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1. NATURAL SCIENCES, Biochemistry and molecular biology, Biochemical research methods
Recent studies in this laboratory showed that an extracellular cutinase from A. mediterranei (AmCut) was able to degrade the plastics polycaprolactone and polybutylene succinate. Such plastics can be slow to degrade in soils due to a lack of efficient polyester degrading organisms. AmCut also showed potential for the biocatalytic synthesis of esters by reverse hydrolysis.
The gene for AmCut has an upstream leader sequence whose transcript is not present in the purified enzyme. In this study, we show using predictive modelling, that this sequence codes for an N-terminal signal peptide that directs transmembrane expression via the Sec secretion pathway.
E. coli is a useful host for recombinant enzymes used in biocatalysis due to the ease of genetic manipulation in this organism, which allows tuning of enzymes for specific applications, by mutagenesis. When a truncated AmCut gene (lacking its signal peptide) was expressed in E. coli, all cutinase activity was observed in the cytosolic fraction. However, when AmCut was expressed in E. coli along with its native signal peptide, cutinase activity was observed in the periplasmic space and in the culture medium. This finding revealed that the native signal peptide of a Gram-positive organism (A. mediterranei) was being recognised by the Gram-negative (E. coli) Sec transmembrane transport system. AmCut was transported into E. coli’s periplasmic space from where it was released into the culture medium. Although the periplasmic targeting was surprising, it is not unprecedented due to the conservation of the Sec pathway across species. It was more surprising that AmCut was secreted from the periplasmic space into the culture medium. This suggests that extracellular AmCut translocation across the E. coli outer membrane may involve non-classical secretion pathways. This tuneable recombinant E. coli expressing extracellular AmCut may be useful for degradation of polyester substrates in the environment; this and other applications are discussed.
Tan, Y., Kinsella, G.K., Henehan, G.T. and Ryan, B.J. (2022). Extracellular secretion of a cutinase with polyester-degrading potential by E. coli using a novel signal peptide from Amycolatopsis mediterranei. World Journal of Microbiology and Biotechnology, 38, Art. 60. DOI: 10.1007/s11274-022-03246-z