(7ba) Enabling C1-Based Bioconversion through Metabolic Engineering

Single-carbon (C1) substrates, such as synthesis gas and methanol, are attractive feedstocks for biochemical processes, as they are widely available, can be produced renewably, and do not compete with food supply. However, their use in industrial bioprocessing remains limited, primarily because microbes that utilize these substrates are poorly characterized biochemically, and limited tools exist for their genetic modification. This leaves the metabolic engineer with a choice: to develop genetic tools to enable engineering in the desired host, or to import the relevant catabolic pathway into a more tractable organism, such as Escherichia coli. My research program will explore both options within the context of developing strains for the conversion of C1 substrates into value-added chemicals and fuels.

Clostridium ljungdahlii is an acetogen that grows autotrophically on synthesis gas (CO, H2, and CO2) using the Wood-Ljungdahl pathway, and is a promising candidate for non-photosynthetic CO2 fixation. In my PhD work, I extended its primitive genetic tools by developing a CRISPRi system for the targeted knockdown of specific genes. Constitutive downregulation of several genes with putative roles in energy conservation and carbon flux by up to 30-fold was demonstrated, and the associated phenotypes analyzed. Optimization of the promoter controlling dCas9 expression allowed for inducible knockdown, paving the way for dynamic metabolic control strategies to redirect carbon flux in engineered strains. To demonstrate this concept, several variants of a heterologous pathway for the biosynthesis of 3-hydroxybutyrate (3HB) were constructed, to probe 3HB production in the wild-type background and with various CRISPRi plasmids. Downregulation of phosphotransacetylase led to a doubling of both titer and yield of 3HB. The CRISPRi system represents a valuable contribution to the metabolic engineering field for its ability to redirect carbon flux, and is also useful to the microbiology community to probe gene function to answer open questions in the biochemistry underlying the Wood-Ljungdahl pathway.

To explore the alternative approach of importing a single-carbon catabolic pathway into a tractable host, again in my PhD work, E. coli was engineered to metabolize methanol. Screening various candidates of the three heterologous pathway enzymes enabled robust incorporation of 13C-labeled methanol into central carbon metabolism. To further improve methanol assimilation, a kinetic-thermodynamic modeling framework was developed and combined with novel isotopic tracing experiments to probe potential pathway limitations. Flux leakage from the cyclical ribulose monophosphate (RuMP) pathway was identified as the primary bottleneck, as this led to the build-up of the toxic intermediate formaldehyde and ablation of the thermodynamic driving force for methanol oxidation. Strategies were developed to re-wire central metabolism accordingly, which restored the driving force. Through the use of deuterated isotopic tracers and the analysis of the corresponding kinetic isotope effect, the kinetics of the first enzyme of the pathway - methanol dehydrogenase (MDH) - were identified as the next limitation. These results represent the first systematic analysis of flux limitations in E. coli engineered for methanol metabolism, and provide clear targets for further metabolic engineering to enable synthetic methylotrophy.

Through these research experiences, I developed the ability to work with non-traditional metabolic engineering hosts such as C. ljungdahlii, and learned advanced methodologies for analyzing heterologous metabolic pathways, such as 13C dynamic labeling. In my research program, I plan to extend the use of these techniques. For example, the CRISPRi system for C. ljungdahlii should allow for a high-throughput generation of libraries of knockdowns that can be assessed for production phenotype and tracked and enriched via NGS technologies. The techniques developed for the analysis of C1 assimilation in E. coli can be extended to substrates beyond methanol, and provide fundamental insight into thermodynamic and kinetic control and promiscuity in central carbon metabolism. Finally, I will combine the expertise I have gained in these areas, for example by applying isotopic tracer analysis to metabolic engineering of C. lljungdahlii.

Teaching Interests:

My chemical engineering background, combined with multidisciplinary training in chemistry, microbiology, biochemistry and molecular biology, has equipped me to teach a variety of courses. I would be particularly interested in teaching the following courses in Chemical Engineering:

1) Bioprocess Engineering - This course would introduce the core chemical engineering components of bioprocessing, including enzyme kinetics, growth kinetics, bioreactor design, and oxygen and heat transport in bioreactors, through a case-study approach designed to introduce students to the career options available in bioprocessing.

2) Metabolic Engineering - This course would introduce the theoretical and computational approaches used in modern metabolic engineering, including metabolic control analysis (MCA), metabolic flux analysis, and flux balance analysis. The course would draw examples from recent and classic literature on these topics.