An efficient cre‐based workflow for genomic integration and expression of large biosynthetic pathways in Eubacterium limosum
Abstract 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. To overcome this problem, we harnessed chassis‐independent recombinase‐assisted genome engineering (CRAGE) to develop a workflow for facile integration of large gene clusters (>10 kb) into the human gut acetogen Eubacterium limosum . We then integrated a non‐ribosomal peptide synthetase gene cluster from the gut anaerobe Clostridium leptum , which previously produced no detectable product in traditional heterologous hosts. Chromosomal expression in E. limosum without further optimization led to production of phevalin at 2.4 mg/L. These results further expand the molecular toolkit for a highly tractable member of the Clostridia, paving the way for sophisticated pathway engineering efforts, and highlighting the potential of E. limosum as a Clostridial chassis for exploration of anaerobic natural product biosynthesis.
- Sponsoring Organization:
- USDOE
- OSTI ID:
- 2391068
- Journal Information:
- Biotechnology and Bioengineering, Journal Name: Biotechnology and Bioengineering Journal Issue: 10 Vol. 121; ISSN 0006-3592
- Publisher:
- Wiley Blackwell (John Wiley & Sons)Copyright Statement
- Country of Publication:
- United States
- Language:
- English
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