Development of new tools for de novo polyketide synthase design

Natural products are diverse chemical compounds, produced chiefly by bacteria and fungi, that confer a survival advantage
on the producing strain often by antagonising the growth of competing organisms. Among such natural products,
polyketides are a particularly successful drug class, with >40 marketed examples, the top six with peak annual sales of >$1
billion. Many companies have tried to improve upon natural diversity, with limited success. Previous methods used to
engineer polyketide synthases (PKS), the proteins which generate these products, can be slow, are complex to design, and
have frequently poorly productive and yielding.We aim to develop new synthetic biology tools for de novo synthetic
generation of productive polyketide synthases (PKS), with broad potential for application in therapeutic discovery and
development and in other areas where natural products have been successful.
Researchers at our industrial partner Isomerase Therapeutics have discovered a groundbreaking technique for using
recombination to rapidly generate novel and productive PKS. We believe that analysing these recombination events and
identifying potential recombination hotspots could lead to new tools and techniques for rational design of new polyketide
natural products. The Cambridge research team will use our extensive expertise in genome sequencing and analysis of
antibiotic-producing bacteria and collaborate with Isomerase to obtain genome sequence of up to 60 strains in which
recombined PKS have been shown to give rise to novel truncated or elongated polyketide products. By careful sequence
comparisons between such rearranged PKS genes, we aim to establish the identity of those 'hotspot' regions in which
recombination favours a successful outcome; and to establish the extent to which these hotspot regions vary between
different PKS. This would open the way to the construction of an in silico database of PKS gene fragments defined by
hotspot boundaries, a potentially valuable asset in the future design and construction of novel PKS from scratch.

Researchers at our industrial partner Isomerase Therapeutics have discovered a groundbreaking technique for using recombination to rapidly generate novel and productive PKS. We believe that analysing these recombination events and identifying potential recombination hotspots could lead to new tools and techniques for rational design of new polyketide natural products. The Cambridge research team will use our extensive expertise in genome sequencing and analysis of
antibiotic-producing bacteria and collaborate with Isomerase to obtain genome sequence of up to 60 strains in which recombined PKS have been shown to give rise to novel truncated or elongated polyketide products. By careful sequence comparisons between such rearranged PKS genes, we aim to establish the identity of those 'hotspot' regions in which recombination favours a successful outcome; and to establish the extent to which these hotspot regions vary between
different PKS. This would open the way to the construction of an in silico database of PKS gene fragments defined by hotspot boundaries, a potentially valuable asset in the future design and construction of novel PKS from scratch. The sequencing of actinomycete genomes presents significant technical problems, in part due to their size (up to 11 Mbp) , their very high G+C content, and especially the presence of extensive repeat regions. In particular, we will need carefully to distinguish between the successive extension modules of the PKS genes whose recombination is under investigation. We have developed in-house protocols for combining the outputs of both short-read (Illumina) and longer-read (454 and more recently PacBio) instruments in assemblies to the required level of accuracy for this project.

As described in proposal submitted to TSB