(7bb) Engineering Metabolism for Carbon Conservation and Cellulosic Biofuel Production

My research interest is to apply metabolic engineering for solving two major limitations in biofuel and biochemical production: (1) intrinsic carbon lost from central metabolic pathways and (2) degradation of recalcitrant biomass. To address these limitations, my research specifically focuses on rewiring central metabolic pathways for carbon conservation, and engineering an isobutanol pathway in a thermophilic cellulolytic microorganism, Clostridium thermocellum.

Rewiring central metabolic pathway for carbon conservation

Acetyl-coenzyme A (acetyl-CoA) is a two-carbon metabolite (C2 metabolite) and an important metabolic precursor to a variety of industrially relevant compounds including biofuels. An intrinsic limitation of acetyl-CoA derived biochemical production is the carbon loss when forming acetyl-CoA. Most organisms use some glycolytic variation, commonly the Embden-Meyerhof Pathway (EMP), to initially degrade sugar into pyruvate. Pyruvate, a C3 metabolite, is then decarboxylated to form acetyl-CoA, losing carbon to the environment. This decarboxylation limits the carbon yield to only two molecules of acetyl-CoA from one molecule of hexose, thus inhibiting the economics of any associated bioprocess. A synthetic sugar catabolism pathway, termed non-oxidative glycolysis (NOG), was recently developed to address this problem. NOG combines a phosphoketolase dependent cleavage of sugar phosphates and a carbon rearrangement cycle to directly generate C2 units per sugar in a redox neutral manner. To further expand the applications using NOG, an Escherichia coli strain was constructed to rely solely on NOG for sugar catabolism through rational metabolic rewiring, recombinant expression, and evolution. In this engineered and evolved bacteria, the major EMP pathway was deleted, and sugar was first converted to C2 metabolite (acetyl-CoA) through NOG prior to pyruvate formation. Under anaerobic conditions, this strain was able to convert 82% of the glucose carbon to acetate (derived from acetyl-CoA), while the maximum acetate yield from EMP is 67%. Therefore, this strain offers significant potential to be engineered for the production of a variety of acetyl-CoA derived compounds.

Consolidated bioprocessing of cellulose to isobutanol using Clostridium thermocellum

Biomass recalcitrance—resistance to degradation—currently limits the use of lignocellulose for biofuel production. Consolidated bioprocessing (CBP), in which cellulose hydrolysis and fermentation occur simultaneously in one pot without added cellulases, is a potential approach to improve lignocellulose utilization. Owing to its high cellulose deconstruction rate, Clostridium thermocellum is a promising thermophilic CBP host that grows at 50-60^oC. The elevated temperature also promotes cellulose degradation, reduces contamination, and minimizes cooling costs. Metabolic engineering in C. thermocellum is usually hampered by enzyme toxicity during cloning, time-consuming pathway engineering procedures, and slow turnaround in production tests. Here, we present a streamlined approach to engineer C. thermocellum to produce isobutanol directly from cellulose. Essential isobutanol pathway genes under different promoters were cloned to create various plasmid constructs in E. coli. Then, these constructs were transformed and tested in C. thermocellum. Among these engineered strains, the best isobutanol producer was selected and the production conditions were optimized. Moreover, acetohydroxy acid synthase (Ahas), the first gene in the isobutanol pathway, was determined as the limiting step during the production by enzyme assay. With further Ahas overexpression, 9.7 g/L of isobutanol was produced within 120 h directly from cellulose using our engineered strain. Our study demonstrates the feasibility of CBP of lignocellulose to biofuel and biochemical compounds using C. thermocellum.

Teaching Interests:

My teaching interests include the all of the core classes (particularly transport phenomena and thermodynamics) of the chemical engineering discipline, as well as hands-on lab classes and research mentoring. I developed my teaching ability from multiple experiences: as a volunteer lecturer, as a teaching assistant, and as a project lead.

Teaching experience

As a graduate student at UCLA, I have provided a review of transport phenomena for students studying for the preliminary oral exam for Ph.D. candidacy. I also served as teaching assistant for thermodynamics. As a teaching assistant I delivered a one hour discussion for the whole class, conducted office hours every week, and graded homework and exams. Since thermodynamics is more abstract than other chemical engineering subjects, I always advised the students to link the problems to thermodynamic properties instead of just starting with an equation. I believe the purpose of education is to help the students develop critical thinking skills to simplify and solve problems, in addition to covering the course content.

Mentoring experience

The experience of mentoring students on research is one of my motivations to pursue a faculty career. In particular, helping students to develop their abilities, expand their competencies and achieve their potential gives me an incredible sense of fulfillment. My mentorship started at UCLA, where I established my own research team and have worked closely with 4 technicians and 9 undergraduate students in the past five years. Now, many of my former students are pursuing their advanced degree (M.D or Ph.D.) or working in related companies like DuPont and Gilead Sciences.