Engineering the Osmotic State of <i>Pseudomonas putida</i> KT2440 for Efficient Cell Disruption and Downstream Processing of Poly(3-Hydroxyalkanoates)

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dc.contributor.authorPoblete-Castro, Ignacio
dc.contributor.authorAravena-Carrasco, Carla
dc.contributor.authorOrellana-Saez, Matias
dc.contributor.authorPacheco, Nicolas
dc.contributor.authorCabrera, Alex
dc.contributor.authorManuel Borrero-de Acuna, Jose
dc.date.accessioned2025-01-23T19:54:27Z
dc.date.available2025-01-23T19:54:27Z
dc.date.issued2020
dc.description.abstractIn the last decade, the development of novel programmable cell lytic systems based on different inducible genetic constructs like the holin-endolysin and lysozyme appears as a promising alternative to circumvent the use of costly enzymes and mechanical disrupters for downstream processing of intracellular microbial products. Despite the advances, upon activation of these systems the cellular disruption of the biocatalyst occurs in an extended period, thus delaying the recovery of poly(3-hydroxyalkanoate) (PHA). Herein the osmotic state of Pseudomonas putida KT2440 was engineered by inactivating the inner-membrane residing rescue valve MscL, which is responsible mainly for circumventing low-osmolarity challenges. Then the major outer membrane porin OprF and the specific porin OprE were overproduced during PHA producing conditions on decanoate-grown cells. The engineered P. putida strains carrying each porin showed no impairment on growth rate and final biomass and PHA yield after 48 h cultivation. Expression of both porins in tandem in the mutant strain KT Delta mscL-oprFE led to a slight reduction of the biomass synthesis (similar to 10%) but higher PHA accumulation (%wt) relative to the cell dry mass. Each strain was then challenged to an osmotic upshift for 1 h and subsequently to a rapid passage to a hypotonic condition where the membrane stability of the KT Delta mscL-oprFE suffered damage, resulting in a rapid reduction of cell viability. Cell disruption accounted for >95% of the cell population within 3 h as reported by colony forming units (CFU), FACS analyses, and transmission electron microscopy. PHA recovery yielded 94.2% of the biosynthesized biopolymer displaying no significant alterations on the final monomer composition. This study can serve as an efficient genetic platform for the recovery of any microbial intracellular compound allowing less unit operation steps for cellular disruption.
dc.fuente.origenWOS
dc.identifier.doi10.3389/fbioe.2020.00161
dc.identifier.issn2296-4185
dc.identifier.urihttps://doi.org/10.3389/fbioe.2020.00161
dc.identifier.urihttps://repositorio.uc.cl/handle/11534/100686
dc.identifier.wosidWOS:000525072600001
dc.language.isoen
dc.revistaFrontiers in bioengineering and biotechnology
dc.rightsacceso restringido
dc.subjectPseudomonas putida
dc.subjectcell lysis
dc.subjectporins
dc.subjectMscL
dc.subjectosmotic stress
dc.subjectpoly(3-hydroxyalkanoates)
dc.subjectPHA recovery
dc.subject.ods12 Responsible Consumption and Production
dc.subject.odspa12 Producción y consumo responsable
dc.titleEngineering the Osmotic State of <i>Pseudomonas putida</i> KT2440 for Efficient Cell Disruption and Downstream Processing of Poly(3-Hydroxyalkanoates)
dc.typeartículo
dc.volumen8
sipa.indexWOS
sipa.trazabilidadWOS;2025-01-12
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