柚皮素是一种植物多酚，因其有趣的生物活性（即抗癌、抗氧化和抗炎）而被广泛研究。由于其潜在的应用和克服工业需求的尝试，人们对其异源生产的兴趣日益浓厚。产生柚皮素的微生物生物合成途径由酪氨酸解氨酶（TAL）、4-香豆酸辅酶A连接酶（4CL）、查耳酮合酶（CHS）和查耳酮异构酶（CHI）组成。在此，我们的目标是通过对途径进行逐步验证和优化，在大肠杆菌中高效地从头生产柚皮素。为此，我们首先在三种不同的大肠杆菌菌株中表达来自不同来源的两种 TAL 基因。最高的对香豆酸产量 (2.54 g/L) 是在来自约氏黄杆菌 (FjTAL) 的携带 TAL 的酪氨酸过量生产 M-PAR-121 菌株中获得的。随后，该平台菌株被用来表达不同来源的4CL和CHS基因的不同组合。通过表达 FjTAL 与拟南芥 (At4CL) 的 4CL 和西葫芦 (CmCHS) 的 CHS (CmCHS) 组合，可实现最高的柚皮素查尔酮产量 (560.2 mg/L)。最后，对不同的 CHI 进行了测试和验证，并通过表达紫花苜蓿 (MsCHI) 的 CHI 与其他先前选择的基因相结合，产生了 765.9 mg/L 的柚皮素。据我们所知，该滴度相当于迄今为止在大肠杆菌中报道的最高的柚皮素从头生产量。要点： • 选择最佳酶和菌株组合用于从头生产柚皮素。 • 经过遗传和操作优化后，产生了 765.9 mg/L 的柚皮素。 • 这种从头生产是迄今为止在大肠杆菌中报道的最高水平。© 2024。作者。

Naringenin is a plant polyphenol, widely explored due to its interesting biological activities, namely anticancer, antioxidant, and anti-inflammatory. Due to its potential applications and attempt to overcome the industrial demand, there has been an increased interest in its heterologous production. The microbial biosynthetic pathway to produce naringenin is composed of tyrosine ammonia-lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). Herein, we targeted the efficient de novo production of naringenin in Escherichia coli by performing a step-by-step validation and optimization of the pathway. For that purpose, we first started by expressing two TAL genes from different sources in three different E. coli strains. The highest p-coumaric acid production (2.54 g/L) was obtained in the tyrosine-overproducing M-PAR-121 strain carrying TAL from Flavobacterium johnsoniae (FjTAL). Afterwards, this platform strain was used to express different combinations of 4CL and CHS genes from different sources. The highest naringenin chalcone production (560.2 mg/L) was achieved by expressing FjTAL combined with 4CL from Arabidopsis thaliana (At4CL) and CHS from Cucurbita maxima (CmCHS). Finally, different CHIs were tested and validated, and 765.9 mg/L of naringenin was produced by expressing CHI from Medicago sativa (MsCHI) combined with the other previously chosen genes. To our knowledge, this titer corresponds to the highest de novo production of naringenin reported so far in E. coli. KEY POINTS: • Best enzyme and strain combination were selected for de novo naringenin production. • After genetic and operational optimizations, 765.9 mg/L of naringenin was produced. • This de novo production is the highest reported so far in E. coli.© 2024. The Author(s).