Abstract：
Ergothioneine (EGT) is a rare thiohistidine derivative with exceptional antioxidant properties. The blood level of EGT is considered highly reliable predictors for cardiovascular diseases and mortality, yet animals lack the ability to synthesize this compound. Free plasmids have been previously used to overexpress genes involved in the EGT biosynthetic pathway of Mycolicibacterium neoaurum. Here, we tentatively introduced a putative transporter gene mfsT1 into high-copy plasmids and sharply increased the ratio of extracellular EGT concentration from 18.7% to 44.9%. Subsequently, an additional copy of egtABCDE, hisG, and mfsT1 was inserted into the genome with a site-specific genomic integration tool of M. neoaurum, leading a 2.7 times increase in EGT production. Co-enhancing the S-adenosyl-L-methionine regeneration pathway, or alternatively, the integration of three copies of egtABCDE, hisG and mfsT1 into the genome further increased the total EGT yield by 16.1% (64.6 mg/L) and 21.7% (67.7 mg/L), respectively. After 168-h cultivation, the highest titer reached 85.9 mg/L in the latter strain with three inserted copies. This study provided a solid foundation for genome engineering to increase the production of EGT in M. neoaurum.
摘要：
麦角硫因（EGT）是一种罕见的硫代组氨酸衍生物，具有出色的抗氧化性能。血液EGT水平被认为是心血管疾病和死亡率的高度可靠的预测因子。然而动物缺乏合成这种化合物的能力。游离质粒先前已用于过表达与新耳分枝杆菌EGT生物合成途径有关的基因。这里,我们初步将一个推定的转运蛋白基因mfsT1导入高拷贝质粒,并将细胞外EGT浓度的比例从18.7%急剧增加至44.9%.随后，egtABCDE的额外副本，HISG,mfsT1被插入到基因组中的位点特异性基因组整合工具的新牛分枝杆菌，导致EGT产量增加2.7倍。共同增强S-腺苷-L-蛋氨酸再生途径，或者，整合了三个egtABCDE副本，hisG和mfsT1进入基因组进一步增加了16.1%(64.6mg/L)和21.7%(67.7mg/L)的总EGT产量，分别。经过168小时的培养，在具有三个插入拷贝的后一个菌株中，最高滴度达到85.9mg/L。这项研究为基因组工程提供了坚实的基础，以增加新耳分枝杆菌中EGT的产量。
