Method for Biosynthesizing Compounds Using Lignocellulose Derivatives

Abstract

The present invention discloses a method for biosynthesizing compounds using lignocellulose derivatives, comprising the following steps: A. Modifying Escherichia coli to obtain a biocatalyst; B. Using lignocellulose derivatives as starting materials to synthesize compounds through the biocatalyst; the lignocellulose derivatives include at least one of p-coumaric acid and ferulic acid; the compounds include at least one of gastrodin, arbutin, salidroside and their derivatives hydroquinone, tyrosol, hydroxytyrosol and homovanillyl alcohol. The present invention modifies Escherichia coli by genetic engineering means, constructs 3 new enzymatic reaction pathways, and uses lignocellulose-derived aromatic compounds p-coumaric acid ( p -coumaric acid) and ferulic acid to efficiently synthesize various high-value-added compounds including gastrodin, arbutin, salidroside, etc., and the product yields all reach grams or more.

Description

Technical Field

[0001] This application is a divisional application of the invention patent with the application number "202011364142.8" and the invention title "Method for Biosynthesizing High-Value-Added Compounds Using Lignocellulose Derivatives".

[0002] The present invention belongs to the field of microbial technology and relates to a method for biosynthesizing compounds using lignocellulose derivatives; in particular, it relates to a method for biosynthesizing compounds using lignocellulose derivatives as raw materials based on a biocatalyst obtained by genetic engineering means. Background Art

[0003] Gastrodin, that is, 4-(hydroxymethyl)phenyl beta-D-glucopyranoside, has been identified as the main active ingredient of Gastrodia elata and is widely used in the treatment of various diseases, such as dizziness, headache, convulsion, and vertigo. In addition, Gastrodin also has other biological activities, such as antioxidant, anti-inflammatory, anti-anxiety, anti-obesity, anti-epileptic, and has the characteristics of memory improvement and neuroprotection.

[0004] Currently, the synthesis of Gastrodin mainly includes chemical synthesis and direct extraction from Gastrodia elata. Chemical synthesis usually faces problems such as poor specificity, many steps, many by-products, and excessive pollutants generated during the reaction process; extraction from Gastrodia elata plants has problems such as limited growth environment, low extraction efficiency, and high cost. In recent years, the use of renewable glucose to synthesize a variety of natural products by constructing recombinant strains has attracted increasing attention. Chinese invention patent 201910882020.9 discloses a method for synthesizing Gastrodin through a heterologous metabolic pathway. This method uses recombinant Escherichia coli including pCDFDuet-aroG*-ppsA-pgm-galU and pETDuet-ubiC-CAR-Sfp-ugt73b6 FS two expression vectors, by constructing a new p-hydroxybenzyl alcohol synthesis pathway, regulating the metabolic flux from glucose to tyrosine, obtaining a p-hydroxybenzyl alcohol yield of 240 mg / L, and introducing a highly efficient UDP-glucosyltransferase mutant, with the highest Gastrodin yield of 265 mg / L.

[0005] Hydroquinone (HQ) is an important intermediate commonly used in the commercial preparation of the chemical, pharmaceutical, and polymer industries. Hydroquinone widely exists in higher plants in the form of glycosylation (arbutin), and it is also a pheromone of termites. Currently, the industrial production of hydroquinone mainly uses benzene as the starting material, usually involving strong acids and heavy metals, which causes serious environmental problems. At the same time, benzene, as a petroleum-based raw material, is not a renewable resource. According to the literature (Chemo- and Regioselective Dihydroxylation of Benzene to Hydroquinone Enabled by Engineered Cytochrome P450 Monooxygenase, Angewandte Chemie International Edition, 2019, 58, 764 - 768.), using benzene as the starting material, the mutant strain A82F / A328F obtained by modifying the P450-MB3 monooxygenase can catalyze the dihydroxylation of 10 mM benzene to synthesize 9.2 mM hydroquinone.

[0006] Arbutin, a glycosylated hydroquinone, exists in plants such as bearberry and wheat. It is a skin whitening agent and also has biological activities such as antibacterial, anti-inflammatory, and antioxidant. As a mild, safe, and effective reagent, arbutin has been widely used in the medical and cosmetic industries. Arbutin is usually obtained by plant extraction methods, but this method is not only complex but also has a low yield. Shen Xiaolin et al. modified the metabolic pathway of Escherichia coli to accumulate p-hydroxybenzoic acid, and then heterologously expressed 4-hydroxybenzoate 1-hydroxylase (MNX1) from Candida parapsilosis CBS604 and 4-hydroxybenzoic acid 1-hydroxylase (MNX1) from Rauvolfia serpentinaArbutin synthase (AS), using 30 g / L glucose as the raw material, can obtain 4.19 g / L of arbutin. Chinese Patent 201510107788.0 discloses a method for enriching and purifying arbutin from blueberries. This method first uses a high-voltage pulsed electric field extraction device to extract α-arbutin, then uses a membrane device and macroporous adsorption resin for the primary purification of α-arbutin, and then uses simulated moving bed chromatography separation technology to separate and purify α-arbutin to obtain a high-purity α-arbutin product. Chinese Patent 201510991581.4 discloses a method for synthesizing arbutin by enzymatic conversion of starch substances and hydroquinone. This method uses starch substances and hydroquinone as raw materials, adds biological enzymes for enzymatic conversion reaction; after removing the enzymes from the reaction solution, yeast is added to ferment to remove glucose in the reaction solution, and then bacteria are removed to obtain arbutin. Chinese Patent 201510335885.5 discloses a method for synthesizing arbutin by biological fermentation. This method uses liquefied flour, bran, calcium carbonate, potassium dihydrogen phosphate and urea as the fermentation substrate, adds hydroquinone thereto, and is prepared by fermentation using Aspergillus oryzae.

[0007] Salidroside, also known as tyrosol 8-O-glucoside, is a biologically active tyrosine-derived phenolic natural product that widely exists in the medicinal plant genus Rhodiola. In addition to its anti-fatigue and anti-hypoxia effects in traditional medicine, Rhodiola extracts and salidroside also show anti-cardiovascular disease and anti-cancer functions. However, the currently commercially available pure salidroside mainly undergoes a long purification process 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. This method first pulverizes the Rhodiola medicinal material, uses water as the solvent, assisted by ultrasonic extraction, and filters to obtain a filtrate; subsequently, it is collected through a macroporous resin column and concentrated under reduced pressure; then it is dissolved in absolute ethanol and separated by simulated moving bed chromatography to obtain a fraction rich in salidroside; finally, the salidroside product is obtained through concentration, crystallization, centrifugation and drying. Chinese Patent 201810753390.8 discloses a method for synthesizing salidroside by fermentation. This method integrates the ketodecarboxylase gene skdc with the trc promoter and the glycosyltransferase gene sugt2 with the tac promoter into the chromosome of SyBE-002447 to obtain the SDR1 strain. Using glucose as the precursor, the SDR1 strain is fermented for 36 h to obtain 0.7 g / L of salidroside.

[0008] Tyrosol (p - hydroxybenzyl alcohol) is a phenolic compound that naturally exists in various foods such as olive oil and wine. Tyrosol and its derivatives have been widely used in the pharmaceutical field (such as bioactive compounds), chemical industry (such as fine chemicals), and other industrial fields. For example, tyrosol can be used to prepare commercial drugs such as betaxolol, metoprolol, and selective β - blockers for the treatment of hypertension, angina, heart failure, and glaucoma. Hydroxytyrosol (HT, 3,4 - dihydroxybenzyl alcohol) is a natural polyphenolic antioxidant mainly present in olive leaves and their fruits, as well as in olive oil industrial by - products and industrial wastewater. In addition, hydroxytyrosol is a bioactive substance closely related to human health that has attracted much attention and has functions such as antibacterial, anti - inflammatory, inhibiting melanin precipitation, and scavenging free radicals. At present, the batch production of hydroxytyrosol is not yet mature, and there are mainly two methods: 1) preparing hydroxytyrosol by acidolysis or enzymatic hydrolysis of oleuropein in olive mill wastewater, olive leaves, and virgin olive oil; 2) preparing hydroxytyrosol by chemical synthesis methods. Through plant extraction, although it is a waste utilization, a large amount of organic reagents are used in the middle; the chemical synthesis method has long reaction steps, many by - products, and is not environmentally friendly, which does not conform to the concept of green and sustainable synthesis. Chinese Patent Invention 201710195462.7 discloses a method for extracting hydroxytyrosol from olive leaves. This method first takes olive leaves, removes impurities, washes, dries, and crushes them; then, high - pressure micro - jet super - fine crushing is carried out to obtain a paste - like slurry; subsequently, microwave extraction is carried out; then, the extract is subjected to macroporous adsorption resin column chromatography and eluted with an ethyl acetate solution; the eluate is concentrated and dried to obtain hydroxytyrosol. Chinese Patent Invention 201910882020.9 discloses a method for synthesizing hydroxytyrosol by enzymatic reaction. This method uses L - dopa as a substrate and synthesizes hydroxytyrosol in Escherichia coli recombinant bacteria that simultaneously express L - phenylalanine dehydrogenase, α - ketoacid decarboxylase, and alcohol dehydrogenase. Chinese Patent Invention 202010582557.6 discloses a method for constructing Escherichia coli with high - yield hydroxytyrosol. This method co - transfers plasmid pACYC - HpaBC, plasmid pET - LAAD - ARO10, and plasmid pRSF - PAR into Escherichia coli BL21(DE3) competent cells to obtain recombinant Escherichia coli, which catalyzes the synthesis of hydroxytyrosol with tyrosine as a substrate. Tyrosine and L - dopa are relatively expensive and do not belong to the category of renewable resources. Summary of the Invention

[0009] The object of the present invention is to overcome the deficiencies of the prior art and provide a method for biosynthesizing compounds using lignocellulose derivatives.

[0010] The present invention modifies Escherichia coli by genetic engineering means to construct three new enzymatic reaction pathways, and uses lignocellulose-derived aromatic compounds, such as p -coumaric acid and ferulic acid, to efficiently synthesize various high-value-added compounds including gastrodin, arbutin, salidroside, etc., and the product yields all reach grams and above.

[0011] The object of the present invention is achieved by the following technical solutions:

[0012] The present invention provides a method for biosynthesizing compounds using lignocellulose derivatives, comprising the following steps:

[0013] A. Modify Escherichia coli to obtain a biocatalyst;

[0014] B. Use the lignocellulose derivative as a starting material to synthesize compounds through the biocatalyst.

[0015] The lignocellulose derivative includes at least one of p-coumaric acid and ferulic acid;

[0016] The compounds include at least one of gastrodin, arbutin, salidroside and their derivatives hydroquinone, tyrosol, hydroxytyrosol, and homovanillyl alcohol.

[0017] Preferably, in step A, the biocatalyst is E. 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) and E. coli (BLPad-StyAB-RostyC-SlPAR1-HpaBC).

[0018] Preferably, the method for obtaining E. coli (Fcs-Ech-SlPAR1-UGT73B6 FS ) is: by E. coliOverexpression from Pseudomonas putida Fcs and Ech from KT2440, from Solanum lycopersicum SlPAR1 and from Rhodiola mutant enzyme UGT73B6 from plants FS Achieved.

[0019] Preferably, the E. coli (Fcs - Ech - Vdh - MNX1 - AS) is obtained by: overexpressing in E. coli Fcs, Ech and Vdh from P. putida KT2440, MNX1 from yeast Candida parapsilosis CDC317 and arbutin synthase AS from Rauvolfia serpentina ;

[0020] The E. coli (Fcs - Ech - Vdh - MNX1) is obtained by: overexpressing in E. coli Fcs, Ech and Vdh from P. putida KT2440, MNX1 from yeast Candida parapsilosis CDC317.

[0021] Preferably, the E. coli (BLPad - StyAB - RostyC - SlPAR1 - UGT85A1) is obtained by: overexpressing in E. coli decarboxylase BLPad from Bacillus licheniformis CGMCC7172, StyAB from Pseudomonas sp. strain VLB120, RostyC from Rhodococcus opacus 1CP, SlPAR1 from Solanum lycopersicum and glycosyltransferase UGT85A1 from Arabidopsis thaliana ;

[0022] The E. coli (BLPad - StyAB - RostyC), E. coli (BLPad - StyAB - RostyC - SlPAR1), E. coli (BLPad - StyAB - RostyC - YqhD), E. coli (BLPad - StyAB - RostyC - YahK) are obtained by: overexpressing in E. coli decarboxylase BLPad from Bacillus licheniformis CGMCC7172, StyAB from PseudomonasStyAB of sp. strain VLB120, derived from Rhodococcus opacus RostyC of Rhodococcus opacus , derived from E. coli YqhD or YahK of BL21(DE3) or derived from Solanum lycopersicum SlPAR1 of Solanum lycopersicum ;

[0023] The E. coli (BLPad-StyAB-RostyC-SlPAR1-HpaBC) is obtained by overexpressing in E. coli decarboxylase BLPad derived from Bacillus licheniformis CGMCC7172, StyAB of VLB120, derived from Pseudomonas sp. strain RostyC of Pseudomonas , derived from Rhodococcus opacus SlPAR1 of Rhodococcus opacus and HpaBC derived from Solanum lycopersicum BL21(DE3). E. coli

[0024] Preferably, when the biocatalyst is E. coli (Fcs-Ech-SlPAR1-UGT73B6 (Fcs-Ech-SlPAR1-UGT73B6 FS ), the lignocellulose derivative used in step B is p-coumaric acid, and the synthesized compound is gastrodin.

[0025] Preferably, 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 compound is arbutin;

[0026] When the biocatalyst is E. coli (Fcs-Ech-Vdh-MNX1), the lignocellulose derivative used in step B is p-coumaric acid, and the synthesized compound is hydroquinone.

[0027] 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 compound is salidroside;

[0028] When the biocatalyst is E. coli (BLPad-StyAB-RostyC), E. coli (BLPad-StyAB-RostyC-SlPAR1), E. coli (BLPad-StyAB-RostyC-YqhD), E. coliWhen (BLPad-StyAB-RostyC-YahK), the lignocellulose derivative used in step B is p-coumaric acid or ferulic acid, and the synthesized compound is tyrosol or homovanillyl alcohol;

[0029] The biocatalyst is E. coli When (BLPad-StyAB-RostyC-SlPAR1-HpaBC), the lignocellulose derivative used in step B is p-coumaric acid, and the synthesized compound is hydroxytyrosol.

[0030] The present invention also provides a biocatalyst for the biosynthesis of compounds. The biocatalyst is a modified Escherichia coli, specifically including E. 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) and E. coli (BLPad-StyAB-RostyC-SlPAR1-HpaBC).

[0031] Preferably, the compound includes at least one of gastrodin, arbutin, salidroside and their derivatives hydroquinone, tyrosol, hydroxytyrosol and homovanillyl alcohol.

[0032] Preferably, the E. coli (Fcs-Ech-SlPAR1-UGT73B6 FS ) is obtained by overexpressing Fcs, Ech derived from E. coli KT2440, SlPAR1 derived from Pseudomonas putida and the mutant enzyme UGT73B6 derived from the plant Solanum lycopersicum in Rhodiola . FS It is achieved.

[0033] Preferably, the E. coli (Fcs-Ech-Vdh-MNX1-AS) is obtained by: inE. coli Overexpression in P. putida KT2440 Fcs, Ech and Vdh, derived from yeast Candida parapsilosis MNX1 from CDC317 and Rauvolfia serpentina Arbutin synthase AS is achieved;

[0034] described E. coli (Fcs-Ech-Vdh-MNX1) was obtained by E. coli Overexpression in P. putida KT2440 Fcs, Ech and Vdh, derived from yeast Candida parapsilosis MNX1 implementation of CDC317.

[0035] Preferably, the E. coli (BLPad-StyAB-RostyC-SlPAR1-UGT85A1) was obtained by: E. coli Overexpression in Bacillus licheniformis The decarboxylase BLPad of CGMCC7172 is derived from Pseudomonas sp. strain VLB120's StyAB is derived from Rhodococcus opacus 1CP of RostyC, derived from Solanum lycopersicum SlPAR1, and implementation derived from Arabidopsis thaliana glycosyltransferase UGT85A1;

[0036] described E. coli (BLPad-StyAB-RostyC), E. coli (BLPad-StyAB-RostyC-SlPAR1), E. coli (BLPad-StyAB-RostyC-YqhD), E. coli (BLPad-StyAB-RostyC-YahK) is obtained by: E. coli Overexpression in Bacillus licheniformis The decarboxylase BLPad of CGMCC7172 is derived from Pseudomonas sp. strain StyAB of VLB120 is derived from RostyC of Rhodococcus opacus 1CP, which is derived from E. coli YqhD or YahK of BL21(DE3) or derived from Solanum lycopersicum SlPAR1 implementation;

[0037] described E.coli (BLPad-StyAB-RostyC-SlPAR1-HpaBC) was obtained by: E.coli Overexpression inBacillus licheniformis The decarboxylase BLPad of CGMCC7172 is derived from Pseudomonas sp. strain StyAB of VLB120 is derived from RostyC of Rhodococcus opacus 1CP, which is derived from Solanum lycopersicum SlPAR1 and SlPAR1 derived from E. coli HpaBC implementation of BL21(DE3).

[0038] Compared with the existing technology, the present invention has the following beneficial effects:

[0039] (1) The present invention obtains a highly efficient biocatalyst through genetic engineering, which can rapidly catalyze the substrates p-coumaric acid and ferulic acid to synthesize a variety of compounds including gastrodin, arbutin and salidroside.

[0040] (2) The present invention utilizes the constructed biocatalyst to achieve efficient utilization of aromatic compounds (p-coumaric acid and ferulic acid) derived from lignocellulose through bioconversion, which is more advantageous than the lengthy steps of fermenting glucose.

[0041] (3) The yield of the target compound obtained by the method of the present invention is in the range of grams or more, and the conversion rate is mostly above 90%, which has great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0043] Figure 1 To construct a biocatalyst for the synthesis of gastrodin;

[0044] Figure 2 It is the result of biotransformation synthesis of gastrodin;

[0045] Figure 3 The result diagram of biosynthesis of hydroquinone:

[0046] Figure 4 Diagram for the construction of a biocatalyst for the synthesis of arbutin;

[0047] Figure 5 This figure shows the results of comparing the synthesis of arbutin by different biocatalysts;

[0048] Figure 6 This is the result diagram of biotransformation synthesis of arbutin;

[0049] Figure 7 Construct a diagram for the biocatalyst for tyrosol synthesis;

[0050] Figure 8 Results of the synthesis of tyrosol by different biocatalysts

[0051] Figure 9 Schematic diagram of the biosynthesis of tyrosol and homovanillyl alcohol

[0052] Figure 10 Results of the biosynthesis of salidroside

[0053] Figure 11 Results of the biosynthesis of hydroxytyrosol

[0054] Figure 12 Schematic diagram of the biosynthesis of gastrodin

[0055] Figure 13 Schematic diagram of the biosynthesis of arbutin and its derivative hydroquinone

[0056] Figure 14 Schematic diagram of the biosynthesis of salidroside and its derivatives tyrosol, homovanillyl alcohol and hydroxytyrosol Detailed implementation manners

[0057] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those of ordinary skill in the art can make several modifications and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0058] The methods of the following embodiments include:

[0059] Step 1: Modify Escherichia coli to obtain an efficient biocatalyst ( E.coli (Fcs-Ech-SlPAR1-UGT73B6 FS )) and successfully synthesize gastrodin starting from p-coumaric acid;

[0060] Step 2: Modify Escherichia coli to obtain efficient biocatalysts ( E.coli (Fcs-Ech-Vdh-MNX1-AS) and E.coli (Fcs-Ech-Vdh-MNX1)), and successfully synthesize arbutin and its derivative hydroquinone starting from p-coumaric acid;

[0061] Step 3: Modify Escherichia coli to obtain multiple efficient 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) and E.coli (BLPad-StyAB-RostyC-SlPAR1-HpaBC), etc. Using p -coumaric acid and ferulic acid as starting materials, salidroside, tyrosol, hydroxytyrosol and homovanillyl alcohol were successfully synthesized;

[0062] In step one, the construction of the biocatalyst ( E.coli (Fcs-Ech-SlPAR1-UGT73B6 FS ) was achieved by overexpressing Fcs, Ech from KT2440, SlPAR1 from E.coli and the mutant enzyme UGT73B6 from plant Pseudomonas putida Solanum lycopersicum Rhodiola E.coli

[0063] FS E.coli E.coli (Fcs-Ech-SlPAR1) catalyzes the synthesis of p -hydroxybenzyl alcohol from p -coumaric acid, and then UGT73B6 was introduced FS to construct a new catalyst to catalyze the synthesis of gastrodin from p -hydroxybenzyl alcohol; E.coli P. putida In step two, the construction of the biocatalyst ( E.coli (Fcs-Ech-Vdh-MNX1-AS) and E.coli (Fcs-Ech-Vdh-MNX1)) was achieved by overexpressing Fcs, Ech and Vdh from KT2440, MNX1 from yeast E.coli P. putida Candida parapsilosis CDC317 and arbutin synthase AS from Rauvolfia serpentina E.coli

[0064] E.coli (Fcs-Ech-Vdh-MNX1) was first constructed to catalyze the synthesis of hydroquinone, the precursor of arbutin, from the substrate p -coumaric acid, and then AS was introduced to construct multiple biocatalysts for the efficient synthesis of arbutin. E.coli E.coli In step three, the biocatalyst ( 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), and E.coli (BLPad-StyAB-RostyC-SlPAR1-HpaBC)), etc. are achieved by overexpressing decarboxylase BLPad derived from E.coli in, StyAB derived from Bacillus licheniformis CGMCC7172, RostyC derived from Pseudomonas sp. strain VLB120, glycosyltransferase UGT85A1 derived from Arabidopsis thaliana , HpaBC, YqhD, and YahK derived from E. coli BL21(DE3), and SlPAR1 derived from Solanum lycopersicum . In this process, multiple biocatalysts E.coli (BLPad-StyAB-RostyC), E.coli (BLPad-StyAB-RostyC-SlPAR1), E.coli (BLPad-StyAB-RostyC-YqhD), E.coli (BLPad-StyAB-RostyC-YahK) are first constructed to catalyze the synthesis of tyrosol from p-coumaric acid, the most suitable catalyst is selected, and then UGT85A1 or HpaBC is introduced to further catalyze the synthesis of salidroside and hydroxytyrosol.

[0065] Example 1

[0066] (1) Construction of a biocatalyst for the synthesis of gastrodin (the construction schematic diagram is as Figure 1 shown)

[0067] Plasmids pET28a-Fcs-Ech-SlPAR1 and plasmid pA7a-UGT73B6 FS are constructed and transformed into BL21 (DE3) bacteria to construct a biocatalyst E.coli (Fcs-Ech-SlPAR1-UGT73B6 FS ). The specific construction method is as follows:

[0068] From Pseudomonas putida the KT2440 genome, Fcs and Ech are cloned by PCR and co-constructed with SlPAR1 on the vector pET28a to obtain plasmid pET28a-Fcs-Ech-SlPAR1; UGT73B6 FsIt was constructed onto the vector pA7a to obtain the plasmid pA7a-UGT73B6 FS ; The plasmids pET28a-Fcs-Ech-SlPAR1 and pA7a-UGT73B6 FS were transformed into the host BL21(DE3) by heat shock or electroporation to prepare the biocatalyst E.coli (Fcs-Ech-SlPAR1-UGT73B6 FS ).

[0069] (2) Biotransformation synthesis of gastrodin (synthesis schematic diagram as Figure 12 shown)

[0070] The prepared biocatalyst E.coli (Fcs-Ech-SlPAR1-UGT73B6 FS ) was inoculated into 2 mL of LB for activation. After 10 - 12 h at 37 °C, the activated biocatalyst was transferred to 100 mL of LB at a ratio of 1:100. When the OD 600 reached 0.6 - 0.8, IPTG was added to a final concentration of 0.5 mM, and induced at 22 °C for 10 - 12 h. Then, at 4 °C, centrifuged at 4000 g for 10 min to collect the cells, resuspended in M9Y medium, added the substrates 2 g / L p-coumaric acid and 10 g / L glucose, and transformed at 37 °C for 12 h to obtain 1.45 g / L gastrodin with a conversion rate of 42.5% ( Figure 2 ).

[0071] Example 2

[0072] (1) Construction of a biocatalyst for the synthesis of hydroquinone

[0073] Plasmids pET28a-Fcs-Ech-Vdh and pA7a-MNX1 were constructed and co-transformed into BL21(DE3) to obtain the biocatalyst E.coli (Fcs-Ech-Vdh-MNX1). The specific method is as follows: From the Pseudomonas putida KT2440 genome, Fcs, Ech, and Vdh were cloned by PCR and ligated into the vector pET28a by restriction digestion to construct pET28a-Fcs-Ech-Vdh, and MNX1 was ligated into the vector pA7a by restriction digestion to construct pA7a-MNX1; The plasmids pET28a-Fcs-Ech-Vdh and pA7a-MNX1 were transformed into the host BL21(DE3) by heat shock or electroporation to prepare the biocatalyst E.coli (Fcs-Ech-Vdh-MNX1).

[0074] (2) Biosynthesis of hydroquinone by biotransformation (synthesis schematic diagram is as Figure 13 shown)

[0075] The prepared biocatalyst E.coli (Fcs-Ech-Vdh-MNX1) was inoculated into 2 mL of LB for activation. After 10 - 12 h at 37 °C, the activated biocatalyst was transferred to 100 mL of LB at a ratio of 1:100. When the OD 600 reached 0.6 - 0.8, the inducer IPTG was added to a final concentration of 0.5 mM. After induction at 22 °C for 10 - 12 h, the cells were centrifuged at 4 °C and 4000 g for 10 min to collect the cells, which were resuspended in M9Y medium. Substrates 3 g / L of p-coumaric acid and 10 g / L of glucose were added, and the transformation was carried out at 37 °C for 8 h. The results are as shown in Figure 3, and 1.95 g / L of hydroquinone can be obtained, with a conversion rate of 97% ( Figure 3 ).

[0076] Example 3

[0077] (1) Construction of a biocatalyst for the synthesis of arbutin (construction schematic diagram is as Figure 4 shown) corresponding E.coli to the (Fcs-Ech-Vdh-MNX1-AS) catalyst, among which SArbutin5 has the best effect.

[0078] 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 the T7 promoter, which adds the T7 promoter before the MNX1 gene compared to pA7a-AS-MNX1) were pairwise combined and transformed into BL21(DE3) to obtain 5 biocatalysts SArbutin1, SArbutin2, SArbutin3, SArbutin4, and SArbutin5. The specific construction methods are as follows:

[0079] From Pseudomonas putidaOn the KT2440 genome, Fcs, Ech, and Vdh were obtained by PCR cloning and ligated into the vector pET28a through enzymatic digestion to construct pET28a-Fcs-Ech, and pET28a-Fcs-Ech-Vdh; MNX1 and AS were ligated into the vectors pA7a or pACYC through enzymatic digestion to construct pA7a-MNX1-AS, pA7a-AS-MNX1, pA7a-AS-7-MNX1; Vdh, MNX1, and AS obtained by PCR cloning were ligated into the vector pA7a through enzymatic digestion to construct pA7a-Vdh-MNX1-AS.

[0080] Five kinds of biocatalysts, SArbutin1, were prepared by transforming the plasmids pET28a-Fcs-Ech and pA7a-Vdh-MNX1-AS into the host BL21(DE3) by heat shock or electroporation; SArbutin2 was prepared by transforming the plasmids pET28a-Fcs-Ech-Vdh and pA7a-MNX1-AS into the host BL21(DE3) by heat shock or electroporation; SArbutin3 was prepared by transforming the plasmids pET28a-Fcs-Ech-Vdh and pA7a-AS-MNX1 into the host BL21(DE3) by heat shock or electroporation; SArbutin4 was prepared by transforming the plasmids pET28a-Fcs-Ech-Vdh and pA7a-AS-7-MNX1 into the host BL21(DE3) by heat shock or electroporation; SArbutin5 was prepared by transforming the plasmids pET28a-Fcs-Ech-Vdh and pACYC-AS-MNX1 into the host BL21(DE3) by heat shock or electroporation.

[0081] (ii) Biotransformation synthesis of arbutin (synthesis schematic diagram as Figure 13 shown)

[0082] The five kinds of biocatalysts prepared were inoculated into 2 mL of LB for activation. After 10 - 12 h at 37 °C, the activated biocatalysts were transferred to 100 mL of LB at a ratio of 1:100. When the OD 600 reached 0.6 - 0.8, IPTG was added as an inducer to a final concentration of 0.5 mM, and induced at 22 °C for 10 - 12 h. Then, the cells were centrifuged at ④ °C and 4000 g for 10 min to collect the cell pellets, which were resuspended in M9Y medium, supplemented with 2 g / L of p-coumaric acid and 10 g / L of glucose, and transformed at 37 °C for 12 h. The results of the synthesis of arbutin by the five kinds of biocatalysts are as Figure 5As shown, the results show that SArbutin5 can convert to produce arbutin with the highest yield of 2.34 g / L. Among them, the results of the conversion of the biocatalyst SArbutin5 to synthesize arbutin over time using the above method are as Figure 6 shown. After 24 h, 3.05 g / L arbutin can be obtained, and the conversion rate is 92%.

[0083] Example 4

[0084] (1) Construction of biocatalysts E.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-SlPAR1) for the synthesis of tyrosol (the construction schematic diagram is as Figure 7 shown)

[0085] Construct 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. Combine them in pairs and transform BL21(DE3) to obtain 8 biocatalysts Styrosol 1, Styrosol 2, Styrosol 3, Styrosol 4, Styrosol 5, Styrosol 6, Styrosol 7 and Styrosol 8.

[0086] The specific construction method is as follows: PCR amplify YqhD and YahK on the genome of BL21(DE3), and combine StyAB, RostyC, BLpad and SlPAR1. Connect StyAB and RostyC into the vector pET28a through enzymatic digestion and ligation to obtain pET28a-StyAB-RostyC; connect StyAB, RostyC and BLPad into the vector pET28a through enzymatic digestion and ligation to obtain pET28a-StyAB-RostyC-BLPad; connect StyAB, RostyC and YahK or YqhD or SlPAR1 into the vector pET28a through enzymatic digestion and ligation to obtain pET28a-StyAB-RostyC-YqhD or pET28a-StyAB-RostyC-YahK or pET28a-StyAB-RostyC-SlPAR1; connect BLPad or its combination with YqhD or YahK or SlPAR1 into the vector pA7a through enzymatic digestion and ligation to obtain pA7a-BLPad, pA7a-BLPad-SlPAR1, pA7a-BLPad-YqhD, pA7a-BLPad-YahK.

[0087] The above-mentioned biocatalyst Styrosol 1 was prepared by transforming the plasmid pET28a-StyAB-RostyC-BLpad into the host BL21(DE3) by heat shock or electroporation. Styrosol 2 was prepared by transforming the plasmids pET28a-StyAB-RostyC and pA7a-BLPad into the host BL21(DE3) by heat shock or electroporation. Styrosol 3 was prepared by transforming the plasmids pET28a-StyAB-RostyC-SlPAR1 and pA7a-BLPad into the host BL21(DE3) by heat shock or electroporation. Styrosol 4 was prepared by transforming the plasmids pET28a-StyAB-RostyC-YqhD and pA7a-BLPad into the host BL21(DE3) by heat shock or electroporation. Styrosol 5 was prepared by transforming the plasmids pET28a-StyAB-RostyC-YahK and pA7a-BLPad into the host BL21(DE3) by heat shock or electroporation. Styrosol 6 was prepared by transforming the plasmids pET28a-StyAB-RostyC and pA7a-BLPad-SlPAR1 into the host BL21(DE3) by heat shock or electroporation. Styrosol 7 was prepared by transforming the plasmids pET28a-StyAB-RostyC and pA7a-BLPad-yqhD into the host BL21(DE3) by heat shock or electroporation. Styrosol 8 was prepared by transforming the plasmids pET28a-StyAB-RostyC and pA7a-BLPad-YahK into the host BL21(DE3) by heat shock or electroporation.

[0088] (2) Biotransformation for the synthesis of tyrosol (synthesis schematic diagram is as Figure 14 shown) The prepared 8 kinds of biocatalysts were inoculated into 2 mL of LB for activation at 37 °C for 10 - 12 h. Then, the activated biocatalysts were transferred to 100 mL of LB at a ratio of 1:100. When the OD 600 reached 0.6 - 0.8, IPTG was added to a final concentration of 0.5 mM, and induction was carried out at 22 °C for 10 - 12 h. After that, centrifugation was performed at 4 °C and 4000 g for 10 min to collect the cells, which were resuspended in M9Y medium. 2.0 g / L of p-coumaric acid and 10 g / L of glucose were added, and transformation was carried out at 37 °C for 12 h. The results of the synthesis of tyrosol by the 8 kinds of biocatalysts are as Figure 8As shown, the results show that Styrosol 3 and Styrosol 7 synthesize the most tyrosol, which are 1.63 and 1.64 g / L respectively. In addition, using the biocatalyst Styrosol 7 to catalyze 2.5 g / L of p-coumaric acid, the results are as Figure 9 shown. After 24 h, 2.04 g / L of tyrosol can be obtained, and the conversion rate is 97.4%.

[0089] Example 5

[0090] Biotransformation synthesis of homovanillyl alcohol (synthesis schematic diagram is as Figure 14 shown)

[0091] The biocatalyst Styrosol 7 prepared in Example 4 was inoculated into 2 mL of LB and incubated at 37 °C. After 10 - 12 h, the activated biocatalyst was transferred to 100 mL of LB at a ratio of 1:100. When the OD 600 reached 0.6 - 0.8, the inducer IPTG was added to a final concentration of 0.5 mM. After induction at 22 °C for 10 - 12 h, the cells were centrifuged at 4 °C and 4000 g for 10 min, and the cells were collected and resuspended in M9Y medium, supplemented with 1.5 g / L of ferulic acid and 10 g / L of glucose, and transformed at 37 °C for 12 h. The results of synthesizing homovanillyl alcohol are as Figure 9 shown. After the transformation, 1.18 g / L of the product was obtained, and the conversion rate was approximately 92%.

[0092] Example 6

[0093] (1) Construction of biocatalyst for the synthesis of salidroside

[0094] Construct plasmids pET28a-StyAB-RostyC-7-SlPAR1 and pA7a-BLPad-UGT85A1, and co-transform them

[0095] into BL21(DE3) to obtain the biocatalyst E.coli(BLPad-StyAB-RostyC-SlPAR1-UGT85A1). The specific method is as follows: The gene sequences BLPad, StyAB, RostyC, SlPAR1, and UGT85A1 synthesized by a biological company were obtained by PCR amplification. StyAB, RostyC, and SlPAR1 were digested and ligated into the vector pET28a to obtain pET28a-StyAB-RostyC-SlPAR1, and BLPad and UGT85A1 were digested and ligated into the vector pA7a to obtain pA7a-BLPad-UGT85A1. The plasmids pET28a-StyAB-RostyC-SlPAR1 and pA7a-BLPad-UGT85A1 were transformed into the host BL21(DE3) by heat shock or electroporation to prepare a biocatalyst E.coli (BLPad-StyAB-RostyC-SlPAR1-UGT85A1).

[0096] (2) Biosynthesis of salidroside (synthesis schematic diagram is as Figure 14 shown)

[0097] The prepared biocatalyst E.coli (BLPad-StyAB-RostyC-SlPAR1-UGT85A1) was inoculated into 2 mL LB and incubated at 37 °C for 10 - 12 h. Then, the activated biocatalyst was transferred to 100 mL LB at a ratio of 1:100. When the OD 600 reached 0.6 - 0.8, the inducer IPTG was added to a final concentration of 0.5 mM, and induction was carried out at 22 °C for 10 - 12 h. Then, the cells were centrifuged at 4 °C and 4000g for 10 min to collect the cells, which were resuspended in M9Y medium supplemented with 2 g / L p-coumaric acid and 10 g / L glucose, and transformed at 37 °C for 24 h. At 12 h, an additional 10 g / L glucose was added. The results are as Figure 10 shown, and finally 1.72 g / L of salidroside was synthesized with a conversion rate of 48.3%.

[0098] Example 7

[0099] (1) Construction of a biocatalyst for the synthesis of hydroxytyrosol

[0100] Plasmids pET28a-StyAB-RostyC-SlPAR1 and pA7a-BLPad-HpaBC were constructed and co-transformed into BL21(DE3) to obtain a biocatalyst E.coli(BLPad-StyAB-RostyC-SlPAR1-HpaBC). The specific method is as follows: HpaBC on the BL21(DE3) genome was obtained by PCR amplification, and the gene sequences BLPad, StyAB, RostyC, and SlPAR1 synthesized by a biological company were used. StyAB, RostyC, and SlPAR1 were digested and ligated into the vector pET28a to obtain pET28a-StyAB-RostyC-SlPAR1, and HpaBC and BLPad were digested and ligated into the vector pA7a to obtain pA7a-BLPad-HpaBC. The plasmids pET28a-StyAB-RostyC-SlPAR1 and pA7a-BLPad-HpaBC were transformed into the host BL21(DE3) by heat shock or electroporation to prepare a biocatalyst E.coli (BLPad-StyAB-RostyC-SlPAR1-HpaBC).

[0101] (2) Biosynthesis of hydroxytyrosol (synthesis schematic diagram is as Figure 14 shown)

[0102] The prepared biocatalyst E.coli (BLPad-StyAB-RostyC-SlPAR1-HpaBC) was inoculated into 2 mL LB and incubated at 37 °C for 10 - 12 h. Then, the activated biocatalyst was transferred to 100 mL LB at a ratio of 1:100. When the OD 600 reached 0.6 - 0.8, IPTG was added to a final concentration of 0.5 mM, and induction was carried out at 22 °C for 10 - 12 h. Then, the cells were centrifuged at 4 °C and 4000g for 10 min, and the cells were collected and resuspended in M9Y medium. 2 g / L p-coumaric acid and 10 g / L glucose were added, and conversion was carried out at 37 °C for 8 h. The results are as Figure 11 shown. Finally, 1.83 g / L of hydroxytyrosol was synthesized, and the conversion rate was 97.5%.

[0103] There are many specific application ways for the present invention. The above description is only the preferred embodiment of the present invention. It should be noted that the above examples are only used to illustrate the present invention and do not limit the protection scope of the present invention. For those of ordinary skill in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be regarded as within the protection scope of the present invention.

Claims

1. A method for synthesizing a compound using a lignocellulose derivative, characterized in that: The following steps are involved: A. Transform Escherichia coli to obtain a biocatalyst; B. Synthesizing compounds using lignocellulose derivatives as starting materials via biocatalysts; The lignocellulose derivative is ferulic acid; The compound is homovanillyl alcohol; In step A, the biocatalyst is obtained by: E. coli Overexpression in Bacillus licheniformis The decarboxylase BLPad of CGMCC7172 is derived from Pseudomonas sp. strain VLB120's StyAB is derived from Rhodococcus opacus 1CP of RostyC and derived from E. coli YqhD of BL21(DE3).

2. A biocatalyst for biosynthesis of a compound, characterized in that The method for obtaining the biocatalyst is: E. coli Overexpression in Bacillus licheniformis The decarboxylase BLPad of CGMCC7172 is derived from Pseudomonas sp. strain VLB120's StyAB is derived from Rhodococcus opacus 1CP of RostyC and derived from E. coli YqhD of BL21(DE3); The compound is homovanillyl alcohol.

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