In this study, the effects of growth conditions on archaellation in Methanococcus maripaludis were examined. Cells were grown in a variety of media, including complex, minimal and with formate as the electron donor, with different nitrogen sources, varied salinities and at a variety of growth temperatures. Of the conditions tested, Western blot results showed that major archaellin FlaB2 levels only varied detectably as a result of growth temperature. Whilst the amount of FlaB2 was similar for cells grown at ,35 8C, protein levels decreased at 38 8C and were barely detectable at 42 8C. Quantitative reverse transcription PCR experiments demonstrated that the flaB2 transcript levels were almost undetectable at 42 8C. Electron microscopy confirmed that the FlaB2 levels detected by Western blots corresponded to the state of archaellation, with cells grown at 42 8C being mostly non-archaellated. Unexpectedly, a lower apparent molecular mass for FlaB2 was observed in Western blots of cells grown at temperatures .38 8C, suggestive of a truncation in the attached N-linked tetrasaccharide at higher growth temperatures. MS analysis of archaella isolated from cells grown at 40 8C confirmed that FlaB2 was now decorated with a trisaccharide in which the third sugar was also lacking the attached threonine and acetamidino modifications found in the WT glycan.

The goal of this project was to adapt the Yarrowia lipolytica plasmid based CRISPR/Cas9 system for usage in Lipomyces starkeyi. Lipomyces starkeyi is an oleaginous yeast, which synthesizes and stores high amounts of intracellular lipids. This specific yeast can store lipids at concentrations higher than 60% of its dry cell weight. Due to these high concentrations of lipids, L. starkeyi is a desired organism for the production of biofuels and other oleochemicals. However, there is a lack of knowledge and of genetic tools when trying to engineer the cells to produce these lipids for our use. The genome editing tool, CRISPR/Cas9 is efficient and simple, therefore desirable for the engineering of L. starkeyi. The goal was achieved by replacing the Y. lipolytica promoter with a L. starkeyi promoter, inserting guide RNA, as well as confirming cas9 protein expression.