A highly efficient Cre-based Clostridial workflow for genomic
integration and expression of large biosynthetic pathways
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.