Genetically engineered saccharomycetes for producing baicalein compounds as well as construction method and application of genetically engineered saccharomycetes

A technology for baicalein and compounds, which is applied in the production of baicalein compounds and the field of genetically engineered yeast, can solve the problems of high requirements, consumption of carbon source and energy of host cells, adverse effects and the like

Active Publication Date: 2020-07-24
EAST CHINA UNIV OF SCI & TECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of synthesizing baicalein by engineering strains of Escherichia coli, the precursor of L-phenylalanine needs to be artificially added, and the synthesis efficiency is low
In addition, endotoxins from E. coli potentially lead to more difficult and demanding drug purification processes
[0006] In the prior art, yeast cells express compounds, although there are some explorations, but yeast cells have not been successfully used to synthesize baicalein compounds in this field, especially Pichia pastoris cells
This may be due to the fact that the expression of such compounds requires the introduction of many genes, and the catalytic activity of some genes encoded by the protein itself is not high, so high expression is required to meet the demand for synthetic capacity, and the excessive dosage of exogenous genes leads to a series of physiological problems in yeast cells
For example, there are too many exogenous genes and there are instability problems; protein folding stress has adverse effects on physiology; protein overexpression will consume the carbon and energy sources of the host cells, and will bring a certain metabolic burden to the host
, although Pichia pastoris has been applied to the expression of many exogenous genes, there are still bottlenecks in this field for the stable co-expression of various exogenous genes and the realization of high-efficiency production

Method used

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  • Genetically engineered saccharomycetes for producing baicalein compounds as well as construction method and application of genetically engineered saccharomycetes
  • Genetically engineered saccharomycetes for producing baicalein compounds as well as construction method and application of genetically engineered saccharomycetes
  • Genetically engineered saccharomycetes for producing baicalein compounds as well as construction method and application of genetically engineered saccharomycetes

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Embodiment 1, the construction of expression plasmid

[0115] 1.P GAP Construction of plasmids expressing each gene

[0116] The genes RtPAL, AtCPR1 and SbUBGT were fully synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and directly constructed on the vector plasmid pGAP Zα (Invitrogen). Plasmid names: pGAP_RtPal, pGAP_AtCPR1pGAP_SbUBGT. The original plasmids of other genes (Sb4CL, SbCHS-2, SbCHI, SbFNSII-2, SbF6H, SbF8H, SbPFOMT5) were all from Shanghai Chenshan Plant Science Research Center, Chinese Academy of Sciences. Design specific primers, use the original plasmid of each gene as a template, and use the corresponding primers in Table 1 to amplify related genes derived from Scutellaria baicalensis (Sb4CL, SbCHS-2, SbCHI, SbFNSII-2, SbF6H, SbF8H, SbPFOMT5) by PCR ; Use AsuII and KpnI to double-digest plasmid pGAP Zα, and use the seamless assembly kit to integrate each gene into plasmid pGAP Zα respectively. P GAP Downstream, after the sequence is correct,...

Embodiment 2

[0139] Embodiment 2, preparation of target recombinant yeast

[0140]1. PCR verification of recombinant strains

[0141] Each DNA fragment and the corresponding gRNA plasmid were electrotransformed into Pichia pastoris GS115-Δku70 (refer to 201910403132.1). After recovery, they were spread on YND solid medium, and after 5 days of cultivation, fresh colonies were picked and cultivated in YPD liquid medium. The yeast genome extraction kit extracts the genome of each transformant, and identifies the integration of each gene synthesized by baicalein into the Pichia pastoris genome by cloning PCR reaction.

[0142] Cloning PCR reaction conditions:

[0143] (1) Initial denaturation at 95°C for 5 minutes;

[0144] (2) Denaturation at 95°C for 30s, annealing at 50°C for 30s, extension at 72°C for 1min / kb, and 30 cycles of reaction;

[0145] (3) Finally extend at 72°C for 7min.

[0146] 2. Acquisition of recombinant bacteria containing 7 foreign genes

[0147] Design specific prim...

Embodiment 3

[0154] Embodiment 3, 250ml Erlenmeyer flask fermentation process of recombinant engineering bacteria

[0155] Baicalein-producing strain: it contains 7 exogenous genes: RtPAL, Sb4CL, SbCHS-2, SbCHI, SbFNSII-2, SbF6H, AtCPR1.

[0156] The strain producing baicalin: it contains 8 foreign genes: RtPAL, Sb4CL, SbCHS-2, SbCHI, SbFNSII-2, SbF6H, AtCPR1, SbUBGT.

[0157] The strain producing wogonin: it contains 2 foreign genes: SbF8H and SbPFOMT5.

[0158] Cultivate the recombinant bacteria to the logarithmic phase in the liquid YPD medium at 30°C with a rotational speed of 200r / min, collect the bacteria and wash them twice with sterile water, then transfer to a 250mL Erlenmeyer flask containing 50mL of YPD liquid medium (the strains that produce baicalein or baicalin do not need to add additional precursors, and the strains that produce wogonin also add 200 μM chrysin), and cultivate them at 30°C with a rotation speed of 200r / min (the strains that produce baicalein or baicalin are...

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Abstract

The invention relates to genetically engineered saccharomycetes for producing baicalein compounds as well as a construction method and application of the genetically engineered saccharomycetes. A series of exogenous genes are introduced into yeast engineering bacteria through a gene recombination technology to obtain the yeast engineering bacteria capable of producing baicalein, baicalin or wogonin. The yeast engineering bacteria have the characteristics of a low metabolic background, strong heterologous expression ability, no need of additional addition of a precursor, capability of synthesizing a final product in a whole-cell manner, easiness in separation of the final product and the like, and a novel thought is provided for industrial production of flavonoid drugs.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a genetically engineered yeast for producing baicalein compounds, its construction method and application. Background technique [0002] Flavonoids widely exist in higher plants and ferns, belong to the secondary metabolites of plants, and are important active ingredients of many traditional Chinese herbal medicines. Due to the particularity and diversity of their structures, flavonoids have a variety of biological functions: treating cardiovascular and cerebrovascular diseases, lowering blood lipids, lowering cholesterol, inhibiting thrombus and expanding coronary arteries; Sensitivity, strengthen the function of thyroid C cells to secrete calcitonin, and finally inhibit bone resorption to treat osteoporosis; remove excess free radicals in the metabolic process, and protect liver cells from damage by free radicals, electrophilic compounds and poisons ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/29C12N15/31C12N15/81C12P17/06C12R1/84
CPCC07K14/39C07K14/415C12N15/815C12P17/06
Inventor 蔡孟浩钱芷兰陈鑫洁赵清
Owner EAST CHINA UNIV OF SCI & TECH
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