Enhancing the flux capability of alternative isoleucine biosynthetic pathway in e coli for the conversion of citramalate to 2 oxobutanoate

Abstract

Pathway engineering for production of high demand products has always received considerable attention. One such important intermediate with robust demand in pharma and food industry is 2- oxobutanoate (2- OB). Being an active metabolic intermediate, 2- OB has been used majorly for the synthesizing three high demand, low value products namely, L-2-Aminobutyric acid (9 $/ kg), propionic acid (1 $/ kg) and isoleucine (5 $/ kg). Hence, there exists a dire need to produce 2- OB at very cheap price of around 3-4 $/ kg for the technology to be co mMercially viable. The production of the 2- OB occurs in majority of organisms using two routes- the normal pathway for isoleucine production which produces 2-OB via threonine and the alternative isoleucine pathway which produces the same via citramalate route. The alternative isoleucine biosynthesis route has been least explored with minimal product titres thereby reflecting the challenges associated in channelizing the citramalate flux to 2-OB and products downstream. Hence, the focus of the current study was to identify the challenges associated with 2-OB production using alternative isoleucine biosynthetic pathway and develop appropriate toolsets that would guide efficient production of 2- OB at a costing of 2-3 $/kg of product. E. coli strains have been metabolically engineered in order to channelize the glycolytic flux towards production of 82 g/L and 54 g/L citramalate suggesting that there are successful strategies for converting glucose to citramalate production and if a chassis is developed to completely pull this flux to 2- OB and products downstream, it would result in a very promising, metabolically engineered 2-OB producer. newline