Artificial Consortium of Three E. coli BL21 Strains with Synergistic Functional Modules for Complete Phenanthrene Degradation

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are highly toxic and persistent organic pollutions that can accumulate in the environment. In this study, an aromatic ring cleavage module, a salicylic acid synthesis module, and a catechol metabolism module were respectively constructed in three Escherichia coli BL21 strains. Subsequently, the engineered strains were cocultured as an artificial consortium for the biodegradation of phenanthrene, a typical PHA. Single factor experiments and response surface methodology were used to identify the optimal degradation conditions, including an inoculation interval of 6 h, inoculation ratio of 1:1:1, and IPTG concentration of 2 mM. Under these conditions, the 7-day degradation ratio of 100 mg/L phenanthrene reached 72.67%. Moreover, the engineered Escherichia coli BL21 strains showed good phenanthrene degradation ability at substrate concentrations 10 mg/L up to 500 mg/L. Enzyme activity assays combined with gas chromatography–mass spectrometry measurements confirmed that the three engineered strains behaved as a synergistic consortium in the phenanthrene degradation process. Based on the analysis of the key metabolites, the engineered bacteria were supplemented at 7-day intervals in batches so that each engineered strain maintained its optimal degradation ability. The 21-day degradation ratio finally reached 90.66%, which was much higher than what was observed with simultaneous inoculation. These findings suggest that the three engineered strains with separate modules constructed in this study offer an attractive solution for removing PAHs from the environment.

Engineering Pseudomonas putida To Produce Rhamnolipid Biosurfactants for Promoting Phenanthrene Biodegradation by a Two-Species Microbial Consortium

“…Our laboratory has successfully designed and constructed engineered E. coli M1, M2, and M3 following the principle of “division-of-labor” in previous work (see Table S1 in Text S1 in the supplemental material) ( 30 ). It should be noted here that we used these three engineered bacteria simultaneously in a 1:1:1 inoculation ratio in this study; this was referred collectively as E. coli M123.…”

“…After having determined the growth and productivity of engineered P. putida , we cocultured the engineered P. putida with the previously constructed E. coli M123, which could degrade phenanthrene to build two-species microbial consortia. As previously mentioned, it should be noted that E. coli M123 is also a consortium consisting of E. coli M1, M2, and M3, which all have different metabolic functions ( 30 ). As anticipated, the three consortia containing engineered P. putida increased the degradation of 100 mg/L phenanthrene compared to E. coli M123 alone ( Fig.…”

“…Accordingly, the degradation ratio stopped increasing at IPTG concentrations above 0.8 mM and even decreased at inducer concentrations that were too high. In our previous study, the best degradation of phenanthrene was achieved by E. coli M123 with an IPTG concentration at 2 mM ( 30 ). However, as shown above, the optimized IPTG concentration for the two-species microbial consortia was 0.8 mM, since the engineered P. putida we constructed was not induced by IPTG in this work but instead had some toxic effects on the cells of engineered P. putida .…”

“…Within the rapid development of emerging biological disciplines such as synthetic biology, increasing attention was paid to the design and development of artificial microbial consortia in order to overcome barriers in the degradation of PAHs. In previous research, our laboratory used a modular construction strategy to develop a consortium consisting of three engineered E. coli strains ( 30 ), whereby metabolic pathways were rationally segmented and allocated. Although this approach reduced the metabolic burden for individual cells and the consortium was able to degrade PAHs, the low water solubility was still a limiting factor for biodegradation.…”

An intelligent synthetic bacterium with sound-integrated ability for chronological toxicant detection, degradation, and lethality

“…Regarding degradation, analysis of the catabolic pathways in natural strains facilitates the migration of functional genes to artificial cells that do not possess efficient or complete degradation abilities ( 5, 7 ). For example, an artificial consortium of three E. coli BL21(DE3) strains with synergistic functional modules was designed to completely degrade phenanthrene ( 8 ), and the pathway of engineered E. coli DH5α was linked with a natural pentachlorophenol degrader to mineralize hexachlorobenzene ( 9 ). Additionally, restraining their proliferation is one of the primary challenges for genetically modified microorganisms.…”