Department of Chemical and Biochemical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854, USA.
Center for Biotechnology and Interdisciplinary Sciences, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA.
Appl Microbiol Biotechnol. 2020 Jun;104(11):4849-4861. doi: 10.1007/s00253-020-10576-1. Epub 2020 Apr 13.
Flavonoids are a large family of plant and fungal natural products, among which many have been found to possess outstanding biological activities. Utilization of engineered microbes as surrogate hosts for heterologous biosynthesis of flavonoids has been investigated extensively. However, current microbial biosynthesis strategies mostly rely on using one microbial strain to accommodate the long and complicated flavonoid pathways, which presents a major challenge for production optimization. Here, we adapt the emerging modular co-culture engineering approach to rationally design, establish and optimize an Escherichia coli co-culture for de novo biosynthesis of flavonoid sakuranetin from simple carbon substrate glucose. Specifically, two E. coli strains were employed to accommodate the sakuranetin biosynthesis pathway. The upstream strain was engineered for pathway intermediate p-coumaric acid production, whereas the downstream strain converted p-coumaric acid to sakuranetin. Through step-wise optimization of the co-culture system, we were able to produce 29.7 mg/L sakuranetin from 5 g/L glucose within 48 h, which is significantly higher than the production by the conventional monoculture-based approach. The co-culture biosynthesis was successfully scaled up in a fed-batch bioreactor, resulting in the production of 79.0 mg/L sakuranetin. To our knowledge, this is the highest bioproduction concentration reported so far for de novo sakuranetin biosynthesis using the heterologous host E. coli. The findings of this work expand the applicability of modular co-culture engineering for addressing the challenges associated with heterologous biosynthesis of complex natural products. KEY POINTS: • De novo biosynthesis of sakuranetin was achieved using E. coli-E. coli co-cultures. • Sakuranetin production by co-cultures was significantly higher than the mono-culture controls. • The co-culture system was optimized by multiple metabolic engineering strategies. • The co-culture biosynthesis was scaled up in fed-batch bioreactor.
类黄酮是植物和真菌天然产物的一个大家族，其中许多已被发现具有出色的生物活性。利用工程微生物作为异源生物合成类黄酮的替代宿主进行了广泛的研究。然而，目前的微生物生物合成策略大多依赖于使用一种微生物菌株来容纳长而复杂的类黄酮途径，这对生产优化提出了重大挑战。在这里，我们采用新兴的模块化共培养工程方法，合理设计、建立和优化大肠杆菌共培养物，从头生物合成来自简单碳底物葡萄糖的类黄酮樱花素。具体来说，使用两种大肠杆菌菌株来容纳樱花素生物合成途径。上游菌株被工程化用于途径中间产物对香豆酸的生产，而下游菌株将对香豆酸转化为樱花素。通过逐步优化共培养系统，我们能够在 48 小时内从 5g/L 葡萄糖中生产 29.7mg/L 的樱花素，明显高于传统基于单培养的方法的产量。共培养生物合成在分批补料生物反应器中成功放大，生产出 79.0mg/L 的樱花素。据我们所知，这是迄今为止使用异源宿主大肠杆菌进行从头樱花素生物合成的最高生物生产浓度。这项工作的发现扩展了模块化共培养工程在解决复杂天然产物异源生物合成相关挑战方面的适用性。 要点： • 使用大肠杆菌-大肠杆菌共培养物实现了樱花素的从头生物合成。 • 共培养物的樱花素产量明显高于单培养对照。 • 通过多种代谢工程策略优化了共培养系统。 • 共培养生物合成在分批补料生物反应器中放大。
