The role of sigma factors in production of cyanobacterial natural products: Escherichia coli as a heterologous expression host

Cyanobacteria represent an underexploited source of bioactive natural products of interest to the pharmaceutical industry. Lack of development of these compounds as drug leads is due mainly to myriad practical problems with the producing strains including long doubling times, genetic intractability, and low compound yields. Previous attempts at heterologous expression in quick-growing, genetically amenable hosts such as Escherichia coli and Streptomyces spp. have encountered mixed success, especially with more complex natural products from non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) pathways. The most successful heterologous expression host to date is the filamentous freshwater cyanobacterium Anabaena sp. strain PCC 7120. Here, we examine the ability of heterologously expressed sigma factors identified in the genome of Anabaena sp. strain PCC 7120 to assist E. coli in the recognition of and transcription from native cyanobacterial promoters. A reporter gene expression assay examining the expression of chloramphenicol acetyltransferase from the promoter regions from complex cyanobacterial gene clusters was used to show that sigma factor All7608 is capable of recognizing multiple cyanobacterial promoters in E. coli. Attempts to produce lyngbyatoxin A from its native cyanobacterial gene cluster were unsuccessful, possibly hindered by deficiencies in recognition of cyanobacterial ribosomal binding sites by native E. coli translational machinery

Assessment of <i>Anabaena</i> sp. Strain PCC 7120 as a Heterologous Expression Host for Cyanobacterial Natural Products: Production of Lyngbyatoxin A

Cyanobacteria are well-known producers of natural products of highly varied structure and biological properties. However, the long doubling times, difficulty in establishing genetic methods for marine cyanobacteria, and low compound titers have hindered research into the biosynthesis of their secondary metabolites. While a few attempts to heterologously express cyanobacterial natural products have occurred, the results have been of varied success. Here, we report the first steps in developing the model freshwater cyanobacterium <i>Anabaena</i> sp. strain PCC 7120 (<i>Anabaena</i> 7120) as a general heterologous expression host for cyanobacterial secondary metabolites. We show that <i>Anabaena</i> 7120 can heterologously synthesize lyngbyatoxin A in yields comparable to those of the native producer, Moorea producens, and detail the design and use of replicative plasmids for compound production. We also demonstrate that <i>Anabaena</i> 7120 recognizes promoters from various biosynthetic gene clusters from both free-living and obligate symbiotic marine cyanobacteria. Through simple genetic manipulations, the titer of lyngbyatoxin A can be improved up to 13-fold. The development of <i>Anabaena</i> 7120 as a general heterologous expression host enables investigation of interesting cyanobacterial biosynthetic reactions and genetic engineering of their biosynthetic pathways