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

Abstract

In 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 mscL-oprFE led to a slight reduction of the biomass synthesis (∼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 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.

Industrial biotechnology of Pseudomonas putida: advances and prospects

“…Findings thereof, together with improvements of genetic engineering tools, are of particular value to complete our holistic understanding of P. putida and ease the development of superior industrial strains. Moreover, it should be stressed that the capability of producing value-added chemicals from alternative feedstocks (Kohlstedt et al 2018;Poblete-Castro et al 2014;Salvachúa et al 2020a;Wierckx et al 2012) is a superior feature to pass into the envisaged circular bioeconomy. Beyond the wide product range already demonstrated for P. putida, a promising upcoming approach is re-designing the biochemical portfolio by introducing complete synthetic pathways (bio-bricks) to access new-tonature products, such as halogenated molecules and boron-containing structures in the near future (Nieto-Domínguez and Nikel 2020;Nikel and de Lorenzo 2018).…”

“…However, these methods are also marked with possible environmental drawbacks, high costs, or degradation of the polymer. Recently, a recovery of nearly 94% of the synthesized mcl -PHA after 3 h could be shown with cell disruption through a programmable cell lysis system in P. putida KT2440 engineered to respond to osmotic state (Poblete-Castro et al 2020a ). Taken together, recent achievements will pave the way to further reduce process costs at the level of raw material selection and downstream processing.…”

Recombinant DNA technology as a tool for improving production of polyhydroxyalkanoates by the natural producers

“…The choice of isolation and purification steps is also related to the application of the biopolymer, because some steps may alter PHA properties or add undesirable biomass-derived compounds such as lipoproteins or toxins to the final product (Koller et al 2013). Modification of the producer's genome by molecular biology tools may facilitate the process of isolating PHA granules from the cell (Borrero-de Acuña et al 2017;Poblete-Castro et al 2020) or reduce the number of isolation steps (Boynton et al 1999;Rodríguez Gamero et al 2018). In recent years, the goal is to develop a programmable cell lysis system that allows the isolation of intracellular metabolites by autolysis of the production organism (Hajnal et al 2016;Tamekou Lacmata et al 2017;Borrero-de Acuña et al 2017).…”

Dehydrogenation Mechanism of Three Stereoisomers of Butane-2,3-Diol in Pseudomonas putida KT2440

“…P. putida KT2440, the first Gram-negative soil bacterium which was certified as generally recognized as safe (i.e., GRAS) ( Nelson, 2002 ; Nelson et al, 2002 ), has attracted substantial attention as a new workhorse for bioindustries ( Nikel and de Lorenzo, 2018 ; Weimer et al, 2020 ). Based on the toolbox for genetic engineering of P. putida KT2440 ( Poblete-Castro et al, 2020 ; Sun et al, 2020 ; Weimer et al, 2020 ; Zhou et al, 2020 ), it has been engineered for heterologous production of diverse value-added products ( Nikel and de Lorenzo, 2013 ; Johnson and Beckham, 2015 ; Dvořák and de Lorenzo, 2018 ; Sánchez-Pascuala et al, 2019 ; Bator et al, 2020 ; Bentley et al, 2020 ; Tiso et al, 2020 ) and environmental remediation ( Franden et al, 2018 ). In addition, the intrinsically high metabolic diversity gives P. putida KT2440 a wide substrate spectrum for chemical production ( Nelson et al, 2002 ; Wu et al, 2011 ).…”