title: Engineering Enzymes and Pathways for Alternative CO2 Fixation and Glyoxylate Assimilation
title: Entwicklung von Enzymen und Stoffwechselwegen zur Alternativen Assimilation von CO2 und Glyoxylat
titleType: AlternativeTitle
Creators: name: Pfister, Pascal
Publisher: name: Philipps-Universität Marburg
Year: 2023
Subjects: Life sciences
Biowissenschaften, Biologie

description: Natural CO2 fixation is mainly associated with the Calvin-Benson-Bassham (CBB) cycle found in many photoautotrophic organisms, e.g. cyanobacteria. The CBB cycle as well as its key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) evolved in a atmosphere, that contained mainly CO2 and merely any O2. With emerging oxygenic photosynthesis and the oxygenation of the atmosphere, RuBisCO became increasingly inefficient. Its inefficiency to discriminate between both substrates, CO2 and O2, led to the evolution of carbon concentrating mechanisms (CCMs) and photorespiration. The latter is a metabolic route to remove the toxic side product of the oxygenase reaction, 2-phosphoglycolate (2PG) and recycle it into useable metabolites. During canonical photorespiration, at least one molecule of CO2 would be released per two molecules of 2PG, reducing on biomass production at a notable margin. Among a variety of different approaches to mitigate this problem, examples for two of them will be discussed in this thesis. Synthetic photorespiration will be adressed via two chapters on the nature-inspired 3-hydroxypropionate (3OHP) bypass. Synthetic CO2 fixiation will be features in one chapter about substrate selectivity in the new-tonature crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle. Photosynthetic organisms not always completely recycle photorespiratory 2PG, but also dephosphorylate and excrete glyoxylate to the surrounding medium. Other bacteria, like the thermophile Chloroflexus aurantiacus can feed on these acids and evolved a pathway, the 3OHP bi-cycle to metabolize them without the loss of CO2. This inspired a synthetic photorespiration pathway, the 3OHP bypass. The first attempt to introduce this pathway into the cyanobacterium Synechococcus elongatus were performed by Shih et al. Chapter 3 features the continued efforts to improve the 3OHP bypass in S. elongatus. A improved selection scheme, based on a carboxysome knockout strain and the pathway based detoxification of propionate were utilized to evolve a part of the 3OHP bypass in a turbidostat setup. The high CO2 requiring strain improved its tolerance from 0.5% to 0.2% within 125 days. Among the 3OHP bi-cycle enzymes are some catalysts with unique properties, like the intramolecular CoA transferase, mesaconyl-C1-C4-CoA CoA transferase (Mct). Chapter 4 is dedicated to a structural analysis on why this enzyme can be exclusively intramolecular. It has a narrow active site, that allows the CoA moiety of mesaconylCoA to blocks external acids from entering. A protein structure with trapped intermediates and kinetic analysis with external acids support this claim. Additionally we investigated a promiscuous succinic semialdehyde dehydrogenase (SucD) that is featured in synthetic CO2 fixation pathways, as described in chapter 2. SucD from Clostridium kluyveri is promiscuous to other CoA esters and especially active with mesaconyl-C1-CoA, another intermediate of the CETCH cycle. This side reaction will slowly drain mesaconyl-CoA from the pool of intermediates and lead to the accumulation of mesaconic semialdehyde. The specificity was addressed by solving the crystal structure of CkSucD and closing the active site by the substitution of an active site lysin to arginine. The mutation decreased site activity from 16% to 2%, but the overall efficiency decreased. In another SucD from Clostridium difficile, the same mutation had a comparable effect, changing the sidereaction from 12% to 2%, while conserving the overall efficiency. The designed enzyme is a wortwhile replacement for future iterations of the CETCH cycle.