Glucose derepression induction promoter and terminator, and applications of promoter and terminator

A promoter and glucose technology, applied in the field of genetic engineering, can solve the problem that the promoter cannot be used

Active Publication Date: 2017-01-11
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many researchers have isolated the ADH2 promoter of Saccharomyces cerevisiae or other ascomycete yeasts, or other glucose derepression-inducible promoters for their own genetic engineering operations (Hintz WE1, Lagosky PA. Biotechnology (NY). 1993,11(7):815-818.; Weinhandl K, Winkler M, Glieder A, et al. Microb Cell Fact.2014, 13:5.; Shen MW, Fang F, Sandmeyer S, et al.Yeast.2012 ,29(12):495-503.; Lee KM, DaSilva NA.Yeast.2005,22(6):431-440.; Li Wei, Li Yuyang. Acta Genetica Sinica, 1997,24(6):561-568. ; Qiao Xuefeng, Lu Junjie, Xiao Ciying, et al. Journal of East China University of Science and Technology (Natural Science Edition), 2013,39(5):559-564), but this type of promoter cannot be applied to Rhodosoporidium, Genetic engineering manipulations for gene expression, genetic engineering and strain improvement in Sporidiobolus, Sporobolomyces and Rhodotorula

Method used

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  • Glucose derepression induction promoter and terminator, and applications of promoter and terminator
  • Glucose derepression induction promoter and terminator, and applications of promoter and terminator
  • Glucose derepression induction promoter and terminator, and applications of promoter and terminator

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082]Embodiment 1: the extraction of Rhodosporidium toruloides (Rhodosporidium toruloides) CGMCC 2.1389 total RNA

[0083] Rhodosporidium toruloides (R. toruloides) CGMCC 2.1389 (purchased from China General Microbiological Culture Collection Center (CGMCC)) was inoculated into 10 mL YEPD liquid medium (glucose 20.0 g / L, Yeast extract 10.0g / L, peptone 20.0g / L, pH 6.0), cultured on a shaker at 30°C for 24h, then transferred the bacterial solution to 100mL YEPD liquid medium at a volume ratio of 1:50, and Incubate in a shaker at 30°C for 14 hours to reach the logarithmic growth phase. Centrifuge at 5000rpm for 4min at 4°C to collect the cells, quickly freeze the cells with liquid nitrogen, and grind to break the wall (Yang F, Tan HD, Zhou YJ, etal.Mol.Biotechnol.2010,47(2):144–151 .). Total RNA was extracted using the TakaRa RNAiso kit and following its standard procedures.

[0084] The RNA was subjected to 1.5% (mass / volume concentration) agarose gel electrophoresis, observ...

Embodiment 2

[0085] Embodiment 2: Rhodosporidium toruloides CGMCC 2.1389cDNA first strand synthesis and ADH2 degenerate PCR

[0086] Using the total RNA of Rhodosporidium toruloides CGMCC 2.1389 as a template, the first strand of cDNA was synthesized by reverse transcription. First, mix 1.0 μL total RNA (about 2 μg), 1.0 μL primer SMART IV: 5′-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3′ and 1.0 μL oligo dT-linker primer CDSⅢ / 3′: 5′-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T) 30N-1 N-3′, 2.0 μL of DEPC-treated water (diethyl pyrocarbonate-treated water, purchased from Dalian TakaRa Company), was added to the PCR tube and mixed evenly, kept at 72°C for 2 minutes, immediately placed on ice for 2 minutes, and 2.0 μL of 5×first-strand buffer (Clontech), 1.0 μL of DTT (20 mM), 1.0 μL of dNTP (10 mM), and 1.0 μL of powerscript reverse transcriptase (Clontech) were added to the system, and mixed well. Extend the reaction at 42°C for 60 minutes, and end the reaction at 4°C, and store it at -20°C for later use...

Embodiment 3

[0088] Example 3: Amplification of Rhodosporidium toruloides CGMCC 2.1389RtADH2 CDS

[0089] Genomic DNA of Rhodosporidium toruloides CGMCC 2.1389 was extracted by glass bead wall breaking method (Chapter 13 of the third edition of the Molecular Biology Experiment Guide, written by Osper et al., translated by Yan Ziying et al., published by Science Press). The prepared genomic DNA was measured by NanodropND-1000, and the OD was measured 260 / OD 280 =1.85, indicating that the quality of genomic DNA is very good. The concentration was 280ng / μL, totaling 500μL, and the genomic DNA samples were frozen at -20°C for later use.

[0090] According to the cDNA sequence of alcohol dehydrogenase (RtADH2) induced by glucose deblocking obtained in Example 2, a pair of gene-specific primers were designed, ADH2-p1:5'-atgagcaaccctcaaatccccaaggaaggc-3' and ADH2-p2:5'-ttagaagttcttgaggacgatgcggcc -3', using the genomic DNA of Rhodosporidium toruloides CGMCC 2.1389 as a template, PCR amplifica...

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Abstract

A promoter and a terminator which can be widely applied to gene expression, genetic engineering operation and strain improvement of Rhodosoporidium, Sporidiobolus, Sporobolomyces and Rhodotorula in red yeast through amplifying the upstream and downstream sequences of Rhodosporidium toruloides glucose derepression induction alcohol dehydrogenase Adh2 genome DNA and carrying out biological information analysis and functional verification, the nucleotide sequence of the promoter is represented by SEQ ID NO:1, and the nucleotide sequence of the terminator is represented by SEQ ID NO:2. The invention also relates to a DNA expression box or a recombinant vector containing above elements, a method using the correlated elements to construct Rhodosoporidium, Sporidiobolus, Sporobolomyces and Rhodotorula gene engineering strains, and corresponding strains.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a promoter terminator of Rhodosporidium toruloides and its application, including a transformation method necessary for the construction of genetic engineering strains and the like. Background technique [0002] Microorganisms are one of the most widely distributed species in nature. They have excellent biosynthetic ability and can synthesize almost all organic chemicals on earth. Compared with multicellular organisms, although the metabolic pathways of microorganisms are relatively simple, the production of their compounds is efficient and fast, with the characteristics of mild reaction conditions, strong controllability, and easy large-scale production, which can be used as an excellent cell factory. [0003] Some microorganisms in nature can store more than 20% of the dry cell weight of oil in the cell under certain conditions (such as nitrogen source d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63C12N1/21C12N1/19C12N15/81
Inventor 赵宗保张素芳马斯佳焦翔
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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