ACIB-Austrian Centre of Industrial Biotechnology, Krenngasse 37, 8010, Graz, Austria.
Institute of Biotechnology and Biochemical Engineering, TU Graz, NAWI Graz, Petersgasse 12, 8010, Graz, Austria.
Biotechnol J. 2020 Nov;15(11):e2000063. doi: 10.1002/biot.202000063. Epub 2020 Aug 7.
Catalyst development for biochemical cascade reactions often follows a "whole-cell-approach" in which a single microbial cell is made to express all required enzyme activities. Although attractive in principle, the approach can encounter limitations when efficient overall flux necessitates precise balancing between activities. This study shows an effective integration of major design strategies from synthetic biology to a coherent development of plasmid vectors, enabling tunable two-enzyme co-expression in E. coli, for whole-cell-production of cellobiose. An efficient transformation of sucrose and glucose into cellobiose by a parallel (countercurrent) cascade of disaccharide phosphorylases requires the enzyme co-expression to cope with large differences in specific activity of cellobiose phosphorylase (14 U mg ) and sucrose phosphorylase (122 U mg ). Mono- and bicistronic co-expression strategies controlling transcription, transcription-translation coupling or plasmid replication are analyzed for effect on activity and stable producibility of the whole-cell-catalyst. A key role of bom (basis of mobility) for plasmid stability dependent on the ori is reported and the importance of RBS (ribosome binding site) strength is demonstrated. Whole cell catalysts show high specific rates (460 µmol cellobiose min g dry cells) and performance metrics (30 g L ; ∼82% yield; 3.8 g L h overall productivity) promising for cellobiose production.
生化级联反应的催化剂开发通常遵循“全细胞方法”，即通过单个微生物细胞表达所有必需的酶活性。尽管从原理上讲很有吸引力，但当需要精确平衡活性以实现有效的整体通量时，该方法可能会遇到限制。本研究展示了从合成生物学中有效整合主要设计策略，以协调质粒载体的开发，从而能够在大肠杆菌中进行可调节的双酶共表达，用于细胞二糖的全细胞生产。通过蔗糖和葡萄糖的平行（逆流）双酶磷酸解级联有效地转化为纤维二糖，需要酶共表达来应对纤维二糖磷酸酶（14 U mg ）和蔗糖磷酸酶（122 U mg ）的比活性存在巨大差异。分析了控制转录、转录翻译偶联或质粒复制的单顺反子和双顺反子共表达策略对全细胞催化剂的活性和稳定生产能力的影响。报道了依赖 ori 的质粒稳定性的 bom（可移动性基础）的关键作用，并证明了 RBS（核糖体结合位点）强度的重要性。全细胞催化剂表现出高比速率（460 µmol 细胞二糖 min g 干细胞）和性能指标（30 g L ；约 82%产率；3.8 g L h 总生产力），有望用于生产细胞二糖。
