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Saccharomyces cerevisiae engineering bacterium for producing rapeseed sterol and construction method and application thereof

A technology of Saccharomyces cerevisiae and construction method, which is applied in the field of metabolic engineering, and can solve problems such as high price of brassicasterol and scarce sources of brassicasterol

Pending Publication Date: 2020-03-24
HEBEI LANSHENG BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The scarcity of sources of brassicasterol makes the price of commercially available brassicasterol very expensive

Method used

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  • Saccharomyces cerevisiae engineering bacterium for producing rapeseed sterol and construction method and application thereof
  • Saccharomyces cerevisiae engineering bacterium for producing rapeseed sterol and construction method and application thereof
  • Saccharomyces cerevisiae engineering bacterium for producing rapeseed sterol and construction method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Construction of Δ7-Red Yeast Expression Vector

[0063]According to the published sterol Δ7-reductase gene (Δ7-red, ATU49398) sequence, the Δ7-red gene (SEQ ID No.1) was artificially synthesized, and the plasmids containing the Δ7-red gene were named pGSI-Δ7-Red2. Primers Fhis49398(SEQ ID No.5) / Rhis49398(SEQ ID No.6) were designed, pGSI-Δ7-Red2 was used as template, KOD PlusTaq was used as polymerase, and PCR was carried out according to the following procedure: 94°C 2min; 94°C 20sec, 55°C 30sec at ℃, 1min at 72℃, 30 cycles; 7min at 72℃; store at 4℃. After the reaction, take 10 μl for electrophoresis, the result is as follows figure 1 shown. It can be seen from the results that a band with a size of 1293bp appeared on the swimming lane, which was consistent with the expected size, indicating that the Δ7-red gene had been amplified successfully.

[0064] The pESC-Trp (50 ng / μl) plasmid was digested with EcoRI and SpeI (Baoric Medical Biotechnology (Beijing) Co., Ltd.)...

Embodiment 2

[0071] Construction of Yeast Expression Vector Containing GDH Gene

[0072] According to the published glucose dehydrogenase sequence (glucose dehydrogenase, GDH, M12276.1), the GDH gene (SEQ ID No.3) was artificially synthesized to obtain the plasmid pGSI-GDH containing the GDH gene. Design primers FBamH12276 (SEQ ID No.7) and RSal12276 (SEQ ID No.8), use pGSI-GDH as template, KOD Plus Taq as polymerase, and perform PCR according to the following procedure: 94°C for 2min; 94°C for 20sec, 60°C 30sec, 30sec at 72°C, 30 cycles; 7min at 72°C; store at 4°C. After the reaction, take 10 μl for electrophoresis, the result is as follows image 3 shown. A specific band with a size of about 750bp appeared on the swimming lane, which was consistent with the theoretical size of GDH, indicating that the GDH gene was successfully cloned.

[0073] The pESCTrp-Δ7Red2 (40ng / μl) recombinant plasmid was digested with BamHI and SalI (Baoric Medical Biotechnology (Beijing) Co., Ltd.) according ...

Embodiment 3

[0079] Construction of Yeast Engineering Strain Containing Δ7-red Gene and GDH Gene

[0080] Extract pESCTrp-Δ7Red2-GDH plasmid. The above plasmid was introduced into Saccharomyces cerevisiae W303-1B by LiAc-mediated method, and cultured upside down at 28°C for 2-3 days, and the engineered bacteria grew clones. Eight clones were picked for plasmid extraction. Using the extracted plasmid as a template and Fhis49398 / Rhis49398( as primers, perform PCR according to the following procedures and systems: 94°C for 2min; 94°C for 20sec, 55°C for 30sec, 72°C for 1min, 30 cycles; 72°C for 7min; 4°C for storage. After the reaction, take 10 μl for electrophoresis. It can be seen from the results that clones 1, 2, 4, 5, and 6 have bands with a size of 1.3 kb, which is consistent with the expected size, indicating that the Δ7-red gene has been introduced into Saccharomyces cerevisiae ( Figure 5 ). Then, using FBamH12276 and RSal12276 as primers and the extracted yeast plasmid as a temp...

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Abstract

The invention provides a saccharomyces cerevisiae engineering bacterium for producing rapeseed sterol and a construction method and application thereof, which belong to the technical field of metabolic engineering. The saccharomyces cerevisiae engineering bacterium contains sterol delta 7-reductase genes and glucose dehydrogenase genes. The saccharomyces cerevisiae engineering bacterium provided by the invention can be used for reducing ergosterol into rapeseed sterol. According to the invention, sterol delta 7-reductase and glucose dehydrogenase are co-expressed in the saccharomyces cerevisiae engineering bacterium; and NADPH formed by the glucose dehydrogenase can be used as a cofactor, and the sterol delta 7-reductase is used for reducing inherent ergosterol of saccharomyces cerevisiaeinto rapeseed sterol, so that the purpose of biologically preparing rapeseed sterol is achieved.

Description

technical field [0001] The invention belongs to the technical field of metabolic engineering, and in particular relates to a brassicasterol-producing Saccharomyces cerevisiae engineered bacterium and its construction method and application. Background technique [0002] Brassicasterol (Brassicasterol, CAS No. 474-67-9) is an important steroidal compound, which can be used to synthesize pesticides such as brassinolide, and has very important value. Brassicasterol is mainly obtained through plant extraction, but due to the low content of brassicasterol in plants, it is difficult to separate and purify. The chemical synthesis of brassicasterol has been successful, but there is no report of industrial production. The scarcity of sources of brassicasterol makes the commercially available brassicasterol very expensive. Contents of the invention [0003] In view of this, the object of the present invention is to provide a brassicaster-producing Saccharomyces cerevisiae engineer...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/53C12N15/81C12P33/00C12R1/865
CPCC12N9/001C12N9/0006C12Y101/9901C12Y103/01072C12N15/81C12P33/00
Inventor 郭庆春孔建强董志鹏苑立刚褚倩倩
Owner HEBEI LANSHENG BIOTECH CO LTD
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