Development of a Recombineering System for the Acetogen with Cas9 Counterselection for Markerless Genome Engineering.

is a Clostridial acetogen that efficiently utilizes a wide range of single-carbon substrates and contributes to metabolism of health-associated compounds in the human gut microbiota. These traits have led to interest in developing it as a platform for sustainable CO-based biofuel production to combat carbon emissions, and for exploring the importance of the microbiota in human health. However, synthetic biology and metabolic engineering in have been hindered by the inability to rapidly make precise genomic modifications.

An efficient cre-based workflow for genomic integration and expression of large biosynthetic pathways in Eubacterium limosum.

Acetogenic Clostridia are obligate anaerobes that have emerged as promising microbes for the renewable production of biochemicals owing to their ability to efficiently metabolize sustainable single-carbon feedstocks. Additionally, Clostridia are increasingly recognized for their biosynthetic potential, with recent discoveries of diverse secondary metabolites ranging from antibiotics to pigments to modulators of the human gut microbiota. Lack of efficient methods for genomic integration and expression of large heterologous DNA constructs remains a major challenge in studying biosynthesis in Clostridia and using them for metabolic engineering applications.

Deletion of biofilm synthesis in Eubacterium limosum ATCC 8486 improves handling and transformation efficiency.

Eubacterium limosum is an acetogenic bacterium of potential industrial relevance for its ability to efficiently metabolize a range of single carbon compounds. However, extracellular polymeric substance (EPS) produced by the type strain ATCC 8486 is a serious impediment to bioprocessing and genetic engineering. To remove these barriers, here we bioinformatically identified genes involved in EPS biosynthesis, and targeted several of the most promising candidates for inactivation, using a homologous recombination-based approach.

Expanding the genetic engineering toolbox for the metabolically flexible acetogen Eubacterium limosum.

Acetogenic bacteria are an increasingly popular choice for producing fuels and chemicals from single carbon (C1) substrates. Eubacterium limosum is a promising acetogen with several native advantages, including the ability to catabolize a wide repertoire of C1 feedstocks and the ability to grow well on agar plates. However, despite its promise as a strain for synthetic biology and metabolic engineering, there are insufficient engineering tools and molecular biology knowledge to leverage its native strengths for these applications.

Synthetic or natural? Metabolic engineering for assimilation and valorization of methanol.

Single carbon (C1) substrates such as methanol are gaining increasing attention as cost-effective and environmentally friendly microbial feedstocks. Recent impressive metabolic engineering efforts to import C1 catabolic pathways into the non-methylotrophic bacterium Escherichia coli have led to synthetic strains growing on methanol as the sole carbon source. However, the growth rate and product yield in these strains remain inferior to native methylotrophs.