AVT - Biochemical Engineering, RWTH Aachen University, Aachen, Germany.
Institute of Applied Microbiology - iAMB, Aachen Biology and Biotechnology - ABBt, RWTH Aachen University, Aachen, Germany.
Biotechnol Prog. 2023 Jan;39(1):e3293. doi: 10.1002/btpr.3293. Epub 2022 Sep 8.
Poly-γ-glutamic acid (γ-PGA) is a biopolymer with a wide range of applications, mainly produced using Bacillus strains. The formation and concomitant secretion of γ-PGA increases the culture broth viscosity, while enzymatic depolymerisation and degradation of γ-PGA decreases the culture broth viscosity. In this study, the recently published ViMOS (Viscosity Monitoring Online System) is applied for optical online measurements of broth viscosity in eight parallel shake flasks. It is shown that the ViMOS is suitable to monitor γ-PGA production and degradation online in shake flasks. This online monitoring enables the detailed analysis of the P promoter and γ-PGA depolymerase knockout mutants in genetically modified Bacillus subtilis 168. The P promoter becomes active under phosphate starvation. The different single depolymerase knockout mutants are ∆ggt, ∆pgdS, ∆cwlO and a triple knockout mutant. An increase in γ-PGA yield in g /g of 190% could be achieved with the triple knockout mutant compared to the P reference strain. The single cwlO knockout also increased γ-PGA production, while the other single knockouts of ggt and pgdS showed no impact. Partial depolymerisation of γ-PGA occurred despite the triple knockout. The online measured data are confirmed with offline measurements. The online viscosity system directly reflects γ-PGA synthesis, γ-PGA depolymerisation, and changes in the molecular weight. Thus, the ViMOS has great potential to rapidly gain detailed and reliable information about new strains and cultivation conditions. The broadened knowledge will facilitate the further optimization of γ-PGA production.
聚-γ-谷氨酸（γ-PGA）是一种具有广泛应用的生物聚合物，主要由芽孢杆菌菌株生产。γ-PGA 的形成和伴随的分泌会增加发酵液的粘度，而 γ-PGA 的酶解和降解会降低发酵液的粘度。在本研究中，最近发表的 ViMOS（粘度在线监测系统）被应用于八个平行摇瓶中的发酵液的光学在线测量。结果表明，ViMOS 适合在线监测摇瓶中 γ-PGA 的生产和降解。这种在线监测使我们能够详细分析经过基因改造的枯草芽孢杆菌 168 中 P 启动子和 γ-PGA 解聚酶缺失突变体。P 启动子在磷酸盐饥饿时变得活跃。不同的单解聚酶缺失突变体是 ∆ggt、∆pgdS、∆cwlO 和三重缺失突变体。与 P 参考菌株相比，三重缺失突变体可使 γ-PGA 的产率提高 190%。cwlO 缺失突变体也增加了 γ-PGA 的产量，而 ggt 和 pgdS 的其他单缺失突变体则没有影响。尽管发生了部分解聚，但仍有部分 γ-PGA 发生了降解。在线测量数据与离线测量数据相吻合。在线粘度系统直接反映了 γ-PGA 的合成、γ-PGA 的解聚以及分子量的变化。因此，ViMOS 具有快速获取有关新菌株和培养条件的详细可靠信息的巨大潜力。更广泛的知识将有助于进一步优化 γ-PGA 的生产。
