Analysis of transcriptional profile from Pseudomonas sp. LFM046 and theirs recombinants producing biopolymer / polyhydroxyalkanoates from glucose based on DNA Microarray and RNA-Seq.

Abstract

The Pseudomonas sp. LFM046 have been studied as biological platform for the production of biodegradable polymers belonging to the polyhydroxyalkanoate (PHA) family, at the Laboratory of Bioproducts, University of São Paulo (LBUSP). The full genome of Pseudomonas sp. LFM046 was obtained (Cardinali-Rezende et al. 2015) and the annotation of their genes were manually refined to enhance the quality of the genome scale metabolic model that has been constructed. This strain produces an copolymer of medium-chain-length 3-hydroxyalkanoates (PHAMCL - 6 to 14 carbons), containing saturated monomers of 6, 8, 10 and 12 carbons and one monomer of 12 carbons with its unsaturated carbon 5. The objective of this proposal is to perform the analysis of the transcriptional profile of Pseudomonas sp. LFM046 and their recombinants in different culture conditions. The analysis of the transcriptome will allow a qualitative and quantitative evaluation, under different growth conditions and polymer accumulation, of the expression of the genes involved in the metabolic pathways of glucose degradation: Pentose-Phosphate pathway, Entner-Doudoroff pathway and TCA cycle and others. This information will help us to understand the metabolism of this strain and would restrict the number of mathematically possible flux distributions predicted for the same experimental condition, enhancing the accuracy of the metabolic model that has been developed at LBUSP with the help of bioinformatics group from Dr. Marie France Sagot lab at INRIA and the University of Lyon, France. The genome by itself doesn't allow the analysis of the metabolic regulation effect in vivo, but such effect is measurable in the gene expression levels. The transcriptome analysis will be carried out using RNA sequencing (RNA-Seq) and DNA microarray. The final results will be made available for the scientific community by presentation of the work in national and international events, publications in high-impact journals and submission of the cDNA sequences of Pseudomonas sp. LFM046 to databases. The experiments and interpretation of the data will be performed under supervision of Dr. Steinbuchel, at University of Munster in German. His lab combines specialized group in microbial metabolism, molecular biology, genetics and bioprocess engineering and will significantly broaden the knowledge on the metabolic pathways involved in the production of PHA. In addition this in turn would accelerate the development of new strains by synthetic/systems biology approaches, even for bioproducts different from PHA, since any metabolic profile required by a new precursor biomolecule would be assessable in the metabolic model.