Co-Production of Platform Chemicals 1, 3 Propane Diol and 3-Hydroxy Propionic Acid from Waste Glycerol By Clostridium Pasteurianum

Clostridium are a family of obligate anaerobic and spore forming bacteria found in diverse environments with roles in clinical pathogenesis but also known for their fermentative abilities. In the early 20^th century, fermentation of starch into acetone, butanol and ethanol (later known as ABE fermentation) by Clostridium acetobutylicum was one of the first commercial applications of a microbial biocatalyst for production of industrial chemicals.

Interest in Clostridium pasteurianum has been reignited in recent years as it is one of the few species of Clostridium which is able to ferment glycerol – a waste by-product of the biodiesel industry – into industrially relevant chemicals n-butanol and 1,3-propanediol (1,3 PDO). 3-Hydroxypropionic acid (3HP) is another industrially relevant chemical for which biological production routes have been pursued as it can easily be dehydrated into acrylic acid. Acrylic acid and 1,3 PDO have forecasted global market values in excess of $13 and $0.5 billion by 2020 respectively.

Using synthetic biology and rational metabolic engineering approaches, we describe the genetic manipulation of C. pasteurianum for the co-production of 1,3 PDO and 3HP from waste glycerol. Aspects of glycerol metabolism have been investigated towards gaining a better understanding of variable gene expression of C. pasteurianum during glycerol fermentation. Heterologous expression of enzymes required for production of 3HP are introduced. Both Co-A dependent and Co-A independent pathways for 3HP production from glycerol are explored. Genes involved in production of unwanted by-products are removed towards enhancing product yield. Effects of genetic modifications on metabolite equilibriums and redox balance. Strain engineering strategies by CRISPR and allelic exchange are employed. Generic lessons learned with broad range applicability for production of other bio commodities using different microbial platforms are discussed.