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Method for biosynthesizing high-added-value compound by utilizing lignocellulose derivatives

A lignocellulose, high value-added technology, applied in the field of microorganisms, can solve the problems of long reaction steps, many by-products, and unsuitable for green and sustainable synthesis in chemical synthesis.

Active Publication Date: 2021-03-12
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Through plant extraction, although it is waste utilization, a large amount of organic reagents will be used in the middle; the chemical synthesis method has long reaction steps, many by-products, is not environmentally friendly, and is not suitable for the concept of green and sustainable synthesis.

Method used

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  • Method for biosynthesizing high-added-value compound by utilizing lignocellulose derivatives
  • Method for biosynthesizing high-added-value compound by utilizing lignocellulose derivatives
  • Method for biosynthesizing high-added-value compound by utilizing lignocellulose derivatives

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] (1) Constructing a biocatalyst for the synthesis of gastrodin (the construction schematic diagram is as follows figure 1 shown)

[0066] Construction of plasmid pET28a-Fcs-Ech-SlPAR1 and plasmid pA7a-UGT73B6 FS , transform it into BL21(DE3) bacteria, construct biocatalyst E.coli(Fcs-Ech-SlPAR1-UGT73B6 FS ). The specific method of construction is as follows:

[0067] From the genome of Pseudomonas putida KT2440, Fcs and Ech were cloned by PCR, and co-constructed with SlPAR1 on the vector pET28a to obtain the plasmid pET28a-Fcs-Ech-SlPAR1; UGT73B6 Fs Constructed on vector pA7a to obtain plasmid pA7a-UGT73B6 FS ; Plasmid pET28a-Fcs-Ech-SlPAR1 and plasmid pA7a-UGT73B6 FS Transformed into the host BL21 (DE3) by heat shock or electric shock, the biocatalyst E.coli (Fcs-Ech-SlPAR1-UGT73B6 FS ).

[0068] (2) biotransformation and synthesis of gastrodin (synthetic schematic diagram as Figure 12 shown)

[0069] The prepared biocatalyst E.coli (Fcs-Ech-SlPAR1-UGT73B6 FS...

Embodiment 2

[0071] (1) Construct biocatalyst to synthesize hydroquinone

[0072] Plasmids pET28a-Fcs-Ech-Vdh and plasmid pA7a-MNX1 were constructed and transformed into BL21(DE3) together to obtain biocatalyst E.coli (Fcs-Ech-Vdh-MNX1). The specific method is as follows: From the genome of Pseudomonas putida KT2440, Fcs, Ech, and Vdh were cloned by PCR to obtain pET28a-Fcs-Ech-Vdh, which was ligated into the vector pET28a by enzyme digestion, and pET28a-Fcs-Ech-Vdh was obtained, and pA7a was constructed by ligation of MNX1 into the vector pA7a by enzyme digestion. -MNX1; the plasmid pET28a-Fcs-Ech-Vdh and the plasmid pA7a-MNX1 were transformed into the host BL21(DE3) by heat shock or electric shock to prepare the biocatalyst E.coli (Fcs-Ech-Vdh-MNX1).

[0073] (2) biotransformation to synthesize hydroquinone (synthesis schematic diagram as Figure 13 shown)

[0074] Inoculate the prepared biocatalyst E.coli (Fcs-Ech-Vdh-MNX1) into 2mL LB for activation. After 10-12h at 37°C, transfer th...

Embodiment 3

[0076] (1) Constructing a biocatalyst for the synthesis of arbutin (the schematic diagram of the construction is as follows Figure 4 shown) corresponds to the E.coli (Fcs-Ech-Vdh-MNX1-AS) catalyst, among which SArbutin5 has the best effect.

[0077]Construction of 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. Compared with pA7a-AS-MNX1, the T7 promoter is added before the MNX1 gene) to transform BL21(DE3) in pairs to obtain five biocatalysts SArbutin1, SArbutin2, SArbutin3, SArbutin4 and SArbutin5 . The specific method of construction is as follows:

[0078] From the genome of Pseudomonas putida KT2440, Fcs, Ech and Vdh were obtained by PCR cloning and ligated into the vector pET28a to construct pET28a-Fcs-Ech, pET28a-Fcs-Ech-Vdh; MNX1 and AS were ligated into the vector pA7a or pACYC was constructed to obtain pA7a-MNX1-AS, pA7a-AS-MNX1, pA7a-AS-7-MNX1; PCR cloned Vdh...

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Abstract

The invention discloses a method for biosynthesizing a high-added-value compound by using lignocellulose derivatives, which comprises the following steps: A, modifying Escherichia coli to obtain a biocatalyst; B, synthesizing a high-added-value compound by taking a lignocellulose derivative as an initial raw material through a biocatalyst, wherein the lignocellulose derivative comprises at least one of p-coumaric acid and ferulic acid, and the high-added-value compound comprises at least one of gastrodin, arbutin, salidroside and derivatives thereof such as hydroquinone, tyrosol, hydroxytyrosol and styracitol. According to the method, escherichia coli is modified through a genetic engineering means, three new enzymatic reaction ways are constructed, and various high-added-value compounds including gastrodin, arbutin, salidroside and the like are efficiently synthesized by utilizing lignocellulose derived aromatic compounds p-coumaric acid and ferulic acid, wherein the product yield reaches gram-level or above.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and relates to a method for biosynthesizing high value-added compounds by using lignocellulose derivatives; in particular, it relates to a biosynthesis method based on biocatalysts obtained by genetic engineering means and using lignocellulose derivatives as raw materials approach to high value-added compounds. Background technique [0002] Gastrodin, namely 4-(hydroxymethyl)phenyl beta-D-glucopyranoside (4-(hydroxymethyl)phenyl beta-D-glucopyranoside), has been identified as the main active ingredient of gastrodia elata and is widely used It is used to treat various diseases, such as dizziness, headache, convulsions and vertigo. In addition, gastrodin also possesses other bioactive effects, such as antioxidant, anti-inflammatory, anxiolytic, anti-obesity, anti-epileptic, and memory-improving and neuroprotective properties. [0003] At present, the synthesis of gastrodin is mainly throug...

Claims

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Application Information

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IPC IPC(8): C12P19/44C12P7/42C12P7/26C12P7/22C12N1/21C12N15/70C12R1/19
CPCC12P19/44C12P7/22C12P7/26C12P7/42C12N15/70C12N15/52C12N9/0073C12N9/1051C12N9/0071C12N9/88C12N9/0006C12Y114/13064C12Y204/01218C12Y204/01203C12Y114/14011C12R2001/19Y02A50/30
Inventor 肖毅赵明涛
Owner SHANGHAI JIAO TONG UNIV
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