Dataset Information

Machine Learning-Supported Enzyme Engineering toward Improved CO₂-Fixation of Glycolyl-CoA Carboxylase.

ABSTRACT: Glycolyl-CoA carboxylase (GCC) is a new-to-nature enzyme that catalyzes the key reaction in the tartronyl-CoA (TaCo) pathway, a synthetic photorespiration bypass that was recently designed to improve photosynthetic CO₂ fixation. GCC was created from propionyl-CoA carboxylase (PCC) through five mutations. However, despite reaching activities of naturally evolved biotin-dependent carboxylases, the quintuple substitution variant GCC M5 still lags behind 4-fold in catalytic efficiency compared to its template PCC and suffers from futile ATP hydrolysis during CO₂ fixation. To further improve upon GCC M5, we developed a machine learning-supported workflow that reduces screening efforts for identifying improved enzymes. Using this workflow, we present two novel GCC variants with 2-fold increased carboxylation rate and 60% reduced energy demand, respectively, which are able to address kinetic and thermodynamic limitations of the TaCo pathway. Our work highlights the potential of combining machine learning and directed evolution strategies to reduce screening efforts in enzyme engineering.

SUBMITTER: Marchal DG

PROVIDER: S-EPMC10729300 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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Machine Learning-Supported Enzyme Engineering toward Improved CO<sub>2</sub>-Fixation of Glycolyl-CoA Carboxylase.

Marchal Daniel G DG Schulz Luca L Schuster Ingmar I Ivanovska Jelena J Paczia Nicole N Prinz Simone S Zarzycki Jan J Erb Tobias J TJ

ACS synthetic biology 20231120 12

Glycolyl-CoA carboxylase (GCC) is a new-to-nature enzyme that catalyzes the key reaction in the tartronyl-CoA (TaCo) pathway, a synthetic photorespiration bypass that was recently designed to improve photosynthetic CO<sub>2</sub> fixation. GCC was created from propionyl-CoA carboxylase (PCC) through five mutations. However, despite reaching activities of naturally evolved biotin-dependent carboxylases, the quintuple substitution variant GCC M5 still lags behind 4-fold in catalytic efficiency com ...[more]

PMID: 37983631

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Project description:The pathway of autotrophic CO2 fixation was studied in the phototrophic bacterium Chloroflexus aurantiacus and in the aerobic thermoacidophilic archaeon Metallosphaera sedula. In both organisms, none of the key enzymes of the reductive pentose phosphate cycle, the reductive citric acid cycle, and the reductive acetyl coenzyme A (acetyl-CoA) pathway were detectable. However, cells contained the biotin-dependent acetyl-CoA carboxylase and propionyl-CoA carboxylase as well as phosphoenolpyruvate carboxylase. The specific enzyme activities of the carboxylases were high enough to explain the autotrophic growth rate via the 3-hydroxypropionate cycle. Extracts catalyzed the CO2-, MgATP-, and NADPH-dependent conversion of acetyl-CoA to 3-hydroxypropionate via malonyl-CoA and the conversion of this intermediate to succinate via propionyl-CoA. The labelled intermediates were detected in vitro with either 14CO2 or [14C]acetyl-CoA as precursor. These reactions are part of the 3-hydroxypropionate cycle, the autotrophic pathway proposed for C. aurantiacus. The investigation was extended to the autotrophic archaea Sulfolobus metallicus and Acidianus infernus, which showed acetyl-CoA and propionyl-CoA carboxylase activities in extracts of autotrophically grown cells. Acetyl-CoA carboxylase activity is unexpected in archaea since they do not contain fatty acids in their membranes. These aerobic archaea, as well as C. aurantiacus, were screened for biotin-containing proteins by the avidin-peroxidase test. They contained large amounts of a small biotin-carrying protein, which is most likely part of the acetyl-CoA and propionyl-CoA carboxylases. Other archaea reported to use one of the other known autotrophic pathways lacked such small biotin-containing proteins. These findings suggest that the aerobic autotrophic archaea M. sedula, S. metallicus, and A. infernus use a yet-to-be-defined 3-hydroxypropionate cycle for their autotrophic growth. Acetyl-CoA carboxylase and propionyl-CoA carboxylase are proposed to be the main CO2 fixation enzymes, and phosphoenolpyruvate carboxylase may have an anaplerotic function. The results also provide further support for the occurrence of the 3-hydroxypropionate cycle in C. aurantiacus.

Project description:Autotrophic Crenarchaeota use two different cycles for carbon dioxide fixation. Members of the Sulfolobales use the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle, whereas Desulfurococcales and Thermoproteales use the dicarboxylate/4-hydroxybutyrate cycle. While these two cycles differ in the carboxylation reactions resulting in the conversion of acetyl-CoA + 2 CO2 to succinyl-CoA, they have a common regeneration part in which succinyl-CoA is reconverted to two acetyl-CoA molecules. This common part includes crotonyl-CoA conversion to acetoacetyl-CoA, which has unequivocally been shown in Ignicoccus hospitalis (Desulfurococcales) and Pyrobaculum neutrophilus (Thermoproteales) to be catalyzed by a bifunctional crotonase/3-hydroxybutyryl-CoA dehydrogenase. It is a fusion protein consisting of an enoyl-CoA hydratase and a dehydrogenase domain. As the homologous bifunctional protein is present in Sulfolobales as well, its common functioning in the conversion of crotonyl-CoA to acetoacetyl-CoA was proposed. Here we show that a model autotrophic member of Sulfolobales, Metallosphaera sedula, possesses in addition to the bifunctional protein (Msed_0399) several separate genes coding for crotonyl-CoA hydratase and (S)-3-hydroxybutyryl-CoA dehydrogenase. Their genes were previously shown to be transcribed under autotrophic and mixotrophic conditions. The dehydrogenase Msed_1423 (and not the bifunctional protein Msed_0399) appears to be the main enzyme catalyzing the (S)-3-hydroxybutyryl-CoA dehydrogenase reaction. Homologs of this dehydrogenase are the only (S)-3-hydroxybutyryl-CoA dehydrogenases present in all autotrophic Sulfolobales, strengthening this conclusion. Two uncharacterized crotonase homologs present in M. sedula genome (Msed_0336 and Msed_0384) were heterologously produced and characterized. Both proteins were highly efficient crotonyl-CoA hydratases and may contribute (or be responsible) for the corresponding reaction in the HP/HB cycle in vivo.

Dataset Information

Machine Learning-Supported Enzyme Engineering toward Improved CO₂-Fixation of Glycolyl-CoA Carboxylase.

Publications

Machine Learning-Supported Enzyme Engineering toward Improved CO<sub>2</sub>-Fixation of Glycolyl-CoA Carboxylase.

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Dataset Information

Machine Learning-Supported Enzyme Engineering toward Improved CO2-Fixation of Glycolyl-CoA Carboxylase.

Publications

Machine Learning-Supported Enzyme Engineering toward Improved CO<sub>2</sub>-Fixation of Glycolyl-CoA Carboxylase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Machine Learning-Supported Enzyme Engineering toward Improved CO₂-Fixation of Glycolyl-CoA Carboxylase.