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Downstream key effector sch9 of Aureobasidium pullulans TOR pathway and its application

A kind of Aureobasidium pullulans and effector technology, applied in the biological field to achieve the effect of increasing yield

Active Publication Date: 2021-05-04
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether the key effector sch9 in the downstream of the TOR pathway is involved in regulating the synthesis of metabolites has not yet been elucidated.

Method used

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  • Downstream key effector sch9 of Aureobasidium pullulans TOR pathway and its application
  • Downstream key effector sch9 of Aureobasidium pullulans TOR pathway and its application
  • Downstream key effector sch9 of Aureobasidium pullulans TOR pathway and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1. Cloning of Aureobasidium pullulans TOR signaling pathway downstream effector sch9 gene

[0030] According to the whole genome sequencing results of Aureobasidium pullulans, the amplification primers for the downstream effector sch9 gene of the TOR signaling pathway were designed using bioinformatics software, and the upstream primer was sch9-F: 5'-ttcttttctctttcttttcccatggacccctcacatccttctt-3' (SEQ ID NO.1); the downstream primer is sch9-R: 5'-tgacataactaattacatgacttagacgtccatgccgtcgttgaa-3' (SEQ ID NO.2), and then Aureobasidium pullulans (A.pullulans CCTCC M2012223) genomic DNA is used as a template for PCR amplification, PCR amplification conditions were: pre-denaturation at 94°C for 5 minutes; 30 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s; post-extension at 72°C for 10 min; and incubation at 25°C for 10 min. Then the PCR product was subjected to agarose electrophoresis, and the target fragment was re...

Embodiment 2

[0031] Example 2, Aureobasidium pullulans TOR signaling pathway downstream effector sch9 gene knockout

[0032] (1) Construction of Aureobasidium pullulans TOR signaling pathway downstream effector sch9 gene knockout vector

[0033]The 20 bp sequence 5'-gtcaccgactacatgtccgg-3' (SEQ ID NO.6) in the middle 1396bp-1415bp of the sch9 gene coding region was selected as the sch9 targeting sequence. Using a one-step cloning kit (ClonExpress TM MultiS One Step Cloning Kit) cloned the gRNA expression cassette containing sch9 targeting sequence gRNA(sch9) (Psnr52-gRNA(sch9)-Tsup4) and neoR expression cassette (PtrpC-neoR-TtrpC) sequentially and simultaneously into p426-loxp-cas9 - The Sac II site of gRNA, that is, the plasmid vector p426-loxp-cas9-neoR-gRNA constructed to simultaneously express the gRNA targeting sch9 and the Cas9 protein performing the cutting function can be screened and transformed in the yeast-like fungus Aureobasidium pullulans (sch9). The specific principle is...

Embodiment 3

[0052] Example 3, Aureobasidium pullulans TOR signaling pathway downstream effector overexpression

[0053] (1) Construction of sch9 overexpression vector

[0054] Amplified from the pBARGPE1 vector with primers PgpdA-F / Pro-R ( Figure 4 In A, the strong promoter PgpdA whose publication number is the Chinese patent of CN104926930A) amplifies the sch9 gene nucleotide sequence (2413bp) from the source of Aureobasidium pullulans strain with primer sch9-F / sch9-R, and uses primer Ter -F / Tcyc1-R amplified from p414-TEF1p-cas9-CYC1t( Figure 4The terminator Tcyc1 in B). Among them, the 5' end of PgpdA-F was introduced into the linearized vector pk2-hyg digested by EcoR I (see Agrobacterium-mediated genetic transformation of Aureobasidium pullulans and high-efficiency screening of polymalic acid high-yielding strains. Tu Guangwei et al. Biological Engineering Journal. 2015.5.25) the 22bp sequence at the upstream end, so that the 5' end of PgpdA has a completely consistent sequence ...

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Abstract

The invention discloses the key effector sch9 downstream of the Aureobasidium pullulans TOR pathway and its application. The genome sequence of the effector sch9 is shown in SEQ ID NO.3, the encoded nucleotide sequence is shown in SEQ ID NO.4, and the encoded amino acid The sequence is shown in SEQ ID NO.5; the study of gene function found that after knocking out the sch9 gene, the biomass of Aureobasidium pullulans decreased, and the yield of polymalic acid increased significantly, about 10‑30%; after overexpressing the sch9 gene, The yield of the metabolite polymalic acid decreased, but the yield of exopolysaccharide was significantly increased by about 200‑300%. Therefore, the gene dosage level of the effector sch9 would regulate the conversion of the metabolite anabolism flow, which was beneficial to the increase of Aureobasidium pullulans multi-target Metabolite synthesis is instructive.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to sch9, a key effector downstream of the Aureobasidium pullulans TOR pathway, and also relates to the application of regulating the synthetic metabolic flow of polymalic acid and exopolysaccharide by regulating the dose level of sch9 gene. Background technique [0002] Aureobasidium pullulans is a yeast-like fungus that can metabolize polymalic acid, pullulan, pullulan and other metabolites. Polymalic acid (Polymalic acid, PMA) is a new type of fully biodegradable polyester polymer. Due to its good water solubility, biodegradability and biocompatibility, it can be used in biomedical materials, nutritional enhancement Pharmaceuticals, food packaging and other fields, with broad application prospects. In addition, polysaccharides are also metabolized to produce a class of polymers, which have broad application prospects in food thickening, drug packaging and other fields. [0003] Aure...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/37C12N15/31C12N15/80C12P19/04C12P7/62C12R1/645
CPCC07K14/37C12N15/80C12P7/625C12P19/04
Inventor 邹祥张园冯莹莹王攀李姗姗
Owner SOUTHWEST UNIV
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