Abstract
Introduction
Limonene is a monoterpene with diverse applications in food, medicine, fuel, and material science. Recently, engineered microbes have been used to biosynthesize target biochemicals such as limonene.
Objective
Metabolic engineering has shown that factors such as feedback inhibition, enzyme activity or abundance may contribute to the loss of target biochemicals. Incorporating a hypothesis driven experimental approach can help to streamline the process of improving target yield.
Method
In this work, time-series intracellular metabolomics data from Escherichia coli cultures of a wild-type strain engineered to overproduce limonene (EcoCTs3) was collected, where we hypothesized having more carbon flux towards the engineered mevalonate (MEV) pathway would increase limonene yield. Based on the topology of the metabolic network, the pathways involved in mixed fermentation were possibly causing carbon flux loss from the MEV pathway. To prove this, knockout strains of lactate dehydrogenase(LDH) and aldehyde dehydrogenase-alcohol dehydrogenase (ALDH-ADH) were created.
Results
The knockout strains showed 18 to 20 folds more intracellular mevalonate accumulation over time compared to the EcoCTs3 strain, thus indicating greater carbon flux directed towards the MEV pathway thereby increasing limonene yield by 8 to 9 folds.
Conclusion
Ensuring high intracellular mevalonate concentration is therefore a good strategy to enhance limonene yield and other target compounds using the MEV pathway. Once high intracellular mevalonate concentration has been achieved, the limonene producing strain can then be further modified through other strategies such as enzyme and protein engineering to ensure better conversion of mevalonate to downstream metabolites to produce the target product limonene.