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Seamless and orthogonal expression of genetic parts in polyhydroxyalkanoate (PHA)-producing bacterial chassis for plastic bio-upcycling applications

Authors :
Matthlessa Matthew Minggu
Nur Anisza Hanoum Naseron
Hazlam Shamin Ahmad Shaberi
Nor Azlan Nor Muhammad
Syarul Nataqain Baharum
Ahmad Bazli Ramzi
Source :
MethodsX, Vol 11, Iss , Pp 102434- (2023)
Publication Year :
2023
Publisher :
Elsevier, 2023.

Abstract

Polyhydroxyalkanoate (PHA)-producing bacteria represent a powerful synthetic biology chassis for waste bioconversion and bio-upcycling where PHAs can be produced as the final products. In this study, we present a seamless plasmid construction for orthogonal expression of recombinant PET hydrolase (PETase) in model PHA-producing bacteria P. putida and C. necator. To this end, this study described seamless cloning and expression methods utilizing SureVector (SV) system for generating pSV-Ortho-PHA (pSVOP) expression platform in bioengineered P. putida and C. necator. Genetic parts specifically Trc promoter, pBBR1 origin of replication, anchoring proteins and signal sequences were utilized for the transformation of pSVOP-based plasmid in electrocompetent cells and orthogonal expression of PETase in both P. putida and C. necator. Validation steps in confirming functional expression of PETase activity in corresponding PETase-expressing strains were also described to demonstrate seamless and detailed methods in establishing bioengineered P. putida and C. necator as whole-cell biocatalysts tailored for plastic bio-upcycling. • Seamless plasmid construction for orthogonal expression in PHA-producing bacteria. • Step-by-step guide for high-efficiency generation of electrotransformants of P. putida and C. necator. • Adaptable methods for rapid strain development (Design, Build, Test and Learn) for whole-cell biocatalysis.

Details

Language :
English
ISSN :
22150161
Volume :
11
Issue :
102434-
Database :
Directory of Open Access Journals
Journal :
MethodsX
Publication Type :
Academic Journal
Accession number :
edsdoj.745a7c41d8494d57b5f1f258c307c004
Document Type :
article
Full Text :
https://doi.org/10.1016/j.mex.2023.102434