Heterologous expression of a global transcriptional regulatory protein in a non-oleaginous Rhodococcus erythropolis strain to improve lipid production.

GALLEGOS A.L.; HERNÁNDEZ M.A.; ACOSTA V.; SILVA R.A.; ALVAREZ H.M.

Congreso; LVII Reunión Anual de SAIB y el XVI Sociedad Argentina de Microbiología General (SAMIGE); 2021

Lipid accumulation is a well-studied process that occurs in many bacteria, such as those of the genus Rhodococcus, due its biotechnological applications. In a previous study, we identified and characterized a pleiotropic transcriptional regulator called NlpR (Nitrogen Lipid Regulator) that simultaneously modulates carbon and nitrogen metabolisms in the oleaginous R. jostii strain RHA1 in response to nitrogen limitation. In this study we analyzed the effect of the heterologous expression of NlpR from R. jostii RHA1 on lipid accumulation in the non-oleaginous strain; Rhodococcus erythropolis ATCC 15960. Bioinformatic analyses demonstrated that nlpR-orthologous gene is also present in R. erythropolis, exhibiting 81% identity (98% query cover) in comparison to the nlpR gene of R. jostii. In addition, a conserved synteny of nlpR locus was observed in genomes of both rhodococcal species. We analyzed the occurrence of putative DNA binding sites for NlpR in R. erythropolis genome. “NlpR box” motifs were found in the upstream region of several genes involved in nitrogen and lipid metabolisms, including nark (nitrate/nitrite uptake), nirD (nitrite reductase small subunit), eukaryotic like-acetyl-CoA carboxylase gene, fasI (fatty acid synthase Complex I) and 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT), among others. To analyze the possible effect of NlpRRHA1 on lipid accumulation in R. erythropolis ATCC 15960, the inducible expression vector pTipQC2/nlpRRHA1 was transferred into the ATCC 15960 cells.Thin layer chromatography analysis of cell lipid extracts demonstrated that the heterologous expression of NlpRRHA1 promoted an increase of neutral lipid fractions, including triacylglycerols (TAG), diacylglycerols and free fatty acids in comparison to the control cells carrying the empty inducible vector. In addition, quantitative gas chromatography analysis revealed an increase of 1.9-fold in total fatty acid content (8.97% CDW) in ATCC15960 pTipQC2/nlpRRHA1 in comparison to the control cells, after cultivation in minimal salt medium with glucose (1%, w/v) and nitrogen-limiting conditions (0.1 g/L of ammonium). Unexpectedly, the heterologous expression of NlpRRHA1 in R. erythropolis ATCC 15960 promoted the production of a co-polymer of 3-hydroxybutyrate-co-3-hydroxyvalerate (12.04% CDW), whereas the control cells produced only traces of the copolymer. In contrast, nlpRRHA1 overexpression in R. jostii RHA1 increased only the total fatty acid content in cells and neutral lipid fractions (TAG, DAG, MAG), but it did not promote the PHA biosynthesis. These results demonstrated that the pleiotropic transcriptional regulator NlpR can be considered an interesting tool for genetic modification of rhodococcal species to improve lipid production. Deregulation of cell metabolism by NlpR expression can produce differential phenotypic effects among rhodococcal species.