Systematic analyses and rational engineering of fast CO2 fixation pathways in living cells

Introduction

Biological CO2 fixation is the primary process responsible for biomass and food production and a key player in the atmospheric CO2 balance. Almost all biological CO2 fixation is carried out by a single pathway: the Calvin cycle. Despite the dominance of this pathway in nature, it seems relatively inefficient due to high energy costs and poor enzyme kinetics.

Exploration of Synthetic Pathways

An exciting option to improve this efficiency is the exploration of potentially more efficient synthetic CO2 pathways. However, a key challenge in identifying promising synthetic CO2 fixation pathways is the limited availability of kinetic data on relevant enzymes.

Limitations of Current Data

In addition, kinetic data are usually measured in vitro and hence not always representative of the performance in living cells.

Methodology

In FASTFIX, I will develop and use a novel method to quantify the kinetics of enzymes within living cells. I will do this by making the growth rate of engineered Escherichia coli cells directly dependent on the kinetics and levels of the enzymes of interest.

Measurement Techniques

By measuring the growth rates and enzyme levels through absolute quantitative proteomics, the in vivo kinetics of the enzymes can be determined. This approach will be used to generate a complete overview of the kinetics of enzymes involved in promising synthetic CO2 fixation pathways.

Systematic Analysis

This will enable an unprecedented systematic analysis of the kinetics of synthetic CO2 fixation pathways. Based on this analysis, I will select the most promising pathway design.

Engineering and Demonstration

Enabled by the in vivo kinetics data, I will then employ a novel forward-engineering method to effectively engineer and demonstrate the performance of the full pathway in E. coli.

Expected Outcomes

The realization of a fast, energy-efficient synthetic CO2 fixation pathway in living cells will be a major milestone. The anticipated results will be promising for efficient CO2-based biotechnological production and, in the longer term, may increase agricultural yields and help to more efficiently mitigate humanity’s CO2 footprint.