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INTRODUCTION

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.

DISCUSSION

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.