Functional replacement of isoprenoid pathways in Rhodobacter sphaeroides

Area of Science:

• Synthetic biology
• Metabolic engineering
• Microbial biotechnology

Background:

• Metabolic engineering utilizes synthetic biology to introduce bypasses for enhanced cellular production.
• Endogenous metabolic pathways are often regulated by host mechanisms, limiting efficiency.
• Isoprenoid biosynthesis is crucial for many cellular functions and valuable compounds.

Purpose of the Study:

• To replace the essential 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in Rhodobacter sphaeroides with a heterologous mevalonate (MVA) pathway.
• To assess the functional replacement and its impact on isoprenoid (sesquiterpene) production.
• To determine if the heterologous pathway offers improved biosynthetic performance compared to the native pathway.

Main Methods:

• Genetic engineering of Rhodobacter sphaeroides to introduce the mevalonate (MVA) pathway.
• Disruption and replacement of the endogenous 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway.
• Cultivation of engineered strains with [4-¹³C]glucose and analysis of the reporter molecule amorpha-4,11-diene.

Main Results:

• Successful replacement of the essential MEP pathway with a heterologous MVA pathway in R. sphaeroides.
• The engineered strain exclusively utilizing the MVA pathway was functional for sesquiterpene production.
• Increased expression of MVA pathway enzymes led to higher sesquiterpene yields than the control strain.

Conclusions:

• Substitution of an essential endogenous pathway with a heterologous one can enhance microbial biosynthetic performance.
• The mevalonate (MVA) pathway serves as an effective orthogonal replacement for the MEP pathway in R. sphaeroides.
• This strategy holds potential for optimizing cell factories for the production of valuable compounds like sesquiterpenes.