Enhancing Isoprenol Phosphorylation Through Combined Engineering of Mevalonate Kinase

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Enzyme Engineering

Background:

• Efficient terpenoid precursor supply is crucial for metabolic engineering.
• Native terpenoid biosynthesis pathways are complex.
• The two-step isoprenol phosphorylation pathway offers a streamlined alternative but is limited by poor Kinase-1 efficiency.

Purpose of the Study:

• To identify and engineer a novel Kinase-1 enzyme for the two-step isoprenol phosphorylation pathway.
• To improve the catalytic efficiency of the enzyme toward isoprenol.
• To enhance the production of terpenoids in Escherichia coli.

Main Methods:

• Bioinformatic screening of potential Kinase-1 candidates.
• Functional evaluation using lycopene production in E. coli.
• Directed evolution to introduce beneficial mutations.
• Molecular docking to understand substrate accommodation.
• Structure-guided rational design to optimize enzyme variants.

Main Results:

• Mevalonate kinase (MvaK) from Kitasatospora griseola was identified as a promising Kinase-1 alternative.
• Two rounds of directed evolution improved lycopene production threefold.
• Rational design identified the E33A/H34L variant, achieving a 10-fold increase in lycopene titer (83.0 ± 1.1 mg/L).

Conclusions:

• MvaK can be effectively engineered as a Kinase-1 for terpenoid biosynthesis.
• Combining bioinformatic mining, directed evolution, and rational design is a powerful strategy for improving enzyme activity on non-native substrates.
• This approach significantly enhances terpenoid production in engineered microorganisms.