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- All Subjects: Metabolic Engineering
- Creators: Chemical Engineering Program
- Creators: Wang, Xuan
- Member of: Barrett, The Honors College Thesis/Creative Project Collection
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.
Strain optimization was specifically studied by enhancing inorganic carbon uptake in synechococcus sp. 7002. It is desired to have both high flux and high affinity transport for the rapid and efficient uptake of HCO3- for enhanced cell growth. The results found that the regulatory gene for carbon transporters in synechococcus genome was successfully deleted. Increasing the toxicity limits of 2-Phenylethanol was done by using adaptive laboratory evolution (ALE). ALE is a widely used practice in biotechnology studies to gain insights on mechanisms of molecular evolution and to better define the functionality of microbial cell factories. It was found that after growing E. coli BW25113 under selective conditions the genome evolved for a higher fitness medium with an increased concentration of 2-Phenylethanol. Overall, two key tools used in bioprocess engineering were successful studied to gain a better insight on the future of biochemical production industry.
Flavonoids are important biomolecules with a variety of pharmaceutical and agricultural applications. Currently, isolating these compounds is done by plant extraction, however this process is hindered by large land and energy requirements. Previous groups have aimed to overcome these challenges by engineering microbes to produce these important compounds, however this is largely bottlenecked by the lack of intercellular malonyl-CoA availability. To remedy this, the genes matB and matC have been identified as coding for malonyl-CoA synthase and a putative dicarboxylate carrier protein, respectively. Other works have successfully engineered two variants, Streptomyces coelicolor and Rhizobium trifolii, of these genes into Escherichia coli, however this has yet to be accomplished in Gram-positive Corynebacterium glutamicum. Additionally, other groups have neglected to attempt tuning these genes with respect to one another by inserting in front of different inducible promoters. This study has successfully assembled two plasmids containing the Streptomyces coelicolor and Rhizobium trifolii variants of both matB and matC. Preliminary fermentations and GCMS results confirmed that little to none naringenin was produced without the matB-matC module. Additionally, preliminary fermentations revealed that the DelAro1 and DelAro3 strains can be used to reduce metabolism of aromatics like naringenin.