Heterologous Expression of Cryptomaldamide in a Cyanobacterial Host.

Filamentous marine cyanobacteria make a variety of bioactive molecules that are produced by polyketide synthases, nonribosomal peptide synthetases, and hybrid pathways that are encoded by large biosynthetic gene clusters. These cyanobacterial natural products represent potential drug leads; however, thorough pharmacological investigations have been impeded by the limited quantity of compound that is typically available from the native organisms. Additionally, investigations of the biosynthetic gene clusters and enzymatic pathways have been difficult due to the inability to conduct genetic manipulations in the native producers. Here we report a set of genetic tools for the heterologous expression of biosynthetic gene clusters in the cyanobacteria PCC 7942 and () PCC 7120. To facilitate the transfer of gene clusters in both strains, we engineered a strain of that contains homologous sequences for chromosomal recombination at a neutral site and devised a CRISPR-based strategy to efficiently obtain segregated double recombinant clones of . These genetic tools were used to express the large 28.7 kb cryptomaldamide biosynthetic gene cluster from the marine cyanobacterium () JHB in both model strains. did not produce cryptomaldamide; however, high-titer production of cryptomaldamide was obtained in . The methods developed in this study will facilitate the heterologous expression of biosynthetic gene clusters isolated from marine cyanobacteria and complex metagenomic samples.