Step change in the efficiency of centrifugation through cell engineering: co-expression of Staphylococcal nuclease to reduce the viscosity of the bioprocess feedstock

• Published in: Biotechnology and bioengineering Vol. 104; no. 1; pp. 134 - 142
• Main Authors: Balasundaram, B, Nesbeth, D, Ward, J.M, Keshavarz-Moore, E, Bracewell, D.G
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01-09-2009; Wiley; Wiley Subscription Services, Inc
• Subjects: Biological and medical sciences; Biotechnology; cell disruption; cell engineering; Cells; Centrifugation - methods; clarification; Deoxyribonucleic acid; DNA; E coli; Escherichia coli; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli Proteins - isolation & purification; Fundamental and applied biological sciences. Psychology; Micrococcal Nuclease - genetics; Micrococcal Nuclease - metabolism; nuclease; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Staphylococcus aureus; Staphylococcus infections; Viscosity; Viscosity; Enzyme; Coexpression; Esterases; cell engineering; clarification; Staphylococcus; cell disruption; Efficiency; Bacteria; Hydrolases; Micrococcales; Micrococcaceae; Nuclease; Operating process
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.22369

Cell engineering to enable step change improvements in bioprocessing can be directed at targets other than increasing product titer. The physical properties of the process suspension such as viscosity, for example, have a major impact on various downstream processing unit operations. The release of chromosomal DNA during homogenization of Escherichia coli and its influence on viscosity is well-recognized. In this current article we demonstrate co-expression of Staphylococcus aureus nuclease in E. coli to reduce viscosity through auto-hydrolysis of nucleic acids. Viscosity reduction of up to 75% was achieved while the particle size distribution of cell debris was maintained approximately constant (d₅₀ = 0.5-0.6 μm). Critically, resultant step change improvements to the clarification performance under disc-stack centrifugation conditions are shown. The cell-engineered nuclease matched or exceeded the viscosity reduction performance seen with the addition of exogenous nuclease removing the expense and validation issues associated with such additions to a bioprocess. The resultant material dramatically altered performance in scale-down mimics of continuous disc-stack centrifugation. Laboratory scale data indicated that a fourfold reduction in the settling area of a disc-stack centrifuge can be expected due to a less viscous process stream achieved through nuclease co-expression with a recombinant protein. Biotechnol. Bioeng. 2009; 104: 134-142