[CCoE Notice] Cullen College Dissertation Defense Announcement - Ehsan Bahrami Moghadam- ChBE

[Hutchinson, Inez A]

[Dissertation Defense Announcement at the Cullen College of Engineering]

Directed Evolution of Protein-Based Sensors for Anaerobic Biological Activation of Methane and Short-chain Alkanes



Ehsan Bahrami Moghadam



June 6, 2024; 3:00PM

Location: Chemical Engineering Senior Room, S144E, Engineering Building 1



Committee Chair:
Patrick Cirino, Ph.D.



Committee Members:
Richard Willson, Ph.D. | Mehmet Orman, Ph.D. | Mehmet Sen, Ph.D. | Yuheng Lin, Ph.D.



Abstract

           Metabolic and protein engineering are powerful approaches to design customized enzymes and microorganisms capable of novel and/or enhanced production of target compounds. In this context, the combination of gene library generation and high-throughput screening comprise a highly effective means to identify improved biocatalysts, or components thereof. Genetically-encoded biosensor/reporter systems developed from small molecule-responsive transcription factors (TFs) have proven to be useful tools for metabolite detection and metabolic monitoring, toward the design of new biocatalysts. In this study, interest lies in microbial alkane degradation pathways, which provide biological routes for converting these hydrocarbons into higher value products. The functional expression of a methyl-alkylsuccinate synthase (Mas) system in Escherichia coli, enabling heterologous anaerobic activation of short-chain alkanes (C3-C6), was recently reported. To date, no system for methane activation has been identified, and putative enzyme systems for activating ethane, propane, and butane are yet to be functionally expressed. Here, the objective was to design a TF-based sensor/reporter system for high-throughput screening of alkylsuccinate synthase activity on various short-chain alkanes. While there are no known regulatory proteins that control gene expression in response to an alkylsuccinate, ItcR from Yersinia pseudotuberculosis exhibits high sensitivity to methylenesuccinate (IA), showing ~100-fold lower response to the structurally similar methylsuccinate (MS). Thus, the focus was on employing protein engineering techniques to alter the inducer specificity of ItcR toward MS firstly, and subsequently other, longer chain alkylsuccinates. The resulting variants represent the first generation of sensors for endogenous molecular reporting of alkylsuccinate biosynthesis, by placing reporter and/or selection genes under control of the ItcR cognate promoter (Pccl). Random mutagenesis followed by fluorescence-activated cell sorting (FACS), combined with site-directed mutagenesis and further protein characterization yielded several MS-responsive ItcR variants. Structural modeling and analysis of the ItcR ligand binding pocket provided mechanistic insights into the altered molecular recognition. Subsequent rounds of directed evolution targeted responsiveness to ethylsuccinate (ES) and methylpentylsuccinate (MPS) and yielded multiple variants with improved response to these compounds. The most sensitive MPS biosensor developed was effectively utilized for detecting the biosynthesis of this compound in the previously established heterologous host capable of hexane activation. Structural analysis and biochemical characterization of WT-ItcR and its variants are presented, providing insights for further biosensor design and applications for engineering microbes capable of converting short alkanes into value-added products.

[Engineered For What's Next]


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