(7bg) Design of Synthetic C1 Carbon Assimilation Pathways

My research interests will focus on improvement of C1 carbon assimilation, including CO₂ equivalents, formic acid, methanol and methane. I will use metabolic engineering tools to increase utilization of these C1 resources to produce useful longer chain products in common microbial platforms, or to enhance overall photosynthetic efficiency in crop systems.

C1 compounds can be produced from various sources. For example, significant amounts of methane and CO₂ are produced from anaerobic digestion of organic wastes resulting from agriculture, animal husbandry and food processing. Methanol is an important intermediate in utilization of natural gas to synthesize other feedstock chemicals. Formic acid can be produced from electrochemical reduction of CO₂, or as a byproduct in biomass pretreatment as well as some chemical synthesis. However, these C1 compounds have a common challenge in subsequent increase in carbon chain length. Chemical approaches to build C-C bonds are feasible, but require high temperature, pressure and large capital investment. Thus, biological conversion is an important step towards utilization of these C1 resources to produce useful longer chain products.

Nature has evolved several distinct pathways to assimilate various C1 compounds to form metabolites necessary for growth. Methylotrophs can grow on methane or methanol as the sole carbon and energy source through the endogenous ribulose monophosphate pathway (RuMP) or serine cycle. Some lithoautotrophs, such as Ralstonia eutropha, can utilize formic acid as the carbon and energy source. In addition, cyanobacteria can fix CO₂ into 3-phosphoglycerate through the Calvin-Benson-Bassham (CBB) cycle. However, engineering on these less-characterized microorganisms involves various degrees of difficulties. One solution is to engineer model organisms, such as E. coli, to utilize C1 compounds.

Previous studies have reported some natural C1 carbon assimilation pathways displayed several limitations when constructed into other host platforms. For example, in the most prevalent CBB cycle, the oxygenation reaction of Rubisco can cause carbon loss through photorespiration of the byproduct, glycolate. Improvement of Rubisco specificity and activity beyond the native level has proven to be ineffective. For methanol condensation via the RuMP, the toxicity of formaldehyde and the metabolite competition with the host metabolic pathways restrict its methanol assimilation efficiency.

To enhance the efficiency of C1 compounds assimilation, I designed a synthetic reverse glyoxylate shunt-glycerate (rGS-Glycerate) pathway to complement the deficiency of the CBB cycle for carbon-efficient acetyl-CoA synthesis, and also constructed a reverse glyoxylate shunt-serine (rGS-Serine) cycle, modified from the natural serine cycle in methylotrophs, for various C1 compounds assimilation.

The designed rGS-Glycerate pathway is capable of converting one C3 metabolite to two acetyl-CoA via fixation of an additional CO₂ equivalent. More important, it can also convert the CBB cycle byproduct, glycolate, into stoichiometric amount of acetyl-CoA without net carbon loss. I demonstrated the effect of the rGS-Glycerate pathway for efficient acetyl-CoA synthesis in vitro and in E. coli. Further, I implemented the pathway into a photosynthetic organism, Synechococcus elongatus, and showed it promoted initial growth and accumulated ketoisocaproate, an acetyl-CoA derived product, which suggests the rGS-Glycerate pathway can complement endogenous photosynthesis system for improved acetyl-CoA biosynthesis. The ultimate goal of the rGS-Glycerate pathway is to be constructed into plant systems, especially in C3 plants, in order to increase carbon fixation efficiency and further improve the production of the acetyl-CoA derived compounds in seeds.

The rGS-Serine cycle exhibits a broad capacity to assimilate various C1 compounds, which can simultaneously assimilate one inorganic carbon (CO₂ equivalents) and one reduced C1 carbon (formic acid or its upstream C1 compounds) to produce an acetyl-CoA. We demonstrated the feasibility of the cycle by complementation of growth and isotope labeling in E. coli. The rGS-Serine cycle opens the possibility for co-utilization of various C1 compounds as raw materials to synthesize a variety of products that E. coli has been engineered to produce. In the future, the rGS-Serine cycle can be developed for methane/CO₂ co-utilization to digest waste resources to produce useful products.

Teaching Interests:

My teaching interests are inseparably conjoined with my research ambitions. I strongly believe that research and teaching should always collaborate closely and that these activities complement each other in a researcher’s career. An intrinsic objective of any research activity is to gain a deep and clear understanding of the underlying principles. Teaching requires the arrangement of one’s knowledge on a subject in a clear and well-organized structure. Learning how to teach a subject well is the first step in becoming well-versed in that area. Furthermore, in my experience, teaching is not a one-way process of imparting knowledge; even with a less skilled group of students, the teacher undergoes a continuous process of learning new ideas and perspectives over the same body of knowledge. I will emphasize that I am highly motivated to impart my skills and knowledge to a wider audience in order to obtain a high level of self-satisfaction and achievement.