pIn-cat-gsdh-RA 10, and 5524-pmao-gsdh-RA 10-LA 5611-gsdh-RA 12-by using restriction enzyme NarI, respectively. Then, an integrated fragment containing the left arm, right arm, kana antibiotic gene, pmaao gene and gsgdh gene was obtained. And transferring the integration fragment into prepared escherichia coli electrotransformation competent cells in an electrotransformation mode to perform lambda-Red homologous recombination, and then screening positive strains and performing colony PCR verification. The temperature sensitive pKD46 plasmid was removed by overnight incubation at 37 ℃. The following strains were finally obtained: e.colli BL21 (dkgB), e.colli BL21 (eaeH), e.colli BL21 (lysO), e.colli BL21 (blu), e.colli BL21 (tam), e.colli BL21 (glsB), e.colli BL21 (rcsB), e.colli BL21 (mntH), e.colli BL21 (nupG), e.colli BL21 (pitB), e.colli BL21 (rbsA) and e.colli BL21 (pgi).
4. Determination of the yield of phenylpyruvate and determination of the glucose dehydrogenase Activity of strains with integration of the pmaao and gsgdh genes at different sites
The E.coli BL21 chromosome different sites were integrated into pmaao and gsgdh. The strain was inoculated in a test tube overnight and then in 50mL fresh LB medium at 1% of the inoculum size, and placed in a shaking table for 24 hours at 25℃under 200 revolutions. After 24 hours, the cells were collected after centrifugation at 8000rpm at 4℃for 10min. The whole cell catalytic reaction system takes 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate solution with pH of 7.5 as a buffer solution for the reaction, and comprises the following steps: 50g/L wet cells, 35 ℃, 3g/L L-phenylalanine, conversion time of 5h. After conversion, the phenylpyruvate yield was determined by High Performance Liquid Chromatography (HPLC). Take 20mL, OD 600 The bacterial liquid was 3.5, centrifuged at 8000rpm for 10min at 4℃and the cells were collected. Using 20mL, 35mM Tris-HCl solution with pH8 as buffer solution, using ultrasonic breaker to break cells, centrifuging at 8000rpm for 10min to obtain crude enzyme supernatant, adding appropriate amount of crude enzyme solution into the solution containing 3mM NAD + And 140mM glucose mixtures, the absorbance change was measured at 366nm to determine glucose dehydrogenase activity in units of glucose dehydrogenase activity per ml of cell suspension (see, for specific procedures, paper: glucose dehydrogenase from Bacillus subtilis expressed in Escherichia coli I: purification, characterization and comparison with glucose dehydrogenase from Bacillus megaterium. Biochimica et biophysica acta1991,1076: 298-304), as shown in Table 3.
Table 3 integration of the phenylpyruvate yields, glucose dehydrogenase Activity and cell wet weight of the strains at various sites
| Strain | Chromosome position (bp) | Phenylpyruvic acid (g/L) | gsgdh Activity (U/mL) | Thallus wet weight (g/L) |
| E.coli BL21(dkgB) | 229167～229970 | 0.16 | 1.54 | 9.13 |
| E.coli BL21(eaeH) | 314357～315244 | 0.07 | 0.58 | 9.84 |
| E.coli BL21(lysO) | 913958～914857 | 0.18 | 1.38 | 9.25 |
| E.coli BL21(bluF) | 1214264～1215475 | 0.26 | 3.03 | 9.92 |
| E.coli BL21(tam) | 1607346～1608104 | 0.24 | 3.12 | 9.66 |
| E.coli BL21(glsB) | 1612325～1613251 | 0.31 | 3.42 | 10.35 |
| E.coliBL21(rcsB) | 2316177～2316827 | 0.15 | 2.41 | 9.74 |
| E.coli BL21(mntH) | 2511468～2512706 | 0.27 | 2.83 | 9.76 |
| E.coli BL21(nupG) | 3105714～3106970 | 0.41 | 4.03 | 10.33 |
| E.coli BL21(pitB) | 3134872～3136371 | 0.32 | 2.41 | 10.45 |
| E.coli BL21(rbsA) | 3933778～3935283 | 0.11 | 1.85 | 10.06 |
| E.coli BL21(pgi) | 4233758～4235407 | 0.19 | 2.13 | 9.78 |
| E.coli BL21 | - | - | - | 8.84 |
As shown in Table 3, the strains having L-amino acid oxidase and glucose dehydrogenase integrated at glsB, nupG and pitB in E.coli BL21 were significantly higher in the amount of phenylpyruvate and glucose dehydrogenase activity than the other strains, and the strains having L-amino acid oxidase and glucose dehydrogenase integrated grew better.
5. Preparation of E.coli electrotransformation competence
(1) E.coli BL21 (DE 3) into which pKD46 had been introduced was inoculated in 50mL shake flasks containing 20mL of LB medium and incubated overnight at 25℃at 200 rpm/min.
(2) The culture was inoculated in 50mL of LB medium at 1% of the inoculum size, incubated at 25℃until OD600 reached about 0.15 (about 2-3 hours), 500. Mu.L of 10M L-arabinose was added to the medium, and the incubation was continued at 25℃until OD600 reached about 0.6.
(3) The bacterial solution was transferred to a 50mL pre-chilled centrifuge tube and placed on ice for 5min at 5000rpm/min and centrifuged at 4℃for 5min.
(4) The supernatant was discarded, 10mL of pre-chilled deionized water was added to suspend the cells, and the cells were centrifuged at 5000rpm/min at 4℃for 5min.
(5) The supernatant was discarded, 10mL of pre-chilled 10% glycerol was added to suspend the cells, and the cells were left on ice for 5min at 5000rpm/min and centrifuged at 4℃for 5min and repeated 2 times.
(6) The supernatant was discarded, 1.5mL of pre-chilled 10% glycerol was added, the cells were gently suspended, and 100. Mu.L was then dispensed into 1.5mL centrifuge tubes and stored in a refrigerator at-80℃for further use.
6. Electric conversion
The steps are as follows:
(1) The integrated or knocked-out gene cassettes were added to 100. Mu.L of BL21 (DE 3) competent centrifuge tubes, into which pKD46 was introduced, respectively, and mixed gently for 10min in an ice bath.
(2) The mixed solution is put into an electric rotating cup for electric shock conversion: the voltage of conversion is 1.8KV, and the electric conversion time is 4.5-6.0 ms.
(3) 1mL of SOC culture medium is rapidly added into the electric shock cup, gently blown and evenly mixed, transferred into an aseptic 1.5mL EP tube, and resuscitated for 2-3 hours at a constant temperature of 30 ℃ in a water bath shaker at 200 rpm/min. Inverted culturing at30 deg.c for 16-20 hr.
(4) Positive clones were picked and verified by sequencing.
Example 2
This example shows the integration of different combinations of L-amino acid oxidase (pmaao, cmaao) and glucose dehydrogenase (gsgdh, hmgdh, bsgdh) at the selected optimal integration site nupG, the different combinations of integrated strains were inoculated in test tubes for overnight incubation, then in 50mL fresh LB medium at 1% inoculum size, and placed in shaking tables for incubation at 25℃for 24 hours at 200 revolutions. After 24 hours, the cells were collected after centrifugation at 8000rpm at 4℃for 10min. The whole cell catalytic reaction system takes 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate solution with pH of 7.5 as a buffer solution for the reaction, and comprises the following steps: 50g/L wet cells, 35 ℃, 3g/L L-phenylalanine, conversion time of 5h. After conversion, the phenylpyruvate yield was determined by High Performance Liquid Chromatography (HPLC). Take 20mL, OD 600 The bacterial liquid was 3.5, centrifuged at 8000rpm for 10min at 4℃and the cells were collected. A reaction buffer solution of 20mL, 35mM, and pH8 Tris-HCl was subjected to ultrasonic disruptionCrushing cells with a chopper, centrifuging at 8000rpm for 10min to obtain crude enzyme supernatant, and adding appropriate amount of crude enzyme solution into the supernatant containing 3mM NAD + And 140mM glucose mixtures, the absorbance change was measured at 366nm to determine glucose dehydrogenase activity in units of glucose dehydrogenase activity per ml of cell suspension (see, for specific procedures, paper: glucose dehydrogenase from Bacillus subtilis expressed in Escherichia coli I: purification, characterization and comparison with glucose dehydrogenase from Bacillus megaterium. Biochimica et biophysica acta1991,1076: 298-304), as shown in Table 4.
TABLE 4 phenylpyruvate yield, glucose dehydrogenase Activity and thallus wet weight of different Gene combinations
As shown in Table 4, the amount of phenylpyruvic acid and glucose dehydrogenase activity produced by transformation of the strain integrated with L-amino acid oxidase pmaao and glucose dehydrogenase bsgdh at nupG in E.coli BL21 were significantly higher than those of other strains, and the wet weight of the cells was slightly higher than those of the other strains under the same conditions.
Example 3
This example shows the multi-copy integration of L-amino acid oxidase pmaao and glucose dehydrogenase bsgdh at the selected optimal integration sites glsB, nupG, pitB, and the production of L-phenylalanine to phenylpyruvate and the activity of glucose dehydrogenase after the multi-copy integration of pmaao and bsgdh at the multiple sites were tested.
Genes of different copy numbers were integrated sequentially at the sites selected in example 1, multicopy integration according to FLP recombination method (rapid and reliable strategy for chromosomal integration of gene(s) with multiple copies.scientific Reports 2015,5.). Integration of E.coli BL21 into pmaao and bsgdhThe strain after copy number was inoculated in a test tube for overnight culture, then inoculated in 50mL of fresh LB medium at an inoculum size of 1%, and placed in a shaking table for 24 hours at 25℃under 200 rotations. After 24 hours, the cells were collected after centrifugation at 8000rpm at 4℃for 10min. The whole cell catalytic reaction system takes 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate solution with pH of 7.5 as a buffer solution for the reaction, and comprises the following steps: 10g/L wet cells, 35 ℃, 3g/L L-phenylalanine, conversion time 2h. After conversion, the phenylpyruvate yield was determined by High Performance Liquid Chromatography (HPLC). Take 20mL, OD 600 The bacterial liquid was 3.5, centrifuged at 8000rpm for 10min at 4℃and the cells were collected. Using 20mL, 35mM Tris-HCl solution with pH8 as buffer solution, using ultrasonic breaker to break cells, centrifuging at 8000rpm for 10min to obtain crude enzyme supernatant, adding appropriate amount of crude enzyme solution into the solution containing 3mM NAD + And 140mM glucose mixtures, the absorbance change was measured at 366nm to determine glucose dehydrogenase activity in units of glucose dehydrogenase activity per ml of cell suspension (see, for specific procedures, paper: glucose dehydrogenase from Bacillus subtilis expressed in Escherichia coli: purification, characterization and comparison with glucose dehydrogenase from Bacillus megaterium. Biochimica et biophysica acta1991,1076: 298-304), as shown in Table 5.
TABLE 5 integration of pmaao and bsgdh copy numbers at various positions and the phenylpyruvate yield and glucose dehydrogenase activity of the integrated strain
Note that: "-" indicates that the locus does not integrate the expression pmaao and bsgdh genes.
As shown in Table 5, E.coli BL1.10 integrates 9 copies of pmao and bsgdh at glsB, 6 copies of pmao and bsgdh at nupG, 9 copies of pmao and bsgdh at pitB, and 2.22g/L of phenylpyruvate yield and 25.69U/mL of glucose dehydrogenase activity.
Example 4: comparison of cell growth conditions
The E.coli BL1.10 strain constructed in examples 2 to 3 was inoculated in a test tube for overnight culture, and then inoculated in 50mL of fresh LB, 2 XYT (Shanghai Soy pal) and M9 medium in an inoculum size of 1%, and placed in a shaking table for 24 hours at 25℃and 200 rpm. After 24 hours, the cells were collected after centrifugation at 8000rpm at 4℃for 10min. The wet weights of the cells are shown in Table 6.
TABLE 6 wet weight of cells after shaking culture
| Strain | LB(g/L) | 2×TY(g/L) | M9(g/L) |
| E.coli BL1.10 | 10.22 | 15.85 | 8.52 |
| E.col BL21(DE3) | 9.53 | 14.11 | 7.74 |
As can be seen from Table 6, the growth of the integrated cells was best performed in 2 XTY medium, followed by LB medium and finally M9 medium. The strain after chromosomal integration of the L-amino acid oxidase grew better in each medium than the original strain E.col BL21 (DE 3).
Example 5: comparison of expression of lactate dehydrogenase on different plasmids by BL21 Strain integrating pmaao and bsgdh with the starting Strain
The lactate dehydrogenase gene derived from lactobacillus fermentum was synthesized in its original sequence (GeneBank: MG 581696) and ligated between BamHI and SalI sites of each plasmid shown in Table 7, and then expressed in each cell.
The induction expression method comprises the following steps: transferring recombinant Escherichia coli into LB fermentation medium (peptone 10g/L, yeast powder 5g/L, naCl g/L) at a volume ratio of 2%, and obtaining cell OD 600 After reaching 0.6-0.8, IPTG was added at a final concentration of 0.4mM, and the system was cultured at 20℃for 8 hours with induction, with a concentration of 50mL. After the induction expression was completed, 30mL of the fermentation broth was collected by centrifugation at 8000rpm for 10 minutes at 4 ℃. The collected cells were resuspended in 30mL of sterile water and after disruption, the lactate dehydrogenase crude enzyme activity was determined using phenylpyruvate as substrate according to the methods described in the literature (Identification of a L-Lactate dehydrogenase with, 4-dihydroxyphenylpyruvic reduction activity for l-Danshensu production. Process Biochemistry2018, 72:119-123.). The results of measurement of the lactate dehydrogenase activity in the fermentation broth are shown in Table 7.
TABLE 7 expression of lactate dehydrogenase Activity in different integration pmaao and bsgdh hosts by each plasmid
It is apparent from Table 7 that the strains integrated with pmao and bsgdh, when overexpressing lactate dehydrogenase, had lactate dehydrogenase activities in the fermentation broth of the original strain E.col BL21 (DE 3) that were all lower to a different extent than the strain E.coli BL1.10 integrated with pmao and bsgdh. It was demonstrated that integration of the pmaao and bsgdh genes facilitates expression of other heterologous genes.
Example 6: high density culture comparison
The culture medium composition was as shown in the literature (Simple fed-batch technique for high cell density cultivation of Escherichia coll. Journal of Biotechnology 1995, 39:59-65) and the final cell mass of each strain after fermentation culture at 25℃for 24 hours were as shown in Table 8.
TABLE 8 comparison of cell densities of different integrative strains
| Strain | Thallus wet weight (g/L) |
| E.coli BL1.10 | 136.48 |
| E.col BL21(DE3) | 103.53 |
As is evident from Table 8, the cells were greatly improved as compared with the ordinary medium in example 3 at all times after the high-density culture using the nutrient-rich medium, and the strain integrated with pmaao and bsgdh had a higher wet weight than E.col BL21 (DE 3) strain.
Example 7: comparison of different E.coli starting strains
Multicopy pmao and bsgdh integration was performed in E.coli BL21, JM109, DH 5. Alpha., top10, respectively, i.e., the following integration was performed in E.coli BL21, JM109, DH 5. Alpha., top10, respectively: 9 copies of pmaao and bsgdh were integrated at chromosome glsB, 6 copies of pmaao and bsgdh were integrated at nupG, and 9 copies of pmaao and bsgdh were integrated at pitB. LB (tryptone 10g/L, yeast powder 5g/L, sodium chloride 10 g/L) medium was used for cultivation at 25℃for 24 hours at 200 rpm. Strains with different copy numbers of integrated pmaao and bsgdh were inoculated in test tubes for overnight culture, then inoculated in 50mL of fresh LB medium at an inoculum size of 1%, and placed in a shaking table for 24 hours at 25 ℃ under 200-rotation conditions. After 24 hours, the amount of final wet cells was measured. Then, the cells were collected by centrifugation at 8000rpm at 4℃for 10min. The whole cell catalyzed 5mL reaction system takes 50mM sodium dihydrogen phosphate-disodium hydrogen phosphate solution with pH of 7.5 as a buffer solution for the reaction, and comprises: 10g/L wet cells, 35 ℃, 3g/L L-phenylalanine, conversion time 2h. After conversion, phenylpyruvate yields are shown in Table 9.
TABLE 9 Wet cell mass and phenylpyruvate yield of different integrated pmaao and bsgdh host strains
| Host strains | OD 600 | Phenylpyruvic acid (g/L) |
| BL21 | 3.84 | 2.21 |
| JM109 | 3.36 | 1.94 |
| DH5α | 3.58 | 2.05 |
| Top10 | 3.75 | 1.88 |
Example 8: application of strain integrated with L-amino acid oxidase and glucose dehydrogenase
(1) The lactate dehydrogenase gene from lactobacillus fermentum was fully synthesized according to the original sequence (GeneBank: MG 581696) and ligated between the BamHI and SalI sites of the pColdII plasmid, and the plasmid was transferred into E.coli BL1.10 host cells, which were then used as whole cell catalysts, and then transformed with L-dopa as a substrate.
Preparation of cells: e.coli BL1.10 strain transformed into the plasmid was inoculated in a test tube for overnight culture, then inoculated in 50mL of fresh LB medium at an inoculum size of 1%, and placed in a shaking table for culture at 25℃for 24 hours under 200 rpm. After 24 hours, the cells were collected after centrifugation at 8000rpm at 4℃for 10min.
The whole cell catalytic reaction system comprises: the wet cells were transformed at a final concentration of 50g/L at 35℃for 6 hours under conditions of 100mM L-dopa, pH7.5, 200 revolutions. The concentration of tanshinol after transformation was determined as shown in Table 10.
TABLE 10 production of tanshinol after whole cell biocatalysis
As can be seen from Table 10, the strain can well produce tanshinol.
(2) The amino acid decarboxylase pmkdc (GeneBank: KY 441412.1) and alcohol dehydrogenase pmadh (GeneBank: MG 736298) derived from proteus mirabilisJN458 were synthesized and then ligated between the NdeI/SacI and BamHI/SalI sites of the pColdII plasmid, respectively, and the plasmid was transferred into E.coli BL1.10 host cells, which were then used as whole cell catalysts, and transformed with L-dopa as a substrate.
Preparation of cells: e.coli BL1.10 strain transformed into the plasmid is inoculated in a test tube for overnight culture, then inoculated in 50mL of fresh LB culture medium with an inoculum size of 1%, and placed in a shaking table for culture at 25 ℃ for 24 hours under the condition of 220 revolutions. After 24 hours, the cells were collected after centrifugation at 8000rpm at 4℃for 10min.
The whole cell catalytic reaction system comprises: the wet cells were transformed at a final concentration of 50g/L at 35℃for 6 hours under conditions of 100mM L-dopa, pH7.5, 200 revolutions. The concentration of hydroxytyrosol after conversion is determined as shown in Table 11.
TABLE 11 production of hydroxytyrosol after whole cell biocatalysis
| Substrate(s) | Hydroxytyrosol yield (mM) |
| L-dopa | 54.43 |
As can be seen from Table 11, the strain is capable of producing hydroxytyrosol well.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of Jiangnan
<120> Escherichia coli having amino acid oxidase and glucose dehydrogenase integrated therein
<130> BAA200345A
<160> 56
<170> PatentIn version 3.3
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Claims (7)
1. A genetically engineered bacterium is characterized in that an L-amino acid oxidase gene and a glucose dehydrogenase gene with 3-12 copy numbers are integrated on a glsB, nupG, pitB site of an escherichia coli chromosome; the glsB is located at chromosome 1612325 ～ 1613251; the L-amino acid oxidase is derived fromProteus mirabilisATCC 29906 orCosenzaea myxofaciensATCC 19692; the glucose dehydrogenase is derived fromGluconobacter suboxydans ATCC 621、Haloferax mediterraneiATCC 33500 orBacillus subtilisATCC 13952; the integration is to integrate the L-amino acid oxidase gene and glucose dehydrogenase gene through an integration box; the conformable frame includes: integration site left arm-L-amino acid oxidase gene, glucose dehydrogenase gene-integration site right arm.
2. The genetically engineered bacterium of claim 1, wherein the escherichia coli isE.coli BL21、E. coliJM109、E.coliDH5 alpha orE. coliTop10。
3. The genetically engineered bacterium of claim 1 or 2, further expressing genes involved in metabolic pathways in which L-amino acid oxidase and glucose dehydrogenase are involved.
4. The genetically engineered bacterium of claim 3, wherein the related gene is expressed by pET20B, pColdII, pETDuet-1 or pacycdat-1.
5. A method for constructing the genetically engineered bacterium of any one of claims 1 to 4, comprising the steps of:
(1) Constructing an L-amino acid oxidase and glucose dehydrogenase gene integration frame; the integration frame is as follows: integration site left arm-L-amino acid oxidase gene, glucose dehydrogenase gene-integration site right arm; the integration site is glsB, nupG, pitB;
(2) Transforming the L-amino acid oxidase and glucose dehydrogenase gene integration frame constructed in the step (1) into an E.coli electrotransformation competent cell; the escherichia coli isE. coli BL21、E. coli JM109、E. coliDH5 alpha orE. coliTop10。
6. A whole cell catalyst comprising the genetically engineered bacterial cell of any one of claims 1-4; the cells are obtained by inoculating the cells into a culture medium and culturing the cells at 22-30 ℃ to be more than or equal to 16h.
7. The use of the genetically engineered bacterium of any one of claims 1-4 in the food and biological field to participate in a reaction with an amino acid as a substrate.
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