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- All Subjects: Metabolic Engineering
- Creators: Varman, Arul
allow E. coli to consume xylose and glucose, two ubiquitous and industrially relevant microbial feedstocks, simultaneously was implemented and systematically evaluated for its effects on L-phenylalanine (Phe; a precursor to many microbially-derived aromatics such as 2PE) production. Ultimately, by incorporating this mutation into a Phe overproducing strain of E. coli, improvements in overall Phe titers, yields and sugar consumption in glucose-xylose mixed feeds could be obtained. While upstream efforts to improve precursor availability are necessary to ultimately reach economically-viable production, the effect of end-product toxicity on production metrics for many aromatics is severe. By utilizing a transcriptional profiling technique (i.e., RNA sequencing), key insights into the mechanisms behind styrene-induced toxicity in E. coli and the cellular response systems that are activated to maintain cell viability were obtained. By investigating variances in the transcriptional response between styrene-producing cells and cells where styrene was added exogenously, better understanding on how mechanisms such as the phage shock, heat-shock and membrane-altering responses react in different scenarios. Ultimately, these efforts to diversify the collection of microbially-produced aromatics, improve intracellular precursor pools and further the understanding of cellular response to toxic aromatic compounds, give insight into methods for improved future metabolic engineering endeavors.
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.