CRISPR-Cas, a highly effective tool for genome editing in Clostridium saccharoperbutylacetonicum N1-4(HMT)

Abstract

The solventogenic clostridia have long been known for their ability to convert sugars from complex feedstocks into commercially important solvents. Although the acetone-butanol-ethanol process fell out of favour decades ago, renewed interest in sustainability and ‘green’ chemistry has re-established our appetite for reviving technologies such as these, albeit with 21st century improvements. As CRISPR-Cas genome editing tools are being developed and applied to the solventogenic clostridia, their industrial potential is growing. Through integration of new pathways, the beneficial traits and historical track record of clostridial fermentation can be exploited to generate a much wider range of industrially relevant products. Here we show the application of genome editing using the endogenous CRISPR-Cas mechanism of Clostridium saccharoperbutylacetonicum N1-4(HMT), to generate a deletion, SNP and to integrate new DNA into the genome. These technological advancements pave the way for application of clostridial species to the production of an array of products.

Using an Endogenous CRISPR-Cas System for Genome Editing in the Human Pathogen Clostridium difficile

“…As a priority choice, a point mutation design could be achieved by introducing changes at the first or second position of PAMs. Combining the changes within PAMs and the seed region could even increase the efficiency of editing, as reported for other endogenous CRISPR editing tools (21,25). We have previously shown that a nonfunctional PAM and mutation in the first position of protospacer within the seed region abolished or considerably impaired CRISPR interference (15).…”

“…Another advantage of this approach is that there is no need to heterologously express potentially toxic Cas proteins inside bacterial or archaeal cells. The genome editing approach based on an endogenous CRISPR-Cas system was successfully applied in several prokaryotic organisms using CRISPR-Cas subtype I-A and III-B or subtype I-B in archaea, Sulfolobus islandicus (21) and Haloarcula hispanica (22), respectively, and subtype I-B in several clostridial species, C. pasteurianum (23), C. tyrobutyricum (24), and C. saccharoperbutylacetonicum (25).…”

Engineering solventogenic clostridia for commercial production of bio‐chemicals

“…In this experiment, the butanol concentration in the media was gradually increased over a period of several weeks and glycerol stocks were taken at different time points throughout the experiment. These cultures were then tested in bottle screens (method in [10]), to assay for improved sugar uptake rates (Fig. 1).…”

“…This is a significant research area in the clostridial community with a number of examples being published, e.g. C. acetobutylicum [25], C. beijerinckii [25, 26], C. saccharoperbutylacetonicum [10, 27, 28], C. tyrobutyricum [29], C. pasteurianum [30], and C. ljungdahlii [31].…”

“…However applying them to industrial strain development has been challenging due to the transfer of antibiotic resistance markers into the genome (ClosTron), or the complexity of generating auxotrophs prior to integrating new DNA (ACE). By hi‐jacking the endogenous CRISPR‐Cas machinery we can overcome these limitations and demonstrate how a single method can be used to make very precise changes in the genome, from single nucleotide changes and in‐frame deletions through to integration of new material [10].…”