Abstract

Autotrophic CO2 fixation is a fundamental metabolic process that enables microorganisms to inhabit carbon-limited environments. Multiple pathways mediate this process, with variants distributed across diverse taxa and some genes shared among pathways, making their identification from genomic data challenging. Here, we present a curated resource comprising pathway-specific KEGG Orthology marker genes and a lightweight, rule-based tool AutoFixMark for predicting the presence of seven known CO2 fixation pathways in microbial genomes. To support marker gene identification and benchmarking, we compiled two reference datasets: (i) 347 manually curated chemolithoautotrophic genomes from 16 phyla, and (ii) a set of 15 well-characterized chemolithoautotrophic genomes used for defining pathway-specific marker genes. Using these marker genes, we developed AutoFixMark and evaluated its performance against two existing tools, METABOLIC and gapseq. Benchmarking results show that AutoFixMark achieves high precision and recall, particularly for pathways that are underrepresented in current tools. All curated gene sets, prediction rules, the AutoFixMark program, and benchmark datasets are publicly available, providing valuable resources for assessing autotrophic carbon fixation potential in microbial genomes.