Engineering cellular dephosphorylation boosts (+)-borneol production in yeast

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• (+)-Borneol is a valuable compound from the Chinese Pharmacopoeia, facing supply challenges from plant extraction.
• Microbial biosynthesis offers a sustainable alternative, but low production yields hinder its viability.
• Optimizing the mevalonate pathway alone has not overcome the low yield of (+)-borneol.

Purpose of the Study:

• To address the low production of (+)-borneol via microbial biosynthesis.
• To investigate and overcome the limitations in the dephosphorylation of bornyl diphosphate (BPP).
• To enhance (+)-borneol synthesis by engineering dephosphorylation and balancing cellular metabolism.

Main Methods:

• Systematic identification and characterization of endogenous and heterologous phosphatases.
• Engineering the dephosphorylation of BPP to minimize side reactions and competition.
• Balancing cellular dephosphorylation metabolism, particularly lipid metabolism.
• Optimizing the mevalonate (MVA) pathway in Saccharomyces cerevisiae.
• Fed-batch fermentation in shake flasks for production assessment.

Main Results:

• Identified two endogenous and seven heterologous phosphatases, increasing (+)-borneol production by up to 152%.
• Engineered BPP dephosphorylation and optimized the MVA pathway, resulting in a 33.8-fold increase in (+)-borneol production.
• Achieved a record production of 753 mg/L of (+)-borneol via fed-batch fermentation.

Conclusions:

• Rewiring dephosphorylation metabolism is crucial for high-level microbial production of (+)-borneol.
• Balancing cellular dephosphorylation metabolism is essential for efficient terpenoid biosynthesis.
• This study provides a foundation for the sustainable and efficient biosynthesis of (+)-borneol and other terpenoids.