Metabolic addiction, an organism that is metabolically addicted with a compound to maintain its growth fitness, is an underexplored area in metabolic engineering. Microbes with heavily engineered pathways or genetic circuits tend to experience metabolic burden leading to degenerated or abortive production phenotype during long-term cultivation or scale-up. A promising solution to combat metabolic instability is to tie up the end-product with an intermediary metabolite that is essential to the growth of the producing host. Here we present a simple strategy to improve both metabolic stability and pathway yield by coupling chemical addiction with negative autoregulatory genetic circuits. Naringenin and lipids compete for the same precursor malonyl-CoA with inversed pathway yield in oleaginous yeast. Negative autoregulation of the lipogenic pathways, enabled by CRISPRi and fatty acid-inducible promoters, repartitions malonyl-CoA to favor flavonoid synthesis and increased naringenin production by 74.8%. With flavonoid-sensing transcriptional activator FdeR and yeast hybrid promoters to control leucine synthesis and cell grwoth fitness, this amino acid feedforward metabolic circuit confers a flavonoid addiction phenotype that selectively enrich the naringenin-producing pupulation in the leucine auxotrophic yeast. The engineered yeast persisted 90.9% of naringenin titer up to 324 generations. Cells without flavonoid addiction regained growth fitness but lost 94.5% of the naringenin titer after cell passage beyond 300 generations. Metabolic addiction and negative autoregulation may be generalized as basic tools to eliminate metabolic heterogeneity, improve strain stability and pathway yield in long-term and large-scale bioproduction.

代谢成瘾是一种通过化合物代谢成瘾以维持其生长适应性的有机体，是代谢工程中一个未充分探索的领域。具有高度工程化途径或遗传回路的微生物在长期培养或放大过程中往往会经历代谢负担，导致退化或流产的生产表型。对抗代谢不稳定性的一个有前景的解决方案是将最终产品与对生产宿主的生长至关重要的中间代谢物联系起来。在这里，我们提出了一种简单的策略，通过将化学成瘾与负自动调节遗传电路相结合来提高代谢稳定性和途径产量。柚皮素和脂质在产油酵母中以反向途径产量竞争相同的前体丙二酰辅酶 A。脂肪生成途径的负自动调节，在 CRISPRi 和脂肪酸诱导型启动子的支持下，丙二酰辅酶 A 重新分配以促进黄酮类化合物的合成，并将柚皮素的产量提高 74.8%。通过类黄酮感应转录激活剂 FdeR 和酵母杂交启动子来控制亮氨酸合成和细胞生长适应性，这种氨基酸前馈代谢回路赋予了类黄酮成瘾表型，可选择性地丰富亮氨酸营养缺陷型酵母中产生柚皮素的化蛹。工程酵母可以持续 90.9% 的柚皮素滴度长达 324 代。没有类黄酮成瘾的细胞恢复了生长适应性，但在细胞传代超过 300 代后失去了 94.5% 的柚皮素滴度。代谢成瘾和负性自动调节可以概括为消除代谢异质性的基本工具，