US 2013/0171698 A1Published application

Heterologous expression of fungal polyketide synthetic gene in yeast

AssigneesNational University Corporation Okayama University, Shizuoka Prefecture Public University Corporation

InventorsKenji Watanabe, Hisao Moriya

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Abstract

The present invention relates to a method of removing an intron contained in a gene from a eukaryotic gene, and linking only the exon sequences to prepare an expression vector comprising the linked sequences. Specifically, the invention relates to a method of preparing an expression vector containing linked exon sequences comprising amplifying exon sequences by PCR as one or more fragments from a giant fungal gene containing an intron, and linking the fragments together with a restriction enzyme-treated vector using the gap repair cloning method; a method of preparing an expression vector containing a full-length cDNA sequence by synthesizing and linking cDNA fragments from a fungal giant gene; a transformant having introduced therein an expression vector prepared by the method; a protein produced by the transformant; and a method of preparing a compound produced by the protein using the expression vector.

Claims18

Dependent claims

A method of preparing an expression vector by linking exon sequences of a eukaryotic gene containing an intron or from the genome sequence of a presumed eukaryotic gene containing an intron to form the expression vector containing the linked sequences, said method comprising the steps of:

(a) amplifying exon sequences from a genome extracted from a eukaryote by PCR to prepare multiple fragments, wherein the forward primer used in the PCR has, in order from the 5′ end to the 3′ end, a sequence complementary to the sequence of the 3′ terminal part of the sense strand of a fragment to which the amplified fragment is to be linked, or a sequence complementary to the sequence of the 3′ terminal part of the sense strand of a restriction enzyme-treated terminal part of the vector, and a sequence complementary to the sequence of the 5′ terminal part of the sense strand of the fragment to be amplified, and wherein the reverse primer has, in order from the 5′ end to the 3′ end, a sequence complementary to the sequence of the 3′ terminal part of the antisense strand of a fragment to which the amplified fragment is to be linked, or a sequence complementary to the sequence of the 3′ terminal part of the antisense strand of a restriction enzyme-treated terminal part of the vector, and a sequence complementary to the sequence of the 5′ terminal part of the antisense strand of the fragment to be amplified, whereby a sequence homologous to a terminal part of a fragment to be linked to the fragment to be amplified or a sequence homologous to a restriction enzyme-treated terminal part of the vector are added to the end of the fragment to be amplified;
and
(b) simultaneously transforming a budding yeast or fission yeast with the fragments obtained in the step (a) and a restriction enzyme-treated vector to obtain the expression vector containing fragments linked to the fragments and fragments linked to the vector that are joined via homologous recombination,
wherein the gene or genome sequence of the presumed gene encodes a polyketide synthase gene or nonribosomal peptide synthetase gene.

1 more element

A method of preparing an expression vector comprising a full-length cDNA sequence from a eukaryotic gene containing an ignitron or of the genome sequence of a presumed eukaryotic gene containing an intron, said method comprising the steps of:

(a) synthesizing cDNA fragments from mRNA extracted from a eukaryote and amplifying the cDNA fragments by PCR, wherein the forward primer used in the PCR has, in order from the 5′ end to the 3′ end, a sequence complementary to the sequence of the 3′ terminal part of the sense strand of a fragment to which the amplified fragment is to be linked, or a sequence complementary to the sequence of the 3′ terminal part of the sense strand of a restriction enzyme-treated terminal part of the vector, and a sequence complementary to the sequence of the 5′ terminal part of the sense strand of the fragment to be amplified, and wherein the reverse primer has, in order from the 5′ end to the 3′ end, a sequence complementary to the sequence of the 3′ terminal part of the antisense strand of a fragment to which the amplified fragment is to be linked, or a sequence complementary to the sequence of the 3′ terminal part of the antisense strand of the restriction enzyme-treated terminal part of the vector, and a sequence complementary to the sequence of the 5′ terminal part of the antisense strand of the fragment to be amplified, whereby a sequence homologous to a terminal part of a fragment to be linked to the fragment to be amplified or a sequence homologous to a restriction enzyme-treated terminal part of the vector are added to the end of the fragment to be amplified;
and
(b) simultaneously transforming a budding yeast or fission yeast with the cDNA fragments obtained in the step (a) and a restriction enzyme-treated vector to obtain the expression vector containing fragments linked to the fragments and fragments linked to the vector that are joined via homologous recombination,
wherein the gene or genome sequence of the presumed gene encodes a polyketide synthase gene or nonribosomal peptide synthetase gene.

1 more element

Dependencies are inferred from the claim text.

Facts

Application: 13/816,826Filed 11 Aug 2011
Priority: JP 2010-18127913 Aug 2010
Published: 4 Jul 2013
Term ends: Not estimatedThe source records don’t support an estimate.

Classification8 codes

C12P 19/34
Nucleotides: polynucleotides, e.g. nucleic acids, oligoribonucleotides
C12N 15/52
DNA or RNA fragments; Modified forms thereof ; Non-coding nucleic acids having a biological activity: genes encoding for enzymes or proenzymes
C12N 15/80
Vectors or expression systems specially adapted for eukaryotic hosts: for fungi
C12N 15/815
For yeasts: for yeasts other than Saccharomyces