Underground isoleucine biosynthesis pathways in E. coli

Area of Science:

• Microbial Metabolism
• Synthetic Biology
• Enzymology

Background:

• Enzyme promiscuity is a key driver for the evolution of novel metabolic functions.
• Underground metabolism refers to alternative biochemical pathways that can be activated under specific conditions.
• E. coli possesses a complex metabolic network that can be reprogrammed for new functions.

Purpose of the Study:

• To investigate E. coli's capacity to utilize underground metabolism for compensating essential gene deletions.
• To identify novel biosynthetic pathways for isoleucine production in E. coli.
• To explore the conditions under which these alternative pathways are activated.

Main Methods:

• Systematic deletion of threonine deaminases in E. coli to block isoleucine biosynthesis.
• Cultivation of the engineered strain under aerobic and anaerobic conditions.
• Metabolomic analysis to identify novel 2-ketobutyrate biosynthesis routes.

Main Results:

• Aerobic conditions activated a novel pathway for 2-ketobutyrate biosynthesis via cystathionine γ-synthase (MetB) promiscuous cleavage of O-succinyl-L-homoserine.
• Anaerobic conditions enabled 2-ketobutyrate biosynthesis from propionyl-CoA and formate, mediated by pyruvate formate-lyase.
• The anaerobic pathway contributed significantly to isoleucine production in wild-type E. coli when propionate was supplied.

Conclusions:

• E. coli can effectively harness underground metabolism to bypass essential pathway deletions, demonstrating significant metabolic plasticity.
• Promiscuous enzyme activities are crucial for the emergence of alternative metabolic routes, providing survival advantages.
• This study highlights the potential of metabolic engineering to exploit and engineer alternative pathways for biotechnological applications.