Development and Analysis of Precursor Production Strains for Chemical Production

Microbes have been engineered to produce a variety of chemicals, including biofuels, commodity chemicals, and therapeutics. Production can be enhanced by connecting biosynthesis pathways to host metabolism and optimizing the pathway’s expression. However, another important step in engineering microbes to produce desired chemicals is to increase production of their chemical precursors. In this work, we evaluated what precursors are needed to synthesize a variety of chemicals, and then designed and engineered strains to produce one of these precursors—pyruvate.

We systematically evaluated what potential non-native products could be produced by Escherichia coli (using both native and heterologous pathways) and then evaluated their distance (in terms of metabolic reactions steps) from central metabolic precursors. Using a genome-scale metabolic model of E. coli and a set of potential heterologous reactions (from the KEGG database), ~1,800 non-native products could potentially be produced in E. coli using heterologous enzymes. Of these 284 have reported commercial applications. The set of 284 chemicals were subsequently analyzed to identify how many heterologous reactions were needed to enable their production and whether they were within five reaction steps of a central metabolic precursor. This analysis identified that of the six central metabolic precursors considered, pyruvate was the closest precursor to the most non-native valuable products.

Since pyruvate has industrial applications and is within 5 reactions steps of many valuable non-native products, we sought to develop a strain of E. coli that could produce pyruvate at high yields. A high-yield pyruvate producing strain has great potential for creating additional strains to produce a variety of other valuable chemicals. Guided by a genome-scale metabolic model of Escherichia coli, we identified different strategies for enhancing production of pyruvate from glucose. The targeted gene deletions minimize acetyl-CoA production, undesired product (acetate and lactate) formation, and NAD(P)H formation. We constructed a number of strains and five of them achieved yields of more than 0.9 g pyruvate per g substrate (94% theoretical yield) under aerobic conditions.

Further genetic modifications were made to evaluate whether additional products (derived from pyruvate) could also be made using these strains. To produce ethanol, pyruvate formate-lyase (PflB) was deleted, and pyruvate decarboxylase (Pdc) and alcohol dehydrogenase II (AdhB) from Zymomonas mobilis were over-expressed in the engineered pyruvate strains. These genetically modified strains fermented glucose to ethanol with up to yields of 0.43 g ethanol per g substrate (~84% of theoretical yield).

These results illustrate how computational models can be used to prioritize precursor-based strategies and identify genetic modifications to enhance precursor production. We also showed that precursor over-production strains can be further modified to produce other valuable products. Current efforts are focused on engineering the pyruvate production strains to produce other native and non-native products, as well as developing other precursor over-production strains.