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

Aug 3, 2026

Glycolyl-CoA carboxylase (GCC) is a new-to-nature enzyme that catalyzes a central step in the tartronyl-CoA (TaCo) synthetic photorespiration bypass designed to enhance photosynthetic CO2 fixation. GCC was created from propionyl-CoA carboxylase (PCC) via five mutations, yielding variant GCC M5 with high activity but reduced catalytic efficiency and futile ATP hydrolysis during CO2 fixation. A machine learning-supported workflow reduces screening burden and identifies improved GCC variants with higher carboxylation rates and lower energy demand, addressing kinetic and thermodynamic limits. The study demonstrates combining machine learning with directed evolution to streamline enzyme engineering.

LanguageEnglish

CategoryResearch & Report

FormatPDF

Pages10

Reading Time25 min

AccessFree to download

Abstract

Introduction

Photorespiration and CO2 fixation limits

TaCo pathway and key enzyme GCC

GCC creation from PCC via mutations

Results and discussion

Workflow for ML-supported directed evolution

Improvements in carboxylation and energy demand

Remaining efficiency gap and futile ATP hydrolysis

This article is licensed under CC-BY 4.0 [pubs.acs.org/synthbio](pubs.acs.org/synthbio) Letter Machine Learning-Supported Enzyme Engineering toward Improved CO2‑Fixation of Glycolyl-CoA Carboxylase Published as part of ACS Synthetic Biology virtual special issue “AI for Synthetic Biology”. Daniel G. Marchal, Luca Schulz, Ingmar Schuster, Jelena Ivanovska, Nicole Paczia, Simone Prinz, Jan Zarzycki, and Tobias J. Erb * Cite This: [https://doi.org/10.1021/acssynbio.3c00403](https://doi.org/10.1021/acssynbio.3c00403) Read Online Downloaded via MPI BIOPHYSIK on November 2 1, 2023 at 08:23:17 (UTC) . See [https://pubs.acs.org/sharingguidelines](https://pubs.acs.org/sharingguidelines) for options on how to legitimately share published articles. | ACCESS | Metrics & More | | Article Recommendations | | *sı Supporting Information | | --- | --- | --- | --- | --- | --- | 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. KEYWORDS: photorespiration, CO2 fixation, machine learning, directed evolution, enzyme engineering, glycolyl-CoA carboxylase ■ INTRODUCTION Photosynthesis plays a crucial role in the global carbon cycle by converting CO2 to organic compounds that feed virtually all life on Earth. However, one limiting factor in photosynthesis is the carbon conversion efficiency of the Calvin−Benson − Bassham cycle and in particular its key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Besides fixing CO2, Rubisco also captures O2 as a side reaction.1 This undesired reaction with O2 yields 2-phosphoglycolate, which needs to be recycled in a process called photorespiration, resulting in the loss of previously fixed carbon. To circumvent the loss of carbon during photorespiration, we recently developed the tartronyl-CoA (TaCo) pathway, a synthetic carboxylation module, which additionally fixes CO2 during photorespiration.2 Theoretical and experimental data show that the TaCo pathway indeed improves carbon yield during photosynthesis.2−4 The key enzyme in the TaCo pathway is a new-to-nature enzyme, glycolyl-CoA carboxylase (GCC), that we established through structure-guided approaches and large-scale screening of mutagenesis libraries of propionyl-CoA carboxylase (PCC) from Methylorubrum extorquens.2 PCC is a biotin-dependent carboxylase that consists of two subunits. The α-subunit comprises a biotin carboxylase domain and a biotin-carboxyl-carrier protein (BCCP) domain. The β-subunit comprises only a carboxyl transferase domain. The enzyme forms an α6β6 dodecameric complex, where the β-subunits arrange in a central core of two trimeric layers, while the α-subunits sit on top of the core and face outward.5 The biotin cofactor that is essential to catalysis is covalently linked to a lysine residue in the BCCP domain of the α-subunit and acts as a flexible arm that transfers the carboxyl group derived from HCO3 − between the active sites of the α - and

FAQs

What is GCC and why is it important for the TaCo pathway?

Glycolyl-CoA carboxylase (GCC) catalyzes the key carboxylation reaction in the tartronyl-CoA (TaCo) pathway, a synthetic bypass intended to improve photosynthetic CO2 fixation during photorespiration.

How was GCC M5 originally engineered from PCC?

GCC was constructed from propionyl-CoA carboxylase (PCC) through five mutations, producing a quintuple substitution variant called GCC M5.

What improvements does the machine learning-supported workflow enable?

The workflow reduces screening efforts and identifies new GCC variants with increased carboxylation rates and substantially reduced energy demand, helping address kinetic and thermodynamic limitations in the TaCo pathway.

Aug 3, 2026

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