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- Creators: Ravel, Maurice, 1875-1937
Efforts were then made to optimize the expression of the C6T Fab in E. coli. Both the periplasmic secretion pathway and the effect of trigger factor were tested. Four expression systems were tested, consisting of one of two signal sequences (either DsbA directing through the SRP-dependent co-translational pathway or stII directing through the sec-dependent post-translational pathway) and one of two expression strains (BW25113 (tig+) containing trigger factor and KTD101 (Δtig) lacking trigger factor). Plasmids were constructed allowing the C6T Fab to be expressed and secreted using both pathways, and transformed into both strains. It was predicted that the protein expression could be optimized by employing the co-translational pathway in cells lacking trigger factor (i.e. the Δtig-DsbA expression system). However, this system severely decreased cell growth post-induction. It was found that both the lack of trigger factor and the employment of the co-translational pathway both significantly decrease cell growth post-induction. It is theorized that the increase in protein expression and secretion rate stresses the cell to a point where it is unable to maintain normal cell function and growth.
Lignin is a naturally abundant source of aromatic carbon but is largely underutilized in industry because it is difficult to decompose. Under the current study we engineered Corynebacterium glutamicum for the depolymerization of lignin with the goal of using it as raw feed for the sustainable production of valuable chemicals. C. glutamicum is a standout candidate for the depolymerization and assimilation of lignin because of its performance as an industrial producer of amino acids, resistance to aromatic compounds in lignin, and low extracellular protease activity. Three different foreign and native ligninolytic enzymes were tested in combination with three signal peptides to assess lignin degradation efficacy. At this stage, six of the nine plasmid constructs have been constructed.