Abstract：
Advancements in synthetic biology have facilitated the microbial production of valuable plant metabolites. However, constructing complete biosynthetic pathways within a single host organism remains challenging. To solve this problem, modular co-culture systems involving host organisms with partial pathways have been developed. We focused on Escherichia coli, a general host for metabolite production, and Pichia pastoris (Komagataella phaffii), a novel synthetic biology host due to its high expression of biosynthetic enzymes. Previously, we reported the co-culture of E. coli cells, which produce reticuline (an important intermediate for various alkaloids) from glycerol, with P. pastoris cells, which produce the valuable alkaloid stylopine from reticuline. However, Pichia cells inhibited E. coli growth and reticuline production. Therefore, we aimed to improve this co-culture system. We investigated the pre-culture time before co-culture to enhance E. coli growth and reticuline production. Additionally, we examined the optimal concentration of Pichia cells inoculated for co-culture and methanol addition during co-culture for the continuous expression of biosynthetic enzymes in Pichia cells. We successfully established an improved co-culture system that exhibited an 80-fold increase in productivity compared to previous methods. This enhanced system holds great potential for the rapid and large-scale production of various valuable plant metabolites.
摘要：
合成生物学的进步促进了有价值的植物代谢物的微生物生产。然而,在单一宿主生物体内构建完整的生物合成途径仍然具有挑战性。为了解决这个问题,已经开发了涉及具有部分途径的宿主生物体的模块化共培养系统。我们专注于大肠杆菌，代谢物生产的一般宿主，和巴斯德毕赤酵母（Komagataellaphafii），一种新型的合成生物学宿主，由于其高度表达的生物合成酶。以前,我们报道了大肠杆菌细胞的共培养，它们从甘油中产生网状蛋白（各种生物碱的重要中间体），带有巴斯德毕赤酵母细胞，从网织蛋白中产生有价值的生物碱苯乙烯。然而,毕赤酵母细胞抑制大肠杆菌生长和网织蛋白产生。因此,我们旨在改善这种共同文化体系。我们研究了共培养前的预培养时间，以提高大肠杆菌的生长和网状细胞的生产。此外,我们检查了在毕赤酵母细胞中连续表达生物合成酶的共培养过程中接种的毕赤酵母细胞和添加甲醇的最佳浓度。我们成功地建立了一种改进的共培养系统，与以前的方法相比，其生产率提高了80倍。这种增强的系统具有快速和大规模生产各种有价值的植物代谢物的巨大潜力。
