Mutagenesis and selective breeding of a high producing ?-poly-L-lysine strain

Abstract

ϵ-Poly-L-lysine (ϵ-PL) is an L-lysine linear homopolymer, which is produced by bacteria belonging to the Streptomycetaceae family and by ergot fungi. However, the production of ϵ-PL by the wild bacteria strain is very low, which limits its utilization. In most bacteria including the Streptomyces genus, L-lysine is a precursor of ϵ-PL and is biosynthesized by the L-aspartate pathway. Aspartokinase (Ask) is the first key enzyme in this pathway and is subject to complex regulation such as the feedback inhibition by the end product amino acids. In addition, phosphoenolpyruvate carboxykinase is feedback-regulated by L-aspartate. To reduce these feedback inhibitions and to improve ϵ-PL productivity, resistant mutants were produced using sulfaguanidine (SG) + glycine+ L-lysine+ DL-3-hydroxynorvaline (AHV) as selective markers. Using the interaction between ϵ-PL and the charged dye in the solid culture medium, hundreds of colonies were simultaneously screened in a quick and effective manner. Finally, one ϵ-PL-producing strain, Streptomyces diastatochromogenes L9, was selected. The productivity of this strain during flask fermentation was 0.77 g/L, which was 15% higher than that of the original strain. Moreover, its fermentation performance and genetic characteristics were very stable.

Keywords

ϵ-poly-L-lysine / plasma / AHV / Streptomyces diastatochromogenes / fermentation

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Acknowledgments

This work was supported by the National Science and Technology Program of China (2011BAD23B05-3) and the National Basic Research Program of China (Gtant No. 2007CB714305). We are grateful to Tsinghua University for providing us with useful experimental instrumentation.

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2014 Higher Education Press and Springer-Verlag Berlin Heidelberg