Genes Influencing Antibiotics
Commercial production of antimicrobial products by large-scale submerged fermentation began with penicillin in the 1940s. Since then, hundreds of natural products have been produced for medicine and agriculture including antibiotics, anti-cancer agents, immunosuppressants and activepharmaceutical ingredients (Bérdy 2005). Traditional strain improvement that leads to improved production levels is an empirical stepwise process performed through multiple labor-intensive cycles of random mutation and screening using higher antibiotic production as the screening criterion (Demain and Adrio 2008). Typically the strongest positive mutation steps are found during the first few cycles of the process followed by cycles that, despite a larger screening effort, produce smaller increases in yield (Barrick and Lenski 2013). Forthis project, in vitro transposition (Goryshin and Reznikoff 1998) was used to generate random mutations. Experiments were performed directly in a Saccharopolyspora erythraea mutB strain (FL2302) and a sample of genes was knocked out comprising ∼7% of the genes in the chromosome. One of the mutations, in the cwh1 gene, had the ability to be scaled-up making it of special interest for further study.
This study used targeted mutagens to explore the genetics behind the process of strain improvement. Traditionally, strain improvement mutations are created by chance and chosen on phenotypic performance alone, with no knowledge of genotype needed; however, no benefit is passed to future strain improvement programs. The transposon-based strategy used in this study allows the characterization of the high-performing genotypes so that these mutations might be rationally incorporated into other desired genetic backgrounds. By using transposon mutagenesis it was possible to see that ∼3%; mutations generated had an effect on erythromycin yield. In a typical actinomycete such as S. erythraea with over 7000 genes, this might mean that a saturating random mutagenesis could yield as many as 210 unique strain improvement mutations. Only one locus, cwh1, was found that could be scaled-up to a 100-fold increase in fermentation volume (0.25– 25 ml). Mutations in cwh1 produced visible changes in growth on solid media, which is consistent with the predicted function of Cwh1 in cell wall biogenesis. If the cwh1 mutation affects the early stages of cell wall biosynthesis, then the yield improvement phenotype could result from the diversion of cell wall precursors such as NDP-rhamnose, from cell wall biosynthesis into erythromycin biosynthesis.
Activation of Antibiotics
During the golden age of antibiotic drug discovery in the 1940’sand ‘50’s hundreds of new antibiotics were found from soil-screening programs. This method is no longer cost effective due to the rediscovery of old antibiotics and the low probability of finding new antibiotics (Baltz 2005).
However it is becoming increasingly apparent that the majority of actinomycetes have silent antibiotic gene clusters (Bentley 2002). Some clusters can be activated to produce novel biologically active compounds (Laureti 2011). This could mean that a cache of actinomycete antibiotics has yet to be found.New methods for finding novel antibiotics
Today new antibiotics can be discovered from DNA sequence data, which would be followed by expression of the relevant gene cluster in a heterologous host (Bachmann 2014). This method, however, suffers from inherent problems in heterologous gene cluster expression (Jensen 2014). The approach described in this study focuses instead on characterizing the activation process for silent gene clusters. Once general principles for activation are developed, the process can be scaled up for new large scale screening programs.
The results from this project illustrate that activation of silent antibiotic gene clusters can occur by unexpected methods. Here we describe two genes from Saccharopolyspora erythraea that can activate actinorhodin production when expressed from a high copy number plasmid. The first gene is an enzyme of tyrosine metabolism (SACE_0905) and the second gene is a cell-wall associated peptidase (SACE_1669). Both genes can highly activate actinorhodin production in S. lividans when expressedfrom high-copy plasmids. Manipulation of the SACE_0905 gene in S. erythraea also resulted in pigmentation effects. It remains to be seen if overexpression of these two genes will find general application in antibiotic discovery.