Metabolic Engineering for Lignin Valorization and Martian In Situ Resource Utilization

Metabolic engineering is a vital tool to help move away from petroleum dependence in chemical production. By taking advantage of native and engineered metabolic pathways in readily culturable microorganisms, we can produce many of the compounds we need renewably and sustainably. Towards this, this thesis first studied the use of metabolic engineering for the production of the commodity chemical adipic acid. A literature review of metabolic pathways towards adipic acid showed distinct advantages to using lignin-derived aromatics as the starting material. To further explore this, Escherichia coli was engineered to produce adipic acid from the lignin derived monomer, catechol, and novel branched adipic acid analogs from alkyl-substituted catechols, providing insight into the substrate specificity and activity of pathway enzymes. Apart from lignin, carbon dioxide is also a promising non-sugar feedstock, allowing for application of metabolic engineering in extreme environments. To further explore this, a biotechnology-enabled process for the production of 2,3-butanediol from Martian CO2 was designed using cyanobacteria to fix the CO2 into sugars and an engineered E. coli to convert the sugars into 2,3-BDO. Process analysis highlighted biological and material improvement targets for increasing feasibility of Martian application, as well as the distinct advantage in O2 production that is gained using a biotechnology-enabled process. Finally, this work reviews the use of cell-free systems (CFSs) for the production of chemicals, energy, and therapeutics, highlighting recent advancements in production of membrane proteins in CFSs. In summary, this thesis provides a multi-level view and analysis of metabolic engineering applications for renewable chemical production.Ph.D

Confirmation of a double-hit model for the NF1 gene in benign neurofibromas.

Neurofibroma is a benign tumor that arises from small or large nerves. This neoplastic lesion is a common feature of neurofibromatosis type 1 (NF1), one of the most common autosomal dominant disorders. The NF1 gene codes for a protein called "neurofibromin." It possesses a region that shares a high homology with the family of GTPase-activating proteins, which are negative regulators of RAS function and thereby control cell growth and differentiation. The evidence points to the NF1 gene being a tumor-suppressor gene. NF1 patients also have an increased incidence of certain malignant tumors that are believed to follow the "two hit" hypothesis, with one allele constitutionally inactivated and the other somatically mutated. Recently, somatic loss of heterozygosity (LOH) has been described for neurofibromas, and mutations in both copies of the NF1 gene have been reported for a dermal neurofibroma. The aim of our study was the analysis of the NF1 locus in benign neurofibromas in NF1 patients. We performed LOH analysis on 60 neurofibromas belonging to 17 patients, 9 of them with family history of the disease and 8 of them sporadic. We have analyzed five intragenic NF1 markers and six extragenic markers, and we have found LOH in 25% of the neurofibromas (corresponding to 53% of the patients). In addition, we found that in the neurofibromas of patients from familial cases the deletions occurred in the allele that is not transmitted with the disease, indicating that both copies of the NF1 gene were inactivated in these tumors. Therefore, the recent reports mentioned above, together with our findings, strongly support the double inactivation of the NF1 gene in benign neurofibromas

Cloning the mouse homolog of the human cystic fibrosis transmembrane conductance regulator gene.

The cystic fibrosis transmembrane conductance regulator is encoded by the gene known to be mutated in patients with cystic fibrosis. This paper reports the cloning and sequencing of cDNAs for the murine homolog of the human cystic fibrosis transmembrane conductance regulator gene. A clone that, by analogy to the human sequence, extends 3' from exon 9 to the poly(A) tail was isolated from a mouse lung cDNA library. cDNA clones containing exons 4 and 6b were also isolated and sequenced, but the remainder of the mRNA proved difficult to obtain by conventional cDNA library screening. Sequences spanning exons 1-9 were cloned by PCR from mouse RNA. The deduced mouse protein sequence is 78% identical to the human cystic fibrosis transmembrane regulator, with higher conservation in the transmembrane and nucleotide-binding domains. Amino acid sequences in which known cystic fibrosis missense mutations occur are conserved between man and mouse; in particular, the predicted mouse protein has a phenylalanine residue corresponding to that deleted in the most common human cystic fibrosis mutation (delta F508), which should allow the use of transgenic strategies to introduce this mutation in attempts to create a "cystic fibrosis mouse"