Pathways to sustainability: a quantitative comparison of aerobic and anaerobic C1 bioconversion routes
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Gasparrini, William↗
Engineering topics
Woolston, Benjamin M. (ORCID:0000000265702236)
Publications and source records attributed to Woolston, Benjamin M. (ORCID:0000000265702236).
Deletion of biofilm synthesis in Eubacterium limosum ATCC 8486 improves handling and transformation efficiency
Abstract 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. Deletion of a single genomic region encoding homologues for epsABC, ptkA, and tmkA resulted in a strain incapable of producing EPS. This strain is significantly easier to handle by pipetting and centrifugation, and retains important wild-type phenotypes including the ability to grow on methanol and carbon dioxide and limited oxygen tolerance. Additionally, this strain is also more genetically tractable with a 2-fold increase in transformation efficiency compared to the highest previous reports. This work advances a simple, rapid protocol for gene knockouts in E. limosum using only the native homologous recombination machinery. These results will hasten the development of this organism as a workhorse for valorization of single carbon substrates, as well as facilitate exploration of its role in the human gut microbiota.
Sanford, Patrick A.↗
Expanding the genetic engineering toolbox for the metabolically flexible acetogen Eubacterium limosum
Abstract 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. To capitalize on the natural advantages of this organism, here we extended its limited engineering toolbox. We evaluated the copy number of three common plasmid origins of replication and devised a method of controlling copy number and heterologous gene expression level by modulating antibiotic concentration. We further quantitatively assessed the strength and regulatory tightness of a panel of promoters, developing a series of well-characterized vectors for gene expression at varying levels. In addition, we developed a black/white colorimetric genetic reporter assay and leveraged the high oxygen tolerance of E. limosum to develop a simple and rapid transformation protocol that enables benchtop transformation. Finally, we developed two new antibiotic selection markers—doubling the number available for this organism. These developments will enable enhanced metabolic engineering and synthetic biology work with E. limosum.
59 BASIC BIOLOGICAL SCIENCES↗