背景:白屈菜红碱是一种重要的生物碱,用于农业和医药。然而,它的结构复杂性和自然界中的低丰度阻碍了大量化学合成或从植物中提取。这里,我们使用遗传重编程重建并优化了酿酒酵母中白屈菜红碱的完整生物合成途径。
结果:能够生产白屈菜红碱的第一代菌株Z4是通过七种植物来源的酶的异源表达获得的(McoBBE,TfSMT,AmTDC,EcTNMT,PSMSH,EcP6H,和PsCPR)在酿酒酵母W303-1A中的表达。当将该菌株在补充了100µM(S)-鱼网碱的合成完全(SC)培养基中培养10天时,它产生高达0.34µg/L白屈菜红碱。此外,通过整合多拷贝限速基因(TfSMT,AmTDC,EcTNMT,PSMSH,EcP6H,PsCPR,INO2和AtATR1),剪裁血红素和NADPH工程,并通过异源表达MtABCG10来促进产品运输,以增强白屈菜红碱生物合成的代谢通量,导致白屈菜红碱产量增加近900倍。结合栽培过程,白屈菜红碱在0.5L生物反应器中以每升12.61mg的滴度获得,比第一代重组菌株高37,000倍以上。
结论:这是在酵母细胞工厂中产生白屈菜红碱的植物来源途径的第一个异源重建。应用组合工程策略已显着提高了酵母中白屈菜红碱的产量,并且是使用微生物细胞工厂合成功能产物的有希望的方法。这一成就强调了代谢工程和合成生物学在彻底改变天然产物生物合成方面的潜力。
BACKGROUND: Chelerythrine is an important alkaloid used in agriculture and medicine. However, its structural complexity and low abundance in nature hampers either bulk chemical synthesis or extraction from plants. Here, we reconstructed and optimized the complete biosynthesis pathway for chelerythrine from (S)-reticuline in Saccharomyces cerevisiae using genetic reprogramming.
RESULTS: The first-generation strain Z4 capable of producing chelerythrine was obtained via heterologous expression of seven plant-derived enzymes (McoBBE, TfSMT, AmTDC, EcTNMT, PsMSH, EcP6H, and PsCPR) in S. cerevisiae W303-1 A. When this strain was cultured in the synthetic complete (SC) medium supplemented with 100 µM of (S)-reticuline for 10 days, it produced up to 0.34 µg/L chelerythrine. Furthermore, efficient metabolic engineering was performed by integrating multiple-copy rate-limiting genes (TfSMT, AmTDC, EcTNMT, PsMSH, EcP6H, PsCPR, INO2, and AtATR1), tailoring the heme and NADPH engineering, and engineering product trafficking by heterologous expression of MtABCG10 to enhance the metabolic flux of chelerythrine biosynthesis, leading to a nearly 900-fold increase in chelerythrine production. Combined with the cultivation process, chelerythrine was obtained at a titer of 12.61 mg per liter in a 0.5 L bioreactor, which is over 37,000-fold higher than that of the first-generation recombinant strain.
CONCLUSIONS: This is the first heterologous reconstruction of the plant-derived pathway to produce chelerythrine in a yeast cell factory. Applying a combinatorial engineering strategy has significantly improved the chelerythrine yield in yeast and is a promising approach for synthesizing functional products using a microbial cell factory. This achievement underscores the potential of metabolic engineering and synthetic biology in revolutionizing natural product biosynthesis.