Project
Increasing the Conservation of Energy by Microorganisms
Efficient microbial processes are crucial to producing bulk chemicals sustainably. However, many of these processes cannot compete with current unsustainable production methods due to low product yields. High product yields are typically achieved in fermentation processes, but microorganisms cannot always harvest sufficient energy from these processes to sustain maintenance and growth. Increasing energy conservation in microorganisms is key to developing efficient microbial production processes. In this project, we will implement different mechanisms in Escherichia coli to enhance metabolic energy conservation.
Background
Microbial cell factories are becoming an increasingly important strategy for the sustainable production of bulk chemicals and fuels. Currently, many of these processes are not efficient enough to compete with fossil-based production methods. High product yields are crucial for these processes to become economically competitive. Generally, fermentation processes give higher yields than aerobic, respiratory processes, as more electrons of the substrate end up in the final product. However, even though many substrate/product combinations have a negative Gibbs free energy (ΔG’), cells cannot always harvest sufficient energy for cell growth and maintenance. This limits the possibility of anaerobic growth. Therefore, aerobic respiration is used instead, which allows the cells to harvest more energy, but often comes at a severe cost for the product yield.
Project description
To overcome the need for aerobic respiration, we aim to introduce novel energy conservation mechanisms in our host organism Escherichia coli.This could increase ATP production, which will enable us to produce our products with a high yield during fermentation.
Approach
To increase the energy conservation in E. coli, we identified key metabolic conversions, with a sufficiently low ΔG’, from which E. coli currently does not harvest energy. We will specifically target the conversion of pyruvate to acetyl-CoA to produce additional ATP. This could be achieved by changing the cofactor specificity of the pyruvate dehydrogenase (PDH) to use NADP+ instead of NAD+. This will result in elevated NADPH and decreased NADH concentrations inside the cell. These elevated NADPH concentrations could then lead to the regeneration of NADP+ via the reverse action of a membrane-bound transhydrogenase. This complex will simultaneously pump protons out of the cell, generating a proton motive force (pmf), while transferring electrons to NAD+. This pmf can contribute to the production of ATP and could, in theory, increase energy conservation from glycolysis by 25%. As an alternative strategy, we aim to incorporate a PDH that uses ferredoxin as a cofactor. Reduced ferredoxin can be regenerated by a heterologous proton-translocating ferredoxin:NAD+ oxidoreductase Rnf. This complex also pumps protons out of the cell, to create a pmf from which ATP can be harvested. Also in this scenario, 25% more energy could be conserved from glycolysis.
Various molecular biology techniques will be used to generate these more energy-efficient strains, including Golden Gate cloning, recombineering, and CRISPR-Cas-guided engineering. In addition, the cofactor levels and ratios will be determined by using different in vivo biosensors, and analytical tools, such as HPLC, will elucidate what products are formed during fermentation.