Biosynthetic systems producing fungal indole alkaloids
The biosynthesis of fungal bicyclo[2.2.2]diazaoctane indole alkaloids with a wide spectrum of biological activities have attracted increasing interest. Their intriguing mode of assembly has long been proposed to feature a non-ribosomal peptide synthetase, a presumed intramolecular Diels-Alderase, a variant number of prenyltransferases, and a series of oxidases responsible for the diverse tailoring modifications of their cyclodipeptide-based structural core. Until recently, the details of these biosynthetic pathways have remained largely unknown due to lack of information on the fungal derived biosynthetic gene clusters. Herein, we report a comparative analysis of four natural product metabolic systems of a select group of bicyclo[2.2.2]diazaoctane indole alkaloids including (+)/(−)-notoamide, paraherquamide and malbrancheamide, in which we propose an enzyme for each step in the biosynthetic pathway based on deep annotation and on-going biochemical studies.
1. A protein in the prenylated indole alkaloid pathway, wherein the protein is a MalG protein having an amino acid sequence that is 98% or more identical to SEQ. ID NO: 15, comprises at least one amino acid substitution, insertion or deletion relative to SEQ ID NO: 15 and has MalG activity.
2. The MalG protein of claim 1 further comprising a chlorinated tryptophan loaded onto the second thiolation (T) domain of the protein.
3. A polynucleotide encoding a protein in the prenylated indole alkaloid pathway, wherein the polynucleotide encodes a MalG protein having an amino acid sequence that is 98% or more identical to SEQ ID NO: 15, comprises at least one amino acid substitution, insertion or deletion relative to SEQ ID NO:15 and has MalG activity.
4. A host cell transformed with the polynucleotide of claim 3 .
5. An expression vector comprising the polynucleotide of claim 3 .
6. A host cell transformed with the expression vector of claim 5 .
7. A method for producing prenylated indole alkaloid or a metabolic intermediate for producing a prenylated indole alkaloid comprising the step of growing a host cell comprising the polynucleotide of claim 3 under conditions to express the protein and producing a prenylated indole alkaloid or the metabolic intermediate for producing a prenylated indole alkaloid.
8. The method of claim 7 further comprising the step of isolating the prenylated indole alkaloid or the metabolic intermediate of the prenylated indole alkaloid.
9. The method of claim 8 wherein the host cell is a prokaryote.
10. The method of claim 9 wherein the host cell is selected from the group consisting of Escherichia coli, Streptomyces lavendulae, Myxococcus xanthus , and Pseudomonas fluorescens.