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.
Polyketides are a wide ranging class of natural microbial products highly relevant to the pharmacological industry. As chemical synthesis of polyketides is quite challenging, significant effort has been made to understand the polyketide synthases (PKSs) responsible for their natural production. Native to Streptomyces, the aln biosynthetic gene cluster was recently characterized and encodes for an iterative type I polyketide synthase (iT1PKS). This iT1PKS produces both , and ,-double bond polyketides named allenomycins; however, the basis in which one bond is chosen over the other is not yet clear. The dehydratase domain, AlnB_DH, is thought to be solely responsible for catalyzing double bond formation. Elucidation of enzyme programming is the first step towards reprogramming AlnB_DH to produce novel industrially relevant products. The Nannenga lab has worked as collaborators to the Zhao lab at the University of Illinois at Urbana-Champaign to unravel AlnB_DH’s structure and mechanism. Here, mutant constructs of AlnB_DH are developed to elucidate enzyme structure and provide insight into active site machinery. The primary focus of this work is on the development of the mutant constructs themselves rather than the methods used for structural or mechanistic determination. Truncated constructs were successfully developed for crystallization and upon x-ray diffraction, a 2.45 Å resolution structure was determined. Point-mutated constructs were then developed based on structural insights, which identified H49, P58, and H62 as critical residues in active site machinery.