Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Institute of Biochemical Engineering, University of Stuttgart, Stuttgart, Germany.
Microb Biotechnol. 2024 Nov;17(11):e70034. doi: 10.1111/1751-7915.70034.
Microorganisms in large-scale bioreactors are exposed to heterogeneous environmental conditions due to physical mixing constraints. Nutritional gradients can lead to transient expression of energetically wasteful stress responses and as a result, can reduce the titres, rates and yields of a bioprocess at larger scales. To what extent these process parameters are impacted is often unknown and therefore bioprocess scale-up comes with major risk. Designing platform strains to account for these intermittent stresses before introducing synthesis pathways is one strategy for de-risking bioprocess development. For example, Escherichia coli strain RM214 is a derivative of wild-type MG1655 that has had several genes and whole operons removed from its genome based on their metabolic cost. In this study, we engineered E. coli strain RM214 (referred to as WG02) to produce octanoic acid from glycerol in batch-flask and fed-batch bioreactor cultivations and compared it to an octanoic acid-producing E. coli MG1655 (WG01). In batch flask cultivations, the two strains performed similarly. However, in carbon limited fed-batch bioreactor cultivations, WG02 provided a greater than 22% boost to biomass compared to WG01 while maintaining similar titres of octanoic acid. Reducing the biomass accumulation of WG02 with nitrogen limited fed-batch cultivation resulted in a 16% improvement in octanoic acid titre over WG01. Finally, in a scale-down system consisting of a stirred tank reactor (representing a well-mixed zone) and plug flow reactor (representing an intermittent carbon starvation zone), WG02 again improved octanoic acid titre by almost 18% while maintaining similar biomass concentrations as WG01.
由于物理混合的限制，大规模生物反应器中的微生物会暴露于不均匀的环境条件下。营养梯度会导致能量浪费的应激反应短暂表达，从而导致生物过程在更大规模下的产率、速率和得率降低。这些过程参数受到多大程度的影响往往是未知的，因此生物过程的放大存在很大的风险。在引入合成途径之前，设计平台菌株来应对这些间歇性应激是降低生物过程开发风险的一种策略。例如，大肠杆菌 RM214 菌株是野生型 MG1655 的衍生物，其基因组中已经去除了几个基因和整个操纵子，原因是它们的代谢成本。在这项研究中，我们对大肠杆菌 RM214 菌株（称为 WG02）进行了工程改造，使其能够在分批摇瓶和补料分批生物反应器培养中从甘油生产辛酸，然后将其与产生辛酸的大肠杆菌 MG1655（WG01）进行了比较。在分批摇瓶培养中，这两个菌株的表现相似。然而，在碳限制补料分批生物反应器培养中，与 WG01 相比，WG02 的生物量提高了 22%以上，同时保持了类似的辛酸产量。通过氮限制补料分批培养减少 WG02 的生物量积累，使辛酸产量比 WG01 提高了 16%。最后，在由搅拌釜式反应器（代表充分混合区）和推流反应器（代表间歇性碳饥饿区）组成的缩小系统中，WG02 再次将辛酸产量提高了近 18%，同时保持了与 WG01 相似的生物量浓度。
