ABSTRACT
Pseudomonas bharatica CSV86T displays the unique property of preferential utilization of aromatic compounds over simple carbon sources like glucose and glycerol and their co-metabolism with organic acids. Well-characterized growth conditions, aromatic compound metabolic pathways and their regulation, genome sequence, and advantageous eco-physiological traits (indole acetic acid production, alginate production, fusaric acid resistance, organic sulfur utilization, and siderophore production) make it an ideal host for metabolic engineering. Strain CSV86T was engineered for Carbaryl (1-naphthyl-N-methylcarbamate) degradation via salicylate-catechol route by expression of a Carbaryl hydrolase (CH) and a 1-naphthol 2-hydroxylase (1NH). Additionally, the engineered strain exhibited faster growth on Carbaryl upon expression of the McbT protein (encoded by the mcbT gene, a part of Carbaryl degradation upper operon of Pseudomonas sp. C5pp). Bioinformatic analyses predict McbT to be an outer membrane protein, and Carbaryl-dependent expression suggests its probable role in Carbaryl uptake. Enzyme activity and protein analyses suggested periplasmic localization of CH (carrying transmembrane domain plus signal peptide sequence at the N-terminus) and 1NH, enabling compartmentalization of the pathway. Enzyme activity, whole-cell oxygen uptake, spent media analyses, and qPCR results suggest that the engineered strain preferentially utilizes Carbaryl over glucose. The plasmid-encoded degradation property was stable for 75-90 generations even in the absence of selection pressure (kanamycin or Carbaryl). These results indicate the utility of P. bharatica CSV86T as a potential host for engineering various aromatic compound degradation pathways.IMPORTANCEThe current study describes engineering of Carbaryl metabolic pathway in Pseudomonas bharatica CSV86T. Carbaryl, a naphthalene-derived carbamate pesticide, is known to act as an endocrine disruptor, mutagen, cytotoxin, and carcinogen. Removal of xenobiotics from the environment using bioremediation faces challenges, such as slow degradation rates, instability of the degradation phenotype, and presence of simple carbon sources in the environment. The engineered CSV86-MEC2 overcomes these disadvantages as Carbaryl was degraded preferentially over glucose. Furthermore, the plasmid-borne degradation phenotype is stable, and presence of glucose and organic acids does not repress Carbaryl metabolism in the strain. The study suggests the role of outer membrane protein McbT in Carbaryl transport. This work highlights the suitability of P. bharatica CSV86T as an ideal host for engineering aromatic pollutant degradation pathways.
ABSTRACT
Hierarchical utilization of substrate by microbes (utilization of simple carbon sources prior to complex ones) poses a major limitation to the efficient remediation of aromatic pollutants. Aromatic compounds, being complex and reduced in nature, appear to be a deferred choice as the carbon source in the presence of a plethora of simple organic compounds in the environment. The soil bacterium Pseudomonas bharatica CSV86T displays a unique carbon source utilization hierarchy. It preferentially utilizes aromatics over glucose and co-metabolizes them with succinate or pyruvate (Basu et al., 2006, Applied and Environmental Microbiology, 72â:â22226-2230). In the present study, the substrate utilization hierarchy for strain CSV86T was tested for additional simple carbon sources such as glycerol, acetate, and tri-carboxylic acid (TCA) cycle intermediates like α-ketoglutarate and fumarate. When grown on a mixture of aromatics (benzoate or naphthalene) plus glycerol, the strain displayed a diauxic growth profile with significantly high activity of aromatic utilization enzymes (catechol 1,2- or 2,3-dioxygenase, respectively) in the first-log phase. This suggests utilization of aromatics in the first-log phase followed by glycerol in the second-log phase. On a mixture of an aromatic plus organic acid (acetate, α-ketoglutarate or fumarate), the strain displayed a monoauxic growth profile, indicating co-metabolism. Interestingly, the presence of glycerol, acetate, α-ketoglutarate or fumarate does not repress metabolism/utilization of the aromatic. Thus, the substrate utilization hierarchy of strain CSV86T is aromatics=organic acids>glucose/glycerol, which is unique as compared to other Pseudomonas species, where degradation of aromatics is repressed by glycerol, glucose, acetate or organic acids, including TCA cycle intermediates. This novel substrate utilization hierarchy appears to be a global metabolic phenomenon in strain CSV86T, thus implying it to be an ideal host for metabolic engineering as well as for its potential application in bioremediation.
