Synergetic Engineering of Multiple Pathways for De Novo (2S)-Naringenin Biosynthesis in Saccharomyces cerevisiae

(2S)-Naringenin is a high-value flavonoid precursor with different biological properties. However, current metabolic engineering strategies do not fully use intracellular carbon sources for the synthesis of (2S)-naringenin. Inadequate precursors and the loss of carbon metabolic flux severely limit the microbial heterologous synthesis of (2S)-naringenin. In this work, a systems engineering strategy was employed for the high-level production of (2S)-naringenin. First, the synthetic pathways of shikimic acid and aromatic amino acids were optimized. Carbon flux was redirected into the (2S)-naringenin biosynthetic pathway through the concentrated utilization of aromatic amino acids and the introduction of a heterologous phosphoketolase pathway. Then, byproduct accumulation was reduced by replacing endogenous enoyl-CoA reductase with a plant homologous enzyme. Subsequently, overexpression of the endogenous malonyl-CoA synthesis pathway genes and introduction of the acetaldehyde dehydrogenase (acylating) pathway promote (2S)-naringenin production. The regulation of the subcellular organelle carbon trafficking system resulted in the production of (2S)-naringenin reaching 986.2 mg/L in shake flasks. Finally, the (2S)-naringenin titer reached 3420.6 mg/L after fermentation in a 5 L bioreactor, the highest titer ever reported. This study not only provides a possibility for the efficient bioproduction of flavonoids but also provides ideas for the biosynthesis of natural products through the centralized utilization of intracellular carbon sources.