CN115786221B - Recombinant bacterium for producing 3, 4-dihydroxyphenethyl alcohol and construction method and application thereof - Google Patents
Recombinant bacterium for producing 3, 4-dihydroxyphenethyl alcohol and construction method and application thereof Download PDFInfo
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- 241000894006 Bacteria Species 0.000 title claims abstract description 46
- 238000010276 construction Methods 0.000 title claims abstract description 14
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a recombinant bacterium for producing 3, 4-dihydroxyphenethyl alcohol, a construction method and application thereof. Wherein the recombinant bacterium contains exogenous genes; exogenous genes include genes encoding oxygenases, alcohol dehydrogenases, O-demethylases and glucose dehydrogenases. The recombinant bacteria take eugenol as a substrate and can be converted into 3, 4-dihydroxyphenethyl alcohol. The eugenol has the advantages of wide source, simple preparation process, low price, high activity, strong optical specificity and the like, is an ideal substrate, and therefore, the recombinant bacterium is utilized to convert and produce the 3, 4-dihydroxyphenylethanol, has high production efficiency, green and environment-friendly performance, low cost and good industrial application prospect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a recombinant bacterium for producing 3, 4-dihydroxyphenylethanol, and a construction method and application thereof.
Background
3, 4-Dihydroxyphenylethanol, also known as hydroxytyrosol, is a natural polyphenol compound with high fat solubility and water solubility. It is a natural antioxidant, most abundant in olives, since olive oil is the main ingredient of a healthy mediterranean diet, and therefore the properties of 3, 4-dihydroxyphenylethanol have been approved and are well-established by the European food and safety agency. It has various biological pharmacological activities such as anticancer, antibacterial, antiinflammatory and neuroprotection, and is a promising compound in the pharmaceutical industry. In addition, the 3, 4-dihydroxyphenethyl alcohol can also be applied to food additives and cosmetics industry. However, the price of commercially available 3, 4-dihydroxyphenylethanol is high, which limits its wide application.
3, 4-Dihydroxyphenylethanol exists in olives mainly in the form of polyphenol, can be extracted from olives or olive processing wastewater, but has limited sources of raw materials, and has long period and low yield of extracting 3, 4-dihydroxyphenylethanol, which greatly increases the preparation cost; there are various starting materials available for the chemical synthesis of 3, 4-dihydroxyphenethyl alcohol, such as 3, 4-dihydroxyphenylacetic acid, catechol, 3, 4-dihydroxybenzaldehyde, tyrosol and the like. However, these raw materials are expensive, and have severe conditions, complicated steps, low yields, and are not suitable for large-scale industrial production. The bioconversion method has the advantages of high specificity, environmental protection, mild reaction conditions, no need of multi-step separation and purification and the like, and has been widely paid attention to at present.
At present, scholars at home and abroad report that various biological preparation routes of 3, 4-dihydroxyphenylethanol are provided, satoh et al construct a 3, 4-dihydroxyphenylethanol synthesis route in escherichia coli, L-tyrosine is converted into levodopa by murine tyrosine hydroxylase, and 3, 4-dihydroxyphenylethanol is generated by the actions of dopa decarboxylase, tyramine oxidase and alcohol dehydrogenase, but tetrahydrodishin (MH 4) is required as an auxiliary factor in the process, the circulation efficiency of MH4 is low, and the synthesis efficiency of 3, 4-dihydroxyphenylethanol is severely limited. Espin and the like use mushroom tyrosinase to convert tyrosol into 3, 4-dihydroxyphenylethanol, the mushroom tyrosinase has activities of monophenolase and bisphenolase, 3, 4-dihydroxyphenylethanol can be further oxidized into quinone substances, and ascorbic acid is required to be added to reduce the quinone substances into 3, 4-dihydroxyphenylethanol, so that the production cost is greatly increased. Brouk and the like, 2-phenethyl alcohol is taken as a substrate, the activity of the substrate is improved by about 190 times through modification of p-toluene-4-monooxygenase, the conversion condition is optimized, and under the optimal condition, the yield of 3, 4-dihydroxyphenethyl alcohol is only 133m g/L. Chinese patent CN104805110 introduces ARO10 gene and HpaBC gene into host bacteria to obtain recombinant colibacillus, and converts glucose as substrate to prepare 3, 4-dihydroxyphenethyl alcohol with 3, 4-dihydroxyphenethyl alcohol yield of 349.05mg/L. The preparation route of the 3, 4-dihydroxyphenylethanol generally has the defects of low production efficiency, high cost, few substrate sources and the like, so that the establishment of a preparation method of the 3, 4-dihydroxyphenylethanol with higher efficiency is urgent.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a recombinant bacterium for producing 3, 4-dihydroxyphenyl ethanol, a construction method and application thereof, and the recombinant bacterium can be used for producing 3, 4-dihydroxyphenyl ethanol, so that the yield of 3, 4-dihydroxyphenyl ethanol can be greatly improved.
The invention is realized in the following way:
In a first aspect, the present invention provides a recombinant bacterium for producing 3, 4-dihydroxyphenethyl alcohol, the recombinant bacterium comprising an exogenous gene; exogenous genes include genes encoding oxygenases, alcohol dehydrogenases, O-demethylases and glucose dehydrogenases.
The inventor of the present invention found through research that eugenol can be converted into 4-hydroxy-3-methoxy phenylacetaldehyde by oxygenase (COO) and Fe 2+, ethanol dehydrogenase (ADH) and NADH can convert 4-hydroxy-3-methoxy phenylacetaldehyde into 4-hydroxy-3-methoxy phenethyl alcohol, 4-hydroxy-3-methoxy phenethyl alcohol reacts under the action of O-demethylase (O-DEMETHYLASES, abbreviated as Odem) to generate 3, 4-dihydroxyphenethyl alcohol, glucose Dehydrogenase (GDH) is also added in the reaction process for cyclic regeneration of coenzyme NADH, and the reaction principle is shown in the following diagram:
According to the reaction principle, the inventor introduces genes encoding oxygenase, alcohol dehydrogenase, O-demethylase and glucose dehydrogenase into host bacteria to obtain recombinant bacteria, and uses the recombinant bacteria as a biocatalyst to convert eugenol into 3, 4-dihydroxyphenethyl alcohol.
In some embodiments, the oxygenase comprises CsCOO and GbCOO; csCOO has an amino acid sequence shown as SEQ ID NO. 1; gbCOO has the amino acid sequence shown in SEQ ID NO. 2.
In the present invention CsCOO is derived from Caulobactersegnis, genbank accession No. ADG12013.1; gbCOO is derived from Gordoniabronchialis and Genbank accession number ACY23219.1. After obtaining amino acid sequences of oxygenases CsCOO and GbCOO, the inventors performed codon optimization according to E.coli preference, and synthesized two optimized nucleotide sequences by a total synthesis method.
In some embodiments, the nucleotide sequence of CsCOO is as shown in seq id No. 8; the nucleotide sequence of GbCOO is shown as SEQ ID NO. 9.
In some embodiments, the alcohol dehydrogenase comprises AbADH and EsADH; abADH has an amino acid sequence shown as SEQ ID NO. 3; esADH has the amino acid sequence shown in SEQ ID NO. 4.
In the present invention AbADH is derived from Actinobacteriabacterium and Genbank accession number KPI21124.1; esADH is derived from Escherichia coli and Genbank accession number UKM85078.1. After obtaining the amino acid sequences of alcohol dehydrogenases AbADH and EsADH, the inventors performed codon optimization according to the preference of E.coli, and synthesized two optimized nucleotide sequences by a total synthesis method.
In some embodiments, the nucleotide sequence of AbADH is as shown in seq id No. 10; the nucleotide sequence of EsADH is shown as SEQ ID NO. 11.
In some embodiments, the O-demethylase comprises SsOdem and SmOdem; ssOdem is shown as SEQ ID NO. 5; smOdem has the amino acid sequence shown in SEQ ID NO. 6.
In the present invention SsOdem is derived from sphingobiumsp, genbank accession No.
BBD02022.1; smOdem is derived from Stenotrophomonasmaltophilia and Genbank accession number AAV53699.1. After obtaining the amino acid sequences of O-demethylases SsOdem and SmOdem, the inventors performed codon optimization according to E.coli preference, and synthesized two optimized nucleotide sequences by a total synthesis method.
In some embodiments, the nucleotide sequence of SsOdem is as shown in seq id No. 12; the nucleotide sequence of SmOdem is shown as SEQ ID NO. 13.
In some embodiments, the glucose dehydrogenase comprises BsGDH; bsGDH has the amino acid sequence shown in SEQ ID NO. 7.
In the present invention BsGDH is derived from Bacillus subtilis and Genbank accession number BAA09024.1. After obtaining the amino acid sequence of glucose dehydrogenase BsGDH, the inventors performed codon optimization according to E.coli preference, and synthesized two optimized nucleotide sequences by a total synthesis method.
In some embodiments, the nucleotide sequence of BsGDH is as shown in seq id No. 14.
In some embodiments, the host of the recombinant bacterium is escherichia coli.
In some embodiments, the E.coli is selected from any one of EscherichiacoliBL, escherichiacoliDH5 alpha, and EscherichiacoliXL-Blue.
In some embodiments, the recombinant bacterium contains a dual promoter expression vector, including the pCDFDuet-1 plasmid and the pACYCDuet-1 plasmid.
In a second aspect, the present invention also provides a method for constructing the recombinant bacterium, which includes: genes of oxygenase, alcohol dehydrogenase, O-demethylase and glucose dehydrogenase are connected to a double-promoter expression vector, and then the obtained recombinant expression vector is introduced into escherichia coli to obtain recombinant bacteria.
In the present invention, four enzymes are co-expressed in combination by selecting one enzyme from among the above-mentioned oxygenase, alcohol dehydrogenase, O-demethylase and glucose dehydrogenase. The mode of introducing the plasmid carrying the genes encoding the enzymes may be any one in which two encoding genes exist on the same plasmid, four genes may exist on different plasmids, or other modes of introducing, and the present invention is not limited thereto.
More preferably, the genes encoding the 4 enzymes are co-expressed using the pCDFDuet-1 and pACYCDuet-1 plasmids.
The combination of the genes encoding 4 enzymes may be any two of the gene combinations encoding the enzymes, and the preferable combination is a combination of the genes encoding oxygenase and alcohol dehydrogenase, and genes encoding O-demethylase and glucose dehydrogenase. A more preferred combination is one in which the pCDFDuet-1 plasmid has genes encoding oxygenase and alcohol dehydrogenase linked thereto, and the pACYCDuet-1 plasmid has genes encoding O-demethylase and glucose dehydrogenase linked thereto.
In some embodiments, cleavage sites EcoRI and HindIII are added to both ends of the nucleotide sequence of the oxygenase; adding enzyme cutting sites NdeI and XhoI at two ends of the nucleotide sequence of the alcohol dehydrogenase; adding enzyme cutting sites EcoRI and HindIII at two ends of the nucleotide sequence of the O-demethylase; the nucleotide sequence of glucose dehydrogenase was flanked by cleavage sites NdeI and XhoI.
In some embodiments, the fermentation culture method of the recombinant bacterium comprises: inoculating the seed solution of the recombinant bacteria into a fresh LB culture medium with an inoculum size of 2%, culturing at 37 ℃ and 200rpm until the bacterial concentration OD600nm reaches 0.7, adding 0.5mM IPTG and 0.1 g/L ferrous chloride, and inducing at 28 ℃ for 15 hours to obtain the product for producing 3, 4-dihydroxyphenylethanol.
In a third aspect, the present invention also provides a method for producing 3, 4-dihydroxyphenethyl alcohol, comprising: and adding the recombinant bacteria into a solution containing eugenol for whole cell transformation to obtain the 3, 4-dihydroxyphenethyl alcohol.
Whole cell transformation refers to a process of chemical transformation using whole biological organisms (i.e., whole cells, tissues, or even individuals) as a catalyst. In the present invention, the whole cell transformed organism is mainly a microorganism-recombinant bacterium, and the nature thereof is to be catalyzed by an enzyme in the recombinant bacterium.
In some embodiments, the whole cell transformed production system comprises: the wet weight of the recombinant bacteria is 1-100 g/L, the concentration of eugenol is 1-100 g/L, the concentration of NADH is 0-1 g/L, and the concentration of glucose is 20-80 g/L.
In some embodiments, the pH in the whole cell transformed production system is from 6.0 to 9.0.
In some embodiments, the temperature of the whole cell transformed production system is 15-40 ℃.
In some embodiments, the reaction time of the whole cell transformed production system is 10-48 h.
In a fourth aspect, the invention also provides application of the recombinant bacterium in the fields of food, cosmetics and medicines.
The invention has the following beneficial effects:
The invention provides a novel recombinant bacterium for producing 3, 4-dihydroxyphenethyl alcohol, which can be used for converting eugenol as a substrate to generate the 3, 4-dihydroxyphenethyl alcohol. The eugenol has the advantages of wide source, simple preparation process, low price, high activity, strong optical specificity and the like, is an ideal substrate, and therefore, the recombinant bacterium is utilized to convert and produce the 3, 4-dihydroxyphenylethanol, has high production efficiency, green and environment-friendly performance, low cost and good industrial application prospect.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The plasmids used in the present invention and E.coli were purchased from Novagen.
Example 1
This example is E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet
The construction method of the recombinant escherichia coli of-SsOdem-BsGDH comprises the following specific steps:
(1) Double-digestion is carried out on the totally synthesized CsCOO recombinant plasmid and the pCDFDuet-1 vector through restriction enzymes EcoRI and HindIII, double-digestion is carried out on the totally synthesized AbADH recombinant plasmid and the pCDFDuet-1 vector through restriction enzymes NdeI and XhoI, and CsCOO and AbADH are connected to the pCDFDuet vector through T4DNA ligase, so that the recombinant plasmid 1 is obtained;
(2) The recombinant plasmid 2 was obtained by double-digestion of the fully synthesized SsOdem recombinant plasmid and pACYCDuet-1 vector with restriction enzymes EcoRI and HindIII, double-digestion of the fully synthesized BsGDH recombinant plasmid and pACYCDuet-1 vector with restriction enzymes NdeI and XhoI, respectively, and ligation of SsOdem and BsGDH to the pCDFDuet vector with T4DNA ligase.
(3) Different recombinant plasmids 1 and 2 were transformed into E.coli BL21 (DE 3) competence to obtain recombinant E.coli.
Example 2
This example is E.coli BL21 (DE 3)/pCDFDuet-GbCOO-AbADH + pACYCDuet
Construction of recombinant E.coli from SsOdem-BsGDH, the procedure was as in example 1.
Example 3
This example is E.coli BL21 (DE 3)/pCDFDuet-CsCOO-EsADH + pACYCDuet
Construction of recombinant E.coli from SsOdem-BsGDH, the procedure was as in example 1.
Example 4
This example is E.coli BL21 (DE 3)/pCDFDuet-GbCOO-EsADH + pACYCDuet
Construction of recombinant E.coli from SsOdem-BsGDH, the procedure was as in example 1.
Example 5
This example is E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet
Construction of recombinant E.coli from SmOdem-BsGDH, the procedure was as in example 1.
Example 6
This example is E.coli BL21 (DE 3)/pCDFDuet-GbCOO-AbADH + pACYCDuet
Construction of recombinant E.coli from SmOdem-BsGDH, the procedure was as in example 1.
Example 7
This example is E.coli BL21 (DE 3)/pCDFDuet-GbCOO-EsADH + pACYCDuet
Construction of recombinant E.coli from SmOdem-BsGDH, the procedure was as in example 1.
Example 8
This example is E.coli BL21 (DE 3)/pCDFDuet-GbCOO-EsADH + pACYCDuet
Construction of recombinant E.coli from SmOdem-BsGDH, the procedure was as in example 1.
Example 9
The example is an induction culture of recombinant E.coli, comprising the following steps:
recombinant E.coli was inoculated into LB medium containing 50 mg/L streptomycin and 50 mg/L chloramphenicol, and cultured at 37℃and 200. 200 rpm for 12 h to obtain a seed solution. Inoculating the seed solution into fresh LB culture medium with 2% inoculum size, culturing at 37deg.C and 200 rpm to bacterial concentration OD600 nm reaching 0.7, adding 0.5mM IPTG and 0.1 g/L ferrous chloride, inducing 15 h at 28deg.C, centrifuging at 8000 rpm for 10 min, discarding supernatant, washing twice with 0.9% physiological saline, centrifuging, and collecting the product.
Example 10
This example shows comparison of transformation capacities of various recombinant E.coli
After the recombinant E.coli constructed in examples 1 to 8 was cultured according to the culture method of example 9, the recombinant E.coli was collected and resuspended in 50 mL systems, the final cell concentration was 100 g/L, the eugenol concentration was 100 g/L, glucose 80 g/L, NADH 1 g/L, pH=8.5, and reacted at 30℃on a shaker at a rotation speed of 200 rpm for a conversion time of 48 h. After the conversion was completed, the yield of 3, 4-dihydroxyphenethyl alcohol was determined by HPLC.
TABLE 1 comparison of the yields of 3, 4-dihydroxyphenethyl alcohol corresponding to various recombinant bacteria
| Recombinant bacterium | 3, 4-Dihydroxyphenethyl alcohol (g/L) | Substrate conversion (%) |
| Example 1 | 71.2 | 71.2 |
| Example 2 | 43.5 | 43.5 |
| Example 3 | 38.6 | 38.6 |
| Example 4 | 66.7 | 66.7 |
| Example 5 | 85.1 | 85.1 |
| Example 6 | 50.1 | 50.1 |
| Example 7 | 42.2 | 42.2 |
| Example 8 | 70.7 | 70.7 |
As can be seen from Table 1, recombinant bacteria constructed by introducing the oxygenase, alcohol dehydrogenase, O-demethylase and glucose dehydrogenase genes of different sources in the invention can be used as biological oxidants to convert eugenol into 3, 4-dihydroxyphenethyl alcohol, and the substrate conversion efficiency is high and can reach 85.1% at most.
Example 11
According to the method of induced expression in example 9, E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet-SmOdem-BsGDH was used to collect cells after the induction of expression, and the cells were transformed at a shaking table rotation speed of 200rpm for 48 hours at a temperature of 30℃in a 50mL system with a wet cell weight of 1 g/L, eugenol 1 g/L, glucose 20 g/L, NADH 0.5 g/L, and pH=8.5. As a result of HPLC measurement, the yield of 3, 4-dihydroxyphenethyl alcohol was 0.9 g/L, and the substrate conversion rate was 90.0%.
Example 12
According to the method of induced expression in example 9, E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet-SmOdem-BsGDH was collected after the induction of expression, and cells were transformed at 30℃for 10 hours at 200rpm in a 50mL system with a wet cell weight of 100 g/L, eugenol of 30 g/L, glucose of 80 g/L, NADH 1 g/L, pH=6.0. As a result of HPLC determination, the yield of 3, 4-dihydroxyphenethyl alcohol was 27.1 g/L, and the substrate conversion rate was 90.3%.
Example 13
According to the method of induced expression in example 9, E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet-SmOdem-BsGDH was collected after the induction of expression, and cells were transformed at 30℃for 20 hours at 200rpm in a 50mL system with a wet cell weight of 50 g/L, eugenol 30 g/L, glucose 80 g/L, NADH 1 g/L, pH=9.0. As a result of HPLC determination, the yield of 3, 4-dihydroxyphenethyl alcohol was 26.3 g/L, and the substrate conversion rate was 87.7%.
Example 14
E.coli BL21 (DE 3)/pCDFDuet according to the method of inducible expression in example 9
After the induced expression of-CsCOO-AbADH + pACYCDuet-SmOdem-BsGDH is finished, the thalli are collected, and in a 50mL system, the wet weight of the cells is 50 g/L, the eugenol is 40 g/L, the glucose is 80 g/L, the NADH is 1 g/L, the pH=8.5, the temperature is 15 ℃, the rotation speed of a shaking table is 200rpm, and the transformation time is 36h. As a result of HPLC determination, the yield of 3, 4-dihydroxyphenylethanol was 36.2 g/L, and the substrate conversion was 90.5%.
Example 15
According to the method of induced expression in example 9, E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet-SmOdem-BsGDH was collected after the completion of induced expression, and cells were transformed at 40℃for 36h at 200rpm in a 50mL system with a wet cell weight of 60 g/L, eugenol of 50 g/L, glucose of 80 g/L, NADH 1 g/L and pH=8.5. As a result of HPLC determination, the yield of 3, 4-dihydroxyphenethyl alcohol was 44.6 g/L, and the substrate conversion was 89.2%.
Example 16
According to the method of induced expression in example 9, E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet-SmOdem-BsGDH was collected after the induction of expression, and cells were transformed in a 50mL system at a wet cell weight of 50 g/L, eugenol 50 g/L, glucose 60 g/L, NADH 0.6 g/L, pH=7.0, temperature of 30℃and shaking rotation speed of 200rpm for 48 hours. As a result of HPLC determination, the yield of 3, 4-dihydroxyphenethyl alcohol was 45.2 g/L, and the substrate conversion rate was 90.4%.
Example 17
According to the method of induced expression in example 9, E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet-SmOdem-BsGDH was used to collect cells after the induction of expression, and the cells were transformed in a 50mL system at a wet cell weight of 50 g/L, eugenol 50 g/L, glucose 50 g/L, pH=8.5, a temperature of 25℃and a shaking rotation speed of 200rpm for 48 hours. As a result of HPLC determination, the yield of 3, 4-dihydroxyphenylethanol was 43.2 g/L, and the substrate conversion was 86.4%.
Example 18
According to the method of induced expression in example 9, E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet-SmOdem-BsGDH was collected after the induction of expression, and cells were transformed at 30℃for 30h at 200rpm in 50mL system with 30 wet cell weight g/L, 10: 10 g/L eugenol, 20: 20 g/L glucose, 1: 1 g/L NADH, pH=8.5. As a result of HPLC determination, the yield of 3, 4-dihydroxyphenethyl alcohol was 8.9 g/L, and the substrate conversion was 89.0%.
Example 19
According to the method of induced expression in example 9, E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet-SmOdem-BsGDH was collected after the induction of expression, and cells were transformed at 30℃for 48 hours at 200rpm in a 50mL system with a wet cell weight of 80 g/L, eugenol 70 g/L, glucose 60 g/L, NADH 1 g/L, pH=7.5. As a result of HPLC measurement, the yield of 3, 4-dihydroxyphenethyl alcohol was 60.1 g/L, and the substrate conversion rate was 85.9%.
Example 20
According to the method of induced expression in example 9, E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet-SmOdem-BsGDH was collected after the induction of expression, and cells were transformed at 30℃for 48 hours at 200rpm in a 50mL system with a wet cell weight of 70 g/L, eugenol 60 g/L, glucose 60 g/L, NADH 1 g/L, pH=7.0. As a result of HPLC determination, the yield of 3, 4-dihydroxyphenethyl alcohol was 54.2 g/L, and the substrate conversion rate was 90.3%.
Example 21
According to the method of induced expression in example 9, E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet-SmOdem-BsGDH was used to collect cells after the induction of expression, and the cells were transformed in a 50mL system at a wet cell weight of 40 g/L, eugenol of 30 g/L, glucose of 40 g/L, NADH of 0.3 g/L, pH=8.5, temperature of 25℃and shaking rotation speed of 200rpm for 48 hours. As a result of HPLC determination, the yield of 3, 4-dihydroxyphenethyl alcohol was 27.5 g/L, and the substrate conversion rate was 91.7%.
Example 22
According to the method of induced expression in example 9, E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet-SmOdem-BsGDH was used to collect cells after the induction of expression, and the cells were transformed at a temperature of 30℃for 36h at a shaking table rotation speed of 200rpm in a 50mL system with a wet cell weight of 20 g/L, eugenol 20 g/L, glucose 30 g/L, NADH 0.5 g/L and pH=8.5. As a result of HPLC determination, the yield of 3, 4-dihydroxyphenylethanol was 18.2 g/L, and the substrate conversion was 91.0%.
Example 23
According to the method of induced expression in example 9, E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet-SmOdem-BsGDH was collected after the induction of expression, and cells were transformed at a temperature of 35℃at 200rpm for 36h in a 50mL system with a wet cell weight of 30 g/L, eugenol 35 g/L, glucose 40 g/L, NADH 1 g/L, pH=8.0. As a result of HPLC determination, the yield of 3, 4-dihydroxyphenylethanol was 32.0 g/L, and the substrate conversion was 91.4%.
Example 24
According to the method of induced expression in example 9, E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet-SmOdem-BsGDH was collected after the induction of expression, and cells were transformed at a shaking table rotation speed of 200rpm for 36h at a temperature of 30℃in a 50mL system at a wet cell weight of 10 g/L, eugenol 10 g/L, glucose 20 g/L, NADH 0.5 g/L and pH=8.5. As a result of HPLC determination, the yield of 3, 4-dihydroxyphenethyl alcohol was 9.2 g/L, and the substrate conversion rate was 92.0%.
Example 25
According to the method of induced expression in example 9, E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet-SmOdem-BsGDH was induced to express and then cells were collected, and in a 50mL system, the wet cell weight was 5 g/L, eugenol 6 g/L, glucose 20 g/L, NADH 0.2 g/L, pH=8.0, temperature was 30℃and shaking table rotation speed was 200rpm, and transformation time was 30h. As a result of HPLC determination, the yield of 3, 4-dihydroxyphenethyl alcohol was 5.5 g/L and the substrate conversion was 91.7%.
Comparative example 1
According to the method of induced expression in example 9, E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet-SmOdem-BsGDH was collected after the induction of expression, and cells were transformed at 30℃for 72 hours at 200rpm in a 50mL system with 150 g/L wet cell weight, 120 g/L eugenol, 120 g/L glucose, and g/L NADH, pH=10.5. As a result of HPLC determination, the yield of 3, 4-dihydroxyphenethyl alcohol was 2.1 g/L, and the substrate conversion was 1.8%.
Comparative example 2
According to the method of induced expression in example 9, E.coli BL21 (DE 3)/pCDFDuet-CsCOO-AbADH + pACYCDuet-SmOdem-BsGDH was collected after the induction of expression, and cells were transformed at a temperature of 50℃and a shaking rotation speed of 200rpm for a transformation time of 72 h in a 50mL system at a wet cell weight of 110 g/L, eugenol 110 g/L, glucose 100 g/L, NADH 1 g/L and pH=8.5. As a result of HPLC determination, the yield of 3, 4-dihydroxyphenethyl alcohol was 3.5 g/L, and the substrate conversion was 3.2%.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (12)
1. A recombinant bacterium for producing 3, 4-dihydroxyphenethyl alcohol, which is characterized in that the recombinant bacterium contains exogenous genes; the exogenous genes include genes encoding oxygenase, alcohol dehydrogenase, O-demethylase and glucose dehydrogenase;
the oxygenase is CsCOO or GbCOO; the amino acid sequence of CsCOO is shown as SEQ ID NO. 1; the amino acid sequence of GbCOO is shown as SEQ ID NO. 2;
the alcohol dehydrogenase is AbADH or EsADH; the amino acid sequence of AbADH is shown as SEQ ID NO. 3; the amino acid sequence of EsADH is shown as SEQ ID NO. 4;
The O-demethylase is SsOdem or SmOdem; the amino acid sequence of SsOdem is shown as SEQ ID NO. 5;
The amino acid sequence of SmOdem is shown as SEQ ID NO. 6; the glucose dehydrogenase includes BsGDH; the amino acid sequence of BsGDH is shown as SEQ ID NO. 7;
The host of the recombinant bacterium is escherichia coli.
2. The recombinant bacterium according to claim 1, wherein the nucleotide sequence of CsCOO is as shown in seq id No. 8; the nucleotide sequence of GbCOO is shown as SEQ ID NO. 9.
3. The recombinant bacterium according to claim 1, wherein the nucleotide sequence of AbADH is as shown in seq id No. 10; the nucleotide sequence of EsADH is shown as SEQ ID NO. 11.
4. The recombinant bacterium according to claim 1, wherein the nucleotide sequence of SsOdem is as shown in seq id No. 12; the nucleotide sequence of SmOdem is shown as SEQ ID NO. 13.
5. The recombinant bacterium according to claim 1, wherein the nucleotide sequence of BsGDH is as shown in seq id No. 14.
6. The recombinant bacterium according to claim 1, wherein the escherichia coli is selected from any one of EscherichiacoliBL, escherichiacoliDH5 α and EscherichiacoliXL-Blue.
7. The recombinant bacterium according to claim 1, wherein the recombinant bacterium comprises a dual promoter expression vector comprising a pcdfdurt-1 plasmid and a pACYCDuet-1 plasmid.
8. The method for constructing a recombinant bacterium according to any one of claims 1 to 7, comprising: and connecting genes of the oxygenase, the alcohol dehydrogenase, the O-demethylase and the glucose dehydrogenase to the double-promoter expression vector, and then introducing the obtained recombinant expression vector into the escherichia coli to obtain the recombinant bacterium.
9. The method according to claim 8, wherein the recombinant bacterium contains both pCDFDuet-1 plasmid and pACYCDuet-1 plasmid.
10. The construction method according to claim 9, wherein genes encoding oxygenase and alcohol dehydrogenase are linked to the pCDFDuet-1 plasmid, and genes encoding O-demethylase and glucose dehydrogenase are linked to the pACYCDuet-1 plasmid.
11. A method for producing 3, 4-dihydroxyphenethyl alcohol, which is characterized by comprising the steps of adding the recombinant bacterium of any one of claims 1-7 into a eugenol-containing solution for whole cell transformation to obtain 3, 4-dihydroxyphenethyl alcohol;
in the whole cell transformation production system, the pH is 6.0-9.0; the temperature of the whole cell transformation production system is 15-40 ℃; the reaction time of the whole cell transformed production system is 10-48 h.
12. The method of claim 11, wherein the whole cell transformed production system comprises: the wet weight of the recombinant bacteria is 1-100 g/L, the concentration of eugenol is 1-100 g/L, the concentration of NADH is 0-1 g/L, and the concentration of glucose is 20-80 g/L.
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