Abstract
Objective
To construct Pseudomonas aeruginosa PA14 derivatives that overproduce rhamnolipids (RL) by blocking the synthesis of the carbon-storage polymer polyhydroxyalkanoates (PHA) and by overexpressing the rhlAB-R operon that encodes for enzymes of RL synthesis and the RhlR transcriptional regulator.
Results
In contrast to previous results showing that overexpression of rhlAB-R genes in two P. aeruginosa strains (PAO1 and ATCC 9027) is sufficient to overproduce RL, we show that a PA14 derivative overexpressing the rhlAB-R operon did not increase the synthesis of these biosurfactants. In addition, PA14 mutants deficient in PHA production did not overproduce RL either. However, if the rhlAB-R genes were expressed in a mutant that is completely impaired in PHA synthesis, a significant increase in RL production was observed (59%). These results show that RL production in PA14 is limited both by the availability of fatty acid precursors and by the levels of the RhlA and RhlB enzymes that are involved in the synthesis of mono-RL.
Conclusions
The limitation of RL production by P. aeruginosa PA14 is multifactorial and diverse from the results obtained with other strains. Thus, the factors that limit RL production are particular to each P. aeruginosa strain, so strain-specific strategies should be developed to increase their production.
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Acknowledgements
UGG and MPSA are doctoral student of Programa de Maestría y Doctorado en Ciencias Bioquímicas, Universidad Nacional Autónoma de México (UNAM), this study was performed in partial fulfillment of the requirements for UGG doctorate degree. UGG (CVU-422007) and MPSA (CVU-741217) received a fellowship from CONACYT. We acknowledge Abigail González-Valdez for technical assistance. This work was supported in part by grant IN200416 from Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica, (Dirección General de Asuntos del Personal Académico -UNAM) and grant 252269 from Consejo Nacional de Ciencia y Tecnología (CONACYT).
Supporting information
Supplementary Fig. 1—shows the standard curve use to determine PHA weight from absorbance at 235 nm.
Supplementary Fig. 2—presents the PA14 strain growth curve in PPGAS medium.
Supplementary Fig. 3—shows the PHA production of PA14 derivatives carrying mutations in genes involved in the synthesis of this fatty acid polymer, and the experiments showing that the phaC1 gene of the phaC2::MAR2xT7 mutant (ID 54085) is not functional since it is unable to complement the phaC1::MAR2xT7 mutant (ID 32531) for PHA synthesis.
Supplementary Table S1—shows the oligonucleotides used to construct the PA14 ΔphaG::aac(3)IV mutant and the pUCP20 derived plasmids expressing phaC1wt and phaC1m.
Supplementary Table S2—shows the results obtained from the analysis of the image of thin-layer chromatography presented in Fig. 2, using the ImageJ software.
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Gutiérrez-Gómez, U., Soto-Aceves, M.P., Servín-González, L. et al. Overproduction of rhamnolipids in Pseudomonas aeruginosa PA14 by redirection of the carbon flux from polyhydroxyalkanoate synthesis and overexpression of the rhlAB-R operon. Biotechnol Lett 40, 1561–1566 (2018). https://doi.org/10.1007/s10529-018-2610-8
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DOI: https://doi.org/10.1007/s10529-018-2610-8