CN112481336A - Method for biosynthesizing high value-added compound by utilizing lignocellulose derivative - Google Patents
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Abstract
The invention discloses a method for biosynthesizing a compound with high added value by utilizing a lignocellulose derivative, which comprises the following steps: A. transforming escherichia coli to obtain a biocatalyst; B. synthesizing a compound with a high added value by taking a lignocellulose derivative as an initial raw material through a biocatalyst; the lignocellulose derivative comprises at least one of p-coumaric acid and ferulic acid; the high value-added compound comprises at least one of gastrodin, arbutin, salidroside and derivatives thereof, hydroquinone, tyrosol, hydroxytyrosol and benzoin alcohol. The invention transforms escherichia coli through a genetic engineering means, constructs 3 new enzymatic reaction ways, efficiently synthesizes various compounds with high added values including gastrodin, arbutin, salidroside and the like by utilizing aromatic compounds derived from lignocellulose, namely p-coumaric acid and ferulic acid, and the product yield reaches gram or more.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and relates to a method for biosynthesizing a compound with high added value by utilizing a lignocellulose derivative; in particular to a method for biologically synthesizing a high value-added compound by using a lignocellulose derivative as a raw material based on a biocatalyst obtained by a genetic engineering means.
Background
Gastrodin (4-hydroxymethyl phenyl beta-D-glucopyranoside), identified as the main active ingredient of gastrodia elata, is widely used for treating various diseases, such as dizziness, headache, convulsion, vertigo, etc. In addition, gastrodine also has other bioactive effects, such as antioxidant, antiinflammatory, anxiolytic, anti-obesity, anti-epileptic, and memory improving and neuroprotective properties.
At present, gastrodin is mainly synthesized by chemical synthesis and direct extraction of gastrodia elata. Chemical synthesis usually faces the problems of poor specificity, multiple steps, more byproducts, excessive pollutants generated in the reaction process and the like; the extraction of the gastrodia elata plants has the problems of limited growth environment, low extraction efficiency, high cost and the like. In recent years, the synthesis of various natural products by constructing recombinant strains using renewable glucose has attracted more and more attention. Chinese patent 201910882020.9 discloses a method for synthesizing gastrodin by heterogenous metabolic pathway. The method utilizes recombinant Escherichia coli including pCDFDuet-aroG-ppsA-pgm-galU and pETDuet-ubiC-CAR-Sfp-ugt73b6FSTwo expression vectors are used for regulating and controlling metabolic flow from glucose to tyrosine by constructing a new p-hydroxy benzyl alcohol synthesis path to obtain the p-hydroxy benzyl alcohol with the yield of 240mg/L, and introducing a high-efficiency UDP glucosyltransferase mutant, wherein the highest yield of gastrodin is 265 mg/L.
Hydroquinone (HQ), an important intermediate, is commonly used in the commercial production of chemical, pharmaceutical and polymer industries. Hydroquinone is widely present in higher plants in glycosylated form (arbutin), and is also a pheromone of termites. At present, benzene is mainly used as a starting material in the industrial production of hydroquinone, and strong acid, heavy metal and the like are usually used, so that serious environmental problems are caused. Meanwhile, benzene is used as a petroleum-based raw material and is not a renewable resource. According to the literature (Chemo-and Regioselective hydrolysis of Benzene to Hydroquinone activated by Engineered Cytochrome P450 Monoxygene, Angewandte Chemie International Edition,2019,58,764 and 768), it is reported that the mutant strain A82F/A328F obtained by modifying P450-MB3 Monooxygenase can catalyze the Dihydroxylation of 10mM Benzene to synthesize 9.2mM Hydroquinone.
Arbutin (Arbutin), a glycosylated hydroquinone, is found in the plants, rubus ursinus, wheat, etc. It is a skin whitening agent, and has biological activities of resisting bacteria, inflammation and oxidation. Arbutin, a mild, safe and effective agent, has been widely used in the medical and cosmetic industries. The arbutin is usually obtained by a plant extraction method, but the method is not only complicated in process but also low in yield. Darlin et al, transform Escherichia coli through metabolic pathway to accumulate p-hydroxybenzoic acid, and then heterologously express 4-hydroxybenzoic acid 1-hydroxylase (4-hydroxybenzoate 1-hydroxylase, MNX1) derived from Candida parapsilosis CBS604 and Arbutin Synthase (AS) derived from Rauvolfia serpentina, and can obtain 4.19g/L of arbutin by using 30g/L of glucose AS raw material. Chinese patent 201510107788.0 discloses a method for enriching and purifying arbutin from blueberries, which comprises the steps of firstly extracting alpha-arbutin by using high-voltage pulse electric field extraction equipment, then carrying out primary purification of the alpha-arbutin by using membrane equipment and macroporous adsorption resin, and then separating and purifying the alpha-arbutin by using a simulated moving bed chromatographic separation technology to obtain a high-purity alpha-arbutin product. Chinese invention patent 201510991581.4 discloses a method for synthesizing arbutin by converting starch substance and hydroquinone by enzyme method, which comprises adding biological enzyme into starch substance and hydroquinone as raw materials to perform enzyme conversion reaction; after the enzyme of the reaction liquid is removed, yeast is added for fermentation to remove glucose in the reaction liquid, and then the arbutin is prepared after sterilization. Chinese patent 201510335885.5 discloses a method for synthesizing arbutin by biological fermentation, which uses liquefied flour, bran, calcium carbonate, potassium dihydrogen phosphate and urea as fermentation substrates, adds hydroquinone into the fermentation substrates, and uses aspergillus oryzae for fermentation.
Salidroside (Salidroside), also known as tyrosol 8-O-glucoside, is a tyrosine-derived phenolic natural product with biological activity, and widely exists in the medicinal plant Salidroside. Besides the anti-fatigue and anti-anoxia effects of rhodiola rosea in traditional medicine, rhodiola rosea extract and salidroside also show anti-cardiovascular disease and anti-cancer functions. However, commercially available pure salidroside currently goes through a lengthy decontamination process mainly starting from its native plant, which is an important bottleneck hindering salidroside as a potential therapeutic agent. The chinese patent 201310463903.9 discloses a method for extracting salidroside from rhodiola rosea. The method comprises the steps of firstly, crushing rhodiola rosea medicinal materials, taking water as a solvent, carrying out ultrasonic-assisted extraction, and filtering to obtain filtrate; then collecting by a macroporous resin column, and concentrating under reduced pressure; dissolving with anhydrous ethanol, and separating by simulated moving bed chromatography to obtain a component rich in salidroside; and finally, concentrating, crystallizing, centrifuging and drying to obtain the salidroside product. The Chinese patent 201810753390.8 discloses a method for synthesizing salidroside by fermentation. The method obtains the SDR1 strain by integrating the genes of a keto decarboxylase gene skdc with a trc promoter and a glycosyltransferase gene sugt2 with a tac promoter onto a SyBE-002447 chromosome. Fermenting the SDR1 strain with glucose as precursor for 36h to obtain 0.7g/L salidroside.
Tyrosol (Tyrosol, p-hydroxyphenylethanol), a phenolic compound, occurs naturally in a variety of foods, such as olive oil and wine. Tyrosol and its derivatives have been widely used in the medical field (e.g., bioactive compounds), the chemical industry (e.g., fine compounds), and other industrial fields. For example, tyrosol can be used to prepare commercial drugs such as betaxolol, metoprolol, and selective beta blockers for the treatment of hypertension, angina, heart failure, and glaucoma. Hydroxytyrosol (HT, 3, 4-dihydroxyphenylethanol), a natural polyphenolic antioxidant, is mainly present in olive leaves and fruits thereof, and also in olive oil industrial by-products and industrial waste water. In addition, hydroxytyrosol is a biological active substance which is concerned about human health, and has the functions of resisting bacteria, resisting inflammation, inhibiting melanin precipitation, scavenging free radicals and the like. At present, the mass production of hydroxytyrosol is not mature, and two main modes exist: 1) preparing hydroxytyrosol by acidolysis or enzymolysis of oleuropein in the wastewater, olive leaves and primary olive oil of olive plants; 2) the hydroxytyrosol is prepared by a chemical synthesis method. By plant extraction, although waste utilization, a large amount of organic reagents are used in the process; the chemical synthesis method has long reaction steps, more byproducts and unfriendly environment, and is not suitable for the concept of green sustainable synthesis. Chinese patent 201710195462.7 discloses a method for extracting hydroxytyrosol from olive leaves. Firstly, removing impurities and cleaning olive leaves, drying and crushing the olive leaves; then, carrying out high-pressure micro-jet superfine grinding for treatment to obtain paste slurry; subsequently, microwave extraction is carried out; then, the extract enters macroporous absorption resin column chromatography, and ethyl acetate solution is used for elution; concentrating the eluate, and drying to obtain hydroxytyrosol. Chinese patent 201910882020.9 discloses a method for synthesizing hydroxytyrosol by enzymatic reaction, which takes L-dopa as a substrate and expresses L-phenylalanine dehydrogenase, alpha-keto acid decarboxylase and alcohol dehydrogenase simultaneously in Escherichia coli recombinant bacteria to synthesize hydroxytyrosol. Chinese patent 202010582557.6 discloses a method for constructing Escherichia coli with high yield of hydroxytyrosol. The method comprises the steps of transferring a plasmid pACYC-HpaBC, a plasmid pET-LAAD-ARO10 and a plasmid pRSF-PAR into a competent cell of escherichia coli BL21(DE3) together to obtain recombinant escherichia coli, and catalytically synthesizing hydroxytyrosol by taking tyrosine as a substrate. Tyrosine and L-dopa are expensive and do not fall into the renewable resource category.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for biosynthesizing high value-added compounds by utilizing lignocellulose derivatives.
The invention transforms escherichia coli through a genetic engineering means, constructs 3 new enzymatic reaction ways, efficiently synthesizes various compounds with high added values including gastrodin, arbutin, salidroside and the like by utilizing aromatic compounds derived from lignocellulose, namely p-coumaric acid and ferulic acid, and the product yield reaches gram or more.
The purpose of the invention is realized by the following technical scheme:
the invention provides a method for biosynthesizing a high value-added compound by utilizing a lignocellulose derivative, which comprises the following steps:
A. transforming escherichia coli to obtain a biocatalyst;
B. synthesizing a compound with a high added value by taking a lignocellulose derivative as an initial raw material through a biocatalyst;
the lignocellulose derivative comprises at least one of p-coumaric acid and ferulic acid;
the high value-added compound comprises at least one of gastrodin, arbutin, salidroside and derivatives thereof, hydroquinone, tyrosol, hydroxytyrosol and benzoin alcohol.
Preferably, in step A, the biocatalyst is E.coli (Fcs-Ech-SlPAR1-UGT73B 6)FS) One of e.coli (Fcs-Ech-Vdh-MNX1-AS), e.coli (Fcs-Ech-Vdh-MNX1), e.coli (BLPad-StyAB-rosetc-SlPAR 1-UGT85a1), e.coli (BLPad-StyAB-rosetc-par sl 1), e.coli (BLPad-StyAB-rosetc-YqhD), e.coli (BLPad-StyAB-rosetc-YahK), and e.coli (BLPad-StyAB-rosetc-SlPAR 1-HpaBC).
Preferably, the E.coli (Fcs-Ech-SlPAR1-UGT73B 6)FS) The obtaining method comprises the following steps: coli by overexpressing Fcs, Ech from Pseudomonas putida KT2440, SlPAR1 from Solanum lycopersicum and the mutase UGT73B6 from the plant RhodiolaFSAnd (5) realizing.
Preferably, the method for obtaining the E.coli (Fcs-Ech-Vdh-MNX1-AS) comprises the following steps: by overexpressing in e.coli Fcs, Ech and Vdh derived from p.pudida KT2440, MNX1 derived from yeast Candida parapsilosis CDC317 and arbutin synthase AS derived from Rauvolfia serpentina;
the method for obtaining the E.coli (Fcs-Ech-Vdh-MNX1) comprises the following steps: this was achieved by overexpressing Fcs, Ech and Vdh derived from p.putida KT2440, MNX1 derived from the yeast Candida parapsilosis CDC317, in e.coli.
Preferably, the method for obtaining the E.coli (BLPad-StyAB-RostyC-SlPAR1-UGT85A1) comprises the following steps: (ii) by overexpressing in e.coli the decarboxylase BLPad derived from Bacillus licheniformis CGMCC7172, stylab derived from Pseudomonas sp.strain VLB120, rosetc derived from Rhodococcus opacus 1CP, SlPAR1 derived from Solanum lycopersicum, and the glycosyltransferase UGT85a1 derived from Arabidopsis thaliana;
the method for obtaining E.coli (BLPad-StyAB-RostyC), E.coli (BLPad-StyAB-RostyC-SlPAR1), E.coli (BLPad-StyAB-RostyC-YqhD) and E.coli (BLPad-StyAB-RostyC-YahK) is as follows: by overexpressing in e.coli the decarboxylase BLPad derived from Bacillus licheniformis CGMCC7172, StyAB derived from Pseudomonas sp.strain VLB120, RostyC derived from Rhodococcus opacus 1CP, YqhD or YahK derived from e.coli BL21(DE3) or SlPAR1 derived from Solanum lycopersicum;
the method for obtaining the E.coli (BLPad-StyAB-RostyC-SlPAR1-HpaBC) comprises the following steps: this is achieved by overexpressing in E.coli the decarboxylase BLPad derived from Bacillus licheniformis CGMCC7172, StyAB derived from Pseudomonas sp.strain VLB120, RostyrC derived from Rhodococcus opacus 1CP, SlPAR1 derived from Solanum dipliscum and HpaBC derived from E.coli BL21(DE 3).
Preferably, the biocatalyst is E.coli (Fcs-Ech-SlPAR1-UGT73B 6)FS) And (C) in the step (B), the lignocellulose derivative adopted is p-coumaric acid, and the synthesized high-added-value compound is gastrodin.
Preferably, when the biocatalyst is E.coli (Fcs-Ech-Vdh-MNX1-AS), the lignocellulose derivative adopted in the step B is p-coumaric acid, and the synthesized high value-added compound is arbutin;
when the biocatalyst is E.coli (Fcs-Ech-Vdh-MNX1), the lignocellulose derivative adopted in the step B is p-coumaric acid, and the synthesized compound with high added value is hydroquinone.
Preferably, when the biocatalyst is e.coli (BLPad-StyAB-RostyC-SlPAR1-UGT85a1), the lignocellulose derivative used in step B is p-coumaric acid, and the synthesized high value-added compound is salidroside;
when the biocatalyst is E.coli (BLPad-StyAB-RostyC), E.coli (BLPad-StyAB-RostyC-SlPAR1), E.coli (BLPad-StyAB-RostyC-YqhD) or E.coli (BLPad-StyAB-RostyC-YahK), the lignocellulose derivative adopted in the step B is p-coumaric acid or ferulic acid, and the synthesized high value-added compound is tyrosol or high-interest vanillyl alcohol;
and when the biocatalyst is E.coli (BLPad-StyAB-RostyC-SlPAR1-HpaBC), the lignocellulose derivative adopted in the step B is p-coumaric acid, and the synthesized high value-added compound is hydroxytyrosol.
The invention also provides a biocatalyst for biosynthesis of high value-added compounds, wherein the biocatalyst is modified escherichia coli, and specifically comprises E.coli (Fcs-Ech-SlPAR1-UGT73B 6)FS) One of e.coli (Fcs-Ech-Vdh-MNX1-AS), e.coli (Fcs-Ech-Vdh-MNX1), e.coli (BLPad-StyAB-rosetc-SlPAR 1-UGT85a1), e.coli (BLPad-StyAB-rosetc-par sl 1), e.coli (BLPad-StyAB-rosetc-YqhD), e.coli (BLPad-StyAB-rosetc-YahK), and e.coli (BLPad-StyAB-rosetc-SlPAR 1-HpaBC).
Preferably, the high value-added compound comprises at least one of gastrodin, arbutin, salidroside and derivatives thereof, hydroquinone, tyrosol, hydroxytyrosol and high-interest cuminol.
Preferably, the E.coli (Fcs-Ech-SlPAR1-UGT73B 6)FS) The obtaining method comprises the following steps: coli by overexpressing Fcs, Ech from Pseudomonas putida KT2440, SlPAR1 from Solanum lycopersicum and the mutase UGT73B6 from the plant RhodiolaFSAnd (5) realizing.
Preferably, the method for obtaining the E.coli (Fcs-Ech-Vdh-MNX1-AS) comprises the following steps: by overexpressing in e.coli Fcs, Ech and Vdh derived from p.pudida KT2440, MNX1 derived from yeast Candida parapsilosis CDC317 and arbutin synthase AS derived from Rauvolfia serpentina;
the method for obtaining the E.coli (Fcs-Ech-Vdh-MNX1) comprises the following steps: this was achieved by overexpressing Fcs, Ech and Vdh derived from p.putida KT2440, MNX1 derived from the yeast Candida parapsilosis CDC317, in e.coli.
Preferably, the method for obtaining the E.coli (BLPad-StyAB-RostyC-SlPAR1-UGT85A1) comprises the following steps: (ii) by overexpressing in e.coli the decarboxylase BLPad derived from Bacillus licheniformis CGMCC7172, stylab derived from Pseudomonas sp.strain VLB120, rosetc derived from Rhodococcus opacus 1CP, SlPAR1 derived from Solanum lycopersicum, and the glycosyltransferase UGT85a1 derived from Arabidopsis thaliana;
the method for obtaining E.coli (BLPad-StyAB-RostyC), E.coli (BLPad-StyAB-RostyC-SlPAR1), E.coli (BLPad-StyAB-RostyC-YqhD) and E.coli (BLPad-StyAB-RostyC-YahK) is as follows: by overexpressing in e.coli the decarboxylase BLPad derived from Bacillus licheniformis CGMCC7172, StyAB derived from Pseudomonas sp.strain VLB120, RostyC derived from Rhodococcus opacus 1CP, YqhD or YahK derived from e.coli BL21(DE3) or SlPAR1 derived from Solanum lycopersicum;
the method for obtaining the E.coli (BLPad-StyAB-RostyC-SlPAR1-HpaBC) comprises the following steps: this is achieved by overexpressing in E.coli the decarboxylase BLPad derived from Bacillus licheniformis CGMCC7172, StyAB derived from Pseudomonas sp.strain VLB120, RostyrC derived from Rhodococcus opacus 1CP, SlPAR1 derived from Solanum dipliscum and HpaBC derived from E.coli BL21(DE 3).
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention obtains high-efficiency biocatalyst by means of genetic engineering, and can rapidly catalyze substrates to coumaric acid and ferulic acid to synthesize various high value-added compounds including gastrodin, arbutin and salidroside.
(2) The invention utilizes the constructed biocatalyst to realize the high-efficiency utilization of aromatic compounds (coumaric acid and ferulic acid) from lignocellulose through biotransformation, and has more advantages compared with the tedious step of glucose fermentation.
(3) The yield of the target compound obtained by the method is more than or equal to gram, the conversion rate is more than 90 percent, and the method has great application potential.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a diagram showing the construction of a biocatalyst for the synthesis of gastrodin;
FIG. 2 shows the result of synthesizing gastrodin by biotransformation;
FIG. 3 is a graph showing the results of the biosynthesis of hydroquinone:
FIG. 4 is a diagram showing the construction of a biocatalyst for the synthesis of arbutin;
FIG. 5 is a graph comparing the results of arbutin synthesis with different biocatalysts;
FIG. 6 is a graph showing the results of the biotransformation of arbutin;
FIG. 7 shows the construction of a biocatalyst for the synthesis of tyrosol;
FIG. 8 is a graph showing the results of different biocatalyst conversions for tyrosol synthesis;
FIG. 9 is a schematic representation of the biosynthesis of tyrosol and of interest;
FIG. 10 is a graph showing the results of biosynthetic salidroside;
FIG. 11 is a graph showing the results of biosynthesis of hydroxytyrosol:
FIG. 12 is a schematic diagram of biosynthetic gastrodin;
FIG. 13 is a schematic diagram of the biosynthesis of para-arbutin and its derivatives hydroquinone;
FIG. 14 is a schematic diagram of biosynthetic salidroside and its derivatives tyrosol, benzoin alcohol and hydroxytyrosol.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
The method of the following example includes:
step one, transforming escherichia coli to obtain a high-efficiency biocatalyst (E. coli (Fcs-Ech-SlPAR1-UGT73B 6)FS) Successfully synthesizing gastrodin from p-coumaric acid;
secondly, transforming escherichia coli to obtain efficient biocatalysts (E.coli (Fcs-Ech-Vdh-MNX1-AS) and E.coli (Fcs-Ech-Vdh-MNX1)), and successfully synthesizing arbutin and hydroquinone derivatives thereof by taking p-coumaric acid AS starting raw materials;
transforming escherichia coli to obtain a plurality of efficient biocatalysts (E.coli (BLPad-StyAB-RostyC-SlPAR1-UGT85A1), E.coli (BLPad-StyAB-RostyC-SlPAR1), E.coli (BLPad-StyAB-RostyC-YqhD), E.coli (BLPad-StyAB-RostyC-YahK), and E.coli (BLPad-StyAB-RosyC-SlPAR 1-HpaBC)) and the like), and taking p-coumaric acid and ferulic acid as starting materials to successfully synthesize salidroside, tyrosol, hydroxytyrosol and high-level linalool;
in step one, the biocatalyst (E. coli (Fcs-Ech-SlPAR1-UGT73B 6)FS) Coli by overexpressing Fcs, Ech from Pseudomonas putida KT2440, SlPAR1 from Solanum lycopersicum and the mutant enzyme UGT73B6 from plant RhodiolaFSAnd (5) realizing. In the process, E.coli (Fcs-Ech-SlPAR1) is firstly constructed to catalyze p-coumaric acid to synthesize p-hydroxybenzyl alcohol, and then UGT73B6 is introducedFSConstructing a new catalyst to catalyze p-hydroxybenzyl alcohol to synthesize gastrodin;
in the second step, the construction of the biocatalysts (e.coli (Fcs-Ech-Vdh-MNX1-AS) and e.coli (Fcs-Ech-Vdh-MNX1)) was achieved by overexpressing Fcs, Ech and Vdh derived from p.pudida KT2440, MNX1 derived from the yeast Candida parapsilosis CDC317 and arbutin synthase AS derived from Rauvolfia serpentina in e.coli. In the process, E.coli (Fcs-Ech-Vdh-MNX1) is firstly constructed to catalyze a substrate to coumaric acid to synthesize arbutin precursor substance, benzenediol, and then AS is introduced to construct a plurality of biocatalysts to efficiently synthesize arbutin.
In step three, the biocatalysts (e.coli (BLPad-StyAB-roseyc-SlPAR 1-UGT85a1), e.coli (BLPad-StyAB-roseyc-SlPAR 1), e.coli (BLPad-StyAB-roseyc-YqhD), e.coli (BLPad-StyAB-roseyc-YahK), and e.coli (BLPad-StyAB-roseyc-par 1-HpaBC)) and the like were achieved by overexpressing in e.coli Bacillus (CGMCC 7172 decarboxylase BLPad, Pseudomonas sp.strn ab 120, Rhodococcus yaphys, Rhodococcus 1CP, Rhodococcus rhodophyse derived from Arabidopsis c, rhodophyse derived from rosepala, and sola derived from Solanum glycoside (Solanum, solarium, r. In the process, a plurality of biocatalysts, namely E.coli (BLPad-StyAB-RostyC), E.coli (BLPad-StyAB-RostyC-SlPAR1), E.coli (BLPad-StyAB-RostyC-YqhD) and E.coli (BLPad-StyAB-RostyC-YahK) are firstly constructed to catalyze p-coumaric acid to synthesize tyrosol, an optimal catalyst is selected, and UGT85A1 or HpaBC is introduced to further catalyze and synthesize salidroside and hydroxytyrosol.
Example 1
(1) Constructing biocatalyst for synthesizing gastrodin (construction schematic diagram is shown in figure 1)
Construction of plasmid pET28a-Fcs-Ech-SlPAR1 and plasmid pA7a-UGT73B6FSThen the strain is transformed into BL21(DE3) strain to construct a biocatalyst E.coli (Fcs-Ech-SlPAR1-UGT73B 6)FS). The specific method comprises the following steps:
fcs and Ech are obtained by PCR cloning from a genome of Pseudomonas putida KT2440, and are constructed on a vector pET28a together with SlPAR1 to obtain a plasmid pET28a-Fcs-Ech-SlPAR 1; UGT73B6FsThe plasmid pA7a-UGT73B6 is obtained by constructing on a vector pA7aFS(ii) a Plasmid pET28a-Fcs-Ech-SlPAR1 and plasmid pA7a-UGT73B6FSTransforming into a host BL21(DE3) by a heat shock or electric shock method to prepare the biocatalyst ER1-UGT73B6FS)。
(2) Synthesizing gastrodin by biotransformation (the synthetic scheme is shown in figure 12)
Coli (Fcs-Ech-SlPAR1-UGT73B 6)FS) Inoculating into 2mL LB for activation, at 37 deg.C for 10-12h, transferring the activated biocatalyst into 100mL LB at a ratio of 1:100, and allowing OD to reach600When the concentration reaches 0.6-0.8, adding an inducer IPTG to the final concentration of 0.5mM, inducing for 10-12h at 22 ℃, centrifuging for 10min at 4 ℃ at 4000g, collecting thalli, resuspending in an M9Y culture medium, adding a substrate of 2g/L p-coumaric acid and 10g/L glucose, and converting for 12h at 37 ℃ to obtain 1.45g/L gastrodin, wherein the conversion rate is 42.5% (figure 2).
Example 2
(1) Construction of biocatalyst for synthesizing hydroquinone
Plasmid pET28a-Fcs-Ech-Vdh and plasmid pA7a-MNX1 are constructed and are jointly transferred into BL21(DE3) to obtain biocatalyst E.coli (Fcs-Ech-Vdh-MNX 1). The specific method comprises the following steps: fcs and Ech obtained by PCR cloning from a genome of Pseudomonas putida KT2440 and pET28a-Fcs-Ech-Vdh obtained by construction of Vdh entering a vector pET28a through enzyme digestion connection, and pA7a-MNX1 obtained by construction of MNX1 entering a vector pA7a through enzyme digestion connection; plasmid pET28a-Fcs-Ech-Vdh and plasmid pA7a-MNX1 are transformed into host BL21(DE3) by a heat shock or electric shock method, and the biocatalyst E.coli (Fcs-Ech-Vdh-MNX1) is prepared.
(2) Biotransformation synthesis of hydroquinone (the synthetic scheme is shown in figure 13)
Inoculating the prepared biocatalyst E.coli (Fcs-Ech-Vdh-MNX1) into 2mL LB for activation, transferring the activated biocatalyst into 100mL LB according to the ratio of 1:100 after 10-12h at 37 ℃, and waiting until OD is reached600When the concentration reaches 0.6-0.8, adding inducer IPTG to the final concentration of 0.5mM, inducing at 22 ℃ for 10-12h, centrifuging at 4 ℃ for 10min at 4000g, collecting thalli, resuspending in M9Y culture medium, adding substrate 3g/L p-coumaric acid and 10g/L glucose, and converting at 37 ℃ for 8 h. As a result, as shown in FIG. 3, 1.95g/L hydroquinone was obtained with a conversion of 97% (FIG. 3).
Example 3
(1) The biocatalyst is constructed and used for synthesizing an E.coli (Fcs-Ech-Vdh-MNX1-AS) catalyst corresponding to arbutin (the construction schematic diagram is shown in figure 4), wherein SArbutin5 has the best effect.
Plasmids pET28a-Fcs-Ech, pET28a-Fcs-Ech-Vdh, pA7a-Vdh-MNX1-AS, pA7a-MNX1-AS, pACYC-AS-MNX1, pA7a-AS-MNX1, pA7a-AS-7-MNX1(7 represents a T7 promoter, and compared with pA7a-AS-MNX1, a T7 promoter is added before MNX1 gene) are constructed and combined two by two to transform BL21(DE3) to obtain 5 biocatalysts, SArbutin1, SArbutin2, SArbutin3, SArbutin4 and SArbutin 5. The specific method comprises the following steps:
fcs and Ech are obtained by PCR cloning from a genome of Pseudomonas putida KT2440, and Vdh enters a vector pET28a through enzyme digestion connection to construct pET28a-Fcs-Ech and pET28 a-Fcs-Ech-Vdh; MNX1 and AS are connected into a vector pA7a or pACYC through enzyme digestion to construct pA7a-MNX1-AS, pA7a-AS-MNX1 and pA7a-AS-7-MNX 1; vdh, MNX1 and AS obtained by PCR cloning enter a vector pA7a through enzyme digestion connection to construct pA7a-Vdh-MNX 1-AS.
The 5 biocatalysts, SArbutin1, are prepared by transforming plasmids pET28a-Fcs-Ech and pA7a-Vdh-MNX1-AS into a host BL21(DE3) through a heat shock or electric shock method, SArbutin2 is prepared by transforming plasmids pET28a-Fcs-Ech-Vdh and pA7a-MNX1-AS into host BL21(DE3) by means of heat shock or electric shock, SArbutin3 is prepared by transforming plasmids pET28a-Fcs-Ech-Vdh and pA7a-AS-MNX1 into host BL21(DE3) by means of heat shock or electric shock, SArbutin4 is prepared by transforming plasmids pET28a-Fcs-Ech-Vdh and pA7a-AS-7-MNX1 into host BL21(DE3) by means of heat shock or electric shock, SArbutin5 is prepared by transforming plasmids pET28a-Fcs-Ech-Vdh and pACYC-AS-MNX1 into a host BL21(DE3) by a heat shock or electric shock method.
(2) Biotransformation synthesis of arbutin (the synthetic scheme is shown in FIG. 13)
Inoculating the 5 prepared biocatalysts into 2mL LB for activation, transferring the activated biocatalysts into 100mL LB according to the proportion of 1:100 after 10-12h at 37 ℃, and waiting until OD is reached600When reaching 0.6-0.8, adding inducer IPTG to final concentration of 0.5mM, inducing at 22 deg.C for 10-12 hr, centrifuging at 4 deg.C and 4000gCollecting thallus after 10min, resuspending in M9Y culture medium, adding 2g/L p-coumaric acid and 10g/L glucose, and converting at 37 deg.C for 12 h. The results of 5 biocatalysts for arbutin synthesis are shown in FIG. 5, and it can be seen that SArbutin5 converted into arbutin with the highest yield, 2.34 g/L. Wherein, the result of the change of the conversion of the biocatalyst SArbutin5 into arbutin with the conversion time by the method is shown in FIG. 6, 3.05g/L arbutin can be obtained after 24h, and the conversion rate is 92%.
Example 4
(1) Construction of biocatalysts E.coli (BLPad-StyAB-Rostyr C), E.coli (BLPad-StyAB-Rostyr C-SlPAR1), E.coli (BLPad-StyAB-Rostyr C-YqhD), E.coli (BLPad-StyAB-Rostyr C-YahK), E.coli (BLPad-StyAB-Rostyr C-SlPAR1) for the Synthesis of tyrosol (schematic construction is shown in FIG. 7)
Plasmids pET28a-StyAB-RostyC, pET28a-StyAB-RostyC-BLpad, pET28a-StyAB-RostyC-SlPAR1, pET28a-StyAB-RostyC-YqhD, pET28a-StyAB-RostyC-YahK, pA7a-BLpad, pA7a-BLPad-SlPAR1, pA7a-BLPad-YqhD, pA7a-BLPad-YahK were constructed. The two-by-two combination thereof was transformed into BL21(DE3), yielding 8 biocatalysts, Styrosol 1, Styrosol 2, Styrosol 3, Styrosol 4, Styrosol 5, Styrosol 6, Styrosol 7 and Styrosol 8.
The specific method comprises the following steps: YqhD and YahK on a BL21(DE3) genome are amplified through PCR, StyAB, RostyC, BLpad and SlPAR1 are combined, and the StyAB and RostyC are connected into a vector pET28a through enzyme digestion to construct pET28 a-StyAB-RostyC; the StyAB, the RostyC and the BLPad are connected into a vector pET28a through enzyme digestion to construct pET28 a-StyAB-RostyC-BLPad; StyAB, RostyC, YahK or YqhD or SlPAR1 are connected into a vector pET28a through enzyme digestion to construct pET28a-StyAB-RostyC-YqhD or pET28a-StyAB-RostyC-YahK or pET28a-StyAB-RostyC-SlPAR 1; the BLPad or the BLPad and YqhD or YahK or SlPAR1 enter a vector pA7a through enzyme digestion to construct pA7a-BLPad, pA7a-BLPad-SlPAR1, pA7a-BLPad-YqhD, pA7 a-BLPad-YahK.
The biocatalyst Styrosol 1 is prepared by transforming plasmid pET28a-StyAB-RostyC-BLpad into host BL21(DE3) by heat shock or electric shock, Styrosol 2 is prepared by transforming plasmids pET28a-StyAB-RostyC and pA7a-BLpad into host BL21(DE3) by heat shock or electric shock, Styrosol 3 is prepared by transforming plasmids pET28a-StyAB-RostyC-SlPAR1 and pA7a-BLpad into host BL21(DE3) by heat shock or electric shock, Styrosol 4 is prepared by transforming plasmids pET 28-StyAB-RostyC-Yqhd and 467-BLpad into host BL 3 (DE3) by heat shock or electric shock, Styrosol 5-RostyC-Blpad into host BL 599 (DE3) by heat shock or electric shock, Styrosol 5-RostyC-BLpad is prepared by transforming plasmid pET 28-RosyAB 9 into host BL 599 (DE 1) by heat shock or electric shock BL21(DE3) was prepared, Styrosol 7 was prepared from plasmids pET28 a-StyAB-RostyrC and pA7a-BLPad-yqhD by heat shock or electric shock transformation into host BL21(DE3), and Styrosol8 was prepared from plasmids pET28 a-StyAB-RostyrC and pA7a-BLPad-YahK by heat shock or electric shock transformation into host BL21(DE 3).
(2) Bioconversion synthesis of tyrosol (the synthesis scheme is shown in FIG. 14), inoculating 8 prepared biocatalysts into 2mL LB, activating at 37 deg.C for 10-12h, transferring the activated biocatalysts into 100mL LB at a ratio of 1:100, and allowing OD to stay600When the concentration reaches 0.6-0.8, adding inducer IPTG to the final concentration of 0.5mM, inducing at 22 ℃ for 10-12h, centrifuging at 4 ℃ for 10min at 4000g, collecting thallus, resuspending in M9Y culture medium, adding 2.0g/L p-coumaric acid and 10g/L glucose, and transforming at 37 ℃ for 12 h. The results of tyrosol synthesis by 8 biocatalysts are shown in FIG. 8, and it can be seen that tyrosol 3 and Styrosol 7 synthesize the most tyrosol, respectively 1.63 and 1.64 g/L. In addition, 2.5g/L p-coumaric acid was catalyzed by the biocatalyst, Styrosol 7, and as a result, 2.04g/L tyrosol was obtained after 24 hours, and the conversion rate was 97.4%, as shown in FIG. 9.
Example 5
Bioconversion to synthesize interest alcohol (the synthesis scheme is shown in figure 14)
The biocatalyst Styrosol 7 prepared in example 4 was inoculated into 2mL LB for activation at 37 ℃ for 10-12h, and then the activated biocatalyst was transferred into 100mL LB at a ratio of 1:100 until OD reached600When the concentration reaches 0.6-0.8, adding inducer IPTG to final concentration of 0.5mM, inducing at 22 deg.C for 10-12 hr, and cooling to 4 deg.CAnd centrifuging at 4000g for 10min, collecting thallus, resuspending in M9Y culture medium, adding 1.5g/L ferulic acid and 10g/L glucose, and converting at 37 deg.C for 12 h. The results of the synthesis of the higher linalool are shown in FIG. 9, with 1.18g/L of product obtained after the end of the conversion, with a conversion of about 92%.
Example 6
(1) Construction of biocatalyst for synthesizing salidroside
Plasmids pET28a-StyAB-RostyC-7-SlPAR1 and pA7a-BLPad-UGT85A1 were constructed and co-transformed into BL21(DE3) to obtain biocatalyst E.coli (BLPad-StyAB-RostyC-SlPAR1-UGT85A 1). The specific method comprises the following steps: PCR amplification is carried out to obtain gene sequences BLPad, StyAB, RostyC, SlPAR1 and UGT85A1 synthesized by the biological company, StyAB, RostyC and SlPAR1 are cut and connected into a vector pET28a to obtain pET28a-StyAB-RostyC-SlPAR1, and BLPad and UGT85A1 are cut and connected into a vector pA7a to obtain pA7a-BLPad-UGT85A 1. Plasmids pET28a-StyAB-RostyC-SlPAR1 and pA7a-BLPad-UGT85A1 were transformed into host BL21(DE3) by heat shock or electric shock to prepare biocatalyst E.coli (BLPad-StyAB-RostyC-SlPAR1-UGT85A 1).
(2) Bioconversion synthesis of Salidroside (the synthetic scheme is shown in FIG. 14)
Inoculating the prepared biocatalyst E.coli (BLPad-StyAB-RostyC-SlPAR1-UGT85A1) into 2mL LB for activating at 37 ℃ for 10-12h, then transferring the activated biocatalyst into 100mL LB according to the ratio of 1:100 until OD is reached600When the concentration reaches 0.6-0.8, adding inducer IPTG to the final concentration of 0.5mM, inducing at 22 ℃ for 10-12h, centrifuging at 4 ℃ for 10min at 4000g, collecting thallus, resuspending in M9Y culture medium, adding 2g/L p-coumaric acid and 10g/L glucose, and converting at 37 ℃ for 24 h. At 12, 10g/L glucose was additionally fed. As a result, as shown in FIG. 10, salidroside was finally synthesized at 1.72g/L with a conversion of 48.3%.
Example 7
(1) Construction of biocatalysts for Hydroxytyrosol Synthesis
Plasmids pET28a-StyAB-RostyC-SlPAR1 and pA7a-BLPad-HpaBC were constructed and co-transformed into BL21(DE3) to obtain biocatalyst E.coli (BLPad-StyAB-RostyC-SlPAR 1-HpaBC). The specific method comprises the following steps: HpaBC on a BL21(DE3) genome and gene sequences BLPad, StyAB, RostyrC and SlPAR1 synthesized by a biological company are obtained by PCR amplification, StyAB, RostyrC and SlPAR1 are subjected to enzyme digestion and connected to enter a vector pET28a to obtain pET28 a-StyAB-RostyrC-SlPAR 1, and HpaBC and BLPad are subjected to enzyme digestion and connected to enter a vector pA7a to obtain pA7 a-BLPad-HpaBC. Plasmids pET28a-StyAB-RostyC-SlPAR1 and pA7a-BLPad-HpaBC were transformed into host BL21(DE3) by heat shock or electric shock, and biocatalyst E.coli (BLPad-StyAB-RostyC-SlPAR1-HpaBC) was prepared.
(2) Biosynthesis of hydroxytyrosol (the synthetic scheme is shown in figure 14)
Inoculating the prepared biocatalyst E.coli (BLPad-StyAB-RostyC-SlPAR1-HpaBC) into 2mL LB to activate 37 ℃ for 10-12h, then transferring the activated biocatalyst into 100mL LB according to the ratio of 1:100 until OD is reached600When the concentration reaches 0.6-0.8, adding inducer IPTG to the final concentration of 0.5mM, inducing at 22 ℃ for 10-12h, centrifuging at 4 ℃ for 10min at 4000g, collecting thallus, resuspending in M9Y culture medium, adding 2g/L p-coumaric acid and 10g/L glucose, and converting at 37 ℃ for 8 h. As a result, as shown in FIG. 11, hydroxytyrosol was synthesized at 1.83g/L with a conversion of 97.5%.
The invention has many applications, and the above description is only a preferred embodiment of the invention. It should be noted that the above examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications can be made without departing from the principles of the invention and these modifications are to be considered within the scope of the invention.
Claims (10)
1. A method for biosynthesizing high value-added compounds by utilizing lignocellulose derivatives is characterized by comprising the following steps:
A. transforming escherichia coli to obtain a biocatalyst;
B. synthesizing a compound with a high added value by taking a lignocellulose derivative as an initial raw material through a biocatalyst;
the lignocellulose derivative comprises at least one of p-coumaric acid and ferulic acid;
the high value-added compound comprises at least one of gastrodin, arbutin, salidroside and derivatives thereof, hydroquinone, tyrosol, hydroxytyrosol and benzoin alcohol.
2. The method for biosynthesizing a high added-value compound from a lignocellulose derivative as recited in claim 1, wherein in step A, said biocatalyst is E.coli (Fcs-Ech-SlPAR1-UGT73B 6)FS) One of E.coli (Fcs-Ech-Vdh-MNX1-AS), E.coli (Fcs-Ech-Vdh-MNX1), E.coli (BLPad-StyAB-RostyC-SlPAR1-UGT85A1), E.coli (BLPad-StyAB-RostyC-PAR Sl 1), E.coli (BLPad-StyAB-RostyC-Yqhd), E.coli (BLPad-StyAB-RostyC-Yahk) and E.coli (BLPad-StyAB-RostyC-SlPAR 1-HpaBC).
3. The method for the biosynthesis of high value-added compounds using lignocellulose derivatives as recited in claim 2, wherein said e.coli (Fcs-Ech-SlPAR1-UGT73B 6)FS) The obtaining method comprises the following steps: coli by overexpressing Fcs, Ech from Pseudomonas putida KT2440, SlPAR1 from Solanum lycopersicum and the mutase UGT73B6 from the plant RhodiolaFSAnd (5) realizing.
4. The method for biosynthesizing a high added-value compound by utilizing a lignocellulose derivative AS recited in claim 2, wherein said E.coli (Fcs-Ech-Vdh-MNX1-AS) is obtained by the following steps: by overexpressing in e.coli Fcs, Ech and Vdh derived from p.pudida KT2440, MNX1 derived from yeast Candida parapsilosis CDC317 and arbutin synthase AS derived from Rauvolfia serpentina;
the method for obtaining the E.coli (Fcs-Ech-Vdh-MNX1) comprises the following steps: this was achieved by overexpressing Fcs, Ech and Vdh derived from p.putida KT2440, MNX1 derived from the yeast Candida parapsilosis CDC317, in e.coli.
5. The method for the biosynthesis of high added value compounds using lignocellulose derivatives as recited in claim 2, wherein said E.coli (BLPad-StyAB-RostyC-SlPAR1-UGT85A1) is obtained by: (ii) by overexpressing in e.coli the decarboxylase BLPad derived from Bacillus licheniformis CGMCC7172, stylab derived from Pseudomonas sp.strain VLB120, rosetc derived from Rhodococcus opacus 1CP, SlPAR1 derived from Solanum lycopersicum, and the glycosyltransferase UGT85a1 derived from Arabidopsis thaliana;
the method for obtaining E.coli (BLPad-StyAB-RostyC), E.coli (BLPad-StyAB-RostyC-SlPAR1), E.coli (BLPad-StyAB-RostyC-YqhD) and E.coli (BLPad-StyAB-RostyC-YahK) is as follows: by overexpressing in e.coli the decarboxylase BLPad derived from Bacillus licheniformis CGMCC7172, StyAB derived from Pseudomonas sp.strain VLB120, RostyC derived from Rhodococcus opacus 1CP, YqhD or YahK derived from e.coli BL21(DE3) or par1 derived from Solanum lycopersicum;
the method for obtaining the E.coli (BLPad-StyAB-RostyC-SlPAR1-HpaBC) comprises the following steps: this is achieved by overexpressing in E.coli the decarboxylase BLPad derived from Bacillus licheniformis CGMCC7172, StyAB derived from Pseudomonas sp.strain VLB120, RostyrC derived from Rhodococcus opacus 1CP, SlPAR1 derived from Solanum dipliscum and HpaBC derived from E.coli BL21(DE 3).
6. The method for biosynthesizing a high added-value compound from a lignocellulose derivative as recited in claim 2, wherein said biocatalyst is eFS) And (C) in the step (B), the lignocellulose derivative adopted is p-coumaric acid, and the synthesized high-added-value compound is gastrodin.
7. The method for biosynthesizing a high added-value compound from a lignocellulose derivative according to claim 2, wherein when the biocatalyst is e.coli (Fcs-Ech-Vdh-MNX1-AS), the lignocellulose derivative used in step B is p-coumaric acid, and the synthesized high added-value compound is arbutin;
when the biocatalyst is E.coli (Fcs-Ech-Vdh-MNX1), the lignocellulose derivative adopted in the step B is p-coumaric acid, and the synthesized compound with high added value is hydroquinone.
8. The method for biosynthesizing a high added-value compound from a lignocellulose derivative as recited in claim 2, wherein when the biocatalyst is e.coli (BLPad-StyAB-rosetyc-SlPAR 1-UGT85a1), the p-coumaric acid used in step B, the synthesized high added-value compound is salidroside;
when the biocatalyst is E.coli (BLPad-StyAB-RostyC), E.coli (BLPad-StyAB-RostyC-SlPAR1), E.coli (BLPad-StyAB-RostyC-YqhD) or E.coli (BLPad-StyAB-RostyC-YahK), the lignin derivative adopted in the step B is p-coumaric acid or ferulic acid, and the synthesized high value-added compound is tyrosol or high-interest cumarol;
and when the biocatalyst is E.coli (BLPad-StyAB-RostyC-SlPAR1-HpaBC), the lignin derivative adopted in the step B is p-coumaric acid, and the synthesized high value-added compound is hydroxytyrosol.
9. The biocatalyst for biosynthesis of high value-added compounds is characterized by being modified escherichia coli, and specifically comprising E.coli (Fcs-Ech-SlPAR1-UGT73B 6)FS) One of e.coli (Fcs-Ech-Vdh-MNX1-AS), e.coli (Fcs-Ech-Vdh-MNX1), e.coli (BLPad-StyAB-rosetc-SlPAR 1-UGT85a1), e.coli (BLPad-StyAB-rosetc-par sl 1), e.coli (BLPad-StyAB-rosetc-YqhD), e.coli (BLPad-StyAB-rosetc-YahK), and e.coli (BLPad-StyAB-rosetc-SlPAR 1-HpaBC).
10. The biocatalyst for the biosynthesis of high value-added compounds according to claim 9, wherein said high value-added compounds comprise at least one of gastrodine, arbutin, salidroside and its derivatives hydroquinone, tyrosol, hydroxytyrosol and benzoin alcohol.
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| CN113462626B (en) * | 2021-06-24 | 2024-06-07 | 上海交通大学 | Expression optimization of multi-subunit enzymes for monolignol synthesis of high value-added compounds |
| CN113897325A (en) * | 2021-11-05 | 2022-01-07 | 江南大学 | Recombinant escherichia coli for producing salidroside and construction method and application thereof |
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| EP4349843A4 (en) * | 2021-11-10 | 2024-04-17 | Shandong Henglu Biotech Co Ltd | Alpha-salidroside, and preparation method therefor and application thereof |
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| CN114438149A (en) * | 2022-01-18 | 2022-05-06 | 上海交通大学 | Method for synthesizing benzylisoquinoline alkaloid by constructing biological cascade reaction |
| CN114891820A (en) * | 2022-05-28 | 2022-08-12 | 湖北大学 | Bacillus licheniformis for efficiently synthesizing hydroxytyrosol, construction method and application |
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