Streamlined identification of strain engineering targets for bioprocess improvement using metabolic pathway enrichment analysis

Abstract

Metabolomics is a powerful tool for the identification of genetic targets for bioprocess optimisation. However, in most cases, only the biosynthetic pathway directed to product formation is analysed, limiting the identification of these targets. Some studies have used untargeted metabolomics, allowing a more unbiased approach, but data interpretation using multivariate analysis is usually not straightforward and requires time and effort. Here we show, for the first time, the application of metabolic pathway enrichment analysis using untargeted and targeted metabolomics data to identify genetic targets for bioprocess improvement in a more streamlined way. The analysis of an Escherichia coli succinate production bioprocess with this methodology revealed three significantly modulated pathways during the product formation phase: the pentose phosphate pathway, pantothenate and CoA biosynthesis and ascorbate and aldarate metabolism. From these, the two former pathways are consistent with previous efforts to improve succinate production in Escherichia coli. Furthermore, to the best of our knowledge, ascorbate and aldarate metabolism is a newly identified target that has so far never been explored for improving succinate production in this microorganism. This methodology therefore represents a powerful tool for the streamlined identification of strain engineering targets that can accelerate bioprocess optimisation.

On‐line targeted metabolomics for real‐time monitoring of relevant compounds in fermentation processes

“…The three aromatic amino acids– l ‐phenylalanine, l ‐tryptophan and l ‐tyrosine–have a common precursor, chorismate, from the shikimate pathway, which in turn requires erythrose‐4P from the PPP. Interestingly, the RTMet data also shows that the MS signals of erythrose‐4P and other sugar phosphates involved in the PPP rapidly increase soon after the beginning of the succinate production phase, such as 6‐phosphogluconate and fructose‐6P, indicating a metabolic shift in glucose consumption from being predominantly channeled down glycolysis in the aerobic batch phase, to a higher consumption via the PPP in the absence of oxygen, as it has been observed in previous LC‐MS experiments (Cortada‐Garcia et al, 2023 ). The metabolic reason explaining this shift could be an increased demand for reducing power during succinate production, which can be supported with NADPH formation via the oxidative PPP.…”