Daoan Wang , Jiamin Chen , Yang Wang , Guocheng Du , Zhen Kang
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引用次数: 9
摘要
Ectoine是一种天然的大分子保护剂,由一些极端微生物合成。它提供对辐射介导的氧化损伤的保护,并被广泛用作制药和化妆品中的生物活性成分。为了满足其日益增长的商业需求,我们设计了大肠杆菌菌株,用于高产量生产外泌碱。通过质粒将来自天然外泌碱产生菌Halomonas elongata的ectABC基因簇引入不同的大肠杆菌(E.Coil)菌株中,并通过工程大肠杆菌BL21(DE3)在烧瓶培养中产生0.8g L-1的外泌碱。随后,我们设计了基因簇的核糖体结合位点,以微调基因ectA、ectB和ectC的表达,从而将外泌碱产量提高到1.6 g L-1。谷氨酸棒杆菌天冬氨酸激酶突变体(G1A,C932T)和H.伸长天冬氨酸半醛脱氢酶进一步组合过表达以增加前体的供应后,在烧瓶培养中,外泌碱的滴度达到5.5g L-1。最后,工程菌株在补料分批培养中以0.25g/g葡萄糖的转化率产生60.7g L-1胞外碱。
Engineering Escherichia coli for high-yield production of ectoine
Ectoine is a natural macromolecule protector and synthesized by some extremophiles. It provides protections against radiation-mediated oxidative damages and is widely used as a bioactive ingredient in pharmaceutics and cosmetics. To meet its growing commercial demands, we engineered Escherichia coli strains for the high-yield production of ectoine. The ectABC gene cluster from the native ectoine producer Halomonas elongata was introduced into different Escherichia coli (E. Coil) strains via plasmids and 0.8 g L-1 of ectoine was produced in flask cultures by engineered E. coli BL21 (DE3). Subsequently, we designed the ribosome-binding sites of the gene cluster to fine-tune the expressions of genes ectA, ectB, and ectC, which increased the ectoine yield to 1.6 g L-1. After further combinatorial overexpression of Corynebacterium glutamicum aspartate kinase mutant (G1A, C932T) and the H. elongate aspartate-semialdehyde dehydrogenase to increase the supply of the precursor, the titer of ectoine reached to 5.5 g L-1 in flask cultures. Finally, the engineered strain produced 60.7 g L-1 ectoine in fed-batch cultures with a conversion rate of 0.25 g/g glucose.