Providing octane degradation capability to Pseudomonas putidaKT2440 through the horizontal acquisition of oct genes located on an integrative and conjugative element

Abstract

The extensive use of petrochemicals has produced serious environmental pollution problems; fortunately, bioremediation is considered an efficient way to fight against pollution. In line with Synthetic Biology is that robust microbial chassis with an expanded ability to remove environmental pollutants are desirable. Pseudomonas putida KT2440 is a robust lab microbe that has preserved the ability to survive in the environment and is the natural host for the self‐transmissible TOL plasmid, which allows metabolism of toluene and xylenes to central metabolism. We show that the P. putida KT2440 (pWW0) acquired the ability to use octane as the sole C‐source after acquisition of an almost 62‐kb ICE from a microbial community that harbours an incomplete set of octane metabolism genes. The ICE bears genes for an alkane monooxygenase, a PQQ‐dependent alcohol dehydrogenase and aldehyde dehydrogenase but lacks the electron donor enzymes required for the monooxygenase to operate. Host rubredoxin and rubredoxin reductase allow metabolism of octane to octanol. Proteomic assays and mutants unable to grow on octane or octanoic acid revealed that metabolism of octane is mediated by redundant host and ICE enzymes. Octane is oxidized to octanol, octanal and octanoic acid, the latter is subsequently acylated and oxidized to yield acetyl‐CoA that is assimilated via the glyoxylate shunt; in fact, a knockout mutant in the aceA gene, encoding isocitrate lyase was unable to grow on octane or octanoic acid.

Enhanced chaotrope tolerance and (S)‐2‐hydroxypropiophenone production by recombinant Pseudomonas putida engineered with Pprl from Deinococcus radiodurans

“…This feature has made it a popular choice for degrading pollutants in waste streams, such as benzene, toluene, ethylbenzene and xylene (BTEX) (Kour et al., 2022 ; Shim & Yang, 1999 ). The high tolerance of certain strains of P. putida is attributed to mechanisms such as modifications in the membrane composition, including a shift in the conformation of membrane unsaturated fatty acids from cis to trans , the presence of efflux pumps that secrete the chaotropic chemicals outside the cell and rapid NADPH supply in response to environmental stresses (Duque et al., 2022 ; Hallsworth et al., 2003 ; Nikel et al., 2021 ; Vallon et al., 2015 ). Additionally, P. putida has gained attention as a microbial host in industrial biotechnology (Nikel & de Lorenzo, 2018 ; Timmis, 2002 ; Volke et al., 2022 ; Volke, Calero, et al., 2020 ; Weimer et al., 2020 ).…”

SynBio 2.0, a new era for synthetic life: Neglected essential functions for resilience

“…These circuits are designed in a manner somewhat similar to that of microprocessors (Buecherl & Myers, 2022) and implemented in model organisms selected for their well‐known physiology (Bernhards et al, 2022). The most common are bacteria such as Escherichia coli (Lynch et al, 2022), Bacillus subtilis (Zhao et al, 2022) and more recently Pseudomonas putida (Duque et al, 2022). In a minor but conceptually important trend, intended to illustrate how we can access the minimal set of functions that could (should) be assembled de novo to make a cell alive, Mycoplasmas' chassis have also been used (Talenton et al, 2022).…”