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Brewer yeast gene engineering strains for producing miltiradiene, and construction method and application of brewer yeast gene engineering strains

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: 2014-05-28
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 Escherichia 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 milligrams of subtanshinone dienes can be obtained (Wei Gao et al., 2009, Organic Letters, 11:5170-5173)

Method used

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  • Brewer yeast gene engineering strains for producing miltiradiene, and construction method and application of brewer yeast gene engineering strains
  • Brewer yeast gene engineering strains for producing miltiradiene, and construction method and application of brewer yeast gene engineering strains
  • Brewer yeast gene engineering strains for producing miltiradiene, and construction method and application of brewer yeast gene engineering strains

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] The cloning of genetic elements is divided into the following five steps:

[0067] (1) Yeast DNA extraction

[0068] 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, 5ul β-ME), and incubate at 30°C for 1h After centrifugation; cells were resuspended in 500ul TENTS 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 1h; phenol / chloroform extraction Extract 2 times; add 3 times the volume of EtOH to the supernatant, 1 / 10 times the volume of 3M NaAc, and place in a refrigerator at -20°C for 2 hours; centrifuge at 13,000g at 4°C for 10 minutes, discard the supernatant, a...

Embodiment 2

[0085] 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 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).

[0086] Primer List 4

[0087]

[0088]

[0089] Embodiment 3, the plasmid construction that carries gene ele...

Embodiment 3

[0090] The construction of plasmids carrying genetic elements is divided into the following two steps

[0091] (1) Construction of plasmid carrying tHMG1-UPC2.1 gene

[0092] The construction of the plasmid carrying the tHMG1-UPC2.1 gene has the following 8 steps:

[0093] The first step: construction of pRS406-δDNA-URA3 plasmid.

[0094] 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 10ul, dNTP (10mM each dNTP) 1ul, DNA template 20ng, primers Sac1-A-δDNA1 a...

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Abstract

The invention discloses a brewer yeast gene engineering strains for producing miltiradiene, and a construction method and application of the brewer yeast gene engineering strains. The construction method for the gene engineering strains comprises the following steps of introducing exogenous SmCPS (salvia miltiorrhiza copalyl pyrophosphate synthase) and SmKSL (salvia miltiorrhiza kaunene synthase like) into original brewer yeast to obtain recombinant brewer yeasts for producing the miltiradiene, wherein the recombinant brewer yeasts are recorded as ZD-T-000 and ZD-T-010; improving the activity of one or more proteins in 3-hydroxy-3-methylglutaryl coenzyme A reductase, a terpene control element protein UPC2, geranylgeranyl pyrophosphate synthase and farnesyl phosphate synthase on such a basis to construct 24 high-miltiradiene yield recombinant brewer yeast strains (ZD-T-001 to ZD-T-008, ZD-T-011 to ZD-T-018 and ZD-T-021 to ZD-T-028). The brewer yeast ZD-Tans-001 can be used for producing 487.91mg / L miltiradiene after being fermented for 6 days under an aerobic condition.

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). This type of compound (such as cryptotanshinone, tanshinone IIA, tanshinone IIB, dihydrotanshinone) has very good application prospects in anti-inflammatory, cardiovascular and cerebrovascular, anti-tumor and other aspects. At present, among its related products, 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...

Claims

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Application Information

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Patent Type & Authority Applications(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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