An extracellular lipase from Amycolatopsis mediterannei is a cutinase with plastic degrading activity

Tan, Yeqi; Henehan, Gary; Kinsella, Gemma K.; Ryan, Barry J.

doi:10.1016/j.csbj.2021.01.019

Cited by 35 publications

(15 citation statements)

References 58 publications

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“…The AmCut gene was known from previous studies (Tan et al 2021 ). It was a 930 bp gene with GenBank ID of ADJ49206.…”

Section: Resultsmentioning

confidence: 99%

“…Previous studies in this laboratory had cloned a truncated AmCut gene lacking the N-terminal signal peptide and showed that the enzyme was expressed as a soluble protein in E. coli cytosol (Tan et al 2021 ). To further examine the role of the signal peptide on AmCut activity, the entire AmCut gene, including the putative signal peptide, was cloned (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The truncated AmCut gene was expressed, as outlined previously (Tan et al 2021 ), without the signal peptide (see Fig. 6 ).…”

Section: Resultsmentioning

confidence: 99%

“…In a previous paper, 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.…”

Section: Introductionmentioning

confidence: 99%

“…In the course of these studies, a novel extracellular cutinase (AmCut) from the Gram-positive organism Amycolatopsis mediterranei (AmCut) was identified with potential applications in biotechnology. AmCut was originally identified as a lipase; however, subsequent structural and substrate specificity studies showed it was more appropriately designated as a cutinase (Tan et al 2021 ). Lipases and cutinases are closely related acyl hydrolases (Chen et al 2013 ).…”

Section: Introductionmentioning

confidence: 99%

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Extracellular secretion of a cutinase with polyester-degrading potential by E. coli using a novel signal peptide from Amycolatopsis mediterranei

Tan

Henehan

Kinsella

et al. 2022

World J Microbiol Biotechnol

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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

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“…The AmCut gene was known from previous studies (Tan et al 2021 ). It was a 930 bp gene with GenBank ID of ADJ49206.…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…The truncated AmCut gene was expressed, as outlined previously (Tan et al 2021 ), without the signal peptide (see Fig. 6 ).…”

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning