(2dd) Synthetic, Orthogonal Metabolic Pathways for Sustainable Bioconversion and Biomanufacturing of Industrially Relevant Chemicals

I am interested in solving global challenges, especially climate change and pollution, using engineering biology. My undergraduate and graduate research focused on biological utilization of one-carbon (C1) molecules which could be derived from greenhouse gases like CO₂ and methane. Specifically, the goal was to build metabolic pathways that assimilate C1 molecules and implement them into a model organism, E. coli, that does not natively utilize these molecules. Challenging part was to address the competition between the heterologous pathways and the native regulatory network originally evolved for efficient use of multi-carbon sources like sugars. In addition, many pathways that aim for non-native substrate utilization and product formation involve toxic intermediates or products which necessitate sophisticated regulation tools to ensure stable operations. Experiences with handling such issues sparked my interest in understanding and developing genomic and transcriptomic regulation systems in various cell environments.

Many synthetic pathways involve enzymes with previously unknown functions, which require extensive searching of protein database to identify ones with desired activities. It is likely that the desired functionality relies on the enzyme promiscuity leading to low affinity and kinetics. Therefore, understanding of enzyme structure and catalytic mechanisms is important to help discover better candidates through sequence and structure analysis or rational engineering of catalytic residues. Although our lab does not have expertise in this area, I was able to get a hand in the field of protein structure analysis and engineering with help of novel tools like AlphaFold. Recently, there have been many breakthroughs in this field thanks to the interdisciplinary efforts encompassing robotics and machine learning for high throughput processing, which made me believe that there is no limit in engineering proteins to accomplish higher rate, stability, and specificity or even create an entirely novel functionalities that are crucial for solving global challenges. I believe there is enormous opportunity in the cross-section between my expertise in metabolic engineering and the field of genetic regulations and protein engineering. Most importantly for my postdoctoral research, I want to find a lab that shares a vision with me in solving global challenges using engineering biology.

Research Abstract

Metabolic engineering aims to alter cellular metabolism from generating energy and maintaining homeostasis for cell growth to producing molecules of interest. Traditional approaches focused on redirecting metabolic flux from host metabolism to specific biosynthetic pathways. While this approach has been successful in biomanufacturing of diverse products, extensive engineering is required to overcome intrinsic metabolic inefficiencies associated with competition between product-forming and growth-sustaining reactions. Moreover, engineering solution varies by organisms as every organism has unique metabolism and regulatory mechanisms. To address these issues, we propose orthogonal metabolic pathways that are decoupled from the host metabolism, enabling precise metabolic control and easy platform transfer to different organisms and platforms. Orthogonal metabolic pathways build upon enzymes with non-native functions identified via genome mining and protein engineering. In this presentation, I will introduce two synthetic pathways developed in our group, reverse β-oxidation (rBOX) pathway and formyl-CoA elongation (FORCE) pathway, and how they are engineered (rBOX) or designed (FORCE) to be orthogonal to the host metabolism.

Reverse β-oxidation (rBOX) pathway is an iterative carbon elongation pathway which operates upon the non-decarboxylative Claisen condensation reaction between two β-ketoacyl-CoA residues, allowing elongation of two-carbon (C2) and longer units with various functionalities every iteration. Our group has demonstrated numerous products with diverse functional groups using this platform. However, there were issues with the precise control of gene expression and metabolic flux due to the crosstalk between rBOX pathway intermediates and native β-oxidation enzymes. To address this, we engineered an “rBOX-deficient” E. coli strain that eliminated genes responsible for the crosstalk. We also constructed novel TriO vector system which allows independent gene expression control of individual rBOX pathway genes to debottleneck metabolic flux and prevent byproduct formation. By making the rBOX pathway orthogonal to the host metabolism and introducing modular gene expression control tool, we were able to accomplish significantly higher titer and yield of the rBOX products reaching over 90% of the theoretical maximum yield. Moreover, we demonstrated the cross-platform capabilities of the orthogonal rBOX pathway in the cell-free platform as well as in an anaerobic autotroph utilizing carbon monoxide and hydrogen instead of sugars as carbon sources.

Whereas rBOX operates via condensation of C2 and longer carbon units, formyl-CoA elongation (FORCE) pathway utilizes C1 residue, formyl-CoA, as an elongation unit. The pathway builds upon the recently identified 2-hydroxyacyl-CoA synthase (HACS) activity, which catalyzes the condensation between formyl-CoA and various aldehydes including C1 aldehyde, formaldehyde. The condensation product, 2-hydroxyacyl-CoA, can be further converted to yield various products functional groups including 2-hydroxyacids, 1,2-diols and alcohols. As formyl-CoA is not a common metabolite, FORCE pathways can be completely decoupled from the host metabolism simply by deleting metabolic nodes that connect pathway intermediates to the host metabolism. The orthogonality of the pathway was demonstrated in glycolate production in growing culture where cell growth was supported by native nutrients while glycolate was produced solely by methanol validated through C13 labeling experiment. FORCE pathway is the first demonstration of C1 utilization pathway that can operate independently from host cell metabolism which opens the possibility for this pathway to be incorporated in various other platforms and organisms.