Final Report Summary - ZHOU_PFL_POLYKETIDES (Genome mining in Streptomyces violaceusniger, a prolific antibiotic producer)
Genome Mining in a prolific antibiotic-producing bacterium
The overarching aim of this project was to investigate the ability of the unusually "talented" bacterium Streptomyces violaceusniger DSM4137 to produce potentially valuable antibiotics and other specialised metabolites using giant assembly-line multienzymes called modular polyketide synthases. Its central objective was to contribute to our understanding of the biosynthetic potential of this organism, and accelerate its exploitation, by studying how to transplant the giant gene clusters intact into other bacterial strains whose ability to support enhanced polyketide antibiotic production is already known. When this project was initiated no straightforward method of doing this was available.
Results
The researcher systematically cloned intact assembly-line biosynthetic gene clusters from a newly-constructed P1-phage based Bacterial Artificial Chromosome (P1-BAC or PAC) library of DSM4137 chromosomal DNA. He characterised candidate clones in Escherichia coli and used them to transfer intact clusters (up to 120 kbp in size) into model strains S. coelicolor and S. lividans. He fermented promising transformants and analysed them for production of the predicted natural product.
In this way he showed successful production of the antifungal marginolactone azalomycin, the polyether nigericin which selectively kills cancer stem cells (at twice the level of production of the parent strain) and the antibiotic macrodiolide elaiophylin in the genome-reduced, ribosome-engineered organism Streptomyces coelicolor and/or the closely-related Streptomyces lividans; and of the neuroprotectant meridamycin in an industrial strain of the erythromycin-producing bacterium Saccharopolyspora erythraea. whose genome had been reduced by excision of the resident erythromycin genes to provide a clean background. This technology cannot however yet be considered routine, for considerable time had to be invested to optimise the conditions for conjugation from E. coli into the actinomycete hosts and the instability of the large PAC clones led to substantial deletion of the inserted clusters in many cases. As a part of this work he assisted in the completion of the genome sequence of the DSM4137 strain and of Streptomyces cinnamonensis (overproducer of monensin), joining them to the handful of large actinomycete genomes (sizes 7-11 Mbp) finished to this gold standard, and providing an important framework for their use as alternative expression hosts for polyketide clusters.
In addition, the researcher carried out the first in-depth analysis of the enzymology of macrodiolide formation by the chain-terminating thioesterase/cyclase (TE) domain of the elaiophylin polyketide synthase. This has led to establishing the molecular mechanism of diolide formation involving "retrotransfer" of chains from the TE to an adjacent monomer on the neighbouring acylcarrier protein (ACP) domain. He was able to show that symmetrical non-natural diolides could be formed starting from chemically-synthesised monomers; and that hybrid asymmetric diolides could also be formed by starting with mixtures of the appropriate monomers.
Impact
achievements of the researcher in this project, especially in functional transplantation of clusters to new host strains, point the way to overcoming a severe bottleneck in the development of a genuine synthetic biology of natural products, in which modular biosynthetic multienzymes are designed and fabricated to order to produce a specified target molecule. He also revealed novel enzymology of diolide cyclases that will contribute to the expanding modular toolkit for pathway engineering and refactoring. The manipulation and refactoring of antibiotic biosynthetic pathways represents one of the promising early fruits of the emerging and heterogenous field of synthetic biology. It is therefore of the utmost importance that the work is carried out vigorously and held to the highest standard, because success or failure in this will colour attitudes both within scientific and non-scientific circles to synthetic biology in general.
Contacts
Dr Yongjun ZHOU: yz417@cam.ac.uk OR zhouyongjun66@gmail.com
Professor Peter LEADLAY FRS FRCS: pfl10@cam.ac.uk
The overarching aim of this project was to investigate the ability of the unusually "talented" bacterium Streptomyces violaceusniger DSM4137 to produce potentially valuable antibiotics and other specialised metabolites using giant assembly-line multienzymes called modular polyketide synthases. Its central objective was to contribute to our understanding of the biosynthetic potential of this organism, and accelerate its exploitation, by studying how to transplant the giant gene clusters intact into other bacterial strains whose ability to support enhanced polyketide antibiotic production is already known. When this project was initiated no straightforward method of doing this was available.
Results
The researcher systematically cloned intact assembly-line biosynthetic gene clusters from a newly-constructed P1-phage based Bacterial Artificial Chromosome (P1-BAC or PAC) library of DSM4137 chromosomal DNA. He characterised candidate clones in Escherichia coli and used them to transfer intact clusters (up to 120 kbp in size) into model strains S. coelicolor and S. lividans. He fermented promising transformants and analysed them for production of the predicted natural product.
In this way he showed successful production of the antifungal marginolactone azalomycin, the polyether nigericin which selectively kills cancer stem cells (at twice the level of production of the parent strain) and the antibiotic macrodiolide elaiophylin in the genome-reduced, ribosome-engineered organism Streptomyces coelicolor and/or the closely-related Streptomyces lividans; and of the neuroprotectant meridamycin in an industrial strain of the erythromycin-producing bacterium Saccharopolyspora erythraea. whose genome had been reduced by excision of the resident erythromycin genes to provide a clean background. This technology cannot however yet be considered routine, for considerable time had to be invested to optimise the conditions for conjugation from E. coli into the actinomycete hosts and the instability of the large PAC clones led to substantial deletion of the inserted clusters in many cases. As a part of this work he assisted in the completion of the genome sequence of the DSM4137 strain and of Streptomyces cinnamonensis (overproducer of monensin), joining them to the handful of large actinomycete genomes (sizes 7-11 Mbp) finished to this gold standard, and providing an important framework for their use as alternative expression hosts for polyketide clusters.
In addition, the researcher carried out the first in-depth analysis of the enzymology of macrodiolide formation by the chain-terminating thioesterase/cyclase (TE) domain of the elaiophylin polyketide synthase. This has led to establishing the molecular mechanism of diolide formation involving "retrotransfer" of chains from the TE to an adjacent monomer on the neighbouring acylcarrier protein (ACP) domain. He was able to show that symmetrical non-natural diolides could be formed starting from chemically-synthesised monomers; and that hybrid asymmetric diolides could also be formed by starting with mixtures of the appropriate monomers.
Impact
achievements of the researcher in this project, especially in functional transplantation of clusters to new host strains, point the way to overcoming a severe bottleneck in the development of a genuine synthetic biology of natural products, in which modular biosynthetic multienzymes are designed and fabricated to order to produce a specified target molecule. He also revealed novel enzymology of diolide cyclases that will contribute to the expanding modular toolkit for pathway engineering and refactoring. The manipulation and refactoring of antibiotic biosynthetic pathways represents one of the promising early fruits of the emerging and heterogenous field of synthetic biology. It is therefore of the utmost importance that the work is carried out vigorously and held to the highest standard, because success or failure in this will colour attitudes both within scientific and non-scientific circles to synthetic biology in general.
Contacts
Dr Yongjun ZHOU: yz417@cam.ac.uk OR zhouyongjun66@gmail.com
Professor Peter LEADLAY FRS FRCS: pfl10@cam.ac.uk