Abstract
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 GST-tagged AmCut gene (lacking its signal peptide) was expressed in E. coli, all cutinase activity was observed in the cytosolic fraction. However, when GST-tagged AmCut was expressed in E. coli along with its native signal peptide, cutinase activity was observed in both the periplasmic space and the culture medium. This finding revealed that the native signal peptide of a Gram-positive organism (AmCut) 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. Surprisingly, the presence of a bulky GST tag at the N-terminus of the signal peptide did not hinder transmembrane targeting. Although the periplasmic targeting was unexpected, 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.Graphical abstract
Highlights
Ongoing studies in this laboratory are focussed on the discovery of thermostable, solvent tolerant enzymes with biotechnological applications in areas such as biofuels synthesis, feedstock degradation and green synthesis of industrially useful compounds (e.g. Uhoraningoga et al 2021; Priyanka et al 2019; Kumar et al 2017)
We showed that the signal peptide functions to direct the expression of Amycolatopsis mediterranei cutinase (AmCut) to the periplasmic space of E. coli
The Amycolatopsis mediterranei cutinase (AmCut) gene showed a high degree of similarity to a number of related enzymes
Summary
Ongoing studies in this laboratory are focussed on the discovery of thermostable, solvent tolerant enzymes with biotechnological applications in areas such as biofuels synthesis, feedstock degradation and green synthesis of industrially useful compounds (e.g. Uhoraningoga et al 2021; Priyanka et al 2019; Kumar et al 2017). We reported the modelled 3-D structure of AmCut and showed it to have an open, surface exposed, active site and that it was capable of degrading specific polyester plastics such as polycaprolactone and polybutylene succinate but not polylactide or polyethylene terephthalate (Tan et al 2021). Extracellular expression of this enzyme in E. coli offers the intriguing possibility of creating an organism capable of secreting a thermostable, solvent tolerant cutinase for degradation of polyester compounds. Such an organism may be readily tuned to tailor its specificity for specific substrates due to the ease of gene manipulation in E. coli
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