Engineering nonphosphorylative metabolism for the biosynthesis of sustainable chemicals

Abstract

Lignocellulosic biomass is one of the largest sources of organic carbon on Earth with the potential to replace fossil fuels for the production of transportation fuels and chemicals. The two biggest challenges facing biosynthesis is the limited natural metabolic capacity of microorganisms and the effective utilization of lignocellulosic biomass. To overcome the first obstacle, over the past several decades researchers have successfully expanded the natural metabolic pathways of microorganisms to allow biosynthesis of a wide array of compounds with applications as advanced biofuels, industrial chemicals, and pharmaceuticals. Most industrial fermentations convert glucose, the major sugar present in biomass, into a value added chemical but are unable to utilize pentose sugars which make up ~30% of a typical biomass feedstock. To improve the overall economics of fermentation process, it is important to ensure that all major sugars present in the feedstock are efficiently converted to target chemicals. This work addresses both these challenges by establishing a novel alterative pathway called nonphosphorylative pathway in Escherichia coli which enables the utilization of underutilized pentose sugars such as D-xylose and L-arabinose using fewer steps and with higher theoretical yields than conventional glycolysis and pentose phosphate pathways (PPP). This nonphosphorylative pathway can convert D-xylose and L-arabinose to 2-ketoglutarate (2-KG), an important TCA cycle intermediate, using less than 6 steps. A growth selection platform based on 2-ketoglutarate (2-KG) auxotrophy was designed in E. coli to confirm the functionality of nonphosphorylative metabolism in host organism. The growth selection platform was also used to mine nonphosphorylative gene clusters from other organisms with improved activity. The pathway was then expanded to allow biosynthesis of two commercially important chemicals, 1,4-butanediol (BDO) and γ-aminobutyric acid (GABA). To improve production titers and yields of the process, protein engineering was used to reduce by-product formation and metabolic engineering was used to eliminate competing pathways and increase carbon flux towards the target compound. Furthermore, to improve uptake of pentoses by E. coli, pentose transporter was overexpressed to allow better carbon utilization. This nonphosphorylative metabolism serves as an efficient platform for biosynthesis and can be extended to produce a variety of compounds derived from TCA cycle including, but not limited to, L-glutamate, mesaconate, 5-aminolevulinic acid, and glutaconate. While the nonphosphorylative pathway has been successfully used for conversion of simple pentose sugars into important chemicals like BDO and GABA, the breakdown of biomass into these pentoses is the bigger challenge. This work also briefly addresses this challenge by comparing different acid hydrolysis treatment conditions to breakdown arabinoxylans in wheat bran into sugars - glucose, D-xylose, and L-arabinose - which can then be used in fermentation via nonphosphorylative metabolism.