Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - NOVCARBFIX (Analysis, Design and Experimental Evolution of Novel Carbon Fixation Pathways)

Teaser

Carbon fixation is a prerequisite for accumulating biomass and storing energy in most of the living world. As such, it supplies our food and dominates land and water usage by humanity. In agriculture, where water and nutrients are abundant, the rate of carbon fixation often...

Summary

Carbon fixation is a prerequisite for accumulating biomass and storing energy in most of the living world. As such, it supplies our food and dominates land and water usage by humanity. In agriculture, where water and nutrients are abundant, the rate of carbon fixation often limits growth rate. Therefore increasing the rate of carbon fixation is of global importance towards agricultural and energetic sustainability.
What are the limits on the possible rate of carbon fixation? Attempts to improve RuBisCO, the key enzyme in the Calvin-Benson cycle, have achieved only limited results. My lab focuses on trying to overcome this global challenge by building synthetic pathways for carbon fixation. We create a computational framework that designs and scores pathways and creates step-wise selection strategies for in-vivo experimental implementation. Our most promising synthetic carbon fixation pathways are found to utilize the highly effective carboxylating enzyme, PEP carboxylase. We experimentally test these pathways in the most genetically tractable context by constructing an E. coli strain that depends on atmospheric CO2 fixation. We will gradually incorporate the pathways, initially as essential reaction steps for biomass production, and finally as the sole carbon input of the cell.
As a stepping-stone towards this challenging goal, we will construct an autotrophic E. coli strain that uses the Calvin-Benson cycle. We systematically convert this synthetic biology grand challenge into a gradual evolutionary ladder with independently selectable steps. We recently achieved key steps in the ladder, such as semiautotrophic growth, serving as powerful proofs of concept.
The proposed research will advance our understanding of evolutionary plasticity. It also paves the way for a hybrid rational-design/experimental-evolution approach to revisit and advance the capacity of metabolism for agricultural productivity and renewable energy storage.

The ultimate goal of this research effort is to implement the most promising synthetic carbon fixation pathways in natural photosynthetic organisms. A photosynthetic organism carrying a synthetic carbon fixation pathway will probably not have a selective advantage in nature, but can hold great promise from a biotechnological point of view, increasing crop yields under controlled and optimized agricultural conditions. It could also serve for efficient production of valuable chemicals. In general, we envision that success in this proposal will build the interest and serve as a starting point for a concerted effort of the scientific community in realizing improvements through improved carbon fixation cycles.

Work performed

Our main work revolve around engineering carbon fixation in E. coli: from heterologous RuBisCO expression to the Calvin-Benson-Bassham cycle. Carbon fixation is the gateway of inorganic carbon into the biosphere. Our ability to engineer carbon fixation pathways in living organisms is expected to play a crucial role in the quest towards agricultural and energetic sustainability. Recent successes to introduce non-native carbon fixation pathways into heterotrophic hosts offer novel platforms for manipulating these pathways in genetically malleable organisms. In this direction, we focus on past efforts and future directions for engineering the dominant carbon fixation pathway in the biosphere, the Calvin-Benson cycle, into the well-known model organism Escherichia coli. We explore how central carbon metabolism of this heterotrophic bacterium can be manipulated to allow directed evolution of carbon fixing enzymes. Finally, we highlight future directions towards synthetic autotrophy.

Final results

We design and validate an evolutionary path from heterotrophy to autotrophy. We are happy to report the success of this approach. Upon mass spectrometry analysis with labeled CO2 we validated the full operation of the Calvin-Benson cycle in E. coli. This is a unique implementation of a fully functional carbon fixation pathway in a non-native autotroph. Deep sequencing and proteomics serve to systematically delineate this extraordinary evolutionary process recapitulated in the lab. Our strain will achieve more CO2 fixation of its biomass than anything achieved before.
We now investigate selection regimes that enable fully autotrophic E. coli, where all the biomass comes from fixed CO2 and energy is generated by oxidizing compounds that do not incorporate into biomass.