By Liz Jenkinson and  Mandy Harding

In 1915 biochemist Chaim Weizmann patented a new technology for the production of solvents using microbial fermentation. His discoveries exploited the solventogenic capabilities of Clostridium acetobutylicum to produce acetone, butanol and ethanol (ABE) from starch-based feedstocks.  During the wars, production of acetone by this method was crucial in the manufacture of munitions and over the next few years ABE process facilities were established globally. Alas, by the 1980’s, the last ABE plants closed their doors (with the exception of a handful in China which clung on until the 2000’s) as this fermentation production method, primarily for butanol and acetone, was unable to compete with petrochemically derived counterparts. Fast-forward to 2016, and after 10 years of research and development and 18 months of plant engineering, the team at Green Biologics celebrated as the first tankers of bio-butanol left Central Minnesota Renewables (CMR), our company’s first ABE plant and the first to operate in the US since the 1940’s.

green biologics

So what’s changed? Firstly, technology. At Green Biologics we have access to hundreds of clostridial microbes characterised in terms of genetic information and feedstock utilisation that work in conjunction with our Advanced Fermentation Process (AFP™). Secondly, the marketplace. Acetone and butanol produced by microbial fermentation contain fewer contaminants than petro-derived equivalents giving us performance advantages in high value markets. And thirdly, there is much greater awareness and a desire to seek alternative, non-fossil fuel sources for everyday chemicals. In combination, this has enabled us to produce acetone and butanol at scale competitively.

The ultimate aim of Green Biologics is to become a renewable chemicals company, which means having the capability to diversify our product portfolio. Our experience with clostridia tells us that they are robust, industrial microbes with a unique anaerobic biochemistry, and they have the capacity to use a wide range of sustainable and/or renewable feedstocks. This makes them ideal platform strains for engineering products beyond butanol. However, this is no easy task. The clostridial genetic toolkit lacks the decades of research that have gone into model strains such as E. coli and until fairly recently, engineering any changes into the genome required stamina and an impressive dedication to the cause. All that changed with the emergence of CRISPR/Cas technology.

At Green Biologics we have developed and patented CLEAVE™, which is based on native clostridial systems rather than applying non-endogenous CRISPR/Cas9. The success of our technology has enabled us to generate strains engineered for enhanced performance in a matter of weeks rather than months (or occasionally years). We have succeeded in making strains that carry a single base pair change, all the way through to complex deletions, and have demonstrated the power of CLEAVE by introducing a new pathway to generate a strain capable of making a non-native high value product. The advantages of CLEAVE are 1) its specificity, the desired modification is the only modification we get, and 2) our ability to layer genetic changes whilst removing plasmid DNA results in markerless, process ready strains. Currently, our industrial ABE process uses lab-evolved clostridial microbes optimised for their performance in the AFP™. In the future, new chemical processes will use CLEAVE engineered strains carrying targeted genetic changes.