Flavonoids have notable biological activities and have been widely used in the medicinal and chemical industries. However, single-copy integration of heterologous pathway genes limits the production of flavonoids. In this work, we designed and constructed single-step integration of multiple flavonoid (2S)-naringenin biosynthetic pathway genes in S. cerevisiae. The efficiency of the naringenin metabolic pathway gene integration into the rDNA site reached 93.7%. Subsequently, we used a high titer p-coumaric acid strain as a chassis, which eliminated feedback inhibition of tyrosine and downregulated the competitive pathway. The results indicated that increasing the supply of p-coumaric acid was effective for naringenin production. We additionally optimized the amount of donor DNA. The optimum strain produced 149.8 mg/L of (2S)-naringenin. The multi-copy integration of flavonoid pathway genes effectively improved (2S)-naringenin production in S. cerevisiae. We further analyzed the copy numbers and expression levels of essential genes (4CL and CHS) in the (2S)-naringenin metabolic pathway by qPCR. Higher copy numbers of the (2S)-naringenin metabolic pathway genes were associated with greater 4CL and CHS transcription, and the efficiency of naringenin production was higher. Therefore, multi-copy integration of genes in the (2S)-naringenin metabolic pathway was imperative in rewiring p-coumaric acid flux to enhance flavonoid production.

黄酮类化合物具有显着的生物活性，已广泛应用于医药和化工行业。然而，异源途径基因的单拷贝整合限制了类黄酮的产生。在这项工作中，我们设计并构建了酿酒酵母中多个类黄酮 ( 2S )-柚皮素生物合成途径基因的单步整合。柚皮素代谢途径基因整合到rDNA位点的效率达到93.7%。随后，我们使用高滴度对香豆酸菌株作为底盘，消除了酪氨酸的反馈抑制并下调了竞争途径。结果表明，增加p的供给-香豆酸对柚皮素的产生有效。我们还优化了供体 DNA 的数量。最佳菌株产生149.8 mg/L (2 S )-柚皮素。类黄酮途径基因的多拷贝整合有效地提高了酿酒酵母中( 2S )-柚皮素的产量。我们通过qPCR进一步分析了（2S）-柚皮素代谢途径中必需基因（4CL和CHS）的拷贝数和表达水平。(2 S )-柚皮素代谢途径基因的较高拷贝数与较大的4CL和CHS 相关转录，柚皮素生产效率更高。因此，( 2S )-柚皮素代谢途径中基因的多拷贝整合对于重新连接对香豆酸通量以提高类黄酮产量是必不可少的。