Method for selectively culturing microorganism using phosphite dehydrogenase gene as marker

摘要

A simple and inexpensive method for selectively culturing a microorganism which method makes it possible to selectively culture a microorganism of interest even without using a sterilization operation or an antibiotic substance is provided. The method according to the present invention selectively culturing a microorganism includes the step of culturing, in a culture medium containing phosphorous acid as a sole phosphorous source, a recombinant microorganism into which a phosphite dehydrogenase gene has been introduced.

主权项

1. A method for selectively culturing a recombinant microorganism, the method comprising the step of culturing, in a culture medium containing phosphorous acid as a sole phosphorous source and containing no antibiotic substance, a recombinant microorganism transformed with a plasmid comprising a phosphite dehydrogenase gene,
wherein the culturing is for 15 hours or longer under both sterile conditions and unsterile conditions, wherein the recombinant microorganism is further transformed with a phosphite transporter gene,
wherein the phosphite transporter gene consists of polynucleotides encoding RsPtxA, RsPtxB, and RsPtxC, respectively, RsPtxA, RsPtxB, and RsPtxC constituting polypeptides of a phosphite transporter;
RsPtxA is a protein consisting of the amino acid sequence of SEQ ID NO: 3 or a protein consisting of the amino acid of SEQ ID NO: 3 with a deletion, substitution, or addition of ten or less amino acids in the amino acid sequence of SEQ ID NO: 3 and functioning as the phosphite transporter when associated with RsPtxB and RsPtxC;RsPtxB is a protein consisting of the amino acid sequence of SEQ ID NO: 4 or a protein consisting of the amino acid of SEQ ID NO: 4 with a deletion, substitution, or addition of ten or less amino acids in the amino acid sequence of SEQ ID NO: 4 and functioning as the phosphite transporter when associated with RsPtxA and RsPtxC; andRsPtxC is a protein consisting of the amino acid sequence of SEQ ID NO: 5 or a protein consisting of the amino acid of SEQ ID NO: 5 with a deletion, substitution, or addition of ten or less amino acids in the amino acid sequence of SEQ ID NO: 5 and functioning as the phosphite transporter when associated with RsPtxA and RsPtxB, wherein the phosphite transporter gene used to transform the microorganism comprises a ribosome binding region upstream of said polynucleotide encoding RsPtxA, wherein said ribosome binding region is a Ralstonia sp. strain 4506 ribosome binding region that is upstream of RsPtxA gene in the chromosome of Ralstonia sp. strain 4506, and wherein guanine (G) located eight nucleotides upstream of the start codon of the RsPtxA gene of Ralstonia sp. strain 4506 is replaced with adenine (A) in said ribosome binding region.