CN112481336B - Method for biosynthesis of compounds using lignocellulose derivatives - 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 a 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, multiple 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 reproducible glucose has attracted increasing attention. Chinese patent 201910882020.9 discloses a method for synthesizing gastrodin by a heterologous metabolic pathway. The method uses recombinant Escherichia coli including pCDFDuet-aroG-ppsA-pgm-galU and pETDuet-ubiC-CAR-Sfp-ugt73b6 FS Two 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 265mg/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 Monooxogene, angewandte Chemie International Edition, 2019, 58, 764-768.) it is reported that the mutant strain A82F/A328F obtained by modifying P450-MB3 Monooxygenase can catalyze the Dihydroxylation of 10 mM Benzene to synthesize 9.2 mM Hydroquinone.
Arbutin (Arbutin), a glycosylated hydroquinone, is found in the plants, rubus ursinus, wheat, etc. It is a skin whitening agent, and has antibacterial, antiinflammatory and antioxidant biological activities. 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. Transformation of Escherichia coli by metabolic pathway to accumulate p-hydroxybenzoic acid, and heterologous expression of p-hydroxybenzoic acidCandida parapsilosis4-hydroxybenzoic acid 1-hydroxylase (MNX 1) of CBS604 and a derivative thereofRauvolfia serpentinaThe Arbutin Synthase (AS) takes 30 g/L glucose AS raw material to obtain 4.19 g/L arbutin. Chinese patent 201510107788.0 discloses a method for enriching and purifying arbutin from blueberries, which comprises the steps of firstly extracting alpha-arbutin by adopting high-voltage pulse electric field extraction equipment, then carrying out primary purification of alpha-arbutin by utilizing 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 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, monopotassium 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. 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 the filtrate by a macroporous resin column, and concentrating the filtrate under reduced pressure; dissolving with anhydrous alcohol, and separating by simulated moving bed chromatography to obtain component rich in salidroside; and finally, concentrating, crystallizing, centrifuging and drying to obtain the salidroside product. Chinese patent 201810753390.8 discloses a method for synthesizing salidroside by fermentation. The method comprises the step of integrating a keto decarboxylase gene skdc with a trc promoter and a glycosyltransferase gene sugt2 with a tac promoter onto a SyBE-002447 chromosome to obtain an SDR1 strain. Fermenting the SDR1 strain with glucose as precursor for 36h to obtain 0.7g/L salidroside.
Tyrosol (Tyrosol, p-hydroxyphenylethanol), a phenolic compound, is naturally present 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 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 ultramicro crushing 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 invention patent 201910882020.9 discloses a method for synthesizing hydroxytyrosol through 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 construction method of Escherichia coli for 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 (DE 3) 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 by means of genetic engineering, constructs 3 new enzymatic reaction paths, and uses aromatic compound (p-coumaric acid) derived from lignocellulosep-coumaric acid) and ferulic acid (ferulic acid) to efficiently synthesize various compounds with high added values including gastrodin, arbutin, salidroside and the like, 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 isE.coli (Fcs-Ech-SlPAR1-UGT73B6 FS )、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)、E.coli (BLPad-StyAB-RostyC-SlPAR1)、E.coli (BLPad-StyAB-RostyC-YqhD)、E.coli(BLPad-StyAB-RostyC-YahK) andE.coli(BLPad-StyAB-RostyC-SlPAR 1-HpaBC).
Preferably, theE.coli (Fcs-Ech-SlPAR1-UGT73B6 FS ) The obtaining method comprises the following steps: by being arranged onE.coliWherein the overexpression is derived fromPseudomonas putidaFcs and Ech of KT2440 derived fromSolanum lycopersicumAnd SlPAR1 and plant-derived productsRhodiolaThe mutant enzyme UGT73B6 FS And (5) realizing.
Preferably, theE.coli The method for obtaining (Fcs-Ech-Vdh-MNX 1-AS) comprises the following steps: by being atE.coliWherein the overexpression is derived fromP. putidaFcs, ech and Vdh of KT2440 from yeastCandida parapsilosisMNX1 from CDC317 and MNX derived fromRauvolfia serpentinaThe arbutin synthase AS of (1);
the above-mentionedE.coli The method for obtaining (Fcs-Ech-Vdh-MNX 1) comprises the following steps: by being atE.coliIn the overexpression comes fromP. putidaFcs, ech and Vdh of KT2440 from yeastCandida parapsilosisMNX1 implementation of CDC 317.
Preferably, theE.coliThe method for obtaining (BLPad-StyAB-RostyC-SlPAR 1-UGT85A 1) comprises the following steps: by being arranged onE.coliIn the overexpression comes fromBacillus licheniformisDecarboxylase BLPad of CGMCC7172, derived fromPseudomonas sp. strainStyAB from VLB120, rostyC from Rhodococcus opacus 1CP, from Rhodococcus opacus 1CPSolanum lycopersicumAnd the implementation is derived fromArabidopsis thalianaThe glycosidase of (1), UGT85A1;
the above-mentionedE.coli (BLPad-StyAB-RostyC)、E.coli (BLPad-StyAB-RostyC-SlPAR1)、E.coli (BLPad-StyAB-RostyC-YqhD)、E.coliThe method for obtaining (BLPad-StyAB-RostyC-YahK) is as follows: by being arranged onE.coliWherein the overexpression is derived fromBacillus licheniformisDecarboxylase BLPad of CGMCC7172, derived fromPseudomonas StyAB of strain VLB120, derived fromRhodococcus opacusRostyC of 1CP, derived fromE. coliYqhD or YahK of BL21 (DE 3) or fromSolanum lycopersicumThe SlPAR1 implementation of (3);
the describedE.coliThe method for obtaining (BLPad-StyAB-RostyC-SlPAR 1-HpaBC) comprises the following steps: by being arranged onE.coliWherein the overexpression is derived fromBacillus licheniformisDecarboxylase BLPad of CGMCC7172, derived fromPseudomonas sp. strainStyAB of VLB120, derived fromRhodococcus opacus 1CP RostyC, derived fromSolanum lycopersicumSlPAR1 and derivatives fromE. coliHpaBC implementation of BL21 (DE 3).
Preferably, the biocatalyst isE.coli (Fcs-Ech-SlPAR1-UGT73B6 FS ) And D, in the step B, the adopted lignocellulose derivative is p-coumaric acid, and the synthesized high-added-value compound is gastrodin.
Preferably, the biocatalyst isE.coli (Fcs-Ech-Vdh-MNX 1-AS), the lignocellulose derivative adopted in the step B is p-coumaric acid, and the synthesized high value-added compound is arbutin;
the biocatalyst isE.coli (Fcs-Ech-Vdh-MNX 1), the lignocellulose derivative adopted in the step B is p-coumaric acid, and the synthesized high value-added compound is hydroquinone.
Preferably, the biocatalyst isE.coli(BLPad-StyAB-RostyC-SlPAR 1-UGT85A 1), the lignocellulose derivative adopted in the step B is p-coumaric acid, and the synthesized high value-added compound is salidroside;
the biocatalyst isE.coli (BLPad-StyAB-RostyC)、E.coli (BLPad-StyAB-RostyC-SlPAR1)、E.coli (BLPad-StyAB-RostyC-YqhD)、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 benzoin alcohol;
the biocatalyst isE.coli(BLPad-StyAB-RostyC-SlPAR 1-HpaBC), the lignocellulose derivative adopted in 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 comprisesE.coli (Fcs-Ech-SlPAR1-UGT73B6 FS )、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)、E.coli (BLPad-StyAB-RostyC-SlPAR1)、E.coli (BLPad-StyAB-RostyC-YqhD)、E.coli(BLPad-StyAB-RostyC-YahK) andE.coli(BLPad-StyAB-RostyC-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 alcohol.
Preferably, theE.coli (Fcs-Ech-SlPAR1-UGT73B6 FS ) The obtaining method comprises the following steps: by being arranged onE.coliWherein the overexpression is derived fromPseudomonas putidaFcs and Ech of KT2440 derived fromSolanum lycopersicumOf SlPAR1 and of plant originRhodiolaThe mutant enzyme UGT73B6 FS And (5) realizing.
Preferably, theE.coli The method for obtaining (Fcs-Ech-Vdh-MNX 1-AS) comprises the following steps: by being atE.coliWherein the overexpression is derived fromP. putidaFcs, ech and Vdh of KT2440 from yeastCandida parapsilosisMNX1 from CDC317 and MNX derived fromRauvolfia serpentinaThe arbutin synthase AS;
the above-mentionedE.coli The method for obtaining (Fcs-Ech-Vdh-MNX 1) comprises the following steps: by being atE.coliIn the overexpression comes fromP. putidaFcs, ech and Vdh of KT2440 from yeastCandida parapsilosisMNX1 implementation of CDC 317.
Preferably, theE.coliThe method for obtaining (BLPad-StyAB-RostyC-SlPAR 1-UGT85A 1) comprises the following steps: by being atE.coliWherein the overexpression is derived fromBacillus licheniformisDecarboxylase BLPad of CGMCC7172, derived fromPseudomonas sp. strainStyAB of VLB120, derived fromRhodococcus opacus1CP RostyC, derived fromSolanum lycopersicumSlPAR1, and implementation derived fromArabidopsis thalianaThe glycoside transferase UGT85A1;
the above-mentionedE.coli (BLPad-StyAB-RostyC)、E.coli (BLPad-StyAB-RostyC-SlPAR1)、E.coli (BLPad-StyAB-RostyC-YqhD)、E.coliThe method for obtaining (BLPad-StyAB-RostyC-YahK) is as follows: by being arranged onE.coliWherein the overexpression is derived fromBacillus licheniformisDecarboxylase BLPad of CGMCC7172, derived fromPseudomonas sp. strainStyAB from VLB120, rostyC from Rhodococcus opacus 1CP, styAB from Rhodococcus opacus 1CPE. coliYqhD or YahK of BL21 (DE 3) or derived fromSolanum lycopersicumThe SlPAR1 implementation of (a);
the above-mentionedE.coliThe method for obtaining (BLPad-StyAB-RostyC-SlPAR 1-HpaBC) comprises the following steps: by being arranged onE.coliIn the overexpression comes fromBacillus licheniformisDecarboxylase BLPad of CGMCC7172, derived fromPseudomonas sp. strainStyAB from VLB120, rostyC from Rhodococcus opacus 1CP, styAB from Rhodococcus opacus 1CPSolanum lycopersicumAnd SlPAR1 and derivatives thereofE. coliHpaBC implementation of BL21 (DE 3). Compared with the prior art, the bookThe invention has the following beneficial effects:
(1) The invention obtains the high-efficiency biocatalyst by means of genetic engineering, and can rapidly catalyze substrates p-coumaric acid and ferulic acid to synthesize various high value-added compounds containing 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 derivative 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 invention.
The method of the following embodiment comprises:
step one, transforming escherichia coli to obtain a high-efficiency biocatalyst (E.coli (Fcs-Ech-SlPAR1-UGT73B6 FS ) Successfully synthesizing gastrodin from p-coumaric acid;
step two, transforming escherichia coli to obtain high-efficiency biocatalyst (E.coli (Fcs-Ech-Vdh-MNX 1-AS) andE.coli (Fcs-Ech-Vdh-MNX 1)), and using p-coumaric acid as a starting material, arbutin and hydroquinone derivative thereof are successfully synthesized;
step three, transforming the escherichia coli to obtain a plurality of high-efficiency biocatalysts (E.coli (BLPad-StyAB-RostyC-SlPAR1-UGT85A1)、 E.coli (BLPad-StyAB-RostyC)、E.coli (BLPad-StyAB-RostyC-SlPAR1)、E.coli (BLPad-StyAB-RostyC-YqhD)、E.coli(BLPad-StyAB-RostyC-YahK) andE.coli(BLPad-StyAB-RostyC-SlPAR 1-HpaBC)) and the like, and p-coumaric acid and ferulic acid are used as starting materials to successfully synthesize salidroside, tyrosol, hydroxytyrosol and benzoin alcohol;
in the first step, the biocatalyst (A)E.coli (Fcs-Ech-SlPAR1-UGT73B6 FS ) Is constructed byE.coliWherein the overexpression is derived fromPseudomonas putidaFcs and Ech of KT2440 are derived fromSolanum lycopersicumAnd SlPAR1 and plant-derived productsRhodiolaThe mutant enzyme UGT73B6 FS And (5) realizing. In this process, first, a construction is madeE.coli (Fcs-Ech-SlPAR 1) catalyzes p-coumaric acid to synthesize p-hydroxybenzyl alcohol, and UGT73B6 is introduced FS Constructing a new catalyst to catalyze p-hydroxybenzyl alcohol to synthesize gastrodin;
in the second step, the biocatalyst: (E.coli (Fcs-Ech-Vdh-MNX 1-AS) andE.coli (Fcs-Ech-Vdh-MNX 1)) is constructed byE.coliWherein the overexpression is derived fromP. putidaFcs, ech and Vdh of KT2440, derived from yeastCandida parapsilosisMNX1 from CDC317 and MNX derived fromRauvolfia serpentinaThe arbutin synthase AS. In this process, first, a construction is madeE.coli (Fcs-Ech-Vdh-MNX 1) to catalyze the substrate to the coumaric acid to synthesize the arbutin precursor substance, namely benzenediol, and then introduce AS to construct a plurality of biocatalysts to efficiently synthesize arbutin.
In step three, the biocatalyst (A)E.coli (BLPad-StyAB-RostyC-SlPAR1-UGT85A1)、E.coli (BLPad-StyAB-RostyC)、E.coli (BLPad-StyAB-RostyC-SlPAR1)、E.coli (BLPad-StyAB-RostyC-YqhD)、E.coli(BLPad-StyAB-RostyC-YahK) andE.coli(BLPad-StyAB-RostyC-SlPAR 1-HpaBC)) and the like byE.coliIn the overexpression comes fromBacillus licheniformisCGMCC7172 decarboxylase BLPad derived fromPseudomonas sp. strainStyAB from VLB120, rostyC from Rhodococcus opacus 1CP, from Rhodococcus opacus 1CPArabidopsis thalianaThe glycoside transferase UGT85A1 is derived fromE. coliHpaBC, yqhd and Yahk of BL21 (DE 3) and derivatives thereofSolanum lycopersicumThe SlPAR1 implementation of (1). In this process, a plurality of biocatalysts are first constructedE.coli (BLPad-StyAB-RostyC)、E.coli (BLPad-StyAB-RostyC-SlPAR1)、E.coli (BLPad-StyAB-RostyC-YqhD)、E.coli(BLPad-StyAB-RostyC-YahK) catalyzing p-coumaric acid to synthesize tyrosol, selecting out the most suitable catalyst, and then introducing UGT85A1 or HpaBC 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 plasmidpA7a-UGT73B6 FS Transforming into BL21 (DE 3) bacteria to construct biocatalystE.coli (Fcs-Ech-SlPAR1-UGT73B6 FS ). The specific method comprises the following steps:
fromPseudomonas putidaOn a KT2440 genome, obtaining Fcs and Ech by PCR cloning, and constructing the Fcs and the Ech on a carrier pET28a together with SlPAR1 to obtain a plasmid pET28a-Fcs-Ech-SlPAR1; UGT73B6 Fs Constructing on a carrier pA7a to obtain a plasmid pA7a-UGT73B6 FS (ii) a Plasmid pET28a-Fcs-Ech-SlPAR1 and plasmid pA7a-UGT73B6 FS Transforming into host BL21 (DE 3) by heat shock or electric shock method to obtain biocatalystE.coli (Fcs-Ech-SlPAR1-UGT73B6 FS )。
(2) Synthesizing gastrodin by biotransformation (the synthetic scheme is shown in figure 12)
The prepared biological catalystE.coli (Fcs-Ech-SlPAR1-UGT73B6 FS ) Inoculating the activated biocatalyst into 2 mL LB for activation, transferring the activated biocatalyst into 100 mL LB according to the proportion of 1 600 When reaching 0.6-0.8, adding inducer IPTG to final concentration of 0.5 mM, inducing at 22 deg.C for 10-12h, at 4 deg.C, 4000gCentrifuging for 10 min, collecting thallus, resuspending in M9Y culture medium, adding substrate 2 g/L p-coumaric acid and 10 g/L glucose, and converting at 37 deg.C for 12 hr to obtain 1.45 g/L gastrodin with conversion rate of 42.5% (FIG. 2).
Example 2
(1) Construction of biocatalyst for synthesizing hydroquinone
The plasmid pET28a-Fcs-Ech-Vdh and the plasmid pA7a-MNX1 are constructed and are jointly transferred into BL21 (DE 3) to obtain the biocatalystE.coli (Fcs-Ech-Vdh-MNX 1). The specific method comprises the following steps: fromPseudomonas putidaOn a KT2440 genome, obtaining Fcs, ech and Vdh by PCR cloning, entering a vector pET28a through enzyme digestion connection to construct pET28a-Fcs-Ech-Vdh, and entering a vector pA7a through enzyme digestion connection to construct pA7a-MNX1; the plasmid pET28a-Fcs-Ech-Vdh and the plasmid pA7a-MNX1 are transformed into a host BL21 (DE 3) by a heat shock or electric shock method to prepare the biocatalystE.coli (Fcs-Ech-Vdh-MNX1)。
(2) Biotransformation synthesis of hydroquinone (the synthetic scheme is shown in figure 13)
The prepared biological catalystE.coli (Fcs-Ech-Vdh-MNX 1) is inoculated into 2 mL of LB for activation, the activated biocatalyst is transferred into 100 mL of LB according to the proportion of 1 600 When reaching 0.6-0.8, adding inducer IPTG to final concentration of 0.5 mM, inducing at 22 deg.C for 10-12h, at 4 deg.C, 4000gCentrifuging for 10 min, collecting thallus, resuspending in M9Y culture medium, adding substrate 3 g/L p-coumaric acid and 10 g/L glucose, and converting at 37 deg.C for 8 hr. As a result, as shown in FIG. 3, 1.95 g/L hydroquinone was obtained with a conversion of 97% (FIG. 3).
Example 3
(1) Constructing biocatalyst for synthesizing arbutin (construction schematic shown in FIG. 4)E.coli (Fcs-Ech-Vdh-MNX 1-AS) catalyst, SArbutin5 of which is the most effective.
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 a T7 promoter is added in front of an MNX1 gene relative to pA7a-AS-MNX 1) are constructed, and the two are combined and transformed into BL21 (DE 3) to obtain 5 biocatalysts, namely SArbutin1, SArbutin2, SArbutin3, SArbutin4 and SArbutin5. The specific method comprises the following steps:
fromPseudomonas putidaOn a KT2440 genome, fcs and Ech are obtained by PCR cloning, and Vdh enters a vector pET28a through enzyme digestion connection to construct pET28a-Fcs-Ech and pET28a-Fcs-Ech-Vdh; MNX1 and AS enter a carrier pA7a or pACYC through enzyme digestion connection to construct pA7a-MNX1-AS, pA7a-AS-MNX1, pA7a-AS-7-MNX1; vdh, MNX1 and AS obtained by PCR cloning enter a vector pA7a through enzyme digestion connection to construct pA7a-Vdh-MNX1-AS.
5 biocatalysts SArbutin1 is prepared by transforming plasmids pET28a-Fcs-Ech and pA7a-Vdh-MNX1-AS into a host BL21 (DE 3) by a heat shock or electric shock method, SArbutin2 is prepared by transforming plasmids pET28a-Fcs-Ech-Vdh and pA7a-MNX1-AS into the host BL21 (DE 3) by a heat shock or electric shock method, SArbutin3 is prepared by transforming plasmids pET28a-Fcs-Ech-Vdh and pA7a-AS-MNX1 into the host BL21 (DE 3) by a heat shock or electric shock method, SArbutin4 is prepared by transforming plasmids pET28a-Fcs-Ech and pA7a-AS-7-MNX1 into the host BL21 (DE 3) by a heat shock or electric shock method, and SArbutin5 is prepared by transforming plasmid pET28 a-Fcs-Ech-MNX 1 and pA7a-AS-MNX1 into the host BL21 (DE 3) by a heat shock or electric shock method.
(2) Biotransformation synthesis of arbutin (the synthetic scheme is shown in FIG. 13)
Inoculating the prepared 5 biocatalysts into 2 mL LB for activation, transferring the activated biocatalysts into 100 mL LB according to the proportion of 1 600 When the concentration reaches 0.6-0.8, adding an inducer IPTG to a final concentration of 0.5 mM, inducing at 22 ℃ for 10-12h, centrifuging at 4 ℃ for 10 min at 4000 g, collecting thalli, resuspending in an M9Y culture medium, adding 2 g/L p-coumaric acid and 10 g/L glucose, and converting at 37 ℃ 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 of 2.34 g/L. Wherein, the result of the change of the conversion of the biocatalyst SArbutin5 into arbutin along with the conversion time by adopting the method is shown in figure 6, 3.05 g/L of arbutin can be obtained after 24 hours, and the conversion rate is 92%.
Example 4
(1) Construction of biocatalystsE.coli (BLPad-StyAB-RostyC)、E.coli (BLPad-StyAB-RostyC-SlPAR1)、E.coli (BLPad-StyAB-RostyC-YqhD)、E.coli (BLPad-StyAB-RostyC-YahK)、E.coli (BLPad-StyAB-RostyC-SlPAR 1) for tyrosol synthesis (schematic construction scheme 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-SlPAR1, pA7a-BLpad-YqhD, pA7a-BLpad-YahK were constructed. The two-by-two combinations thereof were transformed into BL21 (DE 3) to obtain 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 (DE 3) genome are amplified by PCR, combined with StyAB, rostyC, BLpad and SlPAR1, and the StyAB and RostyC are connected by enzyme digestion and enter a vector pET28a to construct pET28a-StyAB-RostyC; styAB, rostyC and YahK or YqhD or SlPAR1 are connected by enzyme digestion and enter the carrier pET28a to construct pET28a-StyAB-RostyC-BLPad to construct pET28a-StyAB-RostyC-YqhD or pET28a-StyAB-RostyC-YahK or pET28a-StyAB-RostyC-SlPAR1; and (3) carrying out enzyme digestion connection on the BLPad or the BLPad and YqhD or YahK or SlPAR1 to enter a vector pA7a to construct pA7a-BLPad, pA7a-BLPad-SlPAR1, pA7a-BLPad-YqhD, pA7a-BLPad-YahK.
The biocatalyst Styrosol 1 is prepared by transforming plasmid pET28 a-StyAB-RostyryC-BLpad into host BL21 (DE 3) by heat shock or electric shock, styrosol 2 is prepared by transforming plasmids pET28 a-StyAB-RostyrC and pA7a-BLpad into host BL21 (DE 3) by heat shock or electric shock, styrosol 3 is prepared by transforming plasmids pET28 a-StyAB-RostyrC-SlPAR 1 and pA7a-BLpad into host BL21 (DE 3) by heat shock or electric shock, styrosol 4 is prepared by transforming plasmids pET28 a-StyAB-RostyrC-Yqhd and pA7a-BLpad into host BL21 (DE 3) by heat shock or electric shock, styrosol 5 is prepared by transforming plasmid pET28 a-StyAB-YastyC-Yayd and pA7a-BLpad into host BL21 (DE 3) by heat shock or electric shock, styrosol 5 is prepared by transforming plasmid pET28 a-StyAB-YaatyC-Yapad 7a-Blpad 3 into host BL 3, and StyB 7a-BLpad 3 by heat shock or electric shock.
(2) Bioconversion synthesis of tyrosol (the synthesis schematic diagram is shown in fig. 14), 8 prepared biocatalysts are inoculated into 2 mL of LB to be activated for 37 ℃, after 10-12h, the activated biocatalysts are transferred into 100 mL of LB according to the proportion of 1 600 When reaching 0.6-0.8, adding inducer IPTG to the final concentration of 05 mM, inducing at 22 deg.C for 10-12h, centrifuging at 4 deg.C for 10 min at 4000 g, collecting thallus, resuspending in M9Y culture medium, adding 2.0g/L p-coumaric acid and 10 g/L glucose, and converting at 37 deg.C 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, 2.5 g/L p-coumaric acid was catalyzed by the biocatalyst, styrosol 7, and as a result, 2.04 g/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 2 mL LB for activation at 37 ℃ for 10-12h, and then the activated biocatalyst was transferred into 100 mL LB at a ratio of 1 600 When the concentration reaches 0.6-0.8, adding inducer IPTG to final concentration of 0.5 mM, inducing at 22 deg.C for 10-12h, centrifuging at 4 deg.C for 10 min at 4000 g, collecting thallus, resuspending in M9Y culture medium, adding 1.5 g/L ferulic acid, 10 g/L glucose, and converting at 37 deg.C for 12 h. The results of the synthesis of higher linalool alcohol are shown in FIG. 9, with 1.18 g/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 used together
Conversion into BL21 (DE 3) to give the biocatalystE.coli(BLPad-StyAB-RostyC-SlPAR 1-UGT85A 1). The specific method comprises the following steps: PCR amplification is carried out to obtain synthetic gene sequences BLPad, styAB, rostyC, slPAR1 and UGT85A1 of the biological company, styAB, rostyC and SlPAR1 are connected into a carrier pET28a in an enzyme cutting mode to obtain pET28a-StyAB-RostyC-SlPAR1, BLPad and UGT85A1 are connected into a carrier pA7a in an enzyme cutting mode to obtain pA7a-BLPad-UGT85A1. Plasmids pET28a-StyAB-RostyC-SlPAR1 and pA7a-BLPad-UGT85A1 are transformed into a host BL21 (DE 3) by a heat shock or electric shock method to prepare the biocatalystE.coli (BLPad-StyAB-RostyC-SlPAR1-UGT85A1)。
(2) Bioconversion synthesis of Salidroside (the synthetic scheme is shown in FIG. 14)
The prepared biological catalystE.coli(BLPad-StyAB-RostyC-SlPAR 1-UGT85A 1) is inoculated into 2 mL LB to be activated at 37 ℃, after 10-12h, the activated biocatalyst is transferred into 100 mL LB according to the proportion of 1 600 When the concentration reaches 0.6-0.8, adding inducer IPTG to the final concentration of 0.5 mM, inducing at 22 ℃ for 10-12h, centrifuging at 4 ℃ for 10 min at 4000 g, collecting thalli, resuspending in M9Y culture medium, adding 2 g/L p-coumaric acid and 10 g/L glucose, and converting at 37 ℃ for 24 h. At 12, 10 g/L glucose was additionally fed. As a result, as shown in FIG. 10, salidroside was finally synthesized at 1.72 g/L with a conversion of 48.3%.
Example 7
(1) Construction of biocatalysts for the Synthesis of hydroxytyrosol
Plasmids pET28a-StyAB-RostyC-SlPAR1 and pA7a-BLPad-HpaBC are constructed and are jointly transformed into BL21 (DE 3) to obtain the biocatalystE.coli(BLPad-StyAB-RostyC-SlPAR 1-HpaBC). The specific method comprises the following steps: the HpaBC on the BL21 (DE 3) genome and gene sequences BLPad, styAB, rostyrC and SlPAR1 synthesized by a biological company are obtained by PCR amplification, styAB, rostyrC and SlPAR1 are cut and connected into a vector pET28a to obtain pET28 a-StyAB-RostyrC-SlPAR 1, and the HpaBC and BLPad are cut and connected into a vector pA7a to obtain pA7a-BLPad-HpaBC. The plasmids pET28a-StyAB-RostyC-SlPAR1 and pA7a-BLpad-HpaBC are transformed into a host BL21 (DE 3) by a heat shock or electric shock method to prepare the biocatalystE.coli (BLPad-StyAB-RostyC-SlPAR1-HpaBC)。
(2) Biosynthesis of hydroxytyrosol (the synthetic scheme is shown in figure 14)
The prepared biological catalystE.coli(BLPad-StyAB-RostyC-SlPAR 1-HpaBC) is inoculated into 2 mL LB for activation at 37 ℃, and after 10-12h, the activated biocatalyst is transferred into 100 mL LB according to the proportion of 1 600 When the concentration reaches 0.6-0.8, adding inducer IPTG to final concentration of 0.5 mM, inducing at 22 deg.C for 10-12h, centrifuging at 4 deg.C for 10 min at 4000 g, collecting thallus, resuspending in M9Y culture medium, adding 2 g/L p-coumaric acid, 10 g/LGlucose, at 37 ℃ for 8h. As a result, as shown in FIG. 11, hydroxytyrosol was synthesized at 1.83 g/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 (3)
1. A method for the biosynthesis of a compound using a lignocellulose derivative, comprising the steps of:
A. transforming escherichia coli to obtain a biocatalyst;
B. synthesizing a compound by taking a lignocellulose derivative as an initial raw material through a biocatalyst;
the lignocellulose derivative is p-coumaric acid;
the compound is one of salidroside, tyrosol and hydroxytyrosol;
in step A, the biocatalyst isE.coli (BLPad-StyAB-RostyC-SlPAR1-UGT85A1)、E.coli(BLPad-StyAB-RostyC-SlPAR1)、E.coli (BLPad-StyAB-RostyC-YqhD)、E.coli (BLPad-StyAB-RostyC-YahK)、E.coli (BLPad-StyAB-RostyC-SlPAR1-HpaBC)；
The biocatalyst isE.coli(BLPad-StyAB-RostyC-SlPAR 1-UGT85A 1), the lignocellulose derivative adopted in the step B is p-coumaric acid, and the synthesized compound is salidroside;
the biological catalyst is,E.coli (BLPad-StyAB-RostyC-SlPAR1)、E.coli (BLPad-StyAB-RostyC-YqhD)、E.coli(BLPad-StyAB-RostyC-YahK), the lignocellulosic derivative used in step B is p-coumaric acid, and the synthetic compound is tyrosol;
the biocatalyst isE.coli(BLPad-StyAB-RostyC-SlPAR 1-HpaBC), the lignocellulosic fiber used in step BThe vitamin derivative is p-coumaric acid, and the synthesized compound is hydroxytyrosol;
the above-mentionedE.coliThe method for obtaining (BLPad-StyAB-RostyC-SlPAR 1-UGT85A 1) comprises the following steps: by being atE.coliWherein the overexpression is derived fromBacillus licheniformisDecarboxylase BLPad of CGMCC7172, derived fromPseudomonas sp. strainStyAB of VLB120, derived fromRhodococcus opacusRostyC of 1CP, derived fromSolanum lycopersicumSlPAR1 and fromArabidopsis thalianaThe glycoside transferase UGT85A1;
the describedE.coli (BLPad-StyAB-RostyC-SlPAR1)、E.coli (BLPad-StyAB-RostyC-YqhD)、E.coliThe method for obtaining (BLPad-StyAB-RostyC-YahK) is as follows: by being arranged onE.coliWherein the overexpression is derived fromBacillus licheniformisDecarboxylase BLPad of CGMCC7172, derived fromPseudomonas sp. strainStyAB of VLB120, derived fromRhodococcus opacusRostyC of 1CP, derived fromE. coliYqhD or YahK of BL21 (DE 3) or derived fromSolanum lycopersicumThe SlPAR1;
the describedE.coliThe method for obtaining (BLPad-StyAB-RostyC-SlPAR 1-HpaBC) comprises the following steps: by being atE.coliWherein the overexpression is derived fromBacillus licheniformisDecarboxylase BLPad of CGMCC7172, derived fromPseudomonas sp. strainStyAB of VLB120, derived fromRhodococcus opacusRostyC of 1CP, derived fromSolanum lycopersicumAnd SlPAR1 and derivatives thereofE. coliHpaBC implementation of BL21 (DE 3).
2. The biocatalyst for the biosynthesis of compounds is characterized in that the biocatalyst is modified escherichia coli and specifically comprisesE.coli (BLPad-StyAB-RostyC-SlPAR1-UGT85A1)、E.coli (BLPad-StyAB-RostyC-SlPAR1)、E.coli (BLPad-StyAB-RostyC-YqhD)、E.coli(BLPad-StyAB-RostyC-YahK) andE.coli(BLPad-StyAB-RostyC-SlPAR 1-HpaBC);
the above-mentionedE.coliThe method for obtaining (BLPad-StyAB-RostyC-SlPAR 1-UGT85A 1) comprises the following steps: by being arranged onE.coliZhongchao watchDerived fromBacillus licheniformisDecarboxylase BLPad of CGMCC7172, derived fromPseudomonas sp. strainStyAB of VLB120, derived fromRhodococcus opacusRostyC of 1CP, derived fromSolanum lycopersicumSlPAR1 and fromArabidopsis thalianaThe glycoside transferase UGT85A1;
the above-mentionedE.coli (BLPad-StyAB-RostyC-SlPAR1)、E.coli (BLPad-StyAB-RostyC-YqhD)、E.coliThe method for obtaining (BLPad-StyAB-RostyC-YahK) is as follows: by being atE.coliWherein the overexpression is derived fromBacillus licheniformisDecarboxylase BLPad of CGMCC7172, derived fromPseudomonas sp. strainStyAB of VLB120, derived fromRhodococcus opacusRostyC of 1CP, derived fromE. coliYqhD or YahK of BL21 (DE 3) or derived fromSolanum lycopersicumThe SlPAR1;
the above-mentionedE.coliThe method for obtaining (BLPad-StyAB-RostyC-SlPAR 1-HpaBC) comprises the following steps: by being atE.coliWherein the overexpression is derived fromBacillus licheniformisDecarboxylase BLPad of CGMCC7172, derived fromPseudomonas sp. strainStyAB of VLB120, derived fromRhodococcus opacus1CP RostyC, derived fromSolanum lycopersicumSlPAR1 and derivatives fromE. coliHpaBC implementation of BL21 (DE 3).
3. The biocatalyst for the biosynthesis of a compound according to claim 2, wherein said compound comprises salidroside and at least one of tyrosol and hydroxytyrosol.
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| CN113897325B (en) * | 2021-11-05 | 2023-06-02 | 江南大学 | Recombinant escherichia coli for producing salidroside as well as construction method and application thereof |
| CN115819479A (en) * | 2021-11-10 | 2023-03-21 | 山东恒鲁生物科技有限公司 | Alpha-salidroside and preparation method and application thereof |
| CN114381484B (en) * | 2021-12-09 | 2024-04-19 | 山东大学 | Application of UGT85A1 or RrUGT3 in catalyzing various substrates to generate glycoside compounds |
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