Genome-wide CRISPRi screen identifies enhanced autolithotrophic phenotypes in acetogenic bacterium Eubacterium limosum.

Acetogenic bacteria are a unique biocatalyst that highly promises to develop the sustainable bioconversion of carbon oxides (e.g., CO and CO2) into multicarbon biochemicals. Genotype-phenotype relationships are important for engineering their metabolic capability to enhance their biocatalytic performance; however, systemic investigation on the fitness contribution of individual gene has been limited. Here, we report genome-scale CRISPR interference screening using 41,939 guide RNAs designed from the E. limosum genome, one of the model acetogenic species, where all genes were targeted for transcriptional suppression. We investigated the fitness contributions of 96% of the total genes identified, revealing the gene fitness and essentiality for heterotrophic and autotrophic metabolisms. Our data show that the Wood-Ljungdahl pathway, membrane regeneration, membrane protein biosynthesis, and butyrate synthesis are essential for autotrophic acetogenesis in E. limosum. Furthermore, we discovered genes that are repression targets that unbiasedly increased autotrophic growth rates fourfold and acetoin production 1.5-fold compared to the wild-type strain under CO2-H2 conditions. These results provide insight for understanding acetogenic metabolism and genome engineering in acetogenic bacteria.

Engineering Acetogenic Bacteria for Efficient One-Carbon Utilization.

C1 gases, including carbon dioxide (CO2) and carbon monoxide (CO), are major contributors to climate crisis. Numerous studies have been conducted to fix and recycle C1 gases in order to solve this problem. Among them, the use of microorganisms as biocatalysts to convert C1 gases to value-added chemicals is a promising solution. Acetogenic bacteria (acetogens) have received attention as high-potential biocatalysts owing to their conserved Wood-Ljungdahl (WL) pathway, which fixes not only CO2 but also CO. Although some metabolites have been produced via C1 gas fermentation on an industrial scale, the conversion of C1 gases to produce various biochemicals by engineering acetogens has been limited. The energy limitation of acetogens is one of the challenges to overcome, as their metabolism operates at a thermodynamic limit, and the low solubility of gaseous substrates results in a limited supply of cellular energy. This review provides strategies for developing efficient platform strains for C1 gas conversion, focusing on engineering the WL pathway. Supplying liquid C1 substrates, which can be obtained from CO2, or electricity is introduced as a strategy to overcome the energy limitation. Future prospective approaches on engineering acetogens based on systems and synthetic biology approaches are also discussed.

Systems Biology on Acetogenic Bacteria for Utilizing C1 Feedstocks.

With a presence of the Wood-Ljungdahl pathway, acetogenic bacteria are capable of converting C1 feedstocks into biomass and various metabolites, receiving industrial interest in microbial production of biochemicals derived from C1 substrates. To understand C1 feedstock fermentation using acetogenic bacteria, most of the studies have focused on revealing their carbon assimilation and energy conservation systems. Despite the determination of the essential mechanisms, a fundamental understanding of acetogenic bacteria and the associated complex regulatory systems remains unclear and is needed for rational strain design. For this purpose, systems biology is a suitable approach for investigating genome, transcription, translation, regulation systems, and metabolic flux, providing a glimpse of the relationship between the genotype and phenotype of the organisms. This chapter will cover recent systems biology applications on acetogenic bacteria and discuss the cellular responses during C1 feedstock fermentation along with the regulatory systems that orchestrate cellular processes.

Development of highly characterized genetic bioparts for efficient gene expression in CO2-fixing Eubacterium limosum.

Acetogenic bacteria demonstrate industrial potential for utilizing carbon dioxide (CO2) for biochemical production using the Wood-Ljungdahl pathway. However, the metabolic engineering of acetogenic bacteria has been hampered by the limited number of available genetic bioparts for gene expression. Here, we integrated RNA sequencing, ribosome profiling, differential RNA sequencing, and RNA 3'-end sequencing results of Eubacterium limosum to establish genetic bioparts, such as promoters, 5' untranslated regions, and transcript terminators, to regulate transcriptional and translational expression of genes composing of biosynthetic pathways. In addition, a transformation method for the strain was developed to efficiently deliver the obtained genetic bioparts into cells, resulting in a transformation efficiency of 2.5 × 105 CFU/µg DNA. Using this method, the genetic bioparts were efficiently introduced, and their strengths were measured, which were then applied to optimize the heterologous expression of acetolactate synthase and acetolactate decarboxylase for non-native biochemical acetoin production. The strategy developed in this study is the first report on integrating multi-omics data for biopart development of CO2 or syngas utilizing acetogenic bacteria, which lays a foundation for the efficient production of biochemicals from CO2 or syngas as a carbon feedstock under autotrophic growth conditions.