UK-2 biosynthetic gene and method for improving UK-2 productivity using the same
The genomic DNA of Streptoverticillium sp. 3-7, which produces UK-2, was analyzed to identify a region expected to be a UK-2 biosynthetic gene cluster. Moreover, by colony hybridization, DNAs in the region were successfully isolated. Further, the DNAs were used to prepare a strain in which the genes present in the region were disrupted. The strain was found not to produce UK-2. It was verified that the genomic region was the UK-2 biosynthetic gene cluster. Furthermore, Streptoverticillium sp. 3-7 was transformed by introduction of a vector in which the isolated UK-2 biosynthetic gene cluster was inserted. It was also found out that the UK-2 productivity by the transformant was improved about 10 to 60 times or more in comparison with that of the parental strain. Moreover, it was revealed that 2 copies of the UK-2 biosynthetic gene cluster were present per cell in these transformants, respectively.
1. A method for producing a derivative of UK-2, comprising the steps of:
culturing a bacterium, and collecting UK-2 from a culture of the bacterium; and
synthesizing a derivative of UK-2 represented by any one of the following formulae (1) from the collected UK-2
in the formula (1),
R represents any one of a 2-methylpropanoyl group, a trans-2-methyl-2-butenoyl group, a 3-methylbutanoyl group and a 2-methylbutanoyl group;
R 1 represents any one of a C 1-6 alkyl group, a benzyl group, a C 1-10 alkylcarbonyl group(the C 1-10 alkylcarbonyl group may be substituted with any one of a carboxyl group, a benzyloxycarbonyl group, a C 1-4 alkyloxycarbonyl group and benzyloxycarbonylamino group), a benzoyl group, a C 1-4 alkyloxycarbonyl group, a (C 1-4 )alkyloxycarbonyl(C 1-4 )alkyl group, a benzyloxycarbonyl(C 1-4 )alkyl group may be substituted with a nitro group, a C 1-6 alkylsulfonyl, di(C 1-6 )alkylphosphoryl group, a diphenylphosphoryl group and a substituent represented by the following formula (2);
in the formula (2),
Q is selected from the group consisting of H, CH 3 , CH 2 CH 3 , CF 3 , Ph, CH═CH 2 and a cyclopropyl;
M is selected from the group consisting of H, CH 3 , CH 2 CH 3 , CF 3 , Ph, CH═CH 2 and a cyclopropyl;
T is selected from the group consisting of O, OC(O), OC(O)O, S, SC(O), SC(O)O and a substituent represented by the following formula (3);
G is selected from the group consisting of H, C 1-6 alkyl group, a C 1-6 alkyloxy C 1-6 alkyl group, a C 2-6 alkenyl group, a C 2-6 alkynyl group, a C 3-6 cycloalkyl group, an aryl group and a heteroaryl group;
G and M may form an isobenzofuran ring optionally having an oxo group;
M and Q may form a 3-8 membered carbocyclic system,
wherein the bacterium is a bacterium transformant comprising a nucleic acid, in which UK-2 biosynthesis is induced and UK-2 production is increased in comparison to a parental strain bacterium, and
the nucleic acid is an isolated nucleic acid that induces UK-2 biosynthesis and improves UK-2 productivity, and is at least one nucleic acid selected from the group consisting of the following (a) to (q):
(a) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 3, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 3, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 2;
(b) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 5, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 5, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 4;
(c) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 7, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 7, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 6;
(d) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 9, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 9, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 8;
(e) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 11, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 11, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 10;
(f) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 13, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 13, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 12;
(g) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 15, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 15, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 14;
(h) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 17, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 17, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 16;
(i) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 19, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 19, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 18;
(j) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 21, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 21, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 20;
(k) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 23, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 23, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 22;
(1) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 25, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 25, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 24;
(m) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 27, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 27, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 26;
(n) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 29, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 29, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 28;
(o) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 31, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 31, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 30;
(p) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 33, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 33, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 32; and
(q) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 35, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 35, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 34.
2. A method for producing a UK-2A derivative, comprising the steps of:
culturing a bacterium, and collecting UK-2A from a culture of the bacterium; and
synthesizing a UK-2A derivative represented by any one of the following formulae (4) to (7) from the collected UK-2A,
wherein the bacterium is a bacterium transformant comprising a nucleic acid, in which UK-2 biosynthesis is induced and UK-2 production is increased in comparison to a parental strain bacterium, and
the nucleic acid is an isolated nucleic acid that induces UK-2 biosynthesis and improves UK-2 productivity, and is at least one nucleic acid selected from the group consisting of the following (a) to (q):
(a) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 3, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 3, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 2;
(b) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 5, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 5, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 4;
(c) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 7, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 7, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 6;
(d) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 9, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 9, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 8;
(e) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 11, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 11, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 10;
(f) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 13, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 13, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 12;
(g) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 15, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 15, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 14;
(h) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 17, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 17, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 16;
(i) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 19, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 19, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 18;
(j) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 21, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 21, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 20;
(k) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 23, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 23, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 22;
(l) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 25, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 25, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 24;
(m) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 27, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 27, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 26;
(n) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 29, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 29, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 28;
(o) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 31, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 31, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 30;
(p) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 33, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 33, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 32; and
(q) a nucleic acid encoding a protein comprising an amino acid sequence of SEQ ID NO: 35, a nucleic acid encoding an amino acid sequence having a homology of 95% or more with an amino acid sequence of SEQ ID NO: 35, or a nucleic acid hybridizing under stringent conditions to a nucleic acid comprising a base sequence of SEQ ID NO: 34.