Saccharomyces cerevisiae genetically engineered bacteria producing hypotanshinone diene and its construction method and application

A technology of hypotanshinone diene and genetically engineered bacteria, which is applied to the field of Saccharomyces cerevisiae genetically engineered bacteria producing hypotanshinone diene and its construction and application, which can solve problems such as unsatisfactory results

Active Publication Date: 2016-12-14
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The pyruvate / phosphoglyceraldehyde pathway exists in microbial Escherichia coli. In the previous study, Gao Wei et al. used the E. coli expression system reported in the literature to co-express the genes SmCPS and SmKSL with the commercial double expression vector pACYCDuet, but the effect Not ideal, only milligram-level subtanshinone dienes can be obtained (Wei Gao et al., 2009, Organic Letters, 11:5170-5173)

Method used

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  • Saccharomyces cerevisiae genetically engineered bacteria producing hypotanshinone diene and its construction method and application
  • Saccharomyces cerevisiae genetically engineered bacteria producing hypotanshinone diene and its construction method and application
  • Saccharomyces cerevisiae genetically engineered bacteria producing hypotanshinone diene and its construction method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1, the cloning of gene element

[0057] The cloning of genetic elements is divided into the following five steps:

[0058] (1) Yeast DNA extraction

[0059] Pick bacterial plaque (Saccharomyces cerevisiae BY4742) in YPD liquid medium (recipe: 1% Yeast Extract (yeast extract), 2% Peptone (peptone), 2% Dextrose (glucose), 30°C, 200rpm, culture 24h. 10000g , collect the bacteria in a 1.5ml centrifuge tube for 5 minutes, wash twice with water, resuspend the bacteria in yeast wall-breaking solution (25ul yeast wall-breaking enzyme, 470ul sorbitol buffer, 5u lβ-ME), and incubate at 30°C for 1h After centrifugation; cells were resuspended in 500ulTENTS buffer (10mM Tris-HCl, pH 7.5; 1mM EDTA, pH8.0; 100mM NaAc; 2% triton-100; 1% SDS), in a water bath at 60°C for 1 hour; extracted with phenol / chloroform Extract 2 times; add 3 times the volume of EtOH to the supernatant, 1 / 10 times the volume of 3M NaAc, and place it in the refrigerator at -20°C for 2 hours; centrif...

Embodiment 2

[0076] Embodiment 2, the cloning of regulatory element

[0077] Using Saccharomyces cerevisiae genomic DNA as a template, use the primers in primer list 4 to amplify the promoters of PGK1 (750bp), TEF1 (450bp) and ADH1 (1500bp), and the terminator ADH1t (158bp). The amplification system is: NewEngland Biolabs Phusion 5Xbuffer 10ul, dNTP (10mM each dNTP) 1ul, DNA template 20ng, primer (10uM) 1ul, Phusion High-Fidelity DNA Polymerase (2.5U / ul) 0.5ul, add distilled water to the total volume 50ul. Amplification conditions are pre-denaturation at 98°C for 2 minutes (1 cycle); denaturation at 98°C for 10 seconds, annealing for 10 seconds (see primer list 4 for annealing temperature), extension at 72°C for 1.5 minutes (32 cycles); extension at 72°C for 8 minutes (1 cycle). The amplified product was cloned into the pEASY-Blunt cloning vector. Transformation, sequencing verification (method is the same as step 2 of Example 1).

[0078] Primer List 4

[0079]

Embodiment 3

[0080] Embodiment 3, the plasmid construction that carries gene element

[0081] The construction of plasmids carrying genetic elements is divided into the following two steps

[0082] (1) Construction of plasmid carrying tHMG1-UPC2.1 gene

[0083] The construction of the plasmid carrying the tHMG1-UPC2.1 gene has the following 8 steps:

[0084] The first step: construction of pRS406-δDNA-URA3 plasmid.

[0085] The plasmid pEASY-Blunt-δDNA1 obtained in Step 2 of Example 1 was digested with BstE1 and Sac1; the plasmid pEASY-Blunt-δDNA2 obtained in Step 2 of Example 1 was digested with BstE1 and Kpn1. Purify the two target fragments by tapping rubber, add 50ng each to the ligation system: 2ul 10XT4ligation Buffer (NEB Company), 1ul T4ligase (NEB Company, 400,000 cohesive end units / ml), add distilled water to 20ul, react at room temperature for 2 hours to obtain the ligation product, take 1ul The ligation product was added to the PCR system: NewEngland Biolabs Phusion 5Xbuffer...

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Abstract

The invention discloses a Saccharomyces cerevisiae genetically engineered bacterium producing hypotanshinone diene, a construction method and application thereof. The genetically engineered bacterium provided by the present invention is constructed according to the method comprising the following steps: introducing exogenous Salvia miltiorrhiza copalyl pyrophosphate synthase gene (SmCPS) and Danshen subtanshinone diene synthase gene (SmKSL) in Saccharomyces cerevisiae ) to obtain recombinant Saccharomyces cerevisiae producing hypotanshinone dienes, which are referred to as recombinant Saccharomyces cerevisiae ZD-T-000 and ZD-T-010. On this basis, increase 3-hydroxy-3-methylglutaryl-CoA reductase, terpene regulator protein UPC2, geranylgeranyl pyrophosphate synthase, farnesyl pyrophosphate synthase Activity of one or several proteins, 24 strains were constructed (ZD‑T‑001~ZD‑T‑008, ZD‑T‑011~ZD‑T‑018, ZD‑T‑021~ZD‑T‑028) Recombinant Saccharomyces cerevisiae strains high in subtanshinone diene production. Saccharomyces cerevisiae ZD-Tans-001 constructed by the present invention can produce 487.91 mg / L of hypotanshinone diene in 6 days of fermentation under aerobic conditions.

Description

technical field [0001] The invention relates to a Saccharomyces cerevisiae genetically engineered bacterium producing hypotanshinone diene, a construction method and application thereof. Background technique [0002] Miltiradiene is a fat-soluble diterpene component extracted from the important medicinal plant Salvia miltiorrhiza, and is a common precursor of tanshinone compounds (Wei Gao et al., 2009, Organic Letters, 11:5170-5173). Such compounds (such as cryptotanshinone, tanshinone Ⅱ A, tanshinone Ⅱ B, dihydrotanshinone) have very good application prospects in anti-inflammation, cardiovascular and cerebrovascular, anti-tumor, etc. Currently, there are compound danshen tablets, Composite Danshen Dripping Pills and other flagship products with sales of hundreds of millions of RMB were launched. Sodium tanshinone IIA sulfonate for injection has been widely used in the treatment of coronary heart disease, myocardial infarction and other diseases. According to the current ra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/63C12P15/00C12R1/865
Inventor 张学礼黄璐琦戴住波刘怡
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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