Heterologous Expression of Biosynthetic Gene Clusters from Marine Cyanobacteria

Cyanobacteria are prolific producers of natural products with diverse structures and biological activities. Natural products are important for drug discovery and some of those isolated from cyanobacteria were found to have activities against cancer cells and infectious disease, or to relieve minor pain as anti-inflammatory drugs. However, slow growth rates, resistance to genetic engineering, and low yields limit the thorough investigation of many of these secondary metabolites within their native organisms. In this study, we describe and demonstrate the successful heterologous production of two bioactive secondary metabolites from marine cyanobacteria, cryptomaldamide and columbamide, in Anabaena sp. PCC 7120. This work resulted in the development and validation of new genetic tools and methods for the heterologous expression of large biosynthetic gene clusters. These tools include cloning vectors for transformation associated recombination in yeast and a new engineered strain of Anabaena along with a CRISPR/cpf1-based system that enables genetic engineering of large biosynthetic gene clusters in both Synechococcus elongatus PCC 7942 or Anabaena. This study showed that Anabaena is a more suitable host than S. elongatus for the production of marine cyanobacterial natural products. The heterologous expression of these biosynthetic gene clusters from marine cyanobacteria represents a significant opportunity to produce valuable compounds at a larger scale and for the further study of the mechanisms involved in the synthesis of these compounds

Heterologous Expression of Biosynthetic Gene Clusters from Marine Cyanobacteria

Cyanobacteria are prolific producers of natural products with diverse structures and biological activities. Natural products are important for drug discovery and some of those isolated from cyanobacteria were found to have activities against cancer cells and infectious disease, or to relieve minor pain as anti-inflammatory drugs. However, slow growth rates, resistance to genetic engineering, and low yields limit the thorough investigation of many of these secondary metabolites within their native organisms. In this study, we describe and demonstrate the successful heterologous production of two bioactive secondary metabolites from marine cyanobacteria, cryptomaldamide and columbamide, in Anabaena sp. PCC 7120. This work resulted in the development and validation of new genetic tools and methods for the heterologous expression of large biosynthetic gene clusters. These tools include cloning vectors for transformation associated recombination in yeast and a new engineered strain of Anabaena along with a CRISPR/cpf1-based system that enables genetic engineering of large biosynthetic gene clusters in both Synechococcus elongatus PCC 7942 or Anabaena. This study showed that Anabaena is a more suitable host than S. elongatus for the production of marine cyanobacterial natural products. The heterologous expression of these biosynthetic gene clusters from marine cyanobacteria represents a significant opportunity to produce valuable compounds at a larger scale and for the further study of the mechanisms involved in the synthesis of these compounds

Heterologous Expression in Anabaena of the Columbamide Pathway from the Cyanobacterium Moorena bouillonii and Production of New Analogs

Columbamides are chlorinated acyl amide natural products, several of which exhibit cannabinomimetic activity. These compounds were originally discovered from a culture of the filamentous marine cyanobacterium Moorena bouillonii PNG5-198 collected from the coastal waters of Papua New Guinea. The columbamide biosynthetic gene cluster (BGC) had been identified using bioinformatics, but not confirmed by experimental evidence. Here, we report the heterologous expression in Anabaena (Nostoc) PCC 7120 of the 28.5 kb BGC that encodes for columbamide biosynthesis. The production of columbamides in Anabaena is investigated under several different culture conditions, and several new columbamide analogs are identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR). In addition to previously characterized columbamides A, B, and C, new columbamides I-M are produced in these experiments, and the structure of the most abundant monochlorinated analog, columbamide K (11), is fully characterized. The other new columbamide analogs are produced in only small quantities, and structures are proposed based on high-resolution-MS, MS/MS, and 1H NMR data. Overexpression of the pathway's predicted halogenases resulted in increased productions of di- and trichlorinated compounds. The most significant change in production of columbamides in Anabaena is correlated with the concentration of NaCl in the medium

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 Synechococcus elongatus PCC 7942 and Anabaena (Nostoc) PCC 7120. To facilitate the transfer of gene clusters in both strains, we engineered a strain of Anabaena that contains S. elongatus homologous sequences for chromosomal recombination at a neutral site and devised a CRISPR-based strategy to efficiently obtain segregated double recombinant clones of Anabaena. These genetic tools were used to express the large 28.7 kb cryptomaldamide biosynthetic gene cluster from the marine cyanobacterium Moorena (Moorea) producens JHB in both model strains. S. elongatus did not produce cryptomaldamide; however, high-titer production of cryptomaldamide was obtained in Anabaena. The methods developed in this study will facilitate the heterologous expression of biosynthetic gene clusters isolated from marine cyanobacteria and complex metagenomic samples