We obtained the complete genome sequence of the psychrotolerant extremophile Pseudomonas sp. MPC6, a natural Polyhydroxyalkanoates (PHAs) producing bacterium able to rapidly grow at low temperatures. Genomic and phenotypic analyses allowed us to situate this isolate inside the Pseudomonas fluorescens phylogroup of pseudomonads as well as to reveal its metabolic versatility and plasticity. The isolate possesses the gene machinery for metabolizing a variety of toxic aromatic compounds such as toluene, phenol, chloroaromatics, and TNT. In addition, it can use both C6- and C5-carbon sugars like xylose and arabinose as carbon substrates, an uncommon feature for bacteria of this genus. Furthermore, Pseudomonas sp. MPC6 exhibits a high-copy number of genes encoding for enzymes involved in oxidative and cold-stress response that allows it to cope with high concentrations of heavy metals (As, Cd, Cu) and low temperatures, a finding that was further validated experimentally. We then assessed the growth performance of MPC6 on glycerol using a temperature range from 0 to 45°C, the latter temperature corresponding to the limit at which this Antarctic isolate was no longer able to propagate. On the other hand, the MPC6 genome comprised considerably less virulence and drug resistance factors as compared to pathogenic Pseudomonas strains, thus supporting its safety. Unexpectedly, we found five PHA synthases within the genome of MPC6, one of which clustered separately from the other four. This PHA synthase shared only 40% sequence identity at the amino acid level against the only PHA polymerase described for Pseudomonas (63-1 strain) able to produce copolymers of short- and medium-chain length PHAs. Batch cultures for PHA synthesis in Pseudomonas sp. MPC6 using sugars, decanoate, ethylene glycol, and organic acids as carbon substrates result in biopolymers with different monomer compositions. This indicates that the PHA synthases play a critical role in defining not only the final chemical structure of the biosynthesized PHA, but also the employed biosynthetic pathways. Based on the results obtained, we conclude that Pseudomonas sp. MPC6 can be exploited as a bioremediator and biopolymer factory, as well as a model strain to unveil molecular mechanisms behind adaptation to cold and extreme environments.

Extreme environments are a unique source of microorganisms encoding metabolic capacities that remain largely unexplored. In this work, we isolated two Antarctic bacterial strains able to produce poly(3-hydroxyalkanoates) (PHAs), which were classified after 16S rRNA analysis as Pseudomonas sp. MPC5 and MPC6. The MPC6 strain presented nearly the same specific growth rate whether subjected to a temperature of 4 °C 0.18 (1/h) or 30 °C 0.2 (1/h) on glycerol. Both Pseudomonas strains produced high levels of PHAs and exopolysaccharides from glycerol at 4 °C and 30 °C in batch cultures, an attribute that has not been previously described for bacteria of this genus. The MPC5 strain produced the distinctive medium-chain-length-PHA whereas Pseudomonas sp. MPC6 synthesized a novel polyoxoester composed of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate-co-3-hydroxyoctanoate-co-3-hydroxydecanoate-co-3-hydroxydodecanoate). Batch bioreactor production of PHAs in MPC6 resulted in a titer of 2.6 (g/L) and 1.3 (g/L), accumulating 47.3% and 34.5% of the cell dry mass as PHA, at 30 and 4 °C, respectively. This study paves the way for using Antarctic Pseudomonas strains for biosynthesizing novel PHAs from low-cost substrates such as glycerol and the possibility to carry out the bioconversion process for biopolymer synthesis without the need for temperature control.

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.

Engineered P. putida H sets a benchmark in lignin‐based PHA. Balancing the catechol degrading pathways enables improved PHA production. A DO‐stat fed‐batch process permits the efficient conversion of lignin hydrolysates into biomass and biopolymer.

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