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Method for heterologous biosynthesis of tonquinol, caffeol and ferulenol

A technology of coumaryl alcohol and coffee alcohol, applied in the biological field, can solve the problems of high cost, harsh reaction conditions, and large environmental pollution of chemical synthesis methods

Inactive Publication Date: 2016-08-31
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the chemical synthesis method can use phenylpropionic acid to realize the synthesis of these three compounds, but the cost of the chemical synthesis method is high, the reaction conditions are harsh, the environment is polluted, and the separation of the products is difficult.

Method used

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  • Method for heterologous biosynthesis of tonquinol, caffeol and ferulenol
  • Method for heterologous biosynthesis of tonquinol, caffeol and ferulenol
  • Method for heterologous biosynthesis of tonquinol, caffeol and ferulenol

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Effect test

specific Embodiment 1

[0034] In vitro addition of coumaric, caffeic and ferulic acids to produce coumaryl, cafestol and ferulic alcohol

[0035] Aromatic compounds are naturally synthesized by many bacteria. The pathway for the synthesis of aromatic amino acids is called the shikimate pathway.

[0036] The first step in the shikimate pathway is the condensation of PEP and E4P to produce DHAP and phosphoric acid. The synthesis of DHAP is catalyzed by DHAP synthase. The second step reaction in the shikimate pathway is the elimination of the DHAP phosphate group, yielding 3-dehydroquinic acid (DHQ). This reaction is catalyzed by DHQ synthase. The third step reaction of the shikimate pathway is the dehydration of DHQ to form 3-dehydroshikimic acid (3-DHS), which is catalyzed by DHQ dehydratase. The fourth step reaction in the shikimate pathway is the reduction of 3-DHS to form shikimate. In E. coli, this reaction is catalyzed by the NADP-dependent shikimate dehydrogenase. In the fifth step reacti...

specific Embodiment 2

[0042] Construction of Metabolic Pathway for Microbial Heterologous Synthesis of Coumaryl Alcohol

[0043] As an important precursor of lignin synthesis, coumaryl alcohol, tyrosine synthesized by shikimate pathway, is screened from bacteria, fungi or protein engineered tyrosine ammonia lyase (TAL), coumaroyl coenzyme A ligase (4CL), cinnamoyl-CoA reductase (CCR), and alcohol dehydrogenase (ADH) catalyze the formation of coumaryl alcohol. Use PCR to obtain the corresponding gene fragments, then digest the fragments and vectors with endonucleases, perform gel recovery or column recovery on the digested fragments, and then insert the target gene into the plasmid pCS27 (middle copy) to obtain pCS - TAL-TPTA (Table 1).

[0044] Take 1 μL of the constructed high-copy plasmid pZE-CA4 and 2 μL of the medium-copy plasmid pCS-TAL-TPTA by electrotransfection into Escherichia coli, then take 200 μL of the bacterial solution and spread it on the antibiotic plate containing Amp and Kan, an...

specific Embodiment 3

[0048] Construction of metabolic pathway for heterologous synthesis of cafestol by microorganisms

[0049] Obtain hpaBC by PCR, then digest the fragment and vector with endonuclease, recover the digested fragment from gel or column, and then insert the target gene into the plasmid pCS27 (medium copy) to obtain the pCS-HpaBC recombinant plasmid (Table 1).

[0050] Introduction of heterologous metabolic pathways into caffeol-producing microorganisms

[0051] Take 1 μL of the constructed high-copy plasmid pZE-luc and 2 μL of the medium-copy plasmid pCS-HpaBC by electrotransfection into Escherichia coli, and then take 200 μL of the bacterial solution and spread it on a plate containing the corresponding antibiotic, and incubate overnight at 37°C.

[0052] Fermentation of coffee alcohol-producing microorganisms

[0053]Pick a single colony from the plate in the genetic engineering of Escherichia coli, transfer it to 3 mL of liquid LB with corresponding resistance, and cultivate i...

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Abstract

The invention relates to the technical field of biology and discloses a method for heterologous biosynthesis of tonquinol, caffeol and ferulenol. The metabolic pathway for production of tonquinol, caffeol and ferulenol is added into hosts. More precisely, tyrosine ammonia lyase, cinnamoyl-coA reductase, alcohol dehydrogenase and coumaric acid coA ligase are efficiently expressed in the host for producing tonquinol, 4-hydroxyphenylacetic acid3-monooxygenase is added to produce caffeol, coffee acyl coenzyme AO-methyl transferase or caffeic acid O-methyl transferase is added to produce ferulenol. Genes coded by the enzyme are guided into the host, and the production host of tonquinol, caffeol and ferulenol can be generated by means of glucose. The invention further discloses a method for co-culture of different hosts, and the caffeol and the ferulenol are produced more efficiently.

Description

technical field [0001] The present invention relates to the field of biotechnology. More precisely, the present invention relates to the metabolic pathway and co-cultivation method of heterologous biosynthesis of coumaryl alcohol, cafestol and ferulic alcohol. By enhancing the expression of upstream shikimate pathway enzymes, Escherichia As a host cell, coli finally increased the production of coumaryl alcohol, cafestol and ferulic alcohol. Background technique [0002] As bulk chemicals, coumaryl alcohol, cafestol and ferulic alcohol have a wide range of applications. These three compounds are direct precursors of lignin synthesis, can be used to synthesize lignin, and can clarify the role of enzymes in the lignin synthesis process. On the other hand, derivatives of coumaryl alcohol, cafestol, and ferulic alcohol have different physiological and pharmacological effects. In 2007, Gazak and Walterova discovered that silymarin, a derivative of ferulic alcohol, has anticancer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/22C12R1/19
CPCC12P7/22
Inventor 袁其朋孙新晓陈振娅刘少奇陈芳
Owner BEIJING UNIV OF CHEM TECH
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