Developing cellulolytic as a platform for the production of valuable products in consolidated bioprocessing of cellulose.

Background: Both industrial biotechnology and the use of cellulosic biomass as feedstock for the manufacture of various commercial goods are prominent features of the bioeconomy. In previous work, with the aim of developing a consolidated bioprocess for cellulose bioconversion, we conferred cellulolytic activity of , one of the most widely studied "nonconventional" oleaginous yeast species. However, further engineering this strain often leads to the loss of previously introduced heterologous genes due to the presence of multiple sites when using -recombinase to remove previously employed selection markers.

Expressing accessory proteins in cellulolytic to improve the conversion yield of recalcitrant cellulose.

Background: A recently constructed cellulolytic is able to grow efficiently on an industrial organosolv cellulose pulp, but shows limited ability to degrade crystalline cellulose. In this work, we have further engineered this strain, adding accessory proteins xylanase II (XYNII), lytic polysaccharide monooxygenase (LPMO), and swollenin (SWO) from in order to enhance the degradation of recalcitrant substrate.

Results: The production of EG I was enhanced using a promoter engineering strategy.

Conferring cellulose-degrading ability to to facilitate a consolidated bioprocessing approach.

Background: , one of the most widely studied "nonconventional" oleaginous yeast species, is unable to grow on cellulose. Recently, we identified and overexpressed two endogenous β-glucosidases in , thus enabling this yeast to use cello-oligosaccharides as a carbon source for growth. Using this engineered yeast platform, we have now gone further toward building a fully cellulolytic for use in consolidated bioprocessing of cellulose.

Development of cellobiose-degrading ability in Yarrowia lipolytica strain by overexpression of endogenous genes.

Background: Yarrowia lipolytica, one of the most widely studied "nonconventional" oleaginous yeast species, is unable to grow on cellobiose. Engineering cellobiose-degrading ability into this yeast is a vital step towards the development of cellulolytic biocatalysts suitable for consolidated bioprocessing.

Results: In the present work, we identified six genes encoding putative β-glucosidases in the Y.

Impact of an N-terminal extension on the stability and activity of the GH11 xylanase from Thermobacillus xylanilyticus.

To understand structure-function relationships in the N-terminal region of GH11 xylanases, the 17 N-terminal amino acids of the GH11 xylanase from Neocallimastix patriciarum (Np-Xyn) have been grafted onto the N-terminal extremity of the untypically short GH11 xylanase from Thermobacillus xylanilyticus (Tx-Xyn), creating a hybrid enzyme denoted NTfus. The hybrid xylanase displayed properties (pH and temperature optima) similar to those of the parental enzyme, although thermostability was lowered, with the Tm value, being reduced by 5°C. Kinetic assays using oNP-Xylo-oligosaccharides (DP2 and 3) indicated that the N-extension did not procure more extensive substrate binding, even when further mutagenesis was performed to promote this.