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Application of ribosome sigma factor, mutant thereof and protein obtained by encoding in increasing yield of lipstatin

A sigma factor and ribosome technology, applied in the field of microbial genetic engineering, can solve the problem that the ribosomal sigma transcription factor has not been reported.

Active Publication Date: 2021-02-09
SICHUAN UNIVERSITY OF SCIENCE AND ENGINEERING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

For example, overexpressing frr in Streptomyces avermitilis can increase the production of avermectin (Li L etal, (2010) Over expression of ribosome recycling factor causes increased production of avermectin in Streptomyces avermitilis strains.J Ind Microbiol Biotechnol 37(7):673–679 ), but the ribosomal σ transcription factor that affects the synthesis of riprestatin has not been reported

Method used

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  • Application of ribosome sigma factor, mutant thereof and protein obtained by encoding in increasing yield of lipstatin
  • Application of ribosome sigma factor, mutant thereof and protein obtained by encoding in increasing yield of lipstatin
  • Application of ribosome sigma factor, mutant thereof and protein obtained by encoding in increasing yield of lipstatin

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Experimental program
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Effect test

preparation example Construction

[0037] (2) Preparation of standard products

[0038] Configure liprestatin standard (0.125g / L, 0.25g / L, 0.5g / L, 1g / L, 2g / L)

[0039] (3) HPLC detection conditions

[0040] Chromatographic column: YMC-Pack ODS-A; detection wavelength: 205nm; mobile phase: acetonitrile: water = 90:10; flow rate: 1.0mL / min; column temperature: 40°C.

[0041] (4) Drawing of liprestatin standard curve

[0042] The standard products with different concentrations were detected by HPLC according to the above conditions, and the peak area riprestatin concentration standard curve was drawn. Take the measured peak area A as the ordinate, and the liprestatin mass concentration C (g / L) as the abscissa, and draw the riprestatin standard curve. See Figure 5 , get the regression equation y=5E+6x+101960, R 2 =0.996, the absorbance has a good linear relationship with the mass concentration. Calculate the sample yield based on the liprestatin standard curve after the liquid phase

Embodiment 1

[0043] The acquisition of embodiment 1 bacterial strain

[0044]The frozen strains were diluted and spread on the ISP3 plate medium, placed in a 30-degree Celsius hydration incubator and cultured upside down. After the colonies grew out, a single colony was picked out and re-cultivated and passed to the second generation. Take a well-grown fresh plate, wash the spores on the lower surface with 10mL sterile water, place them in a conical flask equipped with glass beads, shake and culture at 28°C and 200r / min for 2-3h, filter and serially dilute to make a spore concentration of about 1×10 7 Individuals / mL single spore suspension is ready for use.

Embodiment 2

[0045] Example 2 strain genome analysis

[0046] The strain Streptomyces toxytricini was sent to Huada Genomics Company for whole-genome sequencing, and its ribosomal factors were compared and analyzed through genome sequencing and bioinformatics analysis, and 15 different ribosomal factors were found. Through screening, it was found that overexpression of ribosomal σ transcription Factors A and B can increase the production of riprestatin.

[0047] Based on the ribosomal sigma transcription factors A and B obtained through screening, the mutant gene of ribosomal sigma factor A and the mutant gene of ribosomal sigma factor B were respectively obtained by nucleotide substitution, and their nucleotide sequences are shown in SEQ Shown in ID NO.1 and SEQID NO.3;

[0048] At the same time, corresponding amino acid mutants are obtained, specifically:

[0049] Mutant of ribosomal sigma factor A: the 19th amino acid of the amino acid sequence encoded by ribosomal sigma transcription...

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Abstract

The invention discloses application of a ribosome sigma factor, a mutant thereof and protein obtained by encoding in increasing the yield of lipstatin. The ribosome sigma factor is a ribosome sigma factor A or a ribosome sigma factor B, the nucleotide sequence of the ribosome sigma factor A is shown as SEQ ID NO.5, and the nucleotide sequence of the ribosome sigma factor B is as shown in SEQ ID NO. 7; and the mutants of the ribosome sigma factors A and B are further comprised, the nucleotide sequence of the mutant of the ribosome sigma factor A is shown as SEQ ID NO. 1, and the nucleotide sequence of the mutant of the ribosome sigma factor B is shown as SEQ ID NO. 3. According to the application, the overexpression of the ribosome sigma factors A and B obtained by screening and the mutantsof the ribosome sigma factors A and B can improve the yield of lipstatin.

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering, and specifically relates to a ribosomal sigma factor and its mutant and the application of the encoded protein in increasing the output of riprestatin. Background technique [0002] Orlistat, the tetrahydro derivative of liprestatin, is currently certified by the US FDA, EU EMA and China SFDA as an obesity treatment drug that does not suppress appetite and does not act on the central nervous system. More than 40 million people around the world take it And successfully lose weight, it is currently the best-selling weight loss product. As a long-acting specific pancreatic lipase inhibitor, it can prevent the hydrolysis of triglycerides into free fatty acids and monoacylglycerols that can be absorbed by the small intestinal mucosa, thereby reducing calorie intake and controlling body weight. And liprestatin was originally isolated from Streptomyces toxytricini. It has a β-prop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N1/21C12P17/02C12R1/465
CPCC12N9/127C12P17/02C12Y207/07048C12N15/76C12N2800/101
Inventor 黄胜雄颜一军刘兴勇马小燕
Owner SICHUAN UNIVERSITY OF SCIENCE AND ENGINEERING
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