Combinatorial biosynthesis
Polyketides and nonribosomally formed peptides are particularly diverse classes of natural products that represent a rich source of commercially significant pharmaceuticals including anticancer antibiotics (penicillins, erythromycin) and immunosuppressants (cyclosporins, rapamycin).
The biosynthesis of nonribosomal peptides and complex polyketides occurs on giant multifunctional enzymes through the repeated use of the same types of reaction, peptide-bond formation in the case of nonribosomal peptides and Claisen condensation in the case of polyketide biosynthesis. On these “mega-synthetases” a new set of enzyme activities (a module) is used to accomplish each cycle of chain extension. The structural diversity in complex polyketides is achieved by modifying newly added extension units with a few different types of redox reaction, while nonribosomal peptide synthetases can modify their substrates – proteinogenic and non-proteinogenic amino acids – by, for example,. N-methylation, hydroxylation, or epimersation. Further structural complexity is achieved in both groups with additional, more product-specific enzymatic reactions.
The modular architecture of nonribosomal peptide synthetases and polyketide synthases and the co-linear relationship between the protein domains and modules involved in synthesis and the different portions of the end product suggests that a new combination or modification of modules should give rise to new products. While it is most likely that exactly those mechanisms have led to the generation of novel synthetases during the course of evolution, approaches for the rational design of new multi-enzymes are faced with several difficulties. First of all, an extremely low productivity most likely due to unfavourable insertion positions for the introduction of new modules and domains. In addition the newly combined synthetases are likely to not communicate correctly due to problems in protein-protein interactions.
So far attempts to generate new compounds by rational combination of domains or modules have been somewhat disappointing. Even if a novel molecule does get produced the yields are usually far too low to allow for an efficient production of the novel compound.
Cyano Biotech´s approach to combinatorial biosynthesis of cyanobacterial natural products.
Our proprietary combinatorial technology makes use of specific features of cyanobacterial biosynthetic gene clusters for natural products to circumvent these problems, allowing for the generation of new compounds in higher yields.
The high degree of relationship among cyanobacterial strains reflects a high similarity of analogues of the natural products produced and their respective synthetases. Detailed comparisons of different strains allow for the identification of evolutionary processes as, for example, the appearance of new enzymes by mutation and recombination events. These features make cyanobacteria a much more suitable system for combinatorial biosynthesis than other natural products producers. Cyano Biotech´s capacious in-house database of cyanobacterial NRPS and PKS gene sequences allows us to analyse the underlying principles of the natural evolution of these biosynthetic systems.
Our platform technology for the genetic manipulation of cyanobacteria is the basis for our biocombinatorial approach.
Apart from recombining the structure of the NRP/PK backbone we further increase diversity of products by directed manipulation of modifying enzymes, e.g. glycosylases and methylases.
Our Know-How in the field of cultivation of cyanobacteria enables the generation of new biosynthetic pathways in the producer organism or closely related strains, ensuring an optimised environment for the production of large amounts of modified natural products.
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