Optimising solvent production in Clostridium saccharoperbutylacetonicum N1-4(HMT)

The ever-increasing population and resource demand are putting a stress upon the planet's

resources. This increased demand places an even greater need today for the exploration

of alternative, greener fuels that can aid in the alleviation of the traditionally used fossil fuels.

One such method is in the production of acetone, butanol and ethanol (ABE) by the bacteria

genus Clostridium spp. These gram-positive anaerobic bacteria were first characterised in

the late 19th centaury and have been used throughout the 20th and 21st centauries for their

solvent producing capability, most notably in the supply of weapons grade acetone during

the first world war. After falling out of favour in the last half of the 20th century due to

competition with cheaper and more readily available petrochemicals; interest in ABE

production via Clostridium spp. has been on the rise in recent years as the ABE

fermentation is investigated for its potential as a greener more renewable source of fuel

production. As interest in ABE fermentation has been on the rise in recent years, so too

has our understanding of the genus as a whole. Traditionally C. Acetobutylicum first

described by Chaim Weizmann in the early 20th centaury has been the industrial strain of

choice. However, as the overall understanding of the strains has improved other strains

have been explored for their industrial relevance. These are largely split into two

characterisations, autotrophs who are able to fix CO2 and CO, converting them acetyl-CoA

for solvent production and heterotrophs who are able to metabolise hexose sugars in

solvent production. The strain used in this study is Clostridium saccharoperbutylacetonicum

N1-4(HMT). Clostridium saccharoperbutylacetonicum N1-4(HMT) is a heterotrophic

Clostridium species first described by (Hongo and Ogata, 1969) .

Herein we have utilised CLEAVEÔ, this is a CRISPR/Cas system developed by Green

biologics ltd. CLEAVEÔ was used for the deletion of the gene gapN from the genome of

Clostridium saccharoperbutylacetonicum N1-4(HMT). GapN is a cytosolic nonphosphorylating

NADP-dependant GAPDH that catalyses the irreversible oxidation of

glyceraldehye-3-phospate (G3P) to 3-phospholycerate. Deletion of gapN causes a

reduction in acid production, an increased rate of solvent production to pre-toxic

concentrations, as well as an increase in ATP and ratio of NADH:NAD+. Additionally, the

deletion of gapN results in an increase in formic and lactic acid production that is believed

to be as a result of pyruvate accumulation in response to the earlier shift into

solventogenesis in gapN deletion strain.