3-Phosphate Glyceraldehyde Dehydrogenase Promoter and Terminator and Their Applications

A technology of glyceraldehyde phosphate dehydrogenase and promoter, which is applied in the field of genetic engineering, can solve the problems of no T. dermatosus promoter sequence report, restrictions on strain transformation, and no promoter, etc., so as to promote strain improvement and Effects of Metabolic Engineering Research

Active Publication Date: 2018-09-14
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

But so far, no promoter sequence suitable for Trichosporium dermatitis has been reported, nor is there any endogenous promoter suitable for Lipoyces stariae, which restricts the targeted strain transformation
[0006] Although the 3-phosphate glyceraldehyde dehydrogenase promoters of Rhodosporidium toruloides and Rhodotorula glutinosa have been isolated for their own genetic engineering operations (FEMS Yeast Res.2014, 14(4): 547-555 ; Appl Microbiol Biotechnol.2013, 97 (2): 719-729.), but this promoter has not been seen to be used in Trichosporium dermatosa, and other specialties outside the Basidiomycota, such as genes such as Ascomycota spp. Reports on Engineering Operations

Method used

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  • 3-Phosphate Glyceraldehyde Dehydrogenase Promoter and Terminator and Their Applications
  • 3-Phosphate Glyceraldehyde Dehydrogenase Promoter and Terminator and Their Applications
  • 3-Phosphate Glyceraldehyde Dehydrogenase Promoter and Terminator and Their Applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1: Extraction of total RNA from Lipomyces starkeyi NRRL Y-11557

[0088] L. starkeyi NRRL Y-11557 (purchased from the American Agricultural Research Culture Collection (NRRL), Bacterial Foodborne Pathogens & Mycology Research, 1815N.University Street IL 61604.Peoria, Illinois) was inoculated into 50ml YEPD by slant In the liquid medium (glucose 20.0g / l, yeast extract 10.0g / l, peptone 20.0g / l, pH 6.0), culture on a shaker at 30°C for 24h, and then transfer the bacterial solution to Received into 100ml YEPD liquid medium, cultured on a shaker at 30°C for 12h to reach the logarithmic growth phase. Centrifuge at 5000 rpm for 4 minutes at 4°C to collect the cells, freeze the cells quickly with liquid nitrogen, and grind to break the wall. Total RNA was extracted using the TakaRa RNAiso kit and following its standard procedures.

[0089] The RNA was subjected to 1.5% agarose gel electrophoresis, observed and identified using a fluorescence-ultraviolet analyzer, and ...

Embodiment 2

[0090] Example 2: Synthesis of first strand of L. stardii NRRL Y-11557cDNA and degenerate PCR of GPD

[0091] The first-strand cDNA was synthesized by reverse transcription using the total RNA of L. stariae NRRL Y-11557 as a template. 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 5×first-strand buffer (Clontech), 1.0 μl DTT (20 mM), 1.0 μl dNTP (10 mM), 1.0 μl powerscript reverse transcriptase (Clontech) were added to the system, and mixed. 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.

[0092] Design and synthesize two degenerate...

Embodiment 3

[0093] Example 3: Amplification of Lipospora starii NRRL Y-11557 Genomic DNA

[0094] Genomic DNA of L. stariae NRRL Y-11557 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 Nanodrop 1000, and the OD was measured 260 / OD 280 =1.85, indicating that the quality of genomic DNA is very good. The concentration was 120ng / μl, 500μl in total, and the genomic DNA samples were frozen at -20°C for future use.

[0095] According to the cDNA sequence of glyceraldehyde phosphate dehydrogenase obtained in Example 2, a pair of gene-specific primers were designed, GPD-p1: 5'-ATGCTTAACCTCAAAGTATCTGTT-3' and GPD-p2: 5'-TTAGAACTTCTGCTCGACATCT-3', Using the genomic DNA of L. studlii NRRLY-11557 as a template, PCR amplification was performed according to conventional methods to obtain a PCR prod...

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Abstract

In the present invention, by amplifying the upstream and downstream sequences of 3-glyceraldehyde phosphate dehydrogenase genome DNA of Lipomyces starii, and performing biological information analysis and functional verification, the invention can be widely used in oleaginous yeast Lipomyces (Lipomyces) and silk A promoter and a terminator for gene expression, genetic engineering operation and strain improvement in Trichosporon, the nucleotide sequences of which are SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The present invention also relates to a DNA expression cassette or a recombinant vector containing these elements, and a method for constructing a genetically engineered bacterial strain of the genus Liposaccharomyces or Trichosporon by using the relevant elements and corresponding bacterial strains.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a promoter terminator of Lipomyces starkeyi 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 deficien...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/70C12N15/81C12N1/21C12N1/19C12R1/645
Inventor 赵宗保林心萍张素芳王雪颖
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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