Laboratory of Medicinal Cell Biology, Kobe Pharmaceutical University.
Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University.
Biol Pharm Bull. 2023;46(10):1494-1497. doi: 10.1248/bpb.b23-00473.
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.
合成生物学的进步促进了有价值的植物代谢产物的微生物生产。然而，在单个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，已经开发出了涉及具有部分途径的宿主生物的模块化共培养系统。我们专注于大肠杆菌，一种用于代谢产物生产的通用宿主，以及毕赤酵母（Komagataella phaffii），由于其生物合成酶的高表达，它是一种新型的合成生物学宿主。之前，我们报道了大肠杆菌细胞与毕赤酵母细胞的共培养，大肠杆菌细胞从甘油中产生藜芦碱（各种生物碱的重要中间体），毕赤酵母细胞从藜芦碱中产生有价值的生物碱石蒜碱。然而，毕赤酵母细胞抑制了大肠杆菌的生长和藜芦碱的生产。因此，我们旨在改进这个共培养系统。我们研究了共培养前的预培养时间，以增强大肠杆菌的生长和藜芦碱的生产。此外，我们还研究了共培养时接种毕赤酵母细胞的最佳浓度和共培养过程中甲醇的添加，以实现毕赤酵母细胞中生物合成酶的连续表达。我们成功建立了一种改进的共培养系统，与以前的方法相比，生产力提高了 80 倍。这个增强的系统具有快速大规模生产各种有价值的植物代谢产物的巨大潜力。
