Multi-level flux optimization at the key regulatory node for enhanced production of naringenin using acetate in engineered Escherichia coli

Abstract

Abstract Background Microbial production of naringenin has received much attention due to its pharmaceutical applicability and potential as a key molecular scaffold for various flavonoids. In particular, the oxaloacetate (OAA) node is a key regulatory node for the naringenin biosynthesis from acetate, acting as a critical linkage that reroutes tricarboxylic acid (TCA) cycle intermediates via anaplerosis of the glyoxylate cycle to the naringenin biosynthetic pathway. In this context, to efficiently produce naringenin from acetate, it is crucial to precisely regulate the carbon flux of the OAA-PEP regulatory node through appropriate pckA expression control, as the excessive overexpression of pckAcan cause the extensive loss of OAA and metabolic imbalance. However, considering the crucial impact of pckAon naringenin biosynthesis, the conventional strategy of single-level gene expression is limited in its ability to cover the large and balanced solution space, which could result in suboptimal naringenin production. Results This study conducted multi-layer fine-tuning of pckA expression for the precise exploration of optimal naringenin production from acetate in the large and balanced solution space. Specifically, a combinatorial expression library was generated at both transcriptional and translational levels through promoters with different strengths and rationally designed 5′-UTR variants with discrete translation efficiency. Additionally, we identified the effect of multi-level regulation of pckA expression by validating the correlation between PCK activity and naringenin production. As a result, the flux-optimized strain demonstrated a significant increase in naringenin production, with a 49.8-fold increase (and a 73.8-fold increase in naringenin yield on acetate) compared to the unoptimized strain, producing 122.12 mg/L naringenin with 20.65 mg naringenin/g acetate, which is a comparable result against those from conventional substrates. Conclusions Collectively, we demonstrated the significance of multi-level expression control at the key regulatory node in the metabolic pathway, covering the large and balanced solution space for precise flux rebalancing. This study proposes a platform strain for the biosynthesis of various flavonoids that can be derived from naringenin using acetate.