Assessment of Anabaena 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 Anabaena sp. strain PCC 7120 (Anabaena 7120) as a general heterologous expression host for cyanobacterial secondary metabolites. We show that Anabaena 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 Anabaena 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 Anabaena 7120 as a general heterologous expression host enables investigation of interesting cyanobacterial biosynthetic reactions and genetic engineering of their biosynthetic pathways.

Interpretation of tandem mass spectra obtained from cyclic nonribosomal peptides.

Natural and non-natural cyclic peptides are a crucial component in drug discovery programs because of their considerable pharmaceutical properties. Cyclosporin, microcystins, and nodularins are all notable pharmacologically important cyclic peptides. Because these biologically active peptides are often biosynthesized nonribosomally, they often contain nonstandard amino acids, thus increasing the complexity of the resulting tandem mass spectrometry data. In addition, because of the cyclic nature, the fragmentation patterns of many of these peptides showed much higher complexity when compared to related counterparts. Therefore, at the present time it is still difficult to annotate cyclic peptides MS/MS spectra. In this current work, an annotation program was developed for the annotation and characterization of tandem mass spectra obtained from cyclic peptides. This program, which we call MS-CPA is available as a web tool (http://lol.ucsd.edu/ms-cpa_v1/Input.py). Using this program, we have successfully annotated the sequence of representative cyclic peptides, such as seglitide, tyrothricin, desmethoxymajusculamide C, dudawalamide A, and cyclomarins, in a rapid manner and also were able to provide the first-pass structure evidence of a newly discovered natural product based on predicted sequence. This compound is not available in sufficient quantities for structural elucidation by other means such as NMR. In addition to the development of this cyclic annotation program, it was observed that some cyclic peptides fragmented in unexpected ways resulting in the scrambling of sequences. In summary, MS-CPA not only provides a platform for rapid confirmation and annotation of tandem mass spectrometry data obtained with cyclic peptides but also enables quantitative analysis of the ion intensities. This program facilitates cyclic peptide analysis, sequencing, and also acts as a useful tool to investigate the uncommon fragmentation phenomena of cyclic peptides and aids the characterization of newly discovered cyclic peptides encountered in drug discovery programs.