Modulation of global regulatory mechanisms and biosynthetic gene clusters for simplified production of natural products

A method to elicit production of a secondary metabolite by a bacterium, wherein the method comprises the following steps:

• i) deleting a global regulator (GR) gene in said bacterium, and
• ii) optionally activating a biosynthetic gene cluster (BGC) involved in production of said secondary metabolite in said bacterium;
• or wherein the method comprises the following steps:
• i′) essentially activating a biosynthetic gene cluster (BGC) involved in production of said secondary metabolite in said bacterium, and
• ii′) optionally deleting a GR gene in said bacterium;
• and the method further comprises:
• iii) expressing in said bacterium at least one positive selectable marker gene and at least one negative selectable marker gene;
• wherein deleting a GR gene of steps i) or ii′) comprises introducing in said bacterium at least one GR specific siRNA, or a group of sequences comprising a Cas gene, at least one GR specific crRNA array, a pair of GR specific homology regions left and right;
• and/or
• wherein activating a BGC of steps ii) or i′) comprises introducing in said bacterium a group of sequences comprising a Cas gene, at least one BGC specific crRNA array, a pair of BGC specific homology regions left and right, a promoter system, and optionally an enhancer sequence.

A method for screening for a global regulator (GR) gene in a bacterium, the method comprising:

• a) replacing a gene sequence of a first constitutively expressed biosynthetic gene cluster (BGC) with a first fluorescent reporter gene;
• b) replacing a gene sequence of a second constitutively expressed BGC with a second fluorescent reporter gene, wherein the first and second fluorescent reporters emit two non-overlapping fluorescent signals;
• c) obtaining a bacterium indicator strain emitting the first and the second fluorescent signals;
• d) performing random mutagenesis in said bacterium indicator strain to generate random mutations;
• e) screening and identifying non-fluorescent mutant bacteria that do not emit the first and the second fluorescent signal,
• f) isolating said non-fluorescent mutant bacteria,
• g) performing whole genome sequencing of said non-fluorescent mutant bacterium to map the mutations generated in step d);
• h) identifying the mutations generated in a GR gene, wherein said GR gene regulates the activity of the first constitutively expressed BGC and of the second constitutively expressed BGC in said bacterium;
• wherein the replacing of a gene sequence of a BGC at step a) and b) does not disrupt a regulatory element of said BGC;
• wherein in step a) and b) the reading frame is conserved;
• wherein said GR gene regulates production of a target secondary metabolite.

A compound of general formula (I)

• wherein
• Y represents —CH═CH2
• or
• R1, R2, R3, R4, and R5 are independently of each other selected from —H, —NH2, —NHR6, —F, —OH, —OCH3, —OCH2CH3, and —OCH2CH2CH3;
• or R2 and R3 or R3 and R4 together with the carbon atom to which they are bound to form one of the following rings:
• wherein R6 represents —H, —CH3, —CH2CH2CH3, —CH(CH3)2, —CH2(CH2)2CH3, —CH2(CH2)3CH3, —CH2(CH2)4CH3, —CH2(CH2)5CH3, —CH2(CH2)6CH3, —CH2(CH2)7CH3, —CH2(CH2)8CH3, —CH2(CH2)9CH3, —CH2(CH2)10CH3, —CH2(CH2)11CH3, —CH2(CH2)12CH3, —CH2CH(CH3)2, —C(CH3)3, —CH2CH═CH2, —CH2CH═CH(CH3), —CH2CH═C(CH3)2, or —CH2CH═CHCH2CH3;
• with the proviso that when Y represents —CH═CH2, R1, R2, R4, and R5 cannot be —H, and R3 cannot be —OH simultaneously.