Project description:Bacterial cellulose (BC) represents a renewable biomaterial with unique properties promising for biotechnology and biomedicine. Komagataeibacter hansenii ATCC 53,582 is a well-characterized high-yield producer of BC used in the industry. Its genome encodes three distinct cellulose synthases (CS), bcsAB1, bcsAB2, and bcsAB3, which together with genes for accessory proteins are organized in operons of different complexity. The genetic foundation of its high celluloseproducing phenotype was investigated by constructing chromosomal in-frame deletions of the CSs and of two predicted regulatory diguanylate cyclases (DGC), dgcA and dgcB. Proteomic characterization suggested that BcsAB1 was the decisive CS because of its high expression and its exclusive contribution to the formation of microcrystalline cellulose. BcsAB2 showed a lower expression level but contributes significantly to the tensile strength of BC and alters fiber diameter significantly as judged by scanning electron microscopy. Nevertheless, no distinct extracellular polymeric substance (EPS) from this operon was identified after static cultivation. Although transcription of bcsAB3 was observed, expression of the protein was below the detection limit of proteome analysis. Alike BcsAB2, deletion of BcsAB3 resulted in a visible reduction of the cellulose fiber diameter. The high abundance of BcsD and the accessory proteins CmcAx, CcpAx, and BglxA emphasizes their importance for the proper formation of the cellulosic network. Characterization of deletion mutants lacking the DGC genes dgcA and dgcB suggests a new regulatory mechanism of cellulose synthesis and cell motility in K. hansenii ATCC 53,582. Our findings form the basis for rational tailoring of the characteristics of BC.
Project description:A comparison between the coculture of Debaryomyces hansenii with Yarrowia lipolytica and the monoculture of Debaryomyces hansenii, was carried out at two hours of culture (12h and 27h), in order to understand the metabolic changes induced in D. hansenii when grown in coculture with Y. lipolytica . The transcriptomic study was carried out using Agilent 8X15k slides with a custom oligoset designed by V. Loux using the Debaryomyces hansenii ORFeome extracted from genolevures database
Project description:Total RNA versus genomic DNA hybridization on custom arrays designed for all Debaryomyces hansenii genes Total RNA was collected in mid-log phase from Debaryomyces hansenii cells grown in rich medium (abbreviated CM, in house recipe). RNA was then converted to cDNA, Cy3-labeled and hybridized competitively against Cy5 labeled genomic DNA from Debaryomyces hansenii.
