柚皮素是一种植物多酚,由于其有趣的生物活性而被广泛探索,即抗癌,抗氧化剂,和抗炎。由于其潜在的应用和克服工业需求的尝试,对其异源生产的兴趣增加。产生柚皮素的微生物生物合成途径由酪氨酸氨裂解酶(TAL)组成,4-香豆酸辅酶A连接酶(4CL),查耳酮合成酶(CHS),和查尔酮异构酶(CHI)。在这里,我们通过对途径进行逐步验证和优化,以在大肠杆菌中从头高效生产柚皮素为目标.为此,我们首先通过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基因开始。在酪氨酸过量生产的M-PAR-121菌株中获得了最高的对香豆酸产量(2.54g/L),该菌株携带来自约翰逊黄杆菌(FjTAL)的TAL。之后,该平台菌株用于表达来自不同来源的4CL和CHS基因的不同组合。通过表达FjTAL与拟南芥4CL(At4CL)和南瓜CHS(CmCHS)的结合,可以获得最高的柚皮素查尔酮产量(560.2mg/L)。最后,对不同的CHIs进行了测试和验证,通过表达紫花苜蓿(MsCHI)的CHI与其他先前选择的基因结合,产生765.9mg/L的柚皮素。据我们所知,该滴度对应于迄今在大肠杆菌中报道的柚皮素的最高从头生产。关键点:•选择最佳酶和菌株组合用于从头生产柚皮素。•在遗传和运营优化之后,产生765.9mg/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的。
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.