Methods of Identifying and Characterizing Natural Product Gene Clusters

An mNRPS that is encoded by:

• i. a polynucleotide sequence encoding a modified BpsA synthetase,
• ii. a polynucleotide sequence variant of SEQ ID NO:
• 1 wherein said variant comprises at least 70% nucleotide sequence identity with SEQ ID NO:
• 1, or
• iii. a polynucleotide sequence encoding a modified NRPS comprising a modified T-domain, wherein said T-domain is selected from the group consisting of:
• a. a heterologous T-domain,
• b. a homologous T-domain,
• c. an exogenous T-domain,
• d. an endogenous T-domain,
• e. a T-domain encoded by a nucleotide sequence comprising at least 70% sequence identity with the T-domain of any one of SEQ ID NO:
• 1, 2, 4, 6, 8, 10, 12, 14, 16 and 18, and
• f. a T-domain encoded by a nucleotide sequence of the T-domain of any one of the nucleotide sequences selected from the group consisting of SEQ ID NO:
• 1, 2, 4, 6, 8, 10, 12, 14, 16, and 18.

A modified NRPS (mNRPS) having:

• i. an amino acid sequence that specifies a modified BpsA synthetase,
• ii. an amino acid sequence variant of SEQ ID NO:
• 3 wherein said variant comprises at least 70% amino acid sequence identity with SEQ ID NO:
• 3, or
• iii. an amino acid sequence that specifies a modified NRPS comprising a modified T-domain, wherein said T-domain is selected from the group consisting of:
• a. a heterologous T-domain,
• b. a homologous T-domain,
• c. an exogenous T-domain,
• d. an endogenous T-domain,
• e. a T-domain specified by an amino acid sequence comprising at least 70% sequence identity with the T-domain of any one of SEQ ID NO:
• 3, 5, 7, 9, 11, 13, 15, 17 and 19, and
• f. a T-domain encoded by a nucleotide sequence of the T-domain of any one of the nucleotide sequences selected from the group consisting of SEQ ID NO:
• 3, 5, 7, 9, 11, 13, 15, 17 and 19.

A method of characterizing the activity of a PPTase, the method comprising the steps of

• combining said PPTase, a BpsA synthetase and the necessary substrates for phosphopantetheinylation of said BpsA synthetase in the presence of a carrier protein or peptide that acts as a competitor for one or more of the phosphopantetheinylation substrates,
• incubating the resulting reaction,
• adding the necessary substrates for an indigoidine synthesis reaction,
• using a measurement tool to measure the indoigidine level produced by the synthesis reaction, and
• computing the rate of indigoidine produced over a time period,
• wherein the rate of indigoidine production is indicative of the amount of BpsA synthetase converted from apo to holo form and allows determination of the relative rate of carrier protein or peptide modification by the PPTase.

A method of making a modified PPTase, the method comprising

• a. expressing a modified PPTase from a polynucleotide sequence to form an expressed PPTase,
• b. incubating the expressed PPTase with an NRPS, and
• c. detecting the activation of the NRPS,
• wherein activation of the NRPS confirms that said expressed PPTase is a functional modified PPTase.

A method of characterizing the rate of reaction of PPTases, the method comprising the steps of:

• combining a pigment producing enzyme with a PPTase and substrates and co-factors required for both phosphopantetheinylation and pigment production,
• using a measurement tool to measure the pigment level, and
• computing the rate of pigment produced over a time period,
• wherein the change in rate of pigment produced is proportional to the rate of reaction of the PPTase to be characterized.

A method of detecting a chemical modifier of PPTase activity, the method comprising the steps of

• combining a pigment producing enzyme with a PPTase and substrates and co-factors required for both phosphopantetheinylation and pigment production in the presence of a chemical compound to be tested
• using a measurement tool to measure the pigment produced,
• computing the rate of pigment produced over a time period,
• wherein if the rate of the reaction for the PPTase slows in the presence of the chemical, it is a candidate inhibitor, or if the rate of reaction increases, the chemical is a candidate accelerator.

A method of determining the rate of modification of any carrier protein or peptide substrate by any PPTase, the method comprising the steps of

• combining a PPTase, a pigment producing enzyme and the necessary substrates for phosphopantetheinylation in the presence of a range of known concentrations of a carrier protein or peptide that acts as a competitor for one or more of the phosphopantetheinylation substrates which is in limited supply,
• incubating the resulting reaction,
• adding the necessary substrates for the pigment production reaction,
• using a measurement tool to measure the pigment level, and
• computing the rate of pigment produced over a time period,
• wherein the rate of pigment production is indicative of the amount of pigment producing enzyme converted from apo to holo form and allows determination of the relative rate of carrier protein or peptide modification by the PPTase.