(161a) Metabolic Flux Redistribution for Efficient Anaerobic Production of 1,2-Propanediol and 1-Propanol in Escherichia Coli

Metabolic flux redistribution for efficient anaerobic production of 1,2-Propanediol and 1-Propanol in Escherichia coli

Rachit Jain^a and Yajun Yan^b

^aCollege of Engineering, Driftmier Engineering Center, University of Georgia, Athens, GA 30602, USA

Email: rachit@uga.edu

^bBiochemical Engineering Program, College of Engineering, Driftmier Engineering Center, University of Georgia, Athens, GA 30602, USA

Email: yajunyan@uga.edu

Abstract

With the industrial revolution of the last century and years of rapid globalization a surge in energy production, consumption and demand has affected all countries. With most of the world relying on oil and coal as the primary source of energy, its depletion is at hand. The rapid consumption of oil for fuel and chemicals is leading to crisis in two directions – environmental and economic. The release of green house gas carbon dioxide is primarily due to oil derived fuels and coal based energy generation. At this hour the need is to gradually reduce the reliance on oil based chemicals and fuels. Over the years metabolic engineering and synthetic biology have enabled chemical and biological engineers alike to develop and construct novel metabolic pathways. Truly, the production of “green chemicals” holds paramount importance in reducing the number of chemicals manufactured from oil. In establishing biological processes for their manufacture the dependence on oil can be greatly reduced and hence the expenses on importing oil into the country can be decreased.

The manufacture of biofuels and green chemicals has been proposed by researches across the globe by constructing novel synthetic routes. Although researchers report the achievement of either high titer or high yield, only a handful of processes exist with the capability to achieve both from a single strain. In this work we first engineer an efficient method for the production of high value commodity- 1,2-propanediol and further dehydrated it to 1-propanol. To do this we hypothesized a novel fermentative approach to produce 1-propanol by dragging the carbon flux from the native glycolytic pathway in Escherichia coli. Using bio-prospecting approaches we evaluated eight different methylglyoxal synthases, three alcohol dehydrogenases and three dehydratases that are involved in the pathway via enzyme assays.

After the identification of the minimal set of enzymes we (i) constructed a synthetic pathway via 3 steps leading to the production of 1,2-propanediol and enhancing it from 0.28g/L to 1.52g/L in shake flask studies (ii) redirected the native carbon flux into our synthetic pathway and reduced by-products, improving the yield to about 50% of theoretical maximum (iii) demonstrated the manipulation of cellular energetics (NADH availability) to boost the titer to 2.14g/L in wild type E. coli (iv) developed cell adaptation strategies to improve the growth, resulting in 1,2-propanediol titer to biomass ratio of >4:1 after 48 hours and a titer of 2.7g/L (representing a yield >90% of theoretical maximum) after 72 hours from our engineered strains. This work represents the highest titer and yield of 1,2-propanediol production achieved in shake flask studies from Escherichia colitill date. This process holds potential for future scale up by achieving high titer and yield simultaneously from a single strain.

After the establishment of an efficient 1,2-propanediol pathway, we identified the optimal dehydratase for the production of 1-propanol and achieved its production at 0.11g/L (conversion efficiency of 11%). After establishing this novel metabolic route we proceeded to address the conversion efficiency of 1,2-propanediol to 1-propanol. We constructed a fusion diol dehydratase protein in order to regulate the expression level of individual subunits. By doing so the titer further increased to 0.62g/L. By studying the regulation of upstream pathway (1,2-propanediol) and downstream pathway (1-propanol) via operon organization and plasmid copy number we achieved the improvement in tweaking the cell to improve the conversion efficiency to about 50%.

This work represents metabolic and synthetic biology approaches and demonstrates solutions for the production of bulk chemicals like 1,2-propanediol by developing processes which achieve high titer and high yield simultaneously. This work also highlights protein based engineering and regulation of expression levels of proteins to strike a balance between upstream and downstream pathways to improve production of chemicals (1-propanol) via novel metabolic routes.